Effects of Fusarium solani on AMPK phosphorylation and IL-6 expression in corneal epithelial cells

Objective

To 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.

Methods

The 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).

Results

After 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).

Conclusions

In corneal epithelial cells, AMPK phosphorylation is found, which is enhanced after fungal spores stimulation, and the secretion of IL-6 increases.

Fungi; Keratitis; Epithelial cells; Interleukin-6; Adenine ribonucleotide activated protein kinase; Phosphorylation