| Objective1. To establish rats model of the early stage of type 2 diabetic nephropathy.2. To investigate the effects of Yi-Qi-San-Ju-Gu-Shen-Fang(YQS) on microalbuminuria, glucose and lipid metabolism, and insulin resistance of the early stage of type 2 diabetic nephropathy in rats.3. To investigate the effects of YQS on histopathologic change and diabetic nephropathy related proteins in renal tissue of early type 2 diabetic nephropathy rats.4. To investigate the effects of YQS on renal renin-angiotensin system(RAS) of early type 2 diabetic nephropathy rats.5. The aim of the present study is to investigate the effects of YQS on early diabetic nephropathy in relation to ameliorating insulin resistance and inhibiting renal RAS.Methods1. To establish rats model of the early stage of type 2 diabetic nephropathySixty-five Sprague-Dawley rats (male, weight 180-220g) were randomly divided into two dietary regiments consisting of 10 and 55 rats by feeding either normal pellet diet (NPD) or high fat diet (HFD). After 4 weeks of dietary manipulation, the 55 HFD-fed rats were injected intraperitoneally (i.p.) with low-dose STZ (35 mg/kg), while the 10 NPD-fed rats were injected i.p. vehicle citrate buffer (pH 4.4) in a dose volume 1 ml kg-1.3 days after the STZ or vehicle injection, non-fasting blood glucose (NFBG) was measured in whole blood collected from the tail vein. Rats with NFBG level?16.67 mmol/L were considered type 2 diabetic model.7 days after the STZ or vehicle injection, fasting blood was collected from retro-orbital plexus of the rats under light ether anesthesia using capillary tubes, 24h urine was selected, Intraperitoneal glucose tolerance test (IPGTT) and Insulin tolerance test (ITT) were performed. Analyze body weight, glucose, lipid profile, insulin, HOMA-IR, glucose tolerance, insulin sensitivity, urinary microalbumin excretion rate (UAER), urinary microalbumin creatinine ratio (ACR), and serum creatinine for assessing the early type 2 diabetic nephropathy rats model with insulin resistance. Investigation lasted 9 weeks to assess the stability of the rats model. The rats were fed on their respective diets until the end of the study.2. To investigate the effects of YQS on microalbuminuria, glucose and lipid metabolism, and insulin resistance of the early stage of type 2 diabetic nephropathy in ratsThe model rats further divided into 6 subgroups:the type 2 diabetes model group, low-dose, moderate-dose and high-dose YQS groups (0.47,1.41,2.82 g/100g per day, respectively), Losartan group (20 mg/kg per day), and rosiglitazone group (4 mg/kg per day). Age-matched NPD-fed rats served as controls. Administration of YQS, losartan or rosiglitazone lasted 8 weeks.Fasting blood was collected from retro-orbital plexus of the rats under light ether anesthesia using capillary tubes, IPGTT and ITT were performed, and 24h urine was selected at 4 weeks and 8 weeks after treatment (viz.5 weeks and 9 weeks after the STZ or vehicle injection). At the end of the study, rats were sacrificed under anesthesia and skeletal muscle and kidney tissues were geted.Analyze body weight, glucose, lipid profile, insulin, HOMA-IR, glucose tolerance, insulin sensitivity, urine albuminuria excretion rate (UAER), urine albuminuria creatinine ratio (ACR), and serum creatinine. The cytoplasm and plasma membrane GLUT-4 expression in skeletle muscle tissues of control group, model group, high-dose YQS group, Losartan group, and Rosiglitazone group were studied by Western blot analysis.3. To investigate the effects of YQS on histopathologic change and diabetic nephropathy related proteins in renal tissue of early type 2 diabetic nephropathy ratsKidney sections were stained with HE and PAS for histopathologic analysis.The expression of nephrin, VEGFR2, Phospho-VEGFR2(Tyr951), Phospho-VEGFR2(Tyr1175), TNF-a and IL-6 in kidney tissues of control group, model group, high-dose YQS group, Losartan group, and Rosiglitazone group were studied by Western blot analysis.4. To investigate the effects of YQS on renal renin-angiotensin system(RAS) of early type 2 diabetic nephropathy ratsThe expression of Ang?and AT1 in kidney tissues of control group, model group, high-dose YQS group, Losartan group, and Rosiglitazone group were studied by Western blot analysis.Results1. To establish rats model of the early stage of type 2 diabetic nephropathy with insulin resistanceThere was significant elevation of serum levels of fasting glucose (12.82±1.08mmol/L vs.3.60±0.31mmol/L, P<0.01) insulin, HOMA-IR, body weight, UAER (52.59±6.61ug/24h vs.5.01±0.56ug/24h, P< 0.01), ACR (17.57±3.37ug/umol vs.8.94±1.19ug/umol, P<0.01), and total cholesterol (8.40±0.49mmol/L vs.2.14±0.06mmol/L, P<0.01), triglyceride (2.24±0.12mmol/L vs.0.78±0.05mmol/L, P<0.01), low-density lipoprotein cholesterol (7.84±0.51mmol/L vs.0.72±0.04mmol/L, P<0.01) and decrease in high-density lipoprotein cholesterol (0.57±0.03mmol/L vs.1.27±0.03mmol/L, P<0.01) in the low-dose streptozotocin and high-fat diet induced early type 2 diabetic nephropathy rats model group when compared with the normal pellet diet group. And serum creatinine was not changed significantly. IPGTT and ITT resluts demonstrated the model rats presented insulin resistance. These results indicated the successful induction of early type 2 diabetic nephropathy rat model with insulin resistance.In the whole period of study, fasting serum glucose, lipid profile, UAER, and ACR of the model rats were significantly higher (except HDL-c was reduced) compared to the NPD group, serum creatinine was not changed, and model rats presented insulin resistance. This indicated that the model was stable.2. To investigate the effects of YQS on microalbuminuria, glucose and lipid metabolism, and insulin resistance of the early stage of type 2 diabetic nephropathy in ratsAdministration of YQS induced dose- and time-dependent amelioration in microalbuminuria, glucose and lipid metabolism, and insulin resistance.After 8 weeks treatment, compared with the model group, high-dose YQS (2.82 g/100g per day) group reduced UAER (29.64±6.32ug/24h vs.51.50±6.54ug/24h, P <0.05), ACR (8.52±0.65ug/umol vs.17.21±3.32ug/umol, P<0.01) significantly, similarly with Losartan group; improved the disturbance of glucose and lipid metabolism:reduced serum glucose (12.16±1.00mmol/L vs.17.65±2.22mmol/L, P <0.01), similarly with Rosiglitazone group, reduced triglyceride (0.87±0.15mmol/L vs.1.99±0.26mmol/L, P< 0.01), total cholesterol (3.31±0.52mmol/L vs. 6.50±1.04mmol/L, P<0.01), low-density lipoprotein cholesterol (2.47±0.50mmol/L vs.6.00±1.07mmol/L,P<0.01) significantly, and increased high-density lipoprotein cholesterol (0.84±0.08mmol/L vs.0.50±0.03mmol/L,P<0.01) significantly, more effective than Losartan or Rosiglitazone group; the body weight was not changed significantly; amliorated insulin resistance:decreased HOMA-IR score, IPGTT results demonstrated the glucose intolerance was attenuated, ITT results demonstrated the insulin sensitivity was elevated, enhanced GLUT-4 translocate from the cytoplasm to the plasma membrane in skeletal muscle tissues, similarly with Rosiglitazone group.3. To investigate the effects of YQS on histopathologic change and diabetic nephropathy related proteins in renal tissue of early type 2 diabetic nephropathy ratsAfter 8 weeks treatment, glomerular basement membranes (9.80±1.02um vs. 13.83±2.37um, P<0.05) and extracellular matrix (0.23±0.03 vs.1.06±0.22, P<0.01) was significantly decreased in the high-dose YQS (2.82 g/100g per day) group compared with the model group.Compared with the model group, high-dose YQS group increased the expression of nephrin, decreased the expression of VEGFR2, Phospho-VEGFR2(Tyr951), Phospho-VEGFR2(Tyr1175), TNF-?and IL-6 in kidney tissues.4. To investigate the effects of YQS on renal renin-angiotensin system(RAS) of early type 2 diabetic nephropathy ratsCompared with the model group, the expression of Ang?and AT1 in kidney tissues were reduced in high-dose YQS group.Conclusions1. High fat diet with STZ 35 mg/kg injection induced an early stage of type 2 diabetic nephropathy rat model with insulin resistance. This model could be used in medicine screening and the mechanisms study.2. YQS treatment could ameliorate microalbuminuria of early stage of type 2 diabetic nephropathy in rats, similarly with losartan treatment. YQS treatment could lessen thickened glomerular basement membranes and accrementition of extracellular matrix; increase the expression of nephrin; decrease the exprssion of VEGFR2, p-VEGFR2(Tyr951), p-VEGFR2(Tyr1175), TNF-?and IL-6 in kidney tissues of the early type 2 diabetic nephropathy rats. YQS treatment could inhibit renal renin-angiotensin system by reducing the expression of Ang?and AT1 in kidney tissues of the early type 2 diabetic nephropathy rats. Administration of YQS induced dose- and time-dependent beneficial effects on early type 2 diabetic nephropathy in rats. High dose exhibited best effect.3. YQS treatment could improve the disturbance of glucose and lipid metabolism of early stage of type 2 diabetic nephropathy in rats. Reduce glucose level similarly with rosiglitazone, mediate dyslipidemia effect was more effective than that of losartan or rosiglitazone treatment. YQS treatment could ameliorate insulin resistance, enhance GLUT-4 translocate from the cytoplasm to the plasma membrane in skeletal muscle tissues, similarly with rosiglitazone, but did not cause any increase in body weight.In a word, YQS could attenuate microalbuminuria, ameliorate the disturbance of glucose and lipid metabolism, improve renal histopatholofic changes, and regulate related proteins of type 2 diabetic nephropathy in rats. The beneficial effects may be in relation to ameliorating insulin resistance and inhibiting renal renin-angiotensin system, similarly with rosiglitazone and losartan. It demonstrated the multiple-aspects and multiple-targets character of Chinese medicine. Our research could be an experiment support for prevention and treatment of diabetic nephropathy. |
PDF Full Text Request