摘要
目的:研究雷公藤内酯醇(TPL)对皮质神经元缺氧/复氧(H/R)损伤的影响,并基于Toll样受体4/核因子-κB(TLR4/NF-κB)信号通路探讨其作用机制.方法:分离并体外培养SD大鼠乳鼠皮层神经元,通过缺氧4h复氧24h制备H/R损伤皮质神经元模型,设正常对照组、模型组、TPL(25mg/L)组、TPL(25mg/L)+TAK242(TLR4 抑制剂,1mg/L)组和 TPL(25mg/L)+LPS(TLR4 激动剂,0.1mg/L)组.各组分别给药干预24h后,采用MTT法检测神经元活力,流式细胞术检测神经元凋亡,ELISA法检测培养液中炎症因子(TNF-α、IL-1β、IL-6]含量,Western blot法检测神经元TLR4/NF-κB 信号通路相关蛋白[TLR4、NF-κB p65、p-NF-κB p65、B淋巴细胞瘤-2 基因(bcl-2)、bcl-2 相关 X 蛋白(Bax)、半胱氨酸蛋白酶-3(Caspase-3)、激活型Caspase-3(cleaved Caspase-3)]表达.结果:与模型组比较,TPL组皮质神经元活力显著升高、凋亡率显著降低(P<0.05);培养液中TNF-α、IL-1β、IL-6 含量显著降低(P<0.05);TLR4 表达量及p-NF-κB p65/NF-κB p65、Bax/bcl-2、cleaved Caspase-3/Caspase-3 表达比值均显著降低(P<0.05).TAK242 可显著增强TPL对H/R损伤皮质神经元活力、凋亡、炎症因子含量、TLR4/NF-κB信号通路相关蛋白表达的调控作用;而 LPS 则显著逆转 TPL 对 H/R 损伤皮质神经元各检测指标的调控作用.结论:TPL可以通过抑制 TLR4/NF-κB 通路活化减轻炎症反应和神经元凋亡,进而对皮质神经元H/R损伤起到保护作用.
Abstract
Objective:To investigate the effects of Triptolide(TPL)on hypoxia/reoxygenation(H/R)injury of cortical neurons and explore its mechanism based on toll-like receptor 4/nuclear factor-κB(TLR4/NF-κB)signaling pathway.Methods:The cortical neurons of SD suckling rats were isolated and cultured in vitro,and the H/R damaged cortical neuron model was prepared by hypoxia for 4h and then reoxygenation for 24h.The normal control group,model group,TPL(25mg/L)group,TPL(25mg/L)+TAK242(TLR4 in-hibitor,1mg/L)group,TPL(25mg/L)+LPS(TLR4 agonist,0.1mg/L)group were set up.24h after ad-ministrating separately,the neuronal activity was detected by MTT,the neuronal apoptosis was detected by flow cytometry.The content of inflammatory factors(TNF-α,IL-1β,IL-6)were detected by ELISA.The TLR4/NF-κB signaling pathway related proteins[TLR4,NF-κB p65,p-NF-κB p65,B-lymphoblastoma-2 gene(bcl-2),bcl-2 associated X protein(Bax),Caspase-3,cleaved Caspase-3]were detected by West-ern blot.Results:Compared with the model group,the activity of cortical neuron in TPL group was increased while the apoptosis rate was decreased(P<0.05).The content of TNF-α,IL-1β,IL-6 in culture medium were significantly decreased(P<0.05).The expression of TLR4 and the expression ratio of p-NF-κB p65/NF-κB p65,Bax/bcl-2,cleaved Caspase-3/Caspase-3 were significantly decreased(P<0.05).TAK242 could significantly enhance the regulatory effects of TPL on the activity,apoptosis,inflammatory factors con-tent and the expression of TLR4/NF-κB signaling pathway related protein in H/R damaged cortical neurons.And LPS could significantly reversed the regulatory effects of TPL on H/R damaged cortical neurons.Conclu-sion:TPL can reduce inflammation and apoptosis by inhibiting the activation of TLR4/NF-κB pathway,and thus play a protective role in H/R injury of cortical neurons.
维普
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