CN101791304A - Xyloketal B在制备治疗线粒体损伤疾病的药物中的应用 - Google Patents
Xyloketal B在制备治疗线粒体损伤疾病的药物中的应用 Download PDFInfo
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Abstract
本发明公开了xyloketal B在制备线粒体损伤疾病的药物中的应用。该化合物可以抑制线粒体膜电位降低,抑制线粒体活性氧簇生成,保护线粒体的完整性并抑制细胞色素c的释放,可以保护线粒体免受损伤。本发明发掘了xyloketal B新的医疗用途,开拓了一个新的应用领域,且xyloketal B安全无毒,药理作用强,有很好的药用前景。
Description
发明人:王冠蕾,裴中,庞冀燕,林永成,钱艳,许忠良,李玲,黄如训,赵嘉,刘捷,陈文亮,李振兴
技术领域
本发明涉及xyloketal B在医疗领域中的应用,具体涉及xyloketal B在制备治疗线粒体损伤疾病的药物中的应用。
背景技术
xyloketal B是从南海红树林真菌Xylaria sp.2508中分离到的代谢产物。Xylaria sp.2508的代谢产物包括xyloketal A、B、C和D,其中,xyloketal B的化学结构如下:
由于已发现该化合物可以明显的非竞争性抑制乙酰胆碱酯酶活性,而乙酰胆碱酯酶抑制剂在早老性痴呆,即阿尔茨海默病(Alzheimer’disease,AD)的治疗中具有重要意义,故该化合物常被用作AD治疗药物以及学习记忆功能改善药物的活性成分。
细胞凋亡是多种生理、病理因素参与的细胞启动自身调控机制而发生的一种主动死亡的形式,在维护机体内环境稳定方面发挥着极为重要的作用。细胞凋亡机理复杂,不同病变涉及的信号通路亦不尽相同,一般认为细胞凋亡涉及Caspase介导的二条通路:死亡受体介导通路(death receptor-mediated pathway)及线粒体依赖通路(mitochondria-dependent pathway)。前者与膜死亡受体激活相关;后者与Bcl-2/Bax调控的线粒体释放细胞色素c相关。线粒体损伤在多种疾病过程中的重要性已成为共识。如高血压过程中,多种病理刺激(如AngII,过量的ROS)能够造成线粒体损伤(如线粒体膜电位下降,膜通透性增加,细胞色素c释放等),诱发和加重高血压过程中细胞和组织损害。稳定线粒体膜,减少ROS生成就是抗细胞凋亡的一条途径。细胞凋亡过高或过低都会对机体产生不利影响。大量证据表明由血管平滑肌细胞凋亡引起的血管壁损伤在动脉粥样硬化、冠脉狭窄、高血压等疾病的病理过程中扮演着十分重要的角色。在以上各种疾病过程中,都可出现活性氧分子(Reactive oxygen species,ROS)成分产生和分解不平衡,ROS主要包括H2O2,O2-和-OH等。现已证实活性氧可诱发血管平滑肌细胞凋亡致血管壁损伤,因此以H2O2作为诱导因素被公认能够模拟体内血管病变过程中的高氧化应激状态,可作为筛选防治线粒体损伤相关疾病的药物的体外细胞模型。
发明内容
本发明的目的在于提供xyloketal B在制备治疗线粒体损伤疾病的药物中的应用。
为了实现上述目的,本发明采用如下技术方案:
发明人经过长期研究发现,xyloketal B可以保护细胞线粒体功能,从而对线粒体依赖途径介导的细胞凋亡具有抑制作用,因此可以将xyloketal B用于制备治疗线粒体损伤疾病的药物。
通过进一步研究发现,由于xyloketal B对细胞凋亡有抑制作用,可保护线粒体功能,还能显著提高H2O2诱导的血管平滑肌细胞存活率,可对抗氧化应激诱导的血管平滑肌细胞损伤,因此,本发明还将xyloketal B应用于制备治疗血管氧化应激损伤药物中。
其中,还可以将xyloketal B制备成药物领域可以接受的各种剂型,如片剂、胶囊等等。
与现有的xyloketal B的应用相比,本发明具有如下有益效果:
(1)本发明xyloketal B在治疗线粒体损伤疾病中疗效显著,尤其适合于治疗因线粒体损伤引起的血管氧化应激损伤相关的疾病。
(2)本发明xyloketal B应用于制备治疗线粒体损伤疾病的药物,安全无毒,药理作用强,预示着很好的药用前景。
附图说明
图1为xyloketal B对H2O2诱导VSMCs凋亡的影响;
图2为xyloketal B对H2O2诱导Caspase-3蛋白激活的影响;
图3为xyloketal B对H2O2诱导cytochrome c释放的影响;
图4为xyloketal B对H2O2诱导线粒体膜电位(ψm)的影响;
图5为xyloketal B对VSMCs的胞内H2O2生成的影响;
图6为xyloketal B对OGD引起PC12细胞线粒体形态学的影响。
具体实施方式
以下通过实施例来进一步阐述本发明,但实施例不对本发明做任何限定。
实施例1xyloketal B对H2O2诱导大鼠主动脉平滑肌细胞损伤的作用
(一)材料与方法
1.药物
xyloketal B,白色粉末。溶于DMSO,贮存浓度80mmol/L,保存于4℃备用。
2.大鼠主动脉血管平滑肌细胞分离、培养及鉴定
放血处死大鼠,于无菌条件下取出主动脉,置于无菌PBS缓冲液中,剪开动脉后,小心去除内皮,PBS冲洗后,用眼科弯头镊轻轻撕下平滑肌层,放入加了2-3滴胎牛血清(FBS)的培养皿中,剪碎平滑肌组织,用弯头吸管贴壁种于培养瓶的下壁,翻转培养瓶,加入含20%血清的DMEM培养基,6-8小时后,待组织块贴牢后,翻转培养瓶,使组织块浸于培养液中。可见平滑肌细胞匍匐长出,约10日后传代培养,细胞鉴定用α-actin。细胞传至8-14代用于试验,以H2O2处理细胞24h作为诱导凋亡的模型。
3.细胞活性检测——MTT法
细胞以5×104个/ml密度接种于96孔板,培养24h后,加入不同浓度受试化合物预孵30min后,用200μM H2O2处理细胞,并设阴性对照组(negative control)、阳性对照组(H2O2)、给药组(xyloketal B),每组4个复孔,分别继续培养24h。然后每孔加入MTT 20μL(5mg/ml),置37℃、5%CO2培养箱中孵育4h后去除培养基,加入二甲基亚砜(DMSO)100μl溶解结晶体,待结晶体完全溶解后,于酶标仪上在波长为570nm处测定吸光度值,A值的大小反映细胞的存活情况。细胞存活率(%)=(给药组A值-阴性对照组A值)/(阳性对照组A值-阴性对照组A值)×100%。MTT被线粒体中的琥珀酸脱氢酶代谢后,产生深紫色的甲臜结晶,用DMSO溶解后,通过酶标仪测定570nm波长处的吸光度,其吸光度值的大小与活细胞数成正比,可反映细胞存活率。
4.Hoechst 33258核染色法检测细胞凋亡
细胞接种于35mm培养皿,受试化合物预孵30min并用200μM H2O2处理大鼠VSMCs 24h后,以PBS洗涤细胞,甲醇∶冰醋酸(3∶1,V/V)固定10min,然后用蒸馏水洗细胞2次,晾干,加入Hoechst 33258(5mg/L)染色5min,用蒸馏水洗2次,室温下晾干,荧光显微镜下(激发波长360nm,发射波长450nm)观察并随机拍照。Hoechst33258是一种DNA荧光染料,可以插入并结合于DNA链的A-T区,受波长200-380nm的紫外线激发,释放420nm的蓝色荧光。荧光显微镜下,活细胞核呈弥散均匀荧光,细胞凋亡时,细胞核或细胞质内可见浓染致密的颗粒块状荧光,核出现不同程度的固缩,可见DNA荧光碎片(凋亡小体)。
5.统计分析
数据资料以均数±标准差(x±s)表示。统计学处理由计算机运用SPSS 11.0软件进行,组间差异比较用方差分析,P<0.05表示差异有统计学意义。
(二)实验结果
1.MTT法检测细胞存活率
按文献建立H2O2体外诱导培养大鼠主动脉平滑肌细胞(VSMCs)调亡模型。结果显示,200μM H2O2处理VSMCs后,其存活率为65±7%,较正常对照组明显下降(P<0.01,n=4),而预先给予xyloketal B 10μM、20μM、40μM时,再予200μM H2O2处理,VSMC存活率为63±2%、82±3%、90±6%。预孵20μM、40μM xyloketal B后细胞存活率较H2O2组明显上升。以上结果提示xyloketal B可浓度依赖性对抗H2O2对VSMCs的损伤(图1)。
2.xyloketal B对细胞Hochest33258核染色的影响
采用Hochest 33258荧光染色观察细胞凋亡的形态学变化,200μM H2O2处理后,细胞核体积缩小,荧光染色增强,染色质呈致密浓染的块状或颗粒状荧光以及多个明显凋亡小体形成。预先给予xyloketal B(20μM)共孵育后,与H2O2处理组相比,可见细胞荧光强度明显减弱,凋亡小体数目明显减少。以上结果与MTT实验结果相符,支持xyloketal B可抑制H2O2诱导VSMCs细胞凋亡。
本实施例结果表明,xyloketal B可显著提高H2O2诱导的血管平滑肌细胞存活率,且呈现剂量依赖效应;xyloketal B可以使H2O2诱导的血管平滑肌细胞凋亡的形态学改变明显减轻;以上结果说明xyloketal B具有对抗氧化应激诱导的血管平滑肌细胞损伤的作用。
实施例2xyloketal B对抗H2O2诱导的大鼠主动脉平滑肌细胞凋亡机制的研究
(一)材料与方法
1.药物
xyloketal B,白色粉末。溶于DMSO,贮存浓度80mmol/L,保存于4℃备用。
2.细胞培养及模型制备:同实施例1。
3.细胞线粒体跨膜电位(ψm)检测
细胞接种于35mm培养皿,受试化合物预孵30min并用200μM H2O2处理24h后,以PBS洗涤细胞,按照线粒体膜电位检测试剂盒说明书(Beyotime,China)稀释JC-1染料后,加入1ml JC-1染色工作液,充分混匀。37℃孵育20min。孵育结束后,吸除上清,用PBS洗涤细胞两次,加入1ml细胞培养液,激光共聚焦显微镜下通过JC-1荧光指示剂来检测ψm的改变。JC-1染料红色,绿色荧光的激发波长分别为530nm和488nm。Image J 1.37测定红色和绿色荧光强度,两者的荧光强度比值表示ψm的变化。正常细胞ψm较高,JC-1聚集在线粒体内以多聚体的形式存在,在激发光为525nm时呈现红色荧光;对于凋亡细胞,ψm降低,JC-1不能聚集在线粒体的基质中,以单体的形式存在,在激发光为490nm时呈现绿色荧光。镜下选取10个视野,计算平均荧光密度,以红色荧光/绿色荧光光密度比值表示线粒体膜电位的高低,比值下降表示线粒体膜电位下降。
4.细胞cytochrome c变化的检测
细胞接种于35mm培养皿,受试化合物预孵30min并用200μM H2O2处理24h后,移去培养基,用含4%多聚甲醛的固定液固定细胞10min。固定完毕后,TBS液洗涤两次,每次5min。加入封闭液(含3%BSA,0.2%Triton-100的PBS溶液)封闭60min。分别按兔源caspase-3激活型单克隆抗体(cell signal,CST)1∶100,鼠源cytochrome c单克隆抗体(Santa CruzBiotechnology,Santa Cruz,CA)1∶50的比例稀释一抗并4℃孵育过夜。TBS洗涤3次,每次5min。按照1∶1000比例分别稀释抗兔FITC荧光标记和抗鼠cy-3荧光标记的二抗,室温孵育60min,TBS洗涤3次,每次5min。用Hoechst33258染料染核5min,TBS洗涤2次,每次5min,室温下晾干,激光共聚焦显微镜下观察并随机拍照。
5.Western blot检测相关凋亡因子:
应用相应的抗体,测定相关凋亡因子的表达(包括Bcl-2,Bax,caspase-3等)。按相应方法说明在培养的大鼠主动脉平滑肌细胞上进行。
培养的大鼠主动脉平滑肌细胞经相应的药物处理后,以预冷至4℃PBS洗涤,加入RIPA裂解液(Beyotime)裂解,细胞裂解产物于4℃下12000rpm离心15min。离心后的上清蛋白用BCA法定量后,经SDS-PAGE电泳(12%分离胶,5%浓缩胶)后将蛋白转至PVDF膜上,5%脱脂牛奶封闭后,将相应的一抗(Bcl-2,Bax,caspase-3,1∶1000,Cell Signaling Technology)室温下孵育1~2h或者4℃过夜后,用TBST洗三次后,与相应的二抗稀释液(HRP标记抗兔二抗,1∶1000,Beyotime)室温下孵育1~2h后,用TBST洗三次后,进行化学发光显影(ECL,Amersham)。用凝胶成像系统(ULTRA-LUM,Mega 10)分析目标条带的光密度值。以β-actin作为内参照,比较不同处理后上述凋亡因子蛋白表达的差异。
6.VSMCs胞内H2O2的测定
VSMCs以1×105接种于Petric皿,全培养液培养18h后,无血清培养6h,加入xyloketal B(20μM)预孵30min后,予H2O2(200μM)作用24h后,PBS洗两遍后,加入H2DCF(10μM,sigma)37℃负载20min,在激光共聚焦显微镜系统以FITC波长参数测定荧光强度。每组随机获取3个视野(200×),Image J 1.37计算图像的荧光强度。
7.统计学处理
数据资料以均数±标准差(x±s)表示。统计学处理由计算机运用SPSS 11.0软件进行,组间差异比较用方差分析,P<0.05表示差异有统计学意义。
(二)实验结果
1.Xyloketal B对H2O2诱导Caspase-3蛋白激活的影响;
200μM H2O2处理组与对照组相比,激活型Caspase-3蛋白表达明显增强(P<0.01),预先给予xyloketal B(20μM)共孵育后,激活型Caspase-3蛋白表达较H2O2组明显下降,约下降30%。以上结果提示xyloketal B可抑制H2O2诱导的激活型Caspase-3蛋白含量(图2)。
2.Xyloketal B对H2O2诱导的VSMCs胞内Bcl-2和Bax的影响
200μM H2O2处理24h后,Bcl-2表达明显降低,而Bax表达升高,H2O2处理组Bax/Bcl-2比值与对照组相比显著升高。预先孵育20μM xyloketal B后,再给予H2O2处理,发现20μMxyloketal B可显著升高胞内Bcl-2表达水平,降低Bax表达,并且显著降低Bax/Bcl-2比值。单独孵育20μM xyloketal B对Bax和Bcl-2蛋白质表达水平以及Bax/Bcl-2比值均无影响。提示xyloketal B通过升高抗凋亡蛋白Bcl-2水平参与抗氧化应激损伤作用。
3.Xyloketal B对H2O2诱导cytochtome c释放的影响
200μM H2O2处理VSMCs后,cytochtome C荧光从细胞线粒体向胞浆弥散。H2O2处理组胞浆弥散型cytochtome c荧光强度较正常对照组明显增强(P<0.01),而预先给予xyloketal B(20μM)后,胞浆弥散型cytochtome c荧光强度较H2O2组明显下降,约下降40%。提示xyloketal B可抑制H2O2诱导的线粒体cytochtome C释放(图3)。
4.Xyloketal B对H2O2诱导线粒体膜电位(ψm)的影响
H2O2组线粒体ψm显著下降,明显低于对照组(H2O2:0.63±0.06 vs Con:1.14±0.16,P<0.01,n=6);20μM xyloketal B处理组线粒体ψm显著高于H2O2组(P<0.01,n=6)。提示xyloketal B可稳定线粒体ψm,减轻H2O2诱导VSMC线粒体损伤(图4)。
5.Xyloketal B处理后VSMCs胞内H2O2的测定
200μM H2O2可引起胞内H2DCF荧光强度明显增强(增强约200%)。预先孵育20μMxyloketal B后,发现xyloketal B可抑制H2O2引起的胞内H2O2生成增多,抑制率为65%(图5)。
实施例3Xyloketal B通过保护线粒体功能减弱PC12氧气葡萄糖剥夺-再灌注损伤诱导线粒体损伤
(一)材料与方法
1.药物
Xyloketal B,白色粉末。溶于DMSO,贮存浓度80mmol/L,保存于4℃备用。
2.细胞培养及模型制备
采用PC12(大鼠肾上腺髓质嗜铬细胞瘤细胞)作为体外研究神经元的细胞体系,85%1640(GIBCO,8108029)+10%马血清(GIBCO,720129)+5%胎牛血清(MOREGATE,27825123)于37℃5%CO2培养箱孵育,待细胞生长至70%-80%可于实验。
使用离体氧气葡萄糖剥夺-再灌注模型模拟在体缺血缺氧-再灌注模型:把生长状态良好的PC12接种于96孔板,待其生长至70%-80%时将其正常培养基换成缺氧液(2mM CaCl20.221g,3mM KCl 0.224g,156mM NaCl 9.116g,1.25mM KH2PO4 0.17g,10mM HEPES 2.383g,2mM MgSO40.492g加入去离子水达到1L容量,过滤后使用),再将其置于一个2L容器中,向内通入95%N2+5%CO2混合气体30分钟,随后将容器密闭并放入37℃5%CO2培养箱中4-6小时,以使细胞损伤达40%左右为准。缺氧后把装有细胞的96孔板从密闭容器中取出并换入各自的正常培养基,恢复氧气和葡萄糖供应24小时后进行实验指标的检测。
3.DAPI染色
待PC12处理结束后,用4%的多聚甲醛固定15min,用DAPI(2.5μg/ml)染核20min,在荧光显微镜下拍摄细胞图像观察细胞核形态。
4.线粒体ROS生成的检测
细胞预孵xyloketal B 30min后,缺氧缺糖处理4h,然后细胞用Mitosox(Invitrogen)(2.5μM)负载10min,为了确定mitosox定位与线粒体,细胞同时负载MitoTracker Green(0.2μM,Invitrogen),用HBSS洗3遍后,在荧光显微镜下40倍拍摄图像,荧光强度用SpectraMaxGEMINI EM fluorescent plate reader(Molecular Devices)分析,以MitoSOX和Mitotracker荧光强度的比值表示线粒体ROS的生成。
5.蛋白氧化检测
使用protein-oxidation detection kit(OxyBlot;Chemicon)检测蛋白质氧化羧基修饰,这原理基于羧基与2,4-二硝基苯肼(2,4-dinitrophenylhydrazine,DNPH)反应的衍生物可以用免疫印迹法检测。5μL蛋白样品(20μg)加入5μL 12%SDS和10μL 1×DNPH溶液,室温置15分钟后,在12%SDS-PAGE胶中电泳,然后电转至PVDF膜中,用5%脱脂奶粉室温封闭1h,用抗DNP兔多克隆抗体4℃过夜(1∶150;Chemicon),孵育HRP-标记抗兔IgG后,荧光发光法检测,该试剂盒还提供了阳性和阴性对照以确保特异性。
6.线粒体形态学、线粒体膜电位检测以及Drp1的表达
细胞处理后,用0.2μM Mitotracker Green染色25min,然后在激光扫描共聚焦显微镜下观察线粒体形态学改变(60×)。用Rhodamine-123(Invitrogen)检测线粒体膜电位的改变,细胞处理后,用10μM Rhodamine-123和0.2μM Mitotracker Green负载25min,然后在SpectraMaxGEMINI EM fluorescent plate reader中读取荧光强度值,线粒体膜电位的改变用两者荧光强度的比值来表示。由于Drp1可以通过促进线粒体断裂而促进细胞凋亡,因此还用western blot检测该蛋白表达的改变。
(二)实验结果
1.Xyloketal B对细胞核形态学改变的影响
当PC12细胞OGD 4h后,给予再灌注24h,发现细胞核发生皱缩或者浓缩,而给予xyloketalB预处理后,可以显著抑制OGD引起的细胞核形态学改变(图6)。
2.Xyloketal B对OGD引起PC12细胞线粒体ROS生成、线粒体膜电位以及蛋白氧化的影响
当细胞OGD 4h后,再灌注24h,发现细胞线粒体ROS生成显著增加(1.76±0.01 vs.对照组1.23±0.08)、线粒体膜电位显著降低(5.18±0.31 vs.对照组7.72±0.18),而这些损伤可被预敷xyloketal B所减轻(抑制程度分别为27%和30%)。蛋白羧基化为蛋白氧化的敏感性指标,当细胞如上处理后,发现蛋白羧基化程度显著增加,而预敷xyloketal B则可减轻蛋白氧化。
3.Xyloketal B对OGD引起PC12细胞线粒体形态学和Drp1蛋白表达的影响
当细胞OGD 4h后,再灌注24h,发现细胞线粒体发生明显断裂,对照组线粒体呈梭形杆状,而给予xyloketal B预敷后,OGD引起的线粒体形态学改变明显受到抑制。
当细胞OGD 4h后,再灌注24h,western blot检测发现蛋白羧基化程度明显加重,而这为预敷xyloketal B后所减轻。
Claims (2)
1.xyloketal B在制备治疗线粒体损伤疾病的药物中的应用。
2.如权利要求1所述xyloketal B的应用,其特征在于xyloketal B可用于制备治疗血管氧化应激损伤的药物。
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102702221A (zh) * | 2012-06-04 | 2012-10-03 | 中山大学 | Xyloketal B 类似物及其制备方法和应用 |
CN104945410A (zh) * | 2015-05-22 | 2015-09-30 | 济南新斯诺生物技术有限公司 | 一种不对称催化合成四氢呋喃[2,3-b]苯并吡喃或四氢吡喃[2,3-b]苯并吡喃的方法 |
CN108186666A (zh) * | 2018-01-29 | 2018-06-22 | 西北大学 | 一种dna纳米带在抗细胞凋亡上的应用及其制备方法 |
CN116942697A (zh) * | 2023-08-14 | 2023-10-27 | 上海市第六人民医院 | 普鲁士蓝在制备治疗内质网应激相关疾病药物中的应用 |
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2009
- 2009-08-27 CN CN200910042203A patent/CN101791304A/zh active Pending
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102702221A (zh) * | 2012-06-04 | 2012-10-03 | 中山大学 | Xyloketal B 类似物及其制备方法和应用 |
CN102702221B (zh) * | 2012-06-04 | 2015-03-25 | 中山大学 | Xyloketal B 类似物及其制备方法和应用 |
CN104945410A (zh) * | 2015-05-22 | 2015-09-30 | 济南新斯诺生物技术有限公司 | 一种不对称催化合成四氢呋喃[2,3-b]苯并吡喃或四氢吡喃[2,3-b]苯并吡喃的方法 |
CN104945410B (zh) * | 2015-05-22 | 2017-03-15 | 济南新斯诺生物技术有限公司 | 一种不对称催化合成四氢呋喃[2,3‑b]苯并吡喃或四氢吡喃[2,3‑b]苯并吡喃的方法 |
CN108186666A (zh) * | 2018-01-29 | 2018-06-22 | 西北大学 | 一种dna纳米带在抗细胞凋亡上的应用及其制备方法 |
CN108186666B (zh) * | 2018-01-29 | 2020-02-21 | 西北大学 | 一种dna纳米带在抗细胞凋亡上的应用及其制备方法 |
CN116942697A (zh) * | 2023-08-14 | 2023-10-27 | 上海市第六人民医院 | 普鲁士蓝在制备治疗内质网应激相关疾病药物中的应用 |
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