Fibromyalgia (FM) is a chronic syndrome whose main symptom is the widespread pain caused by both allodynia and hyperalgesia [1
]. Nonetheless, the FM clinic includes a myriad of physical disturbances like joint stiffness, muscular twitches, burning skin, headaches or gastrointestinal alterations, as well as several psychological disorders and cognitive difficulties [2
]. Consequently, this syndrome can be considered as a highly disabling condition that affects to 0.2–6.6% of the general population [3
], with an estimated healthcare cost over 5000€ per patient every year [4
]. Nowadays, neither a healing treatment nor an accurate biomarker-based diagnostic have been unveiled. As a result, the efforts of the scientific community are still focused on depicting the molecular disturbances that might be contributing to the appearance and chronification of such a wide range of symptoms.
There are many factors related to the etiopathogenesis of FM. It has been proposed that central sensitization has a key role in the chronic pain suffered by these patients. This sensitization could be mainly linked to an abnormal performance of ascending and descending neurological pathways involved in pain processing [1
]. Additionally, it has been observed that peripheral silent C nociceptors have a marked hyperexcitability that could be related with aberrant pain perception in FM patients [7
]. Apart from neurological causes, there are a number of molecular disturbances that have been studied in different models. In this context, the role of oxidative stress has been recursively highlighted by scientific literature. In FM patients, there is an increased oxidative stress [8
] and a compromised antioxidant defence in terms of low levels of reducing compounds such as ceruloplasmin or copper, and a lower activity of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase [9
]. Additional studies have also pointed to the involvement of oxidative stress in FM, since an impaired mitochondrial metabolism in leukocytes and fibroblasts from FM patients has been found [10
]. Oxidative stress is highly related to inflammation. In FM, inflammation is described as a ‘neurogenic’ [13
] or as a ‘low-grade’ based systemic symptom [14
]. Both circumstances have been confirmed in cerebrospinal fluid and plasma from patients by an inflammation-related protein panel [15
], pointing to protein-related techniques as key partners in this complex syndrome. However, it has not been fully addressed yet how these conditions contribute to the appearance and development of FM symptoms.
Although there is a plethora of drugs currently used for FM treatment, none of them seem to be completely effective by itself. As a result, a combination of pharmacological and nonpharmacological treatments seems to be the more usual approach [16
]. Even more, treatments that do not use drugs at all are strongly recommended by several organisms and researchers [17
]. In line with this hypothesis, nutritional interventions and supplementations have already been used in the management of FM in order to improve the quality of life of these patients. Given that FM has been related to a deficiency in essential metal ions and vitamins, the regular intake of antioxidant compounds could be beneficial to a certain extent [19
]. Therefore, balanced diets with high levels of antioxidant components, such as the Mediterranean Diet, could have a potential restoration effect in FM. In this nutritional pattern, the main source of fat is Extra Virgin Olive Oil (EVOO), a vegetable oil with an overwhelming list of proven benefits on human health [20
]. In fact, EVOO consumption in FM patients improved their quality of life and health self-perception, as well as decreased oxidative stress markers, such as protein carbonylation and lipid peroxidation [21
]. The main actors in the anti-inflammatory and antioxidative effects of EVOO are its phenolic compounds, especially hydroxytyrosol (HT) [22
]. This amphipathic phenol is mainly originated by the hydrolysis of secoiridoids during fruit ripening [23
] and its concentration in olive oil differs markedly depending on the cultivar, being especially high in ‘Picual’ (up to 600 mg/kg) [24
]. The nutraceutical properties of HT have been demonstrated in a wide range of pathologies like cancer, cardiovascular diseases and diabetes [24
], most of them linked to its outstanding anti-inflammatory and antioxidant effects [25
Since FM is a multifaceted syndrome, the use of omics techniques in its research could be extremely advantageous. In fact, some studies applying genomics [28
], transcriptomics [29
] and proteomics [30
] published in the last decade have been crucial to unravel a huge number of genes and molecular pathways involved in FM.
Given the reported inflammation and oxidative imbalance in FM patients, as well as the nutraceutical properties showed by HT, in this study we evaluated whether this phenol could be an effective ally in the management of FM. For that purpose, and using a proteomic approach in primary fibroblast cultures, we began by elucidating FM-associated molecular changes in order to analyse whether a nutritional relevant dose of HT can ameliorate them.
In the present work, we carried out, for the first time, an nLC-MS/MS study in primary dermal fibroblasts from a healthy volunteer and from a FM patient. Although this study was a case report, it aimed to be a first approach for future studies with more patients. Our results unveiled that fibroblast from FM showed a differential expression in proteins involved in the turnover of extracellular matrix (ECM) and oxidative metabolism that could explain the inflammatory status of these patients. Besides, a number of these proteins were normalized by the treatment with HT, which seems to support that an HT-enriched diet could be highly beneficial in the management of FM.
Among the 816 detected protein in both cultures, 357 showed differential expression in the fibroblast from the patient, composing a proteomic signature of FM. Of these proteins, 154 were upregulated and 203 downregulated in the culture from the patient. The PANTHER GO-Slim analysis of these proteins pointed to several processes linked to cytoskeleton arrangement (‘actin filament depolymerisation’), muscle-skeletal system development (‘connective tissue’, ‘cartilage’ and ‘bone development’) and mostly cell metabolism (‘ribosomal small subunit biogenesis’, ‘translation’ and their associated processes, ‘peptides’ or ‘amides biosynthetic processes,’ etc.). Among them, ‘actin filament depolymerisation’ was the term with the highest Fold Enrichment and the lowest FDR. Three proteins, overexpressed in the fibroblasts from the FM patient were annotated in this process: Alfa-adducin (ADDA), and alpha-1 and alpha-2 subunits of the F-actin capping protein (CAZA1 and CAZA2). Whereas ADDA attaches to actin filament extremes to protect them from depolymerisation [43
], CAZA 1 and CAZA 2 proteins belong to the capZ protein complex that facilitates their assembly [44
]. The homeostasis of actin filament has a crucial role in organelle distribution and dynamics. Indeed, actin depolymerisation participates in mitochondrial fission [45
], a process whose impairment is linked to metabolic stress conditions [46
]. Besides, the KEGG analysis showed that up to 115 FM-affected proteins participated in metabolic pathways. Hence, both results could explain, to some extent, those metabolic alterations described in FM [47
]. In this line, ‘fatty acid β-oxidation’ and ‘tricarboxylic acids cycle’ were two mitochondrial metabolic routes activated in FM fibroblast, both of them with a significant z-score. The fibroblasts from FM patient showed four proteins overexpressed in this route: Alpha (ECHA) and beta (ECHB) subunits from hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex, hydroxyacyl-CoA dehydrogenase (HCDH) and acetyl-CoA acyltransferase 2 (THIM). The trifunctional multienzyme complex catalyses the last three steps in fatty acid β-oxidation [50
]. When processing long chain fatty acids, enoil-CoA hydratase and 3-hidroxyacil-CoA deshydrogenase activities are developed by the alpha and beta subunits of this complex [51
]. However, in short-chain fatty acid metabolism, these activities are managed by HCDH and THIM, respectively [52
]. Acetyl-coA molecules produced in β-oxidation are further catabolized in the tricarboxylic acid cycle. In this regard, four proteins of this cycle were overexpressed in the fibroblasts from the FM patient: Citrate synthase (CISY), malate dehydrogenase 2 (MDHM), oxoglutarate dehydrogenase (ODO1) and dihydrolipoamide S-succinyltransferase (ODO2). The imbalance in these two relevant mitochondrial routes is indicative of an altered energy metabolism in FM. According to PANTHER analysis, some other key processes related with the metabolism of cells were also affected in the fibroblast from the FM patient: transfer RNA processing, protein translation and vesicle-associated transport from endoplasmic reticulum (ER) to Golgi apparatus (‘COPII-coated vesicle budding’). All these processes are somehow linked to protein processing, since once they are assembled in ER, proteins are glycosylated in the Golgi apparatus prior to being allocated to cell membrane, lysosomes or even being secreted. As a whole, these results unveil that protein synthesis and post-translational processing could be abnormal in FM.
Muscle-skeletal system also appeared to have some disrupted processes in the fibroblasts from the FM patient according to PANTHER analysis. ‘Skeletal system development’ and ‘bone morphogenesis’ are essential for a proper and fully functional muscle-skeletal system. Both terms are narrowly related with chondrocyte differentiation, cartilage-forming cells that are required in maintenance of bone-muscle junctions. Most of the proteins participating in these three routes are collagens: Alpha-1 and -2 chains of collagen type I (CO1A1 and CO1A2), alpha-1, -2 and -3 chains of collagen type VI (CO6A2, CO6A3 and CO6A1), and alpha-1 chain of collagen type XII (COCA1). All of them showed lower expression in fibroblasts from the FM patient when compared with those from the healthy volunteer. Type I collagen is ubiquitously present in human body, being the main component of organic mass from bones (up to 90%) and responsible for connective tissue formation and integrity [53
]. In this line, when type I collagen synthesis is affected, the development of muscle-skeletal pathologies such as osteogenesis imperfecta is common [54
]. The degradation of this collagen by matrix metalloproteinase 14 (MMP14) is crucial for ECM turnover [55
]. This metalloproteinase showed a higher expression in the fibroblasts from the FM patient, a situation that could worsen collagen deficiency in FM. An altered ECM dynamic is related with acute and chronic inflammatory processes such as arthritis [56
], a chronic disease with a large number of symptoms shared with FM. Inflammation is a key process in the response to endogenous/exogenous threats in which crosstalk among the immune system, coagulation pathway and the nervous system is essential. This response is managed by inflammatory mediators that can be classified as plasma-derived and cell-derived mediators [58
]. In this context, there is evidence that collagen type I metabolites, together with those of elastin, activate the production of pro-inflammatory cytokines, contributing to the inflammatory situation [59
]. In fact, the KEGG analysis on differentially expressed protein showed that 39 of those proteins participated in necroptosis, an hybrid type of cell death linked to inflammation [61
], reinforcing its role in FM. On the other hand, it is known that collagen type I is associated with collagen type VI, a widely distributed collagen that showed lower expression in the fibroblasts from the FM patient. Type VI collagen is the main responsible for preserving the integrity of cartilage and bone, and its deficiency is related with several muscle-skeletal pathologies such as osteoarthritis, Bethelm and Ulrich myopathies, and cartilage-associated affections like Ehlers Danlos-like syndromes [62
]. This last syndrome is considered a rare disease that affects skin, joints and blood vessels [66
], and shares some symptoms like joint pain, anxiety, depression and chronic fatigue with FM. In fact, it was proposed that FM could be a nonclassified variant of hypermobile Ehlers-Danlos syndrome [67
]. Furthermore, some mutations in the gene-codifying type XII collagen seem to be responsible for the lower expression of this protein and have also been linked to the development not only of Ehlers-Danlos syndrome but also of a Ulrich-like myopathy related with an impairment in ECM [68
]. In fact, ECM-cell interactions seem to be altered in FM, since KEGG analysis showed up to 33 proteins participating in focal adhesion among the differentially expressed proteins in FM. Precisely, an adequate integrity of this structure relays on a proper ECM-cell cytoskeleton interaction, a highly specified junction in which integrins have a key role. These proteins and their associated kinases (ILK) participate in signalling processes between ECM and cells in a bidirectional manner. On the one hand, joining of talin to integrins allow their anchoring to ECM through collagen or fibronectin [70
], all of them with lower expression levels in the fibroblast from the FM patient. On the other hand, ILK can rearrange actin filaments, managing several cell functions such as proliferation or motility [71
]. According to IPA analysis, ILK signalling had a significant lower value of z-score (- 2.14) in fibroblasts obtained from the patient, reflecting the differences in this molecular cascade between the healthy volunteer and the FM patient. Thus, 12 (ACTB, COF1, COF2, CTNB1, FINC, FLNC, MYH9, MYL6, MYL9, MYPT1, NACAM and PARVA) out of 16 proteins annotated in this route had lower levels in FM culture when compared with healthy volunteer one. This finding, added to the previous finding about collagens levels confirmed by western blot, once again indicates the presence of alterations in the cytoskeleton as ECM-cell interactions in FM fibroblasts.
GP6 signalling was the last process which was pointed by IPA analysis as inhibited in FM fibroblasts. The glycoprotein VI is a transmembrane collagen receptor exclusively expressed in platelets that facilitates their aggregation in blood coagulation [74
]. Thus, taking into account that our model is a dermal fibroblasts culture, this result has no biological relevance at all, although it could be explained by the drastic fall in the levels of collagen. In this line, this alteration is a key process to be addressed in the future in the platelets of our patient, given that we already observed an abnormal coagulation cascade in FM patients [30
Once alterations of the energy metabolism and of ECM turnover were confirmed in fibroblasts from FM patient, we proceeded to test the possible modulatory effect of the phenolic compound HT on the proteome of these cells. On the one hand, the plasma concentration of HT after the intake of EVOO was highly variable depending of several factors such as the volume of intake or the olive cultivar. However, it can be assumed that, in humans, peak plasma concentrations of HT after the ingestion of 25 mL of EVOO can be around 0.1 µM [76
]. On the other hand, HT is a compound of great interest for the nutraceutical industry due to its nonmutagenic and nongenotoxic profile of HT [77
]. It has been described that the plasma concentration of HT reaches values of 2.83 µg/mL (around 18 µM) after the daily intake of two gastroresistant capsules (15 mg of HT per unit) for three weeks [79
]. With this background, in this study, we decided to evaluate the effect of a dose in between those mentioned above (1.5 µM), which was relevant from a nutraceutical point of view and previously used in the literature [34
]. After the nLC-MS/MS analysis of fibroblasts, we focused on those proteins from FM fibroblasts that, after HT treatment (FM+HT), showed similar levels to those expressed in fibroblasts from healthy volunteer (C). That is, those proteins that showed statistically significant differences in the FM/C comparison but not when comparing FM+HT versus
C. With this criterion, 91 out of 357 proteins differentially expressed in fibroblast from FM patient were normalized with HT treatment and further analysed through IPA.
The most significant route normalized by HT was ‘remodelling of epithelial adherens junction’. Nine proteins participating in this route (ACTB, ARF6, ARPC1B, CLIP1, CTNNA1, CTNNB1, TUBB6, TUBB and ZYX) responded to HT treatment, and CTNNB1 was even confirmed by ELISA. Although these cell-to-cell junctions are typically related with epithelial cells, they can also be found among fibroblasts, especially in those tissues forming syncytia like the fibrous connective tissue [80
]. In fact, IPA Network tool
confirmed that most of the proteins affected by HT treatment were related with connective tissue, given that ‘connective tissue development and function’ was one of the terms used to define the most relevant network. Additionally, epithelial adherens junctions are also essential structures in the maintenance of the integrity of intestinal epithelial barrier, whose disruption are closely related with inflammatory pathologies such as Crohn disease or even FM [81
]. Even though the phenolic compound curcumin has been able to protect the integrity of this cell-to-cell junction [83
], little is known about the effect of phenolic compounds from EVOO on epithelial adherens junctions in humans. However, a study on a murine model revealed the ability of HT to repair the disrupted intestinal barriers [84
]. Hence, further research is needed to ascertain whether the restorative effect of HT on the intestinal barrier could be linked to its effect on epithelial adherens junctions, and how it could help to ameliorate the proinflammatory component of FM and other inflammatory pathologies.
Besides, the 91 proteins normalized by HT were analysed by KEEG database. By this approach, we found that these proteins were also involved in: (i) Metabolic routes, (ii) ER protein processing, (iii) actin cytoskeleton regulation, (iv) thermogenesis and (v) proteoglycans, all of them involved one way or another in processes that were previously mentioned in FM/C comparison. Among the molecules participating in metabolic routes, we found proteins involved in some relevant processes, such as oxidative phosphorylation (f –ATPK– and B1 –AT5F1– subunits from ATP synthase F0
complex), tricarboxylic acid cycle (citrate synthase –CISY–) or carbohydrates (glutamine fructose-6-phosphate transaminase –GFPT1–; 1,4-alpha-glucan branching enzyme –GLGB–; glucose 6-phosphate isomerase –G6PI–) and lipids metabolisms (Very long chain 3-oxoacyl-coA reductase –DHB12–). Besides, additional processes, like heme group metabolism (Biliverdin reductase A –BIEA–), nucleotide synthesis (Tetrahydrofolate synthase C1 –C1TC–; Adenilosuccinate synthetase –PURA2–; transaldolase –TALDO–) or protein maturation (Catalytic subunit from Oligosaccharyltransferase enzymatic complex –STT3B–), were similarly responsive to HT treatment. This widespread effect of HT on different metabolic routes is a proof of its ability to restore the previously mentioned metabolic alterations found in FM. In fact, some of these normalizing effects on cell metabolism have been demonstrated in murine models [85
] and human endothelial cells [86
HT treatment also normalized the ER protein processing, affecting up to eight proteins (CRYAB, DNAJA2, ERO1A, E2AK2, SC23A, SC31A, STT3B and SAR1A). This organelle is directly responsible for the synthesis, assembly and maturation of more than a third part of all the proteins in the cell [87
]. When unfolded proteins are accumulated within the ER, a molecular cascade is activated in order to counteract the deleterious effect linked to this alteration, worsen by inflammatory processes [88
]. The regulatory effect exerted by HT on this organelle was previously tested on human hepatocarcinoma cells [89
] and animal models [90
]. Since not only ER but also the vesicle-associated transport by COPII between the ER and Golgi apparatus seem to be affected in FM, the regulatory effect exerted by HT on organelle could be beneficious in ameliorating protein processing within the cells. As well as the ER and its associated transport, actin cytoskeleton was also regulated in FM fibroblasts under HT treatment. Concretely, five proteins (ACTB, ARC1B, COF1, COF2 and MYPT1) were normalized, a quite interesting result taking into account that this structure was one of the most affected in the FM/C comparison. To our knowledge, HT effect on actin cytoskeleton has not been studied until now, with scarce studies regarding how other phenolic compound could alter its conformation [91
]. However, actin cytoskeleton plays a crucial role on cell anchoring to ECM, and HT could regulate ECM dynamics by inhibiting its remodelling processes [92
]. In this regard, HT exerted a direct effect on several proteins involved in ‘Cell death and survival, cellular movement, connective tissue development and function’ and, even more, in those related to ‘Connective tissue disorders’ like catenin β (CTNB1), confirmed by ELISA. Besides, HT treatment normalized the levels of five proteins (ACTB, CD63, CTNB1, MYPT1 and KAPCA) related with proteoglycans pathway in the fibroblasts from FM patient. Proteoglycans are cell surface/ECM macromolecules, composed by one or more glycosaminoglycans covalently joined to a membrane or secreted protein [52
]. These molecules are closely related with muscle-skeletal system pathologies such as Schwartz-Jampel syndrome or osteochondritis dissecans, whose patients suffer from muscle weakness or joint problems [93
]. Even more, proteoglycans have been proposed as serum biomarkers for several inflammatory-related syndromes like different types of arthritis or FM [95
]. Altogether, these results about the effect of HT on ECM and proteoglycans reinforce the point to this potential treatment in FM. Nonetheless, it would be necessary further investigation to assess the particular effect of HT on ECM metabolism and its link to their anti-inflammatory properties.
The KEGG analysis also unveiled the effect of HT on thermogenesis pathway and its normalizing effect on five proteins of this route (ACTB, ATPK, AT5F1, KAPCA and KGP1). It has been demonstrated that thermoregulation is an inefficient process in FM patients [97
]. As a result, these patients have an abnormal low body temperature, especially around trigger points [98
]. This points are distributed through the body following a very similar pattern to that followed by brown adipose tissue deposits [99
] and, curiously, thermogenesis-activating stimuli such as stress and cold also worsen FM symptoms [100
]. Some authors have pointed that a disruption in ECM-adipocyte receptors interaction could be responsible for an impaired thermogenesis [102
]. According to our in vitro
model, the interaction between ECM and cell receptors were compromised in FM fibroblasts and, besides, the mitochondrial metabolism was impaired in these cells. Taking together the effect that the HT exert on these processes could somehow improve some of the alterations found in FM.