| Objective: Renal interstitial fibrosis(RIF)is a inevitable pathological process which is variety of chronic kidney disease progress to end-stage renal disease(ESRD),which molecular mechanism is the excessive deposition of extracellular matrix(ECM),resulting in structural changes and functional decline of renal tubular and glomerulus.In the process of renal interstitial fibrosis,there is a series of cellular and molecular events leading to renal interstitial cells and collagen component of aggregation,the renal tubular atrophy and expansion,renal units,progressive destruction of the occasional.These cytokines,chemokines,and inflammatory cells joint mediated renal inherent fibroblasts(FB)and transdifferentiation of renal tubular epithelial cells for Myofibroblast(MFB),namely between the epithelial-mesenchymal transition,EMT.MFB,expression of alpha Smooth Muscle Actin(a-SMA),is the main effect of RIF cells,mainly secreted and ⅠⅢ collagen.Transforming Growth Factor beta 1(TGF-β1)is one of the important fibrosis Factor,many studies have confirmed its downstream Factor extrace-llular signal regulating kinase(ERK),and which is involved in cell proliferation,differentiation,transdifferentiation and apoptosis,playing a key role in the process of inducing EMT.The efficacy of Xuebijing active ingredients includes anti-inflammatory,anti-oxidation,scavenging of free radicals,anti-platelet aggregation,and the protection of vascular endothelium.It has been showed by many modern studies that Xuebijing can delay pulmonary fibrosis and liver fibrosis,but it is rarely reported in the application of renal interstitial fibrosis.The experiment detect the expression of TGF-β1,p-ERK1/ 2,α-SMA,Collagen-Ⅲin unilateral ureteral occlusion rats,to simulate the process of renal interstitial fibrosis,exploring whether Xuebijing provide advanced clinical prevention and treatment of renal fibrosis through anti-inflammatory and antioxidant delay the progress of renal interstitial fibrosis.Method: To choose clean level male 54 SD rats,weight 180-220 g,adaptive feeding 1 week,randomly divided into three groups,that is sham group,model group,Xuebijing group,with 18 rats in each group to build unilateral ureteral obstruction model.All groups would be fasting for 12 hours before surgery.All rats is received Chloral hydrate(0.4ml/100g)by intraperitoneal injecting.After disinfection,the incision was performed on the left of the midline of the ventral midline,and extended along the midline of the abdomen.The kidney was exposed and the ureter was gradually separated along the lower part of the kidney.The ureter was ligated from the proximal kidney with 3-0 suture lines and cuted off to prevent retrograde infection and then sutured to the skin layer by layer.The sham operated rat underwent the same surgery procedure except for the obstruction and cut of the left ureter.Normal diet and activity after operation.The three groups were given medicine 1 days before operation,and group XBJ was injected with Xuebijing injection 4 ml(kg/d),and group Sham and UUO group were intraperitoneally injected with equal volume of normal saline.Six rats were randomly selected to be executed on the 3rd,7th and 14 th day,removed from the obstruction kidney,which was divided into four parts: A quarter was stored in the fixed liquid,and the remaining three parts are kept in the refrigerator at-80℃.HE and Masson staining were used to observe the pathological changes of renal tissue in each group.The expression level of TGF-beta 1,alpha-SMA,p-ERK1/2 and Collagen-Ⅲ in each group was analyzed by immunohistochemistry.Results: 1 Pathomorphological observation of renal tissue 1.1 Naked eye observationIn group Sham,the kidneys were fresh and normal in size,and no atrophy in renal cortex and medulla.In group UUO,the volume of kidneys and the hydronephrosis were the most obvious.The color and size of kidneys in group XBJ were all lighter than those in group UUO.1.2 Microscopy observation 1.2.1 HE staining: In group Sham,there were no abnormal in the rats renal tissue structure;In group UUO,it can be seen that renal interstitial inflammatory cell infiltration,renal tubule dilation,epithelial fine degeneration necrosis,basement membrane thickening,creases,and kidney interstitial thickening,and gradually increase with time.Compared with group UUO,in group XBJ,changes were similar,but still better than those in group UUO.1.2.2 Masson staining: In group Sham,no obvious abnormalities were observed in renal tissue structure.On the third day,there was slight dilation of renal tubules in group UUO and XBJ.On the seventh days and 14 days of Group UUO: renal tubular dilatation and blue interstitial collagen fiber accumulation were observed,and aggrav-ated with prolonged obstruction time.Compared with groupUUO in the same period,the deposition degree of renal interstitial collagen fibers In group XBJ was relatively mild,but it was still significantly heavier than that of group Sham.2 Immunohistochemical results 2.1 TGF-β1In group Sham,only trace in amount of TGF-β1 expression was detected in mesangial cell and tubular epithelial cells,no statistical difference between the third,seventh,fourteenth days after the operation(P>0.05).In group UUO,TGF-β1 was mainly expressed in renal tubular epithelial cells,and increased gradually with the prolongation of obstruction time(P<0.05)on the 3rd,7th,14 th day,and the level of group Sham was lower than that at the same time(P<0.05).In group XBJ,TGF-β1 is mainly expressed in renal tubular epithelial cells,and its level were decreased significantly compared with group UUO(P<0.05),but higher than the same period in group Sham(P<0.05).2.2 p-ERK1/2Group Sham: p-ERK1/2 mainly expressed the nucleus of renal tubular epithelial cells.There was no statistical difference in the 3rd,7th,14 th day expression level.Group UUO: p-ERK1/2 increased as time went by,but expression in group Sham at the same phase was lower(P < 0.05).Group XBJ: The expression of p-ERK1/2,on the same time point,was significant lower than group UUO(P<0.05),but higher than group Sham(P<0.05).2.3 α-SMAIn group Sham,there was no obvious expression of alpha-SMA in renal tubule epithelial cells and renal interstitium,and there was no statistical difference at third,seventh,fourteenth days after operation(P>0.05).Compared with group Sham,the expression of groupUUO was increased as obstructed time went by.In group XBJ,the level was less than group UUO,but higher than group Sham at the same time(P <0.05).2.4 Collagen-ⅢIn group Sham,Collagen-Ⅲhas a small amount of expression in renal interstitium and tubular epithelial cells and There was a significant difference on the 3rd,7th,14 th day(P>0.05).In group UUO,Collagen-Ⅲ,in renal tubular epithelial cells and renal interstitial,expression level increased obviously,and gradually increased with time in the 3rd,7th and the 14 th day,but expression in group Sham was lower at the same time.The CollagenⅢ in renal tubule epithelial cells and renal interstitium increased significantly.The level was significantly lower than that of group UUO in the same period(P<0.05),but still higher than the expression in group Sham(P< 0.05).Conclusion:1.In the UUO model,the expression of TGF-β1,p-ERK1/2,alpha-SMA,and Collagen-Ⅲ were gradually increased with the prolongation of the obstruction time.TGF-β1/ERK1/2 pathway,alpha-SMA,and Collagen-Ⅲ may be participated in the development of renal interstitial fibrosis.2.In the Xuebijing Intervention,the expression of TGF-β1,p-ERK1/2,-SMA,Collagen-Ⅲ was significantly decreased,and renal interstitial fibrosis reduced.Xuebijing has an anti fibrotic effect.Its mechanism may be achieved by cutting down the TGF-β1/ERK1/2 signaling pathway and reducing the expression level of alpha-SMA and Collagen-Ⅲ. |
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