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Experimental Research on the Prevention and Treatment of
来源:Beijing University of Chinese Madcine 100029 | 作者:sjzxads | 发布时间:2012-7-18 访问人数: 192

Experimental Research on the Prevention and Treatment of Shufeng Xuanfei Prescriptions on the Rat Chronic Serous Glomerulonephritis

ABSTRACT

Teng-yu  zhang-qiuju  Jiang-yan  Tu-hengjing

(Beijing University of Chinese Madcine  100029)

Chronic Glomerulonephritis (CGN), which can be caused by multiple reasons, is a series of immunological diseases with similar symptoms, different patho-manifestations and prognosises. CGN gradually progresses with a long course, progressive renal damage and some common clinical characteristics, such as proteinuria, hematuria, hypertension, edema and/or azotemia, etc. It has been showed that CGN, accounting about 64.1% of ESRD, is the most popular cause of End Stage of Renal Diseases (ESRD).

OBJECTIVE:

The pathogenesis of CGN is still not clear, but more and more outcomes on CGN develop it. It is reported that nuclear factor-κB (NF-κB) plays an important role during the course of CGN. NF-κB can specially bind to the κB binding sites which lie in the promoter or enhancer of many genes, and participate in some transcriptional control of genes relevant to immunological and inflammatory reaction. In many conditions, there are several κB binding sites in the transcriptional control region of the inflammatory mediators and cytokines participating in the glomerular inflammatory reaction. Thus these mediators and cytokines can be controlled by NF-κB.

TCM lays emphasis on BianZheng LunZhi, which may be available to the therapeutics of CGN. Professor Chaoqin Zhao, one of the most famous nephrologists in China, is good at using “wind herb”. In this topic, we planed to exert experimental research on professor Zhao’s empirical compound prescriptions, and to classify them on the basis of his medical theories. We hope to partly elucidate their mechanisms of action, and to provide theoretical basis for the clinical medication.

METHODS:

The experimental research can be divided into 3 parts. In the first part, we assorted 68 male Wistar rats randomly, weighing 150 ± 10g, into 5 groups, which are normal group, model group, prescription groupⅠ, prescription groupⅡand prescription groupⅢ. Normal group has 8 rats, and the other 4 groups have 12 rats respectively. We exerted pre-immunization on the rats except normal group, and give them an 8-week formal immunization while starting the dosage after 1 week. We kept the rats’ urine of 24 hours, then detected the urine protein and made a qualitative & quantitative analysis per-week. At the end of the 2nd, 5th and 8th week during the formal immunization, taking the blood directly via the heart, we got the serum after centrifugalization, then detected the BUN、Cre、Alb、TG and Cho respectively. At the end of the 8th week, we killed the rats and took the kidneys of each one, then put the right kidneys into 20% formaldehyde solution for a week. After dehydrolysis, waxing, imbedding and slicing, we performed the H.E. Staining, so as to compare the pharmacodynamic influence on the Rat Chronic Serous Glomerulonephritis of each prescription.

In the second part, we detected the NF-κB p65 subunit of the nephridial tissue with the SP staining method of immunohistochemistry, then carry out the image analysis to the staining results, so as to observe the influence on the e­xpression of NF-κB p65 of each prescription, and initially discuss the mechanism of Shufeng Xuanfei prescriptions on the Rat Chronic Serous Glomerulonephritis.

In the third part, after the formal immunization, we detected the TNF-α, IL-8 and IL-10 in the serum with the method of ELISA. And we grinded the left kidneys with 0.9% NS according to the ratio of 1: 9 (W/V), by which we made the nephridial homogenate. We also use the method of ELISA to detect the TNF-α in the nephridial homogenate. In this part, we planed to compare the influence on the Rat Chronic Serous Glomerulonephritis of each prescription from the cytokine levels.

RESULTS:

The result of the first part showed that the renal index of model group was significantly higher than that of normal group (P<0.01), and the renal index of prescription groupⅠ& Ⅲ was obviously lower than that of model group (P<0.05) after the formal immunization. It also indicated that the 24hrs’ urine protein of model group and each prescription group is obviously higher than that of normal group (P<0.01 or P<0.05) from the 2nd week of the formal immunization. From then on, the urine protein of model group and prescription groupⅡ went on climbing, and at about the 6th week came to the climax. However, as time went by, the urine protein of prescription groupⅠ& Ⅲ went down. The difference between them and normal group became quiet (P>0.05), but the difference between them and model group became more significant (P<0.01). The result of serous Alb revealed that the serous Alb of model group and prescription groupⅡ was obviously lower than normal group (P<0.01 or P<0.05), while prescription groupⅠ& Ⅲ could prevent its loss in some degree. Other serological items also indicated that as time went by, the serous BUN、Cre、Cho and TG of model group became obviously higher than that of normal group (P<0.05), and the serous BUN、Cre、Cho and TG of prescription group Ⅰ& Ⅲ became significantly lower than that of model group (P<0.01). In addition, after the formal immunization, H.E. staining showed us the manifestations of model group, such as the glomerular capillary wall diffusively thickening, splanchnopleural epithelial cells overgrowing, Bowman's space becoming narrow or disappeared, abundant inflammatory cells infiltrating, renal tubular epithelial cell swelling, obvious protein casts and leucocytosis in renal tubules. However, the manifestations of prescription groups were relatively slight, especially prescription group Ⅰ. And prescription group Ⅱ, the less.

The second part showed that NF-κB p65 in nephridial tissue expressed in both cytoplasm and cell nucleus. As for normal group, the cytoplasm showed dilute brown, and the cell nucleus occasionally presented brown-yellow particles. However, in model group, there were more brown-yellow particles than normal group both in cytoplasm and cell nucleus; and at the glomerulus, the brown-yellow particles mostly entered the cell nucleus. Concerning to the prescription groups, NF-κB p65 mainly expressed in cytoplasm, and the color in cell nucleus was diluter than model group, especially prescription groupⅠ& Ⅲ. It can also be confirmed by the image analysis (P<0.01 or P<0.05).

The third part showed, the TNF-α (serum or homogenate), IL-8 and IL-10 of model group were all higher than normal group (P<0.01). TNF-α of prescription groupⅠ& Ⅲ were obviously lower than that of model group (P<0.05). Serous IL-8 of of prescription group Ⅲ was obviously lower than that of model group (P<0.05). However, the difference of serous IL-10 between prescription groups and model group was not significant (P>0.05).

CONCLUSION:

As the first part indicates, prescription Ⅲ & Ⅰ (Shufeng Xuanfei prescriptions) can obviously improve the renal function and the permeability of glomerular basement membrane (GBM), inhibit the hyperlipemia during CGN and lighten the immunological & inflammatory damage. So maybe we can come to the conclusion that Shufeng Xuanfei prescriptions are effective on preventing the loss of urine protein and reducing the deposition of immune complex (IC), by which the glomerular damages are initiated.

From the second part, we can see Shufeng Xuanfei prescriptions can obviously inhibit the activation and nuclear translocation of NF-κB p65, thus weaken the biological effects of downstream genes.

The third part indicates, Shufeng Xuanfei prescriptions can relieve the immunological damage caused by cytokines which promote inflammation, and maybe have good effect on correcting the network imbalance of cytokines participating CGN. Respecting the non- significant difference of the serous IL-10 between prescription groups and model group, we cannot confirm that Shufeng Xuanfei prescriptions are effective on up-regulating anti-inflammatory cytokines, maybe continuing in-vitro experiments are available.

All of above show that Shufeng Xuanfei prescriptions are better than Liangxue Huayu prescription on the prevention and treatment of Rat Chronic Serous Glomerulonephritis.

KEYWORDS:

Chronic Glomerulonephritis(CGN); Serum Disease; Shufeng Xuanfei; Experimental Research; Nuclear Factor-κB (NF-κB)