The Interventional Effect Of Herbs Of Supplement Qi And Nourish In Resolve Masses And Dredge Meridians On Diabetic Nephropathy And The Effect On TGFβ/Smad Signal Transduction System

Objectives: Diabetic nephropathy(DN)is a part of micovasclor complication and the most important reason of end stage renal disease. The researchers keep working on the reason and treatment of DN. It is difficult to prevent and cure DN because numerous biotic activator participate in the process of DN. The medical doctor give the DN patients only with symptomatic treatment such as control hyperglycosemia and hypertension, low-protein diet and so on. The traditional Chinese herbs play an important role in preventing DN because of its advantage. We invented an effective traditional medicine, according to the pathogenesis which is at the early stage of DN and clinical experience for treating DN. This medicine has the functions of promoting circulation of qi and yin, dispersing blood stasis and dredging collateral which was composed of astragali, figwort root, carapax amydae, angle worm, ect. The clinical data showed that the prescription had good effect on the early stage of DN. This study aim to observe the curative effect of herbs of supplement qi and nourish yin resolve masses and dredge meridians(ZY) and investigate the mechanism in TGFβ/Smad signal transduction system in DN rats. All the above that have been done was to provide the foundation in order to generalization and exploitation.Motheds:1 Effect of ZY on the treatment in DN rats:Forty-six male Sprague-Dauley rats were selected in this experiment. According to the weight, the animals were randomly divided into normal group(10 rats)and DM model group(36 rats). 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 DM model were randomly divided into three groups: 12 in the model group, 12 in benazepril group and 12 in ZY group. After one week of DM models were made successfully, the drugs started be given to the rats according to adult dosage 20 times, once daily. The rats of ZY group were given the ZY 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. All animals were killed and the blood and the renal tissue were obtained. 24 hour urinary protein quantitative, glycohemoglobin(HbA1C), total cholesterol(TC), triglyceride (TG), serum creatinine (Scr), urea nitrogen (BUN) were assayed with the automaticbiochemistry analyzer. Pathomorphology change and the glomerulosclerosis index were observed.2 Effect of ZY on TGFβand receptor in DN rats:DM model was duplicated the same as the above.After 24 week of DM models made successfully, the renal tissue were obtained. The Col-Ⅳ,transforming growth factor-beta , transforming growth factor-beta typeⅠreceptor, transforming growth factor-beta typeⅡreceptor were measured by immunohistochemical method and RT-PCR.3 Effect of ZY on TGFβ/Smad signal transduction system in DN rats:DM model was duplicated the same as the above.After 24 week of DM models made successfully, the renal tissue were obtained. The smad2,smad3 and smad7 were measured by immunohistochemical method and RT-PCR.Results:1 Effect of ZY on the treatment in DN rats:1.1 The 24 hour urinary protein quantitative of each groupThe 24 hour urinary protein quantitative of the rats in model group, benazepril group and ZY 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 ZY group was obviously lower (P <0.01). There was no difference between benazepril group and ZY group (P >0.05).1.2 The HbA1C of each groupCompared with normal group, the HbA1C of the rats in model group, benazepril group and ZY group was significantly higher (P <0.01), but there was no difference among model group, benazepril group and ZY group (P >0.05).1.3 The TC and TG of each groupThe TC of the rats in model group, benazepril group and ZY group was significantly higher than that of normal group (P <0.01), compared with model group, the TC of ZY group was obviously lower (P <0.01), there was no difference between benazepril group and model group (P >0.05). The TG of the rats in normal group, benazepril group and ZY group was significantly lower than that of model group (P <0.01), there was no difference among normal group, benazepril group and ZY group (P>0.05).1.4 The Scr and BUN of each groupCompared with model group, the Scr of the rats in normal group, benazepril group and ZY group was significantly lower (P<0.01), and benazepril group was obviously higher than that of normal group (P<0.05). There was no difference between normal group and ZY group (P>0.05). The BUN of the rats in normal group, benazepril group and ZY group was significantly lower than that of model group (P<0.01). Compared with normal group, the BUN of benazepril group and ZY group was significantly higher (P<0.01), and there was no difference between benazepril group and ZY group (P>0.05).1.5 The pathomorphology change of kidneyObservations under light microscope:In the normal group: the structure of renal glomerulus was integrated, glomerular capillary basement membrane, mesenterium and base material were normal. In the model group: glomerular hypertrophy, thickening of glomerular capillary basement membrane, mesangial cell proliferation, mesangial matrix increased, mesangial area broadening and tubulointerstitial fibrosis were seen. vacuolar degeneration, analosis and hyaline cast were seen in some renal tubule. The benazepril group and ZY group also showed different degree of pathological changes, but the pathological degree was obviously lighter than the changes of the model group.Observations under transmission electron microscopy: In normal group: the structure of glomerular capillary basement membrane was clear and complete, microcirculatary endothelial cell, foot processes was normal. In the model group: most of the glomerular capillary basement membrane showed significant homogeneous thicking, partial basement membrane showed elevation in the shape of hills; microcirculatary endothelial cell significantly confluence and the most of window structure disappeared; the fusion of the foot processes was extensive. The benazepril group and ZY group also showed the similar pathological changes, but the pathological degree was lighter than that in model group.The glomerulosclerosis index in each group: The glomerulosclerosis index in model and treatment groups were higher than that in normal group (P<0.05);Compared with model group, the glomerulosclerosis index in benazepril group and ZY group was significantly lower (P<0.05), and the glomerulosclerosis index in benazepril group was higher than that in ZY group(P<0.05).2 Effect of ZY on TGFβ1 and receptor in DN rats2.1 Expression of CollegeⅣexamined by immunohistochemistry:In normal group, Col-Ⅳhas the basal expression. Compared with normal group, the expression of Col-Ⅳin model group was stronger(P<0.01). Compared with model group, the expression of Col-Ⅳin benazepril group and ZY group was significantly lighter (P<0.01), and there was no difference between benazepril group and ZY group (P >0.05).2.2 Expression of TGFβ1 in renal tissueThe results detected by immunohistochemistry showed: The expression of TGFβ1 in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβ1 in benazepril group and ZY group was significantly decreased (P <0.01), and the TGFβ1 in benazepril group was higher than that in ZY group(P<0.05).The results detected by RT-PCR showed: The expression of TGFβ1 in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβ1 in benazepril group and ZY group was significantly decreased (P<0.01), and the TGFβ1 in benazepril group was higher than that in ZY group(P<0.05).2.3 Expression of TGFβtypeⅠreceptor in renal tissueThe results detected by immunohistochemistry showed: The expression of TGFβtypeⅠreceptor in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβtypeⅠreceptor in benazepril group and ZY group was significantly decreased (P <0.01), and the TGFβtypeⅠreceptor in benazepril group was higher than that in ZY group(P<0.05).The results detected by RT-PCR showed: The expression of TGFβtypeⅠreceptor in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβtypeⅠreceptor in benazepril group and ZY group was significantly decreased (P <0.01), and the TGFβtypeⅠreceptor in benazepril group was higher than that in ZY group(P<0.05).2.4 Expression of TGFβtypeⅡreceptor in renal tissueThe results detected by immunohistochemistry showed: The expression of TGFβtypeⅡreceptor in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβtypeⅡreceptor in benazepril group and ZY group was significantly decreased (P<0.01), and there was no difference between benazepril group and ZY group (P>0.05).The results detected by RT-PCR showed: The expression of TGFβtypeⅡreceptor in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβtypeⅡreceptor in benazepril group and ZY group was significantly decreased (P <0.01), and there was no difference between benazepril group and ZY group (P >0.05).3 Effect of ZY on TGFβ/Smad signal transduction system in DN rats3.1 Expression of p-Smad2/3 protein in renal tissueCompared with normal group, the expression of p-Smad2/3 in model group was stronge(rP<0.01). Compared with model group, the expression of p-Smad2/3 in benazepril group and ZY group was significantly lighter (P <0.01), and there was no difference between benazepril group and ZY group (P >0.05).3.2 Expression of Smad7 protein in renal tissue by WesternblottingCompared with normal group, the expression of Smad7 in model group was lower(P<0.01). Compared with model group, the expression of Smad7 in benazepril group and ZY group was significantly stronger (P<0.01), and there was no difference between benazepril group and ZY group (P >0.05).3.3 Expression of Smad2 and Smad7mRNA by3.3.1 Smad2mRNACompared with normal group, the expression of Smad2 in model group was stronger(P<0.01). Compared with model group, the expression of Smad2 in benazepril group and ZY group was significantly lighter (P<0.01), and there was no difference between benazepril group and ZY group (P >0.05).3.3.2 Smad7mRNACompared with normal group, the expression of Smad7 in model group was lower(P<0.01). Compared with model group, the expression of Smad7 in benazepril group and ZY group was significantly stronger (P<0.01), and there was no difference between benazepril group and ZY group (P >0.05).Conclusions:1. Herbs of supplement qi and nourish yin resolve masses and dredge meridians could decreased the DN rat's proteinuria, improve the lipid metabolism, decrease Scr and BUN2. Herbs of supplement qi and nourish yin resolve masses and dredge meridians could lessen the damage of nephridial tissue.3. Herbs of supplement qi and nourish yin resolve masses and dredge meridians could decrease the ColIV deposition in order to slow down the progress of glomerulosclerosis.4. Herbs of supplement qi and nourish yin resolve masses and dredge meridians could decreased TGF-β1and receptorⅠ,Ⅱwhich could resist renal fibrosis and protect renal founction.5. Herbs of supplement qi and nourish yin resolve masses and dredge meridians could inhibit the expression of smad2, smad3 and promote the expression of smad7which could delay the process of DN.