The inhibitory mechanism of Cordyceps sinensis on cigarette smoke extract-induced senescence in human bronchial epithelial cells

Abstract

Objectives: Cellular senescence is a state of irreversible growth arrest induced either by telomere shortening (replicative senescence) or stress. The bronchial epithelial cell is often injured by inhaled toxic substances, such as cigarette smoke. In the present study, we investigated whether exposure to cigarette smoke extract (CSE) induces senescence of bronchial epithelial cells; and Cordyceps sinensis mechanism of inhibition of CSE-induced cellular senescence.

Methods: Human bronchial epithelial cells (16HBE cells) cultured in vitro were treated with CSE and/or C. sinensis. p16, p21, and senescence-associated-galactosidase activity were used to detect cellular senescence with immunofluorescence, quantitative polymerase chain reaction, and Western blotting. Reactive oxygen species (ROS), PI3K/AKT/mTOR and their phosphorylated proteins were examined to testify the activation of signaling pathway by ROS fluorescent staining and Western blotting. Then, inhibitors of ROS and PI3K were used to further confirm the function of this pathway.

Results: Cellular senescence was upregulated by CSE treatment, and C. sinensis can decrease CSE-induced cellular senescence. Activation of ROS/PI3K/AKT/mTOR signaling pathway was enhanced by CSE treatment, and decreased when C. sinensis was added. Blocking ROS/PI3K/AKT/mTOR signaling pathway can attenuate CSE-induced cellular senescence.

Conclusion: CSE can induce cellular senescence in human bronchial epithelial cells, and ROS/PI3K/AKT/mTOR signaling pathway may play an important role in this process. C. sinensis can inhibit the CSE-induced senescence.

Keywords: COPD; Cordyceps sinensis; ROS/PI3K/AKT/mTOR signaling pathway; senescence.

Figure 1

16HBE cells were incubated with CSE at different doses and time points. Notes: Cell survival rate was assayed using the MTT assays to evaluate cell viability (*P<0.05, vs control at respective time points). Data are expressed as mean ± SE. Results represent three independent experiments. Abbreviations: CSE, cigarette smoke extract; HBE, human bronchial epithelial; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide; SE, standard error.

Figure 2

Cellular senescence induced by CSE stimulation. Notes: (A) 16HBE cells were stimulated with different doses of CSE for 24 hours, protein expressions of p16 and p21 were detected by Western blotting (*P<0.05, **P>0.05). (B) 16HBE cells were stimulated by 2% CSE for different time durations and protein expressions of p16 and p21 were detected by Western blotting (*P<0.05). Data are expressed as mean ± SE. Results represent at least three independent experiments. Abbreviations: CSE, cigarette smoke extract; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBE, human bronchial epithelial; SE, standard error.

Figure 3

Cellular senescence affected by CSE and Cordyceps sinensis. 16HBE cells were stimulated with 2% CSE and/or C. sinensis (100 mg/L) (2 hours before adding CSE) for 24 hours. Notes: (A) The protein expressions of p16 and p21 were detected by Western blotting (*P<0.05). (B) The senescent cells in CSE and CSE + C. sinensis groups were examined by SA-β-gal staining. SA-β-gal positive cells were enumerated by counting over 400 cells in three independent fields (*P<0.05). (C) The expression of p16 and p21 in CSE and CSE + C. sinensis groups was determined by immunofluorescence cytochemistry. (D) The expression of p16 and p21 in CSE and CSE + C. sinensis groups was detected by qPCR (*P<0.05). Data are expressed as mean ± SE. Results represent at least three independent experiments. Abbreviations: CS, Cordyceps sinensis; CSE, cigarette smoke extract; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBE, human bronchial epithelial; mRNA, messenger RNA; qPCR, quantitative polymerase chain reaction; SA-β-gal, senescence-associated β-galactosidase; SE, standard error.

Figure 4

ROS fluorescence in 16HBE cells. Notes: 16HBE cells were stimulated with 2% CSE and/or Cordyceps sinensis (100 mg/L) for 24 hours, and stained with ROS fluorescence. Results represent three independent experiments. Abbreviations: CSE, cigarette smoke extract; DAPI, 4′,6-diamidino-2-phenylindole; HBE, human bronchial epithelial; ROS, reactive oxygen species.

Figure 5

Expressions of PI3K, AKT, p-AKT, mTOR, p-mTOR in 16HBE cells. Notes: (A) 16HBE cells were stimulated with different doses of CSE for 24 hours, cells were collected and total proteins were extracted and analyzed by Western blotting (*P<0.05). (B) Then 16HBE cells were stimulated with 2% CSE for different time durations, total proteins were extracted and analyzed (*P<0.05). (C) 16HBE cells were stimulated with 2% CSE or/and Cordyceps sinensis (100 mg/L) for 24 hours, total proteins were extracted and analyzed by Western blotting (*P<0.05, **P>0.05). Data are expressed as mean ± SE. Results represent three independent experiments. Abbreviations: CS, Cordyceps sinensis; CSE, cigarette smoke extract; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBE, human bronchial epithelial; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide-3-kinase; SE, standard error.

Figure 6

The effect of mTOR pathway activation and cellular senescence by blocking ROS. 16HBE cells were stimulated with 2% CSE, 100 mg/L Cordyceps sinensis or/and 10 mM NAC. Notes: (A) 16HBE cells were stained with ROS fluorescence. (B) Total protein was collected to detect the expressions of AKT, p-AKT, mTOR, and p-mTOR by Western blotting (*P<0.05). (C) The protein expressions of p16 and p21 were detected by Western blotting (*P<0.05). (D) The senescent cells were examined by SA-β-gal staining (*P<0.05). Data are expressed as mean ± SE. Results represent at least three independent experiments. Abbreviations: CS, Cordyceps sinensis; CSE, cigarette smoke extract; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBE, human bronchial epithelial; mTOR, mammalian target of rapamycin; NAC, N-acetylcysteine; SA-β-gal, senescence-associated β-galactosidase; ROS, reactive oxygen species; SE, standard error.

Figure 7

The effect of mTOR pathway activation and cellular senescence by blocking PI3K. 16HBE cells were stimulated with 2% CSE, 100 mg/L Cordyceps sinensis or/and 10 μM Ly294002. Notes: (A) 16HBE cells were stained with ROS fluorescence. (B) Total protein was collected to detect the expressions of AKT, p-AKT, mTOR, and p-mTOR by Western blotting (*P<0.05, **P>0.05). (C) The protein expressions of p16 and p21 were detected by Western blotting (*P<0.05). (D) The senescent cells were examined by SA-β-gal staining (*P<0.05). Data are expressed as mean ± SE. Results represent at least three independent experiments. Abbreviations: CS, Cordyceps sinensis; CSE, cigarette smoke extract; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBE, human bronchial epithelial; mTOR, mammalian target of rapamycin; ROS, reactive oxygen species; SE, standard error.