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biomedgrid · 4 years ago

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Biomed Grid | Automatic Generation of Drug Concentration Gradient by a Modified Ink-jet Printer and its Application in Drug Screening

Abstract

Manual preparation of drug concentration gradient is labor-intensive and limits the efficiency of a drug screening to some extent, especially at large scale. In this study, we reported an automatic method to generate drug concentration gradient in 24-well plate. We loaded drug solution in a modified hot ink-jet printer, designed printing pattern in the WORD software, customized the concentration gradient in the WORD software by RGB coding, and finally printed the drug concentration gradient in the 24-well plate. A drug concentration gradient can be automatically generated within 5s in arbitrary pattern. Based on this flexible technology, we developed an ink-jet printer-based drug screening assay. Concentration gradient of 5-fluorouracil, as well as Mcf-7 cells, were printed in the 24-well plate, and the inhibition rate of Mcf-7 cells was measured by MTT method. This was the first time that hot inkjet printing technology was used in drug screening.

Keywords: Bioprinting; Drug screening; Concentration gradient; Ink-jet printer; RGB

Introduction

Preparation of a concentration gradient of drug candidate is a prerequisite to study cell-drug interaction quantitatively. Currently, this step is manually operated in the laboratory, which is labor intensive and annoying. Automation is desirable in practice. Derringer et al. developed an automatic method to generate concentration gradient [1], and Ye et al. [2] used this method in drug screening [2,3], however, this method was only applicable in microfluidic device, not in 96-well or 24-well plate, thus its applications were limited. Previously, we established drug screening device based on biomedical engineering methods [4]. In this study, we developed a method to generate concentration gradient in plate automatically, using normal commercial ink-jet printer and WORD software. We also used the same printer to print the cell suspension, thus we finally developed a drug screening assay based on hot ink-jet printing. Hot ink-jet printing technique is useful for printing cells in biomedical engineering. It works by the principle in which the heating element turns the “biological inks” into bubbles at bottom of the nozzle instantly and the pressure generated makes a certain amount of inks free from the surface tension there and pushed from the nozzle. As an approach for cell printing, hot ink-jet printer has been used in tissue engineering [5], biological microarray construction [6], stem cell research [7] and gene transcription [8]. However, there are few reports in its application in drug screening at cellular level

In this paper, a hot ink-jet printer was custom-modified as an automatic dispensing tool for the engineering of cell arrays and generation of concentration gradient of drugs in a 24-well plate. By means of this apparatus, cell/drug composite arrays can be automatically generated for drug screening. RGB value was used as the output gauge of drug concentrations for the first time. A Mcf-7 cell/5-fluorouracil composite array was engineered to demonstrate the applicability of the hot ink-jet printer in the drug screening.

Experimental

Materials

Hoechst 33342 fluorescence dye and 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Kubolai inc. Beijing. Propidium iodide was obtained from Jingchun technology inc. Shanghai. 5-fluorouracil was purchased from Sigma- Aldrich, United Kingdom. Phosphorate buffer saline (pH7.4) was prepared and sterilized in lab. DMEM cell culture medium and fetal bovine serum from HyClone, New Zealand, was used in this study. Methanol of HPLC grade from local company was used. Ultrapure water was obtained with Millipore water purification system.

Instruments

Canon PIXMA ip 1980 ink-jet printer with PG830 ink box from Canon, Japan, was modified in this study. Inverted fluorescence microscope IX71 from Olympus, Japan, was used. Agilent 1200 HPLC was from Agilent, USA. Synergy H1 microplate reader was from BioTek, USA (Figure 1). A Canon PIXMA ip 1980 printer and a Canon PG830 ink box for biological printing after modification. The white plate was a 24-well plate used as “paper”.

Modification of the Printer and the Cartridge

A Canon PIXMA ip 1980 hot-injet printer was modified as follows the front cover, paper support, rear tray, paper thickness lever and paper guide were dissembled sequentially while cartridge holder and paper sensor being kept intact. The printer after modification was shown as in Figure 1 (left part). Before use, the body of the printer was sterilized and kept inside an ultraviolet-lighted laminar hood overnight. A Canon PG830 ink box was modified as follows: after the top cover of the ink box was ripped from the main body of the box, the sponge inside the ink box was discarded and the metal filter at the bottom of the box was dismantled with the ink reservoir exposed, followed by a thorough cleanup. The ink box after modification was shown as in Figure 1 (right part). Prior to use, the box was emerged in 75% ethanol and air dried in a laminar airflow, followed by being exposed with ultraviolet light for at least 30 mins.

Figure 1: A Canon PIXMA Ip 1980 Printer and a Canon PG830 Ink Box.

Printing Concentration Gradient of 5-Fluorouracil in the 24-Well Plate

10ml 8mg/ml 5-fluorouracil solution was precisely obtained when 0.08g 5-fluorouracil was dissolved in PBS solution in a 10 ml volumetric flask and filtered through a 0.22 μm diameter membrane to remove possible bacteria. This working solution was loaded in the ink box. The printing pattern was designed in the WORD software with designated RGB values. Clicked the “print” button in the WORD software and the concentration gradient of 5-fluorouracil was automated created in the 24-well plate within 5s. We collected the drug solution printed in each well and analyzed their concentrations with HPLC. In a typical setup, a SB-C18 HPLC column (4.6mm diameter, 250mm long) was used and the sample was detected by ultraviolet absorption with a single wavelength of 265 nm. The temperature of the column was set at 35℃. The volume of each sample was 10 μl and the mobile phase was made of methanol and water (5:95) with a flow rate of 1.0ml/min.

Preparation and Printing of Cell Suspension

Mcf-7 cells of the third generation with a high viability was chosen as the main raw material for preparation of the cell suspension. Prior to being loaded into the ink box, a Mcf-7 cell suspension were prepared by pancreatic digestion, centrifuging, collection, counting and re-suspending. The Mcf-7 cell suspensions were prepared at four different concentrations, 3×106, 6×106, 9×106 and 1.2×107/ml. Following the pattern of a round spot with the diameter of 5 mm, the cell suspension with different densities were printed into a 24-well plate with the modified printer as above. Observation was made with an inverted microscope equipped with a X4 zoom objective.

Counting of Printed Cell Numbers and Viability Analysis

400 μl 10% NCBS culture media was added into the plate loaded with the printed Mcf-7 cells to keep their viability. And 10μl 0.4mg/ml Hoechst 33342 solution was added into the plate at 37℃ for 35mins in the dark, followed by the addition of 10 μl 0.2mg/ ml PI solution for another 10 mins. Then the cell suspension in the plate were collected and centrifuged. The cells from the bottom of centrifuge tube were collected and washed with PBS solution for 3 times. Then the cells washed were re-suspended in 1 ml cell culture medium to form a new cell suspension. 20μl new cell suspension was pipetted out and added dropwise onto a cell counter. Counting of cell number and observation of cells were conducted with an inverted fluorescence microscope with a X 10 zoom objective.

Procedure of Drug Screening

Mcf-7 cells with a high viability was selected for construction of cell/drug arrays. The Mcf-7 cell suspension were prepared by pancreatic digestion, centrifuging, collection, counting, and resuspension with a concentration of 9×106/ml. The Mcf-7 cell suspension was loaded into sterilized a PG830 ink box and printed into 5-mm-diameter round spots in a 24-well plate to form a Mcf-7 cell array. 400μl 10% NCBS culture media was immediately loaded into the plate. After being cultured at 37℃ under 5% CO2 for 24hrs, 8 mg/ml 5-fluorouracil solution were printed into the plate which were already loaded with Mcf-7 cells, with different RGB codes. As a result, cell/drug composite arrays (n=3) were formed. After being at 37℃, 5% CO2 for another 48 hrs, 40μl 5mg/ml MTT solution were loaded into the plate and incubated at 37℃ for 4hrs. Then the supernatant was discarded and 500 μl DMSO was added. The plate was shaken for 15mins to achieve DMSO thoroughly dissolved. Then 200 μl supernatant was collected and transferred into a 96-well plate and the adsorption under 490 nm wavelength was recorded.

Results

Printed Concentration Gradient of 5-Fluorouracil

The printing pattern was designed in WORD software, as shown in Figure 2. Each round spot, corresponding to a well in 24-well plate in location, was 5mm in diameter. These round spots varied in RGB values. We printed drugs following the pattern in Figure 2 and measured the concentration of drug in each well. We plotted the drug concentration in each well, as shown in Figure 3. This technology has five advantages compared to manual method

a) It is automatic;

b) We can obtain arbitrary concentration gradient by customizing the RGB values (Figure 3 A-D);

c) The RGB code value was linearly related to the concentration of printed 5-fluorouracil. This can be expressed as Y = -0.7691X+53.975 with R2 being equal to 0.9878 (Figure 3A);

d) It is fast, and a concentration gradient can be generated within 5s;

e) THE hot inkjet printer was cheaper than a micropipette which is used for manual preparation. (Figure 2)

Figure 2: The Printing Pattern Designed in The WORD Software.

Figure 3: Different Types of Drug Concentration Gradient Printed in the 24-Well Plate.

The printing pattern designed in the WORD software. Each row was designed for generation a specific drug concentration gradient. In the first row, the RGB values from left to right were (20,20,20), (30,30,30), (40,40,40), (50,50,50), (60,60,60), (70,70,70). In the second row, the RGB values from left to right were (70,70,70), (20,20,20), (70,70,70), (70,70,70), (20,20,20), (70,70,70). In the third row, the RGB values from left to right were (20,20,20), (20,20,20), (50,50,50), (50,50,50), (70,70,70), (70,70,70). In the fourth row, the RGB values from left to right were (70,70,70), (70,70,70), (50,50,50), (50,50,50), (20,20,20), (20,20,20). (Figure 3) On the top of each plot was the printing pattern designed in the WORD software, and the number below was RGB value. (Figure 4) (from left to right: 3×106/ml, 6×106 /ml,9×106 /ml), scale bar: 500 μm.

Printing of Mcf-7 Cell Suspension

Figure 4: The Bright Field Images of Printed Round Spots of Mcf-7 Cells with Varied Cell Concentration.

Figure 4 shows the printed pattern of Mcf-7 cells, with different concentration of Mcf-7 suspension loaded in the printer. When the concentrations were 3×106 /ml and 6×106 /ml, the printed round spots were incompletely constructed with sparse distributions of cells. It might be caused by the low concentration of cell suspension. When the concentration was 1.2×107/ml, few inks were injected from the nozzle and the printed round spot was hard to be identified. It might be attributed to the high concentration of the cell suspension which jammed the nozzle. However, when the concentration was 9×106/ml, the printed round spot was full of well-distributed cells. It showed that the cell suspension with a concentration of 9×106/ml is the best-performed one and should be adopted in the experiment.

After counting, the average number of cells in each round spot in the cell array was 3×104. After printing, the viability of the cells was the main gauge which reflects the validity of the cell printing process. Hot ink-jet printing was suspected to cause heat damage or mechanical damage on cells [8]. Hoechst33342 is a membrane permissive dye and emits blue fluorescence under 350 nm excitation after combining with DNA in cells. And PI is only capable of penetrating into dead cells. After PI combines with DNA in cells and emits red fluorescence under 543 nm excitation. Therefore, we can investigate the viability of cells when coupling Hoechst33342 and PI dyes after cell printing. As seen in Figure 5, the number of dead cells which emit red fluorescence is negligible and it supports that the negative effect of our cell printing technique on the viability of printed cells can be ignored (Figure 5).

Figure 5: Microscopic observations of printed cells dyed by Hoechst33342(left) and PI (right), under excitations of 350 nm and 543 nm wavelengths, respectively. Scale bar: 500 μm.

Drug Screening Assay Based on Printing

Table 1: Absorbance by MTT and relative inhibition rate (n = 3).

The concentration of the printed 5-fluorouracil can be pretuned with the RGB code setup from the WORD software. A Mcf-7 cell/5-fluorouracil composite array was formed after incubation for 48 hrs. And the inhibition rate of the experimental group at varied concentrations and the control group were tested with MTT method. And the statistics of their optical absorbance at different RGB values and the relative inhibition rates were listed in Table 1. It can be seen from Table 1 that within the range we investigated, the biological viability of the cells in the composite array increased as the RGB value ramped up. In our experimental setup in which RGB code value was kept between 20 and 70, the concentration of the printed 5-fluorouracil increased as the RGB value dropped. In another word, a low RGB value yielded a cell/drug composite array with a relatively high concentration and dose of 5-fluorouracil. Therefore, as the RGB value was lowered, a stronger interaction between Mcf-7 and 5-fluorouracil was achieved, and a higher inhibition phenomenon occurred. This suggested that the Mcf-7 cell/5-fluorouracil composite array established by our method can be used for the inhibition study of 5-fluorouracil at different concentrations and it possessed the potential of evaluating in vitro cellular viability.

Discussion

Cell printing technique has demonstrated remarkable advantages as following :

a) High precision, cells can be precisely localized and distributed in three dimensions [9];

b) Rapid speed, tissue samples with biological viability can be constructed in a short time;

c) High automation grade, it has the possibility to be industrialized at a large scale [10];

d) Simple to be established and operated, it is straightforward to be modified or upgraded [11].

In our experiment, RGB tuning was used for controlling the output doses of 5-fluorouracil and the hot inkjet technique was adopted to build cell/drug composite arrays. And the viability of the cells in the composite arrays were evaluated. Inspired by the model from Boland ‘s group [11,12], a Canon hot inkjet printer and an ink box were successfully modified into a biological printer. And a Mcf- 7 cell suspension with a concentration of 9×106 cells/ml showed the best printing performance in cell distribution and density on the array. The printed cell array demonstrated patterns with the most complete morphology. By testing the concentration of drug solutions with HPLC, we were able to identify the concentration of 5-fluorouracil on the printed matrix produced from different RGB code value. The results show that high-definition Mcf-7 cell arrays can be constructed with our modified printer and ink box. And moreover, the concentration of 5-fluorouracil arrays can be tuned by changing the RGB color code value. The viability of cells by printing is the major indicator for the feasibility of the printing operation. We used Hoechst33342 and PI to dye the printed cells and applied excitations of 350 nm and 543 nm, respectively. The results showed that hot inkjet printing has little damage on the Mcf-7 cells, and they are in consistency with the literatures [13,14]. Also, we used MTT method to identify the inhibition rate of 5-fluorouracil on Mcf- 7 in the cell/drug array. When RGB code value was 30, the dose of the 5-fluorouracil was 30.09±2.69 μg/ml with a relative inhibition rate of 49.02%. These results supported the feasibility of applying the cell/drug printing method in investigating in vitro cellular viability and it provides new possibility of drug screening based on biological printing.

Conclusion

In this study, we developed an automatic method to generate drug concentration gradient in 24-well plate based on hot inkjet printer. The total time was less than 5s. Arbitrary concentration gradient can be easily generated by RGB coding. The cost of printing equipment was very low. This method is expected to make drug screening experiment more user-friendly.

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juniperpublishersophthalmology · 4 years ago

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Juniper Publishers- JOJ Ophthalmology

A Review of the Data on the Recently Approved Xen Surgical Gel Stent in the Management of Glaucoma- Juniper Publishers

Abstract

The cornerstone of glaucoma surgery includes trabeculectomy and tube shunting procedures, which utilize an ab externo approach to divert aqueous humor from the anterior chamber to the sub conjunctival space. The XEN Gel Stent is a 6.0mm tube consisting of porcine-derived collagen that similarly creates a non-physiologic shunt but through an ab interno approach. The XEN gel stent has the potential to effectively lower intraocular pressure and medication use with lower complication rates than traditional glaucoma surgery. This mini-review surveys the data of the XEN implant in current literature.

Introduction

Glaucoma maintains a significant disease burden worldwide. It is the most common cause of irreversible blindness, affecting over 64 million individuals [1]. Therapy is focused on lowering intraocular pressure (IOP) by a variety of methods, including topical medications, laser, and incisional surgeries. These surgical interventions-traditionally trabeculectomy or tube shunt surgery-rely on creating an additional subconjunctival reservoir for aqueous humor (AH) drainage and subsequent resorption. However, the three-year results of the Tube Versus Trabeculectomy Study found failure rates of 15 and 28 percent, respectively. Serious postoperative complications such as persistent corneal edema, endophthalmitis, and chronic or recurrent iritis were also reported [2].

There has been a recent proliferation in procedures and medical devices that provide similar IOP-lowering effects to trabeculectomy or tube shunt surgery with fewer complications. One such product is the XEN Glaucoma Treatment System (Allergan, Inc., Irvine, CA, USA), which consists of the XEN Gel Stent and XEN Injector. The XEN stent is derived from porcine collagen, measuring 6.0mm long with inner diameters of 140|im, 63|im, or 45|im, although the 45|im stent is currently recommended. Previous studies in animal models have demonstrated no significant inflammatory response to implantation and no signs of degradation of the stent itself [3]. It was recently approved by the United States FDA in November 2016 for use in refractory glaucoma, including those with a history of failed prior surgical treatment, primary open-angle glaucoma (POAG), and pseudoexfoliative glaucoma with open angles that are inadequately controlled on maximal medical therapy.

The XEN stent is inserted into the angle through the scleral spur via an ab interno approach with its disposable injector. Ideally, the device should extend 3.0mm posteriorly from the limbus and into the subconjunctival space and 2.0mm anteriorly into the anterior chamber (AC). Once placed in an aqueous environment, the device hydrates, becoming soft and flexible, helping to maintain its position. By taking advantage of the resistance to flow in a cylinder as determined by the Hagen- Poiseuille equation, the XEN45 produces a pressure gradient 7.56mm Hg at the physiologic flow rate of AH within the AC, at 2.5 microliters/minute [4]. Thus, this stent can theoretically reduce the risk of hypotony seen in IOP-lowering surgeries despite being a valve less device. The fact that it is inserted ab interno obviates the need from conjunctival dissection, theoretically reducing the potential for conjunctival fibrosis and leaving the ophthalmologist with the option to perform ab externo surgeries if necessary in the future.

XEN Gel Stent in the Literature

Several studies were performed evaluating the XEN Gel Stent with multiple inner diameters. Sheybani, Dick, and Ahmed reported on the results of 49 eyes of 49 patients treated with the XEN140 stent (140um internal diameter) without the use of mitomycin C (MMC) [5]. Of the 49 patients, 22 received previous glaucoma surgery, and nine (18%) had a prior laser trabeculoplasty. Complete success was defined as an IOP <18mm Hg and a greater than 20% reduction of IOP at the primary endpoint of 12 months without glaucoma medications. Criteria for treatment failure included visual acuity less than or equal to light perception, need for additional glaucoma surgery, or a less than 20% reduction of IOP at 12 months. This study revealed that the mean IOP reduced from 23.1±4.1mm Hg preoperatively to 14.7±3.7mm Hg at 12 months, a 36.4% decrease. In all, 40% met the criteria for complete success and 88.9% for partial success. Three patients (6%) failed the study criteria and required additional surgery. The most common complication was needling (47%), with nearly half of the cases occurring within the first month.

Another early study by Sheybani and Ahmed used the XEN140 and XEN63 stent (63um internal diameter) without MMC in patients undergoing phacoemulsification [6]. Of the 37 eyes, 47.1% were considered complete successes while 85.3% were qualified successes. Mean IOP significantly decreased from 22.4±4.2mm Hg preoperatively to 15.4±3.0mm Hg at 12 months, and mean medications were significantly reduced from 2.5±1.4 to 0.9±1.0.

One of the first reports involving the exclusive use of the XEN45 stent (45um internal diameter) involved 31 eyes receiving phacoemulsification and MMC treatment at the time of implantation. Mean IOP and medication use were reduced significantly from 20.8±4.6mm Hg to 13.1±3.6mmHg at 12 months and 2.7±1 to 0.9±1.1 at 12 months, respectively, and without significant complications [7].

Pérez-Torregrosa et al. [8] were among the first to describe the efficacy of the XEN45 in patients with mild and moderate glaucoma (defined as a mean deviation between 0 and -12dB on Humphrey 24-2 perimetry) [8]. Additionally, subjects included in this study had pressures<30 while being managed on two or more medications. Twelve months after concomitant phacoemulsification and XEN45 implantation, 27 of 30 (90%) subjects met the successful treatment criteria of IOP≤18mm Hg with no glaucoma medications. The authors reported several intra operative complications, including sub conjunctival hemorrhage with MMC instillation (36.6%), and minor hemorrhage intra camerally (86.6%) and at the scleral exit point (90%). A total of six (20%) stents required relocation, and one eye required re-implantation of the device. One subject was excluded from analysis due to extensive subconjunctival hemorrhage after MMC injection and another due to extrusion of the device into the subconjunctival space intra operatively.

Two studies have addressed the effect of simultaneous implantation and phacoemulsification versus the XEN alone. One such study involved 567 eyes, of which 54% underwent XEN implantation only, and 46% the combined implantation and phacoemulsification [9]. There were no inclusion or exclusion criteria regarding the grade of POAG, and data from all three diameters of XEN stents were not separated. The data revealed a mean preoperative IOP of 21.9±4.2mm Hg that was significantly decreased at 12 months (15.7mm Hg), 24 months (15.0mm Hg), and 36 months (13.2mm Hg) of follow-up. Likewise, a significant decrease in medications by 74, 77, and 74 percent from the mean of 2.7 was observed at 12, 24, and 36 months, respectively. The percentage of patients who were converted to another procedure was 4% by 12 months, 5% by 24 months, and 5% by 36 months. After analysis, it was determined that there was no statistical difference in mean IOP or medications between eyes receiving the implant alone and the combination procedure. Likewise, a 75 patient cohort demonstrated no significant difference in IOP reduction between the standalone and combination procedures at 12 months [10]. The most frequent complications from this study were needling (15.4%) and hypotony defined as an IOP <6mm Hg on postoperative day 1 (12.6%).

The most recent article involving the XEN stent evaluated its use in eyes with suboptimal IOP and medication intolerance, medication noncompliance, or maximum therapy with no history of glaucoma surgery [11]. A total of 13 eyes underwent XEN45 implantation in addition to phacoemulsification if previously phakic. At the 12-month end point. 41.7% were complete successes, with a>20% drop in IOP and discontinuation of all glaucoma medications, while an additional 25% met the IOP goal but remained on at least one medication. Among the reported complications, four eyes required needling, two eyes developed choroidal detachment and hypotony requiring systemic steroids and atropine treatment, one implant extruded, and two eyes required subsequent trabeculectomy.

Discussion

The current literature available for the XEN Gel Stent demonstrates its effect on IOP and medication reduction in addition to reducing severe intra operative and postoperative complications. The data also suggests a lower early failure rate than those published from trabeculectomy and tube shunt surgery. There remains little data on long-term outcomes of the XEN stent at this time, inherent with a newly approved procedure. Thanks to its earlier implementation outside the USA, data are available for the XEN from mild to refractory glaucoma. However, this data should be evaluated in light of the fact that some studies were performed using the XEN140 and XEN63 stents, which are no longer recommended by the manufacturer.

Of the complications encountered with the XEN stent, subconjunctival hemorrhage and needling were most common, and extrusion of the device was the most common serious complication. It should be noted that the rates of needling varied widely in the literature, from 15.4% to 47%. This could be due in part to varying degrees of experience with the XEN stent and injector as well as different technical approaches such as the use and dose of anti fibrotic used at the time of implantation.

It is well known that cataract extraction provides some improvement in IOP [12]. Thus, the efficacy of the XEN stent may be confounded by those studies in which patients also underwent phacoemulsification. In fact, higher preoperative IOP, older age, and greater anterior chamber depth have all been shown to correlate to the amount of IOP improvement with phacoemulsification in medically managed glaucoma patients [13]. However, two studies with the XEN stent reported no significant difference in IOP reduction and medication use postoperatively between XEN implantation combined with phacoemulsification and XEN implantation alone. Indeed, this is one relationship that will be unlikely to be discerned until more data from more eyes is available for analysis.

Looking forward, several clinical trials in various stages of progress will look to further elucidate the properties of the XEN device and its role in the management of glaucoma. One trial - NCT02036541 - is a Phase 3 trial currently past its primary completion date that will further assess the XEN45 in patients with refractory glaucoma. Another is a parallel assignment, Phase 4 trial for XEN45 in moderate POAG patients (NCT02006693). Yet another study, soon to begin enrollment, will assess the in vivo effects of various interventions on outflow at Schlemm's canal, of which the XEN stent is part of the surgical branch (NCT02807935).

Conclusion

At present, there are multiple surgical options available for effective glaucoma management. The XEN Gel Stent is one such intervention that provides advantages over traditional glaucoma surgery and newer minimally invasive glaucoma surgery (MIGS) procedures while lowering IOP and medication dependence. The device is well tolerated by the ocular tissues and can be placed via an ab interno approach but still creates a non- physiologic subconjunctival shunt or bleb to increase aqueous outflow without the use of a valve system. Since it involves little manipulation of the conjunctiva, implantation of the XEN stent does not preclude future conjunctival surgeries if necessary. Questions remain as to what long-term outcomes of the XEN Gel Stent will demonstrate, as well as which types of glaucoma patients stand to benefit the most from the surgery. Larger-scale studies are needed to resolve these questions and confirm the initial optimistic results.

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juniperpublishersjcmah · 5 years ago

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Different Drugs in Modulating Gut Microbiome of Colitis Mice Abstract Background: The gut microbiome plays a key role in Inflammatory Bowel Disease (IBD), which has spurred the development of novel therapeutics aimed at restoring microbial community structure. Saccharomyces boulardii is a new probiotic that has not been commonly used to treatment IBD. Although the traditional Chinese herbal medicine Sijunzi Tang (SJZT) decoction has been widely used to alleviate the symptoms of ulcerative colitis (UC), its underlying mechanism remains unknown. Methods: The efficacy of four drugs named S. boulardii, SJZT, Dangshen (Codonopsis pilosula) polysaccharide or S. boulardii in combination with Dangshen polysaccharide were test during treatment with Dextran Sulphate Sodium (DSS)-induced mice colitis. Weight loss, colonic histology were measured. Furthermore, the gut microbiome of mice before and post colitis treatments were performed by 16S rRNA-based microbiome analysis. Results: All four drugs profoundly inhibited weight loss, colon shortening, and ameliorated histological damage as well as DSS-induced dysbiosis in mice. Beneficial bacteria-especially Short Chain Fatty Acid (SCFA)-producing genera were remarkably enriched in the treated mice. Roseburia, Anaerovorax and Lactobacillus were selectively enriched by S. boulardii, while Butyrivibrio, Quinella and Anaerotruncus were selectively enriched by SJZT. Most notably, Dangshen polysaccharide selectively enriched Bifidobacterium, Arthrobacter, Akkermansia, Anaerovorax, Roseburia, Prevotella, Dialister, Megamonas, Faecalibacterium and Subdoligranulum. Conclusion: S. boulardii, Sijunzi decoction, and its main component, Dangshen polysaccharide, are effective in alleviating DSS-induced colitis. Modulating the gut microbiome may be the common mechanism whereby these compounds improve colonic health. Furthermore, Dangshen polysaccharide is a powerful prebiotic that selectively promotes probiotic growth, especially SCFA-producing bacteria. Keywords: Saccharomyces boulardii; Polysaccharide; Gut microbiome; Ulcerative colitis Abbrevations: IBD: Inflammatory Bowel Disease; UC: Ulcerative Colitis; CD: Crohn’s Disease; CAMS: Complementary and Alternative Medicines; SJZT: Sijunzi Tang; DSS: Dextran Sulfate Sodium; OTUS: Operational Taxonomic Units Go to Introduction Inflammatory Bowel Disease (IBD) is the term used to describe chronic intestinal inflammatory conditions, including Ulcerative Colitis (UC) and Crohn’s Disease (CD). While IBD is especially common among western populations, the incidence of IBD in China has increased rapidly in recent years because of the widely adopted westernized lifestyle [1]. The etiology and pathogenesis of IBD are not fully understood, and effective therapeutics are lacking. A healthy, balanced intestinal microbiome plays a key role in maintaining whole-body homeostasis. Significant alterations to the gut microbiome, referred to as dysbiosis, are present in most chronic inflammatory disease. Intestinal dysbiosis is a defining feature of IBD, with some even considering it to be causative. Indeed, manipulating the gut microbiome has been shown to greatly alleviate the symptoms of IBD [2]. Pro- and prebiotics are two widely adopted therapeutic strategies that are also great examples of Complementary and Alternative Medicines (CAMs) being used in clinic. Traditional Chinese medicines have also shown remarkable curative effects as CAMs in IBD patients. Sijunzi Tang (SJZT) decoction is a classical herbal formula composed of Dangshen [Codonopsis pilosula (Franch.) Nannf], Baizu (Atractylodes macrocephalae Koidz), Fuling [Poria cocos（Schw. Wolf] and Gancao (Glycyrrhiza uralensis Fisch). Related researches claimed that SJZT were efficiently in treating UC patients in China. Its mechanism of action however remains unknown. Recent study suggests that Chinese herbal medicines could potentially be used to modulate the intestinal microbiome. Treatment with Gegen Qinlian decoction for example promotes the growth of beneficial bacteria, especially Faecalibacterium prausnitzii, to alleviate type II diabetes [3]. We therefore hypothesized that SJZT decoction could also relieve IBD symptoms by altering the gut microbiome. Furthermore, based on the theory of Chinese medicine, water- soluble polysaccharides are the key compounds in decoctions. Dangshen polysaccharide, a macromolecular substance that is hard to digest, consists of monosaccharides like D-galactose, D-rhamnose and D-arabinose and their derivatives, a backbone of (1→3)-linked-β-D-galactopyranosyl, (1→2,3)-linked-β-Dgalactopyranosyl and (1→3)-linked-α-D-rhamnopyranosyl residues [4]. Based on the fact that herbal polysaccharides such as Lentinula edodes [5] and Ganoderma lucidume [6] are very effective at modulating the gut microbiome, we hypothesized that non-digestible Dangshen polysaccharide might favor the growth of specific, beneficial microbes, i.e. act as a prebiotic. The fungi Saccharomyces is a new probiotic that is resistant to antibiotics and low pH and grows very well at 37°C [7]. Although widely used in preventing and treating diarrhea, it is rarely used to treat IBD. Most studies on S. boulardii in animal colitis models focus mainly on immune-related mechanisms [8-9]. Recently administration of S. boulardii has been reported to exert its therapeutic effects by altering the gut microbiome in obese and type 2 diabetic mice [10]. Whether S. boulardii can change microbial community structure to relieve IBD symptoms remains to be seen. The aim of this study was to determine the effect of Sijunzi decoction, Dangshen polysaccharides and Saccharomyces boulardii on microbial diversity and composition during the treatment of DSS-induced acute colitis in mice, using high-throughput 16S-based sequencing. Go to Material And Methods polysaccharide preparation S. boulardii sachets were purchased from Laboratoires BIOCODEX (Bio flor®, France). In order to remove any contaminants a pure S. boulardii culture was obtained by inoculating the lyophilized yeast in YPD broth with shaking at 37°C for 18 hours; S. boulardii was then harvested and concentrated tenfold to 109CFU/ ml. The yeast was centrifuged and re-suspended in PBS solution for oral administration. The four dried raw materials used to prepare the SJZT decoction (Dangshen, Baizu, Fuling, and Gancao) were all purchased from Xukang pharmaceutical co., ltd (Lanzhou, China). The quality were all met the requirement of Chinese Pharmacopoeia (2015 Version). The voucher specimens number were DS-20150721, BZ-20150701, FL-20150718, GC-20150726 respectively. All specimens were identified by senior engineer Pingrong Yang and deposited in the Herbarium of Chinese patent medicine test laboratory of Gansu Institute of Drug Control, Lanzhou, China. Four materials mixed in a ratio of 9:9:9:6 and herbs were decocted twice with boiling water (10 times of the material’s weight) for 60min then filtered, pooled and concentrated to 1g/ ml. Mice were orally administered 10ml/kg (convert from clinical dose). Quality control of the SJZT decoction was performed by high performance liquid chromatography (HPLC) analysis of glycyrrhizic acid and liquiritin from Gancao, according to an established method [11]. Briefly, chromatographic separation was performed on a Zobax SB-C18 column (4.6mm×150mm, 5μm); the mobile phase consisted of methyl alcohol -0.5% acetic acid; the flow rate was 1ml/min; the column temperature was 30°C; liquiritin was detected at 276nm, and glycyrrhizic acid at 250nm. The crude polysaccharide extract was obtained from Dangshen by water decoction and alcohol precipitation. Total carbohydrate content was then determined by phenol-sulfuric acid assay based on a standard curve determined from D-glucose at six different concentrations (μg/ml). Animal experiments and treatment design The present animal study was conducted according to protocols approved by the Ethics Committee of Animal Experiments of Lanzhou University. Female C57BL/6 mice were purchased from the Gansu university of Chinese medicine. After acclimatization for one week, mice were housed individually in plastic cages at constant temperature (21±2°C), with an alternating 12h light/ dark cycle, and animal chow and water were provided ad libitum. mice were divided into six groups and treatments lasted for 30 days A. Dangshen polysaccharide (dissolved in PBS solution, 300mg/ kg) B. Saccharomyces boulardii (resuspended in PBS to a concentration 109CFU/ml) C. Dangshen + S. boulardii, (Dangshen polysaccharide dissolved in an S. boulardii suspension) D. Sijunzi (SJZT decotion (1g/ml) at 10ml/kg; dose selection was based on that used in clinic patients) E. Colitis (DSS, PBS solution) F. Normal (no DSS, PBS solution) After 21 days of treatment, acute colitis was induced by administering 2.5% DSS solution to groups A-E for 7 days, followed by distilled water for two days. All drugs (groups A-D) were adminis tered throughout the study (see experimental design in Figures). Fresh fecal samples were collected immediately upon defecation on days 1(blank) and 21(before colitis induction), placed in sterile centrifuge tubes and stored at-80°C. On day 30 (post-colitis) mice were sacrificed and intestinal fecal samples were collected from the colon. To evaluate the therapeutic effects of the four different treatments, mice body weight was monitored daily, and following a final assessment of colon length, inflamed colon tissues were treated with formalin and stained with hematoxylin and eosin. Inflammation was graded according to an established system [12]. DNA isolation and illumina pyrosequencing of the V3 16S rRNA gene region Microbial DNA was extracted from fecal samples using the Qian Gen@ Stool DNA kit (Beijing, China) according to the manufacturer’s instructions. DNA concentration and purity were quantified on a Nano Drop 1000 spectrophotometer (Thermo Scientific) and DNA integrity was confirmed by agarose gel electrophoresis. Universal primers were selected to span the V3-V4 hypervariable region of the 16S rRNA gene: 338F (5’-ACTCCTACGGGAGGCAGCA- 3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). In brief, PCRs were performed in 20μl reactions containing 5x Fast Pfu Buffer (4μl), 10 ng template DNA, 2μL dNTPs, 0.8μl forward and reverse primer (5μl), 0.4μl Fast Pfu Polymerase and ddH2O to 20μl. The PCR parameters were: 95°C for 3 min, 27 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 45 seconds, followed by a final extension of 72°C for 10 min. PCR products were detected on a 2% agarose gel and quantified by Quanti Fluor™-ST and all samples were pooled at mean concentrations. Library preparation and pyrosequencing were performed on an Illumina Mi Seq PE 300 platform by Shanghai Majorbio Technology Company. Bioinformatic and statistical analyses High quality sequences were assigned to each sample (demultiplexed) and clustered as Operational Taxonomic Units (OTUs) at a threshold of ≥ 97% similarity. OTU abundance data was used to calculate alpha diversity (Shannon index), richness and rarefaction estimates using Mothur [13]. Community structure was assessed using Uni Frac Principal Coordinate Analysis (PCoA), Nonmetric Multidimensional Scaling (NMDS) based on Bray-Curtis distance, and hierarchical clustering, using R packages vegan. Heatmaps were created using Mothur and R package heatmap [14] to visualize treatment-specific changes in the microbiome. All results are presented as the mean (±SE). Group differences were assessed by ANOVA and the Mann-Whitney test in SPSS19.0. P-values < 0.05 were considered significant. Go to Results Preparation of probiotic, Sijunzi decoction, Dangshen polysaccharide An S. boulardii culture was prepared from a commercial product and typical morphologic characteristics of yeast was observed (Figure 1A). Dangshen polysaccharide constituted 38.7% of the crude, brown Dangshen polysaccharide powder (Figure 1B). HPLC-based quality control of the Sijunzi decoction was performed using glycyrrhizic acid and liquiritin as indicator components (Figure 1C & 1D). The content of glycyrrhizic acid in SJZT (1g/ml) was 13.5μg/ml while that of liquiritin was 5.8μg/ml. Click here to view Large Figure 1 Different treatments alleviate DSS-induced colitis in mice An outline of the animal experiments and treatment design is shown in Figure 2. Before induction of colitis, four different treatments were administered orally for three weeks. Body weight was measured daily (from day 1) and all groups showed a steady increase in body weight. However, Dangshen and especially SJZT attenuated weight gain compared vs normal group (SJZT P<0.05). By day 20, body weight was increased in the S. boulardii and D+S groups (Figure 3A). After induction of colitis (2.5% DSS solution for 7 days) the four treatments were continued administered until the end of study. All treatments significantly inhibited weight loss by day 30 relative to the DSS-only group (P<0.05), especially S. boulardii and D+S significantly (P<0.001) (Figure 3B). Total colon length decreased in all DSS-treated mice relative to controls, while treatment groups A-D showed a slight increase in colon length relative to the DSS-only group (P<0.05, Figure 3C & 3D). Click here to view Large Figure 2 Click here to view Large Figure 3 The degree of colonic inflammation was further confirmed by histological analysis. While control mice had intact surface epithelia, stroma, cryptal glands, and submucosae, DSS-treated mice showed surface epithelium damage, cryptal gland disruption and infiltration of lymphocytes. All treatment groups had significantly lower histology scores than DSS-treated mice (P<0.05). In the S. boulardii and Dangshen + S. boulardii groups, surface epithelium and cryptal glands were more intact than in the Dangshen and SJZT groups (Figure 4A). There was no obvious infiltration of inflammatory cells in any of the treated groups, especially not in the D+S group (Figure 4B). Click here to view Large Figure 4 Gut microbiome structure in response to different treatments in DSS-induced colitis Sequencing the V3-V4 16S hypervariable region produced 2387053 high quality sequences from fecal samples collected on days 1, 21 and 30, with a mean of 43400±1901 sequences per sample. The mean number of OTUs per sample was 373. Rarefaction and diversity analysis show that the majority of the gut microbial diversity was captured at the current sequencing depth, with few new OTUs anticipated at increased sequencing depths (Figure 5A). All treatments increased gut microbiome richness in mice by day 21 relative to baseline levels at day 1 (Figure 5B), especially D+S (P<0.01), Dangshen and S. boulardii (P<0.05), and to a lesser extent SJZT (P>0.05, Figure 5C). Post-treatment (day 30), all groups had slightly more unique OTUs than DSS-only mice, but this was not significant (Figure 5D). In terms of diversity, the two polysaccharide-rich groups (Dangshen, D+S) had significantly higher Shannon indices than control mice before colitis (Figure 5E P<0.05), while the SJZT group appeared unchanged. Acute colitis rapidly decreased diversity however, the four treatments all significantly increased diversity (Figure 5F, P<0.05). The overall gut microbiome structure in response to the various treatments were further analyzed by uniFrac distance-based PCoA, which revealed that control mice had distinct microbiomes compared to all the other groups. All four treatments, except for a few outlier samples, modulated the colitis-associated microbiome, and the different treatments tended to form separate clusters (Figure 6A). NMDS analysis (Bray-Curtis distance) supported the PCoA-based result and further showed that bacterial communities in the Dangshen group are more homogenous than other groups, followed by the SJZT group (Figure 6B). These findings were confirmed by hierarchical clustering analysis and suggest that the four treatments alter the microbiome in the inflamed colon (Figure 6C). Click here to view Large Figure 5 Click here to view Large Figure 6 Gut microbiome composition in response to different treatments in DSS-induced colitis At baseline (day 1), Firmicutes, Bacteroidetes and Proteobacteria were the three, Firmicutes, Bacteroidetes and Proteobacteria are the three most abundant phyla, with Firmicutes and Bacteroidetes accounting for about 91% of reads. Following treatment, both Dangshen (74.97% vs 79.35%) and Sijunzi (75.43% vs 79.82%) groups had increased proportions of Bacteroidetes. Meanwhile, Bacteroidetes were decreased in the S. boulardii (81.98% vs 65.48%) and D+S (75.48% vs 70.97%) groups. Firmicutes were less abundant in the Dangshen (13.10% vs 12.88%) and SJZT groups (16.86% vs 10.64%), while the S. boulardii (11.70% vs 16.37%) and D+S (12.62% vs 16.52 %) groups had an increased proportion of Firmicutes. Proteobacteria was only decreased in the Dangshen group (9.03% vs 6.82%), and increased in all three other groups, especially in the S. boulardii group, where it was increased more than three-fold (4.62% vs 17.14%, P<0.05. Verru comicrobia was significantly decreased (P<0.01) in all treatment groups (Figure 7A). DSS administration led to a remarkable decrease in Firmicutes (17.77% vs 2.36%, P<0.05) and Proteobacteria (5.18% vs 2.52%, P<0.05), with a significant increase in Bacteroidetes (59.65% vs 78.98%, P<0.05) relative to controls. These changes could however be mitigated by the treatments, especially by Dangshen polysaccharide which increased the proportion of Firmicutes (group A: 31.54%, group C: 32.90% vs DSS-only: 17.77%, P<0.05) and decreased the proportion of Bacteroidetes (group A: 57.11%, group C: 61.66% vs DSS-only: 78.98% P<0.05), Figure 7B. Relative to the DSS-only group, the Dangshen group had a significantly higher proportion of Proteobacteria (9.65% vs 2.52%, P<0.05), while Verrucomicrobia was slightly increased by Dangshen and SJZT (0.54%, 0.047% vs 0, P<0.05). Click here to view Large Figure 7 The Firmicutes to Bacteroidetes ratio (F/B) is used as an indicator of the gut microbial composition and was used to evaluate colitis remission and relapse. Post colitis treatment, F/B ratios decreased in the Dangshen and SJZT groups and increased in the S. boulardii and D+S groups (Figure 7C). Induction of colitis (DSS-only)significantly decreased the F/B ratio relative to Normal mice (0.55 vs 0.24, P<0.05), while all four preventative treatments increased the F/B ratio (Figure 7D), which was significant in the Dangshen and D+S groups (P<0.05), where F/B ratios were close to that of controls (0.57, 0.56 vs 0.55, respectively). We next compared community differences at family level (Figure 8A & 8B), which again highlighted the modulating effect of preventative treatment on gut microbial composition. Relative to day 1, all four treatments decreased the proportion of S24-7, especially Dangshen (53.27% vs 30.52%, P<0.05) and S. boulardii (44.37% vs 23.50%, P<0.05). Prevotellaceae was increased in all four treatment groups, and significantly so in the Dangshen (16.27% vs 45.67%, P<0.05) and D+S (18.05% vs 42.84%, P<0.05) groups. S. boulardii promoted Helicobacteraceae growth (3.54% vs 14.87%, P<0.05); SJZT decreased Rikenellaceae (5.37% vs 1.39%, P<0.05), Porphyromonadaceae (1.52% vs 0.72, P<0.05) and Bacteroidaceae (1.92% vs 0.84%, P<0.05); and D+S decreased Verrucomicrobiaceae (1.16% vs 0.001%, P<0.05). Compared with control mice, DSS-induced colitis led to a drastic decrease in S24-7 (35.07% vs 15.18%, P<0.05), Lactobacillaceae (4.98% vs 0.21%, P<0.05) and Helicobacteraceae (3.17% vs 0.27%, P<0.05), while Prevotellaceae (19.27% vs 50.37%, P<0.05) and Bacteroidaceae (1.34% vs 8.67%, P<0.05) were obviously increased. In the treatment groups, S24-7 recovered in the SJZT group compared vs DSS-only (15.18% vs 30.25%, P<0.05); Prevotellaceae was inhibited by all four treatments, especially by Dangshen and SJZT (P<0.05), with Prevotellaceae levels returning to baseline in the SJZT group. Verrucomicrobiaceae was restored in the Dangshen and SJZT groups, while Dangshen and S. boulardii increased Helicobacteraceae abundance compared vs DSS-only (5.23%, 2.22% vs 0, P<0.05). Specific treatment-associated taxa in colitic mice To further compare treatment-specific changes to gut microbial the top 60 most abundant genera (omitting a few rarely reported genera) were selected for comparison between groups (Figure 9). Mice with DSS-induced acute colitis (Group E) showed signs of dysbiosis, marked by a significant decrease in beneficial bacteria and an increase in pathogenic bacteria. Lactobacillus (5.07% vs 0.17%, P<0.05), Bacillus (0.38% vs 0, P<0.05) and Lactococcus (0.32% vs 0, P<0.05) were all decreased in DSS-only vs control mice, while potentially harmful IBD-associated bacteria including Bacteroides (1.37% vs 8.97%, P<0.05), Paraprevotella (0.16% vs 1.78%, P<0.05), Escherichia_Shigella (0.03% vs 0.13%, P<0.05), and Alistipes (0.67 % vs 2.40%, P<0.05) were all notably increased in DSS-only treated mice. Preventative treatment could however alter colitis-associated dysbiosis. Lactobacillus, a commonly used probiotic, was significantly increased in the two S. boulardii-containing groups (P<0.05) yet was only slightly increased in the Dangshen and SJZT groups relative to the DSS-only group, but still far from the levels in the normal group. Bifidobacteriaceae_ unclassified, Bifidobacterium and a new probiotic, Arthrobacter, were all significantly increased in the Dangshen group, and increased (without significance) in the D+S group. Bacillus and Lactococcus could not however be restored in any of the preventative treatment groups. Akkermansia - a new beneficial microbe that plays a key role in combating metabolic disorders [15] - was significantly increased in SJZT and Dangshen groups (Table 1). Group differences in potentially harmful bacteria are summarized (Table 2). All preventative treatments led to a decrease in the relative abundance of Paraprevotella, Parasutterella, and Prevotellaceae_ uncultured; Bacteroides was significantly decreased only in the Dangshen group (P<0.05); Escherichia_Shigella was significantly decreased in all treatment groups except SJZT (P<0.05); Desulfovibrio was decreased in the S. boulardii and SJZT groups; Interestingly, there were significant increases in certain SCFA-producing bacteria, with 9 of the 13 identified genera significantly enriched in at least one treatment group relative to the DSS-only group (D) (Table3); Butyrivibrio, Quinella, and Anaerotruncus were significantly enriched in the SJZT group (P<0.05). Allobaculum Blautia and Odoribacter were marginally increased in all treatment groups (P>0.05); Faecalibacterium, Megamonas, Prevotella, Subdoligranulum and Dialister were increased in the Dangshen and D+S groups, but not in the S. boulardii-only group, which suggests that Dangshen polysaccharide (and not S. boulardii) may be mitigating these effects; Roseburia was markedly increased in all treatment groups, especially in the Dangshen and S. boulardii groups, which also promoted Anaerovorax growth. Click here to view Large Figure 8 Click here to view Large Figure 9 Click here to view Large Table 1 Click here to view Large Table 2 Click here to view Large Table 3 Go to Discussion The human gut microbiome plays a vital role in maintaining body homeostasis. Changes in bacterial compositional (i.e. dysbiosis) can profoundly impact human health, which makes the microbiome an important therapeutic target. Both bacterial diversity and abundance is altered in human IBD; however, whether dysbiosis is a consequence or cause of IBD remains controversial. The success of manipulating the gut microbiome to cure or alleviate the symptoms of IBD has encouraged the use of probiotic and prebiotic therapies. IBD-directed therapies however have variable success rates and a high risk of severe side effects with long-term use. Therefore, in recent years, the use of CAMs have become widespread due to their effectiveness and absence of side effects [16]. Besides probiotics and prebiotics, Chinese traditional medicines may be a viable microbiome-manipulating therapeutic option. In this study, we clearly demonstrated the effectiveness of four different CAMs in relieving DSS-induced colitis. Although most Chinese herbal medicines and their active polysaccharides (e.g. Astragalus membranaceus, Rheum rhabarbarum polysaccharide) have anti-inflammatory effects in a mice colitis model [17], we are the first to report the use of Dangshen [Codonopsis pilosula (Franch.) Nannf] polysaccharide in the treatment of colitis in mice. In fact, Dangsheng polysaccharide was even more effective than the herbal medicine SJZT, which confirms that Dangshen polysaccharide is the key active ingredient in SJZT. In addition, we found that S. boulardii significantly alleviates the symptoms of colitis, which is in agreement with previous research [8]. S. boulardii in combination with Dangshen polysaccharide, was more effective than S. boulardii alone, which suggests a possible synergistic or synbiotic effect. Following 21 days of preventive treatments (before induction of colitis), a high proportion of Bacteroidetes were present in the Dangshen and SJZT groups. Besides Dangshen polysaccharide, the herbal decoction also contains other plant polysaccharides. On the other hand, Bacteroidetes are well equipped for carbohydrate metabolism [18] so it is not surprising that Bacteroidetes are increased in these groups. Enrichment with Bacteroidetes in polysaccharide-treated mice could be related to polysaccharide degradation in the gut. Furthermore, mice body weight was increased in the two S. boulardii-containing groups compared with the Dangshen and SJZT groups. This was associated with increased F/B ratios in S. boulardii-containing groups, which has been associated with enhanced energy harvesting [19]. The F/B ratio is also a key index in evaluating IBD remission and relapse [20]. All treatments inhibited the DSS-induced decrease in the F/B ratio, with significantly increased F/B ratios in the Dangshen and D+S groups (P<0.05) to values similar to that of the normal (non-DSS group). Modulating F/B ratios in DSS-induced colitis may represent an important underlying mechanism of these treatments. Another interesting feature is that Proteobacteria was significantly increased, especially in the S. boulardii group after 21 days of treatment yet increased only in the Dangsheng group post-colitis (day 30). Proteobacteria can induce a specific IgA response to regulate maturation of the immune maturation [21]. It has also been reported that S. boulardii stimulates intestinal IgA production [22], and that total polysaccharide extracted from SJZT can restore IgA production in a cyclophospha mide-induced lymphoid tissue injury model [23]. Therefore, the ability of S. boulardii and Dangshen polysaccharide to induce IgA may promote the growth of Proteobacteria. The most important characteristic of a good prebiotic is to selectively stimulate the growth of host-beneficial bacteria such as Bidifobacteria and Lactobacilli. Bifidobacterium, Bifidobacteriaceae_unclassified and Lactobacillus were only seen in the Dangshen and D+S group. Arthrobacter produces hydrolytic enzymes (endoinulinases [24] that degrade inulin into common prebiotic fructooligosaccharides. Arthrobacter arilaitensis can produce β-fructofuranosidase, which is used to synthesize the prebiotic kestose [25]. Most Arthrobacter species are probiotics and both Arthrobacter agilis and Arthrobacter citreus have been used to treat IBD [26]. In this study, Arthrobacter growth was facilitated by Dangshen polysaccharide, which supports its prebiotic value. Bacterially-derived SCFAs are major products of prebiotic metabolism. We found that Anaerovorax, Roseburia, Prevotella, Megamonas, Dialister, Faecalibacterium and Subdoligranulum were remarkably increased in the Dangshen group. Apart from Anaerovorax and Roseburia, the other taxa were specifically increased in the Dangshen group. Prevotella is associated with consumption of a diet rich in fiber because of its outstanding ability for cellulose and xylan hydrolysis. A long-term fiber-rich diet therefore promotes the growth of Prevotella, along with increased production of SCFAs [27]. Megamonas and Faecalibacterium are known producers of SCFA, which are decreased in IBD patients [28]. F. prausnitzii, the representative species of the Faecalibacterium genus, is a commonly administered intestinal probiotic in recent years. It produces SCFAs and increases production of IL-10 and TGF-β to control colonic inflammation by regulating the Th17/Treg balance. The common prebiotic inulin greatly facilitates Faecalibacterium growth [29]. In this study, when colitic mice were treated with Dangshen polysaccharide, Prevotella, Megamonas and Faecalibacterium all increased to > 1% (2.67%, 1.05%, 1.36% respectively), which supports the value of Dangshen polysaccharide as a powerful prebiotic. Other SCFA-producing bacteria (Butyrivibrio, Quinella and Anaerotruncus) were seen only in the SJZT group, which suggests the presence of small active molecules or medicinal polysaccharides other than Dangshen polysaccharide that promote the growth of these beneficial microbes. The exact composition of SJZT however requires further study. Roseburia, which produces SCFAs and is now used to treat UC [30], was increased in all four treatment groups. Odoribacter was reduced in DSS-induced colitis, and Odoribacter splanchnus, a typical representative of the genus Odoribacter, is a known producer of acetate and propionate [31]. S. boulardii mildly promoted Odoribacter growth. Anaerovorax metabolizes putrescine to acetate and butyrate [32] and Anaerovorax was remarkably increased in the S. boulardii group. Taken together, these results suggest that S. boulardii is an effective probiotic that facilitates the growth of the SCFA-producing bacteria: Roseburia, Odoribacter and Anaerovorax. Lactobacillus was also increased significantly in the S. boulardii group. Other yeasts have been reported to stimulate the growth of lactic acid strains in fermented products [33]. Our result therefore suggests that S. boulardii also has the ability to promote Lactobacillus growth in vivo. Akkermansia plays a key role in preventing obesity and metabolic disease. A. muciniphila is decreased significantly in diabetes patients. Recent research suggests that metformin may act by facilitating Akkermansia proliferation [34]. Akkermansia is also decreased in IBD patients and a previous study reported that extracellular vesicles derived from Akkermansia muciniphila protect against DSS-induced colitis [35]. In addition, administration of common oligofructose-containing prebiotics completely restored Akkermansia levels in both genetically and high fat-fed obese mice [15]. In this study, we found that Dangshen polysaccharide restored Akkermansia abundance in acute colitic mice, which suggests that Dangshen polysaccharide may be useful as a prebiotic in the prevention of obesity and diabetes. Desulfovibrio is the most important member of the sulphate-reducing bacteria. It breaks down SCFA and amino acids to produce H2S that harm intestinal epithelial cells. Desulfovibrio is increased in the inflamed colon [36], which can be counteracted by S. boulardii and SJZT. Bacteroides was increased in acute colitis, hich is in agreement with a previous study [37]. However, only Dangshen polysaccharide was able to control this increase in Bacteroides, while S. boulardii even promoted its growth. This is in agreement with a previous report where Bacteroides was dramatically increased by S. boulardii administration in type 2 diabetic mice [10]. This may be due to the ability of Bacteroides to utilize t cell walls as, which is made up of β-glucans, as an energy source [10]. However, it remains unclear why SJZT increased the proportion of Bacteroides. Prevotellaceae_uncultured and Parasutterella, which are enriched in IBD patients [38] and in colorectal cancer tissue [39], were inhibited by all four treatments at varying degrees. Escherichia- Shigella is a common pathogenic bacterium that was induced by DSS administration. All treatments, except SJZT, inhibited Escherichia-Shigella growth. Taken together, the common mechanism, shared by all four treatments, was to inhibit harmful bacterial growth. Go to Conclusion Chinese traditional herbal medicine SJZT, Dangshen polysaccharide and S. boulardii were all effective in alleviating DSS-induced colitis. Promoting beneficial and inhibiting pathogenic bacteria may be the shared mechanism of these potential CAM drugs in improving colonic health. Go to Acknowledgments We thank Professor Hongyu Li for providing help in data analysis. This work was supported by Jiangsu Science and Technology Major Project (BA2016036) and Gansu Science and Technology Major Project (17ZD2FA009). Go toConflict Of Interest The author has no conflicts of interest to declare

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