银杏内酯K调节缺氧诱导因子-1α通路抑制氧糖剥离再灌注诱导的神经血管单元损伤

Ginkgolide K inhibited neurovascular unit injury against oxygen-glucose deprivation and reperfusion by regulating hypoxia-inducible factor-1αpathway

目的确定银杏内酯K(ginkgolide K,GK)对缺血性脑卒中诱导的神经血管单元损伤的保护作用及调节缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)通路机制。方法采用小鼠小胶质细胞BV-2及人脐静脉细胞融合细胞EA.hy926氧糖剥离再灌注(oxygen-glucose deprivation and reperfusion,OGD/R)模型模拟脑缺血引起的神经血管单元损伤。细胞OGD 4 h后再灌注并给予GK干预。给药24 h时检测BV-2细胞上清中炎症因子水平及EA.hy926细胞损伤情况;Western blot检测BV-2细胞给药1 h后p-Akt、p-Erk和6 h后HIF-1α水平,并应用LY294002验证,同时检测EA.hy926细胞给药1 h后p-Akt及6 h后HIF-1α等蛋白变化。结果GK明显抑制BV-2细胞上清TNF-α、IL-6、IL-1β分泌,激活p-Akt、抑制p-Erk并下调HIF-1α,且LY294002共同干预后,GK调节作用下降;同时,增加EA.hy926细胞活力和抑制LDH释放,上调p-Akt、HIF-1α、HO-1、VEGF并下调cleaved caspase-3/9水平。结论GK可多效应减少缺血性脑卒中诱导的神经血管单元损伤,其机制可能与针对不同细胞HIF-1α调节作用不同有关。

Aim To investigate the protective effects of ginkgolide K(GK)on neurovascular unit injured by ischemic stroke and the potential mechanism associated with hypoxia-inducible factor-1α(HIF-1α)pathway.Methods The BV-2 cells and EA.hy926 cells suffered from oxygen-glucose deprivation and reperfusion(OGD/R)were applied to mimic the injury of neurovascular unit induced by cerebral ischemia in vitro.After 4 h OGD insult,BV-2 cells and EA.hy926 cells received reperfusion and treated with GK.The levels of inflammatory cytokines in the supernatant of BV-2 cells were detected,while the protective effects of GK on EA.hy926 cells were also evaluated after GK administration for 24 h.The p-Akt and p-Erk expressions were examined by Western blot after 1 h of GK treatment,while HIF-1αwas detected after 6 h of GK treatment.In addition,PI3K inhibitor LY294002 was applied to further verify the potential mechanisms underlying the beneficial effects of GK.The expressions of p-Akt after 1 h of GK treatment,and the protein levels of HIF-1αpathway after 6 h of GK treatment were also analyzed by Western blot.Results GK significantly inhibited the levels of TNF-α,IL-6 and IL-1βin supernatant of BV-2 cells injured by OGD/R,through increasing p-Akt and decreasing p-Erk expressions,and then affecting HIF-1αpathway.In addition,LY294002 reduced the regulatory effect of GK.Furthermore,GK significantly improved viability and inhibited the release of LDH in supernatant of EA.hy926 cells suffered from OGD/R,and up-regulated the expressions of p-Akt,HIF-1α,HO-1 and VEGF,while cleaved caspase-3/9 was inhibited.Conclusions GK exerts multi-effects on reducing neurovascular unit injury induced by ischemic stroke,and the potential mechanism might be associated with the different regulatory effects of HIF-1αin different cells.