| Background & Objectives:A complex network of cytokines and cells are involved both at the early stage called alveolitis and the latter course in pulmonary fibrosis.Many cytokines and kinases involved in the formation of pulmonary fibrosis are mostly released by lung parenchymal cells and infiltrating inflammatory cells.They can stimulate fibroblast to proliferate,collagen synthesis,such as increased extracellular matrix(ECM) degradation decrease in alveolar walls and cause inter-quality fibrosis.Lung fibroblasts play an important role in the formation of the interstitial cells,they are the main source of ECM.Recently,renin- angiotensin- aldosterone system(RAAS) become on of the hot spot of investigation for fibrosis.It has been known that renin-angiotensin-aldosterone system(RAAS) plays a key role in the fibrogenesis of local tissue. AngiotensinⅡ(AngⅡ),the principal effector molecule of the RAAS,exert local effects on cell growth and fibrogenesis,angiotensinⅡ(AngⅡ) is the most important effector of this system,it can induce the activation and proliferation of lung fibroblasts In recent years,much attention has been focused on the novel relationship between activation of RAAS and fibrosis of lung.AngⅡcan stimulate lung fibroblasts and increase the production of ECM by interaction with AngiotensinⅡtype1 receptor(AT-1 receptor),leading to lung fibrosis.However,the singnal transduction mechanisms underlying effects of AngⅡon lung fibrogenesis remain fully be elucidated.There are a few reports about the investigation of this field can be found so far.The ACE2-Ang(1-7)-Mas receptor axis is a newly discovered component of RAAS,which have a negative effect on the ACE-AngⅡ-AT-1R-axis,can protect lung,heart,kidney from injury.All that indicates ACE2-Ang(1-7)-Mas receptor axis is a new target for the treatment of lung fibrosis.Materials and MethodsThe present study aims to investigate the signal transduction mechanism underlying effects of AngⅡand Ang(1-7) on the signal pathway of MAPK、RhoA-Rock、Smad、and NF-κB in human lung fibroblasts,and the effects of ACE2-Ang(1-7)-Mas receptor axis on pulmonary fibrosis.This study tried to clarify the promotion of AngⅡon lung fibroblasts,Ⅰ-type collagen synthesis and fibrogenic cytokine TGF-β1,CTGF expression,and the regulation of Ang(1-7) on AngⅡ.At the same time lenti-ACE2 virus vector are established to infected HFL-1, then ACE2,TGF-β1,CTGF,α1-Ⅰcollagen expression are also measured.The protein level were detected by Western blotting.The mRNA level were detected by Real-time RT-PCR and QuantiGene.ResultsThe effect of AngⅡand Ang(1-7) on fibrosis-related signaling pathway and downstream factors in lung fibroblast.1.MAPK pathway:AngⅡcan induce phosphorylation of ERK1/2,P38, JNK,which can be inhibited by irbesartan,Ang(1-7).Mas receptor antagonist A779 can block the inhibitory effect of Ang(1-7).Ang(1-7) can induce phosphorylation of ERK1/2;but did not induce phosphorylation of JNK,and may even inhibit the phosphorylation of P38.2.RhoA-Roek pathway:AngⅡcan stimulate RhoA expression,the effect can be inhibited by Irbesartan,SB203580,PD98059 and Ang(1-7).Mas receptor antagonist A779 can block the inhibitory effect of the latter.Ang(1-7) can stimulate RhoA expression.3.Smad pathway:After AngⅡintervention for 20 min,p-Smad3 nucleus translocation are observed;at the same time,pSmad3,Smad4 expression were significantly increased,which can be inhibited by Irbesartan.SB203580 inhibited AngⅡ-induced pSmad3,Smad4 expression;PD98059 inhibited AngⅡ-induced expression of pSmad3;Ang(1-7) inhibited AngⅡ-induced p-Smad3 expression; A779 can not block the inhibitory effect of the latter.Ang(1-7) can also induce expression and nuclear translocation of p-Smad3.4.NF-κB pathway:AngⅡincreased p-IκKα/βprotein expression,reduced protein expression of IκBα,enhanced the DNA binding activity of NF-κB and nuclear translocation,which can not be inhibited by SB203580,but can be inhibited by irbesartan,PD98059 and Ang(1-7).A779 can block the inhibition of the latter.Ang (1-7) alone can reduce cytoplasmic protein expression of IκBα,enhance the DNA binding activity of NF-κB and nuclear translocation,but the corresponding p-IκKα/βdid not significantly increased.After intervention of AngⅡfor 48 hours,increased expression of ICAM-1 mRNA was observed,and the action can be inhibited by Irbesartan,SB203580,PD98059,NAC and Ang(1-7).A779 can block the inhibitory effect of the latter.Ang(1-7) alone can increase ICAM-1 mRNA.5.TGF-β1:After intervention of AngⅡfor 24 hours,increased expression of TGF-β1 mRNA was observed,and the action can be inhibited by Irbesartan, SB203580,Ang(1-7),and ROCK inhibitor Y27632.A779 can not block the inhibitory effect of the latter.Ang(1-7) alone can not increase TGF-β1 mRNA.6.CTGF:AngⅡcan increase the protein level of CTGF,which can be blocked by Irbesartan,SB203580,PD98059,Y27632 and Ang(1-7).A779 can block the inhibitory effect of the latter.Ang(1-7) alone can increase the protein level of CTGF.7.α1-Coll:AngⅡcan increase the expression ofα1-Col 1mRNA,which can be blocked by Irbesartan,SB203580,PD98059 and Ang(1-7).Y27632 can not block the role.Ang(1-7) alone cannot increase the level ofα1-Col 1mRNA.The effect of lenti-ACE2 virus infection on factor TGF-β1,CTGF,α1-Ⅰcollagen and ICAM-1.1.Lenti-ACE2 virus vector were successfully transfected into human embryonic lung fibroblasts which is confirmed by inverted fluorescence microscope and the protein level of ACE2 by western blot.2.TGF-β1:After lenti-ACE2 and empty lentivirus vector are transfected into HFL-1,the expression of TGF-β1 mRNA has no change.AngⅡseparately can not stimulate lenti-ACE2 virus-infected cells to secrete TGF-β1 mRNA,while after incubation of A779,AngⅡcan stimulate the cells to secrete TGF-β1 mRNA.3.α1-Coll:.After lenti-ACE2 and empty lentivirus vector are transfected into HFL-1,the expression ofα1-Coll mRNA increased significantly.AngⅡseparately can not stimulate lenti-ACE2 vector-infected cells to secreteα1-Coll,while after incubation of A779,AngⅡcan stimulate the cells to secreteα1-Coll.4.CTGF:CTGF protein level in empty lentivirus vector-infected cells was significantly higher than that of conrtol and lenti-ACE2 vector-infected cells.5.ICAM-1:ICAM-1 mRNA level in empty lentivirus vector-infected cells was significantly higher than that of conrtol and lenti-ACE2 vector-infected cells. Conclusions1.AngⅡcan stimlute expression of fibrosis-related cytokine such as TGF-β1 mRNA,CTGF,and collagen typeα1-Ⅰby combining to AT1R.In the same process, MAPK,RhoA-Rock,Smad,NF-κB signaling pathway were activated.TGF-β1 mRNA through the P38 MAPK,RhoA-Rock pathway,CTGF through the P38 MAPK, ERK MAPK,RhoA-Rock pathway,α1-typeⅠcollagen through the P38 MAPK,ERK MAPK pathway.2.The signaling pathways were activated by AngⅡ.P38,one of the MAPK singaling pathway is involved in regulation of RhoA-Rock,Smad signaling pathway activation;P38-specific inhibitor SB203580 could inhibit the activation of these pathways.ERK is involved in RhoA-Rock,Smad,NF-κB signaling pathway activation,ERK1/2 specific inhibitor PD98059 inhibited the activation of these pathways.3.Ang(1-7) have a dual role.It can activate the ERK MAPK,RhoA-Rock,Smad, NF-κB pathway,at the same time,it shows the antagonistic effect on AngⅡ.Ang (1-7) can inhibit AngⅡ-activated MAPK,RhoA-Rock,NF-κB pathway through the Mas receptor,and Smad pathway.4.Ang(1-7)’s dual role is also reflected both in incremental expression of CTGF and ICAM-1 and in the antagonistic effect on AngⅡ-induced TGF-β1 mRNA, CTGF,α1-Coll mRNA and ICAM-1 mRNA expression.5.lenti-ACE2 virus can protect human embryonic lung fibroblasts by decreasing the expression of CTGF and ICAM-1 mRNA caused by virus,but also by decreasing the expression of AngⅡ-induced TGF-β1 mRNA,α1-typeⅠcollagen,while Mas receptor inhibitors A779 may block the action,suggesting that ACE2 cleavage AngⅡto Ang(1-7),the latter play an antagonism role in the action induced by AngⅡ. |
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