miR-150-5p抑制TLR-5/NF-κB p65信号通路对大脑中动脉阻塞模型大鼠的神经保护效应

文题释义： 缺血性脑梗死：临床常见的一种中枢神经系统疾病，是由于脑组织局部供血动脉血流的减少或停止，造成脑组织缺血、缺氧导致脑组织坏死，具有较高的死亡率和致残率，严重威胁人类的健康和生存质量。 微小RNA：是一类长度为18-25 nt的非编码单链小分子RNA，广泛存在于从病毒到人类的各种生物中，能够与mRNA互补结合调控蛋白编码基因的表达，与细胞的生物学行为密切相关。 背景：缺血性脑梗死的病变组织中发生炎症反应，且miR-150-5p表达明显下降，miR-150-5p是否经Toll样受体5/核因子κB途径抑制炎症因子释放并减轻缺血性脑梗死组织损伤尚不清楚。 目的：探讨微小RNA-150-5p(miR-150-5p)在大鼠缺血性脑梗死中的作用及初步机制。 方法：①构建大脑中动脉阻塞模型大鼠，建模后将大鼠分为5组：对照组、miR-150-5p agomir 组、agomir control、miR-150-5p antagomir组和antagomir control组；②对照组大鼠给予生理盐水脑室注射，后4组大鼠脑室分别给予miR150-5p agomir(miR150-5p激动剂)、agomir阴性对照、miR150-5p antagomir (miR150-5p抑制剂)和antagomir阴性对照；③干预7 d后进行神经功能缺损程度(mNNS)评分，磁共振检测法测量脑梗死体积，苏木精-伊红染色观察脑组织病理学变化，qRT-qPCR法、ELISA法和免疫印迹法检测分别检测脑组织中miR-150-5p、白细胞介素6、肿瘤坏死因子α、Toll样受体5和核因子κB p65表达情况，通过检索生物信息学网站Targetscan预测miR-150-5p与Toll样受体5的结合位点，荧光素酶试验验证二者的靶向关系。 结果与结论：①与对照组比较，miR-150-5p agomir组大鼠神经功能损伤评分、脑组织中白细胞介素6、肿瘤坏死因子α水平及Toll样受体5、核因子κB p65蛋白表达明显降低(P < 0.05)；miR-150-5p antagomir组生理评分及生化指标均明显升高(P < 0.05)；②苏木精-伊红染色显示，对照组神经细胞排列紊乱、轮廓模糊不清，神经细胞明显变性坏死；上述病理变化在miR-150-5p agomir组明显减轻，而在miR-150-5p antagomir组中明显加重。而agomir control 组和antagomir control组与对照组各指标比较差异均无显著差异(P > 0.05)；③TargerScan网站预测结果和荧光素酶报告基因分析显示，miR-150-5p与Toll样受体5存在靶向结合位点；④结果证实，miR-150-5p可抑制缺血所致脑损伤过程中Toll样受体5/核因子κB p65炎性信号通路，降低炎性反应，从而减轻神经功能损害，发挥保护作用。 ORCID: 0000-0003-4635-0842(谢媛媛) 中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Mi R-150-5p protects rats from middle cerebral artery occlusion by inhibiting the Toll-like receptor-5/nuclear factor-kappa B p65 signaling pathway

BACKGROUND:There is an inflammatory response in the lesion tissue of ischemic cerebral infarction,and the expression of miR-150-5p is significantly decreased.Whether miR-150-5p inhibits the release of inflammatory factors and alleviates the injury of ischemic cerebral infarction tissue through the Toll-like receptor-5/nuclear factor-κB pathway remains unclear.OBJECTIVE:To investigate the role and preliminary mechanism of miR-150-5p in ischemic cerebral infarction in rats.METHODS:(1)The rat models of middle cerebral artery occlusion were constructed and the rat models were divided into five groups:control,miR-150-5p agomir,agomir control,miR-150-5p antagomir and antagomir control groups.(2)The rats in the control group was given the intracerebroventricular injection of normal saline,and the rats in the latter four groups were given the intracerebroventricular injection of miR-150-5p agomir(miR-150-5p agonist),agomir negative control,miR150-5p antagomir(miR150-5p inhibitor)and antagomir negative control,respectively.(3)After 7 days,the brain was graded by modified neurological severity score,the cerebral infarct volume was measured by MRI,and the histopathological changes were observed by hematoxylin-eosin staining.The expression levels of miR-150-5p,interleukin-6,tumor necrosis factor-α,Toll-like receptor-5 and nuclear factor-κB p65 in brain tissues were detected by qRT-PCR,ELISA and western blot assay,respectively.The target relationship between miR150-5p and Toll-like receptor-5 was verified by luciferase assay by retrieving the bioinformatics website Targetscan to predict the binding sites of miR-150-5p and Toll-like receptor-5.RESULTS AND CONCLUSION:(1)Compared with the control group,the modified neurological severity score,and levels of interleukin-6,tumor necrosis factor-α,Toll-like receptor-5 and nuclear factor-κB p65 proteins were significantly decreased in the miR-150-5p agomir group(P<0.05).The physiological score and biochemical indexes in the miR-150-5p antagomir group were significantly increased(P<0.05).(2)Hematoxylin-eosin staining results showed that the nerve cells in the control group were disordered and ill-defined,and the nerve cells were obviously degenerated and necrotic.The above pathological changes were significantly alleviated in the miR-150-5p agomir group and aggravated in the miR-150-5p antagomir group.There were no significant differences in the indexes between agomir control,the antagomir control and control groups(P>0.05).(3)TargerScan website prediction results and luciferase reporter gene analysis results showed that miR-150-5p and Toll-like receptor-5 had a targeted binding site.(4)These results imply that miR-150-5p can inhibit the inflammatory signaling pathway of Toll-like receptor-5/nuclear factor-κB p65 in brain injury caused by ischemia and reduce the inflammatory response,thereby alleviating the damage of nerve function and playing a protective role.