他克莫司通过上调脊髓背角AMPA受体的突触表达诱发疼痛  

Tacrolimus Induces Pain by Upregulating the Synaptic Expression of AMPA Receptors in the Spinal Cord Dorsal Horn

作　　者：徐嘉钰 李静 李媛 王康丽 曾祥茹 索占伟[1] XU Jiayu;LI Jing;LI Yuan;WANG Kangli;ZENG Xiangru;SUO Zhanwei(Department of Molecular Pharmacology,School of Pharmacy,Lanzhou University,Lanzhou 730000,China)

机构地区：[1]兰州大学药学院分子药理研究所,兰州730000

出　　处：《四川大学学报（医学版）》2024年第1期132-138,共7页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金项目(No.82073822)资助。

摘　　要：目的研究长期使用他克莫司(tacrolimus,or FK506)对小鼠痛行为的影响,并通过检测FK506对小鼠脊髓背角α-氨-3-羟基-5-甲基-4-异恶唑丙酸(α-amino-3-hyroxy-5-methyl-4-isoxazolepropionic acid,AMPA)突触表达和受体磷酸化的影响,探讨其诱发疼痛的机制。方法①将24只小鼠随机平均分为两组,FK506组每天腹腔注射FK506构建疼痛模型,Saline组给予生理盐水,每天均注射1次,连续注射7 d。一部分小鼠(每组6只)连续9 d监测小鼠机械性缩足阈值(paw withdrawal threshold,PWT)、热痛觉潜伏期(paw withdrawal latency,PWL)以及自发性痛行为学的变化,检测模型是否构造成功。另一部分小鼠(每组6只)在注射第7天诱导出明显的疼痛症状后,急性分离脊髓背角,利用免疫印迹法检测AMPA受体亚基GluA1、GluA2的突触表达和磷酸化水平。②FK506+CaN组和FK506+Saline组小鼠,每组6只,在疼痛模型鼠造模完成后给予一次鞘内注射重组钙调蛋白依赖性磷酸酶(calcineurin,CaN)(33 U)或生理盐水,60 min后测定各小鼠的PWT和PWL值,以检测CaN在FK506诱发疼痛中的作用。③另选小鼠18只,将小鼠随机平均分为3组:Control组(腹腔注射Saline后,鞘内注射Saline)、FK506+Saline组(腹腔注射FK506后,鞘内注射Saline)、FK506+CaN组(腹腔注射FK506后,鞘内注射CaN),60 min后,分离脊髓进行免疫印迹实验,检测CaN在FK506调节AMPA受体中的作用。结果①在腹腔连续注射FK506后,小鼠的PWT、PWL值均显著下降,在第7天分别降至对照组的22.3%±0.05%、66.6%±0.05%(P<0.01),并出现明显的自发性痛行为,表现为舔足时间显著增加(P<0.01),疼痛模型制造成功。免疫印迹显示,FK506组脊髓背角GluA1、GluA2亚基的总蛋白表达水平与Saline组差异无统计学意义,但可特异性诱导突触中GluA1的数量增加,GluA1的第845位和第831位丝氨酸的磷酸化水平均显著升高(P<0.05)。②与FK506+Saline组相比,FK506+CaN组小鼠的PWT值和PWL值均升高(P<0.05)�Objective To investigate the effects of long-term administration of tacrolimus(also known as FK506)on the pain-related behaviors in mice and to study the underlying mechanism of pain induced by FK506 via measuring the effect of FK506 on the synaptic expression and phosphorylation of alpha-amino-3-hyroxy-5-methyl-4-isoxazolepropionic acid(AMPA)receptor in the spinal cord dorsal horn of mice.Methods 1)A total of 24 mice were evenly and randomly assigned to two groups,a FK506 group and a Saline group.The FK506 group was given daily intraperitoneal injection of FK506 and the Saline group received normal saline.Both groups received injection once a day for 7 days in a row.Some of the mice(n=6 in each group)were monitored for the changes in the paw withdrawal threshold(PWT),the paw withdrawal latency(PWL),and the spontaneous pain behaviors to establish the pain model.The other mice(n=6 in each group)of each group underwent isolation of the dorsal horn when obvious pain symptoms were induced on day 7 of injection.Then,immunoblotting was performed to determine the synaptic expression and phosphorylation levels of GluA1 and GluA2 subunits of AMPA receptors.2)The mice were randomly divided into two groups,FK506+calcineurin(CaN)group and FK506+Saline group(n=6 in each group).After the pain model was constructed,the mice were given intrathecal injection of recombinant CaN(also know as 33 U)or normal saline.Then,60 minutes later,the PWT and the PWL of the mice were measured to investigate the role of CaN in FK506-induced pain.3)Another18 mice were selected.The mice were randomly and evenly assigned to three groups,a control group(receiving intraperitoneal injection of normal saline followed by intrathecal injection of normal saline),FK506+Saline group(receiving intraperitoneal injection of FK506 followed by intrathecal injection of normal saline)and FK506+CaN group(receiving intraperitoneal injection of FK506 followed by intrathecal injection of CaN).Then,60 minutes later,the spinal cords were isolated and subjected to immuno

关 键 词：他克莫司 脊髓背角 AMPA受体 GluA1亚基 磷酸化 疼痛 

分 类 号：R965[医药卫生—药理学]