miRNA-23a-3p和TLR4在急性脑出血患者血清中的表达及临床意义.pdf

Hainan Med J,Aug.2023,Vol.34,No.15海南医学2023年8月第34卷第15期miRNA-23a-3p和TLR4在急性脑出血患者血清中的表达及临床意义贺琦，王燕西安宝石花长庆医院神经内科，陕西西安710201【摘要】目的探究微小RNA-23a-3p(miR-23a-3p)、Toll样受体4(TLR4)在急性脑出血(ACH)患者血清中的水平及其临床意义。方法选取2020年1月至2022年3月西安宝石花长庆医院收治的110例ACH患者为ACH组，并纳入同期110例健康体检者为对照组。采用实时荧光定量逆转录-聚合酶链反应(qRT-PCR)法测定两组受检者的血清miR-23a-3p表达水平，酶联免疫吸附法(ELISA)测定两组受检者的血清核因子B(NF-B)、TLR4、肿瘤坏死因子-(TNF-)水平；采用Pearson法分析ACH患者血清miR-23a-3p、TLR4水平与TNF-、NF-B的相关性；比较不同预后ACH患者一般资料及血清miR-23a-3p、TLR4、TNF-、NF-B 水平；采用受试者工作特征曲线(ROC)分析血清miR-23a-3p、TLR4 水平对ACH患者预后的评估价值。结果ACH 组患者血清miR-23a-3p、TLR4、TNF-、NF-B水平分别为2.290.67、(32.9410.98)g/L、(51.2821.37)pg/mL、(17.346.19)ng/mL，明显高于对照组的1.010.30、(17.465.82)g/L、(20.318.47)pg/mL、(8.503.04)ng/mL，差异均有统计学意义(P0.05)；ACH患者血清miR-23a-3p、TLR4水平与TNF-、NF-B均呈正相关(r=0.377、0.538、0.403、0.572，P0.05)，且血清miR-23a-3p表达水平与TLR4也呈正相关(r=0.581，P0.05)；预后不良组ACH患者NIHSS评分、脑出血体积、血清miR-23a-3p、TLR4、TNF-、NF-B水平明显高于预后良好组，差异均有统计学意义(P0.05)；血清miR-23a-3p、TLR4预测ACH患者预后的曲线下面积(AUC)分别为0.857、0.868，截断值分别为2.37 g/L、34.52 g/L，敏感度分别为76.9%、78.8%，特异性分别为87.9%、86.2%；miR-23a-3p、TLR4联合预测ACH患者预后的AUC为0.941，敏感度为90.4%，特异性为89.7%。结论ACH患者血清miR-23a-3p、TLR4水平较高，miR-23a-3p与TLR4呈正相关，且miR-23a-3p、TLR4联合检测可较好地预测ACH患者预后，为临床预测ACH患者预后提供新策略。【关键词】急性脑出血；微小miR-23a-3p；Toll样受体4；预后；相关性【中图分类号】R743.34【文献标识码】A【文章编号】10036350（2023）15213505Expression and clinical significance of miRNA-23a-3p and Toll-like receptor 4 in serum of patients with acutecerebral hemorrhage.HE Qi,WANG Yan.Department of Neurology,Xian Baoshihua Changqing Hospital,Xian 710201,Shaanxi,CHINA【Abstract】ObjectiveTo explore the levels of microRNA-23a-3p(miR-23a-3p)and Toll-like receptor 4(TLR4)in the serum of patients with acute cerebral hemorrhage(ACH)and its clinical significance.MethodsA totalof 110 ACH patients admitted to Xian Baoshihua Changqing Hospital from January 2020 to March 2022 were selectedas the ACH group,and 110 healthy subjects in the same period were included as the control group.Real-time quantita-tive reverse transcription-polymerase chain reaction(qRT-PCR)method was used to determine the expression level of se-rum miR-23a-3p in the two groups.Enzyme-linked immunosorbent assay(ELISA)was used to determine serum nucle-ar factor-B(NF-B),TLR4,and tumor necrosis factor-(TNF-)levels in the two groups.Pearson method was usedto analyze the correlation between serum miR-23a-3p,TLR4 levels and TNF-,NF-B in ACH patients.The generaldata and serum levels of miR-23a-3p,TLR4,TNF-,and NF-B in ACH patients with different prognosis were com-pared.Receiver operating characteristic(ROC)curve was used to analyze the serum miR-23a-3p,TLR4 levels in theevaluation of the prognosis of ACH patients.ResultsThe serum levels of miR-23a-3p,TLR4,TNF-and NF-B inthe ACH group were 2.290.67,(32.9410.98)g/L,(51.2821.37)pg/mL,(17.346.19)ng/mL,respectively,whichwere significantly higher than 1.010.30,(17.465.82)g/L,(20.318.47)pg/mL,(8.503.04)ng/mL in the controlgroup(P0.05).The serum levels of miR-23a-3p and TLR4 in ACH patients were positively correlated with TNF-andNF-B(r=0.377,0.538,0.403,0.572,P0.05),and the expression levels of serum miR-23a-3p were also positively cor-related with TLR4(r=0.581,P0.05).The NIHSS score,cerebral hemorrhage volume,serum miR-23a-3p,TLR4,TNF-and NF-B levels of ACH patients in the poor prognosis group were significantly higher than those in the goodprognosis group,and the differences were statistically significant(P0.05).The area under the curve(AUC)of serummiR-23a-3p and TLR4 for predicting the prognosis of ACH patients was 0.857 and 0.868,with the cut-off value of 论著 doi:10.3969/j.issn.1003-6350.2023.15.002基金项目：陕西省西安市科研项目(编号：2020yb69)。第一作者：贺琦(1978)，男，副主任医师，研究方向：脑梗死、脑出血、癫痫、头痛、眩晕、帕金森病、痴呆、周围神经疾病。通讯作者：王燕(1985)，女，副主任医师，硕士，研究方向：脑梗死、脑出血、癫痫、头痛、眩晕、帕金森病、痴呆、周围神经疾病，E-mail：。2135海南医学2023年8月第34卷第15期Hainan Med J,Aug.2023,Vol.34,No.15急性脑出血(acute cerebral hemorrhage，ACH)是一种颅内出血性疾病，其发病率高，预后差1-2。因此，寻找与ACH发病及预后转归相关的指标，对及时干预，改善ACH 患者预后有积极意义。有研究认为，ACH发病与炎症反应、微小RNA(microRNA，miRNA)表达失调、信号传导异常等相关3-4。研究发现，微小RNA-23a-3p(microRNA-23a-3p，miR-23a-3p)在ACH患者中呈高表达，与血肿体积、ACH患者预后相关，可能参与并影响 ACH 患者预后5。此外，研究发现脑出血患者Toll样受体4(Toll-like receptor 4，TLR4)水平较高，是影响脑出血预后的危险因素，且具有评估 ACH 患者预后的潜在价值6。但 miR-23a-3p、TLR4在ACH患者中的相关性，及两者联合预测ACH患者预后的价值尚未明确。基于此，本文通过测定miR-23a-3p、TLR4在ACH患者血清中的水平，旨在分析miR-23a-3p与TLR4的相关性以及miR-23a-3p、TLR4联合检测对ACH患者预后的预测价值。1资料与方法1.1一般资料选取2020年1月至2022年3月西安宝石花长庆医院诊治的 ACH 患者 110 例作为ACH 组。纳入标准：(1)符合 中国脑出血诊治指南(2019)7中有关ACH的诊断标准；(2)首次发病，入院就诊0.05)，具有可比性。本研究经本院医学伦理委员会批准，所有受试者及其亲属对本研究知情同意。1.2检测方法1.2.1样本收集收集ACH患者入院治疗前，及健康者体检当日35 mL空腹外周血，自然凝集，分离出血清(4 000 r/min离心10 min)，分装，存于-70冰箱。1.2.2血清miR-23a-3p表达水平测定采用实时荧光定量逆转录-聚合酶链反应(real-time quantita-tive reverse transcription-polymerase chain reaction，qRT-PCR)法测定血清miR-23a-3p表达水平。首先，按 miRNeasy Serum/Plasma Kit(217184，北京索莱宝科技有限公司)说明书提取血清样本总RNA，且在提取 RNA 时加内参 cel-miR-39。其次，利用 miScriptReverse Transcription Kit(218061，杭州开泰生物技术有限公司)获得 cDNA。最终，应用 miScript SYBRGreen PCR Kit(218075，北京欣华绿源科技有限公司)配制反应体系，qRT-PCR仪(StepOne TM，美国ABI公司)将cDNA扩增、检测。qRT-PCR扩增参数：96.58 min；94 30 s，64.5 40 s，70 20 s，40 个循环。miR-23a-3p 上 游引物 序列 为 5-ACACTCCAGCT-GGGATCA-3，下游引物序列为5-CTCAACTGGTGTCGT-3，cel-miR-39 上游引物序列 为 5-CAGAGT-CACCGGGTGTAAAT-3，下游引物序列为5-CCAGT-GCGTGTCGTGGAGTC-3。血清miR-23a-3p相对表达量以2-CT法计算。1.2.3血清肿瘤坏死因子-(TNF-)、TLR4、核因子B(NF-B)水平测定采用酶联免疫吸附法(en-zyme linked immunosorbent assay，ELISA)测定血清TNF-、TLR4和NF-B水平。采用人TNF-ELISA试剂盒(70-EK182HS-96，杭州联科生物技术股份有限公司)、人 TLR4 ELISA试剂盒(CSB-E12954h-1，上海恒斐生物科技有限公司)、人 NF-B ELISA 试剂盒(XFH11333，上 海 信 帆 生 物 科 技 有 限 公 司)配 制TNF-、TLR4、NF-B的标准样品溶液，解冻血清，酶标仪(美国Awareness公司，Chromate 4300)测定TNF-、TLR4、NF-B标准品溶液及血清样本在450 nm处的吸光度，绘制TNF-、TLR4、NF-B的标准回归曲线，结合TNF-、TLR4、NF-B的标准回归方程，计算血清TNF-、TLR4、NF-B水平。1.3治疗与随访ACH患者均依据 中国脑出血诊治指南(2019)7给予ACH患者降颅内压、吸氧、脑保护剂等对症治疗。对110例ACH患者随访6个月，对患者进行改良Rankin量表(modified Rankin scale，mRS)评估，并依据mRS评分将其分为预后良好组(58例，mRS评分3分)、预后不良组(52例，mRS评分3分)。此外，收集整理两组ACH患者美国国立卫生研究院卒中量表(National Institute of Health Stroke Scale，NIHSS)、年龄、高脂血症、性别、高血压、血小板分布宽度、身体质2.37 g/L and 34.52 g/L,the sensitivity of 76.9%and 78.8%,respectively,and the specificity of 87.9%and 86.2%,re-spectively.The AUC of miR-23a-3p combined with TLR4 in predicting the prognosis of ACH patients was 0.941,withthe sensitivity of 90.4%and the specificity of 89.7%.ConclusionACH patients have high levels of serum miR-23a-3pand TLR 4,and miR-23a-3p is positively correlated with TLR4.The combination of miR-23a-3p and TLR4 can betterpredict the prognosis of patients with ACH,and provide a new strategy for clinical prediction of the prognosis of patientswithACH.【Key words】Acute cerebral hemorrhage;MicroRNA-23a-3p;Toll-like receptor 4;Prognosis;Correlation2136Hainan Med J,Aug.2023,Vol.34,No.15海南医学2023年8月第34卷第15期量指数(body mass index，BMI)、糖尿病等资料。1.4统计学方法应用SPSS25.0软件进行数据统计学分析。计量资料均符合正态分布，以均数标准差(x-s)表示，组间比较采用t检验；计数资料比较采用2检验，理论频数5时，采用Fisher精确概率法分析；ACH患者血清miR-23a-3p、TLR4水平与TNF-、NF-B的相关性采用Pearson法分析；采用受试者工作 特 征 曲 线(receiver operating characteristic curve,ROC)评估血清mmiR-23a-3p、TLR4预测ACH患者预后的价值。以P0.05表示差异有统计学意义。2结果2.1两组受 检 者的 血清 miR-23a-3p、TLR4、TNF-、NF-B水平比较与对照组相比，ACH组患者血清miR-23a-3p、TLR4、TNF-、NF-B水平均升高，差异有统计学意义(P0.05)，见表1。2.2ACH 患者血清 miR-23a-3p、TLR4 水平与TNF-、NF-B的关系经Pearson法分析结果显示，ACH 患者血清 miR-23a-3p、TLR4 水平与 TNF-、NF-B 均呈正相关(r=0.377、0.538、0.403、0.572，P0.05)，见表2；ACH患者血清miR-23a-3p表达水平与TLR4呈正相关(r=0.581，P0.05)，见图1。2.3不同预后 ACH 患者的一般资料及血清miR-23a-3p、TLR4、TNF-、NF-B 水平比较与预后良好组相比，预后不良组ACH患者NIHSS评分、脑出血体积、血清miR-23a-3p、TLR4、TNF-、NF-B水平均升高，差异有统计学意义(P0.05)，见表3。表1两组受检者的血清miR-23a-3p、TLR4、TNF-、NF-B 水平比较(x-s)Table 1Comparison of serum levels of miR-23a-3p,TLR4,TNF-and NF-B between the two groups(x-s)组别对照组ACH组t值P值例数110110miR-23a-3p1.010.302.290.6718.2870.001TLR4(g/L)17.465.8232.9410.9813.0650.001TNF-(pg/mL)20.318.4751.2821.3714.1300.001NF-B(ng/mL)8.503.0417.346.1913.4440.001表 2ACH患者血清 miR-23a-3p、TLR4 水平与 TNF-、NF-B 的相关性Table 2Correlation of serum miR-23a-3p and TLR4 levels withTNF-and NF-B in ACH patients指标miR-23a-3pTLR4r值0.3770.538P值0.0010.001r值0.4030.572P值0.0012.37时，ACH患者发生不良预后概率较高，提示miR-23a-3p不仅与ACH患者预后有关，还可能作为ACH患者预后预测的辅助指标。TLR4是Toll样受体家族的一员，是一种膜蛋白，其主要分布于神经小胶质细胞，可诱导炎症反应，与脑出血的神经损伤密切相关14-15。研究发现，TLR4在脑出血中呈高水平，经麦角甾苷治疗后，TLR4水平下降，TLR4可能通过促进炎症反应进而影响脑出血病理过程16；另外，周喜燕等17也研究发现脑出血大鼠脑组织中TLR4表达水平升高，TLR4可能促进脑组织损伤，增加血脑屏障通透性，其与神经功能显著相关。本研究中ACH患者血清TLR4水平高于健康者，与既往研究16-17类似，提示TLR4可能参与ACH发病进程，推测TLR4可能通过影响炎症反应，进而影响ACH发生发展，其具体机制有待深入探讨。进一步研究发现，预后不良的 ACH 患者血清 TLR4高于预后良好者，提示TLR4可能与ACH患者预后相关，推测高水平TLR4通过促进炎症反应进而影响ACH患者预后。随后的ROC曲线分析显示，血清TLR4预测ACH患者预后的AUC为0.868，当血清TLR4水平34.52 g/L时，ACH患者发生不良预后风险较大，提示TLR4对ACH患者预后有一定预测价值，测定血清TLR4水平有助于临床预测ACH患者的预后情况。近年来，关于miRNA与下游靶基因的3-非翻译区结合，抑制翻译或降解mRNA，进而调控疾病进展方面的研究越来越多。miR-23a-3p靶向TLR4调控疾病进程的研究目前已有报道，如Gao 等18研究发现，miR-23a-3p可以通过调节TLR4表达，进而影响脓毒症的炎症反应过程，Chen等19发现结核患者中miR-23a-3p 的 下 调 通 过 靶 向 IRF1/SP1 的 TLR4/TNF-/TGF-1/IL-10信号传导抑制单核细胞的功能和吞噬作用，目前关于miR-23a-3p/TLR4在ACH中的作用报道较少，故本研究分析了 ACH 患者血清miR-23a-3p表达水平与TLR4的关系，结果显示，血清miR-23a-3p 表 达 水 平 与 TLR4 呈 正 相 关，提 示miR-23a-3p可能与TLR4相互作用进而影响ACH发生发展，但其机制需结合基础实验加以证实。进一步研究显示，血清miR-23a-3p、TLR4联合预测ACH患者预后的 AUC 为 0.941，敏感度为 90.4%，特异性为89.7%，提示miR-23a-3p、TLR4联合可提高对ACH患者预后的预测价值，为临床判定ACH患者预后提供新思路。综上所述，ACH患者血清miR-23a-3p、TLR4水平较高，miR-23a-3p与TLR4呈正相关，且miR-23a-3p、TLR4均与ACH患者预后相关，两者联合检测可提高预测ACH患者预后的价值，为临床预测ACH患者预后提供新思路。但后期仍需深入探讨miR-23a-3p、TLR4在ACH中的机制，为诊治ACH、评估ACH患者预后增加说服力。图2血清miR-23a-3p、TLR4预测ACH患者预后的ROC曲线Figure 2ROC curve of serum miR-23a-3p and TLR4 in predictingprognosis of patients with ACH2138Hainan Med J,Aug.2023,Vol.34,No.15海南医学2023年8月第34卷第15期参考文献1Li AH,Zhao XC,Meng XJ.Changes and clinical significance of se-rum GSN and Nogo-A levels in patients with acute cerebral hemor-rhage J.Shandong Medical Journal,2020,60(25):14-17.李爱华,赵新春,孟宪举.急性脑出血患者血清GSN,Nogo-A水平变化及临床意义J.山东医药,2020,60(25):14-17.2Liu J,Guo MS,Feng ZW.Serum levels of TN-C and LOX-1 in pa-tients with acute cerebral hemorrhage and their clinical significanceJ.China Journal of Emergency Resuscitation and Disaster Medi-cine,2020,15(1):46-50.刘娟,郭明升,冯志伟.急性脑出血患者血清TN-C,LOX-1水平及其临床意义J.中国急救复苏与灾害医学杂志,2020,15(1):46-50.3Yuan JX,Li CC,Xiu B.Relationship of serum level of TNF-andNGF with degree of brain edema and prognosis after acute cerebralhemorrhage J.Chin J Mod Med,2020,30(19):35-39.苑建鑫,李萃萃,修波.血清肿瘤坏死因子-,神经生长因子与急性脑出血后脑水肿程度及预后的关系J.中国现代医学杂志,2020,30(19):35-39.4 Wang Z,Lu G,Sze J,et al.Plasma miR-124 is a promising candidatebiomarker for human intracerebral hemorrhage stroke J.Mol Neuro-biol,2018,55(7):5879-5888.5 Shao Zhihui,Zuo Peng,Yu Zheng,et al.ReIationship between serummiR-23a-3p expression and prognosis in patients with acute cerebraIhemorrhage J.China Journal of Emergency Resuscitation and Di-saster Medicine,2019,14(10):937-940.邵志辉,左鹏,于正,等.急性脑出血患者血清微RNA-23a-3p表达与预后的关系J.中国急救复苏与灾害医学杂志,2019,14(10):937-940.6Jia LJ,Li WQ,Yu LP.Relationship between the levels of TLR4,TNF-and IFN-in peripheral blood and prognosis in patients withcerebral hemorrhage J.Hainan Medical Journal,2019,30(20):2601-2603.贾丽娟,李文强,鱼丽萍.脑出血患者外周血TLR4、TNF-、IFN-水平与预后的关系J.海南医学,2019,30(20):2601-2603.7Neurology Branch of Chinese Medical Association,CerebrovascularDisease Group,Neurological Branch,Chinese Medical Association.Hemorrhage(2019)J.Chin J Neurol,2019,52(12):994-1005.中华医学会神经病学分会,中华医学会神经病学分会脑血管病学组.中国脑出血诊治指南(2019)J.中华神经科杂志,2019,52(12):994-1005.8Xin Y,Zhang X,Li XT,et al.Relationship between serum cy-clophilin a and prodnosis in patients with acute cerebral hemorrhageJ.Chinese Journal of Coal Industry Medicine,2020,23(3):51-55.辛宇,张翔,李夏婷,等.急性脑出血患者血清亲环蛋白A水平与预后的关系J.中国煤炭工业医学杂志,2020,23(3):51-55.9Zhou YX,Song DD,Fang JJ.The relationship between serum com-plements C3,C4,brain edema volume and clinical prognosis in pa-tients with acute cerebral hemorrhage J.Chinese Journal of HealthLaboratory Technology,2020,30(17):2099-2101.周夷霞,宋丹丹,方建江.急性脑出血患者血清补体C3,C4与脑水肿体积及临床预后的关系J.中国卫生检验杂志,2020,30(17):2099-2101.10 Wang WX,Springer JE,Hatton KW.MicroRNAs as biomarkers forpredicting complications following aneurysmal subarachnoid hemor-rhage J.Int J Mol Sci,2021,22(17):9492-9498.11 Hu YL,Wang H,Huang Q,et al.MicroRNA-23a-3p promotes theperihematomal edema formation after intracerebral hemorrhage viaZO-1 J.Eur Rev Med Pharmacol Sci,2018,22(9):2809-2816.12 Bukauskas T,Mickus R,Cereskevicius D,et al.Value of SerummiR-23a,miR-30d,and miR-146a Biomarkers in ST-ElevationMyocardial Infarction J.Med Sci Monit,2019,25(5):3925-3932.13 Kong Y,Li S,Zhang M,et al.Acupuncture ameliorates neuronal celldeath,inflammation,and ferroptosis and downregulated miR-23a-3pafter intracerebral hemorrhage in rats J.J Mol Neurosci,2021,71(9):1863-1875.14 Famakin BM,Vemuganti R.Toll-like receptor 4 signaling in focal ce-rebral ischemia:a focus on the neurovascular unit J.Mol Neurobi-ol,2020,57(6):2690-2701.15 Wang Q,Luo Q,Zhao YH,et al.Toll-like receptor-4 pathway as apossible molecular mechanism for brain injuries after subarachnoidhemorrhage J.Int J Neurosci,2020,130(9):953-964.16 Lai X,Xiong Y,Zhou J,et al.Verbascoside attenuates acute inflam-matory injury in experimental cerebral hemorrhage by suppressingTLR4 J.Biochem Biophys Res Commun,2019,519(4):721-726.17 Zhou XY,Li B,Yu M.The relationship between the expression of in-flammatory factor TLR4 and the permeability of blood-brain barrierin rats with intracerebral hemorrhage J.Chinese Journal of Integra-tive Medicine on Cardio-/Cerebrovascuiar Disease,2019,17(19):2945-2947.周喜燕,李彬,于淼.脑出血大鼠炎性因子TLR4的表达与血脑屏障通透性的关系探讨J.中西医结合心脑血管病杂志,2019,17(19):2945-2947.18 Gao Z,Huang D.lncRNAGAS5-mediated miR-23a-3p promotes in-flammation and cell apoptosis by targeting TLR4 in a cell model ofsepsis J.Mol Med Rep,2021,24(1):510-51819 Chen YC,Lee CP,Hsiao CC,et al.MicroRNA-23a-3p down-regula-tion in active pulmonary tuberculosis patients with high bacterial bur-den inhibits mononuclear cell function and phagocytosis throughTLR4/TNF-/TGF-1/IL-10 signaling via targeting IRF1/SP1 J.Int J Mol Sci,2020,21(22):8587-8603.(收稿日期：2022-11-19)2139