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 Tian-Si-Yin in treating Vascular Dementia based on network pharmacology and experimental verification

关键词:  天丝饮  网络药理学  血管性痴呆  铁死亡  AMPK/Nrf2信号通路

Key words:  Tian-Si-Yin  network pharmacology  vascular dementia  ferroptosis  AMPK/Nrf2 signaling pathway

摘要:

目的 运用网络药理学方法探讨天丝饮治疗血管性痴呆(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GO和KEGG富集分析。采用改良2-VO法制备VD大鼠模型,苏木素-伊红(HE)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(ROS)水平；生化比色法检测海马组织中Fe2+、丙二醛(MDA)和谷胱甘肽(GSH)含量；借助Western blot及q-PCR实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,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,P<0.01),上调Mfn1、Mfn2、GPX4、SLC7A11、FTH1蛋白及mRNA的表达(P<0.05,P<0.01),下调Drp1、Fis1、ACSL4、COX-2蛋白及mRNA的表达(P<0.05,P<0.01)。天丝饮处理提升了AMPK磷酸化水平,上调了Nrf2、HO-1蛋白及mRNA的表达,促进了AMPK/Nrf2 途径的活化(P<0.05,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:

ABSTRACT: OBJECTIVE To study the mechanism of Tian-Si-Yin in treating Vascular dementia（VD） by network pharmacology. METHODS To search the active ingredients and corresponding targets of Tian-Si-Yin through the TCMSP databases, Then obtained the targets of VD through the OMIM and GeneCards database. Constructed protein interaction network map by String online database. At the same time GO and KEGG enrichment analysis of key targets were carried out by using R language. Modified 2-VO method was used to build the VD rat model.The pathomorphological changes of rat hippocampal neurons by hematoxylin-eosin(HE)staining. Transmission electron microscopy was used to observe the mitochondrial ultrastructure in the hippocampal tissue. Flow cytometry to detect changes in mitochondrial membrane potentialthe and the level of reactive oxygen species (ROS).A biochemical colourimetric assay was used to detect the levels of Fe2+,malondialdehyde(MDA),and glutathione（GSH）in hippocampal tissue. Western blotand q-PCR experiments were used to clarify the protein expression and gene transcription of the relevant targets.RESULTS A total of 13 active ingredients of Tian-Si-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 Tian-Si-Yin could 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 Fe2+,MDA, and ROS levels (P<0.05,P<0.01),Up-regulated the expression of Mfn1,Mfn2, GPX4,SLC7A11, FTH1 protein and mRNA (P<0.05,P<0.01), down-regulated the expression of Drp1,Fis1,ACSL4, COX-2 proteins and mRNA (P<0.05,P<0.01).Tian-Si-Yin treatment increased the phosphorylated levels of AMPK, upregulated the expression of Nrf2, HO-1 protein and mRNA,therefore promoting the activation of the AMPK/Nrf2 pathway (P<0.05,P<0.01). However, the co-treatment of compound C partially reversed the therapeutic effects of Tian-Si-Yin on ferroptosis.CONCLUSION Tian-Si-Yin improves mitochondrial dynamics imbalance and inhibits ferroptosis by activating the AMPK/Nrf2 pathway, thereby improve cognitive function in VD rats.