AuthorsRoberto Risitano, Monica Currò, Santa Cirmi, Nadia Ferlazzo, Pietro Campiglia, Daniela Caccamo, Riccardo Ientile, Michele Navarra, Giovanni Li Volti
AbstractPlant polyphenols exert anti-inflammatory activity through both anti-oxidant effects and modulation of pivotal pro-inflammatory genes. Recently, Citrus bergamia has been studied as a natural source of bioactive molecules with antioxidant activity, but few studies have focused on molecular mechanisms underlying their potential beneficial effects. Several findings have suggested that polyphenols could influence cellular function by acting as activators of SIRT1, a nuclear histone deacetylase, involved in the inhibition of NF-κB signaling. On the basis of these observations we studied the anti-inflammatory effects produced by the flavonoid fraction of the bergamot juice (BJe) in a model of LPS-stimulated THP-1 cell line, focusing on SIRT1-mediated NF-κB inhibition. We demonstrated that BJe inhibited both gene expression and secretion of LPS-induced pro- inflammatory cytokines (IL-6, IL-1β, TNF-α) by a mechanism involving the inhibition of NF-κB activation. In addition, we showed that BJe treatment reversed the LPS-enhanced acetylation of p65 in THP-1 cells. Interestingly, increasing concentrations of Sirtinol were able to suppress the inhibitory effect of BJe via p65 acetylation, underscoring that NF-κB–mediated inflammatory cytokine production may be directly linked to SIRT1 activity. These results suggest that BJe may be useful for the development of alternative pharmacological strategies aimed at reducing the inflammatory process.
Stimulation by pathogen-specific ligands, including bacterial lipopolysaccharide (LPS), leads cells of the innate immune system, such as monocytes and macrophages to produce several pro- and anti-inflammatory cytokines .
Accumulating evidences suggest that chronic inflammation represents the main contributing factor to several chronic degenerative pathologies, including cardiovascular diseases, neurological disorders and cancer –.
Nuclear factor-kappa B (NF-κB) has been reported to play a pivotal role in inflammatory response through the induction of inflammation-related cytokines (i.e. IL-6, IL-1β, TNF-α) and enzymes such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) , . The best-studied and most prevalent form of NF-κB exists as a heterodimer composed of p50 and RelA/p65 subunits. In unstimulated cells, NF-κB is sequestered in the cytoplasm through its assembly with its inhibitory proteins, which are members of the IκB family , . In response to various stimuli, such as cytokines, DNA-damaging agents, bacterial wall or viral proteins, IκB dissociates, via phosphorylation by the IκB kinase (IKK), and the activated transcription factor can translocate into the nucleus where is able to induce a large number of target genes involved in cell growth, apoptosis, cell adhesion and inflammation , .
According to several findings, the reversible acetylation of RelA/p65 subunit can modulate NF-κB signaling depending on the acetylation status of specific lysine residues . In particular, acetylation of lysine 310 is required for full transcriptional activity of RelA and for activation of NF-κB complex . Interestingly, SIRT1, a nuclear NAD+-dependent histone deacetylase (HDAC), may play a role in regulating inflammation by directly deacetylating the RelA/p65 protein at lysine 310. Recent studies show how the catalytic activity of SIRT1 can be modulated both positively and negatively through the binding by some agonists. Resveratrol, a polyphenolic compound found in red wine, has been identified as a potent pharmacological activator of SIRT1 . On the other hand, Sirtinol was the first synthetic inhibitor extensively used in literature , .
Many anti-inflammatory drugs (SAID and NSAID) currently used in therapy target NF-κB and COX-2, but because of their frequent and serious associated side effects, result in high rates of morbidity , . Therefore, during the last years there has been a growing interest towards the anti-inflammatory properties of some natural drugs and their bioactive compounds, like polyphenols (PP).
PP are organic molecules that constitute a numerous and heterogeneous family of secondary metabolites of plant cells, where they exert a protective action against ultraviolet radiation and oxidative stress . PP are present in many edible plants and thus represent an integral part of human supply . Several studies have shown that high regular intake of some phenolic compounds in the diet plays a preventive action against several human diseases, such as cardiovascular pathologies, atherosclerosis, osteoporosis, allergies, diabetes, neurodegenerative diseases and cancer , . Although the mechanisms by which these natural compounds exert their benefits are not fully understood, anti-inflammatory effects of PP have been attributed primarily to their antioxidant activity, because they were known to scavenge and prevent the formation of reactive oxygen and nitrogen species (ROS and RNS, respectively) , , which represent important hallmarks of inflammation. Furthermore, during recent years, numerous studies have suggested that PP could influence cellular function by direct interaction with several receptors, modulation of intracellular signaling and transcription of gene involved in different pro-inflammatory pathways , .
Citrus bergamia Risso et Poiteau (Bergamot) is a typical fruit of the Southern Italy, belonging to the family of Rutaceae. Bergamot is cultivated almost exclusively on the Ionian coast of the province of Reggio Calabria (Italy), where there are climatic and environmental conditions particularly suitable for its cultivation. Bergamot fruit is mainly used to extract the essential oil obtained from the peel, much employed in the fragrance industry and which have been experimentally studied to evaluate its potential neuroprotective activity . On the contrary, bergamot juice (BJ), obtained from the endocarp of the fruit, is considered just a secondary and discarded product. Studies performed by Miceli et al.  have shown that a chronic administration of BJ is effective to prevent the diet-induced hyperlipidemia in rat, suggesting a relationship between the beneficial effect and its antioxidant properties. Moreover, a clinical research showed that the bergamot-derived polyphenolic fraction, given orally in patients suffering from metabolic syndrome, produces significant reduction of serum cholesterol, triglycerides and glycaemia , strengthen the finding obtained in animal model. More recently, we demonstrated that BJ is also able to inhibit important molecular pathways related to cancer-associated aggressive phenotype, thus reducing growth, adhesion and migration in different in vitro  and in vivo models . Finally, studies performed on colorectal cancer cell line verified that the antiproliferative effect of BJ is due to its flavonoid fraction which is able to act by multiple mechanisms depending on the concentration .
However, to date, the anti-inflammatory potential of BJ has never been evaluated. Therefore, the present study was designed to assess the modulating effects of flavonoid fraction of BJ (BJe) on the expression of inflammation-related cytokines. Considering the involvement of NF-κB pathway on the production of these pro- inflammatory mediators, we focused on the possibility that BJe may regulate NF- κB activation and examined underlying mechanisms associated with NF-κB/SIRT1 crosstalk in LPS-stimulated THP-1 monocytes, a human leukemia monocytic cell line, that have been widely used as a model to study the inflammatory cell response.
Article Link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178028/