| Objective:The common pathophysiological process from chronic kidney disease(CKD)to end-stage renal disease(ESRD)is renal interstitial fibrosis.At present,there is no effective and feasible treatment for renal interstitial fibrosis.Inflammatory response is the initiating factor of renal interstitial fibrosis.The essence of renal interstitial fibrosis is tubular atrophy and excessive accumulation of extracellular matrix.Myofibroblasts are the main effector cells that secrete extracellular matrix,and their numbers are parallel to the degree of renal interstitial fibrosis.The origin of myofibroblasts involves many mechanisms such as fibroblast activation,epithelial mesenchymal transition(EMT)and bone marrow-derived fibroblast transformation.In recent years,it has been found that Janus kinase(Jak)/ signal transducers and activators of transcription(Stat)signaling pathway plays an important role in renal diseases.At the same time,blocking Stat3 activation can inhibit fibroblast activation and improve UUO associated renal interstitial fibrosis.Ruxolitinib is a selective inhibitor of Jak1 and Jak2,which is used in the treatment of myelofibrosis.The clinical study found that after the treatment of Ruxolitinib,the level of pro-inflammatory cytokines in the blood of myelofibrosis patients was significantly reduced,and the degree of myelofibrosis in some patients was improved.In this study,we used unilateral ureteral obstruction(UUO)mice as renal interstitial fibrosis model in vivo and the activated fibroblasts in vitro to investigate the therapeutic effect and molecular mechanism of Ruxolitinib on renal interstitial fibrosis.The purpose of this study is to provide a new clinical drug for the treatment of renal interstitial fibrosis and to solve practical clinical problems.Methods: 1.The UUO model was established by wild-type C57BL/6 mice.Shamoperation group(Sham),UUO group(UUO)and Ruxolitinib treatment group(UUO+RUX)were set up respectively.Blood and renal tissue samples were collected at 2 weeks after operation which is the time point of UUO associated renal interstitial fibrosis.Serum creatinine was detected by enzyme method.The degrees of renal tubular injury and renal interstitial fibrosis were assessed by glycogen staining(PAS)and Masson trichrome staining.Collagen I ? III and Fibronectin were detected by immunohistochemistry staining and western blot to analyze the extracellular matrix deposition.Tissue inhibitor of metalloproteinase-1(Timp-1)was detected by western blot to analyze the extracellular matrix degradation.?-smooth muscle actin(?-SMA)was detected by immunohistochemistry staining and western blot to analyze the activation of myofibroblasts.E-cadherin was detected by immunohistochemistry staining and western blot to analyze EMT,and also including Snail and Twist were detected by western blot.Cleaved-cysteinyl aspartate specific protease 3(Cleaved-caspase-3)were detected by immunohistochemistry staining and western blot to analyze apoptosis,and also including TUNEL staining.Total superoxide dismutase(T-SOD)was detected by xanthine oxidase method,and malondialdehyde(MDA)was detected by thiobarbituric acid(TBA)to analyze the oxidative stress.F4/80 was detected by immunohistochemistry staining,and monocyte chemoattractant protein-1(MCP-1)mRNA level was detected by real-time PCR to analyze the recruitment and infiltration of macrophages.Nuclear factor kappa bp65(NF?B-p65)and p-NF?B-p65 were detected by Western blot,mRNA levels of interleukin-1 ?1(IL-1?1),interleukin-6(IL-6),and tumor necrosis factor-?(TNF-?)were detected by real-time PCR to analyze the inflammatory response of renal tissue.The signal pathway associated proteins including Stat3,p-stat3,extracellular signal regulated kinase(ERK),p-ERK,mammalian target of rapamycin(mTOR),p-mTOR,Akt,p-Akt and yes associated protein(Yap)were detected by western blot to study the molecular mechanism.2.Fibroblasts NRK-49 F were activated by recombinant human TGF-?1.NRK-49 F were classified into three groups: control group(CON),TGF-?1-induced group(TGF-?1),TGF-?1 and Ruxolitinib treatment group(TGF-?1+RUX).Collagen I,III,Fibronectin and ?-SMA were detected by western blot to analyze the activation of fibroblasts and the secretion of extracellular matrix.P-Stat3,Stat3,p-Erk,Erk,p-mTOR,mTOR,p-Akt,Akt and Yap were detected by western blot to study the molecular mechanism.The cell viability of fibroblasts was detected by MTT.3.The EMT model of renal tubular epithelial cell NRK-52 E was induced by recombinant human TGF-?1.NRK-52 E were classified into three groups: control group(CON),TGF-?1-induced group(TGF-?1),TGF-?1 and Ruxolitinib treatment group(TGF-?1+RUX).E-cadherin,Snail and Twist were detected by western blot.Results:1.The UUO group mice showed tubular atrophy,interstitial fibrosis,interstitial inflammatory cell infiltration and formation of protein tubules.The degree of renal tubule injury and the area of renal interstitial fibrosis in UUO+RUX treatment group were significantly lower than those in UUO group.2.Collagen I,III and Fibronectin proteins were almost not expressed in the kidney of Sham group,but these proteins were significantly increased in UUO group,and these proteins were significantly decreased in UUO+RUX treatment group compared with UUO group.Timp-1 protein in UUO group was higher than that in Sham group,while Timp-1 protein in UUO+RUX treatment group was lower than that in UUO group.3.In Sham group,?-SMA protein was only expressed in the arterial wall of renal tissue.In UUO group,large amounts of ?-SMA positive proteins were strongly expressed in the renal interstitium.The positive area of ?-SMA protein in UUO+RUX treatment group was significantly smaller than that in UUO group.4.Collagen?,?,Fibronectin and ?-SMA proteins of NRK-49 F cells in the TGF-?1 group were significantly higher than those in the Control group,and these proteins in TGF-?1+RUX treatment group were significantly lower than those in TGF-?1 group.The proliferation ability of NRK-49 F cells in TGF-?1 group was higher than that in Control group,while the proliferation ability in TGF-?1+RUX treatment group was lower than that in the TGF-?1 group.5.Compared with Sham group,E-cadherin protein in UUO group was decreased,Snail and Twist proteins were increased.While compared with UUO group,E-cadherin protein in UUO+RUX treatment group was increased,Snail and Twist proteins were decreased.6.Compared with the Control group,E-cadherin protein of NRK-52 E cells in TGF-?1 group was decreased,Snail and Twist protein were increased.While compared with TGF-?1 group,E-cadherin protein in TGF-?1+RUX treatment group was increased,Snail and Twist were decreased.7.Compared with Sham group,macrophage infiltration in UUO group was significantly increased,while compared with UUO group,macrophage infiltration in UUO+RUX treatment group was decreased.The MCP-1 mRNA in UUO group was significantly higher than that in Sham group,while MCP-1 mRNA in UUO+RUX treatment group was significantly lower than that in UUO group.8.The p-p65 and p65 proteins in UUO group were higher than those in Sham group,while p-p65 in UUO+RUX treatment group was lower than that in UUO group.The TNF-?,IL-1? and IL-6 mRNA levels in renal tissue of UUO group were significantly higher than those in Sham group,while those in UUO+RUX treatment group were significantly lower than those in UUO group.9.The number of cleaved-caspase-3 positive cells in UUO group was significantly higher than that in Sham group,which were mainly concentrated in renal tubular epithelial cells,while the number of cleaved-caspase-3 positive cells in UUO+RUX treatment group was lower than that in UUO group.The cleaved-caspase-3 protein in UUO group was significantly higher than that in Sham group,while the cleaved-caspase-3 protein in UUO+RUX treatment group was lower than that in UUO group.The apoptosis of TUNEL positive renal epithelial cells in UUO group was more than that in Sham group,while apoptosis of TUNEL positive renal epithelial cells in UUO+RUX treatment group was less than that in UUO group.10.The T-SOD level in UUO group was lower than that in Sham group,and the TSOD level in UUO+RUX treatment group was higher than that in UUO group.The MDA level in UUO group was higher than that in Sham group,and the MDA level in UUO+RUX treatment group was lower than that in UUO group.11.The p-STAT3,STAT3,p-ERK and ERK proteins in UUO group were significantly higher than those in Sham group,while p-STAT3 and p-ERK proteins in UUO+RUX treatment group were significantly lower than those in UUO group.The pSTAT3 and p-ERK proteins of NRK-49 F cells in TGF-?1 group were higher than those in Control group,while the p-STAT3 and p-ERK proteins in TGF-?1+RUX treatment group were lower than those in TGF-?1 group.12.The p-Akt,Akt,p-mTOR,mTOR and Yap protein in UUO group were significantly higher than those in Sham group,while p-Akt,p-mTOR and Yap proteins in UUO+RUX treatment group were significantly lower than those in UUO group.The pAkt,p-mTOR and Yap protein of NRK-49 F cells in TGF-?1 group were significantly higher than those in Control group,while p-Akt,p-mTOR and Yap protein in TGF-?1+RUX treatment group were lower than those in TGF-?1 group.Conclusions:1.Ruxolitinib can improve renal tubular injury and reduce accumulation of extracellular matrix in UUO mice.2.Ruxolitinib can inhibit the activation of fibroblasts in UUO mice and cultured fibroblasts in vitro,and also alleviate the EMT of renal tubular epithelial cells in UUO mice and cultured renal tubular epithelial cells in vitro.3.Ruxolitinib can reduce the renal inflammatory response and oxidative stress damage in UUO mice,and reduce the apoptosis of renal tubular epithelial cells in UUO mice.4.Ruxolitinib can inhibit the activation of Stat3,Erk and Akt / mTOR / Yap signaling pathway in UUO mice and activated fibroblasts cultured in vitro.5.Ruxolitinib can improve renal interstitial fibrosis in UUO mice,and it is expected to be a new drug for the treatment of fibrotic nephropathy. |
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