Objective: To explore the mechanism of Yifei Jianpi recipe for improving cigarette smoke- induced inflammatory injury and mucus hypersecretion in cultured human bronchial epithelial cells.
Methods: Serum samples were collected from 40 SD rats treated with Yifei Jianpi recipe (n=20) or normal saline (n=20) by gavage. Cultured human bronchial epithelial 16HBE cells were stimulated with an aqueous cigarette smoke extract (CSE), followed by treatment with the collected serum at different dilutions. The optimal concentration and treatment time of CSE and the medicated serum for cell treatment were determined with CCK-8 assay. The expressions of TLR4, NF-κB, MUC5AC, MUC7, and muc8 at both the mRNA and protein levels in the treated cells were examined with RT- qPCR and Western blotting, and the effects of TLR4 gene silencing and overexpression on their expressions were assessed. The expressions of TNF-α, IL-1 β, IL-6 and IL-8 in the cells were detected using ELISA.
Results: At the optimal concentration of 20%, treatment with the medicated serum for 24 h significantly lowered the mRNA and protein expressions of TLR4, NF- κB, MUC5AC, MUC7, and MUC8 in CSE- exposed 16HBE cells, and these effects were further enhanced by TLR4 silencing in the cells. In 16HBE cells with TLR4 overexpression, the expressions of TLR4, NF-κB, MUC5AC, MUC7, and MUC8 were significantly increased after CSE exposure and were lowered following treatment with the medicated serum (P < 0.05). The medicated serum also significantly lowered the levels of TNF-α, IL-1β, IL-6 and IL-8 in CSE-exposed 16HBE cells (P < 0.05).
Conclusions: In the 16HBE cell model of chronic obstructive pulmonary disease (COPD), treatment with Yifei Jianpi recipe-medicated serum improves inflammation and mucus hypersecretion possibly by reducing MUC secretion and inhibiting the TLR4/NF-κB signaling pathway.
目的: 探究益肺健脾方通过TLR4/NF-κB信号通路改善人支气管上皮细胞（16HBE）炎性损伤和黏液高分泌的机制。
方法: 将40只SD大鼠随机分为两组：空白血清组、含药血清组，20只/组，对空白血清组和含药血清组大鼠分别以生理盐水、益肺健脾方药液灌胃，末次灌胃后对大鼠进行麻醉并于无菌状态下腹主动脉采血收集益肺健脾方含药血清；自制气体抽吸泵制备香烟烟雾溶液（CSE），刺激人支气管上皮细胞16HBE，并用CCK8法筛选CSE和益肺健脾方含药血清干预慢性阻塞性肺疾病细胞模型的最佳作用浓度以及最佳作用时间；qRT-PCR法检测TLR4基因沉默和过表达对16HBE中TLR4 mRNA的表达；Western blot法和qRT-PCR法检测益肺健脾方对TLR4沉默和过表达后TLR4、NF-κB、MUC5AC、MUC7、MUC8 mRNA和蛋白表达的影响；ELISA法检测细胞炎症因子TNF-α、IL-1β、IL-6和IL-8的表达。
结果: 选择20%含药血清作用24 h作为益肺健脾方最佳给药浓度及给药时间；与CSE组相比，CSE+含药血清组的TLR4、NF-κB、MUC5AC、MUC7、MUC8 mRNA及相应蛋白的表达显著降低；与CSE+含药血清组相比，CSE+siRNA-TLR4+含药血清组的TLR4、NF-κB、MUC5AC、MUC7、MUC8 mRNA及相应蛋白的表达降低；与CSE+Lv-TLR4组相比，CSE+Lv-TLR4+含药血清组的TLR4、NF-κB、MUC5AC、MUC7、MUC8 mRNA及相应蛋白的表达显著降低；与CSE+含药血清组相比，CSE+Lv-TLR4+含药血清组的TLR4、NF-κB、MUC5AC、MUC7、MUC8mRNA及相应蛋白的表达显著升高（P < 0.05）；与CSE组相比，CSE+含药血清组的TNF-α、IL-1β、IL-6和IL-8炎症因子水平下降（P < 0.05）；
结论: 益肺健脾方可干预TLR4/NF-κB信号通路，作用于COPD细胞模型，降低MUC的分泌抑制COPD气道炎症和黏液高分泌，降低与NF-κB信号通路相关的TNF-α、IL-1β、IL-6和IL-8炎症因子的表达，其机制可能与TLR4/NF-κB信号通路的基因及MUC蛋白表达有关。
Keywords: TLR4/NF-κB; Yifei Jianpi recipe; airway inflammation; chronic obstructive pulmonary disease; mucus hypersecretion.