Cell metabolomics to reveal the mechanism of saponins and alkaloids on prostate inflammation via COX and LOX pathways

Abstract

Inflammation initiates and progresses due to overexpression of the arachidonic acid cascade mediators. Cyclooxygenase (COX) and lipoxygenase (LOX) are considered to be the primary targets, and the aim of this study was to assess the anti- inflammatory activity of several herbal compounds as dual COX-LOX inhibitors on prostatic diseases. Compounds Timosaponin AⅡ, timosaponin AⅢ, berberine and demethyleneberberine were selected according to our previous studies. Human prostate cancer cells (PC-3) were cultured and the cell viability was detected by CCK-8 method at different concentrations. Ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was applied to monitor the changes of AA metabolites after treatment. Western blot and qPCR were used to detect COX-2 and LOX-5 at protein and mRNA levels, respectively. Additionally, the levels of inflammatory factors (IL-6, IL-1β, TNF-α) in each group were detected by ELISA. After drug interference, 13 altered metabolites associated with the COX/LOX pathway, including PGE₂, 5-HETE, LTB₄, etc. were identified in cell medium. Quantitative metabolomics analysis showed that the candidate compounds could significantly decreased the concentrations of dual target related metabolites to varying degrees (P < 0.01). These compounds could also suppressed COX-2 and LOX-5 expression at the protein and mRNA levels simultaneously. Moreover, the levels of cytokine IL-6, IL-1β and TNF-α were also significantly reduced in the treated group compared to controls (P < 0.05 or P < 0.01). Our findings revealed the four active compounds were potential COX/LOX dual-target inhibitors, which inhibited a range of inflammatory responses by interfering with AA metabolism and down-regulating the levels of COX and LOX metabolites.