The Protective Effect Of The Combination Of Triptolide And Irbesartan On The Podocytes In Type 2 Diabetic Rat Model And Its Mechanism

PART ONE: The study of type 2 diabetic rat model and nephropathyBackground and Objective:Diabetic nephropathy is the major cause of end-stage renal disease(ESRD) in the worldwide.The majority of these patients are affected by type 2 diabetes.Animal models of diabetes lay particular emphasis on large dose streptozocin induced type 1 diabetes.For this reason,it is important to establish animal models which simulate the common manifesitation of type 2 diabetes in human population,but the methods of animal models of type 2 diabetes differ,and their characteristic differ too.The study aims at study the feasibility of the high-sucrose-high-fat diet and injected with the low dose STZ induced the model of type 2 diabetic rat,and observed the characteristic of nephropathy.Methods:Wistar rats were fed with regular chow or high-sucrose-high-fat diet. After 8 weeks,rats fed with high-sucrose-high-fat diet were injected with a low dose of STZ(30mg.kg^-1) into abdominal cavity to induce hyperglycemia.Pats fed with regular chow receive vehicle.Determine systolic blood pressure,serum glucose, fasting seruminsulin,serum fat,serum creatinine,blood urine nitrogen,urinary albumin excretion and kidney construction by optical microscope and electron microscope.Results:The rats fed with high-sucrose-high-fat diet and injected with a low dose STZ showed increase in body weight,systolic blood pressure,blood glucose,fat and serum insulin levels in comparison with normal control group,while insulin seneitivity index(ISI) was reduced.At the 8 week after DM,kidney weight/body weight,urinary albumin excretion,blood urea nitrogen,serum creatinine,mean glomerular area,mean mesangial matrix area,mean mesangial matrix area/mean glomerular area ratio were significantly increased as compared with normal control group,but the capillary plexus area/mean glomerular area ratio was reduced. Ultrastructure of kidney damaged.Conclusion:Rats fed a high fat diet and given a low dose of STZ develop type 2 diabete with insulin resistance,hyperinsulinemia,moderate hyperglycemia, hyperlipidemia,hypertension and proteinuria,kidney pathology lesions.We present a nongenetic rat model of type 2 diabetes mellitus and nephropathy.The Type 2 diabetic model rats were successed.PART TWO: The protective effect of the combination of triptolide and irbesartan on the podocytes in type 2 diabetic rat model and its mechanismBackground and Objective:Diabetic nephropathy is one of the most common complications,and also is the major cause of end-stage renal disease(ESRD) in the worldwide.Recently,the researches show that diabetic nephropathy is inflammatory disease induced by metabolic disorder and macrophages have been thought to play a central role in the progression of diabetic nephropathy.Inflammation of renal tissue relates to glomerular filtration barrier injury and proteinuria in diabetic nephropathy. Podocytes locate in the exothecium of filtration barrier.Podocyte injury participates in the early stage of diabetic nephropathy.The studies indicated immune depressant-Tripterygium wilfordii can decrease proteinuria,but its mechanism is not still clear. The study aims to investigate the protective effect of the combination of triptolide and irbesartan on the podocytes in type 2 diabetic rat model,and evaluates its machanism.Methods:Wistar rats were fed with high-sucrose-high-fat diet for 8 weeks,and rats were injected with a low dose of STZ(30mg.kg^-1) into abdominal cavity to induce type 2 diabetic rat model.DM rats were randomly divided into 4 groups:2 diabetic model group(DM),triptolide treatment group(DT),irbesartan treatment group(DI) and triptolide combined with irbesartan treatment group(DTI).In addition,the normal rats served as a normal control group(NC).All the rats were received daily gavage respectively for 8 weeks.The urinary albumin excretion(UAL),body weight(BW), kidney weight(KW),KW/BW,glucemia,urea nitrogen,creatinine,Ccr,total cholesterol,triacylglycerol were detected with correlative methods and the pathological changes of kidney were also detected with optic microscope and transmission electron microscope.The expressions of Nephrin,Podocin and BMP-7, CTGF,TGF-β₁ mRNA and proteins were detected by immunohistochemistry, real-time PCR and Western blot.Results:(1) Rats fed a high fat diet and given a low dose of STZ develop type 2 diabete with insulin resistance,hyperinsulinemia,moderate hyperglycemia, hyperlipidemia,hypertension and proteinuria,kidney pathology lesions.(2) Compared to NC,UAL in 24 hours,KW and Ccr were increased significantly (P＜0.01).Kidney lesion was severe,and GSI,RIFI were increased than NC (P＜0.01).The expressions of Nephrin,Podocin and BMP-7 mRNA and proteins were down-regulated in glumorular,and CTGF,TGF-β₁ mRNA and proteins were up-regulated significantly(P＜0.01).Correlation analysis showed that there were negative correlation between UAL and Nephrin,Podocin,BMP-7 mRNA(r₁=-0.825, r₂=-0.769,r₃=-0.784,P＜0.01) and there were positive correlation between UAL and CTGF,TGF-β₁ mRNA(r₁=0.537,r₂=0.628,P＜0.01).Nephrin correlated positively with BMP-7 mRNA(r=0.737,P＜0.01),and correlated negatively with CTGF,TGF-β₁ (r₁=-0.675,r₂=-0.802,P＜0.01).Podocin correlated positively with BMP-7 mRNA(r=0.641,P＜0.01),and correlated negatively with CTGF,TGF-β₁(r₁=-0.508, r₂=-0.752,P＜0.01).(3) Decrease UAL occurred in all treatment groups.A better improvement on the ultrastructure of the podcytes and alleviated the damage of kidney(P＜0.01). Meanwhile,the expressions of Nephrin,Podocin and BMP-7 were up-regulated remarkably in renal tissue,and CTGF,TGF-β₁ were down-regulated remarkably in renal tissue.These changes were the best in DTI(P＜0.05).Conclusion:There was podocyte damage in the early stage of DN,which related to inflammatory reaction and cytokine disbalance.Renoprotection of triptolide on type 2 diabetic rats may be mediated,at least partly,suppressing inflammatory reaction and balancing cytokine and attenuating podocyte injury.PART THREE: The protective effect of triptolide on the renal tubule-interstitium in type 2 diabetic rat model and its machanismsBackground and Objective:Recently,the researches indicated that diabetic nephropathy was inflammatory disease induced by metabolic disorder and macrophages have been thought to play a central role in the progression of diabetic nephropathy.Mycophenolate mofetil(MMF),an anti-inflammatory agent,has been shown to suppress macrophage infiltration and to improve renal injury in streptozotocin-induced diabetic kidneys.We examined whether triptolide,which acted through immunosupres- sive mechanisms,inhibits progression of diabetic nephropathy in 2 type diabetic model rats.The study aimed to investigate the protective effect of triptolide on renal tubule-interstitium in type 2 diabetic rat model, and evaluate its machanism.Methods:Wistar rats were fed with high-sucrose-high-fat diet for 8 weeks,and rats were injected with a low dose of STZ(30mg.kg^-1) into abdominal cavity to induce type 2 diabetic rat model.DM rats were randomly divided into 2 groups:type 2 diabetic model group(DM),triptolide treatment group(DT).In addition,the normal rats served as a normal control group(NC).All the rats were received daily gavage respectively for 8 weeks.The urinary albumin excretion(UAL),bodyweight(BW), kidneyweight(KW),KW/BW,glucemia,urea nitrogen,creatinine,total cholesterol, triacylglycerol were detected with correlative methods and the pathological changes of kidney were also detected with optic microscope and transmission electron microscope.The expression of BMP-7,CTGF,OPN and TGF-β₁ mRNA and proteins were detected by immunohistochemistry,real-time PCR and Western blot.Results:(1) Rats fed a high fat diet and given a low dose of STZ developed type 2 diabetes with insulin resistance,hyperinsulinemia,moderate hyperglycemia, hyperlipidemia,hypertension and proteinuria,kidney pathology lesions.(2) Compared to NC,UAL,KW and KW/BW were increased significantly (P＜0.01).The expression of BMP-7 mRNA and proteins were down-regulated in renal tubule-interstitium,and CTGF,OPN and TGF-β₁ mRNA and proteins were up-regulated significantly(P＜0.01).Correlation analysis showed that there were negative correlation between UAL and BMP-7 mRNA(r=-0.784,P＜0.01) and there were positive correlation between UAL and CTGF,OPN and TGF-β₁ mRNA (r₁=0.537,r₁=0.502,r₃=0.628,P＜0.01).The number of ET-lpositive cells were directly correlated with RIFI(r=0.585,P＜0.01),also correlated with mRNA expression of OPN,TGF-β₁ in renal interstitium(r₁=0.492,P＜0.05,r₂=0.406,P＜0.05). mRNA expression of TGF-β₁ correlated positively with CTGF(r₁=0.662,P＜0.01),and correlated negatively with BMP-7(r=-0.604,P＜0.01).The ratio of atrophic renal tubule and interstitium fibrosis all correlated positively with CTGF,OPN and TGF-β₁ (P＜0.05),and correlated negatively with BMP-7(P＜0.05).(3) Decrease in UAL occurred in triptolide treatment group.Triptolide treatment alleviated the damage of renal tubule-interstitium(P＜0.01).Meanwhile,the expression of BMP-7 was up-regulated remarkably in renal tubule-interstitium,and CTGF,OPN and TGF-β₁ were down-regulated remarkably in renal tubule-interstitium.Conclusion:Renoprotection of triptolide on renal tubule-interstitium in type 2 diabetic rats may be mediated,at least partly,suppressing macrophage accumulation, down-regulated the expression of CTGF,OPN and TGF-β₁ in renal tissue,meanwhile up-regulated the expression of BMP-7.Triptolide had protective effect on diabetic nephropathy by multitarget,polyols pathway.