Dihydromyrieetin Protects The Oxidative Damage Induced By Hydrogen Peroxide Solution Through Inhibition Of Endoplasmic Reticulum Stress

OBJECTIVE:To investigate the effect of dihydromyricetin?DMY?on the oxidative damage induced by hydrogen peroxide solution?H₂O₂?in human normal hepatocyte line L02 cells,to explore the possible mechanism from endoplasmic reticulum stress?ERS?and to provide new ideas and strategies for prevention and treatment of liver injury.METHODS:Human normal hepatocyte line L02 cells were cultured in DMEM culture medium for the experiment.After L02 cells were pretreated with different doses of DMY?10,20,40,80,160 and 320 ?g/m L?for 24 h,the cells were treated with H₂O₂ for 12 h.The cell morphology was observed by optical microscope.Cell viability was tested by 3-?4,5-Dimethylthiazol-2-yl?-2,5-diphenyltetrazolium bromide?MTT?assay.The level of lactate dehydrogenase?LDH?in cell culture solution was measured by colorimetric method.Apoptosis of L02 cells was tested by Flow cytometry.The level of reactive oxygen species?ROS?in cells was was measured by Flow cytometry.The level of malondialdehyde?MDA?in cells was was tested by colorimetric method.The endoplasmic reticulum stress?ERS?protein markers including glucose-regulated protein 78?GRP78?and CCAAT/enhancerbinding protein homologous protein?CHOP?in the cerebral cortex were measured by Western blot.The second step,pretreated the L02 cells with the inducer of ERS tunicamycin?2?g/m L?for 30 min,then treated with 80?g/m L DMY for 24 h,and then treated with H₂O₂ for 12 h.Cell viability was tested by MTT assay.Apoptosis of L02 cells was tested by Flow cytometry.And the level of LDH in cell culture solution was measured by colorimetric method.RESULTS :1.DMY?10,20,40,80,160 and 320 ?g/m L?did not affect the morphology of L02 cells.After L02 cells were treated with H₂O₂,obvious changes in cell morphology was observed.Most of the cells were round some cells exhibited morphological characteristics of apoptosis.The chromosomes were aggregated at the periphery of the cell nuclear membrane,cytoplasm was contracted and cells were diminished.DMY?40,80 and 160 ?g/m L?improved the change of morphology of L02 cells induced by H₂O₂.2.DMY?10,20,40,80,160 and 320 ?g/m L?did not affect the cell viability of L02 cells compared with control group.Compared with H₂O₂ group,the cell viability was significantly increased in DMY?40,80,160 and 320 ?g/m L?+ H₂O₂ group?all P<0.05?.There were no significant difference about cell viability of L02 cells among DMY?10,20 ?g/m L?+ H₂O₂ group and control group?all P>0.05?.3.Compared with control group,the apoptosis rate of L02 cells was significantly increased,the levels of LDH in cell culture solution and MDA and ROS in cells were significantly increased in H₂O₂ group?all P<0.05?.Compared with H₂O₂ group,the apoptosis rate of L02 cells was significantly decreased,the levels of LDH in cell culture solution and MDA and ROS in cells were significantly decreased in DMY?80 ?g/m L?+ H₂O₂ group?all P<0.05?.4.Compared with control group,the protein expression of GRP78 and CHOP in cells were significantly up-regulated in H₂O₂ group?all P<0.05?.Compared with H₂O₂ group,the protein expression of GRP78 and CHOP in cells was significantly down-regulated in DMY?80 ?g/m L?+ H₂O₂ group?all P<0.05?.5.Compared with H₂O₂+DMY?80 ?g/m L?group,the cell viability of L02 cells was significantly decreased,the apoptosis rate of L02 cells and the levels of LDH in cell culture solution were significantly increased in H₂O₂+DMY?80 ?g/m L?+ Tunicamycin?2?g/m L?group?all P<0.05?.CONCLUSIONS:DMY protects the oxidative damage induced by hydrogen peroxide solution in human normal hepatocyte line L02 cells through inhibition of endoplasmic reticulum stress.