48 Sprague-Dawley rats (half male and half female, 200 ± 20g) were purchased from the Laboratory Animal Center of Henan Province (Zhengzhou, China).
The components of ECC-BYF Ⅲ were purchased from Manster biotechnology co., LTD (Chengdu, China). NAC (Flumucil, as positive control in animal experiment) was purchased from Zambon Pharmaceutical Co., LTD, (Hainan, China). Luteolin (purity: ≥ 98%, 491-70-3) was provided by Manster biotechnology co., LTD, (Chengdu, China).
COPD model and administration
After acclimatized for 7 d, rats were randomized into the normal, model, ECC-BYF Ⅲ and NAC groups (half male and half female in each group). The COPD model was established with tobacco smoke exposure and bacterial infection from week 1 to week 8. Briefly, the model rats were exposed to tobacco smoke (smoke concentrations, 3000±500 ppm, 1.0 mg of nicotine, 11 mg CO and 10 mg of tar per cigarette; Hongqiqu® filter cigarettes, Zhengzhou, China) 40 min, twice a day, and Klebsiella pneumoniae (0.1 ml, 6×108 CFU/mL; Bacterial strain: 46114; National Center for Medical Culture Collection, Beijing, China) for once every 5 days.
From 9 to 16 weeks, the rats of treatment groups were orally gavaged with ECC-BYF Ⅲ (dosage: 6.48 mg/kg, q.d) or NAC (dosage: 54 mg/kg, q.d). Meanwhile, normal rats were orally gavaged with saline once a day. The dosage of ECC-BYF Ⅲ and NAC were decided and adjusted weekly by the following formula: D rat = D human × (K rat / K human) × (W rat / W human)2/3 (D: dosage; K: body shape index; W: body weight). At week 16, the rats were sacrificed and samples were obtained.
Pulmonary function analysis
Pulmonary function was measured in unrestrained rats with Whole Body Plethysmograph (WBP) system (Buxco Inc., Wilmington, NC, USA) every 4 weeks. The relevant continuous ventilatory parameters, including Tidal volume (VT), minute volume (MV), peak expiratory flow (PEF), and expiratory flow at 50% tidal volume (EF50) were calculated meanwhile. A FinePointeTM pulmonary function test system (Buxco Inc, USA) was used to measure the parameters of forced vital capacity (FVC) and forced expiratory volume at 0.3s (FEV 0.3).
The lung tissues were sliced into 3-4 mm sections, fixed in 10% paraformaldehyde solution for 72 h, and then mounted in paraffin-embedded 3 micron sections. The sections were stained with hematoxylin-eosin (H&E) staining. Whereafter, the stained tissue sections were taken utilizing optical microscopy and a photographic system (Olympus Optical Co., Ltd., Japan), and six images were taken for each section. And the alveolar mean linear intercept (MLI, µm) and the mean alveolar number (MAN, /mm2) were counted with the counting tool of Adobe Photoshop CC software to evaluate the degree of emphysema.
The concentration of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and matrix metalloproteinase (MMP)-9 in bronchoalveolar lavage fluid (BALF) were quantified by enzyme-linked immunosorbent assay (ELISA) kits (Boster Bio-Engineering Co., Ltd., Wuhan, China). Meanwhile, total superoxide dismutase (T-SOD), malondialdehyde (MDA)] and glutathione peroxidase (GSH-PX) in lung tissue or cells were measured with kits (Elabscience, Wuhan, China) according to the manufacturer’s protocol.
The expression of p21 in lung, especially in bronchus was detected by immunofluorescence. After dewaxing and dehydration treatment, the lung tissue slices were added with a 5% BSA for 30 min, and then anti-p21 antibody was incubated (1:1000 dilution; Cell signaling technology) overnight at 4 ℃. On the following day, slices were incubated with Cy3-conjugated affinipure goat Anti-Rabbit IgG (H+L) (proteintech, Wuhan, China) for 50 min. At least six images were randomly taken for each section and the red staining area was calculated by CaseViewer software.
Moreover, BEAS-2B cells cultured in 24-well culture slides were fixed with 4% paraformaldehyde for 15 min and blocked with sheep serum protein mixed with 0.3% TritonX-100 for 2 h at room temperature. The primary antibody LC3B (GTX17380, Gene Tex), TOM20 (11802-1-AP, proteintech) and the secondary antibody were incubated according to the manufacturer’ instruction. Finally, confocal laser scanning microscopy was used to picture and assess the mitophagy.
Preparation of cigarette extract
The mainstream cigarette smoke was sucked into 50 ml syringe, and then slowly injected into the serum-free DMEM medium. The optical density at 320 nm wavelength was measured with spectrophotometer, and adjusted to 1.8-2.0 with DMEM medium. Subsequently, the prepared CSE solution was filtered through a filter (0.22 µM) to obtain 100% CSE solution.
Cell culture and treatment
The BEAS-2B (ATCC), a human bronchial cell line, were cultured in DMEM culture medium (Solarbio, Beijing, China) with 10% fetal bovine serum (FBS). In short, after being maintained with FBS-free medium for 3 h, the cells were pre-incubated with Nrf2 inhibitor for 2 h, and then incubated with ECC-BYF Ⅲ at different concentrations (35 µg/ml, 17.5 µg/ml, 8.75 µg/ml) for 3 h, 10% cigarette smoke extract (CSE) was added subsequently. The cells were collected after 6 h.
The experiment of SA-β-Gal staining was carried out following the manufacturer’s instruction (G1580, Solarbio). The proportion of stained cells to total BEAS-2B cells was calculated.
BEAS-2B cells were fixed with 2.5% glutaraldehyde (P1126S, Solarbio, Beijing, China) overnight at 4 ℃. Samples formulation and electron microscope photography were completed by the electron microscope Center of Henan University of Traditional Chinese Medicine. Mitochondria and autophagosomes were assessed.
The cells were lysed in a RIPA lysis mixing protease inhibitor and phosphatase inhibitor. The concentration of protein was determined by BCA protein assay kit (PC0020, Solarbio, Beijing, China). The protein samples were separated in 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk in TBST at room temperature for 1h and incubated with the primary antibodies overnight at 4 ℃. The specific primary antibodies were β-actin (1:1000 diluted; proteintech), p21 (1:1000 diluted; CST), p16(1:1000 diluted; proteintech), PINK1 (1:1000 diluted; proteintech), PARK2 (1:1000 diluted; proteintech), Mfn2 (1:1000 diluted; CST), Drp1 (1:1000 diluted; CST), Nrf2 (1:1000 diluted; Gene Tex), HO-1 (1:1000 diluted; CST). Subsequently, the membranes were incubated with secondary antibodies of HRP-conjugated goat anti-mouse and anti-rabbit (1:5000 diluted, proteintech) for 1 h at room temperature. After Washed three times with TBST, the bands were visualized with enhanced chemiluminescence ECL reagent. The interest protein bands intensities were adjusted with β-actin control intensities.
All data were processed by SPSS 22.0 software and graphed with Graphpad prism 10.0. Data were presented as the means ± standard deviation. Statistically significant differences were assessed by one-way ANOVA followed by the Tukey's test where appropriate. Values of P < 0.05 were considered statistically significant.