Mechanism Of Regulating Smads Signaling Pathway To Inhibit Renal Interstitial Fibrosis By Shenluotong

PartⅠ: Effect of Shenluotong on histomorphology of rats with renal interstitial fibrosisObjective: This experiment established obstruction kidney interstitial fibrosis animal model by unilateral ureteral obstruction(UUO). It adopted Shenluotong intervention treatment, and it compared with the aldosterone receptor blocking agent eplerenone as the control drug to observe the changes of renal function and the renal pathological morphology of experimental animals and to investigate the Chinese herbs recipe mechanisms of inhibition of renal interstitial fibrosis.Methods: 1 Animal grouping and model preparation: Rats were free to eat and drink, and temperature was controled in the condition(22±2)℃. 48 Wistar rats were randomly divided into the Sham group(n=12), UUO group(n=12), EPL group(n=12), and SLT group(n=12). After adaptive feeding 1WK, except the Sham group’rats, the other rats were established model of unilateral ureteral obstruction according to the method of Iwai T. After anesthesia with 10% chloral hydrate, the rats were sliced through the left middle abdominal skin, separated the left ureter, ligated respectively on ureteral 1/3 and middle 1/3 ligation with silk, cut off the ureter, then sutured in skin by layer to layer. The rats in Sham group were only separated the left ureter, but not ligated and cutted off the ureter. At 3 days after operation, renal pathology of modeling rats showed a significant increase with infiltration of inflammatory cells, as well as at 5 days after operation, pathological changes showed renal collagen deposition. Therefore this model was successfully copied. Five rats died after UUO operation. Rats were supplemented with spare rats. 2 Give medicine: After operation, the rats were treated with 100mg/kg Eplerenone in EPL group and 26g/kg in SLT group; others in Sham and UUO group administered with the same volume of saline once a day. Ten days later, rats in each group were weighed and then were executed decollation.The kidneys were cut out of the side of the obstruction. After removal of the membrane, the partial kidney tissues were fixed with 4% formaldehyde to prepare for the renal pathological examination. 3 Index detection and method:We did daily observation and record on each rat’s mental state, activity, fur changes, drinking water and food intake. Each rat body weighted, and urine volume(24h) were weighted; we weighed mice and the left kidney, and then calculated rat kidney weight/body weight ratio(KW/BW); parts of left kidney were prepared respectively for HE, MASSON, Sirius red staining, and optical microscope to observe the pathological changes. 4Statistics processing: The data were expressed as mean ± SD.The data were counted by SPSS16.0 software. Before comparison of each group, tests of normality and homogeneity of variance were given. If the data can achieve such criteria, the results were analyzed with One way ANOVA.Results: 1 General conditions of rats in each group and the results of the determination of the ratio of renal weight/body weight in the obstructed side: General conditions:Rats in Sham group were in good general condition, and had no significant changes before surgery.After UUO, the rats in each model group were in poor general condition: poor hair gloss, poor response to external stimuli, diet significantly reduced. Comparison of renal weight/body weight in the obstructed side: The kidney weight of each model group was significantly increased compared with Sham group after operation(P<0.01),but weight is significantly lower(P<0.01). The ratio of renal weight/body weight was significantly higher in each model group than that in Sham group.Compared with the UUO group rats, the weight of the rats in the EPL group and the SLT group were significantly decreased(P<0.01, 或 P<0.05),but body weight was significantly increased(P<0.01, 或 P<0.05).Therefore, the ratio of renal weight/body weight in the obstructed side was significantly lower than that in the UUO group(P<0.01, 或 P<0.05). 2 Pathological changes of kidney in rats with obstructive side: HE staining showed: In Sham group, there was no abnormal change in renal tissue structure, the structure of renal tubular epithelial cells was not changed, the arrangement was neat, there was no increase and no edema in renal interstitial, and only a small amount of inflammatory cell infiltration was found.The renal tissue structure of UUO group was significantly changed:the structure of renal tubular epithelial cells was abnormal,the changes of degeneration and necrosis were observed, even partial loss. A large number of inflammatory cell infiltration were observed in renal interstitial.Renal tubular showed obvious dilation. Renal tubular epithelial cell of EPL group and SLT group were degeneration, necrosis and the degree of renal tubular expansion slightly was less than the UUO group, the number of inflammatory cell infiltration was significantly reduced.Masson staining showed that a little collagen deposition in glomerular/ tubular basement membrane and renal interstitium in Sham group. The structure was disordered and tubular interstitial collagen increased significantly in UUO group. There were some degree of collagen deposition in treatment groups, but it was significantly reduced compared with the UUO group. Sirius red staining showed that, in Sham group, glomerular and tubular’s structure was normal, and there were very small amounts of collagen expression in renal interstitium. The renal tubules of UUO group were obviously dilated, there were a large number of collagen components in the kidney.The increase of interstitial collagen deposition was in the kidney of the group of the EPL group and the SLT group, but compared with the UUO group was significantly reduced.PartⅡ:Effect of Shenluotong on the expression of TGF-β1 and CTGF in rats with renal interstitial fibrosisObjective: This experiment established obstruction kidney interstitial fibrosis animal model by unilateral ureteral obstruction(UUO), Shenluotong intervention treatment is adopted, and the aldosterone receptor blocking agent eplerenone as the control drug. Immunohistochemistry, Western Blot and RT-PCR were used to detect the expression of TGF-β1 and CTGF in rat kidney. Observe the regulation effect on TGF-β1, CTGF of eplerenone and Shenluotong, to explore the Chinese herbs recipe mechanisms of inhibition of renal interstitial fibrosis.Methods: The methods of animal grouping, model establishment, medication and statistics were same as PartⅠ, the experiment lasted 10 days. At the end of the experiment, the kidney samples were collected. Immunohistochemistry, Western Blot and RT-PCR were used to detect the expression of TGF-β1 and CTGF in rat kidney. The statistical method was same as PartⅠ.Results: 1 The results of Real time-PCR: Compared with the rats in Sham group, the m RNA expression of TGF-β1 and CTGF were enhanced obviously in UUO group(P<0.01, P<0.05). Compared with the rats in UUO group, the high expression of TGF-β1 m RNA and CTGF m RNA was down-regulated respectively in both EPL and SLT group, more obvious in EPL group(P<0.05). 2 The results of Immunohistochemistry:In Sham group, α-SMA mainly expressed in the blood vessels, other parts of the expression was not obvious;PCNA positive cells had a small amount of expression; TGF-β1, CTGF mainly expressed in renal tubular epithelial cells and glomerular, showing weak expression. In UUO group, PCNA positive cells were significantly increased, mainly in the dilated distal tubular epithelial cells; α-SMA expression was also significantly enhanced, mainly in the renal tubular interstitial and epithelial cells.TGF-β1 and CTGF were mainly expressed in renal tubular epithelial cells and glomerular.Compared with UUO group, the positive cells of PCNA in EPL group and SLT group were significantly decreased; the range and intensity of expression of α-SMA, TGF-β1 and CTGF were significantly decreased. 3 The results of Western Blot: Compared with the rats in Sham group, the protein expression of TGF-β1 and CTGF were enhanced obviously in UUO group(P<0.01, P<0.05). Compared with the rats in UUO group, the protein expression of TGF-β1 and CTGF were decreased significantly in both EPL group and SLT group,more obvious in EPL group(P<0.01).PartⅢ: Effect of Shenluotong on Smads signaling pathway in rats with renal interstitial fibrosisObjective: This experimentestablished obstruction kidney interstitial fibrosis animal model by unilateral ureteral obstruction(UUO), Shenluotong intervention treatment is adopted, and the aldosterone receptor blocking agent eplerenone as the control drug. Immunohistochemistry, Western Blot and RT-PCR were used to detect the expression of Smad3、Smad4、Smad7 in rat kidney. Observe the regulation effect on Smad3、Smad4、Smad7 of eplerenone and Shenluotong, to explore the Chinese herbs recipe mechanisms of inhibition of renal interstitial fibrosis.Methods: The methods of animal grouping, model establishment, medication and statistics were the same to PartⅠ, 10 days. At the end of the experiment, the kidney samples were collected. RT-PCR were used to detect the expression of Smad4 m RNA, Smad7 m RNA. Immunohistochemistry and Western Blot were used to detect the expression of Smad3、Smad4、Smad7 in rat kidney.The statistical method is the same as PartⅠ.Results: 1 The results of Real-time PCR: Compared with the rats in Sham group, the m RNA expression of Smad4 were enhanced obviously in UUO group(P<0.01); the expression of Smad7 m RNA was obviously weakened in UUO group(P<0.01). Compared with the rats in UUO group, the m RNA expression of Smad7 were down-regulated significantly in both EPL group and SLT group, especially in EPL group(P<0.05); the expression of Smad7 m RNA was obviously enhanced in both EPL group and SLT group(P<0.05). 2 The results of Radioimmunoassay:The Smad3 and Smad4 of the Sham group were weakly expressed, and the expression of Smad7 was enhanced.The expression of Smad3 and Smad4 in UUO group was significantly enhanced, and the expression of Smad7 was decreased, which was mainly expressed in the cytoplasm of renal tubular epithelial cells. Compared with UUO group, the scope and intensity of expression of Smad3 and Smad4 in both EPL group and SLT group were significantly decreased.The range and intensity of Smad7 expression were significantly enhanced. 3 The results of Western Blot: Compared with the rats in Sham group, the protein expression of Smad3、Smad4 were enhanced obviously in UUO group(P<0.01); Smad7 protein expression was significantly decreased(P<0.01). Compared with the rats in UUO group, the protein expression of Smad3、Smad4 were down-regulated significantly, especially in EPL group(P<0.01);Smad7 expression was up-regulated in both EPL group and SLT group, especially in EPL group(P<0.01).Conclusions: 1 The method of unilateral ureteral ligation can be used to successfully replicate the animal model of renal interstitial fibrosis in rats with obstructive nephropathy. Renal pathology of UUO rats showed a large number of inflammatory cell infiltration and collagen deposition, which is consistent with the pathological changes of renal interstitial fibrosis.Shenluotong can reduce the number of inflammatory cells and collagen deposition in the renal tissue of UUO rats, and alleviate the pathological damage degree of renal interstitial fibrosis in UUO rats, thus delaying the progression of renal interstitial fibrosis and chronic renal failure. 2 Shenluotong and eplerenone can inhibit TGF-β1 signaling pathway and the expression of CTGF, which can inhibition of renal tubular epithelial cell phenotype transformation and reduce the proliferation of fibroblast cells, reduce the synthesis of ECM and increase its degradation, thereby reducing the accumulation of ECM. Thus reduce the degree of renal interstitial fibrosis and slow down the development of renal interstitial fibrosis. 3 TGF-β1/Smads signaling pathway is a common pathway of renal interstitial fibrosis..In the UUO kidney model, the TGF-β1/Smads signaling pathway was obviously activated, and the formation of renal interstitial fibrosis was induced. Shenluotong can inhibit the level of Smad3 and inhibit the combination with Smad4, while up-regulate the expression of Smad7, and blocking the transmission of TGF-beta1 signal,so as to prevent the occurrence and development of renal interstitial fibrosis in UUO rats.