The Early Mechanism Of Liver Fibrosis Mediated By Hepatic Ischemia-reperfusion Injury And Its Effect On TGF-β-Smad Pathway

Objective The mouse liver is subjected to ischemic reperfusion treatment,and a more stable preparation of hepatic fibrosis can be selected by different time of ischemia.Methods 18 C57 male mice were divided into normal control group(CON group,n=6),HI70 min group(70 min ischemia,8 weeks reperfusion,n=6)and HI90 min group(Ischemia 90 min,reperfusion 8 weeks,n=6).The model of hepatic ischemia-reperfusion injury in mice was treated with half hepatic occlusion.The left lateral lobe,middle left lobe,and middle lobe of the mice were subjected to ischemia.They were subjected to ischemia for 70 min and 90 min,and then sacrificed after 8w of reperfusion.The content of serum alanine aminotransferase(ALT)was measured,the content of hydroxyproline(Hyp)in liver tissue was measured,and the degree of hepatic fibrosis was observed by HE staining.Results1.Serum ALT levels: Compared with the CON group,the serum ALT level in the HI90 min group was significantly higher(P<0.05),while the serum ALT level in the HI70 min group was higher than that in the CON group but there was no statistically significant difference between the two groups(P>0.05);Compared with the HI70 min group,the serum ALT level in the HI90 min group was significantly higher,with a significant difference(P<0.05).2.Liver tissue Hyp content determination: Compared with the CON group,the Hyp level in the liver tissue of the HI90 min group was significantly higher(P<0.05),while the Hyp level in the HI70 min group was higher than that in the CON group but there was no significant difference between the two groups(P>0.05);Compared with the HI70 min group,the Hyp content in the liver tissue of the HI90 min group was significantly increased,with a significant difference(P<0.05).3.Morphological observation of liver tissue: HE staining showed that the hepatic lobule structure in the CON group was intact,the hepatocyte cords were arranged neatly,the liver cells were uniform in size,and there was no change in degeneration,necrosis,and fibrosis.In the HI70 min group,the structure of the hepatic lobule was destroyed,with focal necrosis and nucleus dissolution.Only a small amount of fibrosis around the sinuses was seen,but there was no obvious fibroblast proliferation.HI90 min group showed extensive lesions,multifocal hyalinization,collagenization in hepatic parenchyma,capillaryization of hepatic sinusoids,hyperplasia of fibrous tissue in portal area,inflammatory cell infiltration,and disappearance of local hepatic lobules.Conclusion Hepatic injury after 90 min reperfusion of hepatic insufficiency of liver blood was severe,collagen fiber deposition was obvious,liver fibrosis was more stable than the formation of ischemic 70 min,so chose liver ischemia.After 90 min reperfusion,hepatic fibrosis was induced in C57 mice.Objective Through an animal model of hepatic fibrosis induced by hepatic ischemia-reperfusion injury,the early mechanism of hepatic fibrosis formation and its effect on TGF-β-Smad pathway were explored.Methods 36 C57 male mice were divided into normal control group(CON,n=6)and(model group,n=30),and the model group was divided into HIR0 h group according to 0h,3h,6h,72 h and 8w after reperfusion.HIR3 h group,HIR6 h group,HIR72 h group and HIR8 w group,6 in each group.The model of hepatic ischemia-reperfusion injury in mice was treated with half hepatic occlusion.The left lateral lobe,left middle lobe and middle lobe of the mice were subjected to ischemia and ischemia for 90 min.The animals were kept and then sacrificed according to time points.ALT,AST content;Determination of liver tissue superoxide dismutase(SOD),Malondialdehyde(MDA)content;Fluorescent quantitative PCR detection of interleukin 6(IL-6)in liver tissue),interleukin 1b(IL-1b),tumor necrosis factor α(TNF-α),α-smooth muscle actin(α-SMA),The content of transforming growth factor-β(TGF-β),the expression of α-SMA and TGF-β1、smad3、smad7 in liver tissue were detected by Western blot,and the degree of hepatic fibrosis was observed by HE and MASSON staining.Results1.Serum levels of ALT,AST changes:Compared with the CON group,the levels of ALT and AST in HIR0 h,HIR3h,HIR6 h,HIR72h and HIR8 w groups increased significantly(P<0.05),especially in the HIR3 h and HIR6 h groups.However,with the prolongation of reperfusion time,serum ALT and AST levels decreased after 72 hours and 8 weeks,but were still higher than those of the control group(P<0.05).The difference was significant and statistically significant.2.Determination of liver SOD and MDA content: Compared with the CON group,the levels of SOD in the HIR0 h,HIR3h,HIR6 h,HIR72h and HIR8 w groups decreased to varying degrees(P<0.05),while the MDA content increased to varying degrees(P<0.05).The decrease of SOD Content and increase of MDA content were most obvious in the HIR8 w group.The difference is significant and statistically significant.3.Determination of liver tissue IL-6,IL-1b,TNF-α m RNA expression: RT-PCR results showed that compared with CON group,the expression of IL-6,IL-1b and TNF-α m RNA in liver tissue of HIR0 h,HIR3h,HIR6 h,HIR72h and HIR8 w groups increased to varying degrees,among which HIR0 h and The increase in HIR3 h group was significant compared with other groups.However,the expression of IL-6,IL-1b and TNF-α m RNA in liver tissue of HIR6 h group increased only slightly.The difference was statistically significant(P<0.05).4.Determination of liver tissue α-SMA、TGF-β1 m RNA expression: RT-PCR results showed that the expression levels of TGF-β1 and α-SMA m RNA in HIR0 h,HIR3h,HIR6 h,HIR72h and HIR8 w groups were increased in different degrees compared with CON group,and the HIR72 h group and HIR8 w group were increased.Obvious to other groups.However,the levels of TGF-β1 and α-SMA in the HIR6 h group were only slightly increased.The difference was statistically significant(P<0.05).5.Liver tissue α-SMA,TGF-β1 protein content: Western blot results showed that compared with the CON group,HIR0 h,HIR3h,HIR6 h,HIR72h and HIR8 w groups had increased levels of TGF-β1 and α-SMA protein expression in different degrees.Among them,HIR72 h group and HIR8 w group increased.Obvious to other groups.Howeve,the levels of TGF-β1 and α-SMA in the HIR6 h group were only slightly increased..6.Determination of protein levels of TGF-β1,Smad3 and Smad7 in liver tissue :Western blot results showed that compared with CON group,the expression of Smad3 and Smad7 protein in liver tissue of HIR0 h,HIR3h,HIR6 h and HIR72 h groups increased to varying degrees,and All of them increased in parallel,but the Smad3 protein expression was decreased while the expression of Smad3 protein was increased in the HIR8 w group.The expression of TGF-β1 protein increased only slightly at 0 h after hepatic ischemia-reperfusion,but the protein expression of Smad3 and Smad7 in liver tissue increased significantly in parallel.After reperfusion 3,6 and72 hours,TGF-β1 in liver tissue was increased.The expression of TGF-β1 protein increased,and the protein expression of Smad3 and Smad7 also increased in parallel.However,the expression of Smad3 and Smad7 protein decreased at 0 hours after reperfusion,which was in consistent with that of TGF-β1 protein.7.Liver histomorphology observation:HE staining showed that the hepatic lobule structure in the CON group was intact,the hepatocyte cords were arranged neatly,the liver cells were uniform in size,and there was no change in degeneration,necrosis and fibrosis.In the HIR0 h group,after 90 min ischemia,the hepatic hemorrhage was very obvious;in the HIR3 h group,the hemorrhage was reduced but the blood vessel wall was thickened;the blood vessel wall in the HIR6 h group was thicker and the cells appeared autolysis;the HIR72 h group showed a large area of infarction and congestion with a small amount of inflammatory cell infiltration and fibroblast proliferation;HIR8w group has structural disorder of hepatic lobules,disorganized hepatic cords,hepatocyte swelling,loose cytoplasm,balloon-like degeneration,focal hepatic necrosis,and moderate amount of portal area.Inflammation cell infiltration.MASSON staining showed that there was no obvious collagen deposition in the normal group,a small amount of collagen deposition in the HIR0 h group,HIR3 h group,and HIR6 h group;more collagen deposition occurred in the portal area and sinuses in the HIR72 h group;the portal area and sinus weeks appeared in the HIR8 w group.Different degrees of collagen deposition.Conclusion In the early stages of hepatic ischemia-reperfusion,hepatocytes are extensively damaged,the release of inflammatory factors is significantly increased,and the production of cytokines and free radicals is increased.These factors promote each other and cause and cause each other,prompting massive activation of hepatic stellate cells,thereby initiating early stages.The occurrence of liver fibrosis.These factors continue to play a role in the progression of liver fibrosis and promote the further development of liver fibrosis.Liver ischemia-reperfusion induced liver fibrosis has a certain relationship with the activation of TGF-β-Smad pathway.