| 基于TLR4/PI3K/Akt/NF-κB信号通路探讨热毒宁注射液抑制脂多糖刺激的BEAS-2B细胞炎症因子表达的作用机制 |
| 投稿时间：2024-04-25 点此下载全文
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| 引用本文：康建英,颜丽珊,顾春宇,邱新宇,王亦巍,罗赣,张翼.基于TLR4/PI3K/Akt/NF-κB信号通路探讨热毒宁注射液抑制脂多糖刺激的BEAS-2B细胞炎症因子表达的作用机制[J].中国现代中药,2024,26(11):1938-1946 |
| DOI：10.13313/j.issn.1673-4890.20240425003 |
| 摘要点击次数: 288 |
| 全文下载次数: 0 |
| 作者中文名 | 作者英文名 | 单位中文名 | 单位英文名 | E-Mail |
| 康建英 |
KANG Jian-ying |
北京中医药大学 中药学院，北京 102488 |
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing102488, China |
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| 颜丽珊 |
YAN Li-shan |
北京中医药大学 中药学院，北京 102488 |
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing102488, China |
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| 顾春宇 |
GU Chun-yu |
北京中医药大学 中药学院，北京 102488 |
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing102488, China |
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| 邱新宇 |
QIU Xin-yu |
北京中医药大学 中药学院，北京 102488 |
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing102488, China |
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| 王亦巍 |
WANG Yi-wei |
北京中医药大学 中药学院，北京 102488 |
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing102488, China |
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| 罗赣 |
LUO Gan |
北京中医药大学 中药学院，北京 102488 |
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing102488, China |
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| 张翼* |
ZHANG Yi |
北京中医药大学 中药学院，北京 102488 |
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing102488, China |
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| 基金项目:国家自然科学基金青年基金项目（82003957） |
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| 中文摘要:目的 基于Toll样受体4（TLR4）/磷脂肌醇3-激酶（PI3K）/蛋白激酶B（Akt）/核转录因子-κB（NF-κB）信号通路探究热毒宁注射液（RDN）对脂多糖（LPS）刺激的人支气管上皮细胞BEAS-2B炎症因子表达的影响及机制。方法 采用高效液相色谱法（HPLC）测定RDN冻干粉中栀子苷和绿原酸含量；采用分子对接技术评估栀子苷和绿原酸与TLR4的结合能力。通过体外实验构建LPS（1 μg·mL–1）刺激的BEAS-2B细胞气道炎症模型；采用噻唑蓝（MTT）法检测LPS诱导的BEAS-2B细胞活力；通过实时荧光定量聚合酶链式反应（qRT-PCR）法检测LPS诱导的BEAS-2B细胞炎症因子mRNA的表达；通过免疫印迹法（WB）检测LPS诱导的BEAS-2B细胞TLR4/PI3K/Akt/NF-κB信号通路相关蛋白质的表达；采用免疫荧光法（IF）检测转录因子c-Jun和p65的入核情况。结果 RDN冻干粉中栀子苷和绿原酸的质量分数分别为127.00、70.26 mg·g–1；栀子苷和绿原酸均能与TLR4结合，结合能分别为–7.0、–7.9 kcal·mol–1。质量浓度为12.5~400.0 μg·mL–1时，RDN对LPS刺激的BEAS-2B细胞活力无显著的抑制作用；质量浓度为200.0~400.0 μg·mL–1时，RDN能明显下调白细胞介素-1β（IL-1β）、IL-6、趋化因子CXC配体2（CXCL2）、CXCL5、趋化因子配体5（CCL5）、CCL20 mRNA的表达。此外，RDN能降低TLR4/PI3K/Akt/NF-κB信号通路相关蛋白质PI3K、Akt、TANK结合激酶1（TBK1）、IκB激酶（IKK）α/β、NF-κB抑制因子α（IκBα）、p65、p38、c-Jun N-末端激酶（JNK）、胞外信号调节激酶（ERK）和c-Jun的磷酸化水平，同时抑制LPS激活的BEAS-2B细胞p65和c-Jun的入核。结论 RDN能下调LPS诱导的BEAS-2B细胞炎症因子mRNA的表达，其机制与RDN阻断TLR4/PI3K/Akt/NF-κB信号通路有关。 |
| 中文关键词:热毒宁注射液 气道炎症 人支气管上皮细胞 Toll样受体4/磷脂肌醇-3-激酶/蛋白激酶B/核转录因子-κB信号通路 脂多糖 |
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| Mechanism of Reduning Injection in Inhibiting the Expression of Inflammatory Mediators in BEAS-2B Cells Stimulated by Lipopolysaccharide Based on TLR4/PI3K/Akt/NF-κB Signaling Pathway |
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| Abstract:Objective To investigate the effect and mechanism of Reduning Injection (RDN) on the expression of inflammatory factors in human bronchial epithelial cells (BEAS-2B) stimulated by lipopolysaccharide (LPS) based on the Toll-like receptor 4 (TLR4)/phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/nuclear factor-κB (NF-κB) signaling pathway.Methods The content of geniposide and chlorogenic acid in RDN freeze-dried powder was determined by high-performance liquid chromatography (HPLC). Molecular docking was used to assess the binding abilities of geniposide and chlorogenic acid to TLR4. An airway inflammation model in BEAS-2B cells was established by stimulating the cells with LPS (1 μg·mL–1) in vitro. The viability of LPS-induced BEAS-2B cells was detected using the methyl thiazolyl tetrazolium (MTT) assay. The mRNA expression of inflammatory mediators in LPS-induced BEAS-2B cells was measured by quantitative real-time PCR (qRT-PCR). The expression levels of proteins related to the TLR4/PI3K/Akt/NF-κB signaling pathway in LPS-induced BEAS-2B cells were determined by Western blot (WB). The nuclear translocation of transcription factors c-Jun and p65 was detected by immunofluorescence (IF) assay.Results The mass fractions of geniposide and chlorogenic acid in RDN freeze-dried powder were 127.00 and 70.26 mg·g–1, respectively. Both geniposide and chlorogenic acid were able to bind to TLR4, with binding energies of –7.0 and –7.9 kcal·mol–1, respectively. RDN at concentrations of 12.5-400.0 μg·mL–1 did not significantly inhibit the viability of LPS-stimulated BEAS-2B cells. RDN at concentrations of 200.0-400.0 μg·mL–1 significantly downregulated the expression of IL-1β, IL-6, CXCL2, CXCL5, CCL5, and CCL20 mRNA. Additionally, RDN reduced the phosphorylation levels of proteins related to the TLR4/PI3K/Akt/NF-κB signaling pathway, including PI3K, Akt, TANK-binding kinase 1 (TBK1), IκB kinase α/β (IKKα/β), inhibitor of NF-κB α (IκBα), p65, p38, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and c-Jun. It also inhibited the nuclear translocation of p65 and c-Jun in LPS-stimulated BEAS-2B cells.Conclusion RDN downregulates the mRNA expression of inflammatory factors in LPS-induced BEAS-2B cells, and this effect is associated with the inhibition of the TLR4/PI3K/Akt/NF-κB signaling pathway. |
| keywords:Reduning Injection airway inflammatory BEAS-2B cells TLR4/PI3K/Akt/NF-κB signaling pathway lipopolysaccharides |
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