Experimental Study Of PD-1/PD-L1 Inhibitors Accelerating Liver Regeneration By Regulating Macrophage Polarization

Objective1.To investigate the effect of resection size on residual liver regeneration and macrophage polarization after partial hepatectomy in rats.2.To study the effect and mechanism of blocking PD-1/PD-L1 interaction on macrophage polarization and hepatocyte proliferation.3.To verify the regulatory effect of blocking PD-1/PD-L1 interaction on hepatic macrophage polarization and residual liver regeneration after extended partial hepatectomy in rats.Methods1.The liver segment resection model of rats was constructed by using accurate liver lobe resection.Liver regeneration in rats was calculated with the expressions of ki-67 and PCNA by WB and IHC.The expression of macrophage markers CD68,iNOS and CD 163 in liver tissue after hepatectomy in rats was analysised by IHC and IF,the expression of macrophage secretion factors IL-6 and PD-L1 in serum after hepatectomy in rats was analysised by ELISA.2.Macrophages were cultured and induced into different phenotypes and treated by PD-1/PD-L1 inhibitors.The expression of macrophage marker CD68,iNOS and CD163 in was analysised by WB,and the content of PD-L1 and IL-6 in macrophage supernatant was analysised by ELISA.A co-culture system of macrophages and hepatocytes was constructed.The effect of macrophages on the proliferation activity of hepatocytes was detected by CCK-8,and the effect of macrophages on the expression of protein in the proliferation signaling pathway of hepatocytes was detected by WB.3.A rat model of extended hepatectomy and PD-1/PD-L1 inhibitor BMS-1 treatment was implemented.The expression of macrophage marker in liver tissue was analysised by IHC,and liver regeneration was evaluated by calculating response liver regeneration indicators.Results1.The liver regeneration response was inhibited after extended partial hepatectomy in rats,the LBW decreased POD1 and POD3,the KGR peak was delayed.The expression of M2 macrophage marker in liver tissue was increased,the level of IL-6 expression in serum was decreased,and the level of PD-L1 expression in liver tissue and blood was increased.All difference was statistically significant(P<0.05).2.The concentration of IL-6 in the supernatant of M1 macrophage culture medium was significantly increased,and the concentration of PD-L1 in the supernatant of M2 macrophage culture medium was significantly increased.PD-1/PD-L1 inhibitors could significantly increase the expression of iNOS and decrease the expression of CD 163 in M2 macrophage protein,and increase the content of IL-6 and decrease the content of PD-L1 in the supernatant of cell culture medium.All difference was statistically significant(P<0.05).CCK-8 and WB indicate that macrophage polarization to M1 type can promote hepatocyte proliferation,macrophage polarization to M2 type can inhibit hepatocyte proliferation,and PD-1/PD-L1 inhibitors can induce macrophage polarization conversion to M1 type and promote hepatocyte proliferation.WB indicate that macrophages activate IL-6-STAT3/ERK/AKT signaling pathways in the process of promoting hepatocyte proliferation.3.After treatment with PD-1/PD-L1 inhibitor BMS-1,the expression of M1 phenotype macrophage marker in liver tissue was increased and liver regeneration was significantly improved after extended partial hepatectomy.Conclusions1.After partial resection of expanded liver,residual liver regeneration was inhibited and macrophage M2 polarization increased.2.PD-1/PD-L1 inhibitors regulates macrophage M2 to M1 transformation and promote hepatocyte proliferation by activating the IL-6-STAT3/ERK/AKT signaling pathway.3.PD-1/PD-L1 inhibitors can increase the polarization of M1 type macrophages and promote liver regeneration after extended partial hepatectomy.