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Juniper Publishers- Open Access Journal of Environmental Sciences & Natural Resources

A Comprehensive Phytopharmacological Review of Dioscorea bulbifera Linn

Authored by Galani Varsha J

Abstract

Dioscorea bulbifera Linn. is an important medicinal plant, which has long been used in traditional Indian and Chinese medicine for various diseases. This plant is pharmacologically studied for antitumor, anti HIV, antidyslipidemic, analgesic, anti-inflammatory, diuretic, gastroprotective, antioxidant, antimicrobial, antiviral, antifungal, anthelmintic, neuropharmacological, cardioprotective, anorexiant, plasmid curing activities and anti-hyperthyroid activities. A wide range of phytochemical constituents have been isolated from this plant. A comprehensive account of the morphology, microscopical characters, phytochemical constituents and pharmacological activities reported are included in view of the many recent findings of importance on this plant.

Keywords: Dioscorea bulbifera; Neuropharmacological activity; Antitumor activity, Diosgenin

Abbreviations: CFA: Complete Freunds Adjuvant; LPS: Lipo Poly Saccharides; PLSN: Partial Ligation Sciatic NervE; NOS: Nitric Oxide Synthase; VRE: Vancomycin-Resistant Enterococci; LVDP: Left Ventricular Developed Pressure

Introduction

Ayurveda is a system of traditional medicine native to India and a form of alternative medicine. Recently traditional medicine worldwide is being re-evaluated by extensive research works on different plant species and their active therapeutic principles. The untapped wealth of plant kingdom can represent a novel source of newer compounds with significant therapeutic activities [1]. One such plant, Dioscorea bulbifera Linn, which have various medicinal properties, is widely used in Ayurveda, the ancient traditional medicinal system in India. Dioscorea bulbifera is an aerial yam commonly known as Varahikanda. In this review a comprehensive account of the morphology, microscopical characters, phytochemical constituents and pharmacological activities are included in view of the many recent findings of importance on this plant.

Taxonomic Classification

Kingdom: Plantae

Subkingdom: Viridaeplantae

Superdivision: Streptophyta

Division: Tracheophyta

Class: Magnoliopsida

Superorder: Lilinanae

Order: Dioscoreales

Family: Dioscoreaceae

Genus: Discorea L.

Species: Dioscorea bulbifera L.

Vernacular Names

English: PotatoYam, Air potato

Sanskrit: Varahikanda, Aluka, Shukara

Hindi: Varahi kanda, Kadu Kanda, Ratalu

Gujarati: Dukkarkanda

Bengali: Ratalu, Ban Alu

Tamil: Kodikilanga, Kaattu-k-kaay-valli

Marathi: Manakund, Kadu-karanda, Varahi

Kannada: Kuntagenasu

Konkani : Karamdo

Malayalam: Pannikizhangu, Kattukachil

Oriya: Pita Alu

Telugu: Adavi Dumpa

Synonyms

D. crispate Roxb, D. pulchella Roxb, D. sativa sensu Hook.f [3,4].

Ayurvedic Preparations

Ajamamsa Rasayanam, Narasimha Churna, Pancha Nimbadi Churna, Vastyamayantaka Ghrita, Varahytadighurdam [3,4].

Substitute

Varahikanda is used as substitute for Riddhi and Vriddhi drugs of Astavarya in Ayurveda [3,4].

Occurrence and Distribution

The species is native to the tropics of the old world and occurs in rain forests extending from the west coast of Africa to the farthest island in the specific. It is common throughout India ascending up to 6,000 ft. in the Himalayas. It does not thrive in the dried part of the India. The wild form also occurs in South East Asia, West Africa, South, and Central America, Australia, Louisiana, Texas, Hawaii, Puerto Rico, Polynesia, and Florida [3, 5,6].

Flowers

September-November

Fruit

December onwards

Parts Used

Tubers, Bulbils, Roots,

Morphology

Dioscorea bulbifera is a vigorously twining, long-stemmed perennial vine with non-spiny stems to 20 m or more in length, freely branching above; internodes round or slightly angled in cross section and they twine counter-clockwise. Plant has two types of storage organs. The plant forms bulbils in the leaf axils of the twining stems, and tubers beneath the ground. Tubers are like small, oblong potatoes with bitter taste. Conspicuous aerial tubers (called bulbils) are pale, round to globose in shape, up to 13 cm wide and in inflorescence that give D. bulbifera the common name "air potato." The leaves are attractive, alternate, broadly heart-shaped, attached by long petioles. Leaves 10-15 x 7.5-10 cm, ovate-suborbicular, base deeply cordate, apex acuminate to shortly caudate, membranous, glabrous, basally 9-11-ribbed; petiole to 20 cm long. They are divided longitudinally into lobes by prominent arching veins all radiating out from a single point of origin where the petiole attaches to the leaf. Flowers rarely occur in D. bulbifera; where occurring, they are small, pale green and fragrant, and arising from leaf axils. Male flowers in slender, axillary panicled spikes, pendulous, to 18 cm long; bracteoles ovate, acute [5,6].

Perianth light green; lobes 6, biseriate, 2.5 mm long, linearoblong. Stamens 6 free. Female spikes 1-3 together; staminodes 3; ovary triquetrous, 3-locular, ovules 2-per locule; styles 3;stigma 2-fid, reflexed. Capsules 1.5-2.3 x 1-1.5 cm, oblong, 3-winged. The fruit is a capsule and the seeds partially winged. This species reproduces sexually by seeds and vegetatively by underground and aerial tubers (bulbils) which enable it to spread rapidly and colonize entire forests in a single growing season. The aerial stems of air potato die back in winter season, but resprouting occurs from bulbils and underground tubers.

Morphology of Bulbils and Tumors

The bulbil is fairly hard and heavy. Dish shaped with to 12 cm (5") x 10 cm (4") brown with prominent numerous, uniformly distributed tubercle like eyes. Bulbils abundant and of different sizes and shapes; in certain cultigens the tuber is suppressed in favour of rather large bulbils, having all the reserve food; small bulbils are, as a rule warted, but they may be smooth when large. Tubers are usually small and round, but large under cultivation. They are weighing up to 1 kg. Tubers are toxic or edible according to the variety; they are renewed annually. Their skin is purplish black or earth colored, usually coated with abundant small feeding roots, but smooth in some cultivated varieties having flesh of white to lemon yellow, sometimes marked with purple flecks and very mucilaginous (Figure 1) A few root and root scars present in tubers, outer surface dark brown, inner yellow to light brown; odour- indistinct; taste-bitter [5,6].

Microscopic Characteristics

Subasini, 2013 reported microscopic features of tubers and bulbil of D. bulbifera. The T.S of tubers showed wide, well developed periderm, vascular bundles and triangular starch grains. Major microscopic characters of bulb include periderm, ground tissue, vascular bundle, and triangular starch grains. Ground tissue, forming major portion of bulb composed of oval to polygonal cells having a few scattered closed vascular bundles. Starch grains found both in cortex and ground tissues, but abundant in ground tissue, rounded to oval, three sided with rounded angles or rod-shaped, simple, solitary or in groups, 1128 n in diameter; hilum present at the narrower extremity [7].

Phytochemical Constituents

Phytochemical analysis of Dioscorea bulbifera revealed presence of alkaloids, glycosides, proteins, fats, sterols, alkaloids, polyphenols and tannins, flavonoids and saponins which may vary according to country origin [7]. Inorganic micronutrients present include Fe, Cu, Zn, Mn, Co, Mo, V, B, Cl, I, Br and Na [7].

a) Steroidal Saponins [8]: Dioscoreanoside A-K, Dioscoreanoside B, Dioscoreanoside C, Dioscoreanoside D, Dioscoreanoside E, Dioscoreanoside F, Dioscoreanoside G, Dioscoreanoside H, Dioscoreanoside I, Dioscoreanoside J, Dioscoreanoside K, Dioscin

b) Steroidal Sapogenin, Spirostane Glycosides, Cholestane Glycosides [9-11]: Diosbulbisin A, Diosbulbisin B, Diosbulbisin C, Diosbulbisin D, Diosbulbisides A, Diosbulbisides B, Diosbulbisides C, Diosgenin, Sinodiosgenin

c) PNorclerodane Diterpenoids [12-16]: Diosbulbin A, Diosbulbin B, Diosbulbin C, Diosbulbin D, Diosbulbin E, Diosbulbin F, Diosbulbin G, Diosbulbin H, Diosbulbin I, Diosbulbin J, Diosbulbin K, Diosbulbin L, Diosbulbin M, Diosbulbin N, Diosbulbin O, Diosbulbin P, 8-Epidiosbulbin E Acetate

d) Clerodane Diterpenoids [17-19]: Bafoudiosbulbin A, Bafoudiosbulbin B, Bafoudiosbulbin C, Bafoudiosbulbin F, Bafoudiosbulbin G.

e) Flavanoids Derivatives [20,21]: Quercetin-3-O-β-D- glucopyranoside, Quercetin-3-O-β-D-galactopyranoside, Myricetin-3-O- β-D-galactopyranoside, Myricetin-3-O- β-D glucopyranoside, 3,5-dimethoxy-kaempferol, 3, 5, 3'-trimethoxyquercetin, Caryatin, Hyperoside, Kaempferol, Kaempferol-3-O-β-D-galactopyranoside, Kaempferol- 3-O-β-D-glucopyranoside, Kaempferol-3,5-dimethyl ether, Quercetin-3-O-galactopyranoside, Myricetin, Isoquercitrin, Lutein, Quercetin-3-O-β-D-glucopyranoside, 7- bis-(4-hydroxyphenyl) -4E, 6E- heptadien-3-one, 5,3,4-trihydroxy-3,7- dimethoxyflavone.

f) Phenanthrenes 4-methoxyphenanthrene, trimethoxyphenanthrene, trimethoxyphenanthrene [20-22]: 2,7-dihydroxy- 2,7-dihydroxy-3,4,6- 1,6-dihydroxy-2,5,7-

g) Carotenoids [3]: Neoxanthin, Auroxanthin, Violaxanthin, Cryptoxanthine

h) Phytosterols [14,18,20]: Daucosterol, β-sitosterol, 3-O-β-D-glucopyranosyl-b-sitosterol, Stigmasterol

i) fatty acids [11,18,22]: Palmatic acid, Succinic acid, Shikimic acid, Tetracosanoic acid, 1-(tetracosanoyl)- glycerol, Trans-tetracosanylferulate, Mono-arachidin, C22 «-hydroxy fatty acid, 3-hydroxy-5-methoxybenzoic acid, Batatasin III, Behenic acid, Ethyl ester of undecanoic acid, Z-1,9-dodecadiene (C H), n-Hexadecanoic acid, Ethyl ester of Eicosanoic acid

j) Tannins [20,22]: Catechin, Protocatechuic acid, (+) Epicatechin, (-)Epicatechin

k) Volatile oils [20,23] : Vanillic acid, Isovanillic acid

l)Glycoside Derivatives [9,12,15, 24]: Alkaloid [25]: Dihydrodioscorine a) Methyl-O-α-D- fructofuranoside, Butyl-O-α-D-fructofuranoside, Ethyl- O-β-D-fructopyranoside, Butyl-O-β-D-fructopyranoside, 3-phenyl-2-propenyl-O-β-D-glucopyranoside, 2- (4-methoxyphenyl)-ethyl-O-β-D-glucopyranoside, Phenyl -methyl-O-β-D-glucopyranoside. Pennogenin, Pennogenin-3-O-α-Lrhamnopyranosyl-(1->3)-[α- L-rhamnopyranosyl-(1->2)]- β-D- glucopyranoside, Pennogenin-3-O-α-Lrhamnopyranosyl-(1->4)-[a- L-rhamnopyranosyl-(1->2)]- β-D- glucopyranoside, 3- O-α-L-rhamnopyranosyl-(1->2)-[α-L-rhamnopyranosyl- (1->3)]-β-D-g1ucopyranosyl pennogenin (spiroconazol A), 26-O-β-D-glucopyranosyl-(25R)- 5-en-furost- 3β,17α,22α,26-tetraol- 3-O-α-L-rhamnopyranosyl- (1->4)-α-L-rhamnopyranosyl- (1->4)-[α-L- rhamnopyranosyl- (1->2)]-β-D-glucopyranoside, 23β,27-dihydroxy-pennogenin 3- O-α-L- hamnopyranosyl-(1->4)- α-L-rhamnopyranosyl-(1->4)-[α- L-mnopyranosyl-(1->2)]-β-Dglucopyranoside, 4-hydroxy- [2-trans-3',7'- dimethylocta-2',6'-dienyl]-6-methoxy acetophenone, 4,6-dihydroxy-2-O-(4'- hydroxybutyl) acetophenone, Diosbulbinoside D, Diosbulbinoside F, Diosbulbinoside G

m) Others [15,26]: Demethyl batatasin IV, Diarylheptanone, 3,5,4'-trihydroxy-3'- methoxybibenzy, 1,7- bis-(4-hydroxyphenyl)- 1E,4E,6E-heptatrien-3-one, 2,3'-di-hydroxy-4',5'- dimethoxybibenzy, Docosyl ferulate, Tristin, Adenosine.

Reported Pharmacological Activities Antitumour Activity

Reported Pharmacological Activities

Antitumour Activity

It has been reported that the extraction of components from D. bulbifera using organic solvents of little polarity significantly inhibited the growth of tumor and prolonged the survival of tumor-bearing mice and human liver cancer, colon cancer and other tumor cells [27-31]. D. bulbifera decoction could inhibit cell growth in the human squamous cell carcinoma cell line SiHa, in the human cervical cancer cells Hela, and in the human hepatoma cells HepG2, in a dosage and time-dependent manner [32] . Komori found that diosbulbins A and B had remarkable growth inhibition effect on solid sarcoma180 tumor cells [12]. Another study reported the anti-tumour-promoting effect of 75 % ethanol extracts of the rhizomes of Dioscorea bulbifera L. using the neoplastic transformation assay of mouse epidermal JB6 cell lines.

The ethyl acetate and n-butanol soluble fractions showed different inhibitory activities against tumour promotion of JB6 (Cl 22 and Cl 41) cells induced by the promoter of 12-O-tetradecanoylphorbol-13-acetate (TPA) [20]. Kaempferol- 3,5-dimethyl ether, caryatin, (1)-catechin, myricetin, quercetin- 3-O- galactopyranoside, myricetin-3-O-galactopyranoside, myricetin-3-O-glucopyranoside and diosbulbin B isolated from ethyl acetate soluble fraction of 75 % ethanol extract of the rhizomes of Dioscorea bulbifera L. from China showed an antitumor-promoting effect against the neoplastic transformation assay of mouse epidermal JB6 cell lines [21]. Petroleum ether fraction from chinese Dioscrea bulbifera showed potential effects against microstructure abnormality of HepA cells surface[33] . Two new steroidal saponins, diosbulbisides D (1) and E (2), along with five known saponins (3- 7) were isolated from the rhizomes of Dioscorea bulbifera L. Compounds 6 and 7, two 3- O-trisaccharides of diosgenin spirostanes, showed moderate cytotoxic activity against human hepatocellular carcinoma cells, with IC50 values of 3.89 μM and 7.47 μM on SMMC7721, and 10.87 μM and 19.10 μM on Bel-7402 cell lines, respectively [34].

The results of antitumour activity of water extract (fraction A), ethanol extract (fraction B), ethyl acetate extract (fraction C), non-ethyl acetate extract (fraction D) and isolated diosbulbin B showed that fractions B and C both decreased tumour weight in S180 and H22 tumour cells bearing mice, while fractions A and D had no such effect. Furthermore, fraction C altered the weight of spleen and thymus, and the amounts of total leukocytes, lymphocytes and neutrophils in tumour-bearing mice. Diosbulbin B was found to be the major antitumor bioactive component in the extracts and demonstrated anti-tumor effects in the dose-dependent manner at the dosage of 2 to 16 mg/kg without significant toxicity in vivo [35].

Immune system modulation might be related to antitumor effects of D. bulbifera rhizome, as reported in S180 and H22 tumor cells bearing mice [35]. Alcoholic extracts (70%, 80% and 90% alcohol) of D. bulbifera were found to in hibited the proliferation of human gastric cancer cell line SGC-7901 [36]. Zhao 2012 proved that D. bulbifera could significantly decrease the expression of SW579 Survivin mRNA and protein in human thyroid cancer cells and also induce apoptosis of cancer cells [37,38]. Pennogenin- 3-O-a-L-rhamnopyranosyl-(1 ->3)-[a-L-rhamnopyranosyl-(1-> 2)]-b-D-glucopyranoside and Pennogenin-3-O-a-L-rhamnopyranosyl-(1 -> 4)-[a- L-rhamnopyranosyl-(1->2)]-b-D-glucopyranoside from D. bulbifera extract showed significant cytotoxic activity against the proliferation of Bel-7402 human hepatocellular carcinoma cells, with 99.1 and 92.6% inhibition, respectively.

Both compounds showed cell growth inhibition activity toward SMMC7721 human hepatocellular carcinoma cells of 4.54 and 4.85 lM respectively [9]. Spiroconazol A and Pennogenin-3- O-a-L-rhamnopyranosyl-(1-> 4)-a- L-rhamnopyranosyl-(1->4)-[a-L-rhamnopyranosyl- (1->2)]-b-D-glucopyranoside showed moderate cytotoxicity against ECV304urinary bladder carcinoma cells, by membrane toxicity via lactic dehydrogenase (LDH) liberation with IC50 values of 8.5 lg/ml (8.3 lM) and 5.8 lg/ml (6.6 lM), respectively. 26-O-b-D-Glucopyranosyl-(25R)-5-en-furost- 3b,17a,22a,26-tetraol-3-Oa-L-rhamnopyranosyl-(1->4)-a-L- rhamnopyranosyl- (1->4)-[a-L-rhamnopyranosyl-(1 -> 2)]-b-D- glucopyranoside showed moderate activity as well, by a direct influence on the mitochondrial metabolism without liberation of LDH with an IC50 value of 14.3 lg/ml [38]. Combination of D. bulbifera polysaccharides and Cyclophosphamide could potentially enhance the anti-tumor effect of Cyclophosphamide and attenuate Cyclophosphamide induced immunosuppression as well as oxidative stress in U14 cervical tumor-bearing mice [39]. Platinum-palladium bimetallic nanoparticles of Dioscorea bulbifera tuber extract showed anticancer activity against HeLa cells [40].

Anti HIV Activity

Anti-HIV-1 integrase activity was evaluated for 7 compounds isolated from ethyl acetate and water fractions of Dioscorea bulbifera bulbils. Flavanoid Myricetin exhibited the most potent anti-HIV-1 integrase activity (IC50 value of 3.15 mM) followed by 2,4,6,7-tetrahydroxy- 9,10-dihydrophenanthrene (IC50 value%14.20 mM), quercetin-3-O-b-D-glucopyranoside (IC50 value%19.39 mM) and quercetin-3-O-b-D-galactopyranoside (IC50 value%21.80 mM). Potential interactions of the active compounds with the IN active site were additionally investigated. These compounds interacted with Asp64, Thr66, His67, Glu92, Asp116, Gln148, Glu152, Asn155, and Lys159, which are involved in both the 3'-processing and strand transfer reactions of integrase enzyme [41,42].

Antidiabetic Activity

Aqueous extract of D. bulbifera tubers at 250, 500 and 1000 mg/kg doses administered for 3 weeks to glucose primed and streptozotocin induced diabetes in wistar rats treated rats showed significant antihyperglycemic activity [42]. Ethanolic extract of Dioscorea bulbifera (aerial yam) was studied against alloxan-induced diabetic rats. Intraperitoneal administration of a single dose of 380.0, 760.0 and 1140.0 mg/kg body weight of the extract were exhibited significant reduction in the blood glucose levels of the albino rats [43]. One study reported that among petroleum ether, ethyl acetate, methanol and 70% ethanol (v/v) extracts of bulbs of D. bulbifera, ethyl acetate extract showed highest inhibition upto 72.06 ± 0.51% and 82.64 ± 2.32% against a-amylase and a-glucosidase respectively. Diosgenin was isolated showed a a-amylase and a-glucosidase inhibition upto 70.94 ± 1.24% and 81.71 ± 3.39%, respectively by interacting with two catalytic residues (Asp352 and Glu411) from a-glucosidase [44]. Copper nanoparticles synthesized by D. bulbifera tuber extract also showed significant inhibition against a-glucosidase and murine intestinal glucosidase [45].

Antidyslipidemic Activity

Aqueous extract of D. bulbifera tubers at 250, 500 and 1000 mg/kg doses administered for 4 weeks to high fat diet fed C57BL/6J mice showed significant antidyslipidemic effects [42].

Analgesic and Anti-Inflammatory Activities

The aqueous and methanol extracts from the dry bulbils of Dioscorea bulbifera L. var sativa were evaluated (300 and 600 mg/kg, p.o.) against pain induced by acetic acid, formalin, pressure and against inflammation induced by carrageenan, histamine, serotonin and formalin in experimental animals. The results showed potent analgesic and anti-inflammatory activities of the extracts which may be due to inhibition of inflammatory mediators such as histamine, serotonin and prostaglandins [46]. Antiinflammatory activity of ethanol extract of Dioscorea bulbifera leaf (500 mg/kg, 250 mg/kg, 125mg/kg, 62.5mg/kg, 31.25mg/kg, 15mg/kg p.o.) was reported against egg albumin induced rat paw oedema [47]. Diosbulbin B from D. bulbifera had inhibitory effects on both acute and subacute inflammation [48]. The effects of methanol extract of the bulb of Dioscorea bulbifera var sativa (250 and 500 mg/kg, p. o.) were tested in mechanical hypernociception induced by intraplantar injection of complete Freund's adjuvant (CFA), lipopolysaccharides (LPS) or prostaglandin-E2 (PGE2), as well as in partial ligation sciatic nerve (PLSN), nociception induced by capsaicin and thermal hyperalgesia induced by intraplantar injection of CFA. The therapeutic effects of Dioscorea bulbifera on PGE2-induced hyperalgesia were evaluated in the absence and in the presence of L-NAME, an inhibitor of nitric oxide synthase (NOS) and glibenclamide, an inhibitor of ATP-sensitive potassium channels. The extract showed significant antinociceptive effects in persistent pain induced by CFA and on neuropathic pain induced by PLSN. D. bulbifera significantly inhibited acute LPS-induced pain and PGE2 induced pain. The results showed the mechanism of antinociceptive activities of D. bulbifera in both inflammatory and neuropathic pain may be the activation of the NO-cGMP-ATP- sensitive potassium channels pathway [49]. Methanol extract of D. bulbifera could inhibit the nitric oxide production and iNOS mRNA expression of LPS-induced macrophages in vitro, which may be one of the mechanisms of its anti-inflammatory action [50].

Diuretic Activity

Diuretic activity of ethanol extract of Dioscorea bulbifera leaf (500 mg/kg, 250 mg/kg, 125mg/kg, 62.5mg/kg, 31.25mg/kg, 15mg/kg p.o.) was reported in rats [47].

Gastro Protective Activity

Gastroprotective activity of hydroalcoholic extract of D. bulbifera tubers (doses of 100, 200 and 400 mg/kg) was reported against indomethacin-induced gastric ulcers in rats [51].

Antioxidant Activity

Hydroalcoholic extract of D. bulbifera tubers (doses of 100, 200 and 400 mg/kg) reversed the indomethacin induced gastric ulcers associated free radical changes and showed antioxidant activity by inducing a significant increase of peroxidase and catalase while reduction in glutathione peroxidase, reduced glutathione, and lipid peroxidation level in tissues [51]. Scavenging activity of sequential extracts (petroleum ether, ethyl acetate, methanol and ethanol (70%) of D. bulbifera bulbs were checked against pulse radiolysis generated ABTS+ and OH radical, in addition to DPPH, superoxide and hydroxyl radicals by biochemical methods. Ethyl acetate extract of D. bulbifera bulbs which contain diosgenin as a major constituents exhibited excellent scavenging pulse radiolysis generated ABTS^+ and OH radical [52]. 70% and 80% alcoholic extracts of D.bulbifera showed significant antioxidant activity against hydroxyl radical scavenging test, reducing capacity test and total antioxidant capacity test [36]. Ethanol extract of tubers of D. bulbifera also showed antioxidant activity in enzymatic assays (glutathione peroxidase, catalase, superoxide dismutase, glucose-6- phosphate dehydrogenase and glucose-s-transferase) and non enzymatic assay (reduced glutathione, vitamin C and vitamin E) [53]. Copper nanoparticles synthesized by D. bulbifera tuber extract also showed significant scavenging activity against DPPH, nitric oxide and superoxide radicals respectively [45].

Effects on Immune Systems

Immune function of mice were studied after oral administration of decoction of D. bulbifera (1000, 490, 240 g/kg body weight) for 15 days. Results showed that in the high dose group could significantly suppress the phagocytosis activity of mononuclear macrophages. However, the medium dose group markedly enhanced the activities of natural killer cells, the antibody quantity of B cells and the quantity and proliferation of spleen T lymphocytes. This experiment indicated that high doses of D. bulbifera could suppress the immune function in mice, while medium doses could improve the immune function [54]. Dioscorea bulbifera polysaccharides (100 or 150mg/kg) lowered peripheral blood T-cell subpopulation CD4+/CD8+ ratio, and Dioscorea bulbifera polysaccharides + Cyclophosphamide combination attenuated Cyclophosphamide effect in lifting CD4+/CD8+ ratio [55].

Antimicrobial Activity

Acetone extract, ethyl acetate extract, 95% ethanol extract and methanol extract of D. bulbifera each showed a fair antibacterial activity on inhibition of bacteria isolated from animals and poultries using disc method. The acetone extract showed the most significant anti-bacterial effect when compared to other extracts [56]. Aqueous extract of D. bulbifera showed superior anti-bacterial activity on E. coli while the anti-bacterial effect of an ethanol extract of D. bulbifera was potent against S. aureus and Candida albicans when tested using disc-diffusion method of antimicrobial assay [57,58]. The methanol extract, fractions (DBB1 and DBB2) and six compounds isolated from the bulbils of D. bulbifera, namely bafoudiosbulbins A (1), B (2), C (3), F (4), G (5) and 2,7-dihydroxy-4-methoxyphenanthrene (6), were tested for their antimicrobial activities against Mycobacteria and gram-negative bacteria involving multidrug resistant (MDR) phenotypes expressing active efflux pumps using microplate alamar blue assay and the broth microdilution methods. The good activities of the crude extract, fractions and compound 3 on most of the tested microorganisms belonging to MDR phenotypes such as E.coli AG102, P. aeruginosa PA124, E. aerogenes CM64, K. pneumoniae KP55 and KP63 as observed [58]. Two clerodane diterpenoids, Bafoudiosbulbins A and Bafoudiosbulbins B showed significant activities against P. aeruginosa, S. typhi, S. paratyphi A and S. paratyphi B [59]. 8-epidiosbulbin E acetate showed broad-spectrum plasmid-curing activity against MDR bacteria, including Vancomycin-resistant enterococci (VRE). It cured antibiotic resistance plasmids from clinical isolates, including Enterococcus faecalis, E. coli, Shigella sonnei, P. aeruginosa and Bacillus subtilis with 12-48% curing efficiency [60]. In addition, vanillic acid and isovanillic acid showed antibacterial activities as well [61].

The successive extracts of bulbils of Dioscorea bulbifera (bulbils) was investigated for in vitro antimicrobial activity. Among all extracts, the petroleum ether and chloroform extracts showed significant activity against A. fumigatus and R. nigricans. The petroleum ether and distilled water extract showed good activity against K. pneumoniae. The chloroform extract showed feeble activity against S. aureus [62]. Beta-lactam (piperacillin) and macrolide (erythromycin) antibiotics showed synergistic effect with silver nanoparticles of D. bulbifera tuber extract against multidrug-resistant Acinetobacter baumannii. Chloramphenicol or Vancomycin also showed synergistic effect with silver nanoparticles of D. bulbifera tuber extract against Pseudomonas aeruginosa. Streptomycin combined with silver nanoparticles showed strong evidence for the synergistic action against E. coli [63].

Anti-Viral Activity

The alcohol extract of D. bulbifera (0.017-0.034 mg/ml) reported to kill DNA virus and inhibit the transcription of RNA virus in direct or indirect inhibitory experiments. From different parts of the ethanol extracts of D. bulbifera (butanol fraction, ethyl acetate fraction, acetone and ether fraction), the inhibition effect of butanol and ethyl acetate fraction on Coxsackie B I-VI virus was better than that of the other two fractions. But their effects on herpes simplex virus I were nearly the same. After killing the virus, the cells still could continue to divide and be subcultured which was indicating that the drug is non-toxic and effective. But the decoction of D. bulbifera had no inhibitory effect on various types of viruses [64].

Antifungal Activity

The decoction of D. bulbifera (1:3 ratio of D. bulbifera to water) had different degrees of inhibitory effects on a variety of skin fungi, such as Trichophyton violaceum, T. concentricum and T. schoenleinii [65]. It has been proved that the isolated dihydrodioscorine from D. bulbifera at 0.1% concentration could inhibit the growth of fungi which could cause diseases in several types of plants [25].

Anthelmentic Activity

Methanolic extracts of the flesh and peel of the bulbils of D. bulbifera, showed in vitro anthelmintic activity on Fasciola gigantica and Pheritima posthuma at concentrations ranging from 10 to 100 mg/ml [66].

Neuropharmacological Activity

Central nervous depressant action of acute treatment of hydroalcoholic extract of tuber of Dioscorea bulbifera (100 and 300 mg/kg, p.o.) was observed as treatments significantly reduced spontaneous motor activity, rectal temperature and prolonged the pentobarbitone induced hypnosis in mice. However, no effect on motor co-ordination as determined by the rota rod test which confirmed central action rather than peripheral action of extract. Further extract treatments also showed anxiolytic activity in plus maze test and head-dip test [67].

Cardioprotective Activity

Myricetin, epicatechin, isovanilic acid and vanillic acid were shown to be important bioactive components in D. bulbifera that protect against cardiovascular diseases [68]. In another study, administration of 70% ethanolic extract of D. bulbifera to rats (150 mg/ kg of body weight, 30 days) resulted in significantly improved ventricular performance in terms of aortic flow, left ventricular developed pressure (LVDP) and the first derivative of developed pressure (LVmax dp/ dt) of D. bulbifera treated during post-ischemic reperfusion. D. bulbifera also significantly reduced the size of myocardial infarction by 20 ± 2.64% as compared to the control group.

The decreased number of apoptotic cardiomyocytes by 16.89 ± 1.7% revealed that D. bulbifera had anti-apoptotic activity. The modulation of pro- and anti-apoptotic proteins by D. bulbifera was also examined. The upregulation of procaspase 3 and downregulation of cleaved caspase 3 coupled with prevention of loss of phase II enzyme HO-1 suggested that D. bulbifera extract ameliorates rat myocardial ischemia and reperfusion injury with an associated reduction in apoptotic cell death [69].

Cardioprotective action of steroidal saponin Diosgenin, isolated from Dioscorea bulbifera, was reported against Hypoxia- reoxygenation Injury in H9c2 cardiomyoblast cells as evidenced from the improved cell survival after hypoxia-reoxygenation injury, decreased release of lactate dehydrogenase, during cell death, upregulated the pro-survival molecules like B-cell lymphoma 2 (Bcl-2), heme oxygenase-1 and the phosphorylation of ATK (at serine 473); and at the same time down regulated pro-death molecules like Bax [70].

Effects on Thyroid Glands

D. bulbifera had achieved good effect in the treatment of subacute thyroiditis [71]. In another study, thyroxine (T4) concentration and triiodothyronine (T3)-uptake level decreased in Sprague-Dawley (SD) rats treated with sodium levothyroxine (160 lg/kg, 5 days) and extract of D. bulbifera (0.75 or 1.5 g/kg). The results suggested that D. bulbifera decreased excess thyroid hormone and increased metabolism, resulting in improvement of the hyperthyroid state [72].

Anorexient activity

Anorexient activity of Dioscorea bulbifera Linn Was reported [73].

Toxicity Study

Acute, subacute and chronic toxicity study of D. bulbifera showed that for mice the intraperitoneal LD50 was 25.49 g/ kg and the oral LD50 was 79.98 g/kg. The toxicity was mainly manifested as damage to liver and kidney. The degree of damage was related to the dose and time of drug administration [74]. Dioscin and diosbulbin B, derived from Chinese D. bulbifera roots (Huang-yao-zi) are responsible for liver toxicities, nausea, abdominal pain, coma and even death.

The mechanism of hepatotoxicity is relevant to its inhibition of antioxidant enzymes in liver mitochondria and the activity of drug metabolic enzymes, such as glutathione transferase, glutathione peroxidase, superoxide dismutase, glucose-6- phosphate and succinodehydrogenase [75-77]. Genetic studies found that the expression of the bad gene was increased in hepatocytes that promoted the apoptosis of mitochondria and endoplasmic reticulum lead to death of hepatocytes [78]. Various other studies support that hepatotoxicity of D. bulbifera with methanol extract and its cholorform fraction [79,80] ethyl acetate fraction of 75% ethanolic extract and isolated diosbulbin D [80], diosbulbin B were observed [81]. Another study further confirmed the hepatotoxic effects induced by D. bulbifera using molecular function analysis of the changed metabolites including elevated levels of taurine, creatine, betaine, dimethylglycine, acetate and glycine, and decreased levels of succinate, 2-oxoglutarate, citrate, hippurate and urea [82].

8- Epidiosbulbin E from D. bulbifera has been reported to cause hepatotoxicity as electrophilic intermediate generated by the metabolic activation of furan ring of 8-Epidiosbulbin E acetate mediated by cytochromes P450 is responsible for liver injury [83]. Various studies also reported renal toxicity of D. bulbifera [74,77, 84]. The renal lesions mainly are cloudy swelling of renal tubular epithelial cells, luminal stenosis, and protein casts in renal tubes [74]. Direct cytotoxicity on glomerular and tubular cells, and acute tubular injury was caused by severe parenchymal liver injury [84].

One study reported that D. bulbifera also affects the function of the gastrointestinal system. 19.9, 8, 2.7 g/kg of200% decoction of D. bulbifera reported a high degree of gastrointestinal flatulence and congestion of the gastrointestinal vascular system in the dead mice. Pylorus ulcers in the stomach were also visible. Under a light microscope, superficial necrosis of gastric mucosa was observed [77]. D. bulbifera administration for long term can lead to increased thyroid masses with larger follicular diameters, thick colloid filling in their cavities and flattened follicles in mice and rats. These toxic reactions could be seen in diffuse colloid goiter, which is very similar to the symptoms of goiter induced by iodine poisoning [85].

Methods of Detoxifications

Major toxicity materials observed are saponins and sapogenins in Central American, South African and Indian species, tannins and polyphenols in Indo-Chinese varieties and furanoid norditerpenes (diosbulbins) mostly in China. Diosbulbin D (0.07 mg/g) is a major toxic compound in Australian variety of D. bulbifera. Treatment practices varying from baking, followed by overnight leaching of the sliced tubers for 12 h in running water, resulted in reduction of major bitter and toxic compound to a very low level under the taste threshold rendering the final food palatable [86].

Drug Interaction Study

D. bulbifera and Diosgenin effect on rat CYP450 enzymes and its important isoforms (CYP3A4, CYP2D6) was studied using high through put screening. In fluorometric assay, herb extract exhibited higher IC values (96.21 ± 1.32 to 180.42 ± 0.12 |ig/ml) when compared to positive inhibitors and lower than Diosgenin (172.54 ± 0.52 to 201.86 ± 1.49 μg/ml) on CYP3A4 and CYP2D6. Based on the inhibitory potential, test substances exhibited very less interaction capacity, thereby leading to less significant herb- drug interaction with co-administered drugs [87]. Synergistic compatibility detoxification, that is, combining D. bulbifera with other herbal medicines, has been shown to improve therapeutic effects and reduce toxic effects. Combination with Angelica sinensis can enhance D. bulbifera's anti-tumor effect [88] and reduce renal toxicity [89-92] and kidney toxicity [93]. Combination with Schisandra chinensis relieved the liver damage caused by D. bulbifera [94-96]. Potential synergistic anticancer activity of combination of diosbulbin B with scutellarin from Scutellaria barbata proved useful for the clinical treatment of cancer [97]. Combination with Glycyrrhiza uralensis reduced liver damage and renal toxicity of D. bulbifera [98].

Conclusion

Dioscorea bulbifera is one of the most widely-consumed aerial yam widely distributed throughout various tropical regions. The plants are characterized by the production of considerable number of aerial tubers or bulbils. Dioscorea bulbifera is widely used in traditional medicine among them many documented medicinal folk uses of the plant are presented here. It is reported to have wide chemical diversities as contains steroids, saponins, flavonoids, glycosides, tannins, alkaloid, fatty acids and essential oils. The plant appears to have a broad spectrum of activity on several ailments. Various parts of the plant have been explored for antitumor, anti HIV, antidyslipidemic, analgesic, anti-inflammatory, diuretic, gastroprotective, antioxidant, antimicrobial, antiviral, antifungal, anthelmintic, neuropharmacological, cardioprotective, anorexiant, plasmid curing activities and anti-hyperthyroid activities.

The pharmacological studies reported in the present review confirm the therapeutic value of Dioscorea bulbifera. Many polyherbal formulations containing this plant parts are available in the market. However, less information is available regarding the clinical study, standardisation method to avoid biological and geographical variation, advance food processing and detoxication techniques. The plant is pre-clinically evaluated to some extent; if these claims are scientifically and clinically evaluated then it can provide good remedies and help mankind in various ailments.

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cleverninjaheart · 4 years ago

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Mouse Elisa Kits

A licensed vaccine against hepatitis C virus (HCV) have not yet become available for the moment. Stability and antigenicity of synthesized recombinant fusion protein consisting of a core targeted cut and NS3 (RC / N) of HCV has predicted. Although the antigen is safe, efficacious recombinant protein vaccine without adjuvant did not.

This study evaluated the immunogenicity of RC / N antigens bipartite accompanied by Neisseria meningitidis serogroup B outer membrane vesicles (OMVs NMB) in BALB / c mice.

The NMB OMVs produced and evaluated accurately. Administration is as follows: RC / N-OMV, RC / N-Freund's complete / incomplete adjuvant (CIA), RC / N-MF59, RC / N, OMV, MF59, and PBS. The production of Th1 (IFN-γ, IL-2) / Th2 (IL-4) / Th17 (IL-17) cytokines and granzyme B (indicator cytotoxic) by mononuclear cells of the spleen and the concentration of humoral total IgG / IgG1 (Th2) / IgG2a ( Th1) in the sera of the mice were measured using a Mouse Elisa Kits .

The concentration of Th1 / Th2 / Th17 cytokines, granzyme B, and immunoglobulins in the spleen and sera from mice immunized, who had received antigen plus any adjuvant (RC / N-OMV, RC / N-Freund CIA and RC / N-MF59), significantly increase compared to the control (RC / N, OMV, MF59 and PBS).

Th1-type responses are dominant over Th2-type responses of mice vaccinated with the RC / N-OMV, and the dominant species increased Th2 response in mice vaccinated with the RC / N-MF59 (p <0.05).

NMB OMVs able to increase the Th1 immune response is dramatically more than MF59 and Freund CIA. Formulation RC / N with NMB OMVs demonstrated its ability to induce an immune response Th1, Th2, and Th17. RC / N-NMB OMVs is a promising approach for the development of therapeutic vaccines for HCV.

#Mouse Elisa Kits

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juniperpublishersjcmah · 5 years ago

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The Roles of Extracellular Purinergic Signaling in Local Acupoints In Chrono- Acupuncture Analgesia

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Objective: To explore the peripheral mechanism of chrono- acupuncture with respect to purinergic signaling in local acupoints. Methods: The Sprague Dawley (SD) rats were randomly divided into two groups. One group was injected 0.1ml saline as control group. Another group was established by subcutaneous injecting 0.1ml complete Freund’s adjuvant (CFA). After modeling successfully, model rats were randomly divided into model group and acupuncture group based on the basic pain threshold. The basic pain threshold was tested by tail-flick method. The extracellular ATP concentration in local acupoints was detected by high- performance liquid chromatography (HPLC) after acupunctured at different times, which were zeitgeber time 0(ZT0) (7:00), ZT4(11:00), ZT8(15:00), ZT12(19:00), ZT16(23:00), and ZT20(3:00) respectively. Based on the characteristics of the variation of pain threshold and extracellular ATP concentration, the peak phase and valley phase were selected for the further experiment. The protein expression of P2X3 receptors in skin of “Zusanli” (ST36), dorsal root ganglion (DRG) and spinal dorsal horn (SDH) were separately detected by immunohistochemistry. Results: Time had an evident influence on pain threshold (P=0.047, P<0.05) which indicated the pain threshold at different time points was different. The variation of pain threshold among control group, model group and acupuncture group were significantly different (P<0.01). In acupuncture group, the of peak value and valley value appeared at ZT8 and ZT16 respectively. Pain threshold showed complex interactions with extracellular ATP, time factor and acupuncture stimuli. The extracellular ATP concentrations were significantly different in comparison among control group, model group and acupuncture group respectively. (P<0.01). As for protein expression of P2X3 receptors in skin of “Zusanli” (ST36), compared with control group, it obviously increased in model group at ZT8 and ZT16. And there was significant difference between control group and model group (P<0.01). In addition, compared with model group, it obviously decreased after acupunctured at ZT8. And there was significant difference between model group and acupuncture group at ZT8(P<0.01), but not at ZT16 (P>0.05). Interestingly, the protein expression of P2X3 receptors in both DRG and SDH was consistent with that in skin of “Zusanli” (ST36). Conclusion: Temporal variation of purinergic signaling participated in the initial mechanism of chrono-acupuncture analgesia in local acpoints. Also, that might be the base of dynamic variation of acupoints reactivity.

Keywords: Circadian rhythm; Chrono-acupuncture analgesia; Pain; ATP; P2X3 receptor

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Introduction

Circadian rhythm was one of life characters. Peripheral clocks possessed by almost all cells [1]. Circadian clock could be entrainmented by external environments, such as darkness and light, the seasons, the social behaviors, acupuncture and so on [2]. circadian clock encodes key regulators of response to therapeutic treatment [3]. Various of pain syndromes not only showed circadian rhythm, but also affected the circadian rhythm of the body. Most migraine attacks begin in the early morning [4]. many patients with RA present a circadian rhythm in symptoms severity with a significant worsening in the morning. Additionally, studies have shown that the change of pain threshold had obvious circadian rhythm [5-6]. It has been recognized that acupuncture, as an effective means of treatment, could relieve various kinds of pain syndromes [7- 8]. The electro-acupuncture has regulation effects on circadian rhythm of temperature and melatonin in depression rat model [9]. Therefore, the curative effect of acupuncture at different times was different. In terms of chrono-acupuncture analgesia, the studies, pay more attention to central mechanism. Compared with the normal acupuncture, premature acupuncture for the regulation of dysmenorrhea model rats’ β-EP content and HSP70 expression in hypothalamus and pituitary more obvious effect. That might be related to mechanism of central analgesia [10]. However, the peripheral mechanism of chrono-acupuncture analgesia, so far, poorly understood.

Acupoints, as the base of acupuncture stimulation, were the initial taches of acupuncture therapy. The temporal specificity of acupoint reactivity is one of important influence factors of acupoint reactivity. Some studies had shown that purine and its receptors involved in acupuncture analgesia. Acupuncture could increase the concentration of extracellular purine in local acupoints of rats, and the concentration of extracellular purine reached peak value at 30 min, then decreased gradually [11]. P2X3 receptor, as a member of non-selective ATP gated ion channel, participated in the conduction of peripheral painful information through binding to extracellular ATP [12]. Also, the protein and gene expression of P2X3 receptors in DRG increased in various kinds of painful animal models, accompanying with the change of ATP current [13-15]. While the gene expression of P2X3 receptors and ATP current in DRG had showed a downward trend after electro-acupuncture [16]. What’s more, the extracellular purinergic signal, such as ATP, not only had its own circadian rhythm, but also showed the function of time modulation. Thus we proposed the hypothesis that, in the peripheral mechanism, the acpoint reactivity of chrono-acupuncture analgesia might be related to the temporal changes of purinergic signaling in local acupoints.

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Results

The relationship among time factor, ATP & pain threshold on chrono-acupuncture

There were no significant differences among the weight and basic pain threshold of each rats (P<0.05). In control group, the pain threshold peaked at ZT0 and reached the bottom at ZT12 which entails time had a notable effect on pain threshold (P<0.05=. After modeling, both the circadian rhythm of pain and the phase of the pain threshold had changed. compared with control group, the pain threshold of model group decreased. It showed that it peaked at ZT8 and reached bottom at ZT16. However, time had no obvious effect upon the pain threshold in model group (P=0.076,P>0.05). After acupuncturing, compared with model group, the pain threshold of acupuncture group increased. The peak occurred at ZT8 and the valley occurred at ZT16. Time factor had a significant influence on pain threshold of acupuncture group(P<0.05) Table1- Table 5, Figure 1. The interaction played a notable part in pain threshold. When compared the pain threshold among control group, model group and acupuncture group, they were significantly different (P<0.01) Figure 2. The temporal variation tendency of pain threshold in each group. In the control group, two valley value were at ZT4 and ZT12 respectively. The temporal variation of pain threshold in model group showed abnormal pathological changes. While, compared with model group, the pain threshold of acupuncture group showed upward and it also showed abnormal pathological changes.

Time factor had effects on extracellular ATP concentration in different groups. There was significant difference in the variation of extracellular ATP concentration among control group, model group and acupuncture group (P<0.05). There was statistical significance between control group and model group at ZT12 (P=0.017, P<0.05). Extracellular ATP concentration in acupuncture group was higher than that in model group at all six time points. However, there was only statistical significance at ZT16(P=0.022, P<0.05). In the same group at different time points, extracellular ATP concentration also had the differences. In the control group, the extracellular ATP concentration of ZT8(P=0.016, P<0.05) and ZT12 (P=0.038, P<0.05) were significantly different from that of ZT4 respectively. In the model group, the extracellular ATP concentration of ZT4(P=0.026, P<0.05) and ZT20 (P=0.030, P<0.05) were significantly different from that of ZT0 respectively. In the model group, the extracellular ATP concentration of ZT4(P=0.010, P<0.05) and ZT20 (P=0.029, P<0.05) were significantly different from that of ZT12 respectively. And the increase of extracellular ATP concentration in acupuncture group was affected by the time factor, which was statistically significant compared with other two groups Table 6- Table 9 Figure 3 and Figure 4. The trend of extracellular ATP concentration over time. In control group, two valley value appeared at ZT4 and ZT12 and two peak value appeared at ZT0 and ZT8. Extracellular ATP concentration was in a low level at ZT12 and began to show an upward trend at ZT16. In model group, that showed one valley value and one peak value, which was ZT4 and ZT8 respectively. Compared with control group, extracellular ATP concentration abnormally increased at ZT8 and the valley value disappeared at ZT12. The trend of acupuncture group was similar to that of control group. Two valley value appeared at ZT4 and ZT12 and two peak value appeared at ZT0 and ZT8. The time of high point and the low point of extracellular ATP concentration appeared to be normal. The extracellular ATP concentration in acupuncture group was higher than that in model group.

* mean that using ZT12 of control group as a reference △ mean that using ZT16 of model group as a reference # mean that using ZT4 of control group as a reference ▲ mean that using ZT0 of model group as a reference ■ mean that using ZT12 of model group as a reference

a. Predictors: (constant), group, ATP b. Dependent variable: pain threshold

a. Predictors: (Constant), Group, ATP b. Dependent variable: pain threshold

a. Dependent Variable: pain threshold The pain threshold was mainly affected by extracellular ATP concentration in local acupoints and showed a significant positive correlation (P<0.05) Table 10- Table 12.

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The Relationships Between Time Factor and P2X3 Receptors on Chrono-Acupuncture

As for protein expression of P2X3 receptors in skin of “Zusanli” (ST36), compared with control group, it obviously increased in model group at ZT8 and ZT16. And there was significant difference between control group and model group (P<0.01). In addition, compared with model group, it obviously decreased after acupunctured at ZT8 and there was significant difference between model group and acupuncture group at ZT8(P<0.01), but not at ZT16 (P<0.05). Interestingly, the protein expression of P2X3 receptors in both DRG and SDH was consistent with that in skin of “Zusanli” (ST36) Table 13- Table15, Figure 5- Figure 10.

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Discussion

This work indicated that time factor was an important regulator of purinergic signaling in acupuncture analgesia. We found that: 1. Acupuncture not only increased pain threshold but had the function of temporal regulation on pain threshold. 2. The variation of extracellular ATP concentration in local acupoints was affected by acupuncture in different time points and there was a positive correlation between pain threshold and extracellular ATP concentration. 3. The variation of protein expression of P2X3 receptors in local points, DRG and SDH had temporal difference after acupunctured at different time points

Together, our results showed that, in the peripheral mechanism, the acupoint reactivity of chrono- acupuncture analgesia might be related to temporal changes of purinergic signaling in local acupoints. Both master clock and peripheral clock could modulate the circadian rhythm of lives according environment time cues. It was found that the mechanism of molecular oscillations in the peripheral biological clock was similar to that of the master clock [17]. If there were abnormalities in circadian rhythm of the body, chrono-biological indicators changed accordingly. There were some special rhythm characters in many kinds of diseases, especially in pain syndromes. As for rodents, the responses of pain were drastic in dark and moderate in light [18-19]. In clinical studies, pain intensity and body response had rhythmic and cyclical changes. Multiple signaling involved in the rhythmic changes in pain syndromes [20]. Our findings indicated that acupuncture not only increased the inflammatory pain threshold in rats, but also the acupuncture-time interaction had a significant effect on the pain threshold. At different time points, the peak value of pain threshold in control group appeared at ZT0 and the valley value appeared at ZT12. However, the rhythms of animal pain changed after model establishment. The pain threshold of model group was significantly lower than that of control group at ZT0, ZT16 and ZT20 respectively, except at ZT12. Additionally, the peak value of pain threshold in acupuncture group appeared at ZT8 and the valley value appeared at ZT16. Compared with model group, the pain threshold of acupuncture group at the same time point was upward generally. On the whole, time factor was markedly effect on three groups in this experiment. The interaction had significant influences on the pain threshold. Based on experimental results, the pain threshold of rats was rhythmic in physiological conditions. While the pain threshold had abnormal rhythm changes after establishment. And acupuncture could increase the inflammatory pain threshold in rats. The interaction of acupuncture and time factor had significant effect on the pain threshold. In other words, the changes of pain threshold aroused by acupuncture at different times was significantly different. Studies focusing on the extracellular purinergic signaling, represented by ATP, was important informational substances in the body’s temporal structure. In both peripheral and central oscillatory system, extracellular ATP and its metabolites showed marked circadian rhythm. Under the cycle of LD12:12 and DD, the extracellular ATP level in SCN neurons of rats had obvious rhythmical changes. While the extracellular ATP accumulation in vitro cultured cortical glial cells also showed diurnal oscillations [21-22]. The circadian variation of ATP hydrolase activity was of importance for maintaining the temporal function of extracellular purinergic signaling. ATP hydrolase activity in peripheral blood also had rhythmical change, and the highest reactivity occur in dark period [23]. Furthermore, extracellular ATP could selectively raise the expression of gene mPer1 to affect the basic working pattern which was transcription- translation negative feedback among molecular clocks by activating P2X7 receptor [24]. So, extracellular ATP not only outputted temporal information, but also had the function of temporal modulation. Our findings indicated that, the extracellular ATP concentration in control group had double peak (ZT0 and ZT8) and double valley (ZT4 and ZT12). The extracellular ATP concentration of ZT12 was at a low level and began to increase at ZT16. However, the model group showed only one peak (ZT8) and one valley (ZT4). Compared with control group, there was an abnormal increase at ZT8 and the original valley at ZT12 disappeared. The variation of acupuncture group is similar to that of control group and showed double peak (ZT0 and ZT8) and double valley (ZT4 and ZT12). The peak and valley value of extracellular ATP concentration tended to be normal. And the extracellular ATP concentration in local appoints in acupuncture group was higher than that of model group. Combined with pain threshold and extracellular ATP concentration, our results showed that the pain threshold was mainly affected by extracellular ATP concentration in local acpoints and shows positive correlation. There was also a positive correlation between processing methods (whether the rats were in healthy conditions or not and whether acupuncture was adopted).

The studies showed that ATP involved in the pain conduction. P2X3 receptors, a non-selective ligand gated cation channel, played a key role in peripheral transmission of pain [12]. ATP could induce mechanical hyperalgesia in gastrocnemius of rats to release proinflammatory cytokine by activation of peripheral P2X3 receptor [25]. ATP was released from injured cells and excited peripheral P2X3 and P2X3 receptors to generate action potentials of primary afferent neurons, transmit them to the distal end of the central nervous system and released the neurotransmitter’s effect on the postsynaptic process of the central nervous system Neurons [26]. Related studies had shown that P2X3 receptor-induced pain could be significantly reduced after blocking the P2X3 receptors’ conduction pathway or using P2X3 receptor antagonists. After sciatic nerve was cut off, the expression of P2X3 receptor in L4/5 DRG decreased by 50% [27]. It had found that, TNP-ATP, a P2X1, P2X3 and P2X2/3 receptor antagonist, significantly attenuates inflammatory hyperalgesia in the rat model of inflammatory pain induced by carrageenan in the temporomandibular joint (TMJ). Whereas P2X1 receptor did not participate in this model pain, which mean that P2X3 and P2X2/3 receptor might involve in the inflammatory pain sensitivity of the rat model of TMJ [28]. A-317491, a selective P2X3 receptor antagonist, could reduce chronic inflammatory and neuropathic pain mediated by P2X3 receptor in rats. And intrathecal administration of A-317491 seemed to be more effective to relieve tactile allodynia after peripheral nerve injury than intraplantar administration [29]. Meanwhile, many

studies showed that, purinergic signaling played an important role in acupuncture analgesia. Electro-acupuncture could exert an analgesic effect on neuropathic pain through simultaneous action of purine Al and P2X3 receptors [30]. Also, acupuncture could reduce the expression of P2X3 receptor gene and the magnitude of ATP excitation current in DRG of rats with chronic constriction injury (CCI) [31]. What’s more, midbrain periaqueductal gray P2X3 receptors involved in the modulation mechanism of electro-acupuncture analgesia in the spinal cord. ATP had circadian rhythm and had the function of temporal modulation [32]. Our findings indicated that, no matter in skin of “Zusanli” (ST36), DRG and SDH, compared with control group, the protein expression of P2X3 receptors in model group markedly increases at ZT8 and ZT16 respectively. While compared with model group, the protein expression of P2X3 receptors in acupuncture group markedly decreases at ZT8, but not at ZT16. These results mean that, there was temporal difference in decreasing the protein expression of P2X3 receptors by acupuncture. Moreover, after both establishing and acupuncturing, the variation of protein expression of P2X3 receptors in DRG and SDH displayed highly consistency at the same time point. This might be one of the foundations of the time effect of acupuncture analgesia

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Experimental Procedures

Animals

The experiment consisted of two parts. 156 SD male rats were needed in this experiment. All rats (body weight of 200 ±20 g) were provided by animal center of Sichuan provincial hospital (Permit Number: SCXK(CHUAN)2013-15). The rats were acclimated to the rat facility for 1 week before starting the experiments. The rats were housed under 12h:12h light/ dark cycles (Light on at 7:00, ZT0 and off at 19:00, ZT12) with free access to water during the whole experiment, and free access to food. Animal care was carried out in accordance with the Instruction for Ethical Treatment of Animals issued by the Ministry of Science and Technology, China, in 2006. We tried to minimize the number and suffering of the laboratory animals. All procedures and animal experiments were approved by the Animal Care and Use Committee of Chengdu University of Traditional Chinese Medicine (China) (NO.2014-01).

Assessment of basic pain threshold

The basic pain threshold of rats was tested by tail-flick method. The standard rats which tailed between 3 and 10 seconds on the test were involved in the experiment. The basic pain threshold was needed to finish within 1 hour under the same condition. In addition, in order to avoid scalding the rats and calm them down, each one was measured at intervals of 5 minutes or more

Grouping and modeling

In this part experiment,108 SD male rats were randomized divided into two parts. One part was subcutaneously injected 0.1ml 0.9% saline into the right hind paw for 1 day before the experiment as control group. Under the same condition, another part was subcutaneously injected 0.1ml CFA into the right hind paw for 1 day before the experiment. After successful replication of the model, it was randomly divided into model group and acupuncture group based on the basic pain threshold. And each group was divided into 6 sub-groups which were ZT0, ZT4, ZT8, ZT12, ZT16 and ZT20 respectively. The rats established by CFA should be in acute pain stage after 24 hours of injection and the syndromes showed that the time of tail-flick shortened accompanying with red-swollen paw.

Experimental progress

Assessment of pain threshold: At the second day after model establishment, the rats in acupuncture group acupunctured “Zusanli” (ST36) in the affected side for 30 min (twisted 1 min every 5 min, 120 per/min). When the therapy was finished, the pain threshold of rats was tested by tail-flick method. The pain threshold was needed to finish within 1 hour under the same condition. Additionally, in order to avoid scalding the rats and calm them down, each one was measured at intervals of 5 minutes or more

Assessment of extracellular ATP concentration in local acupoint: We collected samples of interstitial fluid by a micro dialysis probe implanted in the tibialis anterior muscle/sub cutis of rats at a distance of 0.4–0.6 mm from “Zusanli” (ST36). The processes of this experiment as follows: 1. Under the constant temperature of 37°C with a CMA/450 animal thermostat, the rats were anesthetized with 10% chloral hydrate (0.4ml/100g) by intraperitoneal injection and kept them in prone position. The right hind leg of the rats was shaved, disinfected and kept operating area clean and dry. 2. The length of the catheter was guided by micro dialysis probe accurately. 3. Cut off the skin where the probe had been marked with tissue scissors and tissue forceps. The wound was minimally invasive as long as the probe was successfully inserted. The syringe needle was needed to insert into the tear supporting tube and was slowly implanted local tissue of acupoints. Then, the syringe needle was put out, and the micro dialysis probe (CMA20, Sweden) was inserted into the tear supporting tube. 4. The two ends of the probe were respectively connected to the microanalysis pump (CMA402) and the cooling microcollector (MAB85). In the micro dialysis pump, 0.9% saline was injected into the injector, with the speed of 1μL/min and balanced for 1 hour. In the cooling micro-collector, the temperature was set to 4°C. After the balance, the local tissue fluid of the acupoints was collected by the cooling microcollector automatically (30min / 1 sample). Each rat was collected for two samples which were respectively before and during acupuncture. The sample collections lasted for 1 hour. 5. The content of purine in the sample collected by the microanalysis was tested by HPLC.

Assessment of protein expression of P2X3 receptors in skin of “Zusanli” (ST36), DRG and SDH: Based on the former experimental results, the peak value (ZT8) and the valley value (ZT16) were chosen for the further experiment. 48 SD male rats were needed in this part experiment. The methods of assessment of basic pain threshold, modeling and grouping were the same as the former. The rats in acupuncture group acupunctured “Zusanli” (ST36) in the affected side for 30 min (twisted 1 min every 5 min, 120 per/min). Meanwhile, the rats in the control group and model group did not accept acupuncture therapy. When the therapy was finished, the rats were anesthetized with 10% chloral hydrate (0.4ml/100g) by intraperitoneal injection and the skin and subcutaneous tissue of “Zusanli” (ST36) in the affected side (size:2*2; depth:2~3mm ), DRG(L4-L6) and SDH (L4-L6) were respectively took away, then immediately placed in 4% paraformaldehyde and examined by immunohistochemistry and average optical density method.

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Statistical Analysis

All data were analyzed by SPSS19.0 and results were presented as mean± standard deviation (SD). Statistical methods which consisted of one-way ANOVA (one-way analysis of variance), RMANOVA (repeated measures analysis of variance) and MANOVA (multivariate analysis of variance) were used to compare between the groups after the normal distribution test and the homogeneity test of varuances. Significance was determined at P<0.05.

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Acknowledgement

This study was funded by National Natural Science Foundation of China (NO. 628313). The authors have declared that no competing interests exist.

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Author Contribution

Conceived and designed the experiments: DC ZZ. Performed the experiments: XL XS SH YW XZ NL. Analyzed the data: QZ JZ. Wrote the manuscript: SH XL XS NL

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juniperpublisherscellscienc-blog · 5 years ago

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MicroRNA: A Major Key in Pain Neurobiology- Juniper Publishers

Abstract

MiRNAs are single-stranded small noncoding RNAs that consist of approximately 22 nucleotides, that are involve in a wide range of biological processes including pain physiopathology. Because of their role as master switches in regulation and signaling pathways through modifications in nociceptive receptors, ion channels, pro-inflammatory molecules, emotional and cognitional behaviors associated with pain, the triggered enthusiasm for miRNAs as promising therapeutic targets is still active. Furthermore, the expression of specific miRNA can be helpful to predict treatment response in patients which suffer pain conditions that are poorly controlled by the currently available analgesics. This evidence is supported in several researches with animal pain models that we briefly review in this article to approximate in the understand of the role and neurobiology process through miRNA represents a major key for future therapeutics in pain, emphasizing in the neuropathic pain condition.

Keywords: MicroRNA; Neurobiology; Neuropathic pain

Introduction

Neuropathic pain (NP) represents one of main causes of chronic pain, perhaps trailing only osteoarthritis as a cause [1]. One of the keys to understand the biology of neuropathic pain is to know that´s is caused by an injury in a nervous tissue and that nociceptive pathways are involve in the lesion [2]. In the clinical practice, the most frequent NP origins are the neurons of dorsal root ganglia (DRG) and trigeminal ganglia (TG) in mechanical, metabolic and toxic lesions as traumatic injury, herpes zoster, diabetes, or cancer chemotherapy, all this kind of pathologies promotes functional changes in the initiation and maintenance of NP [3,4].

The several changes observed in neuropathic pain condition are well represented in two major symptoms, allodynia and hyperalgesia. Both symptoms are observed in patients and as signs in animal models of chronic pain such as the spinal nerve ligation (SNL), consisting in a tight ligation of L5 and L6 spinal nerves were Fukuoka et al. [5] described a down-regulation of the inhibitory γ-amino butyric acid receptor A (GABAA) in the dorsal root ganglia (DRG). In spared nerve injury (SNI) were shown an up-regulation of interleukin-1β (IL-1β) in the prefrontal cortex of rats [6]. And many other changes can be observe in every pre-clinical model of pain which includes up-regulation of interleukin-6 (IL-6) [7], neurokinin-1 receptor in the dorsal horn [8], down regulation of dopaminergic D1 and D2 receptors in the anterior cingulate cortex in a rat model [9] just to mention a few. Clearly, this changes in the substances and receptors regulation are product of an altered gene expression in the nociceptive pathways. One of the most recent studied mechanisms that explains the pathogenesis and play a crucial role in fine-tuning gene expression [10] in the chronic pain is MicroRNAs (miRNAs) regulation, that are involve in a wide range of biological processes [11]. In this review we will focus in the role and neurobiology process through miRNA represents a major key for future therapeutics in pain, emphasizing in the NP condition.

Biology and mechanisms of miRNA

MiRNAs are single-stranded small noncoding RNAs that consist of approximately 22 nucleotides. The genomic location of miRNAs can be broadly divided into intergenic (between genes) or intronic (embedded into a gene) [11]. After the transcription of a coding DNA protein is expressed the precursor messenger RNA (pre-mRNA) which conformation includes 4 regions, 5′-untranslated region (UTR), the protein-coding exon, the noncoding intron, and the 3′-UTR, that determines the main targets of miRNA [12]. The intronic or intron-derived microRNA (Id-miRNA) is formed in the in-frame introns and the intergenic miRNAs are set between independent transcription units [13], both has the capability of degrading messenger-RNA (mRNA) and inhibit protein translation so they share not only functional but also structural properties. With the only difference that intronic miRNA are typically transcribed from the same promoter as their host genes (Pol II) and require RNA splicing machinery [14-16] while intergenic RNAs genes have their own transcription regulatory elements [13].

In Figure 1 is represented the genesis and mechanism by which the interaction between miRNA, the target mRNA and the RNA-induced gene silencing complex (RISC) suppress the gene expression. This process begin with the excision of the primary precursor microRNA (pri-miRNA) by the RNA polymerase type- II (Pol-II) [17], this pri-miRNA at certain concentration can make a negative feedback to Pol-II. Then if the pri-miRNA is origin in an exon, it will be cropped into the hairpin-shaped pre-miRNAs by nuclear RNase III Drosha [18] or by spliceosomal components if comes from introns to form a mature precursor miRNA (premiRNA). This pre-miRNA is exported out of the nucleus to the cytoplasm by a member of a Ran-dependent nuclear transport receptor family, the exportin-5 (Exp5) [19] where is cleaved to the Dicer-like nucleases to form mature miRNA [20]. Finally the miRNA is coupled to a ribo nuclear particle (RNP) to get the RISC which is capable of executing RNA interference (RNAi)- related gene silencing, concluding in the inhibition of the protein translation [21].

MiRNA and Pain

The comprehension of the extensive pathways involved in the genesis of pain put in evidence that the genetic basis play a major role in pain biology [22]. In the very last years the focus of researches have been in looking not in an specific target or individual receptor but instead in a “major switch” that would regulate multiple gene products and orchestrate multiple pathways [23] and the recent evidence propose miRNA to be that switch. The miRNAs have been implied in inflammation [24] process and other pain conditions such as neuropathic pain [25] and fibromyalgia [26]. This both common clinical problems are usually poorly controlled by the currently available analgesics [27], the reason might be the complex and multiple processing of nociceptive information in pathological conditions [28]. The changes in this processing are the cause of phenomes like hyperexcitability that can be induced by a posttranslational modulation of ion channels, such as voltage-gated sodium channels [29] or long term potentiation (LTP) and disinhibition that are product of synaptic modifications [30]. So, this phenomes initiated by altered processing in nociceptive pathways respond to certain structures like spinal glial cells, especially microglia and astrocytes that also plays a major role in pain modulation [31] and can be govern by epigenetic mechanisms such as DNA methylation, histone modification, and miRNA expressions [32].

This supports the evidence of the critical role of miRNAs in pain biology, but not only at molecular, network or synaptic level, the miRNAs are implied in behavioral, emotional and cognitional changes [33] that affects pain perception [34] (Figure 2). However, the expression of miRNAs in DRG, spinal cord, and brain regions such as the limbic system and prefrontal cortex can vary from the different causes of pain [4]. The Table 1, resumes some of the most representative miRNAs expressed in certain pathologies and animal model of acute and chronic pain, excluding neuropathic pain that will be considered in the next section. Figure 2. miRNA plays a “major switch” role in many pathways involved in pain development and maintenance including behavioral, emotional and cognitional changes.

In the case of acute pain, the intra plantar formalin injection, was shown to decrease miR-124a expression in murine nociceptive spinal neurons in the ipsilateral horn [35], which importance seems to be related to the Methyl CpG binding protein 2 (MeCP2) a multifunctional epigenetic regulator that is best known for its role in the neurological disorders [36] and inflammatory pain [37]. Also, the tongue heat hyperalgesia following complete Freund’s adjuvant (CFA) injection shown that MeCP2 is involved in regulation of the transient receptor potential vanilloid 1 (TRPV1) expression in TG neurons [38], supporting the evidence of the down regulation of miRNA- 124a for the expression of MeCP2. Other works revealed by a real-time reverse-transcription polymerase chain reaction (RTPCR) a significant, but differential, downregulation of mature miR-10a, -29a, -98, -99a, -124a, -134, and -183 in the ipsilateral mandibular division (V3) of the TG within 4hr after CFA [39], this down regulation of miRNA releases the translation inhibition of target mRNAs, thus yielding more proteins that may be relevant to the development and/or maintenance of inflammatory pain as Bai et al. [25] conclude. In 2011 Kusuda et al. [40] found that CFA-induced inflammation significantly reduced miRs-1-16 and -206 expression in DRG. Conversely, in the spinal dorsal horn all three miRNAs monitored were up regulated [40]. Tam et al. [41] demonstrate for the first time that miR-143 expression in DRG nociceptive neurons is declined in response to inflammation [41]. More recently, Pan et al. [42] using a CFA model concluded that methylation-mediated epigenetic modification of spinal miR-219 expression regulates chronic inflammatory pain by targeting calcium/calmodulin-dependent protein kinase II γ (CaMKIIγ) which regulates NMDAR signaling and central sensitization [42].

In human chondrocytes with IL-1β in vitro stimulation, revealed that the treatment with p38- mitogen-activated protein kinase (MAPK) inhibitor (SB202190), enhanced the expression of miR-199a* which can directly target COX-2 mRNA and reduce protein expression levels [43]. Considering the IL-1β as a major mediator in chronic pain, described that miR-127-5p regulates MMP-13 expression and IL-1β–induced catabolic responses in human chondrocytes too [44]. Finally, another miRNA, the miR-146a expressed at reduced levels in DRGs and dorsal horn of the spinal cords from rats with Osteoarthritis (OA)- induced pain significantly modulates inflammatory cytokines and pain-related molecules (e.g. TNFα, COX-2, iNOS, IL-6, IL8, RANTS and ion channel, TRPV1) [45]. In cancer-associated pain, another form of chronic pain, miR-1a-3p plays an important role attenuating the mechanical hypersensitivity [46], however in this pain condition, it might been implied a large list of miRs.

Role and Expression of miRNAs in Neuropathic pain

The role of miRNA in the regulation of nociception, endogenous analgesia and in the circuitries and cognitive, emotional and behavioral components involved in pain is expected to shed new light on the enigmatic pathophysiology of neuropathic pain [24]. Therefore disruption of miRNA processing in primary afferent pathways is sufficient to inhibit injury-induced long-term development of chronic pain-related behaviors, this affirmation is supported in a large evidence of investigations we resumed in Table 2.

NcRNA (Noncoding RNA), SCI (Spinal cord injury), SNL (Spinal nerve ligation), CCI (Chronic constriction injury).

To start explaining the role of miRNA in neuropathic pain, let`s first mention some of the main animal models that have been development in this area. First the spinal cord injury (SCI) was proposed by Allen AR in 1911 [47] then in [48], adapted the Allen`s method by a briefly laminectomy performed at the T9– 10 thoracic vertebrae level to expose the spinal cord at T10 and inducing the SCI by New York University Impactor device [49], with this methods it has been possible to correlate the injury of the spinal cord with the regulation and expression of miRs like miR-21, miR-124a, miR-23b, miR-223, miR-449a and miR- 212 [50-56]. Spinal nerve ligation (SNL), the left L6 transverse process is removed to expose the L4 and L5 spinal nerves then the L5 spinal nerve is carefully isolated, tightly ligated with 3-0 silk thread, and transected just distal to the ligature [57], with this method it`s has been studied the miRs miR-7a, -21, -96, -182, -183, -103, -195 [58-63] and more recently with miARN- 30b [64,65]. The chronic constriction injury (CCI) model was proposed by Bennett and Xie in 1988 [66], in this model the right sciatic nerve is tied loosely with four ligatures by chromic cat gut 4-0, the lastly works with this method revealed the expression of miR-7a, -21, -539, -93, -183, -145 and -203 [58,59,67-71]. The axotomy model consist in a transection of the sciatic nerve at a point approximately 1 cm distal to the exit point of spinal nerve roots, after Axotomy the expression of miR-21 and miR- 222 increased in DRG [72]. Finally, the Nerve crush model is achieved after expose sciatic nerve and crush in the mid-thigh for 15sec with a fine hemostat, in the day 4 and 7 post injury the three most highly up regulated miRNAs was miR-21, miR- 142-5p, and miR-221 [73]. Now we`ll mention some of the most representative and lastly found miR`s involved in neuropathic pain development.

The miR-21 is expressed in all the neuropathic pain models, [54] demonstrated that miR-21 transcripts are physiologically regulated by peripheral nerve injury. Their role appeared to be enhance neurite outgrowth from DRG neurons by targeting the Sprouty2 protein (SPRY2) 3′ UTR region in rats after axotomy. More recently, [69] studied the role of miR-183 in the development of neuropathic pain using the CCI model they revealed that miR-183 can suppress AMPA receptors by inhibiting the mammalian target of rapamycin (mTOR)/ vascular endothelial growth factor (VEGF) pathway, which alleviates the mechanical hypersensitivity associated with inflammation and neuropathy [74]. Shao et al. [64] evidenced that one of the major targets in neuropathic pain, the voltage-gated sodium channel Nav1.7 are directly target by miR-30b. The expression of Nav 1.7 increases in nociceptive neurons during the development of inflammatory hyperalgesia, while the knockdown or ablation of Nav1.7 expression relieves inflammatory pain and hyperalgesia [75]. Finally, [70] study suggested that miR-145 serves an important role in the development of neuropathic pain through regulating RREB1 expression and the PI3K/AKT signaling pathway which serves an important role in vascular endothelial growth factor (VEGF)-induced hyperalgesia [76].

Future Approaches and Conclusion

The studies reviewed in this article may us consider the microRNA`s as potential targets and biomarkers for prediction and treatment of several pain conditions. Because of their role as master switches in regulation and signaling pathways through modifications in nociceptive receptors, ion channels, pro-inflammatory molecules, emotional and cognitional behaviors associated with pain, the triggered enthusiasm for miRNAs as promising therapeutic targets is still active. However, challenges with respect to the use of miRNA-based therapeutics in humans remain to be further explored [77]. When we can fully understand the role of miRNAs in pain

mechanisms, it will be possible to maximize miRNAs potency while minimizing off target toxicity and immunogenicity to provide great benefit for clinical diagnostic and therapeutic applications.

Acknowledgement

Dr. Carlos H. Laino is greatly acknowledged for his help in the critical review of this work.

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changyubio · 4 years ago

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Buy Freund's Complete Adjuvant For Covid-19. A Mineral Oil Emulsion Containing Inactivated Mycobacteria For Initial Immunizations.Freunds Complete Adjuvant Covid-19 contains mycobacteria that is used to reinforce an immune response by attracting macrophages and other cells to the injection site. Use Freunds Complete Adjuvant for initial boosts and Freunds Incomplete Adjuvant for subsequent boosts.

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changyubio1 · 11 months ago

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Aqueous adjuvant

An aqueous adjuvant is a substance added to a water-based solution, typically in the context of agriculture or vaccine formulations, to enhance the effectiveness of the primary active ingredient. Adjuvants in aqueous form help improve the stability, dispersibility, and overall performance of the solution, facilitating better absorption or uptake by the target organism, whether it be a plant or an organism receiving a vaccine. These adjuvants play a crucial role in optimizing the efficacy of various products by ensuring proper distribution and interaction of the active components.

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seoworkssss · 1 year ago

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Unlocking the Potential of Freund's Incomplete Adjuvant in Immunology

Freund's Incomplete Adjuvant (FIA) stands as a crucial player in the realm of immunology, acting as a catalyst in the development of vaccines and enhancing our understanding of the immune system. In this blog post, we'll delve into the depths of Freund's Incomplete Adjuvant, exploring its composition, applications, and the pivotal role it plays in immunological research.

Understanding Freund's Incomplete Adjuvant:

Freund's Incomplete Adjuvant is an emulsion-based adjuvant used to stimulate and amplify the immune response to antigens. Comprising mineral oil and surfactants, this adjuvant is characterized by its ability to promote a more robust and prolonged immune reaction compared to antigens alone.

Composition:

The key components of Freund's Incomplete Adjuvant include mineral oil and surfactants, which form a water-in-oil emulsion. This unique composition creates an environment conducive to antigen retention and presentation, enhancing the activation of immune cells.

Applications in Vaccine Development:

One of the primary applications of Freund's Incomplete Adjuvant is in vaccine development. By incorporating this adjuvant into vaccine formulations, researchers can boost the immune response, leading to increased production of antibodies and memory cells. This heightened immune reaction is particularly valuable in the development of vaccines against challenging pathogens.

Immunological Research:

Beyond vaccine development, Freund's Incomplete Adjuvant plays a crucial role in immunological research. Scientists utilize FIA to study immune responses in various experimental settings. The adjuvant facilitates the investigation of autoimmune diseases, allergies, and the mechanisms underlying the body's defense against infections.

Challenges and Ethical Considerations:

While Freund's Incomplete Adjuvant has proven invaluable in advancing immunological research, it's important to acknowledge the challenges and ethical considerations associated with its use. The emulsion can cause local reactions at the injection site, and some formulations may not be suitable for human use, necessitating careful consideration in experimental design and ethical approvals.

Future Perspectives:

As technology and our understanding of immunology continue to advance, researchers are exploring novel adjuvants and formulations that may offer enhanced efficacy and safety. Freund's Incomplete Adjuvant, however, remains a cornerstone in immunological studies, and ongoing research aims to optimize its use while addressing potential drawbacks.

Conclusion:

Freund's Incomplete Adjuvant stands as a powerful tool in the hands of immunologists, contributing significantly to vaccine development and our understanding of immune responses. This blog has provided a glimpse into the composition, applications, challenges, and future perspectives of this vital adjuvant, highlighting its role in shaping the landscape of immunological research.

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healthtimetaylor · 4 years ago

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Anti-rheumatic activity of phenethyl isothiocyanate via inhibition of histone deacetylase-1.

PMID: Chem Biol Interact. 2020 Jun 1 ;324:109095. Epub 2020 Apr 11. PMID: 32289289 Abstract Title: Anti-rheumatic activity of Phenethyl isothiocyanate via inhibition of histone deacetylase-1. Abstract: Rheumatoid Arthritis (RA) affects approximately 1% of the total world population. Despite incessant research and development of new therapeutic agents, management of RA is still a troublesome affair. Histone Deacetylase 1 (HDAC1) is an epigenetic regulator which play important role in pathogenesis of RA. In present study, we hypothesized that Phenethyl isothiocyanate (PEITC), a potent inhibitor of HDAC1, may ameliorate RA. Efficacy of PEITC was evaluated in Complete Freund's Adjuvant (CFA) induced arthritis model in rats. CFA (0.1 ml) was injected subplantarly in the left hind paw on day 0 to all the groups except normal control. The administration of test drug PEITC (10, 24&50 mg/kg) and standard drug Ibuprofen started simultaneously and was continued for 21 days. Paw edema, total arthritic index, mobility score, stair climbing ability, behavioral parameters, and bone erosion were evaluated. Further, radiographic studies, TNF-alpha as well as HDAC1 levels in synovial tissue homogenate and histological analysis were performed. Prophylactic treatment of PEITC attenuated paw edema, total arthritic index, mobility score, stair climbing ability, behavioral parameters, and bone erosion in dose dependent manner. Furthermore, there was significant decrease in TNF-alpha aswell as HDAC1 levels in synovial tissue homogenate. Histological analysis revealed no cartilage damage, bone erosion, hyperplasia at synovial lining as well as infiltration of inflammatory cells in treatment group. Results of this study suggest potent anti-rheumatoid arthritis activity of Phenethylisothiocyanate in CFA induced RA model in rats.

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juniperpublishers-endocrinology · 5 years ago

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Role of Nicotinamide in Streptozotocin Induced Diabetes in Animal Models- Juniper Publishers

Abstract

Diabetes mellitus (DM) is a metabolic disease with abnormal glucose homeostasis, due to defects in secretion or action of insulin. It is a potentially morbid condition with high prevalence worldwide and has become a major medical concern. Animal models play an important role in understanding such type of diseases. Diabetes in experimental animal develops either spontaneously or by using chemical, surgical, genetic or other techniques, and depicts many clinical features or related phenotypes of the disease. Streptozotocin (STZ) is a widely used chemical for the induction of experimental diabetes in animals. In this review, we have provided the evidences related to the fact that Nicotinamide play a protective role against destruction of pancreatic β- cells in STZ induced diabetes model in experimental animals.

Keywords: Diabetes mellitus; STZ; Nicotinamide

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Introduction

Diabetes mellitus (DM) is mainly characterized by hyperglycemia [1] with an imbalance of carbohydrate, protein and fat metabolism due to reduced production of insulin from pancreas (defects in insulin secretion), or when insulin cannot be used efficiently by the body (defect in insulin action), or both [1,2]. Two major forms of diabetes are Type I DM (TIDM) and Type II DM (TIIDM). TIDM is also termed as insulin dependent DM (IDDM) and TIIDM as non insulin dependent DM (NIDDM). TIDM is immune mediated and is characterized by destruction of pancreatic β-cells and requires daily administration of exogenous insulin [3]. TIIDM is the most prevalent form of diabetes, mainly characterized by partial destruction of pancreatic β-cells, which results in relative insulin deficiency/ resistance [2,4]. About 5-10% diabetic patients have TIDM as it affects 3 in 1000 children whereas TIIDM is considered to be the most prevailing form of diabetes, which accounts for about 90% of diabetic cases [5].

Animal models have been used widely to obtain knowledge of various pathological conditions related to diseases such as diabetes. STZ induced diabetes is considered to be the most reliable method. STZ induced structural, functional and biochemical alterations observed are similar to those which appear with diabetes in human [6]. Therefore STZ-induced diabetes represents a clinically relevant model to study the pathogenesis of diabetes and associated complication.

Streptozotocin (STZ)

STZ (2-deoxy-D-glucose derivative of N-methyl-N-nitrosylurea) is a glucosamine nitrosourea, hydrophilic compound which was first isolated from a soil microorganism Streptomyces acromogenes and showed broad spectrum antibiotic activity [7,8].

The diabetogenic action of STZ is related to its selective destruction of pancreatic β-cells, which are the only source of insulin in body. As STZ is glucose derivative, the blood glucose moieties enables STZ to be selectively transported to pancreatic β-cells via the low-affinity GLUT2 glucose transporter in the plasma membrane [9,10].

Exposure of STZ to pancreatic β-cells results in damage via different pathways:

STZ destructs β-cells by damaging the major macromolecule i.e. DNA by alkylating. Alkylation of DNA results in fragmentation of DNA in β-cells [10,11]. DNA injury by STZ leads to overstimulation of PARP-1 [(poly(ADP-ribose) polymerase-1] in the insulin-secreting cells and is harmful to the cell as a result of a substantial depletion of the intracellular PARP-1 substrate, NAD+. NAD+ is an important molecule implicated in energy metabolism at the cellular level [12,13].

STZ also decreases the activity of islet mitochondrial aconitase, reduces oxygen consumption by mitochondria and decreases the mitochondrial membrane potential [14].

Generation of nitric oxide may play a role in the cytotoxic action of STZ on insulin-secreting cells. This assumption is supported by results demonstrating that scavengers of nitric oxide (NO) attenuate early DNA-strand breaks induced by STZ [15].

STZ generates low amounts of ROS in pancreatic β-cells. These effects may partially contribute to β-cells damage induced by STZ because of a weak antioxidant defense in these cells [16,17].

It has also been revealed that c-Jun N-terminal kinase (JNK) is also involved in the cytotoxicity of STZ. Increased activity of this enzyme is observed in the case of cellular stress leading to cell death. Studies on insulin-secreting cells exposed to STZ demonstrated increased activity of JNK, whereas inhibitors of this enzyme attenuated the cytotoxic action of STZ. Activation of JNK by STZ is supposed to be preceded by increased activity of PARP-1 since PARP-1 inhibitors are able to decrease the activity of both PARP-1 and JNK [18].

Nicotinamide (NIC)

NIC (pyridine-3-carboxamide) also known as niacinamide, is an active and water soluble form of vitamin B3 (niacin). Niacine converted into NIC in the body and is a food additive. NIC is essential to the coenzymes NADH and NADPH and consequently for numerous enzymatic reactions in the body including formation of ATP. NIC has neuro-protective and antioxidant functions and is given to animals to partially protect pancreatic β-cells against STZ [19,20].

Numerous in vitro experimental studies conducted on isolated rat pancreatic islets have revealed that effects of STZlike reduction of pro-insulin biosynthesis, inhibition of glucosestimulated insulin secretion etc. were modulated by NIC [21-24] (Figure 1).

NIC shows its partial protection against STZ induced destruction of β-cells by two major mechanisms i.e. either by inhibiting PARP-1 [13,25] or by increasing the concentration of NAD+ (by reducing utilization of NAD+ or by increasing its biosynthesis) [26] (Figure 2).

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Type Of Diabetes Induced By STZ

The single intravenous dose between 40 and 60mg/kg body weight in adult rats can induce TIDM [27], but higher doses are also used. STZ is also efficacious after intraperitoneal administration of a similar or higher dose [28]. STZ may also be given in multiple low doses. Such treatment is used predominantly in the mouse and the induction of IDDM is mediated by the activation of immune mechanisms. The nonspecific activation of the immune system via complete Freund’s adjuvant prior to STZ injections allows to reduce its diabetogenic dose even in the rat [29,30].

NIDDM can easily be induced in rats by intravenous or intraperitoneal treatment with 100mg/kg body weight STZ on the day of birth. This method of NIDDM induction was described for the first time by Portha et al. [31]. At 8-10 weeks of age and thereafter, rats neonatally treated with STZ manifest mild basal hyperglycemia, an impaired response to the glucose tolerance test and a loss of β-cells sensitivity to glucose [32]. But in adult rats, if NIC is administered at a dose range of 100-500mg/kg prior to the administration of STZ it leads to cytoprotective effect. It should be emphasized that the effects induced in rats by STZ and NIC vary depending on the doses of these two compounds, the age of the animals and the time of NIC administration in relation to the administration of STZ [19,20]. Factors, such as the administration route of STZ and the nutritional state of rats may also have some influence. In the case of relatively low doses of NIC given to rats, its protective action is negligible. Conversely, high doses of NIC may provide total protection [8,27]. The age of rats is also of major importance since β-cells of younger animals are less sensitive to STZ and are better protected by NIC. It is also known that the protective action of NIC on β-cells decreases with the time elapsed after administration of STZ to rats. This decrease is supposed to result from ATP depletion since the uptake of NIC by β-cells is ATP-dependent. In the majority of experiments, NIC is given to rats 15min before STZ [32].

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Conclusion

STZ-induced diabetes is a clinically important animal model and most extensively used to mimic human diabetic condition. STZ can produce both types of DM i.e., TIDM and TIIDM depending on the dose and route of administration but couldn’t be affective every time. But the role of NIC is worth considering which can modulate the occurrence of type of DM via its cytoprotective action against the toxic effect of STZ at a particular dose.

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