Bufei Yishen formula alleviates airway epithelial cell senescence in COPD by activating AMPK-Sirt1-FoxO3a pathway and promoting autophagy

Abstract

The Bufei Yishen formula (BYF) has traditionally been employed to treat patients with COPD, demonstrating significant effectiveness. However, the underlying mechanisms through which BYF alleviates COPD remains unclear. Cellular senescence is crucial in the pathogenesis of COPD. This study aims to investigate whether the therapeutic mechanism of BYF is associated with the reduction of cellular senescence. To evaluate the anti-senescence effects of BYF, a COPD rat model and a cellular senescence model were established. The active compounds and underlying mechanisms of BYF were investigated in vitro. BYF treatment significantly mitigated lung function decline and pathological damage in COPD rats. It significantly inhibited senescence in lung tissue by decreasing the expression of the cell cycle inhibitor p21, DNA damage markers, pro-inflammatory cytokines, and matrix metalloproteinases. BYF4/5, isolated from BYF, demonstrated significant anti-senescence effects in bronchial epithelial cells. Additionally, 67 compounds were identified from BYF4/5, and 770 targets were predicted for these compounds. hesperidin and nobiletin, identified as key compounds in BYF, were found to inhibit cellular senescence and activate the AMPK-Sirt1-FoxO3a pathway and autophagy in 16HBE cells. The data indicate that BYF alleviates COPD by activating the AMPK-Sirt1-FoxO3a pathway and autophagy, thereby inhibiting bronchial epithelial cell senescence.

Keywords: AMPK; Autophagy; Bufei Yishen formula; Cellular senescence; Chronic obstructive pulmonary disease; FoxO3a; Sirt1.

Fig. 1

The Bufei Yishen formula improves pulmonary function and pathological changes in COPD rats. BYF (11.6, 5.8 g/kg) and N-acetylcysteine (NAC, 54 mg/kg) were administered to COPD rats for 9 to 16 weeks. (A) Flow chart of the animal experiment. (B) Lung function including tidal volume (TV), minute volume (MV), peak expiratory flow (PEF), and expiratory flow at 50% tidal volume (EF50). (C) Pathological changes in pulmonary tissue (H&E, × 200) and its quantitative analysis, including mean alveolar number (MAN), mean linear intercept (MLI), and mean small airway wall thickness. Values are expressed as the mean ± SEM (n = 6), *P < 0.05, **P < 0.01 compared with the control group; ^#P < 0.05, ^##P < 0.01 compared with the model group.

Fig. 2

The Bufei Yishen formula inhibits inflammation, oxidative stress, and protease expression in COPD rats. (A) Expression of IL-1β, IL-6 and TNF-α in lung tissue were detected using the immunohistochemical method and quantified as their integral optical density (IOD). (B) The expression of SOD2, MMP-2, and MMP-9 were detected by using immunohistochemical method and quantified as their integral optical density (IOD). Values are expressed as the mean ± SEM (n = 6), *P < 0.05, **P < 0.01 compared with the control group; ^#P < 0.05, ^##P < 0.01 compared with the model group.

Fig. 3

The Bufei Yishen formula inhibits inflammation, oxidative stress, and protease expression in COPD rats. (A) The levels of IL-1β, IL-6, TNF-α, SOD2, MMP-2, and MMP-9 were measured using ELISA. (B) Protein levels of E-CAD, OCC and ZO-1 in lung tissue of rats. Values are expressed as the mean ± SEM (n = 6), *P < 0.05, **P < 0.01 compared with the control group; #P < 0.05, ##P < 0.01 compared with the model group.

Fig. 4

Effect of Bufei Yishen formula on the protein levels of p21 and γ-H2 AX, as well as cellular apoptosis in the lung tissues of COPD rats. Immunofluorescence staining was applied to detect the p21 (A), TUNEL (B), and γ-H2 AX (C) in lung tissues.

Fig. 5

Effect of Bufei Yishen formula on the protein levels of Ki67, PINK1 and SP-A in the lung tissues of COPD rats. Immunofluorescence staining was applied to detect the Ki67 (A), PINK1 (B), and SP-A (C) in lung tissues.

Fig. 6

Effect of Bufei Yishen formula 1 ~ 6 on cigarette smoke extract (CSE)- and LPS-induced 16-HBE cells. (A) Flow chart of BYF isolation. (B) Protein levels of p21 in BYF1-6-treated 16-HBE cells induced by CSE/LPS. (C) The impact of BYF4/5 on cell viability (D) The mRNA levels of p53 and p21. (E) Protein levels of p53 and p21 in BYF4/5-treated 16-HBE cells induced by CSE/LPS. (F) The protein levels of p53 and p21 in 16HBE cells were detected by immunofluorescence staining. Values are expressed as the mean ± SEM (n = 3), *P < 0.05, **P < 0.01 compared with the control group; ^#P < 0.05, ^##P < 0.01 compared with the model group.

Fig. 7

Effect of Bufei Yishen formula 4/5 on senescence in cigarette smoke extract (CSE)- and LPS-induced 16-HBE cells. (A) The mRNA levels of IL-1α, IL-1β and TNF-α. (B) The mRNA levels of CXCR2, CXCL8 and MCP-1. (C) The mRNA levels of MMP-1, MMP-8, and MMP-9. (D) The activity of β-galactosidase. Values are expressed as the mean ± SEM (n = 3), *P < 0.05, **P < 0.01 compared with the control group; ^#P < 0.05, ^##P < 0.01 compared with the model group.

Fig. 8

Network analysis of the anti-senescence mechanisms of Bufei Yishen formula. (A) UHPLC Q-Extractive Orbitrap-MS/MS was used to identify the compounds of the BYF4/5. (B) The compound and target network have been constructed. (C) volcano map (C) and cluster heatmap (D) were applied to showed the DEGs. (E) The PPI network of targets and DEGs was constructed The top 157 core nodes (F) with a degree value ≥ 70 were used for KEGG(https://www.kegg.jp/kegg/kegg1.html) pathway enrichment analysis (G). (H) Sankey diagram were used to shown the components, targets and their associated pathways.

Fig. 9

Nobiletin and hesperidin inhibited cellular senescence and activated AMPK-Sirt1-foxO3a-mediated autophagy. 16-HBE cells were treated with nobiletin or hesperidin for 3 h, and then challenged with CSE/LPS for 24 h. The impact of Nobiletin (A) and hesperidin (C) on cell viability. (B, D) Protein levels of p53 and p21. (E) The mRNA levels of p53 and p21. (F) The activity of β-galactosidase. (G) The mRNA levels of IL-1α, IL-1β and MMP-9..Values are expressed as the mean ± SEM (n = 3), *P < 0.05, **P < 0.01 compared with the control group; ^#P < 0.05, ^##P < 0.01 compared with the model group.

Fig. 10

Nobiletin and hesperidin inhibited cellular senescence and activated AMPK-Sirt1-foxO3a-mediated autophagy. 16-HBE cells were treated with nobiletin or hesperidin for 3 h, and then challenged with CSE/LPS for 24 h. (A-D) Protein levels of phosphorylated AMPK, Sirt1 and FoxO3a. (E) The mRNA levels of HDAC. Values are expressed as the mean ± SEM (n = 3), *P < 0.05, **P < 0.01 compared with the control group; ^#P < 0.05, ^##P < 0.01 compared with the model group.

Fig. 11

Effect of Bufei Yishen formula on the protein levels of AMPK andSIRT1 in the lung tissues of COPD rats. Immunofluorescence staining was applied to detect the AMPK (A), SIRT1 (B), in lung tissues.

Fig. 12

Effect of Bufei Yishen formula on the protein levels of LC3 and FoxO3a in the lung tissues of COPD rats. Immunofluorescence staining was applied to detect the LC3 (A), FoxO3a (B), in lung tissues.