Gingival Mesenchymal Stem Cell-Derived Exosomes Ameliorate Type 1 Diabetes By Inhibiting TLR4-MyD88-NF-κB Pathway

Objective:Type 1 diabetes(T1D)is a hyperglycemic condition caused by the lack of insulin in the patient’s body to regulate blood glucose.Its pathogenesis is mainly due to the irreversible destruction of pancreaticβ-cells caused by the immune system attack,which leads to insufficient insulin secretion.Its pathogenesis is closely related to CD4~+T cell subsets,mainly involving Th1,Th17 and Treg cells.Studies have shown that exosomes secreted by gingival mesenchymal stem cells have immunomodulatory effects,and IL-1βcan promote the secretion of exosomes.Therefore,this study set up the control group,GMSC group,GMSC-GW4869 group,GMSC-EXO group and IL-1β-GMSC-EXO group,with the aim of investigating whether human gingival mesenchymal stem cell-derived exosomes(GMSC-EXO)have an ameliorative effect on T1D and exploring its potential mechanism of action.Methods:In this study,the experiments were divided into two parts,in vivo and in vitro experiments.In vivo experiments:Spontaneous non-obese diabetic NOD/Lt J mice were used as the study subjects for in vivo experiments.The mice were randomly divided into PBS group,GMSC group,GMSC-GW4869 group,GMSC-EXO group and IL-1β-GMSC-EXO group.The mice were injected with PBS,GMSC or GMSC-EXO in the tail vein at week8,and then body weight and blood glucose were measured twice a week.When the mice were in the late stage of the disease(about 32 weeks),they were executed.The expression of inflammation-related proteins(P-TLR4,My D88,TRAF6 and p65)in the pancreas and kidney was detected by Western Blot;HE staining to observe the extent of pancreatic islet and renal lesions;the ratio of CD4~+T cell subsets(Th1,Th17,CD4~+IL-10~+and Treg)in splenocytes was detected by flow cytometry;the level of inflammation-associated cytokines(IFN-γ,IL-17A and IL-10)in serum was detected by ELISA.In vitro experiments:CD4~+T cells were co-cultured with GMSC,GMSC-GW4869,GMSC-EXO,and IL-1β-GMSC-EXO for 3 days.Then the ratio of CD4~+T cell subpopulations(Th1,Th17,CD4~+IL-10~+and Treg)was detected by flow cytometry,and the level of cytokines(IFN-γ,IL-17A and IL-10)in culture supernatants was detected by ELISA;The expression of TLR4-My D88-NF-κB signaling pathway-related proteins(P-TLR4,My D88,TRAF6 and p65)was detected by Western Blot.Results:In vivo experimental data showed that compared with the PBS group,GMSC,GMSC-EXO and IL-1β-GMSC-EXO treatment all had obvious improvement effects on T1D,the infiltration of pancreaticβcells was significantly reduced,and the insulitis was significantly alleviated;the expression of TLR4-My D88-NF-κB signaling pathway proteins(P-TLR4,My D88,TRAF6,and p65)in pancreas and kidney was all down-regulated;the ratio of pro-inflammatory immune cells(Th1,Th17)decreased,and the ratio of anti-inflammatory immune cells(Treg)increased;the levels of cytokines(IFN-γ,IL-17A)decreased,and the level of anti-inflammatory cytokine(IL-10)increased.In vitro data showed that after co-culture of GMSC,GMSC-EXO and IL-1β-GMSC-EXO with CD4~+T cell,the ratio of pro-inflammatory immune cells(Th1,Th17)in CD4~+T cells decreased and the ratio of anti-inflammatory immune cells(Treg)increased;the levels of pro-inflammatory cytokines(IFN-γand IL-17A)in culture supernatants decreased and the levels of anti-inflammatory cytokines(IL-10)increased;the expression of TLR4-My D88-NF-κB signaling pathway proteins(p-TLR4,My D88,TRAF6 and p65)in co-cultured cells was down-regulated.Conclusion:1.GMSC,GMSC-EXO and IL-1β-GMSC-EXO all have ameliorating effects on T1D,and in some aspects GMSC-EXO and IL-1β-GMSC-EXO are more effective than GMSC.2.GMSC-EXO regulates the ratio of CD4~+T cell subsets and the expression of their immune cytokines,i.e.reduces isletitis by inhibiting the production of pro-inflammatory cytokines by CD4~+T cells and promoting the production of anti-inflammatory cytokines by CD4~+T cells.3.GMSC-EXO and attenuate isletitis by inhibiting the TLR4-My D88-NF-κB signaling pathway.In the summary,GMSC-EXO could ameliorate T1D by regulating CD4~+T cell subsets involved in immune response and inhibiting TLR4-My D88-NF-κB pathway.