| Objective:1.To analyze the correlation between the expression level of neutrophil extracellular traps and the severity of liver ischemia-reperfusion injury after liver transplantation.2.To observe whether r TM to reduce hepatocyte apoptosis,protect liver function,and alleviate liver ischemia-reperfusion injury by inhibiting the NETs formation.3.To investigate the regulatory effect of r TM on neutrophil TLR-4 and its downstream related signal pathways,and the underlying mechanism of anti-inflammation and anti-apoptosis in alleviating the pathological process of liver ischemia reperfusion injury.Methods:1.Establish a rat liver transplantation model and isolate neutrophils from liver transplant recipients and normal individuals.Immunofluorescence and ELISA were used to detect the expression of NETs in serum of rats and recipients after liver transplantation.2.HE staining to assess liver damage before and after r TM intervention,microplate method to monitor liver function changes in rats with or without r TM intervention,RT-PCR and Evans blue staining was used to evaluate the degree of liver inflammation and hepatic vascular permeability with or without r TM intervention.Ki67 immunohistochemical staining,TUNEL staining and Western blot were used to detect the expression of apoptosis-related proteins and evaluate the proliferation and apoptosis of hepatocyte in rats model with r TM intervention.3.The TLR-4 and its downstream related pathways were further verified by Western blot in liver transplantation models.Finally,HE staining,TUNEL staining,Evans blue staining and ELISA were used to explore whether r TM and DPI combination therapy have a synergistic effect on alleviating liver IRI in liver transplantation.Results:1.With the extension of time,the liver tissue damage in rats gradually aggravated.The rat serum level of AST,ALT,and TBIL in the I/R group gradually increased compared with the control group.The results of immunofluorescence suggested that the tendency of neutrophils in the I/R group to undergo NETosis was increased,and the generation of NETs increased.ELISA results showed that the expression of DNA/NETs and H3 cit were significantly elevated in serum of rats and expression of DNA/NETs and NE were significantly elevated in serum of liver transplant recipients.2.A massive number of neutrophils were activated and recruited to the liver of rats after liver transplantation.The data showed that liver IRI following liver transplantation was accompanied by obvious inflammation and damage to liver vascular endothelial cells,and increased liver vascular permeability.Immunofluorescence co-localization of NETs showed that the NETs formation in liver transplantation patients was significantly increased compared with that of control groups.3.In vivo and in vitro experiments have confirmed that r TM inhibited NETs formation,??promote rat hepatocyte proliferation,inhibit hepatocyte apoptosis,protect liver function and allievate liver IRI.4.Immunofluorescence and Western blot results suggested that r TM reduced the formation of NETs by inhibiting the activation of TLR-4/MAPK signaling pathway and NADPH-ROS-PAD4 signaling pathway.TLR-4 receptor agonists(LPS)can partially reverse the inhibitory effect of r TM on the formation of NETs,??as well as anti-inflammatory and alleviating liver IRI.The combined treatment of r TM and DPI showed synergistic effect on alleviate liver IRI after liver transplantation.Conclusion:1.Increased NETs formation and/or decreased NETs clearance in rats after liver transplantation is a risk factor for liver IRI after liver transplantation,and may be an effective target for intervention in liver IRI.2.r TM regulates the phosphorylation and activation levels of TLR-4/MAPK and NADPH/ROS/PAD4 signaling pathways,thereby reducing the NETs formation in rats after liver transplantation and alleviating liver inflammation.3.The combined therapy of r TM and DPI showed synergistic effect on alleviating liver IRI.Regulation of multiple targets and pathways is a more effective strategy for hepatic IRI therapy. |
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