Abstract: Objective To investigate the molecular mechanism by which heme oxygenase-1 （HMOX1） regulates ferroptosis in endometriosis. Methods Human immortalized endometrial ectopic epithelial cells （hEM15A） were divided into blank control （Con） group, sh-NC group, and sh-HMOX1 group. The hEM15A cells in the sh-NC and sh-HMOX1 groups were transfected with negative control and interfering plasmids targeting HMOX1, respectively. Normal human endometrial stromal cells （hESCs） were divided into blank control （Con） group, FeSO₄ group, FeSO₄+sh-NC group, and FeSO₄+sh-HMOX1 group. Cells in the FeSO₄, FeSO₄+sh-NC, and FeSO₄+sh-HMOX1 groups were exposed to FeSO₄ （100 μmol/L） solution for 24 hours. The hESCs in the FeSO₄+sh-NC and FeSO₄+sh-HMOX1 groups were transfected with negative control and interfering plasmids targeting HMOX1 prior to FeSO₄ exposure. Cell viability was assessed using the Cell Counting Kit-8 （CCK-8） assay. Lipid peroxidation was measured by flow cytometry. Intracellular Fe²⁺ levels were analyzed by FeRhoNox-1 fluorescence staining. Cell migration ability was evaluated by Transwell assay, and pro-angiogenic activity was assessed by HUVECs tube formation assay. Expression of iron metabolism-related proteins was analyzed by Western blotting. Results Compared with hESCs, HMOX1 protein expression was upregulated in hEM15A cells. Compared with the sh-NC group, the sh-HMOX1 group exhibited decreased hEM15A cell viability, number of migrated cells, number of HUVEC tube formation,lipid ROS levels, Fe²⁺ levels, and ACSL4 protein expression （all P<0.05）, while SLC7A11 and GPX4 protein expression was increased （both P<0.05）. Compared with the Con group, the FeSO₄ and FeSO₄+sh-NC groups showed increased lipid ROS, Fe²⁺ levels, and HMOX1 and ACSL4 protein expression in hESCs （all P<0.05）, whereas SLC7A11 and GPX4 protein expression was decreased （both P<0.05）. Compared with the FeSO₄+sh-NC group, the FeSO₄+sh-HMOX1 group showed decreased lipid ROS, Fe²⁺ levels, and HMOX1 and ACSL4 protein expression in hESCs （all P<0.05）, while SLC7A11 and GPX4 protein expression was increased （both P<0.05）. Compared with the Con group, the FeSO₄ and FeSO₄+sh-NC groups exhibited increased numbers of migrated hESCs and HUVEC tube formation（all P<0.05）. Compared with the FeSO₄+sh-NC group, the FeSO₄+sh-HMOX1 group showed decreased cell viability, number of migrated cells, and number of HUVEC tube formation in hESCs （all P<0.05）. Conclusion Knockdown of HMOX1 may inhibit the progression of endometriosis by regulating ferroptosis, providing new evidence for HMOX1 as a potential therapeutic target for endometriosis.

Key words: endometriosis, ferroptosis, heme oxygenase-1, migration, angiogenesis