高同型半胱氨酸通过抑制SETDB1促进肝脂肪沉积

High homocysteine promotes liver fat deposition by inhibiting SETDB1

目的研究同型半胱氨酸(homocysteine,Hcy)和SET结构域分支型1(SET domain bifurcated 1,SETDB1)在肝脏脂肪沉积中的作用和分子机制。方法选取C57BL/6N雄性小鼠共14只。依据经典的Lieber-De Carli方法和高甲硫氨酸饮食(high methionine diet,HMD)建立酒精性肝病(alcoholic liver disease,ALD)和高同型半胱氨酸血症(hyperhomocysteinemia,HHcy)模型,将6只雄鼠随机分为对照组(pair fed,PF组)和酒精组(alcohol fed,AF组),每组3只,持续喂养5周,分别给予对照饲料和酒精液体饲料建立ALD模型;将8只雄性小鼠随机分为对照饲料饮食组(Chow组)与高甲硫氨酸饮食组(HMD组),每组4只,持续喂养5周建立HHcy模型。在AML12细胞中分别加入3.33μmol/L油酸(oleic acid,OA)、100μmol/L同型半胱氨酸(homocysteine,Hcy),将OA与Hcy共同培养48 h,SETDB1 siRNA干扰AML12细胞6 h后培养至48 h。应用甘油三酯(triglyceride,TG)检测试剂盒和Hcy ELISA检测肝脏、血浆和细胞中TG和Hcy水平,使用qRT-PCR和Western blot检测肝脏及细胞SETDB1 mRNA、蛋白水平及其活性,提取细胞总RNA进行RNA表达芯片分析并富集功能通路。结果ALD小鼠模型出现肝脏脂肪沉积,AF组与PF组比较,血浆和肝脏Hcy水平增高,肝脏SETDB1表达和活性降低(P<0.05)。HMD中出现肝脏脂肪沉积,HMD组与Chow组比较,血浆和肝脏Hcy水平上升,肝脏SETDB1表达和活性降低(P<0.05)。外源Hcy导致AML12细胞SETDB1表达和活性下降,同时细胞内TG含量增加(P<0.05)。敲低SETDB1导致AML12细胞TG含量增加(P<0.05)。结论同型半胱氨酸通过抑制肝脏SETDB1表达和活性促进肝脏脂肪沉积。

Objective To clarify the specific role and molecular mechanism of homocysteine(Hcy)and SET domain bifurcated 1(SETDB1)in hepatic adipose deposition.Methods Fourteen male C57BL/6N mice were selected,alcoholic liver disease(ALD)and hyperhomocysteinemia(HHcy)models were established by using Lieber-De Carli method and high methionine diet(HMD).Six male rats were randomly divided into hyperhomocysteinemia pair fed(PF)group and alcohol fed(AF)group,with 3 mouse in each group.Mouse of the two groups were fed with control feed and alcohol liquid feed respectively for 5 weeks to establish ALD model;8 male mice were randomly divided into Chow group and HMD group,with 4 mouse in each group,HHcy mouse model was established by feeding for 5 weeks.AML12 cells were cultured with 3.33μmol/L oleic acid(OA),100μmol/L Hcy,and OA and Hcy for 48 h,respectively.SETDB1 siRNA interfered with AML12 cells for 6 h and was cultured to 48 h.Triglyceride(TG)detection kit and Hcy ELISA were used to detect TG and Hcy levels in liver,plasma and cells,qRT-PCR and Western blot were used to detect SETDB1 mRNA and protein levels and their activities in liver and cells,and total cell RNA was extracted for RNA expression chip analysis and enrichment of functional pathways.Results Liver fat deposition was observed in ALD mice model,plasma and liver Hcy levels were increased in AF group compared with PF group,and liver SETDB1 expression and activity were decreased(P<0.05).Compared with the Chow group,the HMD group had significantly higher plasma and liver Hcy levels and significantly lower SETDB1 expression and activity in the liver(P<0.05).The expression and activity of SETDB1 was decreased and the intracellular TG content in AML12 cells was increased by exogenous Hcy(P<0.05).The TG content of AML12 cells was increased by lowering SETDB1(P<0.05).Conclusion Homocysteine promotes hepatic fat deposition by inhibiting liver SETDB1 expression and activity.