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黄劲, 张越, 曾华婷, 刘碧霞, 杨锐培, 徐伟龙.多技术整合解析四黄液抗炎作用及其机制[J].湖南中医药大学学报,2026,46(6):1108-1120[点击复制] |
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| 多技术整合解析四黄液抗炎作用及其机制 |
| 黄劲,张越,曾华婷,刘碧霞,杨锐培,徐伟龙 |
| (深圳市平乐骨科医院(深圳市坪山区中医院), 广东 深圳 518001;深圳市中医院, 广州中医药大学第四临床医学院, 广东 深圳 518033) |
| 摘要: |
| 目的 基于药代动力学、非靶向代谢组学、网络药理学及分子对接等整合策略，系统探究四黄液的抗炎药效及其作用机制。方法 采用Swiss ADME平台进行药代动力学预测，评估四黄液主要活性成分的口服生物利用度。将30只小鼠随机分为空白组（生理盐水）、四黄液0.125 g/kg组（四黄液0.125 g/kg）、四黄液0.5 g/kg组（四黄液0.5 g/kg）、四黄液2 g/kg组（四黄液2 g/kg）、四黄液8 g/kg组（四黄液8 g/kg）、四黄液16 g/kg组（四黄液16 g/kg），每组5只，每日灌胃给药，连续10 d，确定四黄液的给药剂量范围。将50只小鼠随机分为空白组（生理盐水）、模型组（生理盐水）、阳性药组（阿司匹林100 mg/kg）、四黄液低剂量组（四黄液1 g/kg）和四黄液高剂量组（四黄液5 g/kg），每组10只，每日灌胃给药，连续10 d。通过二甲苯诱导小鼠耳肿胀试验、角叉菜胶诱导小鼠足部肿胀试验和乙酸诱导小鼠扭体试验评价四黄液的抗炎作用。采用超高效液相色谱-串联质谱进行非靶向代谢组学分析，以鉴定差异代谢物并进行通路富集分析。网络药理学筛选四黄液核心抗炎靶点并进行GO功能注释及KEGG通路富集解析。基于MetaboAnalyst 6.0平台，采用皮尔逊和斯皮尔曼相关系数进行代谢物-蛋白-疾病关联网络分析。分子对接分析四黄液关键活性成分与核心抗炎靶点的结合情况。Western blot验证四黄液对核因子κB（NF-κB）通路的作用。结果 药代动力学评估显示，芦荟大黄素、黄芩素、黄芩苷、小檗碱、黄连碱、巴马汀、大黄酸、槲皮素8种主要活性成分具有良好的口服生物利用度，因此本研究选择口服给药途径。四黄液各剂量组（0.125~8 g/kg）的存活率及体质量与空白组相比，均无显著差异（P>0.05），因此选定1 g/kg和5 g/kg为后续实验给药的低、高剂量组。与空白组比较，模型组小鼠的耳肿胀率和耳质量差异率均升高（P<0.01），扭体次数、水肿程度均加重（P<0.01），肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β和前列腺素E2（PGE2）水平均升高（P<0.01）。与模型组比较，阳性药组及四黄液低、高剂量组小鼠耳部肿胀率和耳质量差异率均降低（P<0.05，P<0.01），水肿程度均减轻（P<0.01），TNF-α、IL-1β和PGE2水平均降低（P<0.05，P<0.01）；阳性药组及四黄液高剂量组小鼠扭体次数减少（P<0.01）。与阳性药组比较，四黄液低、高剂量组小鼠扭体次数、水肿程度均增加（P<0.05，P<0.01），TNF-α、IL-1β和PGE2水平均升高（P<0.01）；四黄液低剂量组小鼠耳质量差异率升高（P<0.05）。与四黄液低剂量组比较，四黄液高剂量组小鼠扭体次数减少（P<0.05），水肿程度缩小（P<0.05），TNF-α、IL-1β和PGE2水平均降低（P<0.01）。非靶向代谢组学共鉴定出29个差异代谢物，通路富集分析显示，花生四烯酸代谢、亚油酸代谢、鞘氨醇代谢及组氨酸代谢是与四黄液抗炎作用密切相关的核心代谢通路。网络药理学筛选出150个抗炎潜在靶点，其中前10的核心靶点为：IL-6、AKT丝氨酸/苏氨酸激酶1、TNF、肿瘤蛋白p53、IL-1β、血管内皮生长因子A、Jun原癌基因、胱天蛋白酶-3、前列腺素内过氧化物合酶2（PTGS2）、基质金属蛋白酶9。KEGG富集分析提示NF-κB、丝裂原活化蛋白激酶、IL-17、TNF及磷脂酰肌醇3-激酶/蛋白激酶B等通路与抗炎作用关联密切。代谢物-蛋白-疾病关联网络揭示花生四烯酸代谢处于核心位置，其代谢产物与NF-κB通路蛋白TNF、IL-1β及NF-κB抑制因子α显著相关。分子对接结果显示，黄连碱与IL-6、IL-1β、TNF、PTGS2靶点的结合能均低于-7 kcal·mol-1，且与靶点氨基酸残基形成稳定氢键。与空白组比较，模型组磷酸化NF-κB p65（p-NF-κB p65）、磷酸化IκB激酶（p-IKK）α、p-IKKβ蛋白表达水平均升高（P<0.01）；与模型组比较，阳性药组和四黄液低、高剂量组p-NF-κB p65、p-IKKα、p-IKKβ蛋白表达水平均降低（P<0.01）；与阳性药组比较，四黄液低剂量组p-NF-κB p65、p-IKKα、p-IKKβ蛋白表达水平均升高（P<0.01）；与四黄液低剂量组比较，四黄液高剂量组p-NF-κB p65、p-IKKα、p-IKKβ蛋白表达水平均降低（P<0.01）。结论 四黄液可能通过调节由花生四烯酸等代谢通路与NF-κB通路所构成的“代谢-免疫”调控网络，直接或间接发挥抗炎作用。 |
| 关键词: 四黄液 抗炎 药代动力学 网络药理学 分子对接 非靶向代谢组学 花生四烯酸代谢 NF-κB通路 |
| DOI：10.3969/j.issn.1674-070X.2026.06.003 |
| 投稿时间：2026-03-13 |
| 基金项目:广东省中医药局项目（20241276）；深圳市坪山区卫生系统研究基金（202283，2024512，2024506）；深圳市中医药学会科研项目（2024107）。 |
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| Multi-technology integration elucidates the anti-inflammatory effects and mechanisms of Sihuang Solution |
| HUANG Jin, ZHANG Yue, ZENG Huating, LIU Bixia, YANG Ruipei, XU Weilong |
| (Shenzhen Pingle Orthopedic Hospital (Shenzhen Pingshan District Hospital of Traditional Chinese Medicine), Shenzhen, Guangdong 518001, China;Shenzhen Traditional Chinese Medicine Hospital, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, Shenzhen, Guangdong 518033, China) |
| Abstract: |
| Objective To systematically explore the anti-inflammatory effects and mechanism of action of Sihuang Solution (SHS) through an integrated strategy combining pharmacokinetics, untargeted metabolomics, network pharmacology, and molecular docking. Methods The oral bioavailability of major active components was predicted using the SwissADME platform. To determine the SHS dosage range, 30 mice were randomly divided into a blank group (saline) and five SHS groups (0.125, 0.5, 2, 8, and 16 g/kg; n=5), administered intragastrically once daily for 10 consecutive days. Subsequently, 50 mice were randomly assigned to blank group (saline), model group (saline), positive control group (aspirin, 100 mg/kg), and low-and high-dose SHS groups (1 g/kg and 5 g/kg) (n=10), treated intragastrically for 10 consecutive days. Anti-inflammatory effects were evaluated using xylene-induced ear edema, carrageenan-induced paw edema, and acetic acid-induced writhing tests. Untargeted metabolomics was performed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to identify differential metabolites and conduct pathway enrichment analysis. Network pharmacology was employed to screen core anti-inflammatory targets and to perform GO and KEGG pathway enrichment analyses. Metabolite-protein-disease correlation analysis was carried out on the Metabo Analyst 6.0 platform using Pearson and Spearman correlation coefficients. Molecular docking was applied to assess the binding of core active components to key anti-inflammatory targets. Western blot was used to validate the effects of SHS on the nuclear factor-κB (NF-κB) signaling pathway. Results Pharmacokinetic evaluation revealed that eight major active constituents—aloe-emodin, baicalein, baicalin, berberine, coptisine, palmatine, rhein, and quercetin—exhibited good gastrointestinal absorption and favorable oral bioavailability, supporting the oral administration route. Subsequently, toxicity evaluation showed that there were no significant differences in survival rate or body weight between the SHS groups (0.125-8 g/kg) and the blank group (P>0.05). Accordingly, 1 g/kg and 5 g/kg were selected as the low and high doses for subsequent experiments. Compared with the blank group, the model group showed significant increases in ear swelling rate, ear weight difference ratio, writhing count, edema severity (P<0.01), and the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and prostaglandin E2(PGE2) (P<0.01). Compared with the model group, the positive control, low- and high-dose SHS groups exhibited marked decreases in ear swelling rate, ear weight difference ratio(P<0.05, P<0.01), edema severity (P<0.01), and the levels of TNF-α, IL-1β, and PGE2 (P<0.05, P<0.01), the positive control and the high-dose SHS groups showed a reduction in the writhing count (P<0.01). Compared with the positive control group, the low-and high-dose SHS groups exhibited significantly increased writhing count and edema severity (P<0.05, P<0.01), as well as elevated levels of TNF-α, IL-1β, and PGE2 (P<0.01). Additionally, the ear weight difference ratio was significantly higher in the low-dose SHS group (P<0.05). Compared with the low-dose SHS group, the high-dose SHS group showed significantly reduced writhing count and edema severity (P<0.05), along with decreased levels of TNF-α, IL-1β, and PGE2 (P<0.01). Untargeted metabolomics identified 29 differential metabolites; pathway enrichment analysis indicated that arachidonic acid metabolism, linoleic acid metabolism, sphingosine metabolism, and histidine metabolism are core pathways closely linked to the anti-inflammatory effects of SHS. Network pharmacology yielded 150 potential anti-inflammatory targets, of which the top ten core targets were identified as: IL-6, AKT serine/threonine kinase 1 (AKT1), TNF, tumor protein p53 (TP53), IL-1β, vascular endothelial growth factor A (VEGFA), Jun proto-oncogene (JUN), caspase-3 (CASP3), prostaglandin-endoperoxide synthase 2 (PTGS2), and matrix metalloproteinase 9 (MMP9). KEGG pathway enrichment analysis suggested that the NF-κB, mitogen-activated protein kinase (MAPK), IL-17, TNF, and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathways are closely associated with the anti-inflammatory effects. The metabolite-protein-disease network placed arachidonic acid metabolism at the center, with its metabolites significantly correlated with the NF-κB pathway proteins TNF, IL-1β, and NF-κB inhibitor α. Molecular docking revealed that coptisine displayed binding energies below-7 kcal·mol-1 with IL-6, IL-1β, TNF, and PTGS2, forming stable hydrogen bonds with key amino acid residues. The protein expression of phosphorylated NF-kB p65 (p-NF-kB p65), phosphorylated IkB kinase (p-IKK-α), and p-IKKβ was significantly upregulated in the model group compared with the blank group (P<0.01) and was downregulated in the positive control, low- and high-dose SHS groups (P<0.01) compared with the model group. Furthermore, the expression levels of these phosphorylated proteins were significantly higher in the low-dose SHS group than in the positive control group (P<0.01), and were lower in the high-dose SHS group compared with the low-dose SHS group (P<0.01). Conclusion SHS may exert direct or indirect anti-inflammatory effects by modulating a "metabolism-immunity" regulatory network composed of metabolic pathways, such as arachidonic acid metabolism and the NF-κB signaling pathway. |
| Key words: Sihuang Solution anti-inflammation pharmacokinetics network pharmacology molecular docking untargeted metabolomics arachidonic acid metabolism NF-κB pathway |
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