| Objective: In this experiment, Diabetes mellitus (DM) animal model was first established, then developed for diabetic nephropathy (DN), and carried on the treatment using the traditional Chinese medicine of Xueqitongshentang. 24 hour urinary protein, glycohemoglobin, blood lipid, renal function, pathomorphology change of kidney and the expression of TGF-β1 and p38MAPK protein or mRNA in renal tissue were observed. This study aim to assess the mechanism of Xueqitongshentang on DN to improve the research about Chinese traditional medicine.Methods: Forty-six male Sprague-Dauley rats were selected with the weight of 200±10g in this experiment. The rats were fed in ordinary ways for one week. After 7 days, the urine glucose and urine protein were detected, the result was negative. According to the weight, the animals were randomly divided into four groups: 10 in normal group, 12 in the model group, 12 in benazepril group and 12 in Xueqitongshen group. Besides the normal group, All the rats were injected with streptozotocin (STZ) in the dose of 60 mg/kg, the normal group were given the corresponding quantity's physiological saline. After 72 hours, measured their blood glucose, took"≥16.7mmol/L"as the DM model standard. According to the blood glucose level, the rats of three DM model were randomly divided into three groups again, the category and the animal number were invariable. After one week of DM models made successfully, the drugs started be given to the rats according to adult dosage 20 times, daily one time. The rats of Xueqitongshen group were given the Xueqitongshentang by gavage, 26g/kg, the benazepril group were administrated with benazepril by gavage, 10mg/kg, the normal group and the model group were given physiological saline by gavage. Each group was given the medicine for 23 weeks. After 24 week of DM models made successfully, all rats were put into metabolism cage to obtain 24 hour urine to detect 24 hour urinary protein quantitative. All animals were killed. Glycohemoglobin(HbA1C), total cholesterol(TC), triglyceride (TG), serum creatinine (Scr), urea nitrogen (BUN) were assayed with the automaticbiochemistry analyzer. Weighed the weight of kidney and calculated hypertrophy index. Pathomorphology change of kidney were observed with light microscope. The mRNA expressions Of TGF-β1 and p38MAPK weresemi-quantitatively evaluated by reverse transcription and polymerase chain reaction (RT-PCR). All the data were expressed as the form of Mean±s. F test were used to analyse the difference between the two group. Statistical analysis was finished with SPSS software.Results:1 The general condition of the rats The rats of normal group were eusitia, whose eye is bright, had an agility reaction, gloss color pattern, thick subcutaneous fat, and its dejecta was granular and the amount of urine was normal. After STZ injection, the rats of other three groups all presented obviously polydipsia, polyphagia, polyuria, and presented slowly the energetic dispirited, the slow reaction, little moves hugs the group. In last week of the experiment, compares with the normal group, the rats of DN model were obviously thin and small, the muscle are few, the above symptom were most remarkable by the model group. There were rat death in treatment period among three DN model groups, the model group was three, the benazepril group was one and the Xueqitongshen group was one . The estimated cause of death was the internal organs failure caused by the metabolic disorder for the hyperglycemia.2 The body weight, right kidney weight and hypertrophy index (kidney weight/body weight) of each groupCompared with normal group, the body weight of the rats in model group, benazepril group and Xueqitongshen group was obviously lighter (P <0.01), there was no significant difference among three DN model groups (P >0.05). The right kidney weight of the rats in all four groups had no significant difference (P >0.05). The hypertrophy index of the rats in model group, benazepril group and Xueqitongshen group was obviously higher than that of normal group (P <0.01), but there was no significant difference among three DN model groups (P >0.05). 3 The 24 hour urinary protein quantitative of each groupThe 24 hour urinary protein quantitative of the rats in model group, benazepril group and Xueqitongshen group was significantly higher than that of normal group (P <0.01), compared with model group, the 24 hour urinary protein quantitative of benazepril group and Xueqitongshen group was obviously lower (P <0.01). There was no statistical difference between benazepril group and Xueqitongshen group (P >0.05).4 The HbA1C of each groupCompared with normal group, the HbA1C of the rats in model group, benazepril group and Xueqitongshen group was significantly higher (P <0.01), but there was no statistical difference among model group, benazepril group and Xueqitongshen group (P >0.05).5 The TC and TG of each groupThe TC of the rats in model group, benazepril group and Xueqitongshen group was significantly higher than that of normal group (P <0.01), compared with model group, the TC of Xueqitongshen group was obviously lower (P <0.01), there was no significant difference between benazepril group and model group (P >0.05). The TG of the rats in normal group, benazepril group and Xueqitongshen group was significantly lower than that of model group (P <0.01), there was no statistical difference among normal group, benazepril group and Xueqitongshen group (P>0.05).6 The Scr and BUN of each group Compared with model group, the Scr of the rats in normal group, benazepril group and Xueqitongshen group was significantly lower (P<0.01), and benazepril group was obviously higher than that of normal group (P<0.05), there was no significant difference between normal group and Xueqitongshen group (P>0.05). The BUN of the rats in normal group, benazepril group and Xueqitongshen group was significantly lower than that of model group (P<0.01), compared with normal group, the BUN of benazepril group and Xueqitongshen group was significantly higher (P<0.01), and there was no statistical difference between benazepril group and Xueqitongshen group (P>0.05).7 The pathomorphology change of kidney by Light microscope observationThe normal group showed that: the structure of renal glomerulus was complete, glomerular capillary basement membrane, mesangial and matrix did not show abnormal change. The model group showed that: glomerular hypertrophy, thickening of glomerular capillary basement membrane, mesangial cell proliferation, mesangial matrix increased, mesangial area broadening,tubulointerstitial fibrosis. The benazepril group and Xueqitongshen group also showed different degree of pathological changes, but the pathological degree was obviously lighter than the changes of the model group.8 Immunohistochemical result of the expression of TGF-β1 in renal tissueTGF-β1 was mainly expressed in glomerulus , weakly expressed in proximal renal tubular and weakly expressed in distal renal tubular, TGF-β1 could express in all four groups. The result of semi-quantitative analysis showed that: compared with the normal group, the model group TGF-β1 had stronger expression (P<0.01), the benazepril group and Xueqitongshen group also was stronger than that of the normal group (P<0.01), compared with the model group, the expression of the two treatment groups was lower (P<0.01), and the expression was not statistical difference between the two treatment groups (P>0.05).9 RT-PCR result of the expression of TGF-β1,p38MAPKmRNA in renal tissueThe result of semi-quantitative analysis showed that: compared with the normal group, the model group TGF-β1mRNA had stronger expression (P<0.01), the benazepril group and Xueqitongshen group also was stronger than that of the normal group (P<0.05), compared with the model group, the expression of the two treatment groups was lower (P<0.05), and the expression was not statistical difference between the two treatment groups (P>0.05). Compared with the normal group, the model group p38MAPKmRNA had stronger expression (P<0.01), the benazepril group and Xueqitongshen group also was stronger than that of the normal group (P<0.01), compared with the model group, the expression of the two treatment groups was lower (P<0.01), and the expression was not statistical difference between the two treatment groups (P>0.05)Conclusions:1 The Xueqitongshentang can reduce DN rat's proteinuria, cut down TC, TG , improve the lipid metabolism and decrease Scr and BUN to protect renal function.2 The Xueqitongshentang can effectively inhibit DN rat's thickening of glomerular capillary basement membrane, mesangial matrix increased, fusion of foot processes of podocytes to delay the pathologic development of kindey.3 The Xueqitongshentang can diminution the expression of TGF-β1,p38MAPKmRNA in DN rat's renal tissue, which suggests that the effect of Xueqitongshentang on DN is related with the approach for blocking TGF-β1/ p38 MAPK signal transduction pathway. |
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