CN115463140B - 海洋来源磷脂在制备防护雷公藤多苷造成肝损伤药物中的应用 - Google Patents
海洋来源磷脂在制备防护雷公藤多苷造成肝损伤药物中的应用 Download PDFInfo
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Abstract
本发明为海洋来源磷脂在制备防护雷公藤多苷造成肝损伤药物中的应用，属于生物医药技术领域，具体为海洋来源磷脂在制备防护雷公藤多苷造成器官损伤药物中的应用，进一步的，海洋来源磷脂在制备防护雷公藤多苷造成肝损伤药物中的应用；本发明发现海洋来源磷脂能够防护雷公藤多苷诱导的肝损伤，可以抑制GTW诱导的肝脏转氨酶升高、miR‑122表达下降，改善GTW诱导的斑马鱼肝脏组织及其超微结构损伤。
Description
技术领域
本发明属于生物医药技术领域，具体涉及海洋来源磷脂在制备防护雷公藤多苷造成肝损伤药物中的应用。
背景技术
雷公藤多苷(GTW)是从植物雷公藤(Tripterygium wilfordii Hook.f.)中提取的活性成分之一，具有免疫抑制、抗炎、抗肿瘤等多种药理作用。在临床上被广泛应用于治疗与免疫功能异常有关的各种疾病，如类风湿性关节炎、红斑狼疮、肾病综合征等疾病。近年来，雷公藤多苷的不良反应和毒副作用受到了越来越多的关注，尤其是对肝脏的损害作用。虽然近年来在降低GTW肝毒性方面取得了各种进展，但保护性研究大多采用与临床药物配伍，如京尼平苷、联苯双酯、保肝解毒颗粒，需要兼顾临床药物的药效与毒性。因此，筛选保护或改善肝功能活性化合物，减轻雷公藤多苷在治疗过程中的药物性肝损害，使治疗过程得以顺利进行，具有重大意义。
磷脂(PL)已被证实对慢性炎症疾病有益，主要归因于其抗炎、抗氧化、抗纤维化、抗凋亡、膜保护和脂质调节活性。据报道，大豆和鸡蛋提取的陆源磷脂可通过降低大鼠肝甘油三酯水平来缓解乳糜酸诱导的脂肪肝。临床试验表明PL治疗对酒精性肝损伤具有保护作用。此外，连续6.5年给予卵磷脂(PL的主要组分)可预防酒精喂养阿拉伯狒狒导致的肝纤维化和肝硬化。
海洋磷脂是指来源于海洋生物的磷脂，含有丰富的ω-3多不饱和脂肪酸(ω-3polyunsaturated fatty acids，ω-3PUFAs)，如二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)等。ω-3PUFAs作为脂质介质前体和转录因子配体，对细胞和组织的生理活性和细胞外信号介导具有重要作用。据报道，ω-3PUFAs，尤其是长链脂肪酸EPA和DHA可降低心血管疾病、癌症、糖尿病、关节炎和中枢神经系统紊乱疾病如精神分裂症、抑郁症和阿尔兹海默症的发病率。海洋磷脂相较于陆地磷脂(大豆磷脂、蛋黄磷脂)含有更多的DHA和EPA，因此拥有比陆地磷脂更好的生物活性，调节代谢和清除生物体内自由基，具有缓解衰老、调节动脉粥样硬化块、提高认知功能、抗炎以及调节血脂等活性。
已有研究表明，植物性(如大豆)或陆源动物性(如脑、肝脏、蛋黄)磷脂可降低酒精性及四氯化碳(CCl4)造成的肝细胞损伤，且对高脂低蛋白食物、乙醇和皮下注射CCl4复合因素诱导的肝纤维化具有修复作用。从鱿鱼性腺中获取的海洋来源磷脂对酒精和硫代乙酰胺诱导的脂肪肝均具有改善作用。此外，一些磷脂复合物对CCl4、d-半乳糖胺与弗氏完全佐剂诱导的急、慢性肝损伤也具有明显的保护作用。但是关于磷脂对药物，尤其是雷公藤多苷造成肝损伤的防护，现有技术未见报道。
药物致肝损伤的机理十分复杂，涉及多个途径，包括激活代谢，氧化应激、诱导线粒体损伤，激活免疫应答，诱导溶酶体损伤、胆汁淤积等。随着中药毒理学研究的不断深入，GTW导致肝毒性的作用机制逐渐被揭示。GTW主要是通过抑制肝药酶表达/活性，降低肝脏代谢能力，改变氨基酸、糖、磷脂和激素等代谢途径，诱导氧化应激以及抑制免疫等途径造成肝损伤。目前，常采用西药(如地塞米松、双环醇)加速GTW代谢、抑制肝细胞凋亡等方式拮抗GTW肝毒性，或将单味中药(如甘草、凤尾草、金钱草、五味子、丹参等)、中药组分(如白芍总苷、阿魏酸钠、茶多酚、番茄红素等)以及中药复方制剂(如保肝解毒颗粒、解毒饮复方、逍遥散、四逆散、复方甘草酸铵等)与GTW配伍减轻其造成的肝损伤。然而，同时摄入多种药物容易增加机体负担，且西药或中药成分可能会导致一定的不良反应。
中国专利文献CN109303790A(申请号：201811471991.6)公开了刺山柑或刺山柑提取物的医院用途，该发明发现刺山柑提取物对雷公藤致肝损伤具有保护作用，且优于市售复方甘草酸苷片，其乙酸乙酯萃取物效果非常显著。该专利文献公开的是利用植物提取物对雷公藤多苷造成的肝损伤具有防护作用。
中国专利文献CN109288056A(申请号：201811180650.3)公开了一种含姜黄素、ω-3多不饱和脂肪酸和磷脂的组合物及其应用，该专利文献公开了黄素、ω-3多不饱和脂肪酸和磷脂的组合物有助于降血脂和保护酒精性肝损伤，三者在一定比例范围内具有协同增效作用，该专利文献公开的是对酒精性肝损伤的防护。
肝脏常规评价指标为谷丙转氨酶(ALT)和谷草转氨酶(AST)，诱导肝损伤的药物能增加AST和ALT含量，AST和ALT含量变化对肝功能变化有预警作用。斑马鱼卵黄囊发生吸收延迟现象，也是斑马鱼肝脏受损的毒性表现。MicroRNA-122(miR-122)在肝细胞中高表达，在调控肝脏生长发育、维持肝脏代谢功能、参与肝细胞应急应答等生命活动过程中发挥重要作用，miRNA-122对药物性肝损伤表现出更高的敏感性，是药物肝毒性的早期预测因子。肝脏组织学和超微结构观察也是评价药物或外源性生物制剂对肝脏影响的重要指标。
此外，跟肝脏功能相关的基因表达变化也能反映肝脏损伤水平。如top2α作为细胞增殖的关键蛋白，在细胞周期中调节DNA的拓扑结构，是细胞分裂过程所必需的。top2α的抑制作用可能影响斑马鱼细胞分裂周期的一个或多个阶段。uhrf1是top2a表达的转录激活因子，是细胞周期进程所必需的，其缺失导致G2/M阻滞，可激活DNA损伤反应通路和凋亡。促炎细胞因子il-1β和il-6是激活的巨噬细胞产生的关键炎症介质，在急性期引发炎症反应。cox2广泛参与外源性或促炎细胞因子诱导的肝脏炎症反应。炎症相关基因表达水平与肝毒性指标(ALT、AST)呈正相关，提示炎症水平与肝细胞损伤水平密切相关。pxr具有调节药物代谢酶和药物转运体的作用。cyp3c1和abcb4参与药物代谢和发挥外排转运体功能。
发明内容
针对现有技术的不足，本发明提供了海洋来源磷脂在制备防护雷公藤多苷造成肝损伤药物中的应用。
本发明采用发育至72hpf(hours post fertilization)的增强绿色荧光蛋白(EGFP)标记肝脏的转基因斑马鱼Tg(L-FABP:EGFP)，发现了鱿鱼性腺磷脂(SPL)对雷公藤多苷(GTW)诱导的肝毒性的保护作用及其机制，实验流程示意图见图1。
本发明以肝脏荧光面积、卵黄囊面积、肝脏转氨酶、miR-122表达和肝脏组织学变化等为评价指标，评价海洋来源磷脂在防护雷公藤多苷造成肝损伤中的作用。
术语说明：
hpe(hours post exposure)：指给药后的小时数。
hpf(hours post fertilization)：指受精后的小时数。
本发明的技术方案如下：
海洋来源磷脂在制备防护雷公藤多苷造成器官损伤药物中的应用。
根据本发明优选的，海洋来源磷脂在制备防护雷公藤多苷造成肝损伤药物中的应用。
根据本发明优选的，所述海洋来源磷脂为鱿鱼性腺磷脂。
进一步优选的，鱿鱼性腺磷脂中，总磷脂质量分数为90％以上。
进一步优选的，所述鱿鱼性腺磷脂的制备方法包括如下步骤：
(1)以鱿鱼生殖腺为原料，用体积分数95％以上的乙醇溶液搅拌提取2-4次，每次提取时间为4-6h；提取液过滤、干燥，得到固形物；
(2)将固形物溶于体积分数95％以上的乙醇溶液，再利用正己烷萃取，收集正己烷层，浓缩后干燥，然后利用正己烷将其配制成0.4-0.6g/mL的溶液，加入9-11倍体积丙酮混匀，静置、沉淀、干燥得到鱿鱼性腺磷脂。
进一步优选的，上述制备方法中，所述原料的含水量按质量分数计为10％以下。
进一步优选的，上述制备方法中，步骤(1)中鱿鱼生殖腺原料与乙醇溶液的质量体积比为1：(8-12)g/mL。
进一步优选的，上述制备方法中，步骤(2)中固形物与乙醇溶液的质量体积比为1:(100～200)g/mL。
进一步优选的，上述制备方法中，步骤(2)中乙醇溶液与正己烷的体积比为1:(7-8)。
更优选的，所述鱿鱼性腺磷脂的制备方法包括如下步骤：以鱿鱼生殖腺为原料，将100g鱿鱼生殖腺用1000mL体积分数95％的乙醇溶液搅拌提取3次，每次提取5h；提取液过滤后，蒸干，溶于50mL体积分数95％乙醇溶液，再利用400mL正己烷萃取，收集正己烷层，浓缩后干燥。之后，利用正己烷将其配制成0.5g/mL的样品溶液，加入10倍体积丙酮混匀，静置24h，沉淀干燥后即得鱿鱼性腺磷脂。
有益效果
本发明发现海洋来源磷脂能够防护雷公藤多苷(GTW)诱导的肝损伤，可以抑制GTW诱导的肝脏转氨酶升高、miR-122表达下降，改善GTW诱导的斑马鱼肝脏组织及其超微结构损伤。本发明为雷公藤多苷药物造成的肝损伤提供了候选保护物质，市场应用前景广阔。
附图说明
图1为本发明研究海洋来源磷脂对雷公藤多苷诱导斑马鱼肝损伤作用的实验流程示意图；
图中：SPL表示鱿鱼性腺磷脂，GTW表示雷公藤多苷；于24、48和72hpe(给药后小时数)对GTW、SPL+GTW处理组斑马鱼进行表型观察，分析相应肝脏和卵黄囊的面积；72hpe时测定谷丙转氨酶(ALT)和谷草转氨酶(AST)活性、非编码小分子RNA(miR-122)的转录水平、观察肝脏组织与超微结构变化，检测相关基因的表达水平。
图2鱿鱼性腺磷脂中各磷脂的成分类型及含量比较图；
图中：cPA表示环磷脂酸，PG表示磷脂酰甘油，CL表示心磷脂，PS表示磷脂酰丝氨酸，LdMePE表示溶血二甲基磷脂酰乙醇胺，PA表示磷脂酸，LPA表示溶血磷脂酸，SM表示鞘磷脂，LPI表示溶血磷脂酰肌醇，dMePE表示二甲基磷脂酰乙醇胺，LPE表示溶血磷脂酰乙醇胺，PI表示磷脂酰肌醇，PE表示磷脂酰乙醇胺，LPC表示溶血磷脂酰胆碱，PC表示磷脂酰胆碱。
图3海洋来源磷脂对雷公藤多苷诱导的斑马鱼肝脏和卵黄囊面积变化的影响图片；
图中：SPL表示鱿鱼性腺磷脂，GTW表示雷公藤多苷；图A为24、48和72hpe肝脏荧光转基因斑马鱼Tg(L-FABP:EGFP)的肝脏形态变化；图B和图C分别为肝脏和卵黄囊面积变化统计分析结果，与空白对照组相比，**P<0.01；与GTW组相比，#P<0.05，##P<0.01，每组选取8条幼鱼用于分析。
图4给药72小时后斑马鱼的相对ALT和AST活性图；
图中：SPL表示海洋来源磷脂，GTW表示雷公藤多苷，ALT表示谷丙转氨酶，AST表示谷草转氨酶；与空白对照组相比，**P<0.01；与GTW组相比，##P<0.01，每组收集60条幼鱼用于检测。
图5给药72小时后斑马鱼miR-122转录水平变化图；
图中：SPL表示鱿鱼性腺磷脂，GTW表示雷公藤多苷，miR表示mircoRNA，即非编码小分子RNA；与空白对照组相比，**P<0.01；与GTW组相比，##P<0.01，每组收集30条幼鱼用于检测。
图6给药72小时后斑马鱼肝脏组织切片HE染色图；
图中：SPL表示鱿鱼性腺磷脂，GTW表示雷公藤多苷；黑色箭头指示空泡，蓝色箭头指示细胞间隙，红色箭头指示局灶性坏死，每组选取15条斑马鱼用于分析，图中比例尺为15μm。
图7给药72小时后斑马鱼肝细胞超微结构变化图；
图中：SPL表示鱿鱼性腺磷脂，GTW表示雷公藤多苷，ER表示内质网，N表示细胞核，M表示线粒体；黄色箭头和红色箭头分别指向空泡和线粒体嵴缺失，每张图片的放大系数见右下角；每组选取15条斑马鱼用于分析。
图8海洋来源磷脂对雷公藤多苷诱导的斑马鱼肝脏损伤相关基因表达变化的影响图；
图中：SPL表示鱿鱼性腺磷脂，GTW表示雷公藤多苷；与空白对照组相比，**P<0.01；与GTW组相比，#P<0.05，##P<0.01，每组收集20条幼鱼用于分析。
图9海洋来源磷脂对雷公藤多苷、乙醇和硫代乙酰胺造成肝损伤防护作用的肝脏形态对比图；
图中SPL表示鱿鱼性腺磷脂，GTW表示雷公藤多苷，Ethanol表示酒精，TAA表示硫代乙酰胺。
图10雷公藤多苷导致的肝损伤与乙醇导致的酒精性脂肪肝、硫代乙酰胺导致的非酒精性脂肪肝的肝脏组织切片HE染色对比图；
图中GTW表示雷公藤多苷，Ethanol表示酒精，TAA表示硫代乙酰胺；黑色箭头指示空泡，红色箭头指示局灶性坏死，黄色箭头指示脂滴。
具体实施方式
下面结合具体的实施例对本发明作进一步阐述，但本发明的保护范围不限于此。
实施例中未注明具体条件的内容，按照常规条件进行；所用试剂或仪器未注明生产厂商的，均为普通市售产品。
主要材料来源
雷公藤多苷(GTW)购自浙江得恩德药厂(批号：0802702)。谷丙转氨酶(ALT)和谷草转氨酶(AST)检测试剂盒均购自南京建成生物工程研究所。
实验例1
鱿鱼性腺磷脂(SPL)的提取制备
鱿鱼性腺磷脂(SPL)由山东省科学院药物筛选技术重点实验室提供。具体的提取方法为：以鱿鱼生殖腺为原料，原料的含水量按质量分数计为10％以下，将100g鱿鱼生殖腺用1000mL体积分数95％的乙醇溶液搅拌提取3次，每次提取5h；提取液过滤后，蒸干，溶于50mL体积分数95％乙醇溶液，再利用400mL正己烷萃取，收集正己烷层，浓缩后干燥称量。之后，利用正己烷将其配制成0.5g/mL的样品溶液，加入10倍体积丙酮混匀，静置24h，沉淀干燥后即得鱿鱼性腺磷脂(SPL)样品，计算样品得率为3.75％。
样品得率＝SPL样品质量/原料质量×100％。
采用钼蓝比色法测定，分析得到SPL样品中总磷脂质量分数为91.3％。基于LC-MS脂质组学技术对SPL脂质成分进行分析，结果显示其各类磷脂的主成分结构类型为包括CL(20:2/16:1/18:2/18:1)、LPC(16:0)、LPE(20:1)、LPI(20:5)、PA(18:0/20:5)、PC(16:0/22:6)、PE(20:1/20:5)、PG(16:0/18:1)、PI(18:0/20:5)、PS(18:0/20:5)、SM(d18:1/14:0)、LPA(16:0)和dMePE(16:0/22:6)，各成分类型的相对含量见图2，相对含量从高到低的前六位分别为PC 49.27±0.16％、PE 26.33±0.3995％、LPC 8.32±0.55％、dMePE 6.20±0.30％、SM 4.50±0.02％和LPE 3.02±0.01％，六种成分的总含量占比超过鱿鱼性腺磷脂(SPL)中总磷脂的97％。
实验例2
实施例1制备的鱿鱼性腺磷脂(SPL)对雷公藤多苷(GTW)造成肝损伤的影响，具体如下：
1.1实验动物
本发明采用性成熟的野生型斑马鱼AB品系和肝脏荧光转基因斑马鱼Tg(L-FABP:EGFP)品系，在黑暗10h照明14h的光周期于28℃条件下饲养，每天定时定量对斑马鱼喂食丰年虾。交配取卵时，取健康成熟的雌雄斑马鱼，按1:2的比例交配，获得受精卵。对受精卵进行消毒和清洗之后，移入斑马鱼胚胎培养水中，在28℃下控光培养。
1.2斑马鱼幼鱼处理分组
在斑马鱼发育至72hpf，体视显微镜下挑选发育正常的斑马鱼幼鱼至6孔板中，随机分为空白对照组：含0.1％DMSO养鱼水；GTW组：5μg/mL；海洋磷脂治疗组6个浓度组：在5μg/mL GTW浓度下联合SPL的6个浓度，浓度依次为0.05、0.1、0.15、0.2、0.25、0.3mg/mL。每组设置3个复孔，每孔20尾仔鱼，每个孔中加入5mL处理液，置于培养箱内培养72h，每天更换新鲜处理液。
1.3斑马鱼幼鱼形态和肝脏观察
于24hpe、48hpe、72hpe，将斑马鱼用质量浓度为0.3‰的三卡因麻醉，然后用3％甲基纤维素固定于载玻片上，将斑马鱼固定侧面体位进行拍照。明场下拍照观察SPL及GTW对斑马鱼整体形态的影响，荧光显微镜下观察SPL及GTW对斑马鱼幼鱼肝脏形态、肝脏荧光面积和卵黄囊面积变化情况。
1.4肝脏转氨酶水平检测
幼鱼经药物处理72h后，取出幼鱼并吸干水，用生理盐水制成质量浓度为10％的组织匀浆，按照产品说明书检测谷丙转氨酶(ALT)和谷草转氨酶(AST)活性。
1.5肝脏组织病理学检测
幼鱼经药物处理72h后，用4％多聚甲醛固定24h，石蜡包埋切片，伊红-苏木精染色，显微镜拍照观察肝脏区域。
1.6肝脏电镜检测
幼鱼经药物处理72h后，用电镜固定液5％戊二醛固定，在透射电镜下观察肝组织超微结构变化。
1.7miR-122成熟体水平检测
幼鱼经药物处理72h后，依照说明书用MiPure Cell/Tissue miRNA Kit(南京诺唯赞)提取斑马鱼组织microRNA(miRNA)，并测定miRNA浓度。使用茎环法逆转录(miRNA 1stStrand cDNA Synthesis Kit(by stem-loop))，对肝脏特异性miR-122进行定量检测。
查询miRbase网站得到miRNA122的成熟体序列(dre-miR-122(MIMAT0001818))，设计茎环逆转录引物和上下游引物，以snU6为内参(U6 snRNA(NR_004394.1GI:161087014))，设计的引物序列见表1。
表1 miR-122和内参snU6的茎环逆转录和上下游引物序列
1.8实时荧光定量PCR测定基因表达量变化
药物处理72h后，将幼鱼组织用匀浆器充分研磨，组织总RNA用纳米磁珠法斑马鱼总RNA提取试剂盒(山东善诺惠众生物科技有限公司)提取。Nanodrop One超微量分光光度计(Thermo)测定总RNA浓度后，用LightCycler 96实时荧光PCR仪测定与肝脏损伤相关的基因表达量。实时荧光PCR扩增反应的条件为95℃预变性5min，1个循环后，每个循环反应95℃10s，退火60℃30s，共40个循环后，溶解曲线95℃15s，60℃60s，95℃15s1个循环。以β-actin为内参，对结果进行相对定量分析。基因引物序列见表2。
表2用于实时荧光定量PCR的基因扩增引物序列
1.9数据分析
采用数据统计软件SPSS 16.0对实验数据进行处理，所有实验数据用mean±SE表示，统计学差异通过ANOVA进行分析，组间比较通过Tukey检验。P<0.05为有显著性差异，P<0.01为有极显著性差异。
2.实验结果
2.1 SPL对GTW诱导的斑马鱼肝脏毒性的影响
斑马鱼肝脏和卵黄囊的面积变化是反映肝功能的检测指标。与空白对照组相比，GTW组肝脏的荧光面积在24hpe、48hpe和72hpe时显著减少(图3中A、B)，而卵黄囊面积在48hpe和72hpe时显著增加(图3中C)。SPL在0.05～0.3mg/mL之间可以一定程度逆转GTW诱导的肝脏毒性，其中0.1mg/mL SPL的保肝活性最高，0.1mg/mL SPL组斑马鱼幼鱼肝脏荧光面积和卵黄囊面积趋于正常。因此，选择0.1mg/mL SPL+GTW共处理，以单独暴露SPL的斑马鱼幼鱼为实验对照，进一步探讨SPL对GTW诱导的肝毒性的保护作用。
2.2 SPL抑制GTW诱导的肝脏转氨酶升高
为了更好地表征SPL对GTW诱导的肝毒性的影响，分析了72hpe下斑马鱼肝毒性的转氨酶的变化。与对照组相比，GTW组的ALT和AST活性显著升高(图4)。而SPL+GTW共处理显著逆转了这一升高，表明SPL对GTW诱导的斑马鱼ALT和AST升高具有抑制作用。
2.3 SPL对GTW诱导的斑马鱼miR-122的表达发生逆转
通过检测miR-122的表达水平来评价SPL的肝保护作用。结果显示，GTW暴露后miR-122表达显著降低。然而，SPL+GTW共处理显著逆转了这种下降(图5)，表明SPL可预防或改善GTW诱导的肝损伤。
2.4 SPL改善了GTW诱导的斑马鱼肝脏组织损伤
由于组织学改变是肝损伤的直接表现，通过组织学切片来证明SPL治疗对GTW诱导的病理改变的保护作用。与对照组相比(图6中A)，GTW组肝组织明显异常，表现为细胞不完整，细胞边界不清晰，胞浆空泡化，局灶性坏死(图6中B)。SPL+GTW共处理后，肝细胞组织病理学改变减少，细胞完整，细胞边界清晰，胞浆分布均匀，空泡减少，局灶性坏死现象得到改善(图6中C)。
2.5 SPL改善了GTW诱导的斑马鱼肝组织超微结构损伤
通过超微结构观察进一步验证SPL对GTW诱导的肝损伤的缓解作用。如图7所示，与空白对照组相比，GTW处理组肝细胞线粒体嵴部分缺失(图7中B a-d)和出现空泡(图7中Bb-c)。经SPL+GTW共处理后，肝细胞线粒体形态正常，细胞内空泡结构明显减少(图7中C)。
2.6 SPL对GTW诱导的斑马鱼肝脏损伤相关基因表达变化的影响
为了揭示SPL对GTW诱导的肝毒性保护作用的机制，检测了给药后72小时(hpe)下细胞周期、炎症反应和代谢/运输相关基因的表达变化。
对于细胞周期相关基因(图8中A)，GTW组中DNA拓扑异构酶IIα(top2α)和泛素样含PHD和环指域1(uhrf1)转录水平显著降低。与GTW组相比，SPL+GTW处理能显著抑制GTW诱导的细胞周期阻滞，表明SPL可减轻GTW诱导的细胞周期阻滞。
炎症因子如白细胞介素-1β(il1β)、白细胞介素-6(il6)、环氧化酶-2(cox2)等在炎症的发生和发展中起着关键作用。发现il1β、il6和cox2基因的表达在GTW组中显著上调(图8中B)。与GTW组相比，GTW+SPL联合处理显著逆转了这些基因的表达，这意味着SPL抑制了炎症因子的过度生成。
另外，发现在GTW组中，孕烷X受体pxr基因表达显著降低，cyp3c1和abcb4基因表达显著增加(图8中C)。同样，SPL+GTW联合处理可使这些基因的表达恢复到对照组水平。
实验例3
实施例1制备的鱿鱼性腺磷脂(SPL)对乙醇(Ethanol)和硫代乙酰胺(TAA)造成肝损伤的影响，具体如下：
1实验方法
1.1实验动物
本实验例所用实验动物及养殖方式同实验例2。
1.2斑马鱼幼鱼处理分组
在体视显微镜下选取发育正常3dpf的斑马鱼幼鱼，移至6孔板中，每孔20条。分别选择0.9％Ethanol(V/V)、10mM TAA诱导斑马鱼酒精性和非酒精性脂肪肝模型。将斑马鱼随机分为空白对照组(含0.1％DMSO养鱼水)、Ethanol组、Ethanol+0.1mg/mL SPL组、TAA组以及TAA+0.1mg/mL SPL处理组，每组设置3个复孔，置于28.5℃培养箱中培养，每天更换处理液。于处理后24h和48h，利用荧光显微镜FSX100(Olympus,Tokyo,Japan)在明场和荧光视野下观察斑马鱼肝脏形态并拍照，并将其与同一时期斑马鱼经5μg/mL GTW、5μg/mL GTW+0.1mg/mL SPL处理相同时间下的肝脏面积进行比较。
2实验结果
实验结果如图9所示，24和48hpe时，与Ethanol组相比，Ethanol+0.1mg/mL SPL组斑马鱼的肝脏面积有一定程度的增加，表明0.1mg/mL SPL对酒精性肝损伤有一定的改善作用；与TAA组相比，TAA+0.1mg/mL SPL组斑马鱼肝脏面积无显著变化，表明0.1mg/mL SPL对TAA导致的肝损伤无修复作用。然而，与0.1mg/mL SPL对Ethanol诱导脂肪肝的改善作用相比，0.1mg/mL SPL对GTW诱导肝损伤在24和48hpe时的肝脏面积明显增大，表明SPL对雷公藤多苷造成肝损伤的改善效果更好。
SPL对酒精诱导脂肪肝的防护改善机制与SPL对雷公藤多苷诱导肝损伤的防护改善机制不同。SPL对酒精诱导脂肪肝的防护改善机制与增加脂质的摄取、运输和代谢有关，减少肝组织脂滴。SPL对雷公藤多苷诱导肝损伤的防护改善机制与恢复细胞周期、抑制过度产生的炎症因子和维持代谢/转运稳态有关。
酒精性或者非酒精性脂肪肝也出现细胞间疏松，胞浆空泡化，但是没有局灶性坏死现象。雷公藤多苷造成的肝损伤肝组织没有脂滴，见图10，进一步证明雷公藤多苷造成的肝损伤机制与酒精性或者非酒精性脂肪肝损伤机制不同，造成肝损伤的症状也有明显差异。
综上，本发明发现海洋来源磷脂能够防护雷公藤多苷诱导的肝损伤；本发明发现，5μg/mL GTW对发育至72hpf的斑马鱼幼鱼有显著的肝毒性，主要表现为GTW导致幼鱼肝脏萎缩，转氨酶水平升高以及肝组织结构发生变化。而SPL+GTW共处理可以通过逆转斑马鱼幼鱼肝脏面积减少、转氨酶(AST和ALT)升高和miR-122下调，显著减轻GTW诱导的肝毒性。进一步研究表明，SPL对GTW诱导的肝损伤的保护作用可能与恢复细胞周期、抑制过度产生的炎症因子和维持代谢/转运稳态有关。
在本发明的研究中，观察到暴露于GTW的斑马鱼幼鱼体内AST和ALT显著增加。但SPL+GTW共处理可显著抑制上述升高，提示SPL对GTW诱导的肝损伤具有保护作用。
在本发明研究中，发现GTW处理组miRNA-122的表达显著降低，表明GTW对miRNA-122转录有抑制作用。SPL+GTW共同处理斑马鱼后，降低的miRNA-122表达显著恢复，表明SPL通过保护miRNA-122调控的生物过程改善GTW诱导的肝毒性。
本发明实验结果发现，斑马鱼幼鱼经GTW处理后的肝脏发生严重的肝脏组织病理学改变，出现肝细胞空泡化和局灶性坏死。
GTW组肝细胞超微结构改变，线粒体嵴缺失，出现空泡。但SPL+GTW共同作用后，其组织病理学和超微结构损伤均减轻，说明SPL对GTW诱导的肝细胞损伤具有保护作用。
本发明进一步测定了一系列涉及细胞周期、炎症反应、代谢和转运的基因的表达。在本发明的研究中，GTW暴露显著下调top2α和uhrf1的表达，可能会阻碍正常的细胞周期，导致肝细胞变性和坏死。然而，在SPL+GTW的共同处理中，这种情况发生了逆转，提示SPL可能重新激活top2α和uhrf1的表达，从而防止DNA损伤，从而促进细胞周期进展。
GTW诱导的肝毒性也与炎症反应有关，实验发现GTW导致炎症相关基因(il-1β、il-6和cox-2)的表达显著升高。因此，发明人推测GTW诱导的il1β表达水平升高可导致il6的过量产生，从而导致斑马鱼肝脏损伤。而SPL+GTW共处理显著抑制了肝脏中il1β、il6和cox2表达的升高，证明SPL可能通过抑制促炎细胞因子的过量产生，有效改善炎症反应发挥抗炎作用。
此外，SPL对GTW诱导的肝毒性的保护作用可能与机体代谢和转运的稳态恢复有关。发明人发现，GTW暴露可以显著下调pxr的转录水平。pxr下调，可降低斑马鱼肝脏的药物代谢和转运功能，导致斑马鱼体内GTW的积累，从而发生药物性肝损伤。相反，cyp3c1和abcb4的转录水平显著升高，cyp3c1和abcb4的上调可能是对机体pxr表达降低的调节作用，在一定程度上减轻GTW引起的药物代谢和转运体功能损伤。在SPL+GTW组中，pxr、cyp3c1和abcb4的表达恢复到正常水平，这表明SPL有助于维持肝脏内代谢和转运的稳态，从而抑制GTW在肝脏细胞内蓄积。因此，这些与药物代谢和转运蛋白相关基因的转录变化可能是SPL缓解GTW诱导的肝损伤的保护机制。
本发明发现雷公藤多苷能够诱导细胞周期阻滞、诱导炎症因子的过度生成，降低药物代谢和转运功能等多方面影响，造成肝损伤。
本发明发现海洋来源磷脂能够防护雷公藤多苷(GTW)诱导的肝损伤，本发明为雷公藤多苷药物造成的肝损伤提供了候选保护物质，市场应用前景广阔。
Claims (6)
1.海洋来源磷脂在制备防护雷公藤多苷造成肝损伤药物中的应用；所述海洋来源磷脂为鱿鱼性腺磷脂；
所述鱿鱼性腺磷脂中，总磷脂质量分数为90%以上；
所述鱿鱼性腺磷脂的制备方法，包括如下步骤：
（1）以鱿鱼生殖腺为原料，用体积分数95%以上的乙醇溶液搅拌提取2-4次，每次提取时间为4-6h；提取液过滤、干燥，得到固形物；
（2）将固形物溶于体积分数95%以上的乙醇溶液，再利用正己烷萃取，收集正己烷层，浓缩后干燥，然后利用正己烷将其配制成0.4-0.6g/mL的溶液，加入9-11倍体积丙酮混匀，静置、沉淀、干燥得到鱿鱼性腺磷脂。
2.如权利要求1所述应用，其特征在于，步骤（1）中所述原料的含水量按质量分数计为10%以下。
3.如权利要求1所述应用，其特征在于，步骤（1）中鱿鱼生殖腺原料与乙醇溶液的质量体积比为1：（8-12）g/mL。
4.如权利要求1所述应用，其特征在于，步骤（2）中固形物与乙醇溶液的质量体积比为1:（100~200）g/mL。
5.如权利要求1所述应用，其特征在于，步骤（2）中乙醇溶液与正己烷的体积比为1:（7-8）。
6.如权利要求1所述应用，其特征在于，所述鱿鱼性腺磷脂的制备方法，包括如下步骤：以鱿鱼生殖腺为原料，将100 g鱿鱼生殖腺用1000 mL体积分数95%的乙醇溶液搅拌提取3次，每次提取5 h；提取液过滤后，蒸干，溶于50 mL体积分数95%乙醇溶液，再利用400 mL正己烷萃取，收集正己烷层，浓缩后干燥，利用正己烷将其配制成0.5 g/mL的样品溶液，加入10倍体积丙酮混匀，静置24 h，沉淀干燥后即得鱿鱼性腺磷脂。
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