Lin Qing, Wang Weili, Yang Liping, Duan Xiaohua
Department of Pharmacology, Yunnan University of Chinese Medicine, Kunming, Yunnan 650500, P.R. China.
Yunnan Key Laboratory of Dai and Yi Medicine, University of Chinese Medicine, Kunming, Yunnan 650500, P.R. China.
Exp Ther Med. 2021 Mar;21(3):252. doi: 10.3892/etm.2021.9684. Epub 2021 Jan 24.
Damage to the blood-brain barrier (BBB) during the process of cerebral ischemic injury is a key factor that affects the treatment of this condition. The present study aimed to assess the potential effects of 4-methoxybenzyl alcohol (4-MA) on brain microvascular endothelial cells (bEnd.3) against oxygen-glucose deprivation/reperfusion (OGD/Rep) using an model that mimics ischemia/reperfusion injury. In addition, the present study aimed to explore whether this underlying mechanism was associated with the inhibition of pro-inflammatory factors and the activation status of the PI3K/Akt signaling pathway. bEnd.3 cells were subjected to OGD/Rep-induced injury before being treated with 4-MA, following which cell viability, lactate dehydrogenase (LDH) release and levels of nitric oxidase (NO) were detected by colorimetry, pro-inflammatory factors including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6, were detected by ELISA. The expression levels of occluding and claudin-5were evaluated by immunofluorescence staining. The expression levels of AKT, phosphorylated (p)-Akt, endothelial nitric oxide synthase (eNOS) and p-eNOS were also measured by western blot analysis. After bEnd.3 cells were subjected to OGD/Rep-induced injury, cell viability and NO levels were significantly decreased, whilst LDH leakage and inflammatory factor (TNF-α, IL-1β and IL-6) levels were significantly increased. Treatment with 4-MA significantly ameliorated cell viability, LDH release and the levels of NO and pro-inflammatory factors TNF-α, IL-1β and IL-6 as a result of OGD/Rep. Furthermore, treatment with 4-MA upregulated the expression of occludin, claudin-5, Akt and eNOS, in addition to increasing eNOS and AKT phosphorylation in bEnd.3 cells. These results suggest that 4-MA can alleviate OGD/Rep-induced injury in bEnd.3 cells by inhibiting inflammation and by activating the PI3K/AKT signaling pathway as a possible mechanism. Therefore, 4-MA can serve as a potential candidate for treating OGD/Rep-induced injury.
脑缺血损伤过程中血脑屏障（BBB）的破坏是影响该病症治疗的关键因素。本研究旨在使用模拟缺血/再灌注损伤的模型，评估4-甲氧基苄醇（4-MA）对脑微血管内皮细胞（bEnd.3）抗氧糖剥夺/再灌注（OGD/Rep）的潜在作用。此外，本研究旨在探讨这一潜在机制是否与促炎因子的抑制以及PI3K/Akt信号通路的激活状态有关。在使用4-MA处理之前，bEnd.3细胞先受到OGD/Rep诱导的损伤，之后通过比色法检测细胞活力、乳酸脱氢酶（LDH）释放和一氧化氮（NO）水平，通过酶联免疫吸附测定（ELISA）检测包括肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-1β和IL-6在内的促炎因子。通过免疫荧光染色评估闭合蛋白和Claudin-5的表达水平。还通过蛋白质免疫印迹分析测量AKT、磷酸化（p）-Akt、内皮型一氧化氮合酶（eNOS）和p-eNOS的表达水平。bEnd.3细胞受到OGD/Rep诱导的损伤后，细胞活力和NO水平显著降低，而LDH泄漏和炎症因子（TNF-α、IL-1β和IL-6）水平显著升高。4-MA处理显著改善了OGD/Rep导致的细胞活力、LDH释放以及NO和促炎因子TNF-α、IL-1β和IL-6的水平。此外，4-MA处理上调了bEnd.3细胞中闭合蛋白、Claudin-5、Akt和eNOS的表达，同时增加了eNOS和AKT的磷酸化。这些结果表明，4-MA可能通过抑制炎症和激活PI3K/AKT信号通路来减轻OGD/Rep诱导的bEnd.3细胞损伤。因此，4-MA可作为治疗OGD/Rep诱导损伤的潜在候选药物。
