同型半胱氨酸通过激活beclin 1调节的自噬激活分子(AMBRA1)促进人肝细胞自噬

Homocysteine promotes human hepatocyte autophagy through activating the activating molecule in beclin-1-regulated autophage(AMBRA1)

目的探讨beclin 1调节的自噬激活分子(AMBRA1)在同型半胱氨酸(Hcy)引起肝细胞自噬中的作用。方法体外培养肝细胞,分为对照组(0μmol/L Hcy处理)和100μmol/L Hcy处理组。Western blot法检测自噬相关蛋白微管相关蛋白1轻链3B(LC3BⅡ、LC3BⅠ)的表达水平;使用(0、25、50、100)μmol/L氯喹(CQ)处理肝细胞,CCK-8法检测CQ对肝细胞增殖的抑制作用,Western blot法检测LC3B和AMBRA1的表达;采用AMBRA1小干扰RNA感染肝细胞后,实时荧光定量PCR和Western blot法检测肝细胞AMBRA1表达干扰效率。敲低AMBRA1后肝细胞用Hcy处理,Western blot法检测LC3B蛋白表达水平,转染表达双荧光蛋白和LC3的腺病毒(mRFP-GFP-LC3),激光共聚焦显微镜观察细胞内自噬流的变化。结果与对照组相比较,Hcy处理组LC3BⅡ/LC3BⅠ的比值升高;50μmol/L CQ对肝细胞增殖的抑制率接近于50%;与对照组相比,Hcy组LC3BⅡ/LC3BⅠ比值、AMBRA1的表达明显升高,而Hcy联合CQ组的LC3BⅡ/LC3BⅠ比值、AMBRA1的表达比Hcy组明显降低;敲低AMBRA1后,肝细胞LC3BⅡ/LC3BⅠ比值下降;与对照组相比,Hcy组自噬体和自噬溶酶体增加,敲低AMBRA1后,自噬体和自噬溶酶体减少。结论Hcy可通过激活AMBRA1促进肝细胞自噬。

Objective To investigate the role of activating molecule in beclin-1-regulated autophage(AMBRA1)in homocysteine(Hcy)-induced hepatocytes autophagy.Methods Hepatocytes were cultured in vitro and divided into control group(0μmol/L Hcy)and Hcy treatment group(100μmol/L Hcy).Western blotting was used to detect the expression of microtubule-associated protein 1 light chain 3B(LC3BⅡ、LC3BⅠ);hepatocytes were treated with 0,25,50,100μmol/L chloroquine(CQ),CCK-8 assay was used to detect the inhibitory effect of CQ on hepatocyte proliferation and Western blotting was performed to detect the expression of LC3B and AMBRA1;After hepatocytes were transfected with AMBRA1 small interfering RNA,real-time fluorescent quantitative PCR and Western blotting were used to detect the interference efficiency of AMBRA1 expression;After the transfected hepatocytes were treated with Hcy,the expression of LC3B was detected by Western blot analysis.mRFP-GFP-LC3 adenovirus was transfected with hepatocytes and the autophagy flow was observed by laser scanning confocal microscopy.Results Compared with the control group,the ratio of LC3BⅡ/LC3BⅠincreased in the Hcy treatment group;the inhibition rate of 50μmol/L CQ on hepatocyte proliferation was close to 50%;compared with the control group,the ratio of LC3BⅡ/LC3BⅠand the expression of AMBRA1 increased significantly in the Hcy group,and the ratio of LC3BⅡ/LC3BⅠand the expression of AMBRA1 in the Hcy combined with CQ group were significantly lower than those in the Hcy group;the ratio of LC3BⅡ/LC3BⅠdecreased after knocking down AMBRA1;compared with the control group,the autophagosomes and autophagolysosomes increased in Hcy group and decreased after knocking down AMBRA1.Conclusion Hcy can promote hepatocyte autophagy by activating AMBRA1.