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Outline
To elucidate the regulatory role and molecular mechanism by which silent information regulator 3 (SIRT3) modulates mitochondrial biogenesis in murine alveolar type II epithelial (MLE-12) cells under oxidative stress.
Oxidative stress was induced in MLE-12 cells by exposure to H2O2. MLE-12 cells were transfected with siRNA specifically targeting Sirt3 to achieve knockdown. Thus, the experiment included 6 groups (n=3): normal control group (NC), scrambled negative control siRNA group (NCsiRNA group), Sirt3 siRNA group (siSIRT3 group), H2O2 injury group (NC+H2O2 group), scrambled negative control siRNA+H2O2 injury group (NCsiRNA+H2O2 group), and Sirt3 siRNA+H2O2 injury group (siSIRT3+H2O2 group). Resveratrol was used to stimulate MLE-12 cells to increase SIRT3 protein expression. Another 10 experiment groups (n=3) were also included, that is, normal control group (NC), scrambled negative control siRNA group (NCsiRNA group), DMSO group (DMSO group), Sirt3 siRNA group (siSIRT3 group), resveratrol-treated group (RSV group), H2O2 injury group (NC+H2O2 group), scrambled negative control siRNA+H₂O₂ injury group (NCsiRNA+H2O2 group), DMSO+H₂O₂ injury group (DMSO+H₂O₂ group), Sirt3 siRNA+H2O2 injury group (siSIRT3+H2O2 group), and resveratrol-treated+H2O2 injury group (RSV+H2O2 group). Mitochondrial superoxide (O2•⁻) were detected using MitoSOX Red mitochondrial superoxide indicator, and ATP production was measured by using luciferase-based luminescence assay. The protein levels of SIRT3, AMPKα1, PGC-1α, TFAM, Nrf1, SOD2 and Acetyl-SOD2 were detected with Western blotting.
Exposure to 500 μmol/L H₂O₂ significantly reduced SIRT3 protein level in MLE-12 cells (P<0.000 1). Following H2O2 treatment, all H2O2-treated groups exhibited a further reduction in ATP level and a more pronounced increase in fluorescence intensity (P<0.000 1). Among these, the siSIRT3+H2O2 group demonstrated the most significant decrease in ATP level (P<0.01) and the greatest enhancement in fluorescence intensity (P<0.000 1) relative to the NCsiRNA+H₂O₂ group. Western blot analysis revealed that Sirt3 knockdown resulted in reduced protein levels of AMPKα1, Nrf1, PGC-1α and SIRT3 (P<0.05), while the protein levels of SOD2, acetylated SOD2 (Acetyl-SOD2), and TFAM were increased (P<0.01) when compared to the NCsiRNA group. Resveratrol treatment increased the SIRT3 protein level in MLE-12 cells (P<0.05), subsequently leading to increased protein level of PGC-1α (P<0.05). In H2O2 treated groups, the protein expression levels of AMPKα1, Nrf1, PGC-1α and SIRT3 were reduced compared to their corresponding control groups (P<0.05). The NC+H2O2 and NCsiRNA+H2O2 groups showed increased protein levels of SOD2, Acetyl-SOD2, and TFAM when compared to their corresponding control groups (P<0.05). Compared to the NCsiRNA+H2O2 group, the siSIRT3+H2O2 group exhibited notably reduced protein levels of AMPKα1, Nrf1, PGC-1α, TFAM, SOD2, Acetyl-SOD2 and SIRT3 (P<0.05). Conversely, the RSV+H2O2 group demonstrated significantly higher PGC-1α and SIRT3 protein levels than both the NC+H2O2 and siSIRT3+H2O2 groups (P<0.05).
SIRT3 enhances mitochondrial biogenesis via the AMPKα1/PGC-1α/Nrf1/TFAM signaling pathway, improves mitochondrial function of lung epithelial cells and alleviates lung injury. Regulation of SIRT3 protein may reduce oxidative damage of type II alveolar cells, suggesting a potential therapeutic target for acute lung injury.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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