Lentivirus-mediated ToxoROP16_?/? In Protection Of CACO-2 Cells Via Inhibition Of M1 Inflammatory Macrophages Experimental Researc

Background: Inflammatory bowel disease is a non-specific chronic intestinal disease of unknown etiology and pathogenesis,including ulcerative colitis and Crohn's disease.In patients with inflammatory bowel disease,CD patients mainly Th1 type immune response,while in UC patients.In the treatment of IBD,traditional treatment drugs due to long-term use,patients have more adverse reactions,higher recurrence rate,and the efficacy of severe patients.In the early stage,the research group induced the Th2 type immune response through the soluble antigen of Schistosoma japonicum eggs reduced the experimental colitis was mainly induced by TNBS-induced Th1 type inflammatory reaction in mice.The inflammatory reaction was down-regulated and the inflammation was improved to achieve certain protection.This experiment is based on the previous research,through the study of Toxoplasma related virulence effector molecule ROP16_?/? by activating the alternatively activated macrophages to M2 cells,LPS activates the classical pathway of macrophages to M1 inflammatory cells and Caco-2cells establish an in vitro intestinal IBD inflammation model,and M2 cells down-regulate the inflammation of M1 cells and protection Caco-2 cells.The aim to provide new therapeutic strategies for immunotherapy of IBD.Objective: Toxo ROP16_?/? induced RAW264.7?M??to polarize M2 cells to produce Th2 immune response,LPS induced RAW264.7 cells to polarize M1 cells to produce the predominant of Th1 immune response.Then Th2 reverses the pathological Th1 immune response,down-regulates the inflammatory response,and exerts a protective effect on Caco-2 cells.Methods: M? was divided into five groups,normal control group,LPS-stimulated inflammatory model group,empty lentivirus group,lentiviral stable cells group and M1 ixed M2 culture group.All co-culture with Caco-2 cells in Transwell to estable IBD inflammation model in vitro.The classical pathway of LPS activation induces M?polarize M1 inflammatory macrophages,inducing a Th1-type immune response.Amplification of the ROP16_?/? gene fragment of Toxoplasma gondii,construction of recombinant p EGFP-ROP16_?/? lentiviral plasmid,recombinant lentivirus expressing ROP16_?/?,transfected of M?,inducd M? to M2 macrophages by alternatively activated macrophages.Fluorescence microscopy was used to observe the expression of fluorescent protein in stably transfected cells.The expression of TNF-a,IL-1?,IL-6,TGF-?1,IL-10,i NOS and Arg-1 was detected by q RT-PCR.Western blotting was used to detectd the protein expression of i NOS,Arg-1,ROP16_?/?,Stat3,p-Stat3,Stat6,p-Stat6,PD-L2,caspase3,caspase8 and caspase9.The content of NO was measured in the cell supernatant by Griess.The levels of TNF-a,IL-1?,IL-6,TGF-?1 and IL-10 in the supernatant of the cells were determined by ELISA.Flow cytometry was used to detect apoptosis of Caco-2 cells.Results: The ROP16_?/? recombinant lentivirus was constructed and successfully transformed into M?.The expression of green fluorescent protein was observed by fluorescence microscopy.Western blotting assay showed that the protein expression of i NOS was increased in LPS stimulated inflammatory model group,indicating that M?polarize to M1 cells,and the expression of apoptosis proteins caspase3,caspase8 and caspase9 increased with Caco-2 cells in Transwell co-culture.The differences were statistically significant.Western blotting was used to detect the expression of Arg-1,Stat3,p-Stat3,Stat6,p-Stat6 and PD-L2 in lentivirus-transfected cells,were increased compared with the lentivirus group,indicating M? polarize to M2 cells,mixed with M1,the Arg-1 and PD-L2 protein expression were stabled,the i NOS expression was decreased,mixed culture group and Caco-2 cells were co-cultured,the apoptosis proteins were decreased compared with LPS stimulation model group.ELISA and q RT-PCR showed that the secretion of M1 inflammatory cytokines TNF-a,IL-1? and IL-6 increased,and the relative m RAN expression of i NOS also increased.Lentivirus the levels of M2 cells cytokines TGF-?1 and IL-10 and Arg-1 relative m RNA expression were increased in the stable transfected cells.The secretion of inflammatory cytokines TNF-a,IL-1?,IL-6 and the decrease of i NOS relative to m RAN expression ere decreased in M1 mixed M2 culture group.Flow cytometry was used to detect LPS-stimulated inflammatory model group and Caco-2 cells apoptosis increased in Transwell co-culture,and apoptosis decreased in M1 mixed M2 culture group and co-culture with Caco-2 cells.Conclusions: Toxo ROP16_?/?induced macrophages with M2 phenotype inhibited the apoptosis of Caco-2 cells caused by LPS stimulated macrophages.The findings may be helpful for better understanding of the mechanism and be promising strategy for the novel immunotherapy of the IBD.