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

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

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(1μg·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12.5~400.0μg·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200.0~400.0μg·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。

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 Tolllike 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 highperformance 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(1μg·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 12.5-400.0μg·mL-1 did not significantly inhibit the viability of LPS-stimulated BEAS-2B cells.RDN at concentrations of 200.0-400.0μg·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 kinaseα/β(IKKα/β),inhibitor of NF-κBα(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.