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目的明确Calpain 2及自噬相关基因7(autophagy related gene 7,ATG7)在非酒精性脂肪性肝病中的表达变化及意义。方法应用高脂饮食喂养SD大鼠建立非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)在体模型,并按时相点分为4、8、12周及16周组,同时设立正常饮食组为对照组,每组6只。检测各组大鼠血清ALT、AST、FFA水平,通过HE染色观察肝脏脂肪变情况,利用Real-time PCR及Western blot检测Calpain 2及ATG7的表达变化。结果①HE染色提示肝脏脂肪变程度随高脂饮食喂养时间的延长而加重。NAFLD大鼠模型各时相点血清ALT、AST、FFA水平均较对照组有不同程度的升高,在16周组时分别为(165.95±7.24)U/L、(249.52±4.20)U/L、(0.83±0.05)mmol/L,与对照组比较均有明显的升高(P<0.01)。②以正常饮食组为对照组,肝组织Calpain 2 mRNA相对表达量在高脂饮食喂养后开始上调,在16周时升高最为明显(9.83±0.85,P<0.01);ATG7的mRNA相对表达量在4周组(0.82±0.02)即开始下降,在16周组(0.20±0.03)降至最低,与对照组比较降低显著(P<0.01)。③与对照组比较,Calpain 2蛋白相对表达水平与mRNA表达水平一致,4周组(2.32±0.45)时开始上调,16周组明显上升(9.87±1.20,P<0.01)。而ATG7的蛋白相对表达水平则随肝脏脂肪变性的进展明显下降,16周时降低最为明显(0.18±0.05,P<0.01)。结论脂肪肝发生过程中,Calpain 2的表达上调抑制了自噬相关基因ATG7的表达,进而减弱了自噬对细胞的保护作用,导致肝细胞的损伤加重,自噬可能参与了NAFLD的发生。
Objective To investigate the expression and significance of Calpain 2 and autophagy related gene 7 (ATG7) in non-alcoholic fatty liver disease. Methods SD rats were fed with a high-fat diet to establish non-alcoholic fatty liver disease (NAFLD) in vivo and divided into 4, 8, 12 and 16 weeks according to the time point, Diet group as control group, 6 rats in each group. The levels of ALT, AST and FFA in serum of rats in each group were detected. The hepatic steatosis was observed by HE staining. The expressions of Calpain 2 and ATG7 were detected by Real-time PCR and Western blot. Results (1) HE staining suggested that the degree of hepatic steatosis aggravated with the prolongation of high-fat diet. The levels of ALT, AST and FFA in serum of NAFLD rats at different time points were significantly higher than those in control group (165.95 ± 7.24 U / L, 249.52 ± 4.20 U / L , (0.83 ± 0.05) mmol / L respectively, which were significantly higher than those in the control group (P <0.01). (2) Compared with the normal diet group, the relative expression of Calpain 2 mRNA in the liver tissue began to increase after high-fat diet feeding, and the most significant increase was at 16 weeks (9.83 ± 0.85, P <0.01); the relative expression of ATG7 mRNA was 4 Weekly group (0.82 ± 0.02) began to decline in the 16-week group (0.20 ± 0.03) to a minimum, compared with the control group decreased significantly (P <0.01). (3) Compared with the control group, the relative expression level of Calpain 2 protein was consistent with that of mRNA expression. The expression of Calpain 2 protein was up-regulated in the 4-week group (2.32 ± 0.45) and significantly increased in the 16-week group (9.87 ± 1.20, P <0.01). However, the relative expression of ATG7 protein decreased with the progression of hepatic steatosis and decreased most significantly at 16 weeks (0.18 ± 0.05, P <0.01). Conclusion Upregulation of Calpain 2 inhibits the expression of autophagy-related gene ATG7 in fatty liver and further attenuates the protective effect of autophagy on hepatocytes. The autophagy may be involved in the pathogenesis of NAFLD.