CN113288918B - 益生菌在制备治疗孕期应激对子代损伤药物的应用 - Google Patents
益生菌在制备治疗孕期应激对子代损伤药物的应用 Download PDFInfo
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Abstract
本发明属于生物医药领域,具体涉及益生菌在制备治疗孕期应激对子代损伤药物的应用。本发明通过对孕鼠进行应激,同时给予益生菌干预,子代出生后再进行益生菌干预至成年,检测成年子代尼氏细胞数量、小胶质细胞活化数量、凋亡相关因子表达和炎症因子的表达水平,发现益生菌干预对孕期应激导致的子代焦虑和认知功能障碍有一定的改善作用,降低了子代海马区小胶质细胞的活化,改善了子代神经细胞的损伤与凋亡,同时也缓解了子代体内的炎症反应,说明益生菌对于孕期应激损伤子代具有改善作用,可以用于治疗孕期应激对子代的损伤。
Description
技术领域
本发明属于生物医药领域,具体涉及益生菌在制备治疗孕期应激对子代损伤药物的应用。
背景技术
孕期发生应激性生活事件不仅给怀孕母亲带来精神压力或创伤,产生焦虑、紧张、恐怖和抑郁情绪,而且能够终生影响子代个体的神经发育,导致出生后子代在认知功能、情绪行为等方面损害。
对于孕期应激现有的解决方案主要是临床上通过心理治疗和药物治疗,其中药物治疗主要是采用抗抑郁、抗焦虑、精神类药物等,而这些药物治疗对妊娠其母亲和胎儿会造成一定的影响,母亲容易患有焦虑、抑郁、强迫症等症状,同时也会影响胎儿和新生儿的发育迟缓、早产儿和低体而的概率增加,因此需要对孕期应激采取恰当的,对妊娠期母亲和胎儿均无副作用的治疗方法。
发明内容
为解决上述技术问题,本发明将SD孕鼠分为对照组、应激组和益生菌组,其中应激组怀孕15-20天进行束缚应激实验,益生菌组为进行应激试验的同时给予益生菌,对照组不做任何处理,子代出生后,对照组和应激组正常饲喂,益生菌组正常饲喂同时添加益生菌,成年后取各组子代大鼠的海马组织,进行尼氏染色、免疫组织化学检测和蛋白质印迹法分别检测尼氏细胞数量、小胶质细胞活化数量和凋亡相关因子表达量,并采用酶联免疫吸附剂检测血液中炎症因子的表达水平,可以发现,与正常组相比,应激组尼氏细胞数量显著减少,小胶质细胞活化的数目明显增加,炎症因子含量显著升高,凋亡相关因子含量明显降低,与应激组相比,益生菌组尼氏细胞数量显著增加,小胶质细胞活化的数目减少,炎症因子含量降低,凋亡相关因子含量升高,即益生菌干预对孕期应激导致的焦虑和认知有一定的改善作用,降低了子代海马区小胶质细胞的活化,改善了神经细胞的损伤与凋亡,同时也缓解了子代体内的炎症反应,说明益生菌对于孕期应激损伤子代具有改善作用。
基于上述发现,本发明的目的在于提供益生菌在制备治疗孕期应激对子代损伤药物的应用。
进一步的,所述益生菌为双歧杆菌、乳酸菌、乳球菌、中介链球菌或粪肠球菌中的一种或多种。
进一步的,所述应用具体为:在母代出现孕期应激反应时,以益生菌干预,同时对子代以益生菌干预。
基于同一发明构思的,本发明实施例还提供了一种治疗孕期应激对子代损伤药物组合物,所述药物组合物包含益生菌。
进一步的,所述药物组合物还包括药学上可接受的载体、赋形剂、稀释剂、辅剂和媒介物中的至少一种。
有益效果:
本发明首次通过将益生菌用于干预孕期应激,并在其子代出生后继续益生菌干预,通过检测其检测尼氏细胞数量、小胶质细胞活化数量、凋亡相关因子表达量和炎症因子的表达水平,可知益生菌干预对孕期应激导致的焦虑和认知有一定的改善作用,降低了子代海马区小胶质细胞的活化,改善了神经细胞的损伤与凋亡,同时也缓解了子代体内的炎症反应,说明益生菌对于孕期应激损伤子代具有改善作用,可以用于治疗孕期应激对子代损伤药物,为大脑疾病开发出创新的、无创的以及有效的益生菌疗法提供了新思路。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例提供的益生菌干预孕期应激实验的流程图;
图2为本发明实施例提供的各组子代大鼠海马尼氏染色实验结果图;
图3为本发明实施例提供的各组子代大鼠海马DG区IBA免疫组织化学实验结果;
图4为本发明实施例提供的各组子代大鼠外周血血浆炎症因子IL-1β和TNF-α表达水平;
图5为本发明实施例提供的各组子代大鼠海马Bcl-2、Bax、Caspase3表达量对比图;
图6为本发明实施例提供的各组子代大鼠旷场、高架十字迷宫检测结果;
图7为本发明实施例提供的各组子代大鼠新物体识别及Barnes迷宫检测结果。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合附图及具体实施例进行详细描述,但本发明的保护范围并不限于以下具体实施例。
除非另有定义,下文中所使用的所有专业术语与本领域技术人员通常理解含义相同。本文中所使用的专业术语只是为了描述具体实施例的目的,并不是旨在限制本发明的保护范围。
在本发明实施例中所采用的益生菌为培菲康双歧杆菌三联活菌胶囊,其包含长型双歧杆菌、嗜酸乳杆菌和粪肠球菌及其辅料,活菌数每粒不低于1.0×107CFU。
如图1所示,本发明实施例提供的益生菌干预孕期应激实验的流程图。具体包括如下步骤:
将SD孕鼠随机分成3组,分别为:对照组、应激组和益生菌组,每组6只,在母鼠怀孕的15-20天,对应激组每天进行三次束缚应激,时间分别为:09:00am,13:00pm,17:00pm,每次45min,益生菌组按照应激组的方式进行束缚应激,同时给予益生菌饲喂,饲喂方式为:平均一只一粒双歧杆菌三联活菌胶囊,将益生菌分散于饮用水中,24h换水一次;对照组不做任何处理。
待子代出生后,对照组和应激组正常饲喂,益生菌组正常饲喂同时饲喂益生菌,益生菌饲喂方式与母鼠相同,直至60天后成年。
取子代海马和血液进行神经元、小胶质细胞、凋亡相关因子和炎症因子的检测,另外将子代成年后在一周内进行行为学检测。
实施例1
尼氏细胞数量检测
对成年60天子代海马进行尼氏染色:取各组成年子代鼠海马冷冻切片,每组三只,将冷冻切片贴片在玻片上,玻片放至超净台内晾干一天,玻片依次没进乙醇(100%、95%、75%),待表面干燥后,放入尼式染液中染色5-20min,镜下随时视察组织颜色深浅,双蒸水轻柔洗涤玻片,放至无水乙醇进行分色,数秒钟即可,实时观察分色情况。自来水轻柔冲洗玻片,使用二甲苯Ⅰ脱脂3min,换二甲苯Ⅱ脱脂3min,通过树脂对染好色的大脑组织切片进行封闭片子,使用适量中性树脂滴加到载玻片,将盖玻片从下至上放入,不要过度按压防止空泡生成。在镜下观察细胞的染色情况以及细胞整体的状态,并通过imageJ对尼氏细胞进行统计学分析。
如图2所示,2A-2C:正常组组、应激组、益生菌组成年60天子代海马整体结构;2D-2F:正常组、应激组和益生菌组成年60天子代海马CA1区;2G-2I:正常组、应激组、益生菌成年60天子代海马CA3区;2J-2L:正常组、应激组、益生菌成年60天子代海马DG区,2M-2O:细胞计数统计学分析。结果表明,与正常组相比,应激组神经细胞的数量显著减少,通过益生菌干预,益生菌组尼氏细胞数量增加。Control:对照组,Stress:应激组,Probiotics:益生菌组。*p<0.05,**p<0.01,***p<0.001,****p<0.0001。每组样本均进行了3次独立的重复试验。A-C:比例尺=1mm,D-L:比例尺=50μm。
实施例2
子代鼠海马DG区小胶质细胞活化情况检测
通过IBA免疫组织化学检测海马DG区小胶质细胞活化情况,每组3只,具体步骤如下:以冠状切面,20ul厚度,使用冰冻切片机进行连续切片,组织切好后,转移至24孔板内。采用免疫组织化学方法,组织玻片放入0.01M PBS液中清洗三次,一次10min。随后把海马玻片样本放至3%H2O2内,充分清掉组织中的酶,把海马玻片置入PBS清洗三次,一次10min,彻底清除残留的H2O2。封闭:将海马玻片样本置于封闭液。IBA-1室温过夜。取出海马玻片样本,PBS清洗大脑海马玻片三次,一次10min。二抗孵育,用PBS配制二抗。生物素化羊抗兔IgG正常温度下持续1h,PBS清洗三次,一次10min。加入ABC液,根据ABC液说明书,配制ABC液,将组织切片置入ABC液浸泡,室温1h。将海马玻片用PBS清洗三次,一次10min。根据DAB试剂操作,1滴DAB加入1ml缓冲液,再加PBS稀释后进行染色。染色完成后进行贴片,避免折叠,将贴片自然晾干,进行脱脂,依次在二甲苯I,二甲苯II,无水乙醇(I),无水乙醇(II),乙醇(95%),乙醇(75%)中各浸泡5min。随后把海马玻片在双蒸水下轻柔漂洗5min,稍晾干,将组织玻片投于苏木素里,时长为2min,随后在双蒸水下轻柔漂洗5min。将载玻片按照顺序在乙醇(75%),乙醇(95%),乙醇(100%),乙醇(100%),二甲苯中维持5min,通过树脂对组织切片进行封片。
结果如图3所示,其中3D-3F:子代成年60天后海马DG区IBA免疫组织化学,3A、3D:对照组,3B、3E:应激组,3C、3F:益生菌组,3G:小胶质细胞活化数目计数统计。与正常组相比,应激组海马DG区的小胶质细胞活化的数目明显增加,与应激组相比,益生菌组海马DG区小胶质细胞活化的数目降低。Control:对照组,Stress:应激组,Probiotics:益生菌组,*p<0.05,**p<0.01(A-C:比例尺=200μm;D-F:比例尺=50μm),每组样本均进行了3次独立的重复试验。
实施例3
子代外周血血浆炎症因子IL-1β和TNF-α表达水平检测
具体检测方法为:(1)标准品的稀释。(2)依次打入40ul样品缓冲液于孔道中,随后加入10ul样品(样品稀释5x至10x),样品滴入到孔道的底层位置,轻拿96孔板缓缓甩动摇匀。(3)使用封口膜封上96孔板封后,放置温度箱里,温育程序为30min 37℃。(4)洗涤液需要降低30倍浓度后使用,使用双蒸水对其稀释。(5)温育30min完成后,撕掉封口膜后倒去96孔板中溶液,晾干,随后在每个孔道加入洗涤液进行漂洗,室温停留30s,倒掉洗涤液,该步骤反复5轮,在滤纸上拍干。(6)每个样本洞打进50ul酶标溶剂。(7)使用封口膜将96孔板封好后,将其放置提前设置好的温度箱里,温育程序为30min 37℃。(8)撕掉封口膜,倒掉96孔板中溶液,晾干,打进洗涤液于每个孔道,室温停留30s,倒掉溶液,该步骤执行5轮,在滤纸上拍干。(9)在96孔板有样品的孔道中先加入50ul显色剂A后,马上在每个孔道中再加入50ul显色剂B,手拿酶标板轻轻摇动摇匀,该步骤在37℃不见光操作,反应时长10min。(10)加入50ul终止液于96孔板中,终止反应。(11)操作10步骤15min以内,以对照校准,使用450nm计算得出每个孔道的OD值。
其结果如图4所示,结果表明:与正常组相比,应激组炎症因子TNF-α和IL-1β在子代外周血内的含量显著升高,与应激组相比,益生菌组TNF-α和IL-1β的含量明显降低。Control:对照组,Stress:应激组,Probiotics:益生菌组。*p<0.05,**p<0.01,每组样本均进行了3次独立的重复试验。
实施例4
子代海马Bcl-2、Bax、Caspase3表达检测
具体包括以下步骤:蛋白定量:配制BCA工作液(50:1),混匀待用,打开细菌培养箱温度调至37℃,对BCA标准品进行稀释:准备7个1.5mlEP管及0.9%的生理盐水,首先在其中一个EP管(标记好顺序)中加入40ul BSA标准品(2ug/ul),其余6管分别加入40ul生理盐水,加入40ul BCA标准品至二号EP中(1ug/ul),摇混稀释一倍,打进40ul BCA标准品于二号EP中至三号EP中(0.5ug/ul),混匀稀释一倍,按照排序依次稀释,充分混匀后将稀释好的7个EP管中的BCA标准蛋白各25ul分别加入至酶标板中,稀释蛋白样本:以0.9%的生理盐水为溶剂稀释5倍,加入25ul蛋白样本至96孔板中,随后按顺序打进200ul提前制作好的BCA于在每个孔道内,盖上板子后手拿96板轻轻摇动混匀,设置孵育条件:温度37℃,时长30min。测出BCA标准品及每个样本的吸光值,通过OD值计算样品浓度。
结果如图5所示,5A:蛋白质印记条带图。5B-5D:统计学分析柱状图。结果表明,与正常组相比,应激组Bcl-2的含量显著减少,与应激组相比,益生菌组Bcl-2的表达量明显增高,与正常组相比,应激组Bax及Caspase3的含量显著增高,与应激组相比,益生菌组Bax及Caspase3的表达量显著减少。Control:对照组,Stress:应激组,Probiotics:益生菌组,****p<0.0001,每组样本均进行了3次独立的重复试验。
实施例5
子代焦虑水平测试:
旷场实验:实验场地四个边长为1m,高为0.4m,整个旷场被分割成25个白色的小格子。实验动物测试前,动物被放置于测试房间内,进行时长为30min的适应期。实验动物测试开始时,实验动物被放置在旷场的正中央格子,要求实验动物每次放置的位置保持不变,让其在旷场内进行时长为5min的自由探索。每只实验动物在5min探索实验完成后被抓回先前的鼠笼内。实验员使用75%的消毒液喷洒、清理、晾干旷场,并准备进行下一轮的动物测试。整个实验进程记录摄像,记录实验测试数据。
高架十字迷宫实验:实验动物测试前,被放置于实验测试场所,进行时长为30min的适应阶段。实验测验时保持周围安静,排除环境给实验动物造成干扰因素。开放区域和封闭区域共同构成了高架十字迷宫(50cm×10cm×40cm,L×W×H),高架十字迷宫与地面的垂直距离为75cm。实验开始时,实验员将动物放置于迷宫的中央区域,动物的头部向着开放区域方向,每次实验测试,动物均放在同一位置。把监控器相关摄影设备打开进行录制,记录实验动物5min进入开放区域和封闭区域的时长、进入开放区域和封闭区域的频率。实验人员在实验过程中离开实验场地时,暂停摄像监控器设备,将测试结束的实验动物重新放回先前的饲养笼内,同时清理干净残留在迷宫中粪便和尿液,使用75%酒精擦拭干净实验动物测试过程接触过的位置及高架十字迷宫的开放区域、闭合区域。剔除在实验中从高架摔落在地上的实验动物。记录测试的实验数据:实验动物爬进开放区域和闭合区域的时长和次数。
结果如图6所示,图6A、6B:旷场实验,图6C、6D:高架十字迷宫。旷场结果显示应激组在旷场中间区格子停留时长显著短于对照组,益生菌组在旷场中间区格子停留时长显著长于应激组,高架十字迷宫结果表明应激组在开放区域停滞的时长及进入开放区域的频率均少于正常组,益生菌组在开放区域停留的时长及进入开放区域的频率均高于正常组。Control:对照组,Stress:应激组,Probiotics:益生菌组,*p<0.05,**p<0.01。
实施例6
子代空间学习记忆测试
新物体识别:大鼠探索的物体要求:直径5cm左右;没有气味,不能被老鼠随意移动,不要光滑。设置三个物体Ⅰ、Ⅱ、Ⅲ,其中Ⅰ和Ⅱ两个物体完全一样,Ⅲ物体区别于(Ⅰ、Ⅱ)两个物体,形状和大小完全不一致。实验动物实验前被放置于实验测试场所,进行时长为30min的环境顺应。保持清净,避免外界环境给实验动物造成干扰。训练过程中,在实验场地的对角线角落放置Ⅰ、Ⅱ两个物体,实验动物放于实验场地的正中心,实验动物距离两物体的距离保持一样。允许实验动物在实验场地内活动10min,测试者在放入实验动物至实验场地中央后,立即开启电脑录像设置,开启后测试者马上离开动物测试场所。实验观察指标:记录实验动物在矿场中接触Ⅰ、Ⅱ两个物体的具体情况,如嘴巴、鼻子或前爪与两个相同物体(Ⅰ、Ⅱ)触碰的次数和探索时间。每只实验动物结束测试后,实验者将实验动物重新放回先前的鼠笼内。实验测试阶段:在第二天同一时间给前一天的实验动物开始测试,实验场地内Ⅱ物体被换掉,Ⅲ物体取代Ⅱ物体的位置,放置的位置和训练阶段所放置的位置保持一致,允许实验动物在实验场地内活动10min,同样打开电脑拍摄设置,开启后实验员马上撤离动物测试场所。记录测试的实验数据:实验动物触碰Ⅰ和Ⅲ的总时长、实验动物对Ⅲ物体的探寻时间。
巴恩斯迷宫实验::实验过程为4日,每日对实验动物执行3次测试。第一天的第一次对大鼠进行测试,将实验大鼠放置于巴恩斯迷宫正中央,用黑色不透明箱子(其中一面透明)将大鼠盖住,黑色不透明箱子与迷宫下方的安全区域之间的方向维持在(90-120°),实验者可以看到箱子透明的一面,盖住箱子15s后,手指轻碰透明一面箱子,测试动物的正面向着实验者方向。打开迷宫正上方的白光灯后,随即掀开棕色不透明的箱子,引导测试大鼠爬进目标洞的区域里,实验动物爬入安全区域后,当即熄灯,让实验大鼠在目标洞区域保持90s的停留时间。在实验动物的测试阶段中,其放置的位置和距离和训练阶段时保持一致,实验动物如果超过180s时长找目标洞或一直未找到目标洞时,便将其引导至目标区域内。15min后进行下一只实验动物的测试。在实验动物的实验完成后,夹掉排泄物,随后使用75%的消毒液对巴恩斯迷宫进行擦洗、清理。实验测试记录:实验动物寻觅到安全区域的时长、实验动物的出错频率(以实验动物头颈伸到非安全洞里面记为犯错次数)。
结果如图7所示,图7A、7B:旷场实验,图7C、7D:高架十字迷宫。旷场结果显示应激组在旷场中间区格子停留时长显著短于对照组,益生菌组在旷场中间区格子停留时长显著长于应激组,高架十字迷宫结果表明应激组在开放区域停滞的时长及进入开放区域的频率均少于正常组,益生菌组在开放区域停留的时长及进入开放区域的频率均高于正常组。Control:对照组,Stress:应激组,Probiotics:益生菌组,*p<0.05,**p<0.01。
通过旷场、高架十字迷宫检测益生菌干预对孕期应激子代焦虑水平,结果表明益生菌干预降低了子代焦虑水平,通过新物体识别及巴恩斯迷宫检测益生菌干预对孕期应激子代空间学习记忆的作用,发现益生菌干预可以提升子代空间学习记忆力。
以上所述是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明所述原理的前提下,还可以作出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (2)
1.培菲康双歧杆菌三联活菌在制备治疗孕期应激对子代损伤药物的应用。
2.根据权利要求1所述的应用,其特征在于,所述应用具体为:在母代孕期应激阶段以益生菌干预,并对子代以益生菌干预。
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益生菌改善孕期应激子代大鼠异常行为和海马损伤;黄中俊等;《中南大学学报(医学版)》;20220430;第47卷(第4期);第443-452页 * |
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