组蛋白去乙酰化酶1对非酒精性脂肪性肝病细胞模型胰岛素抵抗的影响

Influence of histone deacetylase 1 on insulin resistance in a cell model of nonalcoholic fatty liver disease

目的建立高脂诱导的HepG2非酒精性脂肪性肝病(NAFLD)细胞模型,分析组蛋白去乙酰化酶1(HDAC1)表达对NAFLD细胞胰岛素抵抗(IR)的促进作用。方法将HepG2细胞分为对照组、模型组(OA)和抑制剂组[OA+Pyroxamide(HDAC1抑制剂)]。采用CCK-8法绘制HepG2细胞标准生长曲线,并筛选OA和Pyroxamide的最佳药物作用浓度和作用时间;油红O染色比较细胞内脂滴蓄积程度;全自动生化仪检测细胞内ALT、AST、TG、TC含量;RT-qPCR法和Western Blot法分别检测细胞内HDAC1、胰岛素受体底物-1(IRS-1)mRNA和蛋白表达量。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果0.25 mmol/L OA处理24 h为细胞最佳造模浓度和持续时间,20μmol/L处理24 h为Pyroxamide最佳给药浓度和持续时间;模型组相比于对照组ALT、AST、TG、TC含量均明显升高,而抑制剂组相比于模型组ALT、AST、TG、TC含量均显著降低(P值均<0.05)。各组细胞内HDAC1 mRNA和蛋白表达量比较,模型组高于对照组,抑制剂组低于模型组,差异均有统计学意义(P值均<0.05);各组细胞内IRS-1 mRNA和蛋白表达量相比,模型组低于对照组,抑制剂组高于模型组,差异均有统计学意义(P值均<0.05)。结论HDAC1通过抑制IRS-1分子的表达,促进IR,参与了NAFLD的发生与发展,HDAC1抑制剂Pyroxamide通过减轻IR,对肝脏起保护作用。

Objective To investigate the promoting effect of histone deacetylase 1(HDAC1)expression on insulin resistance(IR)in nonalcoholic fatty liver disease(NAFLD)cells by establishing an HepG2 cell model of high fat-induced NAFLD.Methods HepG2 cells were divided into control group,model group(OA),and inhibitor group(OA+pyroxamide[an HDAC1 inhibitor]).CCK-8 assay was used to plot the standard growth curve of HepG2 cells and screen out the optimal drug concentration and action time of OA and pyroxamide;oil red O staining was used to compare the accumulation of lipid droplets in cells;an automatic biochemical analyzer was used to analyze the content of alanine aminotransferase(ALT),aspartate aminotransferase(AST),triglyceride(TG),and total cholesterol(TC)in cells;quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression levels of HDAC1 and insulin receptor substrate-1(IRS-1)in cells.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results OA treatment at a concentration of 0.25 mmol/L for 24 hours was the optimal concentration and duration of cell modeling,and treatment at a concentration of 20μmol/L for 24 hours was the optimal administration concentration and duration of pyroxamide.Compared with the control group,the model group had significant increases in the content of ALT,AST,TG,and TC,and compared with the model group,the inhibitor group had significant reductions in the content of ALT,AST,TG,and TC(all P<0.05).The model group had significantly higher mRNA and protein expression levels of HDAC1 than the control group,while the inhibitor group had significantly lower expression levels than the model group(all P<0.05);the model group had significantly lower mRNA and protein expression levels of IRS-1 than the control group,while the inhibitor group had significantly higher expression levels than the model group(all P<0.05).Conclusion HDAC1 participates in the development and progression of NAFLD by inhibiting the expression of IRS-1 molecule and promoting IR,and the HDAC1 inhibitor pyroxamide can exert a protective effect on the liver by alleviating IR.