| Objective: Renal Interstitial Fibrosis(RIF),characterized by extra accumulation of Extracellular Matrix(ECM),is a common pathologic process of Chronic Kidney Disease(CKD)caused by various protopathy diseases.RIF is extremely complicated pathologic process: Under the impact of injury factors,the activations of the inflammatory and oxidative-stress result in relevant change of cellular cytokines,metabolism and structure in renal cells,which lead to a imbalance between ECM-producing and ECM-degradation and finally to additional accumulation of ECM in renal interstitial.The metabolism-imbalance of ECM comes down to two aspects.On the one hand,various-type cells transdifferentiate to Myofibroblast(MFB)being the effective cell of RIF,secreting collagen-Ⅰ and collagen-Ⅲ and featured with expression α-Smooth Muscle Actin(α-SMA).On the other hand,several cytokines restrain ECM-degradation.Reactive oxygen species(ROS)is a series of small molecule with high chemical-activity,for its unpaired electron.Under the physiological condition,the intracellular level of ROS keeps low,which is necessary for cellular normal metabolism.In pathology situation the increasing ROS not only directly produces peroxidation reaction with protein,lipid and DNA and so on,but also serve as single messenger of some signal transduction pathway to distures cellular normal metabolism and function serving.Among them,the end product of lipid oxidation is malondialdehyde which concentration could reflect the level of ROS.As one member of the family of mitogen activated protein kinase(MAPK),extracellular signal-regulated kinase 1/2(ERK1/2)whose activated form is phospho-extracellular signal-regulated kinase1/2,(p-ERK1/2)mediate signal transduction,which plays a important role in cellular appreciation,differentiation,transdifferentiation and apoptosis.Nuclear factor kappa B,NF-κB(NF-κB)is a family of nucleoprotein participating regulation of gene transcription,containing five hypotypes: NF-κB1(p50/p105)、NF-κB2(p52/p100)、Rel(p65)、RelB and C-κB1.The binding-DNA-sequence exists in many proteinic promoter and enhancer.NF-κB can adjust expression-level of many pro-fibrogenic cytokines,such as monocyte chemoattractant protein(MCP-1),intercellular cell adhesion molecule-1(ICAM-1),vascular cell adhesion molecule 1(VCAM-1)and so on.Edaravone(EDA)is a free-radical scavenger to alleviate cellular oxidative injury,which has been used clinically for the treatment of acute cerebral infarction.Given the role of ROS in RIF,we supposed EDA may delay the progress of RIF.To determine this speculation,this experiment detected the influence of EDA on expression of MDA,p-ERK1/2,NF-κB and collagen-Ⅲ in rat model of Unilateral Ureteral Obstruction(UUO).Method: 54 SPF level healthy male SD rats,weighing 180±20 g,after 1 week adaptive feeding,were randomly divided into three groups with 18 mouses in each one:sham group,UUO group and EDA group.During this experiment,all rats were allowed to eat and drink free.The rats in UUO group received Chloral hydrate(4ml/kg)intraperitoneal injection of anesthesia.Under sterile conditions,we placed a longitudinal incision with distances of 1.5cm to abdominal median line and 1cm to left costal margin,separated tissues and muscles step-wise,exposed left kidney and dissociated the left ureter along the lower pole of left kidney,ligated the bare ureter twice at 1/3 upper of ureter and snipd it between two ligation.Finally,washed enterocoelia with sterile normal saline,determine whether there were important viscera injuryed and andactive bleeding,and closed it.The rats in EDA group received the same operation.The rats in Sham group without ligating and cutting ureter,also received the same operation.The EDA group were given EDA 3.75mg/kg/d via intraperitoneal injection.The Sham group and UUO group were injected with isometric saline.We randomly selected 6 rats from each group respectively,and harvested their obstructed kidneys on 3th,7th,14 th day after surgery.Each obstructed kidney were sliced in to for parts,one part reserved in fixation solution and three part stored at-80℃.Observe renal tissue pathological changes via HE staining and Masson staining and determined level of MDA via spectrophotometry,detect the expression of p-ERK1/2,NF-κB p65 and collagen.All data were expressed as mean ± SE and analyzed with one-way ANOVA by statistical analysis software SPSS 22.0.Probability values less than 0.05 were required for statistical significance.Results:1 HE staining under light microscopySham group: No significant abnormality were observed in glomeruli and renal interstitium on the 3th,7th,14 th day after surgery.UUO groups: On 3th day after surgery,slight renal-tubules-expansion and a few inflammatory cells infiltration were observed.On 7th day,beside inflammatory cells infiltration and renal-tubules-expansion aggravated,degeneration and necrosis of tubular epithelial cells occurred and interstitial width were obviously increased.On 14 th day,tubular atrophy was more serious and tubular basement membrane was diffuse thickening with shrinkage and breakage.EDA group: The similar changes with UUO group were observed at the same time phase,which were better than UUO group.2 Masson staining under light microscopySham group: No significant abnormality were observed on the 3th,7th,14 th day after surgery.UUO group: Slight tubular expansion and increased collagenous fibers in renal interstitium were presented on 3th day after surgery and both of them became worese as obstruction-time prolonged on 7th and 14 th day.EDA group: Tubular expansion and increased collagenous fibers in renal interstitium was deteriorating as time went by but better than UUO group at the same time phase.Sham group: There was no statistical difference(P>0.05)among the levels of MDA on the 3th,7th,14 th day after surgery.UUO group: The levels of MDA was increasing as as obstruction-time prolonged on the 3th,7th,14 th day(P<0.05).Additionally,the levels of MDA was higher than that in Sham group at the same time phase(P<0.05).EDA group: The levels of MDA was increasing as as obstruction-time prolonged on the 3th,7th,14 th day(P<0.05).Additionally,the levels of MDA was higher than that in Sham group at the same time phase(P<0.05).On 3th day,There was no statistical difference(P>0.05)between the levels in UUO group and EDA group.On 7th and 14 th day,the level in EDA group was lower than that in UUO group(P<0.05).4 The immunohistochemical results of p-ERK1/2Sham group: Expression of p-ERK1/2 was mainly detected in renal tubular epithelial cell’s nucleus and there was no statistical difference(P>0.05)among the levels of p-ERK1/2 on the 3th,7th,14 th day after surgery.UUO group: Expression of p-ERK1/2 was mainly detected in renal tubular epithelial cell’s nucleus.Its levels was increasing as obstruction-time prolonged on the 3th,7th,14 th day,(P<0.05).Additionally,the levels of p-ERK1/2 was higher than that in Sham group at the same time phase(P<0.05).EDA group: Expression of p-ERK1/2 was mainly detected in renal tubular epithelial cell’s nucleus.Its levels was increasing as obstruction-time prolonged on the 3th,7th,14 th day,(P<0.05).Additionally,the levels of p-ERK1/2 was higher than that in Sham group(P<0.05)but was lower that in UUO group(P<0.05)at the same time phase.5 The immunohistochemical results of NF-κB p65UUO group: Expression of NF-κB p65 was mainly detected in renal tubular epithelial cell’s nucleus,without statistical difference(P>0.05)among its levels on the 3th,7th,14 th day after surgery.UUO group: Expression of NF-κB p65 was mainly detected in renal tubular epithelial cell’s nucleus.Its levels was increasing as obstruction-time 3 The level of MDA in renal tissue homogenater prolonged on the 3th,7th,14 th day,(P<0.05).Additionally,the levels of NF-κB p65 was higher than that in Sham group at the same time phase(P<0.05).EDA group: Expression of NF-κB p65 was mainly detected in renal tubular epithelial cell’s nucleus.Its levels was increasing as obstruction-time prolonged on the 3th,7th,14 th day,(P<0.05).Additionally,the levels of NF-κB p65 was higher than that in Sham group but was lower than that in UUO group(P<0.05).6 The immunohistochemical results of collagen-ⅢSham group: Slight expression of collagen-Ⅲ was mainly detected in renal tubular epithelial cell,without statistical difference(P>0.05)among its levels on the 3th,7th,14 th day after surgery.UUO group: Expression of collagen-Ⅲ in renal interstitium and tubular epithelial cell increasing as obstruction-time prolonged on the 3th,7th,14 th day,(P<0.05).Additionally,its levels was higher than that in Sham group at the same time phase(P<0.05).EDA group: Expression of collagen-Ⅲ in renal interstitium and tubular epithelial cell increasing as obstruction-time prolonged on the 3th,7th,14 th day,(P<0.05).Additionally,its levels was higher than that in Sham group at the same time phase(P<0.05).On 3th day,There was no statistical difference(P>0.05)between the levels in UUO group and EDA group.On 7th and 14 th day,the levels in EDA group was lower than that in UUO group(P<0.05).Conclusion: 1 Oxidative stress,the signaling pathway of p-ERK1/2 and NF-κB p65 may played a role in the progress of RIF in UUO rat model.2 In UUO rat model,EDA may have an antifibrosis-effect that partially deriving from the mechanism that EDA can downregulate the levels of p-ERK1/2、NF-κB p65、collagen-Ⅲ via antioxidation. |
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