Fig 1.
Hyperosmolarity stimulated ROS production in HCECs.
A. Time course graph displayed a time-dependent increase of DCF fluorescence intensity in HCECs exposed to media with 400 and 450 mOsM for 30–180 min. B. A graph displayed osmolarity-dependent increase of relative folds of ROS production normalized by 312 mOsM control group in HCECs exposed to media with 350, 400 and 450 mOsM for 60, 90 and 120 min. Data showing Mean±SD, n = 5, * P<0.05, ** P<0.01, compared with 312 mOsM normal control. C. Representative images with inserts of enlarged single cell showed ROS-DCF fluorescent positive cells in HCECs exposed to media with 312–450 mOsM for 120 min; the phase images showed equal cell density in 4 groups. Magnification 100x.
Fig 2.
Oxidative biomarkers for cell membrane lipid peroxidation.
A. Western blot showed increased protein levels of 4-HNE and MDA with β-actin as an internal control in primary HCECs exposed to hyperosmotic media with 400 and 450 mOsM for 24 hours. Data showing Mean±SD, n = 3, * P<0.05, ** P<0.01, compared with 312 mOsM normal control. B. Representative immunofluorescent images showed increased immunoreactivities of 4-HNE and MDA in HCECs exposed to different osmolarities.
Fig 3.
Oxidative biomarkers for mitochondrial DNA damage.
A. Western blot showed increased aconitase-2 protein levels with β-actin as an internal control in primary HCECs exposed to hyperosmotic media with 400 and 450 mOsM for 24 hours. Data showing Mean±SD, n = 3, * P<0.05, compared with 312 mOsM normal control. B. Representative immunohistochemical images with insets of enlarged single cell showed increased red brown staining of aconitase-2 and 8-OHdG in HCECs exposed to different osmolarities.
Fig 4.
The increased mRNA and protein levels of oxygenases HMOX1 and COX2 induced by hyperosmotic media in HCECs.
Primary HCECs exposed to media at 312, 400 and 450 mOsM for 4 hours were lysed for mRNA expression by RT-qPCR (A), and the cells treated for 24 hours were performed for immunofluorescent staining (B) or lysed in RIPA buffer for Western blotting (C), to determine the mRNA expression and protein production of HMOX1 and COX2. Data showing mean±SD, n = 5, * P<0.05, ** P<0.01, compared with 312 mOsM normal control.
Fig 5.
The reduced production of anti-oxidative enzymes SOD1 and GPX1 in HCECs exposed to hyperosmotic stress.
Primary HCECs exposed to media at 312, 400 and 450 mOsM for 24 hours were performed for immunofluorescent staining (A), or immunohistochemical staining (B), or lysed in RIPA buffer for Western blotting (C), to determine the protein production of SOD1 and GPX1. Data showing mean±SD, n = 4, V P<0.05, VV P<0.01, compared with 312 mOsM normal control.