| Objective: Role of Huayu Tongluo traditional Chinese medicine in the treatment of diabetes nephropathy is clear. Previous experiments have established that the medicine can activat ACE2—Ang-(1-7)—Mas. In this experiment, Huayu traditional Chinese medicine were broken down into Huayu traditional Chinese medicine and Tongluo traditional Chinese medicine. By observing the general situation of rats, 24 hours urinary protein quantitative, blood biochemical indexes, blood coagulation analysis, renal pathology and kidney tissues of angiotensin converting enzyme- related carboxypeptidase 2 expression level, to contrast the Huayu Tongluo traditional Chinese medicine on diabetic kidney and discuss its possible mechanism, provide experimental foundation and theoretical basis for clinical application.Methods: 60 healthy male Sprague- Dawley rats, weight of 60 g to 80 g, were fed ordinary feed for a week. Determination of random blood glucose and 24 hours urinary protein quantitative were negative for rats will be used in this study. Randomly selected 10 rats as normal control group(C) were given ordinary diet, remaining 50 rats as model making group fed with high sugar and high fat diet. After 4 weeks, all rats fasting water more than 12 hours. Model making group rats were given one-time solution intraperitoneal injection of 35 mg/kg STZ. Normal group rats given intraperitoneal injection of the corresponding volume of citrate buffer solution. After 72 hours, which the Random blood glucose of rat ? 16.7mmol/L was the successful DM model. Then the DM model rats were randomly divided into 4 groups again: 12 rats for model group(M), 11 rats for Irbesartan group(I), 11 rats for Huayu group(H), 11 rats for Tongluo group(T). Each treatment group was given by corresponding drug, normal group and model group was given by corresponding volume of pure water, once a day,for 16 weeks. At the end of 2th week, 4th week, 8th week and 16 th week, rats were collected 24 hours?urine for 24-hour urine total protein determination, measured 24-hour urine, wate intake and food intake. Rats fasting 12 hours later, measured body weight, anesthesiaed animal, collected blood from abdominal aorta for the test of blood index; take kidney tissue used for the test of light microscopy, electron microscopy, immunohistochemistry, RT-PCR and Western-blotting.Results:1 The general condition of the experimental rats The rats of C group weight increase gradually, who has bright eyes, soft and shiny hair, well-developed muscles, and its water intake and food ration and urine output were normal. Rats of M group and treatment groups showed polyphagia, polydipsia, polyuria and loss of weight gradually. Their bones become deformed,hair without burnish,tail vein dark purple obviously. At the end of 16 th week, the rats? symptoms of treatment groups eased. 2 The comparison of rats? 24 hours urinary protein quantity(24h UTP) At different time-points, the 24 h UTP of M group and treatment groups were obviously higher than C group(P<0.05). At the end of 16 th week, compared with the M group, the 24 h UTP of treatment groups decreased obviously(P<0.05). There was no significant difference among the treatment groups(P>0.05). 3 The comparison of rats? random blood glucose Compared with the C group, the random blood glucose of M group and treatment groups increased significantly(P<0.05). No significant difference among the M group and treatment groups(P>0.05) 4 The comparison of rats? serum lipid profile Compared with the C group,the total cholesterol,triglyceride and low density lipid cholesterol of M group and treatment groups increased obviously(P<0.05). Compared with the M group, the total cholesterol in treatment groups decreased significantly(P<0.05). There is no obvious difference among treatment groups(P>0.05). Compared with the M group, the triglyceride and low density lipid cholesterol of T and H groups decreased significantly(P<0.05), but no significant difference between the two groups(P>0.05). 5 The comparison of rats? serum creatinine(Scr) and blood urea nitrogen(BUN) Compared with the C group, the Scr and BUN of M group and treatment groups increased significantly(P<0.05). About rats? BUN,there was no significant difference among the M group and treatment groups(P>0.05). Compared with the M group, the Scr of treatment groups decreased significantly(P<0.05), but no significant difference among the treatment groups(P>0.05) 6 The comparison of rats? Fibrinogen Compared with the C group, the Fibrinogen of M group and treatment groups increased obviously(P<0.05). Compared with the M, T and H group decreased significantly(P<0.05), but no significant difference between the M group I groups(P>0.05). 7 The comparison of rats? pathomorphology 7.1 Light microscopy Rats in C group had normal and clear kidney tissue structure. The rats of M group and treatment groups showed kidney structure disorder, glomerular hypertrophy obviously, renal capule were crack-like changes, even balloon adhesions, the basement membrane thickening, mesangial matrix accumulation. Compared with the M group, each treatment group has a similar pathology, but to a lesser extent. 7.2 Electron microscopy Rats in C group had clear and well-distributed ultrastructures, without fusion of foot processes arranged in order. The rats of M group?s glomerular basement membrane thickening, mesangial matrix increased, podocyte foot processes were fusion. Compared with M group, the rats? symptoms of treatment groups eased significantly.chemistry The experimental results show that ACE2 were expressen in glomerulus and renal tubules. 9 The expression of ACE2 m RNA in renal cortex The determination of the expression of ACE2 m RNA in renal cortex by RT-PCR show:Compared with the C group, M and treatment groups ACE2 m RNA expression was significantly decreased(P<0.05). Compared with the M group, ACE2 m RNA expression in treatment groups were significantly increased(P<0.05). There is no obvious difference among treatment groups(P>0.05). 10 The expressions of ACE2 proteins in renal cortex The determination of the expression of ACE2 proteins in renal cortex by Western-blotting show:The determination of the expression of ACE2 m RNA in renal cortex by RT-PCR show:Compared with the C group, M and treatment groups ACE2 proteins expression was significantly reduced(P<0.05). Compared with the M group, ACE2 proteins expression in treatment groups were significantly increased(P<0.05). There is no obvious difference among treatment groups(P>0.05). 8 Determination of the expression of ACE2 in renal cortex by Immunohisto-Conclusions: 1 Combined with random blood glucose, 24 hours urinary protein quantity and pathological changes of electron microscope and light observation proved that diabetic nephropathy model was successful. The rats' serum lipid profile and Fibrinogen in accordance with the features of blood-stasis-blocking-collaterals syndrome in Chinese medicine. 2 Huayu medicine and Tongluo medicine can improve the general condition of diabetic nephropathy rats, decrease the 24 hours urinary protein quantity, maintain renal function, reduce the pathological damage, show a protective effect in the DN rats kidney. 3 Serum lipid profile and Fibrinogen according to the results that Huayu medicine and Tongluo medicine can regulate lipid metabolism in the DN rats,and can improve its high coagulation state. In this respect Huayu medicine and Tongluo medicine better than Irbesartan. 4 ACE2 expression decreased expression in the kidney of DN rats. Huayu medicine and Tongluo medicine can increase the expression of ACE2. The above shows that Huayu medicine and Tongluo medicine can delay the progress of diabetic nephropathy by ACE2, conjecturing the protection mechanism associate with RAS system block. 5 According to the contrast of the above,there is no difference between T and H group to effect in the expression of renal angiotensin converting enzyme-related carboxypeptidase 2 and take protective effect on kidney in diabetic nephropathy rats. |
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