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.

ETHNOPHARMACOLOGICAL RELEVANCE: Danhong 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 STUDY: Our research aimed at identifying the anti-inflammatory ingredients and mechanism of DHI by combining high-throughput screening (HTS) with network pharmacology analysis.

MATERIALS AND METHODS: The 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.

RESULTS: DHI 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.

CONCLUSIONS: DHI 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.