| With the development of economic material and increasing incidences of type 2 diabetes,diabetic nephropathy(DKD)is one of the most serious chronic complications of diabetes,which is the second leading cause of end-stage renal disease(ESRD)in China.Early manifestations of DN are microalbuminuria,followed by persistent proteinuria,edema,hypertension,renal insufficiency,and ultimately developed to ESRD.Histological features of DN are the deposition of collagen,fibronectin and other extracellular matrix(ECM)components in the glomerular mesangial and tubulointerstitial,eventually leading to irreversible renal fibrosis.Long-term hyperglycemia,lipid metabolic disorders,hemodynamic abnormalities and other factors lead to renal mesangial cell damage,releasing monocyte chemoattractant protein-1(MCP-1),Fkn and other inflammatory factors.These inflammatory factors could activate monocytes/macrophages in renal tissue to promote the ECM deposition;on the other hand,they cause FN,CoIV and other collagen metabolism imbalance,ECM accumulation by stimulating TGF-other collagen metabolism imbalan,leading to renal fibrosis.In recent years,many scholars study the pathology of DKD in molecular level,discover many signaling pathways like AMPK(Adeosine 5'-monophosphate-activated protein kinase)and JNK(c-Jun N-terminal kinase),and find the mediation from abnormal activity of oxidative stress and ER stress onto the development of DKD.However,the mechanism of DKD is still unclear,and the effective method to treat DKD is still unavailable.Glomerular mesangial disease is one of the most prominent pathological changes of diabetic nephropathy.Mesangial cells are the main cells in the glomeruli.They are located in the mesenchymal matrix of the renal capillaries.The morphology is irregular and the nuclei are small and stained.Cells have bulges protruding into the endothelial cells and between the basement membrane,and can also be through the endothelial cell gap into the capillary cavity.Mesangial cells through myosin,actin to contraction,regulation of glomerular capillary diameter,thus affecting its blood flow.Mesangial cells also have endocrine function,secretion of renin and a variety of enzymes,the glomerular blood flow can be local regulation.Mesangial cells undergo phenotypic changes from normal stationary phenotypes to proliferative/secretory phenotypes that may be the cytological basis for the abnormal synthesis of mesangial cells and secretion of ECM.a-SMA will not be expressed in mature mesangial cells,but is a marker of activated mesangial cells under high glucose condition.Mesangial cell phenotype changes are important reasons for the progress of diabetic nephropathy and chronic renal fibrosis.The main manifestations of mesangial cell hypertrophy,abnormal expression of a-SMA,extracellular matrix(ECM)secretion and so on.The expression and activity of MMPs were closely related to the phenotypic changes of mesangial cells.The lower the expression and activity of MMPs,the more extracellular matrix accumulation and the more obvious glomerular fibrosis.Peroxisome Proliferator Activated Receptors(PPARs)are nuclear hormone receptors and transcription factors.It has been found to have three subtypes:PPAR-a,PPAR-?,PPAR-?.A series of studies have shown that PPARs play an important role in regulating lipid metabolism,insulin sensitivity,immune response,cell growth and differentiation.PPARs also participate in metabolic syndrome such as insulin resistance,hyperlipidemia,hypertension,Hardened,microalbuminuria.PPARy is a class of ligand-activated nuclear receptor transcription factor,belonging to the hormone nuclear receptor superfamily.It is highly expressed in adipose tissue and plays an important role in regulating lipid metabolism,insulin sensitivity,inflammation and immune regulation,extracellular matrix regulation and cell growth and differentiation.Anti-diabetic drugs Insulin sensitizers Thiazolidinedione(TZD),such as rosiglitazone and troglitazone,are PPAR-y-specific ligands.TZD binds to PPAR-gamma and regulates the transcription of various genes associated with insulin effects.More and more clinical and evidence-based medical evidence suggests that TZD has great benefits in protecting renal function in patients with diabetic nephropathy and non-diabetic nephropathy,but the mechanism remains unclear.PPAR-? is mainly distributed in some lipolytic metabolic active organs or tissues(such as liver,heart,adipose tissue,skeletal muscle,vascular endothelial cells,atherosclerotic plaques,etc.)are the main transcriptional regulators of fatty acid oxidase genes,Lipid uptake and oxidation,regulation of amino acid metabolism,participation in hemostasis,inflammatory response,lipid metabolism disorders,atherosclerosis and coronary heart disease and other pharmacological physiological processes.Studies have shown that excessive cholesterol and triacylglycerol are related to renal ectopic deposition and DKD-caused glomerulosclerosis,and PPARa agonists fenofibrate can improve hypertrophy in spontaneously hypertensive rats,which may be through the role of lipid lowering,such as its anti-inflammatory and immune regulation.Astilbin can activate PPAR?/? expression in order to improve insulin sensitivity,and can reverse the renal tubular epithelial transdifferentiation,which we speculate that Astilbin can inhibit diabetic nephropathy and renal fibrosis development via activating PPAR?/? expression.Recently,PPAR?/? double agonists have gradually entered the field of vision of researchers,which are being tested clinically and are intended for the treatment of type 2 diabetes with cardiovascular complications.DKD belongs to the scope of traditional Chinese medicine Diabetes,the basic pathogenesis:lack of endowment,spleen and kidney deficiency-based,hot and humid intrinsic,blood stasis as the standard.In recent years,pathogenesis of DN was discussed,such as mitogen-activated protein kinase(MAPK),transforming growth factor/silk/threonine kinase receptor(TGF-?1/Smad),JAK/STAT and other signaling pathways lead to the development of DN,but the DN mechanism is not yet fully understood,the current lack of progress for the treatment of diabetic nephropathy effective treatment.Astilbin is a kind of flavonoids extracted from traditional Chinese medicine Poria cocos.Studies have shown that astilbin have anti-inflammatory and immunomodulatory effects.Recently,it was reported that astilbin can inhibit the chemokine MCP-1 and cytokines IL-6 and IL-18 in rat ischemia-reperfusion model.However,to the best of our knowledge,the investigation on the role of astilbin in diabetic nephropathy has not been reported so far.Our previous studies have found that astilbin can activate PPAR6 a and thus to improve insulin sensitivity and reverse the renal tubular epithelial transdifferentiation,but the effect of astilbin in diabetic nephropathy is not clear.In this study,we established the model of diabetic nephropathy in rats,and elucidated the mechanism and mechanism of indigestin in diabetic nephropathy.In this study,we first utilize cytokine chip technique to analyze the variation of Thl/Treg cells and peripheral blood inflammatory related factors of different diabetes patients;based on involvement of PPAR-?/?.in diabetic nephropathy,building diabetes rat model and in vitro cell test demonstrate the utility and mechanism of astilbin to reduce diabetic nephropathy and provide a new method to treat diabetic nephropathy.Part1 Detection of Thl/Treg cells expression and serum cytokines in Fulminant Type 1 DiabetesObjective:To investigate the role of Thl/Treg cells and various cytokines in Fulminant Type 1 Diabetes.Methods:20 normal controls,20 Type 2 DM,18 Classic type 1 DM,and 20 FTIDM in our hospital were enrolled in the study group.We determined the imbalance of Thl/Treg cells and compared the changes within different cytokines,such as TNF-?,IL-6,IL-1?,IL-8,MIP-1?,IL-12p70,IP-10,IFN-y,IL-9,IL-15,IL-17A,IL-10,MCP-1,MIP-1?,Fractalkine,and MCP-3.Results:We found that CD4+CD25+Foxp3+ cells strikingly decreased in Type 1 DM and Type 2 DM.We also observed the cytokines associated with inflammation(TNF-?,IL-6,IL-1?,IL-8,MIP-1?)were significantly higher in FTIDM cases compared with those in classic type 1 DM cases(p<0.05).Thl associated cytokines(IL-12p70,IP-10,IFN-?,and IL-9)were also significantly increased in FT1DM cases compared with those in classic type 1 DM cases(p<0.05).There were no obvious differences in cytokines status(IL-15,IL-17A,IL-10,MCP-1,MIP-la,Fractalkine,and MCP-3)among controls,type 2 DM,classic type 1 DM and FTIDM cases(p>0.05).Conclusions:Our data offered evidence for the imbalance of Thl/Treg cells and changed cytokines in FTIDM patients,which may have significant impacts on the prevention and treatment of FT1DM patients.Part 2 The expression of PPAR-a/y in renal biopsy of diabetic nephropathyObjective:To observe the expression level of PPAR-?/? in renal biopsy of diabetic nephropathy.To analyze the correlation between PPAR-a/y and renal fibrosis.Methods:a total of 18 patients with diabetic nephropathy were enrolled in this study,including male and female,with a mean age of 72 years(range,9 months)and 8 months.Normal control group(8 cases).Immunohistochemical staining was used to detect the expression of PPAR-a/yin renal biopsy.Analysis of the correlation between PPAR-a/y and CollA1,Fibronectin expression.Results:in the control group,the immunohistochemical staining results showed that renal tissue is visible PPAR-?/? expression,but in the proximal tubules of renal tubular expression most,relatively small collection of distal tubules expressed in glomeruli and mesangial cells express more,diabetic nephropathy renal biopsy PPAR-a/y expression was significantly reduced compared with the the control group,reduced by nearly 50%.Semi quantitative analysis showed that the decrease of PPAR-a/y was negatively correlated with Kidney injury score.Conclusion:the expression of PPAR-a/y in renal tissue of patients with diabetic nephropathy was decreased,and PPAR-a/y was negatively correlated with Kidney injury score in diabetic nephropathy.Part 3 Astilbin in mesangial cells inhibits glucose induced transdifferentiation through activation of PPARa/yObjective:to reveal the effect of astilbin in vitro stimulation of mesangial cells in high glucose induced transdifferentiation.Methods:human mesangial cells were cultured in vitro,astilbin with different concentrations(5.5,15,20,30 mM),astilbin(25 mg/L)and high glucose pretreatment(30 mM).Western blotting and qRT-PCR were used to detect the expression of PPAR-?/?,as well as the expression of MMP-9.Results:(1)high glucose(15 mM)inhibited the expression of PPAR-a/y in the dose-dependent manner(35%,P<0.01),the inhibition peaks with 30 mM high glucose(80%,P<0.01),PPAR-a/y mRNA decreases at high glucose 15 mM(45%,P<0.01),and induced the differentiation of mesangial cells.MMP-9 decreases at 15 mM high glucose(P<0.05),but a-SMA elevates(P<0.05)(2)human mesangial cells were cultured in vitro,with 24h stimulation by 25mg/L astilbin,expression of PPAR-a double(P<0.05);PPAR-y increases 55%(P<0.05)(3)after high glucose stimulation,HG group a-SMA doubles expression compared to control group(P<0.05),but MMP-9 decreases 45%(P<0.05);astilbin can inhibit the increasing expression of a-SMA(30%lower than HG group,P<0.05),and inhibit the decreasing of MMP-9(55%higher than HG group,P<0.01)Conclusion:astilbin by activating the expression of PPAR-?/? inhibit tubular epithelial cell transdifferentiation,suggesting that astilbin play an important role in maintaining the normal function of human mesangial cells.Part 4 The effect of astilbin on diabetic nephropathy in ratsObjective:To study the mechanism of astilbin in diabetic nephropathy rats.Methods:70 weight 200-220g male healthy SD rats(45-50d)(purchased from Yangzhou University experimental animal center),adaptive feeding for 1 week,were randomly divided into normal control group(Cntl,10 rats),diabetic nephropathy group(DN,20 rats),diabetic nephropathy and low astilbin group(DN-LD-Ast group,10),diabetic nephropathy and medium astilbin group(DN-MD-Ast group,10),diabetic nephropathy and high astilbin group(DN-HD-Ast group,10)diabetic nephropathy and rosiglitazone group(DN+RGZ,10).Control group fed with normal diet and DN group fed by high-fat diet.After 12 weeks,left kidney resection,postoperative continue feeding a week.One week after injection of streptozotocin(STZ)40mg/kg intraperitoneal injection,the control group was injected with the same dose of 0.1mmol/L citric acid sodium citrate buffer(PH=4.4).Blood glucose was measured once a week,24 hours urine was collected in metabolic cage,urinary microalbumin was detected,and albumin/creatinine>30mg/g was successfully established in diabetic nephropathy.Astilbin was injected into abdominal cavity with respectively 1.25(LD-Ast),2.5(MD-Ast),5(HD-Ast)mg/kg/d dissolved in normal saline.12 weeks later,the mice were sacrificed and the serum,urine and renal tissue samples were obtained.The serum biochemical tests(cholesterol,triglycerides,LDL,Scr,BUN,Alb),Western blotting and qRT-PCR detection of PPAR-a/y,expression and fibroblast cell marker protein expression of a-SMA,TGF-?1 and Coll IV immunohistochemistry.Under PAS light microscope,observe renal morphological change.Results:(1)blood glucose was significantly increased in DN group,with median 26.24 mmol/L,LD-Ast and MD-Ast group has no difference compared to control group,but HD-Ast can significantly improve blood glucose;DN group rats decreases weights compared to control group,and their water intake and urine volume are ten times of those of control group.microalbuminuria was significantly higher in DN group,microalbuminuria in astilbin group was significantly decreased than DN group.(2)After astilbin interferes,different astilbin amount will cause different effect:LD-Ast and MD-Ast can lower the microalbuminuria level without statistic significance;HD-Ast can significant lower microalbuminuria level(P<0.01).In diabetic nephropathy rat model,TGF-?1 expresses not much in glomerulus;semiquantitative analysis shows that TGF-?1 raises three times in DN group,LD-Ast and MD-Ast can inhibit the elevation of TGF-(31 with 25%lower comparing to DN group.And,HD-Ast can decrease 50%(P<0.05).Compared to control group,CollA1 increases significantly in DN group,and LD-Ast could not influence this index,MD-Ast can partially inhibit its elevation(P<0.05),and HD-Ast can inhibit CollA1(P<0.01).Conclusion:astilbin can inhibit kidney fibrosis in diabetic nephropathy by activating PPAR-a/?expression in vivo. |
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