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. 2022 Jan 24;10(1):e002579. doi: 10.1136/bmjdrc-2021-002579

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© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

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Increased eHsp90 secretion in a vascular endothelial cell model of oxidative injury in vitro. A CCK-8 assay (A) was used to detect cell viability in HUVECs treated with different concentrations of H₂O₂ for 2, 4, 6, 8, and 24 hours (n=3). The levels of MDA (B) and SOD (C) were measured with assay kits (n=3). HUVECs were treated with H₂O₂ (100 µM, 200 µM, and 300 µM) for 24 hours, and then the intracellular levels of ROS (D), mitochondrial ROS (E), and 8-oxoG (F) and the extracellular levels of ET-1 (G) and MCP-1 (H) were detected (n=3). (I) Western blot analysis concentrated medium and total cell extracts from HUVECs cultured with the indicated concentration of H₂O₂. ImageJ software was used to determine the relative intensities of extracellular and intracellular Hsp90α and Hsp90β bands (n=3). (J) Extracellular and intracellular Hsp90α and Hsp90β expressions were measured after H₂O₂ stimulation for different durations (n=3). (K) Western blot analysis was performed to assess the expression of related proteins in HUVECs pretreated with 17AAG and then exposed to H₂O₂ (n=3). (L) qPCR was employed to observe the respective Hsp90α and Hsp90β mRNA levels after HUVECs were stimulated with H₂O₂ and/or 17AAG for 24 hours (n=3). (M) Representative confocal images of Hsp90α subunit localization in endothelial cells after 24 hours of H₂O₂ stimulation are shown (n=3). All data represent mean±SD of three biological replicates. Differences between groups were analyzed by one-way ANOVA and the Dunnett test, according to the data features. *P<0.05,**P<0.01,***P<0.001 ****P<0.0001 versus control. 17AAG,17-allyamino-17-demethoxygeldanamycin; ANOVA, analysis of variance; CCK-8, Cell Counting Kit-8; Ctrl, control; DAPI, 4',6-diamidino-2-phenylindole; eHsp90, extracellular heat shock protein 90; ET-1, endothelin 1; H₂O₂, hydrogen peroxide; Hsp90, heat shock protein 90; HUVECs, human umbilical vein endothelial cells; MCP-1, monocyte chemotactic protein 1; MDA, malondialdehyd; mRNA, messenger RNA; 8-oxoG, 8-oxoguanine; qPCR, quantitative real-time PCR; ROS, reactive oxygen species; SOD, superoxide dismutase.