CN117137975A - 鱼腥草来源细胞外囊泡样颗粒在制备预防或治疗脑卒中药物中的应用 - Google Patents
鱼腥草来源细胞外囊泡样颗粒在制备预防或治疗脑卒中药物中的应用 Download PDFInfo
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Abstract
本发明公开了鱼腥草来源细胞外囊泡样颗粒在制备预防或治疗脑卒中药物中的应用。鱼腥草来源细胞外囊泡样颗粒通过以下步骤提取：将新鲜鱼腥草清洗干净，晾干，榨取鱼腥草汁；将鱼腥草汁连续低速梯度离心后高速离心，离心后取上清，弃沉淀；将取得的上清超速离心，于冷冻超速离心机中，在4℃下超速离心，离心后弃上清取沉淀，沉淀悬浮于磷酸盐缓冲液中；将悬浮液过滤，滤液再次于冷冻超速离心机中，在4℃下再次超速离心，离心后弃上清取沉淀，沉淀悬浮于磷酸盐缓冲液中；再次将悬浮液过滤后得到无菌鱼腥草来源细胞外囊泡样颗粒。本发明表明鱼腥草来源细胞外囊泡样颗粒具有减轻缺血再灌注损伤的作用，可以保护缺糖缺氧复氧模型下的神经元细胞。
Description
技术领域
本发明属于医药技术领域，具体涉及鱼腥草来源细胞外囊泡样颗粒在制备预防或治疗脑卒中药物中的应用。
背景技术
鱼腥草为三白草科蕺菜属(拉丁学名：Houttuynia cordata Thunb)的干燥地上部分，别称为折耳根、猪鼻孔和九莲草等。鱼腥草味辛、微寒、归肺经，具有清热解毒、利尿通淋、止血、祛痰止咳等多种功能。在《本草纲目》《滇南本草》等多部古代草本医书中均有记载。
CN112569288A公开了一种用于脑卒中NMN药物组合物，由包括NMN、鱼腥草提取物以及其他药学上可接受的辅料组成。所述NMN和鱼腥草提取物的重量份数比为1-10：50-400。研究表明，鱼腥草提取物对NMN对神经损伤的保护起到了促进作用，但是单独的鱼腥草提取物对于脑卒中后氧化应激造成的神经损伤效果不显著。
目前有关于鱼腥草来源细胞外囊泡样颗粒的脑卒中作用尚未见报道。
发明内容
针对现有技术中的不足之处，本发明提供一种鱼腥草来源细胞外囊泡样颗粒在制备预防或治疗脑卒中药物中的应用。
为实现上述目的，本发明采用如下技术方案：
鱼腥草来源细胞外囊泡样颗粒在制备脑卒中药物中的应用，所述鱼腥草来源细胞外囊泡样颗粒通过以下步骤提取：
1.1、将新鲜鱼腥草清洗干净，晾干，榨取鱼腥草汁；
1.2、将鱼腥草汁连续离心，在4℃下，低俗梯度离心后高速离心，离心后取上清，弃沉淀；
1.3、将取得的上清超速离心，于冷冻超速离心机中，在4℃下，150000g离心，离心后弃上清取沉淀，沉淀悬浮于1-2mL的磷酸盐缓冲液中；
1.4、将悬浮液过滤，滤液再次于冷冻超速离心机中，在4℃下，再次超速离心，离心后弃上清取沉淀，沉淀悬浮于1-2mL的磷酸盐缓冲液中；
1.5、再次将悬浮液过滤后得到无菌鱼腥草来源细胞外囊泡样颗粒。
优选地，步骤1.2中所述低速梯度离心后高速离心的具体步骤是：1000g离心10分钟，3000g离心20分钟，10000g离心40分钟。
优选地，步骤1.4、步骤1.5中所述过滤选择0.22μm的滤膜进行。
优选地，所述药物的剂型为药剂学上允许的口服剂型、注射剂型或粉针剂型。
优选地，所述药物还包括药学上可接受的辅料，例如稀释剂、调味剂、粘合剂、填充剂、增稠剂、润滑剂或pH调节剂等。
本发明还提供一种预防或治疗缺血性脑卒中的药物，所述药物的有效成分包括有效剂量在100μg/kg-400μg/kg的鱼腥草来源细胞外囊泡样颗粒。
采用大脑中动脉栓塞模型(MCAO)的SD大鼠脑缺血再灌注模型以及缺糖缺氧/复氧(OGD/R)细胞模型，大鼠给在再灌注前给予不同浓度的鱼腥草来源细胞外囊泡样颗粒处理，细胞在缺氧复氧时分别给予鱼腥草来源细胞外囊泡样颗粒处理。结果显示，400μg/kg鱼腥草来源细胞外囊泡样颗粒可以明显降低大鼠的脑梗死面积、改善行为学指标、保护血脑屏障；10μg/mL鱼腥草来源细胞外囊泡样颗粒可以降低细胞凋亡以及铁死亡水平。
相对于现有技术，本发明具有以下有益效果：
本发明创造性地提取鱼腥草来源的细胞外囊泡，并对其生理功效进行了研究，发现其可以对抗大鼠缺血再灌注后脑损伤，可以保护缺糖缺氧复氧模型下的神经元细胞，具有用于制备预防或治疗脑卒中药物的潜力，为预防或治疗脑卒中的方法提供了新的策略。
附图说明
图1为本发明实施例中得到的细胞外囊泡样颗粒的粒径图以及免疫印迹图；
图2为本发明实施例中大鼠尾静脉注射不同浓度鱼腥草来源细胞外囊泡样颗粒脑梗死体积的变化图；
图3a为本发明实施例中大鼠尾静脉注射鱼腥草来源细胞外囊泡样颗粒后脑水肿水平的变化图；
图3b为本发明实施例中大鼠尾静脉注射鱼腥草来源细胞外囊泡样颗粒后伊文思蓝染色水平的变化图；
图4a为本发明实施例中大鼠尾静脉注射鱼腥草来源细胞外囊泡样颗粒后自主运动路径的变化图；
图4b为本发明实施例中大鼠尾静脉注射鱼腥草来源细胞外囊泡样颗粒后总路程与中心区域所占时间的结果统计图；
图5为本发明实施例中细胞给予不同浓度鱼腥草来源细胞外囊泡样颗粒后细胞活性的结果统计图；
图6a为本发明实施例中细胞给予鱼腥草来源细胞外囊泡样颗粒后凋亡水平和铁死亡水平的变化图；
图6b为本发明实施例中细胞给予鱼腥草来源细胞外囊泡样颗粒后凋亡水平和铁死亡水平的统计图。
具体实施方式
以下结合附图和具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
实施例
1.实验材料和仪器
1.1实验材料：新鲜鱼腥草、BCA试剂盒、线栓、2',3',5'-三苯基氯化四氮唑(TTC)、伊文思蓝(EB)、三氯乙酸(TCA)、ECL试剂盒、彩色凝胶快速试剂盒、异氟烷。
1.2实验动物：徐州医科大学动物中心提供的本地SD大鼠。
1.3实验仪器：37℃恒温箱、超净台、酶标仪、电子秤、移液枪、胰岛素针。
2.实验步骤
2.1鱼腥草来源细胞外囊泡样颗粒的提取
新鲜鱼腥草样品的细胞外囊泡通过连续差速离心、超离心分离得到；
2.1.1将新鲜鱼腥草清洗干净，晾干，榨取鱼腥草汁；
2.1.2将鱼腥草汁连续离心，在4℃下，1000g离心10分钟，3000g离心20分钟，10000g离心40分钟，离心后取上清，弃沉淀；
2.1.3将取得的上清超速离心，于冷冻超速离心机中，在4℃下，150000g离心90min，离心后弃上清取沉淀，沉淀悬浮于1-2mL的磷酸盐缓冲液中；
2.1.4将悬浮液用0.22μm的滤膜过滤，滤液再次于冷冻超速离心机中，在4℃下，150000g离心90min，离心后弃上清取沉淀，沉淀悬浮于1-2mL的磷酸盐缓冲液中；
2.1.5再次将悬浮液用0.22μm的滤膜过滤后得到无菌鱼腥草来源细胞外囊泡样颗粒。
根据制造商说明使用BCA试剂盒测定鱼腥草来源细胞外囊泡样颗粒的蛋白质浓度。
2.2大鼠大脑中动脉栓塞模型(MCAO)模型的建立
将购买的大鼠适应性饲养2周后开始造模，随机分为模型组(50只)，正常对照组(5只)。将大鼠禁食12-14小时，使用4％异氟烷麻醉SD大鼠，大鼠麻醉后将其四肢固定，并使用2％异氟烷手术中维持麻醉。75％酒精消毒颈部，手术剪于颈部正中偏右的位置剪开皮肤；使用镊子分离颈部腺体组织和筋膜，暴露左颈总动脉(CCA)、颈外动脉(ECA)和颈内动脉(ICA)；将CCA和ECA与周围组织分离，用缝合线扎紧ECA和CCA的近端，微动脉夹夹紧ICA防止出血；在CCA结扎部位和上端三叉口之间用眼科剪剪开一个小口，注意不要将血管剪断，并将线栓从切口处插入，松开动脉夹并调整进线栓的方向使其进入ICA直到大脑中动脉(MCA)的起始处；根据大鼠体重，线栓插入深度以线栓上黑点过三叉口并略微遇到阻力为止，然后扎紧固定ICA，开始计时缺血时间。缝合皮肤后用碘伏涂抹伤口消毒。在大鼠缺血2h后，拔出线栓至黑点刚出三叉口，但不能完全拔出，再灌注开始。对照组不插入线栓，其他处理同模型组。拔线栓时观察到大鼠向对侧转圈，可初步判定模型成功。
2.3实验动物分组以及给药
在本次实验中共造模成功40只大鼠，将造模成功的大鼠随机的分为：模型组、400μg/kg给药组。在造模成功后各给药实验大鼠便开始尾静脉注射给药。
2.4TTC实验
2.4.1SD大鼠缺血2h，再灌注时尾静脉注射400μg/kg的鱼腥草来源细胞外囊泡样颗粒；
2.4.2再灌注24h后异氟烷麻醉老鼠，迅速断头取脑，取脑过程注意保持大脑完整性；
2.4.3将大脑立即放入-20℃冰箱冰冻30min，大脑变硬即可，去除嗅球、小脑和低位脑干，切成2mm厚的冠状脑片，置于六孔板中，使脑片切片保持一致；
2.4.4加入2％的TTC染液，去离子水配制，用巴氏吸管吹起脑片使其悬浮于染液中，包上锡纸置于37℃烘箱孵育30min，每15min将脑片翻面，使其染色均匀，此过程全程避光操作；
2.5伊文思蓝(EB)染色
2.5.1EB染色：SD大鼠缺血2h再灌注24h后处死，处死前2h尾静脉注射2％的EB溶液，剂量为4mL/kg，注射过程中观察到大鼠眼睛、四肢、尾部变蓝。循环2h后麻醉大鼠，用0.9％生理盐水灌注至流出液体变澄清，断头取脑放入-20℃冰箱冰冻30min，取出后先整体拍照，接着切成2mm厚的冠状脑片，排列拍照，观察EB的渗漏情况。
2.5.2伊文思蓝-三氯乙酸(EB-TCA)标准曲线：
①配制1μg/μL EB：2％ EB 50μL+950μL超纯水
②50％ TCA2.5mL+无水乙醇7.5mL
2.5.3EB定量：拍照完成后，收集缺血侧脑组织并称重。缺血侧脑组织用1mL PBS匀浆，加入等体积的50％三氯乙酸继续匀浆，然后4℃，15000rpm离心20min收集上清于新的EP管中，按1:3加入无水乙醇后用酶标仪检测在620nm处的吸光度。用EB标准曲线的梯度浓度测量EB的含量。
2.6CCK8实验
2.6.1制备细胞悬液，计数。
2.6.2在96孔板中接种细胞悬液，每孔约200μL，做3个重复。
2.6.3将培养板放入培养箱中预培养一段时间(37℃，5％CO2)，细胞贴壁后换液为含鱼腥草来源细胞外囊泡样颗粒的无糖培养基，放入三气培养箱(37℃，1％O2，5％CO2)进行缺糖缺氧处理。
2.6.4缺氧10h后换液为含鱼腥草来源细胞外囊泡样颗粒的完全培养基，放入CO2培养箱进行复氧处理。
2.6.5复氧24h后每孔各加入10μLCCK-8溶液，将培养板放入培养箱中孵育1h。酶标仪测定450nm处的吸光值(OD)。
2.7蛋白质免疫印迹
细胞蛋白提取：细胞裂解液(RIPA：PMSF：磷酸酶抑制剂为100:1:1)，全程在冰上操作，弃去细胞培养基，用预冷的PBS洗涤3次，微量移液器吸净皿中的PBS，每个中皿加入120μL配置好的细胞裂解液，用细胞刮刮取细胞，吸取入预冷的1.5mL EP管中，涡旋震荡60s后冰上静置10min，重复三次，最后14000rpm，4℃离心25min，收集上清，上清液即为提取的细胞总蛋白。
蛋白浓度测定：采用BCA法，以0.5mg/mL的牛血清白蛋白(BSA)为标准蛋白，按试剂盒说明书操作步骤进行测定，每个样品测三个复孔，根据标准曲线计算每个样品的平均浓度，并用RIPA裂解液配平至同一浓度。
Western Blot：
(1)样品处理：将提取的蛋白用5×蛋白上样缓冲液和RIPA配制成同一浓度，金属浴中加热10min使蛋白变性；
(2)配胶：根据目的蛋白分子量按照试剂盒说明书配制不同浓度的分离胶和浓缩胶；
(3)电泳：待胶凝固后安装好电泳槽，吸取等量的蛋白样品上样，恒压电泳。120V跑至溴酚蓝到达玻璃板底部，结束电泳；
(4)转膜：电泳结束后，将PVDF膜浸入甲醇中激活1min，半干转法(1A，15V，25min)将蛋白转至PVDF膜上；
(5)封闭：转膜结束后，将PVDF膜置于封闭液(5％脱脂牛奶)中，摇床慢速摇晃，室温封闭2h；
(6)孵育一抗：封闭结束后，回收封闭液，1×Washing Buffer洗膜(5min×3遍)后，加入稀释后的一抗，4℃孵育过夜；
(7)孵育二抗：次日将一抗拿出室温平衡20min后回收一抗，1×Washing Buffer洗膜(10min×3遍)后，加入稀释后的二抗，室温孵育1h；
(8)显影：回收二抗，1×Washing Buffer洗膜(10min×3遍)；按ECL试剂盒说明书配制曝光液进行显影，曝光的条带用ImageJ软件处理分析其灰度值。
2.8旷场实验
实验在安静的环境下进行，SD大鼠换上干净垫料后，放入箱内底面中心，同时进行摄像和计时。观察5min后停止摄像。清洗方箱内壁及底面，以免上次动物余留的信息(如动物的大小便、气味)影响下次测试结果。更换动物，继续实验。
2.9脑水肿实验
大鼠建立MCAO模型72h后处死取脑并分别记录湿重，放入60℃恒温干燥箱烘干72h后分别记录干重。通过干湿称重法计算各脑组织的含水量，计算公式为：[(湿质量-干质量)/湿质量]*100％
2.10粒径分析
2.10.1运行颗粒粒径测量分析系统。
2.10.2向样品池中倒入分散介质，分散介质液面没过进水口上侧边缘，打开排水阀，当看到排水管有液体流出时关闭排水阀(排出循环系统的气泡)，开启循环泵，使循环系统中充满液体，然后关掉循环泵。
2.10.3软件自动到测试界面后，关闭循环泵和搅拌，抬起搅拌，将适量样品放入样品池中。
2.10.4启动超声，并根据被测样品的分散难易程度选择适当的超声时间；启动搅拌器，并调节至适当的搅拌速速，使被测样品在样品池中分散均匀。
2.10.5启动循环泵，测试软件窗口显示测试数据，当数据稳定时，点击随机按钮存储测试数据。
2.10.6数据存储完毕，打开排水阀，被测液排放干净后关闭排水阀，加入纯水冲洗循环系统，重复冲洗至测试软件窗口粒度分布无显示时说明系统冲洗完毕。
图1为本发明实施例中得到的细胞外囊泡样颗粒的粒径图以及免疫印迹图。由图1可知，粒径大小为139.8nm，在50-150nm之间，符合文献报道的细胞外囊泡粒径范围，浓度为2.7*107粒子/mL。
参照图2、图3a、图3b及图4a、图4b，实验结果表明：通过TTC染色实验可得鱼腥草来源细胞外囊泡样颗粒可以减少梗死面积；通过脑水肿、伊文思蓝染色实验可得鱼腥草来源细胞外囊泡样颗粒可以减轻脑水肿，减少伊文思蓝渗漏，提示鱼腥草来源细胞外囊泡样颗粒可以保护血脑屏障；通过旷场实验可知鱼腥草来源细胞外囊泡样颗粒可以提升自主运动能力，参照图5、图6a、图6b，实验结果表明：通过CCK8实验以及免疫印迹实验可知鱼腥草来源细胞外囊泡样颗粒可以提升氧糖剥夺/复氧模型下神经元细胞的存活率，降低凋亡水平和铁死亡水平。
Claims (6)
1.鱼腥草来源细胞外囊泡样颗粒在制备预防或治疗脑卒中药物中的应用，所述鱼腥草来源细胞外囊泡样颗粒通过以下步骤提取：
1.1、将新鲜鱼腥草清洗干净，晾干，榨取鱼腥草汁；
1.2、将鱼腥草汁连续离心，在4℃下低速梯度离心后高速离心，离心后取上清，弃沉淀；
1.3、将取得的上清超速离心，于冷冻超速离心机中，在4℃下，150000g离心，离心后弃上清取沉淀，沉淀悬浮于1-2mL的磷酸盐缓冲液中；
1.4、将悬浮液过滤，滤液再次于冷冻超速离心机中，在4℃下，再次超速离心，离心后弃上清取沉淀，沉淀悬浮于1-2mL的磷酸盐缓冲液中；
1.5、再次将悬浮液过滤后得到无菌鱼腥草来源细胞外囊泡样颗粒。
2.根据权利要求1所述的应用，其特征在于，步骤1.2中所述低速梯度离心后高速离心的具体步骤是：1000g离心10分钟，3000g离心20分钟，10000g离心40分钟。
3.根据权利要求1所述的应用，其特征在于，步骤1.4、步骤1.5中所述过滤选择0.22μm的滤膜进行。
4.根据权利要求1所述的应用，其特征在于，所述药物的剂型为药剂学上允许的口服剂型、注射剂型或粉针剂型。
5.根据权利要求1所述的应用，其特征在于，所述药物还包括药学上可接受的辅料。
6.一种预防或治疗缺血性脑卒中的药物，其特征在于，所述药物的有效成分包括有效剂量在100μg/kg-400μg/kg的鱼腥草来源细胞外囊泡样颗粒。
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