Acellular Amniotic Membrane Extract Reduces Oxidative Stress, Complement, and Angiogenic Pathways in Retinal Cells

Abstract Background: Oxidative stress (OS) and subsequent mitochondrial dysfunction represent critical early upstream events in the degeneration of human high demand metabolic tissues, such as those in the retina. Under persistent oxidative conditions, the accumulation of reactive oxygen species (ROS) triggers chronic inflammation. In retinal tissues, local complement system orchestrates inflammatory signal release and pro-angiogenic program activation leading to progressive cell failure and abnormal vessel growth. Given the limitations of single-target therapies, multi-target cytoprotective strategies are urgently required. While the human amniotic membrane (AM) exhibits well-established anti-inflammatory and tissue-repair properties and is broadly applied in ophthalmic pathologies, its underlying biochemical mechanisms remain poorly defined. This study investigated whether acellular AM-derived factors could protect human retinal pigment epithelium (RPE) cells, ARPE-19, against hydrogen peroxide-induced injury and modulate downstream complement, inflammatory, and angiogenic signaling. Methods and results: ARPE-19 cells were exposed to H 2 O 2 and treated with acellular AM extract in both pre-treatment and post-treatment protocols. Cell viability was assessed via MTT assay, and intracellular ROS accumulation was quantified. Mitochondrial network connectivity and morphological fragmentation were evaluated through quantitative skeletal imaging analysis. Monolayer integrity and cytoprotection were verified by wound-healing and cell attachment assays. Transcriptional profiles of local complement components (C3 and C5), complement receptors (C3aR and C5aR), pro-inflammatory cytokines (IL-6 and IL-8), and angiogenic factors (HIF-1α and VEGF) were quantified by RT-qPCR. Our results shown that the exposure to H 2 O 2 induced a significant, dose-dependent decrease in cell viability, which fell to 35% at 0.2 mM. AM pre-treatment robustly maintained cell viability above 85%, while post-treatment also offered significant rescue against oxidative damage. Both AM administration protocols successfully halted intracellular ROS accumulation and effectively prevented mitochondrial network breakdown, restoring the elongated, interconnected tubular morphology of active mitochondria. Furthermore, AM factors preserved monolayer integrity, preventing cell detachment and degeneration in wounded areas. At the molecular level, oxidative injury triggered a sharp upregulation of complement mediators (C3, C3aR, C5aR), pro-inflammatory cytokines (IL-6 and IL-8), and pro-angiogenic factors (HIF-1α and VEGF). Notably, both AM treatment regimens significantly suppressed these pathogenic pathways, with a particularly pronounced inhibitory effect on upstream angiogenic signaling (HIF-1α) and complement transcription. Conclusion: These findings demonstrate that acellular AM-derived factors act as potent multi-target modulators that intercept oxidative stress, restore mitochondrial network connectivity, and suppress downstream complement-driven inflammation and angiogenic signaling in an in vitro human cell model. This study provides crucial mechanistic evidence supporting the development of AM-based biochemical therapies for complex degenerative applications.