Study On The Role Of Hydrogen Sulfide In The Pathogenesis Of Knee Osteoarthritis

Part Ⅰ The clinical implication of dynamic changes of hydrogen sulfide in synovial fluid of knee osteoarthritisObjective: to determine the levels of hydrogen sulfide(H2S) in synovial fluid of knee which aspirates from osteoarthritis(OA) and healthy volunteers and the distribution of H2S-producing enzymes in knee and analyse the clinical implication.Methods: we collected the synovial fluids of knee which aspirate from 770 cases of osteoarthritis in the affiliated hospital of Qingdao university and 60 cases of healthy volunteers. We analyzed the levels of H2 S in synovial fluid of knee by zinc trapspectrophotometry; using immunohistochemical to detect the distribution of H2S-producing enzymes in articular cartilage and synovium; Analysis the association of the expression of H2 S in synovial fluid of OA and the radiographic stage of OA.Results: H2 S is present in synovial fluid. The average of H2 S in the synovial fluids of healthy volunteers is 45.72±5.33μM, the average of H2 S in the synovial fluids of OA is 29.02±5.54μM, the difference was statistically significant(p <0.001). The level of H2 S in the synovial fluids of OA is lower when the symptoms of OA is severer. The Spearman correlation analysis showed that the level of H2 S in the synovial fluids ofOA is negative correlated with the radiographic stage of OA(r=-0.795,p<0.001). Cystathionine β synthase(CBS), cystathionine γ-lyase(CSE) were expressed in the knee joint. CBS is more expression in articular chondrocytes than synovial tissue, CSE mainly expressed in the synovial tissue.Conclusion: H2 S is present in synovial fluid. In healthy volunteers, the average synovial fluid H2 S levels were significantly higher than in synovial fluid from OA patients. The level of H2 S in the synovial fluids of OA is negative correlated with the radiographic stage of OA. CBS mainly expressed in the articular cartilage, while CSE is mainly expressed in the synovial tissue.Part Experimental study on hydrogen sulfide against articular cartilage influence of rabbit knee osteoarthritis modelObjective: To investigate the effect of hydrogen sulfide on degeneration of articular cartilage in rabbit knee osteoarthritis model and provide a theoretical basis for the prevention of osteoarthritisMethods: The 32 New Zealand rabbits were randomly divided into four groups, A group: control group, B group: model group, C group: H2 S treatment group, D group: suppression of H2 S groups, each group had eight rabbits. A group received no treatment; B, C, D group was made to Rabbit Knee OA model by a modified Hulth method. Postoperatively group B received knee joint cavity injection of saline, once a week for six weeks. Postoperatively group C received knee joint cavity injection of 1ml 60μM of Na HS, once a week for six weeks. Postoperatively group D received knee joint cavity injection of CBS inhibitors, once a week for six weeks. The animals were sacrificed after 7 weeks. Before the animals were sacrificed, knee synovial fluids were extracted, the Griess and ELISA were used to detect the level of NO and PGE2 in synovial fluid. After the animals were sacrificed and cut articular cartilage, the cartilage were HE stained,Observed and scored the optical microscope sections by Mankin’s scoring system. Ⅱtype collagen in articular cartilage was detected by immunohistochemical methods. We detected the expression of i NOS, COX-2, MMP-13 m RNA in articular cartilage using Real-time PCR method.Results: Light microscope observation, the articular cartilage of group A was normal; Collagen fibers of articular cartilage matrix in group B was disorganized and poorly and severely degraded; the degeneration of group C was reduced; the degeneration of group D was increased. Mankin’s score showed: Compared with group A, B、C and D group scores were increased, the difference was statistically significant(P <0.05). The Mankin’s score of OA can be reduced after H2 S treatment(group C); however, when the H2 S was suppressed the Mankin’s score increased. the difference was statistically significant(P <0.05). The expression of NO and PGE2 were increased in synovial fluid of Osteoarthritis group(group B); The expression of NO and PGE2 were reduced after H2 S treatment(group C); The expression of NO and PGE2 were increased after H2 S was suppressed(group D); the difference was statistically significant(P <0.05). Type Ⅱ collagen staining: Compared with normal group(group A), the positive staining of Type Ⅱ collagen significantly reduced and the distribution of Type Ⅱ collagen wasuneven in osteoarthritis group(group B). H2 S after treatment(group C) collagen Ⅱ positive staining increased; suppression of H2S(group D) significantly reduced typeⅡ collagen positive staining and the superficial layers of the defect occurred; the difference was statistically significant(P <0.05). After H2 S treatment compared with the control group, the m RNA expression of i NOS, COX-2, MMP-13 in cartilage is reduced; when the H2 S was inhibited, it was increased, the difference was statistically significance(P <0.05).Conclusion: 1.H2 S can inhibit the expression of NO and PGE2 in synovial fluid of rabbit knee osteoarthritis. 2. H2 S can suppress degradation of collagenⅡtype in cartilage of rabbit knee osteoarthritis. 3. H2 S can suppress the m RNA expression of i NOS, COX-2, MMP-13 in cartilage of rabbit knee osteoarthritis.Part Ⅲ Hydrogen sulfide inhibits IL-1β-induced chondrocyte damage by NF-κB pathwayObjective: To investigate the protective role of hydrogen sulfide on IL-1β-induced chondrocyte damageMethods: We used an in vitro model of cartilage inflammation to investigate the therapeutic potential of H2 S in OA. Human OA chondrocytes were cultured and pretreated with H2S(0.06–1.5m M) with or without IL-1β(10 ng/ml). NO production was quantified by Griess reagent. PGE2 and MMP-13 productions were quantified by enzymelinked immunosorbent assay. The gene expressions of i NOS, COX-2 and MMP-13 were determined by quantitative real time PCR. Mitogenativated protein kinases(MAPKs) and nuclear factorκB(NF-κB) related signaling molecules were determined by Western blotting.Results: Our data showed that IL-1β markedly stimulated the gen expressions of i NOS,COX-2 and MMP-13 and the productions of NO, PGE2, MMP-13, which could be significantly reversed by H2 S. H2 S could also suppress the IL-1β-triggered activation of ERK/ iκBα/NF-κB signaling pathway.Conclusion: The present study suggests that H2 S could suppress the IL-1β-triggered activation of ERK-iκBα-NF-κB signaling pathway and maybe a potential therapeutic choice in the treatment of OA patients.