柴胡龙骨牡蛎汤通过下调miR-146a-3p和miR-146a-5p抑制TLR4信号通路缓解大鼠癫痫发作

Chaihu Longgu Muli Decoction relieving temporal lobe epilepsy in rats by inhibiting TLR4 signaling pathway through miR-146a-3p and miR-146a-5p

    摘要
  • 摘要:
    目的探讨柴胡龙骨牡蛎汤（CHLGMLD）对颞叶癫痫大鼠的疗效及作用机制。
    方法将80只雄性SD大鼠随机分为5组，分别为对照组、模型组、卡马西平（0.1 g/kg）组、CHLGMLD低剂量（CHLGMLD-L，12.5 g/kg）组和高剂量（CHLGMLD-H，25 g/kg）组，每组各16只。除对照组外，其余4组均建立由锂-匹罗卡品诱导的颞叶癫痫大鼠模型。造模成功后，所有用药组给予相应药物，对照组和模型组给予同等体积蒸馏水治疗4周。记录各组大鼠的自发性癫痫反复发作的次数和持续时间以评价癫痫缓解程度。采用实时荧光定量聚合酶链反应法（qRT-PCR）检测海马齿状回miR-146a-3p和miR-146a-5p的表达。免疫荧光法检测海马齿状回中Toll样受体4（TLR4）、白细胞介素-1受体相关激酶1（IRAK1）、肿瘤坏死因子（TNF）受体相关因子6（TRAF6）、TAK1-结合蛋白（TAB）、核转录因子（NF-κB）和白介素1β（IL-1β）的表达。并对以上因子与miR-146a-3p、miR-146a-5p的表达分别进行相关性分析。
    结果CHLGMLD能降低大鼠癫痫发作频率（P < 0.05）和持续时间（P < 0.01）；CHLGMLD能下调miR-146a-5p和miR-146a-3p的表达水平（P < 0.05），抑制TLR4、IRAK1、TRAF6、TAB、NF-κB、IL-1β 的表达（P < 0.01）。相关性分析显示TLR4、IRAK1、TRAF6、TAB、NF-κB、IL-1β 的表达分别与qRT-PCR检测的miR-146a-3p、miR-146a-5p的表达呈正相关（P < 0.01）。
    结论CHLGMLD可通过下调miR-146a-3p和miR-146a-5p的表达水平，抑制TLR4通路，减轻颞叶癫痫大鼠海马齿状回炎症反应，缓解癫痫发作。

     

    Abstract:
    ObjectiveTo explore the effect and mechanism of Chaihu Longgu Muli Decoction (柴胡龙骨牡蛎汤, CHLGMLD) in rats with temporal lobe epilepsy (TLE).
    MethodsA total of 80 Sprague-Dawley (SD) male rats were randomized into control (CON), model (MOD), carbamazepine (CBZ, 0.1 g/kg), CHLGMLD low dose (CHLGMLD-L, 12.5 g/kg), and high dose (CHLGMLD-H, 25 g/kg) groups, with 16 rats in each group. TLE rat models were established in the four groups with the use of lithium-pilocarpine except for the CON group. After the successful establishment of TLE models, all drugs were administered through gavage, and distilled water was given to rats in the CON and MOD groups for four weeks. The frequency and duration of seizures before and after treatment were recorded for the evaluation of the alleviation degree. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of miR-146a-3p and miR-146a-5p. The expression levels of toll-like receptor 4 (TLR4), interleukin-1 receptor-associated kinase 1 (IRAK1), tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), TAK1-binding protein (TAB), nuclear factor-kappa B (NF-κB), and interleukin-1 beta (IL-1β) in hippocampus were tested by immunofluorescence assay. Correlation analysis between the above factors and expressions of miR-146a-3p and miR-146a-5p were performed separately.
    Results CHLGMLD decreased the frequency (P < 0.05) and duration (P < 0.01) of seizures in rats. CHLGMLD down-regulated the expression levels of miR-146a-5p and miR-146a-3p (P < 0.05), and inhibited the expression levels of TLR4, IRAK1, TRAF6, TAB, NF-κB, and IL-1β (P < 0.01). The correlation analysis revealed that the expression levels of TLR4, IRAK1, TRAF6, TAB, NF-κB, and IL-1β were positively correlated with the expression levels of miR-146a-3p and miR-146a-5p detected by qRT-PCR, respectively (P < 0.01).
    ConclusionCHLGMLD can inhibite the TLR4 signaling pathway by lowering the expression levels of miR-146a-3p and miR-146a-5p to alleviate hippocampal dentate gyrus inflammation in TLE rats, thus relieving seizures.

     

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