背景:跳步飞神(TBFS)公式,广泛用于中医(TCM),能增强慢性阻塞性肺疾病(COPD)患者肺肾气虚型急性加重的疗效,减少急性加重的频率。根据中医理论和长期的实践观察,TBFS已成为COPD相关气管支气管软化症(TBM)的有效治疗方法。
目的:基于小窝蛋白1-p38MAPK信号和细胞凋亡研究TBFS配方治疗COPD相关TBM的机制。
方法:吸烟联合气管脂多糖注射制备大鼠COPD模型。分离COPD大鼠气管或支气管软骨细胞,培养,并用10ng/mLIL-1β处理24小时,以建立COPD相关TBM模型。正常大鼠给予TBFS制备含药血清,和CCK8测定用于筛选最佳含药血清浓度和SB203580剂量。TBFS含药血清和SB203580分别处理为对照,模型,含药血清,阻滞剂,含药血清联合阻滞剂组。流式细胞术和CCK8测定用于检测细胞凋亡和增殖活性。使用甲苯胺蓝染色和免疫组织化学分析软骨细胞蛋白聚糖和II型胶原含量。蛋白质印迹法检测caveolin1,p-p38MAPK的表达,TNF-α,IL-1β,MMP-13,Bax,和Bcl-2蛋白。定量PCR检测小窝蛋白1、p38MAPK的表达,IL-1β,MMP-13,Bax,Bcl-2和miR-140-5p。
结果:COPD大鼠支气管软骨细胞的分离和鉴定表明,10ng/mLIL-1β可以产生稳定的COPD相关TBM模型。通过CCK8方法筛选,第四代支气管软骨细胞被确定为最佳细胞,5μMSB203580和5%低剂量含药血清是最佳干预剂量。各组实验软骨细胞分别处理48h。甲苯胺蓝染色和免疫组织化学分析显示,SB203580和TBFS含药血清联合SB203580可有效增加软骨细胞降解后的蛋白聚糖和II型胶原含量。用SB203580和TBFS含药血清联合SB203580处理的细胞的流式细胞术显示细胞凋亡显着减少,细胞增殖活性增强。蛋白质印迹和qPCR分析显示,含有TBFS药物的血清,SB203580和TBFS含药血清联合SB203580有效抑制小窝蛋白1、p-p38MAPK的表达,MMP-13,IL-1β,TNF-α,和Bax蛋白同时促进Bcl-2蛋白表达。用TBFS含药血清和SB203580治疗可有效抑制MMP-13,p38MAPK的表达,小窝蛋白1和Bax基因,并促进Bcl-2和miR-140-5p基因的表达。
结论:浓度为10ng/mL的IL-1β可以产生稳定的COPD相关TBM细胞模型。TBFS可以提高蛋白多糖和II型胶原的含量,增加细胞活性,减少软骨细胞凋亡。TBFS的作用可能与抑制Caveolin1-p38MAPK信号通路中关键信号分子Caveolin1和p-p38MAPK表达的机制有关,从而降低下游效应产物MMP-13,IL-1β的表达,和TNF-α,同时抑制凋亡基因Bax的表达,提高Bcl-2和miR-140-5p基因的表达。
BACKGROUND: The 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).
OBJECTIVE: To investigate the mechanism of the TBFS formula in treating COPD-associated TBM based on caveolin 1-p38 MAPK signaling and apoptosis.
METHODS: A 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.
RESULTS: The 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.
CONCLUSIONS: A 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.