| OBJECTIVEEstablish diabetic nephropathy (DN) rat model by high-fat diet combined with low-dose streptozotocin (STZ) intraperitoneal injection. To discuss the duration of feedinghigh-fat diet and the effects of the incidence and mortality of DN rats model by differentSTZ dosages. At the same time, to establish the preferred plan of the DN rats model.According to the best modeling scheme，we establish the diabetic nephropathy (DN) ratmodel induced by high-fat diet combined with low-dose streptozotocin (STZ)intraperitoneal injection. To investigate the renoprotective effects and its mechanism ofPuerarin on diabetic nephropathy, to provide theoretical basis for preventive treatmentof DN.METHODS1. Study of the influential factor on the establishment of experimental diabeticnephropathy in ratsMature male Sprague-Dawley (SD) rats were randomly divided into7groups, rats ingroup1(normal, N) was given the standard diet. Group2(high fat, HF) was allowed free access to the high-fat diet. Group3(model, M-A) SD rats were intraperitoneallyinjected low-dose STZ (30mg/kg) after having high-fat diet for four weeks. Group4(model, M-B) SD rats were intraperitoneally injected low-dose STZ (35mg/kg) afterhaving high-fat diet for four weeks. Group5(model, M-C) was intraperitoneallyinjected STZ in a dose of40mg/kg after having high-fat diet for four weeks. Group6(model, M-D) was intraperitoneally injected STZ in a dose of30mg/kg after havinghigh-fat diet for six weeks. Group7(model, M-E) was intraperitoneally injected STZ ina dose of35mg/kg after having high-fat diet for six weeks.After the model established, all animals continued to give the high-fat diet for eightweeks. Observe the general health status, measured the body weight every week.Fasting blood glucose was measured every2week on lateral tail vein blood samples.Eight weeks after the induction of diabetes, the overnight fasted rats were sacrificed.Pancreas and kidney were carefully excised. kidney weight to body weight (KW/BW)ratio was evaluated. The sections of Pancreas tissues for pathological analysis werestained with hematoxylin/eosin staining (HE). Masson staining and HE were used toobserve the pathological changes of kidney.2. Protective effects of Pue on diabetic nephropathy in rats and its mechanismDN model in male SD rats were injected intraperitoneally once in a single dose of35mg/kg after having high-fat diet for six weeks. After injected, all animals continuedto be given high-fat diet for72hours. The rats with fasting blood glucose level of above11.1mmol/L were considered diabetic and only uniformly diabetic rats were included inthe experiment. Diabetic rats were randomly divided into7groups: diabetic rats withoutany drug treatment; diabetic rats treated with Puerarin (Pue) at a dose of50,100or200mg/kg every day; diabetic rats treated with Enalapril at a dose of1mg/kg; XiaokeWan at a dose of0.8g/kg and Simvastatin at a dose of2mg/kg every day served as positive control. Additionally, there were non-diabetic control (NC) rats that neitherreceived STZ nor the high-fat diet and high-fat diet control (HF) rats. Except NC, HFand diabetic rats without any drug treatment were given the same volume distilled waterby oral lavage vehicle, while the other groups were given intragastrically drugs for8weeks. the drug dose was adjusted according to the change of animal weight. All therats were allowed to continue to feed on their respective diets until the end of the study.During the experiment, the rats’ diet, drinking and urination situation were observedand body weight was measured every week. Fasting blood glucose was measured every2week on lateral tail vein blood samples. At the end of the8th week, all rats werehoused individually within metabolic cages and urine was collected from the ratshoused in metabolic cages for24h. The overnight fasted rats were sacrificed. Bloodsamples were obtained from femoral artery, centrifuged, and the supernatant was usedfor measurement of glucose, lipid metabolic parameters and so on. Total urine proteinfor24hours (TUP), urine microalbumin for24hours (UmALB), blood urea nitrogen(BUN), urine creatinine (Ucr), serum creatinine (Scr), high density lipoprotein (HDL),low density lipoprotein (LDL), very low density lipoprotein (VLDL) were measuredrespectively. Separate pancreas, kidney and heart from the rats. The ratio of kidneyweight to body weight (KW/BW) and the ratio of heart weight to body weight (HW/BW)were evaluated. The tissue sections of pancreas were stained by HE. Masson stainingHE and transmission electron microscopy were used to observe the pathologicalchanges of kidney. Fibronectin (FN), type IVcollagen (collagen IV), transforminggrowth factor-β1(TGF-β1) and tumor necrosis factor-α (TNF--α) were detected byimmunohistochemistry. The differences between these groups wereRESULTS1. Study of the influential factor on the establishment of experimental diabeticnephropathy in rats 1.1. Effect of each combination on the weight of SD ratsCompared with the normal control group, the weight of M-C、M-D and M-E groupdecreased significantly.1.2. Effects of each combination on the level of blood glucose and the incidence andmortality of SD ratsCompared with other model groups, the level of blood glucose was reasonable and thedifference was small between each other in the M-E group (6W,35mg/kg). And thereis a high success rate of animal model with the typical syndrome of polyphagia,polydispia, polyuria and emaciation. The hair of these rats were messy and withoutburnish.1.3. Effects of each combination on the kidney weight (KW) and the kidneyCompared with other model groups, the ratio of kidney weight/body weight (KW/BW)increased significantly. The compensatory hypertrophy of kidney was the most obviousin this group.1.4. Effect of each combination on pancreatic histopathology in SD ratsPancreatic histopathological results showed that the shape and size of islet cells wasnormal and the demarcation was clear in NC. Compared with the NC group, thedeveloped DN model groups had severe damage on pancreas and the demarcationbetween islet cells and acinar cells was not clear. And islet significantly atrophied andmixed with the surrounding tissue when compared to the control ones. 1.5. Effects of each combination on renal histopathology in SD ratsRenal histopathological results showed that the outline of glomerular and capillarylumen were clear and mesangial cells without proliferation in normal control rats.Compared with the NC group, there were obvious features of enlargement of swollenand Bowman’s capsule was narrowed In developed DN model groups. Part of glomerulihas sclerosis. Part of renal tubule was hypertrophy and the lumen was narrowed.Masson stain showed that collagen fibers in renal tissue of the developed DN modelgroup were more than the other groups.2. Protective effects of Pue on diabetic nephropathy in rats and its mechanism2.1Effects of Pue treatment on the weight and the level of blood glucose of DN ratsCompared with the normal control group, BW significantly decreased in the diabeticmodel group beginning at the third week. Rats in the Pue treatment group maintainedtheir BW when compared with the diabetic model group.2.2Effect of Pue treatment on the renal function of DN ratsThe level of BUN, LDL and Scr significantly increased, whereas the level of HDLdecreased in the diabetic model group as compared to the normal group. The high levelof BUN, LDL and Scr was markedly decreased by Pue (100mg/kg,200mg/kg)treatment in DN rats, and the low level of HDL was markedly increased by Pue(100mg/kg,200mg/kg) treatment in DN rats.2.3Effects of Pue treatment on the KW/BW ratio and HW/BW ratio of DN ratsCompared with the normal control group, the ratio of KW/BW and HW/BWsignificantly increased in the diabetic model group significantly increased. The highratio of KW/BW and HW/BW was decreased by Pue (100mg/kg,200mg/kg) treatmentin DN rats. 2.4Effect of Pue treatment on pancreatic histopathology of DN ratsIn DN model group, pancreatic islet destroyed evidently, its structure was atrophy anddisintegration, and mixed with the surrounding tissue when compared to the controlones. Pue treatment effectively improved the destruction of pancreatic islet.2.5Effect of Pue treatment on renal histopathology of DN ratsIn the model group, glomerular was hypertrophy markedly. There were obvious featuresof enlargement of swollen and Bowman’s capsule was narrowed and mesangial cellproliferation. Part of glomeruli has sclerosis. Part of renal tubule was hypertrophy andthe lumen was narrowed and there was a large number of collagen fibers in renal tissue.Compared with the model group, administration of Pue treatment was able to amelioratemesangial matrix expansion and to effectively ameliorate pathologic state, meanwhilethe expression of collagen fibers in renal tissue markedly decreased.2.6Effects of Pue treatment on the expression of FN, Collagen IV, TGF-β1andTNF--α in renal tissue of DN rats.Compared with the normal control group, the expression of FN, collagen IV, TGF-β1and TNF-α increased significantly in renal cortex of DN rats, which was effectivelyreduced by Pue (100mg/kg,200mg/kg) treatment.CONCLUSIONS1. Study of the influential factor on the establishment of experimental diabeticnephropathy in ratsDN rats model could be successfully established by injected STZ in a dose of35mg/kgafter having high-fat diet for six weeks, with the characteristics of a stabile plasmaglucose level, a lower auto-remission rate, a higher incidence and lower mortality of DN rats model. The model of M-E group mimiced the pathological changes of kidneyduring the natural course of DN. So the model of M-E group was an ideal DN model.2. Protective effects of Pue on diabetic nephropathy in rats and its mechanismPue has positively preventive and therapeutic effect on experimental DN Model. It notonly could improve renal function and lower blood glucose, but also ameliorate thepathological changes of glomerular and pancreatic islet. In addition, Pue could inhibitthe high expression of FN, collagen IV, TGF-β1and TNF--α, which resulted in theoccurrence and development of DN pathological process. These might be helpful tounderstand the role of Pue on diabetes clinical treatment. The present study offered anin-depth theoretical foundation for developing prevention and treatment on this disease,which exhibited vital clinical significance in delaying the development and progressionof DN. |
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