Low-Intensity Physical Exercise Decreases Inflammation and Joint Damage in the Preclinical Phase of a Rheumatoid Arthritis Murine Model

Abstract

Lifestyle modifications in preclinical Rheumatoid Arthritis (RA) could delay the ongoing pathogenic immune processes and potentially prevent its onset. Physical exercise (PE) benefits RA patients; however, its impact in reducing the risk of developing RA has scarcely been studied. The objective was to describe the effects of low-intensity PE applied at the disease's preclinical phase on the joints of DBA/1 mice with collagen-induced arthritis (CIA). Twelve mice with CIA were randomly distributed into two groups: the CIA-Ex group, which undertook treadmill PE, and the CIA-NoEx, which was not exercised. The effects of PE were evaluated through clinical, histological, transcriptomics, and immunodetection analyses in the mice's hind paws. The CIA-Ex group showed lower joint inflammation and damage and a decreased expression of RA-related genes (Tnf Il2, Il10, Il12a, IL23a, and Tgfb1) and signaling pathways (Cytokines, Chemokines, JAK-STAT, MAPK, NF-kappa B, TNF, and TGF-beta). TNF-α expression was decreased by PE in the inflamed joints. Low-intensity PE in pre-arthritic CIA reduced the severity through joint down-expression of proinflammatory genes and proteins. Knowledge on the underlying mechanisms of PE in preclinical arthritis and its impact on reducing the risk of developing RA is still needed.

Keywords: at-risk arthritis; inflammation; joint damage; microarray; physical exercise; preclinical arthritis.

Figure 1

Experimental design and characteristics of the treadmill physical exercise routine. CFA: Complete Freund’s Adjuvant; IFA: Incomplete Freund’s Adjuvant; CII: Collagen type II.

Figure 2

Effect of physical exercise on transcriptomic down-expression in tarsal joints of mice with collagen-induced arthritis. The lists of the down-expressed genes in the CIA-Ex mice with respect to control (Z-score ≥ 1.5 SD) were analyzed on DAVID bioinformatics resource, STRING database, and Cytoscape software (n = 8 mice per group). (a) The associated KEGG pathways (p ≤ 0.05) in DAVID are shown in the bars that indicate the -Log10 (p-value) and the number of genes on the right side of each bar. (b) The primary clusters of sub-networks found with the Molecular Complex Detection (MCODE) complement (cutoff = 0.4) to obtain the protein–protein interaction (PPI) network. (c) KEGG signaling pathways relevant in arthritis. (d) Amplification of the nodes with the most significant interaction in the selected pathways. FDR: false discovery rate.

Figure 3

Effect of physical exercise on transcriptomic up-expression in tarsal joints of mice with collagen-induced arthritis. The lists of the up-expressed genes in the CIA-Ex mice with respect to control (Z-score ≥ 1.5 SD) were analyzed on DAVID bioinformatics resource, STRING database, and Cytoscape software (n = 8 mice per group). (a) The associated KEGG pathways (p ≤ 0.05) in DAVID are shown in the bars that indicate the -Log10 (p-value) and the number of genes on the right side of each bar. (b) The primary clusters of sub-networks found with the Molecular Complex Detection (MCODE) complement (cutoff = 0.4) to obtain the protein–protein interaction (PPI) network. (c) KEGG signaling pathways relevant in arthritis. (d) Amplification of the nodes with the most significant interaction in the selected pathways. FDR: false discovery rate.

Figure 4

Clinical and histological effects of treadmill physical exercise in the preclinical collagen-induced arthritis in DBA/1 mice. (a) Evaluation of the clinical course of arthritis. Erythema and swelling were evaluated using the semiquantitative scale from 0 to 4 in each paw and the sum of the four paws per mouse was obtained. The mean score and standard deviation were calculated and graphed for each group during the experimental intervention. (b) Representative images of the clinical manifestation of CIA in control and exercised mice. (c) Histological evaluation in the hind paws by H&E staining. Inflammatory infiltrates, synovial hyperplasia, cartilage damage, and bone erosions were evaluated using the semiquantitative scale from 0 to 4. The mean score and standard deviation were calculated and graphed for each group. (d) Representative images of the histological findings in the hind paws using H&E staining. The Mann–Whitney U test was used to determine statistically significant differences when p < 0.05. * p ≤ 0.05; ** p ≤ 0.01.

Figure 5

Effects of treadmill physical exercise on inflammatory cytokine expression in the preclinical collagen-induced arthritis in DBA/1 mice. (a) Relative quantification of inflammatory cytokines on hind paws joints. RNA expression was determined by RT-qPCR relative quantification through the ΔΔCt method using the housekeeping gene RPL13A. (b) TNF-α expression was quantified with the ImageJ program and the IHC toolbox in at least 20 microscopic field images from each study subject. The DAB color was extracted from each image, and the maximum and mean gray values were obtained. Each image’s optical density (OD) was obtained with log10 (maximum gray value/mean gray value). The OD means and standard deviations were calculated and graphed by the study group. (c) Representative images of TNF-α detection in hind paws joints by immunohistochemistry. Immunodetection was carried out using streptavidin-peroxidase conjugated and DAB as the chromogen. The Mann–Whitney U test was used to determine statistically significant differences. * p < 0.05; ** p < 0.01; *** p < 0.001.