Rotating Magnetic Field Mitigates Ankylosing Spondylitis Targeting Osteocytes and Chondrocytes via Ameliorating Immune Dysfunctions

Abstract

Ankylosing spondylitis (AS) is clinically characterized by bone fusion that is induced by the pathological formation of extra bone. Unfortunately, the fundamental mechanism and related therapies remain unclear. The loss of SHP-2 (encoded by Ptpn11) in CD4-Cre;Ptpn11^f/f mice resulted in the induction of AS-like pathological characteristics, including spontaneous cartilage and bone lesions, kyphosis, and arthritis. Hence, this mouse was utilized as an AS model in this study. As one of the basic physical fields, the magnetic field (MF) has been proven to be an effective treatment method for articular cartilage degeneration. In this study, the effects of a rotating magnetic field (RMF; 0.2 T, 4 Hz) on an AS-like mouse model were investigated. The RMF treatment (2 h/d, 0.2 T, 4 Hz) was performed on AS mice from two months after birth until the day before sampling. The murine specimens were subjected to transcriptomics, immunomics, and metabolomics analyses, combined with molecular and pathological experiments. The results demonstrated that the mitigation of inflammatory deterioration resulted in an increase in functional osteogenesis and a decrease in dysfunctional osteolysis due to the maintenance of bone homeostasis via the RANKL/RANK/OPG signaling pathway. Additionally, by regulating the ratio of CD4+ and CD8+ T-cells, RMF treatment rebalanced the immune microenvironment in skeletal tissue. It has been observed that RMF interventions have the potential to alleviate AS, including by decreasing pathogenicity and preventing disease initiation. Consequently, RMF, as a moderately physical therapeutic strategy, could be considered to alleviate the degradation of cartilage and bone tissue in AS and as a potential option to halt the progression of AS.

Keywords: CD4-CKO mice; ankylosing spondylitis; cartilage tissues; inflammation; rotating magnetic field.

Figure 1

The RMF exposure apparatus. (A) The physical parameters of the treatment apparatus, which consists of two antiparallel cylindrical NdFeB (neodymium iron boron) permanent magnets (cross-section drawn). (B) The simulation of magnetic field intensity on the murine podalic exposure plane (vertical view). (C) The simulation of magnetic field intensity on murine dorsal exposure flat (vertical view).

Figure 2

RMF treatment alleviates cartilage degradation and bone fusion in age-related AS-like CD4-CKO mice. (A) Gross photographs of female mice in four groups at 6, 8, 10, and 12 months of age (n = 6). (B) Pathological score (n = 6) of mice. (C) Micro-CT radiographs display the spine’s bone structure and intervertebral discs (n = 6). (D) X-ray films of four groups of mice at 12 months old. Red arrows exhibit joint ankylosis and kyphoscoliosis in 12-month-old AS and AS + RMF mice (n = 6). (E) Femoral bone mineral density (BMD, n = 6) of cortical and subchondral bone in four groups of 12-month-old AS mice. (B,E) Data are presented as the mean ± SEM, * p < 0.05, ** p < 0.01, ns, no significance, by one-way ANOVA.

Figure 3

RMF mitigates athletic ability and age-related articular deterioration in AS mice. (A) The behavior trace images were analyzed via an open field test (n = 6). (B) Average locomotor speed of mice among four groups at 6, 8, 10, and 12 months old (n = 6). (C,D) The movement score of mice at 6, 8, 10, and 12 months old in the treadmill experiment (n = 6). (E,F) The movement score of mice at 6, 8, 10, and 12 months old in the fatigue rotating rod experiment (n = 6). (B,C,E) Figures are presented in the form of the mean ± SEM, * p < 0.05, ** p < 0.01, ns, no significance, by one-way ANOVA. (D,F) Figures are presented in the form of the mean ± SEM, ** p: Sham vs. AS, ## p: AS vs. AS + RMF.

Figure 4

RMF alleviates the articular degradation in AS mice without apparent organ toxicity. (A,B) Safranin O/Fast green and H&E staining (n = 6, scale bar: 200 μm) analysis of murine knee joints. The black arrows display the deteriorated articular cartilage tissue; the blue arrows show ectopic novel cartilage tissue formation in the articular cavity. (C–F) The OARSI score is utilized to determine the articular deterioration of the cartilage tissue. Data are presented as the mean ± SEM (n = 6). * p < 0.05, ** p < 0.01, ns, no significance, by one-way ANOVA. (G) Representative H&E staining images (n = 6, scale bar: 50 μm) of the heart, liver, spleen, lung, and kidney of sham and RMF-treated mice.

Figure 5

RMF mitigates the immune dysfunction of AS mice. (A) Serum levels of IL-5, IFN-γ, IL-17A, IL-22, IL-23, and IL-28 expression (n = 6, CBA detection). (B) Serum levels of IL-17A, IL-22, and IL-23 expression (n = 6, ELISA detection). (A,B) Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, ns, no significance, by one-way ANOVA.

Figure 6

RMF ameliorates the immune malfunction of AS mice with cartilage damage. (A) Cartilage tissues from the treated mice at 12 months were stained with an antibody specific for CD3 (n = 6, scale bar: 100 μm). White arrows label the cartilage layer on the articular surface and the epiphyseal cartilage layer. (B) Flow cytometry detection of the percentages of CD4⁺ and CD8⁺ cells in murine spleens (n = 6). (C,D) The proportion of CD4⁺ and CD8⁺ cells (n = 6). (C,D) Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, by one-way ANOVA.

Figure 7

RMF mitigates skeletal disorders by adjusting the AS mice’s bone redox system and energy metabolism. (A) Candidate signaling pathways of co-targets between AS and AS + RMF mice based on Gene Ontology (GO) enrichment analysis (n = 4). Red frames represent candidate pathways in GO enrichment analysis. (B) Candidate signaling pathways of co-targets between AS and AS + RMF mice based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis (n = 4). Red frames represent candidate pathways in KEGG enrichment analysis. (C) Heatmap of Lamb3, Col4, Thbs2, and Itga11 expression differences in cartilage tissues (n = 4). (D) Heatmap of Acaa1b, Plin1, Fabp4, Pck1, and Ucp1 expression differences in cartilage tissues (n = 4). (E) Heatmap of Nlrc3, Havcr2, Irak3, Cidea, and Axl expression differences in cartilage tissues (n = 4). (F) The expression of Lamb3, Itga11, Axl, Nlrc3, Irak3, Col4a1, Col4a2, Fabp4, Pck1, and Ucp1 was evaluated via qPCR (n = 4). Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, ns, no significance, by one-way ANOVA.

Figure 8

RMF adjusts skeletal lesions by regulating the arachidonic acid metabolism of AS mice. (A,B) The PCA analysis of serum specimens from four groups of mice (n = 4). (C) Differential metabolic classifications of the KEGG enrichment map (n = 4). (D,E) The concentrations of 5−oxoETE, 15−oxoETE, 14, 15−EET, 11, 12−EET, and 8, 9−EET in serum specimens from four groups of mice based on metabolomics analysis (n = 4). (F) Heatmap of 5−oxoETE, 15−oxoETE, 14, 15−EET, 11, 12−EET, and 8, 9−EET in murine serum specimens (n = 4). (D,E) Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, ns, no significance, by one-way ANOVA.

Figure 9

RMF moderates bone and cartilage degradation through the RANKL/RANK/OPG signaling pathways. (A,C) Immunohistochemistry (IHC) detection of OPG and RANK antibodies in bone slices among four groups of mice (n = 4, scale bar: 50 μm). (B,D) The relative OPG and RANK protein expression in bone and cartilage tissues (n = 4). Five regions of interest were randomly selected in each sample. The mean optical density of each region was measured, and the mean value of the five regions’ mean optical density was calculated as the relative positive protein expression. The baseline was the mean optical density of the sham mice, and the relative protein expression in each specimen was calculated as the ratio of the optical density of each tissue versus the baseline. (E) Western blot analysis of OPG and RANK protein expression levels in cartilage tissues of four groups of mice (n = 4). (F,G) The relative OPG and RANK protein expression in bone and cartilage tissues (n = 4). The gray values of the target proteins were first normalized with the loading control (GAPDH). After calculating the mean gray value of the sham mice as a baseline, the relative protein expression was determined by the ratio of the normalized gray value of each sample to the baseline. (B,D,F,G) Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, by one-way ANOVA.