目的 探討蛤蚧定喘丸的抗炎機(jī)制，推測蛤蚧定喘丸發(fā)揮抗炎功效的關(guān)鍵活性成分。方法 依次采用石油醚、無水乙醇、超純水對蛤蚧定喘丸不同極性的化學(xué)成分進(jìn)行提?。焕?、4、8、16、32、64、128 μg·mL^-1的醚提物、醇提物、水提物處理RAW264.7細(xì)胞，分別通過CellTiter-Lumi（CTL）發(fā)光法和鈣黃綠素/碘化丙啶（Calcein/PI）染色法檢測細(xì)胞活力，確定提取物的安全濃度；利用各提取物處理脂多糖（LPS）誘導(dǎo)的RAW264.7細(xì)胞炎癥模型，通過格里斯試劑（Griess）測定一氧化氮（NO）釋放量，通過酶聯(lián)免疫吸附（ELISA）法檢測白細(xì)胞介素-1β（IL-1β）、腫瘤壞死因子-α（TNF-α）分泌，篩選具有抗炎活性的提取物。利用超高效液相色譜-四極桿/靜電場軌道阱高分辨質(zhì)譜技術(shù)（UHPLC-ESI-QE-OrbitrapMS）對有抗炎活性的提取物進(jìn)行物質(zhì)分析，推測抗炎活性物質(zhì)及作用機(jī)制，并利用蛋白免疫印跡法（Western blotting）進(jìn)一步驗(yàn)證其對硫氧還蛋白互作蛋白（TXNIP）/p-核因子κB抑制蛋白α（p-IκBα）/核因子-κB（NF-κB）/NOD樣受體熱蛋白結(jié)構(gòu)域相關(guān)蛋白3（NLRP3）信號通路相關(guān)蛋白表達(dá)的影響。結(jié)果 醚提物、醇提物、水提物在質(zhì)量濃度低于64 μg·mL^-1時均未影響細(xì)胞活力。與模型組相比，醇提物在質(zhì)量濃度大于8 μg·mL^-1時顯著降低NO釋放量（P<0.05），醚提物和醇提物在質(zhì)量濃度高達(dá)64 μg·mL^-1時仍未產(chǎn)生抗炎效果；醇提物顯著降低了TNF-α、IL-1β的釋放量（P<0.05）。醇提物中共分析出36種主要化學(xué)成分。經(jīng)Western blotting實(shí)驗(yàn)驗(yàn)證，與模型組比較，蛤蚧定喘丸醇提物對RAW264.7細(xì)胞炎癥模型內(nèi)NLRP3炎癥小體、β干擾素TIR結(jié)構(gòu)域銜接蛋白（TRIF）、誘導(dǎo)型一氧化氮合酶（iNOS）、單核細(xì)胞趨化蛋白-1（MCP-1）、p-IκBα的表達(dá)均有顯著降低作用（P<0.05），而對TXNIP、Toll樣受體4（TLR4）、絲裂原活化蛋白激酶（MAPK）信號通路蛋白的影響并不顯著。結(jié)論 蛤蚧定喘丸通過抑制TRIF/p-IκBα/NF-κB/NLRP3信號通路以及iNOS、MCP-1的合成發(fā)揮抗炎功效，推測巴馬汀、麻黃堿、偽麻黃堿、小檗堿、甘草素、藥根堿、小檗紅堿為其抗炎活性成分。

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, 128 μg·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)-1β and tumor necrosis factor (TNF)-α was 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 factor κB inhibitory protein α (p-IκBα)/nuclear factor κB (NF- κB)/NOD-like receptor thermal protein domain associated protein 3 (NLRP3) signaling pathway. Results The safe concentration of extracts was below 64 μg·mL^-1. Compared with the model group, at the ethanol extract had a mass concentration greater than 8 μg·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 64 μg·mL^-1. Ethanol extract significantly reduced the release amount of TNF-α and 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-IκBα in 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.

R285.5

江蘇省市場監(jiān)管局科技計(jì)劃項(xiàng)目（KJ2023044）