XUE Hongkun, TAN Jiaqi, LI Qian,*, TANG Jintian,*

(1. Key Laboratory of Particle & Radiation Imaging, Ministry of Education, Department of Engineering Physics, Tsinghua University,Beijing 100084, China; 2. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100080, China)

Abstract: The aim of this study is to evaluate the cytoprotection and potential molecular mechanisms of cyanidin-3-O-glucoside (C3G) on hydrogen peroxide (H2O2)-induced oxidative damage in RAW264.7 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to determine the viability of RAW264.7 cells exposure to H2O2 or C3G. Meanwhile, we measured the antioxidant properties of C3G by determining the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), nitric oxide (NO) release and malondialdehyde (MDA) levels by enzyme-linked immunosorbent assay (ELISA). 2’,7’-dichlorofluorescin diacetate (DCFH-DA) was employed to evaluate the production of intracellular reactive oxygen species (ROS). Finally, the expression levels of related mRNA/protein were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. The results showed that the H2O2-induced decrease in the cell viability of RAW264.7 cells was remarkably suppressed C3G(6.25-25.00 μmol/L). C3G significantly inhibited the H2O2-induced of overproduction of intracellular ROS, NO release and MDA levels, but increased the activities of intracellular SOD and GSH-Px (P ＜ 0.05). In addition, the relative mRNA and protein expression levels of Mst1, Mst2 and Keap1 were up-regulated, while the mRNA and protein relative expression levels of Nrf2 and HO-1 were down-regulated in the 400 μmol/L H2O2-treated group when compared to the vehicle-treated group. However, the above changes were reversed by intervention with C3G. C3G could exert a cytoprotective effect possibly by activating the Mst/Nrf2 signaling pathway and improving the activities of antioxidant enzymes.

Keywords: cyanidin-3-O-glucoside; RAW264.7 cells; oxidative damage; Mst/Nrf2 signaling pathway

The reactive oxygen species (ROS), including hydroxyl radicals, singlet oxygen and superoxide anions, can lead to oxidative damage to DNA, protein and lipid[1]. A growing number of researchers have confirmed that oxidative damage is correlated with the pathological development of many chronic diseases, such as aging, cardiovascular disease, Alzheimer’s, diabetes and cancer[2-3]. In addition,the excessive generation of intracellular ROS destroys the balance between oxidation and antioxidant defense system,which eventually leads to irreversible cell damage and death[4]. Mounting evidence has revealed that antioxidants could prevent or delay ROS-triggered apoptosis, which might be a reasonable way to treat a variety of chronic diseases including cholestasis, chronic hepatitis and so on[5-6].Among various antioxidants, natural substances isolated and purified from natural plants showed advantages over synthetic chemicals because the latter had acute by-effects though strong radical scavenging abilities[7]. Besides, natural antioxidants can prevent body injury by removing excessive ROS, decreasing the malondialdehyde (MDA), inhibiting NO release and enhancing activity of antioxidant enzymes[8].Recently, many researchers had been concentrated on hunting for natural active ingredients to protect the health of the body,which could scavenge excess free radicals and prevent cells from oxidative damage.

Blueberry (Vacciniumspp.) grows in Northeast China, Canada and North America. Wild blueberries are extraordinary popular due to its attractive blue and special fragrance. Cyanidin-3-O-glucoside (C3G) is one of the most abundant anthocyanins monomers in blueberry. In our previous study, C3G was obtained (purity ＞ 99.0%) from blueberry by using AB-8 macroporous resin and Sephadex LH-20[9]. C3G, a natural flavonoid compound and as a typical antioxidant, has been testified to have multiple profitable effects with respect to its anti-aging, anti-oxidative,anti-inflammation and vascular relaxation and so on[10]. Lee et al[11]found that C3G isolated from mulberry fruit protected pancreaticβ-cell from H2O2-induced oxidative damage.Aboonabi et al[12]demonstrated that anthocyanins could markedly decrease the cytotoxicity of human diabetic aortic endothelial cells induced by H2O2. Additionally, Jee et al[13]illustrated that anthocyanins from black soybean could protect human lens epithelial cell line from oxidative damage induced by H2O2by decreasing H2O2toxicity, promoting the expression of Bcl-2 and inhibiting the activation of BAD,BAX and caspase-3[13]. Meanwhile, Song Jian et al[14]found that C3G protected human MDA-MB-231 cells against oxidative stress induced via decreasing cerebral superoxide levels, inhibiting apoptosis inducing factor release from mitochondria, activating the cytochrome c-related cell death pathway. Liu Di et al[15]demonstrated that C3G protected HEK-293 cells against H2O2-induced oxidative stress through reducing intracellular ROS and MDA, as well as activating Nrf2/Keap1 signaling pathway. It is worth noting that previous researches have found that C3G had beneficial effects on liver diseases, which included transient cerebral ischemia model and aging accelerated mouse model[16-17].C3G may be a natural medicine for the treatment of various chronic diseases. RAW264.7 cells are mouse peritoneal macrophages, which are often used as classic cells of oxidative damage[18]. Nevertheless, there is not enough information about the effect of C3G on RAW264.7 cells, and the underlying mechanism of the protective effects of C3G on H2O2-induced oxidative damage remains to be elucidated.

Therefore, we explored the protective effects of C3G on H2O2-induced oxidative damage in RAW264.7 cells and investigated the potential molecular mechanisms of action involved in this process.

1 Materials and Methods

1.1 Materials and reagents

C3G (purity ＞ 99.0% and relative molecular weight 449.38) was obtained from blueberry extracts in our previous study[9]. The dimethyl sulfoxide (DMSO), Dulbecco’s modified Eagle medium (DMEM) , streptomycin solution,penicillin solution and H2O2were purchased from Beijing Shengmu Biotechnology Co. Ltd.; RAW264.7 cells were purchased from National Infrastructure of Cell Line Resource(Beijing); Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co. Ltd.; Trypsin digestive fluid was purchased from Beijing Fubo Biotechnology Co.Ltd.; Radio-immunoprecipitation assay (RIPA) buffer was purchased from GIBCO-BRL Inc.; The test kits of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), MDA,NO, ROS and bicinchoninic acid protein were purchased from Nanjing Jiancheng Bioengineering Research Institute Co. Ltd.;TRIzol reagent and reverse transcription kit were purchased from Beijing Baiaosentai Biotechnology Co. Ltd.; The anti-Mst1/2, anti-HO-1, anti-Nrf2, anti-Keap1 and anti-β-actin were purchased from Shenzhen Zike Biotechnology Co. Ltd..

1.2 Instruments and equipment

Bio-Rad ZE5 flow cytometer was purchased from Bio-Rad Laboratories, Inc., USA; BPN-40RHP/CRH CO2incubator was purchased from Shanghai Yiheng Technology Co., Ltd.; WD-2102B automatic microplate reader was purchased from Beijing Liuyi Biotechnology Co., Ltd.;EVOS M7000 phase contrast microscope was purchased from Thermo Fisher Technology Co., Ltd., USA.

1.3 Method

1.3.1 Cell culture condition

DMEM medium, including 10% (V/V) FBS and 1%(V/V) double antibody (penicillin and streptomycin) to avoid contamination by other bacteria, was employed to culture RAW264.7 cells in a humidified atmosphere CO2incubator (5% (V/V) CO2and 37 ℃). In the experiment, the RAW264.7 cells were inoculated into the culture plate, and the experiment was performed when the cells entered the logarithmic growth period.

1.3.2 Establishment of RAW264.7 cells injury model

The RAW264.7 cells (5 × 104cells/mL) were seeded in 96-well plates (50 μL/well) and cultured overnight in a humidified CO2incubator. The medium was subsequently replaced with different final concentrations (0, 50, 100,200, 300, 400, 500, 600, 700 and 800 μmol/L) of H2O2diluted with the DMEM medium for 0, 3, 6, 9, 12 and 24 h,respectively with 6 parallel wells in each group. After H2O2treatment, 10 µL MTT (5 mg/mL) was added to each well and cultured 4 h. Subsequently, the culture medium was removed, and then 150 µL DMSO was added to each well to dissolve the formed blue formazan crystals. Ultimately,the absorption of each well was determined at 490 nm by a WD-2102B automatic microplate reader. Cell viability is calculated by the following equation[19]. The corresponding H2O2concentration and treatment time were regarded as the best conditions for oxidative damage when the cell viability was 50%[20]. All experimental results are shown as the mean ± standard deviation of three experiments with six wells per treatment group.

1.3.3 Determination of the dosage range of C3G

RAW264.7 cells (5 × 104cells/mL) were grown in 96-well plates (50 μL/well) in a humidified CO2incubator for 24 h, and then intervened with C3G (0, 6.25, 12.50, 25.00,50.00 and 100.00 μmol/L) for 24 h. Simultaneously, we utilized a phase contrast microscope to detect RAW264.7 cells morphology. The following experimental steps are the same as the MTT method to determine cell viability, which is calculated using the above equation.

1.3.4 Effect of C3G on cell viability in H2O2-induced RAW264.7 cells

Briefly, RAW264.7 cells (5 × 104cells/mL) were placed into 96-well plates (50 μL/well) and cultured for 24 h at 37 ℃. According to the results of in section 1.3.3, the cells were treated with 400 μmol/L H2O2for 24 h, followed by exposure to various C3G concentrations (0, 6.25, 12.50 and 25.00 μmol/L) for 24 h at 37 ℃. We utilized MTT assay to measure the viability of RAW264.7 as previously reported[21].Cell viability is calculated by using the above equation.

1.3.5 Determination of ROS production

The effect of C3G on H2O2-induced ROS generation in RAW264.7 cells was monitored by the ROS Assay Kit according to its instructions. In short, RAW264.7 cells(3 × 105cells/mL) were grown in 6-well plates (100 μL/well)and incubated overnight, and then each well plate was exposed to 400 μmol/L H2O2for 24 h. Next, the cells were pretreated with C3G at the concentration of 0, 6.25, 12.50 and 25.00 μmol/L for another 24 h. Then the cells were incubated with 2’,7’-dichlorofluorescin diacetate (DCFH-DA) (100 μL,10 μmol/L) at 37 ℃ for 30 min. After DCFH-DA incubation,RAW264.7 cells were collected and determined by a Bio-Rad ZE5 cell analyzer.

1.3.6 Measurement of antioxidant parameters

RAW264.7 cells (3 × 105cells/mL) were incubated in 6-well plates (100 μL/well) in a humidified CO2incubator for 24 h. Then the cells were stimulated with 400 μmol/L H2O2for 24 h, followed by intervention with various C3G concentrations for 24 h. Subsequently, the cells were homogenized in RIPA buffer (2 mL). Then the mixture was centrifuged at 12 000 ×g, 4 ℃ for 15 min. The supernatant was employed to determine antioxidant parameters. The activities of SOD and GSH-Px, NO release and MDA levels were detected through commercial assay kits according to their instructions.

1.3.7 Reverse transcription polymerase chain reaction

Total RNA was obtained from the RAW264.7 cells by the TRIzol Reagent according to its instructions and transcribed into cDNA through a cDNA Reverse Transcription Kit. The specific primers for Mst1/2, Nrf2, Keap1, HO-1 andβ-actin based on Rattussequences were designed via Primer Premier software (Table 1). We utilized an RNA/DNA calculator to detect RNA concentrations. The volume of total reverse transcription RNA was determined according to the concentration. Relative mRNA expression levels of Mst1/2,Nrf2, Keap1 and HO-1 were analyzed by 2-ΔΔCtby the reverse transcription polymerase chain reaction (RT-PCR) method[2].

Table 1 Primer sequences of targeted genes and β-actin

1.3.8 Western blot analysis

RAW264.7 cells (3 × 105cells/mL) were seeded in 6-well plates and cultured overnight. The cells were treated with 400 μmol/L H2O2for 24 h, followed by intervention with various C3G concentrations (0, 6.25, 12.50 and 25.00 µmol/L)for 24 h. Subsequently, the cells were homogenized and dissolved in 2 mL RIPA buffer of radioimmunoprecipitation test to obtain protein extracts in the presence of protease inhibitor[15]. The bicinchoninic acid protein determination kit was used to examine protein concentration. Equal amounts (20 μg) of extracted proteins were separated on 10%SDS-polyacrylamide gels and transferred to the poly vinylidene fluoride (PVDF) membranes. The electrophoresis parameters were constant current 300 mA. The nonspecific sites were sealed with 5% skimmed milk powders in phosphate buffered saline Tween (PBST, pH 6.8, 0.1%V/V)for 60 min, and then the blots were incubated with anti-Nrf2,anti-HO-1, anti-Keap1, anti-Mst1/2 and anti-β-action (1:1 000 dilution,V/V) in PBST 24 h at 4 ℃.β-actin was performed to confirm equal loading of protein in each lane. The protein expression was determined by Western blot analysis.

1.4 Statistical analysis

All data are shown as the means ± standard deviation.SPSS Statistics 19.0 software was used for statistical analysis. One-way analysis of variance (ANOVA) was used to compare the significance of the means using Tukey’s test at the level of 0.05. Origin 9.0 software was employed for drawing.

2 Results and Analysis

2.1 Concentration and time-dependent viability losses in RAW264.7 cells exposed to H2O2

First, we utilized the MTT assay to examine the time and concentration-dependent losses in viability in RAW264.7 cells exposed to H2O2. As shown in Fig. 1, RAW264.7 cells viability dramatically diminished with the increase in H2O2concentration from 50 µmol/L to 800 µmol/L. The cells viability in the groups which were treated in 400, 500,600 and 800 µmol/L H2O2for 24 h, were (49.55 ± 1.86)%,(38.16 ± 1.57)%, (30.56 ± 1.44)% and (20.05 ± 0.77)%,respectively. The results indicated that H2O2resulted in remarkable damage to RAW264.7 cells in a dose-dependent manner. Meanwhile, 400 µmol/L H2O2treatment for 3-24 h gradually reduced RAW264.7 cells viability. Relevant researches have shown that the corresponding H2O2concentration and treatment time were regarded as the best conditions for oxidative damage when the cell viability was 50%[20]. Therefore, subsequent experiments were treated in 400 µmol/L H2O2for 24 h.

Fig. 1 Effect of H2O2 concentration and treatment time on RAW264.7 cell viability

Fig. 2 Effects of different concentrations of C3G on the cell viability (A)and morphology (B) of RAW264.7 cells

2.2 Determination of the dosage range of C3G

To assess the cytotoxicity of C3G on RAW264.7 cells,the cells were treated with C3G (6.25-100.00 µmol/L)for 24 h. As shown in Fig. 2A, C3G did not cause any cytotoxic effect when the concentration was in the range of 6.25 to 25.00 µmol/L. Morphological observations showed that C3G had no significant effects on cell numbers and morphology (Fig. 2B). Subsequently, the cytotoxicity of C3G to RAW264.7 cells increased prominently with the increase of concentration of C3G (P＜ 0.05). Simultaneously, the cells numbers decreased significantly when C3G concentration ranged from 50.00 µmol/L and 100.00 µmol/L (Fig. 2B).Consequently, 6.25-25.00 µmol/L of C3G were employed for latter experiments.

2.3 Effect of C3G on cell viability in H2O2-induced RAW264.7 cells

Cell viability is the most direct index to reflect the degree of cell damage caused by the external environment.H2O2, a considerable active oxygen molecule with relatively stable properties, is often used as a model drug for oxidative injuryin vitro. As shown in Fig. 3, the cell viability of the oxidative damage model group constructed by H2O2decreased significantly compared to the vehicle-treated group (P＜ 0.05),indicating that the oxidative damage model was successfully developed. Nevertheless, C3G (6.25-25.00 µmol/L)treatment remarkably increased cell viability in a dosedependent manner in H2O2-triggered RAW264.7 cells when compared to the H2O2alone treated group (P＜ 0.05).

Fig. 3 Effect of C3G on the viability of RAW264.7 cells treated with H2O2

2.4 Effect of C3G on H2O2-induced ROS production in RAW264.7 cells

The production of excessive ROS causes cells damage.Hence, we investigated the ability of C3G to inhibit the production of ROS through DCFH-DA as a fluorescent probe. As illustrated in Fig. 4B, the ROS levels markedly increased in RAW264.7 cells after H2O2induction compared to untreated control RAW264.7 cells (P＜ 0.05). However,the increased ROS levels caused by H2O2induction was attenuated in the RAW264.7 cells pretreated with C3G. Our results hinted that C3G could inhibit the generation of ROS in RAW264.7 cells under oxidative conditions.

Fig. 4 Effect of C3G on the intracellular ROS level in H2O2-induced RAW264.7 cells

2.5 Effects of C3G on the activities of SOD, GSH-Px, NO release and the MDA levels in H2O2-induced RAW264.7 cells

It is well known that cellular antioxidant systems,including mainly SOD and GSH-Px, can improve the ability of the cell to deal with oxidative damage caused by H2O2[22-23].NO is a signaling molecule that plays a critical role in the regulation of various functions[24]. Furthermore,MDA was acted as a biomarker of oxidative stress. To clarify whether the protective effect of C3G on H2O2-triggered RAW264.7 cells is owing to antioxidant properties, the activities of SOD and GSH-Px, NO release and MDA levels were determined by commercial kits.As shown in Fig. 5, the activities of SOD and GSH-Px were substantially decreased (P＜ 0.05), while the NO release and MDA levels were substantially increased in 400 µmol/L H2O2treated group compared with the control group (P＜ 0.05). Conversely, intervention with C3G (6.25,12.50 and 25.00 µmol/L) improved the activities of SOD and GSH-Px as well as attenuated the NO release and MDA levels in H2O2-induced RAW264.7 cells. Our results implied that the protective effect of C3G on RAW264.7 cells oxidative damage induced by H2O2was due to an improvement in the cellular antioxidant systems.

Fig. 5 Effect of C3G on antioxidant enzyme activities, NO release and MDA levels in H2O2-induced RAW264.7 cells

2.6 Effect of C3G on the relative mRNA expression levels of Mst1/2, Nrf2, Keap1 and HO-1 in H2O2-induced RAW264.7 cells

The relative mRNA expression levels ofMst1/2,Nrf2,Keap1andHO-1were determined by RT-PCR. As shown in Fig. 6, compared with the vehicle-treated group, the relative mRNA expression levels ofMst1/2andKeap1were prominently increased, while the relative mRNA expression levels ofNrf2andHO-1were dramatically decreased in 400 µmol/L H2O2treated group (P＜ 0.05). Conversely, C3G(6.25, 12.50 and 25.00 µmol/L) intervention significantly decreased the mRNA relative expression levels ofMst1/2andKeap1, while markedly increased the relative mRNA expression levels ofNrf2andHO-1in H2O2-triggered RAW264.7 cells when compared to the H2O2alone treated group (P＜ 0.05).

Fig. 6 Effect of C3G on the relative mRNA expression levels of Mst1 (A),Mst2 (B), Keap1 (C), Nrf2 (D) and HO-1 (E) in H2O2-induced RAW264.7 cells

2.7 Effects of C3G on the relative expression levels of Mst1/2,Nrf2, Keap1 and HO-1 in H2O2-induced RAW264.7 cells

Given the above research, C3G can protect RAW264.7 cells from oxidative damage. We carried further researches to determine its potential molecular mechanism. As shown in Fig. 7, 400 µmol/L H2O2treatment significantly increased the relative protein expression levels of Mst1/2 and Keap1,compared with the vehicle-treated group (P＜ 0.05).Compared with the H2O2alone treated group, the addition of C3G (12.50 and 25.00 µmol/L) substantially decreased the relative protein expression levels of Mst1/2 and Keap1(P＜ 0.05). Moreover, H2O2simulation RAW264.7 cells markedly decreased the relative protein expression levels of Nrf2 and HO-1 when compared to the control group(P＜ 0.05). Conversely, RAW264.7 cells treated with C3G(6.25-25.00 µmol/L) enhanced the relative protein expression levels of Nrf2 and HO-1 (Fig. 7E, F). Our findings indicated that C3G could protect RAW264.7 cells against oxidative damage through activation of the Mst/Nrf2 pathway.

Fig. 7 Effect of C3G on the relative expression levels of Mst/Nrf2 signaling pathway-related proteins in H2O2-triggered RAW264.7 cells

3 Discussion

In this present study, we utilized RAW264.7 cells as a model to explore the protective properties of C3G against oxidative damage. RAW264.7 cells were initially treated with H2O2(400 µmol/L) for 24 h, and then intervented with different concentrations of C3G for 24 h. We found that:1) C3G improved substantially H2O2-triggered RAW264.7 cells viability as authenticated through experiments (MTT assay); 2) C3G inhibited the production of intracellular ROS in RAW264.7 cells; 3) C3G (6.25-25.00 µmol/L) could enhance the activities of SOD and GSH-Px, while the NO release and MDA levels were decreased in H2O2-induced RAW264.7 cells; 4) C3G (12.50-25.00 µmol/L) could remarkablely down-regulated the relative mRNA/protein expression levels of Mst1/2 and Keap1, and up-regulated the relative mRNA/protein expression levels of Nrf2 and HO-1 in RAW264.7 cells. These results indicated that C3G protected RAW264.7 cells from oxidative damage via activation of the Mst/Nrf2 pathway and enhancement of the activities of antioxidant enzymes.

H2O2, as an important active oxygen molecule with relatively stable properties, had been widely used to induce oxidative damagein vitromodels. Thus, we chose H2O2to establish the RAW264.7 cells oxidative damage model in this study. The results found that stimulation of RAW264.7 cells with H2O2concentration from 50 to 400 µmol/L resulted in conspicuous decrease RAW264.7 cells viability(Fig. 1). The viability of RAW264.7 cells decreased to(49.55 ± 1.86)% when RAW264.7 cells were treated with 400 µmol/L H2O2for 24 h. Hence, we chose to use a 24 h exposure of 400 µmol/L H2O2for follow-up experiments.We utilized MTT assay to determine the toxicity of C3G in RAW264.7 cells. Results indicated that C3G (from 6.25 to 25.00 µmol/L) was non-toxic in RAW264.7 cells (Fig. 2A).Furthermore, C3G (6.25-25.00 µmol/L) intervention prominently increased cell viability in a dose-dependent manner in H2O2-triggered RAW264.7 cells when compared to the H2O2alone treated group (Fig. 3). Taken together, these results implied that C3G protected against H2O2-mediated RAW264.7 cells oxidative damage.

ROS is one of the vital factors in the formation and development of various diseases. Cell oxidative damage can produce excessive ROS, which damages human endothelial function as well as promotes cell death and apoptosis[25].MDA is the end product of lipid peroxidation by free radicalsin vivo, The MDA levels indirectly reflect the damage degree of cells attacked by free radicals[26]. In addition, to some extent, NO release can reflect the degree of oxidative damage of cells[27]. Therefore, intracellular ROS production,NO release and MAD levels are the most typical indicators of oxidative damage[28-29]. Accordingly, we utilized the kits of ROS, MAD and NO to determine the production of intracellular ROS, NO release and MAD levels in RAW264.7 cells, respectively. This results presented that the H2O2treated RAW264.7 cells, resulting in significant increase levels of intracellular ROS, NO release and MAD levels. However,C3G (6.25-25.00 µmol/L) pretreatment was found to be effective to prevent H2O2-mediated these events (Fig. 4B,Fig. 5C, D). This hinted that C3G could protect RAW264.7 cells from oxidative damage by inhibiting the generation of ROS, NO release and MAD levels[28]. Liu Di et al[30]also reported that C3G decreased intracellular ROS overexpression and MAD levels, and increased the antioxidant activity against oxidative damage in human embryonic kidney cell line HEK-293. Previous researches confirmed that H2O2caused cell oxidative damage mainly related with it attacking the antioxidant system[31-32]. Antioxidant enzyme defense system plays an indispensable role in scavenging ROS and preventing cell from oxidative damage. Increasing studies have confirmed that the over-expression of SOD and GSH-Px could provide cytoprotective effects against ROS in HepG2 cells, HUVECs and BRL-3A cells[33-35]. These results showed that the oxidative damage induced by H2O2decreased memorably the activities of SOD and GSH-Px (Fig. 5A, B),while ROS levels increased markedly. This phenomenon could be efficiently reversed by intervention with C3G.Our results suggested that C3G diminished the oxidative damage via improving antioxidant enzymes activities in H2O2-triggered RAW264.7 cells.

Mst1 is a member of the sterle-20 protein kinase family.At present, four subtypes have been found including Mst1,Mst2, Mst3 and Mst4. Numerous researches have indicated that Mst1/2 could regulate the function of Keap1[36-37].Keap1 is an important negative regulator of Nrf2in vivo.Nrf2 is a highly conserved basic leucine zipper transcription factor, which is mainly expressed in intestine, lung, liver and kidney[38]. Meanwhile, Nrf2 is considered to be a key transcription factor regulating cells against foreign bodies and oxidative damage[39]. Under normal physiological conditions,the most of Nrf2 is chelated with Keap1 in the cytoplasm,which makes Nrf2 unable to enter the nucleus to play its biological activity[40]. When the body is subjected to oxidative stress, the cysteine residues of Keap1 is modified to change the conformation of Keap1, resulting in decoupling of Keap1 and Nrf2. Following the activated Nrf2 is transferred into the nucleus and specifically combined with the antioxidant responsive elements (ARE), and then a series of downstream antioxidant enzymes proteins (glutamyl cystine ligase catalytic, NAD(P)H: quinone oxidoreductase and HO-1) are expressed to enhance the antioxidant activity of the body to resist the damage caused by oxidative stress. A hesperetin as a polyphenol which is found in citrus could memorably augment the antioxidant HO-1 by the up-regulation Nrf2 and decrease the stability of Keap1[41]. A large number of studies have confirmed that natural polyphenol antioxidant could protect cells from oxidative damage through Mst/Nrf2 signaling pathway[42-44]. The mechanism of antioxidant action for C3G might be related to the activation of Mst/Nrf2 pathway. To further ascertain the potential mechanisms, the effects of C3G on the relative mRNA/protein expression levels of Mst1/2, Keap1, Nrf2 and HO-1 in RAW264.7 cells were investigated via RT-PCR and Western blot analysis, respectively. Our results showed that H2O2dramatically increased the relative mRNA/protein expression levels of Mst1/2 and Keap1, while the relative mRNA/protein expression levels of Nrf2 and HO-1 memorably were decreased in 400 µmol/L H2O2treated group when compared to the vehicle-treated group (Fig. 6, 7),and these effects were suppressed by intervention with C3G.In conclusion, C3G initially down-regulated Mst1/2 proteins,and then activated Mst1/2 proteins changed structure of Keap1, followed by the release Nrf2, and Nrf2 is activated and transferred into the nucleus, which combined with ARE to regulate the expression of downstream antioxidant enzymes (Fig. 8). All in all, these results hinted that C3G protected RAW264.7 cells from H2O2-triggered oxidative damage, which might be related to activation of Mst/Nrf2 signaling pathway and enhancement of the activities of antioxidant enzymes.

Fig. 8 Molecular mechanism of C3G against oxidative damage of RAW264.7 cells induced by H2O2

4 Conclusions

In conclusion, our results indicated that C3G could represses H2O2-induced RAW264.7 cells oxidative damage via the direct reduction of intracellular ROS generation,NO release and MDA levels as well as enhancing activities of SOD and GSH-Px. In these processes, the H2O2-induced effects were suppressed the relative mRNA/protein expression levels of Mst1/2 and Keap1, and up-regulated the relative mRNA/protein expression levels of Nrf2 and HO-1 by treatment of C3G. The cytoprotective effect of C3G might be obtained by activating of Mst/Nrf2 signaling pathway and improving of the activities of antioxidant enzymes.