Study On The Function And Mechanism Of Echinococcus Multilocularis Metacestode Larval Stage Soluble Antigen Acting On Macrophages Through RhoA-MAPK Pathway

Objective: To explore the function and mechanism of E.m metacestode larval stage soluble antigen acting on macrophages through Rho A-MAPK pathway in the early stage of infection(8h).Methods: 1.HE staining was used to observe the pathological changes of liver tissue;2.Immunofluorescence double staining was used to detect the expression of CD11C/CD68(M1/Mφ)and CD206/CD68(M2/Mφ)in liver tissue;3.Immunofluorescence was used to detect the effect of macrophage phenotype transformation on HSC activation(α-SMA);4.RAW264.7 cells,primary mouse peritoneal macrophages and ANA-1 cells were treated with Y-27632(Rho A inhibitor),SP600125(JNK inhibitor),U0126-Et OH(ERK1/2inhibitor)and SB20358(p38 inhibitor)respectively,and the soluble antigen of E.m metacestode was added at an appropriate concentration.After treatment for an appropriate time,the antigen was detected;5.The m RNA levels of Rho A,ROCK1 and ROCK2 genes were detected by q PCR;6.The levels of IL-10,IL-12,Arg-1 and NOS-2 were detected by ELISA;7.The proportions of F4/80+CD16/32+M1 macrophages and F4/80+CD206+M2 macrophages were detected by flow cytometry;8.Western blot was used to detect the expression of Rho A,ERK and p-ERK.Results: 1.HE staining showed that inflammatory cells infiltrated in the liver tissue of fibrosis.2.Compared with the normal group,CD11C/CD68(M1/Mφ)and CD206/CD68(M2/Mφ)were more expressed in AE fibrosis liver tissue.3.immunofluorescence showed that the expression of α-SMA protein decreased when the macrophages were co-cultured with HSC.4.CCK8 assay showed that RAW264.7 cells treated with 20μg/m L E.m metacestode larval soluble antigen had the lowest survival rate after 8h.5.In RAW264.7 cells,E.m metacestode larval soluble antigen may decrease the expression of Rho A and inhibit the expression of ROCK1 and ROCK2 genes.U0126-Et OH and SB20385 can inhibit the expression of Rho A.Y-27632 and SB20385 can promote the expression of ROCK1 and ROCK2 m RNA;In ANA-1 cells,Y-27632,SP600125,U0126-Et OH and SB20385 could promote the expression of Rho A,Y-27632 and SB20385 could promote the gene expression of ROCK1 and ROCK2;In peritoneal macrophages,compared with the control group and E.m model group,Y-27632 and U0126-Et OH could promote the expression of Rho A and ROCK1,SP600125 could inhibit the gene expression of Rho A,ROCK1,while U0126-Et OH and SB20385 could significantly promote the gene expression of ROCK2.6.ELISA detection showed that compared with the control group,the contents of Arg-1,IL-10,IL-12 and NOS-2 in the E.m model group had no significant change.Compared with the control group,U0126-Et OH promoted the expression of IL-10 and inhibited the expression of IL-12 in RAW264.7 cells.In ANA-1 cells,compared with the control group,the content of IL-10 in SP600125,U0126-Et OH and SB20358 experimental groups decreased significantly,while the content of NOS-2 increased.Compared with the control group,the levels of IL-10,Arg-1 and IL-12 were up-regulated.7.Flow cytometry showed that compared with the control group,the proportion of F4/80+CD16/32+M1 macrophages in Y-27632 group was significantly decreased,while the proportion of F4/80+CD16/32+M1 macrophages and F4/80+CD206+M2 macrophages in SP600125,U0126-Et OH and SB20358 groups were up-regulated.In ANA-1 cells,compared with the control group,the proportion of M1 macrophages was down regulated and the proportion of M2 macrophages was up-regulated in Y-27632 group.The proportion of M1 macrophages and M2 macrophages was down regulated in SP600125,U0126-Et OH and SB20358 groups.Compared with the control group,the proportion of M1 macrophages was significantly up-regulated and the proportion of M2 macrophages was down regulated in the E.m model group.Compared with the model group,the proportion of M1 macrophages and M2 macrophages in Y-27632 group and SB20358 group were significantly decreased.8.WB results showed that the expression level of Rho A in E.m treated cells decreased as a whole and reached the highest level at 16h;the expression level of ERK in E.m treated cells increased as a whole and reached the highest level at 8h;the expression level of p-ERK in E.m treated cells increased as a whole and reached the highest level at 24 h.Conclusions: The soluble antigen of Echinococcus multilocularis can stably down-regulate the expression of Rho A and inhibit the expression of ROCK1 and ROCK2 genes through Rho A MAPK pathway.In the early stage of AE,macrophages were mainly polarized to M1,which could inhibit the activation of HSC in mice,and then reduce the liver fibrosis in the early stage of AE;in the late stage of AE,macrophages were mainly polarized to M2,which could promote the activation of HSC in mice,and then aggravate the liver fibrosis around the lesion in the late stage of AE.