Mechanistic study on low expression of Ace2 gene activated senescence-related signals and promoted the progression of silicotic fibrosis in mice

Objective: To observe the effects of low expressed Angiotensin-converting enzyme 2 (Ace2) gene on senescence related signals of alveolar type Ⅱ epithelial cells in silicotic mice. Methods: In March 2022, 20 8-12W SPF male wild-type C57BL/6 mice and 20 Ace2 gene knockdown mice (Ace2(+/-), C57BL/6 background) were randomly divided into wild-type control group, Ace2 low expression group, wild-type silicosis group, Ace2 low expression silicosis group, with 10 mice in each group. In vitro MLE-12 cells were divided into control group, MLN-4760 (ACE2 inhibitor) group, SiO(2) group and SiO(2)+MLN-4760 group. The expression of ACE2, collagen I (Col I), fibronectin 1 (Fn1), α-smooth muscle actin (α-SMA), phosphorylation-ataxia telangiectasia-mutated serine/threonine kinase (p-ATM), phosphorylation-ATM Rad3-related kinase (p-ATR), p-p53, p21 and p16 in mice and MLE-12 cells were detected by Western blotting. The expression and location of β-galactosidase were detected by immunofluorescence, β-galactosidase (SA-β-Gal) staining were used to detect the senescence of MLE-12 cells. Results: HE and VG staining results showed that typical silicon nodules with collagen deposition were formed in the lung of wild-type silicotic mice and Ace2 low expression silicotic mice. Immunofluorescence staining results showed that β-galactosidase was mainly located in alveolar type Ⅱ epithelial cells. Western blot results showed that, compared with wild-type silicosis group, the expression of Col I, α-SMA, p-ATM, p-ATR, p-p53, p21 and p16 in Ace2 low expression silicosis group were significantly up-regulated by 540.71%、26.58%、336.84%、139.58%、152.78%、120.10% and 994.63% (P<0.05). In MLE-12 cells, results of western blot showed that compared with SiO(2) group, the expression levels of p-ATM, p-ATR, p-p53, p21 and p16 in SiO(2)+MLN-4760 group were significantly up-regulated by 168.71%、750.78%、149.51%、554.26% and 254.07% (P<0.05). Immunofluorescence staining results showed that compared with SiO(2) group, β-galactosidase positive cells were strongly up-regulated in SiO(2)+MLN-4760 group, and SA-β-Gal staining results showed that compared with SiO(2) group, the number of senescent cells in SiO(2)+MLN-4760 group increased by 63.18% (P<0.05) . Conclusion: Low expression of Ace2 gene activated senescence related signals of alveolar type Ⅱ epithelial cells and promoted the progression of silicotic fibrosis in mice.