Molecular Mechanistic Studies Of Astragaloside IV Attenuates Human Peritoneal Mesothelial Cells Mesothelial-to-mesenchymal Transition

BackgroundContinuous ambulatory peritoneal dialysis(CAPD)has become widespread in patients with end-stage renal disease because peritoneal dialysis(PD)patients exhibit better survival compared with hemodialysis(HD)patients during the first 2 years of dialysis treatment.However,long-term exposure to PD fluid and recurrent episodes of peritonitis result in morphologic and functional changes of the peritoneal membrane,also known as peritoneal fibrosis.These changes to the peritoneal membrane ultimately contribute to ultrafiltration failure,resulting in technique failure and discontinuation of PD therapy.Consequently,the most important challenge currently faced in PD is the prevention of PF and the long-term preservation of peritoneal membrane structure and function.Mesothelial-to-mesenchymal transition(MMT),a reversible process in which epithelial cells transdifferentiate into cells with mesenchymal characteristics,is widely considered to be a crucial process in fibrosis.MMT in mesothelial cells is a critical step in the pathogenesis of peritoneal fibrosis.It has been reported that several growth factor and cytokine factor signaling pathways(TGFβ1,FGF,EGF,HGF,Wnt/β-catenin,and Notch)participate in MMT.Emerging evidences have indicated that TGFβ1 is the most important signaling molecule of all these factors.Currently,no appropriate methods to block TGFβ1-induced EMT have been approved in clinical practice.Interestingly,most studies thus far have focused on herbal medicine as an alternative treatment.Astragaloside Ⅳ(AS-Ⅳ)is a cycloartane triterpene saponin with a clear formula,which is the main active ingredient of Astragalus membranaceus.Emerging evidences have indicated that AS-Ⅳ shows anti-fibrotic effects on organ fibrosis.However,this macromolecule’s effect on TGFβ1-induced EMT of human peritoneal mesothelial cells has not been clearly delineated.Here,we used TGF-β1-induced EMT in mesothelial cells to investigate the role of AS-Ⅳ in EMT and to elucidate the underlying molecular mechanisms.NLRP3 inflammsome is a large protein complex that mediates caspase-1 activation.It consists of specific members of the NOD-like receptor protein(NLRP)subfamily,an adaptor protein of apoptosis-associated speck-like protein containing a CARD(ASC)and procaspase-1.NLRP3 is one of the most important cytosolic pattern recognition receptor,which can recognize both pathogen associated molecular patterns(PAMPs)and damage-associated molecular patterns(DAMPs).Activation of NLRP3 triggers its oligomerisation,which in turn allows for the recruitment and clustering of the ASC inflammasome adapter and caspase-1.Caspase-1 is then activated,and cleaves the interleukins(ILs)-1β and IL-18 to generate their active secreted forms.NLRP3 is activated by numerous substances indicative of metabolic surplus,such as hyperglycemia,ATP,saturated fatty acids,ceramides,monosodium urate and cholesterol crystals.Several molecular mechanisms have been suggested for NLRP3 activation to induce caspase-1 activation and IL-1β maturation.These include pore formation and potassium(K1)efflux,lysosomal rupture,and mitochondrial reactive oxygen species(ROS)generation.Evidence supports that the aberrant activation of the NLRP3 inflammasome is associated with the pathogenesis of various autoimmune diseases,chronic sterile inflammatory and metabolic diseases,including gout,atherosclerosis and type 2 diabetes.The pathophysiologic effect of activation of NLRP3 inflammasome in peritoneal dialysis related peritoneal mesothelial cells injury remains unknown.As we all know,During peritoneal dialysis treatmemt,the PMCs is exposed to PD solution with poor biocompability and abnormal metabolic environment,which include low PH of solution,lactated buffer,hyperosmosis,high concentration of glucose and degradation product.We have reasons to believe that the long-term exposure to abnormal metabolic environment could be able to active NLRP3 inflammsome and then induce PMCs damage.However the exact molecular mechanism is unclear.Astragaloside Ⅳ(AS-Ⅳ)is a cycloartane triterpene saponin with a clear formula,which is the main active ingredient of Astragalus membranaceus.Emerging evidences have indicated that AS-Ⅳ shows attenuate peritoneal fibrosis.but The exact molecular mechanisms still need to be studied.In the present in vitro study,using PD solution-induced PMCs MMT model,we fully investigated the following:1))whether activation of NLRP3 inflammasome mediates PD solution-induced PMCs MMT;2)whether AS-Ⅳ attenuates PD solution induced PMCs MMT through inhibiting the activation of NLRP3 inflammasome.MethodsPart One:①Human peritoneal mesothelial cells(HMrSV5 cells ATCC)were cultured in DMEM supplemented with 10%(v/v)heat-inactivated fetal calf serum(Invitrogen)and 100 U/ml penicillin/streptomycin(Invitrogen).Cells were maintained at 37℃ in a humidified environment(5%CO2 and 95%air),and the culture mediumwas replaced every 2 days.All experiments were carried out 24-48 h after cells were seeded in culture plates.Cells were permitted to attach for 24 h and to grow to 75%confluence.To induce EMT,HMrSV5 cells were treated with TGF-β1(2 ng/ml)②Cells were treated with various dose of AS-Ⅳ(0,10,50,100,200,and 400μg/ml)for 24h or AS-Ⅳ 400μg/ml for various times(0,12,24,36,48 and 72h).Cell viability was measured in a 96-well plate using a quantitative colorimetric assay.③Cells were divided into a vehicle group(cells treated with 0.1%DMSO),a TGF-β1 group(cells treated with 2 ng/ml TGF-β1),an AS-Ⅳ group(cells treated with 400 μg/ml AS-Ⅳ)and an AS-Ⅳ + TGF-β1 group(cells pretreatedwith AS-Ⅳ at 400 μg/ml 2 h prior to 2 ng/ml TGF-β1).After incubation for 24 h,cells were lysed using RIPA buffer or TRIzol reagent for a western blot assay and a quantitative real-time PCR assay according to the manufacturer’s instructions.④Cells were divided into a vehicle group(cells treated with 0.1%DMSO),a TGF-β1 group(cells treated with 10 ng/ml TGF-β1),an AS-Ⅳ + TGF-β1 group(cells pretreated with AS-Ⅳ at 400 μg/ml 2 h prior to 2 ng/ml TGF-β1）and an NAC + TGF-β1 group(cells pretreated with NAC at 100 nM 2h prior to 2 ng/ml TGF-β1).After incubation for 24 h,DCFH-DA fluorescence in cultured cells was analyzed by fluorescence microscopy.Cells were divided into a vehicle group(cells treated with 0.1%DMSO),TGF-β1 group(cells treated with 2 ng/ml TGF-β1),TGF-β1+ AS-Ⅳ group(cells pretreated with AS-Ⅳ at 400 μg/ml 2 h prior to 2 ng/ml TGF-β1),TGF-β1+Smad7 overexpression(OE)group(after Smad7 enforced expression using a Smad7-overexpression lentivirus,cells were treated with 2 ng/ml TGF-β1)and TGF-β1+ AS-Ⅳ + Smad7 knockdown(KD)group(after Smad7 deletion using a Smad7-inhibitor lentivirus,cells were pretreated with AS-Ⅳ at 400 μg/ml 2 h prior to 2 ng/ml TGF-β1).After incubation for 24 h,the levels of vimentin,Smad7 and GAPDH were analyzed by western blot.The migrating cells were detected by Giemsa staining.Part 2:①Cells were treated with various kind of PD solution(1.5%,2.5%,4.25%)for 24h or 4.25%PD solution for various times(0,6,12,24,48 h).the supernant of cell culture was Collected for ELISA assay and cells were lysed using RIPA buffer for a western blot assay according to the manufacturer’s instructions ② To further elucidate the function of NLRP3 inflammsome in the EMT,we generated stable cell lines using expressing NLRP3 siRNA or their scrambled controls constructs inhibiting NLRP3 activation.Cells were divided into a control group(cells treated with 0.1%DMSO),a scramble RNA group,a NLRP3 siRNA group,4.25% PD solution group and an 4.25% PD + NLRP siRNA group.After incubation for 24 h,cells were lysed using RIPA buffer for a western blot assay according to the manufacturer’s instructions.③Cells were divided into a control group(cells treated with 0.1%DMSO),a AS-Ⅳ(400ug/ml)group,a 10ug/mlAS-Ⅳ+4.25%PD solution group,100ug/mlAS-Ⅳ +4.25%PD solution group,a 200ug/mlAS-Ⅳ +4.25%PD solution group and a 400ug/mlAS-Ⅳ +4.25%PD solution group.cells pretreated with AS-Ⅳ 2 h prior.After incubation with 4.25%PD solution for 24 h,cells were lysed using RIPA buffer for a western blot assay according to the manufacturer’s instructions.ResultsPart 1:①various dose of AS-Ⅳ(0,10,50,100,200,and 400μg/ml)for 24h or AS-Ⅳ 400μg/ml for various times neither enhanced cell proliferation nor apoptosis.②After treatment with 2 ng/ml TGF β1 for 24 h,expression levels of E-cadherin was decreased and vimentin,a-SMA and collagen I was increased markedly.These results were accompanied by enhanced expression of phospho-Smad2/3.Pretreatment with AS-Ⅳ attenuated the activation of Smad2/3 and the expression of EMT and ECM markers in HMrSV5 cells.③Smad7 expression in TGF β1-treated HMrSV5 cells was enhanced by treatment with AS-Ⅳ and was accompanied by decreased expression of phospho-Smad2/3 in a dose-dependent manner.AS-Ⅳ(200 and 400 ug/ml)treatment upregulated the expression of Smad 7 both in mRNA and protein levels in HMrSV5 cells.TGF-β1 reduced the Smad 7 expression in protein level but did not affect the expression of Smad 7 mRNA in HMrSV5 cells,the expression of TGFBR1 and TGFBR2 did not change with either TGF β1 or AS-Ⅳ treatment.④After treatment with 2 ng/ml TGF β1 for 24 h,ROS levels were markedly increased.Both AS-Ⅳ and N-acetylcysteine attenuated this increase.NAC treatment did not affect the TGFβ1-induced activation of Smad2/3 and EMT in HMrSV5 cells·⑤To further elucidate the function of Smad7 in the EMT,we generated stable cell lines using expressing Smad7 and Smad7 inhibitors or their scrambled controls with lentiviral constructs inducing or inhibiting Smad7 expression.Overexpression of Smad7 resulted in decreased EMT in TGF β1-treated HMrSV5 cells.In addition,the effect of AS-Ⅳ on the downregulated expression of vimentin in TGF-β1-treated cells was partially reversed by knockdown of Smad7 in HMrSV5 cells.These results were confirmed by the transwell migration assay.Part 2:Part 2:① After treatment with PD solution with different glucose concentrations(1.5%,2.5%,4.25%)for 24h and 4.25%PD solution for various time(0h,6h,12h,24h,48h),the level of IL-18 in PMCs culture supernants was upregulated in a dose-dependent and time-dependent manner.Meanwhile levels of E-cadherin was decreased and vimentin,a-SMA was increased markedly in a dose-dependent and time-dependent manner.These results were accompanied by the activation of NLRP3 inflammsome(NLRP3、pro-casepase-1、pro-IL-1β、IL-1β was upregulated in dose-dependent and time-dependent manner)②the level of IL-18 in PMCs culture supernants and the expression of NLRP3、pro-IL-1β、IL-1β were decreased.More interestingly,PD solution induced PMCs MMT was partially reversed.③AS-Ⅳ with various doses(0,10,100,200,and 400μg/ml)inhibited partly the activation of NLRP3 inflammasome in a dose-dependent manner,which was induced by 4.25%PD solution.Meanwile 4.25%PD solution induced PMCs MMT was partially reversed.Conclusion①AS-Ⅳ attenuates TGF β1-induced EMT by inhibiting the activation of Smad2/3②AS-Ⅳ attenuates Smad2/3 activation by enhancing expression of Smad 7.Smad7 plays a key role in the inhibitory effect of AS-Ⅳ on TGF β1-induced EMT.③AS-Ⅳ-mediated attenuation of TGF β1-induced EMT is independent of its antioxidant effect.④ The activation of NLRP3 inflammsome mediates PD solution-induced PMCs MMT.⑤ AS-Ⅳ inhibites PD solution-induced PMCs MMT by attenuating the activation of NLRP3 inflammsome.