HO-1 Reduces Ischemia Reperfusion Injury In Rat DCD Liver Transplantation By Inhibiting P38 MAPK Activation

Objective:Ischemia Reperfusion Injury(IRI)often occurs in liver transplantation,liver resection,shock,etc.,especially for liver transplantation,the blood flow has to be blocked during the operation until the blood vessel reconstruction is completed,which leads to IRI.Since IRI in liver transplantation is inevitable and predictable,how to reduce IRI to improve the quality of the donor liver is currently the primary strategy to improve the prognosis and quality of life of patients after liver transplantation.The previous research of our group uncovered that Heme Oxygenase-1(HO-1)plays an important role in anti-inflammatory,antioxidant and anti-stress during the Hepatic Ischemia Reperfusion Injury(HIRI).This study intends to use Adeno Associated Virus(AAV)as a vector to construct an overexpression vector of rat HO-1,and to interferes HO-1 mRNA by shRNA AAV vector,and then construct a rat liver transplantation IRI model.Under the premise of inducing the differential expression of the donor liver HO-1,observe the liver transplantation IRI through the local inflammation and histopathological changes of the transplanted liver,and preliminary exploration of its internal molecular mechanism in order to provide a new idea and method to improve the IRI of liver transplantation clinically.Methods:1 Construct the rat HO-1 AAV vector and HO-1 shRNA AAV vector,and transfer them into SD rats,respectively.Harvest the liver 21 days later,observe the co-expressed green fluorescent protein under a fluorescence microscope,and detect the mRNA expression of HO-1 in liver tissue by qRT-PCR,verify the transfection effect.2 The IRI model of orthotopic liver transplantation in SD-SD rats was constructed on the basis of warm ischemia for 10 min and cold ischemia for 4 hr.The"two-cuff method" was used to anastomose the portal vein and the inferior hepatic vena cava."Stent method" reconstructs the common bile duct.6 hr,24 hr,3 d,and 7 d after reperfusion,wo test the liver function,liver tissue morphology,and changes in HO-1 and inflammatory factors over time,respectively,and the time point of the most obvious change was clarified for followup research.3 Inject the AAV vector into the donor SD rats through the tail vein to induce high and low expression of HO-1 in the donor liver,and construct SD-SD rat orthotopic liver transplantation IRI model on the 21st day after injection.Harvest serum to detect ALT and AST changes,liver tissue for HE staining to observe histopathological changes,detect mRNA expression of HO-1,CHOP,and inflammatory factors TNF-α,IL-1β,and IL-6 in liver tissue,and detect the protein expression of HO-1,p-p3 8 MAPK,p38 MAPK,CHOP and inflammatory factors TNF-α,IL-1β,IL-6 in liver tissue.Results:1 Construct the HO-1 AAV overexpression vector and the HO-1 shRNA AAV vector.The DNA-seq confirmed that the amplified sequence was correct,indicating that the vector was successfully constructed.2 AAV vector can induce the increase or decrease of HO-1 transcriptional activity in SD rat liver by tail vein injection,which is statistically significant from the corresponding empty AAV virus group(P<0.05).3 Successfully constructed SD-SD rat liver transplantation IRI model.4 After liver transplantation ischemia reperfusion,the activity of ALT and AST increased rapidly,and reached a peak at 6-24 hr,then fell to almost normal after 7 days;liver tissue pathological damage has been manifested at 6 hr,and it was most damaged at 24 hr.The most severe,necrotic foci were still visible on the 7th day.It shows that the IRI of liver transplantation occurs rapidly after reperfusion,and the pathological changes lag behind the changes of molecular markers.5 Through preoperative induction of HO-1 in donor liver with AAV,the liver transplantation model can still be efficiently induced or inhibited for high expression of HO-1 6 hr after the establishment of the liver transplantation model.The transcriptional activity and protein of HO-1 in the overexpression group and shRNA interference group were higher or lower than the corresponding no-load AAV group,and the difference was statistically significant(P<0.05).6 At 6 hr after liver transplantation IRI,ALT and AST in the sh-HO-1 group were significantly higher than those in the AAV empty virus group(P<0.05),the ALT and AST in the AAV-HO-1 group were significantly lower than which in the AAV empty virus group(P<0.05).HE staining of liver tissue sections was performed.The necrotic area of sh-HO-1 group was larger and the inflammatory cell infiltration was more obvious under light microscope(P<0.05).Detect the transcriptional activity and protein expression of local inflammatory factors TNF-α,IL-1β and IL-6 in the liver.The increase in sh-HO-1 group and the decrease in AAV-HO-1 group are compared with the corresponding no-load AAV group is statistically significant(P<0.05).At the same time,the increase of HO-1 is accompanied by the decrease of the phosphorylation of p38 MAPK and CHOP expression,suggesting that HO-1 may reduce the activation of p38 MAPK mediated inflammatory signaling pathway and reduce CHOP mediated endoplasmic reticulum stress to realise.Conclusions:1 Successfully construct HO-1 overexpression and HO-1 shRNA AAV vector,which can be transfected to induce or inhibit HO-1 expression in rat liver.2 Successfully establish SD-SD rat orthotopic liver transplantation IRI model.3 Inducing high expression of HO-1 in the donor liver before surgery can reduce the IRI of liver transplantation,while inhibiting HO-1 in the donor liver will aggravate the injury.4 HO-1 may reduce IRI by inhibiting the activation of p38 MAPK mediated inflammation signaling pathway and reducing CHOP mediated endoplasmic reticulum stress.