CN113069462A - 青阳参皂苷m1的新用途 - Google Patents
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Abstract
本发明公开了青阳参皂苷M1的新用途,即其在制备大麻素受体激动剂中的应用,本发明通过分子对接实验结果显示青阳参皂苷M1能与大麻素受体CB1结合;体外细胞实验表明,青阳参皂苷M1对大麻素Ⅰ型受体(CB1)有激动作用;本发明提供的CB1的激动剂青阳参皂苷M1为治疗精神性疾病、神经退行性疾病、疼痛、厌食等的药物研发提供了候选化合物。
Description
技术领域
本发明涉及医药领域,具体涉及青阳参中皂苷M1在制备大麻素受体激动剂中的应用。
背景技术
G蛋白偶联受体(GPCR)是人体内最大的细胞膜表面受体家族,在细胞信号转导过程中发挥核心作用,目前有近40%的上市药物以GPCR为作用靶点。GPCR家族成员具有非常保守的七次跨膜螺旋结构,配体与受体结合后,通过跨膜区的构象变化,将信息传递到细胞内。配体的性质决定GPCR的状态:起激活作用的配体(激动剂)使受体活化,而起抑制作用的配体(拮抗剂)则抑制受体活性的发挥,受体与这两类配体结合后呈现的三维结构可以说是这个受体的“阴阳双面”。
大麻素受体CB1是人体中枢神经系统表达量最高的GPCR之一,它参与和调节包括神经形成与调节及能量平衡与代谢等多种生理过程,其介导的信号通路与多种疾病的发生、发展息息相关,是治疗疼痛、炎症、肥胖以及多发性硬化症及神经退行性疾病等的潜在治疗靶标。ERK1/2 ( extracellular regulated protein kinases)是指细胞外调节蛋白激酶,包括ERK1和ERK2,是将信号从表面受体传导至细胞核的关键激酶。ERK1/2是MAPK家族的一员,是传递丝裂原信号的信号转导蛋白。它正常定位于胞浆,当激活后转位至胞核,调节转录因子活性,产生细胞效应。ERK1和ERK2途径是ERK家族中研究最彻底的成员,多种刺激因子如生长因子、细胞因子、病毒、G蛋白偶联受体的配体以及癌基因等都可激活这两条途径;而CB1作为GPCRs的家族成员,近年来也被证实,其配体可能激活ERK1/2这条信号通路。
青阳参( Gynanchumotoplnylumn Schneid. )为萝摩科鹅绒藤属( GmanchumLinn. )植物,多年生草质缠绕藤本根茎入药主产于云南中部、西北部和东北部地区,是云南的民间药物;其性微温,味甘微苦,具有补气益肾,强筋壮骨,活血散癖,祛痰止咳,除湿解赤的作用;常用于治疗子宫肌瘤、腰肌劳损和跌打损伤等腰痛、肺结核与子宫内膜结核以及各种癫痫,其有效成分为青阳参总苷。
发明内容
本发明提供了一种青阳参皂苷M1的新用途,即青阳参皂苷M1在制备大麻素受体CB1激动剂中的应用,青阳参皂苷M1能够激活大麻素受体CB1;所述青阳参皂苷M1化学结构式如下:
本发明青阳参皂苷M1的用途,是将青阳参皂苷M1用作制备大麻素受体激动剂,即以青阳参皂苷M1为活性成分,应用在制备大麻素受体激动剂中,应用中还可以加入一种或多种药物上可接受的辅料,以改善激动剂吸收效果或便于服用,如制成胶囊或丸剂、粉剂、片剂、粒剂、口服液和注射液等,或者与其他活性成分复配发挥作用。
本发明所述辅料包括药学领域常规的填充剂、稀释剂、粘合剂、赋形剂、吸收促进剂、表面活性剂和稳定剂等,必要时还可加入香味剂、色素和甜味剂等。
本发明通过分子对接的方法,验证了青阳参皂苷M1与CB1结合位点与激动剂WIN55212-2处于同一活性口袋,结合能Score为-7.52kcal/mol,青阳参皂苷M1能与MET-384、MET-103、ALA-380、SER-383等残基形成H氢键相互作用;而Western Blot技术则证实青阳参皂苷M1具有激活CB1使下游ERK1/2磷酸化的作用;本发明提供的CB1的激动剂为治疗精神性疾病、神经退行性疾病、疼痛、厌食等的药物研发提供了候选化合物。
附图说明
图1为实施例1中青阳参皂苷M1与大麻素受体CB1的分子对接3D示意图;
图2为实施例1中青阳参皂苷M1与大麻素受体CB1的分子对接2D示意图;
图3为实施例2中验证稳定细胞系VSV表达图;
图4为实施例2中青阳参皂苷M1处理细胞后CB1下游ERK1/2磷酸化水平及表达量结果示意图图;a图为Western Blot结果图,b图为WB灰度扫描值柱形图。
具体实施方式
下面通过附图和实施例对本发明进一步说明,但本发明保护范围不局限于所述内容,本实施例中方法如无特殊说明的均按常规方法操作,所用试剂如无特殊说明均为常规市售试剂或按常规方法配置的试剂,培养基均为市售产品,按使用说明使用。
实施例1:计算机分子对接
本实施例利用分子对接的计算方法,辅助预测配体分子与靶标之间的相互作用,具体步骤如下:
1、制备及优化小分子
首先使用软件ChemDraw绘制青阳参皂苷M1,保存为sdf格式;在软件MOE操作界面打开配体小分子,处理为3D结构,并使用Minimize功能对配体的结构进行简单优化,得到合理的构象,将优化好的构象新建一个mdb格式数据库;
2、蛋白质受体准备
从靶蛋白PDB数据库中获取大麻素受体CB1蛋白的三维结构(PDB ID:5U09),将蛋白质结构导入到软件MOE,删除水分子以及有机溶剂,只保留蛋白质以及原始底物配体,设置对接参数并选择对接活性口袋为Ligand Atoms;
3、分子对接
对配体和定义好口袋的受体进行分子对接,保存好的分子数据库路径输入到对话框。初对接产生50个构象并输出3个对接结果供参考,其余的参数保留默认设置。评价的标准主要看S,即Score,其值越负代表这个构象的小分子与配体结合更加紧密。
4、对接结果
分子对接验证了青阳参皂苷M1与CB1结合位点与激动剂WIN55、212-2处于同一活性口袋,结合Score-7.52 kcal/mol,M1与MET-384、MET-103、ALA-380、SER-383等残基形成H氢键相互作用,结果图见图1、2。
实施例2:利用Western Blot技术检测青阳参皂苷M1对CB1下游ERK1/2信号通路磷酸化水平及表达的影响
1、构建pcDNA5/FRT/TO-VSV-GluR5-CB1-Turquiose质粒
(1)构建线性化pcDNA5/FRT/TO-VSV-GluR5 -Turquiose载体
根据clontech试剂盒原理,在pcDNA5/FRT/TO-VSV-GluR5-OX1-Turquoise质粒(采用Xu T R , Ward R J , Pediani J D , et al. The orexin OX1 receptor existspredominantly as a homodimer in the basal state: potential regulation ofreceptor organization by both agonist and antagonist ligands[J]. BiochemicalJournal, 2011, 439(1):171.文献中方法构建获得)的基础上设计线性化OX1的上下游引物(上游引物Clon-OX1-liner-F:5’GCGGCCGCCATGGTGAGCAAGGGCGAGGAG’,下游引物Clon-OX1-liner-R为:5’CTTACCAAGGCGGTTCATTTCGATATCGGT’),通过PCR反应得到线性化的载体,胶回收纯化线性化载体;PCR反应体系(50μL)为10μL 5×GXL buffer、4μL dNTP、2μL Clon-OX1-liner-F(10μM)、2μL Clon-OX1-liner-R(10μM)、1μL模板DNA(pcDNA5/FRT/TO-VSV-GluR5-OX1-Turquoise质粒)、1μL GXL primer star GXL Enzyme、30μL ddH2O;PCR反应条件:95℃ 3min;95℃ 10s,60℃ 20s,68℃ 3min,25个循环;72℃ 10min;
(2)扩增人大麻素受体1(CB1)目的片段
Trizol法提取人源HEK293细胞总RNA,采用逆转录酶RevertAid RT以总RNA为模板合成cDNA第一链,反应体系和操作过程为:取2μg Total RNA,依次加入1μLoligo(dT)18Primer、DEPC水加至反应体积为12μL;混匀后,65℃加热变性5min后迅速在冰上冷却5min,然后依次加入4μL 5×Reaction buffer、2μL dNTP mix(10nm)、1μL RevertAid RT,1μLRibolock RNase inhibitor混匀并短时离心,42℃温浴1.0h,取出后70℃加热5min,终止反应,cDNA第一链合成后置于-20℃保存备用。
以合成的第一链cDNA为模板,扩增目的基因CB1;所用上下游引物序列分别为上游引物为:5’AACCGCCTTGGTAAGATGAAGTCGATCCTAGATGG3’及下游引物为:5’CACCATGGCGGCCGCCAGAGCCTCGGCAGACGTG 3’。利用Takara GXLDNA聚合酶扩增出目的基因;反应体系(50μL)为1μL GXL Enzyme、10μL 5×GXL buffer、4μL dNTP、1μL cDNA、1μL Clon-CB1-F(10μM)、1μLClon-CB1-R(10μM)、32μL ddH2O;PCR反应条件:98℃ 3min;98℃ 10s,60℃ 15s,68℃ 50s,72℃ 5min,30个循环;72℃ 5min;PCR结束后,进行琼脂糖凝胶电泳,根据扩增产物的特异性以及大小,胶回收纯化目的片段。
(3)构建pcDNA5/FRT/TO-VSV-GluR5 -CB1-Turquiose质粒
使用试剂盒为In-Fusion HD Cloning Kit;连接反应体系和操作过程为:取3μL目的片段(CB1)、1μL 线性化载体和1μL 5×Infusion HD Enzyme premix,混匀后37℃处理15min;50℃处理15min;冰上放置5min;采用热激转化法将连接产物转入大肠杆菌DH5α中,使用含有氨苄青霉素(ampicillin,Amp)的LB固体培养基筛选阳性克隆,挑选若干个单菌落,摇菌后用扩增CB1的特异引物鉴定出多克隆位点插入CB1的克隆,挑选若干个单菌落,摇菌后用扩增CB1的特异引物鉴定出多克隆位点插入CB1的克隆,将所鉴定的克隆进行测序,最终获得的CB1全长cDNA为1545bp。
通过NCBI BLAST(https://blast.ncbi.nlm.nih.gov/Blast.cgi)对测序所得到的序列进行比对,结果显示,CB1受体基因正确连入,无碱基缺失及突变,表明成功构建pcDNA5/FRT/TO-VSV-GluR5 -CB1-Turquiose质粒。
2、筛选稳定的诱导表达细胞系
将生长状态良好的HEK293细胞用0.25%的胰酶(含0.1%EDTA)消化,加入3mL培养基,用移液器吹打培养瓶壁,使细胞完全在培养基中,放置于50mL离心管中800rpm,3min。离心完毕后,弃上层液体,加入6mL培养基混匀制得细胞悬浮液。从酒精缸取出血球计数板和盖玻片,晾干,用移液枪吸取细胞悬浮液约10μL,从血球计数板的一侧缓缓滴入,再用移液枪吸取细胞悬浮液约10μL,从血球计数板的另一侧缓缓滴入,使至盖玻片与计数板间的空隙被液体充满为止;放在显微镜下按箭头方向计数四角内的细胞总数(每角有16小方格)。根据细胞密度和所需细胞总个数M,得出所需要的细胞悬浮液体积V=M/C。计算得出需要补加的培养基V’=10mL-V。取V’体积培养基和V体细胞悬浮液混匀;取一块六孔板,将混匀的细胞悬液按照每孔2mL加入,加入后将六孔板沿着前、后、左、右四个方向摇动,使细胞均匀分散在孔板种。显微镜下镜观察细胞密度,放置在37℃下CO2恒温培养箱中培养。
待细胞状态良好以及细胞密度达到60-70%时,开始做细胞转染实验;将Opti-MEM培养基、PBS放入37℃水浴锅中预热;取出前一天种植的细胞,用移液管吸掉废液;用预热的PBS清洗细胞,然后放置3mL预热过的Opti-MEM培养基于细胞中,置于37℃恒温培养箱,培养2h。在此期间,取2支干净灭菌1.5mL离心管,放置于生物安全柜内,打开紫外照射;1.5h后,配置溶液A1:240μL Opti-MEM培养基+10 uL lipofectamine 2000/well,溶液A2:同A1,温育5min;溶液B1:245μL Opti-MEM培养基+0.3μg质粒(pcDNA5/FRT/TO)+2.7μg质粒POG44;溶液B2:245μL Opti-MEM培养基+0.3μg质粒(pcDNA5/FRT/TO-VSV-GluR5-CB1-Turquiose)+2.7μg质粒POG44;然后将A1与B1溶液混合,为构建pcDNA5/FRT/TO空载稳定细胞备用;A2与B2溶液混合,为构建CB1-WT稳定细胞系备用。用移液枪将混合物,各自加入到25cm2培养瓶,轻轻晃动培养瓶,使其均匀再放入37℃恒温培养箱中;6-8h后,换成新鲜的DMEM完全培养基(含10% FBS),继续培养。
转染细胞培养36h后,将细胞从25cm2培养瓶全部传代到175cm2培养瓶中培养,此期间,所用的培养基为DMEM(FLP-IN 293)+10%FBS。
转染后的细胞在175cm2培养瓶中密度达到80%,加入Hygromycine B对转染的细胞进行筛选。此时培养基为DMEM(10%FBS,1%P/S,6.6 μL Blasticidin,440μL HygromycinB)。每隔四天换新鲜的培养基培养。16天后,看到培养瓶壁上有明显的细胞群落时,就可以将细胞群用胰酶消化下来,离心后,重新加入新鲜的DMEM培养基(10%FBS,1%P/S,6.6μLBlasticidin,110μL Hygromycin B),放37℃恒温培养箱培养。通过Western Blot实验验证所构建pcDNA5/FRT/TO空载稳定细胞(Vector)和CB1-WT稳定细胞系(Probe)的VSV表达,以瞬时转染的细胞(阳性对照)VSV表达为对照,结果表明成功构建了稳定的诱导表达细胞系,结果见图3。
3、Western-blot法检测青阳参皂苷M1对CB1-WT下游ERK通路的作用
将pcDNA5/FRT/TO空载稳定细胞(Vector)和CB1-WT稳定细胞系(Probe)铺于12孔板中,每种细胞6个孔;待细胞融合度达到70-80%时,用20ng/mL多西环素诱导24h,之后饥饿处理12h;用药物刺激细胞5min后收样,药物设置为溶剂空白组(DMSO 10-8mol/L)、非特异性血清激活组(FBS)、激动剂WIN55,212-2刺激对照组(WIN 10-6M)、M1低浓度(10-12 mol/L) 、M1中浓度(10-10 mol/L)和M1高浓度(10-8 mol/L)三个处理组。
①依据Western Blot实验,用VSV抗体检测探针蛋白的表达,检测所构建CB1-WT稳定细胞系的有效性;激动剂WIN55,212-2刺激能够引起ERK磷酸化水平的升高,进一步检测所构建CB1-WT稳定细胞系的有效性;
②与青阳参皂苷M1处理空载体细胞相比,青阳参皂苷M1处理CB1-WT稳定细胞系能剂量依赖性的明显诱导ERK的磷酸化,两组相比具有显著性差异,表明青阳参皂苷M1可以激活CB1受体,结果见图4。
序列表
<110> 昆明理工大学
<120> 青阳参皂苷M1的新用途
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