基于TLR4/NF-κB通路调控神经炎症探讨菖蒲郁金汤对癫痫复发小鼠的影响

Effects of Changpu Yujintang on recurrent epilepsy mice based on TLR4/NF-κB signaling pathway

关键词:  菖蒲郁金汤  癫痫复发  神经炎症  Toll样受体4/核转录因子κB信号通路

Key words:  Changpu Yujintang  recurrent epilepsy  neuroinflammation  TLR4/NF-κB signaling pathway

摘要:

目的 研究菖蒲郁金汤通过改善神经炎症治疗癫痫复发小鼠的作用机制。方法 取SPF级C57BL/6小鼠12只为空白组，另取癫痫持续状态（SE）小鼠60只随机分为5组，分别为模型组，菖蒲郁金汤低、中、高剂量组和丙戊酸钠组。SE小鼠采用腹腔注射（ip）红藻氨酸（KA）25 mg/kg构建SE模型，并在造模成功后开始给予生理盐水，菖蒲郁金汤低、中、高剂量[2.05、4.1、8.2 g/（kg·mL）]和丙戊酸钠缓释片[0.13 g/（kg·mL）]早晚各灌胃1次，共给药1周，1周后再次ip KA 25mg/kg，苏木素-伊红（HE）染色和尼氏甲苯胺蓝染色观察小鼠海马CA3、CA1区病理变化，酶联免疫吸附实验（ELISA）检测小鼠血清和海马组织中肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）的水平，免疫荧光观察小胶质细胞离子钙结合衔接分子1（Iba-1）和星形胶质细胞胶质纤维酸性蛋白（GFAP）的表达，蛋白免疫印迹法（Western blot）检测Toll样受体4（TLR4）/核转录因子κB（NF-κB）信号通路蛋白表达。结果 与模型组相比，菖蒲郁金汤高剂量组可降低癫痫复发小鼠的发作等级（P < 0.05），菖蒲郁金汤中高剂量组小鼠海马CA3、CA1区偶见锥体细胞固缩体积减小，细胞排列较紧密整齐，尼氏小体的表达增加（P < 0.01），小鼠血清和海马组织中TNF-α、IL-1β、IL-6水平降低（P < 0.01），Iba-1、GFAP阳性细胞数量减少（P < 0.01），海马组织中TLR4、磷酸化NF-κB（P-NF-κB）、磷酸化IκB-α（P-IκB-α）蛋白表达下调（P < 0.01）。结论 菖蒲郁金汤干预可以降低癫痫易感性，改善癫痫的病理改变，抑制癫痫复发小鼠血清和海马组织中炎症因子的释放，抑制小胶质细胞和星形胶质细胞的免疫活性，其作用机制可能与TLR4/NF-κB信号通路有关。

Abstract:

Objective To study the mechanism of Changpu Yujintang(CPYJT) in the treatment of recurrent epilepsy by improving neuroinflammation. Methods Twelve SPF C57BL/6 mice were selected as blank group, and another 60 status epilepticus(SE) mice were randomly divided into 5 groups: model group, low, middle and high dose of CPYJT group and sodium valproate group. SE mice were injected intraperitoneally(ip) Kainic acid(KA) 25 mg/kg to establish SE model. After the model was successfully established, normal saline was given to the mice. The low, middle and high doses of CPYJT [2.05、4.1、8.2 g/(kg·mL)]and sodium valproate sustained-release tablets [0.13 g/(kg·mL)]were given intragastrically for one week. Ip KA 25 mg/kg again 1 week later. The pathological changes of CA3 and CA1 regions of hippocampus in mice were observed by hematoxylin-eosin(HE) staining and Nissl toluidine blue staining, and the levels of tumor necrosis factor-a(TNF-α), interleukin-1 β(IL-1β) and interleukin-6(IL-6) in serum and hippocampus were detected by enzyme-linked immunosorbent assay(ELISA). The expression of microglial calcium binding molecule 1(Iba-1) and astroglial fibrillary acidic protein(GFAP) was detected by immunofluorescence, and the expression of Toll-like receptor 4(TLR4)/nuclear factor kB(NF-κB) signaling pathway protein was detected by Western blot(WB). Results Compared with the model group, the high-dose group of calamus tulip decoction could reduce the seizure grade of mice with recurrent epilepsy(P < 0.05), the pyknosis volume of vertebral cells in hippocampal CA3 and CA1 regions of mice in the middle and high dose of CPYJT group and sodium valproate group occasionally decreased, the arrangement was compact and orderly, the expression of Nissl body was increased(P < 0.01), and the levels of TNF-α, IL-1β and IL-6 in serum and hippocampal tissue were significantly decreased(P < 0.01). The number of Iba-1 and GFAP positive cells decreased significantly(P < 0.01), and the expression of TLR4, P-NF-κB and P-IκB-α protein in hippocampal tissue was significantly down-regulated(P < 0.01). Conclusion CPYJT can reduce the susceptibility to epilepsy, improve the pathological changes of epilepsy, inhibit the release of inflammatory factors in serum and hippocampus of mice with recurrent epilepsy, and inhibit the activation of microglia and astrocytes. Its mechanism is related to TLR4/NF-κB signaling pathway.