关键词: Hepa1-6细胞, miR-384, sirt3/FOXO1信号通路, 活性氧

Abstract: Objective To observe the effect of MiR-384 on sirt3/FOXO1 signaling pathway in HFFA-induced Hepa1-6 cells in vitro. Methods Hepa1-6 cells were cultured in DMEM complete medium containing 10% fetal bovine serum. When the cell fusion was up to 80%,DMEM medium containing 1% fetal bovine serum was given and the final concentrations of OA and PA were added with the final concentrations of the two being 1 mmol/L and 0.5 mmol/L,respectively. The incubation at 37°C lasted for 24 h,and the establishment of nonalcoholic steatohepatitis in vitro was determined. The reactive oxygen species（ROS） in the hepatocytes was assayed for successful model. The cells were transfected with miR-384 mimetic,miR-ctrl,miR-384 inhibitor,miR-384 inhibitor ctrl,si-sirt3 or si-ctrl for 48 h. The levels of ROS in the cells were measured by FCM,and the expression of sirt3,FOXO1 and antioxidant proteins（MnSOD and CAT） in the cells were measured by Western blot. Results Compared with in the normal group （0.66±0.01）,the levels of ROS in miR-384 mimetic group and si-sirt3 group were significantly increased [（37.3±1.13） and （10.4±0.36）,respectively,P<0.01];Compared with in the HFFA group（29.4±0.98）,the levels of ROS in HFFA/miR-384 inhibitor group was significantly decreased [（12.8±0.41）,P<0.01];Compared with the expression of sirt3 protein in Hepa1-6 cells in normal group（0.75±0.04）,the sirt3 in HFFA group was decreased[（0.23±0.01）,P<0.01];Compared with in the HFFA group,that in the HFFA/sirt3 group significantly increased[（0.83±0.03）,P<0.01];Compared with the HFFA/sirt3 group,that in the HFFA/sirt3/miR-384 group significantly decreased[（0.46±0.02）,P<0.01];Compared with in the control group,the expression of sirt3 protein and FOXO1 protein in miR-384 mimetic group significantly reduced in Hepa1-6 cells [（0.2±0.01） and（0.3±0.01）,respectively,P<0.01] and the level of CAT and manganese superoxide dismutase（MnSOD） significantly increased [（2.3±0.05） and（2.4±0.06）,P<0.01],while compared in with HFFA group,the expression of sirt3 protein and FOXO1 protein in Hepa1-6 cells in HFFA/miR-384 inhibitor group increased [（0.5±0.02） and （0.7±0.01）,P<0.01],and the expression of MnSOD and CAT decreased[（1.6±0.04）（2.0±0.03）,P<0.01];Compared with the activities of SOD（327±3.45） and CAT（386±4.03） in NAFLD group the levels of SOD and CAT in the normal group[（425±5.49） and（512±6.04）,P<0.01] and SOD and CAT in the miR-384 inhibitor group [（406±4.79） and（447±5.38）,P<0.01] were significantly increased. Conclusion The expression of miR-384 could lead to the increase of oxidative damage induced by HFFA in Hepa1-6 cells,which might partly be contributed to the inhibition of sirt3/FOXO1 pathway.

Key words: Hepa1-6 cells, miR-384, sirt3/FOXO1 signaling pathway, Reactive oxygen species