| Objective:Extracellular vehicles(EVs)are able to selectively enrich specific proteins,and effectively avoid the shortcomings of short storage cycle of living bacteria and infection to immunodeficiency people,showing great potential in disease treatment.In addition,EVs are also good drug carriers.At present,the effectiveness of probiotics EVs in the treatment of diseases has been reported,but the research on the mechanism is very scarce.This paper explores the preparation and characterization of C.butyricum EVs and its alleviation effect on ulcerative colitis in mice.Methods:(1)Isolation and preparation of C.butyricum EVs.C.butyricum was anaerobically cultured,and C.butyricum EVs were isolated by ultracentrifugation.The morphology was analyzed by transmission electron microscopy,the particle size was analyzed by NTA,and the protein distribution was analyzed by SDS-PAGE gel electrophoresis.(2)To explore the mi.tigation effect of C.butyricum EVs on mice with ulcerative colitis.After the mouse model of ulcerative colitis induced by Oxazalone(OXA)was established,C.butyricum(28 mg/(g·day))and its EVs(1011 particles/day)were administered.The changes of body weight and colon morphology in mice were observed,and the disease activity index(DAI)was calculated.The content of inflammatory factors in serum was detected by ELISA,and the SOD activity and MDA level in serum were detected.The changes of colon physical barrier were analyzed by HE staining,PAS staining and immunohistochemistry.The changes of CD11c and LC3b in colon tissue lamina propria were observed by immunofluorescence(IF)technology,and the differentiation of initial T cells in spleen was analyzed by flow cytometry(FCM).(3)Preliminary study on the molecular mechanism of C.butyricum EVs in alleviating intestinal inflammation.After the isolated mouse dendritic cells(DCs)were treated with EVs at a certain concentration(50μg/mL),the expression of antigen presenting related molecule MHC II and costimulatory molecules CD80 and CD86 in DCs was analyzed by FCM.In addition,the co-culture system of DCs and initial T cells was established,and the activated DCs were analyzed by FCM.Results:(1)Cbutyricum EVs were successfully prepared by ultracentrifugation.C.butyricum EVs showed a typical double-membrane vesicle structure,with a particle size distribution of 100-300 nm.The protein size was concentrated in the 135 kDa attachment,and a small part was concentrated in 100-135 kDa and 48-63 kDa,with a total protein content of 562 mg/mL.(2)C.butyricum EVs can improve OXA-induced ulcerative colitis.The study found that after EVs gavage,the weight loss of ulcerative colitis mice was reduced,the degree of colon swelling was weakened,the disease activity index was decreased,the expression of serum inflammatory factors(IL-4,IFNγ)was decreased,the activity of SOD was increased and the content of MDA was decreased.It can be seen from the tissue diagram that EVs can also protect the integrity of colonic mucosa,prevent the destruction of crypts,maintain the number and morphology of goblet cells,and improve the infiltration of immune cells;(3)C.butyricum EVs participate in immune response.Through immunofluorescence labeling,it was found that the number of DCs in the colon tissue lamina propria decreased after EVs intragastric administration in mice with ulcerative colitis,and EVs had little effect on the autophagy of DCs.In addition,OXA modeling could stimulate the differentiation of initial T cells in mice and induce Th2-mediated enteritis,while EVs treatment could significantly reduce the differentiation of Th2;(4)EVs can inhibit the antigen presentation ability of DCs.The results of flow cytometry showed that the expression levels of MHC Ⅱ,CD80 and CD86 in DCs did not change significantly after EVs stimulated DCs.However,EVs and LPS jointly stimulate DCs,and EVs can significantly inhibit the stimulation of LPS on DCs.Further co-culture of EVs-stimulated DCs with initial T cells showed little effect on the differentiation of initial T cells.However,co-culture of DCs stimulated by EVs and LPS with initial T cells can significantly limit the differentiation of initial T cells into Th2 cells.Conclusion:(1)C.butyricum EVs have a typical double-membrane structure with a particle size of 100-300 nm;(2)C.butyricum EVs can improve the integrity of intestinal mucosal barrier,reduce the level of inflammatory factors,reduce the infiltration of lymphocytes such as CD4+T,and alleviate the intestinal inflammation caused by OXA;(3)C.butyricum EVs can regulate the number of DCs in intestinal lamina propria and their antigen presentation ability,inhibit the stimulation of other antigens on dendritic cells,weaken the antigen presentation ability of dendritic cells,and limit the ability of initial T cells to differentiate into Th2 cells. |
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