Abstract

   Physical exercise (PE) is recommended for Rheumatoid Arthritis (RA),
   but the molecular and biological mechanisms that impact the
   inflammatory process and joint destruction in RA remain unknown. The
   objective of this study was to evaluate the effect of PE on the
   histological and transcriptional changes in the joints of
   adjuvant-induced arthritis (AIA) rat model. AIA rats were subjected to
   PE on a treadmill for eight weeks. The joints were subjected to
   histological and microarray analysis. The differentially expressed
   genes (DEGs) by PE in the arthritic rats were obtained from the
   microarray. The bioinformatic analysis allowed the association of these
   genes in biological processes and signaling pathways. PE induced the
   differential expression of 719 genes. The DEGs were significantly
   associated with pathogenic mechanisms in RA, including HIF-1, VEGF,
   PI3-Akt, and Jak-STAT signaling pathways, as well as response to
   oxidative stress and inflammatory response. At a histological level, PE
   exacerbated joint inflammatory infiltrate and tissue destruction. The
   PE exacerbated the stressed joint environment aggravating the
   inflammatory process, the hypoxia, and the oxidative stress, conditions
   described as detrimental in the RA joints. Research on the effect of PE
   on the pathogenesis process of RA is still necessary for animal models
   and human.

   Keywords: running, exercise, physical activity, rheumatoid arthritis,
   adjuvant-induced arthritis

1. Introduction

   Rheumatoid arthritis (RA) is one of the most common inflammatory
   arthritis affecting 0.5–1.0% of the population [[32]1]. RA is a
   systemic autoimmune disease characterized by inflammation of the
   synovial membrane and periarticular structures. Joint pain, stiffness,
   and swelling are the primary symptoms, which eventually lead to joint
   deformity and functional disability. Extra-articular manifestations,
   including cardiovascular, pulmonary, psychological and skeletal
   disorders are common in RA patients [[33]2]. The etiology and
   pathophysiology of RA remain unresolved; however, genetic and
   environmental factors have been associated with the inappropriate
   immunomodulation that triggers the inflammatory process in the joints
   and the subsequent damage to synovial structures [[34]3]. The treatment
   of RA aims to reduce pain and inflammation in the joints and also to
   preserve their structural integrity and the patient’s functionality.
   Currently, treatment includes a variety of pharmacological agents,
   education programs, joint protection, lifestyle changes, PE and
   surgical intervention as a final step [[35]4,[36]5,[37]6].

   PE is considered in the multidisciplinary management of RA patients.
   The European League Against Rheumatism (EULAR) has established the
   recommendations for physical activity in people with inflammatory
   arthritis [[38]4], and its recommendations for cardiovascular disease
   risk management in RA include regular exercise [[39]7]. It has been
   previously shown that PE improves joint health [[40]8], muscle strength
   [[41]9], cardiovascular fitness [[42]10], vascular function [[43]11],
   and psychological well being [[44]12]. PE also reduces the inflammation
   [[45]13], rheumatoid cachexia [[46]14], and fatigue [[47]15] in RA
   patients.

   Although the systemic benefits of PE in rheumatic patients are well
   recognized, the direct consequences of PE in active synovitis and the
   potential role of PE accelerating joint destruction are not clear. Some
   reports have shown that mechanical load influences the onset and
   worsens the progression of the disease in experimental animal models of
   arthritis [[48]16,[49]17]; therefore, the consequences of PE on
   inflamed joints remain a topic that should be addressed.

   We selected adjuvant-induced arthritis (AIA) because it mimics the
   signs and symptoms of human RA, such as the histopathological changes
   of inflammation, vascular proliferation, and importantly, the
   progression to joint destruction [[50]18]. The AIA model has been
   widely used in the development of antirheumatic drugs, including
   several non-steroid anti-inflammatories, methotrexate [[51]19], and
   also biologic drugs, including tocilizumab [[52]20] and Jak-inhibitors
   like tofacitinib [[53]21].

   To understand the effects of PE in the complexity of the inflamed
   joint, we selected an unbiased approach to maintain a broad
   perspective. We selected the bioinformatic analysis of the
   transcriptome from the tarsal joint through a DNA microarray. The
   microarray simultaneously evaluates the expression levels for a large
   number of genes [[54]22]; and, identifies the most relevant mediators
   in complex processes such as the interplay between PE and inflammation.

   A better understanding of the molecular consequences of PE on the
   inflamed joints may contribute to improving its prescription for RA
   patients for the protection of joint integrity. In this regard, the
   objective of this study was to determine the effect of an exercise
   intervention on the transcriptional expression of genes in the AIA rat
   model using microarray technology.

2. Materials and Methods

2.1. Animals and Study Groups

   Male Wistar rats (300–350g) were used for this study. The animals were
   housed in an animal facility with a 12 h light/dark cycle maintained at
   temperatures between 22 ± 1 °C with food and water provided at libitum.
   The experimental protocol was approved by the Committee of Ethics of
   the Instituto Chihuahuense de Salud-Secretaría de Salud-Facultad de
   Medicina y Ciencias Biomédicas, UACH (protocol number CEI-EXP-140/15).
   The animals were randomly divided into two groups of seven animals
   each: (1) arthritis group and (2) arthritis-exercise group.

2.2. Arthritis Induction

   The AIA model was conducted as previously reported [[55]23]. Rats were
   injected in the footpads with 0.2 mL of Complete Freund’s Adjuvant
   (CFA) (Sigma Chemicals, St. Louis, MO, USA) mixed with phosphate buffer
   saline in the 1:1 ratio. To increase the severity of arthritis, a
   booster injection with 0.1 mL of the emulsion was administered in the
   same way on day 5–post first injection.

2.3. Familiarization

   Animals belonging to the arthritis-exercise group were familiarized
   with the treadmill for three weeks to reduce their stress level during
   the PE. Each daily familiarization session included placing the rats on
   the treadmill switched off for 10 min (visual/olfactory adaptation) and
   then turning on the treadmill at the minimum speed (3 m/min) for 5 min
   (sound/movement adaptation). After the first week of the treadmill
   familiarization, arthritis induction was started and the
   familiarization period continued for two more weeks.

2.4. Physical Capacity Test

   After the familiarization, a physical capacity test (PCT) was conducted
   to establish the maximum speed reached by the animals before the
   intervention of PE. The PCT consisted of a single treadmill session in
   which the rats, after 5 min of warm-up (3 m/min), ran in the band with
   an increase in the speed of 3 m/min every 2 min until they fatigued and
   stopped running. The maximal physical capacity (100%) was defined as
   the maximum speed reached by each animal. The average speed per group
   was calculated, and this was used to establish the speed of the
   exercise sessions.

2.5. Exercise Program

   The rats were exercised three times a week for eight weeks. The
   exercise started with a speed of 20% of the PCT and increases of 13% in
   speed were applied every two weeks until reaching 60%. Each exercise
   session included four phases: (1) acclimatization: rats were placed on
   the treadmill switched off for 5 min; (2) warm-up: rats walked for 5
   min at lowest speed; (3) exercise: rats run for 25 min at the
   corresponding speed; and (4) cool down: rats walked at lowest speed for
   5 min. The exercise sessions were administered at the same time each
   day.

2.6. Histological Analysis

   After exercise intervention, the rats were euthanized, and the tarsal
   joints were immediately dissected. The ankle, the subtalar and the
   navicular cuboid joints were extracted and cut proximally in the distal
   fibula and tibia; and, distally in the metatarsal bones at the
   diaphysis. The muscle was dissected as much as possible and the
   synovial and ligament structures were preserved and fixed in 10%
   formalin phosphate buffer for 48 h and decalcified with 5% nitric acid
   for 24 h [[56]24]. The tissues were dehydrated in graded ethanol, and
   embedded in paraffin, sectioned, and stained with hematoxylin-eosin
   (H&E Merck, Darmstadt, Germany). The histological variables: (a)
   inflammatory infiltrate, (b) synovial hyperplasia, (c) pannus
   formation, (d) synovial vascularity, (e) cartilage damage and (f) bone
   erotion were semi-quantitatively evaluated using a scale of 0 = absent,
   1 = mild, 2 = moderate and, 3 = severe on each animal. The maximum
   score reached per animal for each parameter was 12 when the four joints
   showed a severe level. The scoring average per study group was
   estimated. The histological variables were evaluated bu two experienced
   operators blinded to the different groups.

2.7. DNA Microarray and Bioinformatics Analysis

   The effect of PE on transcriptome was evaluated in the DNA microarray
   by comparing the arthritis-exercise group (experimental group) and the
   arthritis group (reference group). The tarsal joints dissected were
   immediately placed in liquid nitrogen and disrupted with mortar and
   pestle. Total RNA was extracted using RNeasy^® Lipid Tissue Mini Kit
   (QIAGEN, Germantown, MD, USA) according to the manufacturer´s
   instructions. The RNA quality and integrity were verified using a 2100
   Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). For the
   microarray, the RNA of every group was pooled keeping equimolar
   quantities of every individual.

   The microarray analysis was performed at the Institute of Cellular
   Physiology of the National Autonomous University of Mexico (México
   City, México). Briefly, the cDNA was synthesized and labeled for
   subsequent hybridization in the Rn5K microchip containing 5000 rat
   genes. Scanning and signal acquisition was performed using the
   ScanArray 4000 (Packard BioChips Technologies, Billerica, MA, USA). The
   GenArise software was used for the analysis of the microarray scan, and
   the lists of DEGs [Z-score ≥ 1.5 standard deviations (SD)] were
   obtained. To assess the biological relevance of the DEGs, DAVID
   Bioinformatics Resources 6.8 platform ([57]https://david.ncifcrf.gov/)
   was used. Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and
   Genomes) pathway mapper with significant associations (p ≤ 0.5) were
   obtained [[58]25]. Also, the STRING 10.5 database
   ([59]http://string-db.org/) was used to obtain the analysis and
   integration of direct and indirect protein-protein interactions (IPP)
   centered on the functional association [[60]26]. The DEGs identified in
   the microarray were loaded, and the interactions were selected with
   minimal confidence (interaction score > 0.4). The obtained IPP network
   was analyzed more thoroughly to obtain primary clusters of sub-networks
   using the Cytoscape software version 3.7.0 (Bethesda, Rockville, MD,
   USA) with the Molecular Complex Detection (MCODE) complement
   [[61]27,[62]28].

2.8. Statistical Analysis

   The statistical analysis was made in SPSS statistics v22 software (SPSS
   Science Inc., Chicago, IL, USA). Measures of central tendency were
   estimated for each variable. T-test was used to compare the effect of
   PE on histological parameters. Differences were considered significant
   when p < 0.05.

3. Results

3.1. Maximal Physical Capacity

   The maximal physical capacity obtained in the PCT for the
   arthritis-exercise group was 21.28 ± 5.96 m/min. The initial speed and
   its increments along the exercise intervention were based on this
   value.

3.2. Histological Analysis

   The effect of exercise in the joints was histologically evaluated using
   H&E staining. The scores of inflammatory infiltrate, synovial
   hyperplasia, pannus formation, synovial vascularity, and cartilage and
   erosions obtained for each study group are shown in [63]Figure 1. The
   highest scores of all evaluated parameters were reached in the
   arthritis exercise group. However, only hyperplasia, cartilage damage
   and bone erosion were statistically different between the groups.

Figure 1.

   [64]Figure 1
   [65]Open in a new tab

   Effects of exercise on tarsal bone histological parameters in
   adjuvant-induced arthritis rats. (A) Representative images of
   histological findings in the tarsal joints of the study groups at the
   end of the exercise intervention using H&E staining. (B) Joint
   involvement was scored by the semi-quantitative scale (showed in E) to
   describe inflammatory changes and structural remodeling in the tarsal
   joints (7 rats per group). The t-student test was used to compare
   histological measurements between groups. * p < 0.01. (C) Exercised
   rats on a treadmill. (D) Representative images of clinical changes on
   hind paws of adjuvant-induced arthritis (AIA) rats exercised (left) and
   non-exercised (right). (E) Representative images of the inflammation
   and structural joint damage scores in the tarsal joints of AIA rats.
   The 0 (normal) score was established in healthy rats, where the bone
   (bo), cartilage (ca) and synovium (sy) did not show alterations. The
   arthritis scores 1 (mild), 2 (moderate), and 3 (severe) were based on
   the inflammatory changes: the presence of synovial hyperplasia (sh) and
   pannus (pa) and structural remodeling: cartilage damage (cd) and bone
   erosion (be). The images were acquired with a 10× and 40×
   amplification. AIA: adjuvant-induced arthritis.

3.3. Microarray and Bioinformatic Analysis

   The transcriptome-wide microarray analysis identified a pool of DEGs in
   the joints from arthritic exercised rats in comparison to arthritic
   non-exercised rats. A total of 719 genes were differentially expressed
   (Z-score ≥ 1.5 SD), 361 up-regulated ([66]Appendix A), and 358
   down-regulated ([67]Appendix B). [68]AB000928 (Zp1) and [69]X68101
   (Trg) were the most significantly up- and down-expressed genes by PE,
   respectively.

   To understand the related biological functions of the 719 DEGs, we used
   the DAVID database for the enrichment analysis of GO and KEGG pathways.
   The top 10 enriched terms of GO and KEGG pathways are shown in
   [70]Figure 2. Response to organic cyclic compound, response to drug,
   and response to ethanol were among the top enriched biological
   processes. The DEGs were enriched in molecular functions like protein
   binding. The most enriched KEGG pathways included hypoxia-induced
   factor-1 (HIF-1), cyclic adenosine monophosphate (cAMP) and
   mitogen-activated protein kinase (MAPK) signaling pathways.
   Subsequently, we selected the biological processes and KEGG signaling
   pathways known to be relevant in the pathogenesis of RA, which
   included: response to hypoxia, response to oxidative stress,
   angiogenesis and inflammatory/immune response ([71]Table 1).

Figure 2.

   [72]Figure 2
   [73]Open in a new tab

   The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes
   (KEGG) pathway enrichment analysis of differentially expressed genes in
   the DAVID database. The 719 differentially expressed genes by exercise
   were uploaded into the DAVID database for enrichment analysis. The top
   10 GO analysis results of these dysregulated genes were displayed in
   the bar chart: (A) KEGG pathway, (B) cellular component, (C) biological
   process and (D) molecular function. The bars indicated the -Log10 (p
   value) of each GO and KEGG term. The number of genes involved in each
   term is shown on the right side of each bar.

Table 1.

   Differentially expressed genes by physical exercise associated with
   pathogenic processes in Rheumatoid Arthritis.
   Description Effect on Regulation Number of Genes Genes p-Value
   Biological Process Response to hypoxia Up 12 Camk2d, Cat, Cdkn1b, Il18,
   Lepr, Mt3, Nppa, Ptk2b, Rhoa, Tgfbr3, Vegfa, Vhl 3.1 × 10^−3
   Down 17 Alas1, Bnip3, Cited2, Ep300, Smad3, Casp3, Hmbs, Hyou1, Igf1,
   Itga2, Icam1, Il1a, Mmp9, Kcnma1, Th, Ucp2, Ucp3 7.5 × 10^−6
   Oxidation-reduction process Up 19 Akr1a1, Aifm1, Cbr1, Cyp2d2, Cyp2t1,
   Cyp27b1, Cyp4f6, Cyp51, Degs1, Gpx1, Hao2, Ido1, Kif1b, Me1, Oxr1,
   Prdx2, Pah, Txn1, Tpo 1.2 × 10^−2
   Down 26 Haao, Hibadh, Ndufa12, Aldh6a1, Aox1, Cp, Crym, Cyp19a1,
   Cyp1b1, Cyp2a3, Cyp2b12, Cyp2p2c7, Cyp2d3, Dio2, Dcxr, G6pd, Glrx,
   Gpx6, Hsd3b6, Hadhb, Ldhb, Scd, Srd5a2, Suox, Tpo, Th 4.9 × 10^−5
   Hydrogen peroxide catabolic process Up 4 Cat, Gpx1, Prdx2, Tpo 2.5 ×
   10^−3
   Response to oxidative stress Up 8 Akt1, Cat, Gpx1, Mt3, Map2k1, Oxr1,
   Prdx2, Tpo 7.4 × 10^−3
   Cellular response to oxidative stress Up 5 Ggt1, Mt3, Nfe2l2, Prdx2,
   Txn1 3.2 × 10^−2
   Cellular response to hydrogen peroxide Up 5 Anxa1, Aifm1, Il18, Nfe2l2,
   Ppp5c 3.5 × 10^−2
   Response to reactive oxygen species Up 3 Cat, Gpx1, Ptk2b 4.1 × 10^−2
   Inflammatory response Up 12 Akt1, Ccl4, Anxa1, Cxcl3, Csf1, Cyp4f6,
   Crlf2, Hmgb1, Ido1, Il18, Mep1b, Nfe2l2 6.3 × 10^−3
   KEGG signaling pathway HIF-1 signaling pathway Up 14 Akt1, Akt3,
   Camk2a, Camk2d, Cdkn1b, Hk1, Ifngr1, Map2k1, Nppa, Pik3cb, Pik3r1,
   Tceb2, Vegfa, Vhl 2.5 × 10^−7
   VEGF signaling pathway Up 8 Akt1, Akt3, Map2k1, Pik3cb, Pik3r1, Ppp3cb,
   Ppp3r2, Vegfa 2.5 × 10^−4
   Rheumatoid Arthritis Up 6 Atp6v0a1, Atp6v0e1, Cd80, Csf1, Il18, Vegfa
   4.2 × 10^−2
   T cell receptor signaling pathway Up 10 Akt1, Akt3, Dlg1, Lcp2, Map2k1,
   Pik3cb, Pik3r1, Ppp3cb, Ppp3r2, Rhoa 4.5 × 10^−4
   B cell receptor signaling pathway Up 7 Akt1, Akt3, Map2k1, Pik3cb,
   Pik3r1, Ppp3cb, Ppp3r2 3.3 × 10^−3
   Chemokine signaling pathway Up 12 Akt1, Akt3, Ccl4, Gng8, Adcy5, Arrb1,
   Cxcl3, Map2k1, Pik3cb, Pik3r1, Ptk2b, Rhoa 1.3 × 10^−3
   PI3K-Akt signaling pathway Up 17 Akt1, Akt3, Faslg, Gng8, Csf1, Cdkn1b,
   Fgd17, Fgf19, Il3ra, Lpar3, Map2k1, Pik3cb, Pik3r1, Ppp2r2c, Vegfa,
   Ywhae, Ywhag 2.2 × 10^−3
   Jak-STAT signaling pathway Up 10 Akt1, Akt3, Cish, Crlf2, Ifngr1,
   Il3ra, Lepr, Pik3cb, Pik3r1, Thpo 2.2 × 10^−3
   TNF signaling pathway Up 7 Akt1, Akt3, Cxcl3, Csf1, Map2k1, Pik3cb,
   Pik3rc1 2.8 × 10^−2
   Toll-like receptor signaling pathway Up 7 Akt1, Akt3, Ccl4, Cd80,
   Map2k1, Pik3cb, Pik3r1 1.7 × 10^−2
   Wnt signaling pathway Up 10 Wif1, Camk2a, Camk2d, Csnk2a1, Csnk2b,
   Ctnnb1, Fzd4, Ppp3cb, Ppp3r2, Rhoa 3.0 × 10^−3
   Osteoclast differentiation Up 13 Akt1, Akt3, Fcgr2a, Csfr1,
   Ifngr1,Lilrb3l, Lcp2, Mapk2k1, Pik3cb, Pik3r1, Ppp3cb, Ppp3r1, Ppp3r2
   8.6 × 10^−5
   [74]Open in a new tab

   After the analysis on DAVID, the list of the 719 DEGs was analyzed on
   the STRING and Cytoscape-MCODE platforms. The first three clusters
   obtained are shown in [75]Figure 3, [76]Figure 4 and [77]Figure 5. The
   genes from the first three clusters were loaded in STRING to identify
   the associated KEGG signaling pathways; those relevant in RA were
   selected and marked with different colors. The pathways considered as
   relevant included the HIF-1 signaling pathway. The analysis of the
   protein networks unveiled relevant genes dysregulated by PE. Those
   presented multiple functional connections included up-regulated genes
   such as serine-threonine protein kinases Akt1, Akt2, and Akt3; the
   vascular endothelial growth factor A (Vefga), the mitogen-activated
   protein kinase kinase 1 (Mapk1), phosphatidylinositol 4,5-bisphosphate
   3-kinase catalytic subunit beta isoform (Pik3cb), glucokinase (Gck),
   fructose-biphosphatase2 (Fbp2); and also down-regulated genes such as
   interleukin 6 (Il6).

Figure 3.

   [78]Figure 3
   [79]Open in a new tab

   The protein-protein interaction network construction of the cluster
   number one obtained with the differentially expressed genes. The lists
   of the differentially expressed genes (Z-score ≥ 1.5 SD) were analyzed
   on the STRING and Cytoscape platforms. The primary clusters of
   sub-networks were obtained using the Molecular Complex Detection
   (MCODE) complement (cutoff = 0.2). Line thickness indicates the
   strength of data support; colored nodes indicate query proteins and
   first shell of interactors; white nodes indicate the second shell of
   interactors.

Figure 4.

   [80]Figure 4
   [81]Open in a new tab

   The protein-protein interaction network construction of the cluster
   number two obtained with the differentially expressed genes. The lists
   of the differentially expressed genes (Z-score ≥ 1.5 SD) were analyzed
   on the STRING and Cytoscape platforms. The primary clusters of
   sub-networks were obtained using the Molecular Complex Detection
   (MCODE) complement (cutoff = 0.2). Line thickness indicates the
   strength of data support; colored nodes indicate query proteins and
   first shell of interactors; white nodes indicate the second shell of
   interactors.

Figure 5.

   [82]Figure 5
   [83]Open in a new tab

   The protein-protein interaction network construction of the cluster
   number three obtained with the differentially expressed genes. The
   lists of the differentially expressed genes (Z-score ≥ 1.5 SD) were
   analyzed on the STRING and Cytoscape platforms. The primary clusters of
   sub-networks were obtained using the Molecular Complex Detection
   (MCODE) complement (cutoff = 0.2). Line thickness indicates the
   strength of data support; colored nodes indicate query proteins and
   first shell of interactors; white nodes indicate the second shell of
   interactors.

4. Discussion

   Current evidence describing the influence of PE on the pathogenic
   mechanisms of the arthritides is scarce and limited to studies in
   animal models. Most of these studies have focused on animal models of
   osteoarthritis and, in most cases, only clinical and histological
   parameters have been evaluated, limiting our understanding of the PE in
   the pathogenesis of active synovitis. To our knowledge, no previous
   study has assessed the effects of PE on the transcriptome profile in
   the AIA model.

   Our study shows that PE induced several genes linked to RA
   pathogenesis. Specifically, PE exacerbated hypoxia, oxidative stress,
   and the immune/inflammatory response in rats with AIA. The histology
   confirms our transcriptome results; in the joints of the exercised
   animals, we observed an increase in the inflammatory infiltrate and
   more severe scores for bone and cartilage destruction.

   In our opinion, the connection between PE and its potential detrimental
   role at active stages of synovitis should be considered as a relevant
   event in patients with inflammatory arthritides. Current
   recommendations for physical activity and PE in people with
   inflammatory and non-inflammatory arthritis [[84]4,[85]5,[86]7,[87]29]
   have been established mostly on clinical studies in humans, which the
   intimate effect of PE in the arthritic joints at a molecular level has
   not been considered. These recommendations have not been specified
   according to the disease activity; however, as our study shows, likely
   at very active stages of inflammatory arthritides, PE could increase
   the inflammatory process. It is unlikely that PE will be prescribed in
   patients with active arthritis; however, a wide prescription of PE
   could also include the large proportion of patients whom remain with
   some degree of inflammatory activity despite therapy. Probably, the PE
   benefits may not be as clear in those patients with partial disease
   control. Therefore, the disease activity should be considered as an
   element for PE prescription.

   RA pathogenesis remains a primary research field; we now understand
   that the rheumatoid pannus is an altered microenvironment that carries
   a significant array of abnormal immune processes and also several
   metabolic alterations. The increase in hypoxia and oxidative stress are
   recognized as relevant inflammation drivers within the rheumatic
   synovium. Their pathological implications are now sustained by
   experimental results both: in animal models [[88]30,[89]31] and human
   disease [[90]32,[91]33]. Synovial hypoxia promotes inflammation,
   angiogenesis, production of chemokines, and cartilage and bone
   destruction, mediated in part, through HIF-1 that is a potent
   pleiotropic mediator [[92]32,[93]34].

   Our study confirms that PE up-regulates genes linked to hypoxia and
   oxidative stress in the arthritic joints; indeed, the HIF-1 signaling
   was the first enriched pathway in our bioinformatic analysis,
   suggesting that this pathway was the strongest influenced by the PE.
   Downstream HIF-linked genes as the vascular endothelial growth factor
   (VEGF) are also up-regulated. VEGF is critical for the onset and
   perpetuation of vascular proliferation in synovitis
   [[94]35,[95]36,[96]37]. Also, it was previously reported the VEGF
   increase in the serum and synovial fluid of RA patients [[97]35,[98]38]
   as well as in experimental animal models [[99]39].

   Hypoxia could activate the genes encoding nearly every step of
   glycolysis [[100]32]. Abnormal glucose metabolism was observed in the
   synovial fluid and synovium of RA, which is evidenced by increased
   glycolytic enzyme activity [[101]40]. A recent study proposes the
   inhibition of hexokinases as a potential therapeutic strategy in the
   treatment of patients with RA [[102]41]. According to this, the
   glucokinase (Gck) and the hexokinase 1 (Hk1) were up-regulated by PE in
   our study. Additionally, genes involved in the oxidative stress process
   were also dysregulated by PE. It is recognized that in an inflamed
   joint, hypoxia and reperfusion cycles occur, producing an excessive
   amount of reactive oxygen species (ROS) known as oxidative stress
   [[103]33,[104]42].

   In addition to hypoxia and oxidative stress conditions induced by the
   effect of PE, other KEGG pathways were dysregulated ([105]Table 1) such
   as rheumatoid arthritis, T-cell and B-cell receptor signaling,
   chemokine, Jak-Stat, TNF, Toll-like-receptor, WNT and osteoclast
   differentiation signaling pathways. This array of dysregulated pathways
   suggests a widespread effect of PE both in the adaptive and innate
   immune response.

   Other genes participating in several KEGG pathways in our study include
   Akt1, Akt2, Akt3, Map2k1 and PIk3cb which belong to PI3K/AKT/mTOR and
   MAPK pathways that are considered central in RA pathogenesis
   [[106]43,[107]44]. They have been implicated in fibroblast-like
   synoviocyte proliferation and activation. They stimulate the production
   of pro-inflammatory and osteoclastogenic cytokines; besides, the
   inhibition of either pathway has been correlated with the improvement
   of disease activity parameters. Therefore, the use of agents with the
   potential to regulate either PI3K/AKT/mTOR or MAPK pathways has been
   considered as a potential therapeutic strategy [[108]45].

   The molecular results of the PE effect are not easily comparable with
   previous studies due to the low number of reports, which had objectives
   aimed at particular aspects of the disease. However, we can distinguish
   that the negative effect of PE has also been reported in the two most
   recent and complete studies in this field [[109]16,[110]17]. The
   results of these studies suggest a direct link between the degree of
   mechanical stress to inflammation and tissue localization, which
   strengthens previous studies that propose biomechanical factors as
   potential determinants for the topographic pattern of joint disorders
   in arthritis [[111]46]. Cambré et al., suggest that the link between
   mechanical stress and the onset of arthritis is explained by local
   recruitment of Ly6high inflammatory cells elicited by
   mechanostress-induced chemokine induction in resident mesenchyme cells
   [[112]17]. They also provide evidence of the local participation of the
   complement activators to maintain the progression and chronicity of
   arthritis through an impaired resolution [[113]16].

   On the other hand, it has been shown that in healthy rats, PE has a
   positive effect in the joints it induces extracellular matrix
   biosynthesis, cartilage strengthening, and attenuation of inflammatory
   pathways [[114]47]. This suggests that the gene induction pattern of
   mechanosensitive genes seems to be distinct between different arthritis
   models and healthy animals, and likely also in regarding to the degree
   of arthritis activity.

   Consistent with the genetic analysis, our histological findings showed
   that PE had an adverse effect on increasing the inflammatory infiltrate
   and the joint destruction. In experimental models of arthritis, it has
   been confirmed that mechanical stress increase joint inflammation,
   stimulate the progression and chronicity of arthritis and structural
   damage [[115]16,[116]17,[117]48]; besides, the mechanical stress
   decrease the bone quality [[118]16]. In contrast to our findings, other
   studies in the AIA model have shown an anti-inflammatory effect of PE
   in some parameters such as synovial hyperplasia [[119]49,[120]50], and
   joint destruction [[121]51].

   Mode, intensity, frequency, timing, and duration of the PE protocol are
   decisive aspects to the physiological responses and different outcomes
   [[122]52]. Moreover, the severity of arthritis at the starting time of
   PE could be definitory. Studies in the AIA model that started the PE at
   the pre-arthritic stage [[123]49,[124]50] showed beneficial results.
   However, we started the PE at an arthritic stage because we think this
   scenario is more representative of RA patients.

5. Conclusions

   The present study explored the influence of PE in the genetic
   expression and the histology of the actively swollen joints in the AIA
   model. Our results describe an exploratory and preliminary scenario in
   which PE increased the expression of genes with a known pathogenic role
   in RA; these findings were consistent with the histological findings.
   These results suggest that the PE exacerbated the stressed joint
   environment, where the inflammation, hypoxia, and the oxidative stress
   prevail, rendering clear parallels to the RA joints. The molecular
   effect of exercise on active stages of inflammatory arthropathies
   requires further studies in animal models and humans, which could
   contribute with the development of adequate programs for RA patients
   that can ensure a beneficial effect without negative implications at
   the tissue level.

Acknowledgments