The effects of different frequency treadmill exercise on lipoxin A4 and articular cartilage degeneration in an experimental model of monosodium iodoacetate-induced osteoarthritis in rats

Abstract

The aim was to investigate the effects of different frequencies treadmill exercise with total exercise time being constancy on articular cartilage, lipoxin A4 (LXA4) and the NF-κB pathway in rat model of monosodium iodoacetate-induced osteoarthritis (OA). Fifty male Sprague-Dawley rats were randomly divided into five groups (n = 10): controls (CG), knee OA model (OAG), OA + treadmill exercise once daily (OAE1), OA + treadmill exercise twice daily, rest interval between exercise>4h (OAE2) and OA + treadmill exercise three times daily, rest interval between exercise>4h (OAE3). Rats were evaluated after completing the treadmill exercise program (speed, 18 m/min; total exercise time 60 min/day; 5 days/week for 8 weeks). Interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and LXA4 in serum and intra-articular lavage fluid were measured by ELISA. Changes in articular cartilage were evaluated by histology, immunohistochemistry, western blotting and quantitative real-time-PCR. LXA4 in the serum and intra-articular lavage fluid increased in all OAE groups, and histological evaluation indicated that the OAE3 group had the best treatment response. The expression of COL2A1 and IκB-β in articular cartilage increased in all OAE groups vs the OAG group, whereas expression of IL-1β, TNF-α, matrix metalloproteinase (MMP)-13, and NF-κB p65 was reduced in all OAE groups compared with the OAG. Under the condition of 60 min treadmill exercise with moderate-intensity, to fulfill in three times would have better chondroprotective effects than to fulfill in two or one time on monosodium iodoacetate-induced OA in rats. And it may be worked through the anti-inflammatory activity of LXA4 and the NF-κB pathway.

Fig 1. The design of the treatment schedule and intervals of various parameters.

Fig 2. Histological evaluation of tibiofemoral joints.

(A) Histological features of representative tibiofemoral joints sectioned in the sagittal plane and stained with HE and toluidine blue. Mankin and OARSI histological scores are shown for each image. F: femur, T: tibia. (B) Mankin score for tibiofemoral joints. Differences between CG and OAG (*P <0.001), OAG vs. OAE2 and OAE3 groups (⁺P <0.001) were significant, and between OAE2 and OAE3 (^#aP = 0.006) were significant. (C) OARSI histological scores for cartilage of tibiofemoral joints. Differences between CG and OAG (*P <0.001), OAG vs. OAE2 and OAE3 (⁺P <0.001) were significant, and between OAE2 and OAE3 (^#bP = 0.026) were significant. Kruskal-Wallis test, n = 10 rats in each group, means with 95% confidence interval.

Fig 3. ELISA results in each group.

Each treadmill frequency significantly alleviated the serum and articular inflammatory responses induced by MIA, including LXA₄ (A,D), IL-1β (B,E), and TNF-α (C,F). Differences between CG and OAG were significant (*P <0.001), and differences between the OAG group and different frequency treadmill groups were significant (⁺P<0.001, ^+aP = 0.010, ^+bP = 0.002, ^+cP = 0.003, ^+dP = 0.001, ^+eP = 0.002, ^+fP = 0.001, ^+gP = 0.007, ^+hP = 0.024, ⁺ⁱP = 0.027, ^+jP = 0.013). One-way ANOVA, n = 10 rats for each group, means with 95% confidence interval.

Fig 4. Immunohistochemical staining in each group.

Increase in the frequency with same amount of treadmill exercise decreased the MIA-induced degradation of COL2A1 in articular cartilage and reduced the expression of MMP-13 and NF-κB p65. The micrographs showed the intensity of immunohistochemical staining of COL2A1, NF-κB p65, MMP-13, and TIMP-1 in the articular cartilage of each experimental group, and the figures showed the percentages of positively stained cells. Differences between CG and OAG were significant (*P <0.001, *^aP = 0.001), and differences between OAG and the treadmill exercise groups were significant (⁺P<0.001, ^+aP = 0.012). One-way ANOVA, n = 5 rats for each group, mean score with 95% confidence interval.

Fig 5. Western blotting results in each group.

Effect of different frequency with same amount of treadmill exercise on the expression of MMP-13, TIMP-1, NF-κB p65, IκB-β, and COL2A1 expression in knee joint cartilage of rats with MIA-induced OA. Protein expression was determined in western blots of total protein extracted from cartilage tissues as described in Materials and Methods. The data was obtained in three separate experiments β-actin as an internal standard. Differences between CG and the OAG group were significant (*P < 0.001), and differences between the OAG and the treadmill frequency groups were significant (⁺P<0.001, ^+aP = 0.030, ^+bP = 0.001, ^+cP = 0.039, ^+dP = 0.010, ^+eP = 0.038, ^+fP = 0.002,). One-way ANOVA, n = 3 rats for each group, means with 95% confidence interval.

Fig 6. qPCR results in each group.

Effect of different frequencies treadmill exercise on the expression MMP-13 and TIMP-1 mRNA in knee joint cartilage from rats with MIA-induced OA. The gene expression of MMP-13 and TIMP-1 was determined by qPCR as described in Materials and Methods. The data were representative of three separate experiments and β-actin was used as an internal standard. Differences between CG and OAG were significant (*P<0.001) and differences between OAG and the different treadmill frequency groups were significant (⁺P<0.001). One-way ANOVA, n = 9 rats in each group, means with 95% confidence interval.

Fig 7. The LXA₄ and NF-κB pathways play a key role in chondroprotective effect of treadmill exercise.

(A) Moderate-intensity exercise promotes platelet/ neutrophils aggregation in capillaries and adipokine metabolism in the infrapatellar fat pad, which upregulate LXA₄ level in the knee articular cavity. LXA₄ interaction with macrophages, neutrophils, and synovial fibroblasts lead to the downregulation of IL-1β, TNF-α and MMP-13. (B) Chondrocytes, as mechanosensitive cells, can transform mechanical signals. Thus, exercise decreases the levels of IL-1β and TNF-α via action of LXA₄, and mechanical signals transduced by chondrocytes inhibit the expression of MMP-13 and TIMP-1 mRNA to protect cartilage via the NF-κB pathway.