Comparative Studies Of The Binding Of Natural Compounds To The Sub-Pocket Of Keap1 Kelch Domain And Their Activation Of The Cellular Antioxidation Defense

Oxidative stress(OS)is defined as the unstable state formed by the lack of balance between the reactive oxygen/nitrogen species(ROS/RNS)produced by the organisms and their ability to remove these species.Under normal physiological conditions,the ROS in cells is in a dynamic balance through various mechanisms.However,when the level of ROS in the cell is too high,or overwhelms the elimination ability of the antioxidant system,the redox homeostasis in the cell is destroyed.All types of ROS have unpaired valence electrons or unstable bonds,which can easily react with a series of biological molecules such as DNA,RNA,protein and lipids,causing irreversible functional changes or even complete damage to these biological molecules.These further induce oxidative stress and unrepairable damage to cells,leading to cell aging and death,and eventually a series of diseases,including neurodegenerative diseases,cancer,inflammation,diabetes,and cardiovascular diseases.Ellagic acid(EA)a natural antioxidant,is a polyphenol widely found in many vegetables,fruits,and nuts.However,it is of low utilization efficiency in the intestine.Generally,it is decomposed into urolithin A,B and C(UA,UB,and UC)and then absorbed and utilized by the human body.UA,UB,and UC have various functions including antioxidant,anti-inflammatory.But whether they can protect cells from 6-hydroxydopamine(6-OHDA)is unclear.In this research,the protective effects of these natural polyphenol,i.e.,UA,UB and UC,on 6-hydroxydopamine(6-OHDA)inducedPD cells were assessed and the comparison of their binding ability with Kelch-like ECH-associated protein 1 in vitro were studied in detail.The specific work includes:The binding affinities of these three natural drugs to Kelch domain were first evaluated by molecular docking and further assessed vis isothermal calorimetric titration(ITC).The cytotoxicity test was conducted to investigate whether these drugs could recover the cell viability that was reduced by 6-OHDA.In addition,the expression levels of genes and proteins involved in antioxidant signal pathway were studied by real-time quantitative PCR(RT-PCR)and Western blot(WB).Moreover,the changes of cellular ROS level was assessed by the fluorescence probe DCFH-DAMolecular docking results showed that the binding ability of UA to the Kelch domain was similar to that of UC.However,ITC experiments revealed that UA is of the highest binding affinity.Cytotoxicity assay found that all these three natural drugs can increase cell viability,with UA showing the best antioxidative effect and increasing the cell viability by about 33.0%.In addition,results of RT-PCR and WB confirmed that all three natural drugs can activate antioxidant pathways and increase the expression of downstream antioxidant factors.This study confirms that natural compounds UA,UB,and UC can competitively bind to the Keap1 protein and facilitate the Nrf2 dissociation from Keap1,resulting in the accumulation of Nrf2 and its transfer to the nucleus.This activates the Nrf2-ARE signaling pathway,leading to upregulated expression of related antioxidant genes and proteins.Thus,excess cellular ROS was cleared,exerting cell protective effects.In this research,the results from ITC are consistent with those from cell experiments.Both results,however,showed some discrepancies from molecular docking,This comparative study shows new insights into the research of natural drugs.