基于脑微血管内皮细胞保护的注射用丹参多酚酸质量生物标志物研究

Study on quality biomarkers of Salvianolic Acids for Injection based on protection of cerebral microvascular endothelial cells

目的基于血管内皮保护的关键药效环节,发现能够反映注射用丹参多酚酸(SAFI)的质量生物标志物(Qbiomarkers),为完善其质量控制评价提供参考。方法90℃加热处理SAFI 4、18、60 h,得到不同化学质量的SAFI(SAFI4、SAFI18、SAFI60),高效液相色谱(HPLC)法分析紫草酸、丹酚酸B、丹酚酸Y、丹参素钠、原儿茶醛、丹酚酸D、迷迭香酸变化趋势。体外培养小鼠脑微血管内皮细胞(bEnd.3),建立氧糖剥夺/复氧(OGD/R)模型,给予SAFI、SAFI4、SAFI18、SAFI60处理18 h,CCK-8法检测细胞相对存活率,试剂盒法检测上清液乳酸脱氢酶(LDH)、NO水平,划痕实验检测细胞迁移率;通过DIA蛋白质组学分析不同化学质量的SAFI给药后产生的差异蛋白,结合药效学及偏最小二乘回归(PLSR)分析筛选SAFI的Q-biomarkers。结果与SAFI相比,SAFI4、SAFI18、SAFI60样品中的紫草酸、丹酚酸B和丹酚酸Y含量下降,丹参素钠含量上升,原儿茶醛、丹酚酸D、迷迭香酸变化不明显。药效学结果表明,与模型组相比,SAFI、SAFI4及SAFI18组细胞相对存活率显著上升、LDH泄漏量显著下降、NO水平显著下降、细胞迁移率显著升高(P<0.05、0.01、0.001),对细胞具有显著保护作用;而SAFI60组无明显保护作用。蛋白质组学结果表明,18个差异蛋白的含量随药效学和成分的变化而发生一致性上升或下降,与药效进行相关性分析发现Kdr、Cxcl12的变量重要性投影值(VIP)皆大于1,尤其Kdr[血管内皮生长因子受体2(VEGFR2)]与血管内皮功能高度相关,可能是潜在的SAFI的Q-biomarkers。结论通过不同化学质量的SAFI的内皮保护药效及差异蛋白关联分析发现,VEGFR2可以作为SAFI的Q-biomarker。

Objective Based on the key pharmacodynamic link of the protection of vascular endothelial,we found quality biomarkers(Q-biomarkers)that can reflect the quality of Salvianolic Acid for Injection(SAFI),which can provide a reference for the improvement of the quality control of it.Methods Preparation of three SAFI samples with different chemical qualities using the 90℃treated for 4,18,60 h,namely SAFI4,SAFI18 and SAFI60.Analyzing for sodium danshensu,protamine sulfates,salvianolic acid D,rosmarinic acid,lithospermic acid,salvianolic acid B,and salvianolic acid Y using HPLC.Mouse brain microvascular endothelial cells(bEnd.3)were cultured in vitro and established oxygen glucose deprivation/reoxygention(OGD/R)model,treated with SAFI,SAFI4,SAFI18,and SAFI60 for 18 h.CCK8 method was used to detect the relative survival rate of cells,the reagent kit method was used to detect the levels of lactate dehydrogenase(LDH)and NO in the supernatant,and scratch assay was used to detect cell migration rate.Analysis of differential expressed proteins(DEPs)produced after administration of SAFI with different chemical qualities by DIA proteomics,and the quality biomarkers of SAFI were screened in combination with pharmacodynamics and partial least squares regression(PLSR)analysis.Results Compared with SAFI,the content of salvianolic acid B,salvianolic acid Y and lithospermic acid decreased,sodium danshensu increased and protamine sulfates,salvianolic acid D,rosmarinic acid showed no significant changes in SAFI samples of different chemical qualities.The pharmacodynamic results showed that,compared with the model group,the SAFI,SAFI4,and SAFI18 groups showed a significant increase in cell relative survival rate,a significant decrease in LDH leakage,a significant decrease in NO levels,and a significant increase in cell migration rate(P<0.05,0.01,0.001),indicating a significant protective effect on cells;the SAFI60 group showed no significant protective effect.Proteomic analysis showed that there were 89 differentially expressed proteins varied with the changes of pharmacodynamics and composition,of which 18 differentially expressed proteins consistent upward or downward in their content with the changes of pharmacodynamics.Correlation analyses with pharmacodynamics showed that the VIP values of Kdr and Cxcl12 were all greater than one,especially Kdr[vascular endothelial growth factor receptor 2(VEGFR2)]was highly correlated with vascular endothelial function,which may be a potential biomarker of endothelial protective quality for SAFI.Conclusion Endothelial protective efficacy of different chemical qualities of SAFI and differentially expressed proteins association analysis revealed that VEGFR2 can be used as a quality biomarker for SAFI.