细胞自噬在D-氨基半乳糖/脂多糖诱导的小鼠急性肝损伤模型中的保护作用及机制

摘要

目的 利用D-氨基半乳糖(D-GalN)/脂多糖(LPS)诱导的小鼠急性肝损伤模型研究细胞自噬在急性肝损伤中的作用及机制.方法 以C57BL/6小鼠为研究对象,腹腔注射D-GalN/LPS建立小鼠急性肝损伤模型.动物实验分组:对照组、D-GalN/LPS组、雷帕霉素+D-GalN/LPS组、三甲基腺嘌呤(3-MA)+D-GaIN/LPS组、Atg7 siRNA+D-GaIN/LPS组.观察不同分组中小鼠的生存情况,观察肝组织病理变化评价肝损伤情况,全自动生化分析仪检测血清ALT、AST水平,实时荧光定量PCR检测肝组织中TNFα和IL-6基因表达,荧光显微镜观察肝组织中肝细胞凋亡情况.多样本组间比较采用One-way ANOVA分析,进一步两两比较,方差齐时采用LSD-t检验,方差不齐时采用Games-Howell法.结果 与D-GalN/LPS组相比,应用自噬激活剂雷帕霉素干预后,小鼠生存率明显升高(80％ vs 40％),肝脏出血、炎症和坏死明显减轻,肝细胞凋亡明显减少,血清ALT、AST水平明显降低[ALT:(427.4±195.5)U/L vs(977.7±247.3) U/L,P=0.002;AST:(378.2±169.7) U/L vs(1100.0 ±438.0) U/L,P=0.004],肝组织中炎症因子TNFα和IL-6 mRNA表达水平明显降低[TNFα mRNA:0.288 ±0.010 vs 1.136 ±0.267,P=0.003;IL-6mRNA:0.272 ±0.061 vs 0.869±0.317,P=0.010];应用自噬抑制剂3-MA和Atg7 siRNA干预后,小鼠生存率明显降低(0、10％vs 40％),肝脏出血、炎症和坏死明显加重,肝细胞凋亡明显增多,血清ALT、AST水平明显升高[ALT:(1836.0±560.5)、(1654.0±627.6) U/L vs(977.7±247.3) U/L,P值分别为0.006、0.034;AST:(1948.0±645.5)、(1804.0±492.6) U/Lvs (1100.0 ±438.0)U/L,P值分别为0.029、0.033],肝组织中TNFα表达水平明显升高[2.026±0.342、1.994 ±0.286 vs 1.136 ±0.267,P值分别为0.006、0.005].结论 在D-GalN/LPS诱导的小鼠急性肝损伤中,细胞自噬发挥着重要的保护性作用,其调控机制可能与自噬抑制炎症因子和肝细胞凋亡有关.%Objective To investigate the role and mechanism of autophagy in acute liver injury induced by D-galactosamine/lipopolysaccharide (D-GalN/LPS) in mice.Methods C57BL/6 mice were used to establish a mouse model of acute liver injury using intraperitoneally injected D-GalN/LPS.In this animal experiment,the mice were divided into control group,D-GalN/LPS group,rapamycin + D-GalN/LPS group,3-MA + D-GalN/LPS group,and Atg7 siRNA + D-GalN/LPS group.The survival of the mice was observed,and liver pathological changes were observed to analyze liver injury.An automatic biochemical analyzer was used to measure the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST),quantitative real-time PCR was performed to measure the mRNA expression of tumor necrosis factorα (TNFα) and interleukin-6 (IL-6),and a fluorescence microscope was used to observe the apoptosis of hepatocytes.A One-way ANOVA was used for comparison between multiple groups;the least significant difference t-test was used for homogeneity of variance and the Games-Howell method was used for heterogeneity of variance.Results Compared with the D-GalN/LPS group,the rapamycin + D-GalN/LPS group had a significant increase in survival rate (80％ vs 40％),significantly reduced liver hemorrhage,inflammation,and necrosis and apoptosis of hepatocytes,and significant reductions in serum levels of ALT (427.4 ± 195.5 U/L vs 977.7 ± 247.3 U/L,P =0.002) and AST (378.2 ± 169.7 U/L vs 1100.0 ± 438.0 U/L,P =0.004),as well as significant reductions in the mRNA expression of TNFα (0.288 ±0.010 vs 1.136 ±0.267,P =0.003) and IL-6 (0.272 ±0.061 vs 0.869 ±0.317,P =0.010).Compared with the D-GalN/LPS group,the 3-MA + D-GalN/LPS group and Atg7 siRNA + D-GalN/LPS group had significant reductions in survival rate (0％/10％ vs 40％),significantly aggravated liver hemorrhage,inflammation,and necrosis and apoptosis of hepatocytes,and significant increases in serum levels of ALT (1836.0 ±560.5 U/L and 1654.0 ±627.6 U/L vs 977.7 ± 247.3 U/L,P =0.006 and 0.034) and AST (1948.0 ± 645.5 U/L and 1804.0 ± 492.6 U/L vs 1100.0 ± 438.0 U/L,P =0.029 and 0.033),as well as significant increases in the expression of TNFα in liver tissue (2.026 ± 0.342 and 1.994 ± 0.286 vs 1.136 ± 0.267,P =0.006 and 0.005).Conclusion In the mouse model of acute liver injury induced by D-GalN/LPS,autophagy has an important protective effect,and its regulating mechanism may be associated with the inhibitory effect on inflammatory factors and apoptosis of hepatocytes.