基于“水不涵木”及“方证相应”探讨肝阳上亢证对机体的影响机制及实验研究

Mechanism and Experiment Study on Impact of Liver Yang Hyperactivity Syndrome on Body Based on"Failure of Water to Nourish Wood"and"Corresponding of Formula and Syndrome"

目的在中医理论指导下预测肝阳上亢证可能对机体的影响及作用机制并观察该证对大脑皮质的影响。方法在“水不涵木”及“方证相应”理论指导下,借助中医药整合药理学研究平台(Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine,TCMIP)V2.0,以肝阳上亢证、肝阳上亢证根本原因肾阴虚证临床表现和经典方剂天麻钩藤饮联合预测肝阳上亢证可能对机体的作用机制,得到核心网络靶点进行GO及Reactome通路富集分析,构建“疾病-证候-方剂-药物-有效成分-核心网络靶标-通路”关联网络;复制肝阳上亢证大鼠模型,Westernblot法检测大鼠大脑皮质上述预测通路关键蛋白的表达,并使用普鲁士蓝染色观察皮质铁代谢情况、Western blot法检测铁代谢相关蛋白表达。结果共筛选出29个肝阳上亢证相关词条、25个肾阴虚证相关词条,获得核心网络靶标564个,其中核心节点209个,GO富集分析生物过程(biological process,BP)、细胞组分(cellular component,CC)、分子功能(molecular function,MF)分别得到前20个条目,Reactome预测得到血管内皮细胞生长因子(vascular endothelial growth factors,VEGF)受体2(VEGF receptor 2,VEGFR2)、Rapidly accelerated fibrosarcoma(Raf)/丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)、磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)、3,4,5-三磷酸磷脂酰肌醇蛋白(PIP3 protein,PIP3)/蛋白激酶B(protein kinase B,Akt)、信号传导转录激活因子3(signal transducer and activator of transcription 3,STAT 3)、蛋白激酶A(protein kinase A,PKA)、环磷腺苷效应元件结合蛋白(cAMP-responsive element-binding protein,CREB)1等相关的20条通路,并结合实验结果构建出包含11味中药86个中药有效成分210个核心节点28条通路的关联网络,成功复制肝阳上亢证大鼠模型,模型大鼠大脑皮质PKA、CREB、VEGF、PI3K蛋白表达较空白大鼠存在变化,铁代谢相关膜铁转运蛋白(recombinant ferroportin,Fpn)、核因子E2相关因子2(nuclearfactor erythroidderived 2-like 2,Nrf2)、血红素氧合酶1(heme oxygenase-1,HO-1)蛋白增加,转铁蛋白受体(transferrin receptor,Tfr)蛋白表达减少,普鲁士蓝染色显示模型大鼠皮质细胞外存在多个蓝色斑点。结论肝阳上亢证通过多靶标、多通路、多机制对机体产生影响,对大脑皮质PI3K、PKA、CREB、VEGF相关通路有较为明确的作用,还可能促使大鼠皮质细胞内铁向细胞外的转运。

Objective Under the guidance of traditional Chinese medicine theory,to predict the possible mechanism of"liver Yang hyperactivity"on the body,and to verify the effect of this syndrome on cerebral cortex.Methods Under the guidance of"Failure of Water to Nourish Wood"and"Corresponding of Formula and Syndrome",TCMIP V2.0 was used.The possible mechanism of liver Yang hyperactivity on human body was predicted by the symptom phenotype of"liver Yang hyperactivity"and"kidney Yin deficiency"combined with the classical prescription"Tianma Gouteng Decoction".The core network targets were obtained,GO and Reactome pathway enrichment analysis were performed,and the"disease-syndrome-prescription-drug-effective components-core network targets-pathway"correlation network was constructed for visual display.Then we replicated the rat model of"liver Yang hyperactivity",and used Western blot method to detect the expression of key proteins in the above prediction pathway in the rat cortex.Perl staining was used to observe the iron metabolism in the cortex,and Western blot method was used to detect the expression of iron metabolism-related proteins.Results A total of 29"liver Yang hyperactivity"and 25"kidney Yin deficiency"phenotypes were screened,and 564 core network targets were obtained by pharmacological analysis,including 209 core nodes,and 20 items were obtained by Biological Process,Cellular Component and Molecular Function.Reactome predicted 20 pathways related to VEGFR2,Raf/MAPK,PI3K,PIP3/Akt,STAT3,PKA and CREB1.Combined with the experimental results,the association network containing 11 Chinese herbs,86 effective components of Chinese herbs,210 core nodes and 28 pathways was constructed.The rat model of"liver Yang hyperactivity"was successfully replicated.The expression of PKA,CREB,VEGF and PI3K protein in the cerebral cortex of the model rats was changed compared with that of the blank rats,which verified the scientificity of the above prediction results.In addition,iron metabolism related Fpn,Nrf2,HO-1 protein increased,and Tfr protein expression decreased.Perl staining showed that there were multiple blue spots in the cortex of the model rats.Conclusion"Liver Yang hyperactivity"may affect the body through multiple targets,pathways and mechanisms.This syndrome had clear effects on PI3K,PKA,CREB,VEGF-related pathways.This syndrome may promote the transport of iron in rat cortical cells to the outside.