南方医科大学学报

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (7): 1372-1379.doi: 10.12122/j.issn.1673-4254.2025.07.03

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补肺益肾方对香烟烟雾提取物诱导的人支气管上皮细胞损伤的保护作用及其机制

范正媛1,2,3(), 沈子涵3, 李亚1,2,3, 沈婷婷1,2,3, 李高峰3, 李素云2,3()   

  1. 1.河南中医药大学第一附属医院 中药药理（呼吸）实验室河南省呼吸病防治中医药重点实验室，河南 郑州 450000
    2.河南中医药大学第一附属医院 呼吸科，河南 郑州 450000
    3.河南中医药大学呼吸疾病中医药防治省部共建协同创新中心，河南 郑州 450046
  • 收稿日期:2025-03-21 出版日期:2025-07-20 发布日期:2025-07-17
  • 通讯作者: 李素云 E-mail:fanzhengyuan0225@163.com;lisuyun2000@126.com
  • 作者简介:范正媛，博士，副研究员，E-mail: fanzhengyuan0225@163.com
  • 基金资助:
    国家自然科学基金(82405345);国家自然科学基金(82374416);四大慢病重大专项(2023ZD0506700);四大慢病重大专项(2023ZD0506702);张仲景传承与创新专项(GZY-KJS-2022-041-1)

Protective effect of Bufei Yishen Formula against cigarette smoke extract-induced human bronchial epithelial cell damage and its mechanism

Zhengyuan FAN1,2,3(), Zihan SHEN3, Ya LI1,2,3, Tingting SHEN1,2,3, Gaofeng LI3, Suyun LI2,3()   

  1. 1.Chinese Medicine Pharmacology (Respiratory) Laboratory, Henan Key Laboratory of Traditional Chinese Medicine for Respiratory Disease Prevention and Treatment, First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou 450000, China
    2.Department of Respiratory Medicine, First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou 450000, China
    3.Henan Province and Ministry of Education Co-construction Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases, Henan University of Chinese Medicine, Zhengzhou 450046, China
  • Received:2025-03-21 Online:2025-07-20 Published:2025-07-17
  • Contact: Suyun LI E-mail:fanzhengyuan0225@163.com;lisuyun2000@126.com
  • Supported by:
    National Natural Science Foundation of China(82405345)

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摘要：

目的 探讨补肺益肾方对香烟烟雾提取物（CSE）诱导的人支气管上皮BEAS-2B细胞损伤的保护作用及机制。 方法 以CSE诱导的BEAS-2B细胞构建模型，以羧甲司坦（S-CMC）为阳性对照，将细胞分为对照组、CSE组、补肺益肾方（BYF）含药血清低剂量（BYL）组、BYF高剂量（BYH）组、NF-κB抑制剂PDTC组、BYH+PDTC组及S-CMC组。CCK-8法筛选CSE最佳造模浓度、作用时间及BYF含药血清、S-CMC给药浓度；ELISA法检测细胞上清炎症因子水平及细胞MUC5AC、MUC5B表达；透射电镜观察细胞超微结构；流式细胞术检测细胞凋亡率；qRT-PCR及Western blotting法检测细胞TLR4/NF-κB通路相关mRNA和蛋白表达。 结果 与Control组相比，CSE组细胞IL-1β、IL-6及TNF-α的分泌升高（P<0.01），细胞MUC5AC、MUC5B mRNA和蛋白表达升高（P<0.01），细胞出现凋亡小体，早期凋亡率及总凋亡率升高（P<0.01），细胞TLR4，I-κB，NF-κB mRNA和蛋白表达升高（P<0.01），AQP5 mRNA和蛋白表达降低（P<0.01）。与CSE组相比，各给药组可降低细胞上清炎症因子水平，细胞MUC5AC、MUC5B mRNA和蛋白水平，细胞早期凋亡率及总凋亡率，细胞超微结构损伤及TLR4/NF-κB通路相关mRNA和蛋白表达被部分逆转，其中以BYH+PDTC组更明显。 结论 补肺益肾方可通过抑制TLR4/NF-κB信号通路抑制CSE诱导的BEAS-2B细胞凋亡、炎症和黏液高分泌。

关键词: 炎症反应, 香烟烟雾提取物, 补肺益肾方, 黏液高分泌, TLR4/NF-κB信号通路

Abstract:

Objective To evaluate the protective effect of Bufei Yishen Formula (BYF) against cigarette smoke extract (CSE)-induced injuries in human bronchial epithelial BEAS-2B cells and explore the underlying mechanism. Methods BEAS-2B cells exposed to CSE were treated with normal rat serum, BYF-medicated rat serum at low or high doses, pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor), PDTC combined with high-dose BYF-medicated serum, or S-carbomethyloysteine (S-CMC, as the positive control). CCK-8 assay was used to determine the optimal concentration and treatment time of CSE, BYF-medicated serum and S-CMC. The treated cells were examined for inflammatory factor levels in the supernatant and cellular expressions of MUC5AC and MUC5B using ELISA, cell ultrastructural changes with transmission electron microscopy, and cell apoptosis rate using flow cytometry. The expression levels of TLR4/NF‑κB pathway-associated mRNAs and proteins were determined by qRT-PCR and Western blotting. Results CSE exposure significantly increased secretions of IL-1β, IL-6 and TNF-α, mRNA and protein expressions of MUC5AC and MUC5B, and early and total apoptosis rates in BEAS-2B cells, where the presence of apoptotic bodies was detected. CSE also significantly enhanced the mRNA and protein expressions of TLR4, I-κB, and NF-κB and reduced mRNA and protein expressions of AQP5. Treatments of the CSE-exposed cells with BYF-medicated serum, PDTC and S-CMC all significantly lowered inflammatory factor levels, MUC5AC and MUC5B expressions, and early and total cell apoptosis rates, and partly reversed the changes in cellular ultrastructure and mRNA and protein expressions of the TLR4/NF-κB pathway, and the effects were the most conspicuous following the combined treatment with high-dose BYF-medicated serum and PDTC. Conclusion BYF can inhibit cell apoptosis, inflammation and mucus hypersecretion in CSE-induced BEAS-2B cells by inhibiting the TLR4/NF-κB signaling pathway.

Key words: inflammation response, cigarette smoke extract, Bufei Yishen Formula, mucin hypersecretion, TLR4/NF?κB signaling pathway