The Effect And Molecular Mechanism Of Angiotensin-(1-7) On Transformation From Fibroblast To Myofibroblast In Airway Remodeling

Background and objective Asthma is a chronic inflammatory airway disease that affects 300 million people worldwide.Refractory asthma accounts for only 5%to 10%of asthmatic patients,but it is particularly difficult to treat with considerable portion of healthcare expense.It is still a challenge to explore the possible pathogenic mechanisms of refractory asthma.Enhanced attention has been directed to airway remodeling,and transforming growth factor-beta 1?TGF-?1?and collagen type I?Col-??may play critical roles in the formation of airway remodeling.For years,the renin-angiotensin system?RAS?constitutes an monolithic endocrine system involved in cardiovascular regulation and water balance.It has been demonstrated that the elevation of plasma renin and Angiotensin ??Ang??levels is observed in asthmatic patients during severe asthma attack.A series of observations suggest that Ang? could be a putative mediator in asthma by increasing TGF-?1 production and Col-? deposition.As for the RAS,angiotensin converting enzyme?ACE?/Ang?/Ang? receptor type 1?AT1?has been the focus for a long time.The activation of this axis causes deleterious effects,including vasoconstriction,inflammation and fibrosis.During the past decade,ACE2,Angiotensin-?1-7?[Ang-?1-7?]and its specific Mas receptor[ACE2-Ang-?1-7?-Mas axis]comes to us as a counter-regulatory axis against ACE/Ang?/AT1 axis.Ang-?1-7?has been shown to have anti-thrombotic,anti-proliferative,anti-fibrotic and anti-inflammatory properties in heart,kidney and arthritis animal model.Animal studies also demonstrate that Ang-?1-7?,via Mas receptor,acts as an anti-inflammatory pathway in allergic asthma.Thus,we hypothesized that Ang-?1-7?could inhibit TGF-?1 and Col-? expression contributing to the attenuation of airway remodeling.Human lung fibroblasts?HLF?-myofibroblasts?MF?transition is an indicator of the process of airway remodeling.In this study,by using HLF cell line,we investigated if Ang-?1-7?could inhibit Ang?-induced HLF-MF transition,and further explored relevant signaling pathways.The results reported here may provide a novel insight on mechanism of airway remodeling.Methods 1.HLF was treated with 1?M Ang?(10^-8-10^-6mol/l)for 0,12,24,48 h,then was harvested for Western blotting to determine the expression of ACE,ACE2 and AT1.At same time,by using RT-PCR and Western blotting methods,we determined the expression of TGF-?1,?-SMA and Col-?.2.We set up four comparative groups as following:a.controlled group;b.single Ang? stimulation group;c.Ang? and Ang-?1-7?combination group;d.A779 intervention group.By using RT-PCR and Western blotting methods,we determined the expression of TGF-?1,?-SMA and Col-?.3.Based on all groups above,we further explored the possible signaling pathways including p38 MAPK,PI3K,AKT,Smad2/3,JNK,ERK.Results 1.The stimulation with 10^-6mmol/l Ang ? for 48h was considered as the best strategy.Ang? increased ACE,AT1R expression and decreased ACE2 expression.Ang? also significantly induced the expression of?-SMA,TGF-?1 and Col-??all P<0.05?,which indicates HLF-MF transition.2.Ang-?1-7?significantly decreased Ang?-induced?-SMA,TGF-?1 and Col-? expression at both mRNA and protein level?all P<0.05?.Such effect was neutralized by the introduction of A779?Mas receptor antagonist??all P<0.05?.3.Ang-?1-7?also downregulated Ang?-induced TGF-?1/Smad2/3 expression and phosphorylation of p38 MAPK,PI3K and JNK?all P<0.05?.Such effect also was antagonized by A779?all P<0.05?.Conclusions 1.All data demonstrated that both Ang? and Ang-?1-7?participate in HLF-MF transition in vitro;2.Ang-?1-7?could inhibit Ang?-induced expression of?-SMA,TGF-?1 and Col-?.3.Ang-?1-7?could downregulate the expression of TGF-?1/Smad2/3 and decrease the phosphorylation of p38 MAPK,PI3K and JNK,which subsequently inhibit HLF-MF transition.