MK8719激活STAT6并促进抗炎型小胶质细胞活化在缺血性脑损伤大鼠中发挥保护作用的研究

MK8719 can alleviate cerebral ischemic injury in rats through STAT6 activating anti-inflammatory microglia

摘要:

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-Triphenyltetrazolium 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（signal 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（Enzyme 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-1β，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。

Abstract:

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 neuromotor 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 increased 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.