Preliminary Study On The Mechanism Of Mesenchymal Stem Cell Exosome-derived MiR-25 In The Treatment Of Type 1 Diabetes In Mice

Objective:To explore the primary mechanism of mesenchymal stem cells exosome-derived miR-25 in the treatment of type 1 diabetes in mice.Method:1.MSCs was isolated,cultured and identified.Cultured C57BL/6 mice compact bone derived-MSCs was isolated and identified.The cell morphology was observed by Giemsa staining.flow cytometry was used to identifie Cell phenotype.Oil-red O staining was determined the adipogenic differentiation ability,and alkaline phosphatase staining was detected osteogenic differentiation.The expression of adipogenic key transcription factors PPAR-γ,C/EBP-ɑand osteogenic key transcription factors Runx2,Osteocalcin were measured by quantitative real-time fluorescence PCR(q-PCR).2.The murine T1DM model was established.MSCs derived exosomes and their miRNA was isolated and identified.The function of miRNA was tested.Mice were given streptozotocin(STZ)to build a murine T1DM model.The changes in body weight and blood glucose of mice were monitored.The pathological changes of pancreatic tissue and insulin secretion status were analyzed by hematoxylin-eosin staining(HE)staining,immunohistochemistry,and immunofluorescence method.These were the identification standards of T1DM.Next,MSCs(5×10~5/mouse)were injected into caudal vein in T1DM mice and normal mice.Exosome was isolated from peripheral blood serum 48 hours after injection.Transmission electron microscope was used to observe the exosome morphology,and the expression of exosome markers CD63 and TSG101were identified by Western blot.Combined with the previous results of MSCs exosomes miRNA microarray,the exosomal RNA was extracted,and the expression of miR-25 in exosomes was detected by q-PCR.Bioinformatics analysis was used to predict the immune-related target gene of miR-25 as CXCR3.miR-25 mimic was further synthesized.Mouse T lymphocytes activated by CD3 monoclonal antibody(anti-CD3)were transiently transfected.q-PCR and Western blot were used to detect the expression of CXCR3 in activated T lymphocytes overexpressing miR-25.3.The molecular mechanism of mesenchymal stem cell exosome-derived miR-25 in the treatment of T1DM was preliminary explored.Pancreas tissue of T1DM model mice were extracted on day 2,4,6,and 8 after STZ induction.HE staining was used to detect the pathological changes of islet tissue,and immunofluorescence was used to detect insulin secretion and T cell infiltration in pancreatic tissue.The expression of CXCR3 in pancreatic tissue was detected by immunohistochemical methods.The expression of chemokines CXCL9,CXCL10,chemokine receptor CXCR3,and inflammatory factors TNF-αand IFN-γin pancreatic tissue were detected by q-PCR.The expression of inflammatory factors TNF-αand IFN-γwere detected by enzyme-linked immunosorbent assay(ELISA)in peripheral blood serum.Results:1.Mouse MSCs were successfully isolated.Giemsa stained cells by microscope showed fibroblast-like,vortex-like adherent growth.The cell surface markers detected by FACS assay were Sca-1~+CD29~+CD90~+CD34~-CD45~-CD11b~-MHCII~-.Oil red O staining showed that a large number of red lipid droplets appeared after adipogenic differentiation of cells.Alkaline phosphatase staining showed a large number of red alkaline phosphatase stained cells after osteogenic induction differentiation.The expression of adipogentic differentiation transcription factors PPAR-γ,C/EBPαand osteogenic differentiation transcription factors Runx2,Osteocalcin were significantly upregulated by q-PCR.All these results confirmed that the isolated cultured cells were MSCs.2.STZ-induced T1DM mouse model was established successfully.The typical symptoms of diabetes in mice was showed,which was increasing drinking wate r volume,polyuria,losing of body weight,and significant rising of blood glucose(≥16.7nmol).HE staining showed that the islet tissue of the mouse was severely damaged,and immunohistochemistry and immunofluorescence assay showed that the insulin secretion was significantly reduced.Furthermore,MSCs were injected into T1DM mice and normal mice.Exosomes were isolated and purified from mice peripheral blood serum.The results showed that exosomes presented vesicle-like goblet structure under transmission electron microscopy,and the size of exosome was about 100nm.Western blot results showed that the exosome markers CD63 and TSG101 was expressed.The expression of miR-25 in MSCs exosomes was detected by q-PCR;the results showed that miR-25 in serum exosomes was significantly increased in both the normal control group and the T1DM group(P<0.01).Moreover,the bioinformation analysis showed that CXCR3 was the target gene of miR-25.Therefore,anti-CD3 was used to activate mouse T lymphocytes.We found that high expression of CXCR3 in activated T lymphocytes was detected by q-PCR assay.miR-25 mimic was synthesis and transient transfected into activated T lymphocytes.The results showed that the miR-25 mimic significantly down-regulated the expression of CXCR3(P<0.01).These data suggested that miR-25 could regulate the immune function of T cells via CXCR3.3.To investigate the molecular mechanism of MSCs exosome-derived miR-25 in the treatment of STZ-induced T1DM in mice,we preliminarily studied the pathological process of STZ-induced T1DM and the progress changes of T cell infiltration into the pancreas.After 5 days of STZ injection,pancreatic tissue HE staining results showed that the decrease in the number of islet cells was positively correlated wit h the induction time of STZ.The islet cells were completely destroyed on the 8th day of induction.Moreover,the infiltration of T lymphocytes in pancreatic tissue tested by immunofluorescence method was found a peak on the 4th day after STZ induction,and then gradually decreased.The number of islets was dramatic decreased depending the induction time,and the change trend was the same as that of HE staining.The results of immunohistochemical assay showed that on the 4th day of STZ induction,CXCR3~+T cells had the most infiltration of pancreatic tissue and a large number of islet cells were damaged,then gradually decreased.The expression of related chemokines and their receptors was detected by q-PCR.The results indicated that CXCL9 was significantly increased on the second day after STZ induction(P<0.01),and its expression was time-dependent.The expression of CXCL10 reached its peak on day 6(P<0.01)and then showed a declining trend.The chemokine receptor CXCR3 expression was reached its peak on the 4th day(P<0.01).The expression of TNF-αand IFN-γin the pancreas was gradually increased,while the expression of TNF-αand IFN-γin the spleen and serum did not change significantly,suggesting that the early inflammatory response for T1DM was mainly in the pancreas.Conclusion:MSCs exosome-derived miR-25 can inhibit the expression of CXCR3 in T lymphocytes.The early inflammatory response of T1DM is related to the expression of CXCR3.These results suggest that MSCs exosome-derived miR-25 can inhibit the expression of CXCR3 in T cells at the early stage of T1DM,thereby reducing or treating T1DM.