Preventive Effect And Molecular Mechanism Of Mesenchymal Stem Cells On Young Mice T1DM Model

Objectives:To explore the preventive effect and possible mechanism of MSCs on T1DM young mice.This will provide theoretical and experimental basis for MSCs prevention and treatment of T1DM.Methods:MSCs derived from murine long bones were isolated and cultured.Long bones from 7-day-old C57BL/6 mice were dissected,digested with type II collagenase,and then transferred to culture flasks for adherent culture.The morphology of MSCs from P0 to P3 was observed with inverted microscope.The phenotype of P3 MSCs was detected by fluorescence activated cell sorter(FACS).P3 MSCs were induced to differentiate into adipocytes and osteoblasts.Oil Red O staining was used to determine adipogenic ability,and alkaline phosphatase staining was used to osteoblast differentiation ability.Furthermore,the total RNA of differentiated cells was extracted.The adipogenic transcription factors C/EBP-α,PPAR/γand osteogenesis key transcription factors RUNX2,osteocalcin was evaluated by quantitative real-time reverse transcription-polymerase chain reaction(q RT-PCR).T1DM prevention model in young mice were established.3-week-old mice were randomly diveded to five experimental groups including normal control group(NC group),T1DM control group(DM group),MSCs early prevention group(P1 group),MSCs advanced prevention group(P7 group),and MSCs treatment group(C group).The mice in each group except NC group were given a small dose(50mg/kg/d body weight)streptozotocin(STZ)for 5 consecutive days via intraperitoneal injection.On day 1 and day 7 of the STZ treatment,the mice in the P1 group and P7 group were injected with MSCs(5×10~5cells per mouse)via the tail vein,respectively.Mice in C group were injected with MSCs(5×10~5cells per mouse)at the day 15.The mice in the NC group and DM group were injected with the PBS.Furthermore,the general status(blood glucose,body weight,skin,hair,mental state and activity)of mice in each group were monitored.To determine the histopathological changes,the pancreatic islet tissue and kidney were stained with hematoxylin and eosin(HE staining),Immunohistochemistry staining and Masson staining.q RT-PCR was used to detected the renal fibrosis status in each group.Evaluation of the early prevention effect of MSCs on T1DM in young mice and its possible molecular mechanism.FACS was used to detect the changes of Th1(Type 1helper T)lymphocyte subsets in the spleen.The expression levels of splenic inflammatory factors were assessed by q RT-PCR and Enzyme Linked Immunosorbent Assay(ELISA).Further combined with the previous results of MSCs exosomes mi RNA microarray,the expression of mi R-148a-3p in exosomes was identified.Bioinformatics analysis was used to predict the immune-related target gene of mi R-148a-3p as IFN-γand Fasl.Further infuse MSCs into recipient mice;the expression of mi R-148a-3p in serum was detected by q RT-PCR.The target genes IFN-γand Fasl of mi R-148a-3p were verified by Luciferase Reporter Assay system.Results:1.MSCs isolated from mouse long bone parenchyma displayed a long spindle-shaped and vortex growth under inverted microscope.Cell phenotypes detected by flow cytometry on the third passage of MSCs showed high expression of CD29,CD90,Sca-1,and low expression of CD34,CD45,CD31,and CD11b.The red lipid droplets in MSCs differentiated into adipocytes were determined by Oil Red O staining.Alkaline phosphatase was positive when detecting MSCs differentiated osteoblasts.Compared with the self-differentiation group,the expression of adipogenic key transcription factor C/EBP-α,PPAR-γand osteogenic key transcription factors Runx2,osteocalcin were exhibited higher expression in the induction group by q RT-PCR assay.2.The model of type 1 diabetes in 3-week-old C57BL/6 mice was successfully established.After 5 consecutive days of intraperitoneal injection of 50 mg/kg STZ,the mice showed increase in blood glucose(blood glucose concentration was higher than 16.7 mmol/L for three consecutive days)and decrease in weight gaining rate.HE staining results of islet tissue showed that inflammatory infiltration was observed in the islet damage pathological sections,and the number of islet cells were significantly decreased.The Masson staining results of kidney showed that the degree of the renal fibrosis was significantly higher.These data indicated that the mice treated by STZ treatment were T1DM(the success rate of modeling was as high as 95%).To determine the prevention effect of MSCs in young mice T1DM,MSCs was injected into the recipient mice via tail intravenous injection.The results showed that the progression of T1DM in MSCs prevention group and treatment group were significant reduced,especially in the early prevention P1 group.The T1DM mice presented lower weight trend;blood glucose was down-regulating;pancreatic islet damage was reduced;and renal fibrosis was decreased.3.To explore the mechanism of MSCs prevention effect in the young mice T1DM,the FACS was used to analyze the proportion of Th1 lymphocyte subsets in the MSCs prevention function.The results showed that the percentage of Th1 cell in P1prevention group was significantly lower than that in the T1DM group.QRT-PCR results indicated that the expression of the spleen inflammatory factors TNF-αand IFN-γwas down-regulated after MSCs infusion.Serum ELISA displayed the similar results.After MSCs were injected into the recipient mice,we found that the expression of mi R-148a-3p from mouse serum exosomes was significantly increased.Moreover,luciferase reporting system was used to detect the target gene for mi R-148a-3p.The results showed that mi R-148a-3p could down-regulate the expression of target genes m Fsal and m IFN-γ.Conclusions:Early infusion of MSCs can significantly improve the blood glucose level of T1DM mice,reduce islet damage,and alleviate the process of renal disease induced by T1DM.MSCs exosomes derived mi R-148a-3p could down-regulated the expression of target gene m Fsal and m IFN-γand alleviated the progression of T1DM mice via restrain the inflammatory response.Therefore,early infusion of MSCs has a significant effect on the prevention and treatment of T1DM.