CN115201361A - 一种风湿宁干预类风湿关节炎大鼠的代谢标志物和通路 - Google Patents
一种风湿宁干预类风湿关节炎大鼠的代谢标志物和通路 Download PDFInfo
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Abstract
本发明公开了一种风湿宁干预类风湿关节炎大鼠的代谢标志物和通路，属于代谢组学技术领域。本发明通过采用UHPLC‑Q‑Exactive Orbitrap‑MS技术检测了风湿宁复方用药前后大鼠的血清中活性化合物的变化，筛选了其治疗类风湿关节炎的关键代谢通路，探讨了风湿宁发挥疗效的机制，筛选出D－半乳糖、胆酸、脱氧胆酸等21种差异代谢物，以及牛磺酸和次牛磺酸代谢通路、初级胆汁酸生物合成代谢通路、类固醇激素生物合成代谢通路、半乳糖代谢通路等9条代谢通路，对该疾病的病理生理学理解以及生物学意义的解释，为风湿宁的后续深入研究奠定了基础。
Description
技术领域
本发明属于代谢组学技术领域，具体涉及一种风湿宁干预类风湿关节炎大鼠的代谢标志物和通路。
背景技术
类风湿性关节炎(rheumatoid arthritis，RA)属于自身免疫性疾病，是一种高度流行的慢性病。目前影响着约1％的世界人口，其病理特点为侵蚀性滑膜炎，临床上的主要表现为关节压痛、肿胀、畸形、功能下降等。严重时会使患者的关节功能丧失。研究表明，这类疾病多发生于女性，发病率较男性高2-3倍，且高发年龄为40-60岁。这类疾病严重影响了患者的生理、心理健康。RA属中医“痹证”范畴，中药复方风湿宁为白清佐的治痹名方临床传承使用七十余年，曾作为院内制剂广泛使用，具有祛风散寒、除湿通络、活血止痛的功效，能够有效地改善RA的主要症状及体征积分。该方主要由羌活，独活，青风藤，威灵仙，片姜黄，防风等17味中药构成，其成分庞杂，入血活性成分不明，治疗RA的物质基础及起效机制尚未阐明，因此，探讨这一具体机制，对于现代化研究具有深刻的意义。
代谢组学是一种可以定性和定量分析的有力工具，可以检测相对分子质量在1000Da以下的内源性小分子。研究对象可以为血清、粪便、尿液等生物样本。通过代谢组学可以确定体内受到内源性或外源性干扰后代谢物的变化，进而寻找这些代谢物与机体变化(包括生理、病理)的相关性。由于中医的整体性和代谢组学的特性一致，所以现在被广泛应用于解释中药处方的疗效和可能的机制。代谢组学的分析技术主要包括液相色谱－质谱(LC-MS)、气相色谱－质谱(GC-MS)和核磁共振(NMR)。LC-MS具有灵敏度高、分辨率好、质量准确的特点，因此可用于分析各种代谢物。GC-MS需要对样本进行衍生化预处理，而LC-MS省去了这个繁琐的步骤；同时，LC-MS又比NMR经济实用，对于热稳定性较差，样品难挥发、衍生化难、分子量大的样品，LC-MS表现出了巨大的优势，这些样品都适用于LC-MS。LC-MS是代谢组学研究中应用最广泛的方法，这项技术为我们的研究提供了很大的便利，在中药处方的代谢组学分析中发挥着重要作用。
发明内容
针对目前风湿宁干预类风湿关节炎的物质基础尚未阐明，其治疗类风湿关节炎起效的关键差异代谢物未知，多成分协同作用通过的代谢通路不明确的问题，本发明提供了一种风湿宁干预类风湿关节炎大鼠的代谢标志物和代谢通路的研究方法及实验结果。
为了达到上述目的，本发明采用了下列技术方案：
一种风湿宁干预类风湿关节炎大鼠的代谢标志物和通路，运用超高效液相－四级杆/静电场轨道阱高分辨质谱仪，对采集的血清样本进行检测，结合HDMB 数据库以及二级质谱碎片进行比对，鉴定出不同的代谢标志物，将筛选出来的代谢标志物导入到MetaboAnalyst 5.0数据库中以进行相关代谢途径分析，筛选出相关性高的代谢通路；
所述代谢标志物为D－半乳糖(D-Galactose)、牛磺酸(Taurine)、尿酸(Uricacid)、泛酸(Pantothenic acid)、D-色氨酸(D-Tryptophan)、硫酸吲哚酚(Indoxylsulfate)、对甲酚硫酸盐(p-Cresylsulfate)、牛磺酸十二氧胆酸(Taurochenodeoxycholic acid)、姜辣素(Gingerol)、胆酸(Cholic acid)、脱氧胆酸(Deoxycholic acid)、皮质甾酮(Corticosterone)、12-羟基十二烷酸(12-Hydroxylauricacid)、γ－亚麻酸(Gamma-Linolenic acid)、二十二碳六烯酸(Docosahexaenoic Acid)、硬脂酸(Stearic acid)、棕榈油酸(Palmitoleic acid)、花生四烯酸(Arachidonic acid)、胆固醇硫酸盐(Cholesterol sulfate)、十四酸乙酯(Ethyl myristate)、油酸(Oleicacid)；
所述代谢通路为不饱和脂肪酸的生物合成代谢通路、初级胆汁酸生物合成代谢通路、牛磺酸和次牛磺酸代谢通路、泛酸盐和辅酶A的生物合成代谢通路、类固醇激素生物合成代谢通路、半乳糖代谢通路、花生四烯酸代谢通路、氨基糖和核苷酸糖的代谢通路、嘌呤代谢通路。优选牛磺酸和次牛磺酸代谢通路、初级胆汁酸生物合成代谢通路、类固醇激素生物合成代谢通路、半乳糖代谢通路。
与现有技术相比本发明具有以下优点：
目前对于类风湿关节炎的研究大多集中在自身免疫异常方面，较少论及其在代谢物方面的异常改变。本发明通过采用UHPLC-Q-Exactive Orbitrap-MS技术检测风湿宁复方用药前后大鼠的血清，探讨风湿宁发挥疗效的机制，筛选出D －半乳糖、胆酸、脱氧胆酸等21种差异代谢物，以及牛磺酸和次牛磺酸代谢通路、初级胆汁酸生物合成代谢通路、类固醇激素生物合成代谢通路、半乳糖代谢通路等9条代谢通路，对该疾病的病理生理学理解以及生物学意义的解释，为风湿宁的后续深入研究奠定了基础。
附图说明
图1正离子、负离子模式下大鼠血清总离子流图
图2各组大鼠血清的PCA图；
图3大鼠血清样本OPLS-DA分析图；
图4NC-CIA组，CIA-FSN组的共同差异代谢物示意图；
图5各组大鼠差异代谢物的相对含量变化示意图；
图6风湿宁对CIA大鼠血清代谢通路分析结果示意图。
具体实施方式
本实验采用UHPLC-QE-MS对风湿宁治疗胶原诱导性关节炎大鼠 (collagen-induced arthritis，CIA)进行了血清代谢组学分析，在这项研究中，首先根据我们前期的研究成功诱导出CIA大鼠模型。其次，采用基于LC-MS的代谢组学方法分析RA疾病引起的代谢物水平的改变。最后，识别差异代谢物，并评价风湿宁的治疗效果。此外，还对代谢物进行了通路富集分析。所以，本实验可阐明风湿宁治疗RA的潜在机制，为临床治疗提供有价值的见解。
实施例1
一、实验材料
1.1药物
风湿宁复方：羌活、独活、青风藤、威灵仙、片姜黄、防风、川芎、麻黄、肉桂、三棱、血竭、延胡索、川牛膝、熟地黄、砂仁、生姜和甘草十七味中药均购自晋中市同仁堂药房连锁有限公司。雷公藤多苷片(批号：Z42021212)购自黄石飞云制药有限公司。
1.2试剂
本实施例所用试剂见下表：
表1试剂一览表
1.3仪器
本实施例所用仪器见下表：
表2仪器设备一览表
1.4动物
20只SPF级雌性Wistar大鼠，体重200±25g，均采购于北京维通利华实验动物技术有限公司，许可证号：SCXK(京)2016-0006。所有大鼠均喂养于山西中医药大学，温度为15℃～25℃，相对湿度45％～55％，模拟自然光照12h明暗交替，自由饮水与摄食，每日更换垫料，适应性喂养一周。
二、实验方法
2.1风湿宁方的制备
购得药材总量为223g，加入500mL清水，冷水浸泡1h，加热煎煮1h，四层纱布滤过；药渣中再加入300mL清水，煎煮45min，纱布再滤过，合并2 次滤液，减压浓缩，生药浓度为2g/mL。
2.2造模、分组与给药
将牛Ⅱ型胶原以及完全弗氏佐剂(CFA)等比例混合。在大鼠的尾巴根部、背部、脚掌分别注射0.1mL试剂。空白组大鼠在同一部位注射0.1mL 0.9％NaCl 溶液。7天后免疫加强一次，以上操作在冰上进行。
20只大鼠按体重随机分为空白组(NC)、模型组(CIA)、风湿宁组(FSN)、雷公藤多苷片组(TG)，每组5只。
FSN组是将预先熬好的风湿宁给CIA大鼠灌胃，每日一次，持续7d。同样的，TG组是将雷公藤多苷片(9mg/kg)用生理盐水配置为溶液，然后给大鼠灌胃，每日一次，持续7d。NC组和CIA组每天给予相同量的蒸馏水。以上四组灌胃剂量均为2mL。
2.3血清样本采集
最后一次给药24h后，用10％水合氯醛麻醉处理。所有大鼠的血样均取自腹主动脉，凝结4小时，进行自然沉降。离心(3000rpm，10min)后的上清液收集于试管中，在-80℃的冰箱中保存。
2.4血清样本制备
血清样品在4℃下解冻1h，精密吸取100μL血清样本至EP管中，加入400 μL甲醇，6000rpm涡旋3min，4℃条件下14000r/min，离心10min，吸取上清液转移至新的1.5mL EP管内，常温下用氮气吹干。然后再吸取100μL甲醇进行复溶，再重复上述操作涡旋混匀，离心10min，离心后再次吸取上清液转移至新的EP管中，再次离心，最后移取10μL上清液，进样。
2.5 LC-MS分析条件
2.5.1色谱条件
ACQUITY BEH C18色谱柱(100mm×2.1mm，1.7μm)；流动相乙腈(A) -0.01％(v/v)甲酸水溶液(B)，梯度洗脱(0-1.5min：95％A；1.5-4.5min：75％A； 4.5-7min：70％A；7-11min：45％A；11-12min：15％A；12-13.5min：5％A； 13.5-14min：95％A)；体积流量0.3mL/min，进样量5μL，柱温40℃。
2.5.2质谱条件
采用电喷雾离子源(ESI)，正、负离子同时扫描模式。正离子模式：喷雾电压3.2kV，鞘气流速40arb，辅助气流速5arb，辅助气加热温度350℃；负离子模式：喷雾电压2.5kV，鞘气流速38arb，辅助气流速10arb，辅助气加热温度300℃。离子传输管温度320℃；S-Lens RFLevel为50；全扫描/数据依赖二级扫描(Full MS/dd-MS2)，扫描范围m/z 100～1000；一级质量分辨率70000 FWHM，二级分辨率17500FWHM，碰撞能量30eV。
2.6数据分析
利用Compound Discoverer 3.3软件(Thermo-Fisher公司，美国)对所得的原始文件进行预处理，提取保留时间、质荷比和峰面积等数据，峰面积归一化后得出的Excel表格导入SIMCA-P 14.1软件(Umetrics公司，瑞典)进行主成分分析(PCA)、以及正交偏最小二乘判别分析(OPLS-DA)。根据VIP＞1和P ＜0.05的条件筛选差异代谢物。最后结合HMDB数据库和质谱的二级碎片离子进行比对，找出差异代谢物，利用MetaboAnalyst 5.0生物学数据库进行差异代谢物的通路富集分析。
三、实验结果
3.1代谢轮廓分析
各组大鼠血清总离子流图(TIC)曲线基本相似，但峰响应值存在一定差异 (如图1所示，A、B空白组；C、D模型组；E、F风湿宁组；A、C、E为负离子模式；B、D、E为正离子模式)。
使用Compound Discoverer 3.3对所获得的原始文件进行归一化处理后，再导入SIMCA-P 14.1软件进行分析。对NC组、CIA组、FSN组、TG组进行无监督模式的主成分分析，得到了PCA图(如图2所示，NC.空白组；CIA.模型组；FSN.风湿宁组；TG.雷公藤多苷片组)。四个组分离趋势显著，说明模型建立成功。FSN组有远离CIA组的趋势，同时有向NC组靠近的趋势，表明风湿宁能调节CIA大鼠体内代谢轮廓趋向健康的状态，从而发挥其治疗效果。
3.2大鼠血清中差异代谢物的筛选与鉴定
从OPLS-DA(正交偏最小二乘判别分析)得分图可以看出CIA组与NC组以及CIA组与FSN组分离显著。说明CIA大鼠代谢轮廓发生了显著变化。采用 OPLS-DA分析后，进行200次模型验证。结果表明，研究模型中的R2值高于 Q2值，Q2回归线与纵轴的截距为负，这决定了模型的有效性和可靠性，并且没有拟合现象。从而能很好的解释以及预测。通过S-plot图并基于VIP＞1，P＜0.05 的条件，筛选出与类风湿关节炎紧密联系的差异代谢物(如图3大鼠血清样本 OPLS-DA分析图所示，NC.空白组；CIA.模型组；FSN.风湿宁组；A:空白组与模型组的OPLS-DA图；B:模型组与风湿宁组的OPLS-DA图；C:空白组与模型组的模型验证图；D:模型组与风湿宁组的验证图E：空白组与模型组的S-Plot 图；F：模型组与风湿宁组的S-Plot图)。
根据筛选出来的代谢物，同时结合HDMB数据库以及二级质谱碎片进行比对，最后共鉴定出21种不同的代谢物(如图4NC-CIA组，CIA-FSN组的共同差异代谢物、表1所示)。选择倍数变化(Fold change,FC)值作为反映CIA和 NC组，FSN组和CIA组血清差异代谢物含量变化的指标。
与NC组相比较，CIA组牛磺酸、尿酸、牛磺酸十二氧胆酸、皮质甾酮、γ -亚麻酸、二十二碳六烯酸、硬脂酸、棕榈油酸、花生四烯酸、十四酸乙酯、油酸这11种差异代谢物水平降低，用药后含量又较之前上升，而D－半乳糖、胆酸、脱氧胆酸这3种代谢物的含量显著上调，给予风湿宁干预后又显著下调(如图5所示，各组大鼠差异代谢物的相对含量变化(x±s，n＝5)与NC组比较*P ＜0.01；与CIA组比较#P＜0.05)。
表3血清差异代谢物鉴定信息
代谢通路分析
将筛选出来的差异代谢物导入到MetaboAnalyst 5.0数据库中以进行相关代谢途径分析，差异代谢物所涉及的代谢途径包括不饱和脂肪酸的生物合成、原代胆汁酸生物合成、牛磺酸和次牛磺酸代谢、泛酸盐和辅酶A生物合成、类固醇激素生物合成、半乳糖代谢、花生四烯酸代谢、氨基糖和核苷酸糖的代谢、嘌呤代谢等9条相关代谢途径。其中牛磺酸和次牛磺酸代谢、初级胆汁酸生物合成、半乳糖代谢、类固醇激素生物合成等4条代谢途径相关性较高，表明这几条通路可能与风湿宁治疗RA有关(如图6代谢通路图和表4通路分析结果)。其他通路上的调节作用差异不大。
表4通路富集分析结果
注：Match status是指路径中化合物的数量与上传数据的匹配数量的比率；Raw P是原始的由富集分析计算出的P值；Impact是由路径拓扑分析计算出的路径影响值。
本实施例采用UHPLC-Q-Exactive Orbitrap-MS技术检测风湿宁复方用药前后大鼠的血清，以探讨风湿宁发挥疗效的机制。风湿宁用药前后的血清样本，总离子流图的轮廓基本相似，但峰的形状和数量还是存在一定的差异。初步说明了风湿宁治疗前后，胶原诱导性关节炎大鼠的血清代谢物存在差异。并且建立了PCA和OPLS-DA模型。PCA、OPLS-DA得分图显示，空白组、模型组和风湿宁组分离度较好，进一步表明了大鼠血清的代谢物小分子有着良好的差异性。
风湿宁用药前后血清中共有的差异代谢物共21种，有可能为RA的潜在生物标志物。将筛选出来的差异代谢物进行通路富集分析后，发现风湿宁发挥药效与牛磺酸和次牛磺酸代谢、初级胆汁酸生物合成、类固醇激素生物合成、半乳糖代谢这4条通路的相关性较高。
牛磺酸的主要功能是抗炎、抗氧化，同时在细胞中也有免疫调节的作用。在我们筛选到的差异代谢物中发现，与空白组相比，CIA大鼠的牛磺酸含量显著降低，提示我们RA的发生可能与牛磺酸代谢紊乱有关，风湿宁给药后，牛磺酸以及牛磺酸十二氧胆酸含量均升高，说明风湿宁可以调节牛磺酸的含量，参与调节牛磺酸和次牛磺酸代谢，并起到抗炎作用，最终达到缓解RA的目的。
胆汁酸代谢紊乱与炎症的发生有关。胆固醇在肝脏中合成了人体内的两种主要胆汁酸，即胆酸(CA)和鹅去氧胆酸(CDCA)。在肝细胞合成后，胆汁酸转化为胆汁盐，它是一种胺偶联物。胆汁盐与磷脂和其他胆汁成分一起被排泄到胆小管中，由此产生的胆汁被运输到胆囊并储存在胆囊中。在我们实验中， CIA大鼠血清中胆酸和去氧胆酸明显增加，说明可能是胆汁酸代谢发生障碍导致的，而在风湿宁用药后，胆汁酸水平出现了回调。
有研究表明，类固醇激素生物合成可作为抗RA的途径之一。肾上腺类固醇激素以及性腺类固醇激素减少会使得RA的发生。正如我们筛选到的差异代谢物皮质甾酮，就是一种肾上腺皮质类固醇，CIA组含量下降，而给予风湿宁治疗后，激活了大鼠体内类固醇激素的生物合成途径，抑制了包括细胞免疫和体液免疫的RA免疫反应，最终起到抑制RA的作用。
半乳糖代谢与肝脏密切相关。Leloir途径(Leloir Pathway)是半乳糖代谢的主要途径。在Leloir途径中，半乳糖首先被半乳糖激酶(GALK)磷酸化为半乳糖-1-磷酸(Gal-1-P)。半乳糖-1-磷酸尿苷转移酶(GALT)，然后将尿苷单磷酸 (UMP)从UDP-葡萄糖(UDP-glc)转移到Gal-1-P，形成葡萄糖-1-磷酸(Glc-1-P) 和UDP-半乳糖(UDP-gal)。在Leloir途径的第三步中，UDP-半乳糖4-差向异构酶(GALE)催化UDP-gal和UDP-glc的相互转化。UDP-gal和UDP-glc都是糖基化反应的糖供体，对糖缀合物的产生很重要。形成的Glc-1-P导致葡萄糖-6- 磷酸(Glc-6-P)和葡萄糖的产生，从而导致能量的产生。
代谢后产生的Glc-6-P可以参与糖酵解途径。一些研究报道RA患者的滑膜组织中的糖酵解活性受到了抑制。通过糖酵解，可以合成乙酸、丙酸、丁酸等短链脂肪酸(SCFAs)。Chen等人通过液质联用，测出人血清中含有丁酸、己酸。 SCFAs经常以游离的形式存在，研究表明在大鼠中，补充SCFA丁酸盐可降低 CIA大鼠的严重程度，它通过增加5-羟色氨衍生代谢物：5-羟基吲哚-3-乙酸 (5-HIAA)的水平，以调节性B细胞(Breg)依赖的方式抑制RA，最终激活芳基烃受体(AhR)，进而缓解RA等自身免疫性疾病的病情。
有研究结果显示半乳糖代谢可能与慢性炎症有关，并且半乳糖的存在对于稳定整个支链碳水化合物链的结构很重要。它的缺失与RA的严重程度相关。因此，半乳糖代谢的扰动可能是RA的一个重要特征。在未用药前D－半乳糖上调，药物干预后D－半乳糖发生下调，我们猜测，风湿宁治疗RA的机制可能是通过消耗半乳糖，发生了半乳糖代谢，然后以己糖途径进入糖酵解，生成的物质如丙酮酸以不同的生物合成途径生成SCFAs，在SCFAs的作用下，最终使症状得以改善。正如有的研究指出，RA发病机制包括能量代谢的异常。所以，我们推测风湿宁治疗RA的原因之一可能是风湿宁激活了半乳糖代谢这一途径，从而间接的改善能量代谢。
综上所述，本发明基于分离效率高、选择性好、检测灵敏度高的高效液相色谱串联质谱检测了风湿宁干预类风湿关节炎大鼠的活性化合物，并通过高通量筛选对大量化合物进行分析，找出了风湿宁干预类风湿关节炎的代谢标志物和通路，为风湿宁的后续深入研究奠定了基础。
本发明说明书中未作详细描述的内容属于本领域专业技术人员公知的现有技术。尽管上面对本发明说明性的具体实施方式进行了描述，以便于本技术领的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
Claims (2)
1.一种风湿宁干预类风湿关节炎大鼠的代谢标志物和通路，运用超高效液相－四级杆/静电场轨道阱高分辨质谱仪，对采集的血清样本进行检测，结合HDMB数据库以及二级质谱碎片进行比对，鉴定出不同的代谢标志物，将筛选出来的代谢标志物导入到MetaboAnalyst 5.0数据库中以进行相关代谢途径分析，筛选出相关性高的代谢通路，其特征在于：所述代谢标志物为D－半乳糖、牛磺酸、尿酸、泛酸、D-色氨酸、硫酸吲哚酚、对甲酚硫酸盐、牛磺酸十二氧胆酸、姜辣素、胆酸、脱氧胆酸、皮质甾酮、12-羟基十二烷酸、γ－亚麻酸、二十二碳六烯酸、硬脂酸、棕榈油酸、花生四烯酸、胆固醇硫酸盐、十四酸乙酯、油酸；
所述代谢通路为不饱和脂肪酸的生物合成代谢通路、初级胆汁酸生物合成代谢通路、牛磺酸和次牛磺酸代谢通路、泛酸盐和辅酶A的生物合成代谢通路、类固醇激素生物合成代谢通路、半乳糖代谢通路、花生四烯酸代谢通路、氨基糖和核苷酸糖的代谢通路、嘌呤代谢通路。
2.根据权利要求1所述的一种风湿宁干预类风湿关节炎大鼠的代谢标志物和通路，其特征在于：所述代谢通路为牛磺酸和次牛磺酸代谢通路、初级胆汁酸生物合成代谢通路、类固醇激素生物合成代谢通路、半乳糖代谢通路。
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