Role Of PM2.5 On Steatosis And Apoptosis In HepG2 Cells And Effects Of Resveratrol

Objectives:The objective of this study was to assess the hepatic steatosis and apoptosis exerted by PM2.5 collected in urban Guangzhou on human hepatocellular line HepG2, while the effect of resveratrol intervention on PM2.5-induced hepatotoxicity was also evaluated.Methods:1) Effects of PM2.5 on steatosis in HepG2 cells:The cell viability was detected by Cell Counting Kit-8 to select the appropriate concentration of PM2.5; HepG2 cells in different groups (0,6.25μg/ml,12.5μg/ml and 25μg/ml PM2.5) were cultured in the corresponding medium for 24h, lipid drops were stained with oil red O; and TG and TCH levels in the total lipid fraction of cells were determined using commercially available analysis kits; Real-time PCR was performed to investigate the mRNA expression of LXRa and SREBP-1c, two key molucules associated with lipid metabolism.2) Effect of PM2.5 on oxidative stress and apoptosis in HepG2 cells:HepG2 cells were exposed to PM2.5 at a concentration of 0,12.5μg/ml,25μg/ml and 50μg/ml.4h after exposure to PM2.5, HepG2 cells were stained with H2DCF-DA to detect total ROS; Commercial enzyme linked immunosorbent assay kits were applied to determine SOD, GSH and MDA levels 6h after exposure to PM2.5; After 4h of incubation, the mRNA expression of SOD1, SOD2, GSH and CAT were investigated by Real-time PCR; The apoptosis rate was determined by Annexin V-FITC/PI double staining after 24h of incubation; The expression of apoptosis-related proteins (Bcl-2, Bax, Cytochrome C, Caspase 9 and Caspase 3) was analyzed by Western Blot 24h after exposure to PM2.5.3) Effect of resveratrol on PM2.5-induced oxidative stress and apoptosis in HepG2 cells:The cell viability was detected by Cell Counting Kit-8 to select the appropriate concentration of resveratrol, and then HepG2 cells were respectively treated with PM2.5 alone or in combination with resveratrol (20μmol/l,40μmol/l, and 80μmol/l). The HepG2 cells cultured in the corresponding medium were treated with H2DCF-DA to observe the effect of resveratrol on ROS production; Total SOD and MDA were determined by commercially available analysis kits in order to evaluate the effect of resveratrol on PM2.5-induced oxidative stress; Real-time PCR was performed to investigate the effect of resveratrol on SOD1, SOD2 and CAT mRNA expression; Cell Counting Kit-8 and Annexin V-FITC/PI double staining was applied to investigate the effect of resveratrol on PM2.5-induced apoptosis; The effect of resveratrol on the expression of apoptosis-related proteins (Bcl-2, Bax, Cytochrome C, Caspase 9 and Caspase 3) was analyzed by Western Blot.Results:1) Effect of PM2.5 on steatosis in HepG2 cells:PM2.5 imparted remarkable decrease in cell viability at concentrations of 50μg/ml and 100μg/ml (P<0.05); Oil red O microscopic examination revealed significantly increased orange-red lipid droplets in PM2.5 groups; The results of enzymatic assays showed, the levels of cellular TG was markedly elevated in a dose-dependent manner in PM2.5 groups when compared with the control group (P<0.05), while the TCH level presented no significant increase (P>0.05); Further gene expression studies by REAL-TIME PCR revealed that PM2.5 significantly activated LXRa and SREBP-1c gene expression compared to control group (P<0.05), which may be one of the reasons for PM2.5-induced hepatic steatosis.2) Effect of PM2.5 on oxidative stress and apoptosis in HepG2 cells:In the fluorescent dye H2DCF-DA experiment, cells in PM2.5 group emitted rather intense fluorescence in a concentration dependent manner when compared with control group (P<0.01); A significant decrease (P<0.01) of SOD enzyme activity and an apperant increase (P<0.01) of MDA activity were found in PM2.5 groups, while no significant change (P> 0.05) was observed in GSH level; Real-time PCR analysis demonstrated a significant downregulated expression of SOD1, SOD2 and CAT mRNA in PM2.5 groups as compared to control (P<0.05); The data collected from Annexin V-FITC/PI double staining experiment showed that cell apoptosis significantly increased in PM2.5 groups in a dose-dependent manner (P<0.05); Further protein expression studies by Western Blot indicated that PM2.5 significantly upregulated the expression of Cytochrome C, Caspase 9, Caspase 3 and bax/bcl-2 in PM2.5 groups when compared with control (P<0.05), which may be associated with PM2.5-induced apoptosis.3) Effect of resveratrol on PM2.5-induced oxidative Stress and apoptosis in HepG2 cells:Resveratrol imparted no significant cytotoxicity (P>0.05) to HepG2 cells at 0～80μmol/l concentration range when incubated for 24h; Microscopic pictures collected from H2DCF-DA staining showed that the ROS generation was significantly mitigated (P<0.01) in resveratrol pretreatment group as compared to PM2.5 used alone, which was consistent with results from the net fluorescence intensity measured by spectrofluorometer; The results of enzyme linked immunosorbent assay showed that the SOD activity was significantly elevated (P<0.01) wheras MDA activity was significantly inhibited (P<0.01) compared to PM2.5 group; SOD1, SOD2 and CAT mRNA expression were significantly upregulated (P<0.01) in resveratrol pretreatment group as compared to PM2.5 group; The data collected from CCK-8 and Annexin V-FITC/PI double staining experiments showed that cell viability was significantly enhanced (P<0.05) whearas cell apoptosis rate was significantly decreased (P<0.01) after resveratrol intervention; Further protein expression studies by Western Blot indicated that the expression of Cytochrome C, Caspase 9, Caspase 3 and bax/bcl-2 significantly reduced in resveratrol pretreatment groups (P<0.05), which maybe one of the mechanisms that resveratrol suppress PM2.5-induced apoptosis.Conclusions:1) Effect of PM2.5 on steatosis in HepG2 cells:PM2.5 is able to induce lipid accumulation, which may be due to upregulation of LXRa and SREBP-1c mRNA expression in a dose-dependent manner.2) Effect of PM2.5 on oxidative stress and apoptosis in HepG2 cells:The intracellular ROS levels in treatment groups with different PM2.5 concentration were significantly enhanced when compared with control group, which may be due to downregulation of SOD1, SOD2 and CAT mRNA expression; When compared with control group,the apoptosis rates of treatment groups with different PM2.5 concentration significantly increased, which may be the results of upregulated expression of Cytochrome C, Caspase 9, Caspase 3 and bax/bcl-2.3) Effect of Resveratrol on PM2.5-indueed oxidative stress and apoptosis in HepG2 cells:Resveratrol exerts therapeutic effects on PM2.5-induced oxidative stress and apoptosis in HepG2 cells,which suggested that resveratrol may prevent PM2.5-indueed apoptosis by inhibiting oxidative stress.