| Diabetic nephropathy(DN)is one of the most common complications of diabetes.The main histological features of DN were glomerular hypertrophy,thickening of glomerular and tubular basement membrane and expansion of the mesangial matrix.In diabetes mellitus,hyperglycemia induces hyperlipidemia,which aggravates the oxidative damage of kidney tissue,further leads to apoptosis and fibrosis,and then develops into end-stage renal disease.Glomerular mesangial cells are intrinsic cells in the glomerulus,which play a key role in the development of DN.High glucose can cause excessive proliferation of glomerular mesangial cells(GMCs),produce reactive oxygen species(ROS),which were accumulated in renal parenchymal cells,leading to apoptosis,matrix remodeling,tissue fibrosis,and eventually glomerulosclerosis.Mesenchymal stem cells(MSCs)are a type of undifferentiated cells with multi-differentiation potential,self-renewal ability,and low immunogenicity.It may be a new method for the treatment of DN,but the underlying mechanism remains unclear.Human umbilical cord mesenchymal stem cells(h UCMSCs)are derived from human umbilical cord tissue.They have many advantages,such as easy to obtain,easy to isolate,high purity,strong ability of proliferation and differentiation in vitro,and no ethical controversy.They have more application prospects than stem cells from other sources.Therefore,we aim to explore the protective effect of h UCMSCs on DN and its mechanism.Some studies have shown that mesenchymal stem cells can reduce apoptosis by activating Nrf2,so as to alleviate lung injury,spinal cord injury and liver injury.Nrf2not only regulates oxidative damage,but also has an anti-apoptotic effect.It is valuable for further discussion whether MSCs improve renal oxidative damage and further inhibit apoptosis and fibrosis by regulating Nrf2.In the first part,in order to prove the renal protective effect of h UCMSCs on type2 diabetes,we conducted experiments in vivo and in vitro.To establish the model of type 2 diabetes,Sprague Dawley rats weighing 180-200g were fed with high-fat diet for 6 weeks,then were intraperitoneally injected with streptozotocin(35 mg/kg).Blood glucose,urine volume and urine protein were monitored.After 8 weeks of STZ injection,the urine volume of diabetic rats increased significantly and urine protein appeared.Diabetic rats were injected with h UCMSCs(2×10~6 cells)by tail vein three times every 10 days.Rats were killed 10 days after the last injection of h UCMSCs.The protective effects of h UCMSCs on DN were observed by western blot,RT-q PCR,immunohistochemistry and TUNEL staining.In vitro,we used the mesangial cells stimulated by high glucose and high fat as the cell model of type 2 diabetes mellitus,and coculture with h UCMSCs using transwell chamber to observe the protective effect of h UCMSCs on mesangial cells stimulated by high glucose and high fat.The second part is to explore the specific mechanism of h UCMSCs on renal protection of type 2 diabetes.Firstly,western blot and RT-q PCR were used to detect the expression level of Nrf2 and its downstream factors.Secondly,to further explore whether the regulation of Nrf2 by h UCMSCs is only fulfilled by increasing its expression or by promoting nuclear translocation,we extracted the nuclear protein from rat kidney tissue and detected the expression of Nrf2 in nucleus and cytoplasm by western blot.The location and level of Nrf2 expression in glomerular mesangial cells were detected by immunofluorescence.In order to prove that Nrf2 is the key factor in the treatment of diabetic nephropathy by h UCMSCs,we used Nrf2 si RNA interference to observe whether the protective effect of h UCMSCs on mesangial cells was weakened after the specific inhibition of Nrf2 expression.To investigate whether h UCMSCs can protect diabetic nephropathy by activating PI3K/AKT/m TOR signaling pathway upstream of Nrf2,we detected the phosphorylation level of PI3K/AKT/m TOR signaling pathway protein by western blot.The results are as follows:1.HUCMSCs reduce blood glucose,urinary protein and renal hypertrophy index in type 2 diabetic ratsIn vivo experiment,we detected the biochemical indexes of each group at the end of the experiment,and found that the blood glucose,urinary microalbumin/creatinine,and renal hypertrophy index of diabetic rats treated with h UCMSCs were significantly lower than those of diabetic rats(P<0.05).2.HUCMSCs improve renal histological changes in type 2 diabetic ratsPathological staining of renal tissue showed that glomerular area and mesangial matrix increased,glomerular basement membrane became thicker,podocyte foot process fusion,glomerular and tubulointerstitium fibrosis in diabetic rats.After treatment with h UCMSCs,the above lesions were significantly reduced.3.HUCMSCs attenuate renal oxidative damage,inflammation,apoptosis and fibrosis in type 2 diabetic ratsCompared with control group,the expression levels of MDA,4-HNE,PAI-1,ICAM-1,TNF-?,Caspase-3,TGF-?,CTGF and collagen?in renal tissue of diabetic rats significantly increased(P<0.05).The levels of MDA and the above proteins in renal tissue of diabetic rats with h UCMSCs treatment were significantly lower than those in diabetic rats(P<0.05).In addition,TUNEL staining showed that the apoptosis level of renal tissue in h UCMSCs treatment group was obviously lower than that in diabetic rats.4.Protective effect of h UCMSCs on glomerular mesangial cells stimulated by high glucose and high fatCompared with the control group,the expression levels of 4-HNE and caspase-3were significantly increased and Bcl2/Bax was significantly decreased(P<0.05);Coculturing with h UCMSCs significantly decreased the expression of 4-HNE and Caspase-3,and up-regulated Bcl2/Bax(P<0.05).5.HUCMSCs promotes the expression of Nrf2 and its downstream factorsIn vivo,the expression of Nrf2 in the renal tissue of diabetic rats treated with h UCMSCs was significantly higher than that of diabetic rats;The expression of Nrf2and downstream factors SOD2,NQO-1 and HO-1 in the diabetic rats treated with h UCMSCs was significantly higher than that in the diabetic group(P<0.05)In vitro,the expression of Nrf2,SOD2,NQO-1 and HO-1 in glomerular mesangial cells stimulated by high glucose and high fat were lower than control group(P<0.05).After coculture with h UCMSCs,the level of Nrf2 and downstream factors was significantly higher than that in high glucose and high fat stimulation group.6.HUCMSCs not only increase Nrf2 expression,but also promote Nrf2 nuclear translocationExperiments in vivo confirmed that after treatment with h UCMSCs,the level of nuclear Nrf2 protein increased significantly,whereas there was no significant difference in cytoplasmic Nrf2 among groups(P<0.05),indicating that h UCMSCs can not only increase Nrf2 expression,but also promote Nrf2 nuclear translocation in diabetic rats.In vitro,the location and level of Nrf2 expression were observed by immunofluorescence.The results showed that Nrf2 was mainly accumulated in the cytoplasm of mesangial cells stimulated by high glucose and high fat.After coculture with h UCMSCs,the level of Nrf2 in mesangial cells increased significantly,and the nuclear aggregation increased.7.Nrf2 plays a key role in the protective effect of h UCMSCs on DNIn vitro,after si RNA interference,compared with the control group,the expressions of 4-HNE and Bax in glomerular mesangial cells stimulated by high glucose and high fat significantly increased(P<0.05),but h UCMSCs could not reduce the expression of these proteins(P>0.05).8.HUCMSCs activate Nrf2 upstream signaling pathway PI3K/AKT/m TORIn vivo,levels of phosphorylated AKT and m TOR in diabetic rats treated with h UCMSCs were significantly higher than those in diabetic group(P<0.05).Experiments in vitro confirmed that levels of phosphorylated AKT and PI3K were upregulated in mesangial cells cocultured with h UCMSCs.In conclusion,we conclude that human umbilical cord mesenchymal stem cells can reduce renal oxidative damage,inflammation,apoptosis and fibrosis in type 2diabetes mellitus;Nrf2 activation is one of the important mechanisms of human umbilical cord mesenchymal stem cells protecting diabetic kidney injury. |
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