Fig 1.
Green-lipped mussel extract reduced pain in MIA-injected rats.
(A) Schedule of rat experimental procedure is shown by a diagram. (B) Pain was analyzed as the difference between the PWL (left) and PWT (right) in vehicle-treated MIA-induced OA rats, and those given GLME orally. Rats with MIA-induced OA (n = 6 per group) were evaluated to day 21. Nociceptive testing was performed using a dynamic plantar esthesiometer; this is an automated version of the von Frey hair assessment tool. (C) Weight-bearing was examined (n = 6 per group) today 28. Data are presented as the means ± SDs of those of three independent experiments. Significant differences were evident between the vehicle- and GLME-treated groups. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the vehicle-treated group.
Fig 2.
Histological evaluation of joints after oral administration of GLME to rats with MIA-induced OA.
Rats were injected with 3 mg of monosodium iodoacetate (MIA) (into the right knee). GLME was given orally every day from day 3 after MIA injection. The joints were resected on day 21 after MIA injection. (A) Knee joints from the OA rats. Joint samples were acquired from the WT, vehicle, GLM (100 mg/kg, 300 mg/kg) and Celecoxib (50 mg/kg) groups and stained with hematoxylin and eosin (original magnification x 200). (B) The OA lesions were graded on a scale of 0–13 using the modified Mankin scoring system that evaluates structure, cellular abnormalities, and matrix staining. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the MIA-injection group. (C) CD4 and CD19 expression levels in the synovia of OA rats as revealed immunohistochemically 21 days after MIA injection. Immunohistochemistry was used to evaluate representative sections of joints from MIA-injected rats given GLM, celecoxib, or the vehicle. Positive cells stain brown; the nuclei were counterstained with hematoxylin. The bar graphs represent the means ± SDs of the numbers of stained cells. *P < 0.05, **P < 0.01 compared to the MIA-injection group.
Fig 3.
Effects of GLME on the expression levels of IL-1β, IL-6, iNOS, and NF-kB in the synovia of OA joints.
(A) Immunohistochemical staining for IL-1β, IL-6, iNOS, and NF-kB after oral administration of GLME daily for 21 days after MIA injection. (B) The positive cells stain brown; the nuclei were counterstained with hematoxylin. Slides of representative sections showing expression of IL-1β, IL-6, iNOS, and NF-kB. *P < 0.05, **P < 0.01 compared to the MIA-injection group.
Fig 4.
Reductions in the levels of catabolic markers and inflammatory cytokines in OA rats and human chondrocytes.
(A, B) Immunohistochemical staining was used to identify MMP1 and MMP3. (C) qRT-PCR analysis of the levels of mRNAs encoding MMP3, MMP13 and MCP-1 in IL-1β (20 ng/mL)-stimulated OA chondrocytes in the presence or absence of (various concentrations of) GLME (10, 100, 250 μg/mL) and Celecoxib (10 μM). (D) IL-6 and MCP-1 levels in cell supernatants were measured via sandwich ELISAs. (E) OA chondrocyte cell viability in the presence or absence of GLME, as revealed by the CCK8 assay. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the vehicle-treated group.
Fig 5.
Reduced levels of necroptosis-related markers in OA rats and human chondrocytes.
(A, B) Representative immunohistochemical staining of RIP1, RIP3, and pMLKL in the synovia of non-OA rats (WT), vehicle-treated MIA-induced OA rats, and GLME-treated MIA-induced OA rats. (C) The levels of mRNAs encoding necroptotic marker genes (as revealed by real-time PCR) in human OA chondrocytes treated with IL-1β (20 ng/mL) and then co-cultured with GLME or celecoxib. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the vehicle-treated group.