| Objective: To investigate the protective effect of Oxymatrine(OM) on human hepatocytes(L-02 cells) exposed to sodium arsenite(Na As O2) and its possible mechanisms. Providing a theoretical basis for seeking a kind of drug which could be widely used in prevention and treatment for arsenic poisoning patients with liver damage. Methods:(1) L-02 cells were treated with different concentrations of Na As O2(25, 50, 100, 200, 500μmol/L) for 24 h,the level of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were determined by biochemical method, cell survival rate was examined by MTT colorimetry to determine the experimental dose of Na As O2. After treatment by Na As O2(the dose is on the basis of the previous step) for 0h, 6h, 12 h, 18 h, 24 h and 36 h, the level of ALT and AST were determined by biochemical method, cell survival rate was examined by MTT colorimetry and the flow cytometry was used to observe the apoptosis of hepatocytes at each time. The cells were treated with different concentrations of OM(25, 50, 100, 200, 500, 1000μg/ml) and cell survival rate was examined by MTT colorimetry.(2) L-02 cells were randomly divided into six groups: control group, OM(200μg/ml)group, Na As O2(100μmol/L) group, Na As O2 with different concentration of OM(50, 100, 200μg/ml) group. The level of ALT and AST in each group were determined by biochemical method. The protein level of GRP78 and CHOP were determined by western blotting. The flow cytometry was used to observe the apoptosis of hepatocytes in each group.(3) L-02 cells were randomly divided into seven groups: control group, OM(200μg/ml) group, Na As O2(100μmol/L) group, Na As O2 with OM group, Na As O2 with 4-PBA(5mmol/L) group, DTT(2.5mmol/L) group, DTT with OM group. ALT and AST level in each group were determined by biochemical method. The protein level of GRP78 and CHOP were determined by western blotting. The flow cytometry was used to observe the apoptosis of hepatocytes in each group. Results:(1) With increasing Na As O2 concentration,the level of AST and ALT in the medium was increased gradually, while the cell proliferation was decreased gradually. The cell survival rate was 76.55% when the Na As O2 concentration was 100μmol/L. The level of AST and ALT, the inhibiting rate of cell proliferation and the late stage of apoptosis rate / necrosis rate were increased with prolongation of exposure time. The early apoptosis rate was reached a peak at 18 h and then decreased gradually. OM had no effect on the cell proliferation of L-02 cells when its concentration was lower than 200μg/ml.(2) There was no obviously difference between control group and OM group. Compared with the control group and OM group, the level of AST and ALT in the Na As O2 group were obviously increased,the protein level of GRP78 and CHOP were markedly higher than those of the control group and OM group(P<0.05), the apoptosis of L-02 cells was also increased in the Na As O2 group(P<0.05). With different concentrations of OM treatment, the above indexes were decreased(P<0.05), the effect was dose dependent, especially in the Na As O2 with OM 200μg/ml group.(3) There was no obviously difference between control group and OM group. Compared with the control group, the level of AST and ALT, the protein level of GRP78 and CHOP, the apoptosis of L-02 cells were obviously increased in the Na As O2 group(P<0.05), and these changes could be reduced by 4-PBA and OM treatment(P<0.05). Compared with the control group, the level of ALT and AST, the rate of apoptosis, expression of GRP78 and CHOP protein in DTT group were significantly increased(P<0.05). OM also ameliorated these changes induced by DTT(P<0.05). Conclusions: Na As O2 could injury L-02 cells and induce endoplasmic reticulum stress.OM could protect L-02 cells exposed to Na As O2 by attenuating endoplasmic reticulum stress and reducing the apoptosis. |
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