Far infrared irradiation suppresses experimental arthritis in rats by down-regulation of genes involved inflammatory response and autoimmunity

Abstract

Introduction: Far-infrared radiation (FIR) is widely used in the treatment of various diseases such as insomnia and cardiovascular risk. Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease in which the therapeutic potential of FIR in RA is unclear.

Objectives: To determine the therapeutic potential and mechanistic actions of FIR in treatment of RA.

Methods: Adjuvant-induced arthritis (AIA) rat models were established to assess the therapeutic potency of FIR in RA treatment. The scoring parameters such as arthritis score, swelling of the hind paw, spleen and thymus indices, micro-CT analysis indices were adopted to estimate the beneficial effects of FIR during RA treatment in AIA model. PCR gene expression arrays were used to analyze inflammatory and autoimmune genes expression profiles in rat synovium. The inflammatory and immunity genes profiling was further analyzed through transcription factor prediction using PROMO. A signaling network map of possible molecular circuits connecting the identified differential genes to the RA's pathogenesis was constructed based on extensive literature reviews, and the major signaling pathways were validated by Western blotting.

Results: Thirty minutes of FIR treatment significantly improved the symptoms of AIA in rats. Gene expression profiling indicated that 27 out of 370 genes were down-regulated by FIR. AP-1, CEBPα, CEBPβ, c-Fos, GR, HNF-3β, USF-1, and USF-2 were predicted as key transcription factors that regulated the identified differential genes. In addition, MAPK, PI3K-Akt, and NF-κB signaling are the major molecular pathways down-regulated by FIR treatment.

Conclusion: FIR may provide beneficial effects on the AIA rat model of arthritis by suppression of the MAPK, PI3K-Akt and NF-κB signaling pathways. Therefore, we believe that FIR may provide an alternative non-pharmacological and non-surgical therapeutic approach for the treatment of RA.

Keywords: Adjuvant-induced arthritis (AIA); Autoimmunity; Far infrared irradiation (FIR); Inflammatory response; Transcription factors.

Graphical abstract

Fig. 1

FIR treatment showed anti-arthritis effect on adjuvant-induced arthritis (AIA) in rats. (A) Setting of FIR spectrum emitting device. The FIR spectrum emitting device was installed on the bracket and was adjusted to 2 cm above the rat paw. After the rats were anesthetized, their limbs were raised to receive FIR treatment. (B) FIR treatment did not affect the body weight of the rats. (C, D) FIR decreased the arthritis score and hindpaw swelling of AIA rats. After arthritis induction, rats were split into 6 groups: Normal control group, AIA control group, Standard treatment group (MTX 7.6 mg/kg), 1 min, 3 min, and 30 min FIR-treated groups. Arthritic inflammatory score and hindpaw volume (mL) were measured every 3 days. The whole experiment lasted 27 days. (E) Representative images of hindpaw swelling in AIA rats after treatment. (F, G) The beneficial effect of FIR on thymus and spleen index of AIA rats. The index of spleen and thymus was expressed as the ratio of wet weight of spleen and thymus to body weight (mg/g). The data are expressed as mean ± SEM (n ≥ 5). * p < 0.05, ** p < 0.01, *** p < 0.001 V.S. the AIA control group. # p < 0.05, ## p < 0.01, ### p < 0.001 V.S. the Normal control group.

Fig. 2

FIR treatment alleviated the destruction of bone and joint tissue in AIA rats. (A) Representative micro-CT images of the hindpaw joint of AIA rats after treatment. After 27 days of treatment, the hindpaws of the rats were dissected and micro-CT analysis of bone erosion was performed (indicated by the yellow arrow). (B) The bone mineral density (BMD), bone volume fraction (BV/TV), bone cortical mineral density (TMD), trabecular number (Tb.N) and total porosity (total porosity) of the hindpaw joint of AIA rats were measured after treatment. (C) Micro-CT score of AIA rats. The micro-CT score was obtained according to five disease-related micro-CT analysis indexes: BMD, BV/TV, TMD, Tb.N and total porosity. (D) Radiological score of AIA rats. The Radiological score was obtained according to micro-CT images. The data are expressed as mean ± SEM (n ≥ 5). * p < 0.05, ** p < 0.01, *** p < 0.001 V.S. the AIA control group. # p < 0.05, ## p < 0.01, ### p < 0.001 V.S. the Normal control group.

Fig. 3

Scatter plot analysis for inflammatory and immunity genes fold regulation from Normal control, AIA control and FIR treatment groups using RT² Profiler PCR Array. (A) The synovial tissues of AIA control group and Normal control group were analyzed and compared by RT² Profiler PCR Array. (B) The synovial tissues of 30 min FIR treatment group and AIA control group were analyzed and compared by RT² Profiler PCR Array. Based on the results, regulatory scatter maps of inflammatory and immunity genes were obtained. Black spots indicate that the genes are not significantly regulated, red spots indicate the genes were up-regulated, and blue spots indicate the genes were down-regulated. The thresholds of regulated genes are ± 2 fold.

Fig. 4

FIR regulated inflammatory and immunity genes expression of articular synovium in AIA rats. RT² Profiler PCR Array was used to analyze the expression of inflammatory and immunity genes in synovium of Normal control group, AIA control group and 30 min FIR-treated group. The average values of Actb, B2m, Hprt1, Ldha and Rplp1 were selected as the reference, and the 2^−ΔΔCT method was used to analyze the gene expression. The gene expression was normalized with the AIA control group. The data are expressed as mean ± SEM (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 V.S. the AIA control group. # p < 0.05, ## p < 0.01, ### p < 0.001 V.S. the Normal control group.

Fig. 5

(A) The regulatory networks of transcription factors and the identified differential genes were visualized in Cytoscape. Blue nodes represent transcription factors, while octagonal nodes represent the identified differential genes. (B) The network map linking the identified differential genes with classical signaling pathways and the pathogenic factors of RA.

Fig. 6

FIR regulated the signaling pathways of Akt, JNK, NF-κB and STAT3 in synovial tissues of AIA rats. (A) Western blotting for phosphorylated AKT (p-AKT) and total AKT. (B) Western blotting for phosphorylated JNK (p-JNK) and total JNK. (C) Western blotting for phosphorylated NF-κB p65 (p-p65) and total p65. (D) Western blotting for phosphorylated STAT3 (p-STAT3) and total STAT3. Tissue lysates were prepared from the synovial tissues of Normal control, AIA control group and FIR treatment group. Quantification of phosphorylated form/total form and total form/β-actin or GAPDH were shown after image J analysis. The data are expressed as mean ± SEM (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 V.S. the AIA control group. # p < 0.05, ## p < 0.01, ### p < 0.001 V.S. the Normal control group. (E) Schematic diagram to show the therapeutic effects and mechanisms of action of FIR in RA treatment.