Activation of NF-kappaB RelA deacetylated at the lysine residues, except the lysine 310, drives pro-apoptotic transcription in noxious brain ischemia. We showed that the sinergistic combination of the histone deacetilase inhibitor MS-275 with the sirtuin 1 activator resveratrol, at very low doses, restores normal RelA acetylation and elicit neuroprotection in mice subjected to transient middle cerebral artery occlusion (tMCAO) and primary cortical neurons exposed to oxygen-glucose-deprivation (OGD). The present study aims at corroborating the neuroprotective potential of the epigenetic treatment in a model of permanent brain ischemia and investigate its effect on post-ischemic inflammation and microglia activation. Male mice subjected to permanent occlusion of the distal MCAO (pMCAO) were treated with vehicle or MS-275 (20 μg/kg) and resveratrol (680 μg/kg) i.p. immediately after the ischemia. Microglia-containing mixed glial cultures were prepared from the brain of 1–3-day-old mice. Primary cortical neurons were prepared from 15-day-old embryonic mice. MS-275 and resveratrol in combination, but not individually, reduced infarct volume and neurological deficits evaluated 48 h after the pMCAO. At 24 h, the treatment inhibited the RelA binding to Nos2 promoter, reduced the elevated expression of Nos2, Il6, Il1b, Mrc1 and Ym1 and the leukocytes infiltration in the ischemic area. The effect was nonpermanent. The treatment did not limit the sustained leukocyte infiltration or Nos2 and Il1b transcription observed at 7 days. Though, it induced alternative activation markers of microglia/macrophages, Arg1, Ym1 and Fcgr2b that could be added to Mrc1, Tgfb1 and Trem2 spontaneously increased at 7 days after ischemia. At 24 hours the drug treatment quenched the microglia/macrophages activation in the ischemic cortical sections, as shown by the recovered ramified morphology and lowered iNOS or CD68 immunoreactivity in Iba1-positive cells. Both microglia and astrocytes in mixed glial cultures, but not pure astrocytes, displayed signs of activation and iNOS-immunoreactivity when treated with a conditioned medium (NCM) from OGD-exposed cortical neurons. The epigenetic drugs limited the OGD-NCM-mediated activation. Our findings indicate that single treatment with MS-275 and resveratrol can reduce stroke-mediated brain injury and inflammation observed 2 days after the pMCAO and put the rational to test repeated administration of the drugs. The anti-inflammatory property of MS-275 and resveratrol combination can be ascribed to both primary direct inhibition of microglia/macrophage activation and secondary glial/macrophages inhibition mediated by neuroprotection.

中文翻译：

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神经保护表观药物在永久性脑缺血小鼠模型中抑制炎症反应和小胶质细胞/巨噬细胞活化

除赖氨酸 310 外，在赖氨酸残基处脱乙酰化的 NF-kappaB RelA 的激活在有害脑缺血中驱动促凋亡转录。我们表明，组蛋白去乙酰化酶抑制剂 MS-275 与 Sirtuin 1 激活剂白藜芦醇的协同组合，在非常低的剂量下，可以恢复正常 RelA 乙酰化，并在遭受短暂大脑中动脉闭塞 (tMCAO) 和暴露的初级皮质神经元的小鼠中引发神经保护作用到缺氧葡萄糖剥夺（OGD）。本研究旨在证实表观遗传治疗在永久性脑缺血模型中的神经保护潜力，并研究其对缺血后炎症和小胶质细胞激活的影响。在缺血后立即用载体或 MS-275 (20 μg/kg) 和白藜芦醇 (680 μg/kg) ip 处理经受远端 MCAO (pMCAO) 永久闭塞的雄性小鼠。从 1-3 日龄小鼠的大脑中制备含有小胶质细胞的混合胶质细胞培养物。从 15 天大的胚胎小鼠制备初级皮质神经元。在 pMCAO 后 48 小时评估 MS-275 和白藜芦醇的组合，但不是单独的，减少梗死体积和神经功能缺损。24 h 时，处理抑制 RelA 与 Nos2 启动子的结合，降低 Nos2、Il6、Il1b、Mrc1 和 Ym1 的升高表达以及缺血区域的白细胞浸润。这种影响是非永久性的。治疗不会限制在 7 天观察到的持续白细胞浸润或 Nos2 和 Il1b 转录。尽管，它诱导了可添加到 Mrc1、Tgfb1 和 Trem2 的小胶质细胞/巨噬细胞的替代激活标志物 Arg1、Ym1 和 Fcgr2b，在缺血 7 天后自发增加。在 24 小时，药物治疗淬灭了缺血皮层切片中的小胶质细胞/巨噬细胞活化，如恢复的分枝形态所示，并降低了 Iba1 阳性细胞中的 iNOS 或 CD68 免疫反应性。当用来自暴露于 OGD 的皮质神经元的条件培养基 (NCM) 处理时，混合胶质细胞培养物中的小胶质细胞和星形胶质细胞，但不是纯星形胶质细胞，都显示出激活和 iNOS 免疫反应性的迹象。表观遗传药物限制了 OGD-NCM 介导的激活。我们的研究结果表明，用 MS-275 和白藜芦醇进行单次治疗可以减少在 pMCAO 后 2 天观察到的中风介导的脑损伤和炎症，并为测试重复给药提供了合理性。MS-275 和白藜芦醇组合的抗炎特性可归因于对小胶质细胞/巨噬细胞活化的初级直接抑制和由神经保护介导的次级神经胶质/巨噬细胞抑制。