| Introduction:Hepatic ischemia/reperfusion injury(IRI),which can cause irreversible damage in liver after transplantation and resection,is a challenging clinical problem.Ischemia-reperfusion injury(IRI)is a very common and intractable problem in clinical work,which has not been solved until now.Liver transplantation is the only effective method for the treatment of end-stage liver disease and liver malignant tumor,but due to the shortage of donors,a large number of patients die when they are in the waiting list.Although living donor liver transplantation has largely alleviated the shortage of donors,serious complications after donor liver transplantation have also been reported,so people are becoming more cautious about living donor liver transplantation.In recent years,people have been trying to use borderline donor livers,such as aging donor livers,fatty livers and cardiac death donor livers,to alleviate the shortage of donors.However,these marginal donor livers are more sensitive to ischemia reperfusion injury,which also leads to the increase of clinical cases of hepatic ischemia reperfusion injury.Hepatic IRI after liver transplantation and hepatectomy is the main reason for the increase of postoperative complications and mortality in patients.About 10% of early transplantation failures are caused by hepatic IRI,and IRI also increases the risk of liver dysfunction and organ rejection.Liver IRI caused by anhepatic ischemia and new hepatic reperfusion is the main cause of liver failure after partial liver resection and liver transplantation.However,up to now,there is no effective method to treat hepatic IRI.Therefore,the study on the pathological mechanism of liver IRI and the establishment of clinically effective and easy-to-use technical means are of great importance for the prevention and treatment of liver IRI.CCN1/ CYR61 is a member of the CCN protein family that regulates various cellular processes,such as cell adhesion,migration,differentiation,proliferation,and apoptosis,and is involved in angiogenesis,inflammation,and fibrous tissue repair by binding to different ligand glyconucleic acid.CCN1 has been identified as an early biomarker of myocardial and renal injury.According to the increase of CCN1 level in hepatic IRI,we speculated that CCN1 may be involved in the regulation of hepatic IRI.CCN1,which is involved in inflammatory response and apoptosis,is upregulated during hepatic ischemia/reperfusion injury,but its underlying mechanism remains unknown.Endoplasmic reticulum(ER)is an organelle responsible for protein synthesis,folding,transport and maturation,which is widely found in eukaryotic cells.By activating the unfolded protein response,ER can resist the cell damage caused by ER stress and restore cell function.It is reported that endoplasmic reticulum stress plays a crucial role in the pathogenesis of hepatic IRI.Intracellular CCN1 induces apoptosis of hepatic stellate cell(HSC)by activating the unfolded protein response triggered by ER stress.Therefore,we planned to establish a hepatic ischemia-reperfusion(IR)model in vivo and a hypoxia/reoxygenation(HR)model in vitro to explore whether CCN1 regulates apoptosis,inflammatory response or endoplasmic reticulum stress through the ERK pathway in liver IRI.Objective :(1)The role of CCN1 in hepatic ischemia reperfusion injury;(2)Whether CCN1 knockdown can play a protective role against hepatic ischemia reperfusion injury;(3)The role of CCN1 in ER stress during hepatic IRI,and whether it regulates the process of hepatic IRI through ER stress;(4)To provide further theoretical basis for hepatic ischemia/reperfusion injury;(5)To provide a new direction for the prevention and treatment of hepatic ischemia reperfusion injury in clinical practice.Method:(1)Ischemia/reperfusion model was established in adult male C57BL/6 mice by blocking the blood vessels in the left and middle lobes of the liver and then reperfusion;(2)Two small interfering RNAs(si RNAs)targeting CCN1 were used to knock down CCN1,and the liver cell function,inflammatory cytokine and apoptosis-related protein expression of the Sham group,IR group,IR+si-NC group,IR+si-CCN1-1 group and IR+si-CCN1-2 group were observed in vivo by histophology,immunofluorescence,Western blot,real-time quantitative PCR,TUNEL and other techniques.(3)Two small interfering RNAs(si RNAs)targeting CCN1 were used to knock down CCN1,and the AML-12 mice liver cells were used in vitro to establish anoxia/reoxygenation model,and the liver cell function,inflammatory cytokine and apoptosis-related protein expression of the Sham group,IR group,IR+si-NC group,IR+si-CCN1-1 group and IR+si-CCN1-2 group were observed in vivo by histophology,immunofluorescence,Western blot,real-time quantitative PCR,TUNEL and other techniques.(4)Means of different groups were compared using student's t-test or one-way analysis of variance with Graphpad Prism 7.p value less than 0.05 indicated significant differences.Results :(1)The expression of CCN1 was up-regulated during hepatic ischemia/reperfusion injury,and the severity of hepatic cell injury and apoptosis was decreased after CCN1 knockdown.(2)After knockdown of CCN1,the levels of pro-inflammatory factors such as TNF-?,IL-6,pro-apoptotic proteins caspase 9,caspase 3,Bax and CHOP decreased,while the expression of anti-apoptotic factor Bcl-2 increased.(3)Knocking down CCN1 can also inhibit the levels of proteins that activate ER stress and ERK pathway activation.(4)Vitro experiments showed that knockdown of CCN1 could inhibit inflammatory response,apoptosis and ER stress,while adding t PA could eliminate this protective effect without changing CCN1 level.Conclusion :(1)CCN1 knockdown has a protective effect on hepatocytes of hepatic IRI;(2)CCN1 knockdown can protect liver cells mainly by inhibiting ERK pathway activation;(3)CCN1 knockdown can inhibit the inflammatory response of hepatic IRI;(4)CCN1 knockdown is associated with the decreased of apoptosis in hepatic IRI by inhibiting ERK pathway. |
PDF Full Text Request