加味柴胡疏肝汤激活RIP1/RIP3/MLKL信号通路抑制癫痫小鼠海马细胞坏死性凋亡  

Modified Chaihu Shugan Decoction(加味柴胡疏肝汤)Activating RIP1/RIP3/MLKL Signaling Pathway and Inhibiting Necrotic Apoptosis of Hippocampal Cells in Epileptic Mice

作　　者：谢苗莹 曾榕 吴琼 陈江玮 梁霜 钟洁[3] 罗玉珍 刁丽梅[3] XIE Miaoying;ZENG Rong;WU Qiong;CHEN Jiangwei;LIANG Shuang;ZHONG Jie;LUO Yuzhen;DIAO Limei(Guangxi University of Chinese Medicine,Nanning 530200,Guangxi,China;Xinyang Central Hospital,Xinyang 464099,Henan,China;First Affiliated Hospital of Guangxi University of Chinese Medicine,Nanning 530200,Jiangsu,China)

机构地区：[1]广西中医药大学,广西南宁530200 [2]信阳市中心医院,河南信阳464099 [3]广西中医药大学附属第一医院,广西南宁530200

出　　处：《中华中医药学刊》2025年第4期144-148,I0030,共6页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(82360875);广西科技厅科技基地和人才专项(桂科AD22035168,桂科AD20238028)。

摘　　要：目的探究加味柴胡疏肝汤通过RIP1/RIP3/MLKL通路对癫痫小鼠的调控作用。方法将60只KM小鼠随机分为空白组、模型组、加味柴胡疏肝汤低剂量组、加味柴胡疏肝汤中剂量组、加味柴胡疏肝汤高剂量组、卡马西平组。采用盐酸匹罗卡品制备癫痫模型,予加味柴胡疏肝汤19.52、39.04、78.08 g/(kg·d),卡马西平30 mg/(kg·d),连续灌胃2周,观察小鼠一般情况;脑电波情况;苏木素-伊红(HE)染色观察病理形态学改变;原位末端转移酶标记技术(TUNEL)检测神经细胞凋亡率;实时定量反转录聚合酶链式反应(qRT-PCR)及蛋白质印迹(Western Blot)检测小鼠海马组织RIP1、RIP3、MLKL、TNF-α、IL-1β、IL-6的mRNA和蛋白表达水平。结果与空白组相比,模型组小鼠体质量明显降低;脑电图可以明显观测到连续发作的痫样波;HE染色可见较多神经元固缩,细胞体积减小,形态呈不规则状,胞核、胞质分界不清,染色加深;凋亡细胞阳性率明显提高;RIP1、RIP3、MLKL、TNF-α、IL-1β、IL-6mRNA和蛋白的相对表达量明显增高。各组经药物治疗之后,小鼠体质量较模型组稍有增加;脑电波痫性放电频次及强度明显减少;加味柴胡疏肝汤中剂量、加味柴胡疏肝汤高剂量组、卡马西平组凋亡细胞阳性率明显低于模型组,差异有统计学意义;加味柴胡疏肝汤高剂量组、卡马西平组的RIP1、RIP3、MLKL、TNF-α、IL-1β、IL-6mRNA和蛋白的相对表达量明显低于模型组,差异具有统计学意义。结论加味柴胡疏肝汤可能通过RIP1/RIP3/MLKL信号通路发挥抗癫痫作用。Objective To investigate the regulatory effect of Modified Chaihu Shugan Decoction(加味柴胡疏肝汤)on epileptic mice through the protein kinases 1(RIP1)/protein kinases 3(RIP3)/mixed lineage kinase domain-like protein(MLKL)pathway.Method Sixty KM mice were randomly divided into normal group,model group,low dose group of Modified Chaihu Shugan Decoction[19.52 g/(kg·d)],medium dose group of Modified Chaihu Shugan Decoction[39.04 g/(kg·d)],high dose group of Modified Chaihu Shugan Decoction[78.08 g/(kg·d)]and carbamazepine group[30 mg/(kg·d)].Epilepsy models were prepared using pilocarpine hydrochloride.After continuous gavage for two weeks,the general condition and electroencephalogram(EEG)of the mice were observed.Hematoxylin-eosin(HE)staining was used to observe pathological and morphological changes.TdT-mediated dUTP Nick-End Labeling(TUNEL)method was used to detect the apoptosis rate of neural cells.Quantitative real time polymerase chain reaction(qRT-PCR)and Western Blot(WB)measured the mRNA and protein expression levels of RIP1,RIP3,MLKL,tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in the hippocampus of the mice.Result Compared with the normal group,the body weight of the model group mice was decreased significantly;EEG can clearly observe continuous epileptic waves;HE staining showed that many neurons were pyknotic,with reduced cell volume and irregular morphology.The boundaries between the nucleus and cytoplasm were unclear,and the staining deepened;The positive rate of apoptotic cells significantly increased;The relative expression levels of RIP1,RIP3,MLKL,TNF-α,IL-1βand IL-6 were significantly increased.After drug treatment,the weight of mice in each group increased slightly compared to that of the model group.The frequency and intensity of epileptic discharges in EEG waves were significantly reduced.The positive rates of apoptotic cells in the medium dose and high dose groups of Modified Chaihu Shugan Decoction and carbamazepine group were significantly lower

关 键 词：加味柴胡疏肝汤 癫痫 RIP1/RIP3/MLKL信号通路 坏死性凋亡 

分 类 号：R289.5[医药卫生—方剂学]