CN117257798A - 小檗碱在预防主动脉夹层动脉瘤进展中的应用 - Google Patents
小檗碱在预防主动脉夹层动脉瘤进展中的应用 Download PDFInfo
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Abstract
本发明公开了的一种小檗碱在预防主动脉夹层动脉瘤进展中的应用,所述小檗碱在制备预防主动脉夹层/动脉瘤进展药物中的应用,所述主动脉夹层/动脉瘤包括β‑氨基丙腈诱导的胸主动脉夹层及动脉瘤,所述小檗碱在制备抑制主动脉夹层/动脉瘤弹性纤维降解药物中的应用,所述小檗碱在制备抑制主动脉夹层/动脉瘤弹性纤维断裂药物中的应用。本发明提供了小檗碱在制备抑制预防主动脉夹层/动脉瘤进展药物中的应用,本研究发现,小檗碱能够改善主动脉夹层/动脉瘤小鼠的生存曲线,抑制成模率,降低主动脉直径的扩张,明显抑制弹性纤维的降解、断裂以及胶原纤维的降解,小檗碱能够明显抑制血管炎症反应,降低血管组织凋亡水平。
Description
技术领域
本发明涉及中医药技术领域,具体是小檗碱在预防主动脉夹层动脉瘤进展中的应用。
背景技术
主动脉夹层(aortic dissection)是指循环血液经过破裂的主动脉内膜和/或中膜进入到主动脉中膜,沿主动脉长轴扩展,将中膜完全撕开,形成假腔的一种病理变化,胸主动脉瘤(thoracic aortic aneurysm)是指胸主动脉部分管壁异常膨大变形呈瘤样突出,主动脉夹层及动脉瘤在病理生理及发病机制上有一定的相似性,以胸痛为主要特征性病变表现,发病隐匿,进展迅速,死亡率高且发病率逐渐增加,是一种严重威胁人类健康的疾病。
主动脉夹层及动脉瘤目前仍以手术及血管内治疗为主,然而由于手术本身的风险性,对于血管早期病变不鼓励手术治疗,近年来,随着诊断技术的提高,一些早期血管病变及小的动脉瘤的检出率明显增加,由于缺乏相应的治疗药物,难以对其进行有效的控制,最终后果仍然是在符合手术指征后进行治疗,甚至是难以预料的主动脉扩张、破裂,给患者造成极大的心里及经济负担。
主动脉夹层及动脉瘤的发病机制目前尚未完全清楚,其危险因素包括高血压、吸烟、遗传以及动脉粥样硬化等,二者的特征性病理变化包括凋亡导致的血管平滑肌细胞的丢失、细胞外基质(ECM)的降解、弹性层的断裂以及炎症细胞的浸润,以赖氨酸氧化酶(LOX)抑制剂β-氨基丙腈(BAPN)诱导的胸主动脉夹层/动脉瘤小鼠模型在帮助理解疾病的病理生理中提供了有效的帮助。
小檗碱(BBR)是从黄连等中草药中提取的出来的一种异喹啉类生物碱,其分子式为C20H18NO4,现以其盐酸盐和硫酸盐为应用的主要形式,此外,现代药理学研究表明小檗碱还具有抗糖尿病、抗炎、抗肿瘤和抗氧化等作用,研究表明,小檗碱能够抑制细胞凋亡,改善动脉粥样硬化,但是小檗碱对主动脉夹层/动脉瘤的保护作用尚无研究。
发明内容
本发明的目的在于提供一种小檗碱在预防主动脉夹层动脉瘤进展中的应用,以解决上述背景技术中提出的问题。
为实现上述目的,本发明提供如下技术方案:
一种小檗碱在预防主动脉夹层动脉瘤进展中的应用,包括以下步骤:所述小檗碱在制备预防主动脉夹层/动脉瘤进展药物中的应用。
作为本发明进一步的方案:所述主动脉夹层/动脉瘤包括β-氨基丙腈诱导的胸主动脉夹层及动脉瘤。
作为本发明再进一步的方案:所述小檗碱在制备抑制主动脉夹层/动脉瘤弹性纤维降解药物中的应用。
作为本发明再进一步的方案:所述小檗碱在制备抑制主动脉夹层/动脉瘤弹性纤维断裂药物中的应用。
作为本发明再进一步的方案:所述小檗碱在制备抑制主动脉夹层/动脉瘤胶原纤维降解药物中的应用。
作为本发明再进一步的方案:所述小檗碱在制备抑制主动脉夹层/动脉瘤组织中MMP9/2的基因转录药物中的应用。
作为本发明再进一步的方案:所述小檗碱在制备促进主动脉夹层/动脉瘤组织中ADAMTS5/8的基因转录药物中的应用。
作为本发明再进一步的方案:所述小檗碱在制备抑制主动脉夹层/动脉瘤组织炎症发生药物的应用。
作为本发明再进一步的方案:所述小檗碱在制备抑制主动脉夹层/动脉瘤组织中性粒细胞浸润药物的应用。
作为本发明再进一步的方案:所述小檗碱在制备抑制主动脉夹层/动脉瘤组织凋亡水平药物的应用。
作为本发明再进一步的方案:所述药物的成分包括小檗碱和辅料。
与现有技术相比,本发明的有益效果是:
本发明提供了小檗碱在制备抑制预防主动脉夹层/动脉瘤进展药物中的应用,本研究发现,小檗碱能够改善主动脉夹层/动脉瘤小鼠的生存曲线,抑制成模率,降低主动脉直径的扩张,明显抑制弹性纤维的降解、断裂以及胶原纤维的降解,小檗碱能够明显抑制血管炎症反应,降低血管组织凋亡水平。
附图说明
图1A为各组实验动物生存曲线,图1B为模型组和小檗碱组成模率的差异;
图2A为各组实验动物主动脉夹层的损伤程度,图2B为各组实验动物主动脉直径的统计值;
图3A为各组实验动物胸主动脉HE染色对比图,图3B为各组实验动物胸主动脉弹性纤维染色对比图,图3C为各组实验动物弹性纤维断裂等级变化;
图4A为各组实验动物流式细胞术检测主动脉组织浸润免疫细胞代表图,图4B为浸润免疫细胞统计图,图4C为各组实验动物流式细胞术检测主动脉浸润中性粒细胞代表图,图4D为浸润中性粒细胞统计图;
图5为各组实验动物胸主动脉组织中IL-6、TNF-α、MCP-1、IL-8 mRNA的相对含量;
图6A为各组实验动物胸主动脉组织中MMP-2、MMP-9 mRNA的相对含量,图6B为各组实验动物胸主动脉组织ADAMTS8、ADAMTS5 mRNA的相对含量;
图7A为各组实验动物主动脉组织BAX、Bcl2和GAPDH的Western Blot图,图7B为Bcl2/GAPDH相对表达量的统计图,图7C为BAX/GAPDH相对表达量的统计图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
如图1-7所示,实施例1:一种小檗碱在预防主动脉夹层动脉瘤进展中的应用,小檗碱在制备预防主动脉夹层/动脉瘤进展药物中的应用,主动脉夹层/动脉瘤包括β-氨基丙腈诱导的胸主动脉夹层及动脉瘤,小檗碱在制备抑制主动脉夹层/动脉瘤弹性纤维降解药物中的应用,小檗碱在制备抑制主动脉夹层/动脉瘤弹性纤维断裂药物中的应用,小檗碱在制备抑制主动脉夹层/动脉瘤胶原纤维降解药物中的应用,小檗碱在制备抑制主动脉夹层/动脉瘤组织中MMP9/2的基因转录药物中的应用,小檗碱在制备促进主动脉夹层/动脉瘤组织中ADAMTS5/8的基因转录药物中的应用,小檗碱在制备抑制主动脉夹层/动脉瘤组织炎症发生药物的应用,小檗碱在制备抑制主动脉夹层/动脉瘤组织中性粒细胞浸润药物的应用,小檗碱在制备抑制主动脉夹层/动脉瘤组织凋亡水平药物的应用,药物的成分包括小檗碱和辅料,上述小檗碱购自上海源叶生物,纯度≥98%,且本发明对药物剂型没有特殊限定,采用小檗碱在医学上可以接受的剂型即可,本发明对药物的制备方法没有特殊限定,采用相应剂型的制备方法即可,本发明对药物中小檗碱的含量没有特殊限定,采用药物中常规活性药物的含量即可。
1、实验材料
1.1实验动物:3周龄雄性SPF级C57BL/6J小鼠,体重约10g,购自北京维通利华实验动物科技有限公司,合格证号:SCXK(京)2021-0006,所有动物饲养于华中科技大学同济医学院实验动物中心,饲养条件为SPF级,光照12h/d,恒温恒湿,针对动物的所有实验条件和操作均符合华中科技大学动物研究所委员会的指导方针,并获得华中科技大学动物保护与利用机构委员会的批准。
1.2实验药物与试剂:小檗碱(上海源叶生物,纯度≥98%),β-氨基丙腈(Sigma,CAS:A3134),抗小鼠CD45.2-FITC 抗体(BD,clone: 104),CD3-APC抗体(BD,clone: 145-2C11),CD19-APC抗体(BD,clone: 6D5),CD11b-BV510抗体(eBioscience,clone: M1/70),Ly6G-APC-Cy7抗体(BD,clone: 1A8),CD4-PE-Cy7抗体(BD,clone: RM4-5),ROR-γt-BV421抗体(BD,clone: Q31-378),Foxp3-PE抗体(BD,Clone: R16-715),死活染料(BD,Cat#564406),小鼠anti-BAX抗体(abmart,CSA:TU333334S),小鼠anti-Bcl2抗体(abmart,CSA:TU323153S)。
1.3实验仪器:流式细胞仪(Beckman),游标卡尺,Nanodrop。
2实验方法
2.1主动脉夹层/动脉瘤模型建立:采用β-氨基丙腈饮水法构建小鼠胸主动脉夹层/动脉瘤模型,按照每只小鼠1g/Kg/day的剂量将β-氨基丙腈溶于饮用水中,连续饮用4周,构建动物模型。
2.2动物分组及给药:小鼠随机分为三组:正常组、模型组及小檗碱组,除正常组外,模型组及小檗碱组按上述构建主动脉夹层/动脉瘤模型,自模型诱导第3周,小檗碱组小鼠每天给予40mg/kg小檗碱灌胃,模型组给予相同体积蒸馏水,直至模型诱导结束。
2.3模型评估:自模型诱导开始,观察小鼠状态,如有死亡小鼠,解剖发现胸腔有积血则视为造模成功;模型诱导结束后,过量戊巴比妥钠麻醉并颈椎脱臼处死小鼠,体式显微镜下暴露小鼠主动脉,游标卡尺测量胸主动脉最宽处直径,以主动脉局部扩张大于其邻近组织直径的50%时视为造模成功(动脉瘤),小鼠胸主动脉大体可见假腔形成也视为造模成功(主动脉夹层),统计各组小鼠生存曲线及成模率。
2.4组织标本:模型诱导4周后,过量戊巴比妥钠麻醉并颈椎脱臼处死小鼠,打开胸腔,自左心室灌注预冷的磷酸盐缓冲液,除净血液,体式显微镜下分离小鼠的主动脉组织,直接用于组织固定、流式细胞术检测或于液氮速冻后-80℃冰箱保存,用于后续PCR和Western Blot检测。
2.5组织石蜡切片:按上述分离主动脉组织后取胸主动脉弓,于4%多聚甲醛中固定24小时,流水冲洗后经不同浓度梯度乙醇逐步脱水,使用二甲苯和无水乙醇进行组织透明,然后将组织置于55-65℃的液体石蜡中浸蜡过夜,取出后用预热的液体石蜡包埋并置于冷冻台塑性,常规石蜡切片4μm,摊片机摊片后,使用防脱载玻片小心捞起完整的切片,烤片机烤干后切片盒保存。
2.6苏木精-伊红(HE)染色:取出上述切片,60℃烘箱烤片至少半小时,将切片依次置入二甲苯及不同浓度梯度乙醇中脱蜡,蒸馏水浸泡清洗后切片依次置于苏木精染液染色,流水冲洗,0.5%盐酸乙醇分化,流水冲洗返蓝以及伊红染料染色,流水浸泡冲洗后再依次经梯度乙醇、二甲苯脱水透明,待组织切片晾干后滴加中性树脂,放置盖玻片,晾干后经显微镜拍照。
2.7 EVG染色:石蜡切片按照上述方式脱蜡至水,加入EVG染液染色30min,水洗后置入三氯化铁分化液分化一下,自来水冲洗,反复操作,显微镜观察控制分化程度在弹性纤维呈现黑色,VG染液复染,水洗后透明封片,显微镜拍照。
2.8动脉组织流式细胞检测:按上述方式分离小鼠主动脉组织,去除周围所有的脂肪组织,充分剪碎动脉组织,使用含胶原酶I(Sigma, Cat# 9001-12-1)的消化液在37℃震荡消化1h,经70μm滤网研磨过滤获得单细胞悬液,在主动脉细胞悬液中加入一定体积的CD45.1小鼠脾单细胞悬液辅助检测,将得到的混合细胞用预冷的磷酸盐缓冲液洗涤离心,沉淀使用表面抗体混悬液进行重悬染色(抗体浓度为1:200),所有抗体在4°C黑暗中孵育30分钟,用抗Cd45.2抗体标记小鼠主动脉源性淋巴细胞,CD19和CD3阴性细胞用于骨髓源性细胞的进一步分析,CD11b抗体和Ly6G抗体用于中性粒细胞的鉴定,固定活性染色(FVS)去除死细胞,染色完成细胞洗净后重悬于FACS缓冲液中,流式细胞术检测。
2.10 PCR分析:将分离的主动脉组织剪碎,使用Trizol 提取组织总RNA,Nanodrop测定浓度后,经逆转录、扩增获得各个样本内参(GAPDH)和目的基因得CT值,以GAPDG作为规范化标准,采用2-ΔΔCT的比较方法分析不同组间目的基因mRNA的相对表达量。
2.11 Western Blot分析:取上述分离的主动脉组织,加入含有蛋白酶和磷酸酶抑制剂的组织裂解液,使用组织匀浆仪匀浆以获得蛋白质样本,高速离心后,收集上清液,BCA法测定浓度,将所有样品调节至均一浓度,添加5×上样缓冲液95°C加热5分钟,在适当电压下经SDS-PAGE凝胶分离蛋白质,然后将蛋白电转至PVDF膜,WB条带经5%脱脂牛奶封闭,适当的一抗4°C过夜,洗膜,加入相应二抗室温孵育1小时,洗膜后采用ECL显色法检测蛋白条带,以GAPDH作为对照,使用ImageJ软件用于定量数据分析。
3、结果
3.1小檗碱抑制主动脉夹层/动脉瘤的形成:如图1,本发明中,根据小鼠死亡时间和大体图评估各组小鼠生存曲线及成模率,数据显示,小檗碱干预后小鼠生存曲线明显优于模型组(图1A);将不同批次模型组小鼠与小檗碱组小鼠的成模率进行比较,发现模型组小鼠成模率明显高于小檗碱组(P<0.001,图1B),这表明小檗碱能够抑制主动脉夹层/动脉瘤的形成。
3.2小檗碱减少胸主动脉的损伤及扩张:如图2,本发明中,根据大体图判断主动脉的损伤程度并使用游标卡尺测量胸主动脉最大直径,大体图显示,模型组主动脉明显扩张、直径增大,伴随腔内积血,表明β-氨基丙腈模型诱导成功,小檗碱组小鼠主动脉的形态学更趋近于正常(图2A),直径统计分析显示,模型组小鼠胸主动脉直径明显增加(P<0.01,图2B),小檗碱组与模型组相比,主动脉直径缩小(P<0.05,图2B)。
3.3小檗碱抑制主动脉夹层/动脉瘤弹性纤维降解和断裂:如图3,本发明中,取模型诱导完成的小鼠胸主动脉石蜡包埋切片后,进行HE染色和EVG染色,观察弹性纤维,,结果显示,与正常组相比,模型组小鼠主动脉结构异常,弹性纤维细而直,并伴有大量弹性纤维碎片,小檗碱组小鼠主动脉更接近正常结构,弹性纤维断裂明显较少(图3A、B),使用弹性纤维碎片等级对各组小鼠弹性纤维破坏进行量化评分,模型组小鼠的评分升高,而小檗碱组小鼠的弹性纤维碎片等级明显低于模型组(P<0.01,图3C),这表明小檗碱能抑制主动脉夹层/动脉瘤小鼠动脉组织弹性纤维降解和断裂。
3.4小檗碱抑制主动脉夹层/动脉瘤血管炎症:如图4和图5,本发明中,使用流式细胞术检测各组小鼠组织浸润免疫细胞的表达,可以发现模型组小鼠主动脉组织CD45+的细胞计数明显增加,说明在主动脉夹层/动脉瘤发生时,炎性细胞浸润增加,小檗碱干预后,组织浸润的免疫细胞显著减少(P<0.01),中性粒细胞浸润与主动脉夹层/动脉瘤的破裂密切相关,流式结果表明,模型组小鼠动脉组织大量中性粒细胞浸润,小檗碱能明显降低中性粒细胞的含量(P<0.001),根据PCR检测结果,与模型组相比,小檗碱能抑制促炎细胞因子IL-6、TNF-α、MCP-1、IL-8的转录水平。
3.5小檗碱抑制主动脉夹层细胞外基质降解并增加基质重塑:如图6,本发明中,通过PCR检测细胞外基质降解调控因子MMP-2、MMP-9及细胞外基质重塑调控因子ADAMTS8、ADAMTS5的转录水平,结果表明,模型组MMP-2、MMP-9的mRNA水平明显增加(P<0.05)而ADAMTS8、ADAMTS5表达下降(P<0.001),小檗碱干预后MMP-2、MMP-9表达下降(P<0.05)而ADAMTS8、ADAMTS5升高(P<0.05),这表明小檗碱不仅抑制了主动脉组织中细胞外基质降解也增加了细胞外基质重塑。
3.6小檗碱抑制主动脉夹层动脉组织凋亡水平:如图7,本发明中,使用BAX与Bcl2的表达评估各组小鼠主动脉组织凋亡水平,BAX表达增加而Bcl2表达下降表明凋亡增加,使用Western Blots检测主动脉夹层组织BAX、Bcl2蛋白的水平,结果表明,模型组BAX表达增加伴随Bcl2下降,而小檗碱处理后BAX表达受到抑制而Bcl2增加(P<0.05),表明小檗碱抑制了主动脉组织的凋亡水平。
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来说,在不脱离本发明原理的前提下,还可以做出若干改进,这些改进也应该视为本发明的保护范围。
Claims (11)
1.一种小檗碱在预防主动脉夹层动脉瘤进展中的应用,其特征在于:所述小檗碱在制备预防主动脉夹层/动脉瘤进展药物中的应用。
2.根据权利要求1所述的一种小檗碱在预防主动脉夹层动脉瘤进展中的应用,其特征在于:所述主动脉夹层/动脉瘤包括β-氨基丙腈诱导的胸主动脉夹层及动脉瘤。
3.根据权利要求1所述的一种小檗碱在预防主动脉夹层动脉瘤进展中的应用,其特征在于:所述小檗碱在制备抑制主动脉夹层/动脉瘤弹性纤维降解药物中的应用。
4.根据权利要求1所述的一种小檗碱在预防主动脉夹层动脉瘤进展中的应用,其特征在于:所述小檗碱在制备抑制主动脉夹层/动脉瘤弹性纤维断裂药物中的应用。
5.根据权利要求1所述的一种小檗碱在预防主动脉夹层动脉瘤进展中的应用,其特征在于:所述小檗碱在制备抑制主动脉夹层/动脉瘤胶原纤维降解药物中的应用。
6.根据权利要求1所述的一种小檗碱在预防主动脉夹层动脉瘤进展中的应用,其特征在于:所述小檗碱在制备抑制主动脉夹层/动脉瘤组织中MMP9/2的基因转录药物中的应用。
7.根据权利要求1所述的一种小檗碱在预防主动脉夹层动脉瘤进展中的应用,其特征在于:所述小檗碱在制备促进主动脉夹层/动脉瘤组织中ADAMTS5/8的基因转录药物中的应用。
8.根据权利要求1所述的一种小檗碱在预防主动脉夹层动脉瘤进展中的应用,其特征在于:所述小檗碱在制备抑制主动脉夹层/动脉瘤组织炎症发生药物的应用。
9.根据权利要求1所述的一种小檗碱在预防主动脉夹层动脉瘤进展中的应用,其特征在于:所述小檗碱在制备抑制主动脉夹层/动脉瘤组织中性粒细胞浸润药物的应用。
10.根据权利要求1所述的一种小檗碱在预防主动脉夹层动脉瘤进展中的应用,其特征在于:所述小檗碱在制备抑制主动脉夹层/动脉瘤组织凋亡水平药物的应用。
11.根据权利要求1-10所述的一种小檗碱在预防主动脉夹层动脉瘤进展中的应用,其特征在于:所述药物的成分包括小檗碱和辅料。
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