Forty-eight Sprague-Dawley rats (24 male and 24 female, 200 ± 20 g) 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). N-acetylcysteine (Flumucil, as a positive control in the animal experiments) was purchased from Zambon Pharmaceutical Co., Ltd., (Hainan, China). Luteolin (purity: ≥ 98%, 491–70-3) was provided by Manster Biotechnology Co., Ltd.
COPD model and administration
After being acclimatized for 7 days, 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) for 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) once every 5 days .
From 9 to 16 weeks, the treatment groups (drug-treated COPD model rats) underwent oral gavage with ECC-BYF III (dosage: 6.48 mg/kg, q.d.) or NAC (dosage: 54 mg/kg, q.d.). Meanwhile, normal rats underwent oral gavage with saline once a day. The dosage of ECC-BYF III and NAC were decided and adjusted weekly based on the following formula: Drat = Dhuman × (Krat / Khuman) × (Wrat / Whuman)2/3 (D, dosage; K, body shape index; W, body weight). At week 16, the rats were sacrificed utilizing 2% sodium pentobarbital (40 mg/kg) 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. A FinePointeTM pulmonary function test system (Buxco Inc., USA) was used to measure the forced vital capacity (FVC), forced expiratory volume at 0.3 s (FEV 0.3), airway resistance (RI) and dynamic lung compliance (Cydn).
The left lobe was fixed in 10% paraformaldehyde solution for 72 h. Then, 3 mm of the lobe near- the lung hilum was cut for dehydration and fixed in paraffin to make wax blocks and subsequently cut into 3 μm - sections for hematoxylin–eosin (H&E) staining. Thereafter, the stained tissue sections were taken using optical microscopy and a photographic system (Olympus Optical Co., Ltd., Japan). Six images were selected for each group and six fields were selected for each image randomly. Moreover, the alveolar mean linear intercept (MLI, μm) and the mean alveolar number (MAN, /mm2) [19, 20] were counted using 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 the bronchoalveolar lavage fluid (BALF) were quantified by enzyme-linked immunosorbent assay kits (Boster Bio-Engineering Co., Ltd., Wuhan, China). Meanwhile, the total superoxide dismutase (T-SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-PX) levels in lung tissues or cells were measured with kits (Elabscience, Wuhan, China) according to the manufacturer’s protocol.
The expression of p21 in lungs, especially in the 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 (CST), MA, USA) overnight at 4 °C. 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 (26 °C). The primary antibody LC3B (GTX17380, Gene Tex), TOM20 (11802–1-AP, Proteintech) and the secondary antibody were incubated according to the manufacturer’s instruction. Finally, confocal laser scanning microscopy was used to image and assess the mitophagy.
Preparation of cigarette extract
The mainstream cigarette smoke was sucked into a 50 mL syringe, and then slowly injected into the serum-free Dulbecco’s modified eagle medium (DMEM). The optical density at 320 nm wavelength was measured with a spectrophotometer, and adjusted to 1.8–2.0 with DMEM. Subsequently, the prepared CSE solution was filtered (0.22 μM) to obtain a 100% CSE solution.
Cell culture and treatment
BEAS-2B (ATCC) cells, a human bronchial cell line, were cultured in DMEM (Solarbio, Science & Technology Co., Ltd., Beijing, China) with 10% fetal bovine serum (FBS). Briefly, after being maintained in 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, 17.5, 8.75 μg/mL) for 3 h, and 10%CSE was added subsequently. The cells were collected after 6 h.
SA-β-Gal staining was conducted following the manufacturer’s instruction (G1580, Solarbio, Science & Technology Co., Ltd.). Briefly, BEAS-2B cells cultured in 6-well culture slides were fixed with 1 mL of β-Gal stationary liquids for 15 min at room temperature (26 °C), washed three times with phosphate-buffered saline (PBS), and then incubated with β-Gal staining at 37 °C overnight. The proportion of the stained cells (green staining cells) to total BEAS-2B cells was calculated.
Reactive oxygen species analysis
BEAS-2B cells were suspended with PBS mixed with the DCFH-DA fluorescent probe (E-BC-K138-F, Elabscience) (10 μM) and incubated at 37 °C for 30 min. The cells were washed twice with PBS and then resuspended with 300 μL of PBS. Thereafter, the fluorescent intensity was measured by the FACS CantoTM II (BD Biosciences, San Jose, CA, USA).
BEAS-2B cells were fixed with 2.5% glutaraldehyde (P1126S, Solarbio, Science and Technology Co., Ltd.) overnight at 4 °C. Sample formulation and electron microscope photography were completed by the electron microscope at the Center of Henan University of Traditional Chinese Medicine. Mitochondria and autophagosomes were assessed.
The cells were lysed in a radioimmunoprecipitation assay lysis mixing protease inhibitor and phosphatase inhibitor. Protein concentration was determined using the BCA protein assay kit (PC0020, Solarbio, Science and Technology Co., Ltd.). The protein samples were separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk in TBST at room temperature for 1 h and incubated with the primary antibodies overnight at 4 °C. 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 Washing three times with TBST, the bands were visualized with an enhanced chemiluminescence reagent. The interest protein band intensities were adjusted with β-actin control intensities. Thereafter, grayscale values were analyzed using Image Lab software.
All data were processed by IBM SPSS Statistics for Windows, Version 22.0 (IBM Corp., Armonk, NY, USA) and graphed with GraphPad Prism 10.0. Data were presented as means ± standard deviation. Significant differences were assessed by one-way analysis of variance followed by Tukey’s test where appropriate. Values of P < 0.05 were considered significant.