Effect of berberine on herpes simplex keratitis through the dimerization of eukaryotic translation initiation factor...

Herpes simplex keratitis (HSK), primarily caused by corneal infection with herpes simplex virus type 1 (HSV-1), is a prevalent sight-threatening ocular disease. Eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2) has emerged as a crucial regulatory target in ocular inflammatory diseases. Berberine (BBR), a natural quaternary ammonium isoquinoline alkaloid, has been shown to exhibit potent inhibitory activity against EIF2AK2. While preliminary in vitro studies have reported the anti-HSV-1 effects of BBR, its therapeutic effects on HSK and underlying biological mechanisms remain to be fully elucidated.

This study aimed to investigate whether BBR could exert protective effects against HSK through the regulation of EIF2AK2.

To evaluate the therapeutic potential of BBR against HSK, human corneal epithelial cells (HCE-T) were infected with HSV-1 for in vitro studies and corneally infected mice were employed for in vivo investigations. An ultraviolet (UV)-triggered recurrent HSK mouse model was established to further explore BBR's effects on HSV-1 latency and reactivation. To elucidate the potential mechanism, molecular docking and microscale thermophoresis (MST) were used to predict and confirm the binding of BBR to EIF2AK2. Molecular dynamics (MD) simulation and co-immunoprecipitation (Co-IP) analyses were performed to determine the impact of BBR on EIF2AK2 dimerization induced by HSV-1. RNA sequencing was conducted to identify downstream effectors in BBR's anti-HSV-1 mechanism. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) technique and adeno-associated virus (AAV) injection were employed to confirm EIF2AK2's role in BBR's efficacy against HSK.

BBR effectively inhibited HSV-1 replication, alleviated HSK severity, and reduced HSK recurrence risk. Notably, loss or knockdown of EIF2AK2 abrogated BBR's protective effect against HSK. Furthermore, MST and molecular docking results confirmed that BBR directly interacts with EIF2AK2. MD and Co-IP analyses further revealed that BBR disrupted HSV-1-induced EIF2AK2 dimerization. This disruption impeded the activation of nuclear factor kappa-B (NF-κB), thereby suppressing viral gene replication and mitigating the inflammatory response.

This study elucidates the role of EIF2AK2 in HSK and demonstrates that BBR-mediated inhibition of EIF2AK2 dimerization could be a novel and promising therapeutic strategy for HSK, providing new perspectives for treating this patient population.