Bromelain enhances nitric oxide bioavailability: Bradykinin’s link to TRPV1/Ca2+ /AMPK/autophagy signaling

Bromelain, a protease enzyme extracted from the pineapple stem, is suggested to protect against atherosclerosis, non-alcohol fatty liver diseases, and coagulation dysfunction. However, the mechanism underlying the vascular protection of bromelain in the cardiovascular system is not fully understood. In this study, we explored the role of the kininogen-bradykinin system in bromelain-mediated nitric oxide (NO) bioavailability in endothelial cells (ECs). NO bioavailability was examined by Griess’s assay, western blot analysis was used to assess protein expression, the level of urea and arginine was evaluated by conventional assay kits. In vivo angiogenesis was performed by Matrigel plug assay. In ECs, bromelain increased NO production by increasing intracellular levels of Ca 2+ , activating AMP-activated protein kinase (AMPK), and phosphorylating endothelial nitric oxide synthase (eNOS). Concurrently, bromelain activated the AMPK-regulated autophagy-urea cycle pathway and increased intracellular levels of L-arginine, the precursor of NO, resulting in an increase in NO biosynthesis. Inhibition of bradykinin receptor B 2 (B 2 R) or transient receptor potential vanilloid 1 (TRPV1) prevented the activation of Ca 2+ -AMPK-eNOS signaling, autophagy-urea cycle pathway, and NO biosynthesis by bromelain in ECs. Mechanistically, bromelain cleaved kininogen into bradykinin and activated B 2 R-TRPV1-Ca 2+ -AMPK-eNOS pathway and autophagy-urea cycle-L-arginine pathway, and these two events may work in concert to promote NO production in ECs. In vivo experiments showed that inhibition of B 2 R, TRPV1, eNOS, or autophagy activity attenuated bromelain-induced angiogenesis in Matrigel. This study presents novel understanding into the molecular mechanisms underlying the vascular protection of bromelain in the cardiovascular system. • The biological actions and mechanisms of bromelain in NO bioavailability in ECs. • The enzyme activity of bromelain links kininogen-bradykinin system to eNOS. • Bromelain activates B 2 R-Ca 2+ -TRPV1-AMPK signaling pathway to increase NO. • Bromelain activates autophagy pathway to increase L-arginine bioavailability. • Bromelain provides benefits on the cardiovascular health and diseases.