Plant polyphenols as inhibitors of NF-κB induced cytokine production—a potential anti-inflammatory treatment for Alzheimer's disease?

Alzheimer’s disease (AD) is a neurodegenerative disorder that impacts the daily lives of many sufferers throughmemory loss as well as behavioral and cognitive changes. AD is themost common form of dementia. One in ten people over the age of 65, and around half of those over 85 have AD. AD can be divided into familial (early-onset) and sporadic (late-onset) cases, with the familial form ( 99% of cases) caused by a variety of genetic (e.g., apolipoprotein E), metabolic and environmental factors. The AD brain is characterized macroscopically by cortical atrophy, caused by degeneration of the cholinergic axonal arborisation and shrinkage of the dendritic tree. Microscopically, amyloid beta peptide deposits (senile plaques) and neurofibrillary tangles are present in affected areas (GilBea et al., 2012). AD is also characterized by chronic neuroinflammation, driven by activation of astroglia and microglia (Rosenblum, 2014). In addition, levels of pro-inflammatory mediators or cytokines which include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors are elevated in the brains of patients with AD (Latta et al., 2014). Furthermore, nuclear translocation of NF-κB and STAT-1α, transcription factors involved in pro-inflammatory gene expression, indicates the presence of a sustained pro-inflammatory process (Lawrence, 2009).


Drug Discovery Difficulties Faced in Alzheimer's Disease
Drug discovery for AD has been strongly focused on β-amyloid (initially plaques, then soluble oligomers), as genetic evidence from the familial cases supported by the hypothesis that β-amyloid must be driving the disease process. Based on the "amyloid cascade hypothesis, " anti-amyloid therapies were hoped to deliver a cure for AD (Robinson et al., 2004). Unfortunately, numerous clinical trials with active and passive amyloid vaccines as well as G-secretase inhibitors have failed (reviewed in Castello et al., 2014). Currently, there are no disease-modifying drugs available for AD. Consequently, alternative therapeutic targets, such as neuroinflammation have been suggested for the prevention and treatment of AD (Shi et al., 2013;Latta et al., 2014). As the expression of many pro-inflammatory cytokines is driven by the transcription factor NF-κB (Hoffmann et al., 2006), we propose that brain-permeable inhibitors of NF-κB signaling have the potential to prevent or slow down the progression of AD.
In unstimulated cells, NF-kB is present in the cytoplasm as an inactive heterodimer composed of two subunits, p50 and p65 (relA). The heterodimer forms a complex with the inhibitory proteins IkB-α or IkB-β, retaining it in the cytoplasm. When stimulated by a PAMP, DAMP or cytokine, it triggers a cascade of signaling events initiated by stimulation of adapter proteins in the cytoplasm. In the case of many TLRs, myeloid differentiation primary response protein 88 (MYD88) is such an adaptor protein (Mogensen, 2009;Figuera-Losada et al., 2014). MYD88 activates specific protein kinases downstream, such as mitogen activated protein kinase (MAPK) and IκB kinase which phosphorylate IkBα. Phosphorylation of IκB-α and its proteasomal degradation leads to its dissociation from the NF-κB complex. NF-κB then translocates into the nucleus, where it binds to the promotor regions of specific genes (Brasier, 2006;Gilmore, 2006;Perkins, 2007). NF-κB then recruits other proteins (coactivators and RNA polymerase), which finally lead to the expression of many chemokines (e.g., IL-8, MIP-1α, MCP1, RANTES and eotaxin), proinflammatory cytokines (e.g., IL-1, IL-2, IL-6 and TNF-α), adhesion molecules (e.g., ICAM, VCAM, E-selectin) as well as inducible pro-inflammatory enzymes (COX-2 and iNOS), which exacerbate and perpetuate the inflammatory process (Barnes, 1997;Ghosh and Karin, 2002;Figuera-Losada et al., 2014).

Plant Polyphenols and their Ability to Prevent Age-Related Degenerative Diseases
Many dietary plants including fruits, vegetables and whole grains contain substantial amounts of polyphenols (Carlsen et al., 2010;Gunawardena et al., 2014). Polyphenols are characterized as compounds with phenolic structural features (Liu, 2004;Tsao, 2010). Dietary polyphenols have been suggested to aid in the prevention of degenerative diseases, including cancer, cardiovascular disease and neurodegenerative diseases such as AD due to their anti-inflammatory and anti-oxidant properties (Liu, 2004;Tsao, 2010). The high diversification of plant polyphenols have led to different ways of categorizing these naturally occurring compounds (Liu, 2004;Tsao, 2010). Polyphenols differ by their source of origin, chemical structure and biological effects (Liu, 2004;Tsao, 2010;Bellik et al., 2012a;Ebrahimi and Schluesener, 2012). They are secondary plant metabolites produced to aid in the defense mechanism against herbivores, insects, ultraviolet radiation, and microorganisms (Yoon and Baek, 2005;Tsao, 2010). More than 8000 plant polyphenols are currently known and among them more than 4000 flavonoids have been identified (Tsao, 2010). Some polyphenols lead to growth inhibition in laboratory animals (Ebrahimi and Schluesener, 2012). However, epidemiological data suggest that intake of small amounts of polyphenols in foods and beverages has a potent effect on reducing chronic diseases (Gotsis et al., 2015). There are studies that have investigated the effectiveness of fruit and vegetable rich diets such as the Mediterranean diet against degenerative diseases, indicating that these diets lead to a reduced incidence of degenerative and agerelated diseases including cancer, cardiovascular diseases and neurodegenerative diseases (Calabrese et al., 2003;Yoon and Baek, 2005;Pan et al., 2010;Sofi et al., 2010;Bellik et al., 2012b;Vetrani et al., 2013;Gunawardena et al., 2014).

Anti-inflammatory Activity of Plant Polyphenols
Many plant polyphenolic compounds including curcumin, apigenin, quercetin, (E)-cinnamaldehyde and (E) -resveratrol have been shown to have anti-inflammatory activities in cell culture studies (Gautam and Jachak, 2009). Molecular targets of plant polyphenols acting as anti-inflammatory compounds include arachidonic acid (AA) dependent pathways and AA independent pathways. In the AA-dependent pathway, the antiinflammatory effect of plant polyphenols is related to their ability to inhibit COX (the isoform Cox-2, also regulated by NF-κB), which converts AA into prostaglandins. AA-independent pathways involve AA signaling through nuclear factor-kappa B (NF-κB) (Yoon and Baek, 2005).
Polyphenols have been shown to interfere at two specific sites in the pathway leading from receptors to NF-kB. Some polyphenols inhibit kinases by inhibiting their phosphorylation or ubiquitination and therefore prevent the subsequent degradation of IkB (Ruiz and Haller, 2006). This prevents NF-κB translocation into the nucleus and transcription of pro-inflammatory cytokines. Additionally, inhibition of the interaction of NF-κB subunits with target DNA has also been proposed as a mode of action of anti-inflammatory polyphenols (Ruiz and Haller, 2006). Both modes of action ultimately lead to the inhibition of expression of various NF-κB regulated proinflammatory proteins (cytokines, chemokines) and enzymes (iNOS, COX-2).

Curcumin is a Prototype Inhibitor of NF-κB Signaling Acting Upstream of IκB
Curcumin occurs naturally in the root of plant Curcuma longa which belongs to the Zingiberaceae family. The root of this plant is powdered and used in cooking for centuries in Asian countries. It has also been used extensively in traditional Indian and Chinese medicine to treat diabetic wounds, hepatic disorders, sinusitis and rheumatism (Chainani-Wu, 2003).
Curcumin has been shown to have various anti-inflammatory properties. Curcumin has a broad cytokine-suppressive antiinflammatory action, it down-regulates the expression of cyclooxygenase-2 (COX-2), lipoxygenase, and inducible nitric oxide synthase (iNOS) enzymes. Curcumin also inhibits the production of the pro-inflammatory cytokines such as TNF-α, IL-1, -2, -6, -8, and -12, and the neurotoxic factors in LPSstimulated monocytes and alveolar macrophages (Abe et al., 1999). Furthermore, the pharmacokinetic properties of curcumin are also favorable. Curcumin crosses the brain-blood-barrier and bioavailable curcumin preparations (orally applied) can achieve therapeutic concentrations in the brain. For example, brain levels of the curcuminoids reached concentrations of up to 3 µM for curcumin, and up to 6 µM for tetrahydrocurcumin (TC). The EC 50 value for iNOS mRNA inhibition in vivo was 1.2 and 0.7 µM for curcumin and TC, respectively.
Inhibition of NF-κB signaling is the best described mechanism of anti-inflammatory action of curcumin (Ambegaokar et al., 2003;Shakibaei et al., 2007). It has been shown that curcumin inhibits the phosphorylation and degradation of IκBα and the subsequent translocation of the p65 subunit of NF-κB to the nucleus (Singh and Aggarwal, 1995). Although the exact target is not known, the study indicates that curcumin interferes with the NF-κB pathway upstream of IκBα phosphorylation (Figure 1). In a further study in intestinal epithelial cells, curcumin has also been shown to block a signal leading to IKK activation (Jobin et al., 1999).

Conclusions
We suggest that inhibitors of NF-kB activation should have the potential to prevent and delay the onset or even treat Alzheimer's disease (Bremner and Heinrich, 2002). However, clinical trials are needed to confirm the effectiveness of some of these plant-based inhibitors of NF-κB pathway as a treatment option for Alzheimer's disease. Nonetheless, in preparation of those trials, critical pharmacokinetic parameters have to be addressed beforehand. Comprehensive investigation models with multidisciplinary approach, including epidemiological, clinical and cellular molecular studies should be employed to ensure that the doses extrapolated from the in vitro data determined in cell cultures have pharmacological relevance (e.g., the tissue concentration matching the EC 50 values). Their efficacy has to be tested in animal models of AD, which exhibits some degree of chronic inflammation generated by the pathological deposits such as beta-amyloid or tau, or in a "pure" animal model of chronic neuroinflammation such as the IL-1β or IL-6 overexpressing mouse (Ebrahimi and Schluesener, 2012;Millington et al., 2014). If these animal studies are successful, and show not only a decrease in NF-κB regulated pro-inflammatory mediators, but also an improvement in cognition and memory, then plant polyphenols might have the potential to be the first disease modifying drugs for the treatment of Alzheimer's disease.