AIMS Ischemic stroke has become one of the main causes of death worldwide. MicroRNAs (miRNAs) have been implicated in cerebral ischemia-reperfusion (I/R) injury and could serve as therapeutic targets. 5-Lipoxygenase (5-LOX) is a key enzyme in the biosynthesis of leukotrienes and has been implicated in inflammatory central nerve system disorders. The objective of this study was to explore the neuroprotective effects of miR-193b-3p against focal cerebral I/R injury in rats by regulating 5-LOX expression. METHODS AND MATERIALS Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion and reperfusion injury. The level of miR-193b-3p expression was observed in the rat cortical peri-infarct region after focal cerebral I/R injury. Bioinformatics analysis was used to predict the binding sites of miR-193b-3p, and a dual-luciferase reporter gene assay was applied to verify the potential interaction between 5-LOX mRNA and miR-193b-3p. Then, rats were injected with a miR-193b-3p agomir (modified and enhanced mimic) or antagomir (modified and enhanced inhibitor) in the right lateral ventricle of the brain. Neurological deficit scores, infarct volumes, neuron damage and 5-LOX enzymatic activity and expression were measured. In an in vitro experiment, cultured PC12 cells were exposed to oxygen-glucose deprivation and reperfusion (OGD/R). OGD/R-induced cells were treated with a miR-193b-3p mimic or inhibitor and 5-LOX siRNA. Cell viability, lactate dehydrogenase release, apoptosis rate and 5-LOX expression were evaluated. RESULTS The level of miR-193b-3p expression was increased in the cortical peri-infarct region of rats with cerebral focal I/R injury. The results of the dual-luciferase reporter gene assay showed that a miR-193b-3p binding site was located in the 3' untranslated region (3'UTR) of 5-LOX mRNA. Neurological deficit scores, infarct volumes and neuronal injury were alleviated by miR-193b-3p agomir treatment but aggravated by miR-193b-3p antagomir. Furthermore, leukotriene B4, cysteinyl-leukotrienes and 5-LOX expression in the cortical peri-infarct region of rats with focal cerebral I/R injury were also downregulated by miR-193b-3p agomir treatment but upregulated by miR-193b-3p antagomir. In PC12 cells, miR-193b-3p mimic significantly decreased OGD/R-induced cell death and reduced lactate dehydrogenase release and 5-LOX expression. In contrast, miR-193b-3p inhibitor exacerbated OGD/R-induced injury in PC12 cells. Additionally, the in vitro effects of miR-193b-3p inhibitor on OGD/R-induced cell injury were partially reversed by 5-LOX siRNA treatment. CONCLUSION MiR-193b-3p has a potentially neuroprotective effect on focal cerebral I/R-induced injury by inhibiting 5-LOX expression.

中文翻译：

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MicroRNA-193b-3p通过抑制5-脂氧合酶的表达减轻大鼠局灶性脑缺血和再灌注损伤。

AIMS缺血性中风已成为全球范围内主要的死亡原因之一。MicroRNA（miRNA）与脑缺血-再灌注（I / R）损伤有关，可作为治疗靶标。5-Lipoxygenase（5-LOX）是白三烯生物合成中的关键酶，与炎症性中枢神经系统疾病有关。这项研究的目的是通过调节5-LOX表达来探讨miR-193b-3p对大鼠局灶性I / R损伤的神经保护作用。方法和材料成年雄性Sprague-Dawley大鼠经历短暂的大脑中动脉阻塞和再灌注损伤。在局灶性脑I / R损伤后的大鼠皮层梗死区域中观察到了miR-193b-3p表达水平。使用生物信息学分析来预测miR-193b-3p的结合位点，并采用双荧光素酶报告基因检测了5-LOX mRNA与miR-193b-3p之间的潜在相互作用。然后，在大脑的右心室向大鼠注射miR-193b-3pagomir（修饰和增强的模拟物）或antagomir（修饰和增强的抑制剂）。测量神经功能缺损评分，梗塞体积，神经元损伤和5-LOX酶活性和表达。在体外实验中，将培养的PC12细胞暴露于氧葡萄糖剥夺和再灌注（OGD / R）。用miR-193b-3p模拟或抑制剂和5-LOX siRNA处理OGD / R诱导的细胞。评价细胞活力，乳酸脱氢酶释放，细胞凋亡率和5-LOX表达。结果miR-193b-3p表达水平在脑局灶性I / R损伤大鼠的皮层梗死区域中升高。双荧光素酶报告基因测定的结果表明，miR-193b-3p结合位点位于5-LOX mRNA的3'非翻译区（3'UTR）。miR-193b-3p agomir治疗可减轻神经功能缺损评分，梗塞体积和神经元损伤，但miR-193b-3p antagomir可加重这种情况。此外，miR-193b-3pagomir处理还下调了局灶性脑I / R损伤大鼠皮质梗死区域的白三烯B4，半胱氨酰-白三烯和5-LOX表达，而miR-193b-3p antagomir上调了白三烯B4，半胱氨酸-白三烯和5-LOX的表达。在PC12细胞中，miR-193b-3p模拟物显着降低了OGD / R诱导的细胞死亡，并减少了乳酸脱氢酶的释放和5-LOX的表达。相反，miR-193b-3p抑制剂会加剧OGD / R诱导的PC12细胞损伤。另外，5-LOX siRNA处理可部分逆转miR-193b-3p抑制剂对OGD / R诱导的细胞损伤的体外作用。结论MiR-193b-3p通过抑制5-LOX表达对局灶性脑I / R损伤具有潜在的神经保护作用。