Tiao-bu-fei-shen formula promotes downregulation of the caveolin 1-p38 mapk signaling pathway in COPD - Associated tracheobronchomalacia cell model

Abstract

Ethnopharmacological relevanceThe Tiao-bu-fei-shen (TBFS) formula, extensively used in Traditional Chinese Medicine (TCM), can enhance therapeutic efficacy and reduce the frequency of acute exacerbations of lung–kidney Qi deficiency in patients with chronic obstructive pulmonary disease (COPD). According to both TCM theory and long-term observation of practice, TBFS has become an effective treatment for COPD-associated tracheobronchomalacia (TBM). Aim of the studyTo investigate the mechanism of the TBFS formula in treating COPD-associated TBM based on caveolin 1–p38 MAPK signaling and apoptosis. Materials and methodsA rat COPD model was prepared by exposure to smoking combined with tracheal lipopolysaccharide injection. The trachea or bronchus chondrocytes from COPD rats were isolated, cultured, and treated with 10 ng/mL IL-1β for 24 h to develop a model of COPD-associated TBM. Normal rats were administered TBFS to prepare drug-containing serum, and CCK8 assays were used to screen the optimal drug-containing serum concentration and SB203580 dose. TBFS drug-containing serum and SB203580 were processed separately for the control, model, drug-containing serum, blocker, and drug-containing serum combined with blocker groups. Flow cytometry and CCK8 assays were used to detect apoptosis and proliferative activity. Toluidine blue staining and immunohistochemistry were used to analyze the chondrocyte proteoglycan and type II collagen content. Western blotting was used to detect the expression of caveolin 1, p-p38 MAPK, TNF-α, IL-1β, MMP-13, Bax, and Bcl-2 proteins. Quantitative PCR was used to detect the expression of caveolin 1, p38 MAPK, IL-1β, MMP-13, Bax, Bcl-2, and miR-140–5p. ResultsThe isolation and identification of bronchial chondrocytes from COPD rats revealed that 10 ng/mL IL-1β can produce a stable COPD-associated TBM model. Screened via the CCK8 method, fourth-generation bronchial chondrocytes were determined as the optimal cells, and 5 μM SB203580 and 5% low-dose drug-containing serum were the optimal intervention doses. The experimental chondrocytes of each group were treated separately for 48 h. Toluidine blue staining and immunohistochemical analysis revealed that TBFS drug-containing serum, SB203580, and TBFS drug-containing serum combined with SB203580 can effectively increase the proteoglycan and type II collagen content after chondrocyte degradation. Flow cytometry of cells treated with SB203580 and TBFS drug-containing serum combined with SB203580 revealed significantly reduced cell apoptosis and enhanced cell proliferation activity. Western blot and qPCR analyses revealed that the TBFS drug-containing serum, SB203580, and TBFS drug-containing serum combined with SB203580 effectively inhibit the expression of caveolin 1, p-p38 MAPK, MMP-13, IL-1β, TNF-α, and Bax proteins while promoting Bcl −2 protein expression. Treatment with TBFS drug-containing serum and SB203580 effectively inhibited the expression of MMP-13, p38 MAPK, caveolin 1, and Bax genes, and promoted the expression of Bcl-2 and miR-140–5p genes. ConclusionsA concentration of 10 ng/mL of IL-1β can generate a stable COPD-associated TBM cell model. TBFS can improve the proteoglycan and type II collagen content, increase cell activity, and reduce the amount of chondrocyte apoptosis. The role of TBFS may be related to mechanisms of inhibiting the expression of the key signaling molecules caveolin 1 and p-p38 MAPK in the caveolin 1–p38 MAPK signaling pathway, thereby reducing the expression of the downstream effector products MMP-13, IL-1β, and TNF-α, while inhibiting the expression of the apoptotic gene Bax and improving the expression of Bcl-2 and miR-140–5p genes.