| Objective:The number of diabetics has reached285million in2010, nearly reached7%of the population around the world, under such a background of a sharp surge in the prevalence of diabetes. It has become another chronic and non-comminicable disease, after the heart and brain vascular diseases.cancer, which have seriously endangered public health. Diabetic nephropathy (diabetic nephropathy, DN) is common chronic microvascular complications in patients with diabetes, and it is also the main reason for end-stage renal failure. The occurrence of DN is recognized that the result of a variety of comprehensive reasons. In recent years, some studies reveal that the incidence of DN in the basis of metabolic disorders and hemodynamic changes, also related to inflammation mechanism.such as transforming growth factor-P(TGF-β1) and fibronectin(FN) are important mediated roles in the development of DN. Therefore, to observed DN expression in renal tissue TGF-β1and FN will help us to understand the pathogenesis of DN.This study is designed to establish rat model of DN and apply Tripterygium glycosides (Tripterygium wilfordii polyglucoside. TWP) to the model of intervention treatment. Whether TWP can reduce urinary protein is need to further confirmed. This paper through observing the changes of experimental animals24hours urine protein, blood count, liver function, renal function, renal pathological morphology and the changes of TGF-β1and FN expression in renal tissue to illustrate some concepts. In addition this study observe how TWP influence DN rat kidney, renal pathological changes.also TGF-(31and FN in protein expression, and discuss the possible protection mechanism. It provide some experimental and theoretical basis for its promotion and application.Methods:136clean grade male SD rats were adopted as the research objects and select16rats of them randomly as prevention normal control group (PNC group) and treatment normal control groups (TNC group), with8rats in each group, fed with regular diet.The other120rats were taken to establish the rat model of T2DM with the method of four weeks’high-sucrose-fat diet in combination with low dose STZ (30mg/kg) injected from tail vein. The DM model rats were randomly divided into prevention model control group (PDM group), prevention TWP low-dose group (PTL group,6mg/kg), prevention TWP middle-dose group (PTM group,12mg/kg) and prevention TWP high-dose group (PTH group,24mg/kg); treatment model control group (TDM group), treatment TWP low-dose group (TTL group,6mg/kg), treatment TWP middle-dose group (TTM group,12mg/kg) and treatment TWP high-dose group (TTH group,24mg/kg), with15rats in each group. The rats of TWP prevention group began intragastric administration immediately after being into DM model and were killed after4weeks, while the TWP treatment group began intragastric administration in the5th week after being into DM model and were killed at the end of the8th week. The PNC, TNC, PDM and TDM group were treated with equal amount of distilled water. The changes of body weight (BW), blood glucose,24-hour urine microalbumin (UMA) were recorded during the experiment. Blood lipids were tested by the means of inner canthus before STZ injection.When the experiment was at the end:Blood specimens were kept for liver and kidney function,blood lipids determination,blood routine examination and kidney weight (KW) were also recorded.; Kidney morphology and ultrastructural changes was observed by light microscopy and electron microscopy;The expression of the cytokine:TGF-β1and FN were observed by RT-PCR and immunohistochemistry personally from the genetic and protein levels in order to investigate the potential mechenism of the related cytokine in diabetic nephropathy and also the effection of the TWP Tablets.Results:(1) We has succeeded in establishing DN model, and the24h urinary protein features and renal pathological changes were in accordance with human characteristics and pathological changes of type2diabetic nephoropathy.(2) TWP could ameliorat the rat general state.During the experiment, the prevention and the treatment group model rats blood glucose maintained above16.7mmol/L, they appeared the symptom of the DM such as the quantum of drinking, eating and urinating inereased,weight loss,symptoms of kidney weight/body weight increase in the proportion (P＜0.05), the triptolide treatment of rats normal circumstances than those in model control has improved;The blood suger and weight in the group after Tripterygium wilfordii administration were not significant changes compared with the model group, but in kidney weight/weight proportion compared with the model group decreased obviously than Tripterygium glycosides administered dose models.(3) At4th week of the prevention gruop, UMA levels of each group was significantly higher than normal control, UMA has declined after Tripterygium glycosides administration, which UMA levels of administered dose group decreased the most obvious (P<0.05). UMA level of each group was significantly higher (P＜0.05) than those4th and8th weeks of treatment model group. At the eighth week, UMA level of the model control and triptolide administration groups were significantly higher than normal control group (P＜0.05), however, the level of UMA of each group of Tripterygium glycosides administration has decreased (P＜0.05), especially the level of the UMA of PTM group decreased significantly (P＜0.05).(4) The AST level of Prevention PTH group rats was significantly higher compared with the rest of the group (P<0.05);but the ALT, SCr, BUN, WBC, LYM levela among the groups was no significant difference (P＞0.05). The LYM level of treatment group after tripterygium glycosides administration was decreased compared with the control group (P＜0.05), the LYM level was no significant difference among the three groups (P＞0.05); The level of ALT, Scr, BUN, WBC have no significant difference among each group in treatment group (P＞0.05).(5) The light microscopy and electron microscopy showed that the normal control rats glomerular volume, mesangial area, the renal capsule, tubular and renal mass showed no obvious abnormalities compared with prevention and treatment groups. The glomerular and renal pathological tubular changes renal pathological lesions as DN of model control rats in the prevention and treatment groups.The pathological lesions of the group after administration of Tripterygium glycosides were improved,which group improved the most obvious is the Tripterygium wilfordii middle dose group.(6) Real-time PCR results showed that TGF-β1and FN mRNA expressions of all the DM groups were significantly higher compared with the control group (P＜0.05); TGF-betal and FN mRNA expression levels of the three groups after Tripterygium glycosides intervention were significantly down-regulated with statistical significance compared with the model control group (P＜0.05); The PTM group significantly declined compared with the rest each group(P＜0.05).(7) Iimmune-histochemistry results showed that TGF-β1and FN mRNA expressions of all the DM groups were significantly higher compared with the control group (P＜0.05); TGF-betal and FN mRNA expression levels of the three groups after Tripterygium glycosides intervention were significantly down-regulated with statistical significance compared with the model control group (P＜0.05); The PTM group significantly declined compared with the rest each group(P＜0.05).Conlusion:(1) We has succeeded in establishing DN model, and the24h urinary protein features and renal pathological changes were in accordance with human characteristics and pathological changes of type2diabetic nephoropathy.(2) The prevention and treatment groups UMA has decreased after Tripterygium glycosides administration(P＜0.05), and in particular the UMA level of Tripterygium wilfordi middle dose group was the most significant decline.Tripterygium glycosides can reduce urinary protein.(3) TGF-betal and FN in the DN rat kidney tissues were significantly higher compared with normal control group, TGF-betal and FN are related with DN.(4) TGF-betal and FN expression were significantly lower compared with the model group after the administration of Tripterygium wilfordii in both prevention and treatment groups, especially these two factors expressions levels in the middle dose group decreased significantly, Tripterygium wilfordii glycosidium may delay the occurrence and development of DN. |
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