β-细辛醚通过抑制 TRPV4 的表达缓解谷氨酸诱导的 Ca^{2+} 超载

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、60 μmol/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 Lipofectamine 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 of β-asarone(15, 30, 60 μmol/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; Lipofectamine 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