To evaluate the potential efficacy of Triptolide (TP) on cerebral ischemia/reperfusion injury (CIRI) and to uncover the underlying mechanism through which TP regulates CIRI. We constructed a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model to simulate CIRI, and established a lipopolysaccharide (LPS)-stimulated BV-2 cell model to mimic the inflammatory state during CIRI. The neurological deficits score (NS) of mice were measured for assessment of neurologic functions. Both the severity of cerebral infarction and the apoptosis level in mouse brain tissues or cells were respectively evaluated using corresponding techniques. The expression levels of Ionized calcium binding adapter molecule 1 (IBA-1), Inductible Nitric Oxide Synthase (iNOS), Arginase 1 (Arg-1), Tumor necrosis factor-α (TNF-α), Interleukin 1 (IL-1), Cysteine histoproteinase S (CTSS), Fractalkine, chemokine C-X3-C motif receptor 1 (CX3CR1), BCL-2-associated X protein (BAX), and antiapoptotic proteins (Bcl-2) were detected using immunofluorescence, qRT-PCR as well as Western blot, respectively. Relative to the Sham group, treatment with TP attenuated the increased NS, infarct area and apoptosis levels observed in MCAO/R mice. Upregulated expression levels of IBA-1, iNOS, Arg-1, TNF-α and IL-1β were found in MCAO/R mice, while TP suppressed iNOS, TNF-α and IL-1β expression, and enhanced Arg-1 expression in both MCAO/R mice and LPS-stimulated BV-2 cells. Besides, TP inhibited the CTSS/Fractalkine/CX3CR1 pathway activation in both MCAO/R mice and LPS-induced BV-2 cells, while overexpression of CTSS reversed such effect. Co-culturing HT-22 cells with TP+LPS-treated BV-2 cells led to enhanced cell viability and decreased apoptosis levels. However, overexpression of CTSS further aggravated HT-22 cell injury. TP inhibits not only microglia polarization towards the M1 phenotype by suppressing the CTSS/Fractalkine/CX3CR1 pathway activation, but also HT-22 apoptosis by crosstalk with BV-2 cells, thereby ameliorating CIRI. These findings reveal a novel mechanism of TP in improving CIRI, and offer potential implications for addressing the preventive and therapeutic strategies of CIRI.

中文翻译：

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雷公藤甲素通过调节Fractalkine/CX3CR1信号通路减轻脑缺血/再灌注损伤

评估雷公藤甲素 (TP) 对脑缺血/再灌注损伤 (CIRI) 的潜在疗效，并揭示 TP 调节 CIRI 的潜在机制。我们构建了大脑中动脉闭塞/再灌注（MCAO/R）小鼠模型来模拟 CIRI，并建立了脂多糖（LPS）刺激的 BV-2 细胞模型来模拟 CIRI 期间的炎症状态。测量小鼠的神经缺陷评分（NS）以评估神经功能。采用相应技术分别评价小鼠脑梗死的严重程度和脑组织或细胞的凋亡水平。离子钙结合接头分子 1 (IBA-1)、诱导型一氧化氮合酶 (iNOS)、精氨酸酶 1 (Arg-1)、肿瘤坏死因子-α (TNF-α)、白细胞介素 1 (IL-1) 的表达水平使用免疫荧光、qRT-PCR 检测半胱氨酸组织蛋白酶 S (CTSS)、Fractalkine、趋化因子 C-X3-C 基序受体 1 (CX3CR1)、BCL-2 相关 X 蛋白 (BAX) 和抗凋亡蛋白 (Bcl-2)以及蛋白质印迹。相对于 Sham 组，TP 治疗减弱了 MCAO/R 小鼠中观察到的 NS、梗塞面积和细胞凋亡水平的增加。 MCAO/R 小鼠中 IBA-1、iNOS、Arg-1、TNF-α 和 IL-1β 表达水平上调，而 TP 抑制 iNOS、TNF-α 和 IL-1β 表达，并增强 MCAO/R 小鼠中 Arg-1 表达。 MCAO/R 小鼠和 LPS 刺激的 BV-2 细胞。此外，TP 抑制 MCAO/R 小鼠和 LPS 诱导的 BV-2 细胞中 CTSS/Fractalkine/CX3CR1 通路的激活，而 CTSS 的过表达则逆转了这种作用。将 HT-22 细胞与 TP+LPS 处理的 BV-2 细胞共培养可增强细胞活力并降低细胞凋亡水平。然而，CTSS的过度表达进一步加剧了HT-22细胞的损伤。 TP 不仅通过抑制 CTSS/Fractalkine/CX3CR1 通路激活来抑制小胶质细胞向 M1 表型极化，而且通过与 BV-2 细胞串扰来抑制 HT-22 凋亡，从而改善 CIRI。这些发现揭示了 TP 改善 CIRI 的新机制，并为解决 CIRI 的预防和治疗策略提供了潜在的启示。