The role of STAT3-targeted therapy created with COLIVELIN in the cross-talk between IL6/JAK2/STAT3 and TGF-β/SMAD2/SMAD3 signaling in a hyperinflammation and ROS-induced in vitro AMD model and its effect on retinal apoptosis.

This study aimed to investigate the therapeutic potential of Colivelin in modulating the cross-talk between the IL-6/JAK2/STAT3 and TGF-β/SMAD2/SMAD3 signaling pathways and its downstream effects on retinal apoptosis in an in vitro AMD model. An in vitro AMD model was established in ARPE-19 human RPE cells using a sublethal combination of lipopolysaccharide and hydrogen peroxide. Apoptosis was quantified via Tali® image cytometry. Gene expression profiling was performed by qRT- PCR. Protein expressions were assessed by Western blot. Formal mediation analysis was employed to quantify pathway-specific mechanistic contributions. The AMD model exhibited significant upregulation of hypoxia-related genes (HIF-1α, VEGF, MMP3, MMP9), pro-inflammatory cytokines (IL-6, TNF-α), and pro-apoptotic markers (BAX, p53, Caspase- 3), accompanied by markedly elevated ROS levels and reduced cell viability. Low-dose Colivelin (1 µM) significantly enhanced STAT3 phosphorylation, restored antioxidant gene expression (GSS, CAT, SOD2), suppressed hypoxia-associated gene expression, and substantially reduced TGF-β receptor, SMAD2, and SMAD3 expression at both transcriptional and protein levels. Formal mediation analysis revealed that 91-98% of Colivelin's anti-apoptotic effect at the therapeutic dose was mediated through STAT3-driven suppression of TGF-β/SMAD2/3 signaling, rather than through direct STAT3 transcriptional activity on apoptotic target genes. Conversely, high-dose Colivelin (10 µM) paradoxically activated SMAD2/3-independent pro-apoptotic cascades, demonstrating a dose- dependent biphasic response. This study provides the first formal mechanistic evidence that Colivelin exerts its cytoprotective effects in AMD primarily through a STAT3 → SMAD2/3 suppression axis. Low-dose (1 µM) Colivelin demonstrated superior and broader therapeutic efficacy compared to Bevacizumab by simultaneously modulating oxidative stress, hypoxia, angiogenesis, and apoptotic signaling pathways. These findings establish Colivelin as a promising multi-target therapeutic candidate for AMD, with its therapeutic window defined by the capacity of STAT3 activation to selectively suppress TGF-β/SMAD-driven apoptotic signaling without engaging compensatory pro-death mechanisms. Rigorous pharmacokinetic optimization and in vivo validation are warranted to advance Colivelin toward clinical translation.