Integrated serum pharmacochemistry and serum pharmacology to investigate the active components and mechanism of Bushen Huoxue Prescription in the treatment of diabetic retinopathy

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.