Computational analysis to define efficacy & molecular mechanisms of 7, 4'- Dihydroxyflavone on eosinophilic esophagitis: Ex-vivo validation in human esophagus biopsies

IntroductionEosinophilic Esophagitis (EoE) is a chronic condition characterized by eosinophilic inflammation of the esophagus which leads to esophageal dysfunction with common symptoms including vomiting, feeding difficulty, dysphagia, abdominal pain. Current main treatment options of EoE include dietary elimination and swallowed steroids. Diet elimination approach could lead to identifying the trigger food(s), but it often requires repeated upper endoscopy with general anesthesia and potentially could negatively affect nutrition intake and growth of the child and individuals' quality of life. Although the swallowed steroid treatment of effective, the EoE will universally recur after discontinuation of the treatment. Digestive Tea formula (DTF) has been used by the Traditional Chinese Medicine (TCM) practice to improve GI symptoms in EoE patients, including abdominal pain, GE reflux, and abnormal bowel movement. Previously, a flavonoid small molecule compound 7, 4 dihydroxy flavone (DHF) from Glycyrrhiza uralensis in DTF inhibited eotaxin, Th2 cytokine and IgE production in vitro and in vivo. MethodThis study comprehensively evaluates the potential therapeutic and immunological mechanisms underlying DHF improvement of symptoms related to EoE using computational modeling, including target mining, gene ontology enrichment, pathway analyses, protein-protein interaction analyses, in silico molecular docking and dynamic simulation followed by ex-vivo target validation by qRT-PCR using cultured human esophagus biopsy specimen with or without DHF from patients with EoE. ResultsComputational analyses defined 29 common targets of DHF on EoE, among which TNF-alpha, IL-6, IL1 beta, MAPK1, MAPK3 and AKT1 were most important. Docking analysis and dynamic simulation revealed that DHF directly binds TNF-alpha with a free binding energy of -7.7 kcal/mol with greater stability and flexibility. Subsequently, in the human esophagus biopsy culture system, significant reduction in levels of TNF-alpha, IL-6, IL-8 and IL1-beta was found in the supernatant of biopsy sample cultured with DHF. Furthermore, the gene expression profile showed significant reduction in levels of TNF-alpha, IL1-beta, IL-6, CCND and MAPK1 in the esophagus biopsy sample cultured with DHF. DiscussionTaken together, the current study provides us an insight into the molecular mechanisms underlying multi-targeted benefits of DHF in the treatment of EoE and paves the way for facilitating more effective EoE therapies.