• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Cyanidin-3-O-Glucoside Protects RAW264.7 Cells against Hydrogen Peroxide-Induced Oxidative Damage

    2021-07-29 03:26:54XUEHongkunTANJiaqiLIQianTANGJintian
    食品科學 2021年13期

    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.

    亚洲国产色片| 又紧又爽又黄一区二区| 一级黄色大片毛片| 老鸭窝网址在线观看| 国产伦人伦偷精品视频| 成年女人永久免费观看视频| 一区二区三区免费毛片| 一级黄片播放器| 少妇人妻精品综合一区二区 | 中文亚洲av片在线观看爽| 亚洲av.av天堂| 可以在线观看的亚洲视频| 大型黄色视频在线免费观看| 俄罗斯特黄特色一大片| 免费黄网站久久成人精品 | 少妇丰满av| 久久精品国产亚洲av天美| 亚洲欧美日韩卡通动漫| 网址你懂的国产日韩在线| 美女 人体艺术 gogo| 日韩欧美 国产精品| 日日摸夜夜添夜夜添av毛片 | 国产精品嫩草影院av在线观看 | 成人毛片a级毛片在线播放| 国产精品电影一区二区三区| 欧美黄色淫秽网站| 男女之事视频高清在线观看| 最新中文字幕久久久久| 美女被艹到高潮喷水动态| 亚洲成人久久爱视频| 国产精品自产拍在线观看55亚洲| 草草在线视频免费看| 最好的美女福利视频网| 国产爱豆传媒在线观看| 午夜精品久久久久久毛片777| 非洲黑人性xxxx精品又粗又长| 国产成人欧美在线观看| 中文字幕久久专区| 成年女人看的毛片在线观看| 久久精品久久久久久噜噜老黄 | 麻豆av噜噜一区二区三区| 免费看日本二区| 成人美女网站在线观看视频| 老司机午夜十八禁免费视频| 亚洲最大成人手机在线| 美女cb高潮喷水在线观看| 性色av乱码一区二区三区2| 一个人观看的视频www高清免费观看| 亚洲精品影视一区二区三区av| 国产欧美日韩一区二区三| 蜜桃亚洲精品一区二区三区| 他把我摸到了高潮在线观看| 少妇高潮的动态图| 在线免费观看的www视频| 美女高潮的动态| 亚洲片人在线观看| 老熟妇乱子伦视频在线观看| 麻豆av噜噜一区二区三区| 成年女人毛片免费观看观看9| а√天堂www在线а√下载| 深夜a级毛片| 一级黄色大片毛片| 可以在线观看的亚洲视频| 露出奶头的视频| 变态另类成人亚洲欧美熟女| 老师上课跳d突然被开到最大视频 久久午夜综合久久蜜桃 | 最新中文字幕久久久久| 国产成人aa在线观看| 99在线人妻在线中文字幕| 不卡一级毛片| 一个人看视频在线观看www免费| 一级黄片播放器| av女优亚洲男人天堂| 又爽又黄无遮挡网站| 精品久久久久久久久久久久久| 国产精品久久久久久久久免 | 一本一本综合久久| 别揉我奶头 嗯啊视频| 级片在线观看| 成人高潮视频无遮挡免费网站| 国产色爽女视频免费观看| 97超级碰碰碰精品色视频在线观看| 婷婷六月久久综合丁香| 十八禁网站免费在线| 国产精品久久久久久人妻精品电影| 精品无人区乱码1区二区| 麻豆一二三区av精品| 亚洲经典国产精华液单 | 国产极品精品免费视频能看的| 久久国产乱子伦精品免费另类| 久久久久久久久久黄片| 国产精品免费一区二区三区在线| 国内少妇人妻偷人精品xxx网站| 亚洲精品一区av在线观看| av在线观看视频网站免费| 少妇丰满av| 无遮挡黄片免费观看| 欧美黑人欧美精品刺激| 精品乱码久久久久久99久播| 在线观看美女被高潮喷水网站 | 亚洲性夜色夜夜综合| av天堂在线播放| 国产精品美女特级片免费视频播放器| 99国产精品一区二区蜜桃av| 亚洲国产精品999在线| 国产一区二区三区视频了| 麻豆久久精品国产亚洲av| 欧美日韩瑟瑟在线播放| 欧美不卡视频在线免费观看| 在线a可以看的网站| 亚洲精品色激情综合| 亚洲综合色惰| 少妇被粗大猛烈的视频| 婷婷精品国产亚洲av在线| 永久网站在线| 国产极品精品免费视频能看的| 免费看a级黄色片| 亚洲av成人av| 亚洲成av人片免费观看| av在线老鸭窝| 嫩草影视91久久| 亚洲av一区综合| 欧美日韩福利视频一区二区| 中文字幕人妻熟人妻熟丝袜美| 国内精品一区二区在线观看| 国产精品国产高清国产av| 午夜免费成人在线视频| 久久久久久大精品| 久久久国产成人免费| 国产综合懂色| 欧美最新免费一区二区三区 | 久久久久久九九精品二区国产| 宅男免费午夜| 亚洲最大成人av| 久久精品影院6| 桃红色精品国产亚洲av| 精品日产1卡2卡| 日本一本二区三区精品| 亚洲人与动物交配视频| 男女之事视频高清在线观看| 久久久久久久久大av| 一区二区三区高清视频在线| 老熟妇乱子伦视频在线观看| 757午夜福利合集在线观看| 亚洲一区高清亚洲精品| 成年女人永久免费观看视频| 男女做爰动态图高潮gif福利片| 日韩欧美 国产精品| 亚洲色图av天堂| 国产免费男女视频| 国产亚洲精品av在线| 少妇高潮的动态图| 欧美最黄视频在线播放免费| 日日摸夜夜添夜夜添小说| 尤物成人国产欧美一区二区三区| 午夜久久久久精精品| 乱码一卡2卡4卡精品| 欧美一级a爱片免费观看看| 成人欧美大片| 国产激情偷乱视频一区二区| 亚洲av成人精品一区久久| 精品国内亚洲2022精品成人| 免费av观看视频| 激情在线观看视频在线高清| 中文字幕久久专区| 白带黄色成豆腐渣| 精品久久久久久,| 久久久成人免费电影| 草草在线视频免费看| 久久久久久久久久黄片| 久久99热6这里只有精品| 亚洲av二区三区四区| 国产高清视频在线观看网站| 99热这里只有精品一区| 国产乱人视频| 99国产精品一区二区蜜桃av| 色综合婷婷激情| 精品国产三级普通话版| 国产成人av教育| 成人高潮视频无遮挡免费网站| 日本熟妇午夜| 色视频www国产| 中亚洲国语对白在线视频| 亚洲精品久久国产高清桃花| 蜜桃久久精品国产亚洲av| 欧美丝袜亚洲另类 | 精品无人区乱码1区二区| 国产精品一区二区三区四区免费观看 | 欧美日韩亚洲国产一区二区在线观看| 久久久久久九九精品二区国产| 免费观看人在逋| 国产色爽女视频免费观看| av中文乱码字幕在线| 欧美黑人巨大hd| 99精品在免费线老司机午夜| 香蕉av资源在线| 欧美xxxx性猛交bbbb| 人妻制服诱惑在线中文字幕| 成年女人永久免费观看视频| 观看免费一级毛片| 五月伊人婷婷丁香| 老司机午夜十八禁免费视频| 亚洲 欧美 日韩 在线 免费| 成人午夜高清在线视频| 亚洲中文日韩欧美视频| 国产真实伦视频高清在线观看 | 国产精品久久视频播放| 亚洲精品粉嫩美女一区| 亚洲最大成人av| 草草在线视频免费看| 午夜福利18| 精华霜和精华液先用哪个| 国内揄拍国产精品人妻在线| a级毛片免费高清观看在线播放| 久久亚洲真实| 老司机午夜十八禁免费视频| 国产 一区 欧美 日韩| 国产伦在线观看视频一区| 亚洲国产高清在线一区二区三| 精品一区二区三区人妻视频| 亚洲第一区二区三区不卡| 一级a爱片免费观看的视频| 免费观看的影片在线观看| 特级一级黄色大片| 成年女人永久免费观看视频| 真实男女啪啪啪动态图| 日本 av在线| 国产精品美女特级片免费视频播放器| 日本成人三级电影网站| 亚洲av一区综合| 在线播放国产精品三级| 婷婷色综合大香蕉| 精品无人区乱码1区二区| 国产亚洲精品久久久久久毛片| 亚洲性夜色夜夜综合| 亚洲第一电影网av| 搡老岳熟女国产| 麻豆国产av国片精品| 国产熟女xx| 美女黄网站色视频| 亚洲人成网站在线播| 老熟妇仑乱视频hdxx| 亚洲aⅴ乱码一区二区在线播放| 成人一区二区视频在线观看| 国产精品久久久久久精品电影| 日本黄色片子视频| 国产精品久久久久久亚洲av鲁大| 欧美成狂野欧美在线观看| 日本熟妇午夜| 偷拍熟女少妇极品色| 精品久久久久久久久久免费视频| 久久这里只有精品中国| 亚洲av不卡在线观看| 亚洲精品亚洲一区二区| 国产欧美日韩一区二区精品| 亚洲国产高清在线一区二区三| 51午夜福利影视在线观看| 午夜精品一区二区三区免费看| 一区福利在线观看| 国产单亲对白刺激| 自拍偷自拍亚洲精品老妇| 麻豆国产av国片精品| 欧美最新免费一区二区三区 | 久久国产精品人妻蜜桃| 最新中文字幕久久久久| 国产精品久久久久久久久免 | 欧美成狂野欧美在线观看| 日韩精品中文字幕看吧| 男女之事视频高清在线观看| 欧美日韩国产亚洲二区| 我要看日韩黄色一级片| 我要搜黄色片| 国产精品,欧美在线| 国产午夜精品久久久久久一区二区三区 | 色哟哟哟哟哟哟| 欧美bdsm另类| 久久中文看片网| 啪啪无遮挡十八禁网站| 国产成人aa在线观看| 国产色爽女视频免费观看| 在线观看舔阴道视频| 欧美日韩瑟瑟在线播放| www日本黄色视频网| 免费搜索国产男女视频| 国产在线精品亚洲第一网站| 人人妻人人澡欧美一区二区| 狠狠狠狠99中文字幕| 国产高清三级在线| 国产精品久久久久久亚洲av鲁大| 亚洲aⅴ乱码一区二区在线播放| 乱码一卡2卡4卡精品| 成人欧美大片| 欧美日韩瑟瑟在线播放| 日本与韩国留学比较| 90打野战视频偷拍视频| 亚洲成a人片在线一区二区| 亚洲无线观看免费| 亚洲国产精品sss在线观看| 中亚洲国语对白在线视频| 亚洲三级黄色毛片| 亚洲乱码一区二区免费版| 免费搜索国产男女视频| 精品久久久久久久久久免费视频| 欧美又色又爽又黄视频| 国产欧美日韩精品亚洲av| 国产亚洲欧美98| 日本成人三级电影网站| 少妇的逼好多水| 久久草成人影院| 亚洲人成网站在线播放欧美日韩| 亚洲内射少妇av| 欧美成人免费av一区二区三区| 亚洲av成人不卡在线观看播放网| 亚洲最大成人手机在线| 亚洲中文字幕日韩| 久久性视频一级片| 夜夜看夜夜爽夜夜摸| 久久天躁狠狠躁夜夜2o2o| 欧美高清成人免费视频www| 国产国拍精品亚洲av在线观看| 丰满人妻熟妇乱又伦精品不卡| 成人国产一区最新在线观看| 好男人在线观看高清免费视频| 国产视频一区二区在线看| 99国产精品一区二区蜜桃av| 伦理电影大哥的女人| 制服丝袜大香蕉在线| 久久久久久久久中文| 麻豆国产97在线/欧美| 男女那种视频在线观看| 久久精品国产清高在天天线| 黄色丝袜av网址大全| 久久亚洲精品不卡| 内地一区二区视频在线| 久久久成人免费电影| 亚洲美女搞黄在线观看 | 久久热精品热| 在线免费观看不下载黄p国产 | 露出奶头的视频| 美女被艹到高潮喷水动态| 亚洲av成人av| 国产av麻豆久久久久久久| 国产av不卡久久| 真实男女啪啪啪动态图| 精品乱码久久久久久99久播| 精品一区二区三区av网在线观看| 免费高清视频大片| 日日摸夜夜添夜夜添小说| 亚洲熟妇熟女久久| 亚洲av中文字字幕乱码综合| 国产黄a三级三级三级人| 国产精品人妻久久久久久| 毛片女人毛片| 日本黄色视频三级网站网址| 午夜福利在线观看吧| 婷婷亚洲欧美| 午夜福利在线观看吧| 亚洲精品一卡2卡三卡4卡5卡| 国产精品久久久久久人妻精品电影| 757午夜福利合集在线观看| 亚洲av一区综合| 欧美+日韩+精品| 亚洲av一区综合| 欧美乱色亚洲激情| 欧美性感艳星| 亚洲国产精品合色在线| 亚洲精品影视一区二区三区av| 国语自产精品视频在线第100页| 岛国在线免费视频观看| 99久久九九国产精品国产免费| 91在线精品国自产拍蜜月| 在线十欧美十亚洲十日本专区| 搡老妇女老女人老熟妇| 欧美+日韩+精品| 男女之事视频高清在线观看| 欧美不卡视频在线免费观看| 97热精品久久久久久| 一级黄色大片毛片| 男人和女人高潮做爰伦理| 亚洲精品在线美女| 久99久视频精品免费| 亚洲 欧美 日韩 在线 免费| 久久6这里有精品| 午夜精品一区二区三区免费看| 精品国产亚洲在线| 欧美一区二区国产精品久久精品| 老司机午夜福利在线观看视频| 国产精品一区二区性色av| 丝袜美腿在线中文| 人人妻人人看人人澡| 久久婷婷人人爽人人干人人爱| 亚洲欧美激情综合另类| 亚洲 欧美 日韩 在线 免费| av视频在线观看入口| 麻豆一二三区av精品| 亚洲,欧美精品.| 欧美极品一区二区三区四区| 大型黄色视频在线免费观看| 日韩高清综合在线| 伊人久久精品亚洲午夜| 亚洲内射少妇av| 男女那种视频在线观看| 在线观看一区二区三区| 99热这里只有是精品在线观看 | 亚洲欧美日韩卡通动漫| 国产亚洲精品综合一区在线观看| 日本精品一区二区三区蜜桃| 亚洲欧美精品综合久久99| 99久久久亚洲精品蜜臀av| 一区二区三区免费毛片| 欧美激情国产日韩精品一区| 国产国拍精品亚洲av在线观看| 欧美成人免费av一区二区三区| 免费看a级黄色片| 能在线免费观看的黄片| .国产精品久久| av在线天堂中文字幕| 人妻丰满熟妇av一区二区三区| 国产主播在线观看一区二区| 热99re8久久精品国产| 熟女电影av网| 国产高清有码在线观看视频| 人人妻人人看人人澡| www.999成人在线观看| 丰满人妻熟妇乱又伦精品不卡| 日韩欧美精品v在线| 别揉我奶头~嗯~啊~动态视频| 亚洲精品一卡2卡三卡4卡5卡| 日本免费a在线| a级一级毛片免费在线观看| 黄色女人牲交| 国产单亲对白刺激| 91字幕亚洲| 亚洲成av人片免费观看| 在线观看免费视频日本深夜| 国产精品久久久久久人妻精品电影| 两性午夜刺激爽爽歪歪视频在线观看| 露出奶头的视频| 国产伦精品一区二区三区四那| 超碰av人人做人人爽久久| 国产精品精品国产色婷婷| 中文字幕久久专区| 宅男免费午夜| 欧美国产日韩亚洲一区| 午夜影院日韩av| 非洲黑人性xxxx精品又粗又长| 三级国产精品欧美在线观看| 一边摸一边抽搐一进一小说| 国产精品国产高清国产av| 噜噜噜噜噜久久久久久91| 国产探花在线观看一区二区| 他把我摸到了高潮在线观看| 亚洲久久久久久中文字幕| 91av网一区二区| 亚洲人成电影免费在线| 神马国产精品三级电影在线观看| 精品99又大又爽又粗少妇毛片 | 国产精品久久久久久精品电影| 亚洲中文字幕日韩| 俺也久久电影网| 我的老师免费观看完整版| 午夜激情福利司机影院| 男女下面进入的视频免费午夜| av在线老鸭窝| 亚洲av日韩精品久久久久久密| 看片在线看免费视频| 舔av片在线| 免费无遮挡裸体视频| 一边摸一边抽搐一进一小说| 91字幕亚洲| 特大巨黑吊av在线直播| 亚洲av美国av| 亚洲午夜理论影院| 欧美在线一区亚洲| 成年女人看的毛片在线观看| 国产视频内射| 欧美午夜高清在线| 国产精品野战在线观看| 国产伦人伦偷精品视频| 在线a可以看的网站| 久99久视频精品免费| 丁香欧美五月| 亚洲不卡免费看| 天堂动漫精品| 好看av亚洲va欧美ⅴa在| 最近最新免费中文字幕在线| 精品人妻偷拍中文字幕| 欧美另类亚洲清纯唯美| 99在线人妻在线中文字幕| 国产精品99久久久久久久久| 一边摸一边抽搐一进一小说| 亚洲av成人不卡在线观看播放网| 内地一区二区视频在线| 国产精品三级大全| 婷婷亚洲欧美| 简卡轻食公司| 久久精品久久久久久噜噜老黄 | 身体一侧抽搐| 国产精品伦人一区二区| 88av欧美| 特级一级黄色大片| 给我免费播放毛片高清在线观看| 又粗又爽又猛毛片免费看| 欧美另类亚洲清纯唯美| 九色成人免费人妻av| 天天一区二区日本电影三级| 男插女下体视频免费在线播放| 午夜福利在线在线| 99久久成人亚洲精品观看| 精品久久久久久久久av| 人妻制服诱惑在线中文字幕| 最近在线观看免费完整版| 亚洲精品日韩av片在线观看| 夜夜躁狠狠躁天天躁| av在线天堂中文字幕| 在线观看免费视频日本深夜| 日本撒尿小便嘘嘘汇集6| 免费av毛片视频| 亚洲av中文字字幕乱码综合| 欧美黑人巨大hd| 大型黄色视频在线免费观看| 亚洲不卡免费看| 久久久久久国产a免费观看| 婷婷亚洲欧美| 国产麻豆成人av免费视频| 国产主播在线观看一区二区| 午夜视频国产福利| 夜夜爽天天搞| 亚洲av成人不卡在线观看播放网| 国产av一区在线观看免费| 淫秽高清视频在线观看| 亚洲av二区三区四区| 三级毛片av免费| 亚洲片人在线观看| 怎么达到女性高潮| 欧美激情在线99| 人妻夜夜爽99麻豆av| 精品日产1卡2卡| 亚洲精品粉嫩美女一区| 成年版毛片免费区| 在线免费观看不下载黄p国产 | 男人的好看免费观看在线视频| 最好的美女福利视频网| 男女那种视频在线观看| 亚洲av第一区精品v没综合| 人妻夜夜爽99麻豆av| 午夜激情福利司机影院| 国产av在哪里看| 精品日产1卡2卡| 国产高清视频在线播放一区| 午夜福利欧美成人| 精品99又大又爽又粗少妇毛片 | 亚洲中文字幕一区二区三区有码在线看| 国产高清有码在线观看视频| 白带黄色成豆腐渣| 69人妻影院| h日本视频在线播放| 午夜免费激情av| 国产精品久久久久久久电影| 久久午夜亚洲精品久久| 91久久精品电影网| 国产日本99.免费观看| 婷婷色综合大香蕉| 久久久久精品国产欧美久久久| 国产成人福利小说| 99国产精品一区二区三区| 18禁裸乳无遮挡免费网站照片| av天堂中文字幕网| 日韩亚洲欧美综合| 亚洲精品在线观看二区| 老司机午夜福利在线观看视频| 国产主播在线观看一区二区| 真实男女啪啪啪动态图| 国产亚洲欧美在线一区二区| 国产精品亚洲美女久久久| 性插视频无遮挡在线免费观看| 永久网站在线| 免费在线观看成人毛片| www日本黄色视频网| 日韩欧美国产在线观看| 国产成人福利小说| 国产成+人综合+亚洲专区| 99久久无色码亚洲精品果冻| 午夜福利在线观看免费完整高清在 | 色精品久久人妻99蜜桃| 精品国产亚洲在线| 亚洲av电影不卡..在线观看| 哪里可以看免费的av片| 网址你懂的国产日韩在线| 97超级碰碰碰精品色视频在线观看| 男女床上黄色一级片免费看| 看十八女毛片水多多多| 色视频www国产| 亚洲熟妇熟女久久| 小说图片视频综合网站| 成年女人毛片免费观看观看9| 床上黄色一级片| 国产老妇女一区| 男人狂女人下面高潮的视频| 国产精品日韩av在线免费观看| 在线天堂最新版资源| 午夜福利成人在线免费观看| 嫩草影院精品99| 悠悠久久av| 中文字幕人妻熟人妻熟丝袜美| а√天堂www在线а√下载| 精品人妻偷拍中文字幕| 九色国产91popny在线| 日韩av在线大香蕉| 欧美日本亚洲视频在线播放| 高潮久久久久久久久久久不卡| 日韩av在线大香蕉| 亚洲乱码一区二区免费版| 免费一级毛片在线播放高清视频| 桃红色精品国产亚洲av|