CN109172584A - 2-apb在制备抗脑血管疾病药物中的应用 - Google Patents
2-apb在制备抗脑血管疾病药物中的应用 Download PDFInfo
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Abstract
本发明公开了2‑氨基乙氧基二苯基硼酸盐(2‑APB)在制备抗脑血管疾病药物中的应用,属于生物医药技术领域。本发明首次在神经元糖氧剥夺模型中研究了2‑APB对缺血性脑卒中的保护作用,且首次提出了2‑APB减少神经元死亡的作用机制与抑制氧化应激及Wnt/Ca2+信号转导通路有关,证明了2‑APB对缺血性神经元具有保护作用,能够减少缺血性脑卒中患者脑损伤。这些进一步补充和丰富了神经元保护机制的相关研究,为研究和开发治疗脑缺血的新药提供了更多的科学依据。
Description
技术领域
本发明涉及2-氨基乙氧基二苯基硼酸盐(2-APB)在制备抗脑血管疾病药物中的应用,特别是涉及2-APB在制备抗缺血性脑卒中的药物中的应用,属于生物医药技术领域。
背景技术
缺血性脑卒中是世界范围内严重危害人类健康和生命安全的常见性疾病,其发病率高、致残率高、死亡率高。已得过脑中风的患者,易再复发,每复发一次,加重一次。临床上一直缺乏有效的治疗措施,因此寻找新型药物和发现新的治疗机理显得十分紧迫和必要。
脑缺血后很多机制参与了脑组织损伤过程,如氧化应激损伤、兴奋性氨基酸毒性作用、线粒体功能障碍以及炎症反应等。其中,氧化应激损伤在脑缺血损伤过程中发挥着重要作用。氧化应激是机体内氧自由基生成增多或清除减少,导致机体内活性氧(reactiveoxygen species,ROS)蓄积引起神经元损伤的过程。大量的临床研究数据表明抑制脑缺血后的氧化应激损伤可以成为潜在的临床干预缺血性脑卒中的措施。
Wnt基因编码的Wnt蛋白家族属于分泌型糖蛋白,它们通过旁分泌或自分泌作用与位于细胞膜上的受体相结合,激活胞内的各级信号转导分子,调节靶基因的表达。Kohn等发现一个Wnt/Ca2+信号传导通路。在该信号中通路Wnt与Frizzled信号介导的蛋白质结合,激活异三聚鸟嘌呤核苷酸结合蛋白的活性,刺激细胞内储存的Ca2+释放,依次激活钙钙调蛋白依赖性蛋白激酶II(CaMKII),增加缺血性神经元损伤。因此,我们推测Wnt/Ca2+信号转导通路可能与脑缺血再灌注损伤有关。
现阶段缺血性脑卒中因为其发病率高,致残率高,死亡率高,引起了世界各地医药研究者的高度注意。但目前为止临床上缺乏安全有效的治疗方法。如果可以提供一种新型药物或新的治疗机理用于缺血性脑卒中,那么对于挖掘更多抗脑血管疾病的新型药物具有重要的意义。
发明内容
本发明的第一个目的是提供2-APB(2-氨基乙氧基二苯基硼酸盐)或其衍生物在制备预防或治疗或辅助治疗脑血管疾病的药物或药物组合物中的应用。
在本发明的一种实施方式中,所述脑血管疾病包括缺血性脑卒中。
在本发明的一种实施方式中,所述2-APB的结构式为:
在本发明的一种实施方式中,所述药物或者药物组合物还包括药学上可接受的赋型剂。
在本发明的一种实施方式中,其特征在于,所述药学上可接受的赋型剂是指任何可用于药学领域的稀释剂、辅助剂和/或载体。
在本发明的一种实施方式中,其特征在于,在药学上可接受的载体是一种或多种在药学上通常使用的填充剂、粘合剂、润湿剂、崩解剂、润滑剂或矫味剂的载体。
在本发明的一种实施方式中,所述药物或药物组合物的剂型包括颗粒剂、胶囊剂、片剂、丸剂和口服液。
本发明的第二个目的是提供一种2-APB及其衍生物在制备预防或治疗或辅助治疗脑血管疾病的食品或保健品中的应用。
本发明的第三个目的是提供2-APB在制备抑制Wnt/Ca2+信号转导通路方面的药物中的应用。
本发明的第四个目的是提供一种用于预防或治疗或辅助治疗脑血管疾病的药物或药物组合物,所述药物或药物组合物以2-氨基乙氧基二苯基硼酸盐(2-APB)或其衍生物为有效成分或主要有效成分。
本发明通过在神经元上构建糖氧剥夺-再复氧模型,模拟脑缺血发生后的神经元损伤,证明了2-APB对糖氧剥夺-再复氧(OGD/R)诱导的原代神经元细胞损伤具有保护作用,在25μM 浓度下,2-APB可以使神经细胞的存活率恢复至90.2%,LDH漏出率降至18.8%,其保护作用与依达拉奉(Eda)相当,优于丁苯酞(NBP),并可以通过抑制Wnt/Ca2+信号转导通路及其抗氧化作用减少神经元凋亡。这进一步补充和丰富了神经元保护机制的相关研究,为研究和开发治疗脑缺血的新药提供了更多的科学依据。
附图说明
图1:不同浓度的2-APB经过不同时间后对原代培养皮层神经元活力的影响;
图2:2-APB对原代培养皮层神经元OGD/R诱导的细胞毒性(A)和LDH释放量(B) 的影响;
图3:2-APB对原代培养皮层神经元中NOX2、Rac1、P40Phox、P47Phox和p67Phox蛋白水平的影响;
图4:以β-actin为内参,NOX2(A),Rac1(B),p40phox(C),p47phox(D)和p67phox(E)蛋白质的相对表达量;
图5:2-APB对培养的皮质神经元中Wnt5a,Frizzled和Dishvelled蛋白质水平的影响;
图6:以β-actin为内参,Wnt5a(A),Frizzled(B)和Dishvelled(C)蛋白质的相对表达量。
具体实施方式
(一)建立OGD/R体外模型:
1.原代皮层神经元细胞的培养
(1)取怀孕15-16天的SD大鼠腹腔注射水合氯醛(350mg/kg)麻醉;
(2)用75%乙醇对孕鼠腹部进行消毒,剪开腹腔,将胎鼠取出,断头,放入预冷的磷酸缓冲液(PBS)中;
(3)左手用精细镊从其双眼插入固定,右手持另一精细镊撕去头皮与颅骨,暴露出大脑,将大脑全部取出,放入预冷的PBS中;
(4)用精细镊小心剥去脑膜、四叠体、髓质等,将大脑皮层转移到预冷的PBS中;
(5)加入0.125%的胰酶,于37℃消化10min,每5min吹打一次;
(6)加入含10%血清的DMEM/F12培养基终止消化,然后继续吹打重悬;
(7)以200目尼龙筛网过滤,1000rpm离心5min收集细胞;
(8)以含10%血清的DMEM/F12培养基调整细胞浓度为1×106/mL,接种于经0.01%多聚赖氨酸包被的细胞培养板,置37℃、5%CO2培养箱中培养;
(9)12h后,更换培养基为含2%B27的Neurobasal培养基,以后每3-4天半量换液1次,细胞培养至7-10天后进行实验。
2.神经元细胞缺氧缺糖再给氧(OGD/R)损伤模型建立
将培养至第7-10天的大鼠皮层神经元细胞换用无糖Earle’s液孵育,并放入37℃三气缺氧培养箱内(1%O2,5%CO2,94%N2)培养30min,取出后全量换液为正常培养液,置于37℃、 5%CO2培养箱中继续培养24h。2-APB(终浓度为1、12.5、25μM)、NBP(终浓度为25μM)和Eda(终浓度为25μM)分别于OGD前2h加入,并维持在OGD/R全过程中。采用溶剂 (0.1%DMSO)作为对照,不进行OGD/R处理的为正常对照组(Control),同样进行OGD/R 处理的为模型对照组(OGD/R)。实验结束后测定相应指标。
(二)LDH的测定和临床意义:
在24孔培养板中进行实验,实验结束后,按照试剂盒说明书方法分别测定细胞内、外LDH的活性,计算漏出率。
LDH漏出率=细胞外LDH活性/(细胞外LDH活性+细胞内LDH活性)×100%。
LDH检测试剂盒是一种基于diaphorase催化的INT显色反应,通过比色法检测细胞毒性时释放的乳酸脱氢酶活性或检测其它样品中的乳酸脱氢酶活性的试剂盒。细胞凋亡或坏死而造成的细胞膜结构的破坏会导致细胞浆内的酶释放到培养液里,其中包括酶活性较为稳定的乳酸脱氢酶(lactate dehydrogenase,LDH)。通过检测从质膜破裂的细胞中释放到培养液中的 LDH的活性,就可以实现对细胞毒性的定量分析。LDH释放被看做细胞膜完整性的重要指标,并被广泛用于细胞毒性检测。
(三)细胞增殖毒性的测定(MTT):
以96孔培养板进行实验。实验结束前,每孔加入5mg/mL MTT溶液20μL,37℃继续培养4h,吸弃培养液,每孔加入150μL DMSO,均匀震荡10min,于酶标仪570nm处测定 OD值。以正常对照组细胞存活率为100%,计算各组细胞存活率。
(四)Western blot测定方法:
(1)在细胞培养皿中加入适量的细胞裂解液,置于冰上孵育30mCin后用细胞刮轻轻刮下后转移到1.5mL的离心管内并置于冰水混合物上,超声裂解后,4℃,12000rpm离心10min,离心后小心吸取上清置于预冷的新离心管中。-20℃保存或立即测定蛋白浓度变性。
(2)用BCA试剂盒测定蛋白浓度,根据测得的蛋白浓度加入适量的裂解液使各组的蛋白浓度一致。
(3)根据各组总样本的体积,按照4:1的比例加入5×Loading Buffer混匀后于100℃条件下水浴5min,取出样本存放于-40℃冰箱。
(4)SDS-PAGE凝胶电泳
①洗净制胶用的玻璃板,用无水乙醇润洗后置于烘箱内烘干,安装备用。
②根据配制SDS-PAGE凝胶试剂盒的说明书,按照配方根据目的蛋白的分子量大小选择合适浓度的分离胶,配好后迅速加入安装好的玻璃板间隙中,并用无水乙醇液封以防止凝胶氧化。室温静置30min后倒掉上层无水乙醇用蒸馏水冲洗数次,随后用滤纸吸干残余水分。
③按照说明书配方配制浓缩胶,配好后迅速加到分离胶上层并立刻插入梳子,室温放置 30min后即可使用。
④将配好的凝胶装配在电泳槽上,在槽内加满1×Running Buffer观察是否漏液,如果不漏拔掉加样孔梳子。
⑤按一定顺序将样本和marker加到加样孔内,每孔的上样量为60ug左右。
⑥将电压设置为70V开始电泳,待溴酚蓝进入分离胶时可将电压调制90V也可保持恒压直至溴酚蓝到达凝胶底部,停止电泳。
(5)转膜:电泳结束后,将SDS-PAGE凝胶从玻璃板中取出,切去浓缩胶及溴酚蓝下面的凝胶。在事先预冷的转膜液中,按从下向上的顺序依次放置海绵、多层滤纸、凝胶、硝酸纤维素膜(NC膜)、多层滤纸、海绵。每放一层注意避免产生气泡,尤其是凝胶与NC膜之间。对应好电极之后插入已倒入转膜液的转膜槽内,外槽与内槽的空隙间用冰袋填充。将外槽置于冰水浴中以缓解转膜过程中产生过多的热量。盖好电极,接通电源,根据分子量大小设置转膜电流为200~300mA,转2h。
(6)转膜结束后,取出NC膜,用TBST漂洗后,放入装有含5%BSA封闭液的盒子中室温孵育1h。
(7)封闭结束后,取出NC膜放入塑料袋中用封口机封好,只留一边用于加入用抗体稀释液稀释好的一抗(NOX2、p67phox Antibody、p40phox Antibody、p47phox Antibody、Wnt5aAntibody、Frizzled5Antibody、Dvl2Antibody),排尽塑料袋内的气泡,封好口,确保塑料袋不漏液,放入4℃冰箱过夜。
(8)取出自封袋,回收抗体稀释液。室温下,将NC膜置于TBST中漂洗三次,每次10min。结束后,根据一抗的来源选择与之相同来源的荧光二抗(稀释比例为1:10000),室温孵育 1h,结束后再用TBST漂洗三次,每次10min。
(9)将洗好的NC膜用Odyssey系统显影获取目的条带的图像,再利用Image J软件对目的条带和内参的面积和灰度值进行采集,统计后用Prism5软件作图分析。
实施例1:2-APB对原代大鼠皮层神经元的影响
原代大鼠皮层神经元细胞培养至7天后,分别加入不同浓度的2-APB(终浓度为1、12.5、 25μM),采用0.1%DMSO作为正常对照组(Control),在相同条件下培养细胞2-24h。MTT 法检测显示,2-APB在1、12.5、25μM浓度范围内对原代大鼠皮层神经元无毒性作用(见图 1)。
实施例2:2-APB对OGD/R损伤后原代大鼠皮层神经元细胞活性的影响
将培养至第7-10天的大鼠皮层神经元细胞换用无糖Earle’s液孵育,并放入37℃三气缺氧培养箱内(1%O2,5%CO2,94%N2)培养30min,取出后全量换液为正常培养液,置于37℃、 5%CO2培养箱中继续培养24h。2-APB(终浓度为1、12.5、25μM)、NBP(终浓度为25μM)和Eda(终浓度为25μM)分别于OGD前2h加入,并维持在OGD/R全过程中。采用溶剂 (0.1%DMSO)作为对照,不进行OGD/R处理的为正常对照组(Control),同样进行OGD/R 处理的为模型对照组(OGD/R)。实验结束后测定相应指标。
如图2所示,与Control组相比,OGD/R处理后,细胞存活率显著下降至44.6%,同时乳酸脱氢酶(LDH)释放显著增加至42.3%,神经元细胞明显受损。2-APB可显著增加原代大鼠皮层神经元细胞存活率并抑制LDH的释放,表现出明显的神经元细胞保护作用。在25μM浓度下,2-APB可以使神经细胞的存活率恢复至90.2%,LDH漏出率降至18.8%,其保护作用与依达拉奉(Eda)相当,优于丁苯酞(NBP)。
实施例3:2-APB对OGD/R损伤后原代大鼠皮层神经元细胞膜上NOX2、Rac1、p40phox、p47phox和p67phox蛋白表达的影响
将培养至第7-10天的大鼠皮层神经元细胞换用无糖Earle’s液孵育,并放入37℃三气缺氧培养箱内(1%O2,5%CO2,94%N2)培养30min,取出后全量换液为正常培养液,置于37℃、 5%CO2培养箱中继续培养24h。2-APB(终浓度为25μM)于OGD前2h加入,并维持在OGD/R全过程中。采用溶剂(0.1%DMSO)作为对照,不进行OGD/R处理的为正常对照组(Control),同样进行OGD/R处理的为模型对照组(OGD/R)。实验结束后测定相应指标。结果如图3和图4所示,OGD/R损伤可诱导原代大鼠皮层神经元细胞内NOX2,Rac1and p40phox蛋白表达上调。与OGD/R组相比,2-APB可以显著性抑制NOX2、Rac1和p40phox蛋白的表达,但对p47phox和p67phox蛋白的表达无明显作用。
因此,2-APB能够减少OGD/R处理后神经元内ROS的含量,发挥抗氧化的作用。2-APB通过抑制Rac1和p40phox的蛋白表达,抑制NOX2的表达,抑制NADPH氧化酶的活化发挥抗氧化保护神经元的作用。
实施例4:2-APB对OGD/R损伤后原代大鼠皮层神经元细胞内Wnt5a、Frizzled、和Dishvelled蛋白表达的影响
将培养至第7-10天的大鼠皮层神经元细胞换用无糖Earle’s液孵育,并放入37℃三气缺氧培养箱内(1%O2,5%CO2,94%N2)培养30min,取出后全量换液为正常培养液,置于37℃、5%CO2培养箱中继续培养24h。2-APB(终浓度为25μM)于OGD前2h加入,并维持在OGD/R全过程中。采用溶剂(0.1%DMSO)作为对照,不进行OGD/R处理的为正常对照组(Control),同样进行OGD/R处理的为模型对照组(OGD/R)。实验结束后测定相应指标。结果如图5和图6所示,表明OGD/R损伤可诱导原代大鼠皮层神经元细胞内Wnt5a、 Frizzled、和Dishvelled蛋白的表达上调。与OGD/R组相比,2-APB可以显著性抑制Wnt5a、 Frizzled、和Dishvelled蛋白的表达。
因此,2-APB能显著抑制OGD/R激活的Wnt5a、Frizzled、和Dishvelled蛋白表达,抑制Wnt/Ca2+信号转导通路。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.2-APB(2-氨基乙氧基二苯基硼酸盐)或其衍生物在制备预防或治疗或辅助治疗脑血管疾病的药物或药物组合物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述脑血管疾病包括缺血性脑卒中。
3.根据权利要求1所述的应用,其特征在于,2-APB或其衍生物通过抑制Wnt/Ca2+信号转导通路及其抗氧化作用减少神经元凋亡。
4.根据权利要求1所述的应用,其特征在于,其特征在于,所述药物或者药物组合物还包括药学上可接受的赋型剂。
5.根据权利要求4所述的应用,其特征在于,所述药学上可接受的赋型剂是指任何可用于药学领域的稀释剂、辅助剂和/或载体。
6.根据权利要求5所述的应用,其特征在于,在药学上可接受的载体是一种或多种在药学上通常使用的填充剂、粘合剂、润湿剂、崩解剂、润滑剂或矫味剂的载体。
7.根据权利要求1-6任一所述的应用,其特征在于,所述药物或药物组合物的剂型包括颗粒剂、胶囊剂、片剂、丸剂和口服液。
8.2-APB或其衍生物在制备预防或治疗或辅助治疗脑血管疾病的食品或保健品中的应用。
9.2-APB在制备抑制Wnt/Ca2+信号转导通路的药品、食品、保健品的应用。
10.一种用于预防或治疗或辅助治疗脑血管疾病的药物或药物组合物,其特征在于,所述药物或药物组合物以2-APB或其衍生物为有效成分或主要有效成分。
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Application publication date: 20190111 |