基于网络药理学和实验验证探讨天丝饮治疗血管性痴呆的作用机制

杨梦琳; 黄玉静; 张运辉; 伍大华; 沈梦玥; 刘霞; 程妍

doi:10.13748/j.cnki.issn1007-7693.20250626

基于网络药理学和实验验证探讨天丝饮治疗血管性痴呆的作用机制

Study on the Mechanism of Tiansi Yin in Treating Vascular Dementia Based on Network Pharmacology and Experimental Verification

摘要

摘要:
目的基于网络药理学及动物实验探讨天丝饮治疗血管性痴呆(vascular dementia，VD)的作用机制。
方法通过TCMSP数据库检索天丝饮的有效成分及相关靶点；采用OMIM和GeneCards数据库搜集VD靶点，通过String在线数据库构建靶蛋白相互作用网络，采用R语言对关键靶点进行基因本体(gene ontology，GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)富集分析。采用改良双侧颈总动脉结扎法制备VD模型。苏木素-伊红染色观察海马区病理形态学变化；透射电镜观察海马组织线粒体超微结构；流式细胞术检测线粒体膜电位变化以及活性氧(reactive oxygen species，ROS)水平；生化比色法检测海马组织中Fe²⁺、丙二醛(malondialdehyde，MDA)和谷胱甘肽(glutathione，GSH)含量；借助Western blotting及RT-qPCR试验明确相关靶点蛋白表达及基因转录情况。
结果共获得天丝饮有效成分13个，与VD相关靶点共103个，KEGG相关信号通路前20条，GO分析前20个生物学过程。动物实验发现，天丝饮能够提高模型大鼠的学习、空间记忆能力(P<0.05或P<0.01)，减轻海马组织的病理形态学及线粒体损伤，提升GSH和线粒体膜电位，降低Fe²⁺、MDA和ROS水平(P<0.05或P<0.01)，上调线粒体融合素1(mitofusion 1，Mfn1)、线粒体融合素2(mitofusion 2，Mfn2)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4，GPX4)、溶质载体家族7成员11(solute carrier family 7 member 11，SLC7A11)、铁蛋白重链 1(ferritin heavy chain 1，FTH1)蛋白及mRNA的表达(P<0.05或P<0.01)，下调动力相关蛋白1(dynamics related protein 1，Drp1)、裂变蛋白1(fission protein 1，Fis1)、酰基辅酶A合成酶长链家族4(acyl-CoA synthetase long-chain family member 4，ACSL4)、环氧合酶 2(cyclooxygenase-2，COX-2)蛋白及mRNA的表达(P<0.05或P<0.01)。天丝饮处理后提升了海马组织中AMPK磷酸化水平，上调了核因子E2相关因子2(nuclear factor erythroid 2-related factor 2，Nrf2)、血红素加氧酶 1(Heme oxygenase 1，HO-1)蛋白及mRNA的表达，促进了AMPK/Nrf2 途径的活化(P<0.05或P<0.01)。然而，Compound C的使用可部分逆转天丝饮对于铁死亡的治疗效果。
结论天丝饮通过激活AMPK/Nrf2通路改善线粒体动力学失衡状态，抑制铁死亡，从而提高VD大鼠的认知功能。

Abstract:
OBJECTIVE To explore the mechanism of Tiansi Yin in the treatment of vascular dementia(VD) based on network pharmacology and animal experiments.
METHODS Searched the effective ingredients and corresponding targets of Tiansi Yin by the TCMSP databases, then obtained the targets of VD through the OMIM and GeneCards database. Constructed protein interaction network map by String online database, and to performed gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis of key targets by using R language. The VD model was established by improved bilateral common carotid artery ligation. The pathomorphological changes of rat hippocampal neurons by hematoxylin-eosin staining. The ultrastructure of mitochondria in hippocampus was observed by transmission electron microscope. The mitochondrial membrane potential and reactive oxygen species(ROS) were detected by flow cytometry. The contents of Fe²⁺, malondialdehyde(MDA) and glutathione(GSH) in hippocampus were detected by biochemical colorimetry assay. Western blotting and RT-qPCR were used to identify the expression of target proteins and gene transcription of the relevant targets.
RESULTS A total of 13 active ingredients of Tiansi Yin were screened,103 targets related to VD, the top 20 KEGG related signaling pathways and GO analysis of the top 20 biological processes. Animal experiments showed that Tiansi Yin improved the learning and spatial memory abilities of model rats significantly(P<0.05, P<0.01), alleviated pathological morphology and mitochondrial damage in hippocampal tissue, increased GSH and mitochondrial membrane potential, reduced the level of Fe²⁺, MDA, and ROS(P<0.05, P<0.01), and up-regulated the expression of mitofusion 1(Mfn1), mitofusion 2(Mfn2), glutathione peroxidase 4(GPX4), solute carrier family 7 member 11(SLC7A11), ferritin heavy chain 1(FTH1) protein and mRNA(P<0.05, P<0.01), down-regulated the expression of dynamics related protein 1(Drp1), fission protein 1(Fis1), acyl-CoA synthetase long-chain family member 4(ACSL4), cyclooxygenase-2(COX-2) proteins and mRNA(P<0.05, P<0.01). The treatment of Tiansi Yin increased the phosphorylated levels of AMPK, upregulated the expression of nuclear factor erythroid 2-related factor 2(Nrf2), Heme oxygenase 1(HO-1) protein and mRNA, and promoted the activation of AMPK/Nrf2 pathway(P<0.05, P<0.01). However, the use of Compound C partially reversed the therapeutic effect of Tiansi drink on ferroptosis.
CONCLUSION Tiansi Yin improved the cognitive function of VD rats by activated the AMPK/Nrf2 pathway to improved mitochondrial dynamic imbalance and inhibited ferroptosis.

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