Sirtuin3 confers protection against acute pulmonary embolism through anti-inflammation, and anti-oxidative stress, and anti-apoptosis properties: participation of the AMP-activated protein kinase/mammalian target of rapamycin pathway

Abstract

An increasing number of studies have suggested that oxidative stress and inflammation play momentous roles in acute pulmonary embolism (APE). Honokiol, a bioactive biphenolic phytochemical substance, is known for its strong anti-oxidative and anti-inflammatory effects, and it served as an activator of sirtuin3 (SIRT3) in the present study. The purposes of the study were to explore the effects of honokiol on APE rats and investigate whether the function of honokiol is mediated by SIRT3 activation. In the study, the rats received a right femoral vein injection of dextran gel G-50 particles (12 mg/kg) to establish the APE model and were subsequently administered honokiol and/or a selective SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl)pyridine (3-TYP; 5 mg/kg) intraperitoneally. The results showed that SIRT3 activation by honokiol attenuated the loss in lung function, ameliorated the inflammatory response and oxidative damage, and inhibited apoptosis in lung tissues of the rats with APE but that this was reversed by 3-TYP. In addition, we found that the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway might be activated by honokiol but restrained by 3-TYP. These results indicated that honokiol was capable of suppressing the adverse effects of APE and that this was diminished by SIRT3 suppression, implying that activation of SIRT3 might serve as a therapeutic method for APE.

Keywords: acute pulmonary embolism (APE); apoptosis; inflammation; oxidative stress; sirtuin3 (SIRT3).

Fig. 1.

Sirtuin3 (SIRT3) expression in the lung tissues of rats with acute pulmonary embolism (APE). (A) The levels of SIRT3 were assessed by real-time quantitative PCR and (B) immunohistochemistry. (C) Immunofluorescence (IF) staining showed the expression of SIRT3 (red) in the lung tissues of APE rats after honokiol and/or 3-TYP treatment. Scale bar=50 µm. Data are presented as the mean ± SD (n=6 in each group). ***P<0.001 versus the control group.

Fig. 2.

Effect of honokiol or 3-TYP on pulmonary function and injury in the rats with acute pulmonary embolism (APE). (A) Arterial partial pressure of oxygen (PaO₂). (B) Fraction of inspiration O₂ (FiO₂). (C) Oxygenation index (PaO₂/FiO₂). (D) Arterial partial pressure of carbon dioxide (PaCO₂). (E) Wet/dry weight ratio (W/D). (F, G) Hematoxylin-eosin (HE) staining was used to observe the pathological changes at 40× (Scale bar=500 µm) and 200× magnification (Scale bar=100 µm) and determine the inflammation grade. Data are presented as the mean ± SD (n=6 in each group). ***P<0.001 versus the control group. **P<0.01 versus the control group. ^###P<0.001 versus the APE group. ^##P<0.01 versus the APE group.

Fig. 3.

Effect of honokiol or 3-TYP treatment on inflammation in the acute pulmonary embolism (APE) rats. (A) Total number of inflammatory cells. (B) Eosinophils. (C) Neutrophils. (D) Lymphocytes. (E) Monocyte macrophages. (F) Measurement of the inflammation-linked indices tumor necrosis factor-α (TNF-α), (G) IL-6, and (H) myeloperoxidase (MPO). Scale bar=50 µm. Data are presented as the mean ± SD (n=6 in each group). ***P<0.001 versus the control group. *P<0.05 versus the control group. ^###P<0.001 versus the APE group. ^##P<0.01 versus the APE group.

Fig. 4.

Effect of honokiol or 3-TYP on oxidative stress and apoptosis in the acute pulmonary embolism (APE) rats. (A) Reactive oxygen species (ROS). (B) Measurement of the oxidative stress-linked indices malondialdehyde (MDA), (C) lactic dehydrogenase (LDH), and (D) superoxide dismutase (SOD). (E) Apoptotic cells were detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) (red), and nuclei were detected by 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (blue). (F-I) Apoptosis-linked indices (cleaved caspase-3, B-cell lymphoma 2 (Bcl-2)-associated X (Bax), Bcl2) were assessed by western blot. Scale bar=50 µm. Data are presented as the mean ± SD (n=6 in each group). ***P<0.001 versus the control group. **P<0.01 versus the control group. *P<0.05 versus the control group. ^###P<0.001 versus the APE group. ^##P<0.01 versus the APE group.

Fig. 5.

Effect of honokiol or 3-TYP on AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in the rats with acute pulmonary embolism (APE). (A, B) The levels of p-AMPK/AMPK were determined by western blot. (C, D) The levels of p-mTOR/mTOR were analyzed by western blot. Data are presented as the mean ± SD (n=6 in each group). ***P<0.001 versus the control group. ^###P<0.001 versus the APE group.

Fig. 6.

Graphical abstract.