氯喹通过抑制NF-κB和MAPK信号通路减轻脂多糖诱导的BV2小胶质细胞炎症反应

Chloroquine attenuates lipopolysaccharide-stimulated BV2 microglial activation by inhibiting NF-κB and MAPK signaling pathways

目的:探究氯喹(CQ)对脂多糖(LPS)刺激的BV2小胶质细胞活化的抑制作用及可能机制。方法:将小鼠BV2小胶质细胞分为对照组、LPS组和LPS+CQ组。LPS+CQ组预先给予CQ(10μmol/L)处理30 min再给予LPS刺激,在LPS组和LPS+CQ组中给予LPS(500μg/L)刺激后,各组细胞分别培养30 min、6 h和24 h。倒置显微镜下观察BV2细胞的形态学改变;RT-qPCR和ELISA法检测白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)的mRNA和蛋白表达水平来评估BV2细胞的活化情况;用免疫荧光染色检测NF-κB蛋白核转移情况;用Western blot检测核因子κB抑制蛋白α(IκB-α)蛋白的表达情况及c-Jun氨基末端激酶(JNK)和p38蛋白的磷酸化水平。结果:LPS刺激后,BV2细胞形态由圆形或椭圆形向多极或纺锤样转变,而预先给予CQ能抑制BV2细胞的形态转变。LPS刺激后,BV2细胞中TNF-α和IL-6的mRNA水平和培养上清液中的蛋白水平明显增加,但预先给予CQ则明显抑制TNF-α和IL-6的mRNA和蛋白表达,可见CQ能减轻LPS诱导的BV2细胞炎症反应。此外,LPS刺激后,BV2细胞内IκB-α蛋白大量降解,细胞核内的NF-κB蛋白显著增多,MAPK信号通路中JNK和p38蛋白磷酸化水平明显升高;而预先给予CQ可显著减少IκB-α蛋白的降解,明显抑制NF-κB蛋白向细胞核内转移,显著降低JNK和p38蛋白的磷酸化水平,可见CQ能抑制LPS诱导下BV2细胞内NF-κB和MAPK信号通路的激活。结论:氯喹显著减轻LPS诱导的BV2细胞炎症反应,其机制与抑制NF-κB和MAPK信号通路有关。

AIM:To investigate the inhibitory effects of chloroquine(CQ)on lipopolysaccharide(LPS)-stimulated BV2 microglial activation and to study the possible mechanism.METHODS:The BV2 microglia were assigned into 3 groups:control group,LPS group and LPS+CQ group.The BV2 microglia were pretreated with or without CQ(10μmol/L)for 30 min and then stimulated with LPS(500μg/L)for 30 min,6 h or 24 h.The morphological changes of the BV2 cells were visualized under microscope.To evaluate the microglial activation,the mRNA and protein levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were measured by RT-qPCR and ELISA,respectively.The nuclear localization of nuclear factor(NF)-κB was examined by immunofluorescence staining.The protein expression of inhibitor of nuclear factorκB(IκB)-αand the phosphorylation levels of c-Jun N-terminal kinase(JNK)and p38 were determined by Western blot.RESULTS:LPS stimulation led to the morphological changes of the BV2 cells from circular or oval shape to spindle shaped or multipolar morphology.Pretreatment with CQ remarkedly prevented the morphological changes of the BV2 cells from normal to activated shape.Stimulation of BV2 cells with LPS led to the increases both at mRNA and protein levels of TNF-αand IL-6.However,the production of these proinflammatory mediators was significantly inhibited by CQ,indicating that CQ remarkably attenuated LPS-stimulated BV2 microglial activation.After exposure to LPS,the protein of IκB-αwas remarkably degraded,the level of NF-κB protein were obviously increased in the nucleus,the phosphorylation of JNK and p38 was markedly up-regulated.However,pretreatment with CQ obviously decreased the degration of IκB-α,remarkably inhibited the translocation of NF-κB from cytoplasm to the nucleus,and markedly decreasd the phosphorylation levels of JNK and p38,indicating that CQ inhibited the activation of NF-κB and mitogen-activated protein kinase(MAPK)signaling pathways induced by LPS in BV2 cells.CONCLUSION:Chloroquine remarkably attenuates the inflammation of LPS-stimulated BV2 microglia via inhibiting NF-κB and MAPK signaling pathways.