含SH2结构域蛋白酪氨酸磷酸酶2表达变化对肝纤维化大鼠肝组织中细胞外信号调节激酶1/2活性的影响

Effects of SHP2 expression changes on ERK1/2 activity in liver tissues of rats with hepatic fibrosis

目的:探讨含SH2结构域的蛋白酪氨酸磷酸酶2(SHP2)表达变化对肝纤维化大鼠肝组织中细胞外信号调节激酶1/2(ERK1/2)活性的影响。方法:健康雄性SD大鼠160只被随机分为五组(对照组、模型组、Ad-GFP组、Ad-SHP2组及Ad-shRNA/SHP组),每组32只。除对照组(腹腔注射0.9%氯化钠溶液)外,其余四组构建四氯化碳诱导的大鼠肝纤维化模型,并将表达绿色荧光蛋白(GFP)的空病毒Ad-GFP、表达野生型SHP2及GFP的腺病毒Ad-SHP2、表达GFP及靶向SHP2的短发夹RNA(shRNA)的腺病毒Ad-shRNA/SHP自大鼠尾静脉分别注射入Ad-GFP组、Ad-SHP2组及Ad-shRNA/SHP2组大鼠,各组分别于造模第2、4、6、8周选取8只大鼠留取肝组织标本。采用HE染色观察大鼠肝组织病理学变化;采用Masson三色染色检测大鼠肝组织胶原沉积;采用实时荧光定量PCR检测大鼠肝组织SHP2、ERK1 mRNA表达;采用免疫组织化学染色检测SHP2蛋白表达;采用Western blot检测ERK1和p-ERK1蛋白表达。结果:大鼠肝纤维化模型成功构建,在各造模时间点,与模型组及Ad-GFP组大鼠肝组织胶原沉积比较,Ad-SHP2组均加重,而Ad-shRNA/SHP2组均减轻。导入野生型SHP2基因后大鼠肝组织的SHP2 mRNA及蛋白表达明显升高(均P<0.05),而导入靶向SHP2的shRNA后大鼠肝组织的SHP2 mRNA及蛋白表达明显降低(均P<0.05)。在各造模时间点(第2、4、6、8周),模型组、Ad-GFP组、Ad-SHP2组及Ad-shRNA/SHP2组大鼠肝组织ERK1 mRNA及蛋白表达以及p-ERK1蛋白表达高于对照组(均P<0.05),但上述四组大鼠肝组织的ERK1 mRNA及蛋白表达差异无统计学意义(均P>0.05);而在各时间点与模型组及Ad-GFP组大鼠肝组织p-ERK1蛋白表达比较,Ad-SHP2组升高(均P<0.05),Ad-shRNA/SHP2组降低(均P<0.05),模型组与Ad-GFP组差异无统计学意义(P>0.05)。结论:大鼠纤维化肝组织中SHP2过表达可通过促进ERK1/2磷酸化增强ERK1/2活性,而SHP2低表达则可通过抑制ERK1/2磷酸化降低ERK1/2活性。

Objective:To investigate the effect of src homology 2-containing protein tyrosine phosphatase 2(SHP2)expression on extracellular signal-regulated kinase1/2(ERK1/2)activity in liver tissue of rats with hepatic fibrosis.Methods:A total of 160 healthy male SD rats were randomly divided into five groups(control group,model group,Ad-GFP group,Ad-SHP2 group and Ad-shRNA/SHP2 group),32 rats in each group.In addition to the control group(intraperitoneal injection of 0.9%sodium chloride solution),the remaining four groups were used to construct a rat liver fibrosis model induced by carbon tetrachloride,and the empty virus Ad-GFP expressing green fluorescent protein(GFP),adenovirus Ad-SHP2 expressing wild type SHP2 and GFP,and adenovirus Ad-shRNA/SHP2 expressing GFP and shRNA targeting SHP2 were injected into the rats in Ad-GFP group,Ad-SHP2 group and Ad-shRNA/SHP2 group through the tail vein of the rats respectively.Liver tissue samples were taken from 8 rats in each group at the 2nd,4th,6th and 8th week of modeling.HE staining was used to observe the pathological changes of liver tissue.Masson trichrome staining was used to observe the collagen deposition in liver tissue.The mRNA expressions of SHP2 and ERK1 in rat liver tissue were detected by real-time fluorescence quantitative PCR.The expression of SHP2 protein was detected by immunohistochemical staining.The protein expression levels of ERK1 and p-ERK1 were detected by Western blot.Results:The rat liver fibrosis model was successfully constructed.At each modeling time point,compared with model group and Ad-GFP group,the collagen deposition of rat liver tissue in Ad-SHP2 group increased,while the Ad-shRNA/SHP2 group decreased.After the introduction of wild-type SHP2 gene,the SHP2 expression of mRNA and protein in rat liver tissue were significantly increased(all P<0.05),while followed by the introduction of shRNA targeting SHP2 that in rat liver tissue were significantly reduced(all P<0.05).At each time point(2nd,4th,6th and 8th week)of modeling,the expression of ERK1 mRNA and protein,as well as p-ERK1 protein,in liver tissue of rats in model group,Ad-GFP group,Ad-SHP2 group,and Ad-shRNA/SHP2 group were higher than those in control group(all P<0.05),but there was no statistically significant difference of ERK1 mRNA and protein expression in liver tissue of rats in the above four groups(all P>0.05).At the above each time point of modeling,compared with the model group and Ad-GFP group,the expression of p-ERK1 protein in the liver tissue of rats in Ad-SHP2 group increased(all P<0.05),while that in Ad-shRNA/SHP2 group decreased(all P<0.05),there was no statistically significant difference between the model group and the Ad-GFP group(P>0.05).Conclusion:Overexpression of SHP2 can enhance ERK1/2 activity by promoting ERK1/2 phosphorylation,while low expression of SHP2 can reduce ERK1/2 activity by inhibiting ERK1/2 phosphorylation in fibrotic liver tissue of rats.