| Objective: Renal interstitial fibrosis(RIF)is the common pathological basis of chronic kidney disease(CKD).Its pathology is based on the excess deposits of extracellular matrix(ECM),resulting in the structural damage in kidney and the gradual decline of renal function.The mechanism of renal interstitial fibrosis is complex,by a variety of cells,cytokines and cell signal transduction pathways involved in the process.The early performance of renal interstitial fibrosis is the state of inflammation including the infiltration of the inflammatory cells such as macrophages and lymphocytes and the increase of cytokines and chemokines.When those cytokines,chemical chemokines and growth factors effect on fibroblasts,FB will present the active hyperplasia and phenotype transformation,to increase collagen synthesis ability,leading to the excessively deposit of the extracellular matrix(ECM)and the remodeling of the kidney,resulting in renal interstitial fibrosis.The study found that the gene expression of a variety of cytokines,chemical chemokines and growth factor is regulate by the transcription mechanism.The nuclear factor-?B(NF-?B)serving as the important transcription factors play a key role in the regulation of various cell signaling transduction pathways.The activation of NF-?B relies mainly on the classic way: In the classic way,After IKK react on I?B?by phosphorylation,I?B? will degradate by the ubiquitin-proteasome to release the NF-?B dimmers.TNF-? and other proinflammatory cytokines can activate the way.IKK is a multicomponent protein kinase complexes(> 700KDa).It consists of four components,that are IKK?/IKK1,IKK?/IKK2 with the catalytic activity and IKK?/NEMO,IKAP with auxiliary activity.IKK? is the key subunits of the classic way to activatethe NF-?B.IKK? react on the Ser32 and Ser36 of the I?B? by phosphorylation to degradate I?B?.Then the activated NF-?B that has dissociated from I?B is transferred to the cell nucleus to activate downstream gene transcription.Fasudil that is a sulfonamide derivatives of isoquinoline series is currently one Rho kinase inhibitors applied to clinic.Fasudil can dilate blood vessels,inhibit free radical generation,decrease inflammatory response,and so on,so it has been widely used in the treatment for cardiovascular and cerebrovascular diseases.Many studies have shown that: In the process of renal interstitial fibrosis,fasudil as the Rho kinase inhibitors can relieve renal interstitial fibrosis by blocking the Rho-ROCK signal transduction pathway.And there are some reports that present it as soquinoline inhibitors reacting on the ATP binding site function of the kinase catalytic domain structure,selectively inhibit other protein kinase.In the experiment we use fasudil in the unilateral ureteral obstruction(UUO)rats,and we observe the relationship between fasudil and IKK?,I?B?,NF-?BP65,col-III.The purpose of this experiment is to discover the possible mechanisms of fasudil used in protecting the kidney.We can find the new therapeutic targets for the treatment of the renal interstitial fibrosis.Method: 54 SPF level healthy male SD rats,body weight 180±20g,were randomly divided into three groups: group A(sham operation group,normal control group),group B(UUO group,model group),group C(fasudil group,rosuvastatin group),with 18 rats in each group.After 1week adaptive feeding group A and B were given the unilateral ureteral obstruction operation,while group A was given the sham operation.We would not provide food for all groups before the operation in 12 hours.Group C were given fasudil 10 mg/kg/d with the intraperitoneal injection on preoperative 1 day,and group A and B were gavaged with isometric saline.The rats were allowed to drink and eat free.We selected 6 rats from each group randomly,and harvested their obstructed kidneys on day3,7,14 after the surgery.After saline irrigation slice the middle 1/4 of thekidneys and place the slices into fixation solution,used for the pathological staining and immunohistochemistry,and take another part of the kidney in liquid nitrogen and transferred to-80? cryogenic refrigerator,used for western blot.The changes were determined in the kidney tissues underwent HE staining,Masson staining and the immunohistochemistry that was to detect the expression of IKK?,I?B?,NF-?BP65 and Collagen Type III,as well as western blot(WB)to detect the expression of NF-?BP65.The result of immunohistochemistry would be analysed by semi-quantitative analysis.Statistical analysis software SPSS 20.0 was applied for the statistical analysis of experimental data which was displayed as mean ± standard deviation,and analyzed with one-way ANOVA,P < 0.05 was considered statistically significant.Results: 1 Pathological changes in kidney tissue of rats1.1 Observed by naked eyesGroup A present the kidney of normal colors and sizes.In the rest of the groups the rat kidney had varying degrees of increase,ureteral dilation and thinning of renal cortex,urinary accumulation as well as ureter,and the change of kidney disease in group B was more significant.1.2 HE staining under light microscopyGroup A: There were no abnormal changes;Group B: A small amount of inflammatory cells infiltration and mild expansion of renal tubules were found after 3days.As well as after 7 days,the inflammatory cells was significantly increased,and tubular epithelial cells presentd more degeneration and necrosis except for the changes of renal dilated tubules,furthermore interstitial fibers increased.After 14 days,visible tubular atrophy was more,tubular basement membrane was damaged;Group C: Changes became worse as time went by,they were better than those in group B,while they were worse than those in group A.1.3 Masson staining under light microscopy:Group A: There were no abnormal changes,and only a small amount of collagen fibers in individual area;Group B: There are increased blue collagenous fibers presented in renal interstitial area and slight tubular expansion on the 3th day after UUO.The changes of renal dilated tubules were worse,and tubular epithelial cells presentd more degeneration and necrosis on the 7th day after UUO,furthermore blue interstitial fibers increased.On the 7th day after UUO blue collagen fibers deposition was very obvious,and interwovened into a network;Group C: The changes became worse as time went by,they were still better than those in group B,however they were worse than those in group A.2 The immunohistochemical results of IKK?,I?B?,NF-?BP65 and Collagen Type III in renal tissueMicroscope,IKK?,I?B?,NF-?BP65,col-III present positive expression in renal tubular epithelial cells and renal interstitial in each group,and we need observe the expression of NF-?BP65 in the cell nucleus.The expression of IKK?,NF-?BP65,col-III were largely detected in tubular epithelial cells and renal interstitial in group B and C,and they are more and more over time.While the changes in the expression level of I?B? was the opposite.There was a significant difference(P<0.05)compared with group A on the each time point.The expression of IKK?,NF-?BP65,col-III decreased and the expression of I?B? increased in group C compared with group B,and there was a significant difference(P<0.05)between them on the same time point.However in group C the expression of IKK?,NF-?BP65,col-III were still much higher than those of group A(P<0.05)on the same time point,and the expression of I?B? was still much lower than that of group A(P<0.05)on the same time point.3 The western blot result of NF-?BP65 in renal tissueThe expression of NF-?BP65 became more and more higher as time went by in group B and C,and present a more expression compared withgroup A on the same time point(P<0.05).However it present less expression in group C than that of group B on each time point(P<0.05).Conclusion: 1 The expression of IKK?,NF-?BP65 and col-III in the obstruction side kidney increased,and positively correlated with prolonged,while I?B? predominated in opposite changes.The experiment presented that the NF-?B signal transduction pathway was activated in the renal interstitial fibrosis,to further promote the progress of renal interstitial fibrosis.2 Fasudil can reduce the expression of NF-?BP65 and col-III in the way that it maybe inhibit IKK? and reduce the phosphorylation and dissociation of I?B?.And then fasudil can play to protect the kidney and delay the progression of renal interstitial fibrosis. |
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