#oxidative stress assay kit

[helvaticacare]

Explore the vital link between nutrition and oxidative stress reduction in human health. Learn how optimizing your diet can combat oxidative stress, enhance well-being, and promote a healthier, more vibrant life. Dive into our expert insights and actionable tips now

[magnificentbouquetperfection]

Gen Storeis part of the Gentaur Genprice group and supplies Quantichrome Assays, Reagents for antibody and Elisa research.

[didanawisgi]

Abstract

Age-related macular degeneration (AMD) is now one of the leading causes of blindness in the elderly population. The antioxidative effects of curcumin on aging retinal pigment epithelial (RPE) cells are still unclear. We conducted an in vitro study to investigate the effects of curcumin on aging RPE cells. A pulsed H2O2 exposure aging model was adopted. Aging RPE cells were treated with curcumin 20 µM, 40 µM, and 80 µM. Apoptosis of RPE cells was analyzed by flow cytometry. The intracellular reactive oxygen species concentration was detected using a specific probe and apoptosis-associated proteins were detected by Western blot. Expression of oxidative biomarkers, including superoxide dismutase, maleic dialdehyde, and glutathione, was detected commercially available assay kits. Compared with normal cells, lower cell viability, higher apoptosis rates, and more severe oxidation status were identified in the aging RPE cell model. Curcumin improved cell viability and decreased apoptosis and oxidative stress. Further, curcumin had a significant influence on expression of apoptosis-associated proteins and oxidative stress biomarkers. In conclusion, treatment with curcumin was able to regulate proliferation, oxidative stress, and apoptosis in aging RPE cells. Accordingly, application of curcumin may be a novel strategy to protect against age-related change in AMD.

[btlbiotechnolabs]

Are you searching for the Assay Kits manufacturers

Assay kits that can be used to conduct biochemical tests. These reagents can be used to measure specific analytes for various purposes in academics and industry. Assay kits are used for life science research, drug discovery and development, and environmental monitoring. Assay kits can be used for various purposes, including studying disease pathways, screening potential drug candidates, and evaluating biopharmaceutical manufacturing processes. Assay kits can assess cell proliferation, signal transduction, cytotoxicity, metabolism, oxidative stress, damage, and metabolism. You can order Assay kits on Biotechnolabs the best Assay kits manufacturers  in India, by visiting our website https://biotechnolabs.com

[biomedres]

Anti-Radical Activity of Low Frequency and Low Amplitude PEMF - BJSTR Journal

Anti-Radical Activity of Low Frequency and Low Amplitude PEMF by Enrico Fermi* in Biomedical Journal of Scientific & Technical Research

https://biomedres.us/fulltexts/BJSTR.MS.ID.006203.php

Many hypotheses have been done to understand how Pulsed Electromagnetic Fields (PEMF) at low frequency (LF) and low intensities/amplitude (LA) impact on biological tissues. One of the responses it is aimed to investigate is the LF LA PEMF capability of promoting an oxidative stress modulation and anti-radical activity. Nonlinear models and, upon all, Schrödinger equations explains what could be the energy activation which drives these changes: the soliton wave. This solution finds its biophysical and biological demonstration in oxidative stress changes, disease or sport related. In vitro test was performed to demonstrate this model. Fibroblast and endothelial cells were exposed to PEMF LFLA 0-300Hz, 100uT emitted from a solenoid for 30 minutes for 21 Days. The assay kit revealed a progressive reduction of oxidative stress after the 8th day, in both cultures. This test was followed by an explorative clinical test report involving n.5 professional running athletes who were exposed for 21 days, once a day, to PEMF LFLA (variable frequencies 0-300Hz and 100uT mean peak) through a multi-solenoid mattress. The photometric analysis done every week (0, 7th, 14th, 21st day) showed a mean reduction of oxidative stress before and after each treatment, and in the overall treatment. The theorical and experimental evidence described in this report illustrate the working principle of PEMF LF LA inducing responses in oxidative stress levels of biological tissues, both in vitro and in vivo. Further studies in wider population need to be done in order to confirm these assumptions.

For more articles Journals of Biomedical Science please click here

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[biotechnolabss]

Oxidative stress is the imbalance between the free radicals and the antioxidants in the body. It can be also said that it is an imbalance between the accumulation and production of Reactive Oxyegen species (ROS). For understanding the oxidative stress in any diseased condition like cancer,  the assessment of oxidants and anti-oxidants is essential. Hence to support the scientist in their research Biotechno Labs are providing kits for assessing these two: Total antioxidant status (TAS) and Total oxidant status(TOS) kits. Both of them are colormetric kits.  

In Total antioxidant assay kit, ABTS is oxidized to green ABTS•+ by appropriate oxidant, which can be reduced to colourless ABTS in the presence of antioxidants. The TAS of the sample can be determined and calculated by measuring the absorbance of ABTS•+ at 660 nm. Trolox is an analog of VE and has a similar antioxidant state to that of VE. Trolox is used as a reference substance for total antioxidant status.In Total oxidant assay kit, under acid conditions, the oxidizing material in the sample can oxidize Fe2+ to Fe3+, which binds highly with xylenol orange to produce a blue-purple complex. When the pH of solution is in the range of 2-3, its maximum absorption wavelength is around 590 nm, and the color depth is proportional to the content of oxidation substances in a certain concentration and a certain time, so as to indirectly calculate the total oxidation state of the sample.

www.biotechnolabs.com #biotechnolabs #science #research #technology #innovation #cancer #scientist #diseaese #oxidants #antioxidnats #kits #oxidativestress #kits

[ivdreagent]

Lactic Acid Assay Kit

A Lactic Acid Assay Kit measures the level of L-lactic acid in dairy products, beverages, and foods. The test kit can measure levels of L-lactic acid in foods and beverages. The method is accurate for measuring L-lactic acid in different products. However, it can also be used to measure other acids such as D-lactic and acetic acids. For a more accurate measurement, use a UV-method.

L-Lactate is a stereoisomer of lactate in human intermediary metabolism. It is present in blood at levels of approximately 1-2 mmol/L, and is formed from the enzyme Lactate Dehydrogenase. It is produced during proliferating cells, as well as during anaerobic conditions. A Lactic Acid Assay Kit does not require any purification, and the kit is not required for samples of lyophilized milk.

This kit is compatible with most buffers, but is not recommended for high-quality results. The K627 buffer is incompatible with D-lactate. It is not necessary to use both K627 and K607 assay kits to obtain accurate results. Both methods can be used for calibrating the kit. The best reagents are available at a variety of price ranges.

This L-Lactic Acid Assay Kit can be used for quantitative determination of L-lactate. Its detection limit is 0.05 mM, which is ideal for research applications. Its sensitivity range is 0.001-10 mM, and its linearity is approximately 2 mM. The L-Lactic Acid Assay kit is sensitive and scalable, and it can detect different concentrations of Lactate.  Lactic Acid Assay Kit details.

The kit uses an enzyme mix that reacts with lactate to produce a product. Its color is proportional to the concentration of L-lactic acid. The dye is not affected by any pretreatment. Its colorimetric version is highly sensitive and is useful for detecting a low concentration of lactate in biological samples. The kits are available at several suppliers. The ELISA-based technique is the most commonly used for assessing lactic acid in human patients.

The L-Lactate Assay Kit has been developed for assessing the concentration of L-lactate in various food products. L-lactate is present in the body in small amounts in both serum and plasma. The L-Lactate Assay Kit is suitable for analyzing a variety of samples. The assay kit is compatible with most laboratory equipment. In addition to this, the L-Lactate Assay kit can also be used for evaluating the level of L-lactate in the body fluid.

The reagents contained in the kit are optimized for 96-well plates. In 384-well plates, the reagent volumes need to be proportionally scaled down. The test can be performed in a laboratory or clinical setting. The LC-LC Assay Kit has a wide range of applications. It is ideal for studies that require the measurement of glucose in blood. The method is suitable for monitoring the concentration of lactate in a small amount of time.

A laboratory may use different reagents to analyze lactic acid. The DLDH over-producing strain, called "tr6," is used in the lactate assay kit. It is essential to select the correct reagents for the assay. The pH range of the DLDH assay should be within the range of pH 7.8 in order to accurately measure the amount of lactate in a sample.

The Cayman's L-Lactate Assay kit offers a fluorescence-based method for measuring L-lactate in biological samples. It can be used to measure intracellular lactate concentrations in cell cultures. The ratio of lactate to pyruvate is a measure of the redox state of a cell and is often used as a tool to assess tissue oxygen demand.

The Lactate-Glo (tm) Assay combines a bioluminescent NADH detection system with oxidation of lactate. It is a combination of an L-lactate-selective lactate dehydrogenase and luciferin. The reaction produces light and is a reliable indication of lactic acid levels. It is important to note that a lactic acid assay should have a pH range of a patient.

The XF Glycolysis Stress Test kit uses a reagent that contains a combination of NAD+ and lactate. It is used to measure the rate of glycolysis in the body. It is a useful tool to assess glucose levels in a patient's blood. If you have diabetes, the XF Glycolysis Stress Test can help you make the necessary changes. In addition to lactic acid, NAD+ and luciferase are also a great indicator of glucose levels in the blood.

[biomedgrid]

Biomed Grid | Evaluation of Oxidative Stress Parameters in Liver in Pentylenetetrazole - Induced Acute and Chronic Epilepsy Model in Rats

Introduction

Epilepsy occurs due to periodic and spontaneous excessive electrical discharges of cerebral, cortical and subcortical neurons. Epilepsy affects about 1% of the global world population [1, 2]. Pentylenetetrazole (PTZ) is a GABA receptor blocker from chemical tetrazole. It is often used to model the occurrence of animal epilepsy and evaluate the efficacy of antiepileptic agents. It is one of the drugs commonly used in the formation of generalized tonic-clonic epileptic seizures [3]. In generalized epilepsy, the formation of reactive oxygen species (ROS) in the brain increases with recurrent seizures [4]. Increased oxidative stress due to increased free radical release has been associated with underlying pathogenesis in the onset and development of epileptic seizures. Therefore, it has been concluded that antioxidant treatment may provide neuroprotective effect by reducing oxidative stress in epilepsy treatment [5]. Increased oxidative stress in the central nervous system has been shown to increase in various experimental epilepsy models and electroshock models [6] such as the amygdala burning model [7], kainic acid model [8], PTZ model [9], sound stimulation (switching) model [10]. The liver is an organ that regulates different functions such as secretion, metabolism, detoxification and storage in the body and is sensitive to oxidative damage [11, 12]. The aim of this study was to evaluate the levels of oxidative injury in chronic and acute epilepsy model by measuring AST and ALT enzyme levels from serum samples and CAD, TBARS and SOD levels in liver tissues.

Materials-and-MethodsSubjects

Male, adult 200-250 g Wistar rats (n = 18) were used in the experiment. Ethics approval was obtained from Sivas Cumhuriyet University Faculty of Medicine Ethics Committee. Animals were divided into six groups:

a) Control Group (Control; n = 6); rats, single dose i.p. saline,

b) Acute Epileptic Model Group (n = 6); rats, single dose i.p. PTZ (45 mg / kg),

c) Chronic Epileptic Model Group (n = 6); rats, repeated i.p. doses PTZ (35 mg / kg) every other day for 15(fifteen) times.

Seizure Model

The chronic epilepsy model is induced by 15 injections of 35 mg / kg PTZ and the acute epilepsy model is induced by single dose PTZ (45 mg / kg). Rats were observed for epileptic seizures for 30 minutes after PTZ injection. The activity was performed as general epileptic seizures beginning with clonus of the forefoot and facial muscles and continuing with tail and neck extensions, tonic flexionextension and loss of straightening reflex and generally extended clonic activity. Transport times and behavioral characteristics of epileptic activities were recorded. Animals were killed by guillotine 24 hours after saline administration or PTZ-induced seizures (single or final seizure).

Tissue Assessment and Methods

Blood was collected before sacrifice from the animals and centrifuged at 2000 rpm for 20 minutes. Serum samples were taken. AST and ALT levels were measured from serum samples using automatic analyzer in Sivas Cumhuriyet University Hospital. After the blood collection, the animals were sacrificed, and the liver tissues of the animals were removed for sampling. It was placed in PBS, which was five times higher than the extracted liver tissue. A manual homogenizer was used to prevent tissue degeneration and all extracted tissues were homogenized on ice in this PBS. Samples were centrifuged at 3000 rpm for 20 minutes and the supernatants were separated. SOD, CAT and TBARS levels in liver supernatants were measured using sandwich-ELISA method according to the manufacturer’s protocol and protein concentration was determined by Bradford protein assay kit.

Statistics

The mean ± SD was used for all data. The SPSS statistical package (SPSS Inc., Chicago, IL) was used to perform statistical analyzes in our study. Statistical analysis was performed to compare changes between individual groups using variance analysis (ANOVA) followed by post ANOVA (Tukey’s HSD) test. The p value was accepted as <0.05 to accept statistically significant difference between the groups.

Results

Table 1 includes serum AST and ALT levels and Chart 1 includes SOD, TBARS and CAT levels in liver tissue in all (3) groups. There was no significant difference between the groups in terms of serum ALT and liver TBARS parameters (p> 0.05). There was no significant difference between chronic epileptic model group and control group (p> 0.05). Serum AST levels of the chronic epileptic model and control group were significantly lower than the acute epileptic model group (p <0.05). SOD levels of the chronic epileptic model group were significantly higher than the acute epileptic model group and the control group (p <0.05). CAT levels of the control group were significantly lower than those of the chronic epileptic model group (p <0.05) (Table 1) (Figure 1).

Figure 1: Liver TBARS, SOD and CAT levels in control, acute epileptic model and chronic epileptic model groups. * significantly different from control group p<0.05.

Table 1:Serum AST and ALT levels in control, acute epileptic model and chronic epileptic model groups

Discussion

Lipid peroxidation (MDA) is caused by free radicals such as NO. Furthermore, glutamine synthase can be directly inactivated for epileptic seizure activation, thereby causing abnormal accumulation of glutamate, the main stimulatory neurotransmitter [13, 3]. In previous studies, it is thought that oxidative stress is an important cause of excitotoxicity and glutamate receptor activation [14]. SOD is one of the important antioxidants against free radicals. In addition, SOD is thought to prevent lipid peroxidation. Normally, lipid peroxidation occurs at low levels in each person and is the result of increased oxidative stress. MDA, the product of lipid peroxidation, has toxic effects on cell membranes and cells [11]. PTZ-induced epileptic seizures are associated with oxidative stress. Because it is a clinical epilepsy model that simulates actual epileptic seizures, it is widely used to create experimental epileptic models [15]. Akbas et al. [16] reported that increased oxidative stress caused by lipid peroxidation in hepatocytes during longterm epileptic seizures may lead to hepatocyte damage [16]. They used an epileptic model PTZ dose. They found that SOD and GSH in the liver and blood were significantly increased in the epileptic model group compared to the control group. We found that SOD levels were significantly lower in the acute epileptic model group and in the control, group compared to the chronic epileptic group (p <0.05). Activation of the nuclear factor erythroid 2-related factor (Nrf2) [17, 18] increases antioxidant defenses in brain cells, and there are mechanisms to prevent the occurrence of nitrogen species and reactive oxygen species. Nrf2 enhances the expression of various endogenous antioxidant enzymes and thus plays a key role in intracellular signaling and redox balance. The regulated ARE genes are activated in astrocytes. As a result, neurons have antioxidant defenses and detoxification. Thus, they protect the cells against oxidative stress [19]. In PTZ-induced animals, an increase in the expression of Nrf2 was observed (data in print), and a marked increase in immunoactivity against enzymes regulated by this factor suggests activation in epileptic seizure models [20]. SOD (CuZnSOD and MnSOD) found in cytosol and mitochondria are found in the antioxidant enzyme systems regulated by Nrf2. H4O can be eliminated by CAT in mitochondria and peroxisomes or neutralized in H4O with GPx in the cytosol [21]; in this paper CAT levels of the control group were significantly lower than those of the chronic epileptic model group (p <0.05).

In previous studies, TBARS, the product of lipid oxidation, was used as an indicator of oxidative stress [22]. In our study there was no significant difference between the groups in terms of serum ALT and liver TBARS parameters (p> 0.05). There was no significant difference between chronic epileptic model group and control group (p> 0.05).

Conclusion

In conclusion, it causes increased oxidative damage and lipid peroxidation in PTZ-induced recurrent and single epileptic seizure models. In addition, antioxidant defense mechanisms have been decreased in these models. Increased oxidative stress, either recurrent or in a single epileptic seizure, causes damage to hepatocytes.

Read More About this Article: https://biomedgrid.com/fulltext/volume5/evaluation-of-oxidative-stress-parameters-in-liver-in-pentylenetetrazole-induced-acute.000966.php

For more about: Journals on Biomedical Science :Biomed Grid | Current Issue

[ka43270]

Oxidative Stress Analysis Market  to Eyewitness Massive Growth by 2026 | COVID19 Impact Analysis and Business Strategy

Market Analysis and Insight: Oxidative Stress Analysis Market

Oxidative Stress Analysis Market By Product (Consumables, Instruments, Services), Technology (Elisa, Flow Cytometry, Chromatography, Microscopy, High- Content Screening, Label- Free Detection), Test Type (Indirect Assays, Antioxidant Capacity Assays, Enzyme- Based Assays, Reactive Oxygen Species-Based Assays), End- User (Pharmaceutical and Biotechnology Companies, Academic Research Institutes, Clinical Laboratories, Contract Research Organizations), Disease (Cardiovascular Disease, Diabetes, Chronic Obstructive Pulmonary Disease, Cancer, Asthma), Geography (North America, Europe, Asia-Pacific, South America, Middle East and Africa) – Industry Trends and Forecast to 2026

Global oxidative stress analysis market is set to witness a substantial CAGR in the forecast period of 2019- 2026. The report contains data of the base year 2018 and historic year 2017. Growth in pharmaceutical industry and rising investments in healthcare sectors are the factor for the growth of this market.

Get a Sample Copy of report @ https://www.databridgemarketresearch.com/request-a-sample/?dbmr=global-oxidative-stress-analysis-market

Competitive Analysis:

Global oxidative stress analysis market is highly fragmented and the major players have used various strategies such as new product launches, expansions, agreements, joint ventures, partnerships, acquisitions, and others to increase their footprints in this market. The report includes market shares of oxidative stress analysis market for Global, Europe, North America, Asia-Pacific, South America and Middle East & Africa.

Market Definition: Global Oxidative Stress Analysis Market

Oxidative stress analysis is used to calculate the amount of stress reagents and markers in the cell. This helps the healthcare providers so they can find the cause and reason for different clinical disorders and help them to customize the specific treatment programs. Assays detect widespread oxidative stress, reactive oxygen species, redox sensors, lipid peroxidation based on fluorescent proteins and concentrations of glutathione. Oxidative stress analysis uses different technologies such as microscopy, high content screening, chromatography, flow cytometry, elisa, label- free detection among others.

Market Drivers

Increasing     R&D investment in biotechnology and pharmaceutical industries will     drive the market growth

Technological     advancement in the high-content screening for drug discovery is also     driving the growth of this market

Rising     government funding for life science research also accelerates the market     growth

Growing     aging population will boost the market growth

Market Restraints

High     cost of instrument will restrict the market growth

Lack     of skilled and trained professionals will also hamper the market growth

Get Detailed Table of Contents @ https://www.databridgemarketresearch.com/request-a-sample/?dbmr=global-oxidative-stress-analysis-market

Segmentation: Global Oxidative Stress Analysis Market

By Product

Consumables

Instruments

Services

By Technology

Elisa

Flow     Cytometry

Chromatography

Microscopy

High-     Content Screening

Label-     Free Detection

By Test Type

Indirect     Assays

Antioxidant     Capacity Assays

Enzyme-     Based Assays

Reactive     Oxygen Species-Based Assays

By End- User

Pharmaceutical     and Biotechnology Companies

Academic     Research Institutes

Clinical     Laboratories

Contract     Research Organizations

By Disease

Cardiovascular     Disease

Diabetes

Chronic     Obstructive Pulmonary Disease

Cancer

Asthma

By Geography

 U.S.

 Canada

 Mexico

 Germany

 Italy

 U.K.

 France

 Spain

 Netherlands

 Belgium

 Switzerland

 Turkey

 Russia

 Rest      of Europe

 Japan

 China

 India

 South      Korea

 Australia

 Singapore

 Malaysia

 Thailand

 Indonesia

 Philippines

 Rest      of Asia-Pacific

 Brazil

 Rest      of South America

 South      Africa

 Rest      of Middle East and Africa

For More Information or Query or Looking for Customization, Visit @ https://www.databridgemarketresearch.com/inquire-before-buying/?dbmr=global-oxidative-stress-analysis-market

Key Developments in the Market:

In     September 2019, Aytu Bioscience, Inc announced the availability of     their  first-in-class seminal oxidative stress test MiOXSYS which is     specially designed for the assessment of male infertility in South Africa.     This is the first vitro diagnostic test which has ability to access the     seminal oxidative stress. The MiOXSYS system is also an excellent tool for     measuring antioxidant treatment requirements and efficacy, enabling a     targeted treatment approach rather than a blanket approach

In     January 2018, Agilent Technologies Inc. announced the acquisition of Cork     so they can expand their cell analysis portfolio with the addition of     easy-to-use assay kits. This acquisition will help the company strengthen     their leadership in kinectic & live cell assay which will help them to     expand them in pharmaceutical and biopharma and in vitro toxicology     screening applications

Key Market Competitors:

Few of the major competitors currently working in the global oxidative stress analysis market are Abcam plc, Enzo Life Sciences, Inc., Merck KGaA, QIAGEN, Thermo Fisher Scientific Inc., BioVision Inc., Cell Biolabs, Inc, OXFORD BIOMEDICAL RESEARCH., Promega Corporation., Quest Diagnostics Incorporated, Array BioPharma., Luminex Corporation., Hycult Biotech, Cayman Chemical, Bioquochem., BioCat GmbH, HCS Pharma, DIACRON, Aytu BioScience, Inc., among others.

Research Methodology: Global Oxidative Stress Analysis Market

Data collection and base year analysis is done using data collection modules with large sample sizes. The market data is analysed and forecasted using market statistical and coherent models. Also market share analysis and key trend analysis are the major success factors in the market report. To know more please request an analyst call or can drop down your enquiry.

The key research methodology used by DBMR research team is data triangulation which involves data mining, analysis of the impact of data variables on the market, and primary (industry expert) validation. Apart from this, other data models include Vendor Positioning Grid, Market Time Line Analysis, Market Overview and Guide, Company Positioning Grid, Company Market Share Analysis, Standards of Measurement, Top to Bottom Analysis and Vendor Share Analysis. To know more about the research methodology, drop in an inquiry to speak to our industry experts.

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[helvaticacare]

Oxidative stress is a leading cause of sexual health complications among men and women. Helvetica Health Care promotes improved life and health.

[sandlerresearch]

Oxidative Stress Assay Market by Product (Consumable, Instruments, Kits, Services), Test Type (Indirect, nucleic acid, Glutathione, ROS), Technology (ELISA, Chromatography), Diseases (CVD, Oncology) End Users (Pharma & Biotech) - Global Forecasts to 2025 published on

https://www.sandlerresearch.org/oxidative-stress-assay-market-by-product-consumable-instruments-kits-services-test-type-indirect-nucleic-acid-glutathione-ros-technology-elisa-chromatography-diseases-cvd-oncology-e.html

Oxidative Stress Assay Market by Product (Consumable, Instruments, Kits, Services), Test Type (Indirect, nucleic acid, Glutathione, ROS), Technology (ELISA, Chromatography), Diseases (CVD, Oncology) End Users (Pharma & Biotech) - Global Forecasts to 2025

“The oxidative stress assay market is projected to grow at a CAGR of 9.7% during the forecast period.”

The oxidative stress assay market is projected to reach USD 1,326 million by 2025 from USD 836 million in 2020, at a CAGR of 9.7%. Growth in the oxidative stress assay market can be attributed to growth in the biopharmaceutical industry and increasing public-sector funding for academic research. Other important factors aiding the growth of this market include technological advancements like HCS for drug discovery, increasing R&D expenditure in biotechnology and pharmaceutical industries, and government support for pharmaceutical and biotechnology industries. On the other hand, the high cost of instruments and shortage of trained and qualified personnel are the major factors restraining the growth of this market.

“The consumables segment accounted for the largest market share in 2020.”

Based on products, the oxidative stress assay market is segmented into consumables, instruments, and software and services. Consumables is the largest and fastest-growing segment in 2020. This large share and high growth can be attributed the recurring requirement of consumables as compared to instruments which is the key factor driving the growth of this segment.

“The cardiovascular diseases segment will continue to dominate the oxidative stress assay market during the forecast period.”

Based on disease type, the oxidative stress assays market is segmented into cardiovascular diseases, diabetes, cancer, respiratory diseases, and other diseases. The cardiovascular diseases segment accounted for the largest share of this market in 2020, primarily due to the high and growing prevalence of cardiovascular diseases across the globe.

“The oxidative stress assay market in the Asia Pacific region is expected to witness the highest growth rate during the forecast period.”

Geographically, the oxidative stress assay market is segmented into North America, Europe, Asia Pacific, and Rest of the World. The Asia Pacific market is expected to grow at the highest CAGR during the forecast period, primarily due to the reason that the Asia Pacific oxidative stress assays market offers significant growth opportunities for market players to expand their presence in this region. Emerging markets such as China and India are witnessing growth due to the large patient population and increasing healthcare expenditure. This has resulted in the increasing acceptance of advanced technologies, such as oxidative stress assays, in the Asia Pacific region.

Breakdown of supply-side primary interviews:

By Company Type: Tier 1: 55%, Tier 2: 20%, and Tier 3: 25%

By Designation: C-level: 35%, Director-level: 25%, and Others: 40%

By Region: North America – 20%, Europe – 25%, APAC – 40%, Rest of the World– 15%

The prominent players in this market are Abcam, plc (UK), Enzo Biochem, Inc. (US), Merck KGaA (Germany), QIAGEN N.V. (Netherlands), Thermo Fisher Scientific, Inc. (US), AMS Biotechnology (Europe), Ltd. (UK), BioVision, Inc. (US), Cell Biolabs, Inc. (US), Oxford Biomedical Research (US), Promega Corporation (US), Cayman Chemical (US), Eagle Biosciences, Inc. (US), Arbor Assays (US), Hycult Biotech (Netherlands), Callegari SRL (Italy), Kamiya Biomedical Company (US), MEGA TIP San.Tic.Ltd.Sti. (Turkey), ImmunoChemistry Technologies, LLC (US), Genox Corporation (US), and Toxys B.V. (Netherlands).

Research Coverage

The report studies the oxidative stress assay market based on test type, technology, disease type, product, end user and region

The report analyzes factors (such as drivers, restraints, and opportunities) affecting the market growth

The report evaluates the opportunities and challenges in the market for stakeholders and provides details of the competitive landscape for market leaders

The report studies micro-markets with respect to their growth trends, prospects, and contributions to the total oxidative stress assay market

The report forecasts the revenue of market segments with respect to four major regions

Reasons to Buy the Report:

The report provides insights on the following pointers:

Market Penetration: Comprehensive information on the oxidative stress assay offered by the key 20 players in the oxidative stress assay market. The report analyzes the oxidative stress assay market by test type, technology, disease type, product, end user and region.

Market Development: Comprehensive information about lucrative emerging markets. The report analyzes the markets for various oxidative stress assay, their adoption, and usage across key geographic regions.

Market Diversification: Exhaustive information about new product launches & approvals, untapped geographies, recent developments, and investments in the oxidative stress assay market

Competitive Assessment: In-depth assessment of market ranking and strategies of the leading players in the oxidative stress assay market.

[btlbiotechnolabs]

Are you searching for the Assay Kits Supplier

Assay kits that can be used to conduct biochemical tests. These reagents can be used to measure specific analytes for various purposes in academics and industry. Assay kits are used for life science research, drug discovery and development, and environmental monitoring. Assay kits can be used for various purposes, including studying disease pathways, screening potential drug candidates, and evaluating biopharmaceutical manufacturing processes. Assay kits can assess cell proliferation, signal transduction, cytotoxicity, metabolism, oxidative stress, damage, and metabolism. You can order Assay kits on Biotechnolabs the best Assay kits supplier in India, by visiting our website https://biotechnolabs.com

[lupine-publishers-lojpcr]

Lupine Publishers |he Anti-Inflammatory Activity of Ferulic Acid on NF-κBDepends on Keap1

Lupine Publishers | LOJ Pharmacology & Clinical Research

Abstract

Nrf2 and NF-κB are the two master transcriptional factors activated by different cellular signals turned to counteract the deleterious effects of pathological cellular processes linked to inflammation and oxidative stress.Several recent studies have highlighted a molecular connection between NF-κB and Keap1/Nrf2 pathways. The Keap1 protein seems to be the central player in this interaction, as it is involved in both IKKβ-NF-κB and Nrf2 modulation. Ferulic acid (FA) is a well-known antioxidant and antiinflammatory agent, able to relieveinflammatory response viaNF-κB/IKK kinase, but until now the complete molecular network under its action is not all clear. Immunoblot data conducted on LPS-treated macrophage-like RAW264.7 cells transfected with si- Keap1 show that the FA anti-inflammatory and modulatory effects on NF-κB are abolished. Luciferase assay conducted in human A549 cell line, in which Keap1 protein is partially inactive, highlights that NF-κB activation induced by LPS is refractory to FA inhibition. This study proved that Keap1 and IKK together are important modulators of NF-κB and their activity is essential for FA anti-inflammatory effectiveness.

Introduction

The process of inflammation leads to the onset of a state of oxidative stress and a series of cascade reactions that are associated with chronic diseases such as cancer, autoimmune disorders, and metabolic diseases [1]. NF-κB is the master transcriptional factor mainly involved in the activation of inflammation and immunity, whose excessive upregulation is associated with many human diseases, including inflammatory disease and cancer [2,3]. External pro-inflammatory stimuli activate NF-κB that induces the expression and the release of a set of inflammatory mediators such as IL-6, a pleiotropic cytokine contributing to switch the acute to chronic phase of inflammation [4,5]. NF-κB is mainly regulated by IKK kinase, which in presence of pro-inflammatory stimuli phosphorylates the inhibitory protein IkB, that dissociates from NF-κB with subsequent nuclear translocation [6]. Recently, many studies have highlighted the interaction between NF-κB/ IKK kinase and Keap1protein [7,8]. Our previous studies showed that Keap1 is a modulator both of Nrf2 and NF-κB pathways [9]. In physiological conditions, Keap1 maintains Nrf2 levels low. In conditions of oxidative stress, Keap1 is oxidized on the reactive cysteine residues and inactivated, so that Nrf2 moves into the nucleus. Nrf2 (nuclear factor erythroid2-related factor2), belongs to the basic leucine zipper (bZIP) transcription factor and heterodimerizes with small Maf proteins [10], and it is the primary player in the inducible cell defense system.

It binds to the promoter region of genes involved in redox regulation, proteostasis, DNA repair, prevention of apoptosis, iron and heme metabolism, and phase I, II, and III drug/xenobiotic metabolism [11]. The activation of this factor is controlled at the transcriptional and post-transcriptional level through the regulation of its stability, the post-transcriptional changes, and the availability of its binding partners [12,13]. In response to different stimuli, Nrf2 moves to the nucleus where it activates the transcription of its target genes, such as HO-1. Nrf2 is a modular protein presenting seven domains of homology to Nrf2-ECH (Neh), each of which performs a different function. In particular, the Neh2 domain binds Keap1 in the Kelch repeats [14]. In addition, Keap1 has five major domains: an N-terminal broad complex, tram track, and bric-a-brac (BTB) domain; a central intervening region (IVR) and a series of six C-terminal Kelch repeats.

The IVR and BTB domains are required for the redox-sensitive regulation of Nrf2 through a series of reactive cysteines. The 624 amino acids of murine Keap1 include 25 cysteines [15,16]. All cysteine residues are conserved and cysteines C257, C273, C288, and C297, located in the intervening region (IVR) domain, give Keap1 the molecular sensor able to respond to such a diverse array of chemical stimuli [17]. Early studies have identified IKKβ and Keap1 as the key drivers of inflammatory response [18,19]. IKK kinase is the main target of several anti-inflammatory molecules and kinase inhibitors are very effective in the control of many diseases [20-23]. In fact, LPS-signalling that is activated in macrophages via TLR4 involves a number of kinases. After binding to its receptor, the signal is transmitted by different adapter proteins: TIRAP (Tollinterleukin 1 receptor domain-containing adapter protein) and MyD88 (Myeloid differentiation primary response gene 88) driving the MyD88-dependent pathway, while TRAM (TRIF-related adaptor molecule) and TRIF (TIR-domain-containing adapter-inducing interferon-β) drive the MyD88-independent pathway.

The MyD88-dependent pathway converges into IKK (IκB kinase) and MAPK (Mitogen-activated protein kinases) activation and subsequent activation of NF-κB and activator protein AP- 1. In LPS-activated THP-1 cells, a human monocytic cell line, has been reported thanks to bioactive compounds such as flavonoids, hydroxycinnamic acids, tannins and in particular ferulic acid (FA), a hydroxycinnamic acid derivative, an effective inhibition of NF-κB activation as well as a decrease in the expression of proinflammatory cytokines TNF-α and IL-1β [24,25]. Furthermore, FA has a wide range of therapeutic effects like anti-inflammatory, antidiabetic, neuroprotective and hepatoprotective properties in others cellular models and in animals [26]. The present study carried out in RAW264.7 cells shows that the ferulic acid exerts its anti-inflammatory activity when Keap1 and IKK kinase are functionally active.

Materials and Methods

The mouse macrophage-like virus-transformed leukemia cell line RAW 264.7 was purchased from American Type Culture Collection (ATCC). RAW 264.7 cells and A549 cell line (provided by Francesca Sardina e Cinzia Rinaldo (IBPM-CNR, Roma, Italy) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% v/v heat-inactivated fetal bovine serum (FBS) and antibiotics (100U/ml penicillin and 100μg/ml streptomycin) at 37 °C in a humidified atmosphere with 5% CO2. FBS, DMEM, penicillin and streptomycin (10,000 U/ml) were purchased from GIBCO (Grand Island, NY). LPS from E. coli serotype O55: B5 and ferulic acid (FA; CAS Number: 537-98-4) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). TRIzol was purchased from Invitrogen. The Quanti Nova RT PCR kit and Quanti Nova Sybr Green PCR kit were obtained from Qiagen (Hilden, Germany). Nitrocellulose Blotting Membrane was purchased from Amersham Protran (Buckinghamshire, UK).

RT-Qpcr

RAW 264.7 cells were cultured (1 ×106 cells/well) in a 6-well plate overnight. Cells were treated with 100 ng/ml LPS or without (negative control) in the presence or absence of 100μM ferulic acid in DMEM supplemented with 10% bovine serum for 4 hours. Cells stimulated with 100ng/ml LPS for 4 hours served as a positive control. After such stimulation, the cells were detached from the wells and washed once with PBS. Total RNA was isolated with TRIzol according to the manufacturer’s instructions and was quantified by UV absorbance spectrophotometry and reverse transcribed with Quanti Nova RT PCR kit. QPCR was performed in triplicate on each cDNA sample for each gene:

IKK-�� NM 010546 F:5’-GCCAGGGAGACTTGATGG-3’ R:5’- GAGGTCTGTGCTTTAGCTGCTT-3’ IL6 NM 031168 F:5’-GAGGATACCACTCCCAACAGACC -3’ R:5’- AAGTGCATCATCGTTGTTCATACA - 3’ HO-1 NM 0125082 F:5’ -GCGAAACAAGCAGAACCCA-3’ R:5’ – GCTCAGGATGAGTACCTCCCA- 3’ HPRT NM 194F:5′-GCTATAAATTCTTTGCTGACCTGCTG-3′ R: 5′-AATTACTTTTATGTCCCCTGTTGACTGG-3′

by using primers set Quantitect from Qiagen. The threshold cycle (CT) values were calculated against the housekeeping gene HPRT. At least three distinct biological samples were examined for each gene and treatment (each one performed in triplicate). The cycling parameters were set as 95 °C for 5min, followed by 40 cycles at 95 °C 15sec and 60 °C for 2-min. The expression was calculated by the 2-ΔΔCt.

Transfection and luciferase reporter assays

A549 cells (3 × 104 cells/well) were seeded in 96-well plates and allowed to adhere for 24 hours. The cells were then cotransfected with pKEAP1Vector, pIL-6FL and Renilla-Luc plasmids (Promega) using the Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. After that, the cell culture medium was removed and replaced with fresh medium containing 100μM of FA for 1-hour, followed by co-incubation with 100ng/ml of LPS for 4 hours. Then, the cells were harvested, the luciferase activity was quantified by using the Dual-Glo Luciferase Assay System (Promega) and was normalised according to Renilla luciferase activity. The level of induction was calculated as the ratio of the normalised luciferase activity of LPS stimulated cells compared to non-stimulated cells and of association between LPS and FA compared to treatment with agents alone. Relative light units were measured in a GloMax 96 microplate luminometer (Promega).

Sirna transfection

The RAW264.7 cells (5×105 cells/well) were seeded in 6-well plates for 24 hours. Briefly, the siRNA pool for Keap1 (Qiagen) and NC-siRNA (Qiagen) were incubated with Lipofectamine RNAiMAX (Promega) in basal media with no serum or antibiotics and allowed to complex for 10 min at room temperature. Then, the complex was added to the cell suspension of each well (final siRNA pool concentration of 10nM). Finally, cells were incubated for 24 hours in a humidified incubator and then used for the analysis.

Western blotting

RAW 264.7 cells (1x107 cells) were cultured in 10-cm dishes (Falcon) and allowed to adhere for 24 hours. After treatment with FA 100μM 1 followed by co-incubation with LPS 100ngr/ml for 4 hours, the cells were washed twice with cold PBS and lysed in lysis buffer (10mM Hepes pH 7.9, 10mM KCl, 1.5mM MgCl2, 0.1mM EGTA pH 7, 0.05 mM DDT, and 1% protease inhibitor cocktail (SIGMA). After vortexing for 10s, the lysates were centrifuged at 1250g for 15min; the supernatants (cytoplasmic extract) were immediately transferred to a clean pre-chilled tube and put on ice. The pelleted nuclei were re-suspended in a hypertonic buffer (5% glycerol, 1.5mM MgCl2, 0.1mM EGTA, 0.05mM DTT, 0.4 M NaCl, 20mM PMSF and 10mM HEPES pH 7.9) and shaken for 60min at 4 °C. The proteins in the supernatants (nuclear extract) were collected by centrifugation at 20817g for 15min, then immediately transferred to a clean pre-chilled tube and put on ice. Whole cell lysates were obtained using RIPA buffer (Cell Signalling Inc. Beverly, MA, USA). The protein concentration of cell lysates was determined by the Bradford method.

An equivalent amount of protein (30μg) from whole or nuclear and cytoplasm fractions, respectively, was separated on 8-16% Tris-Glycine Gel (BioRad) gels by electrophoresis and transferred to a nitrocellulose membrane. The membranes were subsequently incubated for 1 h at room temperature with 3% BSA in TBS buffer (0.1% v/v) to block non-specific binding and incubated with an appropriate primary antibody in 1% BSA in TBST (tween 0.01% v/v). Antibodies polyclonal anti-mouse recognizing p-IKKα/β, IKKα/β, p65 NF-κB, lamin B1, Keap1, Nrf2, Histone H3 and betaactin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Incubation with the secondary antibodies Alexa Fluor 680 goat anti-rabbit (1:2000) and Alexa Fluor 800 rabbit anti-mouse (1:5000) (Molecular Probes, Life Technologies, Carlsbad, CA, USA) was performed for 1h at room temperature. Densitometry analysis was conducted using the Odyssey Infrared Imaging System (Li-COR Bioscience, Nebraska, USA).

Statistics

Statistical analysis and graphical presentation were performed using the statistical package GraphPad Prism Version 8 (GraphPad Software, Inc,USA). Data were expressed as mean ± SD and were evaluated using unpaired t-test calculator or one-way analysis of variance (ANOVA) for multiple comparisons. The differences were considered statistically significant when p values were <0.05.

Results

Keap1 silenced abolishes ferulic acid modulation on NF- κB-IKK pathway in LPS-treated RAW 264.7 cells

Our previous study [9] has proved that in Keap1- silenced RAW264.7 cells, the LPS treatment induced a higher proinflammatory cytokines mRNA expression compared with ncsiRNA transfected cells and moreover FA treatment resulted not able to reduce cytokines mRNA expression induced by LPS. Other authors [27] showed that depletion of Nrf2 induced the activation of NF-κB and a higher expression of TNF-α, IL-1β, IL-6 cytokines. In addition, it was reported that Keap1 is identified as an IKKβ interacting protein, involved in IKKβ phosphorylation [28]. To verify a potential Keap1 role in the FA modulation on NF-κB pathway, we analyzed by Western blot analysis NF-κB-IKK pathway in depleted Keap1 and FA-LPS treated RAW 264.7 cells. Cells were transfected with siRNAs for Keap1 and, after 24h, exposed to 100ng/ml LPS for 4h. One hour before LPS, cells were treated or not with 100μM FA, a known inhibitor of the NF-κB-signalling pathway in LPS-activated RAW 264.7 [29]. Cytoplasmic and nuclear extracts were prepared and analyzed in immunoblotting for the NF-κB p65. Figure 1A (panel a) shows that in Keap1-silenced and LPS-treated cells there is a significant increase of p65 in the nuclear fraction, compared to non LPS treated cells, and in accordance with our previous evidences [9], pre-treatment with FA does not decrease NF-κB translocation. In Figure 1A (panel b) is showed Western blot analysis of NF-κB pathway in nc siRNA transfected and FA-LPS treated RAW 264.7 cells, where FA is able to modulate p65 translocation also when LPS treatment occurs.

IKK kinase activates NF-kB pathway, so we analyzed whether Keap1 silencing had any influence on the kinase expression and activation. Total extract protein was prepared and analyzed in immunoblotting for IKKα/β. As reported in Figure 1B, when LPS treatment occurs, in the nc-siRNA transfected RAW264.7 cells, there is an increase of IKK level protein which decreases when cells were pre-treated with FA (p<0.0005). Instead, in the Keap1- silenced RAW 264.7 cells, neither LPS treatment nor FA have effect. QPCR analysis of IKKβ mRNA expression was conducted on total RNA extracted from Keap1 silenced and LPS treated RAW 264.7 cells. The results showed that IKKβ mRNA expression is lower (1,9-fold LPS vs C, p<0.0001) than the nc-transfected cells, where there is an increase of 8,4 fold LPS vs c (Figure 1C). These data indicate that Keap1-silencing has effect on IKKβ transcription and protein expression. Moreover, we investigated the Keap1 silencing influence on IKKα/β phosphorylation. The results are showed in Figure 1D: in nc-silenced and LPS treated cells, FA inhibits IKK phosphorylation (0,3fold FA+LPS vs 1,08 fold LPS), not such as in Keap1 silenced cells (1,8-fold FA+LPS vs 2 fold LPS). The results show that the Keap1 knockdown results in a much-altered IKK pathway modulation by FA.

 Ferulic acid is effectiveness on NF-κB activation in RAW 264.7cells Keap1 overexpressed

The overexpression of a specific protein is a common method for investigating the specific biological function and the mechanism of action. In order to confirm the data observed previously, we decided to over-express Keap1and to analyze the FA modulation on NF-κB/IKKβ activation. In our previous paper we described the FA effectiveness on inhibition of NF-κB activity [29]. We transfected RAW 264.7 cells with the expression vector for Keap1, let the cells grow for 24 hours, conducted the treatments as already described and extracted total RNA. IL6 is the major inflammatory gene under the NF-κB control, so to examine the effect of Keap1 overexpression on NF-κB modulation by FA, we analyze IL6 mRNA expression in LPS- treated and Keap1- overexpressed cells. Interestingly, FA reduces IL6 mRNA expression such as in RAW 264.7 transfected with pGEM-vector as control and LPS treated cells (Figure 2A). These results show that Keap1 overexpression does not alter neither the NF-κB activation neither the anti-inflammatory effect of FA. To confirm this result, we verify the p65 translocation occurring in LPS treated and Keap1 overexpressed cells in Figure 2A. We conducted an immunoblot analysis on proteins extracted from nuclear and cytoplasmic fractions. Figure 2B shows that LPS induces p65 translocation and FA-modulation occurs (0,5fold FA+LPS vs 6,8-fold LPS p<0.0001), unlike in Keap1 silencing cells. We have also analyzed IKKα/β phosphorylation. As showed in Figure 2C, in Keap1 overexpressed and LPS treated cells, FA is able to modulate IKK phosphorylation. The results show that the Keap1 overexpression does not alter the ferulic acid anti-inflammatory effect on NF-κb pathway.

  A549 cells a useful model to analyze FA effect on NF-kB activities

To confirm the results obtained by molecular approach by silencing the messenger for Keap1, we used A549 cell line. A549 is a lung carcinoma cell line that has an inactivating mutation on Keap1 allele, which translates into a protein with a minor binding capacity towards Nrf2 [30]. This cell line is a useful model to study Keap1 loss of functionality. We transfected A549 cells with pIL6- Luc, containing the IL6 promoter region and examined the effects of FA pre-treatment on luciferase activity and ferulic acid capability to alleviate inflammatory response via NF-κB pathway. Figure 3A shows that the up-regulation of luciferase activity LPS induced is not modulated by pre-treatment with FA (171fold FA+LPS vs 111fold LPS p<0.0001). Immunoblot for IKKα/β in Figure 3B shows that the effectiveness of FA is lost, in fact there is not a significant decrease of IKK protein level when LPS and FA treatment occurs. Moreover, to verify the level of Keap1 protein in A549 cells we conducted an Immunoblot on total proteins extracted from A549 treated or no with FA and LPS. Figure 3C shows that there are not significantly differences in Keap1 level in cells treated or not. These results follow the Keap1 silencing data indicate that Keap1and IKK are involved in the ferulic acid anti-inflammatory

  Ferulic acid maintains its anti-oxidant effectiveness on the Nrf2/HO-1 pathway modulation in Keap1silenced RAW264.7 cells

Nuclear transcription factor erythroid 2-related factor (Nrf2) is the most important transcription factor regulating the coordinated expression of antioxidant enzyme response such as Heme Oxygenase-1 (HO-1). Nrf2 resides in the cytoplasm by forming a complex with Keap1 that regulates its degradation by the proteasome. Our previous study carried out on LPS- treated RAW264.7 cells has shown that pre-treatment with FA determined a down-regulation of HO-1 protein [9]. Thus, to evaluate if FA maintains the antioxidant properties, also in altered Keap1 expression conditions, we analyzed the HO-1 expression in Keap1silenced RAW264.7 cells. As shown in Figure 4, FA down regulated the expression of HO-1 mRNA. These results indicate that FA maintains its antioxidant effect and the capability to modulate Nrf2 activity.

 Discussion

FA, a phenolic phytochemical found in many traditional Chinese medicines such as Angelica Radix and Chuanxiong Rhizoma, appears to be a potential therapeutic agent for treating various inflammatory disorders, although the molecular mechanism underlying the effectiveness is not entirely known. The pharmacological activity of FA in vascular endothelial cells is demonstrated, in fact, capable of inhibiting the expression of adhesion molecules in HUVECs [31]. Therefore, it was demonstrated that free and SLNs-loaded FA recover cell viability in neuroblastoma cells LAN5 [32]. FA treatment, in particular if loaded into SLNs, decreased ROS generation (Figure 5), restored mitochondrial membrane potential [Deltapsi(m)] and reduced cytochrome c release and intrinsic pathway apoptosis activation. In addition, FA modulated the expression of Peroxiredoxin, an anti-oxidative protein, and attenuated phosphorylation of ERK1/2 activated by Abeta oligomers. In our study highlight the Keap1 partnership in the molecular mechanism underlying the well-known anti-inflammatory effect of ferulic acid. Macrophage-like RAW264.7 stimulated by LPS was used in this study in that macrophages play vital roles in regulating inflammatory responses and LPS, the main component of the outer membrane of gram-negative bacteria, binding to the TLR4 receptor promotes inflammation [33]. Many data indicate that the molecular mechanisms underlying inflammation and oxidative stress are interconnected. NF-κB and Nrf2 are the main transcription factors on which a lot of antioxidant and inflammatory genes depend, but also tumorigenesis and apoptosis. There seems to be many the ways in which the two pathways interact, and Keap1 is one of the main [34]. Our results showed that Keap1 protein is the key point of the regulation carried out by FA on NF-κB pathway through IKK kinase. Recently, an ETGE motif (NQE36TGE39) similar to that of Nrf2 (DEE79TGE82) has been identified in IKKβ and so it is reasonable to suppose that Keap1 is an IKK modulator, regulating the ubiquitination and subsequent degradation [12,13,14].

 Moreover, our data highlight that Keap1is able to influence the transcription and protein expression of IKKβ, but the mechanism is not clear. In fact, in Keap1 silenced RAW264.7 cells it is observed a decreased IKKβ transcription and protein expression in RAW264.7 when LPS treatment occurs. Remarkably, Keap1 silencing does not inactivate NF-κB pathway, but the FA modulation is lost. In our previous study, we demonstrated that IKK is an upstream target of ferulic acid in LPS-induced NF-κB activation [29]. It can be hypothesized that Keap1 plays an important role in the NF-κB/ IKK modulation FA mediated. Yet, to confirm the data observed previously, we overexpress Keap1 in RAW264.7 cells and we analyze FA modulation on NF-κB/IKK pathway after treatment with LPS. Immunoblot data confirm that LPS stimulus activates NF-κB/ IKK pathway and FA modulation occurs.

Therefore, Keap1 and IKK kinase proteins are very important for the FA effectiveness. To confirm the results obtained by molecular approach by silencing the messenger for Keap1, we used A549 cell line to examine the biological effect of Keap1 protein mutation on the NF-κB/IKK modulation by FA. A549, in fact, is a lung carcinoma cell line that has an inactivating mutation on Keap1 allele, and the level of the Keap1 protein is the same in non-treated and LPS treated cells. In this cell line the Nrf2 pathway is dysfunctional, because the NRF2-Keap1interaction is altered [30]. The Luciferase assay data show that LPS induces up-regulation of IL-6 promoter in LPS and in FA+LPS treated cells. It is probable so the Keap1 evolvement in NF-κB/IKK FA-modulation. Recent results have revealed that the cysteine residues Cys-151, Cys-273 and Cys-288 have been identified as direct sensors of electrophiles and oxidants [17]. Keap1 acts as a molecular sensor of ROS, NO and oxidant species and the interaction with oxidizing species results in an activation signal towards Nrf2. Nrf2 is an important mammalian member of the CncC family, which has been studied in numerous cellular systems and represents an important target for drug discovery in different diseases. Nrf2 is distributed in the cytoplasm together with Keap1 normally [12,13,14]. Under oxidative stress, Nrf2 was released and migrated to the nucleus, inducing HO-1 expression. HO-1, an antioxidant enzyme in the stress response, catalyzing the degradation of heme, biliverdin, and carbon monoxide (CO), also plays a key role in inflammation and oxidation responses.

Our previous study showed that the pre-treatment with FA carried out on LPS-treated RAW264.7 cells determined a decrease of HO-1 protein level [9]. Until now there are not any data demonstrating IKK involvement in Nrf2 modulation, so to verify the FA effectiveness and IKK/Keap1 involvement we analyze the HO-1 expression in Keap1silenced RAW264.7 cells. The results showed that pretreatment with FA decreased HO-1 mRNA expression in Keap1silenced RAW264.7 cells. Therefore, the results obtained agree with Kim et al [28] who identified Keap1 as IKK interacting protein and highlight that the Keap1 protein underlie the FA effectiveness on NF-κB modulation. The network Keap1-Nrf2-NF-κB/IKKβ is very interesting: it has many implications, being involved in inflammation, chemoresistance and cellular homeostasis [16,17,18]. Further studies are necessary to clarify the role of Keap1 in the NF-κB/ IKKβ dialogue. Our results also show that the Keap1 is an attractive target for therapeutic purposes, especially to improve the efficacy of anticancer and antiinflammatory molecules.

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Research of Oxidative Stress and Serum Thiols as a Criterion for the Antioxidant Barrier in Patients with Heart Failure (NYHA FC III-IV)

Authored by Mariana Georgieva Yordanova*

Aim: To evaluate the status of oxidative stress and antioxidant systems (thiol and biological plasma antioxidant potential) in patients with heart failure, combined with disorders of carbohydrate metabolism and renal failure, and to what extent their values depend on the duration and severity of the disease (by NYHA).

Materials and Methods: Forty subjects (age 66.4±6.8) admitted for exacerbated chronic heart failure (III and IV FC), and 12 of them were examined in the same age range without clinical and echocardiography for HF. The levels of total thiols (SHp), reactive oxygen metabolites (dROMs), plasma antioxidant potential (BAP), creatinine, calculated GFR, glucose and C-reactive protein are analysed. Measurement of SHp, dROMs and BAP assays was performed spectrophotometrically with kits of Diacron International-Italy with an Olympus AU400 biochemical analyser and Carpe diam analyser (Diacron).

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Air Pollution Could Alter Tags on DNA and Increase Risk for Neurodegenerative Disease

Not only is air pollution a major environmental concern, but it poses major health issues. New research suggests that inhaling extremely small particles known as fine particulate matter could add epigenetic marks to DNA. This may potentially lead to stroke, Alzheimer’s disease, Parkinson’s disease, cognitive impairments and neurodevelopmental disorders.

We’re certainly learning more about how our exposure to environmental toxins, especially pollution, affects the chemical tags that attach to our DNA and our health. Previous studies have linked traffic-related air pollution to an increase in histone acetylation, an epigenetic mark found on histone proteins. Similar research uncovered an association between inhaling diesel exhaust fumes and epigenetic changes��that affected around 400 genes.

In a new study published in Toxicology, a team of researchers set out to investigate the role of oxidative stress and DNA hydroxymethylation caused by particulate matter from air pollution, in the development of neurodegenerative disease. 5-hydroxymethylcytosine (5-hmC) is an interesting epigenetic modification and its potential function continues to be explored.

First, they collected actual fine particulate matter from a busy street in Shanghai, China. Then they used a cell culture to administer different doses of the pollution material. As a result of exposing the cells to the pollution, they detected cell cycle arrest, cell apoptosis and cell proliferation inhibition in neuronal cells.

Next, to measure 5-hmC content and investigate the epigenetic impact, the group performed immunofluorescence using a 5-hmC antibody from EpiGentek and found an increase in total 5-hmC. They confirmed the rise in 5-hmC levels using the MethylFlash Hydroxymethylated DNA 5-hmC Quantification Kit (Colorimetric) from EpiGentek.

To look at the epigenetic mark in further detail, they drilled down and investigated the 5-hmC levels in promoter regions of 11 target genes using EpiGentek’s EpiQuik Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit. This assay performs antibody-based capture of hydroxymethylated DNA fragments and enabled the team to detect specific levels of the epigenetic tag on certain genes.

The researchers found that exposure to the pollution increased global DNA hydroxymethylation as well as gene-specific DNA hydroxymethylation of neuronal genes. This mark has been shown to be stimulated by oxidative stress and gives us more insight into which environmental toxins may play a role in the development of neurodegenerative disease.

“We revealed that oxidative stress-mediated neurocytotoxicity and abnormal DNA hydroxymethylation were involved in the mechanisms for the neuronal pathology of [fine particulate matter],” the researchers reported.

These results give us insight into the epigenetic impact of pollution and how it might affect neuronal growth and disease. According to the researchers, future directions for addressing the interactions between environmental toxins and epigenetic marks should focus on in vivo animal models or clinical samples.

SEE ALSO:   Epigenetic Insights on Nutrition, Hormones and Eating Behavior

Bailey Kirkpatrick

Source: Wei H. et al. (2017). Role of oxidative stress and DNA hydroxymethylation in the neurotoxicity of fine particulate matter. Toxicology, 380:94-103. doi: 10.1016/j.tox.2017.01.017

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Advances in Neutrophil Testing In Type 2 Diabetes Mellitus

Authored by Bernhard Otto Boehm

Abstract

Patients with type 2 diabetes mellitus (T2DM) suffer from impaired glucose metabolism which results in low-grade inflammation and activation of the innate immune system. Neutrophils the key effector cells of the innate immune system and heavily implicated in the pathogenesis of T2DM, are promising cell-based inflammatory biomarkers for immune health profiling, provided that they can be rapidly purified and measured with sufficient precision. In this review, we highlight recent advances in neutrophil isolation and functional assay using microfluidics technologies and the potential of their functional phenotype as a novel biomarker of vascular risk in diabetes.

Keywords:  Neutrophils; Diabetes; Point-of-care; Microfluidics; Immunology

Introduction

With the increasing aging population worldwide, metabolic disorders such as diabetes mellitus (DM) and cardiovascular diseases (CVDs) have become the main public health challenges with rising premature morbidity and associated mortality, as well as escalating healthcare costs [1]. DM is characterized by chronic hyperglycemia resulting in increased oxidative stress, inflammation and endothelial dysfunction [2,3]. Patients with CVDs or type 2 diabetes mellitus (T2DM) often exhibit low- grade inflammation, and are assessed based on established cardiovascular risk factors (glycemic control, blood pressure and lipids). Immune health is evaluated by differential leukocyte count and circulating biomarkers (cytokines and C-reactive protein (CRP), which are suboptimal for monitoring stage- dependent pathogenesis, advocating the need to develop new cell-based biomarkers that can quantity specific immune functions in addition to leukocyte enumeration.

Neutrophils, the key effector cells of the innate immune system, play a pivotal role in T2DM and CVDs pathogenesis [4]. Various neutrophil dysfunctions have been reported in T2DM patients including cell stiffening [5,6] impaired chemotaxis [7,8] and phagocytosis which lead to increased susceptibility to bacterial infections [9]. Despite the adverse changes of leukocytes in diabetes, there are currently no specific measurements to assess patient's leukocyte phenotypes or inflammatory status. As distinct neutrophil subsets exhibit functional and phenotypic differences [10] a better understanding of their phenotype and pathophysiological relevance requires novel neutrophil separation tools (independent of surface markers) to improve their predictive capabilities as novel biomarkers [11]. Microfluidics, also known as "lab-on-a-chip" technologies, is a powerful toolbox for rapid sample preparation and detection with its low consumption of sample and reagents, device miniaturization, and single-cell analysis [12]. In this short review, we will highlight recent advances in microfluidics- based neutrophil testing technologies, and the potential of neutrophilfunctional phenotype as biomarkers for diabetes testing.

Discussion

Neutrophil isolation

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes (~50-70%) in humans, with ~2-5x106 neutrophils per mL of whole blood (~109 RBCs). They are short-lived (~5-24hr), prone to activation [13] and should be processed quickly within 2-4 hours of collection. Conventional neutrophil isolation methods include density gradient centrifugation and RBCs lysis, which are laborious (~1- 3hr) and require large blood volume (~10mL). Commercial kits based on magnetic bead-based affinity binding (MACS xpress® Neutrophil Isolation Kit (Miltenyi Biotec) and Easy SepTM neutrophil enrichment kit (STEMCELL Technologies) provide high neutrophil yield and purity by negative selection, but is expensive for large volume processing.

Microfluidics technologies for neutrophil isolation have been developed based on affinity binding to functionalized surfaces using common neutrophil markers (CD66b, P-selectin) [14,15]. However, these methods require on-chip cell analysis as it is non-trivial to elute the purified neutrophils off-chip for downstream assays. Our group has previously developed an efficient size-based cell sorting technique known as Dean Flow Fractionation (DFF) based oninertial focusing phenomenon in micro channels [16]. In DFF systems, fluid flowing through a curvilinear (spiral) channel experiences centrifugal acceleration directed radially outward, leading to the formation of two counter-rotating vortices known as Dean vortices [17]. Besides inertial lift forces (FL) particles experience lateral Dean drag force (FD) due to these transverse Dean flows, which results in superior separation resolution as both forces (FL and FD) scale non-linearly with particle size [18-20].

We first applied DFF technology to isolate circulating tumor cells (CTCs) [21] and microorganisms from whole blood whereby the size ranges of the target cells are distinctly different from RBCs. By exploiting the subtle size differences between major leukocyte subtypes (neutrophils/monocytes~10-12μm lymphocytes~7-9μm), we recently developed a novel DFF spiral micro device to purify neutrophils rapidly from whole blood for functional phenotyping in T2DM [22]. The developed technology enables single-step neutrophil isolation (>90% purity) without immune-labeling, saving both time and cost. In addition, the sorted "untouched" neutrophils are continuously eluted off-chip with simultaneous buffer exchange, facilitating user operation and eliminating the need for centrifugation. Moreover, as the method only requires small blood volumes (finger prick ~50- 100μL) it can be easily integrated with other cellular assays or detection modules for point-of-care (POC) testing (Figure 1).

Neutrophil rolling

During endothelial inflammation, leukocyte adhesion cascade is a multi-step process involving cell rolling, adhesion and transmigration through blood vessel walls to the site of injury [23]. Neutrophil rolling is widely considered a critical step as it can affect cell adhesion with impaired cell tethering or increased rolling speeds [24,25]. Several microfluidics- based cell rolling assays have been reported previously to study rolling behavior under physiological flow conditions (~1-10dynecm-2), but not in disease-specific context [26-28]. In our study, we combined DFF neutrophil sorting method and microfluidics assay to measure neutrophil rolling speed on E-selectin, a cell adhesion molecule expressed by activated endothelium to initiate leukocyte recruitment. This neutrophilendothelial interaction is mediated by several sialyl Lewisx presenting ligands expressed on leukocytes including P-selectin glycoprotein ligand 1 (PSGL1), glycosylated CD44 and E-selectin ligand 1 (ESL1) [29].

In our clinical validation, we observed a significant down regulation of neutrophil PSGL-1 expression in T2DM patients. Using automated cell tracking algorithm, we further showed that rolling trajectories ofT2DM neutrophils were more discontinuous and irregular as compared to healthy neutrophils. Interestingly, diabetic neutrophils had ~20% higher rolling speeds, which correlated with neutrophil activation, PSGL-1 expression, as well as established cardiovascular risk factors (cholesterol, CRP and HbA1c). Taken together, the data support the hypothesis that neutrophil-endothelial interactions are impaired in T2DM patients which can lead to defective neutrophil recruitment, and thus increased patient susceptibility to infection.

Neutrophil chemotaxis

Chemotaxis, a dynamic process where cells sense and move in response to chemical gradients, is traditionally studied using Boyden chamber (transwell), Dunn chamber and micropipette assay [30]. However, these methods suffer from poor reproducibility and ill-defined chemical gradients, which could be overcome by using microfluidics technologies to generate stable and linear chemo attractant gradient in small length scale (~μm) [15]. Moreover, most microfluidic chemotaxis assays only require ~102-3 neutrophils, and facilitate real-time imaging of cell movement at single cell resolution [31]. First performed clinical testing of patients with burn injury using microfluidics, and observed that neutrophils suffered from impaired directionality or slower migration speed, which were associated with degree of burn injury.

Similarly neutrophils from asthmatic patients also displayed significantly slower migration speed as compared to healthy subjects, suggesting its use as a novel diagnostics marker [32]. As impaired neutrophil chemotaxis behavior was reported previously in diabetic patients our group has developed an integrated micro device for neutrophil chemotaxis assay using a drop of blood. The novelty lies in the single-step enrichment of neutrophils using biomimetic cell margination [33] and affinity capture, followed by simultaneous exposure to chemotactic gradient without requiring additional user manipulation [34]. In our preliminary clinical data we also observed signification suppression of chemotaxis behavior in T2DM patient, which can be mitigated by short exposure to metformin in vitro. Besides diagnostics applications, microfluidics chemotaxis assays also enable study of complex chemoattractant gradients with high precision [35], well-controlled spatial and temporal gradients to probe cell migration pattern [36,37], as well as effect of inflammatory mediators in neutrophil-monocyte interactions [38].

Neutrophil extracellular traps (NETs)

First discovered in 2004, formation of neutrophil extracellular traps (NETs) is an innate key defense mechanism against bacterial infections through the release of nuclear and granular contents to contain and kill pathogens [39]. Upon activation or exposure to bacteria, histones undergo citrullination, followed by chromatin decondensation. Nuclear membrane will degrade, leading to DNA release into the cell, and subsequently extrusion out of neutrophils. Secreted NETs (process known as NETosis) then form a sticky scaffold consisting mainly of microbicidal proteases/elastase and cytotoxic molecules (histones). Interestingly, recent work have shown that diabetic neutrophils were more susceptible to NETosis [40], which can mediate delayed wound healing [41].

NETs components (elastase, histones, neutrophil gelatinase- associated lipocalin, and proteinase-3) are also elevated in the blood of patients with diabetic foot ulcers, and were associated with infection or worsening of ulcer [42]. Overall these clinical evidences suggest a major role of NETosis in diabetes pathophysiology and endothelial damage making it a novel biomarker for early detection of diabetes-related vascular or end-organ complications. Compared to chemotaxis development of microfluidics NET osis assay is still at its early infancy with a recent reported assay based on fluorescent imaging of nucleus degradation [43]. Nevertheless given the increasing importance of NETosis and easy quantification using imaging, we expect more development of novel tools to measure NETosis phenotype in POC settings.

Conclusion

Multidimensional neutrophil phenotypic markers will significantly improvetheir predictive capabilitiesasinflammatory biomarkers provided that they can be rapidly purified and measured with sufficient precision. Microfluidics technologies are not only useful for efficient neutrophil purification but they can also be readily developed and integrated into POC testing plat forms to look at the sum effects of diabetes, hypertension and hyperlipidemia. This enables proper identification of high risk patients with appropriate follow up, reduces the risks in different aspects of the endothelial activation pathway and in time, the effects of therapeutics can also be studied in diabetes and other dysmetabolic diseases.

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