CN112043830A - Adar1在治疗非酒精性脂肪肝疾病药物中的应用 - Google Patents
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Abstract
本发明公开了ADAR1在用于治疗非酒精性脂肪肝疾病药物中的应用；所述ADAR1是指过表达ADAR1；所述过表达ADAR1通过抑制NLRP3炎性体及炎症反应，从而抑制非酒精性脂肪肝的形成。过表达ADAR1可有效抑制NLRP3炎性体及炎症反应，从而明显改善非酒精性脂肪肝的形成。也说明可以促进ADAR1过表达的ADAR1促进剂可以用于治疗非酒精性脂肪肝疾病药物。
Description
技术领域
本发明涉及生物医药技术领域，尤其涉及一种ADAR1在治疗非酒精性脂肪肝疾病药物中的应用。
背景技术
非酒精性脂肪肝病(Non-alcoholic fatty liver disease,NAFLD)是一种与胰岛素抵抗和遗传易感密切相关的代谢应激性肝损伤，随着人民生活水平提高及生活方式改变，NAFLD的发病率逐年上升，目前已高达全球总人口的25％，成为全球范围内最常见的慢性肝病，对国民健康和社会发展构成严重威胁。由于其发病率高、发病机制复杂且尚缺乏有效治疗手段，已成为全球代谢领域研究的热点和难点。NAFLD的潜在危害巨大，与肝硬化、肝癌及糖尿病、心血管疾病息息相关，但是目前尚无任何一种药物被FDA批准用于NAFLD的治疗，因此，寻求一种可以用于治疗NAFLD的药物具有重要的临床意义。
发明内容
为了解决上述现有技术的不足，本发明提供ADAR1在治疗非酒精性脂肪肝疾病药物中的应用。
本发明提供的ADAR1在治疗非酒精性脂肪肝疾病药物中的应用是通过以下技术方案实现的：
ADAR1在治疗非酒精性脂肪肝疾病药物中的应用。
进一步地，所述ADAR1是指过表达ADAR1，所述过表达的ADAR1可以通过构建慢病毒质粒或使用ADAR1的促进剂实现。
进一步地，所述过表达ADAR1通过抑制NLRP3炎性体及炎症反应，从而抑制非酒精性脂肪肝的形成。
一种ADAR1促进剂在治疗非酒精性脂肪肝疾病药物中的应用，所述ADAR1促进剂能够促进ADAR1过表达。
本发明的技术方案相对于现有技术而言，具有以下有益效果：
本发明经过实验证明：过表达ADAR1可有效抑制NLRP3炎性体及炎症反应，从而明显改善非酒精性脂肪肝的形成。也说明可以促进ADAR1过表达的ADAR1促进剂可以用于治疗非酒精性脂肪肝疾病药物。
附图说明
附图用来提供对本发明的进一步理解，并且构成说明书的一部分，与本发明的实施例一起用于解释本发明，并不构成对本发明的限制。
图1是过表达ADAR1减少高脂小鼠体重增加幅度及肝指数的示意图；
图2是过表达ADAR1改善高脂小鼠NAFLD形成的示意图；
图3是过表达ADAR1抑制高脂喂养小鼠肝脏组织中炎症因子分泌的示意图；
图4是过表达ADAR1增加高脂喂养小鼠肝脏组织中脂质分解相关基因表达的示意图；
图5是过表达ADAR1抑制高脂喂养小鼠肝脏组织中脂质合成相关基因表达的示意图；
图6是过表达ADAR1抑制高脂喂养小鼠肝脏组织中NLRP3炎性体表达的示意图。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效。本发明中未详细说明工作原理属于现有技术和本领域的公知常识，本技术领域的技术人员应当知晓。
一、实验材料与方法
本发明所使用的实验动物为：8-10周龄C57BL/6雄性小鼠，购自上海斯莱克实验动物有限公司；
本发明所使用的HFD饲料购自江苏南通特洛菲饲料科技有限公司(饲料代码：TP23300)，HFD饮食富含脂肪(60％kcal％)。本发明所使用的引物为引物购自由上海生工设计。
本发明将小鼠分为对照组及高脂组，对照组进一步分为Normal组(空白组)和HFD组(仅喂养HFD组)，高脂组又进一步分为LV-ADAR1组(ADAR1过表达组)及LV-NC组(过表达空载体组)，每组6只，高脂组经HFD饮食诱导16周构建NAFLD模型。
实施例1过表达ADAR1减少高脂小鼠体重增加幅度及肝指数
高脂(HFD)饮食模拟现代西方饮食结构，其主要的能量摄入来自脂肪，饲喂HFD饮食的动物可模拟NAFLD患者的主要组织病理学特征。
本发明所使用的实现ADAR1过表达是通过构建ADAR1慢病毒质粒(LV-ADAR1)来实现的。
利用化学合成的人ADAR1基因(NM_001111)CDS区序列，得到的产物按polyA法在末端加A，使用序列如SEQ ID:1和SEQ ID:2所示的引物对克隆入T载体，T4 DNA连接酶与ADAR1连接，转化、摇菌、测序鉴定。脂质体试剂共转染GV287和包装质粒Helper 1.0和Helper 2.0到293T细胞中，48h后收集细胞培养上清，25000rpm离心1.5h，重组病毒颗粒沉淀重悬于100ul PBS中备用，包装好的成熟病毒及阴性对照分别命名为LV-ADAR1-GFP及LV-GFP。为了测定病毒滴度，使用荧光法系列稀释病毒悬液，感染肝细胞(或巨噬细胞)，感染24h后换液，72h后观察荧光表达情况，荧光细胞数随稀释倍数的增加而减少。根据表达GFP荧光蛋白表达情况，进行滴度分析，计算病毒滴度。
采用ADEasyTM系统构建表达人全长ADAR1蛋白的重组慢病毒载体LV-ADAR1，重组慢病毒进行鉴定、扩增后，测定病毒滴度，实验结束前2周使用尾静脉注射方法分别对小鼠尾静脉进行注射LV-ADAR1和LV-NC，注射病毒滴度为1*108TU。
实验期间每周测定体重，处死后测定肝重，并计算肝指数(肝重/体重)，取肝脏组织做进一步分析。
从图1-a中可看出，与高脂组相比，ADAR1过表达组小鼠体重增加幅度明显减少，从图1-b中可看出，ADAR1过表达组小鼠的肝指数(注：肝指数＝肝重/体重)较高脂组也明显下降。图中，*表示与高脂喂养的模型组小鼠相比p<0.05；#表示与高脂喂养的模型组小鼠相比p<0.05。
实施例2过表达ADAR1改善高脂小鼠NAFLD形成
通过HE染色对上述实施例1的各组小鼠肝脏进行NAS评分，取各组小鼠肝脏组织，经过甲醛固定后，使用酒精脱水，二甲苯透明，经石蜡包埋后使用冰冻切片机进行切片，经二甲苯脱蜡后进行HE染色。
HE染色主要包括苏木精染色，氨水返蓝，伊红染色后使用酒精脱水，二甲苯透明，普通光学显微镜观察拍照，以比较差异各组小鼠肝脏组织脂肪变性、炎症反应及气球样变程度。
其中NAS评分依据为3个NAFLD的主要组织病理学特征的半定量评分指标：脂肪变性评分(0-3分)，炎症反应程度评分(0-3分)，气球样变评分(0-2分)。从结果中可见(图2)，过表达ADAR1的高脂喂养小鼠肝脏NAS评分较HFD组明显降低，表现为肝脏脂滴沉积及炎症反应减轻，说明上调ADAR1表达可明显改善高脂喂养所致的NAFLD病理改变。
实施例3过表达ADAR1抑制高脂喂养小鼠肝脏组织中炎症因子分泌
设计炎症因子IL-1β、IL-6、IL-18、TNF-α的引物，使用Trizol regent(Invitrogenlife Technologies,Carlsbad,CA,USA)试剂盒按照操作手册抽提RNA，使用PrimeScriptTMRT Reagent Kit(Takara，Tokyo，Japan)试剂盒逆转录获得cDNA，使用Thermo ScientificSYBR Green qPCR MasterMixes试剂盒配置反应体系，AB公司Fast 7500PCR仪检测RNA的表达，以分析各组中炎症因子IL-1β、IL-6、IL-18、TNFαmRNA的表达差异，通过Real-time PCR检测各组小鼠肝脏组织中炎症因子表达，从结果中可见(图3)，过表达ADAR1小鼠肝脏组织中IL-1β、IL-18、IL-6、TNF-αmRNA水平较高脂组明显下降，说明上调ADAR1的表达可抑制肝脏炎症因子浸润。
上述炎症因子IL-1β、IL-6、IL-18、TNF-α的引物序列如表1所示：
表1
实施例4过表达ADAR1增加高脂喂养小鼠肝脏组织中脂质分解相关基因表达
由于HE染色中发现过表达ADAR1组小鼠肝脏组织脂肪变性程度减轻，因此，本发明进一步探索了ADAR1是否对脂质代谢具有调控作用。首先，本发明设计脂质分解相关基因PPARα、ACADL、ACADM、ACOX1的引物，使用Trizol regent(Invitrogen life Technologies,Carlsbad,CA,USA)试剂盒按照操作手册抽提RNA，使用PrimeScriptTM RT Reagent Kit(Takara，Tokyo，Japan)试剂盒逆转录获得cDNA，使用Thermo Scientific SYBR GreenqPCR MasterMixes试剂盒配置反应体系，AB公司Fast 7500PCR仪检测RNA的表达，以分析各组中脂质分解相关基因表达的差异。通过Real-time PCR检测各组小鼠肝脏组织中脂质分解相关基因的表达，结果发现过表达ADAR1可使高脂喂养小鼠肝脏组织中脂质分解相关基因(PPARα、ACADL、ACOX1、ACADM)表达升高，使脂质分解增加(图4)，说明ADAR1可通过增加脂质分解的作用减轻肝脏脂肪变性。
上述脂质分解相关基因PPARα、ACADL、ACADM、ACOX1的引物的引物序列如表2所示：
表2
实施例5过表达ADAR1抑制高脂喂养小鼠肝脏组织中脂质合成相关基因表达
本发明设计脂质合成相关基因PPARγ、SCD1、SREBP-1c、FASN的引物，使用Trizolregent(Invitrogen life Technologies,Carlsbad,CA,USA)试剂盒按照操作手册抽提RNA，使用PrimeScriptTM RT Reagent Kit(Takara，Tokyo，Japan)试剂盒逆转录获得cDNA，使用Thermo Scientific SYBR Green qPCR MasterMixes试剂盒配置反应体系，AB公司Fast 7500PCR仪检测RNA的表达，以分析各组中脂质合成相关基因表达的差异。
通过Real-time PCR检测各组小鼠肝脏组织中脂质合成相关基因的表达，结果发现过表达ADAR1可使高脂喂养小鼠肝脏组织中脂质合成相关基因表达下降，从而使脂质合成减少(如图5所示)。
上述脂质合成相关基因PPARγ、SCD1、SREBP-1c、FASN的引物序列如表3所示：
表3
实施例6过表达ADAR1抑制高脂喂养小鼠肝脏组织中NLRP3炎性体表达
NLRP3炎性体是存在于细胞质中可感受危险信号的感受器，相关研究证明NLRP3炎性体在胰岛素抵抗、2型糖尿病、动脉粥样硬化、痛风以及代谢综合征等慢性炎症性疾病中发挥着举足轻重的作用，在NAFLD的发生发展中也起到关键的促进作用。
本发明前期的动物实验显示：高脂饮食可使小鼠肝脏中NLRP3炎性体激活并导致下游促炎性细胞因子IL-1β和IL-18的表达增加，导致肝脏脂肪变性及炎症反应发生，NLRP3炎性体可能是能量代谢异常致炎症反应的调节枢纽，抑制NLRP3炎性体的活性有望阻止NAFLD的进展。研究显示过表达ADAR1可使肝脏组织中的炎症因子IL-1β和IL-18表达下降，而IL-1β和IL-18的表达受到NLRP3炎性体调控，为了更加清楚的理解ADAR1是否是通过抑制NLRP3炎性体而发挥改善NAFLD的作用。
本发明使用Western-blot方法，用蛋白裂解液提取各组肝脏组织全蛋白，经SDS凝胶电泳后进行湿法转膜，孵育NLRP3的相应抗体，漂洗，与对应的二抗孵育后漂洗，采用ECL试剂盒显色拍照，以检测各组中NLRP3蛋白的表达；本发明所使用的二抗为山羊抗兔IgG二抗，购于CST公司，货号：15101。
设计NLRP3的引物，使用Trizol regent(Invitrogen life Technologies,Carlsbad,CA,USA)试剂盒按照操作手册抽提肝脏组织RNA，使用PrimeScriptTM RTReagent Kit(Takara，Tokyo，Japan)试剂盒逆转录获得cDNA，使用Thermo ScientificSYBR Green qPCR MasterMixes试剂盒配置反应体系，AB公司Fast 7500PCR仪检测RNA的表达，以分析各组中NLRP3mRNA表达的差异。结果发现，过表达ADAR1可使高脂喂养小鼠肝脏组织中NLRP3的mRNA和蛋白的表达均下调(图6)。说明ADAR1可作用于NLRP3炎性体，通过抑制NLRP3炎性体并抑制炎症反应，从而达到改善NAFLD的有益作用。
上述NLRP3的引物的序列为：
Forward primer：TGAAGAAGAGTGGATGGGTTTG Reverse primer：CTCACATGTCGTCTGTACATCTT。
序列表
<110> 中南大学
<120> ADAR1在用于治疗非酒精性脂肪肝疾病药物中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 107
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atntnvrsna ggtcgactct agaggatccc gccaccatga atccgcggca ggggtattcc 60
ctctccttgt agtccatacc tactgggcag agataaaagt tcttttc 107
Claims (4)
1.ADAR1在治疗非酒精性脂肪肝疾病药物中的应用。
2.如权利要求1所述的ADAR1在治疗非酒精性脂肪肝疾病药物中的应用，其特征在于，所述ADAR1是指过表达ADAR1。
3.如权利要求2所述的ADAR1在治疗非酒精性脂肪肝疾病药物中的应用，其特征在于，所述过表达ADAR1通过抑制NLRP3炎性体及炎症反应，从而抑制非酒精性脂肪肝的形成。
4.ADAR1促进剂在治疗非酒精性脂肪肝疾病药物中的应用，其特征在于，所述ADAR1促进剂能够促进ADAR1过表达。
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Non-Patent Citations (3)
| Title |
|---|
| GUOLIANG WANG等: "ADAR1 Prevents Liver Injury from Inflammation and Suppresses Interferon Production in Hepatocytes", 《THE AMERICAN JOURNAL OF PATHOLOGY》 * |
| 吴林容: ""腺苷脱氨酶1在非酒精性脂肪性肝病肝细胞线粒体损伤中的保护作用"", 《万方数据知识服务平台》 * |
| 陈柯竹等: "RNA特异性腺苷脱氨酶的生物学作用及其与人类疾病的关系", 《中南大学学报（医学版）》 * |
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