Effect Of M2 Macrophages On Immune Response After DCD Liver Transplantation

Background:Preexperimental confirmation,Macrophage phenotype change affects rejection after liver transplantation in donation after cardiac death(DCD)rats.M1 macrophages accelerate rejection and M2 macrophages alleviate rejection,but the specific mechanism is not clear.Objective:To investigate the mechanism of M2 macrophages attenuating immune response after liver transplantation in DCD rats,and to provide a theoretical basis for its transformation into clinical practice.Method:DCD rat liver transplantation models were divided into 4 groups:Group A:DA→-BN+PBS+WIT0(warm-ischemia-time Omin);Group B:DA→BN+PBS+WIT20;Group C(M2 macrophage group):DA→BN+PBS+WIT20+Adiponectin(M1 macrophage inhibitor)Adiponectin 500μg/kg Intraperitoneal injection,Days 0,1,3,5,and 7 after transplantation;Group D(M1 macrophage group):DA→BN+PBS+WIT20+oxidized ATP(oATP,P2X7 Antagonist,M2 macrophage inhibitor)oATP 0.25mg Intraperitoneal injection,Days 0,1,3,5,and 7 after transplantation.Draw materials:On day 1 and 7 after transplantation,three rats in each group were randomly selected to take venous blood,and liver tissue samples were collected after the rats were killed.The expression levels of CXCL13,TNF-α,TGF-β1,IgG and IgM in serum of rats in each group were compared by ELASA.qPCR was used to compare the mRNA expression levels of TNF-α and TGF-β1 in each group.Immunofluorescence staining was used to detect the surface markers of M1 and M2 macrophages,including inducible nitric oxide synthase(iNOS)and arginase I(Argl),costimulatory molecules CD86,CD40,MHCII,TLR-4,NF-κB signaling pathway and Treg.Results:1.The expression levels of CXCL13,TNF-α,TGF-β1,IgG and IgM in serum of rats in each group were compared by ELASA.The results show that in group WIT20+Adiponectin,the expressions of CXCL13,TNF-a,IgG and IgM were inhibited(P<0.05),while the expression of TGF-β1 was significantly enhanced(P<0.05).In group WIT20+oATP,the expressions of CXCL13,TNF-a,IgG and IgM were significantly increased(P<0.05),TGF-β1 expression was significantly inhibited(P<0.001).2.qPCR was used to compare the mRNA expression levels of TNF-α and TGF-β1 in each group.The results show that in group WIT20+Adiponectin the expression of TNF-αmRNA was significantly inhibited(P<0.05),and the expression of TGF-β1 mRNA was significantly enhanced(P<0.05).In group WIT20+oATP,the expression of TNF-αmRNA was higher than that of the other groups(P<0.05),and the expression of TGFβ1 mRNA was significantly inhibited compared with the other three groups(P<0.05).3.Immunofluorescence staining was used to detect the surface markers of M1 and M2 macrophages,including inducible nitric oxide synthase(iNOS)and arginase I(Argl),costimulatory molecules CD86,CD40,MHCII,TLR-4,NF-κB signaling pathway and Treg.The results show that the M2 macrophage surface marker Arg1 in group WIT20+Adiponectin and the M1 macrophage surface marker iNOS in group WIT20+oATP were significantly increased compared with other groups.In group WIT20+Adiponectin,the expressions of costimulatory molecules CD86,CD40,MHCII,TLR-4 and NF-κB were inhibited to a certain extent,and the production of Treg cells was effectively promoted.In group WIT20+oATP,the expressions of costimulatory molecules CD86,CD40,MHCII and TLR-4,NF-κB signaling pathway were enhanced to a certain extent,but the production of Treg cells was significantly decreased compared with other groups.Conclusion:M2 macrophages can reduce the immune response after liver transplantation in DCD rats.The mechanism may is related to M2 macrophages inhibiting the activation of TLR-4/NF-κB signaling pathway,downregulating the expression of cytokines CXCL13 TNF-α,IgG,IgM and co-stimulatory molecules CD86,CD40,MHCII,and up-regulating the expression of TGF-β1,inducing the production of Treg cells.