Integrating network pharmacology and pharmacological evaluation for deciphering the mechanism of (-)-epigallocatechin-3-gallate alleviating ethanol-inducedendothelial cells injury / 中国药理学与毒理学杂志

ABSTRACT

Objective To investigate the potential therapeutic targets and pharmacological mechanism of (-)-epigal?locatechin-3-gallate (EGCG) based on network pharmacology and experimental verification. METHODS The druggability of EGCG was measured by the traditional Chinese medicine systems pharmacology (TCMSP) server, and potential tar?gets of EGCG were identified by Pharm Mapper and Drug Repositioning and Adverse drug Reaction via Chemical-Pro?tein Interactome (DRAR-CPI). The potential targets were imported into GeneMANIA database to obtain the protein-pro?tein direct interaction network, and target physical interaction, co-expression, prediction, genetic interaction, and shared protein domains. The biological process, molecular functions, cellular components and KEGG signaling pathways of potential targets were analyzed using DAVID database. For further study, ethanol was used to establish a model of endothelial injury in vitro. The cell viability was assayed by MTT method, the cellular apoptosis was stained by Annexin V/PI, and the expression levels of Bcl-2, Bax and cleved-caspase-3 were tested by Western blotting. Then, JC-1 and nuclear translocation of NF-κB experiments were used to study the mitochondrial membrane potential and nuclear trans?location. RESULTS The oral availability of EGCG was 55.09％ (≥ 30％) and drug-like index was 0.77 (≥ 0.18), which were considered pharmacokinetically active. 17 potential targetable proteins of EGCG were predicted by Pharm Mapper and DRAR-CPI. Further research showed that 68.13％ displayed similar co-expression characteristics, 26.11％ physical interactions, and 2.74％ shared the same protein domain. The depth network analysis results showed that the biofunc?tions of EGCG were mainly by regulating glutathione derivative biosynthetic process, glutathione metabolic process, nitrogen compound metabolic process etc.. via drug binding, catalytic activity, glutathione transferase activity, anion bind?ing etc.. in sarcoplasmic reticulum, spindle pole, microtubule cytoskeleton and cytoplasm. KEGG enrichment analysis showed that Glutathione metabolism, IL-17 signaling pathway, EGFR tyrosine kinase inhibitor resistance, PI3K-Akt sig?naling pathway and other pathways were involves in the biofunction of EGCG. The above analyses indicated that EGCG exerts its biofunction through antioxidant and anti-inflammatory mechanisms. The experimental results showed that etha?nol 20.0 mmol·L-1 decreased cell viability, Bcl-2 expression, and increased cell apoptosis, the intracellular ROS, as well as the expression of Bax and cleaved-caspase-3 of human endothelial cells. However, treatment of the cells with EGCG can significantly alleviate ethanol induced endothelial cells injury. Further study showed that EGCG significantly allevi?ates ethanol induced mitochondrial depolarization and nuclear translocation of NF-κB. CONCLUSIONS EGCG exerts pharmacological efficacies on ethanol induced endothelial cell injury through multi-target, multi-function and multi-path?way mode. Protective effect of EGCG on ethanol induced cell injury was mainly through alteration of mitochondrial func?tion and NF-κB translocation. Therefore, EGCG have great potential in protecting against endothelial dysfunction of the persons who are chronically abuse of ethanol. This study also provides a new understanding of EGCG in clinical applica?tion on cardiovascular and cerebrovascular diseases.