【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、60μmol/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。更多还原

【Abstract】 Glutamate treatment can lead to Ca²⁺ overload, and the role and possible mechanism of transient 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 PC12 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 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 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; silencing of TRPV4 inhibited Ca²⁺ overload in cells; overexpression of TRPV4 partially reversed the inhibition 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.更多还原