| Renal fibrosis,characterized by massive interstitial myofibroblast activation and excessive matrix proteins accumulation,is the final common pathway of virtually all kinds of progressive chronic kidney disease(CKD)leading to end stage renal disease(ESRD).[1-4]Mounting evidence has established a crucial role for transforming growth factor-?(TGF-?)in mediating chronic inflammation,activation of myofibroblast,and accumulation of extracellular matrix(ECM).[4-7]TGF-?1 signals are transduced by transmembrane serine/threonine kinase receptors type I(T?RI)and type II(T(3RII)and intracellular mediators known as Smads.[8,9]Upon TGF-?1 stimulation,Smad2 and Smad3 are phosphorylated by T?RI.Phosphorylated Smads heteroligomerize with the common partner Smad4 and then translocate into the nucleus,where they control the transcription of TGF-?-responsive genes through interaction with specific cis-acting elements in the regulatory regions.[10-12]Although both Smad2 and Smad3 are strongly activated in various experimental and human fibrotic kidney diseases,it is now well recognized that Smad3 is the key mediator of TGF-?1-induced ECM production and tissue fibrosis.[13-15]Deletion of Smad3 suppresses fibrogenesis in a number of rodent models,including diabetic nephropathy,[16]obstructive nephropathy,[17,18]and drug toxicity-related nephropathy.[19]On the other hand,conditional knocking out of Smad2 from kidney tubular cells significantly enhanced renal fibrosis via up-regulation of Smad3 signaling.[20]These findings indicate that Smad3 expression and/or phosphorylation might be a potential target for the intervention of renal fibrosis.It is now well accepted that Smad2 and Smad3 are two critical downstream mediators responsible for the biological effects of TGF-?1.In the context of renal fibrosis,Smad2 and Smad3 are strongly activated in both experimental and human kidney diseases,including diabetic nephropathy obstructive kidney diseases,remnant kidney disease,hypertensive nephropathy,drug-associated nephropathy,and immunologically-mediated glomerulonephritis.Many fibrogenic genes,such as(Collal,ColIa2,ColIIIal,ColVa2,ColVIal,and ColVIa3)and tissue inhibitor of MMP-1(TIMP-1)are the downstream targets of TGF-?/Smad3 signaling,suggesting that Smad3 may be a critical mediator of TGF-?/Smad signaling in fibrosis.An essential role for Smad3 in fibrogenesis is confirmed by the findings that deletion of Smad3 from mice suppresses fibrogenesis in a number of rodent models,including diabetic nephropathy,obstructive nephropathy,and drug toxicity-related nephropathy.Furthermore,the use of a Smad3 inhibitor to inhibit endothelial-myofibroblast transition and renal fibrosis in a type-1 diabetic kidney disease demonstrates a therapeutic potential for kidney disease by targeting Smad3 signaling.In the past years,many promising targets for the treatment of renal fibrosis have been validated in various animal models,and even more new targets have been identified.Renal fibrosis,in contrast,remains a largely uncharted territory in clinical trials.The reasons for this are certainly multifactorial and may include long study durations if hard endpoints,the lack of non-invasive markers or diagnostic tools to assess kidney scarring,and thus,monitor therapy.However,the industry has noted the enormous potential market,given the possibility of developing antifibrotic therapy that might be of benefit in many different types of organ fibrosis.Furthermore,there is hope that with a large consortia search for biomarkers and advancing ultrasound,or through MR-based or molecular-imaging techniques.The potential of natural products as the candidates for drug discovery has been well recognized.[21]Resina Toxicodendrl is the dried resin secreted by Toxicodendron venicifluum and has been used as an anti-inflammatory and anti-scarring agent in traditional Chinese medicine for centuries.In the present study,we isolated and purified the major component of Resina Toxicodendri GQ5,a small molecular phenolic compound.We demonstrated that treatment with GQ5 significantly inhibited the progression of interstitial fibrosis in the unilateral ureteral obstruction(UUO)model.We also demonstrated that the anti-fibrotic effect of GQ5 might be mediated by selective inhibition of TGF-?1-induced Smad3 phosphorylation.GQ5 isolated from Resina Toxicodendri is a small molecular phenolic compound,3-[(Z)-Pentadec-8-enyl]catechol.The chemical structure was shown in the Figure as follow.In last study,we have found that GQ5 Ameliorates Renal Interstitial Fibrosis in vitro and vivo.Histology stained with HE detected that GQ-5 could make UUO rat renal interstitial inflammatory cells significantly reduced,fibroblasts was also reduced,renal tubular cell morphology had been restored.Western blot analysis showed that GQ-5 decreased alpha SMA expression,Similar findings were also demonstrated at the protein level by Immunohistochemically analysis.Western Blot and immunohistochemistry confirmed that GQ-5 decreased Fibronectin and Collagen-I expression.Western Blot and immunohistochemistry have revealed that GQ-5 significantly inhibited Smad3 phosphorylation levels,but had no obvious effect on the levels of Smad2 phosphorylation.And in NRK52E,we also have found that GQ-5 could decreased the a-SMA expression in a dose-dependent manner.GQ-5 reduced the up-regulated expression of fibronectin protein as well as ?-SMA ·GQ-5 reduced phosphorylation levels of Smad3 induced by TGF-?,but not the phosphorylated levels of Smad2 and the protein expression levels of Smad4 and Smad7,GQ-5 did not affect the phosphorylation of other signaling pathways,such as MAPK/p38,ERK or phosphoinositide 3-kinase.So we supposed that GQ-5 can ameliotate renal fibrosis by inhibiting TGF-? 1-induced Smad3 phosphorylation.To confirm whether GQ-5 can treat renal fibrosis and inhibit TGF-?1-induced activation of fibroblasts and explore the mechanisms of GQ-5 inhibits TGF-?1-induced smad3 phosphorylation,we have designed experiments as follows.(1)Do experients in GQ-5+UUO day7 of UU014 days rats as same as that have did in GQ-5+UUO 7days rats,and so is it in NRK49F cells.(2)Explore the deeper mechanisms of GQ-5 inhibits renal fibrosis in vitro and vivo.(3)Explore other mechanisms of GQ-5 hinders renal fibrosis,such as kindlin-2,another adapter protein.Part I Explore whether GQ-5 can treat renal fibrosisObjective:In this study,we detected whether GQ-5 can treat renal fibrosis in GQ-5+UUO day7 of UU014 days rats.Method:UUO Rats.The animals were anesthetized with an intraperitoneal(IP)injection.A midline incision was made in the abdominal wall,the left ureter was dissected out and ligated with 4.0 silk at two points along its length.The abdominal wound was approximated with the same silk suture.Three groups of rats comprising 6 animals each(total=18)were included as follows.(1)Sham group:(2)UUO+ Propylene glycol group:the rats received IP injection of Propylene glycol and underwent unilateral ureteral ligation.(3)UUO + GQ-5 day7 group:these rats in addition those in group ? received IP injection of GQ-5 7 days after surgery at a dose of 40mg/kg a day.The rats were killed on day 14 after UUO or Sham operation,and the kidneys were harvested.Western Blot were carried out by using anti-Smad2,anti-Smad3,anti-phospho-Smad2,anti-phospho-Smad3,anti-fibronectin,anti-Collagen-I,anti-Sma4,anti-a-SMA,anti-Smad7,anti-MAPK/p38,anti-ERK,anti-phosphoinositide3,kinase,anti-phospho-p38,anti-phospho-ERK.Result:Western Blot revealed that GQ-5 decreased a-SMA,Fibronectin and Collagen-1 expression.GQ-5 significantly inhibited Smad3 phosphorylation levels,but had no obvious effect on the levels of Smad2 phosphorylation,as well as Smad4,Smad7,P-MAPK/p38,P-ERK or P-phosphoinositide 3-kinase.SummaryIn this study,we proved that GQ-5 can treat renal fibrosis in GQ-5+UUO day7 of UUO 14 days rats.Part? Explore whether GQ-5 can inhibit TGF-?1-induced activation of fibroblastsObjective:In this study,we detected whether GQ-5 can inhibit TGF-?1-induced activation of fibroblasts in NRK-49F cells.Method:Cell culture.NRK49F were grown in DMEM/F12containing 10%fetal bovine serum.When 60-80%cells were stick on the well,they were cultured for12h in free serum medium.Western Blot were carried out by using anti-Smad2,anti-Smad3,anti-phospho-Smad2,anti-phospho-Smad3,anti-fibronectin,anti-Collagen-I,anti-Sma4,anti-a-SMA,anti-Smad7,anti-MAPK/p38,anti-ERK or anti-phos phoino sitide 3-kinase,anti-phospho-p38,anti-phospho-ERK.Result:Western Blot revealed that GQ-5 decreased a-SMA,Fibronectin and Collagen-I expression.GQ-5 significantly inhibited Smad3 phosphorylation levels,but had no obvious effect on the levels of Smad2 phosphorylation,as well as Smad4,Smad7,P-MAPK/p38,P-ERK or P-phosphoinositide 3-kinase.SummaryIn this study,we proved that GQ-5 can inhibit TGF-?1-induced activation of fibroblasts.Part ? Explore the mechanisms of GQ5 Selectively inhibiting TGF-?1-Induced Smad3 Phosphorylation.Objective:To research how GQ-5 Selectively inhibits Smad3 Phosphorylation,we evaluated the efficacy of GQ-5 on the interaction of T?RI with T?RII,Smad3,Smad2 and SARA with T?RI,Smad3,Smad2 in NRK52E cells and UUO 14days rats.MethodCell culture.NRK52E were grown in DMEM/F12 containing 10%fetal bovine serum.When 60-80%cells were stick on the well,they were cultured forl2h in free serum medium.Co-mmunoprecipitation were carried out by using anti-T?RI,anti-Smad3,anti-Smad2,anti-T?RII,anti-SARA.UUO Rats.The animals were anesthetized with an intraperitoneal(IP)injection.A midline incision was made in the abdominal wall,the left ureter was dissected out and ligated with 4.0 silk at two points along its length.The abdominal wound was approximated with the same silk suture.Three groups of rats comprising6 animals each(total=18)were included as follows.(1)Sham group:(2)UUO+ Propylene glycol group:the rats received IP injection of Propylene glycol and underwent unilateral ureteral ligation.(3)UUO+ GQ-5 group:these rats in addition those in group II received IP injection of GQ-5 at a dose of 40mg/kg a day.The rats were killed on day 14 after UUO or Sham operation,and the kidneys were harvested.Co-Immunoprecipitation were carried out by using anti-T?RI,anti-T?RII anti-Smad3,anti-Smad2,anti-SARA.Result:In NRK52E cells,T?RI bound with T?RII,Smad2 and Smad3 upon TGF-?1 stimulation.Treatment with GQ5 significantly blocked the interaction of Smad3 with T?RI,but did not affect the interaction of Smad2 with T?RI.SARA bound with Smad2 and Smad3 upon TGF-?1 stimulation.Treatment with GQ5 significantly blocked the interaction of Smad3 with SARA,but did not affect the interaction of Smad2 with SARA.The same experimental results in UUO 14days rats.SummaryThese data suggests that GQ5 inhibited Smad3 phosphorylation by selectively blocking the interaction of Smad3 with SARA and then blocked Smad3 bond to T?RI in vivo and vitro.In Clonclution1.GQ-5 can treat renal fibrosis in GQ-5+UUO day7 of UUO 14 days rats.2.GQ-5 can inhibit TGF-?1-induced activation of fibroblasts.3.GQ5 inhibited Smad3 phosphorylation by selectively blocking the interaction of Smad3 with SARA and then blocked Smad3 bond to T?RI in vivo and vitro. |
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