高良姜含药血清及主要成分对TRPA1信号通路的影响

Effect of Alpinia officinarum medicated serum and its main components on TRPA1 signaling pathway

目的体外研究高良姜含药血清及主要成分对TRPA1信号通路的影响。方法以稳定表达TRPA1通道的HEK-293T细胞为体外研究模型,分别采用RT-PCR、Western blot、ELISA、激光共聚焦荧光显微镜研究高良姜含药血清对TRPA1 mRNA和蛋白、细胞内cAMP和Ca^(2+)浓度的影响,并初步确认高良姜3类主要成分对TRPA1表达的影响;采用高效液相色谱法检测高良姜含药血清中的主要有效成分。结果与空白血清组相比,高良姜含药血清对TRPA1 mRNA及蛋白的表达均具有一定的抑制作用,能增加细胞中cAMP含量,降低细胞内Ca^(2+)浓度;黄酮类成分20 mg·L^(-1)与二芳基庚烷类40 mg·L^(-1)对TRPA1 mRNA表达有明显的抑制作用;HPLC结果表明高良姜素和二芳基庚烷是高良姜含药血清的主要入血成分。结论高良姜可以下调TRPA1/Ca^(2+)信号通路,提高细胞内cAMP含量,该作用可能是其温中止痛分子机制的重要基础,主要物质基础是黄酮类和二芳基庚烷成分。

Aim To observe the effects of main components of Alpinia officinarum Hance(A.officinarum)on TRPA1 signaling pathway in vitro.Methods TRP1-HEK293T cell model stably expressing TRPA1 channel was constructed as the research object,the effects of A.officinarum medicated serum on the expressions of TRPA1 mRNA and protein were measured by RT-PCR and Western blot separately,the content of cAMP in cells was determined by ELISA,and the intracellular Ca^(2+) concentration was detected by laser scanning confocal fluorescence microscope.The effect of the three main components of galangal on the expression of TRPA1 was preliminarily confirmed.The main effective components in A.officinarum medicated serum were detected by HPLC.Results Compared with the blank serum group,A.officinarum medicated serum could inhibit the expression of TRPA1 mRNA and protein,increase the content of cAMP in cells while decrease intracellular Ca^(2+) concentration significantly.The flavonoid concentration of 20 mg·L^(-1) and diarylheptane concentration of 40 mg·L^(-1) showed significant inhibitory effect on TRPA1 mRNA expression.The results of HPLC showed that galangin and diphenylheptane were the major chemical constituents in A.officinarum containing serum.Conclusions Alpinia officinarum can down-regulate the expression of TRPA1/Ca^(2+) pathway and increase the intracellular cAMP concentration,which suggests that this effect may be an important basis for the molecular mechanism of warming stomach and relieving pain.Flavonoid and diarylheptane are the main material basis of Alpinia officinarum.