UHPLC-ESI-QE-Orbitrap-MS结合TRIF/p-IκBα/NF-κB/NLRP3信号通路的蛤蚧定喘丸抗炎机制与活性成分探讨

Study on anti-inflammatory mechanism and active ingredient of Gejie Dingchuan Pill based on UHPLC-ESI-QE-Orbitrap-MS technology and TRIF/p-IκBα/NF-κB/NLRP3 signaling pathway

目的探讨蛤蚧定喘丸的抗炎机制,推测蛤蚧定喘丸发挥抗炎功效的关键活性成分。方法依次采用石油醚、无水乙醇、超纯水对蛤蚧定喘丸不同极性的化学成分进行提取;利用2、4、8、16、32、64、128μg·mL^(-1)的醚提物、醇提物、水提物处理RAW264.7细胞,分别通过CellTiter-Lumi(CTL)发光法和钙黄绿素/碘化丙啶(Calcein/PI)染色法检测细胞活力,确定提取物的安全浓度;利用各提取物处理脂多糖(LPS)诱导的RAW264.7细胞炎症模型,通过格里斯试剂(Griess)测定一氧化氮(NO)释放量,通过酶联免疫吸附(ELISA)法检测白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)分泌,筛选具有抗炎活性的提取物。利用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱技术(UHPLC-ESI-QE-OrbitrapMS)对有抗炎活性的提取物进行物质分析,推测抗炎活性物质及作用机制,并利用蛋白免疫印迹法(Western blotting)进一步验证其对硫氧还蛋白互作蛋白(TXNIP)/p-核因子κB抑制蛋白α(p-IκBα)/核因子-κB(NF-κB)/NOD样受体热蛋白结构域相关蛋白3(NLRP3)信号通路相关蛋白表达的影响。结果醚提物、醇提物、水提物在质量浓度低于64μg·mL-1时均未影响细胞活力。与模型组相比,醇提物在质量浓度大于8μg·mL-1时显著降低NO释放量(P<0.05),醚提物和醇提物在质量浓度高达64μg·mL-1时仍未产生抗炎效果;醇提物显著降低了TNF-α、IL-1β的释放量(P<0.05)。醇提物中共分析出36种主要化学成分。经Western blotting实验验证,与模型组比较,蛤蚧定喘丸醇提物对RAW264.7细胞炎症模型内NLRP3炎症小体、β干扰素TIR结构域衔接蛋白(TRIF)、诱导型一氧化氮合酶(iNOS)、单核细胞趋化蛋白-1(MCP-1)、p-IκBα的表达均有显著降低作用(P<0.05),而对TXNIP、Toll样受体4(TLR4)、丝裂原活化蛋白激酶(MAPK)信号通路蛋白的影响并不显著。结论蛤蚧定喘丸通过抑制TRIF/p-IκBα/NF-κB/NLRP3信号通路以及iNOS、MCP-1的合成发挥抗炎功效,推测巴马汀、麻黄碱、伪麻黄碱、小檗碱、甘草素、药根碱、小檗红碱为其抗炎活性成分。

Objective To explore the mechanism and key active substances of Gejie Dingchuan Pill for its anti-inflammatory effect.Methods The chemical components with different polarity in Gejie Dingchuan Pill were extracted by petroleum ether,absolute ethanol and ultrapure water in turn.RAW264.7 cells were treated with 2,4,8,16,32,64,128μg·mL−1 ether extract,alcohol extract and water extract,then the cell viability was detected by CellTiter Lumi(CTL)luminescence and Calcein/PI staining to determine the safe concentration of different extracts;Different extracts were used to treat LPS-induced RAW264.7 cells,then the release of nitric oxide(NO)was measured by Griess reagent,the release of interleukin(IL)-1βand tumor necrosis factor(TNF)-αwas detected by enzyme-linked immunosorbent assay(ELISA)to hunt the extracts with anti-inflammatory activity.The extracts with antiinflammatory activity were analyzed by ultrahigh performance liquid chromatography quadrupole/electrostatic field orbitaltrap highresolution mass spectrometry(UHPLC-ESI-QE-Orbitrap-MS),Western blotting was performed further verified the effect of thioredoxin interacting protein(TXNIP)/p-nuclear factorκB inhibitory proteinα(p-IκBα)/nuclear factorκB(NF-κB)/NOD-like receptor thermal protein domain associated protein 3(NLRP3)signaling pathway.Results The safe concentration of extracts was below 64μg·mL−1.Compared with the model group,at the ethanol extract had a mass concentration greater than 8μg·mL−1,there were a significant decrease in NO release(P<0.05).No anti-inflammatory effect was observed at with ether and alcohol extracts reaching a mass concentration of up to 64μg·mL−1.Ethanol extract significantly reduced the release amount of TNF-αand IL-1β(P<0.05).A total of 36 main chemical components were identified in the ethanol extract.The Western blotting experiment verified that the ethanol extract of Gejie Dingchuan Pill could inhibit the expression of NLRP3 inflammatory bodies,TRIF,iNOS,MCP-1 and p-IκBαin LPS-induced RAW264.7 cells(P<0.05),but not affect the expression of TXNIP,TLR4 and MAPK signaling pathway proteins.Conclusion Gejie Dingchuan Pill could play an anti-inflammatory role by inhbiting TRIF/p-IκBα/NF-κB/NLRP3 pathway and the synthesis of iNOS and MCP-1.It is speculated that palmatine,pseudo ephedrine,berberine,liquiritigenin,jatrorrhizine and berberrubine are the key substances for Gejie Dingchuan Pill to exert anti-inflammatory activity.