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目的:观察肉桂醛对0℃冷应激大鼠背根神经节神经细胞胞浆内Ca~(2+)浓度变化,并探讨其对瞬时受体电位通道蛋白A1(TRPA1)和M8(TRPM8)mRNA表达的影响。方法:取大鼠原代背根节神经细胞置于0℃冷应激状态,噻唑蓝(MTT)法检测不同时长冷应激状态下细胞的活性;加入肉桂醛,采用激光共聚焦显微镜观察细胞内外Ca~(2+)分布的变化;另以实时荧光定量PCR(Real-time PCR)检测其TRPA1,TRPM8 mRNA的表达。结果:不同时长的冷应激状态下,细胞存活度无明显差异;冷应激可降低背根神经节(DRG)神经细胞胞浆内Ca~(2+)浓度(P<0.05),一定量的肉桂醛则能使Ca~(2+)浓度显著升高(P<0.01)。Real-time PCR检测显示,长时间冷应激TRPA1 mRNA表达上调(P<0.05);长时间冷应激TRPM8 mRNA表达明显要高于短时冷应激(P<0.05);适当浓度的肉桂醛使冷应激大鼠DRG神经细胞TRPA1 mRNA表达升高(P<0.05,P<0.01),对TRPM8 mRNA的影响尚不明显。结论:冷应激能影响TRPA1,TRPM8 mRNA的表达,降低胞浆内Ca~(2+)浓度,作用机制可能为肉桂醛通过活化TRPA1通道,升高细胞浆内Ca~(2+)浓度。
Objective: To observe the change of intracellular Ca2 + concentration of cinnamaldehyde in neurons of 0 ℃ cold stress rat dorsal root ganglion (DRG) and to investigate the effect of cinnamic aldehyde on the expression of TRPA1 and TRPM8, Effect of mRNA expression. Methods: Primary rat dorsal root ganglion neurons were exposed to cold stress at 0 ℃. MTT assay was used to detect the cell viability under cold stress. Cinnamic aldehyde was added and the cells were observed by laser scanning confocal microscopy The changes of Ca 2+ distribution inside and outside were analyzed. The expression of TRPAl and TRPM8 mRNA was detected by real-time PCR. Results: Under the condition of cold stress for different durations, there was no significant difference in cell viability. Cold stress could decrease the concentration of Ca ~ (2+) in the cytoplasm of DRG neurons (P <0.05) Cinnamic aldehyde could increase Ca 2+ concentration significantly (P <0.01). The expression of TRPAl mRNA in long-term cold stress was significantly higher than that in short-term cold stress (P <0.05) by Real-time PCR. The expression of TRPAl mRNA in long-term cold stress was significantly increased (P <0.05) The mRNA expression of TRPAl in DRG neurons of cold stress rats increased (P <0.05, P <0.01), and the effect on TRPM8 mRNA was not obvious. CONCLUSION: Cold stress can affect the expression of TRPAl and TRPM8 mRNA and decrease the concentration of Ca2 + in cytoplasm. The mechanism may be that cinnamaldehyde increases the intracellular Ca2 + concentration by activating TRPAl channel.