Alterations of autophagy in knee cartilage by treatment with treadmill exercise in a rat osteoarthritis model

Abstract

The aim of the present study was to investigate potential alterations in the articular cartilage in a rat model of monosodium iodoacetate (MIA)‑induced osteoarthritis (OA) with or without treatment with moderate treadmill exercise. A total of 30 male Sprague‑Dawley rats were randomly divided into three groups (n=10), including the control, OA and OA with treadmill exercise (OAE) groups. Rats were evaluated upon completing the treadmill exercise program (speed, 18 m/min; 30 min/day; 5 days/week for 4 weeks). Interleukin (IL)‑1β and IL‑4 levels in the serum and intra‑articular lavage fluid (IALF) were measured by ELISA. Alterations in articular cartilage and synovium were also evaluated by histology, immunohistochemistry, western blotting and reverse transcription‑quantitative polymerase chain reaction. The results revealed that IL‑1β in the serum and IALF decreased in the OAE group, whereas IL‑4 increased, and histological evaluation indicated that the OAE group had a clear treatment response. However, the expression of type II collagen in the articular cartilage increased in the OAE group as compared with the OA group, whereas ADAMTS5 expression decreased. In contrast to light chain 3B (LC3B), the protein expression levels of BECLIN1 and sequestosome 1 (SQSTM1) were increased in the OA group. In addition, a significant increase was observed between OA and OAE groups in LC3B and SQSTM1 protein levels, whereas no change was observed in BECLIN1 levels between the OA and OAE groups in the superficial and deep zones. The results of western blotting demonstrated that LC3II was notably decreased in the OA group and partially increased in the OAE group. The mRNA expression levels of LC3B and SQSTM1 increased in the OA and OAE groups, with a significant difference observed between the two groups, while a concomitant decrease was detected in BECLIN1 levels. In conclusion, 30 min of treadmill exercise had an evident protective effect in the articular cartilage of rats with MIA‑induced OA and may promote autophagy in the articular cartilage.

Figure 1

(A) Treatment schedule for the rats. (B) Body weight during the treadmill exercise. All rats received a standard diet, and body weights were measured weekly. No significant difference was detected between the control and OA group during exercise. Differences between control and OAE, OA and OAE groups in weeks 4 and 5 were significant. ^*P<0.05 and **P<0.01 vs. control group; ⁺P<0.05 and ⁺⁺⁺P<0.001 vs. OAE group. Results are expressed as the mean and 95% confidence interval (one-way analysis of variance; n=10 rats/group). MIA, monosodium iodoacetate; OA, osteoarthritis; OAE, OA with treadmill exercise.

Figure 2

Histological staining and evaluation of the tibiofemoral joint. (A) H&E and toluidine blue staining of sections in the sagittal plane. Images were captured at ×40 magnification, and local magnification of a selected square was used to observe the details for each group (×200 for H&E and ×400 for toluidine blue). (B) Mankin score and (C) OARSI score for the tibiofemoral joint. Differences between the control vs. OA, control vs. OAE, and OA vs. OAE groups were statistically significant. ^***P<0.001, ⁺⁺P<0.01 and ⁺⁺⁺P<0.001 vs. OA group. Results are expressed as the mean and 95% confidence interval (one-way analysis of variance; n=10 rats/group). OA, osteoarthritis; OAE, OA with treadmill exercise; H&E, hematoxylin and eosin.

Figure 3

Type II collagen and ADAMTS5 alterations among the groups. (A) Immunohistochemical staining images and (B) quantified levels of type II collagen. Images were captured at ×40 magnification, and local magnification of a selected square at ×200 was applied. The difference between the control and OA groups was significant (n=5). (C) Western blot analyses and (D) quantified results of the protein levels of type II collagen and ADAMTS5, with β-actin serving as the internal reference. For type II collagen and ADAMTS5, differences between the control vs. OA, control vs. OAE, and OA vs. OAE groups were significant (n=3). Results are expressed as the mean and 95% confidence interval (one-way analysis of variance). ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. control group; ⁺P<0.05 and ⁺⁺P<0.01. OA, osteoarthritis; OAE, OA with treadmill exercise; ADAMTS5, a disintegrin and metalloproteinase with thrombospondin motifs 5.

Figure 4

(A) H&E staining for histological evaluation in synovium, and (B) synovitis score. Images were captured at ×40 magnification, and local magnification of a selected square at ×200 was applied. Differences in synovitis score in the control vs. OA, control vs. OAE and OA vs. OAE groups were statistically significant (n=5). (C) ELISA of IL-1β and IL-4 levels in the serum and IALF. Treadmill exercise reduced the increase in IL-1β and reversed the decrease in IL-4 caused by monosodium iodoacetate injection, in the serum and IALF (n=10). Results are expressed as the mean and 95% confidence interval (one-way analysis of variance). ⁺P<0.05 vs. OA group; ^***P<0.001 vs. control group. OA, osteoarthritis; OAE, OA with treadmill exercise; H&E, hematoxylin and eosin; IALF, intra-articular lavage fluid; IL, interleukin.

Figure 5

Immunohistochemical staining for LC3B, SQSTM1 and BECLIN1. (A) LC3B exhibited a decrease in the SZ and DZ of the cartilage in both the OA and OAE groups, while treadmill exercise increased LC3B expression in OA rats. (B) SQSTM1 expression was increased in the OA and OAE groups, while treadmill exercise evidently increased SQSTM1 level in the SZ and DZ of the cartilage. (C) BECLIN1 expression was increased in the OA and OAE groups in both the SZ and DZ of the cartilage, with no significant difference between the OA and OAE groups. (D) Results are expressed as the mean and 95% confidence interval (one-way analysis of variance; n=5). ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. control group; ⁺P<0.05,⁺⁺P<0.01 and ⁺⁺⁺P<0.001. OA, osteoarthritis; OAE, OA with treadmill exercise; LC3B, light chain 3B; SQSTM1, sequestosome 1; SZ, superficial zone; DZ, deep zone.

Figure 6

Western blot analysis of LC3B protein level, and reverse transcription-quantitative polymerase chain reaction of LC3B, SQSTM1 and BECLIN1 mRNA expression levels. (A) Western blot analyses and (B) quantified levels of LC3B protein, illustrating a marked decrease in the OA and OAE group compared to the control (n=3). No significant difference was detected between the OA and OAE groups. (C) Relative mRNA expression levels of LC3B, BECLIN1 and SQSTM1 (n=4). There was no significant difference between the OA and OAE groups. Results are expressed as the mean and 95% confidence interval (one-way analysis of variance). ^*P<0.05 and ^***P<0.001 vs. control group. OA, osteoarthritis; OAE, OA with treadmill exercise; LC3B, light chain 3B; SQSTM1, sequestosome 1.