| Part 1 The expression and significance of PI3K/Akt and Nrf2 in diabetic rat kidney tissuesObjective:Diabetic nephropathy(DN)is the main microvascular complication of diabetes mellitus,and also the most common cause of end-stage renal disease.DN is characterized by persistent proteinuria and progressive proteinuria accompanied by decreased glomerular filtration rate and impairment of glomerular capillaries and tubulointerstitium.It has been confirmed that the damage and loss of podocytes are the main cause of proteinuria in the early stage of the disease,while the damage of podocytes mainly comes from oxidative stress induced by high glucose.The excessive reactive oxygen species(ROS)produced in oxidative stress exist for a long time,and activate the signaling pathway involved in DN development,leading to apoptosis,inflammation,fibrosis,aging and other events.Nuclear factor E2 related factor 2(Nrf2)is the key factor of cell regulation of oxidative stress,which is the most sensitive signal to eliminate excessive ROS and oxidative stress.It is involved in a variety of cell life activities,including maintaining redox balance,proliferation,metabolism and apoptosis.It has been shown that the signal pathway of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)can regulate Nrf2 activity.AKT phosphorylation further helps Nrf2 to achieve nuclear plasma shuttle,thus enhancing the endogenous antioxidant capacity of cells.The purpose of this part is to study the expression of p-AKT and Nrf2 in the kidney of diabetic rats,and to locate podocytes.The characteristics,interrelation and the effects on apoptosis and oxidative stress of diabetic nephropathy were preliminarily clarified,which provided the theoretical basis of molecular mechanism for further treatment of diabetic nephropathy.Methods:Healthy male SD rats were randomly divided into two groups,one was normal control group(NC),the other was diabetic group(DM).Rats in DM group were given intraperitoneal injection of 1%STZ solution at 65 mg/kg body weight,while rats in NC group were given 0.1 mol/L sterile citric acid sodium citrate buffer.72 hours after injection,blood glucose was measured in the tail vein.If the blood glucose was?16.7 mmol/L and the urine glucose was positive(+++?++++),the diabetes model was established successfully.At the end of 12 weeks after the success of modeling,6 animals in the two groups were randomly collected.Before execution,10%chloral hydrate was injected into abdominal cavity for anesthesia,and blood was taken from heart to detect blood glucose,blood urea nitrogen and blood creatinine.Open the abdominal cavity to take bilateral kidneys,cut part of the kidney tissue and fix it in the OTC embedding for the subsequent frozen section immunofluorescence staining.Some renal tissues were fixed in 4%paraformaldehyde solution for subsequent HE staining.Part of the renal cortex was used to prepare tissue homogenate for protein extraction,and western blot was used to detect the expression of Nox4 protein in renal cortex.Results:1.The biochemical indexes of 12W diabetic rats showed that the levels of blood glucose,creatinine and urea nitrogen were significantly higher than those of the normal control group(P<0.01).2.HE staining showed that the renal structure of normal control group had no obvious change.In diabetic group,glomerulus volume increased,mesangial matrix increased,some glomerular capillary lumen narrowed,interstitial widened and inflammatory cells infiltrated.3.The results of immunofluorescence double staining showed that the expression of Bax and Cleaved caspase-3 in podocytes of diabetic rats was significantly higher than that of normal control group.Nrf2 and p-AKT were significantly decreased in podocytes of diabetic rats.4.Western blot showed that the expression of NOX4 protein in renal cortex of diabetic rats increased significantly(P<0.05).The level of ROS in mitochondria of kidney tissue was detected by Mito Sox red fluorescent dye.The results showed that the level of ROS in mitochondria of podocytes of 12 weeks old rats was significantly higher than that of normal control group.Part 2 The effect and mechanism of PI3K/AKT and Nrf2 on high glucose induced oxidative stress and apoptosis in Mouse podocytesObjective:The pathogenesis of diabetic nephropathy is complex,which has not been clarified.In recent years,a large number of studies have shown that the production of proteinuria in diabetic nephropathy is closely related to the functional and morphological changes of podocytes.Hyperglycemia induced a series of pathological changes in podocytes,including mast cell,apoptosis,epithelial mesenchymal transdifferentiation and podocyte detachment.ROS induced by high glucose is the trigger factor of podocyte injury.Mitochondria are the production center of ROS.Excessive production of ROS stimulates the transport of apoptotic proteins and the release of cytochrome C in mitochondrial respiratory chain,which triggers the mitochondrial mediated apoptosis pathway.In many studies,it has been confirmed that the tumor suppressor gene p53,as an important apoptotic protein,participates in the regulation of apoptosis.P53 can trigger the expression of apoptotic genes,such as puma,Bax,Apaf-1,noxa.In addition,it inhibits the expression of antiapoptotic genes,such as Bcl-2 family(Bcl-2,Bcl-x,bcl-in,Mcl-1);in p53 transcription independent cell apoptosis,it is mediated by mitochondria.It is found that p53 protein can directly mediate the activation of Caspase-3 in cells with only cytoplasmic components(excluding nucleus but including mitochondria),In the follow-up experiment,the activation of Caspase-3 was inhibited after p53 protein was removed by immunoprecipitation.Through the first part of the experimental results,we speculate that p-AKT and Nrf2 are involved in the pathogenesis and progress of diabetes,as well as the process of podocyte anti oxidative stress and antiapoptosis.In this part of the experiment,mouse podocytes will be cultured in vitro to further observe the effect of PI3K/AKT on Nrf2 pathway,and determine whether Nrf2 has an effect on podocyte apoptosis and p53 transcription independent signaling pathway in high glucose environment.Method:1.To detect the effect of PI3K/AKT on Nrf2 pathway expression,differentiated and mature podocytes were divided into normal glucose group(5.5mm glucose,normal glucose,NG),normal glucose+LY294002(20?m),high glucose group(30mm glucose,high glucose Hg),high glucose+LY294002(20?m).LY294002 pretreatment for 2 hours,high glucose stimulation for 48 hours.The expression of AKT,p-AKT,Nrf2,HO-1,nqo-1,SOD2 and GCLC were detected by western blot.The mRNA expression of Nrf2,HO-1,nqo-1,SOD2 and GCLC was detected by QRT PCR.The expression of Nrf2 protein was detected by western blot.The distribution of Nrf2 in podocytes was detected by immunofluorescence.2.To detect the knockdown effect of Nrf2 siRNA on Nrf2 gene,the mature podocytes were divided into two groups:NC group and sinrf2 siRNA group.After 48 hours,the total protein was collected and verified by western blot.3.To detect the effect of knockdown of Nrf2 gene or inhibition of PI3K/AKT pathway on oxidative stress of podocytes in high glucose environment.The mature podocytes were divided into normal glucose group(5.5mm glucose,normal glucose,NG),high glucose group(30mm glucose,high glucose Hg),high glucose+sinrf2,high glucose+LY294002(20 ? m).Sinrf2 was transfected,LY294002 was pretreated for 2 hours,and each group was stimulated by high glucose for 48 hours.ROS was detected by DCFH-DA,Mito Sox red and JC-1 respectively.4.To detect the effect of Nrf2 gene knockdown on podocyte apoptosis in high glucose environment,the differentiated podocytes were divided into normal glucose group(5.5mm glucose,normal glucose,NG),high glucose group(30mm glucose,high glucose Hg),high glucose+negative control(NC),high glucose+sinrf2.After successful transfection,high glucose stimulated 48 hours.The expression of Bax,Bcl-2,cyclochrome C and cleaved caspase-3 was detected by western blot.TUNEL staining was used to detect positive apoptotic cells.5.In order to detect the effect of knockdown of Nrf2 gene on p53 transcription independent signaling pathway in high glucose environment,the experimental cells were divided into normal glucose group(5.5mm glucose,normal glucose,NG),high glucose group(30mm glucose,high glucose Hg),high glucose+negative control(NC),high glucose+sinrf2.After 48 hours of high glucose stimulation,the total protein and nuclear protein were extracted and the expression of p53 was detected by western blot.Mito tracker red probe was used to detect mitochondrial morphology and cellular immunofluorescence,and to locate the distribution of p53 in mitochondria.Result:1.The expression of p-Akt,Nrf2 and its downstream proteins decreased significantly under the stimulation of high glucose.After LY294002 treatment,the expression of p-Akt,Nrf2 and their downstream proteins decreased more significantly,with statistical significance.The mRNA expression of Nrf2 and its downstream genes showed the same trend.In addition,LY294002 could reduce the expression of Nrf2 protein in the nucleus under the stimulation of high glucose(P<0.05),which was consistent with the change of immunofluorescence of Nrf22.The results of DCFH-DA and Mito Sox red staining showed that the fluorescence intensity of HG group was significantly higher than that of NG group.Compared with HG group,the fluorescence intensity of HG+sinrf2 group and HG+LY294002 group increased significantly.JC-1 probe staining showed that compared with NG group,the green fluorescence of JC-1 in HG group was significantly enhanced.After being treated with siNrf2 and LY294002,the green fluorescence was significantly enhanced and the red fluorescence was significantly weakened.3.Compared with HG group,the expression of Bax/Bcl-2 ratio,cyclochrome C and cleaved caspase-3 in HG+siNrf2 group increased significantly.TUNEL staining also indicated that the number of apoptotic cells induced by high glucose increased significantly after Nrf2 knockdown.4.Western blot showed that the expression of p53 protein in the cytoplasm and mitochondria of HG group was significantly higher than that of NG group.Compared with HG group,the expression of p53 protein in mitochondria of Hg+siNrf2 group was significantly higher.The results of immunofluorescence of p53 cells were consistent with those of western blot.Mito tracker red fluorescence probe staining showed that the mitochondria in HG group were significantly shorter,showing swelling and fragmentation,and the fragmentation was more obvious in HG+siNrf2 group than in HG group.In HG+siNrf2 group,the green fluorescence of p53 and the red fluorescence of Mito tracker red probe significantly increased the synthesis of yellow fluorescence.Part 3 The effect of carnosine on oxidative stress and apoptosis of mouse podocytes induced by high glucose through PI3K/AKT and Nrf2 pathwayObjective:carnosine(CA)is a kind of endogenous dipeptide,which has many biological activities,such as buffering physiological acid-base,chelating metal ions and antioxidative stress,inhibiting various inflammatory factors,inhibiting ages,promoting lipid oxidation end products(ales)and inhibiting renin-angiotension system(RAS).It is reported that carnosine can prevent glomerular cell apoptosis and podocyte loss in STZ diabetic rats by reducing the activity of serum carnosine-1.Through the first two studies,PI3K/AKT pathway may be a potential molecular mechanism involved in the protective effect of carnosine on apoptosis induced by high glucose.Therefore,in this part of the experiment.We will study whether carnosine can protect oxidative stress and apoptosis induced by high glucose stimulation through PI3K/AKT and Nrf2 signaling pathway.Method:1.To detect the effect of carnosine on the activity and oxidative stress of podocytes in high glucose environment,the differentiated podocytes were divided into normal glucose group(5.5 mM glucose,normal glucose,NG),high glucose group(30 mM glucose,high glucose Hg),high glucose+carnosine(5 mm carnosine,CA),high glucose+carnosine(10 mm carnosine,CA),high glucose+carnosine(20 mm carnosine,CA),high glucose+carnosine(30 mm)Carnosine,CA)?After 48 hours of stimulation,cell counting kit-8 was used to detect the activity of cells in each group,and dchf-da and Mito Sox red were used to detect the intracellular and mitochondrial ROS levels in each group.2.To detect the effect of carnosine on the apoptosis of podocytes in high glucose environment,the differentiated podocytes were divided into normal glucose group(5.5 mM glucose,normal glucose,NG),high glucose group(30 mM glucose,high glucose Hg),high glucose+carnosine(5 mm carnosine,CA),high glucose+carnosine(10 mm carnosine,CA),high glucose+carnosine(20 mm carnosine,CA).The expression of Bax,Bcl-2,cleaved caspase-3 and nephrin protein was detected by western blot.TUNEL staining was used to detect apoptotic cells.3.To detect the effect of carnosine on PI3K/AKT and Nrf2 pathway of podocytes in high glucose environment.The mature podocytes were divided into normal glucose group(5.5 mM glucose,normal glucose,NG),carnosine(20 mm carnosine,CA),high glucose group(30 mM glucose,high glucose Hg),and carnosine+carnosine(20 mm carnosine,CA).After 48 hours of high glucose stimulation,cells in each group were collected and total protein was extracted.The protein expression levels of Akt,p-Akt,Nrf2 and HO-1 were detected by western blot.The expression of Nrf2 protein was detected.The distribution of Nrf2 in cells was detected by immunofluorescence.4.To detect the effect of PI3K/AKT on Nrf2 pathway expression and carnosine on podocyte apoptosis.The mature podocytes were divided into normal glucose group(5.5 mM glucose,normal glucose,NG),LY294002(20 ? m),high glucose group(30 mM glucose,high glucose Hg),high glucose+carnosine(20 mm carnosine,CA),high glucose+carnosine(20 mm carnosine,CA)+LY294002(20 ? m).LY294002 pretreated cells for 2 hours.Cells were collected 48 hours after high glucose stimulation in each group,and the total protein was extracted.The protein expression levels of AKT,p-AKT,Nrf2,HO-1,Bax,Bcl-2 and cleaved caspase-3 were detected by western blot.TUNEL staining was used to detect apoptotic cells.The mRNA expression of Nrf2 and HO-1 was detected by qRT-PCR.5.To determine the effect of knockdown of Nrf2 gene or inhibition of PI3K/AKT pathway on the inhibition of oxidative stress and apoptosis by carnosine in podocytes in high glucose environment.The differentiated podocytes were divided into:high glucose+carnosine(CA),high glucose+carnosine(CA)+LY294002(20 ?M),high glucose+carnosine(CA)+sinrf2.LY294002 pretreated cells for 2 hours.After successful transfection,cells were collected 48 hours after high glucose stimulation in each group.After extracting the protein,western blot was used to detect the expression of Bax,Bcl-2 and Cleaved caspase-3.TUNEL staining was used to detect apoptotic cells.The intracellular and mitochondrial ROS levels were detected by fluorescence probe DCHF-DA and Mito sox red.Result:1.In the high glucose environment,the cell activity increased in a dose-dependent manner in the range of 5-20mm carnosine concentration,and decreased in the range of 30mM carnosine concentration.Carnosine(5-20mM)can reduce HG induced ROS in podocytes and mitochondria.2.In the range of 5-20mM,TUNEL staining showed that the apoptosis of podocytes induced by high glucose decreased in a dose-dependent manner.Bax/Bcl-2 ratio and Cleaved caspase-3 expression decreased in a dose-dependent manner,while the protein expression of Nephrin increased in a dose-dependent manner.3.Under high glucose environment,the protein expression of Nrf2,HO-1 and p-AKT was significantly lower than that of NG group,while in HG+CA(20mM)group,the protein expression of Nrf2,HO-1 and p-AKT was higher than that of HG group.The results of cell immunofluorescence showed that the fluorescence intensity of Nrf2 in the nucleus of HG group decreased significantly,and that in HG+CA(20mM)group increased significantly.Nrf2 protein expression in HG+CA(20mM)group was significantly higher than that in Hg group.4.TUNEL staining showed that the apoptotic cells in the pretreated HG+CA(20mM)group were higher than those in the pretreated HG+CA(20mM)group.The expression of Bax/Bcl-2 ratio and Cleaved caspase-3 was consistent with TUNEL staining.Compared with HG+CA(20mM)group,the expression of Nrf2 and HO-1 protein decreased significantly(P<0.01).The results of RT-qPCR were consistent with the changes of protein expression.5.ROS fluorescence and apoptotic cells,Bax/Bcl-2 ratio and Cleaved caspase-3 protein expression in mitochondria and cells were significantly increased in the co incubation group of carnosine and high glucose treated with sinrf2 and LY294002.Conclusion:1.The expression of p-AKT and Nrf2 in podocytes of diabetic rats and mice in high glucose environment decreased,and with the increase of apoptosis and oxidative stress,PI3K/AKT and Nrf2 pathway may participate in the occurrence of podocyte injury in diabetic nephropathy.2.PI3K/AKT pathway can resist oxidative stress in diabetic nephropathy by mediating Nrf2 activation,nuclear translocation and expression of HO-1,NQO-1,SOD2 and GCLC3.In the high glucose environment,there is mitochondrial damage in podocytes.The decrease of Nrf2 protein expression and activity and the increase of p53 level may be the mechanism of mitochondrial damage in podocytes.After knockdown of Nrf2 gene,p53 translocated mitochondria increased,and apoptosis was initiated through mitochondrial mediated apoptosis pathway.4.Carnosine can inhibit oxidative stress and apoptosis of podocytes induced by high glucose. |
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