收稿日期: 2023-04-25
网络出版日期: 2023-11-22
基金资助
陕西省重点研发计划(2021ZDLSF04-06);陕西省自然科学基础研究计划(2023-JC-QN-0820);陕西省“秦药”研发重点实验室(2021-QYPT-001);秦创园中医药创新研发转化项目(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(KF2209)
Mechanism of Sophocarpine on improving inflammation and mucous hypersecretion of human bronchial epithelial cells based on TLR4⁃NF⁃κB signaling pathway
Received date: 2023-04-25
Online published: 2023-11-22
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（10 μg/mL LPS）、SC组（10 μg/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 ~ 10 μg/mL、0 ~ 40 μg/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。
关键词： 槐果碱; NCI-H292细胞; TLR4-NF-κB信号通路; 炎症; 黏液高分泌
姜盛楠 , 支文冰 , 张红 , 王晓婷 , 孙婷婷 , 许宗仁 , 陈静 , 李晔 , 刘洋 . 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[J]. 实用医学杂志, 2023 , 39(19) : 2461 -2468 . DOI: 10.3969/j.issn.1006-5725.2023.19.009
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 （10 μg/mL LPS） and SC group （10 μg/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 ~ 10 μg/mL and 0 ~ 40 μg/mL. The results of 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-H292 cells induced by LPS and the expression of NF-κB p65 protein in nucleus （P < 0.01）. Conclusion SC may play an anti-asthmatic role by inhibiting TLR4-NF-κB signaling pathway， improving airway inflammation and mucous hypersecretion.
| 1 | BONSER L R， ERLE D J. Airway mucus and asthma： The role of MUC5AC and MUC5B［J］. J Clin Med，2017， 6（12）： 112. |
| 2 | MIMS J W. Asthma： definitions and pathophysiology［J］. Int Forum Allergy Rhinol， 2015，5 （）： S2-S6. |
| 3 | SOCKRIDER M， FUSSNER L. What is asthma？［J］. Am J Respir Crit Care Med， 2020， 202（9）：25-26. |
| 4 | NAM H H， LEE J H， RYU S M， et al. Gekko gecko extract attenuates airway inflammation and mucus hypersecretion in a murine model of ovalbumin-induced asthma［J］. J Ethnopharmacol， 2022， 282： 114574. |
| 5 | PAPADOPOULOS N G， MILIGKOS M， XEPAPADAKI P. A current perspective of allergic asthma： From mechanisms to management［J］. Handb Exp Pharmacol， 2022， 268： 69-93. |
| 6 | DING F， LIU B， ZOU W， et al. LPS exposure in early life protects against mucus hypersecretion in ovalbumin-induced asthma by down-regulation of the IL-13 and JAK-STAT6 pathways［J］. Cell Physiol Biochem， 2018， 46（3）： 1263-1274. |
| 7 | LIU H， WU X， LUO J， et al. Pterostilbene attenuates astrocytic inflammation and neuronal oxidative injury after ischemia-reperfusion by inhibiting NF-κB phosphorylation［J］. Front Immunol， 2019，10： 2408. |
| 8 | JIANG Z Y， MENG Y， BO L L，et al. Sophocarpine attenuates LPS-induced liver injury and improves survival of mice through suppressing oxidative stress， inflammation， and apoptosis［J］. Mediators Inflamm， 2018， 2018： 5871431. |
| 9 | ZHI W， DU X， LI Y， et al. Proteome profiling reveals the efficacy and targets of sophocarpine against asthma［J］. Int Immunopharmacol， 2021，96： 107348. |
| 10 | CHEN L， XU J， DENG M， et al. Telmisartan mitigates lipopolysaccharide （LPS）-induced production of mucin 5AC （MUC5AC） through increasing suppressor of cytokine signaling 1 （SOCS1）［J］. Bioengineered， 2021，12（1）： 3912-3923. |
| 11 | SUNG Y Y， KIM S H， YUK H J， et al. Siraitia grosvenorii residual extract attenuates ovalbumin-induced lung inflammation by down-regulating IL-4， IL-5， IL-13， IL-17， and MUC5AC expression in mice［J］. Phytomedicine， 2019， 61： 152835. |
| 12 | 黄帅阳，崔红生，候丹，等. PNEC-ILC2神经免疫通路在支气管哮喘中的研究进展［J］. 实用医学杂志， 2022， 38（1）： 120-124. |
| 13 | CHEN X， YANG J， SHEN H， et al. Muc5ac production inhibited by decreased lncRNA H19 via PI3K/Akt/NF-kB in asthma［J］. J Asthma Allergy， 2021， 14： 1033-1043. |
| 14 | GANS M D， GAVRILOVA T. Understanding the immunology of asthma： Pathophysiology， biomarkers， and treatments for asthma endotypes［J］. Paediatr Respir Rev，2020， 36： 118-127. |
| 15 | MA L， CHANG E， RUAN X， et al. The protective effects of Omarigliptin against Lipopolysaccharide （LPS）- induced inflammatory response and expression of mucin 5AC （MUC5AC） in human bronchial epithelial cells［J］. Mol Immunol， 2022， 141： 108-115. |
| 16 | 杨梦， 张伟， 李强， 等. 低氧对人气道上皮细胞NCI-H292黏液合成的影响［J］. 西部医学， 2016， 28（5）： 615-620，624. |
| 17 | SONG W Y， SONG Y S， RYU H W， et al. Tilianin inhibits MUC5AC expression mediated via down-regulation of EGFR-MEK-ERK-Sp1 signaling pathway in NCI-H292 human airway cells［J］. J Microbiol Biotechnol， 2017，27（1）： 49-56. |
| 18 | LEE G H， JIN S W， CHOI J H， et al. Influence of o，p'-DDT on MUC5AC expression via regulation of NF-κB/AP-1 activation in human lung epithelial cells［J］. J Toxicol Environ Health A， 2021， 84（20）： 836-845. |
| 19 | 朱林筠，孙亦鹏，王雄彪. 中药抗哮喘气道炎症机制的研究进展［J］. 上海中医药杂志，2021， 55（3）： 98-104. |
| 20 | DONG Y， ZHANG X， YAO C， et al. Atractylodin attenuates the expression of MUC5AC and extracellular matrix in lipopolysaccharide-induced airway inflammation by inhibiting the NF-κB pathway［J］. Environ Toxicol， 2021， 36（9）： 1911-1922. |
| 21 | XUE K， RUAN L， HU J， et al. Panax notoginseng saponin R1 modulates TNF-α/NF-κB signaling and attenuates allergic airway inflammation in asthma［J］. Int Immunopharmacol， 2020， 88： 106860. |
| 22 | KIM Y D， ChOI Y S， NA H G， et al. Ginsenoside Rb1 attenuates LPS-induced MUC5AC expression via the TLR4-mediated ERK1/2 and NF-κB pathway in human airway epithelial NCI-H292 cells［J］. J Biol Regul Homeost Agents， 2020， 34（2）： 613-618. |
| 23 | SHI X， WANG S， LUAN H， et al. Clinopodium chinense attenuates palmitic acid-induced vascular endothelial inflammation and insulin resistance through TLR4-Mediated NF-κB and MAPK pathways［J］. Am J Chin Med， 2019，47（1）： 97-117. |
| 24 | PAN H H， HSIAO Y P， CHEN P J， et al. Epithelial growth factor receptor tyrosine kinase inhibitors alleviate house dust mite allergen Der p2-induced IL-6 and IL-8［J］. Environ Toxicol， 2019， 34（4）： 476-485. |
| 25 | ZHANG R G， NIU Y， PAN K W， et al. β2-adrenoceptor activation stimulates IL-6 production via PKA， ERK1/2， Src， and Beta-arrestin2 signaling pathways in human bronchial epithelia［J］. Lung，2021，199（6）： 619-627. |
| 26 | SHUKUR W， ALYAQUBI K， DOSH R， et al. Association of Toll-like receptors 4 （TLR-4） gene expression and polymorphisms in patients with severe asthma［J］. J Med Life， 2021， 14（4）： 544-548. |
| 27 | WU Z， MEHRABI NASAB E， ARORA P， et al. Study effect of probiotics and prebiotics on treatment of OVA-LPS-induced of allergic asthma inflammation and pneumonia by regulating the TLR4/NF-κB signaling pathway［J］. J Transl Med， 2022， 20（1）： 130. |
| 28 | 万浩宇，张璐，万海同，等. 基于p38MAPK/NF-κB信号通路的麻黄汤抗哮喘作用机制研究［J］. 中国药理学通报，2021，37（3）： 423-429. |
/
| 〈 |
|
〉 |