基于活细胞固相色谱法联合高分辨质谱及网络药理学的丹参标准汤剂抗神经病理性疼痛作用成分及机制探讨

目的 采用活细胞固相色谱法联合高分辨质谱技术快速筛选鉴定丹参标准汤剂中潜在的抗神经病理性疼痛活性成分,通过网络药理学技术分析其作用机制。方法 小鼠背根神经元细胞与丹参标准汤剂磷酸盐缓冲液(PBS)溶液共孵育,吸附成分采用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱(UPLC-Q-Orbitrap HRMS)鉴别;鉴定得到的活性成分基于网络药理学分析其作用机制:采用中药系统药理学数据库与分析平台(TCMSP)数据库联合 Swiss 数据库寻找成分作用靶点,以"neuropathic pain"为关键词在 Genecards 数据库及 OMIM 数据库检索去重即得疾病靶点,将成分靶点与神经病理性疼痛靶点交集筛选得到共同靶点;将共同靶点导入 STRING数据库进行检索,得蛋白质相互作用(PPI)网络,并导入Cytoscape 3.7.2 绘图,以度值(degree)为标准选取前 10 靶点作为关键靶点;将共同靶点采用 ClusterProfiler 包进行基因本体(GO)、京都基因与基因组百科全书(KEGG)富集分析。结果 共鉴定得到以酚酸类物质为主的潜在活性成分 11 个,分别为香草酸、丹参素、咖啡酸、原儿茶酸、原儿茶醛、阿魏酸、迷迭香酸、紫草酸、丹酚酸 B、异丹酚酸 B、丹酚酸 E。网络药理学分析结果显示活性成分作用靶点174 个,与神经病理性疼痛靶点集交集 67 个;PPI 分析显示信号转导及转录激活蛋白 3(STAT3)、白蛋白(ALB)、原癌基因 c-Jun(JUN)、淀粉样 β 前体蛋白(APP)、基质金属蛋白酶 9(MMP9)、胱天蛋白酶3(CASP3)、toll 样受体 4(TLR4)、丝裂原活化蛋白激酶 1(MAPK1)、环加氧酶 2(PTGS2)、转录因子 p65(RELA)为关键靶点,"药味成分-靶点"网络拓扑分析显示各活性成分可能存在协同作用;GO 富集分析显示与碳酸氢盐转运、对脂多糖反应、对细菌来源分析反应等生物过程,膜筏、膜微区、膜区、分泌颗粒管腔等细胞组成以及碳酸脱水酶活性、水解酶活性、碳氧裂解酶活性等分子功能相关;KEGG 富集显示与氮代谢、糖尿病并发症中的晚期糖基化产物(AGE)-晚期糖基化终末产物受体(RAGE)信号通路、脂质和动脉粥样硬化、缺氧诱导因子 1(HIF-1)信号通路等通路相关;结合文献分析丹参抗神经病理性疼痛起效机制与炎症和免疫反应、神经元恢复及再生、代谢紊乱、病毒、疼痛递质传导、神经元超敏、疼痛阈等相关。结论 应用活细胞固相色谱与高分辨质谱联用技术筛选出丹参标准汤剂中抗神经病理性疼痛活性成分 11 个,桥接网络药理学分析其作用机制可能与炎症和免疫反应、调节代谢紊乱、抑制超敏等相关。

Exploring components and mechanism of anti-neuropathic pain in Salvia miltiorrhiza standard decoction based on live cell immobilized chromatography coupled with high resolution mass spectrometry and network pharmacology

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.