Bioactivity-integrated UPLC/Q-TOF–MS of Danhong injection to identify NF-κB inhibitors and anti-inflammatory targets based on endothelial cell culture and network pharmacology

Abstract

Ethnopharmacological relevanceDanhong injection (DHI) is a traditional Chinese medicine injection that has been widely used in therapy for cardiovascular diseases. However, neither its mechanism nor its active constituents are clearly understood. Aim of the studyOur research aimed at identifying the anti-inflammatory ingredients and mechanism of DHI by combining high-throughput screening (HTS) with network pharmacology analysis. Materials and methodsThe human endothelial cell line EAhy926 was cultured in vitro. Methyl thiazolyltetrazolium (MTT) and lactate dehydrogenase (LDH) assays were performed to detect cell viability. The expression of Bcl-2 and Bax, interleukin-6 (IL-6), inhibitor of nuclear factor kappa-B kinase (IKK), phosphorylated IKK, phosphorylated NF-κB and phosphorylated IκB-α from the supernatant were determined. Then, we constructed an assay system combining ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF–MS) with an NF-κB activity luciferase reporter to screen DHI for essential anti-inflammatory components, and the results were verified using network pharmacology. ResultsDHI could significantly suppress inflammatory responses, and the mechanism may be via an NF-κB-dependent pathway. We found nine potential anti-inflammatory ingredients: danshensu, protocatechuic acid, protocatechuic aldehyde, caffeic acid, hydroxysafflor yellow A, safflor yellow A, salvianolic acid A salvianolic acid B and salvianolic acid C. Among these compounds, the NF-κB inhibitory activity of SAC is reported here for the first time. ConclusionsDHI plays an important role in suppressing inflammatory responses through inhibiting the NF-κB signaling pathway. The potential NF-κB inhibitors in DHI contribute to the cross-talk of multiple targets in anti-inflammation.