基于TLR4-NF-κB信号通路的槐果碱改善人支气管上皮细胞炎症和黏液高分泌的机制  

R285.5Mechanism of Sophocarpine on improving inflammation and mucous hypersecretion of human bronchial epithelial cells based on TLR4-NF-κB signaling pathway

作　　者：姜盛楠 支文冰 张红[1] 王晓婷 孙婷婷[1] 许宗仁 陈静[2] 李晔[1] 刘洋[1] JIANG Shengnan;ZHI Wenbing;ZHANG Hong;WANG Xiaoting;SUN Tingting;XU Zongren;CHEN Jing;LI Ye;LIU Yang(Shanxi Academy of Traditional Chinese Medicine(Shanxi Traditional Chinese Medicine Hospital),Xi'an 710003,China;不详)

机构地区：[1]陕西省中医药研究院(陕西省中医医院),西安710003 [2]陕西中医药大学,陕西咸阳712046

出　　处：《实用医学杂志》2023年第19期2461-2468,共8页The Journal of Practical Medicine

基　　金：陕西省重点研发计划(编号:2021ZDLSF04-06,2022ZDXM-SF-06);陕西省自然科学基础研究计划(编号:2023-JC-QN-0820);陕西省“秦药”研发重点实验室(编号:2021-QYPT-001);秦创园中医药创新研发转化项目(编号:2022-QCYZH-006);陕西中医药大学省级中医体质与疾病防治研究重点实验室(编号:KF2209)。

摘　　要：目的基于TLR4-NF-κB信号通路探讨槐果碱(SC)体外抗炎作用及其抗哮喘的潜在效应机制。方法采用脂多糖(LPS)刺激人支气管上皮细胞NCI-H292诱导体外炎症模型,给予不同浓度SC进行治疗。采用MTT法筛选LPS和SC对NCI-H292细胞的安全浓度;设Control组、LPS组(10μg/mL LPS)、SC组(10μg/mL LPS+不同浓度SC),ELISA法检测细胞中黏蛋白5AC(MUC5AC)的表达和上清液中白细胞介素-6(IL-6)和白细胞介素-8(IL-8)的分泌情况;RT-PCR法检测细胞中MUC5AC、IL-6、IL-8、髓样分化因子(MyD88)和核因子-κB(NF-κB p65)mRNA的表达;Western blot方法检测细胞中Toll样受体4(TLR4)、肿瘤坏死因子受体相关蛋白6(TRAF6)、磷酸化p38(p-p38)和磷酸化p65(p-p65)以及细胞核内NF-κB p65的蛋白表达。结果MTT结果显示,LPS和SC分别在0~10μg/mL、0~40μg/mL浓度范围内对细胞活力无影响;ELISA结果表明,与Control组相比,LPS组细胞中MUC5AC含量极显著升高(P<0.01),细胞上清液中IL-6、IL-8也极显著升高(P<0.01);与LPS组相比,SC组细胞中MUC5AC含量极显著降低(P<0.01),细胞上清液中IL-6、IL-8也极显著降低(P<0.01);RT-PCR结果显示,与Control组相比,LPS组细胞中MUC5AC、IL-6、IL-8、MyD88和NF-κB p65 mRNA表达均极显著升高(P<0.01),而SC组细胞中MUC5AC、IL-6、IL-8 mRNA的表达显著降低(P<0.05),MyD88和NF-κB p65 mRNA的表达极显著降低(P<0.01)。Western blotting结果表明,SC可极显著降低LPS诱导的NCI-H292细胞中TRAF6、TLR4、p-p38和p-p65以及细胞核内NF-κB p65蛋白表达(P<0.01)。结论SC可能通过抑制TLR4-NF-κB信号通路,改善气道炎症和黏液高分泌,进而发挥抗哮喘的作用。Objective To investigate the anti-inflammatory effect of sophocarpine(SC)in vitro and the potential mechanism of its anti-asthma effect based on the TLR4-NF-κB signaling pathway.Methods NCI-H292 cells were stimulated by Lipopolysaccharide(LPS)to induce inflammation model in vitro,and SC was given in different concentrations.The safe concentrations of LPS and SC on NCI-H292 cells were screened by MTT method,and the control group,LPS group(10μg/mL LPS)and SC group(10μg/mL LPS+different concentrations of SC)were set up.The expression of mucin 5AC(MUC5AC)in cells and the secretion of Interleukin-6(IL-6)and Interleukin-8(IL-8)in the supernatant were detected by ELISA.The mRNA expression of MUC5AC,IL-6,IL-8,myeloid differentiation factor 88(MyD88)and nuclear factor-kappa B p65(NF-κB p65)in cells was detected by RT-PCR.The protein expression of TLR4,tumor necrosis factor receptor-associated factor 6(TRAF6),p-p38 and p-p65 in cells and NF-κB p65 in nucleus was detected by Western blotting.Results MTT results showed that LPS and SC had no effect on cell viability in the concentration range of 0~10μg/mL and 0~40μg/mL.The results 19of ELISA showed that the content of MUC5AC in LPS group was significantly higher than that in control group(P<0.01),and the levels of IL-6 and IL-8 in the supernatant of cells were also extremely significant increase(P<0.01).Compared with the model group,the content of MUC5AC in SC group extremely significant reduction(P<0.01),and the content of IL-6 and IL-8 in cell supernatant also significantly reduced(P<0.01).RT-PCR results showed that the expressions of MUC5AC,IL-6,IL-8,MyD88 and NF-κB p65 mRNA in LPS group were extremely significant higher than those in control group(P<0.01),however,the expression of MUC5AC,IL-6 and IL-8 mRNA in SC group decreased significantly(P<0.05),and the expression of MyD88 and NF-κB p65 mRNA extremely significant reduction(P<0.01).Western blotting results showed that SC could extremely significant reduction the expression of TRAF6,TLR4,p-p38 and p-p65 in NCI

关 键 词：槐果碱 NCI-H292细胞 TLR4-NF-κB信号通路 炎症 黏液高分泌 

分 类 号：R285.5[医药卫生—中药学]