Protective Role And Mechanisms Of Osthole On UUQ-induced Renal Fibrosis In Mice

Part one:Protective role of osthole on UUO-induced renal fibrosis in miceAim:Renal fibrogenesis is the final common outcome of various renal diseases,finally leading to chronic kidney disease(CKD).It is estimated that CKD affects more than 10%of world population,while the treatment options are limited.Osthole(OST)is a naturally occurring coumarins,which can be frequently found in plants of the Apiaceae family.It has been showed that OST exhibited a broad range of biological activities,such as anti-inflammatory,anti-oxidation,anti-tumor and vasorelaxation properties.Recent studies demonstrated that OST could exhibit protective role in lung,heart,liver and dermal scar fibrosis both in vivo and in vitro and even has effect on TGF?1/Smad signailing pathway,which play key role in renal fibrogenesis.In kidney,OST ameliorated acute ischemia-reperfusion induced injury,IgA nephropathy and focal segmental glomerulosclerosis.Therefore,in this part we aimed to investigating that whether OST has protective role of on renal fibrosis.Methods:In this present study,classic unilateral ureteral obstruction-induced renal fibrosis mice model was built,during which C57BL/6 mice were subjected to left ureteral obligation operation.OST(40,80 mg/kg/d)were administrated into mice by intragastric given(i.g.)1 day before the UUO and continued afterward once a day.Mice with surgery but not ureteral ligation were used as Control group and were treated with the same volume of OST cosolvent,namely 5%sodium carboxymethyl cellulose solution.HE(Hematoxylin-eosin,HE)and MTC(Masson trichrome,MTC)staining were used to observe renal morphology and the accumulation of collagen deposition at day 7.Then we investigated the protective role of OST on UUO-induced renal fibrosis in mice.Creatinine(Cre)and blood uria nitrogen(BUN)were tested by automatic biochemical analyzer to observe the effect of OST on renal function in UUO mice.Real-time PCR and IHC staining for CD68 were used to measure the mRNA levels of inflammatory factor and macrophage accumulation status in vivo.Furthermore,the TGF?1 and its downstream pathway(Smad and non-Smad signaling)were also investigated in this study to probe the possible mechanisms that involved in OST effect.Results:At day 7,compared to UUO group,OST(40,80 mg/kg/d)treatment significantly improve renal histological architecture,including the progressive dilation of the renal pelvis and reduced total volume of renal medulla and cortex as well as the collagen deposition.In addition,OST diminished inflammatory cytokines mRNA expression and macrophage accumulation in the obstructive kidneys.Moreover,OST inhibited TGF?1/Smad signaling,but not MAPK pathway activation in UUO kidneys,thereby mitigating renal fibrosis.Conclusion:OST may effectively attenuate renal fibrogenesis in UUO-induced mice model.OST administration diminished inflammation and TGF?1/Smad activation in kidney tissue.Based on the preliminary studies from morphology to molecular mechanisms,our study results clearly demonstrated the potential therapeutic role for OST treatment in progressive renal fibrosis,suggesting that OST could be the potential opinion of CKD treatment.Part two:The role of osthole on activation and proliferation of renal fibroblast in UUO-induced mice and NRK49FAim:Fibroblast and myofibroblast(a-SMA positive fibroblast)are the two main cell effectors in renal fibrogenesis,and responsible for the synthesis and accumulation of extracellular matrix(ECM),which leading to loss of renal function.Previous studies showed that OST could decrease ECM components in mouse cardiac fibroblasts and hepatic stellate cell activation as well as depress hypertrophic scar fibroblast prolifertaion.Therefore,we proposed the hypothesis that OST may inhibit renal fibroblast proliferation and activation,thereby ameliorating UUO-induced renal injury.Methods:In vivo,C57BL/6 mice were subjected to UUO surgery and sacrificed at day 7.The way of OST and vehicle administration in mice of OST and Sham groups were the same as the first part study described.Immunofluorescent double staining for a-SMA and ki-67 was used to measure activation levels of renal interstitial fibroblast in kidney sections.In vitro,we use the more available and the most common used rat fibroblast(NRK49F)as the object in this study.NRK49F was cultured and stimulated by TGF?1(5 ng/ml)to establish renal fibrosis model.OST(1 0,20,40 ?M)was pre-incubated to NRK49F before the role of OST on activation and proliferation of renal fibroblast were tested at 12 h and 24 h.Besides,proliferation-and cell cycle-related proteins were measured at protein level.Moreover,immunofluorescence,Western blot and Co-IP methods were also used to test the role of OST on NRK49F activation and the underlying mechanisms.Results:This study revealed that myofibroblast was evidently activated and proliferated in UUO-induced obstructive kidneys,the effect that were reversed by OST.Similarly,in vitro,compared with TGF?1 group,OST significantly inhibited the proliferation of NRK49F with concentration at 40 ?M had the most obvious effect.OST 40 ?M effectively reversed the expression of proliferation related protein PCN A,cyclin D1 and p21 Wafl/Cipl.Meanwhile,compared with TGF?1 group,preincubation with OST inhibited the activation of NRK49F and depressed the expression of ECM components.Our cellular mechanisms result showed that OST selectively inhibited TGF?1/Smad signaling pathway in NRK49F.Conclusion:The results in this study showed that OST could diminished the(myo)fibroblast activation and proliferation.Additionally,OST selectively depressed TGF?1/Smad signaling pathway while had no effect on MAPK pathway during the process of fibroblast activation.The results suggested that the role of OST on fibroblast activation maybe one of the main reasons for alleviating renal fibrosis in UUO mice.Part three:The role of osthole on renal tubular epithelial-to-mesenchymal transition mesenchymal transitionAim:Under the condition of chronic injury,epithelial-to-mesenchymal transition(EMT)of renal tubular not only influences the normal kidney function,but also triggers the secretion of pro-fibrosis or growth cytokines,which set up the local microenvironment for fibrogenesis and worsen the interstitial and fibrosis.It has been demonstrated that OST could reverse EMT in brain,lung and hepatocellular carcinoma cells with downregulating transcriptional factors Snail and Twist level,thus increasing epithelial bio markers E-cadherin and decreasing mesenchymal Vimentin protein expression.As oncogenic“type 3 EMT and organic fibrosis "type 2 EMT" share many of the same important signaling pathways and transcription factors that implicated in the development of EMT,and we had also demonstrated that OST could regulate TGF?1/Smad signing pathway in renal tubular of obstructive kidneys,which play a key role in tubular cell EMT in the first part of study,it is likely that OST attenuated renal fibrosis through suppression of EMT.Methods:In vivo,C57BL/6 mice were challenged to left ureteral obligation operation and sacrificed at day 7.The way of OST and vehicle administration in mice of OST and Sham groups were the same as the first part study described.Western blot and IHC staining methods were used to observe the protein levels of Snail-1,Twist-1,E-cadherin and Vimentin among different mice groups.In vitro,we use TGF?1(10 ng/ml)stimulated-HK-2 cells to establish the cell model of EMT and cell morphology change was visualized by light microscope.Also,EMT-related mRNA and protein level of E-cadherin,Vimentin,Snail-1,Twist-1 as well as inflammatory factors level such as TGF?1,PDGF-B were measured in HK-2 cells to observe the effect of OST on renal tubular EMT program in vitro.Meanwhile,G2/M phase of tubular cells both in mice and HK-2 cell was tested.MAPK,p-Smad3,Akt and ?-catenin signaling pathway were investigated both in vivo and in vitro to probe for the possible potential molecular mechanisms of OST on EMT.Results:In vivo,compared with UUO-injured kidneys,OST treatment significantly improved the expression of E-cadherin and Vimentin,decrease transcription factor Snail-1,Twist-1 protein expression and cell cycle G2/M stage marker phospho-histone H3(p-H3)distribution in renal tissue.Similarly,in vitro,compared with TGF?1-treated HK-2 cells,OST pre-incubation significantly blocked EMT program,decreased cytokines secretion and alleviated G2/M stage arrest.Moreover,in the process of EMT program shown in renal tissue and HK-2 cells,we noticed that p-Smad3,Akt,?-catenin signaling pathway were all inhibited by OST,but not MAPK pathway.Conclusion:OST could obviously inhibit the renal tubular EMT both in vivo and in vitro.Besides,OST has effect on EMT-related pathway,including p-Smad3,Akt and?-catenin.The results of this study revealed that OST suppressed EMT of tubular cell maybe one of the protective mechanisms for alleviating renal interstitial fibrosis.