Protective effects of total fraction of avocado/soybean unsaponifiables on the structural changes in experimental dog osteoarthritis: inhibition of nitric oxide synthase and matrix metalloproteinase-13

Abstract

Introduction: The aims of this study were, first, to investigate the in vivo effects of treatment with avocado/soybean unsaponifiables on the development of osteoarthritic structural changes in the anterior cruciate ligament dog model and, second, to explore their mode of action.

Methods: Osteoarthritis was induced by anterior cruciate ligament transection of the right knee in crossbred dogs. There were two treatment groups (n = 8 dogs/group), in which the animals received either placebo or avocado/soybean unsaponifiables (10 mg/kg per day), which were given orally for the entire duration of the study (8 weeks). We conducted macroscopic and histomorphological analyses of cartilage and subchondral bone of the femoral condyles and/or tibial plateaus. We also conducted immunohistochemical analyses in cartilage for the following antigens: inducible nitric oxide synthase, matrix metalloproteinase (MMP)-1, MMP-13, a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS)4 and ADAMTS5.

Results: The size of macroscopic lesions on the tibial plateaus was decreased (P = 0.04) in dogs treated with the avocado/soybean unsaponifiables. Histologically, in these animals the severity of cartilage lesions on both tibial plateaus and femoral condyles, and the cellular infiltration in synovium were significantly decreased (P = 0.0002 and P = 0.04, respectively). Treatment with avocado/soybean unsaponifiables also reduced loss of subchondral bone volume (P < 0.05) and calcified cartilage thickness (P = 0.01) compared with placebo. Immunohistochemical analysis of cartilage revealed that avocado/soybean unsaponifiables significantly reduced the level of inducible nitric oxide synthase (P < 0.05) and MMP-13 (P = 0.01) in cartilage.

Conclusions: This study demonstrates that treatment with avocado/soybean unsaponifiables can reduce the development of early osteoarthritic cartilage and subchondral bone lesions in the anterior cruciate ligament dog model of osteoarthritis. This effect appears to be mediated through the inhibition of inducible nitric oxide synthase and MMP-13, which are key mediators of the structural changes that take place in osteoarthritis.

Figure 1

Macroscopic appearance of osteoarthritic articular cartilage from femoral condyles and tibial plateaus. Representative pictures of placebo-treated and ASU-treated dogs at 8 weeks after surgery, showing erosion and pitting (circled). A, anterior; L, lateral; M, medial; P, posterior.

Figure 2

Histology of osteoarthritic articular cartilage from the femoral condyles and tibial plateaus. Representative sections of placebo-treated and ASU-treated dogs 8 weeks post-surgery (original magnification ×100). Sections were stained with Safranin-O. ASU, avocado/soybean unsaponifiables.

Figure 3

Subchondral bone and calcified cartilage. Representative sections and data of placebo-treated and ASU-treated dogs 8 weeks post-surgery (original magnification ×100). Sections were stained with haematoxylin/eosin. Data for bone volume and calcified cartilage thickness are represented as box plot and were analyzed using Student's unpaired t-test. P ≤ 0.05 is considered significant. ASU, avocado/soybean unsaponifiables.

Figure 4

iNOS immunostaining. Representative sections and data of the superficial zone of articular cartilage from placebo-treated and ASU-treated dogs (original magnification ×100). Morphometric analysis of iNOS immunostaining data are represented as box plot and were analyzed using Mann-Whitney two-tailed U-test. P ≤ 0.05 is considered statistically significant. ASU, avocado/soybean unsaponifiables; iNOS, inducible nitric oxide synthase.

Figure 5

MMP-13 immunostaining. Representative sections and data of the deep zone of articular cartilage from placebo-treated and ASU-treated dogs (original magnification ×250). Morphometric analysis of MMP-13 immunostaining data are represented as box plot and were analyzed using Mann-Whitney two-tailed U-test. P ≤ 0.05 is considered statistically significant. ASU, avocado/soybean unsaponifiables; MMP, matrix metalloproteinase.