CN114796201A - 佛手柑内酯作为制备治疗缺血性脑卒中药物的应用 - Google Patents
佛手柑内酯作为制备治疗缺血性脑卒中药物的应用 Download PDFInfo
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Abstract
佛手柑内酯作为制备治疗缺血性脑卒中药物的应用。本发明提供佛手柑内酯作为制备治疗缺血性脑损伤药物的应用,佛手柑内酯的分子式C12H8O4,分子量216.19,药物的剂型为口服剂。佛手柑内酯是一种小分子化合物,口服用药,可透过血脑屏障,作用于缺血性脑损伤的病理过程,符合当今研制治疗缺血性脑损伤药物的决策,即首先改善缺血后脑功能,其次抑制小胶质细胞激活从而减轻缺血后炎症反应,通过靶向结合KV1.3通道和CBR1调控小胶质细胞激活。
Description
技术领域
本发明涉及医药技术领域,具体涉及佛手柑内酯作为治疗缺血性脑卒中药物的应用。
背景技术
缺血性脑卒中是世界上最常见的死亡和致残原因之一。受损的细胞碎片和增加的活性氧通过激活小胶质细胞和星形胶质细胞以及吸引循环血液中的白细胞渗入而导致神经炎症。神经炎症的发展受活性氧、细胞因子和趋化因子的调节。小胶质细胞是中枢神经系统的天然免疫细胞,是大脑免疫反应的关键调节器。小胶质细胞的激活发生在神经炎症过程的早期阶段。被激活的小胶质细胞向缺血的大脑皮层迁移,释放促炎症介质,增加血脑屏障通透性,并分泌趋化因子,促进全身性白细胞如中性粒细胞、巨噬细胞和淋巴细胞的通过,从而加重脑损伤。同时,被激活的小胶质细胞吞噬外来生物以及受损的脑细胞以修复脑损伤。因此,调节大脑中的神经炎症反应和小胶质细胞功能是治疗缺血性中风的一个潜在的有效策略。
现在普遍认为,抑制小胶质细胞的激活可以减轻炎症损伤,促进中风恢复。PARP抑制剂可阻断NF-αB的转录活性,阻断小胶质细胞iNOS表达的上调、MMP9的释放、形态学变化以及LPS和其他促炎症介质诱发的神经毒性。HDAC抑制剂丙戊酸和丁酸钠可抑制小胶质细胞的激活,减少小胶质细胞的数量,并抑制缺血脑中的其他炎症标志物。TNF-α是小胶质细胞激活的有效刺激物,有效的TNF-α抑制剂可以抑制小胶质细胞的激活,并通过靶向结合小胶质细胞上的TNF受体来减少梗死体积。因此,急需一种能够靶向作用于小胶质细胞的药物来改善中风后神经炎症。佛手柑内酯(BG)是一种小分子化合物,具有抗炎、镇痛的药理作用,但是其在制备治疗缺血性脑卒中药物的应用方面还未得到认证,因此,本发明提供佛手柑内酯作为制备治疗缺血性脑卒中药物的应用。
发明内容
本发明针对现有技术中的不足,提供佛手柑内酯作为治疗缺血性脑卒中药物的应用。
为实现上述目的,本发明采用以下技术方案:
佛手柑内酯(BG)作为制备治疗缺血性脑卒中药物的应用,佛手柑内酯的结构式如下:
为优化上述技术方案,采取的具体措施还包括:
进一步地,所述治疗是通过抑制小胶质细胞激活,减轻缺血后神经炎症反应实现的。
进一步地,所述治疗是通过佛手柑内酯减弱脂多糖诱导的小胶质细胞的促炎反应实现的。
进一步地,所述治疗是佛手柑内酯通过抑制KV1.3减轻LPS诱导的小胶质细胞的炎症反应实现的。
进一步地,所述治疗是佛手柑内酯通过抑制CBR1蛋白的降解和底物4-HNE的产生进而减轻LPS诱导的小胶质细胞的炎症反应实现的。
进一步地,所述治疗是佛手柑内酯通过调控NF-κB信号通路减轻炎症反应实现的。
进一步地,所述药物的剂型为口服剂。
进一步地,所述口服剂灌胃给药的用量为30mg/kg/次。
本发明的有益效果是:佛手柑内酯是一种小分子化合物,口服用药,可透过血脑屏障,且在剂量范围内,BG未见毒、副作用;佛手柑内酯作用于缺血性脑损伤后神经炎症的病理过程,符合当今研制治疗缺血性脑损伤药物的决策;首先通过梗死面积和行为学,证明BG对缺血性脑损伤具有保护作用,改善缺血后脑损伤面积和神经行为缺陷;其次,BG能够抑制小胶质细胞的激活、抑制小鼠MCAO后脑组织缺血半暗带促炎因子的表达,从而减轻缺血后神经炎症;最后,靶向结合KV1.3通道和CBR1通过NF-κB信号通路调控小胶质细胞的激活。
附图说明
图1是BG对缺血性脑损伤的影响示意图,其中,
A是BG对MCAO小鼠脑梗死体积影响图,
B是BG对MCAO小鼠脑梗死体积影响统计图,
C是BG对MCAO小鼠脑功能改善评分图,
D是BG对MCAO小鼠旋转实验影响图,
E是BG对MCAO小鼠握力实验影响图;
图2是免疫荧光染色检测MCAO小鼠小胶质细胞形态变化图,其中,
A是小胶质细胞免疫荧光染色图,
B是BG对小胶质细胞分支数量的影响,
C是BG对小胶质细胞终端数量的影响,
D是BG对小胶质细胞平均长度的影响,
E是BG对小胶质细胞最大长度的影响;
图3是BG对MCAO小鼠促炎因子mRNA表达水平图,其中,
A是IL-1βmRNA表达水平图,
B是IL-6mRNA表达水平图,
C是TNF-αmRNA表达水平图;
图4是LDH检测BG对原代小胶质细胞和原代神经元毒性影响示意图,其中,
A是BG结构示意图,
B是BG对原代小胶质细胞无药物毒性,
C是BG对原代神经元无药物毒性;
图5是RT-PCR测定LPS刺激后加不同浓度BG原代小胶质细胞促炎因子mRNA表达水平图,其中,
A是IL-1βmRNA表达水平图,
B是IL-6mRNA表达水平图,
C是TNF-αmRNA表达水平图;
图6是ELISA测定LPS刺激后加不同浓度BG原代小胶质细胞促炎因子蛋白表达水平图,其中,
A是IL-1βmRNA表达水平图,
B是IL-6mRNA表达水平图,
C是TNF-αmRNA表达水平图;
图7是Western blot检测LPS刺激后原代小胶质细胞NF-κB信号通路表达水平图,其中,
A是P65蛋白、p-P65蛋白、IκBα蛋白、p-IκBα蛋白表达水平图,
B是p-P65/P65蛋白表达水平统计图,
C是p-IκBα/IκBα蛋白表达水平统计图;
图8是PBFI-AM测定LPS刺激后加最大浓度BG原代小胶质细胞内钾离子浓度变化图;
图9是RT-PCR测定LPS刺激后加不同浓度PAP1原代小胶质细胞促炎因子mRNA表达水平图,其中,
A是IL-1βmRNA表达水平图,
B是IL-6mRNA表达水平图,
C是TNF-αmRNA表达水平图;
图10是RT-PCR测定LPS刺激后加最大浓度PAP1和BG原代小胶质细胞促炎因子mRNA表达水平图,其中,
A是IL-1βmRNA表达水平图,
B是IL-6mRNA表达水平图,
C是TNF-αmRNA表达水平图;
图11是分子对接BG和CBR1蛋白结构图;
图12是Western blot检测LPS刺激后原代小胶质细胞CBR1蛋白表达水平图,其中,
A是CBR1蛋白表达水平图,
B是CBR1蛋白表达水平统计图;
图13是RT-PCR检测LPS刺激后原代小胶质细胞CBR1 mRNA表达水平图;
图14是ELISA检测LPS刺激后原代小胶质细胞4-HNE蛋白水平统计图。
具体实施方式
本发明的技术思路是从动物模型到细胞、分子水平,系统探讨BG对缺血后脑损伤、炎症反应的影响及其分子机制,以证明BG是一种保护缺血性脑损伤的新型药物。首先,从整体角度,验证BG能改善缺血后脑功能,降低脑梗死体积,改善神经行为缺陷,从而证明BG对缺血性脑损伤具有保护作用;其次,从整体到离体水平,用分子生物学等技术证明BG抑制小胶质细胞激活减轻炎症反应的作用;最后,从整体水平,用分子生物学等技术研究BG靶向结合KV1.3通道和CBR1通过IKB/NF-γB信号通路调控小胶质细胞的激活,以证明BG可制备为保护缺血性脑损伤新型药物的应用。
佛手柑内酯(BGGAPTEN,BG)是一种天然的香豆素,也被称为柑橘内酯,结构式如图4A所示,分子式C12H8O4,分子量216.19,白色粉末,规格是100ug/瓶,干燥保存于-20℃。将药物固体粉末混悬于1%的CMC-Na溶液中,配制成保护缺血性脑损伤的口服悬浊液制剂,灌胃注射30mg/kg/次。体外实验中,将佛手柑内酯溶于DMSO溶液中,最大稀释浓度为40μM,该剂量范围内,BG未见毒、副作用。
实施例1
体内研究证明BG对MCA0小鼠具有治疗作用:
以线栓法制作C57L/B6小鼠一侧大脑中动脉梗死模型,即MCAO模型,使用BG灌胃注射治疗。通过梗死面积和行为学,证明BG对缺血性脑损伤具有保护作用。
1)材料和方法
以线栓法建立MCAO小鼠模型,实验随机分为3组:SHAM组,MCAO+CMC-Na组,MCAO+BG(Targetmol,Shanghai,China)组,每组l0只小鼠,灌胃注射。
在MCAO后的第三天,小鼠被颈椎脱臼并被处决。取出完整的大脑并切成约1mm厚的5片。将切片浸泡在2%的2,3,5-三苯基氯化四氮唑(TTC,Sigma-Aldrich)中,并在37℃加热垫上染色15分钟。浅色区域为梗塞区,深色区域为完整的脑区。图像使用数码相机拍摄,使用Image J软件进行面积分析。梗死体积的百分比按照以下公式计算:梗死面积的百分比=(对侧面积-同侧非梗死面积)/(2×对侧面积)×100%。
神经功能缺损程度(mNSS)评分:采用改良神经功能缺损评分量表(The ModifiedNeurological Severity Score,mNSS),对小鼠神经功能缺损程度进行评分。mNSS评分总分为18分,1-6分为轻度损伤,7-12分为中度损伤,13-18分为重度损伤。
2)结果
如图1A、B所示,BG降低MCAO小鼠脑梗死面积,MCAO+CMC-Na组脑梗死面积较大,而BG显著降低MCAO小鼠脑梗死面积。
行为学结果:如图1C-E所示,MCAO后小鼠出现明显的脑功能障碍,而BG可以改善小鼠神经功能缺损。
实施例2
体内研究证明BG抑制小胶质细胞激活并减轻缺血后神经炎症反应:
1)材料与方法
小鼠皮层组织中小胶质细胞的激活状态测定:小鼠MCAO第三天,4%多聚甲醛灌注后取各组小鼠全脑组织,蔗糖梯度脱水处理后冰冻切片机切片。用0.25%的Triton X-100对大脑切片进行破膜处理15分钟,并用2%的BSA进行封闭120分钟。PBS冲洗3次后,将切片与Iba1的一抗(1:500;Abcam)在4℃下孵育过夜。第二天,将脑组织与指定的二抗在室温下避光孵育2小时。用DAPI对细胞核进行了15分钟的染色。荧光显微镜拍摄图像,用Image J软件分析小胶质细胞的形态。
炎症因子mRNA表达水平检测:小鼠MCAO第三天,阿弗汀麻醉下死于颈椎脱位,分离缺血半暗带区脑组织。使用TRIzol试剂组织提取总RNA,并按照制造商的说明使用试剂盒逆转录成cDNA。RT-PCR检测采用SYBRGreen试剂盒。相应的引物序列为:
IL-1β,f:CCATCCTCTGTGACTCATGGG,r:TCAGCTCATATGGGTCCGAC;
IL-6,f:GACAAAGCCAGAGTCCTTCAGAGAG,r:CTAGGTTTGCCGAGTAGATCTC;
TNF-α,f:CCACCACGCTCTTCTGTCTA,r:GATCTGAGTGTGAGGGTCTGG;
GAPDH,f:AGGTCGGTGTGAACGGATTTG,r:TGTAGACCATGTAGTTGAGGTCA。
2)结果
免疫荧光染色结果:如图2所示,MCAO后小鼠皮层组织缺血半暗带区大量小胶质细胞激活,BG处理后可见小胶质细胞激活数量显著下降。Image J对小胶质细胞形态分析结果显示BG处理组小胶质细胞胞体较小,分支数量较多,分支长度较长,说明BG处理组小胶质细胞激活程度较低,该结果进一步证实BG能够抑制小胶质细胞的激活。
RT-PCR结果:如图3所示,BG抑制小鼠MCAO后脑组织缺血半暗带促炎因子的表达。MCAO后脑组织明显出现炎症反应,而BG可显著下调促炎因子IL-1β、IL-6和TNF-α的mRNA表达水平。
实施例3
体外研究证明BG对原代小胶质细胞和原代神经元没有药物毒性:
1)材料和方法
用新生的C57/BL6J小鼠制备原代小胶质细胞和原代神经元。10-12天后,用200rpm振荡培养瓶10min,从星形胶质细胞中分离出小胶质细胞。小胶质细胞培基使用含有90%MEM,10%胎牛血清和1%青霉素/链霉素的培养基,原代神经元使用Neurobasal 50ml、B271ml、GlutaMAX50ul培养基,细胞在37℃,5%CO2的潮湿气氛中培养。
药物毒性检测:乳酸脱氢酶(LDH)释放试验用于测定试验物质的细胞毒性。原代小胶质细胞和原代神经元在96孔板中种板培养,不同浓度的BG处理24h,用未处理细胞的孔作为空白对照,用细胞中加入T-X100的孔测定最大LDH释放量(100%)。将测定液与50uL上清液混合,在室温下以500rpm摇动10分钟。使用酶标仪在490nm处测量光密度(OD)。结果根据背景吸光度进行了校正,并以最大LDH释放的百分比表示。
2)结果
LDH结果:如图4B、C所示,不同浓度的(1μM,5μM,10μM,20μM,40μM)BG对原代小胶质细胞和原代神经元没有药物毒性。
实施例4
体外研究证明BG可减弱脂多糖诱导的小胶质细胞的促炎反应:
1)材料和方法
原代小胶质细胞用不同浓度BG(10、20和40μM)预处理1h,然后用LPS(100ng/mL)刺激3h。RT-PCR实验操作方法同上。ELISA检测同样用不同浓度的BG(10、20和40μM)预处理原代小胶质细胞1h,然后用LPS(100ng/mL)刺激24h后收集上清液,根据制造商的说明(Fomax生物制品),测定IL-1β、IL-6和TNF-α的浓度。使用酶标仪在450nm处测量光密度(OD)。
2)结果
RT-PCR结果:如图5所示,激活的小胶质细胞会释放多种炎症因子加剧损伤。LPS刺激后小胶质细胞IL-1β、IL-6和TNF-α的mRNA表达水平明显升高。BG呈剂量依赖性抑制LPS诱导的IL-1β、IL-6和TNF-α产生。
ELISA结果:如图6所示,LPS刺激后小胶质细胞IL-1β、IL-6和TNF-α的蛋白表达水平明显升高。BG呈剂量依赖性抑制LPS诱导的IL-1β、IL-6和TNF-α产生。
实施例5
体外研究证明BG调控LPS刺激后NF-κB信号通路:
1)材料与方法
原代小胶质细胞用BG(40μM)预处理1h,加入LPS刺激0.5h后,与含有1%蛋白酶抑制剂裂解缓冲液缓冲液均匀反应30min,4℃,15000rpm离心20min,收集上清液。使用BCA试剂盒计算蛋白质浓度。等量的蛋白质用10%的SDS-PAGE分离,并在250mA恒流转移到PVDF膜上,持续100min。用快速封闭液封闭15min,然后与适当的一抗(IκBα,1:1000;p-IκBα,1:1000;NF-κB,1:1000;p-NF-κB,1:1000;β-actin,1:5000)孵育过夜。第二天,用二抗在室温下再孵育2小时。用增强化学发光法检测蛋白条带,用Gel-Pro系统获取图像,用Image J软件分析各条带的强度。β-actin被用来作为内参。
2)结果
Western blot结果:如图7所示,LPS刺激后NF-κB p65磷酸化水平明显升高,IκBα明显磷酸化,同时通过泛素化降解。BG可以抑制NF-κB信号通路磷酸化。
实施例6
体外研究证明BG可部分通过抑制KV1.3减轻LPS诱导的小胶质细胞的炎症反应:
1)材料和方法
将5mm的PBFI-AM原液与等体积的25%Pluronic F-127混合后加入无血清细胞培养基中,后将培基加入原代小胶质细胞进行探针标记。100min后用无血清培养基洗涤多余的PBFI-AM。加入LPS刺激3H后用生理盐水洗涤细胞终止实验。用344和400nm处的激发比和500nm处的激发比来测定细胞内K+的含量。
PAP1是目前针对KV1.3最有效和特异性的小分子抑制剂,ic50为2nmol/L。原代小胶质细胞用不同浓度PAP1(10μM,20μM,50μM)预处理1h,然后用LPS(100ng/mL)刺激3h。原代小胶质细胞用最大浓度的PAP1(50μM)和BG(40μM)预处理1h,然后用LPS(100ng/mL)刺激3h。RT-PCR实验操作方法同上。
2)结果
如图8所示,PBFI-AM结果显示LPS刺激后小胶质细胞钾离子外流,细胞内钾离子浓度下降,BG可以抑制细胞内钾离子外流,表明BG可以部分通过抑制KV1.3通道发挥抑制炎症效果。
如图9所示,RT-PCR结果表明PAP1呈剂量依赖性抑制LPS诱导的IL-1β、IL-6和TNF-α产生。
如图10所示,RT-PCR结果显示最大浓度的PAP1(50μM)和BG(40μM)联合使用能够抑制LPS诱导的IL-1β、IL-6和TNF-α产生,与单独使用最大浓度BG(40μM)抑炎效果相比具有统计学意义,说明抑制KV1.3通道并不能完全阻断BG的抑炎效果,表明BG可以部分通过抑制KV1.3通道发挥抑制炎症效果。
实施例7
体外研究证明BG抑制CBR1蛋白降解和底物4-HNE的产生减轻LPS诱导的小胶质细胞的炎症反应:
1)材料与方法
人源性CBR1(PDB ID:3BHJ)的晶体结构来自蛋白质数据库(https://www.pdb.org/),BG(PubChem CID:2355)的三维结构从PubChem下载。CRR1的晶体被输入Pymol软件进行脱水、氢化和去除配体。蛋白质受体和药物配体被导入AutoDockTools以确定对接网格盒。对接是由Autodock Vina软件进行的。生物大分子及其配体之间的非共价相互作用是由PLIP Web工具(https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index)确定的。AutoDock Vina和PLIP的结果通过PyMOL软件进行了可视化。
Western blot操作方法同上。采用LPS刺激3h后收集小胶质细胞的蛋白质。一抗(CBR1,1:1000;β-actin,1:5000)ELISA和QPCR操作方法同上。
2)结果
如图11所示,分子对接结果显示采用AutodockTools软件将BG与蛋白CBR1进行分子对接分析,结合能为-6.3kcal/mol,表明BG可以与CBR1稳定结合。一般认为配体与受体蛋白结合能越低,构象越稳定,发生相互作用可能性越大,当结合能绝对值<-5.0表示两者较好结合活性。此外,BG分别与CBR1的LYS-197,VAL-230之间形成氢键,使其结合更加稳定。
Western blot结果:如图12所示,LPS后小胶质细胞中CBR1蛋白表达下降,而BG处理后可以抑制CBR1的降解。
如图13所示,RT-PCR结果显示LPS刺激后小胶质细胞中CBR1 mRNA水平降低。BG在mRNA水平不能抑制CBR1的降解。
如图14所示,ELISA结果显示LPS刺激后小胶质细胞4-HNE的蛋白表达明显升高。BG有效抑制LPS诱导的小胶质细胞中4-HNE的产生。
以上仅是本发明的优选实施方式,本发明的保护范围并不仅局限于上述实施例,凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理前提下的若干改进和润饰,应视为本发明的保护范围。
SEQUENCE LISTING
<110> 南京鼓楼医院
<120> 佛手柑内酯作为制备治疗缺血性脑卒中药物的应用
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Claims (8)
2.根据权利要求1所述的应用,其特征在于,所述治疗是通过抑制小胶质细胞激活,减轻缺血后神经炎症反应实现的。
3.根据权利要求2所述的应用,其特征在于,所述治疗是通过佛手柑内酯减弱脂多糖诱导的小胶质细胞的促炎反应实现的。
4.根据权利要求3所述的应用,其特征在于,所述治疗是佛手柑内酯通过抑制KV1.3减轻LPS诱导的小胶质细胞的炎症反应实现的。
5.根据权利要求3所述的应用,其特征在于,所述治疗是佛手柑内酯通过抑制CBR1蛋白的降解和底物4-HNE的产生进而减轻LPS诱导的小胶质细胞的炎症反应实现的。
6.根据权利要求2所述的应用,其特征在于,所述治疗是佛手柑内酯通过调控NF-κB信号通路减轻炎症反应实现的。
7.根据权利要求1所述的应用,其特征在于,所述药物的剂型为口服剂。
8.根据权利要求7所述的应用,其特征在于,所述口服剂灌胃给药的用量为30mg/kg/次。
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CN104815192A (zh) * | 2015-04-30 | 2015-08-05 | 青岛市市立医院 | 一种治疗高血压的药物组合物及其应用 |
CN109793748A (zh) * | 2019-03-31 | 2019-05-24 | 李锋 | 一种防治脑缺血引起炎症反应的药物及其制剂 |
CN110420214A (zh) * | 2019-03-31 | 2019-11-08 | 李锋 | 一种防治脑缺血引起氧化应激反应的药物及其制剂 |
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CN104815192A (zh) * | 2015-04-30 | 2015-08-05 | 青岛市市立医院 | 一种治疗高血压的药物组合物及其应用 |
CN109793748A (zh) * | 2019-03-31 | 2019-05-24 | 李锋 | 一种防治脑缺血引起炎症反应的药物及其制剂 |
CN110420214A (zh) * | 2019-03-31 | 2019-11-08 | 李锋 | 一种防治脑缺血引起氧化应激反应的药物及其制剂 |
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