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方远, 赵晶燕, 蒋庆要, 吴柏合, 刘千柳, 刘向国.六味补气方通过促进细胞自噬抑制肺泡上皮细胞衰老治疗COPD的机制研究[J].湖南中医药大学学报,2025,45(10):1852-1860[点击复制] |
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| 六味补气方通过促进细胞自噬抑制肺泡上皮细胞衰老治疗COPD的机制研究 |
| 方远,赵晶燕,蒋庆要,吴柏合,刘千柳,刘向国 |
| (安徽中医药大学, 安徽 合肥 230012) |
| 摘要: |
| 目的 研究六味补气方对细胞自噬和肺泡上皮细胞衰老的影响及其治疗慢性阻塞性肺疾病（COPD）的机制。方法 按照随机数字表法将大鼠分为正常对照组（N组）、模型组（M组）、桂龙咳喘宁组（GL组）和六味补气方低、中、高剂量组（LL组、LM组、LH组），每组10只。采用烟熏及气管滴入脂多糖（LPS）的方法建立COPD实验动物模型。药物干预后，检测大鼠肺功能；HE染色观察支气管肺组织形态学变化；免疫组化检测肺组织中细胞周期蛋白依赖性激酶（CDK）4和细胞周期蛋白D1（CyclinD1）表达水平；ELISA检测血清和肺泡灌洗液（BALF）中p21和p53含量；Western blot检测肺组织中Beclin-1、自噬相关基因（ATG）14和微管相关蛋白1轻链3（LC3）-Ⅱ/LC3-Ⅰ蛋白表达水平。结果 与N组比较，M组大鼠肺活量（FVC）值、第三秒用力呼气容积（FEV3）值、FEV3/FVC值和呼气峰流速（PEF）值均下降（P＜0.01）；肺组织出现慢性支气管炎及肺气肿的病理学变化；肺组织中CDK4和CyclinD1阳性面积比率以及组织化学评分（H-score）均降低（P＜0.01）；血清及BALF中p21和p53含量均升高（P＜0.01）；肺组织中Beclin-1、ATG14和LC3-Ⅱ/LC3-Ⅰ蛋白表达水平均降低（P＜0.01）。与M组比较，各药物干预组大鼠FVC值、FEV3值、FEV3/FVC值和PEF值均上升（P＜0.05，P＜0.01）；肺组织病理学变化减轻；肺组织中CDK4和CyclinD1阳性面积比率以及H-score均增加（P＜0.01）；血清及BALF中p21和p53含量均下降（P＜0.01）；肺组织中Beclin-1、ATG14和LC3-Ⅱ/LC3-Ⅰ蛋白表达水平均上升（P＜0.01）。与GL组比较，LL组大鼠FVC值、FEV3值、FEV3/FVC值和PEF值均下降（P＜0.01），肺组织中CDK4和CyclinD1阳性面积比率以及H-score均降低（P＜0.01），血清及BALF中p21和p53含量均升高（P＜0.01），肺组织中Beclin-1、ATG14和LC3-Ⅱ/LC3-Ⅰ蛋白表达水平均降低（P＜0.01）；LH组大鼠FVC值、FEV3值、FEV3/FVC值和PEF值均上升（P＜0.05）；LH、LM组肺组织中CDK4和CyclinD1阳性面积比率以及H-score均上升（P＜0.01），血清及BALF中p21和p53含量均下降（P＜0.01），肺组织中Beclin-1、ATG14和LC3-Ⅱ/LC3-Ⅰ蛋白表达水平均上升（P＜0.01）。与LH组比较，LL、LM组大鼠FVC值、FEV3值、FEV3/FVC值和PEF值均下降（P＜0.05）；肺组织中CDK4和CyclinD1阳性面积比率以及H-score均降低（P＜0.01）；血清及BALF中p21和p53含量均升高（P＜0.01）；肺组织中Beclin-1、ATG14和LC3-Ⅱ/LC3-Ⅰ蛋白表达水平均降低（P＜0.01）。结论 六味补气方可加速细胞自噬，延缓肺组织损伤和肺泡上皮细胞的衰老、减轻气道炎症、增强肺功能；其机制可能是六味补气方诱导CDK4和CyclinD1表达，降低p21、p53含量，提升Beclin-1、ATG14和LC3-Ⅱ/LC3-Ⅰ表达，从而加速细胞自噬、抑制细胞衰老所致。 |
| 关键词: 六味补气方 慢性阻塞性肺疾病 肺功能 细胞自噬 细胞衰老 细胞周期 |
| DOI：10.3969/j.issn.1674-070X.2025.10.007 |
| 投稿时间：2025-04-15 |
| 基金项目:安徽省高校杰出青年科研项目（2022AH0200）。 |
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| Mechanism of Liuwei Buqi Formula in alleviating COPD by enhancing autophagy and suppressing alveolar epithelial cell senescence |
| FANG Yuan, ZHAO Jingyan, JIANG Qingyao, WU Baihe, LIU Qianliu, LIU Xiangguo |
| (Anhui University of Chinese Medicine, Hefei, Anhui 230012, China) |
| Abstract: |
| Objective To investigate the effects of Liuwei Buqi Formula (LWBQF) on cellular autophagy and alveolar epithelial cell senescence, and its mechanism in treating chronic obstructive pulmonary disease (COPD). Methods Rats were randomized into the following groups using a random number table (n=10 per group): normal control group (Group N), model group (Group M), Guilong Kechuanning group (Group GL), and low-, medium-, and high-dose LWBQF groups (Groups LL, LM, LH). A COPD experimental animal model was established using cigarette smoke exposure and intratracheal instillation of lipopolysaccharide (LPS). After drug intervention, rat lung function was measured. Morphological changes in bronchial and lung tissues were observed by HE staining. The expression levels of cyclin-dependent kinase (CDK) 4 and cyclin D1 (CyclinD1) in lung tissue was detected by immunohist-ochemistry. The levels of p21 and p53 in serum and bronchoalveolar lavage fluid (BALF) were measured by ELISA. The protein expression levels of Beclin-1, autophagy-related gene(ATG) 14, and microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I ratio in lung tissue were detected by Western blot analysis. Results Compared with Group N, Group M exhibited significant decreases in forced vital capacity (FVC), forced expiratory volume at 3 seconds (FEV3), FEV3/FVC ratio, and peak expiratory flow (PEF) (P＜0.01). It also showed pathological changes of chronic bronchitis and emphysema, reduced positive area ratios and histochemistry scores (H-scores) for CDK4 and CyclinD1 in lung tissue (P＜0.01), increased levels of p21 and p53 in serum and BALF (P＜0.01), and downregulated protein expression levels of Beclin-1, ATG14, and LC3-II/LC3-I ratio in lung tissue (P＜0.01). Compared with Group M, all drug intervention groups exhibited increased FVC, FEV3, FEV3/FVC ratio, and PEF (P＜0.05, P＜0.01), alleviated pathological changes in lung tissue, increased positive area ratios and H-scores for CDK4 and CyclinD1 in lung tissue (P＜0.01), decreased levels of p21 and p53 in serum and BALF (P＜0.01), and upregulated protein expression levels of Beclin-1, ATG14, and LC3-II/LC3-I ratio in lung tissue (P＜0.01). Compared with Group GL, Group LL showed decreased FVC, FEV3, FEV3/FVC ratio, and PEF (P＜0.01), reduced positive area ratios and H-scores for CDK4 and CyclinD1 in lung tissue (P＜0.01), increased levels of p21 and p53 in serum and BALF (P＜0.01), and downregulated protein expression levels of Beclin-1, ATG14, and LC3-II/LC3-I ratio in lung tissue (P＜0.01). Group LH showed increased FVC, FEV3, FEV3/FVC ratio, and PEF (P＜0.05). Groups LH and LM showed increased positive area ratios and H-scores for CDK4 and CyclinD1 in lung tissue (P＜0.01), decreased levels of p21 and p53 in serum and BALF (P＜0.01), and upregulated protein expression levels of Beclin-1, ATG14, and LC3-II/LC3-I ratio in lung tissue (P＜0.01). Compared with Group LH, Groups LL and LM exhibited decreased FVC, FEV3, FEV3/FVC ratio, and PEF (P＜0.05), reduced positive area ratios and H-scores for CDK4 and CyclinD1 in lung tissue (P＜0.01), increased levels of p21 and p53 in serum and BALF (P＜0.01), and downregulated protein expression levels of Beclin-1, ATG14, and LC3-II/LC3-I ratio in lung tissue (P＜0.01). Conclusion LWBQF can accelerate cellular autophagy, delay lung tissue damage and alveolar epithelial cell senescence, alleviate airway inflammation, and enhance lung function. The mechanism may be that LWBQF induces the expression of CDK4 and CyclinD1, reduces the levels of p21 and p53, and upregulates the expression of Beclin-1, ATG14, and LC3-II/LC3-I ratio, thereby promoting autophagy and inhibiting cell senescence. |
| Key words: Liuwei Buqi Formula chronic obstructive pulmonary disease pulmonary function autophagy cell senescence cell cycle |
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