| BackgroundLiver fibrosis is a necessary stage of various kinds of chronic liver disease to cirrhosis,and is characterized by excess accumulation of extracellular matrix in liver.Unlike liver cirrhosis,early stage of liver fibrosis is reversible,therefore,finding a way to inhibit,even to reverse,liver fibrosis is very important for the treatment of chronic liver disease.To explore the mechanism of liver fibrosis and seek effective treatment measures has great realistic meaning for our country,which has great number of people suffering chronic liver disease.Hepatic stellate cells(HSC)play a critical role in the development of liver fibrosis.Under various pathogenic factors,quiescent HSCs will be activated into myofibroblasts.The myofibroblasts can synthesis and secrete much more ECM and tissue inhibitor of metalloproteinase(TIMPs),reducing activity of matriptase and decreasing degradation of ECM..As a result,excessive ECM accumulates in liver,leading to liver fibrosis and cirrhosis.In recent years,more and more researches show that a variety of inflammatory medium receptors exist in HSCs surface,which may have a close relationship with HSCs activation.For large numbers of chronic liver disease,liver inflammation and liver fibrosis exist simultaneously.Activation of HSCs,as an indispensable element in liver fibrosis,also has close correlation with inflammation.Inflammation and inflammatory cytokine play an important role in HSCs activation and proliferation.NLRP3 inflammasome,as an intracelluar multiprotein complex,consists of the NOD-like receptor NLRP3,the adaptor molecule apoptosis-associated speck-like protein(ASC),and the effector molecule cysteinyl aspartate specific proteinase(caspase-1).NLRP3,as pattern recognition receptor,can be activated by various of danger signals in cell,resulting in a large number of precursor caspasel converting into caspasel p10 fragments and downstream interleukin 1 beta(IL-1 beta)and interleukin 18(IL-18)production,which play an important role in body immune response and inflammatory response.Researches have confirmed that components of NLRP3 inflammasome exited in mice primary HSCs.In mice liver fibrosis model induced by CC14 and TTA,NLRP3 inflammasome was activated,and the mRNA levels of TGF? and COL1A was up-regulated in primary HSCs extracted from wide type mice,promoting the development of liver fibrosis.However,when NLRP3 or ASC gene was knock out,the mRNA levels of TGF? and COL1A in HSCs was significantly decreased.All of researches suggest that the inflammation induced by NLRP3 inflammasome play a key role in the development of liver fibrosis.However,its specific mechanism remains to be further research.Numerous studies have demonstrated that local renin angiotensin system(RAS)in liver is closely related to liver fibrosis.In fibrosis liver tissue,Ang?,the main bioactive component of the RAS,have been up-regulated significantly,which can induce HSCs proliferation and the synthesis of collagen,therefore Ang? is an important matter to promote liver fibrosis.In recent years,ACE2 and Ang-(1-7),the new complexity to the RAS,exerts opposite effects to those of Ang?,so they can inhibit liver fibrosis induced by Ang?.Howere,the specific mechanism of RAS to regulate liver fibrosis remains unclear.Whether its effect on liver fibrosis rely on NLRP3 inflammasome.All of these need further researches.ObjectionThis research plan to induce liver fibrosis by intraperitoneal injection of CCl4 into wide type and ACE2 over expression mice.In order to assess the effect of Ang-(1-7)on mice liver fibrosis and NLRP3 inflammasome activation,we measured the content of Ang-(1-7)and NLRP3 inflammasome activation.Through intensive study of the effect of Ang? and Ang-(1-7)on the expression of the components of NLRP3 inflammasome and NLRP3 inflammasome activation in rat primary HSC,we can make clear possible mechanism of liver fibrosis and provide theoretical basis for the pathogenesis of liver fibrosis and effective treatment.MethodsVivo experiments1.18 4-week-old wide type C57BL/6 mice and 18 4-week-old ACE2 over expression C57BL/6 mice were randomly divided into four groups of 9 mice each:WT+oil group,ACE2+/++ oil group,ACE2+/++CCl4 group,WT+CCl4 group.WT+oil group and ACE2+/++ oil group were intraperitoneal injection of olive oil.ACE2?/++CCl4 group and WT+CCl4 group were intraperitoneal injection of CCl4(1ml/kg;diluted 1:4 in olive oil).4 weeks after intraperitoneal injection,liver tissue specimens and blood specimens were obtained.In order to evaluate the content of Ang-(1-7)in ACE2 over expression mice liver,the protein level of ACE2 was detected by Western blot and the content of Ang-(1-7)was measured by ELISA.H&E staining,Masson staining,hydroxyproline assay and the expression of COL1a detected by immunohistochemistry were use to assess liver fibrosis of different groups.For assessing the HSC activation of different groups,the expression of CTGF and a-SMA were measured by Western blot and immunohistochemistry.2.4 weeks after intraperitoneal injection,liver tissue specimens were obtained.In order to evaluate liver inflammation of different groups,the protein levels of NLRP3,ASC,caspase-1 and IL-1?in liver were measured by Western blot and immunohistochemistry.Vitro experiments3.Rat primary HSCs were stimulated by Ang?(10-7 mol/L)or Ang(1-7)(10-7 mol/L)for 0h,0.5h,1h,2h,6h,12h and 24h.After stimulation,the protein levels of COL1A??-SMA and CTGF in cells were measured by Western blot.Repeat the experiment 3 times.4.Rat primary HSCs were stimulated by various concentration of Ang? for 12h,including 0 mol/L,10-9mol/L,10-7 mol/L,10-5 mol/L,2*10-5 mol/L and 4*10-5 mol/L.After stimulation,the protein levels of COL1A??-SMA and CTGF in cells were measured by Western blot.Repeat the experiment 3 times.5.Rat primary HSCs were stimulated by various concentration of Ang-(1-7)for 12h,including 0 mol/L,10-9mol/L,10-7mol/L and 10-5 mol/L.After stimulation,the protein levels of COL1A??-SMA and CTGF in cells were measured by Western blot.Repeat the experiment 3 times.6.Rat primary HSCs were divided into control group,Ang? group,Ang-(1-7)group,Ang?+Ang-(1-7)group,AngII+Ang-(1-7)+A779 group and Ang?+Irb group.After stimulation by different drugs,the protein levels of COL1A??-SMA and CTGF in cells were measured by Western blot.Repeat the experiment 3 times.7.Rat primary HSCs were stimulated by Ang?(10-7 mol/L)for 12h.After stimulation,HSCs were analyzed by triple immunofluorescence staining.The location of the components of NLRP3 inflammasome were observed by confocal microscopy after staining.Repeat the experiment 3 times.8.Rat primary HSCs were transfected with scramble siRNA or NLRP3 siRNA for 72h.HSCs were divided into control group,Ang? group?scramble siRNA group?scramble siRNA+Ang? group?NLRP3 siRNA group and NLRP3 siRNA+Ang?group.After stimulation,the protein levels of NLRP3?COL1A and ?-SMA in cells were measured by Western blot.Repeat the experiment 3 times.9.Rat primary HSCs were divided into control group,Ang? group,and Ang?+Ac-YVAD-cmk(caspase-1 inhibitor)group.After stimulation by different drugs,the protein levels of COL1A in cells were measured by Western blot.Repeat the experiment 3 times.10.Rat primary HSCs were stimulated by Ang?(10-7 mol/L)or Ang(1-7)(10-7 mol/L)for 0h,6h,12h and 24h.After stimulation,the protein levels of NLRP3?ASC?caspase-1 and IL-1? in cells were measured by Western blot.Repeat the experiment 3 times.11.Rat primary HSCs were stimulated by Ang?(10-7 mol/L)for Oh,1h,4h and 24h.After stimulation,the mRNA levels of TLR4?MyD88 and TRAF6 in cells were measured by RT-qPCR.Repeat the experiment 3 times.12.Rat primary HSCs were stimulated by various concentration of Ang? or Ang-(1-7)for 12h,including 0 mol/L,10-9mol/L,10-7mol/L and 10-5 mol/L.After stimulation,the mRNA levels of TLR4?MyD88 and TRAF6 in cells were measured by RT-qPCR,the content of intracellular ROS was detected by microplate reader,the protein levels of TLR4?MyD88?nuclear p65?NLRP3?ASC?caspase-1 and IL-1?in cells were measured by Western blot.Repeat the experiment 3 times,intracellular caspase-1 activity of differ groups were determined by caspase-1 activity assay.Repeat the experiment 3 times.13.Rat primary HSCs were transfected with scramble siRNA or MyD88 siRNA for 72h.HSCs were divided into scramble siRNA group,scramble siRNA+LPS group?scramble siRNA+Ang? group?MyD88 siRNA group?MyD88 siRNA+LPS group and MyD88 siRNA+Ang? group.After stimulation,the protein levels of MyD88?NLRP3 and IL-1? in cells were measured by Western blot.Repeat the experiment 3 times.14.Rat primary HSCs were divided into control group,Ang? group,Ang?-DPI(NAD(P)H oxidase inhibitor)group,Ang?+NAC(peroxidase inhibitor)group,AngII+CAT(H2O2 scaverger)group,and Ang?+TEMPO(Mitochondrial ROS inhibitor).After stimulation by different drugs,the protein levels of NLRP3?caspase-1 and IL-1? in cells were measured by Western blot.Repeat the experiment 3 times.15.Rat primary HSCs were divided into control group,Ang? group,Ang-(1-7)group,AngII+Ang-(1-7)group,AngII+Ang-(1-7)+A779 group and Ang?+Irb group.After stimulation by different drugs,the mRNA levels of TLR4?MyD88 and TRAF6 in cells were measured by RT-qPCR.Repeat the experiment 3 times.16.Rat primary HSCs were divided into control group,Ang? group,Ang-(1-7)group and Ang?+Ang-(1-7)group.After stimulation by different drugs,the protein levels of NLRP3?ASC?caspase-1?IL-1? and nuclear p65 in cells were measured by Western blot.Repeat the experiment 3 times.17.Rat primary HSCs were divided into control group,Ang? group,Ang-(1-7)group and AngII+Ang-(1-7)group.After stimulation by different drugs for Omin,5min,10min,and 15min,the content of intracellular ROS was detected by microplate reader.Repeat the experiment 3 times.Results1.Compared with wide type mice,the protein level of ACE2 in liver tissue was significantly up regulated in ACE2 over expression mice(P<0.05).Compared with WT+oil group,ACE2?/+? oil group,and WT+CCl4 group,the content of Ang-(1-7)in liver tissue was significantly increased in ACE2+/++CCl4 group(P<0.05).Compared with WT+oil group and ACE2+/++ oil group,the liver fibrosis score,collagen area,hydroxyproline,and the protein levels of COL1A,?-SMA and CTGF were obviously increased in WT+CCl4 group(P<0.05).Compared with WT+CCl4 group,the liver fibrosis score,collagen area,hydroxyproline,and the protein levels of COL1A,a-SMA and CTGF were obviously decreased in ACE2+/++CCl4 group(P<0.05).2.Compared with WT+oil group and ACE2?/+? oil group,the protein levels of NLRP3,ASC,caspase-1 and IL-1?in liver tissue were obviously increased in WT+CCl4 group(P<0.05).Compared with WT+CCl4 group,the protein levels of NLRP3,ASC,caspase-1 and IL-lpin liver tissue were obviously decreased in ACE2+/++CCl4 group(P<0.05).3.After treatment with Ang?(10-7 mol/L),the protein levels of COL1A,a-SMA and CTGF in rat primary HSCs were obviously increased in a time-dependent manner detected by Western blot(P<0.05).After treatment with Ang-(1-7)(10-7 mol/L),the protein levels of COL1A,a-SMA and CTGF in rat primary HSCs were obviously decreased in a time-dependent manner detected by Western blot(P<0.05)4.After treatment with various concentration of Ang? for 12h,the protein levels of COL1A,?-SMA and CTGF in rat primary HSCs were significantly increased in a dose-dependent manner detected by Western blot(P<0.05).The peak concentration of Ang? was 4*10-5 mol/L(P<0.05).5.After treatment with various concentration of Ang-(1-7)for 12h,the protein levels of COL1A,a-SMA and CTGF in rat primary HSCs were significantly decreased in a dose-dependent manner detected by Western blot(P<0.05).The peak concentration of Ang-(1-7)was 10-5 mol/L(P<0.05).6.The protein expression of COL1A,?-SMA and CTGF in rat primary HSCs induced by Ang? could be inhibited by Ang-(1-7),however the inhibitory effect of Ang-(1-7)could be reversed by A779(a Mas selective inhibitor)detected by Western blot.Besides that,Irb,an AT1 selective inhibitor,inhibited the effect of Ang?.7.After treatment with Ang?(10-7mol/L)for 12h,cells immunofluorescence staining showed that Ang? induced intracellular NLRP3,ASC,and caspase-1 to co-localized with mitochondrial,mediating the activation process of NLRP3 inflammasome.8.After transfected with scramble siRNA or NLRP3 siRNA for 72h,compared with scramble siRNA+Ang? group,the protein levels of NLRP3,COL1A and a-SMA in HSCs were obviously decreased in NLRP3 siRNA+Ang? group detected by Western blot(P<0.05).9.Western bolt showed that Ac-YVAD-cmk(caspase-1 inhibitor)could inhibit the protein expression of COL1A in HSCs induced by Ang?(P<0.05).10.After treatment with Ang?(10-7 mol/L),the protein levels of NLRP3,ASC,caspase-1 and IL-1? in rat primary HSCs were significantly increased in a time-dependent manner detected by Western blot(P<0.05).After treatment with Ang-(1-7)(10-7 mol/L),the protein levels of NLRP3,ASC,caspase-1 and IL-1? in rat primary HSCs were obviously decreased in a time-dependent manner detected by Western blot(P<0.05).11.After treatment with Ang?(10-7mol/L)for various time,the mRNA levels of TLR4,MyD88 and TRAF6 in HSCs were increased to the peak at 1h detected by RT-qPCR(P<0.05).One hour later,as the stimulus time extended,the mRNA levels of TLR4,MyD88 and TRAF6 were decreased.12.After treatment with various concentration of Ang? for 12h,the mRNA levels of TLR4,MyD88 and TRAF6 in HSCs were increased to the peak at the concentration of 10-7 mol/L detected by RT-qPCR(P<0.05);the intracellular ROS were significantly increased in a dose-dependent manner detected by microplate reader(P<0.05);the protein levels of TLR4,MyD88,nuclear p65,NLRP3,ASC,caspase-1 and IL-1? were significantly increased in a dose-dependent manner detected by Western blot.(P<0.05);the intracellular caspase-1 activity was significantly increased in a dose-dependent manner detected by caspase-1 activity assay(P<0.05).Howere,after treatment with various concentration of Ang-(1-7)for 12h,all results above were reversed.13.After transfected with scramble siRNA or MyD88 siRNA for 72h,compared with scramble siRNA+Ang? group,the protein levels of MyD88,NLRP3 and IL-1?in HSCs were obviously decreased in MyD88 siRNA+Ang? group detected by Western blot(P<0.05).14.Western bolt showed that DPI,NAC,CAT or TEMPO could inhibit the protein expression of NLRP3,caspase-1 and IL-1? in HSCs induced by Ang?(P<0.05).15.The mRNA transcription of TLR4,MyD88 and TRAF6 in rat primary HSCs induced by Ang? could be inhibited by Ang-(1-7),however the inhibitory effect of Ang-(1-7)could be reversed by A779(a Mas selective inhibitor)detected by RT-qPCR.Besides that,Irb,an AT1 selective inhibitor,inhibited the effect of Ang?.16.Western bolt showed that Ang-(1-7)could inhibit the protein expression of nuclear p65,NLRP3,ASC,caspase-1 and IL-1? in HSCs induced by Ang?(P<0.05).17.After treatment with Ang?(10-7 mol/L),the intracellular ROS were significantly increased in a time-dependent manner,and reached to the peak at 15min detected by microplate reader(P<0.05).However,after treatment with Ang?-(1-7)(10-7mol/L),the results above were reversed,and Ang-(1-7)coule inhibit the increase of intracellular ROS induced by Ang? at each time.Conclusion1.The increase of Ang-(1-7)content in ACE2 over expression mice liver tissue can improve liver fibrosis induced by CCl4.2.The increase of Ang-(1-7)content in ACE2 over expression mice liver tissue can inhibit the protein expression of components of NLRP3 inflammasome.3.Ang-(1-7)can block the activation of HSC and synthesis of collagen induced by Angll by inhibiting activation of NLRP3 inflammasome,which improve liver fibrosis. |
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