The Mechanism Of Angiotensin Converting Enzyme 2/Angiotensin(1-7)/Mas Axis To Relieve Pulmonary Fibrosis Through Regulating Autophagy

BackgroundIdiopathic pulmonary fibrosis（IPF）is a chronic and irreversible fatal diseases.Activation of lung fibroblasts followed by large secretion of extracellular matrix and abnormal repair of injury mediated eventually pathological changes of pulmonary fibrosis.Renin angiotensin system（RAS）is the emphasis of this research,which includes the angiotensin converting enzyme（ACE）-angiotensin（Ang）Ⅱ-Ang Ⅱ-type 1 receptor（AT1R）axis and the ACE2-Ang（1-7）-Mas axis.AngⅡ and Ang（1-7）act as the representative polypeptide in the RAS with relatively antagonistic function,playing an important role in maintaining survival such as metabolism of water and electrolyte as well as vascular contraction,etc.However,they have been found to be related to fibrosis in recent years.Our previous studies and recent researches of RAS in organ fibrosis showed that ACE-Ang Ⅱ-AT1R axis increased oxidative stress and induced cardiac muscle,blood vessels,liver and lung fibrosis.However,ACE2-Ang（1-7）-Mas axis played a role in anti-fibrosis through antagonism of ACE-Ang Ⅱ-AT1R axis and reducing oxidative stress.This research had continued to explore the new mechanism of ACE2-Ang（1-7）-Mas axis relieving pulmonary fibrosis and considered autophagy as a new direction.Autophagy is described as a cellular dynamic process that various cargoes were transported to the lysosomes for degradation and reuse,which is not only participates in physiological processes,but also has a close relationship with pathological processes such as oxidative stress,organ fibrosis etc.Autophagy is demonstrated to play a role in alleviation of pulmonary fibrosis.However,ACE2 and Ang（1-7）were exhibited effect of autophagy inhibition mostly in cardiovascular system and brain,and also autophagy induction recently reported.So,it will be focused on this study what the level of autophagy was displayed inACE2-Ang（1-7）-Mas axis and influence of autophagy intervene to oxidative stress and pulmonary fibrosis.ObjectionTo explore what’s the level of autophagy in ACE2-Ang（1-7）-Mas axis,and how autophagy affects oxidative stress and pulmonary fibrosis.Methods1.Separation,purification and determination of primary lung fibroblasts in mice as well as culture in vitro2.ATP concentration detection3.Establishment of animal models4.Staining and immunohistochemistry of lung tissue slices5.Western blotResearch contentsThis study planed to find the effects of Ang（1-7）and ACE2 to autophagy related proteins expression levels.The protein expression levels of autophagy,collagen and NOX4 in lung fibroblasts of mice were detected after intervention of ACE2-Ang（1-7）-Mas axis and autophagy pathway.In vivo,bleomycin-induced pulmonary fibrosis in mice was evaluated by the degree of fibrosis as well as NOX4 and autophagy protein levels,further to verify the mechanism of ACE2-Ang（1-7）-Mas axis to improve pulmonary fibrosis.Conclusions1.Ang（1-7）induced autophagy resulting in reduction of collagen and NOX4 induced by AngⅡ,which was reversed by Mas blocker A779.2.Elevated autophagy in lung fibroblasts overexpressed ACE2(ACE2^+/+)reduced collagen and NOX4 induced by AngⅡ,which was reversed by A779 and autophagy inhibitor.3.ACE2^+/+ mice displayed a protect role in lung fibrosis induced by BLM as milder fibrosis change,oxidative stress and higher autophagy.