MMP13和TIMP2在肝缺血再灌注损伤介导肝纤维化进程中的动态变化及作用

探讨肝缺血再灌注损伤(HIRI)介导肝纤维化进程中基质金属蛋白酶-13(MMP13)和基质金属蛋白酶组织抑制剂2(TIMP2)的动态变化及作用。将70只C57雄性小鼠随机分为假手术组(Sham组)及肝缺血再灌注组(I/R 0 h组、I/R 3 h组、I/R 6 h组、I/R 72 h组、I/R 7 d组及I/R 15 d组)。夹闭肝门静脉、左叶和中叶的肝动脉,缺血90 min后开夹,分别再灌0、3、6、72 h、7 d及15 d,处死小鼠。检测小鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST)的水平;测定肝组织中羟脯氨酸(Hyp)的含量;免疫组化法检测肝组织中α-平滑肌肌动蛋白(α-SMA)表达量;RT-PCR检测肝组织MMP13和TIMP2 mRNA的表达水平;HE染色观察肝纤维化程度。结果显示,与Sham组相比,I/R 0 h、I/R 3 h及I/R 6 h组血清ALT、AST升高(P<0.05),I/R 72 h、I/R 7 d及I/R 15 d组下降(P<0.05)。I/R各组肝组织Hyp含量随再灌注时间延长逐渐升高,其中I/R 72 h、I/R 7 d、I/R 15 d组显著高于Sham组(P<0.05)。免疫组化显示,肝组织α-SMA的蛋白表达量随再灌注时间延长而逐渐增加。RT-PCR显示,I/R各组MMP13 mRNA的表达水平呈先高后低的趋势,与Sham组相比,I/R 0 h、I/R 3 h组显著升高(P<0.05)。而TIMP2的表达为先低后高,与Sham组相比,I/R 72 h、I/R 7 d及I/R 15 d组显著升高(P<0.05)。HE染色结果与上述变化趋势相一致。结果表明,在HIRI介导肝纤维化形成过程中MMP13与TIMP2呈动态的协同作用,共同促进肝纤维化的发生发展,是肝纤维化形成的关键因素。

Dynamic Changes and Effects of MMP13 and TIMP2 in Process of Hepatic Fibrosis Mediated by Hepatic Ischemia Reperfusion Injury

To investigate the dynamic changes and effects of MMP13 and TIMP2 in the process of liver fibrosis mediated by hepatic ischemia reperfusion injury(HIRI).70 male C57 mice were randomly divided into Sham group and hepatic ischemia reperfusion group(I/R0 h group,I/R3 h group,I/R6 h group,I/R72 h group,I/R7 d group and I/R15 d group).The portal vein and hepatic arteries of the left and middle lobes were clamped by clips which were opened after 90 minutes ischemia.Then,the mice were reperfused for 0,3,6,72 hours,7 and 15 days,respectively,and put to death.Serum ALT and AST levels were detected in mice.The content of Hyp in liver tissue was determined.The expression of α-smooth muscle actin(α-SMA) was detected by immunohistochemistry.The expression levels of MMP13 and TIMP2 mRNA in liver tissues were detected by RT-PCR.The degree of liver fibrosis was observed by HE staining.The results of this experiment showed that compared with Sham group,serum ALT and AST of I/R0 h,I/R3 h and I/R6 h groups increased(P<0.05),while serum ALT and AST of I/R72 h,I/R7 d and I/R15 d groups decreased(P<0.05).The content of Hyp in liver tissue of I/R group increased gradually with the extension of reperfusion time,among which I/R72 h,I/R7 d and I/R15 d groups were significantly higher than Sham group(P<0.05).Immunohistochemical results showed that the protein expression of α-SMA increased with the time of reperfusion.RT-PCR showed that the expression level of MMP13 mRNA in I/R groups raised at first,then decreased,and compared with Sham group,the expression levels in I/R0 h and I/R3 h groups increased significantly(P<0.05).While the expression of TIMP2 raised at first,then decreased.Compared with Sham group,the expression of I/R72 h,I/R7 d and I/R15 d was significantly increased(P<0.05).The results of HE staining were consistent with the above trend.In this way,MMP13 and TIMP2 show a dynamic synergistic effect in the process of HIRI mediated liver fibrosis formation,and jointly promote the occurrence and development of liver fibrosis,which is the key factor in the formation of liver fibrosis.