Title : Autophagy impairment-derived SQSTM1 accumulation promotes ferroptosis in corneal epithelial cells through ACSL4 in dry eye
Abstract:
Purpose: To reveal the function of autophagy impairment-derived Sequestosome-1 (SQSTM1) in inducing ferroptosis in experimental dry eye model and investigate the underlying mechanism.
Methods: To induce dry eye animal model, 8-week C57BL/6 mice were subcutaneously injected with scopolamine and exposed to desiccated environment. To build in vitro dry eye model, Human corneal epithelial cells (HCECs) were applied with desiccating stress. Cell viability was examined by CCK8 kit. Intracellular reactive oxygen species(ROS), oxidative lipid, and Fe2+ were detected by H2DCFDA assay kit, C11 Bodipy probe, and FerroOrange probe. Gene expression was screened by RNA-sequencing. Protein expression was evaluated by western-blot and immunofluorescence staining. Cornea defect area was assessed by fluorescein sodium staining. Conjunctiva goblet cells were counted by PAS staining. Tear secretion was measured using phenol red cotton thread.
Results: Desiccating stress induced ferroptosis and SQSTM1 accumulation both in HCECs and C57BL/6 mice. Knocking down of SQSTM1 alleviated ferroptosis in HCECs. On the contrary, overexpressing SQSTM1 promoted ferroptotic changes. Additionally, overexpression of SQSTM1 significantly increased the acyl-CoA synthetase long chain family member 4 (ACSL4). And the targeting inhibition of ACSL4 mitigated dry eye symptom and ferroptosis caused by both SQSTM1 overexpression and desiccating stress.
Conclusions: The accumulating SQSTM1 triggers corneal epithelial cells ferroptosis through ACSL4 in dry eye disease.
