| Background and ObjectionChina is a big country with the higher incidence of chronic liver disease.In the clinical treatment,liver cirrhosis lack effective therapic method.Liver cirrhosis is developed from liver fibrosis.Liver fibrosis,characterized by activation of fibroblasts,necrosis of hepatocytes,and accumulation of excessive extracellular matrix(ECM),is the final consequence of many chronic liver injuries with currently limited therapeutic options.However,liver fibrosis is a reversible prosess.Hence,it is essencial to explore the pathogenesis of liver fibrosis.In recent years,the epithelial-to-mesenchymal transition(EMT)/mesenchymal-to-epithelial transition(MET)is perhaps the most intriguing and controversial concept involved in the mechanism of liver fibrosis.Besides the role of EMT in embryonic development(type 1 EMT)and tumor metastasis(type 3 EMT),EMT has also been well studied in organ fibrosis(type 2 EMT),Type 2 EMT has been classically associated with fibrosis in adult organs such as kidney,liver,lung,heart,and intestine.As a common event during chronic liver diseases,NAPDH oxidase(NOX)/mitochondria derived reactive oxygen species(ROS)contributes to the progression of EMT.Racl and NOX isoform have been evidenced to mediate the trigging of EMT.However,the role of the downstream molecules involved in oxidative stress in modulation of EMT is still unclear.There are numerous downstream molecules of oxidative stress,among which NOD-like receptor,pyrin domain containing-3(NLRP3)inflammasome is identified as a novel mechanism of liver injury and fibrosis hepatocyte.NLRP3 inflammasome consists of NLRP3,the adaptor molecule such as the apoptosis associated speck like protein(ASC),and the effector molecule pro-caspase-1.NLRP3 inflammasome activation promotes the cleavage and activation of caspase-1 resulting in maturation of effector pro-inflammatory cytokines such as pro-interleukin-1?(pro-IL-1?).Renin-angiotensin system(RAS)is a pivotal regulator of hepatic fibrosis.Studies revealed that NOX/mitochondria derived ROS could be induced by Ang ? in the process of liver fibrosis.ACE2/Ang-(1-7)/Mas axis of RAS can be viewed as the principal counter-regulatory mechanism for the ACE/Ang ?/AT1R axis.Emerging evidence proposed that the ACE2/Ang-(1-7)/Mas axis possesses antioxidant capability,which protect against injuries induced by oxidative stress.However,the precise mechanism by which ACE2/Ang-(1-7)/Mas axis attenuates liver fibrosis is unclear.Does Ang-(1-7)attenuate Ang?-induced hepatocytes EMT via inhibiting NLRP3 inflammasome activation?There's no answer until now and further investigation is warranted.Consequently,we hypothesize that Angiotensin(1-7)reversed Ang?-induced hepatocyte EMT by inhibiting NOX-derived ROS-mediated NLRP3 inflammasome/smad pathway.MethodsCell cultureThe normal human hepatocyte cell line(L02)was purchased from the Type Culture Collection of the Chinese Academy of Sciences,Shanghai,China and cultured in RPMI medium 1640 with 20%fetal bovine serum(FBS)at 37? with 5%CO2.At 60-70%of confluence,cells were growth arrested in serum-free medium for 24 h before the experiments.AnimalsRats weighing(250-280 g)were raised in the Animal Experiment Center of Nanfang Hospital.Protocols for animal use were approved by the Ethics of Animal Experiments Committee at the Southern Medical University.Animal Treatment RegimensWe established two animal models.In the first model,male wistar rats were randomly divided into two groups:control group,Ang?-infused group.Each group contained 6 rats.Micro-osmotic pumps were implanted subcutaneously into each every rats,which allowed 28 days of continuous infusion.Rats in sham group were received 28 days infusion of physiological saline.At the same time,rats in Ang?group received 28 days infusion of Ang? at a rate of(25 ?g·kg-1·h-1).In the second animal model,rats were randomly divided into three groups:sham,BDL and BDL+Ang(1-7).In the sham operated group,rats received 28 days infusion of physiological saline and were opened abdominal cavity without bile duct ligation under general anesthesia.Rats in BDL group also received 28 days infusion of physiological saline and were opened abdominal cavity with bile duct ligation under general anesthesia.BDL+Ang(1-7)group rats were received 28 days infusion of Ang(1-7)at a rat of(25 mg/kg-1·h-1),and were opened abdominal cavity with bile duct ligation under general anesthesia.The rats were sacrificed 4 weeks after surgery under lethal anesthesia,and liver samples were collected.Small interference RNA(siRNA)transfection assayCells at 70%confluence were transiently transfected with 50 nM siRNA during 6 hours using Lipo2000.Oligos were obtained from Genepharma(Shanghai,China).The oligo sequences were as follows:unsilencing:GUAAGACACGACUUAUCGC;Human NOX4:GCAGGAGAACCAGGAGAUUTT;Human NLRP3:GCUGCUGAAAUGGAUUGAATT.Specific oligoes with maximal knock-down efficiency were selected among three different sequences for each gene.After 48 h,cells were treated with Ang?,and harvested.Under-agarose Cell SpreadingHepatocytes were cultured with or without Ang? for 72 h.Ang?-treated cells was stained with a green fluorescent cell tracker dye(PKH 67 green fluorescent cell linker kit,sigma)and control cells with red fluorescent dye(PKH 26 red fluorescent cell linker kit).Both cells were mixed with the same population,and placed into the middle hole in an agarose-filled tissue culture dish.The cells were then allowed to migrate from the center hole toward a serum gradient for 48 h,allowing for direct comparion between the Ang?-treated(green)cells and the untreated(red)cells.Histological and immunochemical assessmentThe liver was fixed using an intratracheal instillation of 4%paraformaldehyde and embedded in paraffin.Sections were stained with Masson's trichrome and examined with light microscopy.For immunohistochemical(IHC)analysis,liver specimens were incubated with primary antibody against Col1A1(1:100,Proteintech),NOX4(1:100;Abcam),followed by incubation with streptavidin-peroxidase complex.Peroxidase conjugates were subsequently visualized by utilizing diaminobenzidine(DAB)solution.The sections were then counterstained with hematoxylin and mounted on a cover slipCaspase-1 Activity AssayCaspase-1 activity was measured in liver tissue using a colorimetric assay kit according to the manufacturer's instructions obtained from BioVision(Biovision,CA,USA)RhoA pull-down assayCells were lysed on the culture plate with 0.5 ml lysis/binding/wash buffer with inhibitors.Next,the cells were scraped off the plate.The suspensions were centrifuged for 15 min at 16,000 g at 4?.The clarified cell lysates(500 ?g)were incubated with 400 ?g GST-Rhotekin-RBD bound to glutathionine coupled-agarose beads at 4? for 1 h and then centrifuged at 6000 g for 30 s.After being washed three times with 400 ?l lysis/binding/wash buffer,the rensin was added to 50 ?l of 2×SDS sample buffer that contained 2.5?l mercaptoethanol.Half of each elution was analyzed using SDS-PAGE and detected with Western blotting using the specific RhoA-GTPase primary antibody.Racl pull-down assayActive Rac1 protein was measured in reference to the RhoA pull-down assayImmunoprecipitation analysis of Racl and NOX4Homogenates from Ang?-stimulated lung fibroblast were adjusted to equal protein concentration(1 mg/mL),pre-cleared and then incubated with 2 ?g/mL of anti-Racl antibody overnight at 4?.Racl and NOX4 immune complexes were immunoprecipitated with protein agarose beads for two additional hours.Immunoprecipitates were resolved in SDS-polyacrylamide gels.Immunoblottings of NOX4 immunoprecipitates were undertaken with anti-Rac1 antibody.Hydroxyproline assayConcentration of hydroxyproline was measured according to the manufacturer's instructions(Hydroxyproline Assay Kit,Sigma,USA).Hydrogen Peroxide AssayThe content of H2O2 was analyzed with a hydrogen peroxide assay kit(Beyotime Institute of Biotechnology,jiangsu China)according to the manufacturer protocols.Intracellular ROS detectionIntracellular ROS production was quantified using the oxidation-sensitive probe-dichlorofluoresceindiacetate(DCF-DA)(Applygen,Beijing,China).Briefly,10 mM DCF-DA stock solution(in methanol)was diluted 1000-fold in cell culture medium without serum to yield a 10-?mworking solution.After 24 h of stimulation,the cells in 24-wellplates were washed twice with PBS and incubated in 500?l working solution of DCF-DA at 37? in dark for 1h.The cells were then washed twice with cold PBS and resuspended in the PBS for an analysis of intracellular ROS using a multiwell fluorescence scanner(Spectra Max M5/M5e,Molecular Devices,USA)and microscope(Olympus,Japan).DCF-DA fluorescence was detected at an excitation wavelength of 480 nm and an emission wavelength of 525 nm.Migration assayCell migration assays were performed using Corning cell culture inserts.Microphotographs of five different fields were taken,and the cells were counted.The average number of migrating cells was determined for each experimental condition.Western blotTotal proteins(from liver tissue or cell extracts)were separated by SDS-PAGE,transferred to a PDVF membrane,and incubated with appropriate primary and second antibodies.Levels of proteins were detected by using ODYSSEYR Infrared Imaging System.The relative density of bands was quantified with ?-actin as an internal control.Results are expressed as n-folds over control as mean±SEM of experiments made.Immunofluorescent cytochemistryCells grown on coverslips were fixed in 4%paraformaldehyde and stained with the appropriate primary antibodies,followed by Cy3-conjugated anti-mouse or FITC-conjugated anti-rabbit secondary antibodies.Nuclei were stained with DAPI.Fluorescence was visualized using an OLYMPUS FV10i-W confocal microscope.Controls with no primary antibody showed no fluorescence labeling,and single label controls were performed in the double-labeling experiments.Statistical analysisAll data are presented as the means±SEM deviation.Significant differences were evaluated by ANOVA with the LSD for multiple comparisons.Differences were considered significant at a p-value<0.05.All of the data were analyzed using SPSS 13.0.Results1.Ang? induced EMT state and promoted motility in HepatocytesBy using immunofluorescent staining,we found that the protein expressionof e-cadherin was markedly decreased,while that of Col1A1 was significantly increased in Ang?-treated cells.Similarly,Western blot analysis showed vimentin protein level was elevated after incubation with Ang? for 24 h,while the loss of e-cadherin and albumin in Ang?-treated hepatocytes was occurred at 72 h,which was got out of sync with the change of vimentin(p<0.05).In addition,we found that 10-7 mol/L was the most appropriate concentration of Ang?(p<0.05).We found that Ang?-treated cells(green)moved farther than control cells(red),which indicated that Ang? increased motility of hepatocytes.2.Angll increased NOX-derived ROS in hepatocytes.Intracellular ROS and H2O2 production were increased by Ang? treatment(p<0.05).The H2O2 production were inhibited by DPI and catalase pretreatment(p<0.05).We further found the protein level of the NOX4 was significantly up-regulated in hepatocyte by Ang? treatment(p<0.05).The increase of active Racl protein level was observed in Ang? treated hepatocyte,and Co-IP assay further evidenced the enhanced interaction between NOX4 and Rac1 by Ang?stimulation(p<0.05).3.NOX-derived ROS regulated Ang?-induced EMT in hepatocyte.We observed that pretreatment with DPI,NOX4siRNA,ROS scavenger NAC,catalase,and EHOP markedly suppressed Ang?-increased vimentin and Col1A1 protein levels,while rescued Ang?-induced lose e-cadherin protein(p<0.05).In addition,Western blot showed Ang?-induced activation of P-moesin and active RhoA were inhibited by DPI,EHOP,and catalase(p<0.05).Furthermore,cell migration was inhibited by DPI and EHOP(p<0.05).Collectively,these results confirmed that NOX-derived ROS was required for the Ang?-induced hepatocyte EMT.4.Ang? induced hepatocyte EMT via NOX-derived ROS-mediated NLRP3/smadpathway.Immunofluorescence staining and Western blot confirmed that NLRP3 inflammasome was activated by Ang? treatment.Dual immunofluorescence staining further showed that Ang?-induced ASC translocates from the nucleus into the cytoplasm and co-localizes with NLRP3.NLRP3 gene knockdown suppressed Ang?-induced protein expression of caspase-1-P10 and IL-1?-P17(p<0.05).NLRP3 siRNA and caspase-1 inhibitor YVAD pretreatment decreased Ang?-induced protein expression of vimentin and Coll A1 and rescued the e-cadherin protein(p<0.05).We observed that IL-1? induced hepatocyte EMT,evidenced by increased production of Col1A1 and vimentin and loss of e-cadherin(p<0.05).In addition,IL-1? led to phosphorylation of P-moesin and significant increase in cell migration(p<0.05).The result showed the activation of NLRP3 inflammasome was blocked by pre-treatment with DPI(p<0.05).Transfection with smad4 siRNA suppressed Ang?-induced EMT(p<0.05).NLRP3 knowdown diminished Ang?-induced phosphorylation of smad2/3(p<0.05).Furthermore,IL-1? induced a rapid phosphorylation of smad2/3(p<0.05).5.Ang(1-7)suppressed Ang?-induced EMT by inhibiting NOX-derived ROS-activated NLRP3 inflammasome.Ang(1-7)significantly inhibited the aforementioned Ang? effects,and the inhibitory effects of Ang(1-7)were reversed by the addition of a Mas receptor antagonist,A779(p<0.05).The production of Ang?-induced H2O2 and NOX4 protein levels were inhibited by Ang(1-7)(p<0.05).Similarly,the Ang?-induced increase of active Racl protein expression was abolished by Ang(1-7),which was blocked by A779(p<0.05).In addition,the Ang?-activated NLRP3 inflammasome was significantly inhibited by Ang(1-7)(p<0.05).We found that Ang?-induced activation of NLRP3 inflammasome was blocked by pretreatment of Ang(1-7),and Ang(1-7)inhibited the phosphorylation of Smad2/3(p<0.05).Furthermore,the signaling molecules mediated cell migration(active RhoA protein)were markedly reduced by Ang(1-7)(p<0.05).In keeping with protein levels,Ang?-induced cell migration was reduced(p<0.05).6.Ang? infusion caused increased protein expression of Col1A1 and FSP-1 in hepatocytes,and activated the ROS-correlated NARP3 inflammasome in rat liverCompared with sham group,Ang? treatment caused a marked increase in mesenchymal markers(FSP-1,Col1A1)in albumin-positive cells,indicating that Ang? induced hepatocytes entered into intermediate transitional stage of EMT.We observed a marked decrease in epithelial-specific marker(e-cadherin)and a concomitant increase in the expression of Col1A1 and vimentin in Ang?-infused liver(p<0.05).Compare to sham group,the NOX4 expression in albumin-positive hepatocytes was increased,which was similar to the result in vitro.Futhermore,we observed that Ang? infusion enhanced the content of H2O2 in rat liver(p<0.05).Hence,Ang? infusion elevated the production of NOX-derived ROS in liver tissue.In addition,compared to sham group,Western blot analysis indicated NLRP3 inflammasome and smad2/3 protein expression were up-regulated in Ang? treatment group(p<0.05).7.Ang(1-7)infusion inhibited the production of Col1A1 in hepatocytes and suppressed the activation of ROS-correlated NLRP3 inflammasome pathway in fibrotic liver.ECM accumulation,as measured with Masson's trichrome,increased in BDL rats liver,which was attenuated by Ang(1-7)infusion.The hydroxyproline content,H2O2 content,and caspase-1 activity were markedly increased in the liver of BDL-treated rat,and these effects were suppressed by Ang-(1-7)constant infusion(p<0.05).In addition,the protein levels of components of the NLRP3 inflammasome/IL-1?secretion axis and NOX4 protein level were markedly elevated in BDL induced fibrotic liver,which could be significantly inhibited by Ang-(1-7)infusion(p<0.05).Furthermore,compared with BDL-treated liver,Ang(1-7)-treatment reversed the up-regulation of Col1A1,vimentin and reduced the protein expression of e-cadherin in liver(p<0.05).Double staining showed that the expressions of NLRP3,NOX4,and CollAl in albumin-positive hepatocyte was increased in BDL-induced fibrotic liver,which could be inhibited by Ang(1-7)treatment. |
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