目的 探讨非酒精性脂肪性肝病(NAFLD)中自噬与脂质代谢的相互作用.方法 体外人肝细胞培养(脂肪变性)制备NAFLD细胞模型,雷帕霉素诱导细胞自噬,3-甲基腺嘌呤抑制细胞自噬,MTr比色法测定细胞活力,ELISA法检测各组细胞IG、ALT、AST、LDH、GGT、A1b水平,IF法检测LC3-Ⅱ的定位与分布,Western Blot检测LC3-Ⅱ/LC3-Ⅰ比值.计量资料多组间比较采用方差分析,进一步两两比较采用SNK-q检验.结果 诱导自噬组的吸光度值和细胞活率与脂肪变组比较,明显下降(HL-7702细胞q值分别为4.160、4.110,SK-HEP-1细胞q值分别为4.407、4.032;P值均＜0.05).脂肪变组TG、ALT、AST、LDH、GGT、AIb水平与对照组相比,明显升高(HL-7702细胞q值分别为5.316、3.730、4.013、6.967、6.192、5.531,SK-HEP-1细胞q值分别为4.963、3.603、4.774、7.479、6.319、5.193;P值均＜0.05).诱导自噬组TG、ALT、AST、LDH、GGT、A1b水平与脂肪变组相比,明显降低(HL-7702细胞q值分别为4.978、3.695、3.960、5.130、4.695、3.192,SK-HEP-1细胞q值分别为3.846、5.575、4.184、5.019、4.203、3.049;P值均＜0.05).LC3-Ⅱ在各组肝细胞中的标记值,诱导自噬组最高(HL-7702细胞为90.1％,SK-HEP-1细胞为80.0％),其次是脂肪变组(HL-7702细胞为47.2％,SK-HEP-1细胞为48.4％)及抑制自噬组(HL-7702细胞为30.2％,SK-HEP-1细胞为45.5％).诱导自噬组LC3-Ⅱ/LC3-Ⅰ比值与脂肪变组相比,明显升高(HL-7702细胞q值为6.786,SK-HEP-1细胞q值为5.926;P值均＜0.05).结论 自噬的上调有利于促进肝脏脂肪的清除,而下调则促进脂质的聚积.%Objective To investigate the interaction between autophagy and lipid metabolism in nonalcoholic fatty liver disease (NAFLD).Methods Human hepatocytes (steatosis) were cultured in vitro to establish a cell model of N AFLD.Rapamycin was used to induce autophagy and 3-methyladenine was used to inhibit autophagy.MTT colorimetry was used to measure cell viability.ELISA was used to measure the levels of triglyceride (TG),alanine aminotransferase (ALT),aspartate aminotransferase (AST),lactate dehydrogenase (LDH),gamma-glutamyl transpeptidase (GGT),and albumin (Alb).IF method was used to determine the location and distribution of LC3-Ⅱ.Westem blot was used to measure LC3-Ⅱ/LC3-Ⅰ ratio.An analysis of variance was used for comparison of continuous data between groups,and the SNK-q test was used for further comparison between two groups.Results Compared with the steatosis group,the induced autophagy group had significant reductions in absorbance and cell viability (HL-7702 cells:q =4.160 and 4.110,P ＜0.05;SK-HEP-1 cells:q =4.407 and 4.032,P ＜ 0.05).Compared with the control group,the steatosis group had significant increases in the levels of TG,ALT,AST,LDH,GGT,and Alb (HL-7702 cells:q =5.316,3.730,4.013,6.967,6.192,and 5.531,P ＜0.05;SK-HEP-1 cells:q =4.963,3.603,4.774,7.479,6.319,and 5.193,P ＜ 0.05).Compared with the steatosis group,the induced autophagy group had significant reductions in the levels of TG,ALT,AST,LDH,GGT,and Alb (HL-7702 cells:q =4.978,3.695,3.960,5.130,4.695,and 3.192,P ＜ 0.05;SK-HEP-1 cells:q =3.846,5.575,4.184,5.019,4.203,3.049,P ＜ 0.05).The induced autophagy group had the highest percentage of LC3-Ⅱ-positive HL-7702 cells (90.1％) and LC3-Ⅱ-positive SK-HEP-1 cells (80.0％),followed by the steatosis group (47.2％ LC3-Ⅱ-positive HL-7702 cells and 48.4％ LC3-Ⅱ-positive SK-HEP-1 cells) and the antophagy inhibition group (30.2％ LC3-Ⅱ-positive HL-7702 cells and 45.5％ LC3-Ⅱ-positive SK-HEP-1 cells).The induced autophagy group had a significant increase in LC3-Ⅱ/IC3-Ⅰ ratio compared with the steatesis group (HL-7702 cells:q =6.786,P ＜ 0.05;SK-HEP-1 cells:q =5.926,P ＜ 0.05).Conclusion Upregulation of autophagy can promote the elimination of liver fat,while downregulation of autophagy can promote lipid accumulation.
展开▼
