β-细辛醚通过抑制TRPV4的表达缓解谷氨酸诱导的Ca2+超载
β-Asarone alleviates glutamate-induced Ca2+overload by inhibiting TRPV4 expression
蒋兰兰 1陈向涛 1储涛 1蔡静雯 2刘浩宇 1尹兰香1
作者信息
- 1. 安徽医科大学药学院,安徽合肥 230022
- 2. 安徽医科大学药学院,安徽合肥 230022;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
- 折叠
摘要
谷氨酸处理会导致Ca2+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、60 μmol/L)预处理4 h,40 mmol/L谷氨酸处理实时记录对PC12细胞Ca2+浓度的影响,采用钙成像技术检测Ca2+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Ca2+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Ca2+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Ca2+超载和TRPV4的表达;沉默TRPV4抑制细胞Ca2+超载;过表达TRPV4则部分逆转β-细辛醚抑制谷氨酸诱导的Ca2+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Ca2+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.
Abstract
Glutamate treatment can lead to Ca2+overload,and the role and possible mechanism of tran-sient receptor potential vanilloid 4(TRPV4)in Ca2 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 PC 12 cells as the research object,the effect of β-asarone(15,30,60 μmol/L)pretreatment for 4 h,40 mmol/L glutamate treatment on the Ca2+concentration of PC12 cells was recorded in real time,and the change of Ca2+concentration was detected by calcium imaging technology;TRPV4 mRNA and protein expression was detected by fluorescent quantitative polymer-ase 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 Ca2+overload.The results showed that compared with the control group,glutamate treatment for 5 min could induce Ca2+overload,and the mRNA and protein expression of TRPV4 was significantly increased;compared with the model group,β-asarone could reduce glutamate-induced Ca2+overload and TRPV4 expression in a dose-dependent manner;si-lencing of TRPV4 inhibited Ca2+overload in cells;overexpression of TRPV4 partially reversed the in-hibition of glutamate-induced Ca2+overload by β-asarone.This study demonstrated that glutamate treatment of PC12 cells for 5 min induced Ca2+overload by up-regulating the expression of TRPV4,and β-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)/Ca2+超载Key words
glutamate/excitotoxicity/β-asarone/transient receptor potential vanilloid 4(TRPV4)/Ca2+over-load引用本文复制引用
基金项目
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项目(KJ2021A0234)
出版年
2024
NETL
NSTL
万方数据