CN116747304A - P2y12受体及其拮抗剂的用途 - Google Patents
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Abstract
本发明涉及生物医药领域,特别涉及P2Y12受体及其拮抗剂的新用途。本发明公开了P2Y12受体作为靶点在筛选GABAA受体、NMDA受体类麻醉剂的干预药物中的应用。本发明还公开了P2Y12受体拮抗剂‑‑‑替格瑞洛在制备GABAA受体类麻醉剂的干预药物中的应用,能显著降低GABAA受体类麻醉剂诱导的意识丧失时长。本发明还公开了敲除小胶质细胞中P2Y12受体对GABAA受体麻醉剂戊巴比妥钠、NMDA受体麻醉剂氯胺酮诱导的全身麻醉效果的应用。
Description
技术领域
本发明涉及生物医药领域,特别涉及P2Y12受体及其拮抗剂的新用途。
背景技术
全身麻醉可以诱导可逆的意识和痛觉的丧失,被广泛应用于现代外科学手术及相关医学检查。全身麻醉剂种类繁多,分别针对不同的神经元受体或分子靶点,包括阿片类受体、γ-氨基丁酸A型受体(GABAA受体复合物:GABA受体、苯二氮卓类受体及GABA受体偶联的氯离子通道)、N-甲基-D-天冬氨酸(NMDA)受体、β2肾上腺素(β2A)受体等,发挥全身麻醉的作用。为了实施合适深度的麻醉,临床上常常会联合使用几种全身麻醉剂,通过与多个神经元受体或分子靶点综合作用,产生理想的麻醉效果。虽然,现代麻醉技术基本保障了麻醉过程中的安全性和有效性,但仍存在少部分患者因麻醉复苏较慢而出现一系列并发症,如中枢神经系统永久性损伤、术后谵妄等,会严重影响患者的预后。
现有技术所使用的促麻醉觉醒药物仅针对阿片类和苯二氮卓类麻醉剂,现有的作用于苯二氮卓类受体的麻醉剂的促觉醒药物为氟马西尼;作用于阿片受体的阿片类药物的促觉醒药物为纳洛酮。其他靶点麻醉剂均无有效的拮抗剂。而临床上最常用的全身麻醉剂主要是作用于GABAA受体和NMDA受体,因此,寻找针对性拮抗这两类受体的药物将对麻醉复苏产生重要的作用。
具体而言:目前促进麻醉患者快速觉醒的干预手段十分有限,主要是针对神经元上的一系列受体。例如,阿片类药物(作用于阿片类受体)引起的镇静或呼吸抑制,可使用类阿片拮抗剂纳洛酮促进觉醒或解除呼吸抑制;苯二氮卓类药物(作用于GABAA受体复合物组成之一的苯二氮卓类受体)诱导的麻醉,可以使用选择性的苯二氮卓类受体拮抗剂氟马西尼。但这些促麻醉觉醒药物快速注射后可见焦虑、心悸、恐惧等副作用,同时除了阿片类和苯二氮卓类麻醉剂,其他临床常用的麻醉剂均无有效的拮抗剂。
临床上全身麻醉剂多由针对不同受体的麻醉剂联合使用,而GABAA受体和NMDA受体麻醉剂是最常用的类型之一,此类靶点麻醉剂诱导的全身麻醉复苏慢会引起严重的中枢损伤,但目前缺乏针对此受体诱导的全身麻醉状态的干预药物,因此找寻针对GABAA受体和NMDA受体麻醉剂的干预药物,对于因使用GABAA受体或NMDA受体麻醉剂而处于麻醉状态的病人重新恢复正常的生理活动具有重要临床意义。即,找寻针对GABAA受体和NMDA受体麻醉剂的促觉醒药物对于麻醉病人的状态逆转具有重要意义。
P2Y12受体是嘌呤受体P2家族成员,由342个氨基酸组成的G蛋白偶联受体,在外周中主要表达于血小板,而在中枢神经系统则主要表达在脑中的小胶质细胞上。P2Y12受体的天然激动剂是ADP,前期研究证明ADP作用于血小板上P2Y12受体可引起血小板的聚集与活化,作用于小胶质细胞上可引起其突起的运动和迁移。目前临床上使用P2Y12受体拮抗剂来治疗血栓性心血管病、脑卒中、急性胰腺炎等疾病,其中阿斯利康研发的可逆的P2Y12受体拮抗剂替格瑞洛在临床上广泛使用,有临床研究显示替格瑞洛治疗12个月时,能在不提高出血风险的情况下能显著降低患者心血管死亡/心肌梗死/卒中的发生率。最近的研究显著小胶质细胞P2Y12受体介导的信号对于神经元活动的调节具有重要作用,能够对脑缺血、癫痫等疾病状态下过度活化的神经元起到抑制作用。
CN113267636A的发明还告知了P2Y12受体及其拮抗剂在诊治急性胰腺炎中的应用。
但是目前暂无相关性技术报道P2Y12受体与全身麻醉和麻醉复苏的关联性。
发明内容
本发明要解决的技术问题是提供一种P2Y12受体及其拮抗剂的新用途。
为解决上述技术问题,本发明提供P2Y12受体作为靶点在筛选GABAA受体、NMDA受体类麻醉剂的干预药物中的应用。
本发明还同时提供了P2Y12受体拮抗剂在制备GABAA受体类麻醉剂的干预药物中的应用。
作为本发明应用的改进:所述P2Y12受体拮抗剂为替格瑞洛。
作为本发明应用的进一步改进:干预GABAA受体类麻醉剂诱导的全身麻醉效果。
作为本发明应用的进一步改进:显著降低GABAA受体类麻醉剂诱导的意识丧失时长。
作为本发明应用的进一步改进:GABAA受体类麻醉剂为戊巴比妥钠。
综上,本发明提供了P2Y12受体及其拮抗剂在干预GABAA受体类麻醉剂诱导的全身麻醉效果中的应用。
本发明还同时提供了敲除小胶质细胞中P2Y12受体对GABAA受体麻醉剂戊巴比妥钠、NMDA受体麻醉剂氯胺酮诱导的全身麻醉效果的应用。
本发明,使用临床上已广泛应用的可逆的P2Y12R拮抗剂替格瑞洛或敲除小胶质细胞中p2y12r基因,均可以显著缓解GABAA受体特异性麻醉剂诱导的全身麻醉,表现为小鼠意识丧失时间缩短、麻醉深度降低等。因此本发明为干预GABAA受体类型麻醉剂诱导的全身麻醉提供了新的靶点,在此基础上完成了本发明。
发明人经过广泛而深入的研究,首次意外发现,脑中小胶质细胞特异性P2Y12受体在全身麻醉的维持过程中起着重要的靶点和调控作用。即,P2Y12R在脑中特异性表达于小胶质细胞。使用临床上已广泛应用的可逆的P2Y12R拮抗剂替格瑞洛或敲除小胶质细胞中p2y12r基因,均可以显著缓解GABAA受体特异性麻醉剂诱导的全身麻醉,表现为实验小鼠意识丧失时间缩短、麻醉深度降低等。为后续开发GABAA受体类麻醉剂的干预药物奠定基础。
本发明首次发现P2Y12受体的拮抗或敲除可以显著缓解GABAA受体类麻醉剂诱导的麻醉深度,降低麻醉诱导的意识丧失时间。
本发明采用临床上已广泛应用针对P2Y12受体的拮抗替格瑞洛,本发明的实验证明替格瑞洛可以降低GABAA受体类麻醉剂诱导的麻醉深度,缩短麻醉诱导的意识丧失时间。在注射麻醉剂前的约30分钟,向体内(颅内)注射替格瑞洛即可。
附图说明
下面结合附图对本发明的具体实施方式作出进一步详细说明。
图1为P2Y12受体拮抗剂替格瑞洛能够显著缩短戊巴比妥钠诱导的全身麻醉效果的持续时间;
图1中:
A为实验开展的流程图,包括侧脑室套管埋置的时间、套管给药时间及麻醉记录时间节点;
B为脑室注射P2Y12受体拮抗剂替格瑞洛不影响小鼠进入麻醉的时间;
C为脑室注射P2Y12受体拮抗剂替格瑞洛显著缩短了戊巴比妥钠诱导的全身麻醉维持时间。
图2为小胶质细胞特异性P2Y12受体敲除能够显著缩短GABAA受体和NMDA受体类麻醉剂诱导的全身麻醉效果的持续时间;
图2中:
A为实验开展的流程图,包括对照组和小胶质细胞P2Y12受体条件性敲除组小鼠进行他莫昔芬注射诱导基因敲除、及麻醉记录的时间节点;
B为特异性敲除小胶质细胞中P2Y12受体不影响戊巴比妥钠诱导的麻醉进入时间,而显著缩短戊巴比妥钠诱导的全身麻醉维持时间;
C为特异性敲除小胶质细胞中P2Y12受体不影响氯胺酮诱导的麻醉进入时间,而显著缩短氯胺酮诱导的全身麻醉维持时间。
具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1:P2Y12R受体拮抗剂替格瑞洛使用对GABAA受体麻醉剂的影响
(1)实验动物
八周龄SPF级C57bl/6j雄鼠(12只)购买于北京维通利华实验动物技术有限公司,SPF级环境中饲养动物。
(2)侧脑室套管埋置(常规实验方式)
脑室给药的小鼠均使用8周C57BL6/j雄鼠。所有的小鼠根据体重使用氯胺酮(100mg/kg)和甲苯噻嗪(10mg/kg)的混合麻醉剂腹腔注射进行麻醉。小鼠进入麻醉状态后,小鼠的眼部使用药膏覆盖,剪去小鼠颅骨上方的毛发,使用碘伏进行消毒,再将小鼠固定在立体定位仪上;使用手术剪刀剪开头皮,使用医用棉签将颅骨表面清理干净,然后根据脑图谱定位到侧脑室位置埋置套管。套管埋置坐标为(AP,-0.45mm;ML,1.0mm;DV,-2.0mm;即,脑的立体定位坐标)。为防止套管梗阻,将套管闭孔器插入套管中,然后使用牙科水泥固定套管,待水泥凝固后取下小鼠,饲养恢复7天。在套管给药当天,在实验前30min移除套管中的闭孔器,并将一个连接到汉密尔顿微量注射器的聚乙烯管插入套管中,手动控制微量注射器进行激动剂或者拮抗剂注射,注射时间为2min,注射完成后停针至少1min,使药物得到充分进入扩散。
(3)麻醉剂注射及麻醉时长统计
埋置套管后的动物分为对照组和P2Y12R拮抗剂组,每组6只小鼠。实验当天称量每只动物体重,在注射麻醉剂前30分钟给对照组注射生理盐水2ul、P2Y12R拮抗剂组注射替格瑞洛(用量为80ug替格瑞洛/kg体重,溶于2ul生理盐水),再根据体重腹腔注射GABAA受体麻醉剂戊巴比妥钠(用量为100mg戊巴比妥钠/kg体重,溶于0.2ml生理盐水),动物置于视频记录器下;随后检查每只小鼠丧失翻正反射的具体时间,并通过视频记录每只小鼠恢复翻正反射的具体时间,计算出每只小鼠进入全身麻醉诱导的意识丧失时长。
(4)统计方法
数据以平均值±标准误(SEM)表示,使用Graphpad Prism进行统计显著性分析。两组数据的比较是采用未配对student’s t检验。在两组以上的比较,是采用单因素方差分析(one-way ANOVA)以及Bonferroni’s的检验来计算多组间的统计学差异。在所有统计结果中,n.s.标记没有显著性差异,p<0.05被认为是显著差异,*标记p<0.05,**标记p<0.01,***标记p<0.001。
(5)实验结果
给予8周龄C57bl/6j小鼠脑室注射P2Y12受体拮抗剂替格瑞洛或生理盐水,30分钟后根据每只小鼠的体重,腹腔注射GABAA受体麻醉剂戊巴比妥钠(100mg/kg),置于视频记录仪下记录麻醉过程(图1A)。在麻醉诱导阶段,人工检查每只小鼠丧失翻正反射(loss ofrighting reflex,LORR,即进入全身麻醉)的时间点;以及通过视频确定恢复翻正反射(recovery of righting reflex,RORR,即从全身麻醉中觉醒)时间点,分别计算LORR和RORR时长并进行分析。
实验发现,P2Y12受体拮抗剂替格瑞洛的使用能够显著缩短戊巴比妥钠诱导的全身麻醉RORR时长,进入全身麻醉的LORR无显著统计学差异(图1B-C),说明通过脑中给予P2Y12受体拮抗剂替格瑞洛,可以缩短GABAA受体麻醉剂戊巴比妥钠诱导的全身麻醉时长。
实施例2:小胶质细胞P2Y12受体敲除对GABAA和NMDA受体麻醉剂的影响
(1)实验动物
八周龄雄性P2y12rf/f(对照组)和Cx3cr1CreER/+::P2y12rf/f(小胶质细胞P2Y12受体条件性敲除组)小鼠获赠于南昌大学彭吉云课题组,动物在八周时使用他莫昔芬(用量为100mg他莫昔芬/kg体重,溶于0.2ml生理盐水)每隔48小时灌喂一次,连续灌喂8次来确保小胶质细胞中P2Y12受体的有效敲除。动物正常饲养2weeks后进行后续实验,确保特异性只敲除小胶质细胞的P2Y12受体。
Cx3cr1CreER/+::P2y12rf/f,即《Microglial P2Y12 Receptor Regulates Seizure-Induced Neurogenesis and Immature Neuronal Projections》中的CX3CR1CreER/+:P2Y12fl /fl。
(2)麻醉剂注射及麻醉时长统计
动物分为对照组(n=8)和P2Y12受体条件性敲除组(n=10)。实验当天称量每只动物体重,再根据体重腹腔注射GABAA受体麻醉剂戊巴比妥钠(用量为100mg戊巴比妥钠/kg体重,溶于0.2ml生理盐水)或NMDA受体麻醉剂氯胺酮(用量为100mg氯胺酮/kg体重,溶于0.2ml生理盐水),动物置于视频记录器下;随后检查每只小鼠丧失翻正反射的具体时间,并通过视频记录每只小鼠恢复翻正反射的具体时间,计算出每只小鼠进入全身麻醉诱导的意识丧失时长。
(3)统计方法
数据以平均值±标准误(SEM)表示,使用Graphpad Prism进行统计显著性分析。两组数据的比较是采用未配对student’s t检验。在所有统计结果中,n.s.标记没有显著性差异,p<0.05被认为是显著差异,*标记p<0.05,**标记p<0.01,***标记p<0.001。
(4)实验结果
给予8周龄P2y12rf/f(对照组)和Cx3cr1CreER/+::P2y12rf/f(小胶质细胞P2Y12受体条件性敲除组)进行他莫昔芬灌喂诱导p2y12r基因敲除,在最后一次诱导后两组动物正常饲养两周。由于正常外周单核巨噬细胞会快速更新,饲养两周后更新的外周单核巨噬细胞表达P2Y12受体,而脑中小胶质细胞正常更新速度极慢,因此小胶质细胞中P2Y12受体仍被敲除,从而实现小胶质细胞特异性P2Y12受体的敲除(图2A)。
在麻醉记录当天,分别记录每一只小鼠的体重,随后根据每只小鼠的体重,腹腔注射GABAA受体麻醉剂戊巴比妥钠(100mg/kg),置于视频记录仪下记录麻醉过程(图2B)。在麻醉诱导阶段,人工检查每只小鼠丧失翻正反射(loss of righting reflex,LORR,即进入全身麻醉)的时间点;以及通过视频确定恢复翻正反射(recovery of righting reflex,RORR,即从全身麻醉中觉醒)时间点,分别计算LORR和RORR时长并进行分析,发现特异性敲除小胶质细胞中P2Y12受体能够显著缩短戊巴比妥钠诱导的全身麻醉RORR时长,而进入全身麻醉的LORR无显著统计学差异(图2B)。
等戊巴比妥钠麻醉实验完成三天后(小鼠体内戊巴比妥钠基本被代谢清除),再次记录每一只小鼠的体重,随后根据每只小鼠的体重,腹腔注射NMDA受体麻醉剂氯胺酮(100mg/kg),置于视频记录仪下记录麻醉过程(图2C)。在麻醉诱导阶段,人工检查每只小鼠丧失翻正反射(loss of righting reflex,LORR,即进入全身麻醉)的时间点;以及通过视频确定恢复翻正反射(recovery of righting reflex,RORR,即从全身麻醉中觉醒)时间点,分别计算LORR和RORR时长并进行分析,发现特异性敲除小胶质细胞中P2Y12受体能够显著缩短氯胺酮诱导的全身麻醉RORR时长,而进入全身麻醉的LORR无显著统计学差异。
这些实验结果说明通过特异性敲除脑中小胶质细胞的P2Y12受体,可以缩短GABAA受体麻醉剂戊巴比妥钠和NMDA受体麻醉剂氯胺酮诱导的全身麻醉时长。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (7)
1.P2Y12受体作为靶点在筛选GABAA受体、NMDA受体类麻醉剂的干预药物中的应用。
2.P2Y12受体拮抗剂在制备GABAA受体类麻醉剂的干预药物中的应用。
3.根据权利要求2所述的应用,其特征在于:所述P2Y12受体拮抗剂为替格瑞洛。
4.根据权利要求2或3所述的应用,其特征在于:干预GABAA受体类麻醉剂诱导的全身麻醉效果。
5.根据权利要求4所述的应用,其特征在于:显著降低GABAA受体类麻醉剂诱导的意识丧失时长。
6.根据权利要求5所述的应用,其特征在于:GABAA受体类麻醉剂为戊巴比妥钠。
7.敲除小胶质细胞中P2Y12受体对GABAA受体麻醉剂戊巴比妥钠、NMDA受体麻醉剂氯胺酮诱导的全身麻醉效果的应用。
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