无标记细胞检测方法揭示舒血宁注射液对血栓素A2受体亚型的选择性抑制作用

Selective inhibition of Shuxuening injection on thromboxane A2 receptor subtypes revealed by a label-free cell assay

目的:构建人源血栓素A2受体(Thromboxane A2 receptor,TXR)Label-free细胞检测体系,并采用以检测中药注射剂舒血宁注射液及其活性组分对该受体的作用。方法:构建TXRα、TXRβ表达质粒,及稳定表达TXRα或TXRβ的HEK293细胞系;通过Label-free细胞检测方法,使用TXR特异性激动剂U46619及拮抗剂SQ-29548对TXRα-HEK293和TXRβ-HEK293稳定转染细胞系进行功能鉴定,并利用该体系辨识舒血宁注射液及其总黄酮、总内酯组分对TXRα和TXRβ的作用。结果:基因测序结果表明,成功构建TXRα、TXRβ表达质粒;PCR结果表明,成功建立了TXRα-HEK293和TXRβ-HEK293稳定转染细胞系;Label-free细胞检测结果显示U46619和SQ-29548能够分别特异性激活和拮抗受体,表明TXRα和TXRβLabel-free细胞检测体系构建成功;Label-free细胞检测结果表明舒血宁注射液能够抑制TXRβ,对TXRα无影响。结论:成功建立TXRα和TXRβLabel-free细胞检测体系,并发现舒血宁注射液能够选择性作用于TXRβ,提示舒血宁注射液可通过抑制TXRβ发挥其药理活性。

Objective ： To establish a cell based Label-free detection system for human Thromboxane A2 Receptors （TXRs） , and to evaluate effects of Shuxuening injection and its active components on TXR activation. Methods： Recombinant plasmids encoding TXRα/TXRβ were con- structed, and HEK293 cell lines stably transfected with the receptor subtypes were established. Label-free cell based assay for TXRα/TXRβ function was established and validated by TXR agonist U46619 and antagonist SQ-29548. Shuxuening injection, total flavonoids and total lactones of Ginkgo biloba were analyzed for their effects on TXRα/TXRβ with this system. Results ： DNA sequencing results showed that TXRα/ TXRβ recombinant plasmids were successfully constructed. PCR results showed TXRα/TXRβ stable transfected HEK293 cell lines were es- tablished. Label-free cell based assay results showed that TXR agonist U46619 and antagonist SQ-29548 activated and inhibited TXRα/TXRβ respectively, which indicated the TXRα/TXRβ cell based Label-free detecting system was established. Label-free cell based assay results showed Shuxuening injection inhibited TXRα, but had no effect on TXRα. Conclusion： The human TXRα/TXRβ cell based Label-free de- tecting system is successfully established, and Shuxuening injection selectively affected TXRβ, which presented that the inhibition effect of Shuxuening injection on TXRβ plays a role in its pharmacological function.