| Part one The expression of Sirt3 in the kidney tissues of diabetic rats and high glucose-stimulated HK-2 cellsObjective: A report from International Diabetes Federation reveals that diabetic patients have reached 382 million until 2013.It is predicted that the population will reach 471 million by 2035.At the end of 2013,5.1 million people have died of diabetes,medical expenses for the treatment of diabetes has been as high as 548 billion US dollars.Because the persistent increase in incidence of diabetes in recent years,diabetic has been one of diseases that seriously threaten people's health.Diabetic nephropathy,one of the most common microvascular complications of type 1 and type 2 diabetes,represents the major cause of chronic renal failure and end-stage renal diseases.It is reported that 40%-50% of type 1 diabetes exist renal damage,the data in type 2 diabetes presents 30%.It is therefore a pressing issue to discover new molecular mechanisms and effective treatments of diabetic nephropathy in future research.The pathogenesis of diabetic nephropathy is complex.It consists of hemodynamic,oxidative stress,the activition of cytokine,mitochondrial dysfunction,endoplasmic reticulum stress and so on.Key extracellular stimulations that contribute to damage to the kidney in DN include hyperglycaemia,inflammation and so on.The ultrastructural alterations in kidney include GBM thickening,mesangial extracellular matrix accumulation,glomerulosclerosis,renal tubular interstitial fibrosis,renal tubular atrophy.Besides the glomerular changes,renal tubular play an important role in the development of diabetic nephropathy.The alternations in structure and function of renal tubular accelerate the appearance of proteinuria in the early stage of diabetic nephropathy.The mitochondria are recognized as the main site of energy generation,they regulate the cell survive,metabolic balance and cell death.It has been implicated that the excessive reactive oxygen species(ROS)produced by mitochondria is the primary initiating event in the development of diabetic nephropathy.The ROS can active several signaling pathway related to oxidative stress,cell apoptosis,fibrosis,inflammation that cause renal injuries.Thus,the identification of the mechanism by which they diminish ROS generation in mitochondria and cellular may provide potential therapeutic targets in treating renal damage associated with diabetes.Sirt3,a kind of NAD+-dependent protein deacetylases,is one member of the mammalian sirtuin homologs of the yeast Sir2 gene.Sirt3 is localized mainly in mitochondria and is recognized as a mitochondrial stress sensor that can regulate the acetylation and activity of several mitochondrial enzymes exsisted in metabolism-related pathways,including mitochondrial respiratory chain reaction,tricarboxylic acid cycle,?-oxidation of fatty acids and so on.Sirt3 plays an important role in modulating the oxidative homeostasis,energy metabolism,cell apoptosis in cell.It has been suggested that Sirt3 can counterwork several oxidative stress-related diseases,such as cardiovascular diseases,tumour,neurodegenerative diseases,acute renal injury and so on.However,few studies have assessed the role of Sirt3 in the pathological processes of diabetic nephropathy.We hypothesize that Sirt3 may have ability to influence the progression of diabetic nephropathy.In this part,we will observe the expression and distribution of Sirt3 in diabetic kidney and high glucose-incubated HK-2 cell.The relationship between Sirt3 and diabetic nephropathy will be discussed.Methods: 1 Forty Male SD rats were randomly divided into two groups: normal control group(NC)and diabetic group(DM),every group has twenty.Diabetes model were generated through intraperitoneal injection of 1% streptozotocin(STZ,65 mg/kg).The rats in NC group were injected by the same volume of citric acid-sodium citrate buffer.The blood glucose was detected after 72 h.If the blood glucose concentration of one rat was greater and equal to 16.7 m M and the glucose in urine was positive for three consecutive days,the rat was consided as diabetes.The rats in NC group and DM group were separated as NC 8 week,NC 12 week,DM 8 week,DM 12 week.At 8 and 12 weeks after modeling success,rats in corresponding group were sacrificed.The 24 h urine samples were collected before the rats were sacrificed.The blood in femoral artery was collected.The biochemical indicators such as Blood glucose(BG),Blood urea nitrogen(BUN),Serum creatinine(Scr)were tested separately.The partial renal tissures were fixed in 4% paraformaldehyde for H.E staining,PAS staining and immunohistochemical staining.Protein and RNA extracted from partial renal were used to detected the expression of Sirt3 via Western blot and Real-time PCR.2 HK-2 cell culture and sample collection: The HK-2(human kidney 2)cells,a sort of proximal tubular cell line,were cultured in DMEM supplemented with 10% fetal bovine serum,100 U/ml penicillin and 100 mg/ml streptomycin in a 95% air,5% CO2 atmosphere at 37?.When grown to 80% confluence,the cells were inherited.The HK-2 cells were stimulated with normal concentration D-glucose medium(NG,5.6 mmol/L)and high concentration medium(HG,30 mmol/L)separately.The high glucose were incubated for 0h,6h,12 h,24h,48 h and 72 h respectively,and then the HK-2 cells were harvested.The protein and RNA extracted from HK-2 cells were used to detected the expression of Sirt3 via Western blot and Real-time PCR.The distribution of Sirt3 in HK-2 cell was detected by immunofluorescence.Results: 1 The results from H.E and PAS staining revealed that the structure of kidney tissue in normal control rats was normal;the significant glomeruli hypertrophy,expansion of the mesangial matrix in glomeruli,renal tubular compensatory hypertrophy and tubular vacuolization were seen in kidney tissue of diabetic rats;2 Compared with normal control rat,the protein expression of SOD2 in 12 w diabetic rats was significantly decreased,the ratio of Bax/Bcl-2 was markedly increased(P<0.01);3 Compared with normal control rat,the 24 h urine protein,BUN and Scr levels increased in diabetic rats(P<0.01);4 Compared with normal control rats,the protein and m RNA expression of Sirt3 significantly increased in 8w diabetic rats and decreased in 12 w diabetic rats(P<0.01).The results from immunohistochemical staining showed that Sirt3 was mainly diatributed in proximal tubule cells,seldomly in glomerular and renal interstitial.The immunohistochemical staining results also showed that the expression of Sirt3 was significantly decreased in 12 w diabetic rat kidney compared to normal control rats;5 High glucose changed the protein and m RNA expression in a time-dependent manner in HK-2 cells.Compared with NG group,the protein and m RNA expression was increased in 12 h and significantly decreased in 48h(P<0.01).The immunofluorescence staining revealed that Sirt3 mainly distributed in cytoplasm,seldomly in cell nuclear.Part two The effect of Sirt3 on HG-induced oxidative injuries in HK-2 cell and its molecular mechanismsObjective: Sirt3 is a kind of NAD+-dependent protein deacetylases,belonging to the Sirtuins family.As the main protein deacetylases in mitochondria,Sirt3 regulates the acetylation level and activity of several proteins in mitochondria and influences the biological effects in mitochondria,such as respiratory chain reaction,tricarboxylic acid cycle,fatty acid ?-oxidation,antioxidant pathway and so on.Thus,Sirt3 also influence the metabolic reaction,oxidative stress,cell apoptosis in cell.It is reported that Sirt3 could influence the redox homeostasis by regulating the activity of Complex?,?,?.The accumulation of ROS in mitochondria is associated with cell oxidative injury and the progression of many diseases,such as neurodegenerative diseases,diabetes,cancer and so on.The anti-oxidative pathways in mitochondria could clear the excessive ROS and protect the mitochondria from oxidative injuries.It is reported that Sirt3 could modulate the anti-oxidative pathway in mitochondria.Sirt3 could accelerate the clearance of ROS by increasing the expression and activity of SOD2.Sirt3 could decrease the toxic effects of ROS via interacting the isocitrate dehydrogenase.In addition,Sirt3 could influence the cell apoptosis by regulating the calcium homeostasis in cell.The endogenous apoptotic pathway is dependent on the open of mitochondrial permeablity transition pore.In cardiomyocyte,Sirt3 prevent the open of mitochondrial permeablity transition pore through deacetylating cyclophilin D and thus resiste the cell apoptosis.In part one,we explore that the expression of Sirt3 in diabetic kidney and high glucose-incubated HK-2 cell significantly decreases.The results suggest that Sirt3 is associated with the development of diabetic nephropathy.In this part,we will study the effect of Sirt3 overexpression on oxidative stress and apoptosis in HK-2 cell.The underlying molecular mechanisms about the effect of Sirt3 will be discussed in this part.Methods: 1 The effects of high glucose on HK-2 cell apoptosis and Akt/Fox O signaling pathway: The HK-2 cells were stimulated with normal concentration D-glucose medium(NG,5.6 mmol/L)and high concentration medium(HG,30 mmol/L)separately.The high glucose were incubated for 0h,6h,12 h,24h,48 h and 72 h respectively,and then the protein and RNA of HK-2 cells were extracted.The protein expression of Bax,Bcl-2,Cleaved caspase-3,Bim,Fas L,Akt,p-Akt,Fox O1,p-Fox O1,Fox O3 a,p-Fox O3 a in HK-2 cells were detected by Western blot.2 Sirt3 was overexpressed in HK-2 cells: The Fu GENE transfection reagent and p CMV-Sirt3 plasmid in different proportions were transfected in HK-2 cell.The optimal proportion was selected based on the Western blot and Real-time PCR results.3 To examine the effects of Sirt3 on HK-2 cell and Akt/Fox O signaling pathway,the HK-2 cells were separated as four groups: p CMV-vector control group(control),p CMV-Sirt3 plasmid transfection group(Sirt3),high glucose plus p CMV-vector control group(HG + control),high glucose plus p CMV-Sirt3 plasmid tranfection group(HG + Sirt3).The protein expression of SOD2,catalase,Bax,Bcl-2,Cleaved caspase-3,Bim,Fas L,Akt,p-Akt,Fox O1,p-Fox O1,Fox O3 a,p-Fox O3 a were detected by Western blot;the ROS level in mitochondria and cellular were detected by Mito SOX staining and cell flow cytometer respectively;the cell apoptosis was detected by TUNEL;the nuclear and cytoplasmic protein were extracted separately,the protein expression of Fox O1 and Fox O3 a were examined via Western blot;the distribution of Fox O1 and Fox O3 a were examined by immunofluorescence.4 To examine the relationship between antioxidants and the Akt/Fox O signaling pathway,the HK-2 cells were separated into five groups: normal D-glucose control group(NG,5.6 mmol/L),high concentration group(HG,30 mmol/L),mannitol control group(M,5.5 mmol/L D-glucose plus 24.5 mmol/L D-mannitol),NAC-contained group(N,5 m M),NAC plus HG group(HG + N).The protein expression of Bax,Bcl-2,Cleaved caspase-3,Akt,p-Akt,Fox O1,p-Fox O1,Fox O3 a,p-Fox O3 a were detected by Western blot;the ROS level in cellular was detected by cell flow cytometer.Results: 1 HK-2 cells were transfected with Fugene HD 4 ?l and p CMV6-Sirt3 2 ?g for optimal transfection efficiency;2 Western blot results showed that the protein expression of SOD2 and catalase significantly decreased in HG group compared to NG group,and was reversed by Sirt3 overexpression(P<0.01).The results from Mito SOX staining and cell flow cytometer illustrated that Sirt3 overexpression could decrease high glucose-induced ROS in mitochondria and celluar(P<0.01);3 In HK-2 cell,the ratio of Bax/Bcl-2 and the expression of Cleaved caspase-3,markers of cell apoptosis,were markedly increased after high glucose incubation for 48 h.The Western blot results showed that the significant increase of Bax/Bcl-2 ratio,Cleaved caspase-3,Bim and Fas-L could be reduced in Sirt3 overexpression plus HG group compared with HG group(P<0.01);4 The Western blot results revealed that the protein expression of p-Akt(Ser 473),p-Fox O1(Ser 256)and p-Fox O3a(Ser 253)was significantly decreased in HG group(P<0.01).Compared with HG group,Sirt3 overexpression could markedly upregulated the protein level of p-Akt(Ser 473),p-Fox O1(Ser 256)and p-Fox O3a(Ser 253)(P<0.05).In addition,the protein expression of Fox O1 and Fox O3 a in cell nuclear was significantly decreased in HG plus Sirt3 group compared to in HG group(P<0.01).The immunofluorescence staining illustrated that the expression of Fox O1 and Fox O3 a in cell nuclear was significantly decreased in HG plus Sirt3 group compared to in HG group;5 The Western blot results showed that NAC treatment significantly decreased the ratio of Bax/Bcl-2 and the protein expression of Cleaved caspase-3,and increased the phosphorylation of Fox O1 and Fox O3 a compared with HG group(P<0.01).Part three The protective effects of kaempferol on high glucose-sti mulated HK-2 cell and its mechanismsObjective: Kaempferol,a naturally occurring phytoestrogen and one of the most common dietary flavonoids,has been found commonly existed in many plants including grape fruit,tea,broccoli and so on.A large amount of studies demonstrated that kaempferol has potential to exert a variety of biological effects such as antioxidative,antiapoptotic and antiinflammatory.It has been proposed that kaempferol have protective effect on oxidative stress-related diseases,such as Ischemia-Reperfusion induced myocardium injuries,osteoporosis,obesity and so on.Nevertheless,the effects of kaempferol on diabetic nephropathy and the exact mechanisms have not been studied accurately.Until now,the molecular mechanisms about the biology effect of kaempferol remains unclearly.Recently,a study reported that the expression of Sirt3 could be increased by kaempferol treatment,and thus decreased acetylation of the Sdh A subunit and increased Complex II activity in mitochondria.In this part,we will study the effects of kaempferol in cultured HK-2 cell and the relationship between kaempferol and Sirt3.Methods: 1 To find out the safety and effective concentration of KMP in our experiment,the cell viability under different concentration(0.01 ?mol/L,0.1 ?mol/L,1 ?mol/L,5 ?mol/L,10 ?mol/L,15 ?mol/L,20 ?mol/L,50 ?mol/L KMP)were detected by MTS method.2 The HK-2 cells were separated into five groups: normal D-glucose medium control group(NG,5.6 mmol/L),high concentration group(HG,30 mmol/L),mannitol control medium(M,5.5 mmol/L D-glucose plus 24.5 mmol/L D-mannitol),kaempferol-contained medium(KMP,10 ?mol/L kaempferol),kaempferol plus HG medium(HG + KMP).The protein and m RNA expression of SOD2 and catalase were detected by Western blot and Real-time PCR;the protein expression of Bax,Bcl-2,Cleaved caspase-3,Akt,p-Akt,Fox O3 a,p-Fox O3 a were detected by Western blot;the ROS level in cellular was detected by cell flow cytometer;the SOD activity was examined by the SOD acticity detection kit.Results: 1 The MTS results showed that kaempherol had no significant effect on cell viability when the working concentrations were 0.01,0.1,1,5,10,15,20 ?mol/L.While,50 ?mol/L kaempherol exhibited a marked inhibition effect on the proliferation of HK-2 cells(P<0.05).Compared with control group,the cell viability decreased significantly following stimulation with a high concentration of D-glucose for 48 h.10?M kaempherol co-stimulation with high glucose increased the cell viability compared to a high glucose only(P<0.05).Taken together,the concentration of 10 ?M kaempherol was selected as use concentration in subsequent experiments;2 The Western blot results illustrated that the protein and m RNA expression of SOD2 and catalase were significatly decreased in HG group compared with NG group(P<0.05).KMP reversed the effect of high glucose on SOD2 and catalase(P<0.01).The SOD activity test showed that KMP significantly increased the activity of SOD compared with HG group(P<0.05).The results from cell flow cytometer illustrated that KMP treatment could decrease high glucose-induced ROS in celluar(P<0.05);3 Western blot results showed that high glucose markedly increased the ratio of Bax/Bcl-2 and the protein expression of Cleaved caspase-3,and the effect was restored by KMP treatment(P<0.01);4 Western blot,Real-time PCR and immunofluorescence results showed that the protein and m RNA expression of Sirt3 in HK-2 cells was markedly increased in HG plus KMP group compared to HG group(P<0.01);5 Western blot results showed that the ratio of p-Akt/Akt and p-Fox O3a/Fox O3 a were significantly increased in HG plus KMP group compared with HG group(P<0.05).Conclusions: 1 The decrease of Sirt3 is found in the kidney of diabetic rats and high glucose-incubated HK-2 cells by which suggests that Sirt3 mediate the renal injury in diabetes.2 Sirt3 could protect HK-2 cell from high glucose-induced injury via its anti-oxidative and anti-apoptosis effect.3 Sirt3 regulates HK-2 cell apoptosis through the ROS-sensitive Akt/Fox O signaling pathway.4 Kaempherol could exert its protective effect on HK-2 cell by its anti-oxidative and anti-apoptosis property.5 Kaempherol could fight against high glucose-induced oxidative stress and cell apoptosis through increasing the expression of Sirt3 and regulating ROS-sensitive Akt/Fox O3 a signaling pathway. |
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