| The Osteoarthritis(OA)morbidity is rising as the aging of population in the world.OA causes great pain and even disability,and affects people' work ability seriously.Articular cartilage is injured first when OA happens.It becomes thinner, denatured and destroyed,and joint space becomes narrower.Non-steroidal anti-inflammatory drugs and chondroprotective agents are now used in the therapy of OA.Chondroprotective agents are often polysaccharide,such as polysulfated aminoglycans,polysulfated Fondaparinux sodium and polysulfated glycosaminoglycans.Their effects are to maintain the concentration of proteoglycans in cartilage and stimulate the synthesis of typeⅡcollagen by chondrocyte,thus maintaining the integrity of articular cartilage.Chondroitin sulfate(CS),the main component of articular cartilage,is a glycosaminoglycan with a disaccharide unit of→4)-β-D-GlcUA-(1→3)-β-D-GalNAc-(1→. The content of CS decreases when OA happens and articular cartilage injured.Administration of CS can down-regulate matrix metalloproteinases-9,ease symptoms of OA,and may also decrease incidence of the disease.CS compound recipe has been used clinically for therapy of OA.Now CS is often extracted from animals,the main components of which are 4-sulfate CS(CSA)and 6-sulfate CS(CSC).The main component in human articular cartilage is 4,6-disulfate CS(CSE). In vitro experiments show that the content of 4,6-disulfate CS decreases in cultured articular cartilage when simulating OA,while the addition of 4,6-disulfate CS can stimulate the synthesis of cartilage,suggesting that sulfate group may be import for the treatment of OA.In order to develop chondroprotective agent,PSCS containing 4,6-disulfate structure were prepared by the sulfation of 4-sulfate CS with chlorosulfonic acid-formamide methods.As PSCS is a macromolecule with large amounts of anions, it is difficult to be absorpted after oral administration.So it is necessary to prepare suitable dosages in which the PSCS with easy absorption after oral taken.Therefore, we investigated the PSCS preparation,structural determination,and chondroprotective effect on the OA of rabbit knee.1.Preparation,purification and the properties of PSCS.We prepared the polysulfated CS derivatives with chlorosulfonic acid-formamide methods,and test the results by the determination of the content of SO42-and SO42-/COO- ratio.SO42-was increased from 12.76%to 20.62%;SO42-/COO- ratio was from 1.35 to 3.35.The yield of the reaction was 78.9%.The capillary electrophoresis and HPLC results of PSCS showed a symmetrical sharp peak, suggesting the high purity of PSCS.The physico-chemical properties were determined such as the content of glycuronic acid,aminohexose,molecular weight and specific optical rotation.And its anticoagulant activity is 40usp/mg,suggesting that PSCS has anticoagulant activities.2 Structure of PSCSThe structure of PSCS was analysized by the spectra of UV,IR,1H-NMR and 13C-NMR.UV shows that there is no conjugated double band in its structure,and no protein or nucleic acid in the sample.By comparising its IR with that of CS,it is shown that PSCS has the same major structure as CS,that is the disaccharide unit of→4)-β-D-GlcUA-(1→3)-β-D-GalNAc-(1→,and the absorption at 818cm-1suggests that 6-hydroxy of galactosamine is substituted by sulfate radical.1H-NMR and 13C-NMR spectropy shows that C-2 and C-3 of glucuronic acid are sulfated.The major disaccharide unit of PSCS could be→4)-β-D-GlcUA(2S,3S)-(1→3)-β-D-GalNAc(4S, 6S)-(1→.3.Preparation of oral dosage formThe conditions and method of preparaing PSCS-chitosan nanoparticle were screened.The molecular weight of chitosan is 5kDa,the concentration is 2mg/ml,and pH at 6.0.A quarter of the volume of 1mg/ml PSCS was titrated at a rate of 2s/drop, with the stirring rate of 600 r/min.The diameters and the charges of the nanoparticles are 200 nm and+46mV respectively,with good homogeneity,spherical degree,and good stability.4.The chondroprotective effect of PSCS on rabbit Knee OsteoarthritisThe animal knee OA model was made by intraarticular injection of typeⅡcollagenase to rabbits.PSCS-chitonan nanoparticules were administered by gastric perfusion daily for 4 weeks.The ethology,iconography and pathology studies showed that PSCS-chitosan nanoparticules therapeutic effects for rabbit knee OA.Ethology observation showed that the administration of PSCS can ease the pain of knee OA.X-ray photography showed that the rough articular facet,narrower joint space and doubtful splints exist in the articulations of model group,while the articulations in the PSCS group have smooth articular facet,less narrower joint space and no splints.The pathological sections were assessed with Markin method.The results showed scores of model group,PSCS low dosage group and the PSCS high dosage group are 3.32±2.02, 3.98±1.88,5.76±3.23,respectively.Statistics difference exist between the model group and PSCS low dosage group,suggesting that PSCS has chondroprotective effect even at a low dosage.The concentrations of TNF-αand IL-1βin the serum of the rabbits were detected. The concentration of TNF-αin PSCS low dosage group and model group are 18.76±1.23pg/ml and 8.98±1.98pg/ml,respectively,with significant difference, suggesting that PSCS can decrease the concentration of TNF-αin OA rabbit serum, and has no significant difference with positive control group.The concentration of IL-1βin the PSCS group and control group has no significant difference.The accomplishments of the subject:■Successfully prepared highly purified PSCS,containing the structure of 4,6-bisulfate CS.■Successfully prepared the PSCS-chitinan nanoparticles and screened the conditions.■The PSCS-chitinan nanoparticles were administrated orally for treatment of rabbit knee osteoarthritis and confirmed the protective effects.
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