This study explored the protective function and mechanism of neocryptotanshinone (NEO) on cerebral ischemia. Lipopolysaccharide/γ-interferon(LPS/IFN-γ)was employed to mimic the polarization of mouse microglial cells BV2. After NEO treatment, the M1 polarization level of BV2 cells was identified using flow cytometry (FCM), fluorescent cell staining and enzyme linked immunosorbent assay(ELISA). Moreover, the mouse endothelial cells bEnd.3 were applied to be the study objects, which were intervened with NEO under the hypoxic condition. Thereafter, based on tubule formation assay and fluorescence staining, the tubule formation ability of bEnd.3 cells was detected. By adopting middle cerebral artery occlusion(MCAO) method, we constructed the mouse model of cerebral ischemia. After NEO intervention, the pathological changes of brain tissues were identified, while CD34 expression was measured by immunohistochemical (IHC) staining, nerve injury was detected by Nissl staining, and the changes in neurological behaviors of mice were also detected. Our results showed that NEO suppressed M1 polarization of BV2 cells, which exerted its effect through suppressing NF-κB and STAT3 signals, thereby decreasing the levels of iNOS, CD11b and inflammatory factors. NEO stimulated tubule formation in bEnd.3 cells based on the hypoxic situation, which exerted its effect through activating the Vascularendothelial growth factor-Vascular Endothelial Growth Factor Receptor 2-Notch homolog 1(VFGF-VEGFR2-Notch1) signal. Furthermore, NEO suppressed cerebral ischemia in mice and lowered the ischemic penumbra. NEO also improved the neurological behaviors of mice, increased the CD34 levels and decreased the expression of inflammatory factors. NEO has well protective effect against cerebral ischemia, and its mechanisms are related to suppressing M1 polarization of microglial cells and promoting cerebral angiogenesis, which are the mechanisms of NEO in treating ischemic encephalopathy.

中文翻译：

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新隐丹参酮抗脑缺血损伤的机制：抑制小胶质细胞M1极化，促进脑血管生成

本研究探讨新隐丹参酮（NEO）对脑缺血的保护作用及机制。采用脂多糖/γ-干扰素(LPS/IFN-γ)模拟小鼠小胶质细胞BV2的极化。NEO处理后，使用流式细胞术（FCM）、荧光细胞染色和酶联免疫吸附测定（ELISA）鉴定BV2细胞的M1偏振水平。并以小鼠内皮细胞bEnd.3为研究对象，在缺氧条件下对其进行NEO干预。此后，基于小管形成实验和荧光染色，检测bEnd.3细胞的小管形成能力。采用大脑中动脉闭塞（MCAO）方法构建小鼠脑缺血模型。NEO干预后，鉴定脑组织病理变化，同时通过免疫组织化学（IHC）染色检测CD34表达，通过尼氏染色检测神经损伤，并检测小鼠神经行为的变化。我们的结果表明，NEO 抑制 BV2 细胞的 M1 极化，通过抑制 NF-κB 和 STAT3 信号发挥作用，从而降低 iNOS、CD11b 和炎症因子的水平。NEO基于缺氧情况刺激bEnd.3细胞中的小管形成，通过激活血管内皮生长因子-血管内皮生长因子受体2-Notch同源物1(VFGF-VEGFR2-Notch1)信号发挥作用。此外，NEO 抑制小鼠脑缺血并降低缺血半暗带。NEO还可以改善小鼠的神经行为，增加CD34水平并减少炎症因子的表达。NEO对脑缺血具有良好的保护作用，其机制与抑制小胶质细胞M1极化、促进脑血管生成有关，这是NEO治疗缺血性脑病的机制。