Effective-component compatibility of Bufei Yishen formula protects COPD rats against PM2.5-induced oxidative stress via miR-155/FOXO3a pathway.

PM2.5-aggravated COPD Sprague-Dawley rat

32 male SD rats, 6-9 weeks old, randomized into four groups (n = 8): Control, Model (PM2.5-aggravated COPD), ECC-BYF, NAC. COPD induced over 8 weeks by twice-daily cigarette smoke inhalation (30 min each) plus intranasal Klebsiella pneumoniae every five days, then 8 weeks of whole-body concentrated PM2.5 exposure.

Pulmonary function measured every fourth week to week 16 (noninvasive) and at week 17 (invasive)

PM2.5-aggravated COPD Sprague-Dawley rat - lung tissue

32 male SD rats, 6-9 weeks old, randomized into four groups (n = 8): Control, Model (PM2.5-aggravated COPD), ECC-BYF, NAC. COPD induced over 8 weeks by twice-daily cigarette smoke inhalation (30 min each) plus intranasal Klebsiella pneumoniae every five days, then 8 weeks of whole-body concentrated PM2.5 exposure.

PM2.5-aggravated COPD Sprague-Dawley rat - serum

32 male SD rats, 6-9 weeks old, randomized into four groups (n = 8): Control, Model (PM2.5-aggravated COPD), ECC-BYF, NAC. COPD induced over 8 weeks by twice-daily cigarette smoke inhalation (30 min each) plus intranasal Klebsiella pneumoniae every five days, then 8 weeks of whole-body concentrated PM2.5 exposure.

PM2.5-aggravated COPD Sprague-Dawley rat - BALF

32 male SD rats, 6-9 weeks old, randomized into four groups (n = 8): Control, Model (PM2.5-aggravated COPD), ECC-BYF, NAC. COPD induced over 8 weeks by twice-daily cigarette smoke inhalation (30 min each) plus intranasal Klebsiella pneumoniae every five days, then 8 weeks of whole-body concentrated PM2.5 exposure.

Beas-2B human bronchial epithelial cell line

Primary mouse tracheal epithelial cells at air-liquid interface

Pulmonary function in PM2.5-aggravated COPD rats with ECC-BYF or NAC

Noninvasive (VT, PEF, EF50) and invasive (FVC, FEV0.3, FEV0.3/FVC) pulmonary function in four rat groups. VT, PEF and EF50 decreased continuously from week 1 to week 16 in the model group; all six parameters were significantly lower in the model group than in the healthy control at week 17 (P < 0.01), indicating irreversible airflow limitation. ECC-BYF and NAC administration significantly increased all six parameters relative to the model group (P < 0.01). n = 8 per group. Values are reported only in Fig. 1b-g, so no numeric measurements are recorded here.

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Evidence

Goblet cell hyperplasia in rat airway epithelium (H&E)

Qualitative histology. H&E-stained lung sections from model rats showed interalveolar septa thickening, numerous inflammatory cell infiltration, narrowed airway lumen and goblet cell hyperplasia in the airway epithelium; ECC-BYF- and NAC-treated rats appeared more or less similar to control. Goblet cells were identified by arrow annotation in Fig. 2b and were not counted, so no goblet cell percentage or count is recorded.

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Evidence

T-SOD and MDA in serum of PM2.5-aggravated COPD rats

Total superoxide dismutase (T-SOD, antioxidant) and malondialdehyde (MDA, oxidant) measured in rat serum by kit assay. T-SOD significantly decreased and MDA increased in the model group versus control (P < 0.01), indicating an oxidant/antioxidant imbalance. ECC-BYF and NAC reversed the imbalance; serum T-SOD was significantly higher and MDA significantly lower in ECC-BYF-treated than in NAC-treated rats (P < 0.01). Values reported only in Fig. 3a-b; none recorded.

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Evidence

T-SOD and MDA in bronchoalveolar lavage fluid of PM2.5-aggravated COPD rats

T-SOD and MDA measured in rat BALF by kit assay. The same oxidant/antioxidant imbalance seen in serum was present in BALF: T-SOD decreased and MDA increased in the model group versus control, and ECC-BYF and NAC reversed the imbalance. Values reported only in Fig. 3a-b; none recorded.

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Evidence

ROS fluorescence in PM2.5-exposed Beas-2B cells

Intracellular ROS imaged with DCFH-DA in Beas-2B pretreated for 24 h with 16 ug/mL ECC-BYF, 500 uM NAC or DMSO and then exposed to 100 ug/mL PM2.5 for 24 h. ECC-BYF-pretreated cells showed a significant reduction of ROS fluorescence compared with the PM2.5-exposed model cells. Fluorescence intensity is shown only as images in Fig. 4b; no quantitative value is recorded.

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Evidence

MDA, T-SOD and GSH-Px in PM2.5-exposed Beas-2B across an ECC-BYF dose series

Beas-2B pretreated for 24 h with 2, 4, 8 or 16 ug/mL ECC-BYF, 500 uM NAC or DMSO, then exposed to 100 ug/mL PM2.5 for 24 h. A dose-dependent increase in T-SOD and GSH-Px and a dose-dependent decrease in MDA were observed with ECC-BYF pretreatment compared with the PM2.5 model group. Cell viability (CCK8) changed only slightly, so 2-16 ug/mL ECC-BYF is non-toxic to Beas-2B. Values reported only in Fig. 4c-e; none recorded.

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Evidence

ROS fluorescence in PM2.5-exposed primary mouse tracheal epithelial cells

MTECs isolated from the tracheas of C57BL/6 mice exposed for 8 weeks either to filtered air or to cigarette smoke plus intranasal Klebsiella pneumoniae (COPD model; 16 mice, n = 8 per group), cultured at air-liquid interface, then pretreated with ECC-BYF and exposed to PM2.5. Intracellular ROS increased in MTECs from both normal and COPD mice after PM2.5 exposure, and ECC-BYF pretreatment decreased ROS relative to the PM2.5-exposed cells. Images only in Fig. 4f; no value recorded.

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Evidence

Nrf2 and HO-1 protein in rat lung tissue (Western blot)

Western blot of the antioxidant-response regulators Nrf2 and HO-1 in lung tissue from the four rat groups. Both were increased in the ECC-BYF group compared with the model group, further validating the antioxidant effect of ECC-BYF. Blots only in Fig. 3c; no densitometric value is reported in the text, so no value is recorded.

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Evidence

FOXO3a, SOD2 and CAT expression in rat lung tissue

Both protein (Western blot, Fig. 5c) and mRNA (qPCR, Fig. 5d) expression of FOXO3a, together with its downstream antioxidants SOD2 and CAT, were markedly decreased in lung tissue from the PM2.5-aggravated COPD model group, and were up-regulated by ECC-BYF or NAC administration. Values reported only in figures; none recorded.

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Evidence

FOXO3a protein and mRNA in PM2.5-exposed Beas-2B across an ECC-BYF dose series

Beas-2B pretreated for 24 h with 2, 4, 8, 16 or 32 ug/mL ECC-BYF, then exposed to 100 ug/mL PM2.5 for 24 h. Western blot (Fig. 5a) showed ECC-BYF significantly elevated FOXO3a protein in a dose-dependent fashion, and qPCR (Fig. 5b) confirmed up-regulated FOXO3a mRNA. Values reported only in figures; none recorded.

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Evidence

miR-155-5p in Beas-2B across a PM2.5 dose series

qPCR of miR-155-5p (normalized to U6) in Beas-2B treated with 10, 25, 50 or 100 ug/mL PM2.5 suspension for 24 h. A significant increase of miR-155 was observed after PM2.5 stimulation (Fig. 6b). Values reported only in the figure; none recorded.

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Evidence

miR-155-5p in PM2.5-exposed Beas-2B across an ECC-BYF dose series

qPCR of miR-155-5p in Beas-2B pretreated for 24 h with 2, 4, 8 or 16 ug/mL ECC-BYF and then exposed to 100 ug/mL PM2.5 for 24 h. A dose-dependent decrease of miR-155 was found in response to ECC-BYF treatment (Fig. 6c). Values reported only in the figure; none recorded.

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Evidence

FOXO3a protein in miR-155-overexpressing Beas-2B with or without ECC-BYF

Beas-2B transfected with miR-155 mimic (50 nM) or negative control, pretreated with 16 ug/mL ECC-BYF for 24 h where indicated, and exposed to 100 ug/mL PM2.5 for 24 h. Compared with ECC-BYF-treated cells, miR-155 mimic-pretreated cells showed a significant decrease of FOXO3a protein (Fig. 6d). Values reported only in the figure; none recorded.

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Evidence

SOD2 and CAT mRNA in miR-155-mimic Beas-2B with or without FOXO3a overexpression

qPCR of SOD2 and CAT mRNA in miR-155-mimic-transfected Beas-2B with or without co-transfected pcDNA-FOXO3a (14 ug), pretreated with 16 ug/mL ECC-BYF for 24 h and exposed to 100 ug/mL PM2.5 for 24 h. miR-155 mimic reduced SOD2 and CAT mRNA; FOXO3a plasmid co-transfected cells showed a significant increase in SOD2 and CAT mRNA compared with the miR-155-mimic cells (Fig. 6e). Values reported only in the figure; none recorded.

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Evidence

Increased miR-155 expression leads to decreased FOXO3a expression

miR-155 binds the 3'-UTR of FOXO3a mRNA and suppresses it. Transfecting a miR-155 mimic into Beas-2B significantly decreased FOXO3a protein, and ECC-BYF raised FOXO3a while lowering miR-155 dose-dependently.

Decreased FOXO3a expression leads to increased oxidative stress

FOXO3a drives transcription of the antioxidant enzymes SOD2 and CAT. Lowering FOXO3a with a miR-155 mimic reduced SOD2 and CAT and abolished the antioxidant protection of ECC-BYF, while FOXO3a overexpression partially restored it.

Increased oxidative stress leads to decreased lung function

In PM2.5-aggravated COPD rats the oxidant/antioxidant imbalance in serum and BALF accompanied reduced lung function and lung tissue remodeling, and treatments that suppressed oxidative stress (ECC-BYF, NAC) also improved lung function.

Noninvasive and invasive pulmonary function testing in rats

Unrestrained lung function (tidal volume VT, peak expiratory flow PEF, expiratory flow at 50% tidal volume EF50) measured by whole-body plethysmography every fourth week from week 1 to week 16. At week 17 rats were anesthetized with 3% pentobarbital sodium (35 mg/kg, ip) and FVC, FEV0.3 and FEV0.3/FVC were measured on an invasive pulmonary function test system. n = 8 per group.

Equipment: Whole-body plethysmograph; Invasive pulmonary function test (PFT) system

Hematoxylin-eosin staining and histomorphometry of rat lung

Lung tissues collected at week 17 and stained with hematoxylin-eosin. Light microscopy scored interalveolar septa thickening, inflammatory cell infiltration, airway lumen narrowing and goblet cell hyperplasia in the airway epithelium. Histomorphometry reported mean linear intercept (MLI), mean alveolar number (MAN) and a total lung injury score. n = 6-8 per group.

Equipment: Microtome; Light microscope

Colorimetric kit assays for MDA, T-SOD and GSH-Px

Malondialdehyde (MDA), total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) determined in rat serum and bronchoalveolar lavage fluid, and in Beas-2B lysates, with matched commercial kits according to the manufacturer's protocol (Nanjing Jiancheng Co., Jiangsu, China).

Equipment: Microplate reader; MDA / T-SOD / GSH-Px assay kits (Nanjing Jiancheng Co.)

DCFH-DA immunofluorescence detection of intracellular ROS

Intracellular reactive oxygen species detected with a 2',7'-dichlorofluorescein-diacetate (DCFH-DA) probe in Beas-2B cells (scale bar 100 um) and in primary mouse tracheal epithelial cells (scale bar 200 um); results reported as ROS fluorescence intensity.

Equipment: Fluorescence microscope; DCFH-DA probe

Quantitative real-time PCR for FOXO3a, SOD2, CAT and miR-155-5p

Total RNA isolated with TRIZOL from lung tissues or cultured cells, reverse transcribed with a high capacity cDNA reverse transcription kit, and quantified on a 7500 real-time PCR system with SYBR Green Master Mix. Relative expression analysed by the 2^-ddCT method. miR-155-5p normalized to U6 snRNA; FOXO3a, SOD2 and CAT mRNA normalized to GAPDH.

Western blotting for Nrf2, HO-1, FOXO3a and SOD2

Protein extraction, SDS-PAGE and probing with Nrf2 (Proteintech 16396-1-AP), HO-1 (GeneTex GTX101147), FOXO3a (GeneTex GTX100277) and SOD2 (GeneTex GTX116093) antibodies, all at 1:1000. GAPDH used as internal control.

Equipment: SDS-PAGE apparatus; Chemiluminescence imaging system

Lipofectamine transfection of miR-155 mimic and pcDNA-FOXO3a

Beas-2B cells seeded in six-well plates at 60-70% confluence in serum-free medium and transfected with pcDNA-FOXO3a plasmid (14 ug), miR-155 mimic (50 nM) or the matched negative controls (Guangzhou RiboBio) complexed with Lipofectamine 2000. After 24 h the cells were used for ECC-BYF treatment and PM2.5 exposure.

Equipment: Lipofectamine 2000 (Invitrogen); miR-155 mimic and pcDNA-FOXO3a plasmid (Guangzhou RiboBio)