目的 基于血管內(nèi)皮保護(hù)的關(guān)鍵藥效環(huán)節(jié)，發(fā)現(xiàn)能夠反映注射用丹參多酚酸（SAFI）的質(zhì)量生物標(biāo)志物（Qbiomarkers），為完善其質(zhì)量控制評價提供參考。方法 90℃加熱處理SAFI 4、18、60 h，得到不同化學(xué)質(zhì)量的SAFI（SAFI4、SAFI18、SAFI60），高效液相色譜（HPLC）法分析紫草酸、丹酚酸B、丹酚酸Y、丹參素鈉、原兒茶醛、丹酚酸D、迷迭香酸變化趨勢。體外培養(yǎng)小鼠腦微血管內(nèi)皮細(xì)胞（bEnd.3），建立氧糖剝奪/復(fù)氧（OGD/R）模型，給予SAFI、SAFI4、SAFI18、SAFI60處理18 h，CCK-8法檢測細(xì)胞相對存活率，試劑盒法檢測上清液乳酸脫氫酶（LDH）、NO水平，劃痕實驗檢測細(xì)胞遷移率；通過DIA蛋白質(zhì)組學(xué)分析不同化學(xué)質(zhì)量的SAFI給藥后產(chǎn)生的差異蛋白，結(jié)合藥效學(xué)及偏最小二乘回歸（PLSR）分析篩選SAFI的Q-biomarkers。結(jié)果 與SAFI相比，SAFI4、SAFI18、SAFI60樣品中的紫草酸、丹酚酸B和丹酚酸Y含量下降，丹參素鈉含量上升，原兒茶醛、丹酚酸D、迷迭香酸變化不明顯。藥效學(xué)結(jié)果表明，與模型組相比，SAFI、SAFI4及SAFI18組細(xì)胞相對存活率顯著上升、LDH泄漏量顯著下降、NO水平顯著下降、細(xì)胞遷移率顯著升高（P＜0.05、0.01、0.001） ，對細(xì)胞具有顯著保護(hù)作用；而SAFI60組無明顯保護(hù)作用。蛋白質(zhì)組學(xué)結(jié)果表明，18個差異蛋白的含量隨藥效學(xué)和成分的變化而發(fā)生一致性上升或下降，與藥效進(jìn)行相關(guān)性分析發(fā)現(xiàn)Kdr、Cxcl12的變量重要性投影值（VIP）皆大于1，尤其Kdr ［血管內(nèi)皮生長因子受體2 （VEGFR2）］與血管內(nèi)皮功能高度相關(guān)，可能是潛在的SAFI的Q-biomarkers。結(jié)論 通過不同化學(xué)質(zhì)量的SAFI的內(nèi)皮保護(hù)藥效及差異蛋白關(guān)聯(lián)分析發(fā)現(xiàn)，VEGFR2可以作為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.

R285.5

天津市科技計劃項目（21YDTPJC00240）；天津市研究生科研創(chuàng)新項目（2021YJSS178）