Effect And Mechanism Of Glycyrrhiza Polysaccharides On Glycosaminoglycan Synthesis Of Rat Osteoarthritic Chondrocytes

Part1:The extraction and quality analysis of Glycyrrhiza polysaccharidesObjective:To extact the polysaccharides form crude glycyrrhiza, and take quality analysis for the Glycyrrhiza polysaccharides.Methods:Crude glycyrrhiza was crumbled into powder, centrifugation method was used to remove the insoluble material, Sevag method was used to remove free protein, pigment in glycyrrhiza polysaccharides was adsorpted by activated carbon. And then detected residual protein content of the glycyrrhiza polysaccharides that has been extracted, calculated the conversion factor, recovery rate, and the content of glycyrrhiza polysaccharides in the crude glycyrrhiza, analysis the quality of glycyrrhiza polysaccharides that has been extracted.Results:The conversion factor was1.584, recovery rate was98.59%, the content of glycyrrhiza polysaccharides in the crude glycyrrhiza was2.33%.Conclusion:The extracted glycyrrhiza polysaccharides was hoar solid. The protein in glycyrrhiza was almost removed by Sevag method. Part2:The effects and mechanism of glycyrrhiza polysaccharides on GAG synthesis and expression of GIcAT-1mRNA in rat chondrocytes in vitroObjective:To investigate the effect and mechanism of different concentration of glycyrrhiza polysaccharides on GAG synthesis and expression of GlcAT-1mRNA in rat osteoarthritic chondrocytes in vitro.Methods:Chondrocytes isolated from knee and shoulder joints cartilage of rats were cultured. After monolayers were established, chondrocytes induced by IL-1β were treated with glycyrrhiza polysaccharides of0.1、1、10、100mg/L respectively.4.5mg/L Glucosamine was used as the positive control. The effects on chondrocytes were analyzed using MTT assay for chondrocyte proliferation and1,9-dimethylmethylene blue assay with purified shark chondroitin sulphate (CS) as a standard for GAG content, mRNA expression of GlcAT-1by rat chondrocytes was analyzed using a quantitative multistandard RT-PCR.Results:Glycyrrhiza polysaccharides promoted normal chondrocyte proliferation in all observed concentrations. Glycyrrhiza polysaccharides up-regulated GAG synthesis in all observed concentrations, with the concentration of0.1mg/L was the most effective (P<0.01). All concentrations of glycyrrhiza polysaccharides didn’t enhance the expression of GlcAT-1, but we observed all concentrations of glycyrrhiza polysaccharides groups had a significantly different compared with model control group.Conclusion:The effects and mechanism of glycyrrhiza polysaccharides that promoted normal chondrocyte proliferation, up-regulated GAG synthesis of rat chondrocytes induced by IL-1β in vitro was probably because of the increase of substrate concentration. And also the enhancing of the expression of GlcAT-1probably improve the efficiency of the GAG synthesis. Part3:The protective effect and mechanism of glycyrrhiza polysaccharides in rat osteoarthritic articular cartilage in vivoObjective:To investigate The protective effect and mechanism of glycyrrhiza polysaccharides in rat osteoarthritic articular cartilage in vivo.Methods:70Wistar rats were randomly divided into A、B、C1、C2、C3、C4、D groups. Group A was normal control group. Group D was4.5mg/L Glucosamine used as the positive control. OA models of rats were established by0.2ml intra-articular injections of4%papain solution into both knees in group B、C1、C2、C3、C4、D at the1st,4th and7th day. The knees of rats in group C1、C2、C3、C4respectively received 0.2ml intra-articular injections of glycyrrhiza polysaccharides at concentrations of0.1、1、10、100mg/L at the10th day, group D was given4.5mg/L Glucosamine, group A and B were given physiological saline at the same time, twice weekly for five weeks. Six weeks later, all rats were sacrificed. Observed the morphological changes of articular cartilage both in naked eye and under microscopy, graded according to the general observed score standard. Made articular cartilage into paraffins sections, and deal with toluidine blue staining and HE staining, graded according to the Mankin’s score standard. And detected the content of MDA in serum and synovial, the activity of SOD in serum, and the content of GAG in articular cartilage.Results:The results of both general observed score and Mankin’s score shown that the morphological of articular cartilage in glycyrrhiza polysaccharides groups were obviously improved, particularly in l0mg/L group which has no diffrernce compare with group D. The activity of SOD in serum in all glycyrrhiza polysaccharides groups and group D were obviously increased compare with the model group. The content of MDA in all glycyrrhiza polysaccharides groups and group D were obviously reduced compare with the model group. But the effects in C1、C2still has difference compare with group D(p<0.01). The content of GAG in all glycyrrhiza polysaccharides groups and group D were obviously increased compare with the model group, and the effects in C3、C4has no difference compare with group D.Conclusion:Intra-articular injection of papain in rats to establish OA model was simple and quick, repeatability and stability,with less related complications, which could lead to pathological changes similar to clinical OA. The glycyrrhiza polysaccharides had a protective effect, and10mg/L group shown the strongest protective effect on articular cartilage. The mechanism of protective effect on OA cartilage was probably because of the glycyrrhiza polysaccharides could promote the GAG synthesis, and then promote the generation of cartilage matrix by inhibiting oxygen free radical damage to the cartilage cells.