The Effect Of Leukotriene Receptor Antagonist On Expression Of JAK2,STAT3,IL-6 And Collagen ? In The UUO Model

Objective: Renal interstitial fibrosis is the common pathological manifestations of Chronic Kidney Disease(CKD)caused by various protopathy diseases,which is the final common pathway leading to end-stage renal disease.The pathophysiological changes associated with renal fibrosis include renal tubular interstitial fibrosis and glomerular sclerosis,characterized by inflammatory cells infiltration,cytokine activation,epithelial mesenchymal transition(EMT),fibroblast proliferation,extracellular matrix deposition and cell apoptosis,eventually leading to end-stage renal failure.With the progress of CKD study,we found that number of cytokines and growth factors have been implicated in the pathophysiology of CKD,like transforming growth factor ?1,tumor necrosis factor ?.The Janus kinase family(JAK)/signal transducers and activators of transcription(STAT)signaling pathway.The JAK/STAT signaling pathway is an important cascade for signal transduction for a wide variety of cytokines and growth factors,This pathway regulates the occurrence and development of disease,which activation can promote the progress of various diseases,such as lymphoma,leukemia and a variety of inflammatory diseases.JAK/STAT signaling pathways have been implicated in the pathophysiology of renal disease,such as diabetic nephropathy,obstructive nephropathy,and polycystic kidney disease.The JAK/STAT pathway is closely related to the progression of inflammation.It can not only control the expression of downstream inflammatory genes,but also interacts with other inflammatory pathways,such as nuclear transcription factor kappa B and transforming growth factor ?,and plays an important role in the inflammatory process.In addition,the JAK/STAT pathway is the core pathway for macrophage activation,and through its signal transduction,macrophages can respond to more than 20 inflammatory factors.Leukotrienes(LTs)is a kind of inflammatory mediators with highly biologically activity,synthesized mainly by inflammatory cells,which receptors as important inflammatory mediators are widely found in inflammatory cell granules and other tissues.Few studies have investigated that Multiple pathologic factors stimulate the overexpression of LTs in the kidney,playing an important role in renal injury.Inhibiting the overexpression of LTs may play a beneficial role in the treatment of renal disease.Montelukast,which is a selective cysteinyl-leukotriene(Cys-LT)receptor antagonist,is widely used in the treatment of asthma.The purpose of this experiment was to explore whether leukotriene receptor antagonist can reduce the progress of renal interstitial fibrosis,and the influence of the expression of JAK2,STAT3,IL-6 and collagenin-? rat model of Unilateral Ureteral Obstruction(UUO).in addition,to explore whether JAK/STAT pathway may play a role in the treatment of renal interstitial fibrosis and provide a new therapeutic target for the treatment of renal interstitial fibrosis.Method: 54 clean level healthy male SD rats,weighting 200 g to 240 g,were randomly divided into three groups,each consisting of 18 animals(Group A was sham,group B was rats with UUO and group C was rats with UUO which were given montelukast sodium).One week after acclimatization,rats in each group were fasted for 12 hours before operation.Rats in group A underwent unilateral ureteral ligation and received no treatment.Group B rats underwent sham operation and received no treatment with montelukast sodium.Rats in Group C were subjected to unilateral ureteral ligation and received MON(p.o.10mg/kg/day).At 3/7/14 days after UUO,18 rats were sacrificed by high-dose Chloral hydrate.Left kidney was immediately excised and separated from the surrounding tissues,washed twice with normal saline,and take the middle 1/4 fixed in paraformaldehyde for pathological HE and Masson staining and immunohistochemical staining.The other three fractions were quickly frozen in liquid nitrogen to prevent protein degradation and stored in a-80oC for Western blot.All data were expressed as mean ± SE and analyzed with one-way ANOVA by statistical analysis software SPSS 20.0.Probability values less than 0.05 were required for statistical significance.Results: 1 kidney histopathological changes 1.1 HE staining under light microscopyGroup A: No significant abnormality were observed in glomeruli and renal interstitium on the 3th,7th,14 th day after surgery.Group B: Slight renal tubules expansion and a few inflammatory cells infiltration were observed on 3 days after surgery.On 7th day,beside inflammatory cells infiltration and renal tubules expansion aggravated,tubular epithelial cells fall off and vacuolar degeneration.Tubular atrophy was more serious and tubular basement membrane was diffuse thickening with shrinkage On 14 th day.Group C: Compared with the same period group B,mild tubular degeneration and less severe tubular necrosis were observed,but still significantly heavier than group A.1.2 Masson staining under light microscopyGroup A: There were no obvious abnormality in glomeruli,renal tubules and interstitium on the 3th,7th,14 th day after surgery.Group B: There were extensive renal tubular expansion,renal interstitial expansion and collagen deposition.With the development of the lesion,collagen fiber gradually extended to the renal tubules surrounding and interwovened into a network.Group C: Compared with the same time group B,renal interstitial collagen deposition decreased,but it is still significantly heavier than group A.2 kidney immunohistochemical staining 2.1 P-JAK2Group A: In the renal tubular epithelial cell cytoplasm was weak expressed.Group B: The expression of cytoplasm in rat renal tubular epithelial cells was increased,and the expression was also found in interstitium.With the obstruction extended,the expression of P-JAK2 was increased,and compared with group A were statistically significant(P<0.05).Group C:The expression of P-JAK2 was decreased with group B,but still higher than group A.2.2 P-STAT3Group A: There was little expressed in the renal tubular epithelial cell's nucleus.Group B: The expression of cell's nucleus in rat renal tubular epithelial cells was increased.With the progression of the lesion,the expression of P-STAT3 was increased,and extensive expression was detected in renal tubular epithelial cell's nucleus,and compared with group A there were statistical significance.Group C:The levels of P-STAT3 was higher than that in group A but was lower than that in group B(P<0.05).2.3 IL-6Group A: Slight expression of IL-6 was mainly detected in renal tubular epithelial cell cytoplasm.Group B: Positive expression increased significantly,with the progress of disease,the expression level continued to increase,compared with the same period group A there were statistically significant(P<0.05).Group C: Compared with the same period group B,IL-6 expression level decreased obviously in the 3rd,7th,14 th day after surgery.but was higher than group A.2.4 collagen-?Group A: There was a little expression in the glomerular mesangial cell cytoplasm,interstitial and renal tubular epithelial cell cytoplasm.Group B: The level in renal tubular epithelial and interstitial increased as obstructed time went by,but the expression of tubulointerstitium was not obvious,additionally more than group A.There was a significant difference(P<0.05).Group C: Compared with group B,collagen-III expression level was significantly decreased(P<0.05),but still higher than the same period expression level in group A.3 The results of P-JAK2 and P-STAT3 Western Blot in renal tissueP-JAK2 protein expression in renal tissues: the expression of target protein in group B gradually increased,while compared with A,the expression were increased(P<0.05),In group C it increased gradually with time,however,the level of group C was less than group B,but more than group A(P< 0.05).P-STAT3 protein expression in renal tissues: Compared with group A on the same time point,the expression of group B and C were gradually increased(P<0.05).The expression were 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:1 It was shown that JAK2/STAT3 signaling pathway may be involved in the progression of renal interstitial fibrosis in UUO rats.2 Leukotriene receptor antagonists serves as a novel anti-inflammatory agent that attenuates ureteral obstruction-induced renal interstitial fibrosis,possibly through reducing the expression of inflammatory cytokines,thereby inhibiting the activation of JAK2/STAT3 signaling pathway.