Inhibition, Nuclear factor kappa B (NF-kB)

The NF-κB pathway consists of a series of events including IRAK (IL-1 receptor-associated kinase) signaling, where the transcription factors of the NF-κB family play the key role. The canonical NF-κB pathway can be activated by a range of stimuli, including TNF receptor activation by TNF-a. Upon pathway activation, the IKK complex will be phosphorylated, which in turn phosphorylates IkBa. This NF-κB inhibitor will be K48-linked ubiquitinated and degradated, allowing NF-κB to translocate to the nucleus. There, this transcription factor can express pro-inflammatory and anti-apoptotic genes. Furthermore, negative feedback genes are also transcribed and include IkBa and A20. When the NF-κB pathway is inhibited, its translocation will be delayed (or absent), resulting in less or no regulation of NF-κB target genes. This can be achieved by IKK inhibitors, proteasome inhibitors, nuclear translocation inhibitors or DNA-binding inhibitors (Gupta et al., 2010; Liu et al., 2017). Therefore, inhibition of IL-1R activation suppresses NF-κB.

In addition to the NF-kB pathway, IRAK activates a variety of transcription factors, including Interferon regulatory factor 5 (IRF5), Adaptor protein-1 (AP-1) and cAMP response element binding protein (CREB), resulting in the expression of broad array of inflammatory molecules and apoptosis-related proteins (Jain, 2014).

NF-κB transcriptional activity: Beta lactamase reporter gene assay (Miller et al. 2010)

NF-κB transcription: Lentiviral NF-κBGFP reporter with flow cytometry (Moujalled et al. 2012)

IκBα phosphorylation: Western blotting (Miller et al. 2010)

NF-κB p65 (Total/Phospho) ELISA：

ELISA for IL-6, IL-8, and Cox

The binding of sex steroids to their respective steroid receptors directly influences NF-κB signaling, resulting in differential production of cytokines and chemokines (McKay and Cidlowski, 1999; Pernis, 2007). 17b-estradiol regulates pro-inflammatory responses that are transcriptionally mediated by NF‑κB through a negative feedback and/or transrepressive interaction with NF-κB (Straub, 2007). Progesterone suppresses innate immune responses and NF-κB signal transduction reviewed by Klein et al. (Klein and Flanagan, 2016). Androgen-receptor signaling antagonises transcriptional factors NF-κB(McKay and Cidlowski, 1999).

Evidence for perturbation of this molecular initiating event by stressor

Dex inhibits IL-1β gene expression in LPS-stimulated RAW 264.7 cells by blocking NF-κB/Rel and AP-1 activation (Jeon et al., 2000).

Various inhibitors for NF‐κB, such as dimethyl fumarate, curcumin, iguratimod, epigalocathechin gallate (EGCG), and DHMEQ inhibits lLPS-induced NF-κB activation and LPS-induced secretion of IL-1β (McGuire et al., 2016; Mucke, 2012; Peng et al., 2012; Suzuki and Umezawa, 2006; Wang et al., 2020; Wang et al., 2018; Wheeler et al., 2004).

TAK-242 (Matsunaga et al., 2011) inhibit TLR4 itself. There are several IRAK4 inhibitors (Lee et al., 2017). These molecules block the upstream signal to NF‐κB activation. IRAK4 has recently attracted attention as a therapeutic target for inflammation and tumor diseases (Chaudhary et al., 2015).

LPS treatment induced a significant upregulation of the mRNA and release of IL-1β from retinal microglia. Minocycline inhibited its releases. Thus, minocycline might exert its antiinflammatory effect on microglia by inhibiting the expression and release of IL-1β (Wang et al., 2005).

Caspase-1 inhibition reduced the release of IL-1β in organotypic slices exposed to LPS+ATP. Administration of pralnacasan (intracerebroventricular, 50 μg) or belnacasan (intraperitoneal, 25–200 mg/kg) to rats blocked seizure-induced production of IL-1β in the hippocampus, and resulted in a twofold delay in seizure onset and 50% reduction in seizure duration (Ravizza et al., 2006).

Belnacasan, an orally active IL-1β converting enzyme/caspase-1 inhibitor, blocked IL-1β secretion with equal potency in LPS-stimulated cells from familial cold urticarial associated symdrome and control subjects (Stack et al., 2005).

In LPS-induced acute lung injury (ALI) mice model, LPS induced inflammatory cytokines such as TNF-α, IL-6, IL-13 and IL-1β were significantly decreased by cinnamaldehyde (CA) (Huang and Wang, 2017).

The suppressing capacities of six cinnamaldehyde-related compounds were evaluated and compared by using the LPS-primed and ATP-activated macrophages. At concentrations of 25~100 mM, cinnamaldehyde and 2-methoxy cinnamaldehyde dose-dependently inhibited IL-1β secretion (Ho et al., 2018).

In vitro, CA decreased the levels of pro-IL-1β and IL-1β in cell culture supernatants, as well as the expression of NLRP3 and IL-1β mRNA in cells. In vivo, CA decreased IL-1β production in serum. Furthermore, CA suppressed LPS-induced NLRP3, p20, Pro-IL-1β, P2X7 receptor (P2X7R) and cathepsin B protein expression in lung, as well as the expression of NLRP3 and IL-1β mRNA (Xu et al., 2017).

IL-1Ra binds IL-1R but does not initiate IL-1 signal transduction (Dripps et al., 1991). Recombinant IL-1Ra (anakinra) is fully active in blocking the IL-1R1, and therefore, the biological activities of IL-1α and IL-1β. The binding of IL-1α and IL-1β to IL-1R1 can be suppressed by soluble IL-1R like rilonacept (Kapur and Bonk, 2009). The binding of IL-1β to IL-1R1 can be inhibited by anti-IL-1β antibody (canakinumab and gevokizumab) (Church and McDermott, 2009) (Roell et al., 2010).

IL-1 is known to mediates autoinflammatory syndrome, such as cryopyrin-associated periodic syndrome, neonatal-onset multisystem inflammatory disease and familial Mediterranean fever. Blocking of binding of IL-1 to IL-1R1 by anakinra, canakinumab, and rilonacept have been already used to treat these autoinflammatory syndrome associated with overactivation of IL-1 signaling (Quartier, 2011).

Dex inhibits IL-1β gene expression in LPS-stimulated RAW 264.7 cells by blocking NF‐κB/Rel and AP-1 activation (Jeon et al., 2000).

Inhibition of IL-1 binding to IL-1R or the decreased production of IL-1b leads to the suppression of IL-1R signaling leading to NF‐κB activation.

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