藏红花素减轻皮质神经元缺氧复氧损伤与抑制TLR4/NF-κB信号通路有关

Crocin attenuates hypoxia-reoxygenation injury in cortical neurons by inhibiting TLR4/NF-κB pathway

[目的]探讨藏红花素对皮质神经元缺氧复氧损伤的影响及其分子机制。[方法]分离并培养SD大鼠皮质神经元,设对照组、模型组、藏红花素(50 mg/L)组、藏红花素(50 mg/L)+脂多糖(TLR4激活剂,100μg/L)组。对照组常规培养,模型组建立缺氧4 h复氧24 h模型,藏红花素组和藏红花素+脂多糖组分别给药干预24 h后造模。通过CCK-8法、Annexin V-FITC/PI双染法检测神经元活力和凋亡率,ELISA法检测培养液中炎症因子水平,Western blot检测Toll样受体4(TLR4)、核因子κB p65(NF-κB p65)、p-NF-κB p65、NF-κB抑制蛋白α(IκBα)、p-IκBα、高迁移率族蛋白B1(HMGB1)、cleaved Caspase-3、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)表达。[结果]与对照组比较,模型组大鼠皮质神经元活力明显降低(P<0.05),凋亡率、培养液中TNF-α、IL-1β、IL-6水平、神经元TLR4、HMGB1、cleved Caspase-3表达量及p-NF-κB p65/NF-κB p65、p-IκBα/IκBα、Bax/Bcl-2比值均明显升高(P<0.05)。与模型组比较,藏红花素组皮质神经元活力升高94.97%(P<0.05),凋亡率降低65.80%(P<0.05),培养液中TNF-α、IL-1β、IL-6水平分别降低61.86%、78.34%、63.42%(P<0.05),神经元TLR4、HMGB1、cleaved Caspase-3表达量及p-NF-κB p65/NF-κB p65、p-IκBα/IκBα、Bax/Bcl-2比值分别降低73.43%、52.13%、81.52%、69.70%、60.55%、95.05%(P<0.05)。脂多糖明显逆转藏红花素对缺氧复氧损伤皮质神经元上述各指标的调控作用(P<0.05)。[结论]藏红花素可抑制缺氧复氧损伤皮质神经元凋亡和炎症反应,对缺氧复氧损伤皮质神经元起到保护作用,其机制可能与抑制TLR4/NF-κB信号通路有关。

Aim To investigate the effect of Crocin on hypoxia/reoxygenation injury of cortical neurons and its molecular mechanism.Methods The cortical neurons of SD rats were isolated and cultured,and the control group,model group,Crocin(50 mg/L)group,Crocin(50 mg/L)+lipopolysaccharide(TLR4 activator,100μg/L)group were set.The cortical neurons in control group were routinely cultured;the cortical neurons in model group were given hypoxia 4 h and reoxygenation 24 h;the cortical neurons in Crocin group and Crocin+lipopolysaccharide group were intervened for 24 h,and then the model was established.The neuronal viability and apoptosis rate were detected by CCK-8 method and Annexin V-FITC/PI double staining method.The levels of inflammatory factors in the culture medium were detected by ELISA method.The expression of toll-like receptor 4(TLR4),nuclear factor-κB p65(NF-κB p65),p-NF-κB p65,inhibitorαof NF-κB(IκBα),p-IκBα,high mobility group box protein B1(HMGB1),cleaved Caspase-3,B lymphoma-2 gene(Bcl-2),Bcl-2-associated X protein(Bax)were detected by Western blot method.Results Compared with the control group,the activity of cortical neurons in the model group decreased significantly(P<0.05);the apoptosis rate,the levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6 in the culture medium,the expressions of TLR4,HMGB1,cleaved Caspase-3 and the ratio of p-NF-κB p65/NF-κB p65,p-IκBα/IκBα,Bax/Bcl-2 increased significantly(P<0.05).Compared with the model group,the activity of cortical neurons in Crocin group increased by 94.97%(P<0.05);the apoptosis rate decreased by 65.80%(P<0.05);the levels of TNF-α,IL-1β,IL-6 in the culture medium decreased by 61.86%,78.34%,63.42%(P<0.05);the expressions of TLR4,HMGB1,cleaved Caspase-3 and the ratio of p-NF-κB p65/NF-κB p65,p-IκBα/IκBα,Bax/Bcl-2 decreased by 73.43%,52.13%,81.52%,69.70%,60.55%,95.05%(P<0.05).Lipopolysaccharide significantly reversed the regulatory effects of Crocin on hypoxia/reoxygenation injured cortical neurons(P<0.05).Conclusion Crocin can inhibit the apoptosis and inflammatory response of hypoxia/reoxygenation injured neurons,and has protective effect on hypoxia/reoxygenation injury of cortical neurons,which mechanism may be related to the inhibition of TLR4/NF-κB signaling pathway activation.