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

Figures & data

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.