Wei Jingjing, Xie Yanting, Yue Juan, Dong Junlu, Si Wei, Wang Chunmei, Zhang Hongmin, Wang Liya
Abstract
ObjectiveTo investigate the expression of adenosine 5'-monophosphate-activated protein kinase (AMPK) phosphorylation in corneal epithelial cells and the effects of fungus on AMPK phosphorylation and interleukin-6 (IL-6) production in corneal epithelial cells.
MethodsThe human immortalized corneal epithelial cell line was selected.The safe concentration range of AMPK agonist 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) (100, 300, 500, 1 000 μmol/L) and inhibitor Compound C (10.0, 12.5, 15.0, 17.5, 20.0 μmol/L) on corneal epithelial cells was screened by multi-function real-time unlabeled cell analyzer.Corneal epithelial cells without any treatment were used as the normal control group, and those co-cultured with spores were used as the spore control group.Corneal epithelial cells co-cultured with spores were treated with AICAR and Compound C for 4 hours in the AICAR group and Compound C group, respectively.The expression of phosphorylated AMPK (p-AMPK) and AMPK in corneal epithelial cells was detected by Western blot, and the concentration of IL-6 in the culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA).
ResultsAfter treatment with different concentrations of AICAR for different periods, there was no statistical significance in the cell index of corneal epithelial cells (all at P>0.05). The cell index of corneal epithelial cells was increased with 10.0 μmol/L and 12.5 μmol/L Compound C treatment compared with that of the normal control group.The expression levels of p-AMPK were 0.67±0.15, 2.57±0.12, 3.67±0.58 and 1.50±0.50, respectively, in the normal control group, spore control group, AICAR group and Compound C group, showing a statistically significant difference among them (F=32.820, P<0.001). The expression level of p-AMPK was significantly higher in the spore control group compared with the normal control group (P<0.001). The expression level of p-AMPK in the AICAR group was higher than that in the spore control group, and the expression level of p-AMPK in the Compound C group was lower than that in the spore control group, and the differences were statistically significant (both at P=0.010). There was no significant difference in the relative expression level of AMPK among the four groups (F=0.120, P=0.950). The expression levels of IL-6 concentration in the normal control group, spore control group, AICAR group and Compound C group were (107.81±17.15), (156.32±9.94), (167.96±14.16) and (127.42±19.75)pg/ml, respectively, showing a statistically significant difference among them (F=15.210, P<0.001). The IL-6 concentration of the spore control group was higher than that of the normal control group, and the difference was statistically significant (P<0.001). The IL-6 concentration of the AICAR group was higher than that of the spore control group, but the difference was not statistically significant (P=0.260). The IL-6 concentration of the Compound C group was lower than that of the spore control group, and the difference was statistically significant (P=0.010).
ConclusionsIn corneal epithelial cells, AMPK phosphorylation is found, which is enhanced after fungal spores stimulation, and the secretion of IL-6 increases.
Key words:
Fungi; Keratitis; Epithelial cells; Interleukin-6; Adenine ribonucleotide activated protein kinase; Phosphorylation
Contributor Information
Wei Jingjing
People's Hospital of Zhengzhou University, Department of Ophthalmology, Henan Provincial People's Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Science, Zhengzhou 450003, China
Xie Yanting
People's Hospital of Zhengzhou University, Department of Ophthalmology, Henan Provincial People's Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Science, Zhengzhou 450003, China
Yue Juan
People's Hospital of Zhengzhou University, Department of Ophthalmology, Henan Provincial People's Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Science, Zhengzhou 450003, China
Dong Junlu
People's Hospital of Zhengzhou University, Department of Ophthalmology, Henan Provincial People's Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Science, Zhengzhou 450003, China
Si Wei
People's Hospital of Zhengzhou University, Department of Ophthalmology, Henan Provincial People's Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Science, Zhengzhou 450003, China
Wang Chunmei
People's Hospital of Zhengzhou University, Department of Ophthalmology, Henan Provincial People's Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Science, Zhengzhou 450003, China
Zhang Hongmin
People's Hospital of Zhengzhou University, Department of Ophthalmology, Henan Provincial People's Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Science, Zhengzhou 450003, China
Wang Liya
People's Hospital of Zhengzhou University, Department of Ophthalmology, Henan Provincial People's Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Science, Zhengzhou 450003, China
