CN115317502B - 一种线性硬毛藻提取的粗多糖在制备非酒精性脂肪性肝药物中的应用 - Google Patents
一种线性硬毛藻提取的粗多糖在制备非酒精性脂肪性肝药物中的应用 Download PDFInfo
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Abstract
本发明涉及生物医药技术领域，具体为一种线性硬毛藻提取的粗多糖在制备非酒精性脂肪性肝药物中的应用，包括以下步骤：多糖提取、设置分组、饲养条件、进行建模、建模后检测。本发明通过实验证明，从野生毛藻中用热水提取粗多糖CLH，在高脂诱导的NAFLD小鼠模型中，粗多糖CLH具有抑制肥胖，降低血糖，减少肝脏脂质蓄积，起到对NAFLD的治疗作用，基于CLH治疗NAFLD的良好作用，其可用于NAFLD患者的临床治疗药物的研发，效果显著优于其他试药。
Description
技术领域
本发明涉及生物医药技术领域，具体为一种线性硬毛藻提取的粗多糖在制备非酒精性脂肪性肝药物中的应用。
背景技术
非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)是慢性肝病(如肝纤维化和肝癌)和代谢紊乱(如肥胖、Ⅱ型糖尿病和动脉粥样硬化)的最常见前兆之一[1]。NAFLD患者无法从这种情况中康复，相反，该疾病会继续发展为非酒精性脂肪性肝炎(NASH)、肝细胞纤维化、肝硬化，甚至肝细胞，而且作为代谢综合征的重要组分与心脑血管疾病密切相关，严重影响了人们的身体健康和生活质量，也给社会带来沉重负担。除了饮食和运动等干预措施外，目前还没有可用于治疗NAFLD的靶向药物[2]。
毛藻属属于绿藻门，是生长在河口和海洋环境[3]中的不分枝丝状单细胞绿藻。大约76种已知的毛形属[4]。其中，亚麻属植物具有很高的湿度，干燥后的残渣主要由蛋白质和碳水化合物组成。毛藻主要作为生态调节剂调节河口生境的营养有效性，是一种膳食纤维，含有多种微量元素[5,6]。毛藻在我国分布广泛，在医药和食品研究中均有应用。
最近，我们在中国荣城天鹅湖沿岸采集了一种野生毛藻，经提取、纯化和鉴定了多糖，发现提取的多糖几乎是硫酸阿拉伯半乳聚糖。因此本发明旨在研究线性硬毛藻提取的粗多糖在治疗小鼠非酒精性脂肪性肝病中的作用。
发明内容
针对相关技术中的问题，本发明提出的一种线性硬毛藻提取的粗多糖在制备非酒精性脂肪性肝药物中的应用，以克服现有相关技术所存在的上述技术问题，本发明的目的是从野生毛藻中用热水提取粗多糖CLH，在高脂诱导的NAFLD小鼠模型中，粗多糖CLH具有抑制肥胖，降低血糖，减少肝脏脂质蓄积，起到对NAFLD的治疗作用，基于CLH治疗NAFLD的良好作用，其可用于NAFLD患者的临床治疗药物的研发，效果显著优于其他试药。
为实现上述目的，本发明提供如下技术方案：
本发明提供一种线性硬毛藻提取的粗多糖在制备非酒精性脂肪性肝药物中的应用，包括以下步骤：
(1)多糖提取：将野生毛藻在55℃干燥后粉碎，后脱脂脱盐并浓缩冻干，用热水提取粗多糖得CLH；
(2)设置分组：将野生型雄性C57BL/6J小鼠随机分为对照组、模型组和预防组，每组包含小鼠6只；
(3)饲养条件：分笼饲养于SPF级动物饲养室，每笼3只小鼠，鼠龄为8周，所有小鼠先用普通维持饲料饲养一周，以便使小鼠适应环境；
(4)进行建模：适应环境之后开始进行建模，模型组(HFD)和治疗组(CLH-50、CLH-150)小鼠给予高脂饲料，对照组(NC)给予普通维持饲料，每天一次15g定量补给饲料，保证50g/笼；
高低剂量治疗组小鼠分别给予CLH 150mg/kg/天和50mg/kg/天，溶解在蒸馏水中口服，模型组和对照组给予等体积的蒸馏水；
(5)建模后检测：建模9周后通过腹腔注射葡萄糖进行空腹血糖指标及糖耐量检测，于10周进行摘眼球取血并用断颈法将小鼠处死，检测小鼠血糖血脂四项指标(TC、TG、LDL-c、HDL-c)和肝功指标(AST和ALT)，取小鼠新鲜肝脏组织进行匀浆，检测肝脏内TC和TG的含量，另取肝组织做石蜡和冰冻切片，分别进行H.E.和油红O染色。
优选的，所述步骤(1)中脱脂为藻粉在80℃下用85％乙醇提取3h，提取的次数为三次，所述藻粉与乙醇的料液比为1:20。
优选的，所述步骤(1)中脱盐为提取液旋转蒸发浓缩，加入4体积乙醇沉淀，将沉淀再溶解，与水(7000Da MWCO)透析3天，除去盐。
优选的，所述步骤(2)中的野生型小鼠具体为C57BL/6背景的野生型小鼠。
优选的，所述步骤(2)中的小鼠全部为雄性。
优选的，所述步骤(3)中的饲养条件，具体为：饲养室内温度为22～26℃，湿度为40％～60％，光照条件为12小时光照和12小时黑暗交替。
与现有技术相比，本发明的有益效果是：
本发明为一种线性硬毛藻提取的粗多糖在制备非酒精性脂肪性肝药物中的应用，本发明从野生毛藻中用热水提取粗多糖CLH，在高脂诱导的NAFLD小鼠模型中，粗多糖CLH具有抑制肥胖，降低血糖，减少肝脏脂质蓄积，起到对NAFLD的治疗作用，基于CLH治疗NAFLD的良好作用，其可用于NAFLD患者的临床治疗药物的研发。
附图说明
图1是CLH提取的策略图。
图2是C57BL/6J小鼠的体重趋势图(****：p＜0.001vs NC组；#：p＜0.05vs HFD组)。
图3是C57BL/6J小鼠的空腹血糖结果图(****：p＜0.001vs NC组；##：p＜0.01vsHFD组；####：p＜0.001vs HFD组)。
图4是C57BL/6J小鼠通过腹腔注射葡萄糖耐量结果图。
A为通过腹腔注射葡萄糖后不同时间点小鼠血糖水平统计图，B为各组小鼠糖耐量曲线下面积(area under the curve，AUC)比较图(****：p＜0.001vs NC组；##：p＜0.01vsHFD组)。
图5是C57BL/6J小鼠血脂四项指标结果图。
A是小鼠血清TG结果，B是小鼠血清TC结果，C是小鼠血清HDL-c结果，D是小鼠血清LDL-c结果(*：p＜0.05vs NC组；***：p＜0.001vs NC组；#：p＜0.05vs HFD组)。
图6是C57BL/6J小鼠肝功指标结果图。
A是小鼠血清AST结果，B是小鼠血清ALT结果(**：p＜0.01vs NC组；##：p＜0.01vsHFD组)。
图7是C57BL/6J小鼠肝脏TG和TC结果图。
A是小鼠肝脏TG结果，B是小鼠肝脏TC结果(***：p＜0.001vs NC组；#：p＜0.05vsHFD组)。
图8是C57BL/6J小鼠肝重/体重结果图(****：p＜0.001vs NC组)。
图9是C57BL/6J小鼠肝脏形态图。
图10是C57BL/6J小鼠肝脏H.E.染色X200病理切片图。
图11是C57BL/6J小鼠肝脏油红染色X200病理切片图。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
多糖提取
本发明中的线性硬毛藻取自我国荣城天鹅湖，样品在自来水中彻底洗净后，风干后放在塑料袋中，室温下置于干燥处保存。
将野生线性硬毛藻在55℃干燥后粉碎。
为了去除脂质，藻粉在80℃下用85％乙醇(料液比1:20)提取3h(3次)。脱脂后的残渣在55℃下干燥，用室温水(料液比1:45)提取3h(3次)。
85℃用热水(料液比1:45)进一步提取3h(3次)过滤。
结合提取液，旋转蒸发浓缩，加入4体积乙醇沉淀。
将沉淀再溶解，与水(7000Da MWCO)透析3天，除去盐，浓缩并冻干。提取策略图见图1。
实验用动物及饲养
本发明方法中SPF级C57BL/6野生小鼠购买自南京大学南京生物医药研究院。
本发明方法中的动物饲料为高脂饲料(HFD)和普通维持饲料(ND)，其中高脂饲料购买自北京蕙特比科技有限公司，产品编号为D12492，普通维持饲料购买自江苏省协同医药生物工程有限责任公司，产品编号为1010011。
实施例1
小鼠非酒精性脂肪肝病模型的获得
1.实验动物：选用8周龄，雄性，C57BL/6野生小鼠，体重为(26±2)g。
2.饲养环境：实验动物均饲养于SPF级动物房，饲养室内温度为22～26℃，湿度为40％～60％，光照条件为12小时光照和12小时黑暗交替。
3.分组饲养：将小鼠分为四组，分别为NC、HFD、CLH-50及CLH-150。每笼饲养4只小鼠，小鼠详细分组情况如下表1所示。
所有小鼠先用普通维持饲料饲养1周，之后开始建模，建模时长为10周，每天上午8点添加一次饲料，保证50g/笼，饮水供应充足，NC和HFD水源为蒸馏水，CLH-50和CLH-150组分别为蒸馏水中溶解50mg/kg/天和150mg/kg/天的CLH，为每周称量体重2-3次。
表1
实施例2
小鼠血浆生化指标的测定
1.建模10周后，对所有小鼠禁食12小时，然后将小鼠处死并进行取材，对小鼠腹腔注射4％水合氯醛(0.1ml/10g)进行麻醉，然后进行摘眼球取血，将血置于含有EDTA抗凝剂的采血管中，摇晃均匀，4℃放置10分钟，离心分离血浆和血细胞，离心条件为4℃下1500g离心5分钟，取上层血浆。
2.通过使用试剂盒(南京建成生物工程研究所，中国)和酶标仪将步骤1中的上层血浆TC、TG、LDL-c、HDL-c、ALT和AST的含量进行检测。
本实施例表明HFD组小鼠与NC组小鼠相比TC、TG、LDL-c、HDL-c、ALT和AST均有明显升高，HFD组小鼠相较于CLH-50预防组小鼠TG、TC、ALT和LDL-c均有明显降低，HFD组小鼠较CLH-150与预防组小鼠TC、TG、LDL-c、ALT和AST均有明显降低。这些结果说明CLH对预防NAFLD小鼠的血清脂质恶化上的优势较明显。
实施例3
小鼠空腹血糖水平的测定
将所有待实验的小鼠从上午8:00至下午2:00间禁食(不禁水)，即禁食6小时后开始实验操作。
1.血糖仪准备：检查血糖仪(OMRON)电池，按开关，将试纸正确放入下方插槽，屏幕显示与血糖试纸条相应代码的数字，随后显示滴血图案，提示血糖仪进入待测状态。
2.固定小鼠：右手抓鼠尾，左手持一块毛巾，将毛巾对折，用拇指和食指捏住毛巾对折处，将鼠头部和身体包入手掌内的毛巾，拇指和食指在将鼠尾根部固定。
3.剪尾：眼科剪迅速在距鼠尾末端0.1-0.2cm处剪下鼠尾，待血滴自行流出。
4.血糖检测：将血糖仪试纸边缘轻触血滴，血液浸入试纸，血糖仪倒计时5秒显示读数。
本实施例表明经空腹血糖检测后发现在HFD组小鼠与NC组小鼠相比空腹血糖显著升高(p＜0.001)，HFD组小鼠空腹血糖水平较CLH-50和CLH-150组均有显著下降(p＜0.01，p＜0.001)，表明CLH预防能影响小鼠在HFD饲养状态下的糖代谢稳态并能显著提高小鼠的糖代谢能力。
实施例4
葡萄糖耐量实验(intraperitoneal glucose tolerance test，IPGTT)实验第9周，进行腹腔注射葡萄糖实验(IPGTT)，以评价小鼠机体对糖耐受能力。
1.在测血糖之前，先测量小鼠的空腹体重，根据10μL/g计算葡萄糖的注射体积。
2.先检测葡糖糖注射前即0分钟时空腹血糖，在检测完毕后迅速经腹腔注射葡萄糖液。
3.腹腔注射操作方法：①固定小鼠；抓起小鼠，左手的小指和无名指抓小鼠的尾巴，另三手指抓住小鼠的颈部，使小鼠的头部向下，将小鼠腹部充分暴露。②进针定位及注射：从腹部一侧进针右手持注射器，将尖端与小鼠腹部成45°的夹角，进针，回抽，注射时针头在腹部皮下穿行一小段距离，穿过腹中线后在腹部另一侧进入腹腔，注射完药物后，缓缓拔出针头，并轻微旋转针头，防止漏液。
4.分别于腹腔注射后30分、60分、120分钟时间点剪尾测小鼠血糖值，并记录血糖数值和检测时间。
进一步，通过腹腔注射葡萄糖耐量实验(IPGTT)来评估各组小鼠对葡萄糖的处理能力，在实验第9周，通过注射1.0g/kg体重的葡萄糖后，绘制0分、30分、60分、120分钟的曲线并绘制小鼠血糖曲线下面积(area under the curve，AUC)。
本实施例表明HFD组、CLH-50和CLH-150组的小鼠血糖水平在30分钟时间点剧增达到峰值，随着时间推移至注射后60分钟，三组小鼠血糖水平稍微下降，但仍处于高于NC组血糖水平，在120分钟时恢复至空腹血糖水平。比较各组小鼠血糖曲线下面积(area underthe curve，AUC)，发现HFD组小鼠的AUC显著高于NC组(p＜0.001)，CLH-150预防组小鼠的AUC显著低于HFD组(p＜0.01)。表明CLH对维持糖代谢稳态具有强大的调控能力。
实施例5
肝脏大体外观及肝脏脂质水平的测定
1.终末肝脏组织取材
1)小鼠称重后，迅速脱颈处死。仰卧固定小鼠，用蒸馏水将小鼠胸部，腹部毛发润湿。
2)用一镊子钳夹小鼠腹部正中皮肤，沿腹部正中向头部剪开皮肤至剑突下，向尾端剪开皮肤，逐层暴露皮下筋膜，肌肉等，打开腹腔，充分暴露各脏器。
3)迅速找到并取下小鼠的肝脏，将取下的肝脏标本置于灭菌纱布上，拭干肝脏表面上残留血液，将肝脏置于无菌培养皿中，迅速拍照，称重。
2.小鼠肝组织脂质检测
1)从-80℃冰箱取出肝脏组织样品，称量10mg组织，使用研磨器将肝脏组织研磨成切成碎片并放入含有90uL的无水乙醇的试管中。
2)3500×g离心两次5分钟，得到上清液。使用试剂盒(南京建成生物工程研究所，中国)和酶标仪检测上清液中的TC和TG含量。
本实施例显示HFD小鼠相较于NC组小鼠，肝脏中TG的含量明显升高(P<0.001)，经CLH预防后与HFD组小鼠相比肝脏中TG的含量显著降低(P<0.05)，表明CLH对肝脏脂质起到了一定的预防作用。
实施例6
肝脏组织病理学检测
具体以一只小鼠为例，眼眶取血后，断颈法将小鼠处死，剖开小鼠腹部，取新鲜肝脏一块，分为两份，一份用10％中性福尔马林固定液固定，用于做石蜡切片进行H.E.染色；另一份用于做石蜡切片进行油红O染色。
由病理切片可以看出，NC组小鼠肝组织结构完整，细胞核质饱满，肝细胞内脂滴含量较少。高脂饲料喂养的HFD组小鼠肝脏细胞有较为明显的气球样变和脂肪浸润，脂质小滴含量较对照组明显增加，应发展为轻中度脂肪肝。而CLH干预后肝小叶结构相对正常，肝细胞脂肪变性明显改善，肝细胞坏死较少，减少了肝脏切片中的脂肪堆积且高剂量CLH-150组脂肪堆积较HFD差异更显著。该实施例进一步说明CLH能够很好预防小鼠在HFD饲养状态下的肝脏脂肪变性。
参考文献：
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尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
Claims (1)
1.一种线性硬毛藻提取的粗多糖在制备非酒精性脂肪性肝药物中的应用，其特征在于，粗多糖提取的步骤如下：将野生毛藻在55℃干燥后粉碎，后脱脂脱盐并浓缩冻干，用热水提取得到粗多糖CLH；脱脂为藻粉在80℃下用85%乙醇提取3 h，提取的次数为三次，所述藻粉与乙醇的料液比为1:20；脱盐为提取液旋转蒸发浓缩，加入4体积乙醇沉淀，将沉淀再溶解，与水透析3天，截留分子量为7000Da，除去盐。
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