目的 采用活細(xì)胞固相色譜法聯(lián)合高分辨質(zhì)譜技術(shù)快速篩選鑒定丹參標(biāo)準(zhǔn)湯劑中潛在的抗神經(jīng)病理性疼痛活性成分,通過網(wǎng)絡(luò)藥理學(xué)技術(shù)分析其作用機(jī)制。方法 小鼠背根神經(jīng)元細(xì)胞與丹參標(biāo)準(zhǔn)湯劑磷酸鹽緩沖液(PBS)溶液共孵育,吸附成分采用超高效液相色譜-四極桿-靜電場軌道阱高分辨質(zhì)譜(UPLC-Q-Orbitrap HRMS)鑒別;鑒定得到的活性成分基于網(wǎng)絡(luò)藥理學(xué)分析其作用機(jī)制:采用中藥系統(tǒng)藥理學(xué)數(shù)據(jù)庫與分析平臺(TCMSP)數(shù)據(jù)庫聯(lián)合 Swiss 數(shù)據(jù)庫尋找成分作用靶點(diǎn),以"neuropathic pain"為關(guān)鍵詞在 Genecards 數(shù)據(jù)庫及 OMIM 數(shù)據(jù)庫檢索去重即得疾病靶點(diǎn),將成分靶點(diǎn)與神經(jīng)病理性疼痛靶點(diǎn)交集篩選得到共同靶點(diǎn);將共同靶點(diǎn)導(dǎo)入 STRING數(shù)據(jù)庫進(jìn)行檢索,得蛋白質(zhì)相互作用(PPI)網(wǎng)絡(luò),并導(dǎo)入Cytoscape 3.7.2 繪圖,以度值(degree)為標(biāo)準(zhǔn)選取前 10 靶點(diǎn)作為關(guān)鍵靶點(diǎn);將共同靶點(diǎn)采用 ClusterProfiler 包進(jìn)行基因本體(GO)、京都基因與基因組百科全書(KEGG)富集分析。結(jié)果 共鑒定得到以酚酸類物質(zhì)為主的潛在活性成分 11 個,分別為香草酸、丹參素、咖啡酸、原兒茶酸、原兒茶醛、阿魏酸、迷迭香酸、紫草酸、丹酚酸 B、異丹酚酸 B、丹酚酸 E。網(wǎng)絡(luò)藥理學(xué)分析結(jié)果顯示活性成分作用靶點(diǎn)174 個,與神經(jīng)病理性疼痛靶點(diǎn)集交集 67 個;PPI 分析顯示信號轉(zhuǎn)導(dǎo)及轉(zhuǎn)錄激活蛋白 3(STAT3)、白蛋白(ALB)、原癌基因 c-Jun(JUN)、淀粉樣 β 前體蛋白(APP)、基質(zhì)金屬蛋白酶 9(MMP9)、胱天蛋白酶3(CASP3)、toll 樣受體 4(TLR4)、絲裂原活化蛋白激酶 1(MAPK1)、環(huán)加氧酶 2(PTGS2)、轉(zhuǎn)錄因子 p65(RELA)為關(guān)鍵靶點(diǎn),"藥味成分-靶點(diǎn)"網(wǎng)絡(luò)拓?fù)浞治鲲@示各活性成分可能存在協(xié)同作用;GO 富集分析顯示與碳酸氫鹽轉(zhuǎn)運(yùn)、對脂多糖反應(yīng)、對細(xì)菌來源分析反應(yīng)等生物過程,膜筏、膜微區(qū)、膜區(qū)、分泌顆粒管腔等細(xì)胞組成以及碳酸脫水酶活性、水解酶活性、碳氧裂解酶活性等分子功能相關(guān);KEGG 富集顯示與氮代謝、糖尿病并發(fā)癥中的晚期糖基化產(chǎn)物(AGE)-晚期糖基化終末產(chǎn)物受體(RAGE)信號通路、脂質(zhì)和動脈粥樣硬化、缺氧誘導(dǎo)因子 1(HIF-1)信號通路等通路相關(guān);結(jié)合文獻(xiàn)分析丹參抗神經(jīng)病理性疼痛起效機(jī)制與炎癥和免疫反應(yīng)、神經(jīng)元恢復(fù)及再生、代謝紊亂、病毒、疼痛遞質(zhì)傳導(dǎo)、神經(jīng)元超敏、疼痛閾等相關(guān)。結(jié)論 應(yīng)用活細(xì)胞固相色譜與高分辨質(zhì)譜聯(lián)用技術(shù)篩選出丹參標(biāo)準(zhǔn)湯劑中抗神經(jīng)病理性疼痛活性成分 11 個,橋接網(wǎng)絡(luò)藥理學(xué)分析其作用機(jī)制可能與炎癥和免疫反應(yīng)、調(diào)節(jié)代謝紊亂、抑制超敏等相關(guān)。

Objective To rapidly screen and identify the potential anti-neuropathic pain active components in Salvia miltiorrhiza standard decoction by live cell extraction and liquid chromatography-mass spectrometry, and analyze the mechanism of action by network pharmacology. Methods Using mouse dorsal root neuron cells co-incubated with Salvia miltiorrhiza PBS solution, the adsorption components were identified by UPLC-Q-Orbitrap HRMS, and the components were analyzed based on network pharmacology and their mechanism of action. TCMSP database and Swiss database were used to search for the target of component action. The disease target was retrieved from Genecards database and OMIM database with the keyword "neuropathic pain". The common target was obtained by interscreening the component target and neuropathic pain target. The common targets were imported into the STRING database for retrieval to obtain protein interaction (PPI) network, and then imported into Cytoscape 3.7.2 for drawing. The top 10 targets were selected as key targets according to the degree value. Common targets were analyzed using the ClusterProfiler package for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Results A total of 11 potential active ingredients were identified, namely vanillic acid, danshensu, caffeic acid, protocatechuic acid, protocatechuic aldehyde, ferulic acid, rosmarinic acid, shikonic acid, salvianolic acid B, isotope salvianolic acid B and salvianolic acid E, the network pharmacology analysis showed that there were 174 targets, and 67 intersected with the target set of neuropathic pain. Protein interaction network analysis showed that STAT3, ALB, JUN, APP, MMP9, CASP3, TLR4, MAPK1, PTGS2, RELA as core targets The network topology analysis of "medicine components-target" showed that there may be synergistic effects of S. miltiorrhiza components. GO enrichment analysis showed that it was related to biological processes such as bicarbonate transport, response to lipopolysaccharide, and response to molecule of bacterial origin, membrane rafts, etc. raft, membrane microdomain, membrane region, secretory granule lumen and other cell composition and carbonate dehydratase activity, hydrolase activity, carbonoxygen lyase activity and other molecular functions. KEGG enrichment shows that it was related to nitrogen metabolism, AGERAGE signaling pathway in diabetic complications, lipid and atherosclerosis HIF-1 signaling pathway and other pathways. Combined with literature analysis, its onset mechanism was related to inflammation and immune response, neuronal recovery and regeneration, metabolic disorder, virus, pain transmitter conduction, neuronal hypersensitivity, pain threshold, etc. Conclusion Eleven antineuropathic pain active ingredients in S. miltiorrhiza standard decoction were screened by live cell solid phase chromatography and high resolution mass spectrometry. Bridging network pharmacology was used to analyze the mechanism of action, which may be related to inflammation and immune response, regulation of metabolic disorders, and inhibition of hypersensitivity.

R284.1;R285.5

上海市科委科研計劃項(xiàng)目(15DZ1900100)