| Objective: Diabetic nephropathy (DN) is one of common and serious microvascular complications of diabetes, and is the main cause for end-stage renal disease and diabetes′death. Thus, effectively prevention the occurrence and development of DN is of great significance.This study was established by intraperitoneal injection of STZ to get diabetic rat models, then intervented by tonify-qi and nourish-yin, promote blood circulation (TNPC) recipe. By measuring the blood glucose, total cholesterol, triglycerides, 24-hour urinary albumin excretion rate, renal function, renal pathological changes, and the expression of bax and bcl-2 in renal tissue, the preventment and treatment of DN and possible mechanism were studied, to provide an experimental basis and theoretical basis for clinical application.Methods :Fifty clean healthy male Wistar rats with body weight 200g±10g, feed normal diet for one week to adapt. Urine sugar and urine protein were negative. After 12 hours fasting, 38 rats were taken out randomly to get diabetic models by intraperitoneal injection 45mg/kg of 1% new solution of streptozotocin. The remain 12 were normal control group, injected equal amount of 0.1mmol/L citrate buffer. After 72 hours, models′blood glucose were measured three times in 3 days. If blood glucose≥16.7mmol/L twice, the diabetic models succeeded. Models were divided into 3 groups randomly, i.e. model group, irbesartan-treated group, and Traditional Chinese Medicine (TCM) treatment group, 12 rats respectively. One week later, two treatment groups began ig at the dose of adult′s 10 times once a day, TCM group used TNPC recipe, 32g/(kg·d); Irbesartan group used irbesartan tablets, 25mg/(kg·d) . The normal group and model group were fed with equal amount of saline. After 8 weeks,6 rats taken from each group randomly sacrificed, after 12 weeks ,the remaining 6 sacrificed. On the day before sacrifice, the rats were placed in metabolic cages to collect 24-hour urine, noted urine volume, and measured urinary albumin excretion rate. Without food or water for 12 hours, body weight was weighted, blood glucose was measured from tail blood. The rats were anesthetized by intraperitoneal injection 2% pentobarbital. Sample was taken from femoral artery, so total cholesterol, triglycerides, blood urea nitrogen and creatinine clearance rate were tested. Rats were killed, then the right kidney was taken out, its capsule was removed, blood was siphoned by filter paper. After weighting, kidney's weight was recorded and the hypertrophy index (kidney weight/body weight,KW/BW) was calculated. For routine pathological observation and immunohistochemical detection of bax and bcl-2 expression, 4% paraformaldehyde fixed partial kidney tissue. Semi-quantitative analysis was completed by the pathological image analysis software.Results:1 The general state of trial rats:Normal control group rats′appetite, mental station, reaction and urine volume were normal, with body weight increased significantly. The other three groups manifested significant polydipsia, polyphagia, polyuria and poor mental state, slowly responsive, less dynamic, dull fur and small size after the injection of STZ. After 12 weeks, the mentioned manifestions were more remarkable than after 8 weeks, with the model group the most significant. In model group, two rats died after injection STZ 3 ~ 5 days, the cause maybe high blood sugar which may lead to metabolism disorder, lead to organ failure eventually.2 Urinary albumin excretion rate of 24-hourAfter 8 weeks and 12 weeks, for 24-hour urinary albumin excretion rate, diabetic rats′were significantly higher than the control group′s . The comparation was significant (p<0.01, p<0.05). Irbesartan group and TCM group increased more than model group significantly (p<0.05).There was no statistical significance (p>0.05) between Irbesartan group and TCM group. At 12 and 8 weeks′end, the changes in diabetic group were not significant (p>0.05).3 Blood glucose, total cholesterol and triglycerideDiabetic rats increased more blood glucose, total cholesterol (TC), and triglyceride (TG) compared with cotemporaneous control rats at 8 and 12 weeks′end (p<0.05). The difference between Irbesartan group, TCM group and model group was significant (p<0.05). There was no significant difference (p> 0.05) between irbesartan group and TCM group. Compared 12 weeks with 8 weeks, the blood glucose, TC and TG decreased in the each treated group, but without statistical significant (p<0.05).4 Renal function (BUN and creatinine clearance rate)As for 8 weeks and 12 weeks, diabetic rats′BUN increased while creatinine clearance rate decreased significantly (p<0.01) compared with the control group at cotemporaneous time.In irbesartan group and TCM group BUN decreased and creatinine clearance rate increased more than model group significantly (p<0.01).There was no statistical significance (p>0.05) between Irbesartan group and TCM group. At 12 and 8 weeks′end, BUN decreased and creatinine clearance rate increased in each treated group, but without statistical significant (p>0.05).5 Rats′body weight, right kidney weight, hypertrophy index (KW / BW)For 8 weeks and 12 weeks, diabetic rats′body weight dropped, right kidney weight and hypertrophy index raised, compared with the contemporary control group , the difference was significant (p<0.05). Irbesartan group and TCM group changed more significantly than model group (p<0.05).There was no statistical significance (p>0.05) between Irbesartan group and TCM group. At 12 and 8 weeks′end, the body weight and right kidney weight of model group, Irbesartan group and TCM group changed significantly (p<0.05).As for hypertrophy index changes, there was no significance (p>0.05).6 Light microscopy observation of renal tissue sectionsRoutine pathological observation: Under light microscope, HE and Masson stain. Compared with the control group, diabetic rats increased glomerular volume, mesangial matrix, mild proliferation of mesangial cells, bulb-like degeneration of partial tubular epithelial cells. At 12 weeks′end, the following changes were visible , partial tubular atrophy or expansion, hyaline cast formation, glomerular and tubular basement membrane thickening, matrix widened, fibrous tissue hyperplasia, adhesion of glomerular and capsule .7 Immunohistochemical detection of bax, bcl-2 expression7.1 Optical microscope observationBax expression in renal tissue: For normal control group, it expressed in the epithelial cells′cytoplasm of glomerular podocytes, proximal tubule, distal convoluted tubule and collecting ducts, etc. The diabetic group increased its expression in podocytes, increased more markedly in distal convoluted tubule and tubular-interstitial cells. After 8 weeks′, its expression in the proximal tubule and distal convoluted tubule decreased more in treated group than in the model group. At 12 weeks′end, its expression in all parts of distal convoluted tubule decreased significantly.Bcl-2 mainly expressed in all parts of proximal convoluted tubules and cortex-medullary junction, less expressed in distal convoluted tubule and collecting duct in control group; In diabetic group, its expression reduced in partial proximal convoluted tubule significantly, while no significant change of its expression in partial proximal convoluted tubules. For 8 weeks, the treatment group than in the diabetic group, in the proximal convoluted tubule proximal a slight increase in the distal tubule increased significantly; At 12 weeks, compared with model group, its expression in treated group in the proximal convoluted tubules increased less,but increased significantly in distal tubule. At 12 weeks′end, its expression was strongly positive in proximal convoluted tubules, stronger in cortex-medullary junction.7.2 Semi-quantitative analysis of pathological imagesAs for 8 weeks and 12 weeks, compared with the normal control group, the expression of bax in renal tissue from model group (P<0.01), irbesartan group (P<0.05), TCM group (P<0.05) were significantly higher, while Irbesartan group and TCM group expressed significantly lower than model group (P<0.01); There was no significant difference between TCM group and irbesartan group (P>0.05). Compared 12 weeks and 8 weeks, model group increased (P<0.05), TCM group and irbesartan group decreased (P<0.01), normal group had no statistical significance (P>0.05).Bcl-2 expression in model group, irbesartan group, expression of TCM group was significantly higher (P <0.05) for 8 weeks and 12 weeks; Irbesartan group and TCM group were significantly higher than model group (P <0.01), there was no significant difference between TCM group and irbesartan group (P>0.05). Compared 12 weeks with 8 weeks, model group decreased (P<0.01), TCM group and irbesartan group increased (P<0.05), no statistical significance to the normal group (P>0.05).For 8 weeks and 12 weeks, bax/bcl-2 was higher in model group, TCM group and irbesartan group than normal group (P<0.05).TCM group and irbesartan group were lower than model group (P<0.05), There was no difference between TCM group and irbesartan group (P>0.05). Compared 12 weeks with 8 weeks, bax/bcl-2 had no difference between normal group (P> 0.05), model group (P>0.05), but its reducement in TCM group and irbesartan group (P<0.01) had statistically significance.Conclusions:1 TNPC-recipe can reduce blood glucose, total cholesterol and triglyceride in rats with diabetic nephropathy.2 TNPC-recipe can reduce urinary protein, lower blood urea nitrogen, increased creatinine clearance rate, protect renal function in rats with diabetic nephropathy.3 TNPC-recipe can effectively inhibit glomerular basement membrane thickening, mesangial matrix proliferation, solw down the progress of renal pathology in rats with diabetic nephropathy.4 TNPC-recipe can effectively up-regulate the expression of bcl-2 gene, and down-regulate the expression of bax gene, thereby reduce bax/bcl-2, reduce the renal tissue apoptosis rate, so protect the integrity of the kidney structure and functional stability. With the increasement of treat times, this trend may be more marked. A new way of thinking to prevent and treat of diabetic nephropathy is provided. |
PDF Full Text Request