CKIP-1对非酒精性脂肪性肝病小鼠肝细胞凋亡的影响

Effect of CKIP-1 on hepatocyte apoptosis in nonalcoholic fatty liver disease

目的观察酪蛋白激酶2相互作用蛋白-1(CKIP-1)对非酒精性脂肪性肝病(NAFLD)小鼠肝细胞凋亡的影响并探讨其相关机制。方法实验研究。使用油酸(OA)制备NAFLD细胞模型。将Flag-CKIP-1表达载体以及shRNA-CKIP-1转染肝细胞, 实现CKIP-1的异常表达。采用流式细胞术检测肝细胞凋亡水平。Western blot检测凋亡相关蛋白表达。构建CKIP-1敲除NAFLD小鼠模型, 肝脏病理切片免疫组化检测凋亡相关信号蛋白表达。结果 CKIP-1转染后, 肝细胞脂肪变性的程度明显减轻。与OA组相比, OA+sh-CKIP-1组肝细胞凋亡率明显增加(P<0.01), 而OA+CKIP-1组肝细胞凋亡率明显下降(P<0.05)。CKIP-1抑制NAFLD细胞模型中凋亡相关信号分子蛋白caspase-3和caspase-9的表达(P<0.05、P<0.01), 同时增加Bcl-2/Bax的表达水平(P<0.05)。CKIP-1敲除增加NAFLD小鼠肝细胞凋亡相关信号caspase-3和caspase-9的表达(P<0.01, P<0.05), 同时降低Bcl-2/Bax的表达(P<0.05)。结论 CKIP-1通过上调Bcl-2/Bax凋亡开关, 产生对蛋白酶caspase-3和caspase-9的影响, 从而抑制肝细胞的凋亡, 改善NAFLD细胞的脂肪变性程度。

Objective To explore the effect and underlying mechanism of casein kinase 2 interacting protein-1(CKIP-1)on hepatocyte apoptosis in nonalcoholic fatty liver disease(NAFLD).Methods Experimental study.An NAFLD cell model was established by inducing human hepatoma cell line,HepG2 cells,with oleic acid(OA).Flag-CKIP-1 expression vector and shRNA-CKIP-1 were transfected into HepG2 cells.Flow cytometry was used to detect the effect of CKIP-1 on the activity and apoptosis of NAFLD hepatocytes.The levels of apoptosis-related proteins were detected by Western blot.CKIP-1 knockout mice in C57BL/6 back-ground were fed with either standard or high-fat diet for 8 weeks.Apoptosis-related signal proteins in NAFLD hepatocytes were detected by immunohistochemistry.Results After CKIP-1 was transfected into HepG2 cells,the degree of OA induced cell liposis was significantly reduced(P<0.05).Annexin V-FITC/PI flow cytometry showed that CKIP-1 reduced the apoptosis of steatotic hepatocytes.Overexpression of CKIP-1 could significantly inhibit the expression of caspase-3 and caspase-9 and increase the expression of Bcl-2/Bax(P<0.05).Knockdown of CKIP-1 could increase the expression of caspase-3 and caspase-9(P<0.05).CKIP-1 knockout could further increase the expression of caspase-3 and caspase-9 in NAFLD mice(P<0.01,P<0.05),and further decrease the expression of Bcl-2/Bax(P<0.05).Conclusion CKIP-1 inhibited the apoptosis of steatotic hepatocytes by up-regulating the expression of apoptosis inhibitor gene,Bcl-2/Bax,and affecting the proteases,caspase-3 and caspase-9.