目的 基于特征图谱探究牛膝盐炙、酒炙前后的差异性特征成分，结合网络药理学预测及分子对接验证，筛选牛膝干预膝骨关节炎（KOA）的潜在质量标志物（Q-Marker）。方法 采用HPLC技术建立牛膝、盐牛膝及酒牛膝的特征图谱，结合主成分分析（PCA）与正交偏最小二乘法-判别分析（OPLS-DA）筛选炮制前后的主要差异成分；通过网络药理学预测特征成分治疗KOA的核心靶点与关键通路，绘制“药物-成分-靶点-疾病-通路”网络以筛选抗KOA差异活性成分；结合Q-Marker五原则及分子对接验证结果，筛选潜在Q-Marker。结果 15批牛膝、盐牛膝及酒牛膝的特征图谱分别标定27、28、34个共有峰，经对照品比对，3种牛膝共指认14种共有化学成分；化学计量学分析显示，牛膝盐炙前后的主要差异成分为人参皂苷Ro、姜状三七皂苷R₁、齐墩果酸、金盏花皂苷E、竹节参皂苷I、竹节参皂苷IVa及竹节参皂苷IVa甲酯，酒炙前后的主要差异成分为人参皂苷Ro、竹节参皂苷I、姜状三七皂苷R₁、金盏花皂苷E、β-蜕皮甾酮、齐墩果酸及竹节参皂苷IVa。网络药理学预测表明，核心成分β-蜕皮甾酮、旌节花甾酮D、竹节参皂苷Ⅳa甲酯、齐墩果酸、人参皂苷Ro，可能通过调控白蛋白（ALB）、原癌基因（JUN）、过氧化物酶体增殖物激活受体γ（PPARG）、半胱氨酸天冬氨酸蛋白酶3（CASP3）、雌激素受体α基因（ESR1）、抗凋亡蛋白B细胞淋巴瘤因子-2（BCL2） 6个核心靶标，参与癌症通路、白细胞介素（IL）-17信号通路、前列腺癌等关键信号通路发挥抗KOA作用；分子对接验证进一步证实，上述5个核心成分与JUN、BCL2靶标均具有良好结合活性。结合Q-Marker五原则，最终筛选出β-蜕皮甾酮、人参皂苷Ro、齐墩果酸为牛膝治疗KOA的潜在Q-Marker。结论 建立的HPLC特征图谱可有效区分牛膝及其盐炙、酒炙品；经网络药理学预测、分子对接验证及Q-Marker五原则筛选出的差异活性成分，可作为牛膝治疗KOA的潜在Q-Marker。

Objective To explore the differential characteristic components of raw Achyranthes bidentata, salt-processed A. bidentata, and wine-processed A. bidentata through feature fingerprinting, combined with network pharmacology prediction and molecular docking verification, to screen potential quality Markers (Q-Markers) for the intervention of A. bidentata in knee osteoarthritis (KOA). Methods The characteristic fingerprints of raw A. bidentata, salt-processed A. bidentata, and wine-processed A. bidentata were established by HPLC. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to screen the main differential components before and after processing; Network pharmacology was used to predict the core targets and key pathways of the characteristic components in treating KOA, and a “drug-component-target-disease-pathway” network was drawn to screen the differential active components against KOA; Based on the Q-Marker five principles and the results of molecular docking verification, potential differential Q-Markers were screened. Results The characteristic fingerprints of 15 batches of raw A. bidentata, salt-processed A. bidentata, and wine-processed A. bidentata respectively identified 27, 28, and 34 common peaks. After comparison with reference substances, 14 common chemical components were identified for three A. bidentata. Chemometric analysis showed that the main differential components before and after salt processing of raw A. bidentata were ginsenoside Ro, zingibroside R₁, oleanolic acid, calenduloside E, chikusetsusaponin IVa, chikusetsusaponin IVa methyl, while the important differential components before and after alcohol processing were ginsenoside Ro, chikusetsusaponin I, zingibroside R₁, calenduloside E, β-yecdysone, oleanolic acid, chikusetsusaponin IVa. Network pharmacology prediction indicated that β- yecdysone, cyasterone, chikusetsusaponin IVa, oleanolic acid, ginsenoside Ro in A. bidentata, might participate in anti-KOA by regulating 6 core targets including albumin (ALB), proto-oncogene (JUN), peroxisome proliferator-activated receptor γ (PPARG), cysteine aspartate protease 3 (CASP3), estrogen receptor α gene (ESR₁), and anti-apoptotic protein B-cell lymphoma factor-2 (BCL2) in cancer pathways, interleukin (IL)-17 signaling pathway, and prostate cancer and other key signaling pathways; Molecular docking verification further confirmed that the above 5 core components had good binding activity with JUN and BCL2 targets. Based on the Q-Marker five principles, β-yecdysone, ginsenoside Ro, oleanolic acid were finally selected as potential Q-Markers for the treatment of KOA with A. bidentata. Conclusion The established HPLC characteristic fingerprints can effectively distinguish raw A. bidentata and its salt-processed and wine-processed products; The differential active components screened by network pharmacology prediction, molecular docking verification, and the Q-Marker five principles can be used as potential QMarker for the treatment of KOA with A. bidentata.

R284;R684.3

2023年郑州市协同创新专项（2023XTCX041）；豫财教[2023]47号-达2023年第三批“双一流”创建资金-中医学青年骨干人才引进培养-青年B类资助项目（00104311-2023-71）；国家药品监督管理局中药材及饮片质量控制重点实验室开放课题资助项目（KF202201）；基于单细胞转录组技术关联大分子多糖变化解析牛膝盐炙影响肝-肠细胞动态异质性的炮制机制研究（252300421621）