Fig 1.
Hyperosmotic stress induces a dose- and time-dependent apoptosis in mouse corneal epithelial cells.
Mouse corneal epithelial cells were treated with varying osmolarities (450, 550 or 650 mOsm) by addition of glucose for 24 h. The apoptotic cells were observed under inverted contrast microscopy (A) and detected by staining with FITC-Annexin V/PI and FACS analysis (B). Mouse corneal epithelial cells were treated with 550 mOsm hyperosmotic stress by addition of glucose for 12, 24 or 48h, and the apoptotic cells were investigated by staining with FITC-Annexin V/PI and FACS analysis (C). Hyperosmotic stress treatment induced the apoptosis of mouse corneal epithelial cells in a dose and time-dependent manner (B, C).
Fig 2.
SP protects from hyperosmotic stress-induced apoptosis of corneal epithelial cells.
Mouse corneal epithelial cells were treated with 550 mOsm hyperosmotic stress by addition of glucose with or without 0.1, 1 or 10 μM SP for 24 h. Cell morphology was observed under inverted contrast microscopy (A). The apoptosis was evaluated by FACS analysis followed by FITC-Annexin V/PI staining (B), caspase activity measurement (C), and the detection of Bcl-2-associated death promoter (Bad), BCL2-associated X protein (Bax), apoptosis inducing factor (AIF), Ca2+ and mitochondrial membrane potential (JC-1 staining) (D).
Fig 3.
SP reactivates the phosphorylation of Akt and recovers the redox balance of corneal epithelial cells impaired by hyperosmotic stress.
Mouse corneal epithelial cells were treated with 550 mOsm hyperosmotic stress by addition of glucose with or without 1 μM SP for 24 h. The phosphorylation of Akt was evaluated by Immunofluorescence staining or Western blot (A). The intracellular ROS and glutathione (GSH) levels were detected by staining with the fluorescence probes (B) and measured by the fluorescence intensity (C). The cellular total antioxidant capacity (TAC) was measured by the ABTS assay (C).
Fig 4.
Role of Akt reactivation in the anti-apoptotic effects of SP.
Mouse corneal epithelial cells were treated with 40 μM Akt inhibitor V and 1 μM SP 2 h before the addition of glucose for 24 h. The phosphorylation of Akt was evaluated by Immunofluorescence staining or Western blot (A). The apoptosis was evaluated by FACS analysis followed by FITC-Annexin V/PI staining (B), and the detection of Bad, Bax, AIF, Ca2+ and mitochondrial membrane potential (C). The intracellular ROS and glutathione (GSH) were detected by staining with the fluorescence probes (D) and measured by the fluorescence intensity (E). The cellular total antioxidant capacity (TAC) was measured by the ABTS assay (E).
Fig 5.
Role of redox regulation in the anti-apoptotic effects of SP.
Mouse corneal epithelial cells were treated with 100 μM L-BSO and 1 μM SP 2 h before the addition of glucose for 24 h. The phosphorylation of Akt was evaluated by Immunofluorescence staining or Western blot (A). The apoptosis was evaluated by FACS analysis followed by FITC-Annexin V/PI staining (B), and the detection of Bad, Bax, AIF, Ca2+ and mitochondrial membrane potential (C). The intracellular ROS and glutathione (GSH) were detected by staining with the fluorescence probes (D) and measured by the fluorescence intensity (E). The cellular total antioxidant capacity (TAC) was measured by the ABTS assay (E).
Fig 6.
Role of NK-1 receptor in the anti-apoptotic effects of SP.
Mouse corneal epithelial cells were treated with 1 μM NK-1 receptor antagonist L-733,060 with 1 μM SP 2 h before the addition of glucose for 24 h. The phosphorylation of Akt was evaluated by Immunofluorescence staining or Western blot (A). The apoptosis was evaluated by FACS analysis followed by FITC-Annexin V/PI staining (B), and the detection of Bad, Bax, AIF, Ca2+ and mitochondrial membrane potential (C). The intracellular ROS and glutathione (GSH) were detected by staining with the fluorescence probes (D) and measured by the fluorescence intensity (E). The cellular total antioxidant capacity (TAC) was measured by the ABTS assay (E).