Rosmarinic acid exerts anti-CVB3 effects by inducing HO-1 via the SIRT1/AMPKα/Nrf2 pathway

ABSTRACT Coxsackievirus B3 (CVB3) is the primary pathogen of viral myocarditis (VMC), and there is no specific therapy. Rosmarinic acid (RA) has anti-inflammatory and antioxidant properties, but its anti-CVB3 activity and mechanism remain unclear. This study examined the inhibitory effect of RA on CVB3 replication and VMC, and the underlying mechanism. In CVB3-infected HeLa cells and a mouse VMC model, RA blocked viral replication dose-dependently, with an EC50 of 26.49 μM and a selectivity index of 21.72, which was more effective than ribavirin. Time-of-addition experiments showed that RA interfered with the post-entry replication stage. Mechanistically, RA directly bound sirtuin 1 (SIRT1), with an affinity of −10.2 kcal/mol, increasing its conformational stability and inhibiting ubiquitin-proteasome degradation, thereby reversing CVB3-induced SIRT1 downregulation. Antiviral and anti-inflammatory effects of RA were abolished by SIRT1 knockdown. RA activated the AMPK/Nrf2/HO-1 cascade in a SIRT1-dependent manner. Notably, the HO-1 metabolite biliverdin (BV) stimulated STAT1/2 phosphorylation and interferon-stimulated gene expression. In vivo , RA effectively ameliorated CVB3-induced myocardial injury and restored SIRT1/AMPK/Nrf2/HO-1 expression. In conclusion, RA prevents CVB3 replication and VMC via direct binding and stabilization of SIRT1, activation of the SIRT1/AMPK/Nrf2/HO-1/BV signaling pathway, and stimulation of interferon response through STAT1/2.