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Mitochondrial coenzyme Q attenuates lipopolysaccharide-induced mitochondria-dependent apoptosis in type Ⅱ alveolar epithelial cells via phosphatidylinositol 3-kinase/Akt pathway / 中华危重病急救医学
Chinese Critical Care Medicine ; (12): 378-382, 2022.
Artículo en Chino | WPRIM | ID: wpr-955975
ABSTRACT

Objective:

To investigate the protective effect and potential mechanism of mitochondrial coenzyme Q (MitoQ) on mitochondria-dependent apoptosis in type Ⅱ alveolar epithelial cells induced by lipopolysaccharide (LPS).

Methods:

The type Ⅱ lung epithelial cell line (A549) were cultured with different concentrations of LPS in vitro, a cell model of acute lung injury (ALI) was reproduced, the optimal concentration of LPS was obtained according to the half maximal inhibitory concentration (IC 50). The cells were pretreated with different concentrations of MitoQ to determine the best intervention concentration of MitoQ. The cells were divided into four groups the cells in blank control group were cultured in DMEM; the cells in LPS group were stimulated with 10 mg/L of LPS for 24 hours; the cells in MitoQ+LPS group were pretreated with 1 μmol/L MitoQ for 60 minutes, and then were co-cultured with 10 mg/L of LPS for 24 hours; and the cells in MitoQ+phosphatidylinositol 3-kinase (PI3K) selective inhibitor LY294002+LPS group were pretreated with 1 μmol/L MitoQ and 20 μmol/L LY294002 for 60 minutes, and then were co-cultured with 10 mg/L of LPS for 24 hours. Cell viability was measured using cell counting kit-8 (CCK-8). The cell apoptosis rate was determined by flow cytometry and TdT-mediated dUTP-nick end labeling (TUNEL) method. The protein expression levels of apoptosis protein Bax, anti-apoptotic protein Bcl-2 and PI3K-serine/threonine kinase (Akt) protein PI3K expression and Akt phosphorylation level were detected by Western blotting.

Results:

According to the inhibition rate curve, the IC 50 of LPS on A549 cells was 11.06 mg/L. Therefore, 10 mg/L was selected as the stimulating concentration of LPS. After stimulation with 10 mg/L LPS, the cell viability first increased and then decreased with the increase in MitoQ pretreatment concentration. According to the cell viability curve, 1 μmol/L was selected as the optimum concentration of MitoQ. Compared with LPS group, after pretreated with 1 μmol/L MitoQ, cell mitochondrial dependent apoptosis was significantly attenuated, which was characterized by the apoptosis rate was significantly decreased [flow cytometry (8.73±0.25)% vs. (18.10±0.70)%, TUNEL (12.30±0.82)% vs. (21.43±0.86)%, both P < 0.05], the expression of Bax was significantly down-regulated (Bax/β-actin 0.58±0.03 vs. 1.06±0.10, P < 0.05) and Bcl-2 level was significantly up-regulated (Bcl-2/β-actin 1.03±0.06 vs. 0.53±0.07, P < 0.05), meanwhile the expression of PI3K and Akt phosphorylation level were significantly increased [PI3K protein (PI3K/β-actin) 1.20±0.02 vs. 0.96±0.04, phosphorylated Akt (p-Akt) protein (p-Akt/t-Akt) 1.22±0.08 vs. 0.92±0.04, both P < 0.05]. Pretreatment with LY294002 could inhibit the anti-apoptotic effect of MitoQ on cells, it was characterized by the apoptotic rate was significantly increased as compared with MitoQ+LPS group [flow cytometry (14.50±0.57)% vs. (8.73±0.25)%, TUNEL (16.50±0.53)% vs. (12.30±0.82)%, both P < 0.05], the expression of Bax was significantly up-regulated (Bax/β-actin 0.95±0.03 vs. 0.58±0.03, P < 0.05) and Bcl-2 level was significantly down-regulated (Bcl-2/β-actin 0.62±0.03 vs. 1.03±0.06, P < 0.05), meanwhile the expression of PI3K and Akt phosphorylation level were significantly decreased [PI3K protein (PI3K/β-actin) 0.90±0.05 vs. 1.20±0.02, p-Akt protein (p-Akt/t-Akt) 0.89±0.02 vs. 1.22±0.08, both P < 0.05].

Conclusion:

MitoQ improved LPS induced mitochondria-dependent apoptosis of A549 cells by significantly activating PI3K/Akt signal pathway, which provided a new treatment for LPS induced ALI.

Texto completo: Disponible Índice: WPRIM (Pacífico Occidental) Idioma: Chino Revista: Chinese Critical Care Medicine Año: 2022 Tipo del documento: Artículo

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Texto completo: Disponible Índice: WPRIM (Pacífico Occidental) Idioma: Chino Revista: Chinese Critical Care Medicine Año: 2022 Tipo del documento: Artículo