Study On The Renal Protection Of BMSCs Via Inhibiting TLR4/NF-?B Signaling In Diabetic Rats

Objectives:Diabetic nephropathy(DN)is one of the major complications of diabetic mellitus(DM),and DN develops in approximately 40%of patients who are diabetic,which is the leading cause of end-stage renal disease(ESRD).Bone marrow mesenchymal stem cells(BMSCs)have positive therapeutic effects on DN.However,the mechanisms about protection effect of BMSCs in DN remain unclear.In this study,we aimed to investigate the effect of transplantation of BMSCs on renal fibrosis and function,and explore the possible mechanism of BMSCs on toll-like receptor 4(TLR4)/nuclear factor-KB(NF-?B)signaling in diabetic rat model.Methods:We isolated and cultured BMSCs of rats to the third generation and labeled them with green fluorescent protein(GFP).Sprague-Dawley(SD)rats(n=30)were randomly divided into three groups,rats in the diabetic mellitus group(DM group)and the bone marrow mesenchymal stem cells transplantation group(BMSCs group)were intraperitoneally injected with 1%streptozotocin(STZ)at a dose of 60 mg/kg body weight,and fasting blood glucose(FBG)was measured three days later.The rats with FBG>16.7mmol/L for 3 consecutive days were regarded as diabetic rats.The other 10 rats were intraperitoneally injected with the citrate buffer at a dose of 60 mg/kg body weight as the normal group(NG group).Rats from BMSCs group were injected with BMSCs(4×106cells/200?L)by tail vein injection,the other two groups were injected with the same amount of phosphate buffer solution(PBS)by tail vein injection.And the FBG and body weight of the rats were measured weekly.At the 6th week after BMSCs transplantation,serum creatinine(Scr),blood urea nitrogen(BUN)and the kidney weight were measured,then we calculated the kidney weight/body weight.Paraff-in sections of kidney tissue were prepared,and hematoxylin-eosin(HE)staining,periodic acid-Schiff(PAS)staining and Masson staining were used to observe pathological changes.Immunohistochemistry was used to detect the expression of type ? collagen(Collagen ?)and type ? collagen(Collagen ?).The expression levels of TLR4,NF-?B and monocyte chemoattractant protein-1(MCP-1)in rat kidney were detected by western bloting and immunohistochemistry.The model of high-glucose cultured mesangial cell model was established in vitro.After co-culture with BMSCs,the proliferation of rats' mesangial cells was measured,and the expression levels of TLR4,NF-?B and MCP-1 were assessed by immunohistochemistry and western bloting assayResults:We observed BMSCs transfected successfully with GFP under the fluorescence microscope,and the green fluorescence was also observed in the rat kidneys.The FBG and kidney weight/body weight of the DM group were significantly higher than those of the NG group,and the body weight of the DM group was significantly lower than the NG group(P<0.05).BMSCs transplantation could significantly improve the FBG and body weight of DM rats(P<0.05).The Scr and BUN of the DM group were significantly higher than normal rats(P<0.05);the renal function was improved after BMSCs transplantation,and the levels of Scr and BUN were also decreased(P<0.05)The pathological staining results showed that the kidney structure of the NG group was normal,and the blue collagen deposition in the renal interstitial and perivascular was rare;the glomerular volume of the DM group was hypertrophied,the mesangial matrix increased,the mesangial area widened,and the glomerular basement membrane(GBM)was thickened,the capillary ring was compressed and deformed,and the renal interstitial showed obvious blue collagen fiber deposition;the glomerulus of the BMSCs group increased slightly,and the mesangial matrix changed slightly than the NG group,but it was much better than the DM group.The blue stained collagen deposition in the glomerular stroma was also significantly reduced.The immunohistochemistry indicated that the levels of type ? collagen and type ? collagen in the DM group were significantly higher than those in the NG group(P<0.05);after BMSCs transplantation,the expression of Collagen ? and Collagen ? decreased(P<0.05),which indicated that the renal fibrosis of DM rats was improved.The results of the immunohistochemistry,western bloting and cell experiment in vitro confirmed that the expression of TLR4,NF-?B and MCP-1 in the DM group was significantly higher than that in the NG group(P<0.05),and BMSCs transplantation significantly reduced the levels of TLR4,NF-?B and MCP-1 of rat renal tissue in vivo and vitro(P<0.05)Conclusion:BMSCs transplantation could significantly improve the FBG,body weight and renal function,ameliorate renal fibrosis and pathological injury via inhibiting TLR4/NF-?B signaling in diabetic rats.The present study provides a new therapeutic strategy and an experimental basis for the future treatment of DN via BMSCs transplantation.