Modulation of NF-κB signaling pathway by tocotrienol in neurodegenerative diseases

Abstract

Neurodegenerative diseases (ND) are a form of non-communicable disease that have placed immense social and financial burdens on society. The number of people affected by ND is an ongoing trend due to the global issue of an ageing population. Hence, more effort should be made to treat ND, so alternative options like natural components are considered. Tocotrienol (T3) is a form of vitamin E that possesses many beneficial properties, such as antioxidant, anti-cancer and anti-inflammatory properties. In recent years, many clinical studies revolving around diseases such as neurodegenerative diseases, cardiovascular diseases and osteoporosis appear to suggest that T3 supplementation can be effective in alleviating the symptoms of these diseases. Given that T3 has multiple health benefits and is a potential therapeutic agent for ND, it is important to understand the mechanism through which T3 exerts the beneficial works. One such pathway is the NF-κB signalling pathway, which is closely associated with neuroinflammation and neurodegenerative diseases. Understanding the mechanism of how T3 affects this pathway could be crucial to demonstrate the efficacy of T3 in the management of ND.

Introduction

Neuroinflammation is the body’s natural protective mechanism whereby a reaction to stimuli is generated in the central nervous system, aiming to initiate the healing process by activating the immune cells and cytokines. However, sustained and prolonged neuroinflammation will cause neurotoxicity and lead to neurodegeneration [1]. Neurodegenerative diseases (ND), being the main cause of disability and dependence, have made a huge impact on the sufferers and their caretakers physically and emotionally. These diseases have affected people’s quality of life as well as their personal development, imposing substantial public health burdens globally [2]. The prevalence of NDs has increased with a progressive, growing trend due to the ageing population. According to the WHO, it was estimated that NDs will become the second most common cause of mortality after cardiovascular diseases in 15 to 20 years’ time due to the progressive increase in the elderly population [3]. Neurodegenerative disease is caused by progressive loss and death of neurons and is portrayed by motor and cognitive dysfunctions [4]. It is associated with synapse dysfunction, neural network dysfunction or even deposition of altered proteins in the brain [5]. The two most common NDs are Alzheimer’s disease (AD) and Parkinson’s disease (PD), which affects a large population and places a huge burden on themselves and their caretakers. There are many NDs, and despite various medical technology advancements, clinicians still face challenges in managing patients with NDs due to the complexity and unknown pathogenesis of these diseases [6]. Hence, there are many studies performed with natural compounds acting as alternatives and these compounds are also used as the core ingredients in designing new drugs with desirable efficacy [7, 8]. In this recent review, the pharmacotherapeutic targets of AD is explored and various traditional medicinal plants like Gingko biloba are explored for the potential in the management of AD [9]. These natural compounds, like tocotrienols (T3), have been shown to be effective in several NDs [10]. Given the complexity of neurodegenerative diseases and the urgency of finding new ways of tackling them, this review aimed to investigate the effect of tocotrienol on the NF-κB signalling pathway in neurodegenerative diseases.

The importance of NF-κB and its association with tocotrienol

As T3 belongs to the vitamin E family, there are data showing that having a higher dietary intake or plasma level of any Vitamin E forms can lower the incidence of AD [11]. In the literature, it is reported that T3 possess many bioactive properties, including anti-inflammatory, antioxidant, cholesterol-lowering and anticancer effects [12]. Its neuroprotective ability is achieved by influencing anti-inflammatory mechanisms and preventing disease progression [12]. Among the NDs, a specific inflammatory transcription factor known as the nuclear factor kappa-b (NF-κB) fuels neurodegeneration [13]. The NF-κB is one of the best-understood transcription factors as it is most intensively researched and is well-known for many physiological processes like cell proliferation and growth, as well as inflammation and immune responses [14]. The role of NF-κB in NDs is complex as it can play dual roles, i.e. protecting and exacerbating factors, depending on the type of cells involved and the context of activation. It could help repair and regenerate cells but is also involved in mediating inflammatory pathways [15]. In addition, NF-κB plays an important role in neuroinflammation as its activation will induce a pro-inflammatory state in the brain and induce the release of cytokines, adhesion molecules and chemokines [16]. There are several activation pathways for NF-κB, but the two major pathways are the classical or canonical pathway and the alternative or non-canonical pathway [17]. Due to its complexity and extensive involvement in many diseases, NF-κB is targeted by many pharmaceutical companies for drug development to tackle NDs [15]. As there is a scarcity in the number of drugs available in the market to treat NDs, drugs or natural compounds that target NF-κB, an important signalling factor in neuroinflammation, could serve as an important target in the management of NDs.

There are several in vitro and in vivo studies that proves targeting the NF-κB pathway is the potential effective strategy in treating various diseases especially neurodegenerative diseases, however the molecular mechanism remains unclear. In vitro studies showed that γ-tocotrienol (γTE) inhibits NF-κB activation in leukaemia KBM-5 and other cancer cells whereas in vivo studies showed that proinflammatory cytokines will be inhibited by γTE supplementation in animal and human models [18].

NF-κB signaling pathway in neurodegenerative diseases

The family of NF-κB transcription factors has five DNA binding proteins, which share the N-terminal Rel-homology domain (RHD). The domain consists of a sequence of 300 amino acids used for interaction with the inhibitor molecule protein IκB, DNA-binding, and homologous and heterologous dimerization [15]. These five family members in the mammalian cells are p50/p105 (NF-κB1), p52/p100(NF-κB2), RelA (p65), RelB and cRel [17]. In the inactivated state, the NF-κB is bound to the IκB and is located in the cytosol (Fig. 1). There are three principal types of IκB inhibitory proteins, i.e., IκBα, IκBβ and IκBε, and their role is to keep NF-κB in the cytosol when inactivated [17]. There are two main NF-κB activation pathways: classical and alternative pathways [16].

Fig. 1.

Mechanisms of activation of NF-κB classical pathway. IKB cleavage from NF-κB is required to allow NF-κB to be activated and translocate into the nucleus to start gene transcription

The classical/canonical pathway is the most extensively studied NF-κB activation pathway, and it can be activated by different types of cell surface receptors, such as toll-like receptors (TLRs) and tumour necrosis factor (TNF) receptors, as well as pro-inflammatory mediators like lipopolysaccharide (LPS) and interleukin-1 (IL-1) [16]. There will be an intracellular activation of catalytic kinases complex known as the IκB kinase (IKK) complex in response to stimuli, whereby the IKK will phosphorylate IκB leading to the cleavage of IκB from NF-κB, allowing the free activated NF-κB to translocate to the nucleus, bind to the promoter region of the target gene and start gene transcription [19]. The IκB protein, which is ubiquitinated, will be regulated and later degraded by proteasomes [15].

In the alternative/non-canonical pathway, it is activated by various stimuli such as lymphotoxin beta receptor (LTBR), B cell activator receptor (BAFF-R) and CD40 [19]. As of the latest studies, this pathway is proven to be only effective in the immune system and requires IKKα homodimers to be activated. The main component in this pathway is the NF-kB-inducing kinase (NIK), it is constantly degraded by TNF Receptor-Associated Factor 3 (TRAF3) in the absence of stimuli. Hence, the level of intracellular NIK is low and NF-κB is retained in the cytoplasm and transcription of gene doesn’t occur. In the presence of a stimuli, there will be degradation of TRAF3, this leads to accumulation and stabilisation of NIK [20]. IKKα will be phosphorylated and activated by NIK, IKKα will then phosphorylate p100 which lead to partial proteolysis of p100 and formed active p52. P52 will pair with RelB to form the activated version of NF-κB, which lead to the translocation to the nucleus and gene transcription (Fig. 2) [16].

Fig. 2.

The non-canonical/alternative pathway of NF-κB. The NF-κB -inducing kinase (NIK) is the main deciding factor in this pathway as it needs to activate IKK, which subsequently leads to proteolysis of p100 into p52 and, together with RelB, forms the activated version of NF-κB

In NDs like AD, there will be overexpression of TLRs on microglia and neurons, which leads to the activation of the canonical NF-κB signalling pathway. Microglial activation, with the aim of clearing amyloid plaque in AD, results in the activation of various cytokines and chemokines that induce chronic inflammation [13]. The high levels of inflammatory cytokines can damage the oligodendrocytes, which leads to myelin injury, leaving the neurons more susceptible to amyloid beta plaque neurotoxicity and further progression of AD [13]. The NF-κB also regulates the expression of the BACE1 gene, which is responsible for the up-regulation of miR-125b and the production of beta-amyloid, which causes the level of pro-inflammatory mediators to increase, leading to neurodegeneration [21].

Regarding PD, it has been shown that there is a ten times higher amount of NF-κB-positive dopaminergic neurons in the brains of PD patients compared to healthy people, which suggests that the pathophysiology of PD may be closely related to the NF-κB signalling pathway [22]. Activation of the NF-κB pathway in astrocytes and microglia resulted in a higher number of inflammatory cells being released, which worsened neuronal cell death; hence, in order to maintain the normal functioning of the dopaminergic neurons, excessive microglia activation has to be inhibited [23]. Thus, inhibition of this pathway plays a crucial role in the prevention of dopaminergic neuron loss and neuroinflammation in Parkinson’s disease [24].

In both AD and PD, tocotrienol had been proven to inhibit the NF-κB pathway to prevent neuroinflammation and generation of ROS which worsens the progression of the disease. A study with transgenic mouse model that mimics AD-like phenotype treated with T3 and Tocs showed reduced amyloid deposition and beta plaques as well as reduced brain oxidative stress in hippocampus [25]. In another study whereby rat models are injected with 6-hydroxydopamine (6-OHDA) to mimic PD, T3 also proves it effectiveness to ameliorate neurodegeneration and motor deficits as well as being neuroprotective against dopaminergic neurons [26].

Tocotrienols

Both tocotrienols (T3s) and tocopherols (Tocs) are part of the vitamin E family, which are well-known for their antioxidant property. The T3s and Tocs exist naturally in four forms, i.e. alpha (α), beta (β), delta (δ), and gamma (γ), giving rise to eight vitamin E analogues [12]. In terms of their chemical structure, T3s and Tphs are similar as both have an aliphatic side chain and a chromanol head, but the side chain of T3s is an isoprenoid (unsaturated) while Tocs have a saturated side chain [10]. In comparison to T3s, there are more studies done on Tocs mainly due to their presence in most of the body cells and their high bioavailability from diet [12]. It is known that α-Tocs are useful in sustaining cardiovascular health. However, there are also studies which found that T3s not only exhibit antioxidant properties but also have other health benefits [10]. For instance, T3s protect against cardiovascular diseases via the inhibition of the HMG-CoA reductase enzyme [REF] and anticancer activities by inducing apoptosis [27]. Alpha-T3 is reported to possess neuroprotective properties as it has been proven to reduce inflammation and oxidative stress in the neurons. Hence this analogue may be a good target to evaluate therapeutic efficacy in NDs [28]

Seed oils are rich sources of natural vitamin E and T3s can be found in annatto bean seed, palm oil and rice grain [29]. The most predominant form of T3 is αT3, which can be found in barley and oats, whereas βT3 is mainly found in wheat [30]. Palm oil contains the richest natural form of T3 (α, β, δ and γ), while annatto bean seed oil contains predominantly δT3 (90%) and γT3 (10%) [31].

Tocotrienols’ anti-inflammatory property

The anti-inflammatory properties of T3 have been studied extensively. Inflammation is often associated with the activation of NF-κB, and studies have shown that T3s can suppress the expression of receptors that mediate inflammation, such as IL-1, TNF-α and many more [32]. In addition, the tocotrienol-rich fraction (TRF), the vitamin E fraction from palm oil was reported to inhibit activation of the NF-κB pathway and block the expression of the cyclooxygenase-2 (COX-2) gene, which is important to make prostaglandins (PGE2), which controls inflammation [33]. In another study, it was reported that T3 stabilised IκB by preventing its’ degradation [18]. This is an important anti-inflammatory response as IκB is the protein that retains NF-κB in the cytoplasm, which in turn inhibits the activation of the NF-κB pathway and the transcription of pro-inflammatory mediators. It was proven in a study that gamma-tocotrienol (γTE) managed to abolish TNF-α-induced NF-κB activation in rats by reducing IκBα’s degradation [34]. Other than the anti-inflammatory property, tocotrienols are also equipped with proinflammatory properties, making them dual biological properties [12]. It was reported that cigarette smoking induced oxidative stress and inflammation could be lowered by tocotrienols via the inhibition of translocation of pro-inflammatory transcription factors like STAT3 and NF-κB, as well as enhancement of nuclear factor erythroid-2-related factor 2 (Nrf2) [12].

Antioxidant properties of tocotrienols

The generation of reactive oxygen species (ROS) from molecular oxygen’s incomplete reduction is closely associated with cellular damage, and the balance between ROS production and its removal by the antioxidant defence system is required to maintain the normal physiology of the body [32]. Tocotrienols exert their antioxidant property by induction of antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) [27] (Fig. 3). In AD, T3s influence the expression and activity of these antioxidant enzymes to help the cell to manage oxidative stress [32]. By reducing oxidative stress, T3s also indirectly inhibit oxidative stress-induced pathways like the NF-κB pathway. As the NF-κB pathway can be activated by ROS, T3s can prevent the pathway from being activated by reducing oxidative stress, which in turn reduce production of inflammatory cytokines and other pro-inflammatory mediators [13]. It has been found that the number of methyl groups on the chromanol ring of T3 can influence the strength of antioxidant activity. Hence, the order of antioxidant activity of the T3 analogues from the strongest to lowest antioxidant activity was reported to be αT3 > βT3 > γT3 > δT3 [31].

Fig. 3.

Modulation of the NF-κB pathway by tocotrienol

Another property that makes T3 a useful antioxidant is that T3s are lipid-soluble molecules, which allows these compounds to readily incorporate into the lipid bilayer of cell membranes and prevent lipid peroxidation [35].

The brain consumes a large amount of oxygen as it has higher metabolic needs and, hence, its higher susceptibility to oxidative stress [36]. Given that the brain is rich in polyunsaturated fatty acids (PUFAs), the cell membrane is highly susceptible to lipid peroxidation, where free radicals take away electrons from the lipid layer of the cell membrane, causing cell damage. The unsaturated side chain of T3s allows these compounds to have higher mobility compared to Tocs within the membrane, further enhancing its interaction with the fatty acid molecules. In addition, T3s can prevent the lipid peroxidation chain reaction by donating hydrogen atoms to the lipid radicals and stabilising them [37]. The antioxidant and anti-inflammatory properties of T3s complement each other, as the antioxidant property protects the cells from undergoing ROS-activated tissue damage, hence preventing tissue inflammation [38].

Mitochondrial dysfunction

Mitochondrial dysfunction is another hallmark of AD and PD. In the case of AD, amyloid plaques accumulate in the mitochondria and leads to impaired energy metabolism and increased mitochondria ROS. In neurodegenerative diseases, mitochondria, being the powerhouse of cells will eventually lose it proper functions and leads to cognitive impairments and synaptic dysfunctions through ROS mediated pathway seen in preclinical and clinical studies [39]. T3 has shown to act as a protective shield against mitochondrial dysfunction. Study found that there is improved mitochondrial function on aged mice using γ-T3 as elevation of membrane potential and ATP level are observed [40].

Tocotrienols vs other NF-κB modulators

As we have understood the mechanism at which T3 works on the NF-κB signalling pathway, it is essential to compare T3 over other NF-κB modulators such as steroids and NSAIDS. T3 is advantageous over other NF-κB modulators as they are a part of the vitamin E family and they are made of natural components. Based on the current studies, T3 does not possess any significant side effects, aside from the minimal gastrointestinal effects when ingested orally, side effects such as weight gain and immunosuppression which are seen in steroids are not observed in T3. Another advantage would be the specificity of T3 over other NF-κB modulators in targeting neuroinflammation. T3 can combat neuroinflammation selectively while preserving important immune functions without causing global immune suppression. On the other hand, steroids cause a general immune suppression, it is not feasible to block the NF-κB pathway for prolonged periods since the pathway plays a core role in the host defence response [41].

Future studies

As we ventured through the various beneficial properties of T3, it is crucial to understand that to improve the bioavailability and retention rate of T3 remains as a challenge for it to exert its therapeutic efficacy. The main challenge of T3 is the first pass metabolism. Tocotrienol undergoes first pass metabolism, whereby a significant proportion of the active compounds is cleared by the liver before reaching the systemic circulation, hence the effectiveness is reduced [42]. The possible strategy to this will be developing prodrug derivatives of T3 whereby the inactive compounds are only converted to active forms after absorption and hence the first pass metabolism by the liver is bypassed. Another challenge that T3 faced is its lipophilicity and poor water solubility. T3 is poorly soluble in water and this makes absorption by the gastrointestinal system difficult, eventually hampering their bioavailability when administered via oral route. An alternative delivery system such as a nanoparticle-based delivery system can be considered, whereby T3 is encapsulated, enhancing its retention duration and ensuring its precision to reach the targeted tissue. This system also allows controlled-release which means stable supply of T3 can be supplied overtime, however further studies are still needed as the usage of nanoparticles on humans still arise as a concern [43].

As T3 is still not officially available in the market, factors that will influence its acceptance needs to be considered. When it comes to natural supplements like T3, consumers will want to know its long term safety so they can consume for long term especially for neurodegenerative diseases which are chronic diseases. Consumers who have more advanced stages of neurodegenerative disease would also want to know if side effects would arise if T3 is taken in high doses for a long duration. Hence, further clinical studies in humans should be conducted in larger scale and longer duration to provide reliable data for the consumers to gain more trust in natural remedies like T3. There is currently no established standard for the optimal dosing for the management of neurodegenerative diseases using T3. Various studies use different dosing regimens and optimal dose will differ depending on factors such as the patient's age, comorbids and disease subtypes. The appropriate dosing of T3 is important as inappropriate could limit the therapeutic potential of T3. Another concern that should be taken into consideration will be the cost and accessibility of the drug. If T3 supplements are expensive and inaccessible, consumers will go for cheaper alternatives, hence future studies should investigate the extraction methods of T3 which are cost effective and still ensure its efficacy.

Conclusion

It is undeniable that T3 possesses many health benefits in various systemic diseases; this is particularly evident in NDs, whereby its antioxidant and anti-inflammatory properties have been utilised in the brain as T3s can cross the blood–brain barrier. Due to the lack of approved pharmaceutical drugs for NDs, there has been a shift of focus on searching for alternative natural components to treat these diseases, and T3 has shown great promise on its’ anti-inflammatory and neuroprotective effects. Although more attention has been placed on Tocs in the earlier years, it has already been established that T3s have a higher superiority compared to Tocs. Further studies should be conducted to explore the delivery route of T3s to improve its bioavailability and retention duration and further clinical evidence of T3s are needed to increase the acceptance rate among consumers.

Author contributions

T.R conceptualized the project and worked together with S.B, V.L.L.L & A.K.R to craft the hypothesis and aim of the project. A.S.Y drafted and wrote the manuscript as well as the figures. T.R, S.B, V.L.L.L, J.Y.T & A.K.R were involved in editing, critical manuscript revision, and final manuscript approval and accountability for the work. All authors have read and agreed to the published version of the manuscript.

Funding

This project received funding from the Monash University Malaysia Jeffrey Cheah School of Medicine and Health Sciences (JCSMHS) Seed Grant 2023 [I-M010-SED-000 173].

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare no competing interests.