| Chronic renal failure (CRF) is a common clinical syndrome, it occurs on the basis of chronic kidney disease, appeared to slow renal dysfunction and failure. In recent years, the incidence of chronic renal failure in China, along with the aging of society, hypertension disease, diabetes mellitus and other diseases has increased every year. It showed a clear upward trend. Chronic renal failure is a clinical refractory disease. Most patients often end up on the need dialysis for uremia or kidney transplant to survive. Therefore, in chronic renal failure and end-stage renal failure (uremia), take effective measures to slow down the disease progression that is of great significance. Renal fibrosis is a common approach to chronic renal failure in various kidney diseases. It includes glomerulosclerosis and tubulointerstitial fibrosis, and it is the basic pathological changes of various end-stage renal disease. As other organs fibrosis, renal fibrosis has been one of the hot-spot issues of medical research in the world. In the progress of kidney disease, tubulointerstitial injury has played an important role in the same patients with glomerular lesions. Although the lesions is same, the prognosis can be a big difference. Renal interstitial fibrosis is the extracellular matrix that is mainly deposition and increasing of collagen of typeâ… and typeâ…¢. The main pathological changes were reduced on renal tubular epithelial cells and there was substantial interstitial myofibroblasts. Myofibroblasts can synthesis and secretionâ… ,â…¢collagen to promote the occurrence of renal interstitial fibrosis, which plays an important role in the process of renal fibrosis.â… ,â…¢collagen fibers forming the proliferation is an important factorof renal fibrosis, therefore inhibiting its proliferation and promoting its degradation are both important ways to combat renal interstitial fibrosis. Clinical research has shown that Traditional Chinese Medicine has its unique advantage in the progression of chronic renal failure and to improve the prognosis of patients.Large number of experiments have demonstrated that the Traditional Chinese Medicine hasa good effect on anti-renal fibrosis.Zhenwu decoction comes from the Treatise on Cold-Attack that is written by Zhang Zhongjing, and is the main representative Formula of yang asthenia of Spleen and Kidney in the treatment of CRF. This paper used six types of Chinese Periodical Literature database and pubmed retrieval system, combined with manual retrieval, and identified 57 years ago(1949～2006) journal published 132 article document by the use of Zhenwu decoction prevention of chronic renal failure. After analysising the research literatures of 57 years about Zhenwu decoction to prevention of CRF, found: Zhenwu decoction to the prevention of chronic renal failure has continued since the 1950s, researches gradually increased since the 1980s, and its research content is clinical research primarily. In 91 literatures of clinical research used Zhenwu decoction addition and subtraction side primarily, but also effect of three reports made better by using the original Formula. Experimental study reported less, only 7 literatures. There really have a whole animal experiments method to improve renal function using Zhenwu decoction in rat models of CRF, and have observed serum pharmacology Zhenwu decoction containing serum on glomerular mesangial cell proliferation and the impact on human fetal glomerular mesangial cells linked extracellular matrix protein fibronectin, laminin and typeâ…£collagen role. Has not seen the original Formula of the Zhenwu decoction preventing and controlling CRF in the overall animal experimental research and its the anti-renal interstitial fibrosis were reported.1 ObjectiveUsing model of Traditional Chinese Medicine syndrome of yang asthenia of Spleen and Kidney binding CRF, to observe the protective effect of Zhenwu decoction of CRF in rats, to explore the role of anti-renal fibrosis andâ… ,â…¢collagen fibers affected in renal fibrosis of rat.2 Methods2．1 Experimental animals:Only 65 healthy male Wistar rats, six weeks, cleaning and weighing 200±20g.2．2 Experimental drugs2．2．1 Makes the mold drug: adenine purchased from drug companies, distilled water before adding to the preparation of 20mg/ml suspension.2．2．2 Treatment group drugs: Zhenwu decoction's reference dose group and the national medical institutions of higher education books"Formulaology", that Aconiti Praeparata Radix 9g, Poriacacos wolf 9g, Paeonia lactiflora Pall 9g, Atractylodes macrocephala koida 6g and Zingiber officinale Rosc 9g. 60kg weight as the normal adult dose of this on the 1st dose. According to the document conversion method rat dosage, 60kg weight to normal dosage of 3．5 times smaller dose and 60kg weight 14 times the normal dosage for the high dose. Pieces purchased from Beijing Tongrentang Pieces limited liability company. Wangjing Hospital Pharmacy prepared, which contains a small dose of concentrated liquid volume and 0．245g/ml, 0．98g/ml large dose of raw drug dose concentrated solution.2．2．3 Positive control drug: Niaoduqing particles produced by the Guangzhou Kang medicine Limited, according to the literature, the use of rats in a dose of 3．75g/kg Niaoduqing. Niaoduqing particles will be used for the preparation of 375mg/ml distilled water solution.2．3 Makes the mold method: "single kidney resection with gavage adenine " was establishing a model of CRF in rats. After rats nephrectomy alone on 1 week, 200mg·kg-1·d-1 dose of adenine-scale suspension was administered,28 consecutive days.2．4 Stochastic grouping method: 65 rats were elected randomly according to the weight by the random number table, 10 rats were selected randomly just as a normal group, 10 rats as the sham-operated group, 45 rats were left nephrectomy excisioned. Five rats died after surgery 1 week. The survival of 40 rats were randomly divided into weight based on the random number table, model 10, Zhenwu decoction low-dose group 10, Zhenwu decoction high-dose group 10 and Niaoduqing group 10． 2．5 For medicine method:Normal group, sham-operated group and model group : 2 ml distilled water twice a day up to 28 days. In the treatment groups: Niaoduqing group by3．75g·kg-1·d-1 dose to Niaoduqing solution, Zhenwu decoction high-dose group by aquaculture 9．8g·kg-1·d-1 dose to high dose Zhenwu decoction concentrated solution, and Zhenwu decoction low-dose group by 2．45g·kg-1·d-1 dose to the small dose of concentrated liquid Zhenwu decoction, up to 28 days. According to body weight of rats in each group by each week, the volume of distilled water and dose were adjusted.2．6 Observed and measured methods2．6．1 General: weight, feces, hair, the mental state and situation, and Food intake, water intake.2．6．2 24h urine and 24h urinary protein: every seven days after the rats placed in metabolism cages dedicated 9:00 pm to 9:00 am the next day, and took 24-hour urine. During fasting urine can not help but leave the water. Records of 24 urine and using Coomassie Brilliant Blue measured 24 urinary protein.2．6．3 Blood routine and blood biochemistry: After 4 weeks with 10% chloral hydrate (0．15ml/100g) intraperitoneal injection of anesthesia, blood samples taken from the femoral artery. RBC, Hb detected by blood routine analysis apparatus; Serum creatinine, blood urea nitrogen, serum calcium and phosphorus detected by automatic biochemical analyzer.2．6．4 The renal general observation: kidney sophisticated electronic scales called solitary kidney weight, Unilateral calculated rat kidney weight/body weight×103 value, the naked eye of kidney size, shape, color, texture coating, cortex, medulla other.2．6．5 Pathology staining method: Renal pathology staining of paraffin sections carried on Hemotoxylin and eosin staining, Periodic Acid-Silver Methenamine staining,Masson staining,Sirius red staining, Mallory staining, and they were observed in renal morphological changes.2．6．6 Semi-quantitative grading of tubulointerstitial lesions method: According to Sergio semi-quantitative analysis of semi-quantitative classification standard, HE staining was randomly selected from each of the 20 endoscopic vision for semi-quantitative analysis.2．6．7 Kidney pathology image analysis method: PASM staining pathological image analysis system mesangial integral optical density value of the quantitative comparison, Masson staining area of quantitative comparison of renal interstitial fibrosis. Sirius red staining was observed in polarized light microscopy sections of theâ… andâ…¢collagen. Using image analysis and pathological analysis system calculateâ… and typeâ…¢percentage of the total value of inter-quality vision.2．7 Statistical methods: SPSS (15．0 V) statistical software was elected for statistical analysis. All measurement data to mean±standard deviation ((?)±s ). The differences between the different groups of single-factor analysis of variance. Between 2 groups were compared using LSD test, Wilcoxon test was used at variance missing. Wilcoxon test was used to compare the information hierarchy. P<0．05 was considered statistically significant difference vs. P<0．01 that the difference was statistically significant. 3 Results3．1 General: After makes the mold, the model group rats started to weight loss, the growth inhibition, the same time the hair was dry yellow different, easy peeling and chills, Spiritual dispirited, active reduction, tail and ear pale, and other symptoms of syndrome of yang asthenia of Spleen and Kidney.The treatment group decreased weight, less than normal, it was better than to model group. Performance than the general model of light, but not as the sham-operated group and the normal group health rats.3．2 24h urine and 24h urinary protein: Low-dosage Zhenwu decoction and High-dosage Zhenwu decoction group better than model group in 24h urine. The treatment group compared with 24 urine, Zhenwu decoction high-dose group with the most significant increase. Low-dosage Zhenwu decoction and High-dosage Zhenwu decoction group urinary protein excretion was significantly reduced compared with the model group, P<0．05, High-dosage Zhenwu decoction group is the best between them.3．3 RBC, Hb: Red blood cells of High-dosage Zhenwu decoction group and Niaoduqing group were significantly higher than the model group, P<0．05．RBC between the treatment group compared to P>0．05, no significant difference.Model group and the treatment group was significantly lower than the normal hemoglobin, P<0．05．The treatment groups were significantly higher than model group in hemoglobin, P<0．05, with the most obvious High-dosage Zhenwu decoction group. However,the treatment groups between hemoglobin, P>0．05, no significant difference.3．4 Serum creatinine, blood urea nitrogen, calcium, phosphorus: SCr values of the treatment groups were significantly lower than that in the model group, P<0．05．BUN values of High-dosage Zhenwu decoction group and Niaoduqing group were significantly lower than the model group, P<0．05,but Low-dosage Zhenwu decoction group showed no significant difference compared with the model group. Calcium values of High-dosage Zhenwu decoction group and Low-dosage Zhenwu decoction group increased than model group P<0．05,and Niaoduqing group showed no significant difference compared with the model. Phosphorus values of the treated groups were significantly lower than that in the model group, P<0．05, Zhenwu decoction with the most significant effect of high-dose group.3．5 Kidney general observation: The model group: Right kidney increased significantly, pale color, texture hard Matt. Cortex was thinner, corticomedullary unclear boundaries capsule thickening. High-dosage Zhenwu decoction group and Low-dosage Zhenwu decoction group: a small dose Right kidney mildly increased, light brown, Matt texture harder cortical thinning, corticomedullary line to be identified, coating thickness. Niaoduqing group: Right kidney increased slightly, light brown, with hard Matt cortical thinning, corticomedullary unclear boundaries capsule thickening.3．6 Unilateral renal weight:â‘ Model group and the treatment groups has a marked increase than the normal group and sham-operated group, P<0．05;â‘¡In Low-dosage Zhenwu decoction group and High-dosage Zhenwu decoction group weight of unilateral renal significantly reduced than the model group P<0．05, and Niaoduqing group compared with the model group, P>0．05, with no statistical significance;â‘¢Unilateral renal weight of High-dosage Zhenwu decoction group significantly reduce than Niaoduqing group, P<0．05．Unilateral renal weight/body weight×103:â‘ the treatment groups has a marked increase than the normal group and sham-operated group, P<0．05．②In Low-dosage Zhenwu decoction group and High-dosage Zhenwu decoction group unilateral renal weight/body weight×10 significantly reduced than the model group P<0．05, and Niaoduqing group compared with the model group, P>0．05, with no statistical significance;â‘¢Unilateral renal weight/body weight×103 of High-dosage Zhenwu decoction group significantly reduce than Niaoduqing group, P <0．05．3．7 Renal tissue pathology by optical microscope: Glomerular and tubular structures was normal in normal and sham-operated rats. Model group: large tubular epithelial cell apoptosis and necrosis or vacuolar degeneration, tubular great expansion of tubular. There are a lot of renal interstitial golden-yellow product of purine metabolism crystal deposition severe renal interstitial fibrosis proliferation. This shows a large number of renal interstitial infiltration of inflammatory cells, renal cysts expansion of mesangial cells mild hyperplasia. Niaoduqing group: tubular mild expansion, a small golden-yellow crystalline product of purine metabolism. Mild renal tubular epithelial cells in vacuolar degeneration, mild interstitial fibrosis, renal interstitial infiltration of inflammatory cells was observed, expansion of renal cysts, mesangial cells mild hyperplasia. Low-dosage Zhenwu decoction group: tubular moderate expansion with tubular atrophy, tubular epithelial cell necrosis, renal interstitial with a small amount of golden-yellow product of purine metabolism crystal deposition, moderate interstitial fibrosis, renal cysts expansion mesangial cells mild hyperplasia. High-dosage Zhenwu decoction group: small tubular showed moderate expansion of renal interstitial fibrosis, and renal interstitial with a small amount of golden-yellow crystalline product of purine metabolism, mild renal cysts expansion. There was a mesangial cells.3．8 Semi-quantitative grading tubulointerstitial lesions: In Niaoduqing group, Low-dosage Zhenwu decoction group and High-dosage Zhenwu decoction group tubulointerstitial damage compared with the model group, P<0．01; tubulointerstitial damage of High-dosage Zhenwu decoction group close to the Niaoduqing group, P = 0．05,and more than tubulointerstitial damage of Low-dosage Zhenwu decoction group to the light, P<0．05．3．9 PASM mesangial integral optical density quantitative model of mesangial proliferative: The most obvious model of mesangial proliferative positive goals largest integrated optical density value, compared with the normal group,a significant difference, P<0．05．Mesangial proliferative of the treatment groups lighter than model group, P<0．05, and mesangial proliferative of Low-dosage Zhenwu decoction group and High-dosage Zhenwu decoction group significantly lighter than Niaoduqing group, P<0．05．3．10 Masson staining fibrosis in the area of renal interstitial: The model group has the largest group fibrosis, renal interstitial area of the highest percentage share, compared with the normal group, a significant difference, P<0．05．The area of renal interstitial fibrosis of Low-dosage Zhenwu decoction group and High-dosage Zhenwu decoction group were significantly lower than the model group, P<0．05 ,and in the Niaoduqing group renal interstitial fibrosis in the area although lower than the model group, but no significant differences, P>0．05．The area of renal interstitial fibrosis of Low-dosage Zhenwu decoction group and High-dosage Zhenwu decoction group were significantly lower than Niaoduqing group, P <0．05．3．11 Percentage distribution area of Sirius red staining of collagenâ… andâ…¢:â… andâ…¢collagen distribution larger in the model group, compared with the normal group, a significant difference, P<0．05．In Low-dosage Zhenwu decoction group and High-dosage Zhenwu decoction group and Niaoduqing group renal interstitial of type I and III collagen were significantly smaller than the size distribution in model group, P<0．05．In Low-dosage Zhenwu decoction group and High-dosage Zhenwu decoction group renal interstitial of typeâ… andâ…¢distribution area less than Niaoduqing group. However, no significant difference, P<0．05．4 Conclusion4．1 Zhenwu decoction can improve the overall situation in rats, diuresis, reducing urinary protein of 24 hours and improving renal function. It can ease of renal anemia, regulate calcium metabolism; expansion and inflammatory cell infiltration in renal tubules, and renal interstitial fibrosis lower,â… ,â…¢collagen fibers hyperplasia reduced compensatory renal cysts mesangial expansion and reduce proliferation. This shows that Zhenwu decoction has a protective effec in rats of chronic renal failure using adenine after unilateral nephrectomy.4．2 In renal interstitial of Low-dosage Zhenwu decoction group and High-dosage Zhenwu decoction group,â… and typeâ…¢hyperplasia fall below the model group. Zhenwu decoction shows inhibitâ… ,â…¢collagen fibers proliferation and promote their degradation, thus achieving the anti-renal interstitial fibrosis, which may be Zhenwu decoction on renal interstitial fibrosis and chronic renal failure mechanism of the development process. However, the mechanism to be further in future studies.4．3 Experimental results show that Zhenwu decoction high-dose group is better than Zhenwu decoction low-dose group in rats with chronic renal failure.However, the experiment only use two-dose groups. Zhenwu decoction could not explain the treatment of chronic renal failure effective dose and dose-effect relationships. Aconite is the main drug of toxic medicine in Zhenwu decoction, therefore Zhenwu decoction need further toxicological studies and safety evaluations.
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