Effect Of SHP2 Expression Changes On Collagen Metabollsm In Liver Tissues Of Rats With Hepatic Fibrosis

Objectives To study the effect of the expression changes of src-homology domain 2-containing protein tyrosine phosphatase 2（SHP2）on collagen metabolism in liver tissues of rats with hepatic fibrosis and its mechanism.Methods The rats liver fibrosis model was established by intraperitoneal injection of carbon tetrachloride（CCl₄）,and the recombinant adenovirus Ad-SHP2 carrying wild-type SHP2（PTPN11）gene and green fluorescent protein（GFP）,recombinant adenovirus Ad-sh RNA/SHP2 carrying short hairpin RNA（sh RNA）targeting SHP2 and expressing GFP,and control empty virus Ad-GFP only expressing GFP were introduced into rats.160healthy male SD rats were randomly divided into 5 groups:control group（injection with normal saline only）,model group（injection with CCl₄）,Ad-GFP group（injection with CCl₄ and Ad-GFP）,Ad-SHP2 group（injection with CCl₄ and Ad-SHP2）and Ad-sh RNA/SHP2 group（injection with CCl₄ and Ad-sh RNA）.There were 32 rats in each group,and 8 rats in each group were killed and taken appropriate amount of liver tissues at2 weeks,4 weeks,6 weeks and 8 weeks of modeling.Masson trichromatic staining and HE staining were used to observe the pathological changes of liver tissues in each group.Immunohistochemical staining was used to detect the expression of collagenⅠand collagenⅢin liver tissues of rats in each group.The expressions of SHP2,collagenⅠ,collagenⅢ,matrix metalloproteinase-13（MMP-13）and tissue inhibitor of metalloproteinase-1（TIMP-1）protein and m RNA in liver tissues of rats in each group were detected by Western blot and real-time fluorescence quantitative PCR.Results 1 Masson trichromatic staining and HE staining showed that CCl₄-induced rats liver fibrosis model was successfully established.With the prolongation of modeling time,liver fibrosis in model group and adenovirus treatment group（Ad-GFP group,Ad-SHP2group,Ad-sh RNA/SHP2 group）were gradually aggravated.Making comparison at the same time,the liver fibrosis in Ad-SHP2 group was aggravated,while that in Ad-sh RNA/SHP2 group was alleviated.2 Western blot and real-time fluorescence quantitative PCR were used to detect the expression of SHP2 in liver tissues of rats in each group,the expression of SHP2 protein and m RNA in liver tissues of model group and adenovirus treatment group increased gradually with the prolongation of modeling time.Comparing the expression of SHP2 protein and m RNA in liver tissues of rats at different modeling time points（the 2nd,4th,6th and 8th week）,the results showed that the expression of SHP2 protein and m RNA in model group and adenovirus treatment group was significantly higher than that in control group（P<0.05）,while that in Ad-SHP2 group was significantly higher than that in model group and Ad-GFP group（P<0.05）,and that in Ad-sh RNA/SHP2group was significantly lower than that in model group and Ad-GFP group（P<0.05）,but there was no significant difference in SHP2 expression between Ad-GFP group and model group at each time point（P>0.05）.3 Immunohistochemical staining,Western blot and real-time fluorescence quantitative PCR were used to detect the expression of collagenⅠand collagenⅢprotein and m RNA in liver tissues of rats in each group.The results showed that the expression of collagenⅠand collagenⅢprotein and m RNA in liver tissues of rats in model group and adenovirus treatment group increased gradually with the prolongation of modeling time.Comparing the expressions of collagenⅠand collagenⅢprotein and m RNA in liver tissues of rats in different modeling time points（the 2nd,4th,6th and 8th week）at the same time point,it showed that the expression of collagenⅠand collagenⅢin model group and adenovirus treatment group was significantly higher than that in control group（P<0.05）.Ad-SHP2 group was significantly higher than model group and Ad-GFP group（P<0.05）.Ad-sh RNA/SHP2 group was significantly lower than model group and Ad-GFP group（P<0.05）.However,there was no significant difference between Ad-GFP group and model group at each time point（P>0.05）.4 The expression of MMP-13 protein and m RNA in liver tissues of rats in each group was detected by Western blot and real-time fluorescence quantitative PCR.The results showed that the expression of MMP-13protein and m RNA in liver tissues of rats in model group and adenovirus treatment group first increased and then decreased with the prolongation of modeling time.Comparing the expression of MMP-13 protein and m RNA in liver tissues of rats in different modeling time points（the 2nd,4th,6th and 8th week）,it shows that there is no significant difference between Ad-GFP group and model group at each time point（P>0.05）.At 2 weeks,Ad-SHP2 group was higher than model group and Ad-GFP group（P<0.05）,while Ad-sh RNA/SHP2 group was lower than model group and Ad-GFP group（P<0.05）.At 4 weeks,Ad-SHP2 group and Ad-sh RNA/SHP2 group were lower than model group and Ad-GFP group（P<0.05）,while model group and adenovirus treatment group were higher than control group at 2 weeks and 4 weeks（P<0.05）.At 6 and 8 weeks,Ad-SHP2 group is lower than model group,Ad-GFP group and control group（P<0.05）,Ad-sh RNA/SHP2group is higher than model group,Ad-GFP group and control group（P<0.05）,while model group and Ad-GFP group were higher than control group at 6 weeks and lower than control group at 8 weeks（P<0.05）.5 The expression of TIMP-1 protein and m RNA in liver tissues of rats in each group was detected by Western blot and real-time fluorescence quantitative PCR.The results showed that the expression of TIMP-1 protein and m RNA in liver tissues of rats in model group and adenovirus treatment group increased gradually with the prolongation of modeling time.The expression of TIMP-1 protein and m RNA in liver tissues of rats in different modeling time points（the 2nd,4th,6th and 8th week）was compared at the same time point.the expression of TIMP-1 protein and m RNA in model group and adenovirus treatment group was significantly higher than that in control group（P<0.05）,Ad-SHP2 group was significantly higher than that in model group and Ad-GFP group（P<0.05）,Ad-sh RNA/SHP2 group was significantly lower than model group and Ad-GFP group（P<0.05）.However,there was no significant difference between Ad-GFP group and model group at each time point（P>0.05）.Conclusions 1 Up-regulation of SHP2 expression inhibits the degradation of collagenⅠand collagenⅢin liver tissues of rats with liver fibrosis and promotes the deposition of collagenⅠand collagenⅢ.Down-regulation of SHP2 expression promotes the degradation of collagenⅠand collagenⅢand inhibits the deposition of collagenⅠand collagenⅢin hepatic fibrosis rats.2 The MMP-13/TIMP-1 mechanism is involved in the regulation of SHP2 on and metabolism of collagenⅠand collagenⅢin liver tissues of rats with hepatic fibrosis.3 Down-regulation of SHP2 expression can improve liver fibrosis by promoting the degradation of collagenⅠand collagenⅢand reducing the deposition of collagenⅠand collagenⅢin liver tissues of rats with liver fibrosis.Figure10;Table11;Reference 76...