Nutritional Epigenomics: Bioactive Dietary Compounds in the Epigenetic Regulation of Osteoarthritis

1. Osteoarthritis, a Chronic Disease

Osteoarthritis (OA) is one of the most common disabling chronic progressive diseases in middle-aged and elderly people [2] and, it is among the main public health problems worldwide, due to its high prevalence [3]. It is characterised by deterioration of the articular cartilage, alteration of subchondral bone, formation of osteophytes, joint space narrowing, and inflammation of the synovium [4]. Symptoms generally include severe joint pain, stiffness, joint contractures, muscle atrophy, reduced movement, swelling, tenderness, and variable degrees of local inflammation, limb deformity and crepitus [5]. There are many etiological factors for OA, including genetic predisposition, dietary intake, obesity, sex, aging, traumatic joint injury, mechanical stress, metabolic disease, and sedentary lifestyle [6]. It is important to highlight the synergistic effects of pathologies such as cardiovascular disease and obesity coexisting with OA [7,8].

Pharmacological treatments such as paracetamol, nonsteroidal anti-inflammatory drugs, tramadol, and opioids are used to reduce pain and inflammation, but do not prevent, reverse or cure OA [9]. However, a long-term use of these drugs to relieve OA is associated with substantial gastrointestinal, renal, hepatic, blood, cardiovascular, and cerebrovascular adverse effects [10,11,12]. In this review, we present the importance of a healthy diet in preventing the development or progression of OA and, summarise chondroprotective properties and beneficial epigenetic modifications of bioactive compounds or nutraceuticals against inflammation and catabolic activity in OA.

2. Epigenetics and Osteoarthritis

Over the last 20 years, the study of epigenetics has expanded (especially in the cancer field). However, studies on the importance of epigenetic mechanisms in OA are only now increasing. Roach and collaborators demonstrated the first evidence of how epigenetic changes, such as DNA methylation, may relate to the pathogenesis of OA and, can be potentially reversible [13].

Epigenetics can be defined as heritable changes in gene expression and/or phenotype that can occur without changes in the primary DNA sequence [14]. The epigenome of each cell is unique and can undergo temporal changes in response to environmental factors such as diet, physical activity, smoking, pollutants and disease status [15]. OA is distinguished by unfavourable dynamic regulation of gene transcription in joint tissues due to environmental disturbances; therefore, epigenetics has developed as a new and important area for OA research [16,17,18]. Candidate gene and epigenome-wide studies have demonstrated their association with OA development and progression through epigenetic modifications, and these epigenetic mechanisms can change in response to stimuli and, in some cases, pass on to future generations [19,20,21]. Given the importance of gene expression or silencing, and associated epigenetic modifications, we will briefly mention various epigenetic mechanisms of pro-inflammatory cytokines and metalloproteinases (MMPs) that contribute to cartilage destruction. Three main mechanisms are implicated in epigenetic regulation: (1) DNA methylation changes that covalently alter chromatin structure. In general, DNA hypomethylation enhances gene transcription, and DNA hypermethylation suppresses gene transcription. (2) Post-translational modification of histones that alters chromatin conformation, include methylation of arginine and lysine, acetylation of lysines, phosphorylation of serine and treonine, or sumoylation and ubiquitination of lisyne. (3) Non-coding RNAs regulate gene expression but do not translate into proteins (i.e., microRNAs (miRNAs), long non-coding RNAs) acting both at transcriptional, or post-transcriptional levels [22,23,24].

2.1. DNA Methylation

DNA methylation process is mediated by DNA methyltransferases (DNMTs): DNMT1 (maintenance), DNMT3A and DNMT3B (de novo) and, involves the addition of a methyl group to the 5′ position of cytosine, most commonly occurs in CpG dinucleotides, forming 5-methylcytosine. The hypermethylation by DNMTs leads to transcriptional gene silencing and gene inactivation [22,23].

Nakano and collaborators found that DNMT1 and DNMT3A expression was decreased by IL-1β, while DNMT3A also decreased expression and activity by TNF-α in fibroblast-like synoviocytes [25]. Both DNA methylation and histone modification are involved in the control of TNF-α expression [26]. Hashimoto and collaborators found that the methylation of the −115 CpG site enhances MMP13 promoter activity as opposed to the inhibitory effect of −110 CpG methylation; also, the demethylation of the specific CpG sites at −299 position of the IL1B promoter activity correlates with enhanced IL1B gene expression in human primary chondrocytes [27,28]. Furthermore, Bui and collaborators showed that the -104 CpG site is demethylated in OA cartilage and is accompanied with an elevated MMP13 expression [29]. In articular cartilage, the methylation of cytosines at positions −1,680 and −1,674 blocks COL10A1 expression in chondrocytes, while gene expression is activated during chondrogenesis in cells with partial methylation of these two specific CpG sites [30]. Cheung and collaborators found that DNA demethylation at one or more specific CpG sites in the ADAMTS4 promoter corresponds to increased expression of ADAMTS4 in human OA chondrocytes, which plays a role in aggrecan degradation in OA [31]. In addition, Roach and collaborators showed an association between loss of DNA methylation of CpG sites in the promoters and abnormal expression of MMP3, MMP9, MMP13, and ADAMTS4 by OA chondrocytes [13]. Besides, the sclerotin (SOST) mRNA and protein expression levels are increased in OA chondrocytes, suggesting the SOST promoter is hypermethylated in normal chondrocytes and hypomethylated in OA [32]. An interesting study suggests that hip OA is associated with decreased SOX9 gene and protein expression, showing that methylation of SOX9 promoter was increased in OA cartilage [33]. Imagawa and collaborators reported that COL9A1 promoter activity is significantly decreased by DNA hypermethylation, and could be reversed through inhibition of DNA methylation. In addition, abnormal DNA methylation of the CpG sites in the COL9A1 promoter is associated with decreased expression of SOX9 [34]. Moreover, hypomethylation in the IL8 promoter is correlated with higher IL8 gene expression in OA chondrocytes; it was also shown a significant increase in IL8 promoter activity by the transcription factors NF-κB, AP-1 and C/EBP [35]. de Andrés and collaborators demonstrated the association between an increase of inducible nitric oxide synthase (NOS2) gene expression in OA chondrocytes and, the demethylation of NF-κB responsive enhancer elements [36]. Furthermore, in OA synovial fibroblasts showed DNA hypomethylation and histone hyperacetylation in the IL6 promoter [37].

2.2. Histone Modifications

Methylation/demethylation and acetylation/deacetylation are the main and recurrent histone changes in OA [38]. Two families of enzymes catalyse the modification de histones: histone methyltransferases (HMTs) and histone demethylases (HDMTs), or acetyltransferases (HATs) and histone deacetylases (HDACs) [39]. The majority of these modifications take place at lysine, arginine and serine residues within the histone tails and, regulate key cellular processes such as transcription, replication and repair [40]. Hyperacetylation of histone tails induces transcriptional activation while hypoacetylation is associated with transcriptional repression [41]. HDAC family members have been associated with OA, and HDAC inhibitors (HDACi) can protect chondrocytes, prevent cartilage damage, and possess therapeutic potential against OA [15,42]. Young and collaborators demonstrated that HDACi decreased the expression and activity of MMPs and ADAMTSs [43]. In addition, histone deacetylase-1 (HDAC1) and HDAC2 levels are elevated in both chondrocytes and synovium from OA patients compared to controls [44,45]. Higashiyama and collaborators demonstrated the increased expression of HDAC7 in human OA cartilage that was correlated with elevated MMP13 gene expression, contributing to cartilage degradation [46]. Class III HDACs (sirtuins) are a class of NAD+- dependent histone deacetylases and differ from the class I and II HDACs. Sirtuin 1 (SIRT-1) is a positive regulator of cartilage-specific gene expression in chondrocytes [47]. SIRT-1 activation has the potential to prevent cartilage damage and inhibit its destruction [48,49]. SIRT-1 suppresses protein tyrosine phosphatase 1B (PTP1B) and activates insulin-like growth factor (IGF) receptor pathway, enhancing survival of chondrocytes [50]. Also, decreased expression of COL2A1 mRNA and type II collagen protein correlates with decreased SIRT1 activity [51]. In addition, in OA cartilage, the overexpression of E74-like factor 3 (ELF3) inhibited Sox9/cAMP-response element-binding protein (CREB)-binding protein (CBP)-driven HAT activity and decreased COL2A1 [52]. The disruptor of telomeric silencing 1-like (DOT1L) gene, a HMT, is a protector of cartilage health, thereby is reduced in damaged areas of OA joints; the protective function of DOT1L is attributable to Wnt signalling inhibition [53,54].

2.3. Non-Coding RNA (ncRNAs)

ncRNAs, including small non-coding RNAs (miRNA) and long non-coding RNAs (lncRNAs), have the ability to regulate gene expression at both transcriptional (lncRNAs) and post-transcriptional levels (small and lncRNAs) [55]. lncRNAs are key regulators of gene expression; thus, the overexpression of lncRNA-CIR increased the expression of MMPs, whereas the collagen and aggrecan expression was reduced in OA cartilage [56]. Small ncRNA mainly includes miRNAs, siRNAs and piRNAs. miRNAs have been the most frequently investigated; they are considered an alternative mechanism of post-transcriptional or translational regulation, at post-transcriptional level binds to complementary mRNA, leading to degradation of mRNA or prevention of its translation into a protein [55,57,58,59]. Several miRNAs have showed an altered expression in OA and, are involved in various aspects of cartilage homeostasis and OA pathogenesis [60]. Rasheed and collaborators showed that IL-1β-induced iNOS gene expression is correlated with the down-regulation of miR-26a-5p in human OA chondrocytes [61]. Furthermore, miRNAs such as miR-320, miR-381, miR-9, miR-602, miR-608, miR-127-5p, miR-140, miR-27b, miR-98 and miR-146 have a significant role in the regulation of genes relevant to OA pathogenesis [59]. In another study, the overexpression of miR-27b inhibited IL-1β-stimulated MMP13 gene and protein expression in human OA chondrocytes [62]. Moreover, the overexpression of miR-558 directly inhibited COX2 mRNA and protein expression [63]. Also, miR-199a levels are inversely correlated with COX2 mRNA and protein levels in IL-1β-stimulated human chondrocytes [64]. There is a relationship between HDACs and miRNA in OA, thus overexpression of miR-92a-3p suppressed HDAC2 production and increased the level of histone H3 acetylation of the COMP/ACAN/COL2A1 promoter [65]. Overexpression of miR-193b-3p inhibited HDAC3 expression, enhanced histone H3 hyperacetylation and, increased the expression of SOX9, COL2A1, ACAN, and COMP in chondrocytes [66]. Guan and collaborators showed that miR-146a protects against OA, inhibiting inflammatory factors [67]. In addition, a study demonstrated the significant increase in miR-146a expression induced by the HDAC inhibitors in OA-fibroblast-like synoviocytes [68]. Another study demonstrated that miR-146b is downregulated in the chondrogenic differentiation of human stem cells and upregulated in OA [69]. Overexpression of miR193b-5p inhibited HDAC7 expression and, decreased MMP3 and MMP13 expression [70]. Both miR-199a-3p and miR-193b expressions are upregulated with age and, may be involved in chondrocyte senescence by downregulating anabolic factors such as type 2 collagen, aggrecan, and SOX9, therefore may be involved in cartilage degeneration [71]. In addition, increase of TNFA, IL1B and IL6 gene expression was correlated to miR-149 down-regulation, through the inhibition of post-transcriptional control in human OA chondrocytes [72]. miR-140, the most well studied miRNA in OA, plays a protective role in OA development. It is important for chondrogenesis and osteogenesis, and is highly expressed in normal cartilage, but their expression levels are decreased in OA chondrocytes; its overexpression could inhibit inflammation and cartilage degradation [73,74,75,76,77]. A study showed that miR-140 is expressed specifically in cartilage tissues during mouse embryonic development and, siRNA-140 significantly downregulated the accumulation of Hdac4 protein in fibroblast cells [78]. Further, miR-140-3p and its isomiRs: miR-140-3p.1 and miR-140-3p.2 are abundantly expressed in cartilage [79]. Decreased miR-let7e expression has been suggested as a potential predictor of hip OA [57,80]. The increase of miR-145 levels directly represses SOX9 expression, resulting in the inhibition of COL2A1 and ACAN, with increased expression of RUNX2 and MMP13 in human chondrocytes [81].

3. Inflammation and Diet

Inflammation is a complex biological response of the immune system to pathogens, damaged cells, injury, toxic compounds, and infection. This immune system utilises a large number of specialised cells such as lymphocyte, monocytes and macrophages to restore homeostasis [82,83,84]. Inflammation in OA is an important pathway in its pathogenesis and development [85,86]. Inflammation in OA joints is chronic, low grade, and involves the interplay of the innate immune system and inflammatory mediators [85,87,88]. These include cytokines, chemokines, growth factors, adipokines, prostaglandins, leukotrienes, nitric oxide, and neuropeptides [87,89]. Strikingly, the reduction of this low-grade inflammation is closely linked with a higher adherence to healthier diets such us the Mediterranean diet [90,91,92].

Diet plays an important role in the development or prevention of many chronic diseases [93,94] and, may regulate chronic inflammation, improving quality of life [95,96,97]. Thus, dietary composition is able to modulate epigenetic marks like changes in DNA methylation, histone or chromatin remodelling key inflammatory genes and, ncRNAs that may be causal for the development of chronic diseases or, may be beneficial against inflammation; in this way, can block, retard, or reverse pathologic processes [98,99,100,101,102].

A diet with high dietary inflammatory index (DII) score has been associated with severe pain and lower quality of life in patients with knee OA [103,104]. Another study showed that the energy-adjusted DII (E-DII) score was associated with a high risk of knee OA in the Osteoarthritis Initiative (OAI) cohort [105]. The DII showed to predict inflammatory biomarkers [103,106]. Biomarkers of inflammation, especially serum C-reactive protein (CRP), IL-6, TNF-α and MMPs, have been associated with pain and the progression of OA [107,108,109,110]. Dyer and collaborators showed that biomarkers of inflammation and cartilage degradation related to OA were lower with higher uptake of Mediterranean diet [111]. In addition, several studies have found that better quality of life was associated with a higher adherence to this diet [112,113,114,115]. Veronesse and collaborators, in a large cohort of North Americans from the OAI database, demonstrated that a higher adherence to the Mediterranean diet is associated with better quality of life, which is correlated with less pain, disability, depression, better cognitive performance, and physical functioning [116]. The adherence to Mediterranean diet in these studies was assessed according to the Mediterranean diet score by Panagiotakos [117] based on a food frequency questionnaire [118]. Strikingly, a higher adherence to a Mediterranean diet is associated with lower prevalence of knee OA [119]. A high adherence to this diet increases the antioxidants levels in serum samples with a reduction on oxidative stress biomarkers levels [120,121], such as F2-isoprostane, indicator of oxidative stress in plasma [122]. Moreover, Martín-Núñez and collaborators found a correlation between lower adherence to the Mediterranean diet pattern, and changes in DNA methylation levels and diseases [123].

4. Bioactive Compounds: Health-Protective Benefits

The complex biological activities of plants can promote their abundance in secondary metabolites or bioactive compounds, they are also known as phytonutrients or nutraceuticals. The bioactive compounds are widely known for their unique medicinal properties; they possess antimicrobial [124], anti-inflammatory [125], antiviral [126,127], cardioprotective [128], neuroprotective [129], chemopreventive [130], phytohormone [131], and antioxidant properties [132]. Multiple pathological processes are involved in the pathogenesis of OA, such as inflammation, oxidative stress, apoptosis, autophagy and senescence; hence phytochemical or bioactive compounds have been shown as therapeutic and nutraceutical agents, showing their antiarthritic potential. They mainly exert anti-inflammatory effects through blockade of pro-inflammatory cytokines (IL1-β, IL-6, IL-8, TNF-α), inhibition of NF-κB pathway, antiapoptotic effects, preventing oxidative damage to proteins and DNA (reduction of both reactive oxygen species and reactive nitrogen species), suppressing the production of prostaglandins and leukotrienes, and decreasing levels of MMPs [133,134,135,136,137].

Bioactive phytochemicals have a wide variety of compounds and are classified as phenolics, alkaloids, organosulfur compounds, terpenes and terpenoids, and each class is divided into further classes. They are present in fruits, vegetables and spices, and can modify metabolic, cellular, molecular, and epigenetic processes [138]. Polyphenols represent the largest and ubiquitous group of natural phytochemicals structures, these compounds are present in fruits, vegetables, cereals, tea, dark chocolate, cocoa powder, coffee, extra virgin oil, and wine [139,140,141]. The main groups the polyphenols are flavonoids, phenolic acids, and secoiridoids among others. Just flavonoids comprise more than 10,000 natural compounds including anthocyanidins, proanthocyanidins flavones, flavanones, flavonols, isoflavones and flavan-3-ols [142,143,144,145]. In this review a total of 85 bioactive compounds and nutraceuticals with potential anti-OA properties were analysed for the management, treatment, or prevention of OA in both human (Table 1) and animal (Table 2) studies.

5. Nutritional Epigenomics: Bioactive Compounds in Dietary Balance and Health

Nutritional epigenomics is exceptionally important because hold great potential in the prevention, suppression and therapy of a wide variety of diseases by altering various epigenetic modifications. This novel field involves the lifelong remodelling of our epigenomes, even during cellular differentiation in embryonic and foetal development, by nutritional factors and, describes how the bioactive molecules can influence and modify gene expression at the transcriptional level [336,337,338,339]. For example, DNA methylation depends on the methyl group donors and cofactors found in foods, thus dietary excess or deficiencies in a critical and sensitive period like embryogenesis can alter methylation process, gene expression and therefore the metabolism and physiology of the individual, programming pathologic processes during a lifetime [340,341]. Jirtle and Skinner observed that hypermethylating dietary compounds could reduce the effect of environmental toxicants that cause DNA hypomethylation [342]. An interesting study in Apis mellifera, about the different honeybee phenotype, demonstrated that silencing Dnmt3 gene expression decreased methylation in dynactin p62 gene in larval heads, which lead to an increase of queens and reduction of workers; these epigenetic changes in DNA methylation depended on whether they are fed royal jelly or beebread [343].

Wolff and collaborators showed one of the first evidences that maternal nutrition can impact the epigenome and phenotype of the offspring of dams fed with folate-supplemented diets, the nutrition affected agouti gene expression in A^vy/a mice and caused a wide variation in coat colour ranging from yellow (unmethylated) to light brown (methylated). Pseudoagouti A^vy/a brown mice were lean, healthy, and longer lived than their yellow phenotype siblings (larger, obese, hyperinsulinemic, more susceptible to cancer) [344]. Furthermore, in macaques that were fed a high fat diet during pregnancy (predisposing offspring to metabolic syndrome), foetal offspring had increased H3 acetylation and decreased Hdac1 gene expression in the liver compared to macaques fed with a low-fat diet [345]. An experimental study in Agouti A^vy/a mice fed with genistein (a soy polyphenol), which acts during early embryonic development, showed that genistein-induced hypermethylation persisted into adulthood, by altering epigenome, decreased ectopic agouti expression, and protecting offspring from obesity, diabetes, and cancer across multiple generations [346]. In addition, experimental data have shown that maternal consumption of dietary polyphenols such as resveratrol during preconception, gestation and lactation ameliorated metabolic programming. Resveratrol reduced renal oxidative stress, activated nutrient-sensing signals, modulated gut microbiota, and prevented associated high-fructose intake induced programmed hypertension in the rat offspring [347].

The four primary targets for epigenetic therapy are DNMTs, HDACs, HATs and miRNA; thereby, numerous bioactive compounds such as sulforaphane, tea polyphenols, ellagic acid, genistein, curcumin, hydroxytyrosol, resveratrol, organosulfur compound, oleanolic acid, and alkaloids have been studied as potent agents for regulating epigenetic mechanisms [102,339,348]. Bioactive compounds can influence epigenetic processes through different mechanisms that interfere with the 1-carbon metabolism and affect S-adenosyl methionine (SAM) levels being able to modulate DNA and histone methylation [349]. Many polyphenols, such as quercetin, fisetin, and myricetin, inhibit DNMT by decreasing SAM and increasing S-adenosyl-L-homocysteine (SAH) and homocysteine levels [350].

Global DNA hypomethylation has been associated with hypermethylation and inactivation of specific genes [351], thus hypermethylation of cytidine by DNMTs usually results in transcriptional gene silencing and gene inactivation including tumour suppressor genes, while promoters of transcriptionally active genes typically remain hypomethylated [352]. Genes such as O⁶-methylguanine methyltransferase, retinoic acid receptor β (RARB), the tumour suppressor p16^INK4a, and the DNA repair gene human mutL homologue 1 (hMLH1) were shown to be frequently inactivated by hypermethylation and, polyphenols such as epigallocatechin-3-gallate and genistein from soybean demonstrated to be strong DNMT inhibitors, leading to demethylation and reactivation of methylation-silenced genes [353]. DNMTs do not act alone, also recruit HDACs to synergistically repress gene transcription [354].

The combination of bioactive compounds acting as DNMT inhibitors, together with phytochemicals that can alter histone modifications, and those that can influence miRNAs expression in OA, are all of them potentially more synergistic and significant approaches as therapeutical strategies to prevent and treat various diseases, including cancer [355,356]. In this context of nutriepigenomics, we have particularly analysed the epigenetic mechanisms related to 11 bioactive compounds focusing on prevention or treatment in OA in both human (Table 3) and animal (Table 4) studies.

6. Conclusions

In this review, we analysed the importance of bioactive compounds as epigenetic modulators in the prevention and treatment of OA. The reduction of inflammation, catabolic and oxidative activity is essential in OA treatment. Bioactive compounds or nutraceuticals can directly protect and repair DNA damage, modulating signalling pathways and genes implicated in OA pathogenesis or modifying intra- and extracellular activities. Bioactive compounds are potentially capable of reversing the phenotype of OA chondrocytes. Moreover, the combination of bioactive compounds that act as DNMT inhibitors together with HDAC inhibitors, HAT inhibitors or activators and, miRNA regulators, all of them are potential approaches more synergistic and significant to prevent and treat OA (Figure 1).

Several mixtures have also demonstrated the additive and synergistic potential of bioactive compounds; these mixtures enhanced their chondroprotective properties via anti-inflammatory mechanisms, and reducing oxidative stress. Bioactive compounds are also effective in reducing pain and decreasing the need for NSAIDs, with fewer adverse effects that provide safety and therapeutic efficacy in OA treatment. In addition, new formulations of bioactive compounds have been developed for example with nanoparticles; these phytonutraceuticals possess higher absorption and bioavailability and, could serve as a therapeutic strategy in the prevention and treatment of OA. However, the potential of bioactive compounds as epigenetic regulators in OA has been little studied; further research is needed towards this promising area of research. For this reason, the proposal nutriepigenomic arises and focusses on the ability of numerous bioactive compounds as an alternative to prevent or treat OA.