β-细辛醚通过抑制TRPV4的表达缓解谷氨酸诱导的Ca^(2+)超载  

β-Asarone alleviates glutamate-induced Ca^(2+) overload by inhibiting TRPV4 expression

作　　者：蒋兰兰 陈向涛[1] 储涛 蔡静雯 刘浩宇 尹兰香 JIANG Lanlan;CHEN Xiangtao;CHU Tao;CAI Jingwen;LIU Haoyu;YIN Lanxiang(School of Pharmacy,Anhui Medical University,Hefei 230022,China;Department of Pharmacy,The First Affiliated Hospital of University of Science and Technology of China(Anhui Provincial Hospital),Hefei 230001,China)

机构地区：[1]安徽医科大学药学院,安徽合肥230022 [2]中国科学技术大学附属第一医院(安徽省立医院)药剂科,安徽合肥230001

出　　处：《合肥工业大学学报（自然科学版）》2024年第2期263-269,共7页Journal of Hefei University of Technology：Natural Science

基　　金：国家自然科学基金资助项目(82274124);安徽省高校自然科学研究重点资助项目(KJ2021A0234)。

摘　　要：谷氨酸处理会导致Ca^(2+)超载,瞬时受体电位香草素受体4(transient receptor potential vanilloid 4,TRPV4)在其中的作用及可能机制尚不清楚。β-细辛醚能快速透过血脑屏障,对兴奋性毒性具有较强的神经保护作用。文章以高分化的PC12细胞为研究对象,探究β-细辛醚(15、30、60μmol/L)预处理4 h,40 mmol/L谷氨酸处理实时记录对PC12细胞Ca^(2+)浓度的影响,采用钙成像技术检测Ca^(2+)浓度的变化;采用实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)、Western Blot及免疫荧光技术检测TRPV4的mRNA和蛋白的表达;采用Lipofectiamine 2000脂质体实验转染TRPV4-siRNA和pEX-3-TRPV4,观察沉默和过表达TRPV4对谷氨酸引起Ca^(2+)超载的影响。结果表明:与正常对照组相比,谷氨酸处理5 min可诱导Ca^(2+)超载,显著提高TRPV4的mRNA和蛋白的表达;与模型组相比,β-细辛醚能够剂量依赖性地降低谷氨酸诱导的Ca^(2+)超载和TRPV4的表达;沉默TRPV4抑制细胞Ca^(2+)超载;过表达TRPV4则部分逆转β-细辛醚抑制谷氨酸诱导的Ca^(2+)超载。该研究证明,谷氨酸处理PC12细胞5 min通过上调TRPV4的表达诱导Ca^(2+)超载,β-细辛醚作为TRPV4的拮抗剂,是一种潜在的抑制兴奋性毒性的药物。Glutamate treatment can lead to Ca^(2+)overload,and the role and possible mechanism of transient receptor potential vanilloid 4(TRPV4)in Ca^(2+)overload are still unclear.β-Asarone can quickly penetrate the blood-brain barrier and has a strong neuroprotective effect on excitotoxicity.In this study,taking highly differentiated PC12 cells as the research object,the effect ofβ-asarone(15,30,60μmol/L)pretreatment for 4 h,40 mmol/L glutamate treatment on the Ca^(2+)concentration of PC12 cells was recorded in real time,and the change of Ca^(2+)concentration was detected by calcium imaging technology;TRPV4 mRNA and protein expression was detected by fluorescent quantitative polymerase chain reaction,Western Blot,and immunofluorescence techniques;Lipofectiamine 2000 liposome experiment was used to transfect TRPV4-siRNA and pEX-3-TRPV4 to observe the effect of silencing and overexpression of TRPV4 on glutamate-induced Ca^(2+)overload.The results showed that compared with the control group,glutamate treatment for 5 min could induce Ca^(2+)overload,and the mRNA and protein expression of TRPV4 was significantly increased;compared with the model group,β-asarone could reduce glutamate-induced Ca^(2+)overload and TRPV4 expression in a dose-dependent manner;silencing of TRPV4 inhibited Ca^(2+)overload in cells;overexpression of TRPV4 partially reversed the inhibition of glutamate-induced Ca^(2+)overload byβ-asarone.This study demonstrated that glutamate treatment of PC12 cells for 5 min induced Ca^(2+)overload by up-regulating the expression of TRPV4,andβ-asarone,as an antagonist of TRPV4,is a potential drug to inhibit excitotoxicity.

关 键 词：谷氨酸 兴奋性毒性 Β-细辛醚 瞬时受体电位香草素受体4(TRPV4) Ca^(2+)超载 

分 类 号：R285.5[医药卫生—中药学]