摘要
目的:探讨微小RNA(miR)-181c对脑出血(ICH)大鼠神经的作用及靶向半胱氨酸-天冬氨酸蛋白酶-3(Caspase-3)信号通路调控ICH的分子机制。方法:选取60只SD雄性大鼠,采用随机数字表法分为假手术组(Sham组)、ICH组、表达miR-181c激动剂组(ICH+agomir组)和表达miR-181c抑制剂组(ICH+antagomir组),每组15只。提取各组大鼠脑组织RNA,采用实时荧光定量聚合酶链反应(RT-qPCR)技术检测miR-181c的表达水平;采用苏木精-伊红(HE)染色、尼氏染色、Zea Longa评分标准评估各组大鼠脑损伤及神经功能;采集各组大鼠脑脊液,通过酶联免疫吸附实验(ELISA)检测肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、3-硝基酪氨酸(3-NT)及4-羟基壬烯酸(4-HNE)的水平;提取各组大鼠脑组织蛋白,采用蛋白质免疫印迹实验检测Caspase-3剪切体/Caspase-3前体的表达水平。采用生物信息学分析、荧光素酶报告基因实验分析miR-181c对Caspase-3的靶向调控关系,两组间比较采用独立样本t检验。结果:RT-qPCR结果显示,ICH组miR-181c的表达水平低于Sham组(0.56±0.09比1.00±0.13,t=7.230,P<0.05)。HE染色结果显示,ICH组神经元细胞数量减少、体积变小、排列不齐,出现空泡样改变,核固缩、破裂,ICH+agomir组上述改变减轻。尼氏染色结果显示,ICH+agomir组神经元细胞浆中的尼氏小体数量多于ICH组(98.30±14.12比48.90±9.20,t=9.337,P<0.01),ICH+antagomir组与ICH组比较差异无统计学意义(P>0.05)。ELISA结果显示,ICH+agomir组大鼠脑组织TNF-α、IL-1β、3-NT和4-HNE的水平显著低于ICH组[TNF-α:(3.30±1.25)ng/ml比(8.22±1.66)ng/ml,t=9.120,P<0.01;IL-1β:(9.87±3.02)ng/ml比(15.14±2.93)ng/ml,t=8.183,P<0.01;3-NT:(23.39±3.44)pg/ml比(45.61±2.65)pg/ml,t=7.902,P<0.01;4-HNE:(10.10±1.75)pg/ml比(14.93±2.63)pg/ml,t=5.778,P<0.05]。蛋白质免疫印迹结果显示,ICH组Caspase-3剪切体/Caspase-3前体的表达水平显著高于Sham组(2.63±0.58比1.00±0.10,t=8.190,P<0.01),ICH+agomir组大鼠脑组织Caspase-3剪切体/Caspase-3前体的水平显著低于ICH组(1.52±0.37比2.63±0.58,t=5.398,P<0.05)。生物信息分析结果显示,miR-181c与Caspase-3有互补的核苷酸序列,miR-181c mimics+野生型-Caspase-3组细胞荧光素酶活性显著低于NC mimics+野生型-Caspase-3组(0.52±0.07比1.03±0.08,t=6.334,P<0.01)。结论:miR-181c通过靶向抑制Caspase-3信号通路,减轻大鼠ICH后神经功能损伤和继发性脑水肿,降低脑组织的氧化应激水平和炎性反应,对ICH大鼠具有保护作用。
Objective To investigate the effect of microRNA(miR)-181c on the nerves of rats with cerebral hemorrhage(ICH)and the molecular mechanism of targeting cysteinyl aspartate-specific protease(Caspase)-3 signaling pathway to regulate ICH.Methods Totally,60 SD male rats were divided into sham operation group(sham group),ICH group,miR-181c agonist group(ICH+agomir group)and miR-181c inhibitor group(ICH+antagomir group)by the random number table method.There were 15 rats in each group.RNA was extracted from brain tissue of rats in each group,and the expression level of miR-181c was detected by real-time fluorescent quantitative polymerase chain reaction(RT-qPCR).Hematoxylin-eosin staining,Nissl staining and Zea Longa scoring criteria were used to evaluate the brain injury and neurological function of rats in each group.The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),3-nitrotyrosine(3-NT)and 4-hydroxynonenal acid(4-HNE)in cerebrospinal fluid(CSF)were detected by enzyme-linked immunosorbent assay(ELISA).Western blotting was used to detect the expression of cleaved Caspase-3/pro-Caspase-3.Bioinformatics analysis and luciferase reporter gene experiment were used to analyze the targeted regulation relationship between miR-181c and Caspase-3.Independent sample t test was used for comparison between the two groups.Results RT-qPCR results showed that the expression level of miR-181c in ICH group was lower than that in sham group(0.56±0.09 vs.1.00±0.13,t=7.230,P<0.05).HE staining showed that the number of neurons in the ICH group decreased,the volume became smaller,the arrangement was irregular,and the neurons displayed vacuole-like changes,nuclear pyknosis and rupture,which were alleviated in the ICH+agomir group.Nissl staining showed that the number of Nissl bodies in the neuronal cytoplasm of ICH+agomir group was more than that of ICH group(98.30±14.12 vs.48.90±9.20,t=9.337,P<0.01),and there was no significant difference between ICH+antagomir group and ICH group.ELISA results showed that the levels of TNF-α,IL-1β,3-NT and 4-HNE in brain tissue of ICH+agomir group were significantly lower than those of ICH group[TNF-α:(3.30±1.25)vs.(8.22±1.66)ng/ml,t=9.120,P<0.01;IL-1β:(9.87±3.02)vs.(15.14±2.93)ng/ml,t=8.183,P<0.01;3-NT:(23.39±3.44)vs.(45.61±2.65)pg/ml,t=7.902,P<0.01;4-HNE:(10.10±1.75)vs.(14.93±2.63)pg/ml,t=5.778,P<0.05].Western blotting showed that the expression level of leaved Caspase-3/pro-Caspase-3 in ICH group was significantly higher than that in sham group(2.63±0.58 vs.1.00±0.10,t=8.190,P<0.01).The levels of leaved Caspase-3/pro-Caspase-3 in ICH+agomir group were significantly lower than those in ICH group(1.52±0.37 vs.2.63±0.58,t=5.398,P<0.05).Bioinformatics analysis showed that miR-181c and Caspase-3 had complementary nucleotide sequences.The luciferase activity in miR-181c mimics+WT-Caspase-3 group was significantly lower than that in NC mimics+WT-Caspase-3 group(0.52±0.07 vs.1.03±0.08,t=6.334,P<0.01).Conclusion MiR-181c can reduce neurological damage and secondary brain edema after ICH by targeting Caspase-3 signaling pathway,and reduce oxidative stress and inflammatory response in brain tissue,which has a protective effect on ICH rats.
作者
张霄
吴勇强
叶同
樊凯
郎张峰
Zhang Xiao;Wu Yongqiang;Ye Tong;Fan Kai;Lang Zhangfeng(Emergency Medicine Center,the First Hospital of Shanxi Medical University,Taiyuan 030001,China;Department of Neurosurgery,Xinghualing Central Hospital of Taiyuan,Taiyuan 030001,China)
出处
《中华实验外科杂志》
CAS
2024年第5期991-994,共4页
Chinese Journal of Experimental Surgery
