[Mechanism of angiotensin-converting enzyme 2 overexpression improving collagen synthesis in lung]

Abstract

Objective: To explore the mechanism of angiotensin-converting enzyme 2 (ACE2) overexpression improving collagen synthesis in lung. Methods: Lung fibroblasts of mice over-expressing ACE2 and the wild type (WT) were cultured in vitro and divided into 5 groups: WT-control, WT-AngiotensinⅡ (AngⅡ), ACE2(+ /+) -control, ACE2(+ /+) -AngⅡ and ACE2(+ /+) -AngⅡ+ A779. The protein relative expression levels of ACE2, collagen Ⅰ, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), autophagy-related protein (Beclin1), ubiquitin-binding protein p62 (P62), microtubule-associated proteins light chain 3-Ⅱ (LC3-Ⅱ) were measured by Western blot and triphosadenine (ATP) level was measured by ATP Assay Kit. Fibroblasts over-expressing ACE2 were pretreated with or without the autophagy inhibitor and were separated into 4 groups: ACE2(+ /+) -control, ACE2(+ /+) -AngⅡ, ACE2(+ /+) -AngⅡ+ 3-MA and ACE2(+ /+) -3-MA. In vivo, random allocation was used to averagely divide mice into four groups: WT-control, WT-Bleomycin (BLM), ACE2(+ /+) - control, ACE2(+ /+) -BLM. Wild type and ACE2 over-expressing mice were instilled with bleomycin endotracheally (3.5 mg/kg) or the same volume saline. All mice were sacrificed after 28 days and the lung tissue were used for HE and Masson staining as well as immunohistochemical staining for NOX4, P62 and LC3. Results: The vimentin in lung fibroblasts isolated from mice was proved to be positive by both immunohistochemical and immunofluorescence. The ACE2 protein level of lung fibroblasts over-expressing ACE2 was higher than the wild type (0.202±0.062 and 0.067±0.040, P<0.05). The protein levels of collagenⅠ, NOX4 and NLRP3 in WT-AngⅡ group were obviously higher than the WT-control group (0.861±0.129 and 0.417±0.076, 0.432±0.036 and 0.318±0.058, 0.367±0.125 and 0.045±0.012, all P<0.05). The difference of collagenⅠand NLRP3 between ACE2(+ /+) -AngⅡ group and ACE2(+ /+) -control group had no statistical significance (all P>0.05). CollagenⅠand NOX4 protein level in ACE2(+ /+) -AngⅡ+ A779 group were observably higher than ACE2(+ /+) - AngⅡ group (0.707±0.155 and 0.458±0.108, 0.299±0.038 and 0.149±0.090, all P<0.05). The autophagy related protein levels of Beclin1, P62 and LC3-Ⅱ in ACE2(+ /+) -control group were distinctly higher than WT-control group (0.834±0.051 and 0.274±0.018, 0.467±0.078 and 0.093±0.025, 0.494±0.065 and 0.150±0.054, all P<0.05). However, these protein levels in ACE2(+ /+) -AngⅡ+ A779 group were lower than ACE2(+ /+) -AngⅡ group (1.331±0.203 and 1.565±0.069, 0.298±0.096 and 0.438±0.077, 0.464±0.093 and 0.768±0.071, all P<0.05). ACE2(+ /+) -AngⅡ+ 3-MA group had higher collagenⅠ (0.383±0.125 and 0.032±0.013, P<0.05) and lower LC3-Ⅱ protein level (1.177±0.140 and 1.387±0.183, P<0.05) than AngⅡ group. In bleomycin induced lung fibrosis in mice, ACE2(+ /+) -BLM mice exhibited milder lung fibrosis and lower NOX4 protein level but higher LC3-Ⅱprotein level compared with WT-BLM mice. Conclusion: ACE2 over-expression ameliorated collagen synthesis through enhancing autophagy in lung.

Keywords: Autophagy; Collagen; Oxidative stress; Peptidyl-dipeptidase A; Pulmonary fibrosis.