The Intervention of Sodium Hydrosulfide on Cuproptosis in Pulmonary Arterial Endothelial Cells under Hypoxia
HUANG Man1#, ZHOU Haizhen2#, CHENG Yuan1, HAN Congmei3, YUAN Linbo3, YOU Liyi4*, WANG Wantie1*
This study aims to explore the effects and mechanisms of exogenous hydrogen sulfide donor NaHS (sodium hydrosulfide) on the function and metabolism of HPAECs (human pulmonary artery endothelial cells) induced by hypoxia. Cell identification was performed using immunofluorescence. The optimal concentration of NaHS to inhibit hypoxia-induced HPAEC injury was determined using the CCK-8 assay. ELISA was used to measure the levels of ET-1 (endothelin-1), NO (nitric oxide) and PGI2 (prostacyclin I2) in HPAECs. Fluorescence was used to detect Cu+ concentration in each group of HPAEC. Western blot was used to detect copper transport proteins CTR1, ATP7A, ATP7B and cuproptosis markers FDX1, LIAS, DLAT, DLAT Dimer, DLST. RT-qPCR was used to detect the mRNA levels of FDX1, LIAS, DLAT and DLD. Immunofluorescence was used to observe the ex pression of FDX1 and DLAT. JC-1 mitochondrial membrane potential detection kit was usd for monitoring chang es in cellular mitochondrial embrane potential. ATP levels in cells were detected by chemiluminescence. ELISA was used to detect the content of PDHA1 (pyruvate dehydrogenase E1 subunit alpha 1). Sulfosalicylic acid method was used to measure CA (citric acid) content. The research results showed that theoptimal concentration of NaHS to inhibit hypoxia-induced HPAEC injury was 200 µmol/L. Under hypoxic conditions, the concentration of the en dothelial cell-secreted mediator ET-1 increased, while the concentrations of NO and PGI2 decreased. Intracellular Cu+ levels rose significantly, copper transporter 1 expression was markedly upregulated, and copper effluxproteins ATP7A/B were significantly downregulated. The expression levels of cuproptosis markers FDX1, LIAS, DLAT, and DLST were significantly reduced, while DLAT Dimer expression was significantly increased. The expression levels of cuproptosis FDX1, LIAS, DLAT and DLD genes were all significantly downregulated. At the same time, mito chondrial function indicators JC-1 and ATP levels were significantly decreased, and tricarboxylic acid cycle indica tors PDHA1 and CA contents were significantly reduced. After adding NaHS (200 µmol/L), the abnormal changes in the above indicators were all reversed to varying degrees. The study results indicate that hypoxia can trigger cu proptosis in HPAECs, leading to mitochondrial dysfunction and metabolic disorders; NaHS can improve hypoxia induced HPAEC dysfunction and metabolic disorders by inhibiting cuproptosis.
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