CN117562903A - 一种抑制新生内膜增生改善术后再狭窄的中药单体及应用 - Google Patents
一种抑制新生内膜增生改善术后再狭窄的中药单体及应用 Download PDFInfo
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Abstract
本发明涉及一种抑制新生内膜增生改善术后再狭窄的中药单体及应用;结果表明,体内实验证明去甲乌药碱可以抑制股动脉内皮细胞损伤术后新生内膜的形成,体外实验证明去甲乌药碱促进血管平滑肌细胞的凋亡;而血管平滑肌细胞的异常活化会导致多种血管性疾病的发生,如冠心病支架内再狭窄、静脉疾病以及与移植相关的血管病变,则去甲乌药碱能防治此类血管性疾病,也能在改善股动脉损伤术后再狭窄。
Description
技术领域
本发明涉及中药单体的应用技术领域,具体地说,是关于一种抑制新生内膜增生改善术后再狭窄的中药单体及应用。
背景技术
血管平滑肌细胞的异常活化会导致多种血管性疾病的发生,如冠心病支架内再狭窄、静脉疾病以及与移植相关的血管病变;在生理条件下,血管平滑肌细胞位于血管的中层,处于静止状态;这类细胞高度分化,能够分泌大量的收缩蛋白以维持血管收缩表型和血管张力;同时,血管平滑肌细胞的表型具有一定的可塑性,在血管损伤等病理性刺激下会发生表型转变,由收缩型转变成合成型合成型血管平滑肌能够下调收缩型蛋白的表达,同时上调生长因子、生长因子受体、细胞外基质、细胞外基质蛋白酶和炎症介质引起血管平滑肌异常增生,这种异常增生进一步会引起血管重塑;临床通过抑制血管平滑肌细胞的增殖,拮抗病理性血管重塑治疗动脉狭窄类血管疾病。
目前临床上最常见的治疗冠心病方法为经皮冠状动脉介入治疗,而术后血管的再狭窄很大程度影响了临床疗效;而预防再狭窄的主要方法是通过药物洗脱支架局部给予抗增殖剂,如紫杉醇和雷帕霉素;这种治疗策略是通过抑制支架破坏内皮产生血管平滑肌细胞表型变换、异常增殖、迁移等出现新生内膜的形成;而这些药物对于血管局灶性病变是有效的,却无法治疗新生内膜病变引起的弥漫性血管疾病。
中国专利:CN 113881638A,公开了一种去甲乌药碱半抗原、单克隆抗体、杂交瘤细胞株及应用,属于食品安全免疫检测领域,以去甲乌药碱与丁二酸酐反应得到具有羧基的产物为半抗原,用碳二亚胺法将半抗原与载体蛋白偶联,得到完全抗原,用紫外法测定偶联物的偶联比,并得到一株单克隆抗体杂交瘤细胞株,此细胞株分泌的单克隆抗体,对去甲乌药碱具有较好的特异性和检测灵敏度,IC50值为10ng/mL,可实现对中药材及功能性食品中去甲乌药碱残留量的检测,为功能性食品中去甲乌药碱残留的免疫检测提供了原料,具有实际应用价值。本发明还成功合成了去甲乌药碱人工抗原,合成步骤简洁有效,完全可用于免疫分析中,为以后人们的研究提供了必需的人工抗原。
去甲乌药碱作为附子中强心成分的主要水溶性单体之一,具有强心、抗心肌细胞凋亡和舒张气管平滑肌的作用;但是在改善股动脉损伤术后再狭窄领域缺少相关研究。
发明内容
本发明的第一个目的是,针对现有技术中的不足,提供一种中药单体去甲乌药碱的用途。
本发明的第二个目的是,提供一种由中药单体去甲乌药碱组成的药物。
为实现上述目的,本发明采取的技术方案是:
第一方面,本发明提供了中药单体去甲乌药碱在制备抑制动脉中新生内膜的形成的药物中的应用。
第二方面,本发明提供了中药单体去甲乌药碱在促进血管平滑肌细胞的凋亡制备药物中的应用。
作为一个优选例,所述的甲乌药碱在抑制作用具有剂量依赖性。
第三方面,本发明提供了中药单体去甲乌药碱在制备改善动脉狭窄的药物中的应用。
根据上述任一所述的应用,所述的药物包括中药单体去甲乌药碱和药学上可接受的辅剂。
为实现上述第二个目的,本发明采取的技术方案是:
一种改善动脉狭窄的药物,所述的药物是由中药单体去甲乌药碱和药学上可接受的辅剂制成的。
一种防/治血管平滑肌细胞异常活化致使的血管性疾病的药物,所述的药物是由中药单体去甲乌药碱和药学上可接受的辅剂制成的。
根据上述任一所述所述的药物,所述的药物制剂包括颗粒剂、散剂、胶囊剂、片剂、合剂或口服液。
本发明优点在于:
本发明研究了中药附子中的单体去甲乌药碱,通过各类实验证明了去甲乌药碱能改善股动脉损伤术后再狭窄,且去甲乌药碱剂量依赖性促进血管平滑肌细胞的凋亡,则相应的抑制由血管平滑肌细胞的异常活化会导致多种血管性疾病,如冠心病支架内再狭窄、静脉疾病以及与移植相关的血管病变。
【附图说明】
附图1为去甲乌药碱对股动脉内皮细胞损伤小鼠新生内膜形成的影响。(A,通过HE染色小鼠股动脉组织切片,去甲乌药碱(10mg/kg/天)或生理盐水腹腔注射给药21天后采集图片。图B和C,形态分析表明造模后21天,腹腔注射去甲乌药碱比模型组新生内膜(B)、血管中层(C)厚度明显减少。D,新生内膜增生厚度与平滑肌厚度的比值显示去甲乌药碱组比模型组新生内膜厚度明显减少。E,免疫组化染色后,血管新生内膜(Int),平滑肌层(Med)和血管外层(Adv)结构图。F,免疫组织化学分析显示去甲乌药碱降低细胞增殖与Brdu染色。右上角的小图是假手术样本的对应图像。G,Brdu阳性细胞的定量数据。*p<0.05代表与模型组比较,NS无统计学差异,每组样本(n=8)。)
附图2为去甲乌药碱对血管平滑肌细胞凋亡的作用。(P5/P6血管平滑肌细胞,铺板密度:6×104,在含0.1%FBS下饥饿48H,更换为10%FBS培养基,在不同浓度梯度的去甲乌药碱中刺激48H,用不含EDTA的胰酶消化细胞,弃上清,加入Annexin-V-FITC/PI染料,在流式细胞仪中分选细胞,对处在早期凋亡和晚期凋亡细胞百分比进行统计。*p<0.05,**p<0.01相比于10%FBS组,NS无统计学差异,结果为三个独立实验的平均数±标准差。)
【具体实施方式】
下面结合具体实施方式,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明记载的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
1.实验内容
体内构建金属导丝小鼠股动脉损伤模型,通过对股动脉进行HE染色探讨去甲乌药碱(HG)对小鼠新生内膜形成的影响。
体外培养血管平滑肌细胞,先用0.1%FBS培养基饥饿细胞48H,后加入含10%FBS培养基使血管平滑肌细胞变成增殖状态构建体外细胞模型,加入单体HG培养48H,Annexin-V-FITC/PI双染流式细胞术检测去甲乌药碱对血管平滑肌细胞凋亡的作用。
2.实验方法
2.1体内实验
2.1.1实验动物
12周龄C57BL/6野生型雄性小鼠24只,体重25–30g,在动物房适应性饲养一周后进行造模实验。
2.1.2实验分组
将小鼠随机分成3组:对照组(Sham)、模型组(injury)、实验组(injury+HG)(10mg/kg·d),每组8只。
2.1.3造模方法
用三溴乙醇(30mg/kg-50mg/kg)腹腔注射麻醉,仰卧位固定小鼠的四肢及头部,使股动脉部能够完全暴露,经皮肤消毒后沿双侧腹股沟中点纵行切开皮肤,可见左股动脉。
用直镊和弯镊将股动脉与股静脉及伴行的神经顿性分离,并进一步分离出左股动脉分支,用6-0丝线暂时将股动脉近端,远端和分支固定,刚好能阻碍股动脉血液流动。将股动脉分支用剪刀剪出一个小缺口,用0.38mm金属导丝从分枝穿入股主动脉约1min,损伤股动脉内皮细胞;取出金属导丝,结扎分支,缝合伤口;术后给予抗生素预防伤口感染。
2.1.4实验给药
造模完成后,各组通过腹腔注射进行相应的药物干预21天,实验组给予去甲乌药碱(10mg/kg),对照组、模型组给予等容量生理盐水灌胃。
2.2观测指标
2.2.1石蜡包埋
实验最终日禁食12小时后,腹部麻醉小鼠,脱颈处死。
取股总动脉血管分叉结扎处至其上2cm处组织,置于4%的多聚甲醛溶液中固定24小时后,准备石蜡包埋。
2.2.2组织切片和HE染色
组织切片和HE染色步骤:
①脱水:将固定的组织取出,依次经过:70%酒精→80%酒精→90%酒精→95%酒精→无水乙醇(两次),每级35-45分钟。
②透明:无水乙醇+二甲苯(1:1)30分钟,二甲苯(Ⅰ)10分钟。
③浸蜡:二甲苯+石蜡(1:1)30分钟,石蜡(Ⅰ)40分钟→石蜡(Ⅱ)30分钟。
④包埋:包埋将己浸蜡的组织块放入热融的包埋石蜡中,迅速冷凝,组织块便被蜡包埋。
⑤切片:切片石蜡切片机切片,厚度为5μm。
⑥贴片烤干:切好的石蜡片放在60℃温水上展平后,载玻片捞片,60℃烤箱烘烤过夜,备用。从结扎处起连续石蜡切片厚度为5μm,每隔200μm切8张片子,连续切40张片子,进行染色。
⑦脱蜡:二甲苯Ⅰ、Ⅱ、Ⅲ,各5分钟→无水乙醇,2分钟→90%乙醇,2分钟→80%乙醇,2分钟→70%乙醇,2分钟。蒸馏水略洗。
⑧苏木素染液中染色1分钟30秒,水洗3遍。
⑨1%盐酸乙醇分化5秒,流水漂洗。
⑩2%氨水返蓝15秒,流水漂洗。
伊红染液中染色1分钟,90%乙醇对伊红分色。
脱水,染色后的切片依次置于80%乙醇,90%乙醇,无水乙醇,各2分钟,二甲苯Ⅰ、Ⅱ、Ⅲ中各5分钟。
取出切片,风干组织上的二甲苯,中性树胶封片。
显微镜下进行拍照,用Image pro plus6.0分析软件进行图像分析,用软件测量出内腔面积,内膜面积以及中膜面积,新生内膜厚度由新生内膜面积与中膜面积之比来表示。
2.2.3免疫组化
准备工作:组织切片、DAB试剂盒、胰酶修复液、3%双氧水,PBS缓冲液、苏木素、1%盐酸乙醇、2%氨水、二甲苯、无水乙醇。
①脱蜡:二甲苯Ⅰ、Ⅱ、Ⅲ,各5分钟→无水乙醇,2分钟→90%乙醇,2分钟→80%乙醇,2分钟→70%乙醇,2分钟,蒸馏水清洗。
②组织修复:在组织片上滴入适量胰酶在37℃恒温箱反应10分钟。
③抗原修复后,切片在PBS中,浸泡1分钟,再将3%H2O2滴于样本上,室温放置10分钟,用于消除内源性过氧化物酶的活性。
④水洗2遍,PBS缓冲液2遍。
⑤取出切片,滴加10%BSA封闭液,37℃孵育40分钟。
⑥将切片放在专用孵育盒内滴加一抗(用4%BSA稀释),4℃冰箱过夜。
⑦PBS缓冲液3次×3分钟,充分洗涤,防止非特异性染色。
⑧擦干组织周围液体后滴加辣根酶标羊抗小鼠/兔IgG聚合物,37℃温箱内孵育1小时。PBS缓冲液洗涤3分钟×3次。
⑨显色,配DAB显色剂(在2.5ml DH2O内加入2滴(约100μl)试剂A,1滴试剂B和1滴试剂D混合液,即配成DAB工作液)。室温显色5-20分钟。自来水终止显色。水洗3遍,第一遍的水需集中处理,。
⑩复染,苏木素染液中染色1分钟30秒,水洗3遍。
1%盐酸乙醇分化5秒,流水漂洗。
2%氨水返蓝15秒,流水漂洗。
脱水,切片依次置于80%乙醇,90%乙醇,无水乙醇,各2分钟,二甲苯Ⅰ、Ⅱ、Ⅲ中各5分钟。
中性树胶封片。
荧光显微镜下观察结果,拍照。Imageproplus6.0分析结果。
2.3体外实验
2.3.1血管平滑肌细胞原代取材及培养
材料:SD大鼠、雄性、2只(200-250g)。
准备工作:弯头手术剪,止血钳,眼科镊,直剪,弯镊,35mm培养皿,54mm/100mm培养皿,15ml离心管,移液管,注射器1.5ml,无菌纱布,75%乙醇,舒泰50,HBSS(平衡盐溶液),Dig#1(20mgⅡ型胶原酶310U/mg+HBSS,共10ml),Dig#2(20mgⅡ型胶原酶310U/mg+418μlElastase 9.7mg蛋白/mL,4U/mg蛋白+HBSS,共10ml),10%FBS,20%FBS。
①器材及试剂紫外消毒30min,同时麻醉SD大鼠,腹腔注射舒泰50原液0.2-0.3ml。消毒结束后,75%乙醇消毒腹部被毛并置于消毒后的超净台内,麻醉后将大鼠仰卧固定在操作台上。
②剑突下以V字型剪开大鼠胸腔,为了减少细菌污染,每剪开一层组织即更换一把剪刀,更换后的剪刀置于75%乙醇中浸泡消毒,依次剪开皮肤、胸腔,用镊子夹住剑突将其固定。充分暴露大鼠胸腔后固定胸壁,然后将肺叶拨向大鼠右侧(操作者左侧)并固定,充分暴露胸主动脉(紧贴大鼠脊柱)。钝性分离腹主动脉(分离时尽量将血管外膜的脂肪和结缔组织分离干净),用弯头剪由上到下剪取尽量多的血管。用小锐利剪刀沿脊柱剪断,上至主动脉弓,下肢腹主动脉分支上端。切除时小心不要拉伸血管。
③取出的血管用HBSS(约2ml)清洗3次。全部血管清洗干净后,移入5mlDig#1消化液中,并置于37℃培养箱中消化约30min。
④血管置于10%FBS培养基中终止消化,从血管断口处剥下外膜。纵向剪开血管,用移液管擦去内皮组织。尽量剪碎血管,成1mm3大小,加入5mlDig#2,于无菌培养箱中消化2h,期间可适时取出吹打,使其消化完全;加入10%FBS培养基终止消化,移入15ml离心管中,900rpm,5min,弃上清。
⑤加入8ml 20%FBS培养基吹打均匀,接种至100mm培养皿中孵育;24h后可见少量细胞贴壁,换液,7-9天后形成致密单层细胞层。随后进行传代,传至5-6代用于实验。
2.3.2血管平滑肌细胞体外培养
探讨去甲乌药碱对增殖型血管平滑肌细胞凋亡的作用。血管平滑肌细胞传代至P5/P6,铺板密度:6×104,在含0.1%FBS下饥饿48H,更换为10%FBS培养基,在不同浓度梯度的去甲乌药碱中刺激48H。将上层培养基抽吸到15ml离心管中,2ml PBS洗两次,抽吸到15ml离心管中。加入200μl不含EDTA胰酶,在37℃培养箱中消化1min,加入1ml 10%FBS终止消化,把细胞吹下来移到15ml离心管。1000RPM,5min,离心。弃上清。配制1X AnnexinVBinding Buffer,10X Annexin V Binding Buffer:ddH2O=1:9,600μl:5400μl。加入100μl1X AnnexinV Binding Buffer于15ml离心管,混匀,移至流式管中。每个流式管中加入5μlFITC Annexin V、5μl Propidium Iodide Staining Solution,枪头混匀,室温避光15min。流式仪开机,加入400μl 1X AnnexinV Binding Buffer,混匀,上机。
3.实验结果
3.1体内实验
去甲乌药碱可以抑制股动脉内皮细胞损伤术后新生内膜的形成:通过HE染色进行形态分析,在股动脉内皮细胞损伤模型21天后,结扎损伤引起的新生内膜(图1B)和中膜(图1C)增厚增加显著,血管腔面积减少,新生内膜/中膜面积比升高,与模型组相比较HG新生内膜和中膜面积明显减少,血管腔面积增大,新生内膜/中膜面积比降低,说明HG可以抑制股动脉内皮细胞损伤术后新生内膜的形成。由于血管平滑肌细胞增殖是内膜增生的发展至关重要的一步,我们进行了在股动脉细胞中SM-α-actin与Brdu(细胞增殖相关抗体)平滑肌肌动蛋白抗体的染色(图1F)。结果发现,模型组中Brdu阳性细胞相比假手术组数量显著增加,而在去甲乌药碱治疗后小鼠Brdu阳性细胞数减少约50%(图1G)。
3.2体外实验
去甲乌药碱可以促进血管平滑肌细胞的凋亡:对处在早期凋亡和晚期凋亡细胞百分比进行统计,300μM HG组相较于10%FBS组处于早期凋亡和晚期凋亡细胞百分比明显升高,说明去甲乌药碱可以促进血管平滑肌细胞的凋亡。
以上所述仅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,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (8)
1.中药单体去甲乌药碱在制备抑制动脉中新生内膜的形成的药物中的应用。
2.中药单体去甲乌药碱在制备促进血管平滑肌细胞的凋亡药物中的应用。
3.根据权利要求2所述的应用,其特征在于,所述的甲乌药碱在抑制作用具有剂量依赖性。
4.中药单体去甲乌药碱在制备改善动脉狭窄的药物中的应用。
5.根据权利要求1-4任一所述的应用,其特征在于,所述的药物包括中药单体去甲乌药碱和药学上可接受的辅剂。
6.一种改善股动脉损伤术后再狭窄的药物,其特征在于,所述的药物是由中药单体去甲乌药碱和药学上可接受的辅剂制成的。
7.一种防/治血管平滑肌细胞异常活化致使的血管性疾病的药物,其特征在于,所述的药物是由中药单体去甲乌药碱和药学上可接受的辅剂制成的。
8.根据权利要求8-9所述的药物,其特征在于,所述的药物制剂包括颗粒剂、散剂、胶囊剂、片剂、合剂或口服液。
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