OBJECTIVE: To investigate the role and underlying mechanism of the protein kinase B (Akt)/sirtuin 3 (SIRT3) signaling pathway in hyperoxia-induced acute lung injury (HALI).
METHODS: 1) In vivo experiments: C57BL/6J mice were randomly assigned to a control group (normoxia) or a HALI group (≥95% oxygen), with six mice in each group. After 24 hours of exposure, lung histopathological changes were evaluated by hematoxylin-eosin (HE) staining, and the lung wet/dry weight (W/D) ratio was calculated. Apoptosis-related proteins in total lung lysates (cleaved caspase-3, Bax, and Bcl-2) and mitophagy-related proteins in mitochondrial fractions [p62, Beclin-1, and microtubule-associated protein light chain 3-II/I (LC3-II/I)] were measured by Western blotting. 2) In vitro experiments: Mouse type II alveolar epithelial cells (AEC II) MLE-12 in the logarithmic growth phase were used. Cells were divided into control group, HOgroup (500 μmol/L HOto mimic the HALI), and HO+3-methyladenine (3-MA) group. After 24 hours, cell viability was assessed by cell counting kit-8 (CCK-8), apoptosis was quantified by flow cytometry, and related proteins were detected by Western blotting. To explore the role of mitophagy in HO-induced apoptosis, cells were pretreated 2 hours before modeling with 10 μmol/L 3-MA, a mitophagy inhibitor. In additional experiments, cells were divided into control, HO, HO+Ly294002, and HO+SC-79 groups; 10 μmol/L Ly294002 (Akt inhibitor) or SC-79 (Akt activator) was added 2 hours before modeling to investigate the involvement of Akt signaling in mitophagy-mediated protection against HO-induced apoptosis. SIRT3 was further knocked down to determine whether it functions downstream of Akt in this protective pathway. 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
RESULTS: 1) In vivo results: Under light microscopy, the control group exhibited clear and regularly arranged alveolar structures, whereas the HALI group showed marked inflammatory cell infiltration, edema, and thickened alveolar walls. The lung W/D ratio was higher in the HALI group than that in the control group (11.5±1.1 vs. 5.4±0.8, P < 0.05). Compared with the control group, the HALI group showed increased expression of pro-apoptotic proteins Bax, cleaved caspase-3 [Bax (Bax/GAPDH): 0.49±0.06 vs. 0.18±0.05, cleaved caspase-3 (cleaved caspase-3/GAPDH): 1.71±0.07 vs. 0.63±0.06, both P<0.05], and decreased expression of anti-apoptotic protein Bcl-2 (Bcl-2/GAPDH: 0.39±0.05 vs. 1.03±0.08, P<0.05). Mitophagy-related markers were also altered, with increased LC3-II/I ratio and Beclin-1 [LC3-II/I ratio: 2.16±0.06 vs. 1.10±0.10, Beclin-1 (Beclin-1/GAPDH): 1.04±0.08 vs. 0.42±0.04, both P<0.05], and decreased p62 (p62/GAPDH: 0.41±0.04 vs. 0.99±0.08, P<0.05), suggesting that apoptosis and mitophagy may be involved in HALI progression. 2) In vitro results: Compared with the control group, HOtreatment reduced cell viability, Bcl-2, and p62 [cell viability: (48.45±6.48)% vs. (96.80±2.27)%, Bcl-2 (Bcl-2/GAPDH): 0.57±0.07 vs. 0.82±0.05, p62 (p62/GAPDH): 0.39±0.06 vs. 1.03±0.07, all P<0.05], while increasing apoptosis rate, LC3-II/I ratio, Beclin-1, Bax, and cleaved caspase-3 [apoptosis rate: (16.12±1.76)% vs. (6.54±0.75)%, LC3-II/I ratio: 2.05±0.12 vs. 0.97±0.07, Beclin-1 (Beclin-1/GAPDH): 0.69±0.05 vs. 0.18±0.03, Bax (Bax/GAPDH): 0.53±0.07 vs. 0.29±0.04, cleaved caspase-3 (cleaved caspase-3/GAPDH): 0.63±0.04 vs. 0.19±0.04, all P<0.05]. Compared with the HOgroup, 3-MA pretreatment further aggravated apoptosis [cell viability: (34.00±5.05)% vs. (48.45±6.48)%, apoptosis rate: (22.21±3.05)% vs. (16.12±1.76)%, cleaved caspase-3 (cleaved caspase-3/GAPDH): 1.03±0.09 vs. 0.63±0.04, Bax (Bax/GAPDH): 0.69±0.07 vs. 0.53±0.07, Bcl-2 (Bcl-2/GAPDH): 0.30±0.04 vs. 0.57±0.07, p62 (p62/GAPDH): 0.64±0.05 vs. 0.39±0.06, Beclin-1 (Beclin-1/GAPDH): 0.34±0.04 vs. 0.69±0.05, LC3-II/I ratio: 1.64±0.05 vs. 2.05±0.12, all P<0.05], indicating that inhibition of mitophagy promotes MLE-12 apoptosis. Similar changes were observed after adding the Akt inhibitor Ly294002, whereas the Akt activator SC-79 produced effects opposite to those of 3-MA, suggesting that Akt activation promotes mitophagy and thereby suppresses apoptosis. Compared with the HO+SC-79 group, SIRT3 knockdown (HO+SC-79+siSIRT3) increased apoptosis rate, cleaved caspase-3, Bax, and p62 [apoptosis rate: (10.30±0.96)% vs. (8.82±0.95)%, cleaved caspase-3 (cleaved caspase-3/GAPDH): 0.69±0.05 vs. 0.44±0.05, Bax (Bax/GAPDH): 0.99±0.06 vs. 0.49±0.04, p62 (p62/GAPDH): 0.59±0.06 vs. 0.38±0.04, all P<0.05], and decreased SIRT3, Bcl-2, and Beclin-1 [SIRT3 (SIRT3/GAPDH): 0.48±0.05 vs. 0.68±0.04, Bcl-2 (Bcl-2/GAPDH): 0.64±0.05 vs. 0.78±0.05, Beclin-1 (Beclin-1/GAPDH): 0.68±0.04 vs. 0.79±0.04, all P<0.05]. These findings indicate that SIRT3 knockdown attenuates SC-79-induced mitophagy and enhances apoptosis, supporting SIRT3 as an important downstream effector of the Akt pathway. 2 2 2 2 2 2 2 2
CONCLUSIONS: Activation of the Akt/SIRT3 signaling pathway promotes mitophagy to inhibit alveolar epithelial cell apoptosis, thereby attenuating HALI.
