| Objective: Diabetic nephropathy(DN) is one of the most common microvascular complications of diabetes mellitus(DM), and also a leading cause of morbidity and mortality in diabetic patients. We aimed to probe its possible mechanisms by observing the effects of Huayu-Tongluo recipe on DN and the expression of slit diaphragm protein Nephrin and NEPH1, to compare the actional difference with Huayu recipe and Tongluo recipe, and to provide basis for clinical practice.Methods: Healthy male 80 SD rats, weighing 100 g ± 10 g, after adaptively feeding for one week, were grouped randomly. Ten rats were normal control group(group C) with a normal diet. Other 70 rats were building group fed with high-sugar and high-fat diet throughout the study. After 4 weeks, rats in building group were injected intraperitoneally with STZ(35mg/kg), and rats in group C were received the intraperitoneal injections of citrate buffer. Seventy-two hours after injection the rats showing random blood glucose?16.7 mmol/L were considered as diabetic model rats. The diabetic rats were randomly divided into model group(group M), Huayu- Tongluo group(group Q), Huayu group(group H), Tongluo group(group T), irbesartan group(group I). The corresponding drugs were administered via gavage for 16 weeks. Rats in model group and normal control group received saline in the same volume by the same route. At the end of 16 th week, rats' 24-hour urine was collected for 24-hour urinary protein quantitative determination. Rats were anesthesiaed and blood samples were collected from the abdominal aorta for testing serum biochemical parameters; Pathomorphology changes of kidney were observed with light microscope and electron microscope.And the expression levels of Nephrin and NEPH1 were detected by immunohistochemistry, Western Blot and Real-time PCR.Results: 1 The general status of ratsRats of group C were generally in good condition. Compared with group C, the rats of other groups showed polydipsia, polyphagia, polyuria and loss of weight gradually, which is most significant in group M. The rats? symptoms of treatment group eased during treatment. 2 The body weight, the kidney weight and the kidney hypertrophy index of ratsCompared with group C, the body weight of rats in group M reduced significantly, kidney hypertrophy index increased significantly(P<0.05); Compared with group M, there was no difference of kidney hypertrophy index and body weight of rats in each treatment group(P>0.05).There was no significant difference of kidney weight in all groups(P>0.05). 3 24-hour urinary total protein(24h UTP) of ratsCompared with group C, 24 h UTP of rats in group M significantly increased(P<0.01). Compared with group M, 24 h UTP of rats in each treatment group reduced significantly(P<0.01). No significant difference was found between Q and I groups(P>0.05). The 24 h UTP of rats in group Q was less than H and T groups(P<0.05). 4 The fasting blood glucose(FBG), total cholesterol(TC) and triglyceride(TG) of ratsCompared with group C, FBG of rats in model groups was significantly higher(P<0.01). FBG of every model group showed no significant difference(P>0.05).Compared with group C, the TC and TG of rats in group M were significantly higher(P<0.01). Compared with group M, the TC and TG of rats in all treatment groups decreased significantly(P<0.01). No statistical difference of TC was found among treatment groups(P>0.05). The TG of rats in group Q was less than H, T and I groups(P<0.05). 5 The total protein(TP), albumin(ALB) and serum creatinine(Scr)No significant differences of TP and ALB of rats were found among all groups(P>0.05). Compared with group C, Scr level of rats in group M was significantly higher(P<0.05). Compared with group M, Scr level in group Q decreased significantly(P<0.05), while Scr level in I, H and Tgroups did not decrease notably(P>0.05). 6 The prothrombin time(PT)Compared with group C, the PT of rats in group M significantly decreased(P<0.05). Compared with group M, the PT of rats in group Q was significantly longer(P<0.05), while PT of I, H and T groups did not change notably(P>0.05). 7 The pathomorphologic changes of kidney 7.1 Light microscopy observationsThe glomerular structure of rats in group C was normal. The glomerulus of rats in group M became hypertrophic, part of the renal capsule narrowed, the mesangial matrix increased and the basement membrane thickened. Compared with group M, the pathological changes in treament groups were significantly improved. The pathology of rats in group H presented slightly worse than other treament groups. 7.2 Electron microscopy observationsThe glomerular ultrastructure of rats in group C was normal. The glomerular basement membrane of rats in group M homogeneously thickened, the mesangial matrix increased and the foot process fused. Compared with group M, the pathological changes of all cure groups were significantly less. The pathology of rats in group H presented slightly worse than other treatment groups. 8 Detecting the expression of renal tissue Nephrin and NEPH1 by immunohistochemistry assayThe results showed: Nephrin and NEPH1 mainly expressed in glomeruli. Compared with group C, the expression of Nephrin and NEPH1 of rats in M and all treatment groups decreased. Compared with group M, the expression of Nephrin and NEPH1 of all treament groups increased. 9 Detecting the expression of renal tissue Nephrin and NEPH1 by Western BlotCompared with group C, expressions of Nephrin and NEPH1 proteins in group M significantly decreased(P<0.01). Compared with group M, expressions of Nephrin and NEPH1 proteins in Q, I and T groups increased(P <0.05). But there was no difference in Nephrin and NEPH1 expressions between group H and group M. Compared among treatment groups, Nephrin protein expression in group Q was more than H, T and I groups(P<0.05), while no significant difference of NEPH1 protein expression of rats was found(P>0.05). 10 Detecting the expression of renal tissue Nephrin and NEPH1 m RNA by real-time PCRCompared with group C, Nephrin and NEPH1 m RNA expressions of rats in group M significantly decreased(P<0.01). Compared with group M, Nephrin and NEPH1 m RNA expressions of rats in all treament groups increased(P<0.05). Compared among treatment groups, no significant difference of NEPH1 m RNA expression of rats was found(P>0.05), while Nephrin m RNA expression of rats in Q, T and I groups was more than group H(P<0.05).Conclusions:1 Huayu-Tongluo recipe and decomposed recipes can improve the general status of diabetic nephropathy rats, reduce urine protein, decrese the levels of total cholesterol triglyceride and serum creatinine, prolong the prothrombin time, reduce the pathological changes of DN rats and show protective effects on kidney.2 Huayu-Tongluo recipe and decomposed recipes can reduce the proteinuria and relieve the renal pathological damage by increasing the protein and gene expression of Nephrin and NEPH1.3 Nephrin and NEPH1 expressions in renal tissues are synchronous, which prompts Nephrin and NEPH1 may be closely related on structure and function.4 In the aspect of protective effects on DN, Huayu-Tongluo recipe is superior to Huayu recipe and Tongluo recipe; Compare two decomposed recipes, Huayu recipe and Tongluo recipe may play a role in a certain synergy, and Tongluo recipe is better than Huayu recipe. |
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