WO2023092927A1 - 一种用于缓解应激性高血压血管内皮功能障碍的小rna及应用 - Google Patents

一种用于缓解应激性高血压血管内皮功能障碍的小rna及应用 Download PDF

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WO2023092927A1
WO2023092927A1 PCT/CN2022/086193 CN2022086193W WO2023092927A1 WO 2023092927 A1 WO2023092927 A1 WO 2023092927A1 CN 2022086193 W CN2022086193 W CN 2022086193W WO 2023092927 A1 WO2023092927 A1 WO 2023092927A1
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stress
sponge
vascular endothelial
endothelial dysfunction
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张海锋
邢文娟
马李杰
李凯峰
李泽
王孙云澍
张拓
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中国人民解放军空军军医大学
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  • the invention relates to the fields of molecular biology and medicine, in particular to a small RNA for alleviating stress-induced hypertension vascular endothelial dysfunction and its application.
  • Stress is closely related to human health. Moderate stress can mobilize the defense mechanism, which is beneficial to the body, but chronic stress that continues to be too high will have adverse effects on the body in terms of physiology, psychology, and behavior, and even cause a variety of stress. disease. Studies have shown that continuous high-intensity stress load can cause body damage, affect individual health, reduce work efficiency, and cause fatigue; it can even induce various diseases and extreme events such as sudden death. Studies have shown that 75% to 90% of human diseases are related to stress. Cardiovascular is the primary target organ of damage caused by stress. Various stress animal models (such as noise, electric shock, restraint, damp heat and exhausted swimming, etc.) have confirmed that stress can cause myocardial injury and vascular endothelial dysfunction, leading to or aggravate cardiovascular disease.
  • Hypertensive disease is the most common disease of the cardiovascular system, and the increase in blood pressure caused by various stress factors is called stress hypertension.
  • Various forms of stress factors beyond the scope of individual ability can produce a series of psychophysiological behavioral responses. Therefore, the occurrence and development of stress-induced hypertension play an initiating and maintaining role. To study the relationship between stress and hypertension, It is of great significance to the prevention and treatment of stress hypertension.
  • RNA as a new type of regulatory molecule, plays an important regulatory role in biological processes such as development, cell cycle, tumorigenesis, and metastasis. In recent years, studies have found that miRNA in vivo may serve as a new target for the treatment of certain diseases.
  • the present invention provides a small RNA for alleviating stress-induced hypertension vascular endothelial dysfunction and its application.
  • the present invention provides a new direction for alleviating stress-induced hypertension vascular endothelial dysfunction.
  • the present invention provides a small RNA for alleviating stress-induced hypertension vascular endothelial dysfunction
  • the small RNA is miR-219a-5p sponge, the nucleus of the miR-219a-5p sponge See SEQ ID NO.1 for the nucleotide sequence; the miR-219a-5p sponge counteracts endothelial DDAH1 expression and downstream eNOS phosphorylation by endogenous miR-219a-5p in combination with cellular endogenous miR-219a-5p Inhibitory effect, thereby alleviating stress-induced hypertension vascular endothelial dysfunction.
  • the present invention also provides the application of the small RNA in the preparation of a drug for alleviating stress-induced hypertension vascular endothelial dysfunction.
  • the beneficial effect of the present invention is that the present invention provides a new target and direction for alleviating stress-induced hypertension vascular endothelial dysfunction.
  • Fig. 1 is the blood pressure level, serum hormone and inflammatory factor level and blood vessel miR-219a-5p expression result graph of rats in each group in the embodiment of the present invention
  • Fig. 2 is the result figure of rat mesenteric fretting endothelial function test of the embodiment of the present invention
  • Fig. 3 is a graph showing the secretion level of endothelial cells NO and ADMA and the phosphorylation level of related regulatory molecule eNOS and the expression level of DDAH1 protein according to the embodiment of the present invention
  • Fig. 4 is a graph showing the verification of miR-219a-5p target and the effect of miR-219a-5p sponge according to the embodiment of the present invention.
  • This example provides a small RNA for alleviating stress-induced hypertension vascular endothelial dysfunction
  • the small RNA is miR-219a-5p sponge
  • the sequence of miR-219a-5p sponge is: AGAATTGCGTTTGGACAATCA, see SEQ ID NO for details .1.
  • the miR-219a-5p sponge counteracts the inhibitory effect of the endogenous miR-219a-5p on endothelial DDAH1 expression and downstream eNOS phosphorylation by binding to the endogenous miR-219a-5p of the cell, thereby alleviating stress hyperactivity Blood pressure Vascular endothelial dysfunction.
  • the above-mentioned small RNA can be applied to the preparation of drugs for alleviating stress-induced hypertension vascular endothelial dysfunction.
  • a stress-induced hypertension model was constructed, and a rat chronic stress model was established by the method of noise compound plantar electric shock stimulation (current: intensity 3-5mA, random interval 2-20s, continuous 1-2s ; Noise: 200-2000Hz, 80-100dB. 4h/d, lasted 28d), and studied the blood pressure, serum stress-related hormones and inflammatory markers of rats with chronic stress.
  • noise compound plantar electric shock stimulation current: intensity 3-5mA, random interval 2-20s, continuous 1-2s ; Noise: 200-2000Hz, 80-100dB. 4h/d, lasted 28d
  • A each group rat systolic blood pressure level
  • B each group rat serum norepinephrine (NE) level
  • C serum angiotensin II (AngII) levels of rats in each group
  • D serum TNF ⁇ levels of rats in each group
  • E expression level of vascular miR-219a-5p (control group: Mean 1.00, SEM 0.06; stress group: Mean 3.37, SEM 0.41).
  • the specific implementation method is: after the rat is anesthetized by intraperitoneal injection, the mesenteric arteriole is taken out and placed in pre-cooled physiological saline solution (PSS) middle. 1 Vessel separation and fixation. Under the stereoscope, the perivascular fat tissue was stripped clean, and then cut into small sections with a length of about 1.0 mm. Place the vascular ring in the constant temperature bath of the microvascular perfusion apparatus, and add 5ml of PSS solution to each constant temperature bath. Use a 40 ⁇ m metal wire to pass through the blood vessel and fix it.
  • PSS physiological saline solution
  • the blood vessels are fixed and adjusted to a parallel state (the blood vessels are in a relaxed state without force); 2 heating and ventilation. Turn on the ventilation, continue to infuse a mixture of 5% CO 2 and 95% O 2 , turn on the heating to raise the temperature to 37°C, and turn on the instrument and recording software for data recording; 3
  • Initial preload of blood vessels The blood vessels were stabilized under the initial preload state for 1 hour, and then 60 mmol/L KCl solution was used to act on the blood vessels twice, and the blood vessels with a contraction amplitude difference of ⁇ 10% and a tension greater than 1 mN were selected; 4Balance and vascular activity detection.
  • the blood vessels were preconstricted with phenylephrine (PE) at a final concentration of 10 -6 mol/L, and the next experiment was carried out after the tension curve reached the highest point and became stable.
  • Gradient concentrations of acetylcholine (ACh) (10 -9 -10 -5 mol/L) were added sequentially to detect endothelium-dependent vasodilation. After elution, the same method was used to observe the endothelium-independent relaxation function induced by gradient concentrations of sodium nitroprusside (SNP) (10 -9 -10 -5 mol/L).
  • SNP sodium nitroprusside
  • the microvascular ACh-induced, i.e., endothelium-dependent, vasodilation of rats in the stress group was significantly reduced (P ⁇ 0.01).
  • ACh-induced vasodilation was significantly increased after 5p sponge (P ⁇ 0.05), while SNP-induced vasodilation was not different (P>0.05), suggesting that miR-219a-5p sponge can significantly improve arteriolar endothelial function in stressed rats .
  • the present invention further detected the effect of miR-219a-5p sponge on the NO release level of endothelial cells, using a nitric oxide (NO) detection kit to detect the NO release level.
  • NO nitric oxide
  • the method is as follows: first, nitrate reductase is used to convert nitrate into nitrite, and then Griess reagent is used to convert nitrite into deep purple azo compound, and the total nitrate in the sample is expressed by colorimetry to reflect the content of NO.
  • DDAH1 dimethylarginine dimethylaminohydrolase 1
  • ADMA nitric oxide synthase inhibitor asymmetric methylarginine
  • the inventors further studied the upstream regulatory molecules of NO and found that Hormones can down-regulate the phosphorylation level of eNOS protein and the expression of DDAH1, while the miR-219a-5p sponge can be partially reversed. The results suggested that miR-219a-5p sponge may reduce the accumulation of ADMA by increasing the expression of DDAH1, thereby alleviating its inhibition on eNOS activity and NO production.
  • Table 2 The secretion levels of NO and ADMA in endothelial cells and the phosphorylation level of related regulatory molecules eNOS and the expression level of DDAH1 protein
  • this example is verified by screening with dual luciferase reporter gene.
  • miR-219a-5p sponge can counteract the inhibitory effect of endogenous miR-219a-5p on endothelial DDAH1 expression and eNOS phosphorylation by binding to endogenous miR-219a-5p, thereby improving stress-induced hypertension Vascular endothelial function.

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Abstract

本发明涉及分子生物学和医学领域，公开了一种用于缓解应激性高血压血管内皮功能障碍的小RNA序列及其应用，所述小RNA为miR-219a-5p sponge，所述miR-219a-5p sponge的核苷酸序列见SEQ ID NO.1。

Description

一种用于缓解应激性高血压血管内皮功能障碍的小RNA及应用
本申请要求于2021年11月25日提交中国专利局、申请号为202111412601X、发明名称为“一种用于缓解应激性高血压血管内皮功能障碍的小RNA序列及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及分子生物学和医学领域，具体涉及一种用于缓解应激性高血压血管内皮功能障碍的小RNA及应用。
背景技术
应激与人类健康密切相关，适度的应激可调动防御机制对机体有利，但持续过高强度的慢性应激会在生理、心理和行为等方面对机体产生不良影响，甚至引起多种应激性疾病。研究表明，持续较高强度的应激负荷可造成机体损伤，影响个体健康、降低工作效率、导致疲劳；甚至会诱发多种疾病，发生猝死等极端事件。研究表明，75％～90％的人类疾病和应激有关。心血管是应激所致损伤的首要靶器官，多种应激动物模型(如噪声、电击、束缚、湿热和力竭游泳等)均证实，应激可引起心肌损伤和血管内皮功能障碍，引发或加重心血管疾病。高血压疾病是心血管系统最为常见的疾病，其中由各种应激因素引起的血压升高称为应激性高血压。各种不同形式的应激因素超过个体能力范围后可产生一系列心理生理行为的反应，因此应激所致高血压的发生、发展有着始动和维持作用，研究应激与高血压的关系，对应激性高血压的预防和治疗具有重要的意义。
已有的研究表明，约70-90％人类基因组可以被转录，但是这其中仅有2％的基因组编码大约21,000个蛋白质，表明转录组中非编码RNA占据绝大多数，大量的非编码RNA以“暗物质”形式存在。其中miRNA作为一类新型的调控分子，在发育、细胞周期和肿瘤发生、转移等生物学过程中具有重要的调控作用，近年来研究发现体内miRNA可能作为某些疾病治疗的新靶点。
发明内容
基于以上问题，本发明提供一种用于缓解应激性高血压血管内皮功能障碍的小RNA及应用，本发明为缓解应激性高血压血管内皮功能障碍提供了新的方向。
为解决以上技术问题，本发明提供了一种用于缓解应激性高血压血管内皮功能障碍的小RNA，所述小RNA为miR-219a-5p sponge，所述miR-219a-5p sponge的核苷酸序列见SEQ ID NO.1；所述miR-219a-5p sponge通过结合细胞内源的miR-219a-5p来抵消细胞内源的miR-219a-5p对内皮DDAH1表达及其下游eNOS磷酸化的抑制作用，进而缓解应激性高血压血管内皮功能障碍。
为解决以上技术问题，本发明还提供了所述小RNA在制备用于缓解应激性高血压血管内皮功能障碍的药物中的应用。
与现有技术相比，本发明的有益效果是：本发明为缓解应激性高血压血管内皮功能障碍提供了新的靶点和方向。
附图说明
图1为本发明的实施例中各组大鼠血压水平、血清激素和炎性因子水平及血管miR-219a-5p表达结果图；
图2为本发明的实施例的大鼠肠系膜微动内皮功能检测结果图；
图3为本发明的实施例内皮细胞NO、ADMA分泌水平及其相关调控分子eNOS磷酸化水平和DDAH1蛋白表达水平结果图；
图4为本发明的实施例miR-219a-5p靶点验证及miR-219a-5p sponge的作用结果图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，下面结合实施例和附图，对本发明作进一步的详细说明，本发明的示意性实施方式及其说明仅用于解释本发明，并不作为对本发明的限定。
实施例：
本实施例提供了一种用于缓解应激性高血压血管内皮功能障碍的小RNA，小RNA为miR-219a-5p sponge，miR-219a-5p sponge的序列为： AGAATTGCGTTTGGACAATCA，具体见SEQ ID NO.1。所述miR-219a-5p sponge通过结合细胞内源的miR-219a-5p来抵消细胞内源的miR-219a-5p对内皮DDAH1表达及其下游eNOS磷酸化的抑制作用，进而缓解应激性高血压血管内皮功能障碍。上述小RNA可应用于制备用于缓解应激性高血压血管内皮功能障碍的药物中。
本实施例为了获得上述研究成果构建了应激性高血压模型，通过噪声复合足底电击刺激方法建立大鼠慢性应激模型(电流：强度3～5mA，随机间隔2～20s，持续1～2s；噪声：200～2000Hz，80～100dB。4h/d，持续28d)，并研究了慢性应激大鼠的血压、血清应激相关激素和炎症标志物。
见附图1，其中：A：各组大鼠收缩压水平，B：各组大鼠血清去甲肾上腺素(NE)水平(control组：Mean 7.04，SEM 0.35；stress组：Mean 11.31，SEM 0.42)，C：各组大鼠血清血管紧张素II(AngII)水平(control组：Mean 71.96，SEM 1.43；stress组：Mean 95.79，SEM 4.27)，D：各组大鼠血清TNFα水平(control组：Mean 196.4，SEM 11.6；stress组：Mean 296.8，SEM 8.9)，E：血管miR-219a-5p表达水平(control组：Mean 1.00，SEM 0.06；stress组：Mean 3.37，SEM 0.41)。与同时期Control组比较，**P<0.01，每组n＝15。从图1可看出，与对照组相比，应激组大鼠血清NE、AngII水平显著增加，TNFα水平显著增加；同时，血管miR-219a-5p表达显著增加。
分离应激28天后大鼠肠系膜微动脉，利用离体微血管灌流系统观察血管收缩舒张功能，具体实施方法为：腹腔注射麻醉大鼠后，取出肠系膜微动脉置于预冷的生理盐溶液(PSS)中。①血管分离与固定。体视镜下，将血管周围脂肪组织剥离干净后，剪成长度为1.0mm左右的小段。将血管环置于微血管灌流仪的恒温浴槽中，给每个恒温浴槽加入5ml的PSS溶液。使用40μm金属丝穿过血管，固定。血管固定好调至平行状态(血管处于松弛状态，不受力)；②加热与通气。打开通气，持续通入5％CO 2和95％O 2的混合气，打开加热升温至37℃，打开仪器和记录软件进行数据记录；③血管初始预负荷。血管在初始预负荷状态下稳定1h，随后用60mmol/LKCl溶液作用血管2次，选取收缩幅度相差<10％，且张力大 于l mN的血管；④平衡及血管活性检测。调节血管至最适初长度，平衡1h后，用60mM的KPSS检测血管环活性良好后进行下一步实验。采用终浓度为10 -6mol/L的苯肾上腺素(phenylephrine，PE)预收缩血管，待达到最高点张力曲线稳定后，进行下一步实验。依次加入梯度浓度的乙酰胆碱(Acetylcholine，ACh)(10 -9-10 -5mol/L)检测内皮依赖性血管舒张功能。洗脱后采用同样方法，观察梯度浓度的硝普钠(Sodium nitroprusside，SNP)(10 -9-10 -5mol/L)引起的内皮非依赖性舒张功能。
结果见附图2和表1，其中：A：乙酰胆碱(ACh)诱导的肠系膜微动脉舒张，B：硝普钠(SNP)诱导的肠系膜微动脉舒张。与同浓度下Control组比较，**P<0.01；与同浓度下Stress组比较，#P<0.05，每组n＝6。结果显示，各组大鼠的微动脉随硝普钠(SNP)和乙酰胆碱(ACh)浓度的增加表现出浓度依赖性的舒张反应。其中，与对照组比较，应激组大鼠微血管ACh诱导的，即内皮依赖性的血管舒张程度明显降低(P<0.01)，与应激组比较，应激大鼠微血管转染miR-219a-5p sponge后ACh诱导的血管舒张程度显著增加(P<0.05)，而SNP诱导的血管舒张效应没有差异(P＞0.05)，提示miR-219a-5p sponge可显著改善应激大鼠微动脉内皮功能。
表1 大鼠肠系膜微动内皮功能检测结果
Figure PCTCN2022086193-appb-000001
Figure PCTCN2022086193-appb-000002
为探讨miR-219a-5p sponge改善内皮功能的机制，本发明进一步检测了miR-219a-5p sponge对内皮细胞NO释放水平的影响，采用一氧化氮(NO)检测试剂盒检测NO释放水平。其方法为，首先利用硝酸盐还原酶将硝酸盐转化为亚硝酸盐，而后使用Griess试剂将亚硝酸盐转化为深紫色偶氮化合物，通过比色法表示样品中总硝酸根反映NO的含量。结果见附图3和表2，其中：A：内皮细胞NO释放水平，B：内皮细胞ADMA释放水平，C：内皮细胞磷酸化eNOS水平，D：内皮细胞DDAH1蛋白表达水平。与Control组比较，*P<0.05；与Stress组比较，#P<0.05，每组n＝4。发现与对照组相比，应激激素干预可降低减少内皮细胞NO分泌水平(P<0.05)，增加内源性eNOS抑制物非对称性二甲基精氨酸(ADMA)水平(P<0.05)；而miR-219a-5p sponge转染后，与应激组相比，ADMA水平降低(P<0.05)，内皮细胞NO分泌水平恢复。由于二甲基精氨酸二甲基氨基水解酶1(DDAH1)可导致一氧化氮合酶抑制剂非对称甲基精氨酸(ADMA)的积累，发明人进一步研究NO上游调控分子，发现应激激素可下调eNOS蛋白磷酸化水平，DDAH1表达降低，而miR-219a-5p sponge可部分逆转。结果提示miR-219a-5p sponge可能通过提高DDAH1表达，降低ADMA积累，从而减轻其对eNOS活性和NO生成的抑制。
表2 内皮细胞NO、ADMA分泌水平及其相关调控分子eNOS磷酸化水平和DDAH1蛋白表达水平
Figure PCTCN2022086193-appb-000003
为明确miR-219a-5p参与应激性高血压血管舒张功能障碍的直接靶点，本实施例通过筛选，利用双荧光素酶报告基因进行验证。
一、首先构建野生r-Ddah1-3UTR-wt和突变r-Ddah1-3UTR-mu载体，r-Ddah1-3UTR-wt序列信息:
Figure PCTCN2022086193-appb-000004
r-Ddah1-3UTR-mu序列信息:
Figure PCTCN2022086193-appb-000005
Figure PCTCN2022086193-appb-000006
二、细胞转染：
1.事先准备好用于转染的分到96孔板中的293T细胞和目的质粒，待细胞密度达到50％-70％为宜。
2.将10ul DMEM与0.16ug的r-Ddah1-3UTR目的质粒以及5pmol的rno-miR-219a-5p/Negative Control(NC)充分混匀后室温放置，之后将10ul DMEM与0.3ul的转染试剂充分混匀，室温放置5min。
3.将上述溶液充分混匀，室温放置20min。
4.转染前为细胞换取新鲜培养基，之后将转染混合物加入混匀。37℃，5％CO 2培养。
5.转染6h后换取新鲜培养基，转染48h后收集细胞检测。
三、双荧光素酶报告基因检测(Promega Dual-Luciferase system)
1、将5×PLB(Passive Lysis Buffer)用蒸馏水稀释至1×PLB，以96孔板每孔100ul的量加入，用移液枪吹打打散细胞，置于室温摇床上缓慢摇15分钟后，将细胞裂解液吸至1.5ml离心管，4度12000rpm离心10分钟，取上清移入新的管子；
2、96孔板中加入Luciferase Assay Reagent II(LAR II)(Luciferase Assay Reagent,Progema)工作液100ul；
3、加入20ul细胞裂解液，移液枪吹打混匀2-3次，测定记录Firefly luciferase值，此值为内参值。
4、加入100ul Stop&
Figure PCTCN2022086193-appb-000007
Reagent(Luciferase Assay Reagent, Progema)，移液枪吹打混匀2-3次，测定记录Renilla luciferase值，此即为报告基因发光值。
结果见附图4和表3，结果显示rno-miR-219a-5p可与ADMA产生上游调控基因DDAH1的3’UTR区相互作用，这一作用在DDAH13’UTR突变后消失(r-Ddah1-3UTR-mu)；而miR-219a-5p-sponge的作用可抵消miR-219a-5p对DDAH1基因的抑制作用。
表3 miR-219a-5p靶点验证及miR-219a-5p sponge的作用
Figure PCTCN2022086193-appb-000008
上述结果证明miR-219a-5p sponge通过结合细胞内源的miR-219a-5p来抵消细胞内源的miR-219a-5p对内皮DDAH1表达和eNOS磷酸化的抑制作用，进而改善应激性高血压血管内皮功能。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其它实施例，这些实施例都属于本发明保护范围。

Claims (4)

  1. 一种用于缓解应激性高血压血管内皮功能障碍的小RNA，其特征在于，所述小RNA为miR-219a-5p sponge，所述miR-219a-5p sponge的核苷酸序列见SEQ ID NO.1；
    所述miR-219a-5p sponge通过结合细胞内源的miR-219a-5p来抵消细胞内源的miR-219a-5p对内皮DDAH1表达及其下游eNOS磷酸化的抑制作用，进而缓解应激性高血压血管内皮功能障碍。
  2. 权利要求1所述的小RNA在制备缓解应激性高血压血管内皮功能障碍的药物中的应用。
  3. 根据权利要求2所述的应用，其特征在于，所述药物的作用机理，为miR-219a-5p sponge通过结合细胞内源的miR-219a-5p来抵消细胞内源的miR-219a-5p对内皮DDAH1表达及其下游eNOS磷酸化的抑制作用，进而缓解应激性高血压血管内皮功能障碍。
  4. 一种缓解应激性高血压血管内皮功能障碍的方法，其特征在于，包括以下步骤，对应激大鼠微血管转染miR-219a-5p sponge；所述miR-219a-5p sponge的核苷酸序列见SEQ ID NO.1。
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