鲨鱼软骨硫酸角质素的制备及其壳聚糖纳米粒抗类风湿性关节炎作用的研究
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摘要
类风湿性关节炎(rheumatoid arthritis,RA)是一种常见的以慢性多关节炎症为主要表现的全身性自身免疫性疾病。通过对RA的发病机制研究,抗炎治疗已经成为RA治疗的一个重要原则。近年来,糖胺聚糖(glycosaminoglycans,GAGs)类药物的抗炎作用研究已有报道,本课题选择了鲨鱼软骨中的硫酸角质素进行抗炎作用研究。
硫酸角质素(keratan sulfate,KS)属于糖胺聚糖类化合物,以半乳糖与6-硫酸-N-乙酰氨基葡萄糖通过β1→4糖苷键组成的二糖单位为主要重复双糖单位,双糖之间通过β1→3糖苷键连接形成线形分子,它是鲨鱼软骨多糖成分中唯一不含糖醛酸的糖胺聚糖,来源于鲨鱼软骨的KS与其他来源的KS相比,结构上的差别主要在于其含有更多的硫酸根取代基。最近研究表明,KS寡糖级分可作为抗炎剂、抗过敏剂、免疫调节剂等,KS二糖片段Gal-6-SO_3-(β1,4)-GlcNAc-6-SO_3能通过抑制磷酸蛋白激酶C及磷酸肌醇-3激酶通路,从而抑制IL-12的分泌从而起到抗炎用。
本课题研究了来源于鲨鱼软骨的KS的制备,结构解析,壳聚糖纳米粒的制备及其对RA大鼠的治疗作用,探讨了KS对大鼠RA炎症过程中血清中IL-Iβ和TNF-α含量的影响。
1鲨鱼软骨多糖的分离和纯化
将精制的鲨鱼软骨多糖提取物经Q Sepharose~(TM)Fast Flow强离子交换柱层析进行分离,Sephadex G-10凝胶柱层析脱盐后,获得组成相对单一的组分,通过糖醛酸反应鉴定出KS,并对其进行了分子量、氨基己糖含量、硫酸根含量等理化性质及结构特点研究。
2 KS的结构分析
利用红外光谱(IR)、核磁共振光谱(~1H-NMR、~(13)C-NMR)结果分析确定KS的主要分子结构。红外光谱结果显示,KS结构中含有羟基、羰基和酰胺基结构,并含有硫酸基,其结构中不含有糖醛酸,进一步说明其为KS。核磁共振结果显示,KS多糖分子中除含有Gal及GlcNAc单糖残基外,还含有NeuAc残基。
3 KS-CTS纳米粒的制备
设计正交试验,通过考察纳米粒粒径及Zeta电位值,分析了壳聚糖(chitosan,CTS)分子量,KS、CTS溶液体积比等因素对纳米粒制备的影响。确定最佳实验条件:CTS分子量为5kD,KS、CTS体积比为1:4,pH值为6,KS浓度为1mg/mL,CTS浓度为2 mg/mL,液体混合滴加速度为3s一滴。纳米粒制备中,为减小大鼠灌胃量,在45℃下旋蒸纳米粒溶液使体积缩减一半。
4 KS对大鼠类风湿性关节炎的治疗作用
建立大鼠RA模型,对大鼠体重、足跖肿胀度、X线分析及病理切片确定各组大鼠炎症情况。结果显示,KS能够减轻炎症反应、降低大鼠足跖肿胀度。研究了KS对RA大鼠血清中细胞因子Rat TNF-α、Rat IL-1β的影响,结果显示,模型组大鼠血清中细胞因子Rat TNF-α含量为14.47±4.832 pg/mL,明显高于阴性对照组(4.375±2.611 pg/mL)、阳性对照组(5.217±2.589 pg/mL)和样品组(7.086±3.201 pg/mL)(P＜0.05);模型组大鼠血清中细胞因子Rat IL-1β含量为39.5±17.735 pg/mL,明显高于阴性对照组(7.330±2.733 pg/mL)、阳性对照组(9.038±5.310 pg╱mL)、样品组(15.462±7.281 pg/mL)(P＜0.05)。
本研究取得的主要成果有:
(1)将KS分离纯化后,首次将KS制备成壳聚糖纳米粒,并进行了制备工艺研究。
(2)研究了KS对大鼠RA模型的治疗作用。结果显示,KS能够抑制大鼠血清中Rat TNF-α、Rat IL-1β的含量,从而在一定程度上对大鼠佐剂性关节炎的关键步骤起到一定的抑制作用。
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-SO_3-(β1,4)-GlcNAc-6-SO_3, 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 KS
KS were separated and purified from shark cartilage polysaccharides by Q Sepharose~(TM)Fast 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 KS
Infrared spectra(IR)and NMR spectra(~1H-NMR,~(13)C-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 Preparation
Designing 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 rat
A 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.
硫酸角质素(keratan sulfate,KS)属于糖胺聚糖类化合物,以半乳糖与6-硫酸-N-乙酰氨基葡萄糖通过β1→4糖苷键组成的二糖单位为主要重复双糖单位,双糖之间通过β1→3糖苷键连接形成线形分子,它是鲨鱼软骨多糖成分中唯一不含糖醛酸的糖胺聚糖,来源于鲨鱼软骨的KS与其他来源的KS相比,结构上的差别主要在于其含有更多的硫酸根取代基。最近研究表明,KS寡糖级分可作为抗炎剂、抗过敏剂、免疫调节剂等,KS二糖片段Gal-6-SO_3-(β1,4)-GlcNAc-6-SO_3能通过抑制磷酸蛋白激酶C及磷酸肌醇-3激酶通路,从而抑制IL-12的分泌从而起到抗炎用。
本课题研究了来源于鲨鱼软骨的KS的制备,结构解析,壳聚糖纳米粒的制备及其对RA大鼠的治疗作用,探讨了KS对大鼠RA炎症过程中血清中IL-Iβ和TNF-α含量的影响。
1鲨鱼软骨多糖的分离和纯化
将精制的鲨鱼软骨多糖提取物经Q Sepharose~(TM)Fast Flow强离子交换柱层析进行分离,Sephadex G-10凝胶柱层析脱盐后,获得组成相对单一的组分,通过糖醛酸反应鉴定出KS,并对其进行了分子量、氨基己糖含量、硫酸根含量等理化性质及结构特点研究。
2 KS的结构分析
利用红外光谱(IR)、核磁共振光谱(~1H-NMR、~(13)C-NMR)结果分析确定KS的主要分子结构。红外光谱结果显示,KS结构中含有羟基、羰基和酰胺基结构,并含有硫酸基,其结构中不含有糖醛酸,进一步说明其为KS。核磁共振结果显示,KS多糖分子中除含有Gal及GlcNAc单糖残基外,还含有NeuAc残基。
3 KS-CTS纳米粒的制备
设计正交试验,通过考察纳米粒粒径及Zeta电位值,分析了壳聚糖(chitosan,CTS)分子量,KS、CTS溶液体积比等因素对纳米粒制备的影响。确定最佳实验条件:CTS分子量为5kD,KS、CTS体积比为1:4,pH值为6,KS浓度为1mg/mL,CTS浓度为2 mg/mL,液体混合滴加速度为3s一滴。纳米粒制备中,为减小大鼠灌胃量,在45℃下旋蒸纳米粒溶液使体积缩减一半。
4 KS对大鼠类风湿性关节炎的治疗作用
建立大鼠RA模型,对大鼠体重、足跖肿胀度、X线分析及病理切片确定各组大鼠炎症情况。结果显示,KS能够减轻炎症反应、降低大鼠足跖肿胀度。研究了KS对RA大鼠血清中细胞因子Rat TNF-α、Rat IL-1β的影响,结果显示,模型组大鼠血清中细胞因子Rat TNF-α含量为14.47±4.832 pg/mL,明显高于阴性对照组(4.375±2.611 pg/mL)、阳性对照组(5.217±2.589 pg/mL)和样品组(7.086±3.201 pg/mL)(P＜0.05);模型组大鼠血清中细胞因子Rat IL-1β含量为39.5±17.735 pg/mL,明显高于阴性对照组(7.330±2.733 pg/mL)、阳性对照组(9.038±5.310 pg╱mL)、样品组(15.462±7.281 pg/mL)(P＜0.05)。
本研究取得的主要成果有:
(1)将KS分离纯化后,首次将KS制备成壳聚糖纳米粒,并进行了制备工艺研究。
(2)研究了KS对大鼠RA模型的治疗作用。结果显示,KS能够抑制大鼠血清中Rat TNF-α、Rat IL-1β的含量,从而在一定程度上对大鼠佐剂性关节炎的关键步骤起到一定的抑制作用。
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-SO_3-(β1,4)-GlcNAc-6-SO_3, 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 KS
KS were separated and purified from shark cartilage polysaccharides by Q Sepharose~(TM)Fast 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 KS
Infrared spectra(IR)and NMR spectra(~1H-NMR,~(13)C-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 Preparation
Designing 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 rat
A 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.
引文
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