Investigating The Mechanism Of TGF-β1/Smad3 Signaling Pathway In Liver Ischemia Reperfusion Injury In Mice

Objective:Ischemia reperfusion injury is still an inevitable complication of hepatobiliary surgery,especially affecting liver viability and prognosis during partial hepatectomy and liver transplantation.It is very important to prevent ischemia reperfusion injury for the quality of life of patients after surgery,with the increasing the number of hepatobiliary surgeries.The molecular mechanism of liver ischemia reperfusion injury is very complicated,TGF-β/Smad3signaling pathway is closely related to the pathogenesis of acute liver injury induced by CCL4,stellate activation induced liver chronic fibrosis.However,the mechanism of TGF-β/Smad3 signaling pathway in liver ischemia reperfusion injury is not completely clear.This study is expected to explore the influence and mechanism of TGF-β/Smad3 signaling pathway in acute warm ischemia reperfusion injury and provide new therapeutic targets for the treatment of ischemia reperfusion injury.Methods:The experimental mice were SPF 129S2/Sv Pas Crl wild-type male mice and 129S2/Sv Pas Crl Smad3 gene mice with 6-8 weeks.First,twenty of 129S2/Sv Pas Crl wild-type mice were randomly divided into two groups as ischemic reperfusion injury group（Ischemia reperfusion,IR-WT,n=10）and sham control（Sham operation,SH-WT,n=10）to construct a model between liver ischemia reperfusion injury and sham operation group.Serum alanine transferase（ALT）and histopathological hematoxylin-eosin（HE）staining were used to evaluate the pathological changes of hepatocytes and liver tissue damage;Real-time fluorescence quantitative PCR（q RT-PCR）and Western blot were used to detect the expression of TGF-β1 and Smad3 in liver tissue.Constructed liver ischemia reperfusion injury model of 129S2/Sv Pas Crl Smad3 wild-type mice（Smad3+/+）and 129S2/Sv Pas Crl Smad3 homozygous mutant mice（Smad3-/-）,which were randomly divided into four groups:Smad3+/+mice with liver ischemia reperfusion injury group(IR^Smad3+/+,n=10);Smad3+/+mice with liver sham operation group(Sham^Smad3+/+,n=10);Smad3-/-mice with liver ischemia reperfusion injury group(IR^Smad3-/-,n=5);Smad3-/-mice with sham operation group(Sham^Smad3-/-,n=5).ALT and HE staining were used to evaluate the pathological changes of hepatocytes and liver tissue damage;Apoptosis and proliferation in Smad3+/+and Smad3-/-mice after ischemia reperfusion injury were assessed by Western blot and immunohistochemical method.Immunohistochemical staining and q RT-PCR were used to detect the expression of associated inflammatory cells and to explore the infiltration of inflammatory cells.Experimental results:During liver ischemia reperfusion injury model in29S2/Sv Pas Crl wild-type mice,serum ALT was significantly higher in wild-type mice after liver ischemia reperfusion injury than in sham operation mice（p<0.05）.In addition,HE staining of liver tissues showed that irregular liver lobular structure,disorder of liver cell arrangement,swelling of liver cells,slight congestion of liver sinusoids and inflammatory cell infiltration,high Suzuki score after ischemia reperfusion injury compared to sham operation mice.Further studies revealed that TGF-β1,Smad3 and p-Smad3 protein levels was significantly up-regulated within liver tissue after ischemia reperfusion injury than sham operation mice by Western blot.During liver ischemia reperfusion injury model 129S2/Sv Pas Crl Smad3gene mice,serum ALT was significantly higher in Smad3+/+mice and Smad3-/-mice after liver ischemia reperfusion injury than in sham operation mice（p<0.05）.After liver ischemia reperfusion injury,the serum ALT of Smad3-/-mice was significantly higher than Smad3+/+mice（p<0.05）.In addition,HE staining of liver tissues showed that irregular liver lobular structure,disorder of liver cell arrangement,swelling of liver cells,slight congestion of liver sinusoids and inflammatory cell infiltration,high liver injury Suzuki score after ischemia reperfusion injury compared to sham operation mice.Besides,infiltration of inflammatory cells and Suzuki score was more obvious higher in Smad3-/-than that of Smad3+/+mice after liver ischemia reperfusion injury（p<0.05）.The expression of Cleaved caspase-3 protein in liver tissue of Smad3-/-mice was significantly higher than that of Smad3+/+mice after ischemia reperfusion injury.The number of positive cells by Caspase-3 staining and TUNEL staining in mice after liver ischemia reperfusion injury were more than sham operation mice;besides,the number of positive cells by Caspase-3staining and TUNEL staining in Smad3-/-mice liver tissue was significantly higher than of Smad3+/+mice after liver ischemia reperfusion injury（semi-quantitative analysis,p<0.05）.The number of positive cells by CD45staining and F4/80 staining in mice after liver ischemia reperfusion injury were more than sham operation mice;besides,the number of positive cells by CD45staining and F4/80 staining in Smad3-/-mice liver tissue was significantly higher than of Smad3+/+mice after liver ischemia reperfusion injury（semi-quantitative analysis,p<0.05）.The q RT-PCR results showed that the m RNA expression of B220 and Ly-6G in the ischemia reperfusion injury mice was higher than sham operation mice.after liver ischemia reperfusion injury surgery,Smad3-/-mice expressed higher Ly-6G while B220 expressed lower compared with Smad3+/+mice.However,the opposite result was observed in Smad3-/-mice,which was that the expressions of TLR4,IFN-γ,and TNF m RNA in Smad3-/-mice were lower than in Smad3-/-mice with liver ischemia reperfusion injury compared with Smad3-/-mice by sham operation mice（p<0.05）.Conclusion:（1）Activated the TGF-β/Smad3 signaling pathway in liver ischemia reperfusion injury mice（2）Liver injury,apoptosis,and inflammatory cell infiltration in surrounding tissues increased after ischemia reperfusion injury in Smad3-/-mice.These results suggest that TGF-β/Smad3 signaling pathway may play a role in protecting liver cells from hepatic ischemia reperfusion injury.It also suggests that TGF-β/Smad3 pathway can be used as a therapeutic target for clinical intervention of hepatic ischemia reperfusion injury.