CN116889631A - 促进或激活Skp2基因表达的试剂在制备治疗脓毒症或改善脓毒症预后的药物中的应用 - Google Patents
促进或激活Skp2基因表达的试剂在制备治疗脓毒症或改善脓毒症预后的药物中的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域，具体涉及一种促进或激活Skp2基因表达的试剂在制备治疗脓毒症或改善脓毒症预后的药物中的应用。本发明通过动物实验表明Skp2过表达在脓毒症小鼠模型中能有效降低脓毒症小鼠的死亡率与减轻肺损伤，Skp2基因可作为脓毒症的治疗靶点，改善脓毒症预后。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种促进或激活Skp2基因表达的试剂在制备治疗脓毒症或改善脓毒症预后的药物中的应用。
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(Singer M,Deutschman C S,Seymour C W,et al.The Third International ConsensusDefinitions for Sepsis and Septic Shock(Sepsis-3)[J].JAMA,2016,315(8):801.)。截至2017年，全球报告了4890万例脓毒症患者，脓毒症的发病率约为677.5例/10万人，脓毒症相关死亡人数为1100万例，占全球死亡总人数的19.7％(Rudd K E,Johnson S C,AgesaK M,et al.Global,regional,and national sepsis incidence and mortality,1990-2017:analysis for the Global Burden of Disease Study[J].Lancet,2020,395(10219):200-211.)。脓毒症的发病和进展具有多样性和异质性，同时具有发展隐秘、发病速度快、病程严重等特点。近年来伴随糖尿病、肾衰竭、癌症等慢性病的脓毒症共病患者数量的增加，临床上对脓毒症的及时诊断和治疗存在较大难点。尽管脓毒症病理生理学研究逐步深入，脓毒症患者的标准治疗大部分停留在对症治疗，主要包括广谱抗生素和液体复苏(Evans L,Rhodes A,Alhazzani W,et al.Surviving sepsis campaign:internationalguidelines for management of sepsis and septic shock 2021[J].Intensive CareMedicine,2021,47(11):1181-1247.)，同时早期目标导向治疗(Early Goal DirectedTherapy，EGDT)并未显示出显著效果(Saleh AS.Early,Goal-Directed Therapy forSeptic Shock—A Patient-Level Meta-Analysis[J].New England Journal ofMedicine,2017,377(10):994.)。脓毒症的结局与病原学检查中的病原体结果并非总是相关。COVID-19危重患者的死亡原因主要为呼吸功能衰竭，其共病因素、病理生理学改变(如炎症因子风暴)以及诊断标志物(如降钙素原、白细胞介素水平、白细胞和淋巴细胞数量)等与脓毒症高度相似(Clark I A.Background to new treatments for COVID-19,including its chronicity,through altering elements of the cytokine storm[J].Reviews in Medical Virology,2021,31(5):1-13.)。脓毒症的结局并不总是与病原学检查中的病原体结果相关，病原体感染(包括SARS-CoV-2)仅仅是脓毒症全身反应的导火索。机体免疫系统的激活和炎性细胞因子的产生对于抗感染免疫反应至关重要。免疫系统过度激活会引起促炎细胞因子的循环水平剧增，从而诱发“细胞因子风暴”，进而导致全身炎症、高铁蛋白血症、血流动力学不稳定和多器官衰竭(Karki R,Sharma B R,Tuladhar S,etal.Synergism of TNF-αand IFN-γTriggers Inflammatory Cell Death,TissueDamage,and Mortality in SARS-CoV-2Infection and Cytokine Shock Syndromes[J].Cell,2021,184(1):149-168.)。
炎症反应能利用泛素化来调节蛋白质的活性和稳定性，介导信号复合体的精确的空间和时间激活(Cockram P E,Kist M,Prakash S,et al.Ubiquitination in theregulation of inflammatory cell death and cancer[J].Cell Death&Differentiation,2021,28(2):591-605.)。泛素是一种由76个氨基酸组成的蛋白质，在所有类型的细胞中普遍表达。泛素分子上的赖氨酸残基或甲硫氨酸残基可以作为受体与其他泛素结合，从而导致多聚泛素化的不同连接发挥不同的细胞功能，例如标记底物蛋白水解或调节蛋白质-蛋白质相互作用、蛋白质活性或细胞内蛋白质运输(Gao P,Wu B,Ding Y,etal.circEXOC5 promotes acute lung injury through the PTBP1/Skp2/Runx2axis toactivate autophagy[J].Life Science Alliance,2022,6(1):e202201468.)。泛素系统中有大量调节炎症和细胞死亡的靶点。泛素系统的异常功能会导致炎症途径的异常激活和转录因子的持续抑制，这与脓毒症的机制密切相关。泛素化能够通过参与关键分子的修饰与功能改变，介导炎症信号传导的蛋白质降解引起炎症以及调控信号的转录以介入炎症过程。
泛素系统的激活需要通过泛素激活酶(E1)、泛素结合酶(E2)和泛素蛋白连接酶(E3)酶连激活。E3连接酶的高度可变性在决定底物特异性方面起着关键作用，因此E3连接酶也是泛素化系统中最关键的成分(Pao K,Wood N T,Knebel A,et al.Activity-basedE3 ligase profiling uncovers an E3 ligase with esterification activity[J].Nature,2018,556(7701):381-385.)。Cullin-RING家族是数量最多的E3泛素连接酶组分，以常见的Cullin支架蛋白为特征，F-box蛋白是Skp1/Cullin 1/F-box(SCF)复合体中负责底物识别和连接到SCF复合体其他成分的关键蛋白，Skp2作为SCF复合体中的一员，主要通过其E3连接酶活性发挥其功能。目前Skp2的功能主要集中在细胞周期、衰老、细胞干性与耐药等方面(Cai Z,Moten A,Peng D,et al.The Skp2 Pathway:A Critical Target forCancer Therapy[J].Seminars in Cancer Biology,2020,67:16-33.)，而与炎症相关的研究较少。研究显示，AMPK-mTOR-Skp2介导的自噬调节与乙醇诱导的心肌功能障碍、炎症和细胞凋亡有关(Yang L,Wang S,Ma J,et al.CD74 knockout attenuates alcohol intake-induced cardiac dysfunction through AMPK-Skp2-mediated regulation ofautophagy[J].Biochimica et Biophysica Acta(BBA)-Molecular Basis of Disease,2019,1865(9):2368-2378.)，LPS诱导的心肌功能损伤中，自噬介导的Skp2被显著抑制(LuoY,Fan C,Yang M,et al.CD74 knockout protects against LPS-induced myocardialcontractiledysfunction throughAMPK-Skp2-SUV39H1-mediated demethylation ofBCLB[J].British Journal of Pharmacology,2020,177(8):1881-1897.)。因此，Skp2可能与多个炎症过程和死亡形式相关，可能是调控脓毒症炎症的重要靶点。
许多针对抗炎药物治疗的临床试验强有力地显示，仅依靠抗炎因子进行免疫治疗以抑炎症反应，可能会导致不可预测的结果。由于泛素化分子在不同阶段参与免疫激活、炎症细胞因子的转录、细胞分化和其他功能，因此必须在多个水平上严格控制和调节泛素化，以限制炎症细胞因子风暴的爆发。脓毒症中观察到的泛素化系统失调也表明，强调泛素化在炎症反应中的作用可能是未来控制脓毒症的一种新的治疗方法。
目前对Skp2参与脓毒症的相关研究较少，主要集中与Skp2与癌症和其他代谢性疾病诊疗。如Skp2通过Kindlin-2参与非酒精性脂肪性肝病的调控(Huanqing Ga,LiangZhou,Yiming Zhong et al.Kindlin-2haploinsufficiency protects against fattyliver by targeting Foxo1 in mice[J].Nat Commun,2022,3；13(1):1025.),恶性胸膜间皮瘤中Skp2能够驱动对类化抑制剂和顺铂的敏感性，并可以作一种新的恶性胸膜间皮瘤分层标记物(Iris Chiara Salaroglio,Dimas Carolina Belisari,Paolo Bironzo etal.SKP2 drives the sensitivity to neddylation inhibitors and cisplatin inmalignant pleural mesothelioma[J].J Exp Clin Cancer Res,2022,23；41(1):75.),Skp2作为致癌基因能够参与卡波西肉瘤的发展(Soo Mi Le,Kenneth M Kay,Frank JSlack.Cellular microRNA-127-3p suppresses oncogenic herpesvirus-inducedtransformation and tumorigenesis via down-regulation of SKP2[J].Proc NatlAcad Sci U S A,2021,118(45):e2105428118.),Skp2能在体外和体内诱导癌细胞的增殖和迁移从而参与肝癌发展(Jun-Jie H,Cui Zhou,Xin Luo,et al.Linc-SCRG1accelerates progression of hepatocellular carcinoma as a ceRNA of miR26a toderepress SKP2[J].J Exp Clin Cancer Res,2021,40(1):26.)而目前针对脓毒症中的Skp2调控炎症和治疗的作用尚无研究。
发明内容
鉴于此，本发明的目的在于提供一种促进或激活Skp2基因表达的试剂在制备治疗脓毒症或改善脓毒症预后的药物中的应用，以解决现有治疗脓毒症手段的局限性。
为实现上述目的，本发明所采取的解决方案如下：
第一个方面，本发明提供一种促进或激活Skp2基因表达的试剂在制备治疗脓毒症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Skp2基因表达的试剂包括：
(1)Skp2的mRNA和重组蛋白，和/或
(2)能够特异性增加Skp2基因表达的siRNA、miRNA、IncRNA、gRNA和反义核苷酸中的任意一种；
以及用于递送上述(1)和/或(2)物质的体内或体外递送载体。
优选地，所述药学上可接受的体内或体外递送载体为脂质纳米颗粒。
优选地，所述脓毒症包括脓毒症发展的急性期、免疫平衡期或免疫抑制期，还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。
第二个方面，本发明还提供一种用于治疗脓毒症的药物组合物，所述药物组合物包括药学上可接受的载体和有效量的活性成分，所述活性成分包括如上所述的促进或激活Skp2基因表达的试剂。
优选地，所述促进或激活Skp2基因表达的试剂包括：
(1)Skp2的mRNA和重组蛋白，和/或
(2)能够特异性增加Skp2基因表达的siRNA、miRNA、IncRNA、gRNA和反义核苷酸中的任意一种；
以及用于递送上述(1)和/或(2)物质的体内或体外递送载体。
优选地，所述药学上可接受的体内或体外递送载体为脂质纳米颗粒。
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Skp2过表达在脓毒症小鼠模型中能有效降低脓毒症小鼠的死亡率与减轻肺损伤，因此，Skp2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Skp2基因功能的新发现，通过促进和激活Skp2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Skp2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为脓毒症小鼠肺中Skp2表达受到抑制；图中，A.脓毒症小鼠肺中Skp2表达；Sham:假手术组；CLP:脓毒症造模组。
图2为炎症因子抑制肺上皮中Skp2表达；图中，A.体外模拟脓毒症导致的炎症因子风暴造成肺上皮损伤，体外实验分为四组：对照组(Con.)、LPS组(LPS)、LPS刺激的条件培养基RCM组(RCM)、RCM与LPS共刺激组(RCM+LPS)，Western blot检测Skp2蛋白在各组小鼠肺上皮细胞中的表达量。
图3为Skp2缺失导致脓毒症小鼠死亡率明显增加和肺损伤增加；图中，A.Skp2+/-小鼠脓毒症与对照组脓毒症小鼠生存曲线；B.Skp2+/-小鼠脓毒症与对照组脓毒症小鼠肺部HE染色。
图4为Skp2 LNP递送显著改善脓毒症小鼠肺损伤；图中，A.Skp2在肺中的表达效率；B.Skp2-OE小鼠脓毒症与对照组脓毒症小鼠生存曲线；C.Skp2-OE小鼠脓毒症与对照组脓毒症小鼠H&E染色。
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
本发明提供一种促进或激活Skp2基因表达的试剂在制备治疗脓毒症或改善脓毒症预后的药物中的应用，所述脓毒症包括脓毒症发展的急性期、免疫平衡期或免疫抑制期，还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。
在本发明中，所述促进或激活Skp2基因表达的试剂包括：
(1)Skp2的mRNA和重组蛋白，和/或
(2)能够特异性增加Skp2基因表达的siRNA、miRNA、IncRNA、gRNA和反义核苷酸中的任意一种；
以及用于递送上述(1)和/或(2)物质的体内或体外递送载体。
作为本发明的一种具体实施方式，本发明的体内或体外递送载体可以为脂质纳米颗粒。本发明所述的“脂质纳米颗粒”指具有至少一个纳米量级尺寸的颗粒(如1-1000nm)。脂质纳米颗粒可被包含在药物组合物中，用于将活性剂或治疗剂(在本发明中为促进或激活Skp2基因表达的试剂)递送至目标靶部位(如细胞、组织(如肿瘤组织等患病组织)、器官)。在一些实施方案中，本发明的脂质纳米颗粒包含核酸。此类脂质纳米颗粒通常包含一种或多种辅助脂质分子、一种或多种胆固醇或胆固醇衍生物和/或一种或多种聚合物缀合的脂质分子。所述辅助脂质分子可以是一种或多种中性脂质分子。活性剂或治疗剂(在本发明中为促进或激活Skp2基因表达的试剂)被封装在脂质纳米颗粒的脂质部分中或者由脂质纳米颗粒的一些或所有脂质部分包封的水性空间中，从而保护其免于酶促降解，或不会产生由宿主有机体或细胞的机制诱导的其它不期望的作用，如不良免疫应答。
本领域周知，脂质纳米颗粒的平均直径可为约30nm至约40nm至约150nm、约50nm至约150nm、约60nm至约130nm、约70nm至约110nm、约70nm至约100nm、约80nm至约100nm、约90nm至约100nm、约70nm至约90nm、约80nm至约90nm、约70nm至约80nm、或约30nm、约35nm、约40nm、约45nm、约50nm、约55nm、约60nm、约65nm、约70nm、约75nm、约80nm、约85nm、约90nm、约95nm、约100nm、约105nm、约110nm、约115nm、约120nm、约125nm、约130nm、约135nm、约140nm、约145nm或约150nm，并且脂质纳米颗粒基本上是无毒的。
本发明所述的“基因”指核酸(如DNA或RNA)序列，其包含产生多肽或前体多肽所必需的部分长度或整个长度的编码序列。
本发明发现Skp2敲除小鼠呈现脓毒症死亡率增加的表型。特异性过表达Skp2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。Skp2过表达可以抵抗脓毒症炎症因子损伤。显示Skp2过表达可以通过减轻炎症反应，改善脓毒症预后。
因此，促进或激活Skp2基因表达的试剂可以作为活性成分的组分用于制备治疗脓毒症或改善脓毒症预后的药物组合物。
一般来说，本发明的药物组合物可以在有效量下，通过任何可接受的用于其他类似用途的给药方式进行给药。举例来说，本发明的药物组合物可以口服、非肠道给药、透皮给药、局部给药、直肠给药或鼻内给药。
当用作药物时，本发明通常以药物组合物的形式给药。这些组合物可用制药学领域熟知的方法进行制备，这些组合物包含至少一种活性化合物，在本发明中，该活性化合物为如上所述的促进或激活Skp2基因表达的试剂。在配制本发明提供的组合物时，有效成分通常与医学上可接受的辅料或载体混合，被医学上可接受的辅料或载体稀释或被以胶囊，小袋，纸或其他形式的容器包裹。当所述医学上可接受的辅料或载体作为稀释剂时，可以是固体，半固体，或液体物质，可作为有效成分的运载体、载体或媒介。由此，所述组合物可以是药片、药丸、粉末、锭剂、小袋、胶囊、酏剂、混悬剂、乳剂、溶液、糖浆、喷雾(作为固体或在液体介质里)，软膏，软和硬的明胶胶囊，栓剂，无菌注射溶液，和无菌包装粉末的形式。
一些典型的医学上可接受的辅料或载体包括乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、淀粉、阿拉伯树胶、磷酸钙、海藻酸盐、黄芪胶、明胶、硅酸钙、微晶纤维素、聚乙烯吡咯烷酮、纤维素、无菌水、糖浆和甲基纤维素。另外还可以包括润滑剂(如滑石粉、硬脂酸镁和矿物油)、浸润剂、乳化和悬浊剂、防腐剂(如对羟基苯甲酸甲酯和对羟基苯甲酸丙酯)、甜味剂和增味剂。本发明的药物组合物可以通过具体赋形方式在对病人进行给药之后达到药物活性成分的快速、持续或延时释放，这也是本领域中广泛应用的方法。
活性成分，即本发明中的促进或激活Skp2基因表达的试剂，在药物构成和单位剂型中的数量可根据具体应用情况、特定化合物的活性和预期浓度进行改变或有较大调整。
“治疗”意味着对哺乳动物体内疾病的任何治疗，包括：(1)防止疾病，即造成临床疾病的症状不发展；(2)抑制疾病，即阻止临床症状的发展；(3)减轻疾病，即造成临床症状的消退。
本发明通过动物实验表明Skp2过表达在脓毒症小鼠模型中能够有效降低脓毒症小鼠的死亡率与减轻肺损伤，因此，Skp2基因可作为脓毒症的治疗靶点，改善脓毒症预后。
本发明通过动物实验发现Skp2敲除小鼠呈现脓毒症死亡率增加的表型。特异性过表达Skp2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。Skp2过表达可以通过减轻炎症反应，改善脓毒症预后。
以下结合具体实施例，对本发明的技术方案做进一步的描述，但是本发明的保护范围并不限于这些实施例。凡是不背离本发明构思的改变或等同替代均包括在本发明的保护范围之内。
实施例：
本实施例公开了Skp2基因在治疗脓毒症中的应用。本实施例首先构建了脓毒症小鼠模型，通过检测发现脓毒症小鼠肺中Skp2的表达水平显著降低；随后通过递送含Skp2mRNA的LNP颗粒构建Skp2肺部过表达小鼠，特异性过表达Skp2(Skp2-OE)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。Skp2过表达可以通过其泛素化功能减轻炎症反应，改善脓毒症预后。本实施例探索了Skp2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。
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2/3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.Western Blots检测：
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。
3.构建Skp2mRNA递送的LNP颗粒
3.1mRNA的合成
合成的mRNA包含Skp2 mRNA，合成的mRNA溶解于1mM Sodium Citrate(pH 6.4)。
3.2LNP的包装
脂质纳米颗粒组分溶解于100％乙醇中，其中LNP包含成分ionizable lipids/DSPC/Cholesterol/DMG-PEG2000。将mRNA溶解在50mM乙酸缓冲液(pH 4.5)中，通过Precision Nanosystems nanoassembly将水相(mRNA)和有机相(LNP)成分混合。收集LNPs并在PBS(pH 7.2)中稀释，透析过夜。0.2毫米的无菌过滤器滤过。LNP保存温度为2℃～8℃。采用Malvern Zetasizer动态光散射(DLS)仪测量LNP的粒径和多分散性。RNA包装效率由Quant-it RiboGreen RNA检测试剂盒测定。
4.动物实验
将含Skp2 mRNA的LNP颗粒(Skp2-OE)通过尾静脉注射进小鼠体内，并验证表达效率。
二、实验结果
1.脓毒症小鼠中Skp2表达受到抑制
通过对脓毒症模型小鼠肺中Skp2蛋白质进行含量分析，发现脓毒症模型小鼠肺中Skp2表达显著降低，如图1A所示。
2.炎症因子抑制肺上皮中Skp2表达
通过用脂多糖(LPS)刺激巨噬细胞，收集含炎症因子的条件培养基刺激肺上皮细胞，发现肺上皮细胞中Skp2表达同样明显下调，如图2A所示。
3.Skp2缺失导致脓毒症小鼠死亡率明显增加和肺损伤增加
通过生存曲线分析Skp2+/-小鼠与野生型假手术组和CLP组小鼠死亡率，提示Skp2缺失显著增加小鼠脓毒症死亡率，如图3A所示。通过HE染色发现Skp2+/-脓毒症小鼠肺损伤明显较野生型加重，如图3B所示。
4.Skp2 LNP递送显著改善脓毒症小鼠肺损伤
LNP递送能够促使小鼠肺中显著表达Skp2,如图4A所示。使用LNP肺内递送Skp2mRNA的脓毒症小鼠相比对照LNP脓毒症小鼠死亡率显著降低，如图4B所示。通过HE染色发现，过表达Skp2小鼠与对照组小鼠脓毒症造模后相比，肺损伤明显减轻，如图4C所示。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (7)
1.促进或激活Skp2基因表达的试剂在制备治疗脓毒症或改善脓毒症预后的药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述促进或激活Skp2基因表达的试剂包括：
(1)Skp2的mRNA和重组蛋白，和/或
(2)能够特异性增加Skp2基因表达的siRNA、miRNA、IncRNA、gRNA和反义核苷酸中的任意一种；
以及用于递送上述(1)和/或(2)物质的体内或体外递送载体。
3.根据权利要求2所述的应用，其特征在于，所述体内或体外递送载体为脂质纳米颗粒。
4.根据权利要求1所述的应用，其特征在于，所述脓毒症包括脓毒症发展的急性期、免疫平衡期或免疫抑制期，还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。
5.一种用于治疗脓毒症的药物组合物，其特征在于，所述药物组合物包括药学上可接受的体载体和有效量的活性成分，所述活性成分包括如权利要求1所述的促进或激活Skp2基因表达的试剂。
6.根据权利要求5所述的药物组合物，其特征在于，所述促进或激活Skp2基因表达的试剂包括：
(1)Skp2的mRNA和重组蛋白，和/或
(2)能够特异性增加Skp2基因表达的siRNA、miRNA、IncRNA、gRNA和反义核苷酸中的任意一种；
以及用于递送上述(1)和/或(2)物质的体内或体外递送载体。
7.根据权利要求6所述的药物组合物，其特征在于，所述体内或体外递送载体为脂质纳米颗粒。
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