A Study On The Effect Of Toxoplasma Gondii ROP16 Protein On Polarization And Phenotype Of Macrophage Infection And Its Mechanism

ObjectiveTo investigate the expression of type I(RH strain),type II(ME49 strain)and type III(VEG strain)Toxoplasma gondii ROP16 protein in RAW 264.7 cells and MH-S cells,and the effects of infection on the polarization,cell proliferation,apoptosis and cell cycle of the two cells and their mechanisms of action.Methods1.Recombinant vector overexpressing ROP16Ⅰ/Ⅱ/Ⅲ was constructed using genetic engineering technology,and transfected RAW 264.7 cells and MH-S cells,and stable cell lines overexpressing ROP16 were constructed.2.cck-8 and flow cytometry were used to investigate the effects of ROP16 protein on proliferation,cycle,apoptosis and other cell phenotypes of RAW 264.7 cells and MH-S cells.3.The relative transcription levels of rop16,apoptosis-related factors,cell cycle related factors and cell polarization related factors were detected by RT-qPCR.4.The relative expression levels of ROP16 protein,apoptosis-related protein,cell cycle related protein,cell polarization related protein and signaling pathway related protein in each group were detected by Western-blot.5.Localization of ROP 16 in cells and subcellular colocalization of ROP 16 and pTyr705-STAT3 in cells were detected by immunofluorescence method.Results1.The recombinant vector overexpressing ROP16Ⅰ/Ⅱ/Ⅲ has been successfully constructed,and strong green fluorescence can be observed after transfection of RAW 264.7 and MH-S cells,and rop16 gene transcription and protein expression can be detected.The protein was mainly located in the nucleus and cytoplasm of RAW 264.7 and MH-S.2.cck-8 detection showed that cell proliferation was significantly promoted in the ROP16 overexpression group(all P<0.01).Flow cytometry showed that the apoptosis rate of ROP16 overexpression group was significantly lower than that of blank group and empty vector group(all P<0.01).Compared with blank group and empty vector group,the G0/G1 phase of ROP16 overexpression was significantly decreased,the G2 phase of ROP16 overexpression group(MH-S)was significantly increased,and the S phase of ROP 16 overexpression group was significantly increased(P<0.05 or P<0.01).4.RT-qPCR results showed that,compared with the blank group and the empty vector group,the inhibitory factor Bcl-2,Cyclin D1 and CDK6,the pro-apoptotic factors Bax,Caspase3 and Caspase9,were significantly increased in the ROP16 overexpression group.Cell cycle-related factors p21 and p53 were significantly decreased,and Bax/Bcl-2 was decreased.Polarization factors IL-4,IL-10 and TGF-β were significantly increased in ROP16Ⅰ/Ⅲ overexpression group,while polarization factors IL-1 0,IL-6,IL-12,IL-18 and TNF-α were significantly increased in ROP16Ⅱ overexpression group(P<0.05 or P<0.01).5.Western-blot results showed that,compared with the blank group and the empty vector group,the inhibitory protein Bcl-2,Cyclin D1 and CDK6,the pro-apoptotic protein Bax,Caspase3 and Caspase9,were significantly increased in the ROP16 overexpression group.Cell cycle-related proteinp21 and p53 weresignificantly decreased,and Bax/Bcl-2 was decreased.Signaling pathway proteins pTyr705-STAT3,pTyr641-STAT6 and polarizing protein Arg-1 were significantly increased in ROP16Ⅰ/Ⅲ overexpression group.Signaling pathway proteins pTyr701-STAT1,pNF-κB and polarizing protein iNOS were significantly increased in ROP16Ⅱ overexpression group(P<0.05 or P<0.01).5.Immunofluorescence results showed that ROP16 and pTyr705-STAT3 in the ROP16Ⅰ/Ⅲ overexpression group were co-localized in the nuclei and cytoplasm of the two cells.Conclusion1.ROP16 protein was successfully expressed in RAW 264.7 and MH-S cells,and was mainly localized in the nucleus and cytoplasm around the nucleus of RAW 264.7 and MH-S cells,indicating that the ROP16 stable cell line was successfully constructed.2.ROP16Ⅰ/Ⅱ/Ⅲ protein could promote the proliferation and inhibit the apoptosis of RAW 264.7 and MH-S cells,and induced cell cycle arrest in S/G2 in two cells.3.ROP16Ⅰ/Ⅲ proteins phosphorylated Tyr705-STAT3,Tyr641-STAT6 and shifted RAW 264.7 and MH-S cells toward M2-type polarization,and ROP16Ⅱ proteins phosphorylated Tyr701-STAT1,NF-κB and shifted RAW 264.7 and MH-S cells toward M1-type polarization.