天麻素对毛果芸香碱诱发的癫痫大鼠TLR4/NF-κB信号通路影响的研究

Study on the Effect of Gastrodin on TLR4/NF-κB Signaling Pathway in Rats with Epilepsy Induced by Pilocarpine

目的:探讨天麻素对毛果芸香碱诱发的癫痫大鼠的脑保护作用及其作用机制。方法:72只大鼠随机分为空白组、模型组、阳性药组、天麻素组、Toll样受体4 (TLR4)激活剂脂多糖(LPS)组、天麻素+LPS组,每组12只。采用腹腔注射毛果芸香碱建立癫痫大鼠模型。双抗体夹心酶联免疫法检测大鼠血清和海马组织肿瘤坏死因子-α(TNF-α)与白细胞介素-1β(IL-1β)水平,TUNEL染色法检测海马组织细胞凋亡数,Western blot分别检测海马组织活化半胱氨酸基天冬氨酸酶-3 (CleavedCaspase-3)、B淋巴细胞瘤-2 (Bcl-2)、Bcl-2相关X蛋白(Bax)、TLR4、兔抗大鼠p-核因子-κB亚基p65亲和肽(p-NF-κBp65)和磷酸化核因子κB抑制蛋白α(p-IκB-α)表达。结果:与空白组比较,模型组大鼠脑电频率显著降低(P<0.05),波幅显著升高(P<0.05);血清和海马组织TNF-α和IL-1β水平,海马组织细胞凋亡率、Cleaved-Caspase-3、Bax、TLR4和p-NF-κBp65蛋白明显升高(P<0.05);IL-10水平,Bcl-2和p-IκB-α蛋白表达显著降低(P<0.05)。与模型组比较,阳性药组、天麻素组和天麻素+LPS大鼠脑电频率显著升高(P<0.05),波幅显著降低(P<0.05),血清和海马组织TNF-α和IL-1β水平,海马组织细胞凋亡率、Cleaved-Caspase-3、Bax、TLR4和p-NF-κBp65蛋白明显降低(P<0.05),IL-10水平,Bcl-2和p-IκB-α蛋白表达显著升高(P<0.05);LPS组大鼠脑电频率、血清及海马组织IL-10水平、Bcl-2蛋白显著降低(P<0.05),血清与海马组织TNF-α与IL-1β水平、海马组织细胞凋亡率、Cleaved-Caspase-3、Bax、TLR4、p-NF-κBp65和p-IκB-α蛋白表达明显升高(P<0.05)。与LPS组比较,天麻素+LPS组大鼠脑电频率显著升高(P<0.05),波幅显著降低(P<0.05);血清及海马组织TNF-α与IL-1β水平、海马组织细胞凋亡率、Cleaved-Caspase-3、Bax、TLR4、p-NF-κBp65蛋白表达均明显降低(P<0.05),IL-10水平、Bcl-2及p-IκB-α蛋白表达明显升高(P<0.05)。结论:天麻素可通过抑制TLR4/NF-κB信号通路对毛果芸香碱诱发的癫痫大鼠发挥脑保护作用。

objective:To investigate the cerebral protective effect of gastrodin on rats with epilepsy induced by pilocarpine and its mechanism. Methods:Divided 72 rats randomly into the blank group,the model group,the positive drug group,the gastrodin group,the Toll-like receptor 4(TLR4) and activator lipopolysaccharide(LPS) group and the gastrodin+LPS group,with 12 rats in each group. Established the epileptic rat models by intraperitoneal injection of pilocarpine. Detected the levels of tumor necrosis factor-α(TNF-α) of serum and hippocampus and interleukin-1β(IL-1β) in rats by double antibody sandwich ELISA,the apoptotic number of hippocampal cells by TUNEL staining method,the expression of Cleaved-Caspase-3,Bcell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),TLR4,rabbit anti-rat p-nuclear factor-κB subunits p65 binding peptides(p-NF-κBp65) and phosphor nuclear factor κB inhibitory protein α(p-IκB-α) in hippocampus by Western blot.Results: Compared with the blank group, the electroencephalogram frequency of rats in the model group was evidently decreased(P<0.05) and the amplitude was significantly increased(P<0.05);the levels of TNF-α and IL-1β of serum and hippocampus,the apoptosis rate of hippocampal cells,Cleaved-Caspase-3,Bax,TLR4 and p-NF-κBp65 protein were obviously increased(P<0.05). IL-10 levels and the expression of Bcl-2 and p-IκB-α protein were evidently decreased(P<0.05). Compared with the model group,the electroencephalogram frequency of rats in the positive drug group,the gastrodin group and the gastrodin+LPS group was evidently increased(P<0.05),the amplitude was significantly decreased(P<0.05),the levels of TNF-α and IL-1β of serum and hippocampus,the apoptosis rate of hippocampal cells,Cleaved-Caspase-3,Bax,TLR4 and p-NF-κBp65 protein were obviously decreased(P<0.05) and IL-10 levels and the expression of Bcl-2 and pIκB-α protein were evidently increased(P<0.05);the electroencephalogram frequency of rats, the levels of IL-10 of serum and hippocampus and Bcl-2 protein in the LPS group were significantly decreased(P<0.05), the levels of TNF-αand IL-1β of serum and hippocampus,the apoptosis rate of hippocampal cells,Cleaved-Caspase-3,Bax,TLR4 and the expression of p-NF-κBp65 and p-IκB-α protein were obviously increased(P<0.05). Compared with the LPS group,the electroencephalogram frequency of rats in the gastrodin+LPS group was evidently increased(P<0.05) and the amplitude was significantly decreased(P<0.05);the levels of TNF-α and IL-1β of serum and hippocampus, the apoptosis rate of hippocampal cells, Cleaved-Caspase-3, Bax, TLR4 and the expression of p-NF-κBp65 protein were all obviously decreased(P<0.05),and IL-10 levels and the expression of Bcl-2 and p-IκB-α protein were obviously increased(P<0.05).Conclusion:Gastrodin has cerebral protective effect on rats with epilepsy induced by pilocarpine through inhibiting TLR4/NF-κB signaling pathway.