CN115105595A - Nat10基因在治疗脓毒症中的应用 - Google Patents
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Abstract
本发明公开了NAT10基因在治疗脓毒症中的应用。本发明首先构建了脓毒症小鼠模型，通过检测发现脓毒症小鼠血液、脾脏和BALF中NAT10的表达水平显著降低；随后通过构建NAT10过表达小鼠，特异性过表达NAT10的脓毒症小鼠相比对照组小鼠提高了脓毒症小鼠的存活率并改善了肺损伤。NAT10过表达可以通过ac4C修饰稳定下游基因，I型IFN和包括IL‑6和TNF‑α的细胞因子的分泌减少。显示NAT10过表达可以通过ac4C修饰稳定mRNA的抑制细胞因子产生，减轻炎症反应，改善脓毒症预后。本发明探索了NAT10基因的新功能并为脓毒症的治疗提供了新的靶点和治疗策略。
Description
技术领域
本发明涉及NAT10基因在治疗脓毒症中的应用，属于生物医药领域。
背景技术
脓毒症是机体对感染反应失调导致的危及生命的器官功能障碍，与炎症因子风暴、脏器损伤等表现相关。根据2017年的数据，每年全球脓毒症患者为4890 万例。脓毒症由于发展隐秘、发病速度快、病程严重等特点，大部分患者在诊断脓毒症后进入重症监护病房(intensive care units，ICU)接受治疗，占ICU入院患者的10％到20％，给患者带来了沉重的经济负担。尽管在使用抗生素治疗、液体复苏、脏器功能支持等多种治疗手段，脓毒症死亡率依旧居高不下，2017 年存在1100万例脓毒症相关死亡病例，是全球疾病死亡的主要原因之一(Bourke Tillmann,Hannah Wunsch.Epidemiology and Outcomes.Crit CareClin.2018 Jan；34(1):15-27.)。
脓毒症常常发生在感染后，脓毒症患者常常表现出过度炎症和免疫抑制的迹象。在脓毒症早期，病理过程与先天免疫系统的强烈激活有关，促炎细胞因子包括肿瘤坏死因子、白细胞介素和干扰素的大量释放导致的炎症因子风暴将导致大量免疫细胞活化，并激活补体系统、凝血系统和血管内皮，进而导致血栓生成和器官功能损伤，这是诱发脓毒症死亡的最大原因。阻断或消除这些细胞因子可为暴发性感染的脓毒症患者提供保护(Tom vander Poll,Frank L van de Veerdonk, Brendon P Scicluna et al.Theimmunopathology of sepsis and potential therapeutic targets.Nat RevImmunol.2017Jul；17(7):407-420.)。随着疾病的进展，通过治疗免疫系统将会发生抗炎改变以恢复免疫稳态，部分患者适应性和先天免疫细胞将会出现免疫重编程，进入慢性危重病状态，这些患者经历持续的免疫抑制和炎症，这与持续的急性期反应和细胞蛋白分解代谢有关，称为持续炎症-免疫抑制-分解代谢综合征(Persistent Inflammation,Immunosuppression and Catabolism Syndrome,PICS)。脓毒症的发病机制十分复杂，准确了解发病过程，调节炎症和免疫，将是未来脓毒症治疗的新方向。
目前针对脓毒症治疗的常规手段是抗感染、器官支持等对症治疗技术。免疫制剂能够靶向脓毒症炎症反应的本质，快速精准的控制脓毒症进程。如何治疗脓毒症患者的宿主反应是有争议的。许多抗炎药物治疗的临床试验强烈表明，单纯使用抗炎因子的免疫治疗抑制过度炎症反应，可能会导致不可预知的后果。脓毒症期间免疫细胞基因转录的表观遗传过程紊乱的发现，开启了针对这些过程的新疗法用于平衡免疫功能的可能性。
RNA修饰由专门的、高度保守的酶机制催化，其机制的破坏可导致一系列疾病。近年来基因组研究方法的进展促进了对mRNA分子的各种修饰的研究。 N4-乙酰胞苷(ac4C)是mRNA中广泛的化学修饰之一，在调节mRNA稳定性和 mRNA翻译过程中发挥着关键作用。ac4C的产生都是由N-乙酰转移酶10(NAT10) 酶或其同源物催化的，NAT10是唯一同时具有乙酰转移酶和RNA结合活性的人类酶。NAT10目前已被发现与许多癌症的发生有关，其调节mRNA乙酰化的功能也让mRNA的转录形式变得独特而丰富。mRNA半衰期分析显示，在乙酰化 mRNA队列中，NAT10依赖将促进mRNA的稳定性增加,在体内外增强底物翻译 (Daniel Arango,David Sturgill,Najwa Alhusaini et al.Acetylation of Cytidine in mRNA PromotesTranslation Efficiency.Cell.2018Dec 13；175(7):1872-1886.e24.)。
目前对NAT10功能的相关研究较少，主要集中与NAT10与癌症诊断与治疗的关系，例如NAT10能促进胃癌转移导致不良预后，小分子抑制剂靶向NAT10 可能是未来治疗胃癌的一种潜在策略(Yigan Zhang,Yuanxue Jing,Yinxue Wang et al.NAT10 promotesgastric cancer metastasis via N4-acetylated COL5A1.Signal Transduct TargetTher.2021May 3；6(1):173.)靶向NAT10使乳腺癌细胞对DNA 损伤的化疗和放疗敏感(Hong-Yi Liu,Ying-Ying Liu,Fan Yang et al.Acetylation of MORC2 by NAT10regulates cell-cycle checkpoint control and resistance to DNA-damagingchemotherapy and radiotherapy in breast cancer.Nucleic Acids Res. 2020Apr 17；48(7):3638-3656.)。NAT10的缺失而导致的RNA ac4C修饰的缺失通过降低病毒RNA的稳定性，抑制了HIV-1的复制，NAT10成为抗病毒药物开发的潜在靶点(Kevin Tsai,AnandaAyyappan Jaguva Vasudevan,Cecilia Martinez Campos et al.Acetylation ofCytidine Residues Boosts HIV-1Gene Expression by Increasing Viral RNAStability.Cell Host Microbe.2020Aug 12；28(2):306-312.e6.)。而目前针对脓毒症中的ac4C修饰以及NAT10的作用尚无研究。
发明内容
本发明的目的在于针对现有治疗脓毒症手段的局限，提供NAT10在治疗脓毒症中的应用。
为了达到上述目的，本发明提供了促进或激活NAT10基因表达的物质在制备治疗脓毒症的药物或改善脓毒症预后的药物中的应用。
优选地，所述促进或激活NAT10基因表达的物质包括NAT10重组蛋白，能够特异性增加NAT10基因表达的核酸分子(如siRNA、miRNA、lncRNA、gRNA 和反义核苷酸等)，包含所述核酸分子的重组载体或重组菌。
优选地，所述脓毒症包括脓毒症并发持续炎症-免疫抑制-分解代谢综合征(PICS)、脓毒症相关炎症反应和脓毒症相关脏器损伤以及其他脓毒症相关疾病 (包括脓毒症相关肺炎、脑损伤等脏器功能损害)。
本发明提供了一种用于治疗脓毒症或改善脓毒症预后的药物，所述的药物包括药学上可接受的载体和有效量的活性成分，所述活性成分包括促进或激活 NAT10基因表达的物质。
优选地，所述促进或激活NAT10基因表达的物质包括NAT10重组蛋白，能够特异性增加NAT10基因表达的核酸分子(如siRNA、miRNA、lncRNA、gRNA 和反义核苷酸等)，包含所述核酸分子的重组载体或重组菌。
与现有技术相比，本发明的有益效果在于：
(1)本发明通过动物实验表明NAT10过表达在脓毒症小鼠模型中表现出优良的抗炎症因子生成特性，且能有效降低脓毒症小鼠的死亡率并改善了肺损伤，因此，NAT10基因可作为脓毒症的治疗靶点，改善脓毒症预后；
(2)本发明开拓了NAT10基因功能的新发现，通过促进和激活NAT10功能的方式对免疫的影响方面较小，副作用较少，且针对性和特异性高，应用前景较广；本发明探索了NAT10基因的新功能并为脓毒症的治疗提供了新的靶点和治疗策略。
附图说明
图1为脓毒症小鼠中NAT10表达受到抑制；A:脓毒症小鼠生存曲线，B:脓毒症小鼠NAT10在血液、脾脏和肺泡灌洗液中NAT10 mRNA表达；Sham:假手术组；CLP:脓毒症造模组；*：p<0.05，**：p<0.01；
图2为NAT10过表达脓毒症小鼠死亡率明显降低；A:过表达NAT10小鼠脓毒症造模前后NAT10 mRNA表达；B:过表达NAT10小鼠脓毒症与对照组脓毒症小鼠生存曲线；
图3为NAT10过表达抑制脓毒症小鼠炎症因子生成；A:过表达NAT10小鼠与对照组小鼠脓毒症造模前后血液、脾脏和肺泡灌洗液中IL-1β表达；B:过表达 NAT10小鼠与对照组小鼠脓毒症造模前后血液、脾脏和肺泡灌洗液中IL-18表达； C:过表达NAT10小鼠与对照组小鼠脓毒症造模前后血浆中干扰素、肿瘤坏死因子、白细胞介素表达；
图4为NAT10过表达显著改善脓毒症小鼠肺损伤；A：假手术组和脓毒症小鼠H&E染色与肺损伤评分；B：过表达NAT10小鼠与对照组小鼠脓毒症造模前后肺H&E染色与肺损伤评分；
以上各图中，*表示经过统计学分析，两组间具有显著性差异，P<0.05；** 表示经过统计学分析，两组间具有显著性差异，P<0.01；***表示经过统计学分析，两组间具有显著性差异，P<0.001。
具体实施方式
为使本发明更明显易懂，兹以优选实施例，并配合附图作详细说明如下。
本发明提供N-乙酰转移酶NAT10在治疗脓毒症中的应用。所述的脓毒症包括脓毒症并发持续炎症-免疫抑制-分解代谢综合征(PICS)以及其他脓毒症相关疾病(包括脓毒症相关肺炎等脏器功能损害)。
本发明发现NAT10敲除小鼠呈现细胞因子释放减少的表型。特异性过表达NAT10的脓毒症小鼠相比对照组小鼠提高了脓毒症小鼠的存活率并改善了肺损伤。NAT10过表达可以通过ac4C修饰稳定下游基因，I型IFN和包括IL-6和TNF-α的细胞因子的分泌减少。显示NAT10过表达可以通过ac4C修饰稳定mRNA的抑制细胞因子产生，减轻炎症反应，改善脓毒症预后。
实施例
本实施例提供了NAT10基因在治疗脓毒症炎症反应中的应用：
一、实验方法
1.脓毒症模型构建：
通过盲肠结扎和穿刺(CLP)建立脓毒症模型。小鼠麻醉后，在腹中线切开一个1厘米长的腹部切口，结扎后33％的盲肠，用25号针头穿刺两次。将盲肠放回腹腔，腹部缝合。假手术小鼠在没有CLP手术的情况下接受了相同的开腹方案。
2.PCR检测：
小鼠外周血和脾脏，裂解红细胞，洗涤后备用。小鼠肺泡灌洗液离心后取沉淀细胞。分别使用RNA抽提试剂盒进行抽提。逆转录后通过qRT-PCR检测mRNA 含量。
3.Western Blots检测：
小鼠外周血和脾脏，裂解红细胞，洗涤后备用。小鼠肺泡灌洗液离心后取沉淀细胞。使用RIPA和蛋白酶抑制剂抽提蛋白。通过电泳、转膜、一抗和二抗孵育、条带曝光等步骤检测蛋白质表达情况。
4.ELISA检测：
将提取到的小鼠血液常温放置，离心后取血浆。将血浆稀释后，通过包被、加样、加酶标抗体、底物显色等步骤检测血浆中细胞因子含量。
5.H&E染色与肺损伤评分
小鼠肺组织用PBS洗涤后，用4％多聚甲醛固定，并包埋在石蜡中。石蜡包埋的肺组织切片用H&E染色，并进行组织病理学检查。在光学显微镜下分析染色切片。根据肺水肿、肺泡出血、肺间质增厚和中性粒细胞浸润评分的总和计算组织学损伤评分。以0到3的等级评估每个组织学特征：0、正常(无损伤)； 1、极少(视野中损伤低于25％)；2、轻度(视野中损伤在25％到50％之间)；3、中度(视野中损伤在50％到75％之间)；4、重度(视野中损伤75％以上)。
6.构建NAT10过表达小鼠：
在小鼠6号染色体Rosa26位点插入NAT10基因，利用Cre-LoxP系统对目的基因进行可诱导表达调控。构建Rosa26-loxp-Stop-loxp-NAT10重组载体，在鉴定序列正确与载体构建成功后，将条件性过表达结构插入到Rosa26基因 intron1中，并注射到小鼠受精卵胞质。对载体阳性受精卵进行筛选，并将微注射后存活的受精卵移植到同步交配的假孕母鼠的输卵管内，其中一部分移植卵能够继续生长发育成个体。子代鼠出生后，鉴定子代鼠中NAT10的整合和表达。将鉴定后的子代雌雄鼠进行交配，鉴定F2代小鼠NAT10的表达情况，以确定Rosa26-loxP-stop-loxP-NAT10转基因小鼠品系的建立。所有动物均在12小时明暗循环和特定无病原体条件下饲养。
二、实验结果
1.脓毒症小鼠中NAT10表达受到抑制
通过对脓毒症模型小鼠进行生存分析，发现脓毒症模型小鼠八天存活率为 40％，如图1A所示。通过qRT-PCR检测脓毒症小鼠血液、脾脏和BALF(支气管肺泡灌洗液)中的NAT10的表达水平，结果显示脓毒症小鼠血液、脾脏和 BALF中NAT10的表达水平显著降低，如图1B所示。
2.NAT10过表达脓毒症小鼠死亡率明显降低
通过构建NAT10过表达小鼠，通过qRT-PCR检测脓毒症小鼠NAT10的 mRNA含量发现，在血液、脾脏和肺泡灌洗液中NAT10 mRNA上调，如图2A 所示。通过对NAT10过表达小鼠与对照组小鼠脓毒症模型进行生存分析，发现 NAT10过表达能降低脓毒症小鼠死亡率，如图2B所示，图2B中，假手术对照组和假手术过表达NAT10组曲线重合。
3.NAT10过表达抑制脓毒症小鼠炎症因子生成
通过Western Blots检测过表达NAT10小鼠与对照组小鼠脓毒症造模前后血液、脾脏和肺泡灌洗液中IL-1β和IL-18的表达水平，结果均显示下调，如图3A、 3B所示。通过ELISA检测过表达NAT10脓毒症小鼠与对照组小鼠中血浆中干扰素、肿瘤坏死因子、白细胞介素表达水平，结果同样显示下调，如图3C所示。
4.NAT10过表达显著改善脓毒症小鼠肺损伤
通过H&E染色与肺损伤评分发现，CLP小鼠相比Sham组小鼠出现明显肺损伤，如图4A所示。过表达NAT10小鼠与对照组小鼠脓毒症造模前后肺H&E 染色与肺损伤评分显示，NAT10小鼠的肺损伤明显减轻，如图4B显示。
本发明首次正面证实NAT10对脓毒症小鼠炎症反应存在影响。NAT10可以通过其ac4C修饰功能稳定底物mRNA影响细胞因子的产生。
本发明从炎症因子角度阐述了炎症对脓毒症的影响，过表达NAT10能够抑制细胞因子生成，减轻炎症反应，同时明显降低了脓毒症小鼠的死亡率。
本发明的实验结果表明NAT10过表达在脓毒症小鼠模型中表现出优良的抗炎症因子生成特性，且能有效降低了脓毒症小鼠的死亡率与肺损伤程度，因此， NAT10基因可作为脓毒症的治疗靶点，改善脓毒症预后，在临床上具有良好的应用前景。
上述实施例仅为本发明的优选实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (5)
1.促进或激活NAT10基因表达的物质在制备治疗脓毒症的药物或改善脓毒症预后的药物中的应用。
2.如权利要求1所述的应用，其特征在于，所述促进或激活NAT10基因表达的物质包括NAT10重组蛋白，能够特异性增加NAT10基因表达的核酸分子，包含所述核酸分子的重组载体或重组菌。
3.如权利要求1所述的应用，其特征在于，所述脓毒症包括脓毒症并发持续炎症-免疫抑制-分解代谢综合征、脓毒症相关炎症反应和脓毒症相关脏器损伤。
4.一种用于治疗脓毒症或改善脓毒症预后的药物，其特征在于，所述的药物包括药学上可接受的载体和有效量的活性成分，所述活性成分包括促进或激活NAT10基因表达的物质。
5.如权利要求4所述的用于治疗脓毒症或改善脓毒症预后的药物，其特征在于，所述促进或激活NAT10基因表达的物质包括NAT10重组蛋白，能够特异性增加NAT10基因表达的核酸分子，包含所述核酸分子的重组载体或重组菌。
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