Study on quality biomarkers of Salvianolic Acids for Injection based on protection of cerebral microvascular endothelial cells
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,SAFI 18 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,SAFI 18,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 SAFI 18 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.
quality biomarkersquality control of traditional Chinese medicineproteomicsSalvianolic Acids for Injectionvascular endothelial growth factor receptor 2(VEGFR2)
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