过表达CYLD对氧糖剥夺/复氧大鼠原代皮质神经元中NF-κB信号通路的影响

Effect of CYLD Overexpression on NF-κB Signaling Pathway after Oxygen-Glucose Deprivation/Reoxygenation in Rat Primary Cortical Neurons

脑卒中是目前导致我国人口寿命缩短的最主要原因之一。在缺血性脑卒中的局灶性缺血/再灌注后,受损脑组织中存在多种复杂的病理生理机制,核因子-κB(nuclear factor kappaB, NF-κB)信号通路参与的炎症反应是重要机制之一。相关研究显示,头帕肿瘤综合征蛋白(cylindromatosis, CYLD)可以参与NF-κB信号通路的调节。在脑缺血/再灌注损伤中,上调CYLD表达水平,对氧糖剥夺/复氧后NF-κB信号通路是否存在影响?如何影响?尚未见明确报道,这需要我们进一步探究。该研究通过上调CYLD在SD大鼠原代皮质神经元中的表达水平,观察其对氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation, OGD/R)后神经元中NF-κB信号通路的影响。使用过表达慢病毒感染体外培养的原代皮质神经元,采用免疫荧光实验鉴定神经元, Western blot及RT-qPCR验证CYLD的过表达情况, CCK-8实验检测细胞活力, Western blot检测p-IκBα的蛋白表达情况, RT-qPCR检测NF-κB p65的mRNA表达情况。结果显示,过表达CYLD慢病毒可有效提高神经元中CYLD的表达水平;过表达CYLD后,较对照组相比,神经元在氧糖剥夺/复氧处理后的活力有所增高, p-IκBα的表达水平有所下降,同时NF-κB p65的m RNA表达水平也明显降低。研究结果表明,在原代皮质神经元中过表达CYLD,能减轻氧糖剥夺/再复氧对神经元的损伤、抑制NF-κB信号通路的激活。

Stroke is one of the most leading cause of years of life lost(YLLs) in our population. After cerebral ischemia/reperfusion in ischemic stroke, there are many complex pathophysiological mechanisms in the damaged brain tissue. The inflammatory response in which nuclear factor-κB(NF-κB) signaling pathway participates is one of the most important mechanisms. Studies have shown that cylindromatosis(CYLD) can regulate NF-κB signaling pathway. However, can up-regulation of CYLD expression regulate the NF-κB signaling pathway in neurons after oxygen-glucose deprivation/reoxygenation? How to? So far no study has been reported yet, it needs to be researched. Our study up-regulates the expression of CYLD in neurons to investigate the effect of CYLD on NF-κB signaling pathway after oxygen-glucose deprivation/reoxygenation(OGD/R) in rat primary cortical neurons. Primary cortical neurons were infected by lentiviral vector-mediated overexpression of CYLD. Neurons were identified by immunofluorescence. The protein levels and mRNA levels were detected respectively by Western blot and RT-qPCR to confirm the expression of CYLD. Cell viability was detected by CCK-8 assay. The protein levels of p-IκBα were detected by Western blot. The mRNA levels of NF-κB p65 were detected by RT-qPCR. The results indicated that lentiviral vector-mediated CYLD overexpression can efficiently upregulate CYLD expression in neurons. After OGD/R, neuronal viability was enhanced significantly in CYLD-overexpressing neurons compared to control neurons. Meanwhile, the expression of p-IκBα and NF-κB p65 were decreased. The results suggested that overexpression of CYLD in primary cortical neurons could attenuate neuronal damage and inhibit the NF-κB signaling pathway induced by oxygen-glucose deprivation/reoxygenation.