Abstract

Enterovirus A/B is a significant pathogen responsible for a wide range of human diseases. Currently, effective preventive and therapeutic strategies against these viruses remain limited. Timosaponin BII is the active ingredient of a natural product with potential antioxidant and anti-inflammatory properties. This study aims to investigate the antiviral activity of Timosaponin BII and elucidate its underlying mechanism, thereby providing a scientific basis for the development of novel broad-spectrum anti-enterovirus agent. Firstly, systems pharmacology approaches were employed to integrate the potential targets of Timosaponin BII and predict its mechanism of action. Subsequently, the antiviral activity against representative strains of enterovirus A/B (EV-A71 for enterovirus A and CVB3 for enterovirus B respectively) was verified through cell morphology observation and CCK-8 assay in interferon-deficient Vero cells. Finally, the inhibitory effect on viral replication was assessed using a time-course drug addition experiment in non-interferon-deficient HeLa cells, and the inhibitory effect on viral proteins was detected by Western Blot. Timosaponin BII exhibited significant antiviral activity against enterovirus A and B at the cellular level, with EC 50 values of 0.54 μM against EV-A71 and 2.59 μM against CVB3 at 48 h post-infection. Quantitative real-time PCR (qRT-PCR) analysis revealed that the levels of EV-A71 and CVB3 viral VP1 RNA in the group treated with Timosaponin BII simultaneously with viral infection were lower than those in the viral infection group. Western blot results showed that Timosaponin BII could reduce the expression level of viral protein. Timosaponin BII, a potential broad-spectrum antiviral agent, exhibits antiviral activity against EV-A71 and CVB3. Its antiviral mechanism may involve the synergistic interaction of multiple targets and signaling pathways.