| Background and aims:Diabetes mellitus(DM)is the most common chronic metabolic disease,and diabetic kidney disease(DKD)is one of the most common chronic microvascular complications of diabetes mellitus.At present,diabetic kidney disease has become one of the main causes of end-stage renal disease(uremia),and there is still no good treatment,which is a serious threat to human health.Autophagy is a cellular process that degrades damaged or redundant organelles and proteins and also plays an important role in the development of DKD,and the SIRT1(Silent information regulator of transcription 1)/FOXO1(Forkhead box Transcription factor O1)signaling pathway is related to autophagy.Mesenchymal Stem Cells(MSCs)possess strong self-renewal ability,differentiation potential,immunomodulatory effects,paracrine anti-inflammatory,cytoprotective factors and so on.Studies have shown that the therapeutic effect of MSCs is related to changes in autophagy levels in tissues and host cells.Therefore,we speculated that P-MSCs in DKD may promote autophagy by regulating the SIRT1/FOXO1 autophagy pathway and alleviate kidney injury.In this study,rat DKD model and podocyte injury model induced by high glucose were established to observe whether P-MSCs(Placenta-Derived MSCs,P-MSCs)played a renal protective role by regulating autophagy,and then to explore the potential mechanism of SIRT1/FOXO1 pathway mediated autophagy in the treatment of renal injury and podocyte injury induced by high glucose in diabetic rats by P-MSCs.Methods:DKD model of SD rats was established by one-time intraperitoneal injection of STZ(Streptozotocin),and divided into 4 groups:control group(NC group),diabetes kidney disease group(DKD group),DKD+P-MSCs caudal intravenous injection group(P-MSCs group),DKD+P-MSCs+3-MA(3-Methyladenine)group(P-MSCs+3-MA group).The P-MSCs group was injected with P-MSCs through caudal vein(1×10~6)once a week,3 times in total;the P-MSCs+3-MA group received caudal vein injection of P-MSCs and intraperitoneal injection of 3-MA(15mg/Kg/d)for 8W.Urine,blood and renal tissue samples were collected 8 weeks later.Using automatic biochemical analyzer test thebiochemical function,and the ELISA was used to detect the rat urine albumin/urine creatinine and kidney biopsy special dyeing testing kidney pathological changes.The expression of Podocin and Nephrin were tested of immunofluorescence test,while the renal autophagy related protein(LC3,Beclin 1),SIRT1 and FOXO1 expression levels were tested of immunohistochemical.In vitro,a podocyte injury model induced by high glucose was established.1)To observe the effects of different high glucose concentrations on MPC5,the experiment was divided into four groups:normal group(5.5m M,NG group),high glucose group(30m M,HG-30 group),high glucose group(40m M,HG-40 group)and high glucose group(50m M,HG-50 group);2)To observe the effect of P-MSCs on MPC5 induced by high glucose,the experiment was divided into three groups:normal group(5.5mm,NG group),high glucose group(50m M,HG group),and high glucose(50m M)+P-MSCs group(P-MSCs group);3)To observe the effect of autophagy on MPC5induced by high glucose treated by P-MSCs,the experiment was divided into four groups:normal group(NG group),high glucose group(HG group),high glucose+P-MSCs group(P-MSCs group),and high glucose+P-MSCs+3-MA group(P-MSCs+3-MA group);4)To observe the role of SIRT1/FOXO1 pathway in P-MSCs in the treatment of MPC5 autophagy induced by high glucose,the experiment was divided into two groups:high glucose+P-MSCs+si RNA control group(si-Con group)and high glucose+P-MSCs+si SIRT1 si RNA group(si-SIRT1group).Cell immunofluorescence was used to detect the expression of podocin in high glucose induced podocytes.Western blot was used to detect the expression level of autophagy-related proteins,podocin,SIRT1 and FOXO1,and PCR was used to detect the m RNA expression level of SIRT1.Results:Animal experiments showed that after 8 weeks of P-MSCs treatment,compared with DKD group,the levels of blood glucose(BG),blood triglyceride(TG),blood low density lipoprotein cholesterol(LDL-C),blood urea nitrogen(BUN),and urinary albumin/creatinine ratio(ACR)in P-MSCs group were significantly decreased;The HE and PAS staining results showed that P-MSCs could significantly reduce renal tubule injury and deposition of glomerular mesangial matrix;Immunofluorescence staining results showed that P-MSCs could significantly increase the expression of podocin and nephrin in renal tissue of DKD rats.The immunohistochemical results showed that the expression levels of SIRT1 and FOXO1 were decreased in renal tissue of DKD rats.P-MSCs could significantly up-regulate the expression of SIRT1and FOXO1,and meanwhile increase the expression of LC3-II and Beclin1,and promote autophagy.3-MA inhibited the improvement effect of P-MSCs on renal tubular injury and mesangial matrix deposition in DKD rats,and it inhibited the up-regulation effect of P-MSCs on the expression of SIRT1and FOXO1,and meanwhile inhibit the expression of LC3-II and Beclin1,in other words,it inhibited autophagy.In vitro experiments showed that high glucose significantly decreased the expression of podocin in MPC5;P-MSCs up-regulated SIRT1 and FOXO1,increased Beclin1 and LC3-II expression,and up-regulated the autophagy level of MPC5 in high glucose induced MPC5.The protective effect of P-MSCs was significantly decreased by 3-MA.Silent SIRT1 gene could counteract the increase of Podocin expression in podocytes by P-MSCs,and the expression levels of SIRT1,FOXO1 and autophagy related proteins were all significantly decreased.Conclusion:The results of this study suggest that P-MSCs can significantly improve the glucose and lipid metabolism and pathological injury of renal tissue in DKD rats,and reduce the injury of glomerular podocytes.The mechanism may be through regulating the SIRT1/FOXO1 pathway to increase the level of renal tissue and podocyte autophagy,thereby improving the DKD.The study provides a new idea for the mechanism of P-MSCs in the treatment of diabetic kidney disease. |
