Effective-Component Compatibility of Bufei Yishen Formula (cid:0) Ameliorated COPD By Improving Airway Epithelial Cell Senescence Via Promoting Mitophagy By Nrf2/PINK1 Pathway

Background: Effective-component compatibility of Bufei Yishen formula (cid:0) (ECC-BYF (cid:0)) shows positive effects on stable chronic obstructive pulmonary disease (COPD). Purpose: To investigate the mechanisms of ECC-BYF (cid:0) on COPD rats from the aspect of airway epithelial cell senescence. Methods: COPD model rats were treated with ECC-BYF (cid:0) for 8 weeks and the ecacy was evaluated. Cigarette smoke extract (CSE) induced senescence model of airway epithelial cells were treated with ECC-BYF (cid:0), the related enzymes and proteins involved in oxidative stress and mitophagy were detected. Results: ECC-BYF (cid:0) markedly rescued pulmonary function and histopathological changes, which might be associated with the amelioration of lung senescence, including reduction of malondialdehyde (MDA) and tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and matrix metalloproteinase (MMP)-9, increase of the level of total superoxide dismutase (T-SOD), and decease of p21 level in airway. Furthermore, ECC-BYF (cid:0) suppressed p16, p21 expressions and senescence-associated β-galactosidase (SA-β-Gal) in CSE-induced airway epithelial cells. Moreover, ECC-BYF (cid:0) upregulated the mitophagy-related proteins, including co-localization of TOM20 and LC3B, PINK1, PARK2, and improved mitochondrial function with upregulating mitochondrial mitofusin (Mfn)2 and reducing dynamin-related protein 1 (Drp1) expression. ECC-BYF (cid:0) enhanced the activities of T-SOD and GSH-PX by up-regulating Nrf2, thus inhibiting oxidative stress. After intervention with Nrf2 inhibitor, the regulation effects of ECC-BYF (cid:0) on oxidative stress, mitophagy and senescence in airway


Background
Chronic obstructive pulmonary disease (COPD) is a heterogeneous disorder involving irreversible air ow restriction and chronic abnormal in ammatory reaction to harmful particles or gases [1,2]. COPD has become the third-leading cause of death worldwide, with a high incidence and disability rate, and it is accompanied by signi cant economic and social pressure [1,3]. Traditional Chinese medicine (TCM)therapies have been widely used for stable COPD with no or mild side effects and are gaining increasing attention for their signi cant effects. Bufei Yishen formula (BYF), a Chinese herbal formula, has shown good e cacy on COPD clinical symptoms including reducing the frequency of acute exacerbation by our previous studies [4]. However, the complex ingredients of BYF makes it di cult for elucidating the mechanisms involved and hinders the international promotion. Therefore, ve effective components were identi ed from herbal medicines of BYF based on vivo experiments, combined in a xed ratio as the effective-component compatibility of BYF (ECC-BYF ), which suppressed the in ammation by regulating p65, JNK, and p38 in COPD [5].
Cell senescence in COPD, particularly in alveolar and airway epithelial cells, increases the risk of respiratory tract infection, progressive emphysema aggravation, and may lead to airway remodeling, which can be induced by extracellular or intracellular stimuli such as telomere attrition (replicative senescence), irreparable DNA damage, mitochondrial dysfunction, and oxidative stress (excessive senescence) [2,[6][7][8][9][10][11]. Cell senescence is always accompanied by a complex phenotype, such as altered cell morphology, cell cycle arrest, increased senescence-associated β-galactosidase (SA-β-gal) and the senescence-associated secretory phenotype (SASP), which is primarily mediated by the p53 or p16/p21 pathway [11]. Mitophagy is a type of elimination of irreversibly damaged mitochondria that helps to slow cell senescence. It is regulated by PTEN-induced putative kinase 1 (PINK1) and Parkin (PARK) 2

Histopathology analysis
The lung tissues were sliced into 3-4 mm sections, xed in 10% paraformaldehyde solution for 72 h, and then mounted in para n-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, /mm 2 ) were counted with the counting tool of Adobe Photoshop CC software to evaluate the degree of emphysema.

Immuno uorescence analysis
The expression of p21 in lung, especially in bronchus was detected by immuno uorescence. 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 a nipure 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 xed 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 serumfree 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 ltered through a lter (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 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.

SA-β-Gal Staining
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.
Electron microscopy BEAS-2B cells were xed 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.

Western Blotting
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 di uoride (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 speci c primary antibodies were β-actin 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.

Statistic Analysis
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 signi cant differences were assessed by oneway ANOVA followed by the Tukey's test where appropriate. Values of P < 0.05 were considered statistically signi cant.

Results
Effect of ECC-BYF on pulmonary function and histopathological changes of COPD rats The severity of COPD is determined by pulmonary function and histopathological changes. V T , MV, PEF, and EF50 were all signi cantly lower in COPD rats, as were FVC and FEV 0.3, as shown in Figure 1. Treatment with ECC-BYF III and N-Acetylcysteine (NAC) improved lung function signi cantly. Figure 2 depicts the histopathological changes in COPD, including alveolar and bronchial thickening, emphysema, and in ammatory cell in ltration. Quantitative analysis of MAN and MLI showed that increased MLI was accompanied by decreased MAN. Treatment with ECC-BYF and NAC could effectively rescue the histopathological changes above. These data suggested that ECC-BYF III could attenuate COPD rats.
Effect of ECC-BYF on cell senescence in BEAS-2Bs induced by CSE Previous research has found a high level of p21 expression in lung tissue, particularly in the airways of COPD rats, and we know that cigarette smoke exposure is a major factor that causes elevated p21 expression, as well as airway epithelial cell senescence and COPD airway epithelial dysfunction. The expressions of p21, p16, and SA-β-Gal were detected in CSE-induced BEAS-2Bs to determine the effect of ECC-BYF III on cell senescence. We found that 10% CSE exposure increased the expression of p21 and p16, and SA-β-Gal staining. ECC-BYF treatment demonstrated a signi cant decrease in the expression of p21 and p16 and the number of SA-β-Gal-stained cells (Figure 4). These ndings suggested that ECC-BYF effectively suppressed CSE-induced airway epithelial cell senescence.

ECC-BYF upregulated mitophagy in BEAS-2B cells induced by CSE
Mitophagy de ciency is a critical inducer of the accumulation of damaged mitochondrial and subsequent cell senescence. As a result, we assessed mitophagy in BEAS-2Bs induced by CSE or ECC-BYF treatment. We found that ECC-BYF could signi cantly increase the expression of PINK1, PARK2, and intensi ed the co-localization of TOM20-labeled mitochondria, as shown by the yellow dots and LC3B (green dots) in gure 5B. Furthermore, we found that ECC-BYF treatment obviously improved the swelling of mitochondria, decreased the number of damaged mitochondria, and increased the quantity of autophagosomes and lysosomes. Meanwhile, the expression of Mfn2 increased and Drp1 decreased. Taken together, ECC-BYF exhibited effective role in enhancing mitophagy, subsequently eliminating damaged mitochondria, and improving mitochondrial function ( Figure 5).

ECC-BYF improved CSE induced oxidative stress in BEAS-2Bs
CSE-induced insu cient mitophagy and abundant damaged mitochondria are frequently caused by oxidative stress [21]. After CSE exposure, reactive oxygen species (ROS) increased while the activities of T-SOD and GSH-PX were down-regulated in BEAS-2Bs. ROS was reduced by ECC-BYF III treatment, whereas the activity of T-SOD and GSH-PX was increased in BEAS-2Bs ( Figure 6A). Furthermore, we discovered that ECC-BYF III treatment signi cantly increased Nrf2 and HO-1 ( Figure 6B). The ndings suggested that ECC-BYF III could activate Nrf2 and prevent CSE-induced oxidative stress.

Involvement of Nrf2 signal in ECC-BYF ' s anti-senescence
To investigate the role of Nrf2 in the anti-senescence effect of ECC-BYF III, we treated BEAS-2Bs with Nrf2 inhibitors and/or ECC-BYF III. We discovered that co-treatment with Luteolin (Nrf2 inhibitor) inhibited the effects of ECC-BYF III, which could be re ected by the down-regulation of Nrf2 and HO-1 and higher ROS activity, as well as inhibiting the activation of related proteins (PINK1, PARK2, Mfn2) and signi cantly upregulating the expression of Drp1. Luteolin eventually inhibited ECC-BYF III's ability to reduce the expression of p16 and p21 ( Figure 7). All data demonstrated that ECC-BYF may play a role in ameliorating cell senescence through inhibiting oxidative stress and mitochondrial function through Nrf2 signaling.

Discussion
COPD is a major social problem that endangers public health because of its high morbidity and mortality rates. Traditional Chinese medicine is widely acknowledged for its e cacy in the treatment of COPD. The original BYF, which included twelve Chinese medicines, had a positive effect on COPD clinical symptoms [4,22]. However, the complexity of the ingredients makes elucidating the mechanisms involved di cult. The effective compounds were identi ed from BYF and combined with a xed ratio to produce the ECC-BYF III. 20-S-ginsenoside Rh1, astragaloside IV, icariin, nobiletin, and paeonol are among the ve compounds found in ECC-BYF III [5,23]. ECC-BYF III effectively rescued pulmonary function, lung histopathological changes, and cell senescence-associated secretory phenotype in COPD rats in this study. Furthermore, by inhibiting oxidative stress and promoting insu cient mitophagy, ECC-BYF III may be able to alleviate CSE-induced airway epithelial cell senescence.
Lung senescence, mainly including alveolar and airway epithelial cell senescence, participates in the pathogenesis of COPD [2,6]. In COPD, cigarette smoke is a major driver of cell senescence. The accumulation of senescent cells in lung contributes to the acquisition of SASP, and senescence increases susceptibility to infection, airway remodeling and exacerbation of COPD-emphysema[8, 10,24]. The SASP takes the form of cell cycle arrest, the release of in ammatory cytokines, proteases, and the reduction of antioxidant capacity [25,26]. Indeed, our studies found that the SASP occurred in COPD rats, such as increased p21, TNF-α, IL-6, MMP-9 and MDA activity and T-SOD level decreased signi cantly in lung or BALF. ECC-BYF suppressed pulmonary function reduction and histopathological changes, meanwhile, improved SASP in COPD rats. Thus, we conclude that ECC-BYF exerted distinct effect on COPD rats through ameliorating the airway epithelial cell senescence and the SASP.
The rst barrier against external stimuli are airway epithelial cells. CS typically promotes airway epithelial cell senescence, with elevated p16, p21, and SA-β-Gal expression [27,28]. Our ndings showed that ECC-BYF III can prevent CSE-induced airway epithelial cell senescence. Excessive oxidants/ROS produced by CS exposure can damage biological macromolecules and cause mitochondrial dysfunction [29,30]. Given that mitochondria are the pivotal hub of energy production and the producer of ROS, substantial studies have proved the importance of mitochondrial dysfunction in CS-induced cell senescence [12,31]. Mitophagy is a special form of autophagy where damaged mitochondria are eliminated to maintain the mitochondrial function. Insu cient mitophagy leads to the accumulation of damaged mitochondria, and thus cause the de ciency of energy, which impels cell senescence[28, 32,33]. There are evidence suggesting that PINK1 and PARK2 recruitment are required for mitophagy to ameliorate CSE-induced cell senescence, PINK1 knockdown noticeably reduced the expression of PARK2, and PARK2 knockdown could elevate the expression of p21 and SA-β-Gal staining in response to CSE exposure [12,15,28]. In this study, we found ECC-BYF could inhibit the production of ROS induced by CSE, and enhance mitophagy by increasing the levels of PINK1 and PARK2. Aravamudan Bharathi et al pointed out that CSE induced mitochondrial fragmentation and dysfunction as marked by decreasing Mfn2 and increasing Drp1 [34].
Treatment with ECC-BYF could increase the expression of Mfn2 and reduce Drp1, also alleviate the swelling and cristae disruption of mitochondria in CSE-induced airway epithelial cells with the increase of autophagosomes and lysosomes. This is the result of ECC-BYF III enhancing mitophagy and improving mitochondrial function. As a result, we hypothesized that ECC-BYF III reduced cell senescence by increasing the activity of PINK1 and PARK2. Since Nrf2 is a common anti-oxidant factor and activation of Nrf2 can protect cells from mitophagy de ciency caused by oxidative stress[16, 35,36], we administered cells with Nrf2 inhibitor and discovered that Nrf2 inhibitor suppressed the inhibition effect of ECC-BYF III on ROS production, as well as mitophagy de ciency and cell senescence. Hence, Targeting the Nrf2 pathway to inhibit oxidative stress and intensify mitophagy is the potential mechanism for ECC-BYF to ameliorate cell senescence.

Consent for publication
Not applicable.

Availability of data and material
The datasets used and/or analysed during this study are available from the corresponding author on reasonable request.

Con ict of interests
The authors declare that they have no competing interests concerned with this article.      Immuno uorescent staining of the co-localization of TOM20 (yellow dots) and LC3B (green dots), (magni cation, ×200); (C) Electron microscopy detection of mitochondria and mitophagy in BEAS-2Bs (magni cation, ×30000).  the ECC-BYF treatment group, △ P<0.05 vs. the ECC-BYF treatment group.