Effects Of Human Umbilical Cord Mesenchymal Stem Cell-derived Exosomes On Autophagy Of CD4~+T Cells In Sj(?)gren’s Syndrome Mouse

Objective:Sj(?)gren’s syndrome(SS)is a kind of secretory CD4~+T lymphocyte highly immersion embellish of chronic systemic autoimmune disease,characterized by the excessive activation of CD4~+T cells play a key role in the pathogenesis of SS.Autophagy is to maintain CD4~+T cells,one of the important mechanisms of immune homeostasis and function.The SS treatment can improve symptoms,unable to repair damaged tissue,and the long-term use can lead to infection and malignant diseases such as deputy.Hence the need to find a safe and effective treatment strategy for SS.human umbilical cord-derived mesenchymal stem cells(h UCMSCs)adjustable SS the patient’s immune disorder,promote restoration of the glands,but sources,safety and ethical question limit its clinical application.h UCMSC-derived exosomes(h UCMSC-Exos)can simulate the h UCMSCs immune regulation and tissue repair effect,avoid its limitations.Our previous research has confirmed that h UCMSC-Exos inhibits SS in patients with peripheral blood CD4~+T cell autophagy level,and through the regulation of CD4~+T cell autophagy level and regulating the differentiation.Can then h UCMSC-Exos autophagy level regulation of CD4~+T cells in the body,give play to the role of the treatment.This research aims at h UCMSC-Exos in non-obese diabetic(Non-obese diabetic,NOD)of mice and the regulating NOD mice CD4~+T cell autophagy level issues for further discussion,in order to provide certain theoretical and experimental basis for clinical treatment of SS.Methods:(1)Primary h UCMSCs were cultured from fresh umbilical cord and subcultured to identify the surface phenotypehe and multilineage ability of h UCMSCs.Get h UCMSC-Exos ultracentrifugation,transmission electron microscopy to identify its shape,nanometer particle tracking analysis technology to analyze its particle size,protein imprinting method to identify the surface antigen phenotype.(2)Eighteen 8-week-old spontaneous Sj(?)gren’s syndrome animal model NOD female mice were randomly divided into three groups:PBS intervention group,h UCMSCs intervention group and h UCMSC-Exos intervention group.C57BL/6 mice served as normal control group.(3)After 4 weeks of intervention,the body weight and salivary flow rate of mice were recorded every week,and the submandibular glands of mice were stained with HE to observe the histopathological changes.(4)The expression of LC3Ⅱ,P62,Atg5 and Beclin1 in submandibular gland and spleen were detected by immunohistochemistry.(5)The co-localization of LC3Ⅱ,P62,Atg5,Beclin1 and CD4~+T cells in submandibular gland and spleen was detected by immunofluorescence.(6)CD4~+T cells from mouse spleen were isolated by immunomagnetic beads,and the expression levels of LC3Ⅱ,P62,Atg5 and Beclin1 in CD4~+T cells were detected by Western blot.Results:(1)Compared with the NOD mice in PBS group,the body weight of NOD mice in h UCMSC-Exos group was significantly increased(P<0.0001).The body weight of NOD mice in h UCMSC-Exos group was not significantly higher than that in h UCMSCs group.(2)Compared with C57BL/6 mice,the salivary flow rate of NOD mice treated with PBS decreased significantly(P<0.001).h UCMSC-Exos significantly increased salivary flow in NOD mice(P<0.001).(3)Compared with C57BL/6 mice,the submandibular gland of NOD mice treated with PBS showed more lymphocyte infiltration and more tissue damage.After intervention with h UCMSC-Exos in NOD mice,the degree of lymphocyte infiltration was reduced,the tissue injury was improved,and the pathological score of submandibular gland was reduced(P<0.05).(4)Compared with C57BL/6 mice,the expressions of LC3Ⅱ,Atg5 and Beclin1 in submandibular gland and spleen of NOD mice treated with PBS increased,while the expression of P62 decreased(P<0.001).The expression of LC3Ⅱ,Atg5 and Beclin1 was decreased and the expression of P62 was increased in NOD mice treated with h UCMSC-Exos(P<0.001).(5)Compared with C57BL/6 mice,the expression of LC3Ⅱ,Atg5 and Beclin1 on CD4~+T cells in submandibular gland and spleen of PBS-treated mice increased,while the expression of P62 decreased(P<0.001).The expression of LC3Ⅱ,Atg5 and Beclin1 on CD4~+T cells in submandibular gland and spleen of NOD mice was decreased and the expression of P62 was increased after the mice were pretreated with h UCMSC-Exos(P<0.01).(6)Compared with C57BL/6 mice,the expression level of LC3Ⅱ/Ⅰprotein was increased(P<0.001),the expression level of P62 protein was decreased(P<0.001),the expression level of Atg5 protein was increased(P<0.01),and the expression level of Beclin1 protein was increased(P<0.01)in spleen CD4~+T cells of NOD mice treated with PBS.h UCMSCs-Exos could decrease the expression of LC3Ⅱ/Ⅰprotein(P<0.01),increase the expression of P62 protein(P<0.05),decrease the expression of Atg5 protein(P<0.05)and decrease the expression of Beclin1 protein(P<0.01).Conclusions:(1)h UCMSC-Exos can increase the body weight and salivary flow rate of NOD mice,and reduce the infiltration of lymphocytes in submandibular gland tissue and improve tissue damage.(2)h UCMSC-Exos can inhibit the autophagy of CD4~+T cells in submandibular gland and spleen of NOD mice.