Preparation Of Shark Keratan Sulfate And Its Chitosan Nanoparticle And The Anti-Rheumatoid Arthritis Function

Rheumatoid arthritis(RA)is a common systemic autoimmune disease,which is characterized by chronic polyarthritis symptom.According to the RA pathogenesis studies,anti-inflammatory therapy has become one of important treatment.It is reported that glycosaminoglycans(GAGs)has anti-inflammatory effect in recent years.KS from Shark cartilage was selected as anti-rheumatoid arthritis materials in this study.KS(KS)belong to the family of GAGs,is composed of galactose and 6-sulfuric acid-N acetylgalactosamine,which are connected byβ1→4 glucosidic bond,and one disaccharide are connected to another byβ1→3 glucosidic bond,forming the liner chain polysaccharides.KS is the only glycosaminoglycan that dose not contain glycuronate.Compared to the ones from other source,the main different of shank KS in structure is that it has more sulfuric.According to the recent reports,KS oligosaccharide fraction can be used as active ingredients of anti-inflammatory agents, antiallergic agents,immunomodulators etc.KS disaccharide,Gal-6-SO₃-(β1,4)-GlcNAc-6-SO₃, as anti-inflammatory agents,can suppress IL-12 production in macrophages by inhibiting phosphoprotein kinase C and phosphoinositide 3-kinase pathways.The separation and purification,Structure Determination,Preparation of KS-CTS nanoparticle and effects of KS on RA rat treatment were studied,and the influence of KS on the expression of inflammatory factors(Rat IL-1βand Rat TNF-α)were investigated.1 Separation and purification of KSKS were separated and purified from shark cartilage polysaccharides by Q Sepharose^TMFast Flow strong anion exchange chromatography methods.After desalting by Sephadex G-10 gel filtration chromatography method,some physicochemical properties and construction features such as molecular mass,the content of mucosal aminohexose,sulphate content was detected then.2 Structure analysis of KSInfrared spectra(IR)and NMR spectra(¹H-NMR,¹³C-NMR)were used to analyze the molecular structures of KS.IR spectra showed the presence of Hydroxide radical,carbonyl group,amide group and sulfate group,Carboxy group was not shown in the IR spectra,which indicated that there was no uronic acid in its structure, implying that it was KS.3 Study on Preparing Technology of KS-CTS nanoparticle and PreparationDesigning orthogonal experiment,based on the investigation of the nanoparticle size and Zeta potential,we analyze the influence of chitosan(CTS)Molecular Weight, volume ratio of KS to CTS and others to the preparation of namoparticle.The Optimum experiment results are:the molecular weight of CTS is 5kD,volume ratio of KS to CTS is 3:4,pH is 6,concentration of KS is 1mg/mL and concentration of CTS is 2mg/mL,respectively.Adding KS solution dropwise into the CTS solutions every 3s.To diminish volume,the nanoparticle solution was condensed a half under 45℃。4 Therapeutical effect of KS on RA ratA rheumatoid arthritis rat model was induceded by complete Freund's adjuvant (CFA)and definited by rat weight,Joint Swelling,X-ray analysis and pathological section.It was found that KS have the function of relieving inflammatory reaction and depressing the joint swelling in RA rats.The effects of KS on the content of Rat IL-1βand Rat TNF-αin RA rat blood serum were studied.The results showed that the contents of Rat TNF-αin the blood serum was 14.47±4.832 pg/mL in model group, which was significant higher than the normal control group(4.375±2.611 pg/mL), positive control group(5.217±2.589pg/mL)and sample group(7.086±3.201 pg/mL) (P＜0.05).And the contents of Rat IL-1βin the blood serum was 39.5±17.735 pg/mL in model group,which had significant difference versus the normal control group (7.330±2.733 pg/mL),positive control group(9.038±5.310 pg/mL)and sample group(15.462±7.281 pg/mL)(P＜0.05)In conclusion,the major achievements of this research were as follows:(1)After separation and purification of KS,it was the first time studying on preparing technology of KS-CTS nanoparticle.(2)The effects of KS on RA rat were reported.The results showed that KS had inhibitory effects on Rat TNF-αand Rat IL-1βexpression and activity in RA rat.It demonstrated that KS acted effectively on some key process of inflammation in RA.