| Part I Experiments in vitro Experiment 1. Effects of IL-1β and Ginsenoside Rbl on the metabolism of type II collagen in SW1353 chondrosarcoma cellsObjective:To observe the effect of IL-1β on the protein expressions of CII and MMP-13 of SW1353. To study the effects of GRb1 at different concentrations on the expressions of type II collagen (CII), and its metabolic markers, PIINP, hydroxyproline, CTX-Ⅱ, and MMP-13 of SW1353 cells to screen the optimal concentration.Methods:Human SW1353 chondrosarcoma cells were worked as the study carrier. Except the normal control group, SW1353 chondrosarcoma cells were pre-treated with or without Ginsenoside Rb1 (GRb1) at the concentrations of 20,40,80 μM, then stimulated with IL-1β. (1) SW1353 cells were stimulated with IL-1β, of which the expressions of CII and MMP-13 were verified by Western Blot. (2) To select the optimal concentration of GRb1, SW1353 cells were pre-treated with IL-1β for 1 h, and then treated with four concentrations of GRbl (i.e.0,20,40,80 μM) for 24h, of which the expressions of CⅡ, PIINP, Hyp, CTX-II and MMP-13.Results:Compared with normal control group, the expression of CII was significantly decreased in EL-1β, and the expression of MMP-13 increased significantly. GRb1 has been shown to cause dose-dependent decreases of the expression of MMP-13 and CTX-II but increase of the expression of CII, PIINP and Hyp in IL-1β-induced SW1353 cells. Therefore, the concentration at 80 μM was used in subsequent experiments.Conclusions:The expressions of CII were enhanced, while the expressions of MMP-13 in IL-1β-stimulated SW1353 cells. GRb1 at the incremental concentrations showed a enhance power of CII, PIINP and Hyp, a supress effects of MMP-13 and CTX-CⅡExperiment 2. Effect of GRb1 on the Notch signaling pathway in IL-1β-stimulated SW1353 chondrosarcoma cellsObjective:To observe the effect of GRb1 on the key proteins of Notch signaling pathway in IL-1β-stimulated SW1353 cellsMethods:SW1353 cells being stimulated by IL-1β were treated with the optimal concentration of GRb1, and then were harvested for determining the expressions of Notchl and JAG1, the most critical receptro and ligand, by WB and q-PCR.Results:Compared with IL-1β group, the expressions of Notchl and JAG1 were down-regulated in GRbl group.Conclusions:GRbl could supress IL-1β-stimulated increasements of Notchl and JAG1.Experiment 3. Chondroprotective effects and mechanism of GRb1 on regulating MMP-13 in IL-1β-stimulated SW1353 cellsObjective:To study the chondroprotective effect mechanism of GRb1 via observing the effects of GRb1 at the optimal concentration and the specific signaling pathway inhibitor DAPT on the mRNA expression of MMP-13, Notch1, JAG1 and the protein expressions of CⅡ, MMP-13, Notch1 and JAG1.Methods:IL-1β-stimulated SW1353 cells were respectively treated with GRb1 and the Notch signaling pathway specific inhibitor, DAPT. The mRNA expressions of MMP-13, Notchl and JAG1 were determined by q-PCR, and the protein expressions of CII, MMP-13, Notch1 and JAG1 were verified by ELISA.Results:Compared with EL-1β group, the expression of CⅡ increased but the expressions of MMP-13, Notchl and JAG1 decreased in GRbl-treated group. Compared with DAPT-treated group, the expression of CⅡ increased, while that of MMP-13, Notchl and JAG1 declined.Conclusions:GRbl could supress the decline of IL-1β-stimulated decline of CⅡ and increases of Notchl, JAG1. GRb1 exhibited certain suppressive effect on the active Notch signaling pathway in osteoarthritis.Part Ⅱ Experiments in vitro Experiment 4. Chondroprotective mechanism of GRbl on catilage in a rat osteoarthritic modelObjective:To observe the effects of GRb1 on articular cartilage histomorphology, MMP-13, Notch signaling pathwy in experimental osteoarthritic cartilages and study the possible chondroprotective mechanism of GRb1 in osteoarthritis.Methods:Forty 8-week-old SD rats were obtained from the Animal Center of Chongqing Medical University. Besides ten rats were used as normal controls, the other thirty rats underwent ACLT in the right posterior knee joint to duplicate an experimental model of OA, which were ramdonly divided into three groups (i.e. the Vehicle group, GRbl group and DAPT group). After four weeks, the rats were received weekly intra-articular injections with DMSO (0.3 mL,0.05%) in normal group, GRb1(0.3 mL,80 μM) in GRb1 group, DPAT (0.3 mL,10 μM) in DAPT group. These injections were maintained for six weeks. The articular cartilage histomorphology were analyzed via Grading of H/E staining according to Mankin Scoring system. The expressions of CII, MMP-13, Notch 1 and JAG1 in cartilages of different groups were verified by Immunohistochemistry and western blot. The expressions of PIINP and CTX-II in serum of rats were analyzed by ELISA, and the expression of Hyp was determined by chemical digestion method.Results:(1) H/E staining of knee joint sections showed a smooth surface and a clear laminar structure in the articular cartilage from the normal group, and the Mankin’s score was low. In contrast, in the vehicle-treated group, the cartilage surface was rough with some superficial leakage and ulcers, of which the mankin’s score was much higher. Compared with the vehicle-treated group, GRb1-treated cartilage and cartilage treated with signaling pathway specific Inhibitor showed fewer lesions and lower mankin’s scores compared with vehicle-treated cartilage sections. (2) Compared with the normal group, the expressions of PIINP and Hyp were decreased in the vehicle group, while that of CTX-II was enhanced. Compared with the vehicle group, the expressions of PIINP and Hyp were up-regulated in GRbl group and DAPT group, while CTX-II was down-regulated. (3)In normal cartilage, the positive expression of CII was massive, while the positive expressions of MMP-13, Notch 1 and JAG1 were light. All positive expressions located at cytoplasm and extracellular matrix. Compared with the normal control group, in the vehicle-treated group, the positive expression of CII was fewer, but the expressions of MMP-13, Notchl and JAG1 were much more. Compared with the vehicle-treated group, the positive expression of CⅡ was more, while the expressions of MMP-13, Notchl and JAG1 were much fewer. (4) Cartilages in all groups were verified by WB. Compared with the normal group, the expressions of MMP-13, Notchl and JAG1 were enhanced in the GRb1 and the expression of CII decreased. Compared with the vehicle-treated group, the former three targets were down-regulated. Compare with the pathway inhibitor, the expressions of CII enhanced and the expressions of MMP-13, Notchl and JAG1 were declined in GRbl group.Conclusions:GRbl could protect cartilage from degeneration via decreasing the Mankin’s scores and damages of the articular cartilage in a rat osteoarthritis model. Moreover, Icariin exhibited certain improvement of decreased CII and supressions of increased MMP-13, Notch1 and JAG1. |
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