Alamandine Via MrgD Receptor By Inhibiting NOX4 And Inducing Autophagy Pathway Attenuates Pulmonary Fibrosis

BACKGROUNDAlamandine(ALA)and its receptor Mas associated G protein coupled receptor(MrgD)are recently reported as new components of the renin-angiotensin system(RAS).ALA can be formed by decarboxylation of angiotensin(1-7)(Ang(1-7))or by hydrolysis of proangiotensin A(Ang A)via angiotensin converting enzyme 2(ACE2).ALA acts mainly through the activation of MrgD receptor,and its effects include vasodilation,protection of ischemic reperfusion heart,inhibition of oxidative stress,and alleviation of heart and renal fibrosis.However,the role of ALA in pulmonary fibrosis and whether MrgD receptor is expressed in lung fibroblasts are still unclear.OBJECTIVE1.Analysed the hypothesis of ALA reducing Ang?-induced lung fibroblasts collagen deposition and discussed the related pathways.2.The expression of MrgD receptor in fibroblasts was analyzed by qPCR.3.To observe the role of ALA in bleomycin(BLM)-induced pulmonary fibrosis in mice and explore the relevant mechanisms.METHODS1.Used the third to the fifth generation of C57/B mice primary lung fibroblasts to do the vitro experiment,used Western blot method to detect ?-collagenI,CTGF,LC3B,NOX4,P62 protein levels in each group;qPCR was used to detect the expression level of MrgD mRNA in primary lung fibroblasts.The expression levels of H₂O₂ and HYP in cells were detected by the kit.The level of autophagy flow was detected by immunofluorescence.2.In vivo experiment,the pulmonary fibrosis model of C57/B mice was established by endotracheal bleomycin(BLM).After 28 days,lung tissue was removed,and lung tissue damage was observed by HE staining,collagenous content of the tissue was analyzed by Masson staining,semi quantitative score of pulmonary fibrosis was performed by Ashcroft score and Masson,immunohistochemistry was used to detect the immune strength of LC3B,NOX4 and P62 in the control group,BLM group,BLM+ALA group and BLM+Pirfenidone group,respectively.3.SPSS22.0 software was used for statistical analysis,and P< was used.0.05 indicates statistical significance.RESULTS1.ALA alleviated collagen deposition in lung fibroblasts.ALA(10^-7mol/L)pretreatment lung fibroblasts,then exposed to Ang ?(10^-7 mol/L)24h.ALA can significantly reduced the protein expression levels of ?-collagen I and CTGF in fibroblasts.2.ALA acted through MrgD receptors.qPCR indicated that MrgD mRNA was expressed in lung fibroblasts.The anti-fibrosis effect of ALA was significantly reversed after the addition of MrgD receptor inhibitor D-Pro7-Ang-(1-7).3.ALA alleviated pulmonary fibrosis by inhibiting oxidative stress.Ang ?+ALA group and Ang +NAC group ?-collagen I,NOX4 protein expression quantity and concentration of H₂O₂ significantly lower than Ang? group and Ang?+H₂O₂ group.4.ALA alleviates pulmonary fibrosis by inducing autophagy.Ang?+ALA group and Ang?+rapamycin group LC3B I transformated to LC3B ?higher than Ang? group,and P62,NOX4 protein expression quantity lower than Ang group.The autophagy induced by ALA was reversed after the addition of bafilomycicn A1,a late autophagy inhibitor.We transfected lung fibroblasts with MRFP-GFP-LC3B adenovirus.and then examined and evaluated by confocal microscopy.It was found that the red fluorescence signal of Ang?+ALA group was significantly enhanced,but there was no obvious green fluorescence,and autophagy was activated,which was the same as that of Ang?+rapamycin group.When bafilomycinA1,the late autophagy inhibitor,was added,we found only yellow fluorescence but no red fluorescence in the combined graph,autophagy was inhibited,and the effect of ALA was eliminated by bafilomycinA1.5.ALA alleviates BLM-induced pulmonary fibrosis in mice.HE staining of pathological sections of lung tissue of mice showed that the lung tissue of mice in the control group was generally normal,and no obvious infiltration of inflammatory cells and deposition of collagen fibers in lung interstitium were observed.But the BLM group,most alveolar walls were destroyed and collapsed,alveolar septum was significantly thickened,inflammatory cells were infiltrated,a large number of fibroblasts were proliferated,and the deposition of collagen fibers in the lung interstitium was significantly increased.The degree of alveolar structure destruction and inflammatory cell infiltration and the deposition of collagen fibers in alveolar septum in BLM+ALA group and BLM+Pirfenidone group were significantly lower than the BLM group.The Ashcroft score of pulmonary fibrosis,Masson score and the contents of HYP in BLM group was higher than the control group,BLM+ALA group and BLM+Pirfenidone group.Masson staining showed that there were a small amount of blue-stained colagen fibers in the lung interstitium of mice in the control group,but a large amount of blue-stained collagen fibers in the lung interstitium of mice in the BLM group,while the blue-stained collagen fibers in the BLM+ALA group and the BLM+ Pirfenidone group were significantly less than those in the BLM group.The expression levels of NOX4 and P62 proteins in BLM+ALA group and BLM+Pirfenidone group were lower than those in BLM group,but the opposite could been seen in LC3B group.The contents of H₂O₂ and HYP in BLM group were higher than those in BLM+ALA group and BLM+Pirfenidone group.CONCLUSION1.In vitro,ALA reduce Ang?-induced lung fibroblasts collagen deposition by inhibiting NOX4 inducing autophagy.2.MrgD receptor was expressed on primary lung fibroblasts of mice.3.In vivo,ALA alleviates BLM-induced pulmonary fibrosis by inhibiting oxidative stress inducing autophagy in mice.