| In recent years,the prevalence rate of Diabetes mellitus(DM)is increasing year by year.As we all know,the main harm caused by diabetes mellitus to patients is diabetes complications caused by poor blood sugar control,including acute diabetes complications and chronic diabetes complications.For example,Diabetic kidney disease(DKD)is a disease that is often treated with hypoglycemic,hypotensive,inhibition of(Rein-Angiotensin-Aldosterone-System,RASS)activation,and traditional Chinese medicine.Some patients will have poor clinical efficacy and will not be able to prevent the development of DKD into end-stage renal disease.It brings heavy economic burden and poor quality of life to patients.Therefore,early prevention and control of DKD progression is a great challenge for endocrinologists.Previous studies have found that ectopic lipid deposition and its resulting renal damage have occurred in the kidney in the early stage of DKD,but the specific metabolic mechanism is still unclear.Junctional adhesion molecules L(JAML)is a newly discovered member of the JAMs family.Its expression is significantly elevated in the kidneys of patients with DKD,and is associated with excessive renal lipid deposition and glomerular filtration.It can regulate sterol regulatory element-binding protein-1(SREBP-1)and its downstream key enzymes through the SIRT1/AMPK signaling pathway,thereby reducing lipids in podocytes Deposition and damage.Resveratrol(RSV)is a polyphenolic compound that can be extracted from various plants.Previous basic studies have shown that RSV can regulate the kidney through multiple pathways,including apoptosis,oxidative stress,endoplasmic reticulum stress,inflammation,autophagy and other mechanisms.However,there are few studies on the upstream regulatory mechanisms leading to these reactions.In this study,through clinical renal tissue biopsy,it was observed that lipid metabolism related indexes in DKD patients at different stages were closely related to DKD kidney injury,and there were lipid deposition and abnormal expression of JAML/SIRT1 lipid de novo synthesis pathway in the kidneys of DKD patients.The results of animal experimental platform showed that RSV intervention can improve the blood glucose,blood lipid and kidney function of mice induced by high fat diet,and improve the lipid deposition in the kidney by affecting the JAML/SIRT1 de novo lipid synthesis pathway.The effect and mechanism of RSV on the lipid composition in MPC-5podocyocytes induced by palmitic acid were observed in vitro.This study provides theoretical basis and data support for the application of resveratrol in DKD to improve lipid deposition and damage in the kidneys.Part one Correlation between lipid metabolism and renal injury in patients with different stages of diabetic nephropathyObjective: By observing the changes of lipid metabolism and renal injury in patients with different stages of diabetic nephropathy,to explore the correlation between lipid metabolism and renal injury in patients with different stages of DKD and the possible mechanism of lipid deposition and renal injury in the kidneys.Methods: According to e GFR level,83 DKD patients were divided into DKD-G2 group,DKD-G3 group,and DKD-G4 group.At the same time,56 patients with type 2 diabetes without complications were selected as the control group.Basic information of enrolled patients was collected.The glycosylated hemoglobin,blood lipid,urea,serum creatinine,Superoxidedismutase(SOD),24 h urinary albumin and so on were determined in each group.CKD-EPI formula to estimate e GFR;Pathological changes of renal tissue(HE,PAS,Masson staining)were observed in some patients by renal biopsy.Changes in the levels of JAML,SIRT1,SREBP-1,Ch REBP and ADRP related to lipid synthesis pathways in kidney tissue can be observed by immunohistochemistry.The average optical density value of xanthoxpositive expression can be calculated using Image Pro Plus 6.0 software to analyze statistically.Results:1 Comparison of general clinical data and biochemical indicatorsCompared with the control group,TC,TG,LDL-C,Apo B,Lp(a)gradually increased,HDL-C,Apo A,SOD decreased gradually with the aggravation of the disease in patients with DKD(P < 0.05).2 Analysis of factors related to renal injuryDisease course,Hb A1 C,Apo B,LP(a),TC,LDL-C and TG were negatively correlated with e GFR,while HDL-C,SOD and Apo A were positively correlated with e GFR.TG,LDL-C and Apo B were independent risk factors for e GFR.Disease course,Hb A1 C,TC,TG,LDL-C,Apo B,LP(a)were positively correlated with 24 h urinary albumin,while HDL-C,Apo A,SOD were negatively correlated with 24 h urinary albumin.TG,TC and LP(a)were independent risk factors for 24 h urinary albumin.3 Kidney pathology and immunohistochemical results3.1 Compared with the control group,HE staining and PAS staining of DKD patients with different stages showed glomerular hypertrophy,increased mesangial matrix,narrowed glomerular capsule,and early changes of afferent arteriole hyaline degeneration.Later,the pathological changes were more obvious,and the DKD-G3 and DKD-G4 groups showed local sclerosis-like changes in the glomerulus and the formation of K-W nodules.Masson staining showed renal interstitial fibrosis-like changes in the DKD-G3 and DKD-G4 groups.3.2 Compared with the control group,the expression of ADRP,a lipid component in the kidneys of patients with different stages of DKD,was increased,and the expressions of DKD-G3 and DKD-G4 groups were higher than those of the DKD-G2 group(P < 0.001).3.3 Compared with the control group,the expressions of JAML,SREBP-1,Ch REBP and ADRP in the kidneys of patients with different stages of DKD by immunohistochemistry were significantly increased,and the expression of SIRT1 was significantly decreased,and with the aggravation of DKD,the above expressions were abnormal(P < 0.05 or 0.001).3.4 Spearman correlation analysis results showed that SIRT1 was positively correlated with e GFR,while JAML,SREBP-1,Ch REBP and ADRP were negatively correlated with e GFR.SIRT1 was negatively correlated with 24 h urinary albumen,while JAML,SREBP-1,Ch REBP and ADRP were positively correlated with 24 h urinary albumen.Summary:1.Patients with different stages of DKD have different degrees of abnormal lipid metabolism.The more serious the disease,the more abnormal blood lipid related indicators.2.Renal lipid deposition and the expression of JAML/SIRT1 lipid de novo synthesis pathway were impaired in patients with different stages of DKD.Part two Effects of resveratrol on high-fat-induced nephrolipotoxicity in mice based on JAML/SIRT1 lipid de novo synthesis pathwayObjective: To observe the effects of resveratrol on blood glucose,blood lipid,renal function and JAML/SIRT1 lipid synthesis pathway,lipid deposition and lipotoxicity in C57BL/6J mice induced by high-fat diet.Methods: 6-week-old male C57BL/6J mice(n = 30)were divided into two groups: Control group(n = 10)and high fat group(HFD,n = 20).Mice in Control group were fed conventional diet,and mice in HFD group were fed high fat content diet.After 12 weeks of the above process,20 mice in HFD group were randomly divided into HFD + RSV group(n = 10)and HFD group(n = 10).The mice in the HFD + RSV group were given resveratrol,while Control group and HFD group were given corresponding solvent control.Each group received intervention for a total of 6 weeks.The changes of blood sugar,body weight,blood lipids and renal function indexes of mice were observed,and the renal tissue was stained with HE,PAS,Masson,Oil Red O andBODIPY to observe lipid deposition and pathological changes in renal tissue.m RNA and protein were extracted from each group,and the expressions of JAML,SIRT1,SREBP-1,Ch REBP and ADRP in the kidneys of mice in each group were detected by real-time quantitative polymerase chain reaction(RT-PCR)and Western blot.Results:1 Effects of resveratrol on body weight and IPGTT blood glucose in high-fat-induced miceAfter 12 weeks of high-fat diet intervention,the body weight and blood sugar of the mice in the HFD group were significantly higher than those in the control group.Compared with the HFD group,the body weight and blood sugar of the mice in the HFD + RSV group decreased.2 Effects of resveratrol on blood lipids,renal function,histopathology and lipid deposition in high-fat diet-induced mice2.1 Compared with the Control group,the levels of TC,TG,LDL-C,BUN,and SCr in the HFD group were increased,and the HDL level was decreased.Compared with HFD Group,the levels of TC,TG,LDL-C and BUN in HFD +RSV group were significantly lower,while the levels of HDL-C were significantly higher.2.2 In HFD group,HE-stained glomeruli were enlarged,Mesangium matrix was increased,renal tubule cells were enlarged and swollen,and multiple vacuoles were formed.The injury scores of renal tubules and glomeruli in HFD group were significantly higher than those in Control group.PAS glycogen staining was more obvious.Masson staining showed early mild fibrotic changes.In HFD + RSV group,all the above pathological changes were alleviated,and the injury scores of renal tubules and glomeruli were decreased.2.3 Compared with the HFD group,the positive expression of Oil Red O in the kidney tissue of the HFD + RSV group mice was significantly reduced,and the fluorescence expression of BODIPY lipid staining was reduced.Compared with the HFD group,the glomerular basement membrane thickness and foot process fusion of the mice in the HFD + RSV group were reduced under electron microscope.3 Effects of resveratrol on lipid synthesis pathway in high-fat-induced mouse kidneyCompared with the HFD group,the protein and mRNA expressions of JAML,SREBP-1,Ch REBP and ADRP in the kidney tissue of the HFD + RSV group were decreased,and the activation of SIRT1 was increased.Summary:1.Resveratrol intervention for 6 weeks can reduce the increase of blood glucose,blood lipid and urea nitrogen in C57BL/6J mice induced by high-fat diet.2.Resveratrol intervention for 6 weeks can improve the abnormal expression of JAML/SIRT1 lipid de novo synthesis pathway in the kidneys of C57BL/6J mice induced by high-fat diet,and reduce lipid deposition and damage in the kidneys.Part three To investigate the effect and mechanism of resveratrol on palmitic acid-induced lipid deposition in MPC-5 podocytes by regulating the expression of JAMLObjective: Using silencing si RNA and overexpression plasmid to up-regulate and down-regulate the expression of JAML,to explore whether resveratrol can regulate JAML,activate SIRT1,inhibit the expression of SREBP-1 and Ch REBP,thereby improving palmitic acid-induced lipid deposition in MPC-5 podocytes.Methods:1.The CCK-8 cytotoxicity test was used to select the optimal concentration of palmitic acid(PA)and resveratrol on MPC-5 podocytes.MPC-5 podocytes were divided into groups: Control group,PA group(PA 200μM intervention for 24 h),PA + RSV 10 group(PA 200 μM for 24 h and then RSV 10μM intervention for 24 h),PA + RSV 20 group(PA 200 μM intervention After 24 h,RSV 20 μM intervention was given for 24 h).The content of TG in the cells of each group was detected,and the lipid composition in the cells was observed by Oil Red O and BODIPY staining.The expression position and level of JAML in the cells of each group were observed by immunofluorescence localization staining.The protein and m RNA expressions of JAML,SIRT1,SREBP-1,Ch REBP and ADRP were detected by RT-PCR and Western blot.2.Using siRNA technology to knock down JAML expression,MPC-5podocytes were divided into four groups: Control group,PA group(PA 200μM intervention for 24 h),PA + si RNA-NC group(transfected with blank si RNA for 24 h and then added with PA 200 μM intervention for 24 h),PA +si RNA-JAML group(transfected with JAML si RNA 24 h added PA 200 μM for 24 h).After group intervention,the procedures of RT-PCR and Western blot detection were the same as before.3.The expression of JAML was up-regulated using the transfection technique of overexpressed plasmid,and the MPC-5 podocytes were divided into four groups: Control group,Control + PI-NC group(blank plasmid transfection for 24 h),PI-JAML group(transfection with JAML overexpressed plasmid for 24 h),RSV + PI-JAML group(transfection with JAML overexpressed plasmid for 24 h followed by RSV 20 μM intervention 24 h).After group intervention,the procedures of RT-PCR and Western blot detection were the same as before.Results:1 Effects of different concentrations of palmitic acid and resveratrol on the survival of MPC-5 podocytesThe results of the CCK-8 cytotoxicity experiment showed that the optimal intervention concentration of palmitic acid was 200 μM,and the optimal intervention concentration of resveratrol was 10 μM and 20 μM,so the above concentrations were selected for subsequent experiments.2 The effect of resveratrol on palmitic acid-induced lipid deposition in MPC-5podocytesCompared with the PA group,the lipid components in the MPC-5 podocytes in the PA + RSV 10 group and the PA + RSV 20 group were significantly reduced by oil red O staining and BODIPY staining,and the semi-quantitative analysis showed that the difference was statistically significant.The decrease of intracellular lipid droplets in PA + RSV 20 group was better than that in PA + RSV 10 group.3 Effects of resveratrol intervention on palmitic acid-induced triglyceride levels in MPC-5 podocytesCompared with PA group,the content of TG in MPC-5 podocytes in PA +RSV 10 and PA + RSV 20 groups was significantly decreased,and PA + RSV20 group was better than PA + RSV 10 group.4 The effect of resveratrol on palmitic acid-induced JAML/SIRT1 lipid de novo synthesis pathway in MPC-5 podocytes4.1 Compared with the control group,the expression of JAML in MPC-5podocytes was significantly increased under the intervention of palmitic acid,and the expression was mainly located in the cytoplasm of MPC-5 podocytes compared with the control group,while the expression of JAML decreased after the intervention of resveratrol for 24 h.4.2 Compared with the PA group,the protein and mRNA expressions of JAML,SREBP-1,Ch REBP and ADRP in the MPC-5 podocytes of the PA + RSV 10 and PA + RSV 20 groups decreased,while the expression level of SIRT1 increased.5 The effect of silencing JAML in MPC-5 podocytes on palmitic acid-induced intracellular SIRT1,SREBP-1,Ch REBP and ADRPCompared with the PA group,the protein and mRNA expressions of SREBP-1,Ch REBP and ADRP in the PA + si RNA-JAML group were decreased,and the expression of SIRT1 was increased.6 To observe the effect of resveratrol on JAML and its downstream SIRT1,SREBP-1,Ch REBP and ADRP after transfection with overexpressed JAML plasmidCompared with Control group,the expression of SIRT1 in MPC-5podocytes in PI-JAML group decreased,while the expression of JAML,SREBP-1,Ch REBP,ADRP protein and m RNA in PI-JAML group increased significantly.Compared with PI-JAML group,the expression of SIRT1 protein and m RNA in MPC-5 podocytes in RSV + PI-JAML group increased,while the expression of JAML,SREBP-1,CHREBP and ADRP in MPC-5 podocytes in RSV + PI-JAML group decreased significantly.Summary: Resveratrol can improve palmitate-induced lipid deposition in MPC-5 podocytes by partially regulating the JAML/SIRT1-mediated de novo lipid synthesis pathway of SREBP-1 and Ch REBP.Conclusion:1.Lipid metabolism disorder is closely related to DKD kidney damage.The more lipid deposition,the more serious the DKD.Abnormal expression of JAML/SIRT1 lipid synthesis pathway-related proteins in the kidneys of DKD.2.Resveratrol intervention can improve the levels of body weight,blood glucose,blood lipid and urea nitrogen in mice induced by high fat diet,improve lipid deposition in the kidney,and alleviate diabetic kidney injury.One of the mechanisms is the regulation of JAML/SIRT1 protein and m RNA expression. |
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