| Objective: Renal interstitial fibrosis is the final performance of chronic kidney disease(CKD),which is inevitable progress to end-stage renal disease(ESRD).The main performances of renal interstitial fibrosis are renal tubular epithelial cells-the muscle fiber cell transdifferentiation,expressing muscle fibroblasts markers,such as alpha SMA protein,secretion of extracellular matrix(ECM),produce a large amount of collagen,excessive deposition in renal interstitial.To our knowledge,it is closely related to the development of renal interstitial fibrosis and oxidative stress.Renal production of oxygen free radicals and clear imbalance is one of the important mechanism of chronic renal interstitial fibrosis.Probucol play an important role in treatment and prevention of cardiovascular disease,having strong antioxidant effect.According to research,probucol test by reducing reactive oxygen species(ROS)generation and lipid peroxidation,inhibiting oxidative stress,and by reducing the expression of cytokines(TGF–?,for example)and connective tissue growth factor(CTGF)to protect mesangial cells affected by the high glucose.Multiple signaling pathways in renal interstitial fibrosis can be activated by oxidative stress,which P38 is MAPK signaling pathways,mediated by cell growth,differentiation,proliferation,apoptosis,inflammation and other physiological process,plays an important role in the process of renal interstitial fibrosis.Studies have shown that the oxidative stress activated,with the generation of ROS increasing,MLK3 expression inducing,activate the downstream P38 and make its phosphorylation expression,eventually leading to collagen type ? increased expression.Meanwhile,probucol,one of the antioxidants,can supress oxidative stress and reduce ROS generation.Through the intervention of probucol,this experiment detect the expression of MDA,MLK3,P-P38 and Collagen type ? in unilateral ureteral occlusion rats,exploring how to reduce and delay the renal interstitial fibrosis,possible mechanisms of renal protection effect,to delay the progress of chronic kidney disease(CKD)and provide new therapeutic basement.Method:54 clean level healthy male SD rats,body weight 180±20 g,after 1 week adaptive feeding,were randomly divided into three groups: sham group(group A),model group(group B),probucol group(group C),with 18 rats in each group.All groups would not provide food before 12 hours in the operation day and build unilateral ureteral obstruction model.Each group administrated orally one day before the surgery.The treatment group was given probucol 500 mg/kg/d orally,the sham group and model group were gavaged with isometric saline.The rats were allowed to eat and drink free.Randomly select 6 rats from each group respectively,and harvest their obstructed kidneys on day 3rd,7th,14 th after the UUO surgery.The kidneys were divided into 4 parts after 0.9% saline flushing.A quarter was fixed by 4% neutral formalin and was detected by pathological section to observe the kidney tissues pathological changes and the degree of renal fibrosis,as well as immunohistochemistry to detect the expression of MLK3,P-P38,and Collagen Type ?.Three quarters of kidney was reserved under-80?,to be used for MDA detection and western blot detecting the expression of P-P38.The experimental data would be analysed by statistical analysis software SPSS 20.0 and displayed as mean ± standard deviation,ANOVA test were used for data analysis.P <0.05 was considered statistically significant.Results: 1 Pathomorphological observation of renal tissue: HE staining under light microscopy: In group A,there were no significant change in renal tubular and interstitial;In group B,renal tubular was mild expansion,contraction,vacuoles degeneration,epithelial cells falls off.Inflammatory cells local infiltrating in the renal interstitial was observed;Compared with group B,in group C,changes were still better than those in group B,but they were worse than those in group A,renal tubular interstitial lesions with obstruction after prolonged become progressive worse,the normal structure was gradually disappear;Masson staining under light microscopy: In group A,renal interstitial was not observed obvious collagen expression and abnormal structure;In group B,slight tubular expansion and collagen fibers deposition were seen,gradually extended to the surrounding,intertwined,and the formation of "mesh" structure;In group C,changes were better than those in group B,but worse than group A.The degree of renal tubular expansion,collagen expression level were got worse as obstructed time went by;2 MDA content in the kidney tissues of associates method detection: There was no significant change in group A.In group B,MDA content in renal tissue increased after 3rd days.Compared with group A,as well as postoperative 7th days and 14 th days,MDA content in renal tissue increased progressively(P < 0.05);In group C,MDA expression in the 3rd,7th,14 th day was slightly lighter than group B,but significantly worse than group A;3 The immunohistochemical results of MLK3,P-P38 and Col-? in renal tissue: MLK3:In group A,only trace amount of MLK3 expression was detected in tubular epithelial cells and renal interstitial;However,the expression of MLK3 were largely detected in proximal tubular epithelial cells and renal interstitial in group B.The expression level of MLK3 went up in a time dependent manner,and there was a significant difference(P<0.05)between group A and group B on the same time point;In group C,a large amount of MLK3 expression was detected in tubular epithelial cells and renal interstitial,increased as time went by.The expression level of MLK3 was not as much as that in group B(P<0.05),but still much higher than that of group A(P<0.05);P-P38: In group A,the expression of P-P38 was detected only bassic trace expression in the tubular epithelial cells,renal interstitial and nuclei;Compared with group A,the expression of group B was increased in the tubular epithelial cells,renal interstitial and nuclei as obstructed time went by,and there was a significant difference(P<0.05);In group C,the level increased as obstructed time went by,and was less than group B,but more than group A.There was a significant difference(P<0.05);Col-?: In group A,the expression of Col-? in the tubular epithelial cells and renal interstitial was detected only bassic trace expression;In group B,Col-? significantly increased with the obstruction time,and there was a significant difference(P<0.05)in the 3rd.On the 7th day,and the 14 th day,there was statistically significant difference(P<0.05)compared to group A on the same time point;The level of group C was less than group B,but more than group A,increased as obstructed time went by,and there was a significant difference(P<0.05);4 P-P38 in the kidney tissues detected by Western Blot:In group A,the expression of P-P38 was only bassic trace expression;Compared to group A on the same time point,the expression of group B were apparently increased(P<0.05).The level of group C was increased as obstructed time went by,which was less than group B,but more than group A,and there was a significant difference(P<0.05).Conclusion:The expression of MDA,MLK3,P-P38 and Col-? were increased in unilateral ureteral occlusion rats,and they are relaying on time.It was shown that there was an obvious state of oxidative stress in UUO model of renal interstitial fibrosis,and was closely associated to P38 MAPK signaling pathway activation.Expression of MDA,MLK3,P-P38 and Col-?in the probucol group was lower than the same period UUO group,which implys that,in the process of renal interstitial fibrosis,probucol can inhibit oxidative stress,reduce the expression of MLK3,P-P38,resulting in Col-expression ?desreased,have the effect of delay and reduce the renal interstitial fibrosis.It maybe infer that one of the mechanism of probucol inhibited renal interstitial fibrosis should suppress oxidative stressreduce,which may protect renal by reducing the expression of ROS. |
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