| Background and ObjectiveDiabetic nephropathy(DN),one of the microvascular complacations of diabetes,is a major cause of end-stage renal disease(ESRD).Many pathogenic factors contribute to the initiation and development of DN.Although glomeruli injury,especially the proliferation of measngial cells and the glomerular hypertension,is the main pathological feature of DN,lots of clinical and animal studies have proved that the injury of renal tubular epithelial cells is related to the reduced renal function,and is considered to be a prognostic index of DN.Therefore,the mechanism of renal tubular epithelial cells injury in DN is crucial to identify and effective drugs to early prevention and treatment of DN should be searched.Oxidative stress refers to the situation of imbalance between the production of free radicals and antioxidant defence,leading to potential tissue damage.There is merging evidence showed that oxidative stress plays an important role in the initiation and development of DN.The role of forkhead box protein 03a(FOXO3a)in the pathogenesis of the kidney disease has caused wide public concern over the recent years.FOX03a belongs to a transcription factor family FOXOs.It plays an important role in aging,cell metabolism,insulin resistance,and oxidative stress resistance.It has been demonstrated that FOX03a regulates the antioxidant,superoxide(SOD)and catalase(CAT).An increasing number of studies have been showed that FOXO3a plays an critical role in a variety of kidney disease,including acute kidney injury and DN.Apoptosis,one of the modes of cell death,participates in kidney physiologic remodeling processes and contributes to cell loss and structural damage.For instance,renal proximal tubular cells exhibited apoptosis in diabetic patients as well as in streptozotocin(STZ)-induced diabetic mice.P53 is a tumor suppressing gene,studies have indicated that the expression of P53 in kidney disease is increased,including DN.P53 induces the injury of kidney by increasing the apoptosis of podocyte,glomeruli mesangial cells and renal tubular epithelial cells.Silent information regulator(Sir2)belong to the deacetylase enzymes,which is dependent on nicotinamide adenine dinucleotide(NAD+)for activity.There are seven SIRTs in mammal Sir2.SIRT1 is the closest to yeast Sir2,and is also the most extensively studied to date.In addition to deacetylate the histone proteins,SIRT1 can also deacetylate nonhistone proteins,for instance,FOXO,P53,NF-?B,PGC-1a,PARP and Ku70.So SIRT1 could regulate numerous signal pathway through its activity of deacetylation.SIRT1 take an important role in regulating oxidative stress and apoptosis.SIRT1 can regulate the transcriptional activity and reduce the oxidative stress by combining with FOXO3a directly and deacetylating FOXO3a.But,the mechanism of SIRT1/FOXO3a in resisting hyperglycemia induced oxidative stress injury in renal tubular epithelial cells is poor.SIRT1 ameliorates cell apoptosis by deacetylating the P53 protein at lysine 382 and inactivating its downstream genes.Novel drugs targeting SIRT1 may become available in preventing the initiation and development of DN.Resveratrol is a polyphenolic phytoalexin that abundant in the skins of grapes,and has numerous bioactivity and pharmacological function,including anti-oxidative stress,anti-apoptosis,anti-inflammation,anti-cancer,and cardiovascular protective effects.So it provides effective treatments for many diseases.But the mechanism of resveratrol on delaying the diabetic associated with oxidative stress and apoptosis is complicated.As stated,we hypothesize that the oxidative stress and apoptosis were aggravated because of the decreased expression of SIRT1-induced increased expression of acetylated FOXO3a and acetylated P53 in the kidney of diabetic rats.And resveratrol treatment could suppress the expression of acetylated FOXO3a and acetylated P53 by increasing the expression of SIRT1 and its deacetylation activity,so the oxidative stress and apoptosis injury of diabetic were ameliorated.We determined the expression of SIRT1,SIRT1 deacetylation activity,FOXO3a,acetylated FOXO3a,P53,acetylated P53,oxidative stress indicators and apopotosis indicator.And then we tried to reveal the above mechanism in high glucose(HG)-induced renal tubular epithelial cells in vitro.Methods1.To make clear the inhibition effect and the related mechanism of resveratrol on kidney oxidative stress in diabetic rats,STZ(60 mg/Kg)-induced diabetic rats was used as DM model.In our study,we divided the rats into three groups at random:Normal group,DM group,DM + resveratrol(30 mg·kg-1·d-1)group.After 16 weeks of treatment,urine sample,body weight,blood sample and kidney were used for the following experiment;HK-2 cells were pretreated with resveratrol(25 ?M)or vehicle for 2 h,and then the cells were cultured in medium with either HG(30 mM)for 72 h in vitro.To further evaluate the effect of SIRT1 on HG-induced oxidative stress in HK-2 cells,a SIRT1 siRNA or CON siRNA were transfected into cultured HK-2 cells.Western blot and RT-PCR were performed to detect the transfection efficiency,then HK-2 cells were incubated with HG(30 mM)for 72 h with or without resveratrol(25?M).Then kidney weight-to-body weight ratio(KW/BW),fasting blood glucose(FBG)and creatinine clearance(Ccr)were measured by laboratory methods;the glycogen deposition and fibrosis injury in the rats kidney was detected by PAS staining and masson staining respectively;immunohistochemistry were performed to assess the expression of SIRT1 in rats kidney;special assay kits were used to detect SIRT1 deacetylation activity,SOD activity and MDA content;and the protein expression of SIRT1,FOX03a and CAT in kidney tissues of diabetic rats and HK-2 cells were measured by western blot;acetylated FOX03a protein expression in kidney tissues of diabetic rats renal tubular and HK-2 cells was measured by immunoprecipitation.2.To make clear the effect and mechanism of resveratrol on apoptosis in kidney of diabetic rats,we used STZ(60 mg/kg)-induced diabetic rats as DM model,and the rats were divided into three groups at random:Control group,DM group and DM +resveratrol(30 mg·kg-1·d-1)group.After 16 weeks of treatment,body weight,blood sample and kidney were used for analysis.To determine if HG exerts an influence on apoptosis,HK-2 cells were grown under HG conditions for 0 h,24 h,48 h and 72 h respectively.HK-2 cells were pretreated with resveratrol(25?M)or vehicle for 2 h and then incubated with HG(30 mM)for 72 h in vitro for manifested the effect of resveratrol on HG induced apoptosis.To further evaluate the effect of SIRT1 on HG-induced oxidative stress in HK-2 cells,a SIRT1 siRNA or CON siRNA were transfected into cultured HK-2 cells.Western blot and RT-PCR were performed to detect the transfection efficiency,then HK-2 cells were incubated with HG(30 mM)for 72 h with or without resveratrol(25 ?M).Then kidney weight-to-body weight ratio(KW/BW),FBG,serum creatinine(Scr)and blood urea nitrogen(BUN)were detected by laboratory methods;PAS staining and masson staining were performed to assess the glycogen deposition and fibrosis injury in the rats kidney;immunohistochemistry were performed to assess the expression of SIRT1;special assay kits were used to detect SIRT1 deacetylation activity,caspase-3 and caspase-9 activity;JC-1 staining was used to detected the mitochondrial membrane potential,apoptotic nuclei were detected by Hoechst 33258 staining;and the protein expression of cleaved caspase-3,cleaved PARP,SIRT1,P53 and Ac-P53 in kidney tissues of diabetic rats and HK-2 cells were measured by western blot.Results1.Resveratrol prevents the kidney of diabetic rats against oxidative stress injury via increasing deacetylated FOXO3a by SIRT1.1.1 Resveratrol ameliorated metabolic index and kidney fibrosis of diabetic ratsCompared with the DM rats,FBG did not improved in DM+RSV rats,but the KW/BW was significantly decreased and Ccr was significantly increased.Resveratrol reduced diabetes-induced glycogen deposition in rat kidneys.Compared with DM rats,treatment of reservatrol significantly reduced the glomerulosclerosis.Masson staining indicated that the renal fibrosis in DM rats could be ameliorated by reservatrol treatment.1.2 Effects of reservatrol on SIRT1 expression and oxidative stress indicators in the kidney of DM rats.Compared with normal rats,SIRT1 expression was significantly reduced in the kidney of DM rats,reservatrol treatment significantly increased it.Reservatrol treatment significantly increased the SOD activity,and significantly reduced the MDA content.Western blot results showed that the expression of SIRT1 and CAT in DM+RSV group rats was higher than that in DM group rats,but there was no significant difference in the FOXO3a protein expression between the DM rats and DM+RSV rats.Reservatrol treatment significantly increased the SIRT1 deacetylation activity of diabetic rats.Meanwhile,Immunoprecipitation showed that the expression of acetylated FOXO3a was significantly decreased in the DM+RSV group rats compared with those in the DM group rats.1.3 Reservatrol ameliorated HG-induced oxidative stress injury and elevated SIRT1 deacetylation activity in HK-2 cells.Compared with the HG-cultured cells,reservarol treatment attenuated the HG-induced reduction of SOD in addition to the HG-induced increases of MDA and ROS in HK-2 cells.Western blot results showed that the protein expression of SIRT1 and CAT in HG+RSV group was higher than that in HG group,but there was no significant difference in FOXO3a protein expression between HG group and HG+RSV group.The SIRT1 deacetylation activity was significantly decreased in HK-2 cells cultured under HG conditions,but RSV treatment ameliorated the change.Meanwhile,the results showed that the acetylated FOXO3a expression were higher in the HK-2 cells cultured under HG conditions than those in the HK-2 cells cultured under LG conditions,and the high levels of acetylated FOXO3a was ameliorated by RSV treatment.The results also showed that none of these indicators were significantly influenced by the osmotic pressure.1.4 Effects of SIRT1 on HG-induced oxidative stress in HK-2 cellsSIRTI siRNA decreased the endogenous expression of SIRT1 protein and mRNA by 65%in HK-2 cells.When HK-2 cells were transfected with CON siRNA,the elevated SOD activity and the lessened levels of MDA,ROS and acetylated-FOXO3a induced by HG could be offseted by reservatrol treatment.Compared with the cells that transfected with CON siRNA,the cells that were trasfected with SIRT1 siRNA had decreased SOD activity and increased MDA contene,ROS and acetylated FOXO3a,but reservatrol treatment could not ameliorate those oxidative stress indicators in the cells that were trasfected with SIRT1 siRNA.2.Resveratrol ameliorants the kidney apoptosis of diabetic rats via regulating SIRT1/P53.2.1 Resveratrol ameliorated metabolic index and kidney fibrosis of diabetic ratsCompared with the DM rats,FBG,Scr and BUN did not improved in DM+RSV rats,but the KW/BW was significantly decreased.PAS staining demonstrated that resveratrol reduced diabetes-induced glycogen deposition in rat kidneys.Compared with DM rats,treatment of reservatrol significantly reduced the glomerulosclerosis index(GSI).Masson staining indicated that the renal fibrosis in DM rats could be ameliorated by reservatrol treatment.2.2 Effects of reservatrol on SIRT1 expression and apoptosis indicators in the kidney of DM rats.The immunohistochemistry and western blot results showed that the SIRT1 level was lower in the DM group rats than that of those in the control group rats.Compared with control rats,the activity of caspase-3 and caspase-9 were significantly increased in the kidney of DM rats.Western blot showed that,compared with control rats,the protein expression of cleaved caspase-3?cleaved PARP were higher than that in DM rats.And above apoptosis indicators could be ameliorated by reservatrol treatment.2.3 Reservatrol augmented the SIRT1 deacetylation activity and decreased the protein expression of acetylated p53.Reservatrol treatment significantly elevated the SIRT1 deacetylation activity of diabetic rats.Meanwhile,Western blot showed that the expression of acetylated P53 was significantly decreased in the DM+RSV group rats compared with those in the DM group.2.4 Effects of HG on apoptosis and the SIRT1 deacetylation activity in cultured HK-2 cells.Compared with HGOh group,the results of MTT and JC-1 staining showed that the cell viability and the mitochondrial membrane potential were significantly reduced,the result of Hoechst33258 staining showed that the number of condensed or fragmented nuclei was significantly increased in the cells grown under HG for 72 h.The caspase-3 activity was significantly increased in the cells grown under HG for 48 h and 72 h.Western blot results indicated that the protein expressions of cleaved caspase-3 and cleaved PARP were significantly increased and the SIRT1 expression was significantly decreased in the cells grown under HG for 48 h and 72 h compared with the cells grown under HG for 0 h.The SIRT1 deacetylation activity was significantly decreased after 48 h and 72 h under HG conditions.The acetylated p53 protein levels revealed by western blot showed that the expression of acetylated p53 was significantly raised after 48 h and 72 h under HG conditions,but the expression of P53 were at the same level in cells grown under HG for 0 h,24 h,48 h and 72 h.2.5 Effects of reservatrol treatment on apoptosis indicators and SIRT1 deacetylation activity in HK-2 cells cultured under HG conditions.The results of MTT showed that there were no differences between HK-2 cells cultured under LG conditions that were treated with different concentration of reservatrol(5 ?M,10 ?M,or 25 ?M)in cell viability,but,25 ?M reservatrol significantly increased the cell viability in cells cultured under HG conditions.Compared with HG group,the results of JC-1 staining showed that the mitochondrial membrane potential were significantly increased,the result of Hoechst33258 staining and caspased-3 activity showed that the number of condensed or fragmented nuclei and the caspase-3 activity were significantly decreased in the HG+RSV group.The results of western blot showed that,reservatrol treatment could reduce HG-induced cleaved caspase-3 and cleaved PARP protein levels.Compared with the HG group,the deacetylase activity of SIRT1 was significantly increased and the protein expression of acetylated-p53 were significantly decreased in the HG+RSV group,but the expression of P53 protein have no difference between the two groups.The results also show that the above indicators were not significantly different between the LG and mannitol groups.2.6 Effects of SIRT1 on HG-induced apoptosis in HK-2 cells.The SIRT1 siRNA treatment decreased the expression of SIRT1 protein by 60%and the SIRT1 mRNA by 65%in HK-2 cells.When HK-2 cells were transfected with CON siRNA,the elevated cell division,caspase-3 activity,protein levels of cleaved caspase-3,cleaved PARP and acetylated P53,and also the lessened levels of mitochondrial membrane potential and the deacetylase activity of SIRT1 induced by HG could be offseted by reservatrol treatment.Compared with the cells that transfected with CON siRNA,the cells that were trasfected with SIRT1 siRNA had increased cell division,caspase-3 activity,protein levels of cleaved caspase-3,cleaved PARP and acetylated P53 and decreased mitochondrial membrane potential and the deacetylase activity of SIRT1,and reservatrol treatment was unable to reverse the change of those apoptosis indicators.Conclusion1.Reservatrol could attenuate the development of DN,as revealed by reduction of KW/BW,amelioration of renal function.2.Reservatrol ameliorated kidney fibrosis in STZ-induced diabetic rats.3.The mechanism of reservatrol mediated beneficial effects was related to increase the expression of SIRT1.Reservatrol treatment ameliorated hyperglycemia induced oxidative stress via raising the deacetylase activity of SIRT1,down-regulating the protein expression of acetylated FOXO3a.4.The mechanism of reservatrol-mediated beneficial effects was related to inhibit the expression of acetylated P53.Reservatrol treatment ameliorated hyperglycemia induced apoptosis via down-regulating the protein expression of acetylated P53 in HK-2 cells. |
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