Chebulinic Acid Exerts Anti-rotavirus Effects through the p38MAPK/ERK1/2 Signaling Pathway

Rotavirus (RV) is a leading cause of diarrhea in infants and young children. Drugs effective against RV infection are not yet available in clinical practice. To investigate the anti-RV activity of chebulinic acid (CA) and its potential mechanism against RV.

The anti-RV activity of CA in vitro was evaluated by CCK8 assay, and the effects of CA on VP6 expression for RV RNA synthesis and protein expression were assessed using qRT-PCR、western blotting, and immunofluorescence, respectively. Before mechanistic validation, an in silico network pharmacology screen was performed to build a CA-host target-interaction map; DAVID enrichment flagged the p38/ERK axis as a top hit. Additionally, immunofluorescence and DCFH-DA ROS fluorescent probe were used to assess CA's effects against RV-induced ROS production and its direct ROS-scavenging activity, respectively, and western blotting was employed to evaluate whether CA exerts anti-RV activity by inhibiting the p38MAPK/ERK1/2 signaling pathway. Furthermore, we also evaluated the anti-viral effect of CA in RVinfected 4 days post-fertilization (4dpf) zebrafish model.

Our results indicated that 4-10 μmol/L of CA has the ability to hinder VP6 expression, and it also decreased mitochondrial ROS production. Network pharmacology screening had previously identified 38 CA- RV intersection targets and ranked the p38 MAPK axis as the top-enriched pathway. Further research confirmed that CA downregulated p38MAPK/ERK1/2 phosphorylation levels in response to viral infection. In the RV-infected zebrafish model, CA greatly improved the survival rate. In addition, RV infection resulted in abnormal behavior in zebrafish, and CA was found to substantially decrease the incidence of convulsive behavior. The intestinal tract of zebrafish treated with CA led to the restoration of intestinal morphology and exhibited fewer inflammatory cell infiltrates, with a significantly reduced degree of inflammation compared to the viral group.

This study comprehensively evaluated the anti-RV activity of CA using two RV strains, RV-WA and SA-11, an infected-cell model in vitro, as well as the RV-WA-infected zebrafish model in vivo. As we know, CA, belonging to polyphenolic compounds, possesses notable antioxidant and anti-inflammatory potential; therefore, we investigated how RV-induced oxidative stress affected the host cell apoptosis and mitochondrial membrane potential, and how it activated the p38MAPK/ERK1/2 kinase signaling pathway. Our findings demonstrate that CA can reverse these pathological alterations and thereby exert anti-RV effects, offering a new therapeutic strategy against RV infection.

This study first demonstrated that CA possessed anti-RV properties by inhibiting mitochondrial oxidation-induced apoptosis via the p38MAPK/ERK1/2 kinase signaling pathway. These findings provided a basis for the clinical application of CA as an anti-RV therapy.