基于TLR4/NF-κB通路探讨石斛多糖改善CSE诱导的人支气管上皮细胞炎性损伤  

Dendrobium Polysaccharides Mitigate CSE-induced Inflammatory Damage of Human Bronchial Epithelial Cells via TLR4/NF-κB Pathway

作　　者：王蕾 王雅娟[1] 贺增洋 许凤清 王文斌[2] 王桐生[1] 舒俊生[2] WANG Lei;WANG Yajuan;HE Zengyang;XU Fengqing;WANG Wenbin;WANG Tongsheng;SHU Junsheng(Anhui University of Chinese Medicine,Hefei 230012,China;Technology Center of China Tobacco Anhui Industrial Co.Ltd,Hefei 230088,China;College of Pharmacy,Anhui University of Chinese Medicine,Hefei 230012,China)

机构地区：[1]安徽中医药大学,合肥230012 [2]安徽中烟工业有限责任公司技术中心,合肥230088 [3]安徽中医药大学药学院,合肥230012

出　　处：《中国实验方剂学杂志》2023年第23期64-71,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：安徽省高校自然科学研究项目(KJ2021A0602);校企联合科研项目(202034000034027)。

摘　　要：目的:探讨石斛多糖对香烟烟雾提取物(CSE)损伤的人支气管上皮细胞(16HBE)的炎症因子分泌及TLR4/NF-κB通路活性的影响。方法:体外培养16HBE细胞,设置空白组、CSE损伤组、CSE+石斛多糖组(100、200、400 mg·L^(-1)),CCK-8法检测细胞活力,显微镜观察细胞状态,酶联免疫吸附测定法(ELISA)检测细胞培养上清中白细胞介素(IL)-8、IL-1β、IL-4、IL-13、转化生长因子(TGF)-β含量,实时荧光定量聚合酶链式反应(Real-time PCR)检测Toll样受体4(TLR4)、核转录因子-κB(NF-κB)及IL-4 mRNA的相对表达量,蛋白免疫印迹法(Western blot)检测白细胞介素-4受体(IL-4R)、TLR4、髓系分化初级反应蛋白88(MyD88)、NF-κB、磷酸化NF-κB(p-NF-κB)及核蛋白NF-κB(NEs-NF-κB)蛋白相对表达量,免疫荧光法检测NF-κB核转位水平。结果:与空白组比较,CSE损伤组细胞存活率降低,细胞皱缩,细胞培养上清中IL-8、IL-1β、IL-4、IL-13、TGF-β含量明显增加(P<0.05,P<0.01),TLR4、MyD88、NF-κB、p-NF-κB、NEs-NF-κB、IL-4表达水平显著升高(P<0.01),NF-κB入核现象明显;与CSE损伤组比较,石斛多糖各剂量组细胞存活率显著升高,细胞状态恢复,IL-8、IL-1β、IL-4、IL-13、TGF-β含量及TLR4、MyD88、NF-κB、p-NF-κB、NEs-NF-κB、IL-4表达水平明显降低(P<0.05,P<0.01),NF-κB入核明显减少。结论:石斛多糖对CSE诱导的16HBE细胞损伤具有明显的保护作用,其机制可能与抑制TLR4/NF-κB信号通路活性有关。Objective:To observe the effects of Dendrobium polysaccharides on the secretion of inflammatory cytokines and Toll-like receptor 4(TLR4)/nuclear factor(NF)-κB pathway in 16HBE cells exposed to cigarette smoke extract(CSE).Method:The 16HBE cells were classified into the control,CSE,and CSE+Dendrobium polysaccharides(100,200,400 mg·L^(-1))groups.The cell-counting kit-8(CCK-8)assay was employed to measure the cell viability,and a microscope was used to observe the cell morphology.The enzyme-linked immunosorbent assay was employed to measure the levels of interleukin(IL)-8,IL-1β,IL-4,IL-13,and transforming growth factor(TGF)-βin cell culture supernatants.Real-time PCR was carried out to determine the mRNA levels of Toll-like receptor 4(TLR4),nuclear factor-κB(NF-κB),and IL-4.Western blot was employed to determine the protein levels of interleukin-4 receptor(IL-4R),TLR4,myeloid differentiation primary response protein 88(MyD88),NF-κB,phosphorylated nuclear factor-κB(p-NF-κB),and nucleoproteins nuclear factor-κB(NEs-NF-κB).The immunofluorescence assay was employed to measure the nuclear translocation of NF-κB.Result:Compared with the control group,the CSE group showed elevated levels of IL-8,IL-1β,IL-4,IL-13,and TGF-βin the cell culture supernatants(P<0.05,P<0.01),up-regulated expression levels of TLR4,MyD88,NF-κB,p-NF-κB,NEs-NF-κB,and IL-4(P<0.01),and significant nuclear translocation of NF-κB.Compared with the CSE group,Dendrobium polysaccharides increased the cell survival rate,recovered the cell activity,lowered the levels of IL-8,IL-1β,IL-4,IL-13,and TGF-β,downregulated the expression of TLR4,MyD88,NF-κB,p-NF-κB,NEs-NF-κB,and IL-4(P<0.05,P<0.01),and reduced the nuclear translocation of NF-κB.Conclusion:Dendrobium polysaccharides showed significant protective effects on the 16HBE cells exposed to CSE by inhibiting the TLR4/NF-κB signaling pathway.

关 键 词：石斛多糖 人支气管上皮细胞(16HBE) 香烟烟雾提取物(CSE) 炎症因子 Toll样受体4(TLR4) 核转录因子-κB(NF-κB) 

分 类 号：R2-0[医药卫生—中医学] R22R285.5R289R33