目的 基于高通量基因表達數(shù)據(jù)庫(GEO)、網(wǎng)絡藥理學和分子對接技術(shù)分析歸芍地黃湯對新生血管性年齡相關(guān)性黃斑變性的可能作用靶點和相關(guān)機制,并通過體內(nèi)實驗進行驗證。方法 通過查閱文獻及中國傳統(tǒng)醫(yī)學公共數(shù)據(jù)庫(TCMSP)確定歸芍地黃湯的主要成分及靶標,繪制成分-靶點網(wǎng)絡拓撲圖。通過整合GEO數(shù)據(jù)庫和公共數(shù)據(jù)庫對新生血管性年齡相關(guān)性黃斑變性的疾病靶點進行預測。使用Venny在線平臺確定藥物-疾病的交集靶點,構(gòu)建蛋白質(zhì)相互作用(PPI)網(wǎng)絡圖,利用Cytoscape軟件篩選關(guān)鍵基因(Hub),利用R語言對交集靶點進行基因本體(GO)和京都基因與基因組百科全書(KEGG)富集分析。將歸芍地黃湯核心成分與靶基因之間進行分子對接,最后利用光學相干斷層掃描和免疫組化顯示組織病理學變化,熒光素眼底血管造影觀察脈絡膜新生血管膜滲漏情況,并驗證其對靶基因表達水平的影響。結(jié)果 從GSE29801數(shù)據(jù)集中獲得50個差異基因,篩選得到藥物-疾病交集靶點134個。核心靶點包括白蛋白(ALB)、腫瘤蛋白p53(TP53)、蛋白激酶B1(Akt1)、白細胞介素-6(IL-6)、腫瘤壞死因子(TNF)、半胱氨酸天冬氨酸蛋白酶-3(CASP3)、血管內(nèi)皮生長因子A(VEGFA)、過氧化氫酶(CAT)、表皮生長因子受體(EGFR)、缺氧誘導因子1A(HIF1A)。這些基因主要富集在對外來生物刺激的反應、對營養(yǎng)水平的反應和創(chuàng)口愈合等生物學過程中,參與衰老、氧化應激、免疫炎癥反應和細胞凋亡等相關(guān)途徑。分子對接預測顯示,歸芍地黃湯核心成分與絲裂原活化蛋白激酶8(MAPK8)、VEGFA、TNF-α、IL-6表現(xiàn)出穩(wěn)定的結(jié)合。動物實驗顯示,歸芍地黃湯減輕了激光造模誘導的眼底病變,下調(diào)了VEGF、TNF-α水平,且治療前后BN大鼠p38 MAPK表達水平存在差異。結(jié)論 歸芍地黃湯可以減輕脈絡膜新生血管膜滲漏大鼠視網(wǎng)膜病變,揭示了歸芍地黃湯治療新生血管性年齡相關(guān)性黃斑變性可能與激活MAPK信號通路有關(guān)。

Objective To explore the potential targets and related mechanisms of Guishao Dihuang Decoction in treatment of neovascular age related macular degeneration based on the GEO gene microarray database, network pharmacology and molecular docking technology.Methods The main targets and main component of Guishao Dihuang Decoction were identified by reviewing the literature and public databases of Chinese traditional medicine(TCMSP). Disease targets of neovascular age related macular degeneration were predicted by integrating GEO database and public databases. Drug-disease intersection targets were identified using the Venn online platform and the intersection targets were used to construct protein interaction network maps. Hub genes were screened using Cytoscape software, GO and KEGG enrichment analysis of the intersection targets were performed using R language. Molecular docking was performed between the core components of Guishao Dihuang Decoction and the target genes. Finally, optical coherence tomography and immunohistochemistry were used to reveal histopathological changes, and fluorescein fundus angiography was used to observe the leakage of choroidal neovascularization membrane, and to verify its influence on the expression level of target genes.Results There were 50 differentially expressed genes from the GSE29801 dataset. A total of 134 intersection targets were screened.The core targets included ALB, TP53, AKT1, IL6, TNF, CASP3, VEGFA, CAT, EGFR, and HIF1A. These genes were mainly enriched in biological processes such as response to xenobiotic stimulation, response to nutrient level, and wound healing, and involved in related pathways such as aging, oxidative stress, immune inflammatory response, and apoptosis. The result of molecular docking showed that the core components of Guishao Dihuang Decoction had a stable binding with MAPK8, VEGFA, TNFα, and IL-6. Animal experiments revealed that Guishao Dihuang Decoction alleviated fundus lesions induced by laser modeling, down-regulated the expression levels of VEGF and TNFα, and there was a difference in the expression level of p38MAPK in BN rats before and after treatment.Conclusion Guishao Dihuang Decoction can attenuate retinal lesions in leakage of the choroidal neovascularization membrane rats and preliminarily revealed that the treatment of neovascular age related macular degeneration with Guishao Dihuang Decoction may be related to the activation of MAPK signaling pathway.

R285

福建省自然科學基金資助項目(2020J011046);福建中醫(yī)藥大學附屬人民醫(yī)院中藥制劑研發(fā)項目(ZYZJ2019003)