基于网络药理学和实验验证探讨四物汤养血安神的作用机制*

作者：葛洁倩，王成喆，易 爽，马梓翔，赵 燕

单位：北京中医药大学中医学院，北京 102409

引用：引用：葛洁倩，王成喆，易爽，马梓翔，赵燕.基于网络药理学和实验验证探讨四物汤养血安神的作用机制[J].中医药导报,2025,31(10):41-49,96.

DOI：10.13862/j.cn43-1446/r.20250530.001

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摘要：

目的：探讨四物汤通过调节外周及脑内铁代谢发挥养血安神的作用机制，并通过动物实验进行验证。方法：利用在线数据库检索四物汤的活性成分和疾病靶点并取两者交集，构建“活性成分-靶点-疾病”网络图，对交集靶点进行PPI分析、KEGG富集分析及GO富集分析；采用失血法制备血虚大鼠模型，观察大鼠血虚表征，检测外周血红细胞参数和血清铁（SI）含量变化，qRT-PCR法检测大鼠脾脏组织蛋白激酶B（Akt）mRNA、核因子E2相关因子2（Nrf2）mRNA、血红素氧合酶1（HMOX-1）mRNA相对含量；睡眠监测实验观察大鼠的睡眠潜伏期和睡眠持续时间，蛋白质印迹法（Western blotting）检测海马体Akt、Nrf2、HMOX-1蛋白的表达；酶联免疫吸附试验（ELISA）检测海马体5-羟色胺（5-HT）、多巴胺（DA）、Fe含量；HE染色观察海马体的病理变化。结果：四物汤的有效活性成分为β-谷甾醇、豆甾醇、山柰酚、杨梅酮等；对贫血和失眠起作用的靶点是Akt1、HMOX-1、白介素-6（IL-6）、肿瘤坏死因子TNF；KEGG富集分析显示四物汤治疗贫血和失眠的靶点富集在相同的信号通路，GO富集分析显示四物汤治疗贫血和失眠具有相同的生物过程和分子功能。动物实验显示：与空白组比较，造模后四物组和模型组大鼠红细胞计数（RBC）、血红蛋白（HGB）、红细胞压积（HCT）均明显降低（P＜0.01）。给药后，与模型组比较，四物组大鼠HGB、HCT明显升高（P＜0.05）；与空白组比较，模型组大鼠RBC、HGB、HCT更低；与模型组比较，四物组大鼠脾脏Akt mRNA、Nrf2 mRNA、HMOX-1 mRNA水平和SI含量显著升高（P＜0.01）；与空白组比较，模型组大鼠Nrf2 mRNA、HO-1 mRNA和SI含量更低（P＜0.05）。造模后，与空白组比较，四物组和模型组大鼠睡眠潜伏期显著延长（P＜0.05），睡眠持续时间明显缩短（P＜0.01）。给药后，各组睡眠潜伏期变化差异不明显；与模型组比较，四物组大鼠睡眠持续时间显著延长（P＜0.01）；与空白组比较，模型组大鼠睡眠持续时间更短（P＜0.05）；与模型组比较，四物组大鼠Akt蛋白相对含量具有升高趋势；Nrf2、HMOX-1蛋白的相对含量和脑内铁含量显著升高（P＜0.05或P＜0.01）；与空白组比较，模型组大鼠Nrf2蛋白相对含量更低（P＜0.05），Akt和HMOX-1蛋白相对含量无明显变化，脑内铁含量更低（P＜0.05）；与模型组比较，四物组大鼠海马体5-HT含量显著升高（P＜0.05），DA含量显著下降（P＜0.05）；与空白组比较，模型组大鼠海马体5-HT含量无明显变化，DA含量显著增加（P＜0.01）。四物组和空白组大鼠海马体锥体细胞排列整齐规则，细胞浸润减少，胞质和胞核形态正常；模型组大鼠海马锥体细胞排列紊乱，细胞浸润严重，细胞形态不规则伴有胞核增大、胞质减少。结论：四物汤可通过激活Akt/Nrf2/HMOX-1通路实现铁代谢双重调控，促进外周铁代谢参与血红蛋白合成以养血，提升脑内铁含量为5-HT、DA等神经递质的合成提供物质基础，发挥安神作用。该发现阐明“养血-安神”的协同机制，证实其通过铁代谢桥梁作用实现外周造血与中枢神经保护的系统整合。

关键词：四物汤；Akt/Nrf2/HMOX-1通路；养血安神；铁代谢；网络药理学；大鼠

Abstract：

Objective: To investigate the mechanism of Siwu decoction (SWD) in nourishing blood and tranquilizing by regulating iron metabolism in peripheral tissues and the brain, and to validate it through animal experiments. Methods: Active components of SWD and disease targets were retrieved from online databases with intersection taken to construct a "active ingredient-target-disease" network diagram. PPI analysis, KEGG enrichment analysis, and GO enrichment analysis were performed on the intersecting targets. A blood-deficiency rat model was established using blood loss method. Blood deficiency manifestations, peripheral blood erythrocyte parameters and serum iron (SI) content changes were observed. The qRT-PCR was used to detect the relative content of Akt mRNA, Nrf2 mRNA, and HMOX-1 mRNA in spleen tissue. Sleep monitoring experiments were conducted to observe the sleep latency and duration of rats, and Western blotting was used to detect the expression of Akt, Nrf2, and HMOX-1 proteins in the hippocampus. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of 5-HT, DA, and Fe in the hippocampus. HE staining was used to observe the pathological changes in the hippocampus. Results: The effective active components of SWD included β-sitosterol, stigmasterol, kaempferol, and myricetin. Core targets for anemia and insomnia included Akt1, HMOX-1, IL-6, and TNF. KEGG enrichment analysis showed that the targets of SWD in treating anemia and insomnia were enriched in the same signaling pathway, while GO enrichment analysis showed that SWD had the same biological processes and molecular functions in treating anemia and insomnia. Animal experiments showed that compared with the blank group, RBC, HGB, and HCT levels decreased significantly in the SW group and model group after modeling (P<0.01). After administration, HGB and HCT levels in the SW group were significantly higher than those in the model group (P<0.05), and the model group showed lower RBC, HGB, and HCT levels than the blank group. The SW group exhibited significantly higher levels of Akt mRNA, Nrf2 mRNA, HMOX-1 mRNA, and SI in the spleen compared with the model group (P<0.01), while the model group showed lower Nrf2 mRNA, HO-1 mRNA, and SI levels than the blank group (P<0.05). After modeling, sleep latency was significantly prolonged (P<0.05) and sleep duration significantly shortened (P<0.01) in the SW group and model group compared with the blank group. After administration, sleep latency did not differ significantly between groups, but sleep duration in the SW group was significantly longer than those in the model group (P<0.01), and the model group showed shorter sleep duration than blank group (P<0.05). The SW group showed a trend of increased Akt protein expression, significantly higher Nrf2 and HO-1 protein levels, and higher brain iron content compared with the model group (P<0.05 or P<0.01). The model group showed lower Nrf2 protein levels than the blank group (P<0.05), with no significant changes in Akt and HO-1 proteins and lower brain iron content (P<0.05). Compared with the model group, the content of 5-HT in the hippocampus of rats in the SW group was significantly increased (P<0.05), while the content of DA was significantly decreased (P<0.05). Compared with the blank group, there was no significant change in the content of 5-HT in the hippocampus of rats in the model group, but the content of DA was significantly increased (P<0.01). Hippocampal pyramidal cells in the SW group and blank group were neatly arranged with reduced cell infiltration and normal cytoplasm and nucleus morphology, whereas the model group showed disordered cell arrangement, severe cell infiltration, irregular cell morphology, and enlarged nuclei with reduced cytoplasm. Conclusion: This study reveals that SWD can achieve dual iron metabolism regulation by activating the Akt/Nrf2/HO-1 pathway, promoting peripheral iron metabolism to participate in hemoglobin synthesis for blood nourishment, and increasing brain iron content to provide a material basis for the synthesis of neurotransmitters such as 5-HT and DA, exerting a tranquilizing effect. This discovery elucidates the synergistic mechanism of "nourishing blood and tranquilizing", confirming its systemic integration of peripheral hematopoiesis and central nervous system protection through the bridging effect of iron metabolism.

Key words：Siwu decoction; Akt/Nrf2/HMOX-1 pathway; nourishing blood and tranquilizing; iron metabolism;network pharmacology; rat

发布时间：2026-01-08

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