【作者】 肖玉良；

【导师】 王凤山；

【作者基本信息】 山东大学 ， 微生物与生化药学， 2014， 博士

【摘要】 研究目的硫酸软骨素(chondroitin sulfate, CS)是一类硫酸化的糖胺聚糖,存在于细胞表面和哺乳动物的细胞外基质中,主要分布于软骨、骨、肌腱、神经组织和血管壁中。CS的基本结构是由D-葡糖醛酸和N-乙酰-D-氨基半乳糖以1,3糖苷键连接形成的二糖,二糖之间以β-1,4糖苷键连接而成。CS具有抗炎、免疫调节、心脑血管保护、神经保护、抗氧化、细胞黏附调节等多种药理生理学活性与功能,且长期服用毒副作用小或无毒副作用。在欧洲、美国、东南亚、澳大利亚等国家,CS主要作为膳食补充剂或药品,用于心脑血管疾病、骨关节炎等疾病的预防与治疗。但是,由于其分子量大、亲水基团多、脂溶性差,体内吸收不完全,口服生物利用度低；在CS治疗心脑血管疾病的研究中,其降脂、抗动脉粥样硬化(AS)的疗效和量效关系以及其抑制斑块性的分子机制都尚不明确。为了解决这些问题,本研究拟对CS结构进行修饰改造,采用降低分子量、增加亲脂基团、构建两亲性纳米粒子等方法来改善其口服吸收效果,然后通过整体动物法筛选出肠道吸收效果好的CS衍生物,并采用Caco-2细胞模型对其促肠道吸收作用机制进行探讨；构建ApoE-/-小鼠AS动物模型,考察CS及其衍生物对AS斑块的抑制作用,并对其可能的抗AS作用机制进行探讨。研究方法1.具有两亲性的α-亚麻酸-低分子量硫酸软骨素结合物的制备及表征采用H202氧化降解法对CS进行降解,制备低分子量CS (LMCS),并采用亲脂性的α-亚麻酸(α-LNA)对LMCS进行修饰,合成出一系歹(?)α-LNA取代度不同的两亲性LMCS (α-LNA-LMCS)。采用红外光谱法(FTIR)、核磁共振波谱法(NMR)以及热差-热重分析法(TGA/DSC)对两亲性α-LNA-LMCS的结构进行表征；对两亲性a-LNA-LMCS的自组装及其自组装后的胶束的粒径大小、粒径分布、Zeta电位、表面形态、临界胶束浓度以及生物安全性等性能进行研究。2.CS及其衍生物的体内外肠道吸收及其机制研究采用整体动物模型考察CS及其衍生物的肠道吸收效果,筛选出肠道吸收效果好的α-LNA-LMCS;采用Caco-2细胞模型验证整体模型的研究结果,通过跨膜电阻的变化考察药物种类、浓度以及作用时间对Caco-2单层细胞完整性的影响；激光共聚焦显微镜观察CS及其衍生物在Caco-2细胞内的摄取和转运及其对Caco-2细胞F-肌动蛋白(F-actin)表达的影响,研究α-LNA-LMCS促进LMCS肠道吸收的作用机制。3.CS衍生物的抗动脉粥样硬化活性及其机制研究8周龄ApoE-/-小鼠104只随机分成8组(每组13只),高脂高胆固醇饮食(15%脂肪和0.25%胆固醇),同时进行药物干预,16周后用10%水合氯醛麻醉,心脏取血,制备血浆后生化法测定小鼠血浆中甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)的含量,考察药物对小鼠血脂的影响。ELISA法测定小鼠血浆中IL-6、TNF-α、C反应蛋白(CRP)的含量。通过对小鼠主动脉表面AS病变分析及主动脉根部病灶的苏木素-伊红(H&E)染色、油红O染色、Masson染色、巨噬细胞及平滑肌细胞免疫组化染色考察CS及其衍生物对AS斑块形成的影响；Western Blot法考察小鼠主动脉中p-NF-κB、p-JNK、p-ERK1/2、COX-2、MCP-1、VCAM-1和ICAM-1等蛋白的表达情况。实时定量PCR法考察小鼠主动脉中IL-6、TNF-α、CRP、MCP-1、 VCAM-1、ICAM-1等炎症相关因子基因的表达。从炎症调节方面探讨CS及其衍生物抗AS的分子机制。研究结果1.具有两亲性的a-亚麻酸-低分子量硫酸软骨素结合物的制备及表征合成了一系列α-LNA取代度不同的两亲性α-LNA-LMCS,并对其进行了表征。FT-IR、1H NMR以及TGA/DSC分析结果表明,α-LNA作为侧链通过与D-葡糖醛酸和N-乙酰-D-氨基半乳糖糖环上的羟基形成酯键而连接到LMCS的主链上,α-LNA取代度在0.034-0.123的范围内；激光光散射仪、电位分析仪以及扫描电镜表征结果表明,α-LNA-LMCS胶束外观呈球形结构,水合粒径在78-117nm的范围内,Zeta电位在-30～-25mV之间,粒径大小适宜且粒径分布范围较窄；荧光光谱法考察a-LNA-LMCS自聚集性能的结果表明,a-LNA-LMCS能够在水溶液中形成较稳定的胶束,且a-LNA-LMCS胶束的临界胶束浓度在0.016-0.20mg/mL之间。2.CS及其衍生物的体内外肠道吸收及其机制CS及其衍生物的动物体内肠道吸收研究发现,LNA-LMCS2在血浆中的最高浓度Cmax、达峰时间Tmax、半衰期t1/2、体内总清除率CL以及曲线下面积AUCo-24分别为10.8±0.5mg/L、8.8±2.3h、18.8±3.1h、0.6±0.4L/(h-kg)以及12.0±0.6h,Cmax分别是CS和LMCS的2.8倍和1.6倍,t1/2是CS和LMCS的1.7倍和3.2倍,CL分别是CS和LMCS的0.18倍和0.19倍,AUC0-24分别是CS和LMCS的4.13倍和3.12倍,这些表明LMCS在经a-LNA修饰后肠道吸收效果得到显著的改善。Caco-2细胞模型研究发现,与CS及LMCS相比,LNA-LMCS2表观渗透系数Papp大(p<0.001)、外排率低(p<0.05), LNA-LMCS2可以抑制P-gp的活性,减少药物的外排,通过胞吞作用来促进增加LMCS的肠道吸收。对Caco-2单层细胞膜的跨膜电阻研究发现,LNA-LMCS2作用后,Caco-2单层细胞膜的跨膜电阻明显降低,通透性增加,这表明LNA-LMCS2可能打开了Caco-2单层细胞膜间的紧密连接。激光共聚焦显微镜观察发现,LNA-LMCS2可以影响F-肌动蛋白的微丝及骨架,打开细胞间紧密连接,增强细胞膜的通透性,且对单层细胞膜整体完整性没有影响。体内外实验研究表明,经a-LNA修饰后的LMCS可以通过促进胞吞作用和增加旁路吸收两种方式来提高LMCS的肠道吸收。3.CS衍生物的抗动脉粥样硬化活性及其机制药效学研究发现,8组间的小鼠体重未见统计学差异(p>0.05),这说明给药各组对实验动物的体重没有显著性的影响；与对照组相比,H-LNA-LMCS2组及H-LMCS组ApoE-/-小鼠血液中LDL-C和TG的浓度明显降低(p<0.05)；H-LNA-LMCS2组ApoE-/-小鼠血液中TNF-α、IL-6(?)(?)CRP的浓度显著降低。这些结果表明,LNA-LMCS2可以有效地降低AS模型中ApoE-/-小鼠的血脂浓度,并能够抑制机体的炎症反应。小鼠主动脉脉面病变分析(大体油红O染色)研究表明,与对照组相比,H-LNA-LMCS2组及H-LMCS组的病变面积明显降低,具有明显的统计学意义(p<0.01和p<0.05)；小鼠主动脉窦冰冻切片H&E染色和油红O染色研究表明,H-LNA-LMCS2组和H-LMCS组斑块面积／主动脉窦瓣环面积比值明显减少(p<0.001和p<0.05)；小鼠主动脉窦冰冻切片Masson染色表明,LNA-LMCS2及LMCS对给药各组斑块中胶原纤维含量影响不大,无统计学意义；小鼠主动脉窦冰冻切片免疫组化研究表明,与对照组相比,H-LNA-LMCS2组和H-LMCS组小鼠斑块中巨噬细胞的含量明显降低(p<0.05),平滑肌细胞明显升高(p<0.05)；高剂量的LNA-LMCS2及LMCS可以有效的抑制AS模型中ApoE-/-小鼠主动脉斑块的形成。Western blot及RT-PCR研究结果表明, LNA-LMCS2和LMCS能够通过下调磷酸化细胞外调节蛋白激酶(p-ERK1/2)抑制p-NF-κB的核转位,下调COX-2、 MCP-1、VCAM-1、ICAM-1的表达,降低IL-6、TNF-α、MCP-1、VCAM-1、ICAM-1的mRNA水平。以上结果表明,LNA-LMCS2和LMCS的延缓AS斑块发展的作用可能是通过降脂和抗炎两条途径来实现的。结论和意义1.本研究成功的采用a-LNA通过酯化反应对LMCS进行了修饰,制备出了口服生物利用度良好的两亲性CS衍生物。2.两亲性LNA-LMCS2促进LMCS肠道吸收的作用机制研究表明,LNA-LMCS2可以通过促进胞吞作用和增加旁路吸收两种方式来提高LMCS的肠道吸收。3.高剂量(400mg/kg)的LNA-LMCS2和LMCS对ApoE-/-小鼠具有较好的降脂和抑制促炎酶及促炎因子的作用,可以通过降脂和抗炎两种途径来抑制ApoE-/-小鼠AS斑块形成及发展。4.本研究证明了两亲性LNA-LMCS2是一种具有良好口服生物利用度和抗AS活性的多糖衍生物,并确定了其部分药物活性作用机制。本课题的完成,不但为大分子多糖类药物的口服吸收改善研究提供一定的理论基础,而且为新型CS类抗AS药物的开发和利用奠定了基础,具有较好的应用开发前景。更多还原

【Abstract】 ObjectiveChondroitin sulfate (CS) ubiquitously distributes on cell surfaces and in the extracellular matrix (ECM) of mammalian animals, and is particularly abundant in bones, tendons, blood vessels, nerve tissues, and cartilage. CS, a sulfated glycosaminoglycan (GAG), is composed of repeating disaccharide units of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), which is commonly sulfated at the C-4and/or C-6of GalNAc in mammals. CS has been discovered to have various biological functions including anti-oxidation, anti-inflammation, anti-atherosclerosis, immunoregulation, neuroprotection, regulating cell adhesion and morphogenesis, and low or no toxicity with long term administration. The market for products containing CS and glucosamine is developing in North America, Europe, South East Asia and Australia. In United States, CS is recognized as a "dietary supplement". While in Europe, CS is marketed as symptomatic slow-acting drugs for osteoarthritis and is widely used for the relief of symptoms and pain of arthritic diseases. However, its clinical use has encountered certain limitations because of its poor intestinal absorption, resulting from its high molecular weight, charge density, as well as hydrophilicity. Therefore, intestinal absorption become one of critical factors for the successful application of CS and CS derivatives in the treatment of osteoarthritis and atherosclerosis. In order to get around this drawback, researchers have tried many strategies including adding absorption enhancers, preparing bioadhesive nanoparticles or prodrugs, as well as increasing drug lipophilicity. In this work, we prepared a new amphiphilic CS, a-linolenic acid (a-LNA)-low molecular weight CS (LMCS) conjugate (a-LNA-LMCS), which can form stable nanomicelles in aqueous media. The oral bioavailability of a-LNA-LMCS micelles was evaluated in vivo and in vitro, and the possible transport mechanism of a-LNA-LMCS micelles was determined by using Caco-2cell monolayers. Furthermore, the effect and possible mechanisms of anti-atherosclerosis of CS and its derivative were investigated by using apolipoprotein E-deficient(ApoE-/-) mice.Method1. Preparation and characterization of a-LNA-LMCSLMCS was prepared by using a controlled oxidative depolymerization process in the presence of hydrogen peroxide. Then, LMCS was hydrophobically modified with a-linolenic acid (a-LNA) to obtain a series of amphiphilic CS (a-LNA-LMCSs) with different degree of a-LNA substitution. Structural characterizations of a-LNA-LMCSs were analized by FTIR,1HNMR, TGA/DSC. The physicochemical properties of a-LNA-LMCSs in aqueous media were characterized by transmission electron microscopy (TEM), laser light scattering, zeta potential and fluorescence spectroscopy.2. In vitro and in vivo oral bioavailability and absorption mechanism study of CS and its derivativesThe oral bioavailability of a-LNA-LMCS micelles in vivo was evaluated by determining the a-LNA-LMCS concentrations in plasma levels following oral administration to rats in comparison with CS. Caco-2cell monolayers representing in vitro model of the intestinal epithelial barrier were used to determine the intestinal transport ability of a-LNA-LMCS micelles. Furthermore, confocal laser scanning microscope (CLSM) was used to study the transport mechanism of CS and its derivatives across the intestinal epithelial barrier.3. Anti-atherosclerosis effect and the mechanism study of LMCS and LNA-LMCS2 8-week-age ApoE-/-mice were randomly divided into8groups (13each), treated with atherogenic diet (15%fat and0.25%cholesterol) together with or without tested compounds. After16weeks of administration, mice were anesthetized with10%chloral hydrate, and then the blood samples were collected by cardiac puncture. The levels of triglycerides (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) in blood plasma were measured by an automatic biochemistry analyzer. The plasma levels of IL-6, TNF-a and C-reactive protein (CRP) were detected by enzyme-linked immuno sorbent assay (ELISA) kits. Meanwhile, part of the hearts and aortas were perfusion-fixed with4%paraformaldehyde for histological and morphological staining (Hematoxylin and eosin staining, Oil-Red-O staining, Masson staining) and immunohistochemistry staining (MAMO-2, a-SMA). The remaining part of the hearts and aortas were treated with PBS for real-time polymerase chain reaction (real-time PCR) and Western Blot. Real-time PCR was used to detect the mRNA levels of IL-6, TNF-a, CRP, MCP-1, VCAM-1and ICAM-1. Western blot was used to detect the protein expression levels of p-NF-κB, p-JNK, p-ERK1/2, MCP-1, VCAM-1and ICAM-1.Results1. Preparation and characterization of a-LNA-LMCSA series of α-LNA-LMCSs with different degree of a-LNA substitution were prepared and characterized. The results of FT-IR,1HNMR and TGA/DSC indicated that all samples had successfully undergone the esterification of the hydroxyl groups in the repeating disaccharide of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), which meant that the α-LNA was introduced as the side chains of LMCS. The degree of substitution (DS) of α-LNA-LMCSs ranged from0.034to0.123. TEM observation demonstrated that α-LNA-LMCS micelles were roughly smooth sphere morphology and had a narrow and unimodal size distribution. The mean diameters of α-LNA-LMCS micelles were in the range of78-117nm. The critical aggregation concentrations of α-LNA-LMCS micelles were in the range of0.016-0.20mg/mL. The zeta potential of a-LNA-LMCS micelles were in the range of-30～-20mV, and the high negative charge improved the stability of micelles in aqueous media.2. In vitro and in vivo oral bioavailability and absorption mechanism of CS and its derivativeThe absorptions of CS and its derivatives were evaluated in vivo. The maximum concentration (Cmax) of LNA-LMCS2was10.8±0.5mg/L at8.8±2.3h, which was obviously higher than that of CS and LMCS. LNA-LMCS2showed a much longer circulation time and its elimination t1/2was18.8±3.1h,1.7times and3.2times longer than that of CS and LMCS. The total body clearance (CL) of LNA-LMCS2was0.6±0.4L/(h-kg), which was5.6times and5.3times smaller than that of CS and LMCS, respectively. Moreover, the AUC0-24of LNA-LMCS2after intragastric administration was172.4±20.9mg/(L·h), extremely higher than that of CS,40.0±4.4mg/(L-h) and LMCS,55.2±4.4mg/(L·h), respectively (p<0.001). It was deduced that the oral bioavailability of CS has been significantly improved.Caco-2transport studies demonstrated that the apparent permeability coefficient (Papp) of LNA-LMCS2was significantly higher than that of CS and LMCS (p<0.001), and no significant effects on the overall integrity of the monolayer were observed during the transport process. In addition, the efflux ratios of a-LNA-LMCSs were remarkably lower than that of CS or LMCS (p<0.05), which indicated that a-LNA-LMCS micelles had inhibitory effect on P-glycoprotein (P-gp). The transepithelial electrical resistance (TEER) values of Caco-2cell monolayers incubated with a-LNA-LMCSs decreased significantly compared to that of the control groups incubated with CS and LMCS, indicating that a-LNA-LMCSs had some effects on the opening of intercellular tight junctions. Furthermore, evident alterations in the F-actin cytoskeleton were detected by CLSM observation following the treatment of the cell monolayers with a-LNA-LMCS micelles, which further certified the capacity of a-LNA-LMCS micelles to open the intercellular tight junctions rather than disrupt the overall integrity of the monolayer. Based on these results and the in vivo efficacy study, it was deduced that the modification of LMCS with a-LNA improved its intestinal absorption through the enhancement of the transcellular and paracellular transport across intestinal cells.3. Anti-atherosclerosis effect and the mechanism of LMCS and LNA-LMCS2ApoE-/-mice in8groups fed a atherogenic diet with or without LNA-LMCS2or LMCS did not differ in body weight (p>0.05), which indicated that these drugs had no effect on body weight of the mice. The levels of TC and LDL-C in the high-dose group of LNA-LMCS2or LMCS were significantly lower than that of control group (p<0.05). More importantly, the plasma levels of TNF-a, IL-6and CRP were significantly lower in the high dose group of LNA-LMCS2than those in model control group. These results suggest that LNA-LMCS2is effective in reducing lipid levels and mitigating the inflammatory response on atherosclerosis.By using en face analysis of the arteria aorta, lesion area was significantly smaller in the high dose groups of LNA-LMCS2and LMCS than in the control group (p<0.001and p<0.05, respectively). Atherogenesis level at the aortic sinus was evaluated by H&E and Oil-Red-O staining by ratio of total atherosclerotic lesion area to aortic valve ring area. The mean lesion size at the aortic sinus was smaller in the high dose groups of LNA-LMCS2and LMCS than in the control group (p<0.001and p<0.05, respectively). The result of Masson staining indicated that LNA-LMCS2and LMCS had little effect on the collagen fibers in plaques. Immunohistochemistry staining demonstrated that, compared with the control group, the numbers of macrophages in plaques were significantly less in the high dose groups of LNA-LMCS2and LMCS (p<0.05). At the same time, smooth muscle cells in plaques were significantly higher in the high dose groups of LNA-LMCS2and LMCS (p<0.05) compared than in the control group. All the results demonstrated that LNA-LMCS2and LMCS could decelerate the progression of atherosclerosis in ApoE-/-mice.The results of Western blot showed that LNA-LMCS2and LMCS could reduce the nuclear translocation of NF-κB by inhibiting the activity of ERK1/2, and further decrease the protein expressions of COX-2, MCP-1,VCAM-1and ICAM-1, which is consistent with the findings of RT-PCR. Based on these results, it was deduced that LNA-LMCS2and LMCS inhibited the atherosclerotic plaques through two ways:regulation of the lipid metabolism and anti-inflammation.Conclusions and significance1. A new series of amphiphilic polysaccharides, a-LNA-LMCSs, were successfully synthesized by using a-LNA as the hydrophobic chain. a-LNA-LMCS micelles were found to effectively increase the oral bioavailability of LMCS.2. The transport mechanism of LNA-LMCS2micelles across intestinal epithelial barrier via paracellular pathway and endocytosis was proved for the first time.3. High dose of LMCS and LNA-LMCS2(400mg/kg) can regulate the lipid metabolism and diminish the synthesis of pro-inflammatory enzymes and cytokines, which might be the mechanisms of anti-atherosclerosis effect of CS and its derivatives.4. This study has demonstrated that LNA-LMCS2is a new amphiphilic polysaccharide with good oral bioavailability and anti-atherosclerosis effect, and uncover the fundamental intestinal transport mechanisms and anti-atherosclerosis mechanisms of LNA-LMCS2. The results of this study will not only provide a good strategy to enhancing the intestinal absorption of polysaccharides, but also improve the development of CS based anti-atherosclerosis drug in the future.更多还原