本研究旨在研究补肾活血方(BHP)的活性成分,并基于体内谱效关系,通过血清药物化学和血清药理学的整合进一步阐明了机制。在本文中,采用超高效液相色谱-四极杆-ExactiveOrbitrap-高分辨质谱(UPLC-Q-ExactiveOrbitrap-HRMS)分析血清中吸收的成分。并选择Müller细胞作为靶细胞,进一步研究其机制。细胞纯化后,通过苏木精-伊红染色(HE)染色和免疫荧光分析鉴定生长良好的细胞,如胶质纤维酸性蛋白(GFAP)和谷氨酰胺合成酶(GS)。将对数期细胞分为正常组,模型组和BHP组12个。用50mmol/L葡萄糖和1mmol/L焦亚硫酸钠诱导高血糖和低氧模型。采用酶联免疫吸附试验(ELISA)检测与DR密切相关的5种因子的表达,命名为血管内皮生长因子(VEGF),缺氧诱导因子1-α(HIF-1α),蛋白激酶C-β(PKC-β),血管生成素-2(ANG-2)和转化生长因子-β(TGF-β)。最后,通过偏最小二乘回归(PLSR)研究光谱-效应关系以筛选BHP的活性成分。结果表明,83种代谢成分,在BHP血清中发现了30种原型和53种代谢物。选择12个特征公共峰建立谱-效应关系。重要的是,所有12种BHP血清对VEGF的表达均表现出较强的抑制作用,PKC-β,和ANG-2,以及VEGF的表达,PKC-β,选择ANG-2来建立体内的谱-效应关系。PLSR结果表明,咖啡酸的甲基化和硫酸化含量,丹参素的脱羟基和硫酸化,Daidzein,O-去甲基angolanolanin,cryptotanshinone,丹参酮IIA和原参三醇与Müller细胞的VEGF表达呈负相关;二氢咖啡酸的面积,咖啡酸的甲基化和硫化,丹参素的脱羟基和硫酸化,Daidzein,cryptotanshinone,丹参酮IIA与PKC-β的表达呈负相关;而羟基酪醇硫酸化系数,R-雌马酚,O-去甲基angolanolanin,二氢丹参酮IIA,水合隐丹参酮,原参三醇与ANG-2的表达呈负相关。以上结果表明隐丹参酮,丹参酮IIA,我们未来的研究需要关注大豆黄酮和原人参三醇。此外,该研究思路为研究和确定中药及其配方的药理作用物质基础和Q标记物的筛选提供了可行的途径。
This study aimed to investigate the active components of Bushen Huoxue Prescriptions (BHP), and further clarify the mechanism by the integration of serum pharmacochemistry and serum pharmacology based on spectrum-effect relationship in vivo. In this paper, the components absorbed into serum were analyzed by ultra-high performance liquid chromatography-quadrupole-Exactive Orbitrap-high resolution mass spectrometry (UPLC-Q-Exactive Orbitrap-HRMS). And Müller cells were chosen as target cells to further investigate the mechanism. After cell purification, the well-grown cells were identified by Hematoxylin-eosin staining (HE) staining and immunofluorescence assay, such as glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). The logarithmic phase cells were divided into normal group, model group and 12 BHP groups. The hyperglycemic and hypoxic model was induced by 50 mmol/L glucose and 1 mmol/L sodium disulfite. Enzyme-linked immunesorbnent assay (ELISA) was used to detect the expressions of five factors closely related to DR, named vascular endothelial growth factor (VEGF), hypoxia-inducible factor1-alpha (HIF-1α), protein kinase C-β (PKC-β), angiopoietin-2 (ANG-2) and transforming growth factor-β (TGF-β). Finally, the spectrum-effect relationship was investigated to screen the active components of BHP by partial least squares regression (PLSR). The results showed that 83 metabolic components, containing 30 prototypes and 53 metabolites were found in BHP serum. 12 characteristic common peaks were chosen to establish spectrum-effect relationship. Significantly, all the 12 BHP serum exhibited stronger inhibition on the expression of VEGF, PKC-β, and ANG-2, and the expression of VEGF, PKC-β, ANG-2 was chosen to establish the spectrum-effect relationship in vivo. The results of PLSR revealed that the content of methylation and sulfuration of caffeic acid, dehydroxylation and sulfation of Danshensu, daidzein, O-demethylangolanolin, cryptotanshinone, tanshinone IIA and protopanaxatriol were inversely correlated with VEGF expression of Müller cells; the areas of dihydrocaffeic acid, methylation and sulfuration of caffeic acid, dehydroxylation and sulfation of Danshensu, daidzein, cryptotanshinone, tanshinone IIA were negative correlation with the expression of PKC-β; while the coefficient of hydroxytyrosol sulfation, R-equol, O-demethylangolanolin, dihydrotanshinone IIA, hydrated cryptotanshinone, protopanaxatriol showed negative correlation with the expression of ANG-2. The above results indicated that cryptotanshinone, tanshinone IIA, daidzein and protopanaxatriol need be focused in our future research. In addition, this research idea provides feasible ways to investigate and determine pharmacodyamic material basis and screen the Q-markers of TCM and its formulas.