CN108676013B - 具有自噬激活活性的黄醇酮类化合物、其制备方法及其制药应用 - Google Patents
具有自噬激活活性的黄醇酮类化合物、其制备方法及其制药应用 Download PDFInfo
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- CN108676013B CN108676013B CN201810660648.XA CN201810660648A CN108676013B CN 108676013 B CN108676013 B CN 108676013B CN 201810660648 A CN201810660648 A CN 201810660648A CN 108676013 B CN108676013 B CN 108676013B
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Abstract
Description
技术领域
本发明属于医药生物技术领域,具体而言,涉及一类具有自噬激活活性的黄醇酮类化合物、其制备方法及其制药应用。
背景技术
放线菌(actinomycete)是一类重要的微生物,能够产生结构多样和活性丰富的次级代谢产物,其中链霉素(streptomycin,抗菌药物)、红霉素(erythromycin,抗菌药物)、利福霉素(rifamycin,抗结核病药物)、雷帕霉素(rapamycin,免疫抑制剂;又称为sirolimus,西罗莫司)和道诺霉素(daunomycin或daunorubicin,抗肿瘤药物)等已成为重要的临床药物;但是,很多放线菌的次级代谢产物(包括新结构化合物),其生物(或生理)活性和用途仍有待挖掘和发现。
自噬(autophagy)是细胞维持自身生理稳态的一种机制,是细胞中不被使用的组分和受损细胞器被降解与再利用的一种基本过程。自噬能快速地为细胞提供燃料以应对能量需求(细胞对饥饿的响应);自噬可以激活免疫反应,降解侵入细胞内的病原微生物;自噬也影响着胚胎发育和细胞变异等。因此,自噬对细胞具有非常重要的生理功能,自噬活性必须受到严格控制。
细胞自噬活性异常或失控,会导致人体免疫、病原微生物感染、炎症、肿瘤、心血管病、神经退行性疾病等的发生,而自噬活性调节剂(激活或诱导剂,以及抑制剂)有望成为***、病原微生物感染、免疫性疾病、Ⅱ型糖尿病和神经退行性疾病(例如帕金森症)等的药物。
细胞自噬对人类皮肤的表皮分化也具有重要的调控作用。皮肤表皮角质细胞的终末分化与细胞自噬具有相似的机制,例如它们都需要有控制地对胞内细胞器进行降解,表明细胞自噬在表皮分化过程中扮演了关键角色。已知细胞自噬信号途径参与了表皮角质细胞的炎症反应,以及细胞自噬的激活能够部分地通过炎性小体介导的信号途径诱导机体的抗炎症反应。
细胞自噬是一个多阶段过程,涉及数十种自噬相关蛋白(ATG)的参与。在细胞启动自噬机制时,自噬体和自噬溶酶体的形成均是非常重要的环节。自噬体和自噬溶酶体的形成,与ATG12和LC3B这两个泛素化复合物***的形成和运转密切相关。其中,自噬蛋白ATG4b、ATG5和ATG7参与了ATG12和LC3B这两个泛素化复合物***的形成。因此,细胞中的自噬蛋白ATG4b、ATG5和ATG7等的含量变化,以及LC3B-II/I的比值或LC3B-II水平的变化(通常将LC3B-II作为自噬体形成的标志蛋白)等,是检测细胞自噬活性增高或降低的重要指标。P62作为自噬底物的受体,其水平变化再结合LC3B-II水平变化,可以作为自噬流是否正常的判断标准;而自噬流受阻常出现在一些疾病病理过程中。
细胞自噬过程受到了上述多种蛋白质和/或蛋白质复合物的调控,影响这些蛋白质和/或蛋白质复合物功能发挥的小分子化合物就成为自噬活性调节剂(regulator)。例如,由链霉菌(属于放线菌)产生的雷帕霉素(rapamycin)和衣霉素(tunicamycin),已被证实是自噬激活剂(activator,inducer),它们已分别作为药物新用途(或适应症)进行开发研究或和作为生化试剂得到应用。
某些放线菌菌株能够产生黄色色素类次级代谢产物。其中,有一类化合物的化学结构非常相似,即它们均具有由6-脱氧己糖(6-deoxyhexose,A环)、呋喃(furan,B环)、环庚三烯酮(tropolone,C环)、环戊酮(cyclopentanone,D环)和吡喃(pyran,E环)组成的稠合五环核心结构。这些化合物只是在核心结构上有1-3个侧链取代基和个别手性碳原子构型上有差异。根据这类化合物具有共同的颜色(黄色)和取代官能团(醇羟基和酮基)特点,本发明将这类化合物统称为黄醇酮类化合物(flavolones)。已知的黄醇酮类化合物产生菌有链霉菌Streptomyces(产生原黄醇酮,isatropolones)和马杜拉放线菌Actinomadurasp.5-2(产生特黄醇酮,prerubterolones)等。
Yijun Yan等和Xiaofeng Cai等在对红醇酮类化合物(redolones,属于1类具有由6-脱氧己糖、呋喃、环庚三烯酮、环戊酮和吡啶组成的稠合五环核心结构的红色化合物)生物合成机制的研究中,发现它们是以黄醇酮类化合物为前体,前体分子中的E环开环后与NH3或伯胺化合物R-NH2中的氨基发生环合反应(形成1个吡啶环)而产生的。因此,红醇酮类化合物产生菌也是黄醇酮类化合物产生菌,例如刺孢红链霉菌Streptomyces echinoruberCGMCC 4.1707T和链霉菌Streptomyces sp.KIB-H033(二者均产生玉红醇酮,rubrolones)、马杜拉放线菌Actinomadura sp.5-2(产生特红醇酮,rubterolones)等。
由于黄醇酮类化合物含有环庚三烯酮环,因此它们属于含环庚三烯酮环的天然产物。在自然界中,具有环庚三烯酮环的天然产物很多,其生物活性和用途多样。例如,秋水仙素(colchicine)是一种生物碱,来自百合科植物秋水仙,具有抑制真核细胞有丝***的生物学活性;秋水仙素是一种治疗和预防急性痛风性关节炎的临床药物,此外还被广泛应用于细胞学和遗传学研究以及植物育种。桧木醇(hinokitiol)是一种单萜类化合物,来自松柏科植物,具有良好的抗菌、保湿和忌避害虫效果。与上述含环庚三烯酮环的天然产物相比,黄醇酮类化合物的显著结构特点是含有稠合五环(ABCDE)核心结构;而黄醇酮类化合物的生物活性,除了文献报道的原黄醇酮A具有抗寄生虫(Leishmania donovani,杜氏利什曼虫)活性和特黄醇酮C具有抗炎活性外,对其生物活性还了解很少,因此有待挖掘与发现。
发明内容
本发明的目的在于提供一类具有稠合五环(6-脱氧己糖,A环;呋喃,B环;环庚三烯酮,C环;环戊酮,D环;吡喃,E环)核心结构的黄醇酮类化合物,它们具有激活细胞自噬的生物活性,以及基于黄醇酮类化合物的激活细胞自噬的生物活性将其作为药物或药物组合物中的活性成分,在医药领域中用于治疗或预防肿瘤、病原微生物感染、免疫性疾病、Ⅱ型糖尿病和神经退行性老年疾病等,以及紫外线照射/炎症导致的皮肤损伤或衰老。
本发明首先涉及一组具有自噬激活活性黄醇酮类化合物,其化学结构如下式(Ⅰ)所示:
其核心结构为稠合五环;
其中,A环为6-脱氧己糖、B环为呋喃、C环为环庚三烯酮、D环为环戊酮、E环为吡喃;
取代基R1为-H、-CH3或-CO(CH2CH=CHCH3);
取代基R2为碳原子数1~5的烷基或支链烷基,以及碳原子数1~5并带有1个羟基取代基的烷基或支链烷基,
优选的,
R2=CH3CH2CH2-、CH3CH2CH(OH)-或CH3-;
更优选的,
R1=CH3-或H;
R2=CH3CH2CH2-或CH3CH2CH(OH)-;
最优选的,
R1=CH3-时,R2=CH3CH2CH2-;
R1=H-时,R2=CH3CH2CH(OH)-或CH3CH2CH2-。
本发明还涉及所述的黄醇酮类化合物在制备激活自噬活性的制剂或药物中的用途,所述的激活自噬活性是指:
(1)剂量依赖性地上调细胞自噬蛋白ATG4b、ATG5和ATG7等的水平,激活自噬泛素化***,促进LC3B-I向LC3B-II的转化,促进自噬体的形成和自噬流的畅通;
和(2)剂量依赖性地提高LC3B-II/I的比值和选择性减少载体蛋白P62的水平,增强自噬溶酶体的功能;
所述的药物包括但不限于治疗如下疾病的药物:肿瘤、病毒感染、免疫性疾病、Ⅱ型糖尿病、神经退行性疾病或紫外线照射/炎症导致的皮肤损伤及皮肤衰老。
本发明还涉及一株链霉菌菌株,所述菌株已于2018年4月10日保藏于中国微生物菌种保藏委员会普通微生物中心,地址:北京市朝阳区大屯路中国科学院微生物研究所,邮政编码100101,该菌株的分类名称为Streptomyces sp.,保藏编号为CGMCC No.15540。
本发明还涉及一种通过发酵产黄醇酮类化合物的链霉菌生产所述黄醇酮类化合物的方法,所述的方法包括如下步骤:
(1)种子培养;
(2)固态或液态发酵所述链霉菌;
(3)从发酵产物中分离纯化所述的黄醇酮类化合物。
所述的链霉菌优选为链霉菌CGMCC No.15540或刺孢红链霉菌Streptomycesechinoruber CGMCC 4.1707T。
所述的固态或液态发酵的时间为2~3天。
所述的种子培养的方法为:
将零下80℃冷冻保藏的链霉菌CGMCC No.15540孢子悬液,接种于斜面培养基,28℃培养7-10d,成为富含新鲜成熟孢子的斜面种子;
所述的斜面培养基为:可溶性淀粉10.0g/L,酵母提取物4.0g/L,麦芽提取物10.0g/L,葡萄糖4.0g/L,琼脂15.0g/L,pH6.0-7.0。
所述的固态发酵方法为:
取富含链霉菌CGMCC No.15540新鲜成熟孢子的斜面种子,用无菌水或20%甘油将孢子洗下,振荡分散后取适量孢子悬液均匀涂布于ISP2培养基平板,28℃培养2-4天;
所述的ISP2培养基为:酵母提取物4.0g/L,麦芽提取物10.0g/L,葡萄糖4.0g/L,琼脂15.0g/L,pH6.0-7.0。
所述的液态发酵方法为:
取富含链霉菌CGMCC No.15540新鲜成熟孢子的斜面种子,挖块接种于装有液体发酵培养基的摇瓶中,28℃摇床振荡培养2-4天,收集发酵液,经离心或过滤后得到发酵上清液;
所述的液体发酵培养基为:酵母提取物4.0g/L,麦芽提取物10.0g/L,葡萄糖4.0g/L,pH6.0-7.0。
所述的从发酵产物中分离纯化所述的黄醇酮类化合物的方法为:
1)发酵产物粗提获得一级产物;
2)使用ODS中压色谱柱提纯一级产物,获得粗产物;
3)使用反相HPLC提纯粗产物,分离不同组分,获得黄醇酮类化合物纯品。
所述的发酵产物粗提获得一级产物的方法是:
有机溶剂萃取固态发酵产物或大孔吸附树脂吸附液态发酵产物;
所述的有机溶剂优选为乙酸乙酯;
所述的大孔吸附树脂优选Daion HP-20,使用甲醇-水、乙醇-水或丙酮-水解吸附目标化合物;
所述使用ODS中压色谱柱提纯一级产物的方法中,洗脱***为甲醇-水(含1‰HAc)或乙醇-水(含1‰HAc);
所述的反相HPLC提纯粗产物的方法中,洗脱***为甲醇-水(含1‰HAc)或乙腈-水(含1‰HAc)。
本发明人对从北京市密云区土壤中分离得到的一株链霉菌CGMCC No.15540(该菌株已于2018年4月10日保藏于中国微生物菌种保藏委员会普通微生物中心,地址:北京市朝阳区大屯路中国科学院微生物研究所,邮政编码100101。该菌株的分类名称为Streptomyces sp.,保藏编号为CGMCC No.15540)进行了次级代谢产物研究,经分离纯化和化学结构解析,确定该菌株产生原黄醇酮A和C(isatropolones A and C)。本发明人还对刺孢红链霉菌(Streptomyces echinoruber)CGMCC 4.1707T产生的玉黄醇酮A(echitropolone A)进行了分离纯化和结构鉴定。
本发明人对这些黄醇酮类化合物进行生物活性筛选研究后,发现它们均具有激活细胞自噬的生物活性。由于黄醇酮类化合物具有高度的结构相似性,显然它们都具有激活细胞自噬的生物活性。尤其是考虑到黄醇酮类化合物的细胞毒活性很低,因此基于其激活细胞自噬的生物活性,它们有望作为药物或药物组合物中的活性成分,用于治疗或预防肿瘤、病原微生物感染、免疫性疾病、Ⅱ型糖尿病和神经退行性老年疾病(例如帕金森症)等,以及用于治疗或预防紫外线照射/炎症导致的皮肤损伤或衰老。
为了获得本发明所述的黄醇酮类化合物,以及测定其激活细胞自噬的生物活性,本发明采取了以下技术路线和步骤。
产生黄醇酮类化合物的放线菌的培养与发酵,采用固态发酵模式,或者液态发酵模式。(1)固态模式,将黄醇酮类化合物产生菌的新鲜斜面种子(含丰富成熟孢子)用无菌水或20%甘油洗下,振荡分散得孢子悬液,将孢子悬液均匀涂布于固态发酵培养基,27-30℃培养2-4d,收集发酵培养物。(2)液态模式,将黄醇酮类化合物产生菌的新鲜斜面种子(含丰富成熟孢子)挖块接种于液体发酵培养基中,27-30℃振荡培养2-4d,收集发酵液。
为了提高发酵培养过程中的黄醇酮类化合物产量,在发酵培养基中需要含有丰富的碳源和氮源。碳源包括各类单糖(例如葡萄糖、果糖和核糖)、双糖(例如麦芽糖和蔗糖)和多糖(例如淀粉和糊精)等;氮源、特别是有机氮源,包括但不限于氨基酸、(动物、植物和微生物来源的)多肽和蛋白胨、黄豆饼粉、棉籽饼粉、酵母粉、玉米浆和花生饼粉等。碳源和氮源除了为黄醇酮类化合物产生菌的生长提供所必需的营养物质和能量之外,还用于提供菌体生物合成黄醇酮类化合物分子骨架结构所需的各种前体物等。
黄醇酮类化合物的分离纯化,根据固态或液态两种发酵模式,分别采用以下两种分离纯化流程:
(1)固态发酵,发酵培养物用低级醇或酯(例如甲醇、乙醇、乙酸乙酯等)等溶剂提取,减压旋蒸提取液除去提取溶剂,获得黄醇酮类化合物粗提物;粗提物上ODS中压制备色谱柱分离,洗脱***为甲醇-水或乙醇-水,减压旋蒸除去洗脱溶剂,得到黄醇酮类化合物半纯品;半纯品经制备型反相C18色谱柱HPLC精制,洗脱***为甲醇-水或乙腈-水,减压旋蒸除去洗脱溶剂,获得黄醇酮类化合物纯品。
(2)液态发酵,发酵液经离心或过滤除去菌体后,上清液用大孔吸附树脂吸附,然后用乙醇-水、甲醇-水或丙酮-水***洗脱,得到含有黄醇酮类化合物的洗脱液,减压旋蒸除去乙醇-水、甲醇-水或丙酮-水洗脱溶剂,获得黄醇酮类化合物粗提物;粗提物上ODS中压制备色谱柱分离,洗脱***为甲醇-水或乙醇-水,减压旋蒸除去洗脱溶剂得到黄醇酮类化合物半纯品;半纯品经制备型反相C18色谱柱HPLC精制,洗脱***为甲醇-水或乙腈-水,获得黄醇酮类化合物纯品。
将分离纯化得到的黄醇酮类化合物纯品溶于氘代溶剂中,通过NMR确定其化学结构。或者,将含黄醇酮类化合物纯品(或半纯品、粗提物),通过LC-MS测定,初步确定化学结构。本发明人对链霉菌CGMCC No.15540产生的原黄醇酮A和C的化学结构(见图2)进行了NMR确证(原黄醇酮A和C的NMR谱图见图3-图12、核磁数据见表1);对刺孢红链霉菌CGMCC4.1707T产生的玉黄醇酮A进行了LC-MS确证。
黄醇酮类化合物的激活细胞自噬生物活性,是指它们:
(1)剂量依赖性地上调细胞中的自噬蛋白ATG4b、ATG5和ATG7等的水平,激活自噬泛素化***,促进LC3B-I向LC3B-II的转化,促进自噬体形成和自噬流畅通;
(2)剂量依赖性地提高细胞中的LC3B-II/I的比值并同时促进自噬受体蛋白p62的降解,增强自噬溶酶体的功能。
本发明的有益效果是:具有稠合五环(6-脱氧己糖,A环;呋喃,B环;环庚三烯酮,C环;环戊酮,D环;吡喃,E环)核心结构的黄醇酮类化合物,均具有激活细胞自噬的生物活性。基于黄醇酮类化合物的激活细胞自噬的生物活性,将其作为药物或药物组合物中的活性成分,应用于医药领域中有望治疗人类疾病。所述疾病包括但不限于:肿瘤、病原微生物感染、免疫性疾病、Ⅱ型糖尿病、神经退行性疾病,以及紫外线照射/炎症导致的皮肤损伤或衰老。
本发明还提供了黄醇酮类化合物产生菌的发酵培养方法,所述方法包括如下步骤:(1)固态或液态发酵所述黄醇酮类化合物产生菌;(2)从发酵产物中分离纯化所述的黄醇酮类化合物。所述的黄醇酮类化合物产生菌为链霉菌CGMCC No.15540,或刺孢红链霉菌Streptomyces echinoruber CGMCC 4.1707T等。
其中,步骤(1)所述的固态发酵的发酵培养基为ISP2培养基(酵母提取物4.0g/L,麦芽提取物10.0g/L,葡萄糖4.0g/L,琼脂粉15.0g/L,pH 6.0-7.0),培养温度27-30℃,培养时间2-4d,然后收集发酵培养物。步骤(1)所述的液态发酵的发酵培养基配方为:酵母提取物4.0g/L,麦芽提取物10.0g/L,葡萄糖4.0g/L,pH 6.0-7.0。振荡培养(100-300rpm),温度27-30℃,时间2-4d,然后收集发酵液,发酵液经离心或过滤后收集上清液。
步骤(2)所述的分离纯化方法为:①有机溶剂(例如乙酸乙酯)提取发酵产物;或者采用大孔吸附树脂(例如Daion HP-20)提取发酵上清液,甲醇-水、乙醇-水或乙腈-水醇***解吸附目标产物;经减压旋转蒸发除去提取溶剂***或解吸附***后,获得黄醇酮类化合物粗提物;②粗提物经ODS中压色谱柱分离纯化,洗脱***为甲醇-水(含1‰HAc)、乙醇-水(含1‰HAc)等;经旋转蒸发除去洗脱溶剂***后,获得黄醇酮类化合物半纯品;③半纯品经反相HPLC分离纯化,洗脱***为乙腈-水(含1‰HAc)或甲醇-水(含1‰HAc);经旋转蒸发除去洗脱溶剂***后,获得黄醇酮类化合物纯品。
附图说明
图1黄醇酮类化合物结构通式;
图2黄醇酮类化合物包括原黄醇酮A和C的化学结构;
图3原黄醇酮A的1H核磁谱图;
图4原黄醇酮A的13C核磁谱图;
图5原黄醇酮A的1H-1HCOSY核磁谱图;
图6原黄醇酮A的HSQC核磁谱图;
图7原黄醇酮A的HMBC核磁谱图;
图8原黄醇酮C的1H核磁谱图;
图9原黄醇酮C的13C核磁谱图;
图10原黄醇酮C的1H-1HCOSY核磁谱图;
图11原黄醇酮C的HSQC核磁谱图;
图12原黄醇酮C的HMBC核磁谱图;
图13蛋白质免疫印迹法测定原黄醇酮A的自噬激活活性;
图14蛋白质免疫印迹法测定原黄醇酮C的自噬激活活性。
具体实施方式
实施例1链霉菌CGMCC No.15540培养发酵(产生原黄醇酮A和C)
斜面培养基:可溶性淀粉10.0g/L,酵母提取物4.0g/L,麦芽提取物10.0g/L,葡萄糖4.0g/L,琼脂15.0g/L,pH6.0-7.0;
ISP2培养基:酵母提取物4.0g/L,麦芽提取物10.0g/L,葡萄糖4.0g/L,琼脂15.0g/L,pH6.0-7.0;
液体发酵培养基:酵母提取物4.0g/L,麦芽提取物10.0g/L,葡萄糖4.0g/L,pH6.0-7.0。
1、斜面(种子):
将零下80℃冷冻保藏的链霉菌CGMCC No.15540孢子悬液,接种于斜面培养基,28℃培养7-10d,成为富含新鲜成熟孢子的斜面种子。
2、培养发酵:
2.1、固态培养发酵:将上述链霉菌CGMCC No.15540富含新鲜成熟孢子的斜面种子,用无菌水或20%甘油将孢子洗下,振荡分散后取适量孢子悬液均匀涂布于ISP2培养基平板。平板直径约15cm,每个平板倒入约45ml培养基,28℃培养2-3d,收集发酵培养物,用于分离纯化原黄醇酮A和C。
2.2、液态培养发酵:将上述链霉菌CGMCC No.15540富含新鲜成熟孢子的斜面种子,挖块接种于装有液体发酵培养基的摇瓶(每个500ml摇瓶装有约100ml发酵培养基)中,28℃摇床振荡(200rpm)培养2-3d,收集发酵液,经离心或过滤后得到发酵上清液,用于分离纯化原黄醇酮A和C。
实施例2原黄醇酮A和C的分离纯化
1、发酵产物粗提:
对于固态发酵培养物,取实施例1中固态发酵培养物约3.0L,加入等体积乙酸乙酯提取24-48h,重复提取2-3次,合并乙酸乙酯提取液并减压旋蒸浓缩后,得到约1.5g粗提物。
对于液态发酵培养物,采用离心(3500rpm,15-20min)或过滤等方法首先获得发酵上清液,将发酵上清液经大孔吸附树脂Daion HP-20吸附,然后用不同比例的乙醇-水、甲醇-水或丙酮-水洗脱,洗脱液经减压旋蒸除去洗脱溶剂后得到粗提物。
2、ODS色谱柱制备半纯品:
用适量(10-50ml)甲醇复溶上述1.5g粗提物,与适量(3-5g)ODS填料(粒径50μm,货号AAG12S50,日本YMC公司)拌匀后装入上样柱色谱柱(内径10mm,长度100mm)中,进行ODS分离色谱柱(内径38mm,长度220mm)分离,洗脱液为甲醇-水(含1‰HAc);
首先,15%-70%甲醇-水(含1‰HAc)线性梯度洗脱,流速15.0mL/min,洗脱时间60min;
其次,70%甲醇-水(含1‰HAc)等度洗脱,流速20.0mL/min,洗脱时间60min;
最后,70%-100%甲醇-水(含1‰HAc)线性梯度洗脱,流速20.0mL/min,洗脱时间60min。
每100毫升收集1管,对每管进行TLC分析检测,合并相同组分收集管,最终得到得到9个合并组分(F1-F9)。经LC-MS等分析,确定2个合并组分(F8和F4)分别主要含有原黄醇酮A和C组分;
经减压旋蒸除去洗脱溶剂后,得到原黄醇酮A(10.2mg)和C(27.2mg)半纯品。
3、反相HPLC分离制备纯品:
对上述原黄醇酮A和C半纯品分别进行反相HPLC精制,采用YMC-Pack ODS-A色谱柱(250mm×10mm,S-5μm,12nm;色谱柱货号AA12S05-2510WT,日本YMC公司产品)分离。
对于原黄醇酮A半纯品,采用39%乙腈-水(含1‰HAc)等度洗脱***,流速2.0mL/min,目标峰的保留时间约为38min。
对于原黄醇酮C半纯品,采用32%乙腈-水(含1‰HAc)等度洗脱***,流速2.0mL/min,目标峰的保留时间约为21min。收集目标洗脱峰并通过减压旋蒸除去洗脱溶剂后,分别得到原黄醇酮A(3.4mg)和C(8.5mg)纯品。
原黄醇酮A和C的NMR核磁数据分别如表1所示。
表1原黄醇酮A和C的核磁数据
实施例3原黄醇酮A和C激活细胞自噬的生物活性测定
采用蛋白质免疫印迹法,对经不同浓度的黄醇酮类化合物(原黄醇酮A和C)处理的HepG2肝癌细胞的自噬体泛素化相关蛋白和自噬流标志蛋白进行含量检测,以未经化合物处理的HepG2细胞为对照,通过对蛋白水平变化的综合分析,评价原黄醇酮A和C对细胞自噬活性的影响。原黄醇酮A和C(纯品)的母液浓度均为10mM,检测浓度均设置为1、5、10和20μM。按照以下程序完成细胞自噬活性测定。
1、细胞培养和测试化合物处理:
(1)细胞复苏:从液氮罐中将冻存的人肝癌细胞HepG2细胞取出,迅速置于37℃水浴中,直至冻存细胞完全融化。
(2)细胞培养:将HepG2细胞悬液加入适量MEM(10%FBS)细胞培养液,混匀后置于5%CO2培养箱中,37℃培养。
(3)细胞传代:待细胞达到70%-80%融合后,弃培养液,加入PBS缓冲液洗涤2次,0.05%胰蛋白酶37℃消化2min,加入含血清培养基终止消化;收集细胞悬液置于离心管中,800rpm离心5min,弃上清,加入新培养液重悬细胞,按1:3的比例传代。
(4)黄醇酮类化合物处理细胞:将HepG2细胞培养至80%左右的汇合度,分别给予不同浓度的测试化合物,同时以未给测试化合物的细胞为对照,继续培养24h;吸除培养液,PBS缓冲液洗两次,0.25%胰酶消化收集细胞,PBS缓冲液洗2次。
2、Western blot检测:
(1)蛋白样品制备:采用试剂盒提取细胞总蛋白,测定蛋白浓度;取上样蛋白量加入上样缓冲液,100℃加热10min;涡旋、离心,去上清,可-80℃长期保存或准备上样。
(2)SDS-PAGE电泳:参照《蛋白质技术手册》(汪家政等,2000年)制备SDS-聚丙烯酰胺凝胶。电泳条件:初始电压80V、约30min后样品进入分离胶,然后将电压调至120V进行电泳,直至电泳结束。
(3)转膜:与胶同样大小的6张滤纸、1张硝酸纤维素膜(NC)浸入转移缓冲液中平衡15min;将SDS-PAGE胶板置于转移缓冲液中平衡5min;将凝胶放在6层滤纸之间、与NC膜(其下方)相邻,各层之间去气泡和皱折。在恒流200mA,冰浴条件下转膜1.5h。
(4)抗体杂交:转移后的NC膜在10%脱脂奶粉/TBST中封闭1h;TBST洗膜1-2次,10min/次;分别加入不同一抗与NC膜温育,抗体分别为:
抗人的ATG4a(抗ATG4a,兔IgG;Cell Signaling Technology)、
抗人的ATG4b(抗ATG4b,兔IgG;Cell Signaling Technology)、
抗人的ATG5(抗ATG5,兔IgG;Novus Biologicals)、
抗人的ATG7(抗ATG7,兔IgG;Cell Signaling Technology)、
抗人的P62(抗P62,兔IgG;MBL)
抗人的LC3B(抗LC3B,小鼠IgG;MBL),
以及对照:抗人的GAPDH(三磷酸甘油醛脱氢酶)抗体(anti-GAPDH,中国ZSGB-BIOCo.),
然后放于平缓摇动摇床上,4℃过夜。TBST洗涤。将滤膜置于新的平皿中,加入二抗:
偶联辣根过氧化物酶的山羊抗小鼠IgG,
偶联辣根过氧化物酶的山羊抗兔IgG,
偶联辣根过氧化物酶的兔抗山羊IgG;中国ZSGB-BIO Co.,
然后室温温育1h;TBST洗涤数次;把滤膜置于一新的平皿中,加入化学发光显色液West Pico Chemiluminescent Substrate(Thermo),在膜上加入A液和B液的等比例新混合液,利用天能化学发光成像仪观察结果并记录。
(5)ECL显影:在铝箔包裹的1.5ml离心管中,等体积混合ECL反应试剂盒中的A和B液;将上述反应液滴满膜表面,曝光后保存图像。
3、测定结果:
原黄醇酮A和C均可浓度依赖性地上调HepG2细胞中的自噬蛋白ATG4b、ATG5和ATG7的水平1-3倍,以及上调LC3B-II/I的比值1-3倍,并同时降低自噬蛋白ATG4a和P62的水平(见图13-14),表明它们均具有激活细胞自噬和促进细胞自噬流畅通的生物活性。
最后需要说明的是,以上实施例仅帮助本领域技术人员理解本发明的实质,不用做对本发明保护范围的限定。
参考文献:
[1]Yan Y,Yang J,Yu Z,et al.Non-enzymatic pyridine ring formation inthe biosynthesis of the rubrolone tropolone alkaloids[J].NatureCommunications,2016,7:13083.
[2]Schüep W,Blount J F,Williams T H,et al.Production of a novel redpigment,rubrolone,by Streptomyces echinoruber sp.nov[J].The Journal ofAntibiotics,1978,31(12):1226-1232.
[3]Yan Y,Ma Y T,Yang J,et al.Tropolone ring construction in thebiosynthesis of rubrolone B,a cationic tropolone alkaloid from endophyticStreptomyces[J].Organic Letters,2016,18(6):1254-1257.
[4]Cai X,Shi Y M,N,et al.Structure and biosynthesis ofisatropolones,bioactive amine-scavenging fluorescent natural products fromStreptomyces[J].Angewandte Chemie International Edition,2017,56(18):4945-4949.
[5]Guo H,Benndorf R,Leichnitz D,et al.Isolation,biosynthesis andchemical modifications of rubterolones A-F:rare tropolone alkaloids fromActinomadura sp.5-2[J].Chemistry-A European Journal,2017,23(39):9338-9345.
Claims (2)
1.一组具有自噬激活活性的黄醇酮类化合物在制备激活自噬活性的制剂或药物中的用途,所述的黄醇酮类化合物,其化学结构如下式(Ⅰ)所示,
其中,
R1=H-,R2=CH3CH2CH(OH)-,该化合物为原黄醇酮C;
或R1=H-,R2=CH3CH2CH2-,该化合物为原黄醇酮A;
其特征在于,所述的激活自噬活性是指,
(1)剂量依赖性地上调细胞自噬蛋白ATG4b、ATG5和ATG7的水平,激活自噬泛素化***,促进LC3B-I向LC3B-II的转化,促进自噬体的形成和自噬流的畅通;
和(2)剂量依赖性地提高LC3B-II/LC3B-I的比值和剂量依赖性地降低载体蛋白P62的水平,增强自噬溶酶体的功能。
2.根据权利要求1所述的用途,其特征在于,所述的药物治疗如下疾病:肿瘤、病毒感染、免疫性疾病、Ⅱ型糖尿病、神经退行性疾病、或紫外线照射/炎症导致的皮肤损伤及皮肤衰老。
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