| Objective: Diabetic nephropathy is a microvascular complication of diabetic mellitus, and it has been the most common cause of death for diabetic patients.Its pathologic mechanism has the close relation to high blood sugar, the excrescent of kidney blood dynamics and the household hereditary, etc. The renin-angiotensin system (RAS) has been implicated in the pathogenesis of diabetic nephropathy, In addition to its ability to induce systemic and intraglomerular hypertension and to increase glomerular capillary permeability, AngII is a renal growth factor and causes hypertrophy of mesangial cells and tubular epithelial cells and promotes production of the prosclerotic cytokine transforming growth factor-β1. Transforming growth factor-beta1 (TGF-β1) plays a critical role in the pathogenesis of diabetic nephropathy(DN) through inducing cell hypertrophy of glomerulus and renal tubule, accelerating the accumulation of extracellular matrix(ECM), and promoting glomerulosclerosis and tubulointerstitial fibrosis, etc。According to traditional Chinese medicine theory, diabetic nephropathy is included in categories of "wasting-thirsting" and "lower-wasting", Deficience of kidney-yin has been thought as the importent pathogeny of diabetic nephropathy. At the same time, Liuweidihuang Decoction has been proved to have satisfactory therapeutic effect on DN although the mechanism of its action is still unkown. In this experiment, Diabetes mellitus (DM) animal model was first established, then developed for diabetic nephropathy (DN), and carried on the treatment usingLiuweidihuang Decoction. we observe the change of its blood sugar, renal function, the appearance of kidney and expressions of transforming growth factor-β1,renal tissue rennin activity,the content of angiotensin II,and inquiry the prevention and cure mechanism of Liuweidihuang Decoction, so as to provid scientific experimental foundation for reinforcing kidney toreplenish Yin method of clinical application.Partâ… :Effect of reinforcing kidney toreplenish Yin method on the renal function and pathomorphology of the diabetic nephropathy ratsMethods: 6 rats were selected for normal control group (NC) randomly in the 30 clean healthy male Sprague-Dawley rats. Others were injected the streptozocin (STZ) intraperitoneally after one week according to 60 mg/kg.We regarded the rats as the model rats whose blood sugar≥16.65mmol/L, urine glucose was masculine.The model rats were randomly divided into: model group(DM), Benazepril group (BN), Liuweidihuang Decoction group (LD),n=8. 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 Liuweidihuang Decoction group were given the Liuweidihuang Decoction by gavage, 25g/kg, the benazepril group were administrated with benazepril by gavage, 6.67mg/kg, the normal group and the model group were given physiological saline by gavage. Each group was given the medicine for 8 weeks. After 9 weeks of DM models made successfully, We took rats 24h urine in metabolic cages to measure urinary protein and urinary creatinine (Ucr). Animals were sacrificed to measure fasting plasma glucose (FPG), blood urea nitrogen (BUN), serum creatinine(Scr) and to calculate creatinine clearance rate(Ccr). Some renal tissue was obtained for the pathomorphology.Results: 1. Fasting plasma glucose (FPG),Kidney weight / body weigh(tKW/BW)and the data of urinary protein:The body weight of model groups increased slowly after the injection . The worse hair color, high gloucose and the expanded kidney were showed at the end of test. Five rats died of higher glucose. The FPG of Model group was higher than that of NC group significantly (p<0.01); there was no difference between the BN group and DM group ; LD group was lower than that of DM group significantly (p<0.05). The 24h total urinary protein of DM group was higher than that of NC group significantly (p<0.01); BN group were decreased remarkably compared with that of DM group (p<0.05), LD group was superior to BN group (p<0.05). The KW/BW of DM group was higher than that of NC group significantly (p<0.01); BN group were lower than that of DM group significantly (p<0.05); LD group was lower than that of BN group,but there was nosignificant difference between them. 2. The results of blood urea nitrogen(BUN), creatirine clearance rate (Ccr):The BUN of DM group was higher than that of NC group significantly (p<0.01); BN group was decreased remarkably compared with that of DM group (p<0.05), and that of LD group was superior to BN group (p<0.01), LD group was higher than that of NC group, but there was nosignificant difference between them.The Ccr of DM group were higher than NC group significantly (p<0.01); BN group was lower than that of DM group significantly (p<0.05); The effect of LD group superior to that of BN group, but that had no nosignificant difference. 3.Renal tissue pathomorphology: The staining of HE and PAS of renal tissues,In the NC group, the light microscope observation result showed that: the structure of renal glomerulus was complete, glomerular capillary basement membrane, mesangial and matrix did not show abnormal change. In the DM group,mesangial area of renal glomerulus broadening, even protein substances with PAS positive markebly increasing, extracellular matrix of glomerulus enlarging in number, glomerular basement membrance (GBM) thickening and the blood capillary is compressd,narrowed and vanished, so on. But in the LD group, the changes were better than those in the DM group. In BN group, renal tissue pathomorphology was similar to the LD group. Partâ…¡:Effect of Liuweidihuang Decoction on the renal renin angiotensin system and TGF-β1 in diabetic nephropathy ratsMethods: The methods for test were as the same as those in Partâ… . The levels of renin activity and angiotensinâ…¡of renal tissue homogenate were tested with radioimmunoassay method. The results were adjusted with renal tissue total protein to correct the renin activity and the content of angiotensinâ…¡in each sample. Some samples of renal tissue were fixed in 4% paraform, dehydration, paraffin embedding, and by the means of immunohistochemistry method to observe the expression of TGF-β1. The protein expression of TGF-β1 adopted SP method, The integral optical density and relative areal were measured with pathological image analysis system.Results: 1.The renin activity and the content of angiotensionâ…¡in renal tissue, which could be founded in the experimental animals'kidney samples, were assayed by radiative immune method. The results showed: It was obviously that the renin activity strengthened in DM group against NC group (P<0.05), The data of BN group decreased comparing with that of DM group (P<0.05). The data of LN group was lower than that of BN group (P<0.05);There was no statistic sense between LD group with DM group. Angâ…¡'s content of renal tissue in DM group increased markely,which refered to a significant difference in comparing with group NC(P<0.01). The data of BN group and LD group were decreased comparing with that of DM group(P<0.05). There was no statistic sense between BN group with LD group. 2.The semi-quantitative analysis of TGF-β1: It was seen that the TGF-β1 protein expressed hardly in the renal tissue in NC group observed by light microscope; The expression of TGF-β1mainly localized in the cell cytoplasm of the renal Mesangial area. The expression of TGF-β1in BN group and LD group were both lower than that in Model group significantly. Pathologic image semi-quantitative analysis shows: Compared with group NC, the expression of TGF-β1 in DM group increased separately (P<0.01). Compared with group DM, the expression of TGF-β1 in BN group and LD group decreased (P<0.05).There was no statistic sense between BN group with LD group.Conclusions:1 The results indicated that Liuweidihuang Decoction was benefit for regulating glucose metabolism, improving renal funition.2 The results indicated that Liuweidihuang Decoction was benefit for reducing the urinary protein excretion, inhibiting renal hypertrophy and delaying the advancement of renal phathematology.3 It indicated that Liuweidihuang Decoction had a certain effect on reducing the content of Angâ…¡in renal tissue, which probaboly had correlation with its ability to treat early DN.4 It indicated that Liuweidihuang Decoction had the ability to inhibit extra express of TGF-β1 in drabetic rats'renal tissue, which probaboly had correlation with its ability to treat early DN. |
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