Substance P Inhibits Hyperosmotic Stress-Induced Apoptosis in Corneal Epithelial Cells through the Mechanism of Akt Activation and Reactive Oxygen Species Scavenging via the Neurokinin-1 Receptor

Abstract

Hyperosmolarity has been recognized as an important pathological factor in dry eye leading to ocular discomfort and damage. As one of the major neuropeptides of corneal innervation, substance P (SP) has been shown to possess anti-apoptotic effects in various cells. The aim of this study was to determine the capacity and mechanism of SP against hyperosmotic stress-induced apoptosis in cultured corneal epithelial cells. The cells were exposed to hyperosmotic stress by the addition of high glucose in the presence or absence of SP. The results showed that SP inhibited hyperosmotic stress-induced apoptosis of mouse corneal epithelial cells. Moreover, SP promoted the recovery of phosphorylated Akt level, mitochondrial membrane potential, Ca2+ contents, intracellular reactive oxygen species (ROS) and glutathione levels that impaired by hyperosmotic stress. However, the antiapoptotic capacity of SP was partially suppressed by Akt inhibitor or glutathione depleting agent, while the neurokinin-1 (NK-1) receptor antagonist impaired Akt activation and ROS scavenging that promoted by SP addition. In conclusion, SP protects corneal epithelial cells from hyperosmotic stress-induced apoptosis through the mechanism of Akt activation and ROS scavenging via the NK-1 receptor.

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).