Vitamin D alleviates hypoxia/reoxygenation-induced injury of human trophoblast HTR-8 cells by activating autophagy

Introduction: Attenuation of trophoblast cell dysfunction would be beneficial for retarding pre-eclampsia (PE). Vitamin D has been reported to improve trophoblast cell function in early PE, but the mechanism involved is not fully elucidated. This study is aimed to investigate whether vitamin D alleviates trophoblast cell dysfunction via regulating Autophagy.

Methods: Human trophoblast HTR-8 cells were cultured in hypoxia/reoxygenation (H/R) condition to simulate the oxidative stress state of early PE in vitro. MTT, Transwell and tube formation assays were respectively applied to assess cell proliferation, invasion, and angiogenesis abilities. DCFH-DA staining was performed to detect cellular Reactive Oxygen Species levels. GFP-RFP-LC3 plasmid transfection and transmission electron microscopy were subjected to monitor Autophagy. Enzyme-linked immunosorbent assay and Western blot analysis were used to detect autophagy-related and pyroptosis-associated molecules.

Results: H/R led to severe impairments on the bio-function of HTR-8 cells, as evidenced by the deficiency of cell proliferation, invasion, and angiogenesis abilities, and the increase of cellular ROS production. Simultaneously, H/R inhibited Autophagy and triggered Pyroptosis. 1,25(OH)₂D₃, the hormonally active form of vitamin D, dramatically attenuated H/R-induced trophoblast dysfunction. Also, 1,25(OH)₂D₃ activated Autophagy and inhibited Pyroptosis. Additionally, autophagy-enhancer rapamycin exerted similar protective effect to that of 1,25(OH)₂D₃, whereas autophagy-inhibitor 3-methyladenine blocked the protective effect of 1,25(OH)₂D₃.

Discussion: The mechanism that vitamin D alleviates trophoblast cell dysfunction is associated with Autophagy induction and Pyroptosis inhibition.