Attenuation of Lipopolysaccharide-Induced Inflammatory Responses through Inhibition of the NF-κB Pathway and the Increased NRF2 Level by a Flavonol-Enriched n-Butanol Fraction from Uvaria alba

Abstract

Pathologic hyperreactive inflammatory responses occur when there is excessive activation of a proinflammatory NF-κB pathway and a reduced cytoprotective NRF2 cascade. The noncytotoxic, highly selective COX-2 inhibitory flavonol-enriched butanol fraction (UaB) from Uvaria alba (U. alba) was investigated for its inflammatory modulating potential by targeting NF-κB activation and NRF2 activity. Enzyme-linked immunosorbent assay was initially performed to measure levels of proinflammatory mediators [nitric oxide (NO), prostaglandin E2, and reactive oxygen species (ROS)] and cytokines [tumor necrosis factor-alpha (TNF-α), IL-1β, and IL-6], followed by reverse transcription-polymerase chain reaction and western blotting to determine mRNA and protein expression, respectively. Using immunofluorescence staining combined with western blot analysis, the activation of NF-κB was further investigated. NRF2 activity was also measured using a luciferase reporter assay. UaB abrogated protein and mRNA expressions of inducible nitric oxide synthase (iNOS), COX-2, TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophages, thereby suppressing the production of proinflammatory mediators and cytokines. This was further validated when a concentration-dependent decrease in NO and ROS production was observed in zebrafish (Danio rerio) larvae. UaB also increased NRF2 activity in HaCaT/ARE cell line and attenuated NF-κB activation by inhibiting the nuclear translocation of transcription factor p65 in RAW 264.7 macrophages. Nontargeted LC–MS analysis of UaB revealed the presence of the flavonols quercitrin (1), quercetin (2), rutin (3), kaempferol (4), and kaempferol 3-O-rutinoside (5). Molecular docking indicates that major flavonol aglycones have high affinity toward COX-2 NSAID-binding sites, TNF-α, and TNF-α converting enzyme, while the glycosylated flavonoids showed strong binding toward iNOS and IKKall possessing dynamic stability when performing molecular dynamics simulations at 140 ns. This is the first report to have elucidated the mechanistic anti-inflammatory potential of the Philippine endemic plant U. alba.

WITHDRAWN: Effects of ethyl acetate fraction ofPsychotria vogelianaleaf on liver function, kidney function, and cytokines level of induced rheumatoid arthritis in albino rats

“…By activating NF-kB and AP-1, inhibiting the inflammatory-causing activation of enzymes such as cyclooxygenase-2, lipoxygenase, and inducible NO synthase, and activating phase II antioxidant detoxifying enzymes in rats, flavonoids have been shown to have anti-inflammatory properties through molecular mechanisms 39 . Pro-inflammatory cytokines/chemokines are inhibited by antioxidants in a variety of cell types, such as peripheral blood mononuclear cells, Jurkat T-cells, and RAW macrophages.…”

Study on network pharmacology of Ginkgo biloba extract against ischaemic stroke mechanism and establishment of UPLC‐MS/MS methods for simultaneous determination of 19 main active components

“…The benefits of G. biloba and its preparations in the prevention and treatment of cardiovascular diseases mainly derive from the effective chemical components such as flavonoids and terpenoid lactones. In this study, through data mining and network pharmacology analysis of relevant literature, we found that the organic acid components (protocatechuic acid, caffeic acid, chlorogenic acid, and gallic acid), flavonoid components (quercetin‐3‐O‐glucoside, quercetin‐3‐O‐rutinoside, kaempferol‐3‐O‐rutinoside, kaempferol‐3‐O‐glucoside, isorhamnetin‐3‐O‐rutinoside, isorhamnetin‐3‐O‐glucoside, apigenin‐7‐O‐glucoside, and apigenin), and terpenoid lactones (bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, and ginkgolide J) had different effects such as anti‐inflammatory and anti‐oxidative activities, protecting the nervous system, changing blood rheology, and so on 22–50 . These components may be the potential active substance basis of its anti‐IS effect, so they were included in the scope of analysis and investigation of the active components of GBE against IS.…”

“…Bilobalide, ginkgolide A, and so on can play an anti‐inflammatory role by regulating inflammation‐related pathways and inhibiting the release of inflammatory factors. Quercetin‐3‐O‐rutinoside, kaempferol‐3‐O‐rutinoside, kaempferol‐3‐O‐glucoside, and isorhamnetin‐3‐O‐rutinoside can inhibit the PI3K‐Akt, NF‐κB, and other signalling pathways and reduce cardiovascular and cerebrovascular injuries 37–44 . The ingredients such as caffeic acid, protocatechuic acid, gallic acid, bilobalide, ginkgolide A, quercetin‐3‐O‐rutinoside, kaempferol‐3‐O‐rutinoside, kaempferol‐3‐O‐glucoside, and isorhamnetin‐3‐O‐rutinoside also have anti‐oxidative, free radical‐scavenging, and anti‐apoptotic effects 22–50 .…”

“…Quercetin‐3‐O‐rutinoside, kaempferol‐3‐O‐rutinoside, kaempferol‐3‐O‐glucoside, and isorhamnetin‐3‐O‐rutinoside can inhibit the PI3K‐Akt, NF‐κB, and other signalling pathways and reduce cardiovascular and cerebrovascular injuries 37–44 . The ingredients such as caffeic acid, protocatechuic acid, gallic acid, bilobalide, ginkgolide A, quercetin‐3‐O‐rutinoside, kaempferol‐3‐O‐rutinoside, kaempferol‐3‐O‐glucoside, and isorhamnetin‐3‐O‐rutinoside also have anti‐oxidative, free radical‐scavenging, and anti‐apoptotic effects 22–50 . Consequently, it was deduced that anti‐inflammatory, anti‐oxidative, anti‐apoptotic, and other activities are the main anti‐IS effects of GBE.…”

“…Based on literature data mining, flavonoid‐glycosides and terpenes in G. biloba are the main components of its pharmacological activities such as anti‐inflammatory, antioxidant, blood circulation‐improving, and platelet anti‐aggregatory activities 22–51 . Therefore, the above ingredients were selected for the study of the active ingredients of GBE against IS, as shown in Table 1.…”