Rutin exerts anti-oxidant effects in H2O2-induced microglia oxidative stress

doi:10.21203/rs.3.rs-1417130/v1

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Rutin exerts anti-oxidant effects in H2O2-induced microglia oxidative stress

https://doi.org/10.21203/rs.3.rs-1417130/v1

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Microglia oxidative stress is of significance in central nervous system (CNS) dysfunctions. Rutin, a natural citrus flavonoid glycoside, possesses evident anti-oxidant effects in many organs. However, whether rutin participates microglia oxidative stress remains blurred. In present study, H₂O₂-induced BV-2 microglia oxidative stress (OS) model was used to explore how rutin act in microglia oxidative stress. Our findings suggest rutin supplement limited tumor necrosis factor-α, interleukin-1β, interleukin-6, Nitric oxide (NO) and iNOS secretion from microglia, but elevated interleukin-10 production. More importantly, rutin protected BV-2 microglia against oxidative damage by inhibiting reactive oxygen species (ROS) and malondialdehyde (MDA) production and restoring the activity of antioxidant enzymes superoxide dismutases (SODs) and Glutathione peroxidase (GSH-Px). Moveover, rutin upregulatd Nrf2 and Nrf-2 responsive gene expression, but downregulated keap 1 expression. These data exhibit rutin may alleviate microglia oxidative stress by modulating inflammatory factors and anti-oxidant enzymes in a Nrf2-dependent way.

oxidative stress

rutin

microglia

neuroinflammation

Nrf2/keap1 axis

Reactive oxygen species (ROS) are normal byproducts during everyday cellular metabolism. Oxidative stress occurs due to an imbalance between ROS production and ROS clearance by anti-oxidant system[1]. Mechanistically, Keap1-Nrf2-ARE signal is central in anti-oxidant system. Nrf2 expression in brain decreases with age, which associates with the imbalance of oxidative stress responses. The former study has shown that the mRNA level of NADPH quinine oxidoreductase (NQO1) and glutamine cysteine ligase (GCLC) were downregulated in AD mouse model, resulting in increased Aβ deposition[2]. Later, the same group discovered that Nrf2 intrahippocampal administration improved the capability of spacial learning and memory in AD mice[3], implying the promising role of Nrf-2 in AD and other neurodysfunctions. In addition, the major source of enhanced ROS production in aging brain is microglia, and intense microglial ROS production might at least partly account for age-related CNS dysfunctions[4]. Oxidative damage to nuclear and mitochondrial DNA and RNA were found in AD brain [5–7]. Similarly, in PD patients’ brain tissues, DNA, lipids and protein oxidative injury were also observed[8, 9]. A single cell transcriptome analysis have suggested numerous microglia population were found ROS positive in animal model of multiple sclerosis[10]. Interestingly, ROS tend to be an essential factor for microglia rapid proliferation during neuroinflammation, which was shown by my coworkers in primary microglial cells treated with LPS[11].

In comparison with other cell types, brain cells, especially microglia, are likely to experience OS due to their lipid-rich environment, high oxygen consumption and weak antioxidant defense. Microglial cells are important in brain innate immunity, which mainly function to remove infectious or useless substances in CNS[12]. Upon CNS injury, microglial cells can shift from a surveillant state into an activated state [13], concomitant with altered cellular responses[14]. With respect to our previous study, microglial cells are capable to establish innate immune memory in response to LPS restimulation after various dose of LPS priming. In depth, these microglia built immune tolerance at high dose of LPS priming, but low dose of LPS priming elicited, an opposing effect, immune training[15].

Rutin is a low molecular weight natural citrus flavonoid glycoside which is widely found in vegetables and citrus fruits such as oranges and lemons[16]. Numerous studies have demonstrated that ruitn has significant anti-inflammatory, anti-tumor, and antioxidant effects which make it an important dietary component[17]. Importantly, the neuroprotective effect of rutin has been well reviewed in neurodegerative disease progression[18, 19]. Furthermore, a very recent paper has shown rutin improved tau pathology in animal model of AD[20]. However, oxidative stress-related innate immune cell subsets are heterogeous[10]. The current study will focus on how rutin acts on microglial oxidative stress.

In present study, BV-2 microglia stimulated with H₂O₂, a widely used cell model in investigations of brain oxidative stress injury, were chosen to explore the antioxidant functions of rutin. we hypothesize that rutin may prevent H₂O₂-induced oxidative stress injury via Nrf2/keap1 axis.

Materials

BV-2 microglial cells was obtained from Wuhan University Cell Library (Wuhan, China). Rutin (Cat# R8170) was obtained from Solarbio Science & Technology Company (Beijing, China) with high purity(≥ 98%). Rabbit polyclonal anti-iNOS antibody (Cat# ab15323), mouse monoclonal anti-Keap1 antibody (Cat# ab119403), mouse monoclonal anti-NQO1 antibody (Cat# ab28947), rabbit monoclonal anti-GCLC antibody (Cat# ab190685), rabbit monoclonal anti-HO-1 antibody (Cat# ab62352) and mouse monoclonal anti-β-actin antibody (Cat# ab6276) were purchased from Abcam (Cambridge, UK). Secondary antibodies were obtained from Beyotime (Haimen, China). In addition, we obtained ROS assay kit (Cat# S0033M) and NO assay kit (Cat# S0021S) from Beyotime (Shanghai, China). MDA assay kit (Cat# A003-1), SOD assay kit (Cat# A001-3) and GSH-Px assay kit (Cat# A005-1) were bought from Jiancheng Bioengineering Institute (Nanjing, China) and CCK-8 kit (Cat# CK04) was obtained from Dojindo (Japan). H₂O₂ (Cat# 323381) was provided by Sigma Aldrich (St. Louis, USA).

Cell Culture and Treatment

BV-2 microglia were kept in DMEM/HIGH GLUCOSE medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% FBS (BioInd, Inc., Shanghai, China) and 1% penicillin/streptomycin (Solarbio Science & Technology Co., Ltd., Beijing, China) at 37℃ and 5% CO₂. The medium was changed once every 24 h and cultured for 48 h for subculture. Cells at logarithmic growth stage were selected for subsequent experiments. Cells were pretreated with different concentrations of rutin (12.5, 25 and 50 µg/ml) for 1 h, and then incubated with H₂O₂ (250 µM) for 24 h to induce microglia oxidative stress injury.

CCK-8 assay

BV-2 cell viability was assessed by CCK-8 assay. briefly, in the first part of our study, we cultured BV-2 cells (6×10³ cells/well) for 24 h in 96-well plates with H₂O₂ (50 µM–350 µM). In the next part, the cells (8×10³ cells/well) were first treated with rutin (12.5, 25 and 50 µg/ml) for 1 h followed by stimulation with 250 µM H₂O₂ for 24 h. Thereafter, we added 10 µl CCK-8 reagent into the culture medium for 3 h. At last, the absorbance was measured at 450 nm by microplate reader (Thermo Scientific). Six independent experiments were conducted for each group with three replicates.

ELISA

As we mentioned above, BV-2 cells (1×10⁶ cells/ml) were seeded into 6-well plates followed by 12.5, 25 and 50 µg/ml rutin pretreatment for 1 h and 250 µM H₂O₂ incubation for 24 h. Thereafter, the concentration of inflammatory factors TNF-α (Cat# MM-0132M1), IL-6 (Cat# MM-0163M1), IL-1β (Cat# MM-0040M1) and IL-10 (Cat# MM-0176M1) in supernatants were estimated with ELISA kits (MEIMIAN Co., Ltd., Jiangsu, China) based on the manufacturer’s instructions. Optical density (OD) was read at 450nm by a microplate reader (Thermo Scientific). Five independent experiments were conducted for each group with three replicates.

NO assay

BV-2 cells (3×10⁵ cells/ml) were seeded into 6-well plates and pretreated with 12.5, 25, and 50 µg/mL rutin for 1 h, followed by stimulation with 250 µM H₂O₂ for 24 h. The content of NO in BV-2 cells was evaluated by NO test kit by following the kit instructions. 50 µl cell supernatant was mixed with equal volume of Griess reagent I and Griess reagent II at room temperature. The concentration of NO content was calculated using the NaNO₂ standard calibration curve. The absorbance of the reaction mixtures was measured at 540 nm with a microplate reader (Thermo Scientific). Six independent experiments were conducted for each group with three replicates.

ROS assay

The intracellular ROS in BV-2 cells was detected by ROS assay kit. BV-2 cells (4×10⁴ cells/well) were seeded into 96-well plates and treated with rutin and H₂O₂ as mentioned above. Cells were washed with DMEM (FBS free) for three times and incubated with 10 µl DCFH-DA (1 µM) at 37°C for 20 min in dark. After washed with FBS free DMEM, ROS positive populations were measured by flow cytometer (Thermo Scientific) at 488 nm laser wave length and 525 nm detection wave length. Six independent experiments were performed for each group with three replicates.

MDA, SOD and GSH-Px assay

The MDA, SOD and GSH-Px content in BV-2 cells was determined in the following experiments using commercial kits. Cells (2×10⁶ cells/well) were seeded into 6-well plates and pretreated with 12.5, 25, and 50 µg/mL rutin for 1 h, followed by stimulation with 250 µM H₂O₂ for 24 h. After administration with rutin and H₂O₂, BV-2 microglial cells were homogenized in turn. After centrifugation at 12,000 g for 5 min, the supernatant was collected and quantified using a BCA protein assay kit. The content of MDA, SOD and GSH-Px were measured using assay kits by following individual instructions. MDA content was performed by thiobarbituric acid (TBA) method. Briefly, MDA reacts with TBA to generate an MDA-TBA adduct. 40 µl supernatant, 10 µl testing buffer solution I, 600 µl testing buffer solution II and 200 µl testing buffer solution III were mixed in 1.5 ml tube and heated to 95°C for 40 min, after cooling, the samples were centrifuged at 4000 r/min for 10 min. Then 200 µl of the solution of each sample were added to 96-well plates and measured at 532 nm with a microplate reader (Thermo Scientific). The activity of SOD was measured by WST method, 20 µl supernatant, 20 µl enzyme working solution and 200 µl substrate solutions were mixed and incubated at 37°C for 20 min. The absorbance was measured on a microplate reader at 450 nm. GSH-Px was measured using the GSH assay kit based on the manufacturer’s instruction, the absorbance was measured at 412 nm. All the test were performed by six independent experiments with three replicates.

Quantitative real-time polymerase chain reaction

For quantification of mRNA, 1×10⁷ cells were seeded into 6-well plates and incubated at 37°C (5% CO₂) overnight and treated with rutin and H₂O₂ as mentioned above. An Axyprep total RNA preparation kit (AXYGEN Biosciences, Union City, CA, USA) and PrimeScriptTM RT reagent kit (TaKaRa, Beijing, China) were used to perform total RNA extraction and reverse transcription, respectively. DNase was used to avoid contamination with chromosomal DNA. RNA concentration was determined by Nano-Drop. Relative mRNA expression data was analyzed by fold change (2^−ΔΔCt) and normalized to housekeeping gene GAPDH expression. Murine GAPDH primer was obtained from Sangon Biotech (Shanghai, China). The following murine primer pairs were used: Nrf2: forward: 5′-CAGCCATGACTGATTTAAGCAG-3′, reverse: 5′-CAGCTGCTTGTTTTCGGTATTA-3′; Keap1: forward: 5′-GACTGGGTCAAATACGACTGC-3′, reverse: 5′-GAATATCTGCACCAGGTAGTCC − 3′; NQO-1: forward: 5′-GAAGACATCATTCAACTACGCC-3′, reverse: 5′-GAGATGACTCGGAAGGATACTG-3′; HO-1: forward: 5′-TCCTTGTACCATATCTACACGG-3′, reverse: 5′-GAGACGCTTTACATAGTGCTGT − 3′; GCLC: forward: 5′-CTATCTGCCCAATTGTTATGGC-3′, reverse: 5′-CCTCCCGTGTTCTATCATCTAC − 3′.

Immunofluorescence staining

Coverslips were coated for 2 hours by poly-L-lysine (0.1 mg/ml; Beyotime) at 37°C then placed into a 12-well plate. BV-2 cells were cultured on the coverslips at 7.5×10⁴ cells/well and pretreated with rutin (12.5, 25 and 50 µg/ml) for 1 h, followed by incubation with H₂O₂ (250 µM) for 24 h. The cells were permeabilized with 0.1% Triton X-100 for 15 min following fixation with 4% paraformaldehyde for 30 min at room temperature. We subsequently blocked the cells with 2% bovine serum albumin at 37°C for 1 h and incubated cells with anti-Nrf2 (1:100) antibody at 4°C overnight. We next stained the cells at room temperature with the secondary fluorescent anti-rabbit IgG antibody Alexa Fluor® 488 (1:500, Cell Signaling Technology, Danvers, USA) for 2 h followed by incubation with DAPI for 2 min. The coverslips were mounted and examined under a fluorescence microscope (Olympus, Tokyo, Japan) at 20× magnification.

Western Blot

BV-2 cells (1×10⁶ cells) were collected after pretreating with rutin (12.5, 25 and 50 µg/ml) for 1 h and stimulating with H₂O₂ (250 µM) for 24 h. Total proteins were extracted by RIPA lysis buffer (Solarbio Science & Technology Co., Ltd.), the samples were centrifuged at 12,000 r/min for 10 minutes at 4°C, and quantified by BCA Kit (Generay Biotech Co., Ltd., Shanghai, China). Protein samples were loaded on 8 or 15% SDS-PAGE gel for protein separation, which was then transferred to 0.45-µm PVDF membranes (4.5 cm × 9 cm). The membranes were blocked by blocking buffer (Beyotime, Cat# P0252, Shanghai, China) for 10 min, then incubated with following primary antibodies: iNOS (1:1000), Keap1 (1:2000), NQO1 (1:1000), GCLC (1:1000), HO-1 (1:2000) and β-actin (1:2000) at 4°C overnight. We next incubated the membranes with HRP-conjugated secondary antibody (goat anti-mouse IgG, Cat# A0216, 1:2000, Beyotime; goat anti-rabbit IgG, A0208, 1:2000) for 1 h at room temperature. Finally, protein bands were visualised with ECL substrate and quantified with FujiFilm Multi Gauge Ver. 3.0 software (Fuji Photo Film Co., Tokyo, Japan). The relative quantification of protein band intensity was divided by loading control normalized intensity of corresponding untreated cells.

Statistical Analysis

Data analyses were performed using Prism 5 software (GraphPad Software Inc., San Diego, CA, United States). All data were demonstrated as means ± SEM. All results were analyzed by one-way ANOVA and Post hoc comparisons were made with the Tukey’s Multiple Comparison test. p < 0.05 was defined as statistically significant.

Effects of rutin on the viability of BV-2 cells treated with H₂O₂

We first used CCK-8 assay to evaluate BV-2 cell survival rate. To estimate the best stimulation concentration of H₂O₂ for this assay, BV-2 cells were treated with H₂O₂ at the concentrations between 0 µM and 350 µM for 24 h. Results showed (Fig. 1A) that the cell viability was significantly decreased by approximately 80% at 250 µM. Cells were subsequently subjected to 250 µM H₂O₂ treatment for 1, 3, 6, 12, 24 and 48 h to explore an optimal time point for further experiments. As shown in Fig. 1B, survival rate of BV-2 cells was significant reduced after H₂O₂ administration for 24 h. Therefore, we decided to treat BV-2 cells with H₂O₂ at 250 µM for 24 h for the following investigations. Next, the effect of ruitn on the viability of BV-2 cells treated with H₂O₂ was studied. BV−2 cells were pretreated with rutin at 12.5, 25 and 50 µg/ml for 1 h followed by treatment with H₂O₂ (250 µM) for 24 h (method 1). As shown in Fig. 1C, 50 µg/ml rutin treatment successfully rescued H₂O_2 − induced BV−2 microglia viability reduction, indicating a promising protective effect of rutin in microglia oxidative stress.

Rutin exhibits inhibitory effects on H₂O₂-induced BV-2 cell inflammation

BV-2 cell treatment for this experiment is same as method 1. ELISA was performed to measure the concentrations of IL-6, IL-1β, TNF-α and IL-10 in the supernatants. Results showed,upon H₂O₂ administration, IL-6, IL-1β and TNF-α content were markly enhanced (p < 0.001) and IL-10 concentration was dramatically decreased (p < 0.001) when compared with the unstimulated group (Fig. 2A-D). As expected, the alterations occured above were reversed by rutin pretreatment at certain concentration or concentration ranges (Fig. 2A-D).

Rutin attenuates H₂O₂-induced iNOS and NO production

NO signaling plays a central role in microglia-related behaviours in CNS. To investigate whether rutin could inhibit OS-induced iNOS and NO production, BV-2 cells were treated as method 1. We first examined the iNOS protein expression by weastern blotting and results showed microglia-derived iNOS was decreased by rutin pretreatment (Fig. 3A and Fig. 3B). Thereafter, NO test kit was used to assess the NO content secreted by BV-2 cells. As shown in Fig. 3B, H₂O₂ alone remarkably enhanced NO production compared to untreated group, and rutin significantly suppressed the NO level in H₂O₂-induced BV-2 cells in a concentration dependent fashion (Fig. 3C), which correlated with iNOS level alterations. Therefore, our data above indicated that rutin limited INOS/NO-mediated microglial oxidative stress.

Rutin inhibits H₂O₂-induced BV-2 microglia oxidative stress

ROS release is pivotal during oxidative stress. Therefore, we investigated the capability of rutin to inhibit H₂O₂-induced ROS generation in BV-2 cells using ROS assay kit. The results in Fig. 4A indicated that the H₂O₂ stimulation exhibited an increased ROS level compared to the unstimulated group, which was further significantly restricted by 50 µg/ml rutin pretreatment; however, lower dose (12.5 µg/ml and 25 µg/ml) of rutin pretreatment were not able to prevent the alterations induced by H₂O₂ alone. As we know, MDA levels refects the extent of lipid peroxidation and cellular injury. In our results, MDA level was markly elevated followed by H₂O₂ addition and rutin was capable to antagonize MDA elevation (Fig. 4B). Apart from ROS and MDA, we also examined SOD and GSH-Px activity to evaluate the microglia antioxidant status. As shown in Fig. 4C and 4D, rutin restored the SOD and GSH-Px activity reduced by H₂O₂ in a dose-dependent way.

Rutin alleviated H₂O₂-induced oxidative stress associated with Nrf2-Keap1 signaling

Nrf2 is an important factor in cellular antioxidant defense system. Nrf2-mediated gene transcription is prone to inhibit oxidative stress induced central nervous system injury. Therefore, we next focus on the influence of rutin on Nrf2-Keap1 signaling pathway in BV-2 cells. BV-2 cells were treated as described above. Our data showed that the expression of Nrf2 (Fig. 5A) and NQO1 (Fig. 5C) were significantly increased, whereas Keap1 (Fig. 5B) expression was significantly decreased in rutin pretreatment cells. However, the HO-1 and GCLC mRNA expression were affacted by rutin pretreatment (Fig. 5D and 5E).

In the following experiment, the expression level of Nrf2 was examined by immunofluorescence staining, at the same time, NQO1, HO-1 and GCLC protein levels were determined by western blot. As expected, the immunofluorescence staining results revealed that BV-2 cells displayed decreased Nrf2 immunopositivity after H₂O₂ stimulation compared with that in the control group, which can be enhanced by rutin pretreatment (Fig. 6A). In addition, Keap1 protein expression level was increased by H₂O₂ stimulation, whereas rutin treatment effectively reversed this upregulation in BV-2 cells (Fig. 6B and 6F). We next tested NQO1, HO-1 and GCLC protein expression. Results showed an decreased NQO1 and HO-1 expression following H₂O₂ stimulation, which was also enhanced by rutin pretreatment (Fig. 6C, 6D and 6F). However, GCLC expression was not affacted by rutin addition (Fig. 6E-F). These results suggested that rutin may inhibit the expression of Keap1, accelerate the entry of Nrf2 into the nucleus, promote the downstream NQO1 and HO-1 expression, and thus inhibit the oxidative stress injury of BV-2 cells induced by H₂O₂ .

This present exploration found rutin is capable to increase BV-2 microglial viability under H₂O₂- induced oxidative conditions, indicating rutin is a promising candidate to ease oxidative stress in neurodegenerative diseases. In detail, our study discovered rutin markly reduced oxidative markers ROS and MDA fomation in BV-2 microglia, but enhanced the star antioxidant enzymes including SOD and GSH-Px activities. In additon, rutin effectively inhibits extracellular proinflammatory ingredients TNF-α, IL-1β, IL-6 secretion, but promotes anti-inflammatory factor IL-10 expression of BV-2 microglia upon H₂O₂ stimulation, suggesting the beneficial effects of rutin in oxidative stress induced inflammation. Furthermore, NO and iNOS produced by BV-2 microglia were also significantly downregulated by rutin. Last but not least, rutin pretreatment restored Nrf-2 decrease resulted from H₂O₂ addition, reduced keap1 expression and consequently elevated the downstream gene expression of Nrf-2 signaling, which implies signaling pathway via Nrf-2 is involved in rutin mediated anti-oxidant responses. The best way to explain our results is that H₂O₂ trigger more ROS production by microglial mitochondria, consequently causing increased inflammatory factors and lipid peroxidation marker MDA, in contrast, rutin reversed this process by initiating anti-oxidant system components including Nrf-2, SOD, downstream responsive genes (for example HO-1 and NQO-1) .

Inflammation and oxidative stress prone to co-exist and interact in such a manner that one can be easily exacerbated by the other[21]. It is well documented that an chronic sustained inflammatory-oxidative state existed in age-related diseases, such as cardiac disorders[22]. This view can be explained by our findings. Specifically, our present study found H₂O₂ mediated oxidative stress promotes microgial inflammation with an increase of TNF-α, IL-1β, IL-6 and an decrease of IL-10. On the contrary, our previous study displayed LPS-induced microgial inflammation enhanced NO and iNOS generation[14], which is believed to further induce oxidative stress.

With respect to signaling pathways involved in oxidative stress, CoO ENP -related ROS elevation triggered pathways including PI3K/AKT and Nrf-2-involved signaling[23]. A study focusing on the role of caffeine in AD and PD suggested transcription factor Nrf-2 was enhanced by caffeine, subsequently easing oxidative responses[24]. Unlike caffeine, quercetin facilitate the clearance of oxidative stress and inflammation by modulating Nrf-2 dependent antioxidant responsive elements and NF-κB signal transducer and activator of transcription-1 respectively[25]. Similar to caffeine and quercetin, rutin restrained H₂O₂ responsive oxidative stress accompanied by increased expression of Nrf-2 and decreased keap1 in our study, suggesting Nrf-2/keap1 signaling might be an critical component in rutin-mediated anti-oxidant effect in neuroinflammation. Unforturnately, our study could not conduct rescue experiments to confirm the necessity of Nrf/keap1 pathway in rutin-mediated oxidative stress in microglia. At the same time, we also observed increased level of inflammation-promoting factors, therefore NF-ҡB-signaling might also be responsible for this alteration in our H₂O₂ microglial cell model. Additionally, NO-signaling is associated with inflammation both in atherosclerosis and microglial activation[14, 26]. Our results showed NO upregulation in response to H₂O₂ addition in microglia. Data above implicated a possible NO-signaling participation in inflammatory-oxidative disease environment, like AD and PD.

In summary, rutin has the ability to protect microglia against oxidative-inflammatory state, which may in turn limit microglia-related neurodegerative diseases. Moverover, this study revealed rutin may take part in Nrf-2/keap1 anti-oxidant network in BV-2 microglia. Therefore, our data pave the way for rutin in the drug development of oxidative stress related neurodegerative diesease. However, the detailed modulating way of rutin in microglia oxidative stress require further researches, for instance in vivo experiments.

Author Contributions

The whole experiment was conceived and designed by G.-P.L. and Y.-Y.H. ; literature search and experimental studies were performed by G.-P.L., Y.-Y.H., X.X.; S.-J.L., Y.Y. and Y.-L.Y. ; data acquisition and statistical analysis were conducted by Y.-Y.H. ; manuscript preparation and manuscript editing were completed by G.-P.L. and Y.-Y.H.

Funding

This study was supported by National Natural Science Foundation of China (Grant No. 82104188), Joint fund project of National Natural Science Foundation of China and People’s Government of Guizhou Province (Grant No. U1812403).

Conflict of Interest

The authors declare no competing interests.

Data availability

The data used to support the findings of this study are included within the article. All data generated or analyzed during this study are included in this published article.

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Rutin exerts anti-oxidant effects in H2O2-induced microglia oxidative stress

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Materials

Cell Culture and Treatment

CCK-8 assay

ELISA

NO assay

ROS assay

MDA, SOD and GSH-Px assay

Quantitative real-time polymerase chain reaction

Immunofluorescence staining

Western Blot

Statistical Analysis

Results

Effects of rutin on the viability of BV-2 cells treated with H₂O₂

Rutin exhibits inhibitory effects on H₂O₂-induced BV-2 cell inflammation

Rutin attenuates H₂O₂-induced iNOS and NO production

Rutin inhibits H₂O₂-induced BV-2 microglia oxidative stress

Rutin alleviated H₂O₂-induced oxidative stress associated with Nrf2-Keap1 signaling

Discussion

Declarations

Author Contributions

Funding

Conflict of Interest

Data availability

References

Additional Declarations

Status:

Version 1

Rutin exerts anti-oxidant effects in H2O2-induced microglia oxidative stress

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Materials

Cell Culture and Treatment

CCK-8 assay

ELISA

NO assay

ROS assay

MDA, SOD and GSH-Px assay

Quantitative real-time polymerase chain reaction

Immunofluorescence staining

Western Blot

Statistical Analysis

Results

Effects of rutin on the viability of BV-2 cells treated with H2O2

Rutin exhibits inhibitory effects on H2O2-induced BV-2 cell inflammation

Rutin attenuates H2O2-induced iNOS and NO production

Rutin inhibits H2O2-induced BV-2 microglia oxidative stress

Rutin alleviated H2O2-induced oxidative stress associated with Nrf2-Keap1 signaling

Discussion

Declarations

Author Contributions

Funding

Conflict of Interest

Data availability

References

Additional Declarations

Status:

Version 1

Effects of rutin on the viability of BV-2 cells treated with H₂O₂

Rutin exhibits inhibitory effects on H₂O₂-induced BV-2 cell inflammation

Rutin attenuates H₂O₂-induced iNOS and NO production

Rutin inhibits H₂O₂-induced BV-2 microglia oxidative stress

Rutin alleviated H₂O₂-induced oxidative stress associated with Nrf2-Keap1 signaling