Anti-inflammatory effects of vinpocetine in LPS-stimulated microglia via activation of AMPK

Abstract Microglia are the resident immune cells in the central nervous system (CNS), which play important roles in the repair of neuroinflammatory injury. The present study investigated the anti-neuroinflammatory effects of vinpocetine induced by lipopolysaccharide (LPS) in BV2 microglia. BV2 microglia were pretreated with vinpocetine, and then stimulated with LPS (100 ng/mL). The cytotoxicity of BV2 microglia was assessed by MTT assay. The expression levels of nitrite oxide were measured by Griess assay. Proinflammatory cytokines and mediators were determined by Western blot, ELISA, or quantitative real-time PCR. Vinpocetine significantly decreased the generation of nitric oxide-inducible nitric oxide synthase (iNOS), cyclooxygenase- (COX-) 2 in a dose-dependent manner. In addition, vinpocetine decreased the production of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-6 and IL-1β. Furthermore, it was observed that phosphorylation levels of AMPK (Thr-172) decreased in LPS-stimulated BV2 microglia. Vinpocetine treatment increased AMPK phosphorylation in LPS-stimulated BV2 microglia. AMPK inhibition by siRNA blocked the anti-inflammatory effects of vinpocetine induced by LPS in BV2 microglia. The overall results demonstrate that vinpocetine has anti-inflammatory effects on LPS-stimulated BV2 microglia via inducing phosphorylation of AMPK, suggesting that vinpocetine is a potential therapeutic agent in neuroinflammatory injury.

摘要：小胶质细胞（Microglia）是中枢神经系统（CNS）内的固有免疫细胞，在神经炎性损伤修复中发挥关键作用。本研究探讨了长春西汀（vinpocetine）对脂多糖（lipopolysaccharide, LPS）诱导的BV2小胶质细胞的抗神经炎性效应。将BV2小胶质细胞先用长春西汀预处理，随后以100 ng/mL的脂多糖进行刺激。采用MTT法检测BV2小胶质细胞的细胞毒性，通过Griess法检测一氧化氮的表达水平；采用蛋白质印迹法（Western blot）、酶联免疫吸附实验（ELISA）或实时定量聚合酶链反应（quantitative real-time PCR）检测促炎细胞因子与介质的表达情况。长春西汀可呈剂量依赖性显著降低一氧化氮、诱导型一氧化氮合酶（inducible nitric oxide synthase, iNOS）及环氧合酶-2（cyclooxygenase-2, COX-2）的生成量。此外，长春西汀可减少肿瘤坏死因子α（tumor necrosis factor-α, TNF-α）、白细胞介素-6（interleukin-6, IL-6）及白细胞介素-1β（interleukin-1β, IL-1β）等促炎细胞因子的产生。进一步研究发现，脂多糖刺激的BV2小胶质细胞中腺苷酸活化蛋白激酶（AMP-activated protein kinase, AMPK）的Thr-172位点磷酸化水平降低，而长春西汀处理可上调该磷酸化水平。通过小干扰RNA（siRNA）抑制AMPK的表达，可阻断长春西汀对脂多糖诱导的BV2小胶质细胞的抗炎作用。综上，本研究结果表明，长春西汀可通过诱导AMPK磷酸化发挥抗脂多糖诱导的BV2小胶质细胞炎症反应的作用，提示长春西汀是治疗神经炎性损伤的潜在候选治疗药物。