Study On Ameliorative Effect And Its Mechanism Of APS Combined With HUCMSCs On Renal Lesion In Diabetic Rats

Diabetic nephropathy(DN),one of the chronic microvascular complications of diabetes mellitus,is the major cause of end-stage renal disease.The pathogenesis of this disease mainly includes glucose and lipid metabolism disorders,oxidative stress,hemodynamics,cytokines and abnormal activation of related pathways.Micro RNAs are ubiquitous endogenous,noncoding,single-stranded(ss)RNA transcripts,most frequently of 19~25 nucleotides in length,that act as posttranscriptional regulators of gene expression by blocking protein translation and/or inducing messenger RNA(m RNA)degradation.In recent years,research on micro RNAs has become a hotspot because that may serve as key pathogenic factors in diabetic nephropathy.NOX4,a member of the family of NADPH oxidases,is a known inducer of oxidative stress.In addition,NOX4/PKC/Rho A/ROCK,the mechanism of mutual circulation,plays an important role in the pathogenesis of diabetic nephropathy.As one of the main active ingredient in Astragalus,Astragalus polysaccharides(APS)have many pharmacological functions,such as reduction of blood glucose,improvement of insulin resistance,increasement of insulin sensitivity,reduction of he oxidative stress level,immune enhancement,effect of anti-inflammatory.Human umbilical cord-derived mesenchymal stem cells(HUCMSCs)can be horizontal differentiation,paracrine,and has the effect of regulating immunity.Both of them have made great progress in the treatment of diabetes,however,the study on ameliorative effect of APS combined with HUCMSCs on renal lesion has not reported yet.Objective: To explore the ameliorative effect of APS combined with HUCMSCs on glucose/lipid metabolism,renal function,oxidative stress and renal pathology in diabetic rats,and to investigate its mechanism from the perspective of micro RNA.Methods: Wistar rats were injected intraperitoneally with streptozotocin(STZ)to prepare diabetic model,then they were randomly divided into DN group,APS group(APS 12.5 g/kg,once/day),MSCs group(HUCMSCs 2×106 cells per rat,once/2 weeks),A+M group(APS 12.5 g/kg,once /day;HUCMSCs 2×106 cells per rat,once/2 weeks),Gli group(Glibenclamide 1.5 mg/kg,once/day),Bena group(Benazepril 12 mg/kg,once/day).They were treated for 12 weeks.In addition,Con group was set as normal control group.The ameliorative effect of APS combined with HUCMSCs on renal lesion in diabetic rats was observed by measuring glucose and lipid metabolism,renal function,antioxidant capacity and renal pathology(HE,PAS and immunohistochemistry staining).In addition,the differential expression of micro RNAs in Con,DN and A+M group were obtained by microarray and real-time PCR.The target genes of micro RNAs were predicted by the bioinformatics software and their function were analyzed.The mechanism of APS combined HUCMSCs in ameliorating renal lesion in diabetic rats was explored by detecting the relative expression of target protein and the key proteins in the coupled pathway by immunohistochemistry and western blot.Results:1.The rats in DN group showed the following symptoms: blood glucose was significantly higher than the normal level;urine BUN,UAER,UAlb and serum BUN significantly increased,CCR levels decreased significantly;T-CHO,TG,LDL-C increased significantly,HDL-C decreased(P<0.01).All the indicators suggested that DN rats were disordered in glucose/lipid metabolism and renal function.These indexes in treatment groups were improved in different degrees.The levels of blood glucose,UAER,serum BUN,CCR,T-CHO,TG and LDL-C in A+M group were significantly higher than those in other treatment groups.That suggested APS combined with HUCMSCs could ameliorate the glucose/lipid metabolism and renal function on diabetic rats,and was superior to APS and HUCMSCs alone.2.Compared with the Con group,the levels of T-SOD,T-AOC,CAT and GSH-PX were significantly decreased,and the content of MDA increased significantly in DN group.That indicated DN rats exposed to oxidative stresse.All the indexes in treatment groups were improved in different degrees.Those indexes in A+M group were improved most obviously,were superior to APS and HUCMSCs alone.3.Typical alterations were detected in the glomeruli of diabetic rats in DN group,including glomerular hypertrophy,segmental thickening of glomerular basement membranes,proliferating of mesangial cells,increasing positive staining of glycogen,increasing extracellular matrix accumulation,increasing expression of FN and Col IV.The amelioration of renal pathology was the most significant in the A+M group,including no significant increasing in glomerular volume,no significant proliferation of mesangial cells,no obvious thickening of the basement membrane,decreasing expression of glycogen,no significant increasing of FN and Col IV,no significant increasing of extracellular matrix accumulation.4.The result of microarray showed that 15/6 micro RNAs were up-regulated /down-regulated in renal tissues of DN group compared with Con group;21/19 micro RNAs were up-regulated/down-regulated in renal tissues of A+M group compared with DN group.The result of real-time PCR suggested that rno-mi R-208a-3p and rno-mi R-363-5p were up-regulated in DN group compared with Con group,and were down-regulated in A+M group compared with DN group(P<0.01).The prediction of target genes showed that there were 146 genes associated with rno-mi R-208a-3p and 678 genes associated with rno-mi R-363-5p.Combined with literature,pre-experimental results and GO function analysis,NOX4,one of the target genes of rno-mi R-363-5p,was selected as the research target.5.NOX4,one of the target genes of rno-mi R-363-5p,was found to mediate the NOX4/PKC/Rho A/ROCK signaling pathway.The results of immunohistochemistry and western blot suggested that NOX4,PKCβ,Rho A and ROCK1 were significantly increased in DN group,and were decreased in A+M group.Based on the results we get the following conclusions:1.APS combined with HUCMSCs could significantly ameliorate the glucose/lipid metabolism,renal function,oxidative stress and renal pathology,and the effect was superior to APS and HUCMSCs alone.2.APS combined with HUCMSCs could regulate the expression of rno-mi R-363-5p and rno-mi R-208a-3p on the renal in diabetic rats.3.APS combined with HUCMSCs could inhibit the activity of NOX4 on the renal in diabetic rats,and regulate the NOX4/PKC/Rho A/ROCK signaling pathway,and the effect was superior to APS and HUCMSCs alone.4.The ameliorative effect of APS combined with HUCMSCs on renal lesion in diabetic rats might be achieved by regulating the expression of rno-mi R-363-5p,inhibiting the activity of NOX4,and inhibiting the NOX4/PKC/Rho A/ROCK signaling pathway.Innovations:This is the first time to observe the ameliorative effect of APS combined with HUCMSCs on renal lesion in diabetic rats.In addition,the mechanisms were explored from micro RNAs.The study showed that the ameliorative effect of APS combined with HUCMSCs on renal lesion in diabetic rats might be achieved by down-regulating the expression of rno-mi R-363-5p,inhibiting the activity of NOX4,and inhibiting the NOX4/PKC/Rho A/ROCK signaling pathway.