Protective Effects Of Hongbeiyegen On The Model Of Rats With Renal Interstitial Fibrosis

BackgroundRenal fibrosis (including renal glomerular sclerosis and renal interstitial fibrosis) is almost the common pathways of renal failure from the start to the end, the atrophy of renal tubular and the hyperplasia of renal interstitial fibroblasts and the excessive accumulation of extracellular matrix (ECM) are the characteristic of pathological changes. When the renal disease happen, the inflammatory cells especially the monocytes and the growth factor of renal interstitial fibroblasts (RIF) which synthesis and secretion by the renal tubular epithelial cell, such as transforming growth factorβ1 (TGF-β1) and endothelin and platelet derived growth factor (PDGF), etc, infiltrate the renal interstitium, and act on the mechanocyte by paracrine, let it activate and proliferate or transform to the myofibroblast, express the proteins of a-SMA, and secrete a lot of ECM, contain CⅢand FN, etc. Therefore, inhibiting the activation and proliferation of the renal fibroblasts, and reducing the secretion of ECM can play an important part in preventing or reversing the process of renal interstitial fibrosis.The normal tissue of kidney is replaced by the ECM and accompany with the irreversible damage of renal function which caused by the continues primary or secondary damage of the kidney lead to the RIF, and the atrophy of renal tubular and the hyperplasia of renal interstitial fibroblasts and the excessive accumulation of ECM are the characteristic pathologic changes.According to the present research, there are five factors lead to the mechanism of RIF:Firstly, the activation of interstitial fibroblasts. Most scholars believe that interstitial fibroblasts are the main effector cell of RIF, the activation of interstitial fibroblasts is one of the the key link in RIF. Secondly, the excessive accumulation of ECM. ECM not only plays a very important role in maintaining the normal tissue structure and function of kidney but also in the grow and atomization of the cells, it between the dynamic equilibrium of metabolic and remodeling, the activity of MMPS which degrade the ECM drop and the expression of TIMPS which restrain the degradation of ECM up regulation lead to the disorder of dynamic balance in ECM. The excessive accumulation of ECM in renal interstitium are the main cause of RIF. Thirdly, the effect of vaso-activesubstance, growth factor and cell factor. Such as the Angiotensin, TGF-β1, FGF, PDGF, CTGF, TNF-α, IL-1, ET-1, BMP-7.They take park in the activation of interstitial fibroblasts, cell proliferation, TEMT, increase of ECM. Fourthly, apoptosis. We guess there are some relevance between the disappear of renal tubular epithelial cells and apoptosis. Fifth, TEMT. The normally renal tubular epithelial cells express the keratin which is the sign protein of epithelial, but in pathologic state, it express the vimentin and a-SMA which are the sign protein of interstitial cell.Recently, there are many studies about restraining renal fibrosis in modern medicine, but less of them gain clinical applications. By the contrary, TCM not only take a good clinical applications in controlling chronic renal failure, but also get a good result from the experiment research of renal fibrosis, which give a solid foundation for the clinical.Hongbeiyegen (HBYG) which also call honglingdan, hongluoqun, hongbeiniang Honggubaiquanshugen, belong to euphorbia plants branching from dicotyledon, it has "Sweet taste and cool feeling", it has the effect of "Clear away heat and promote diuresis, activating blood circulation, detoxifying ". Previous studies have shown that HBYG can resist rat liver fibrosis induced by alcohol ang carbon tetrachloride and immunity."Homotherapy for heteropathy" is a theory of traditional Chinese medicine, and the modern medicine studies have demonstrate that the sediment of ECM which cause by every pathogenic factor on the viscera is the same forming mechanism of hepatic fibrosis and renal interstitial fibrosis. According the above theory and the studies we have done, did the HBYG have the effect of antifibrotic in renal fibrosis? Therefore, we observe the affect of HBYG on histopathology, express of TGF-0-1, index of hematology in rats of RIF, and to preliminary elucidate how it effects on. Objective:To research the affect of hongbeiyegen on histopathology, TGF-β1, four index of fibrosis, renal function, and hemorheology on rats with renal interstitial fibrosis following unilateral ureteral obstruction (UUO), and to discussed the Protective effects and mechanism of hongbeiyegen on renal interstitial fibrosis.Method:1. Establishment of the animal model, Grouping and treatment40 specific pathogen free (SPF) SD rats,180-200g body weight, half male and half female, were randomly assigned into five groups after one week’s adaptation living, as follows:the sham operation group, the renal interstitial fibrosis model group, Dahuangzhechongwan (DHZCW) group, normal-dose of Hongbeiy (HBYG-L) group, high-dose of Hongbeiyegen (HBYG-H) group.Rat renal interstitial fibrosis Model induced by unilateral ureteral obstruction, and the sham operation group just dissociated the left ureter, and did not deligation it, then sew up the muscle and skin.The whole operation demand asepsis. The corresponding drug were given in DHZCW group, HBYG-L group, HBYG-H group (1 ml/100g per day), and the sham operation group, the renal interstitial fibrosis model group were given saline. All rats were allowed food and water ad libitum. It kept for 4 weeks. After the experiment, kidney organization was got, and stained by HE and Masson. The pathological changes and the fibrosis proliferation were observed under the light microscope. We got the upper animal serum, measured the BUN and Scr by automatic biochemical analyzer; measured the HA, LN, PCⅢandⅣ-C by radio-immunity r counter according to radioimmunoassay method. We got the blood, measured the blood rheometer which at different shear rate and HCT by blood rheometer. And the expression of TGF-β1 were observed through immunohistochemistry.2. The statistics method.Statistical software SPSS 13.0 was used to analyze the experimental data. The experimental data were indicated by mean±standard deviation(x±s). One-way ANOVA was used to analyze the data. LSD method was used in multiple comparisons between groups when variance was homogeneous. Dunnett’sT3 was used when variance is not homogeneous. Kruskal Wallis was used to analysis the difference of the stage of renal interstitial fibroblasts. Difference is significant when P<0.05.Result1. Pathological result1.1 HE resultThe sham operation group:the structure of kidney was normal, the renal tubule ranked tightly and orderly, the renal tubule basilar membrane was smooth and continuous, and there were not inflammatory cells infiltrated in renal interstitium. The model group:hardly all the renal corpuscle and renal tubule were necrosis, the rest of renal tubule was atrophic or ecstatic; the renal interstitial fibrosis was serious, the inflammatory cells infiltrated. The DHZCW group:most of the renal corpuscle and renal tubule was necrosis, the rest of renal tubule was atrophic or ecstatic; the renal interstitial fibrosis was generic and the inflammatory cells infiltrated generic. The HBYG-L and HBYG-H group:a few of the renal corpuscle and renal tubule were necrosis, part of the renal tubules were ecstatic, most of the renal corpuscle and renal tubule were normal; the renal interstitial fibrosis were lowly and the inflammatory cells infiltrated generic.1.2 Masson resultThe sham operation group:the tintage of renal collagen mainly lied on basement membrane of glomerulus, Bowman sac, mesangium, around the blood capillary which between the renal tubule, and they were narrow in the mesenchyme which round the renal tubule. The model group:the renal tubules were enlarged, the widths of renal interstitium were increased, the blue collagenous fiber was increased, and the colouration deepened. The DHZCW group, HBYG-L group, HBYG-H group:the expansion of renal tubules, broadening of mesenchyme, and deposition of collagen were lower than the model group. The percentage of tintage area in renal interstitium: compared with the sham operation group, the model group, DHZCW group, HBYG-L group, HBYG-H group increased significantly, they had sociodemographic difference (P<0.05); Compared with the model group, the DHZCW group, HBYG-L group, HBYG-H group reduced significantly, they had sociodemographic difference (P<0.05); Compared with the DHZCW group, the HBYG-L and HBYG-H group had no significant difference, they did not have sociodemographic difference (P>0.05).2. Blood index result2.1 Effect of HBYG on serum BUN and Scr in ratCompared with the sham operation group, the serum BUN and Scr at the model group, DHZCW group, HBYG-L group, HBYG-H group increased significantly, they had sociodemographic difference (P<0.05); Compared with the model, the DHZCW group, HBYG-L group, HBYG-H group reduced significantly, they had sociodemographic difference (P<0.05); Compared with the DHZCW group, the HBYG-L and HBYG-H groups had no significant difference, they did not have sociodemographic difference (P>0.05).2.2 Effect of HBYG on serum HA、LN、PCⅢ、Ⅳ-C in ratCompared with the sham operation group, the serum HA、LN、PCⅢ、Ⅳ-C at the model group, DHZCW group, HBYG-L group, HBYG-H group increased significantly, they had sociodemographic difference (P<0.05); Compared with the model, the DHZCW group, HBYG-L group, HBYG-H group reduced significantly, they had sociodemographic difference (P<0.05); Compared with the DHZCW group, the HBYG-L and HBYG-H groups had no significant difference, they did not have sociodemographic difference (P>0.05).2.3 Effect of HBYG on the blood viscosity at different shear rate and HCT in ratCompared with the sham operation group, the blood viscosity at different shear rate and HCT at the model group, DHZCW group, HBYG-L group, HBYG-H group increased significantly, they had sociodemographic difference (P<0.05); Compared with the model, the DHZCW group, HBYG-L group, HBYG-H group reduced significantly, they had sociodemographic difference (P<0.05); Compared with the DHZCW group, the HBYG-L group and HBYG-H group had no significant difference, they did not have sociodemographic difference (P>0.05).3. Immunohistochemistry resultTGF-β1 hardly expressed in the sham operation group, but expressed with varying degrees in renal tubular epithelial cells, glomerulus and fibroblast, it showed as a tawny and granulate deposition, especially at the model group. The semi-quantitative analysis showed that:compared with the sham operation group, the express of TGF-β1 at the model group, DHZCW group, HBYG-L group, HBYG-H group increased significantly, they had sociodemographic difference (P<0.05); Compared with the model group, the DHZCW group, HBYG-L group, HBYG-H group reduced significantly, they had sociodemographic difference (P<0.05); Compared with the DHZCW group, the HBYG-L group and HBYG-H group had no significant difference, they did not have sociodemographic difference (P>0.05).Conclusions:The HBYG can significantly reduce the damage of renal tissue, protect the renal tubular epithelial cells, reduce the fibrosis degree of renal tissue, improve the index of serum BUN, Scr, HA, LN, PCⅢandⅣ-C, reduce the expression of TGF-β1, give a protection in rats of RIF, and it can improve the indexes of hemorrheology, pay an effect in improving the microcirculation which signs that the function of promoting blood circulation and removing blood stasis may be one of the drug efficacy.