CN105535976A - 转录谱印记预测药物ms-275在制备小鼠炎症性肠病药物中的应用 - Google Patents
转录谱印记预测药物ms-275在制备小鼠炎症性肠病药物中的应用 Download PDFInfo
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Abstract
本发明公开了转录谱印记预测药物MS-275在制备小鼠炎症性肠病药物中的应用，属医药卫生领域。该药物由预测药物MS-275与二甲亚砜(DMSO)和生理盐水按照质量比1:22:500组成,该药物能有效减轻小鼠结肠炎症程度，降低死亡率。
Description
技术领域
本发明涉及转录谱印记预测药物MS-275在制备小鼠炎症性肠病药物中的应用
背景技术
炎症性肠病(InflammatoreBowelDisease,IBD)是一组病因尚不十分清楚的慢性非特异性肠道炎症性疾病，包括溃疡性结肠炎和克罗恩病。IBD是北美及欧洲的常见病。近年来随着生存环境及膳食结构的改变，我国IBD发病率呈明显增加趋势，构成日益严重的社会健康问题。深入揭示IBD发生发展的分子机制，并发掘潜在的干预治疗靶点，对提高IBD防治水平有重要意义。
IBD发病机制尚未完全明确，目前主要认为与遗传、环境、免疫等因素有关。目前仍缺乏安全有效的药物诱导及维持缓解。药物临床上多用氨基水杨酸制剂(5-ASA)，糖皮质激素，免疫抑制剂、生物制剂等药物缓解治疗IBD；激素类药物作用虽明确，却难于获得满意疗效，新的生物制剂仅对部分患者有效。因此，开发新型靶向药物对改善IBD患者预后，干预并阻断疾病向肠癌转变，对提高人民健康水平具有深远意义。
转录谱印记(gene-expressionsignatures)比对方法是运用一定数量显著差异表达基因的有序集合代替原始转录谱，通过将细胞或组织在生理、病理、药物治疗等状态下的转录谱，与数据库中的各种基因沉默、miRNA干扰和相关疾病的表达谱进行比对和相关性计算分析，挖掘出与疾病及药物治疗相关的基因干扰印记。项目申请者前期运用GEO等转录谱数据库中数据建立了IBD和1310种药物作用构成的转录谱印记关联网络，通过Affymetrix基因芯片数据与上述转录谱印记数据库进行比对，得出一系列疾病-药物距离值，发现一些新型可能为治疗IBD潜力药物，排名前三分别是MS-275、三氟拉嗪和皮质酮(距离值分别是-0.20166，-0.1795，-0.17228，负值越大越有可能起治疗作用；其中强的松，一种有效治疗IBD急性发作糖皮质激素，距离值为-0.07073)。申请者运用MS-275干预DSS诱导的小鼠急性IBD模型，发现与激素治疗组、模型对照组相比，在小鼠急性发病期间，MS-275疾病活动指数(DAI)评分明显低于其他非空白对照组，结肠炎组织病理评分明显低于其他处理组。以上转录谱印记计算结果及模型动物验证提示：MS-275极可能是有效治疗及诱导IBD缓解新型药物。
基因表达的精确调控是细胞正常增殖分化及机体生长发育的基础；组蛋白乙酰化转移酶(HATs)和组蛋白去乙酰化酶(HDACs)分别通过乙酰化及去乙酰化组蛋白或非组蛋白来协同调节DNA的表达及蛋白的活性状态，隶属表观遗传学的范畴。当HDAC过度表达引起转录因子募集时，就会引起相关蛋白表达受到抑制，从而导致机体处于疾病状态。组蛋白去乙酰酶抑制剂(Histonedeacetylaseinhibitor,HDACi)因能有效诱导基因表达、促进细胞分化、致使肿瘤细胞凋亡及产生细胞周期阻滞的抗肿瘤活性，从而在治疗血液恶性肿瘤上得到应用。近年HDACi类药物作为潜在抗炎药物为治疗IBD提供了新的思路。
MS-275(又名SNDX-275，Entinostat，恩提诺特)隶属合成型酰胺类HDACi，是高度特异的组氨酸乙酰基转移酶抑制剂，选择性亲和I型HDAC(包括HDAC1/3)。MS-275抑制HDAC效力虽不及TSA等一些其他HDACi，但其在体内抗肿瘤效果显著(经过I/II期临床试验)，具有药毒性小，口服有效，有高度的选择性与特异性等优点。MS-275作为特异性高HDAC1/3抑制剂，在治疗IBD尚无报道。在免疫调节方面，MS-275通过抑制HDAC3活性影响调节性T淋巴细胞(Tregs)生成及Foxp3+的表达；此外，HDAC3可影响黏附因子、干扰素、MHCⅡ类分子以及转录因子如STAT1、STAT3和NF-κB相关基因表达，抑制HDAC3可减少TNF-ɑ，IL-1/6等下游促炎症因子的产生，减轻炎症程度。由此，MS-275作为一种特异性强的HDAC1/3抑制剂在治疗缓解免疫相关疾病IBD中具有巨大药物潜力。本发明旨在通过动物实验揭示转录谱预测药物MS-275在预防和治疗DSS所诱导的小鼠急性IBD模型中的重要作用，并探讨其内在机制，并为其在临床上运用提供重要的思路。
发明内容
本发明目的是针对目前治疗IBD药物的缺陷，提供一种转录谱印记预测药物MS-275在制备小鼠炎症性肠病药物中的应用。
本发明的目的是通过以下技术方案来实现的：一种转录谱印记预测药物MS-275在制备小鼠炎症性肠病药物中的应用。
进一步地，该应用具体为，将预测药物MS-275与二甲亚砜(DMSO)和生理盐水按照质量比1:22:500混合均匀后，调节PH至5.0-7.0。
本发明的有益效果是，本发明基于转录谱预测药物MS-275能靶向抑制TNF-ɑ，IL-6产生，有效改善小鼠结肠炎症程度的特点，将其应用于制备小鼠炎症性肠病药物，具有药毒性小，高度的选择性与特异性等特点。
附图说明
图1为DSS诱导的小鼠急性IBD模型结果图；A：模型组及对照组小鼠期间每天的结肠炎疾病活动指数(DAI)；B：模型组及对照组小鼠结肠长度；C：模型组及对照组小鼠远端结肠促炎症因子TNF-ɑ、IL-6、IFN-γ含量；D:两组小鼠结肠组织H&E染色；E：模型组及对照组小鼠结肠炎病理评分。
图2为MS-275减轻IBD模型小鼠DAI结果图；
图3为IBD小鼠模型阴性对照组、模型对照组、激素对照组及MS-275对照组的存活率(图3A)及结肠长度(图3B)比较结果图；
图4为IBD小鼠模型阴性对照组、模型对照组、激素对照组及MS-275对照组的病理结果图；
图5为MS-275降低小鼠结肠炎组织病理评分结果图；
图6为MS-275逆转LPS所致小鼠巨噬细胞产生炎症因子TNF-α、IL-6升高结果图。
具体实施方式
实施例1
(一)材料与方法
1、主要仪器：
7900RealTimePCRSystem(美国AppliedBiosystems公司)；CO2细胞培养箱MODEL3111(美国ThermoScientific公司)；六孔培养板(美国Corning公司)。
2.药品和试剂
胎牛血清(FBS)、DMEM培养液(澳洲GIBCO公司)，甲醇、乙醇、异丙醇、氯仿(国药集团化学试剂有限公司)，小鼠TNF-ɑ、IL-6ELISA试剂盒(美国eBioscience有限公司)，Trizol试剂、RT-qPCR试剂盒(日本Takara公司)；PCR引物合成(上海生工生物工程技术服务有限公司)；脂多糖(LPS，美国Sigma公司)。
(二)细胞实验及处理方案
用含10％FBS的DMEM培养基培养小鼠巨噬细胞RAW264.7。分为4组：阴性对照组(NC组)，药物对照组(MS-275处理组)，单纯刺激组(LPS处理组)及MS-275治疗组(LPS+MS-275)。具体方案为：用2μMMS-275预处理RAW264.7细胞1小时，后用1μg/mlLPS刺激该细胞6小时，提取细胞mRNA及留取细胞培养上清。运用qPCR测定细胞内促炎症因子TNF-α及IL-6mRNA表达水平。ELISA法测定培养上清中相应TNF-α及IL-6蛋白表达量。
(三)RT-qPCR
按照Trizol试剂的说明书提取得到小鼠巨噬细胞总RNA，按照RT试剂盒说明书逆转录得到cDNA，按照qPCR试剂盒说明书检测特定基因的mRNA表达量。实验涉及的引物序列如表3所述。
表3.引物序列
| Gene | Species | Forward primer(5′→3′) | Reverse primer(5′→3′) |
| β-Actin | Mouse | GAAGATCAAGATCATTGCTCCT | TGGAAGGTGGACAGTGAG |
| TNF-α | Mouse | GCCACCACGCTCTTCTGTCT | GTCTGGGCCATAGAACTGAT |
| IL-6 | Mouse | GATGCTACCAAACTGGATATAAC | CTGGCACCACTAGTTGGTTGTC |
(四)数据统计
各组实验数据均以mean±S.E.M.表示，采用GraphPadPrism5软件进行统计分析，采用unpairedStudent’sttest进行两组间比较，P<0.05被认为有显著性差异。其中*表示两组比较P<0.05，**表示两组比较P<0.01，***表示两组比较P<0.001。
(五)实验结果
MS-275有效地抑制LPS刺激RAW264.7产生促炎症因子：通过细胞实验，发现与单纯LSP刺激组相比，在mRNA及蛋白水平，MS-275治疗组能有效抑制RAW264.7细胞中促炎症细胞因子TNF-α、IL-6的产生(如图6)。
基于MS-275这一特性，将其与二甲亚砜(DMSO)和生理盐水按照质量比1:22:500混合均匀后，浓盐酸调节PH至5.0-7.0，配制得到可用于治疗小鼠炎症性肠病的MS-275药物。现通过应用例1对药物的治疗效果进行进一步说明。
应用例1
(一)材料与方法
1、主要仪器：
台式低温高速离心机CL17(美国ThermoFisherScientific公司)；96孔Imark酶标仪(美国BIO-RAD公司)；倒置显微镜CK40-32PH(日本OLYMPUS公司)；电热恒温水槽、隔水式恒温培养箱(上海精宏实验设备有限公司)；电子天平PL2002(瑞士METTLERTOLEDO公司)。
2、药品和试剂
硫酸盐葡聚糖钠(dextransodiumsulfate,DSS纯度≥99％，美国MP生物试剂公司)；MS-275(纯度≥99.96％，美国SELLECK生物试剂公司)，二甲亚砜(DMSO，纯度≥99.7％，美国Sigma公司)；醋酸泼尼松龙注射液(药用级，浙江仙琚制药股份有限公司)；小鼠TNF-ɑ、IL-6、IFN-γELISA试剂盒(美国eBioscience有限公司)；甲醛、乙醇、过氧化氢、二甲苯(国药集团化学试剂有限公司)，其它试剂均为分析纯级别。
(二)实验动物及给药方案
将MS-275、二甲亚砜(DMSO)、生理盐水按照质量比1:22:500混合均匀后，用浓盐酸调节PH至5.0-7.0，得到MS-275药物。
C57BL/6小鼠，雌性，8-10周龄，体重18-22克，由上海西普尔-必凯实验动物有限公司提供，许可证号为SCXK(沪)2013-0016。饲养于SPF级动物房中，室温23±2℃；随机分为4组，每组小鼠各10只，其中3组为DSS诱导建模组，剩余一组为阴性对照组；具体造模方案为：造模组小鼠饮用含3％DSS高压灭菌水，对照组饮用普通灭菌水，隔天更换；第8天全换为新鲜高压灭菌水；第9天颈椎脱臼处死。
将30只DSS诱导的急性小鼠IBD模型分为3组：MS-275药物处理组，激素治疗干预组，模型对照组。具体给药方案如下：
MS-275药物处理组小鼠予以10ml/kg注射量，腹腔内注射MS-275药物，1次/天，于造模第二天给药，持续到第八天，共7次。
激素干预组小鼠予以10ml/kg注射量，腹腔内注射醋酸泼尼松龙(醋酸泼尼松龙的浓度为3mg/kg)，1次/天，于造模第二天给药，持续到第八天，共7次。
模型对照组小鼠予以10ml/kg注射量，腹腔内注射生理盐水+DMSO，1次/天，于造模第二天给药，持续到第八天，共7次。
此外，阴性对照组小鼠予以10ml/kg注射量，腹腔内注射生理盐水+DMSO，1次/天，于造模第二天给药，持续到第八天，共7次。
观察并记录每天各组小鼠精神状态、体重、便血情况，评估小鼠结肠炎疾病活动指数(DAI评分，如表1所示)。造模第9天处死各组小鼠，测量各组小鼠结肠长度，留取结肠用4％多聚甲醛固定，常规石蜡包埋、切片，H&E染色后光镜下作小鼠结肠炎病理评分，如表2所示。
表1.DAI评分项目
| 体重下降(％) | 大便性状 | 肉眼血便 | 计分 |
| 0 | 正常 | 正常(+/-) | 0 |
| 1-5 | 松散 | 1 | |
| 5-10 | 2 | ||
| 10-15 | 稀便 | 肉眼血便(或+++) | 3 |
| >15 | 大量肉眼血便(或++++) | 4 |
注：正常大便为成形大便；松散大便为糊状、半成形、不黏附于肛门的大便；稀便为黏附于肛门的稀水样便。DAI评分为各项评分之和。
表2.小鼠结肠炎病理评分
| 程度 | 病变程度 | 计分 |
| 炎症程度 | 无 | 0 |
| 轻度 | 1 | |
| 中度 | 2 | |
| 重度 | 3 | |
| 病变深度 | 无 | 0 |
| 粘膜层 | 1 | |
| 粘膜及粘膜下层 | 2 | |
| 全层破坏 | 3 | |
| 隐窝破坏 | 无 | 0 |
| 基底1/3隐窝破坏 | 1 | |
| 基底2/3隐窝破坏 | 2 | |
| 仅上皮完整 | 3 | |
| 全部隐窝和表皮破坏 | 4 | |
| 再生指数 | 完全再生或正常组织 | 0 |
| 几乎完全再生 | 1 | |
| 再生，伴隐窝缺损 | 2 | |
| 表面上皮不完整 | 3 | |
| 无修复 | 4 | |
| 病变范围(％) | 1—25 | 1 |
| 26—50 | 2 | |
| 51—75 | 3 | |
| 76—100 | 4 |
(三)结肠组织组织与血清化学检测
结肠组织切片与H&E染色由浙江大学医学院附属第一医院病理切片室完成，小鼠远端结肠促炎症因子TNF-ɑ、IL-6、IFN-γ含量检测方法按照试剂盒的说明书来完成。
(四)数据统计
各组实验数据均以mean±S.E.M.表示，采用GraphPadPrism5软件进行统计分析，采用unpairedStudent’sttest进行两组间比较，P<0.05被认为有显著性差异。其中*表示两组比较P<0.05，**表示两组比较P<0.01，***表示两组比较P<0.001。
(五)实验结果
1、成功构建DSS诱导的小鼠急性IBD模型
饮用含3％DSS诱导C57BL/6系小鼠，构建小鼠急性IBD模型，通过小鼠结肠炎疾病活动指数、结肠长度及结肠炎病理评分标准等综合评价动物模型建模情况。结果如图1所示，相比阴性对照组，急性IBD模型小鼠疾病活动指数升高(图1A)，结肠缩短(图1B)，结肠末端促炎症因子产生增多(图1C)，结肠组织病理黏膜正常结构破坏，隐窝结构紊乱(图1D)，模型组结肠炎病理评分明显高于对照组(图1E)；提示成功诱导小鼠急性结肠炎模型。
2、MS-275药物减轻DSS诱导的急性IBD模型小鼠疾病活动指数
如图1A所示，与空白对照组相比，结肠炎造模组小鼠在第5天DAI明显升高，表现体重下降，水样便，肉眼血便急性中重度结肠炎表现。协同给予20mg/kgMS-275或者3mg/kg醋酸泼尼松龙，如图2所示，发现与激素干预组或模型对照组相比，MS-275处理组小鼠体重未发生明显下降，大便成形，无明显肉眼血便。图2提示MS-275能有效降低DSS诱导的急性IBD模型小鼠疾病活动指数。
3、MS-275药物提高IBD模型小鼠存活率，维持结肠长度
如图3A所示，与空白对照组相比，3％DSS造模组小鼠的死亡率为30％，协同给予20mg/kgMS-275可以有效的逆转DSS诱导结肠炎小鼠模型的死亡率；另外，结肠长度作为衡量结肠验证的有效指标，MS-275处理组小鼠结肠长度也明显长于模型对照组，接近阴性对照组小鼠(如图3B)。上述结果提示MS-275提高IBD模型小鼠存活率，减轻肠道炎症，并维持结肠长度。
4、MS-275药物有效降低小鼠结肠炎组织病理评分
如图4所示，与激素处理组及模型对照组对比，MS-275处理组小鼠结肠粘膜层及粘膜下层中性粒细胞、淋巴细胞等炎性细胞浸润程度明显减轻，即结肠局部炎症程度减轻，此外累及结肠的深度及范围，隐窝破坏均有明显减轻，再生程度趋近于阴性对照组。小鼠结肠炎组织病理评分中，MS-275处理组分数明显低于激素处理组及模型对照组(15.30士2.12VS50.67士1.33VS36.86士3.97，***P<0.001，如图5)。小鼠病理结果提示MS-275有效降低小鼠结肠炎组织病理评分，减轻结肠局部炎症。
Claims (2)
1.一种转录谱印记预测药物MS-275在制备小鼠炎症性肠病药物中的应用。
2.根据权利要求1所述的应用，其特征在于，该应用具体为，将预测药物MS-275与二甲亚砜(DMSO)和生理盐水按照质量比1:22:500混合均匀后，调节PH至5.0-7.0。
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