Flavonoids as Natural Anti-Inflammatory Agents Targeting Nuclear Factor-Kappa B (NFκB) Signaling in Cardiovascular Diseases: A Mini Review

Cardiovascular diseases (CVDs) such as angina, hypertension, myocardial ischemia, and heart failure are the leading causes of morbidity and mortality worldwide. One of the major transcription factors widely associated with CVDs is nuclear factor-kappa B (NFκB). NFκB activation initiates the canonical and non-conical pathways that promotes activation of transcription factors leading to inflammation, such as leukocyte adhesion molecules, cytokines, and chemokines. Flavonoids are bioactive polyphenolic compounds found abundantly in various fruits, vegetables, beverages (tea, coffee), nuts, and cereal products with cardiovascular protective properties. Flavonoids can be classified into six subgroups based on their chemical structures: flavanones, flavones, flavonols, flavan-3-ols, isoflavones, and anthocyanidins. As NFκB inhibitors, these flavonoids may modulate the expression of pro-inflammatory genes leading to the attenuation of the inflammatory responses underlying various cardiovascular pathology. This review presents an update on the anti-inflammatory actions of flavonoids via inhibition of NFκB mechanism supporting the therapeutic potential of these natural compounds in various CVDs.

The inhibition of NFκB pathway has been demonstrated to show beneficial effect in various CVDs including hypertension (Koeners et al., 2016), myocardial infraction (Zhao et al., 2017), and arteriosclerosis . These findings support that targeted inhibition of NFκB appears to be a promising strategy in reducing cardiovascular complications.
Flavonoids are plant polyphenolic compound derivatives from natural origin found in fruits, grains, vegetables, roots, bark, flowers, stems, tea, and wine (Zeinali et al., 2017). Non-plant natural products such as mushrooms and honey, plant extracts, plant juices, plant powders, and essential oils have shown to possess antiinflammatory activities and many of these plant natural products have polyphenols as their major compound (Khalil and Sulaiman, 2010;Azab et al., 2016). However, the protective effects of flavonoid in CVDs via inhibition of NFκB are yet to be reviewed. Therefore, in this mini-review, we focused on the anti-inflammatory actions of flavonoids via inhibition of NFκB mechanism in CVDs.
Flavanones widely present in all citrus fruits, which gives the bitter taste of the juice and its peel. Oranges, lemons, and grapes are rich sources of flavanones and major compounds are hesperitin, naringenin, and eriodictyol (Barreca et al., 2017).
Isoflavones are unique in that they resemble estrogen in structure and, therefore, are classified as phytoestrogens. There are found abundantly in soy products such as tofu, roasted soy nuts, and miso (Marzocchella et al., 2011).

NFкB INDUCeD INFLAMMATION AND CvDS
There are a few cellular redox pathways involved in the development of the chronic inflammatory CVD, which includes NFκB. NFκB is a transcription factor that activates inhibitor of kappa B (IκB) kinase in the cytosol upon being stimulated by inflammatory stimuli (Brasier, 2010). Subsequent signaling pathways via canonical or non-canonical lead to migration of NFκB toward the nucleus and hence initiates the targeting gene such as pro-inflammatory cells, monocytes, macrophages, and T and B cells (Figure 1).
The canonical NFκB pathway responds rapidly to stimuli and activates NFκB, which increases pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α), which results in cell apoptosis. TNF-α receptor signaling plays an important role in the canonical pathway of NFκB in cell death via Jun N-terminal kinases (JNK), p38, and caspase 8 cascades (Ghosh et al., 2009). Furthermore, NFκB also activates angiotensin II, endothelin-1, and phenylephrine as hypertrophic agonist via IκB degradation and p65 nuclear translocation.
A central signaling component of the non-canonical NFκB pathway is NFκB-inducing kinase, which induces p100 phosphorylation through kinase IKKα in a slow manner (Sun, 2017). Ligands of a subset of tumor necrosis factor receptor (TNFR) superfamily members are typical inducers of the noncanonical NFκB pathway (Shih et al., 2011).

Quercetin
Quercetin or 3, 3′, 4′, 5, 7-pentahydroxyflvanone that falls into the category of flavonol is widely found in plants such as Ginkgo biloba, Hypericum perforatum, and Sambucus canadensis as well as vegetables such as apples, berries, grapes, onions, shallots, and tomatoes .
In a clinical study involving patients with chronic systemic inflammation (CSI) in stable coronary artery disease (CAD), quercetin showed anti-inflammatory effects with reduction in indicators of CSI (Chekalina et al., 2018). Quercetin decreased IL-1β and TNF-α levels in blood serum, in addition to decreasing the transcriptional activity of NFкB in blood mononuclear cells (Chekalina et al., 2018). In leptin-induced inflammation model using human umbilical vein endothelial cells (HUVECs), quercetin significantly suppressed the upregulation of Ob-Ra (leptin receptor) expression, ERK1/2 phosphorylation, NFкB, and TNF-α (Indra et al., 2013). Furthermore, in a neonatal rat cardiac fibroblast, quercetin inhibited TNF-α, IL-1β, and IL-6 secretion by inhibiting the activation of NFкB and Akt induced by lipopolysaccharide (LPS) (Tang et al., 2014).
In an in-vivo sodium fluoride-induced hypertensive model, administration of luteolin increased nitric oxide (NO) bioavailability, reversed prolongation of QT and QTc intervals, and reduced the expressions of kidney injury marker 1 (Kim-1), NFκB, and cardiac troponin I (CTnI), which eventually normalized the blood pressure (Oyagbemi et al., 2018a). Previous study in neonatal rat cardiac myocytes exposed to LPS showed luteolin reduced the TNF-α levels in the medium, downregulated the TNF-α mRNA in myocytes, inhibited degradation of IκB-β and nuclear translocation of NFκB, as well as reduced NFκB DNA binding, proposing the therapeutic potential of luteolin the management of inflammation-related myocardial diseases (Lv et al., 2011).

Fisetin
Fisetin or 3, 3c,4c,7-tetrahydroxyflavone is a bioactive molecule found in fruits such as strawberry, apple, persimmon, and grape and vegetables such as onion and cucumber (Arai et al., 2000). Garg et al. (2019) reported the protective effect of fisetin against isoproterol-induced myocardial injury by suppressing myocardial injury markers, creatine kinase-muscle/brain (CK-MB), lactate dehydrogenase (LDH), and inflammatory markers (TNF-α and IL-6) in the blood serum as well as normalization of histological and ultrastructure of the heart. In addition, fisetin regulated the balance between pro-or anti-oxidants and pro-or anti-apoptotics proteins in the myocardial tissue (Garg et al., 2019). These protective effects of fisetin are attributed to the downregulation of receptor for advanced glycation end products (RAGE) and NFκB (Garg et al., 2019).
Fisetin attenuated the development of diabetic cardiomyopathy by attenuating the expression of myocardial NFκB and the proinflammatory cytokines IL-1β, IL-6, and TNF-α in the heart of diabetic rats. These result in reduction of cardiac function markers such as CK-MB, LDH, and cTn as well as normalization heart morphology (Althunibat et al., 2019).
FIGURe 1 | Mechanism of NFκB action. In inactivated state, NF-κβ, which consists of Rel and p50 proteins, is located in the cytosol complexed with the inhibitory protein Iκβα. IκB kinase (IKK) is activated by extracellular signals via membrane receptors. Subsequently, IKK phosphorylates the Iκβα protein resulting in ubiquitination of Iκβα and eventually by the proteasome for Iκβα degradation (canonical pathway). In non-canonical pathway, RelB favors the activation of NF-κβ via RelB. Activated NF-κβ is further translocated into the nucleus for DNA bindings, called response elements (RE). The DNA/NF-κβ complex attracts coactivators and RNA polymerase, which transcribe the DNA into mRNA resulting in a cell transformation.
Apigenin was also shown to improve cardiac dysfunction and fibrosis in diabetic cardiomyopathy. Apigenin blunted the activity of NFκB and downregulated the activity of caspase3 accompanying with decreasing oxidative stress marker, glutathione peroxidase (GSH-Px), malondialdehyde (MAD), and superoxide dismutase (SOD) (Huangjun et al., 2016).

Isoliquiritigenin
Isoliquiritigenin is extracted from root of licorice and has been used traditionally for the treatment of inflammatory or pulmonary diseases (Peng et al., 2015). In HUVECS exposed to TNF-α, isoliquiritigenin blocked the involvement of NFкB at the transcriptional levels, and thus attenuated the downstream expression of VCAM-1, E-selectin, THP-1 monocyte adhesion, IкB-α, and PECAM-1, suggesting the protective effects of isoliquiritigenin through NFкBdependent mechanisms (Kwon et al., 2007). In angiotensin II induced hypertension model, isoliquiritigenin attenuated inflammation cytokines including IL-1β and TNF-α, excessive deposition of extracellular matrix, and oxidative stressinduced apoptosis via nuclear factor E2-related factor 2 (Nrf2) and NFκB pathways .

Rutin
Rutin is a flavonol that presents in buckwheat and citrus fruits. In a sodium fluoride-induced hypertensive rats, administration of rutin reduced blood pressure elevation by enhancing NO bioavailability via down-regulation of NFκB expression and up-regulation of Nrf2 (Oyagbemi et al., 2018b).
In carfilzomib-induced cardiotoxicity in rat, rutin protected against myocardial hypertrophy by upregulating IκB-α and downregulating NFκB expression, resulting in attenuation of β-myosin heavy chain, reduction in B-type natriuretic peptide mRNA expressions, and normalization of cardiac muscle fiber morphology (Imam et al., 2017).
In addition, rutin increased activities of Nrf, decreased activation of NFκB in human embryonic kidney reporter cell line, and preserved relaxation of fetal placental arteries derived from human chorionic plate (Sthijns et al., 2017).
In LPS-induced inflammation in HUVECs, rutin reversed barrier disruption, expression of cell adhesion molecules, and adhesion and migration of monocytes in endothelial cells. The barrier protective effects of rutin were linked to a down-regulation of TNF-α, deactivation of NFκB, and reduced phosphorylation of IκB-α (Lee et al., 2012).
In a rat model of monocrotaline-induced pulmonary arterial hypertension (PAH), chrysin reduced right ventricular systolic pressure and mean pulmonary artery pressure. In addition to suppression of right ventricular remodeling, chrysin abolished increased expression of collagen I, collagen III, and NFκB .
In isoprenaline-induced myocardial injury in rats, chrysin relieved hemodynamic and ventricular dysfunction as well as reduced ultrastructural myocardial damage via inhibition of NFκB, IκKβ expression, and TNF-α level as well as increased peroxisome proliferator-activated receptor-gamma (PPAR-γ) expression (Rani et al., 2016).
In a rat model of myocardial infarction, fibrosis in the interstitial and perivascular regions and expression of collagen was reduced following chrysin treatment (Yang et al., 2018). This effect is associated with increased PPAR-γ expression and decreased NFκB expression via inhibition of IκKβ phosphorylation, leading to reduction of matrix metalloproteinase-2 (MMP-2), MMP-9 levels, and suppression of activator protein 1 (AP-1) level.

Genistein
Genistein under the subgroup of an isoflavone [ 4 ′ , 5 , 7 -t r i hy d r o x y i s o f l a v o n e , 5 , 7 -d i hy d r o x y -3 -( 4hydroxyphenyl)-4-H-1-benzopyran-4-one] is primarily found in soy-based foods, legumes, and red clover. In homocysteineinduced endothelial cell inflammatory injury, genistein prevented endothelial damage via blockade of activation of NFκB, expression of inflammatory cytokine and adhesion molecule, IL-6, and ICAM-1 (Han et al., 2015). Xu et al. (2019) explored the effect of genistein on angiotensin II-induced vascular smooth muscle cell inflammation. Angiotensin II-induced expression of NFκB, C-reactive protein (CRP), MMP-9, phosphorylation of ERK1/2 and p-38, which lead to atherosclerotic inflammatory damage, were reversed following genistein treatment. Furthermore, genistein enhanced expression of PPAR-γ, suggesting cardiovascular protective effect by the isoflavone is through regulation of p38/ERK1/2-PPARγ-NFκB signaling pathway (Xu et al., 2019).

Silymarin
Silymarin is a flavonolignan extracted from the milk thistle. Silymarin augmented relaxation of pulmonary arteries isolated from a lung ischemia-reperfusion (I/R) injury model (Jin et al., 2016). Vascular protective effect of silymarin is due to inhibition of NFκB, thus suppressing the serum concentration of inflammatory cytokines and reducing protein expression of hypoxia inducible factor-1α (HIF-1α) and iNOS.
Silibinin, a major active constituent of silymarin, was able to reduce the abnormal size of cardiac myocytes and prevent hypertrophy by alleviating the production of epidermal growth factor receptor (EGFR) (Ai et al., 2010). Silibilin exerted its anti-inflammatory effect by suppressing the activation of NFκB stimulated by angiotensin II in cardiac myocytes or in the aortic banding male mice. Furthermore, silibilin interfered with the phosphorylation and degradation of IκB-α and activation of IκKβ in vivo.

CONCLUSION
The actions of flavonoids in mitigating inflammation by modulation of NFкB offer potential agents for the treatment of CVDs. However, several of these actions reported in vitro may yet to be fully recognized due to their low bioavailabilities following oral administration (Hollman and Katan, 1998;Thilakarathna and Rupasinghe, 2013). Flavonoids have shown promising results in reducing atherosclerosis in several animal experimental models; however, conflicting results were reported in human clinical trials (Arts and Hollman, 2005;Zordoky et al., 2015). The low bioavailability and clinical efficacy of flavonoids are attributed to their poor absorption, metabolism by the metabolizing enzymes in the intestine and liver, and structural modifications by the colonic bacteria remain as the major problems. Continuous investigation is required to enhance the bioavailability and efficacy of the flavonoids to tap the full potential of these natural agents.

AUTHOR CONTRIBUTIONS
All authors contributed to the writing. KC, DM, and MM conceived, designed, and revised the manuscript.

FUNDING
This study was funded by Government Agency grant GA001-2017, MAHSA University and USIM research project code: PPPI/FPG/0118/051000/15618 Universiti Kebangsaan Malaysia and Universiti Putra Malaysia. The funding agencies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript, which are fully the responsibilities of the authors.