To investigate whether endogenous hydrogen sulfide (H2S) was involved in the pathogenesis of osteoarthritis (OA) and its underlying mechanism, to detect H2S and its synthases expression in knee cartilage in patients diagnosed with different severity of OA, and to explore the transcription and expression of gene MMP-13 in chondrocytes treated with IL-1β or H2S.
Synovial fluids of the in-patients with different severity of OA hospitalized in Peking University First Hospital were collected for measurement of H2S content using methylene blue assay. Articular cartilages of the patients who underwent knee arthroplasty were collected for the cell culture of relatively normal chondrocytes. The chondrocytes were cultured to the P3 generation and H2S molecular probes were used for detection of endogenous H2S generation in the chondrocytes. Immunocytochemistry was used to detect the localization of H2S synthases including cystathionine β-synthase (CBS), cystathionine-γ-lyase (CSE), and mercaptopyruvate sulfurtransferase (MPST) in OA chondrocytes. Western blot was used to quantify the protein expressions of CSE, MPST, and CBS in cartilage tissues of the patients who were diagnosed with OA and underwent knee arthroplasty. The relatively normal human chondrocytes were cultured to passage 3 and then divided into 4 groups for different treatments: (1)the normal control group, no reagent was added; (2)the IL-1β group, 5 μg/L of IL-1β was added; (3)the IL-1β+H2S group, 200 μmol/L of NaHS was added 30 min before adding 5 μg/L of IL-1β;(4)the H2S group, 200 μmol/L of NaHS was added. The transcription and expression of gene MMP-13 in chondrocytes of each group were determined with Real-time PCR and Western blot, respectively. And the total NF-κB p65 and phosphorylated NF-κB p65 in chondrocytes were detected with Western blot.
The content of H2S in the synovial fluid of degenerative knee was (14.3±3.3) μmol/L. Expressions of endogenous H2S and its synthases including CBS, CSE and MPST were present in the cytoplasm of chondrocytes.CSE protein expression in Grade 3 (defined by outerbridge grading) cartilage tissues was significantly increased as compared with that of Grade 1 cartilage tissues (1.67±0.09 vs. 1.26±0.11, P< 0.05). However, no significant difference of CBS or MPST expression among the different groups was observed. The expression of MMP-13 protein in the IL-1βgroup was significantly higher than that in the normal chondrocytes (1.87±0.67 vs. 0.22±0.10, P<0.05), and that in the IL-1β+H2S group was significantly decreased than that in the IL-1β group (0.55±0.11 vs. 1.87±0.67, P< 0.05), and that in the H2S group had no significant difference compared with that in the normal control group. The transcription of MMP-13 protein in the IL-1β group was significantly higher than that in the normal chondrocytes (31.40±0.31 vs. 1.00±0.00, P<0.05), and that in the IL-1β+H2S group was significantly decreased than that in the IL-1β group (24.41±1.28 vs. 31.40±0.31, P<0.05), and that in the H2S group had no significant difference compared with that in the normal control group. The total NF-κB p65 in the IL-1β group was significantly higher than that in the normal chondrocytes (2.13±0.08 vs. 0.73±0.08, P< 0.05), and that in the IL-1β+H2S group was significantly decreased than that in the IL-1β group (1.24±0.13 vs. 2.13±0.08, P<0.05), and that in the H2S group had no significant difference compared with that in the normal control group. The phosphorylated NF-κB p65 in IL-1β group was significantly higher than that in the normal chondrocytes (1.30±0.13 vs. 0.19±0.04, P<0.05), and that in IL-1β+H2S group was significantly decreased than that in the IL-1β group (0.92±0.26 vs. 1.30±0.13, P<0.05), and that in the H2S group had no significant difference compared with that in the normal control group.
H2S affected the cartilage degeneration by partly inhibiting the degradation of extracellular matrix.