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裴方妤, 吴吉, 贺思雨, 雷倩, 赵莎, 张涤, 朱沁泉.基于中医方证代谢组学研究六味地黄丸治疗孤独症谱系障碍肝肾不足证的物质基础[J].湖南中医药大学学报英文版,2026,46(1):51-61.[Click to copy
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| 基于中医方证代谢组学研究六味地黄丸治疗孤独症谱系障碍肝肾不足证的物质基础 |
| 裴方妤,吴吉,贺思雨,雷倩,赵莎,张涤,朱沁泉 |
| (湖南中医药大学第一附属医院, 湖南 长沙 410007;广东省深圳市龙岗区人民医院, 广东 深圳 518000;联勤保障部队第九二二医院, 湖南 衡阳 421002) |
| 摘要: |
| 目的 采用中医方证代谢组学策略研究六味地黄丸治疗孤独症谱系障碍（ASD）肝肾不足证的药效物质基础。方法 采用孕期腹腔注射丙戊酸钠（VPA）法构建ASD大鼠模型，将造模成功的仔鼠随机分为模型组（n＝8）与六味地黄丸组（n＝8），另设空白组（n＝8）及空白给药组（n＝5）。每日上午，六味地黄丸组与空白给药组灌胃六味地黄丸混悬液（3.4 g/kg），模型组与空白组给予等量蒸馏水；每日下午模型组与六味地黄丸组灌胃左甲状腺素钠（150 μg/kg），然后距尾尖约2 cm处夹尾5 min给予情志刺激，以建立肝肾不足证候；灌胃及夹尾均1次/d，共计14 d。给药结束后，通过三箱社交实验评价仔鼠社交行为；HE、尼氏染色观察前额叶皮质组织病理学变化；超高效液相色谱-四极杆-飞行时间-串联质谱法（UPLC-Q-TOF-MS）结合多元统计分析鉴定差异代谢物，并在Metabo-Analyst 5.0平台进行通路富集分析；UPLC-Q-TOF-MS结合提取离子流（EIC）功能与质谱比对，鉴定六味地黄丸的入脑成分；AutoDock Vina软件对六味地黄丸入脑成分与差异代谢物关键酶进行分子对接。结果 三箱社交实验结果显示，在社交能力阶段，与空白组比较，模型组仔鼠与陌生鼠1社交时间更短（P＜0.01）；与模型组比较，六味地黄丸组仔鼠与陌生鼠1社交时间更长（P＜0.05）。在社交新颖性阶段，与空白组比较，模型组仔鼠与陌生鼠2社交时间更短（P＜0.01）；与模型组比较，六味地黄丸组仔鼠与陌生鼠2社交时间增加（P＜0.05）。HE和尼氏染色结果显示，与空白组比较，模型组前额叶出现神经病理损伤；与模型组比较，六味地黄丸组前额叶神经病理损伤情况改善。代谢组学结果显示，ASD肝肾不足证病理状态显著影响六味地黄丸的体内代谢过程；多元统计分析共鉴定出42个差异代谢物，其中嘌呤、2-（2-氨基乙基）吡啶、四癸胺、丁烯酰肉碱、鞘氨醇、吡哆醇（维生素B6）、α-亚麻酸等为六味地黄丸治疗ASD肝肾不足证的关键差异代谢物，涉及鞘脂代谢、维生素B6代谢、亚麻酸代谢、卟啉代谢通路。入脑成分分析结果显示，共鉴定出9种六味地黄丸入脑成分，包括甜菜碱、2-呋喃甲酸、腺苷、丹皮酚、荜茇宁、薯蓣皂苷、齐墩果酸、亚油酸、棕榈酸；分子对接结果显示，差异代谢物关键酶[包括腺苷酸琥珀酸裂解酶（ADSL）、5-磷酸核糖-1-焦磷酸合成酶（PRPS）、脂肪酰胺水解酶（FAAH）、酰胺酶（Acy）、肉碱棕榈酰转移酶1（CPT1）、鞘氨醇激酶2（SphK2）、吡哆醇激酶（PDXK）、脂肪酸去饱和酶2（FADS2）]与薯蓣皂苷、齐墩果酸结合能力最强，其中ADSL、Acy、CPT1、PDXK、FADS2与薯蓣皂苷，CPT1、PDXK与齐墩果酸结合能均小于-10 kcal·mol-1。结论 六味地黄丸治疗ASD肝肾不足证的作用机制可能与调节鞘脂代谢、维生素B6代谢、亚麻酸代谢、卟啉代谢通路相关，其物质基础可能为甜菜碱、2-呋喃甲酸、腺苷、丹皮酚、荜茇宁、薯蓣皂苷、齐墩果酸、亚油酸、棕榈酸，该实验为六味地黄丸制备工艺优化、质量标准建立及创新药物开发奠定了理论基础。 |
| 关键词: 孤独症谱系障碍 六味地黄丸 肝肾不足证 方证代谢组学 分子对接 |
| DOI：10.3969/j.issn.1674-070X.2026.01.007 |
| Received:September 23, 2025 |
| 基金项目:湖南省自然科学基金项目（2024JJ8233）；湖南省中医药科研计划项目（A2023036）；湖南省卫生健康高层次人才项目（20230448）；湖南中医药大学校院联合基金项目（2025XYLH038）；湖南省大学生创新创业训练计划项目（S202410541157）。 |
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| On the material basis of Liuwei Dihuang Pill in treating autism spectrum disorder with liver-kidney deficiency pattern based on TCM formula-pattern metabolomics |
| PEI Fangyu, WU Ji, HE Siyu, LEI Qian, ZHAO Sha, ZHANG Di, ZHU Qinquan |
| (The First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China;Longgang District People's Hospital, Shenzhen, Guangdong 518000, China;No. 922 Hospital of the PLA Joint Logistics Support Force, Hengyang, Hunan 421002, China) |
| Abstract: |
| Objective To investigate the pharmacodynamic material basis of Liuwei Dihuang Pill (LWDHP) on autism spectrum disorder (ASD) with liver-kidney deficiency pattern based on the strategy of TCM pattern-related metabonomics. Methods An ASD offspring rat model was established by intraperitoneal injection of sodium valproate (VPA) during pregnancy. Successfully modeled offspring rats were randomly divided into model group (n=8) and LWDHP group (n=8), with additional blank control group (n=8) and blank medication control group (n=5). Every morning, the LWDHP and the blank medication groups were gavaged with LWDHP suspension (3.4 g/kg), while the model and the blank control groups received an equal volume of distilled water. Every afternoon, the model and the LWDHP groups were gavaged with levothyroxine sodium (150 μg/kg), followed by a 5-minute tail clamp at approximately 2 cm from the tail tip as an emotional stimulation to establish a liver-kidney deficiency pattern. Both gavage and tail-clamp were performed once daily for 14 consecutive days. After treatment, the three-chamber social test was used to evaluate social behavior of offspring rats. HE staining and Nissl staining were performed to observe histopathological changes in the prefrontal cortex. Ultra performance liquid chromatography-quadrupole-time of flight-tandem mass spectrometry (UPLC-Q-TOF-MS) combined with multivariate statistical analysis was employed to identify differential metabolites, and pathway enrichment analysis was conducted using the MetaboAnalyst 5.0 platform. UPLC-Q-TOF-MS combined with the extracted ion chromatogram (EIC) function and mass spectrometry comparison was used to identify the brain-penetrating components of LWDHP. Molecular docking between the brain-penetrating components of LWDHP and key enzymes of differential metabolites was performed using AutoDock Vina software. Results The three-chamber social test results showed that in the sociability phase, compared with the blank control group, offspring rats in the model group spent significantly less time interacting with stranger rat one (P＜0.01); compared with the model group, the LWDHP group spent more time interacting with stranger rat one (P＜0.05). In the social novelty phase, compared with the blank control group, the model group spent significantly less time interacting with stranger rat two (P＜0.01); compared with the model group, the LWDHP group spent more time interacting with stranger rat two (P＜0.05). HE and Nissl staining results showed neuropathological damage in the prefrontal cortex of the model group compared with the blank control group, while this damage in the LWDHP group was alleviated compared to the model group. Metabolomic results indicated that the pathological state of ASD with liver-kidney deficiency pattern significantly affected the in vivo metabolic processes of LWDHP. Multivariate statistical analysis identified a total of 42 differential metabolites, among which purine, 2-(2-aminoethyl) pyridine, tetradecylamine, butenoylcarnitine, sphingosine, pyridoxol (vitamin B6), and α-linolenic acid were identified as key differential metabolites in the treatment of ASD with liver-kidney deficiency pattern by LWDHP, involving sphingolipid metabolism, vitamin B6 metabolism, linolenic acid metabolism, and porphyrin metabolic pathways. Analysis of brain-penetrating components identified nine components of LWDHP, including betaine, 2-furoic acid, adenosine, paeonol, piperlonguminine, dioscin, oleanolic acid, linoleic acid, and palmitic acid. Molecular docking results showed that key enzymes of the differential metabolites including adenylosuccinate lyase (ADSL), phosphoribosyl pyrophosphate synthetase (PRPS), fatty acid amide hydrolase (FAAH), amidase (Acy), carnitine palmitoyltransferase 1 (CPT1), sphingosine kinase 2 (SphK2), pyridoxal kinase (PDXK), and fatty acid desaturase 2 (FADS2) exhibited the strongest binding energy with dioscin and oleanolic acid. The binding energy of ADSL, Acy, CPT1, PDXK, and FADS2 with dioscin, and of CPT1 and PDXK with oleanolic acid were all less than -10 kcal·mol-1. Conclusion The mechanism of action of LWDHP on ASD with liver-kidney deficiency pattern may be related to the regulation of sphingolipid metabolism, vitamin B6 metabolism, linolenic acid metabolism, and porphyrin metabolism pathways. Its material basis may involve betaine, 2-furoic acid, adenosine, paeonol, piperlonguminine, dioscin, oleanolic acid, linoleic acid, and palmitic acid. This study provides a theoretical foundation for optimizing the preparation process, establishing quality standards, and developing innovative drugs for LWDHP. |
| Key words: autism spectrum disorder Liuwei Dihuang Pill liver-kidney deficiency pattern formula-pattern metabolomics molecular docking |
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