Bufalin mitigates corneal damage in herpes simplex keratitis mice via inhibiting viral replication

Herpes simplex keratitis (HSK) is an ocular viral infectious disease with limited therapeutic options and increasing resistance, leading cause of infectious blindness worldwide. Bufalin is a cardiotonic steroid, more specifically a bufadienolide, which can be found in many plant or animal species, but their main sources are skin and parotid gland secretions of toads (such as Bufo bufo gargarizans Cantor), which constitutes the traditional Chinese medicine preparation known as Chan Su (Venenum Bufonis). Despite its broad pharmacological activities, the potential role of bufalin in controlling HSK progression remains to be systematically investigated.

This study systematically evaluates the antiviral and anti-inflammatory activities of commercial bufalin standards along with the underlying mechanisms in an HSK mouse model, aiming to discover lead compounds for the treatment of drug-resistant HSK.

In vivo studies were conducted using an HSK mouse model established via corneal infection with either HSV-1F or the clinically acyclovir (ACV)-resistant strain HSV-1/153. In vitro experiments were conducted in ARPE-19 and HCEC cell lines, commonly used in ophthalmic research. Plaque assay, RT-qPCR, Western blot, CCK-8, sodium fluorescein staining, H&E staining, and other related experiments were employed to evaluate the antiviral and anti-inflammatory activities of bufalin. The ocular and systemic toxicities of topical bufalin were evaluated using in vivo confocal microscopy, TUNEL staining, body weight monitoring, and organ index analysis.

Bufalin and Chan Su alleviates corneal damage in HSK mice, due to its antiviral and anti‑inflammatory activities both in vitro and in vivo. Bufalin not only inhibits the replication of viruses including HSV-1F and HSV-1/153 strains but also reduces inflammatory infiltration and the expression of inflammatory cytokines (IL-1β and IL-6) in the corneas of HSK mice. RNA-seq analysis reveals that bufalin may act as a potential broad-spectrum antiviral agent by upregulating OAS1 and ISG15 expression, which may represent a distinct antiviral mechanism distinct from ACV. No systemic or ocular toxicity was observed with bufalin at therapeutic doses in this study.

These findings identify the natural product bufalin as a potential candidate against drug-resistant HSK by modulating the expression of broad-spectrum antiviral factors OAS1 and ISG15. This study expands the pharmacological application scope of bufalin, and thereby establishes a novel strategic direction for developing plant-derived bufalin and its derivatives as anti‑herpesvirus agents.