Key Laboratory of Pharmacodynamic Mechanism and Material Basis of Traditional Chinese Medicine, Shaanxi Provincial Administration of Traditional Chinese Medicine, Shaanxi University of Chinese Medicine, Xianyang, 712046, China.
Affiliated Hospital of Shaanxi University of Chinese Medicine, Xianyang, 712046, China.
Neurochem Res. 2021 Jul;46(7):1844-1858. doi: 10.1007/s11064-021-03305-1. Epub 2021 Apr 23.
Cerebral ischemia causes severe neurological disorders and neuronal dysfunction. Baicalin (BC), geniposide (GP), and their combination (BC/GP) have been shown to inhibit post-ischemic inflammatory injury by inhibiting the 5-LOX/CysLTs pathway. The aims of this study were to observe the inhibitory effects of BC/GP on the activation of microglial cells induced by oxygen glucose deprivation and reoxygenation (OGD/R) and to investigate whether the 5-LOX/LTB4 pathway was involved in these effects. Molecular docking showed that BC and GP exhibited considerable binding activity with LTB4 synthase LTA4H. BV-2 microglia were transfected with a 5-LOX overexpression lentiviral vector, and then OGD/R was performed. The effects of different concentrations of BC, GP, and BC/GP (6.25 μM, 12.5 μM, and 25 μM) on cell viability and apoptosis of microglia were evaluated by MTT and flow cytometry. The expression of TNF-α, IL-1β, NF-κB, and pNF-κB also was measured by ELISA, Western blots and immunofluorescence. Western blots and qRT-PCR analysis were used to determine the levels of CD11b, CD206, and 5-LOX pathway proteins. Results showed that BC, GP, and BC/GP reduced the apoptosis caused by OGD/R in a dose-dependent manner, and cell viability was significantly increased at a concentration of 12.5 μM. OGD/R significantly increased the release of TNF-α, IL-1β, NF-κB, pNF-κB, and CD11b. These effects were suppressed by BC, GP, and BC/GP, and the OGD/R-induced transfer of NF-κB p65 from the ctytoplasm to the nucleus was inhibited in microglia. Interestingly, the LTB4 inhibitor, U75302, exhibited the same effect. Also, BC, GP, and BC/GP significantly reduced the expression of 5-LOX pathway proteins. These results demonstrated that BC/GP inhibited OGD/R-induced polarization in BV2 microglia by regulating the 5-LOX/LTB4 signaling pathways and attenuating the inflammatory response. Our results supported the theoretical basis for additional in-depth study of the function of BC/GP and the value of determining its unique target, which might provide a new therapeutic strategy for ischemic cerebrovascular disease.
脑缺血导致严重的神经功能障碍和神经元功能障碍。黄芩苷（BC）、栀子苷（GP）及其联合用药（BC/GP）已被证明可通过抑制 5-LOX/CysLTs 通路来抑制缺血后炎症损伤。本研究旨在观察 BC/GP 对氧葡萄糖剥夺再复氧（OGD/R）诱导的小胶质细胞活化的抑制作用，并探讨 5-LOX/LTB4 通路是否参与这些作用。分子对接表明 BC 和 GP 与 LTB4 合酶 LTA4H 具有相当的结合活性。用 5-LOX 过表达慢病毒载体转染 BV-2 小胶质细胞，然后进行 OGD/R。通过 MTT 和流式细胞术评估不同浓度 BC、GP 和 BC/GP（6.25 μM、12.5 μM 和 25 μM）对小胶质细胞活力和凋亡的影响。ELISA、Western blot 和免疫荧光法测定 TNF-α、IL-1β、NF-κB 和 pNF-κB 的表达。Western blot 和 qRT-PCR 分析用于测定 CD11b、CD206 和 5-LOX 通路蛋白的水平。结果表明，BC、GP 和 BC/GP 呈剂量依赖性降低 OGD/R 引起的细胞凋亡，在 12.5 μM 浓度时细胞活力显著增加。OGD/R 显著增加了 TNF-α、IL-1β、NF-κB、pNF-κB 和 CD11b 的释放。BC、GP 和 BC/GP 抑制了这些作用，并且 OGD/R 诱导的 NF-κB p65 从小胶质细胞的细胞质向细胞核的转移被抑制。有趣的是，LTB4 抑制剂 U75302 也表现出相同的效果。此外，BC、GP 和 BC/GP 显著降低了 5-LOX 通路蛋白的表达。这些结果表明，BC/GP 通过调节 5-LOX/LTB4 信号通路和减轻炎症反应，抑制 OGD/R 诱导的 BV2 小胶质细胞极化。我们的研究结果支持了进一步深入研究 BC/GP 功能及其确定独特靶点价值的理论基础，这可能为缺血性脑血管病提供新的治疗策略。
