CN114681464B - 二甲双胍类似物在制备脑缺血再灌注损伤预防和治疗药物中的应用 - Google Patents
二甲双胍类似物在制备脑缺血再灌注损伤预防和治疗药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种二甲双胍类似物在制备脑缺血再灌注损伤预防和治疗药物中的应用。所述的二甲双胍类似物为2‑氨基‑4‑二甲基氨基‑6‑苯基‑3,6‑二氢‑1,3,5‑三嗪，其结构式为本发明的二甲双胍类似物对短暂性缺血急性脑卒中的治疗，与二甲双胍相比，具有更好的抗卒中疗效，不仅大大降低模型的死亡率、梗死面积，减少水肿的面积，而且显著降低了缺血缺氧对脑神经细胞和星形胶质细胞造成的损伤，并且对于缺血再灌注所造成的氧化损伤有更加显著的改善，有望作为脑缺血再灌注损伤预防和治疗药物应用于临床。
Description
技术领域
本发明属于医药技术领域，涉及一种二甲双胍类似物在制备脑缺血再灌注损伤预防和治疗药物中的应用。
背景技术
脑血管病，是继心血管疾病、癌症之后的第三大致死性疾病，并且是首位致残因素。在我国脑血管病的发病率、病死率及致残率有逐年上升的趋势，其中缺血性脑卒中占绝大部分(80～88％)，尤其在脑缺血后再灌注的损伤使其病势急、病死率高、最佳治疗时间窗短、后遗症重，因此成为临床的难治病。目前唯一被FDA批准用于缺血性脑卒中治疗的药物是重组组织型纤溶酶原激活剂(rt-PA)，在脑卒中发生4.5h内应用rt-PA对提高神经功能起到一定促进作用，但由于其治疗窗窄，不到5％的患者从中受益。因此寻找新的缺血性脑卒中治疗药物成为当务之急。
二甲双胍是当前全球应用最广泛的口服降糖药之一。研究发现，缺血性脑卒中对大脑缺血性神经的损伤主要是因为能量供应不足，从而引起星形胶质细胞的糖酵解增加来提供能量，但长期糖酵解过程造成乳酸堆积增加，并且抑制神经元利用乳酸作为能量来源，从而导致神经细胞死亡。单磷酸腺苷(adenosine monophosphate，AMP)激活的蛋白激酶(AMPK)作为能量代谢通路中的关键酶，控制着整个机体的能量代谢。而二甲双胍作为AMPK激动剂，当ATP与AMP比例失衡即AMP比例增加时，二甲双胍通过促进AMPK上苏氨酸残基(Thr-172)的磷酸化而激活AMPK。已有实验证明在中动脉栓塞(Middle cerebral arteryocclusion，MCAO)后，二甲双胍可通过激活AMPK促进小胶质细胞/巨噬细胞由M1向M2极化状态转变，从而促进组织修复，发挥神经保护作用(Jin Q,Cheng J,Liu Y,et.al.Improvement of functional recovery by chronic metformin treatment isassociated with enhanced alternative activation of microglia/macrophages andincreased angiogenesis and neurogenesis following experimental stroke.BrainBehav Immun.2014 Aug；40:131-42.doi:10.1016/j.bbi.2014.03.003.Epub 2014Mar12.PMID:24632338.)。
发明内容
本发明的目的在于提供一种二甲双胍类似物(TD-3)在制备脑缺血再灌注损伤预防和治疗药物中的应用。
实现本发明的目的如下：
本发明所述的二甲双胍类似物，为2-氨基-4-二甲基氨基-6-苯基-3,6-二氢-1,3,5-三嗪，其结构式如下所示：
本发明还提供上述二甲双胍类似物的制备方法，合成路线如下：
具体步骤如下：
将二甲双胍加入异丙醇，搅拌至完全溶解后加入苯甲醛，HCl调节pH＝5～6，80～100℃下回流反应，反应结束后冷却至室温，抽滤洗涤得到2-氨基-4-二甲基氨基-6-苯基-3,6-二氢-1,3,5-三嗪即二甲双胍类似物。
进一步的，本发明提供上述二甲双胍类似物在制备脑缺血再灌注损伤预防和治疗药物中的应用。
本发明所述的脑缺血再灌注损伤包括但不限于局灶性脑缺血(MCAO)再灌注损伤、四动脉结扎致全脑缺血损伤和急性不完全脑缺血损伤等。
本发明通过大鼠中动脉栓塞模型建立脑缺血再灌注动物模型，观察模型死亡率、梗死面积和神经行为学评分、HE染色及免疫荧光化学染色切片以及给药后大鼠皮层损伤情况，并通过对超氧化物歧化酶(SOD)、脂质过氧化物丙二醛(MDA)及还原型谷胱甘肽(GSH)的检测，观察给药后氧化损伤的改善情况。结果均证明TD-3具有显著的抗脑缺血再灌注损伤的作用，可以用于制备脑缺血再灌注损伤的预防和治疗药物。
附图说明
图1为各实验组的动物死亡率、神经行为学评分和脑组织梗死面积统计图。
图2为各实验组的具有代表性的TTC染色切片图。
图3为各实验组的HE染色图。
图4为各实验组的免疫荧光染色图。
图5为各实验组的氧化损伤相关指标图。
具体实施方式
以下通过实施例和附图来进一步描述本发明。
实施例1
二甲双胍类似物的制备方法，具体步骤如下：
在装有电动搅拌器、温度计和回流冷凝管的三口瓶中依次加入二甲双胍和异丙醇。待二甲双胍全部溶解后再加入苯甲醛，用HCl调节pH＝5-6，控制反应溶液的温度为80～100℃，20min后溶液变浑浊伴有白色沉淀生成，继续反应一定时间后停止加热，静止冷却，析出大量白色晶体，抽滤洗涤得到2-氨基-4-二甲基氨基-6-苯基-3,6-二氢-1,3,5-三嗪，即二甲双胍类似物。
实施例2
二甲双胍类似物对大鼠脑缺血再灌注损伤的作用试验，以二甲双胍(TD-1)作为阳性药对照，对二甲双胍类似物(TD-3)进行抗脑卒中的动物药效评价，通过对SD大鼠进行中脑动脉栓塞手术(MCAO)，造成缺血再灌注模型以模拟人体急性缺血性脑卒中，术后2h分别尾静脉注射给以5.0mg/kg的TD-1、TD-3治疗，通过神经行为学评分、脑梗死面积、死亡率、HE染色切片、免疫荧光化学染色切片评价其药效，并且进一步检测血液的生化指标变化以探讨其保护脑卒中的调控机制，具体方法如下。
1.大鼠脑缺血再灌注模型的建立
采用大鼠中动脉栓塞模型(MCAO)建立大鼠脑缺血再灌注模型：大鼠用3％水合氯醛(350mg/kg,ip)麻醉后仰卧固定，常规消毒，在颈正中稍偏左做一纵向长2cm的切口，钝性分离各层组织，暴露右侧颈总动脉(CCA)，分离颈内动脉(ICA)和颈外动脉(ECA)。暂时性夹闭颈总和颈内动脉，将颈外动脉游离，并在颈外动脉上做一纵向切口，将栓线从切口沿颈外动脉经颈总动脉分叉口缓缓推入到颈内动脉以阻断大脑中动脉(MCA)，栓线进入距分叉口的长度约18至20mm。2h后缓缓拔出线栓，实现缺血再灌注。对模型组和两个给药组构建MCAO模型，假手术组只分离血管，不插入栓线。整个手术过程在保温毯上进行，以保证老鼠直肠温度维持在37±1℃。在实验过程中记录实验模型动物的死亡率以及行为状态。
2.分组与给药
适应性饲养7天后，将SD大鼠随机分为4组，每组20只：假手术组(Sham)，模型组(Model)，TD-1组(5.0mg/kg)，TD-3组(5.0mg/kg)，在再灌注的2小时后尾静脉注射(I.P.)治疗给药。Sham组和Model组给予与TD-1组，TD-3组同等剂量的生理盐水。
3.实验结果及分析
(1)神经行为学评分
再灌注24h后，按照Berderson评分法姿势反射测试(postural reflex test)的标准进行神经行为学评分。评分规则为：
0分：未见行为缺陷；
1分：前肢屈曲；
2分：侧推抵抗力下降，伴前肢屈曲，无转圈行为；
3分：同2级行为，伴自发性旋转。
(2)脑组织切片TTC染色
神经行为学评分之后，将大鼠脱颈处死，每组随机取6只脑组织样本，-20℃冷冻30min，取出，将两侧大脑半球切成厚约2mm的冠状切片，浸泡于15ml，2％TTC染液中。37℃避光孵育约30min，此间将脑片的上下面翻转至少一次。4％的甲醛固定液中过夜，拍照。正常脑组织染为深红色，脑梗死区为白色。
图1为各实验组的动物死亡率、神经行为学评分和脑组织梗死面积统计图。从图中可以看出，在TD-1和TD-3治疗后，大鼠造模24h内的死亡率大大下降，神经行为学评分明显降低，大鼠的脑组织梗死面积显著性减小，且TD-3组对梗死面积改善最为明显。
图2为各实验组的具有代表性的TTC染色切片图。正常脑组织染为深红色，脑梗死区为白色。从图中可以直观的看到TD-1和TD-3对于脑梗死区域的改善，具体表现为：相对于Model组，染色切片图中深红色正常区域增加和白色脑梗死区域的减少，并用Image J软件对梗死面积进行统计分析发现，TD-3组的梗死面积更小，表现出较好的治疗效果。
(3)HE染色和免疫荧光化学分析
取脑后，迅速投入到组织固定液中浸泡过夜，进行石蜡包埋，切片机切成5μm厚的连续冠状切片，用HE(hematoxylin-eosin)染色以进行形态学观察。免疫荧光化学分析，测试神经元核心抗原(NeuN)和神经胶质酸性蛋白(GFAP)两种蛋白表达情况，以考察脑组织中神经细胞和星形胶质细胞的状态。
图3为各实验组的HE染色图。显微镜下Model组可见右侧脑皮层大面积梗死，脑组织坏死液化，形成圆形或卵圆形，边界清楚的镂空网状软化灶；神经元大量减少，坏死神经元嗜酸性增强，胞核固缩深染，结构不清，部分坏死神经元溶解消失，伴有小胶质细胞浸润；梗死损伤侵及海马，海马CA3区大量锥体细胞固缩深染，胞核与胞质界限模糊。TP-3组，脑皮层坏死液化的面积相较于Model组有显著缩小，脑损伤的程度较TP-1组更小。
图4为各实验组的免疫荧光染色图。两给药组的免疫荧光结果均趋近于假手术组(Shame)，与HE染色结果类似，表明在TD-1和TD-3治疗后，显著降低了缺血缺氧对脑神经细胞和星形胶质细胞造成的损伤。
(4)SOD、MDA及GSH水平的测定
取血清，冰上复融后，按照试剂盒说明书操作。
图5为各实验组的氧化损伤相关指标图。氧化应激是指机体在遭受各种有害刺激时，体内高活性分子如活性氧自由基和活性氮自由基产生过多，氧化程度超出了机体对于氧化物的清除能力，氧化系统和抗氧化系统失衡，从而导致组织损伤。丙二醛(MDA)是自由基作用于脂质过氧化的产物，具有细胞毒性，会引起蛋白质、核酸等生命大分子的交联聚合。因此，通过对MDA的检测可以了解膜过氧化的程度，并间接测定膜系统的损伤程度。实验结果显示模型组的MDA相对于假手术组呈显著上升趋势，经TD-1治疗后未发生显著下降，而TD-3治疗可使MDA显著下降。超氧化物歧化酶(SOD)是一个重要的机体超氧阴离子自由基清除剂，可以抑制自由基引起的脂质过氧化反应；谷胱甘肽(GSH)是由半胱氨酸、甘氨酸和谷氨酸结合形成的含有巯基的三肽，它的主要生理作用是能够清除掉人体内的自由基。模型组SOD和GSH显著下降，表明SOD和GSH为应对氧化应激而被大量消耗，TD-1和TD-3治疗后SOD与GSH的水平均显著上升。在TD-1和TD-3治疗后，两给药组的各项指标均有向Shame组回归的显著趋势，并且相对于TD-1组，TD-3对于SOD和MDA的改善更为显著，在缺血再灌注中表现出更好的氧化损伤的保护效果。
Claims (3)
1.二甲双胍类似物在制备预防和治疗脑缺血再灌注损伤药物中的应用，其特征在于，所述的二甲双胍类似物为2-氨基-4-二甲基氨基-6-苯基-3,6-二氢-1,3,5-三嗪。
2.根据权利要求1所述的应用，其特征在于，所述的二甲双胍类似物通过以下步骤制备：
将二甲双胍加入异丙醇，搅拌至完全溶解后加入苯甲醛，HCl调节pH=5~6，80~100℃下回流反应，反应结束后冷却至室温，抽滤洗涤得到2-氨基-4-二甲基氨基-6-苯基-3,6-二氢-1,3,5-三嗪即二甲双胍类似物。
3.根据权利要求1所述的应用，其特征在于，所述的脑缺血再灌注损伤为局灶性脑缺血再灌注损伤或急性不完全脑缺血损伤。
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