Abstract
It is hypothesized that the oxidative/nitrosative stress inhibitory effect of a flavanone is governed by its chemical structure. However, the existing cell‐based antioxidant assays primarily focus on single chemical to initiate toxic species production. In this study, a novel cell model using macrophage treated with a combination of PMA and LPS leading to generation of peroxynitrite was proposed to provide a more real physiological condition. Three flavanones (eriodictyol, naringenin, and pinocembrin) with different number of ortho‐dihydroxyl groups on B‐ring were used to provide a more comprehensive evaluation of the role of chemical structure in the new model. Dihydrorhodamine123 assay, protein immunoblotting, immunofluorescence assay, and in silico analysis by molecular docking between the flavanones and IKKβ catalytic kinase domain at the ATP binding site were employed. Results indicated that the generation of peroxynitrite was decreased at 10 µM of flavanones; eriodictyol was the most effective inhibitor. Western blot analysis and confocal fluorescence image also showed that eriodictyol could inhibit iNOS and p47 protein expressions through the inhibition of NF‐kB translocation and performed the maximal inhibition compared to that of the other groups. In addition, the highest CDOCKER energy values of eriodictyol (38.6703 kcal/mol) confirmed that the 3′,4′‐ortho‐dihydroxylation on the B‐ring played a crucial role in binding with IKKβ kinase domain at ATP binding site. Finally, we propose that the ortho‐dihydroxyl groups on B‐ring of flavanone may influence directly the occupation of the ATP binding site of IKKβ kinase domain leading to the abrogation of peroxynitrite formation in the innovative cell model.
Highlights
It is evidenced that free radicals, both reactive oxygen (ROS) and nitrogen species (RNS), play a dual role in both causing oxidative/nitrosative stress, tissue damages, and regulating various physiological functions that are beneficial to the living system (Lushchak, 2014)
This finding is inconsistent with the results reported by Huang et al (2013), where eriodictyol did not show considerable cytotoxicity at 100 μM in U937 cells, or by
Even though a weaker hydrogen-bonding parameter is known for B-ring compared to A-ring (Suresh, Srivastava, & Mishra, 2012), our results showed that the 3′,4′-ortho-dihydroxyl groups in the B-ring of eriodictyol have an important role in inhibiting the formation of peroxynitrite by the formation of a strong hydrogenbonding interaction with the binding domain
Summary
Macrophage has been proven to play an important role in regulating the immune system in response to chronic, autoimmune, and infectious diseases (Navegantes et al, 2017). Their overexpression can induce tissue damage during infection, autoimmune, and chronic diseases. It has been proposed that the simultaneous activation of superoxide (O−2) and nitric oxide (NO) synthesis within a cell induces their diffusion-controlled reaction to produce peroxynitrite (ONOO−), which is a powerful oxidant against the invading agents (Koppenol, 2001; Padmaja & Huie, 1993). ONOO− may be an important mediator of free radical-induced cellular injury. These compounds were used to illustrate the relationship between the structure and peroxynitrite-scavenging activities of flavanones in an innovative cell model
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