Isolation And Identification Of Extracellular Vesicles From Trichinella Spiralis Muscle Larva And Study Of Immunomodulatory Function

Trichinella spiralis uses a variety of complex mechanisms to regulate the host’s immune response,creating an effective anti-inflammatory environment,ensuring its successful parasitization and long-term survival in the host.Extracellular vesicles(EVs)are tiny vesicle-like structures that can be produced and released by almost all types of cells.Studies have shown that various parasites can secrete extracellular vesicles and mediate the host’s immune response during parasite infection.The purpose of this project was to use 2,4,6-trinitro benzene sulfonic acid(TNBS)-induced experimental colitis model in mice to study the immunomodulatory effects of EVs secreted by Trichinella spiralis on the host,and to provide new ideas for the study of the immunomodulatory mechanism of Trichinellosis.Firstly,Trichinella spiralis muscle larvae extracellular vesicles(Ts-EVs)were isolated,and the morphology,size and specific markers of isolated Ts-EVs were identified by transmission electron microscopy(TEM),Western Blot and nanoparticle tracking analysis(NTA).TEM identification showed bilayer vesicle-like structure;Western Blot identification showed that Ts-EVs could specifically react with the specific markers of EVs:CD63 and enolase;Further NTA identification showed that the main peak of particle size was 133nm and the particle size was mainly distributed between 50-250 nm.The results showed that the Trichinella spiralis muscle larvae extracellular vesicles were successfully obtained,and the isolated EVs can be used for further functional identification.Secondly,this study analyzed the intervention effects of Ts-EVs on TNBS-induced experimental colitis in mice.The immune process of Ts-EVs was as follows:the obtained EVs were injected intraperitoneally with Balb/c mice,and then administered rectally to establish TNBS-induced experimental colitis model in mice.The mice were sacrificed on the 3 day of modeling,and the intervention effects of EVs on colitis were analyzed by DAI,macroscopic score and histopathologicl score.Macroscopically,based on indicators such as body weight,DAI score,colon length,colonic hemorrhage and congestion,it was found that Ts-EVs can alleviate clinical symptoms;Further based on microanalysis of HE,PAS staining and MPO activity,Ts-EVs reduced colon histopathological damage such as the infiltration of inflammatory cells were reduced,the number of goblet cells were increased,and MPO activity was reduced.To investigate the effect of Ts-EVs on the intestinal barrier of TNBS-induced colitis mice,the expression of tight junction proteins Occludin and ZO1 in colon tissue was detected by immunofluorescence.The results showed that the expression levels of Occludin and ZO1 in colon tissue of mice in TNBS+Ts-EVs group were significantly higher than TNBS group,suggesting that Ts-EVs could protect TNBS-induced colitis by preventing the destruction of the intestinal barrier.Based on the above experimental results,Ts-EVs can interfere with TNBS-induced experimental colitis in mice.Finally,in this study,local intestinal inflammatory factors and transcription factors were detected by MSD electrochemiluminescence technology and qRT-PCR.The effects of Ts-EVs on T cell differentiation in colitis models were further analyzed by flow cytometry.MSD results showed that TNBS induced the release of all inflammatory cytokines,and preventive treatment with Ts-EVs significantly reduced the expression levels of proinflammatory cytokines IL-1β,IFN-γ,TNF-αand IL-17A,and promoted the expression levels of anti-inflammatory cytokines IL-10,TGF-β,IL-4 and IL-13.In addition,compared with the TNBS group,preventive treatment with Ts-EVs significantly reduced the level of the chemokine MIP-3α.qRT-PCR results showed that compared with the TNBS group,preventive treatment with Ts-EVs significantly inhibited Th1(IFN-γ,T-bet)and Th17(IL-17A,RORγt),but promoted Th2(IL-4,GATA3)and Treg(IL-10,Foxp3)mRNA expression of inflammatory factors and related transcription factors.The above results indicated that Ts-EVs can effectively interfere with the occurrence and development of IBD by regulating the expression of inflammatory factors and transcription factors.Further flow cytometry results showed that compared with the TNBS group,preventive treatment with Ts-EVs significantly reduced Th1(CD4~+IFN-γ~+)and Th17 cells(CD4~+IL-17~+),in which Th1cells were more inhibited,while Th2 cells(CD4~+IL-4~+and IL-13~+)were significantly increased.Moreover,preventive treatment with Ts-EVs promoted a small amount of proliferation of Treg cells.The above results indicated that the ability of Ts-EVs to improve colitis was related to the inhibition of Th1 cell proliferation and the regulation of Th17/Treg cell balance in mesenteric lymph nodes.Based on the above experimental results,Ts-EVs have immunomodulatory effects,and can alleviate TNBS-induced experimental colitis in mice,providing experimental basis and theoretical basis for interaction between Trichinella spiralis and host immune system.