目的:观察氧连接N-乙酰葡萄糖胺(O-linked N-acetylglucosamine,O-GlcNAc)水解酶抑制剂MK8719在脑缺血损伤大鼠中的作用.方法:建立大鼠大脑中动脉梗塞模型模拟脑缺血再灌注损伤,通过2,3,5-三苯基氯化四氮唑(2,3,5-Triphen-yltetrazolium chloride,TTC)染色评估脑梗死体积,并通过改良的神经损伤评分量表评估大鼠的神经运动障碍减轻,HE和尼式染色用于评估脑损伤后组织改变,组织免疫荧光染色用于评估抗炎型小胶质细胞活化情况.体外培养BV2小胶质细胞并建立氧糖剥夺/复糖复氧模型模拟缺血缺氧再灌注损伤,Western blotting检测总蛋白及核蛋白中的信号转导及转录激活蛋白6(sig-nal transducer and activator of transcription 6,STAT6)、磷酸化STAT6及 O-GlcNAc糖基化STAT6表达.酶联免疫吸附检测(En-zyme linked immunosorbent assay,ELISA)评估小胶质细胞释放的炎性因子白细胞介素-6(interleukin-6,IL-6)、白细胞介素-1β(interleukin-1 β,IL-1β)、白细胞介素-10(interleukin-10,IL-10)和转化生长因子-β(transforming growth factor-β,TGF-β)量.结果:MK8719给药组大鼠梗死灶体积减小,神经运动障碍,抗炎型小胶质细胞比例增加;在体外培养的小胶质细胞中,MK8719组的O-GlcNAc糖基化STAT6、磷酸化STAT6及核STAT6表达增加(P＜0.001),促炎因子(IL-6及IL-1β)释放减少(P＜0.001),抗炎因子(IL-10及TGF-β)释放增加(P＜0.001).结论:MK8719对脑缺血损伤大鼠有保护作用,其作用机制可能与STAT6激活诱导的抗炎型小胶质细胞活化有关.
MK8719 can alleviate cerebral ischemic injury in rats through STAT6 activating anti-inflammatory microglia
Objective:To investigate the effects of MK8719(an inhibitor of O-linked N-acetylglucosamine[O-GlcNAc]hydrolase)in a rat model of cerebral ischemic injury.Methods:A rat middle cerebral artery occlusion model was established to simulate cerebral ischemia/reperfusion injury.We assessed cerebral infarct volume with 2,3,5-triphenyltetrazolium chloride staining;evaluated neuro-motor function using the modified Neurological Severity Score;observed cerebral tissue changes after injury with Nissl staining and HE staining;and assessed the activation of anti-inflammatory microglia by immunofluorescence assay.An in-vitro BV2 microglia-based oxygen and glucose deprivation/re-oxygenation model was established to simulate ischemia-hypoxia/reperfusion injury.We measured the levels of total signal transducer and activator of transcription 6(STAT6),nuclear STAT6,phosphorylated STAT6,and O-GlcNAcylated STAT6 by Western blot;and measured the levels of pro-inflammatory interleukin(IL)-6 and IL-1β and anti-inflammatory IL-10 and transforming growth factor-β(TGF-β)released from microglia by enzyme-linked immunosorbent assay.Results:The model rats treated with MK8719 showed a significantly smaller cerebral infarct size,significantly alleviated neurological deficits,and a significantly higher proportion of anti-inflammatory microglia.BV2 cells treated with MK8719 showed significantly in-creased expression of O-GlcNAcylated STAT6,phosphorylated STAT6(P＜0.001),and nuclear STAT6,significantly reduced release of IL-6 and IL-1β(P＜0.001),and significantly increased release of IL-10 and TGF-β(P＜0.001).Conclusion:MK8719 can produce neuroprotective effects in rats with cerebral ischemic injury,which may be mediated by STAT6 activating anti-inflammatory microglia.
cerebral ischemic strokeMK8719signal transducer and activator of transcription 6microglia
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