Effect of reactive oxygen species generation in rabbit corneal epithelial cells on inflammatory and apoptotic signaling pathways in the presence of high osmotic pressure

Abstract

It is generally accepted that high osmotic pressure (HOP) of lacrimal fluid is the core mechanism causing ocular inflammation and injury. However, the association between HOP and the regulation of cell inflammatory response and apoptotic pathways remains unclear. In the present study, we used HOP to interfere with in vitro cultured rabbit corneal epithelial cells, and found that HOP increased the generation of reactive oxygen species (ROS) in rabbit corneal epithelial cells, and increased ROS in turn induced the activation of JNK inflammatory signaling pathway, which further promoted the expression of pro-inflammatory factor NF-κβ and induced the generation of inflammatory factor IL-1β and TNF-α. In addition, HOP-induced ROS in rabbit corneal epithelial cells regulated the CD95/CD95L-mediated cell apoptotic signaling pathway by activating JNK inflammatory signaling pathway. These findings may serve as new theoretical basis and a new way of thinking about the treatment of ocular diseases, especially dry eye.

Figure 1. Culture and identification of RCECs under light microscopy.

Pure RCECs were obtained at the second passage (A). RCECs were detected positive for cytokeratin3/2p (B). A: Original*100. B: Original*200.

Figure 2. Detection of ROS by flow cytometry.

RCECs were cultured in normal osmotic pressure(A), normal osmotic pressure added with NAC (B) or DPI (C), and 90 mM NaCl high osmotic pressure (HOP) group (D), 90 mM NaCl HOP added with NAC (E) or DPI (F). Intracellular ROS levels in RCECs were detected using DCFH-DA. Intracellular ROS levels increased in the HOP group. ROS levels decreases in the HOP added with NAC and DPI group. Statistics (G) showed that the fluorescence intensity in the 90 mM NaCl HOP group was significantly higher than that in the normal osmotic pressure group (P<0.01), and the fluorescence intensity in the HOP group added with ROS inhibitor NAC and DPI decreased to some extent. The decrease was statistically significant in the HOP group added with DPI(P<0.01). ** P<0.01 vs. A, ## P<0.01 vs. D.

Figure 3. Expression of JNK1, JNK2, p-JNK1 and p-JNK2 protein in RCECs.

RCECs cultured in NOP (normal osmotic pressure, 312mOsM) (1), HOP (high osmotic pressure, 500mOsM) (2), HOP+NAC (10 mM) (3), HOP+ DPI (10 µM) (4), HOP+ JNK inhibitor SP600125 (25 µM) (5) for 24 h. There was no significant difference in p-JNK-1/JNK-1 between the HOP group and the NOP group. Compared with the HOP group, p-JNK-1/JNK-1 levels decreased significantly in the HOP group added with ROS inhibitor NAC,DPI and JNK inhibitor SP600125. The p-JNK-2/JNK-2 ratio was increased in the HOP group compared with the NOP group. Compared with the HOP group, p-JNK-2/JNK-2 ratio was significantly decreased in HOP group added with ROS inhibitor NAC, DPI and JNK inhibitor SP600125. (mean±SD, n = 3) ** P<0.01 vs. NOP, # P<0.05 vs. HOP, ## P<0.01 vs. HOP.

Figure 4. RCECs p-JNK (I), phosphorylated-NF-κβ (II) and cultured medium IL-1β(III) levels.

RCECs cultured in NOP (normal osmotic pressure, 312mOsM) (A), HOP (high osmotic pressure, 500mOsM) (B), HOP+NAC (10 mM) (C), HOP+ DPI (10 µM) (D), HOP+ JNK inhibitor SP600125 (25 µM) (E) and HOP+ NF−kB inhibitor Bay11-7082(5 µM) (F) for 24 h. p-JNK expression in the HOP group was significantly higher than that in the NOP group (P<0.01). p-JNK expression in the HOP group added with ROS inhibitor NAC and JNK inhibitor SP600125 was decreased significantly (P<0.01) (I). phosphorylated-NF-κβ expression in the HOP group was significantly higher than that in the NOP group (P<0.01). phosphorylated-NF-κβ expression in the the HOP group added with ROS inhibitor NAC, DPI and JNK inhibitor SP600125 was decreased significantly (P<0.01) (II). IL-1β expression in the HOP group was significantly higher than that in NOP group (P<0.01). IL-1β expression in the HOP group added with ROS inhibitor NAC, DPI, JNK inhibitor SP600125 and NF-κB inhibitor Bay11-7082 was decreased (P<0.01) (III). (mean±SD, n = 3). ** P<0.01 vs. group A, ## P<0.01 vs. group B.

Figure 5. RCECs TNF-α gene expression.

RCECs cultured in NOP (normal osmotic pressure, 312mOsM) (A), HOP (high osmotic pressure, 500mOsM) (B), HOP+NAC (10 mM) (C), HOP+ DPI (10 µM) (D), HOP+ JNK inhibitor SP600125 (25 µM) (E) and HOP+ NF−kB inhibitor Bay11-7082(5 µM) (F) for 24 h. RCECs TNF-a mRNA expression in the HOP group was significantly higher than that in the NOP group (P<0.05). TNF-a mRNA expression in the HOP group added with ROS inhibitor NAC, DPI and NF-kB inhibitor Bay11-7082 was decreased (P<0.05). (mean±SD, n = 3). * P<0.05 vs. group A, # P<0.05 vs. group B.

Figure 6. Expression of CD95 and CD95L protein in RCECs.

A: western blotting analysis showing the presence of CD95 and CD95L protein in RCECs. B, C: Results of statistical analysis of protein levels relative to GAPDH. RCECs cultured in NOP (normal osmotic pressure, 312mOsM) (1), HOP (high osmotic pressure, 500mOsM) (2), HOP+NAC (10 mM) (3), HOP+ DPI (10 µM) (4), HOP+ JNK inhibitor SP600125 (25 µM) (5) for 24 h. CD95 and CD95L expression in the HOP group was significantly higher than that in NOP group (P<0.01). NAC, DPI and JNK inhibitor SP600125 downregulated CD95 expression in RCECs exposed to HOP. NAC and JNK inhibitor SP600125 upregulated CD95L expression in RCECs exposed to HOP, while CD95L expression was decreased significantly in the HOP group added with DPI (P<0.01). (mean±SD, n = 3). ** P<0.01 vs. NOP, ## P<0.01 vs. HOP.

Figure 7. Detection of cell apoptosis.

A-E: TUNEL assay; F-J: Hoechst33342 fluorescent staining assay. A and F: normal osmotic pressure group; B and G: 90 mM NaCl HOP group; C and H: 90 mM NaCl HOP added with NAC group; D and I: 90 mM NaCl HOP added with DPI group; E and J: 90 mM NaCl HOP added with SP600125 group. HOP increased RCECs apoptosis. NAC, DPI and SP600125 decreased cell apoptosis exposed to HOP.

Figure 8. Detection of apoptotic cells with Annexin V assay.

A: normal osmotic pressure group; B: 90 mM NaCl HOP group; C: 90 mM NaCl HOP added with NAC group; D: 90 mM NaCl HOP added with DPI group; E: 90 mM NaCl HOP added with SP600125 group. Statistics (F) showed that the percentage of apoptotic cells in the 90 mM NaCl HOP group was significantly higher than that in the normal osmotic pressure group (P<0.01), and the percentage of apoptotic cells in the HOP group added with NAC, DPI and SP600125 significantly decreased (P<0.01). (mean±SD, n = 3). ** P<0.01 vs. A, ## P<0.01 vs. B.