Zotarolimus alleviates post-trabeculectomy fibrosis via dual functions of anti-inflammation and regulating AMPK/mTOR axis

Objective: Postoperative scar formation is the primary cause of uncontrolled intraocular pressure following trabeculectomy failure. This study aimed to evaluate the efficacy of zotarolimus as an Adjuvant anti-scarring agent in the experimental trabeculectomy.

Methods: We performed differential gene and Gene Ontology enrichment analysis on rabbit follicular transcriptome Sequencing data (GSE156781). New Zealand white Rabbits were randomly assigned into three groups: Surgery only, Surgery with mitomycin-C treatment, Surgery with zotarolimus treatment. Rabbits were euthanized 3 days or 28 days post-trabeculectomy. Pathological sections were analyzed using immunohistochemistry, immunofluorescence, and Masson staining. In vitro, primary human tenon's capsule fibroblasts (HTFs) were stimulated by transforming growth factor-β1 (TGF-β1) and treated with either mitomycin-C or zotarolimus. Cell proliferation and migration were evaluated using cell counting kit-8, cell cycle, and scratch assays. Mitochondrial membrane potential was detected with the JC-1 probe, and Reactive Oxygen Species were detected using the DCFH-DA probe. RNA and protein expressions were quantified using RT-qPCR and immunofluorescence.

Results: Transcriptome Sequencing analysis revealed the involvement of complex immune factors and metabolic disorders in trabeculectomy outcomes. Zotarolimus effectively inhibited fibrosis, reduced proinflammatory factor release and immune cell infiltration, and improved the surgical outcomes of trabeculectomy. In TGF-β1-induced HTFs, zotarolimus reduced fibrosis, proliferation, and migration without cytotoxicity via the dual regulation of the TGF-β1/SMAD2/3 and AMPK/Akt/mTOR pathways.

Conclusion: Our study demonstrates that zotarolimus mitigates fibrosis by reducing immune infiltration and correcting metabolic imbalances, offering a potential treatment for improving trabeculectomy surgical outcomes.

Adenosine monophosphate-activated protein kinase (AMPK); Anti-fibrosis; Glaucoma; Mammalian target of rapamycin (mTOR); Trabeculectomy; Zotarolimus.