| Background:Biliary ischemia-reperfusion injury determines whether biliary-related complications occur in patients after hepatobiliary surgery,especially after liver organ transplantation,biliary epithelial EMT of biliary epithelial plays an important role in the process of biliary ischemia-reperfusion injury.CTHRC1 is an extracellular matrix glycoprotein normally expressed at the epithelial-mesenchymal interface and is widely involved in growth and development,injury repair,organ fibrosis,tumor invasion and metastasis.Currently,the role of CTHRC1 on biliary epithelial cells during biliary IRI and the specific molecular mechanisms involved have not yet been elucidated.Objective:Using a rat biliary ischemia-reperfusion injury model,this study was designed to investigate the mechanism of CTHRC1 on biliary EMT during biliary ischemia-reperfusion injury by examining the expression of CTHRC1,EMT-related proteins and TGF-β1/Smad signaling pathway-related proteins in biliary tissues before and after transgenic induction of Cthrc1 overexpression.To provide new ideas to explore the mechanism of biliary ischemia-reperfusion injury and repair after hepatobiliary surgery.Methods1.A rat biliary ischemic injury model was constructed using Pringle’s method.The ischemic time was set at 30 min,and blood was collected from the inferior vena cava of rats after 24h,48h and 72h of reperfusion,and the blood was centrifuged for 15min after coagulation.2.The rats were divided into sham-operated group,control group and experimental group,the sham-operated group was only operated on and off the abdomen,the control group was constructed by Pringle’s method,the experimental group was introduced the self-fluorescent adeno-associated virus,AAV2/9-CMV-r-Cthrc1-3xflage-Zs Green,by hepatic portal vein injection to induce CTHRC1 overexpression.After 3 weeks,the effect of virus transfection and expression of the target gene were observed,and serum was collected and bile duct tissues were obtained at different reperfusion time loci after reconstitution of the rat biliary ischemic injury model using transgenic rats.3.The bile duct tissue specimens of rats were paraffin-embedded and stained with HE to observe the bile duct injury.IHC was used to detect the expression levels of CTHRC1,E-cadherin,TGF-β1,Smad2/3 proteins in the experimental and control groups,and the IHC pictures were processed,and the MOD values were used to represent the expression intensity of the proteins in the tissues,and Spearman correlation was used to analyze the relationship between CTHRC1,E-cadherin,TGF-β1,Smad2/3 between them.Results1.A multi-group comparison of serum markers in the control group revealed that serum ALP andγ-GT were significantly elevated at 24H(P<0.001),48H(P<0.001)and 72H(P<0.05)of reperfusion,with expression decreasing gradually with time.Serum ALT was significantly elevated at 24H of reperfusion(P<0.001)and expression continued to decrease with time.In the experimental group,serum ALP remained elevated at 24H(P<0.01)and gradually decreased with time of reperfusion.γ-GT and ALT were also elevated at 24H(P<0.001)and gradually decreased with time of reperfusion.The serum ALT,ALP andγ-GT indexes in the experimental group were lower than those in the control group at the same time points of reperfusion(P<0.05).2.The histopathological sections of the bile ducts of rats showed that compared with the bile ducts of the sham-operated group,the bile ducts of the control group had different degrees of epithelial cell damage or loss,disorganized bile duct epithelial cells,dilated bile ducts,thickened bile duct walls,infiltration of surrounding inflammatory cells,and partial loss of peribiliary glands due to heat-ischemia-reperfusion injury,while the bile ducts of rats were injected with AAV2/9-r-Cthrc1-3x via portal vein 3 weeks before heat-ischemia-reperfusion injury.Cthrc1-3xflag-Zs Green(200ul/each),we observed that the bile ducts of rats maintained essentially normal morphology at different reperfusion time points.3.The IHC results showed that The expression intensity of CTHRC1 was significantly higher in the bile ducts of rats with thermal ischemic injury than in normal rats,and the difference between the two was statistically significant(P<0.05).Spearman’s correlation analysis revealed a strong positive correlation between the expression intensity of TGFβ1 and Smad2/3 in the experimental group and the control group(r_s=0.8115,P<0.001),and a weak negative correlation between the expression intensity of CTHRC1 and the two(CTHRC1 and E-ca:r_s=-0.6204,P<0.001;CTHRC1 and E-ca:r_s=-0.6204,P<0.001;CTHRC1 and E-ca:r_s=-0.6204,P<0.001).0.001;CTHRC1 and TGF-β1:r_s=-0.7555,P<0.001).Conclusion1.The Pringle’s method for establishing the rat bile duct IRI model is highly reliable,and the damage caused by the rat bile duct tissue can be recovered within 30 min of ischemia.2.CTHRC1 expression was significantly up-regulated in rat biliary heat ischemia at different reperfusion time points and may play an important role in bile duct ischemia-reperfusion injury and repair process.3.Portal vein injection completed adeno-associated virus transgenic manipulation resulted in stable expression of CTHRC1 in experimental rats.4.CTHRC1 was involved in the bile duct EMT process and attenuated bile duct injury by inhibiting the TGF-β/Smad signaling pathway. |
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