Heme oxygenase-1 alleviates ischemia-reperfusion injury by inhibiting hepatocyte pyroptosis after liver transplantation in rats

Abstract Background:Heme oxygenase-1 (HO-1) is recognized as a key cytoprotective mechanism for inflammation during ischemia-reperfusion injury (IRI). Accumulating evidence suggests that pyroptosis plays an important role in inflammation of IRI. However, the mechanism between HO-1 and pyroptosis in IRI requires further investigation.Methods:The "two-cuff" method was performed to establish a model of liver transplantation (LT) from donors after circulatory death. The HO-1 overexpression and shRNA recombinant adeno-associated virus (AAV) were constructed and transfected into rats. Flow cytometry was used to detect the expression of ROS in cells to evaluate the degree of oxidative stress. An automatic biochemical analyzer was performed to detect serum ALT and AST levels and evaluate liver function. Paraffin sections were stained with HE to observe the degree of pathological damage to the tissues. ELISA was applied to detect the levels of IL-1β and IL-18 in the liver tissues. The tissues were analyzed for the expression of HO-1, pro-caspase-1, p22, full-GSDMD, and cleaved-N-GSDMD by western blot. Immunohistochemistry was used to detect the expression of NLRP3. Results.The model was successfully constructed, and the difference in survival time obtained was statistically significant after HO-1 overexpression and shRNA recombinant AAV transfected into the donor. The expression of HO-1 was time-dependent with IRI. HE and Suzuki score showed that the short-term groups' tissue necrosis was severer than the control. ROS, ALT, and AST expression in the short-term after IRI was significantly higher than control. The expression of pro-caspase-1, p22, full-GSDMD, and cleaved-N-GSDMD trend was consistent with HO-1, increased significantly in short-term while decreasing in long-term. The expression level of NLRP3 was the lowest in the recent six hours. At six hours after IRI, compared with HO-1-shRNA groups, the ROS, IL-1β, IL-18, ALT, and AST in the HO-1 overexpression were significantly reduced, as well as p22 and cleaved-N-GSDMD, were significantly inhibited. Immunohistochemical revealed that the expression of NLRP3 had a higher level in HO-1 overexpression groups.Conclusion.HO-1 improved the survival rate of rats after LT. And HO-1 promoted the recovery of IRI after LT. This study demonstrated that HO-1 could inhibit hepatocyte pyroptosis, thereby reducing IRI after LT.