基于OX1R/PLCβ-1/PKCα/ERK1/2信号通路探讨安寐丹对失眠模型大鼠肝脏神经递质和昼夜节律的影响及机制  

Anmeidan Regulates Hepatic Neurotransmitters and Circadian Rhythm in Rat Model of Insomnia via OX1R/PLCβ-1/PKCα/ERK1/2 Signaling Pathway

作　　者：徐波[1,2] 王平 夏婧[1,2] 谢光璟 叶子靖 秦庆花 程静 XU Bo;WANG Ping;XIA Jing;XIE Guangjing;YE Zijing;QIN Qinghua;CHENG Jing(Engineering Research Center of Chinese Medicine Protection Technology and New Product Development,Ministry of Education,Hubei University of Chinese Medicine,Wuhan 430065,China;Hubei Shizhen Laboratory,Wuhan 430065,China)

机构地区：[1]湖北中医药大学,老年脑健康中医药防护技术与新产品研发教育部工程研究中心,武汉430065 [2]湖北时珍实验室,武汉430065

出　　处：《中国实验方剂学杂志》2024年第15期11-20,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金青年基金项目(82104712);湖北省自然科学基金项目(2022CFB829)。

摘　　要：目的:基于食欲素受体1(OX1R)/磷脂酰肌醇特异性磷酯酶Cβ-1(PLCβ-1)/蛋白激酶Cα(PKCα)/细胞外信号调节激酶1/2(ERK1/2)信号通路探讨安寐丹对失眠大鼠肝脏神经递质及昼夜节律的影响及机制。方法:SPF级SD大鼠60只,随机分为空白组、模型组、苏沃雷生组、安寐丹低、中、高剂量组,各10只;除空白组外,其余各组通过腹腔注射对氯苯丙氨酸(PCPA)进行造模,空白组给予等容生理盐水、苏沃雷生组给予苏沃雷生溶液30 mg·kg^(-1)·d^(-1)灌胃、安寐丹低、中、高剂量组分别给予安寐丹水煎液(4.55、9.09、18.18 g·kg^(-1)·d^(-1));观察各组一般情况、体质量和24 h自主活动情况;采用苏木素-伊红(HE)和马松(Masson)染色观察肝脏病理学改变,酶联免疫吸附测定法(ELISA)检测肝脏神经递质γ-氨基丁酸(GABA)、5-羟色胺(5-HT)、肾上腺素(EPI)、去甲肾上腺素(NE)和乙酰胆碱(ACh)的表达,生化检测肝脏谷氨酸(Glu)的表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测肝脏生物钟基因Per1、Per2、Cry1、Cry2、Bmal1、Bmal2的m RNA表达,蛋白免疫印迹法(Western blot)、Real-time PCR检测肝脏OX1R/PLCβ-1/PKCα/ERK1/2信号通路蛋白及m RNA表达。结果:与空白组比较,模型组体质量下降(P<0.05,P<0.01),狂躁、静止节律紊乱(P<0.01),肝脏肌纤维断裂、水肿伴炎性细胞浸润,GABA、5-HT、EPI、NE和ACh含量降低、Glu含量升高(P<0.01),Per1、Per2、Cry1和Cry2 m RNA表达降低(P<0.01),Bmal1和Bmal2 m RNA表达升高(P<0.01),OX1R、PLCβ-1、PKCα、ERK1/2蛋白及m RNA基因表达均增高(P<0.01);与模型组比较,苏沃雷生和安寐丹低、中、高剂量组可增加失眠大鼠体质量(P<0.05,P<0.01),减少狂躁状态、增加其静止时间和频率(P<0.05,P<0.01),并可上调神经递质GABA、5-HT、EPI、NE、ACh和节律基因Per1、Per2、Cry1、Cry2 m RNA表达(P<0.05,P<0.01),抑制Glu及Bmal1、Bmal2、OX1R、PLCβ-1、PKCα、ERK1/2 m RNA和OX1R、PLCβ-1、PKObjective:To explore the effect and mechanism of the classic famous prescription Anmeidan(AMD)developed in the Qing Dynasty in regulating the hepatic neurotransmitters and circadian rhythm in the rat model of insomnia via the orexin-1 receptor(OX1R)/phosphatidylinositol-specific phospholipase Cβ-1(PLCβ-1)/protein kinase Cα(PKCα)/extracellular signal-regulated kinase 1/2(ERK1/2)signaling pathway.Method:Sixty SPF-grade SD rats were randomized into blank,model,suvorexant(30 mg·kg^(-1)·d^(-1)),and low-,medium-,and high-dose(4.55,9.09,18.09 g·kg^(-1)·d^(-1),respectively)AMD groups,with 10 rats in each group.The rats in other groups except the blank group were modeled by intraperitoneal injection of p-chlorophenylalanine(PCPA)and administrated with corresponding drugs by gavage,and the blank group received an equal volume of normal saline.The general condition,body mass,and 24 h autonomic activity of each group were observed.The pathological changes of the liver tissue were observed by hematoxylin-eosin(HE)staining and Masson staining.The expression of gamma-aminobutyric acid(GABA),5-hydroxytryptamine(5-HT),epinephrine(EPI),norepinephrine(NE),and acetylcholine(ACh)in the liver tissue was detected by enzyme-linked immunosorbent assay.The glutamate(Glu)expression in the liver tissue was detected by the biochemical method.The mRNA levels of biological clock genes Per1,Per2,Cry1,Cry2,Bmal1,and Bmal2 in the liver were determined by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).The protein and mRNA levels of factors in the OX1R/PLCβ-1/PKCα/ERK1/2 signaling pathway in the liver were determined by Western blot and Real-time PCR,respectively.Result:Compared with the blank group,the modeling decreased the body mass(P<0.05,P<0.01)and caused mania and disturbed resting rhythms(P<0.01),hepatic muscle fiber fracture,and edema with inflammatory cell infiltration.In addition,the modeling decreased the GABA,5-HT,EPI,NE,and ACh content,increased Glu content(P<0.01),down-regulated the mRNA levels

关 键 词：安寐丹 失眠 昼夜节律 神经递质 食欲素受体1(OX1R)/磷脂酰肌醇特异性磷酯酶Cβ-1(PLCβ-1)/蛋白激酶Cα(PKCα)/细胞外信号调节激酶1/2(ERK1/2)信号通路 

分 类 号：R2-0[医药卫生—中医学] R33R289R338.63R24