Hypobaric Hypoxia Increased Autophagy and Apoptosis in PC12 Rat Pheochromocytoma Cells More Than Normobaric Hypoxia

Jiaojiao Yin, Yuhang Wang, Bing Li, Xiaoyan Hu, Yao Ma, Chong Zhang, Xiaoqin Ha, Linyan Wang, and Yaozhu Pan. Hypobaric hypoxia increased autophagy and apoptosis in PC12 rat pheochromocytoma cells more than normobaric hypoxia. High Alt Med Biol. 00:00-00, 2025. Purpose: Currently, in vitro studies have focused on hypoxia injury in acute mountain sickness (AMS), but little effort has been made to assess the effects of hypobaric hypoxia. AMS is a neurological disorder, and rat pheochromocytoma (PC12) cells are a model to study neuronal survival and apoptosis. Here, we developed a novel cell culture method that mimics hypobaric hypoxia at high attitude and compared the effects of hypobaric hypoxia and normobaric hypoxia on autophagy and apoptosis of PC12 cells. Methods: PC12 cells were cultured under normal conditions, normobaric hypoxia, and hypobaric hypoxia. Autophagy was observed by transmission electron microscopy and immunofluorescence microscopy. The hypoxia-inducible factor1-α (HIF1-α), LC3, caspase-3, and cleaved caspase-3 expression levels were determined by Western blot. Results: The cell culture chamber mimicking hypobaric hypoxia at high attitude perfectly maintained the air pressure at 41.1 kPa and the oxygen density at 1% (PO2 around 3.08 mmHg). Hypobaric hypoxic treatment of PC12 cells at 0, 4, 8, 16, 24, and 48 hours resulted in an increase in HIF1-α and LC3Ⅱ protein levels, and the ratio of HIF1-α/actin and LC3Ⅱ/actin both peaked at 16 hours (p < 0. 01) when the cell viability was 88.02%. There was a 1.5-fold increase in LC3Ⅱ expression, a 2-fold increase in LC3B-positive spots, and an increase in autophagosome accumulation at hypobaric hypoxia compared to PC12 cells at normobaric hypoxia for 16 hours (p < 0.001). Interestingly, the promotion of autophagy (coculture with rapamycin or 3-MA) in PC12 cells under normobaric hypoxia reduced cleaved caspase-3 expression (the ratio of cleaved caspase-3/caspase-3 decreased, p < 0.01). However, under hypobaric hypoxia, the promotion of autophagy inversely increased cleaved caspase-3 (the ratio of cleaved caspase-3/caspase-3 increased, p < 0.01), and the inhibition of autophagy (hydroxychloroquine [HCQ] coculture) decreased cleaved caspase-3 (the ratio of cleaved caspase-3/caspase-3 decreased, p < 0.01). Conclusions: Compared with normobaric hypoxia cells, hypobaric hypoxia cells cultured in vitro exhibited increased autophagy and apoptosis.