Effect Of Fungi On AMPK Phosphorylation In Corneal Epithelial Cells

ObjectThe purpose of this study was to study the expression of AMPK phosphorylation in corneal epithelial cells and the effect of fungi on AMPK phosphorylation in corneal epithelial cells,and to explore the relationship between AMPK phosphorylation and inflammatory factor IL-6,so as to explore the pathogenesis of fungal keratitis and provide new ideas for the development of new anti-infective drugs.Methods1.Corneal epithelial cells were incubated with AMPK agonist AICAR and AMPK inhibitor Compound C,respectively.The concentration gradients of AICAR and Compound C were 0 μM,100 μM,300 μM,500 μM,1000μM and 0μM,10μM,12.5 μM,15 μM,17.5 μM and 20 μM.To determine the safe concentration of drug use,the effect of drug on cell growth was observed by real-time unlabeled cell multi-function analyzer.2.AICAR was given to stimulate corneal epithelial cells,and the concentration gradient of AICAR was 0 μM,50 μM,500 μM,1000 μM,cultured for 4 h,and then the time gradient of AICAR stimulation was set for 0h,1h,2h,4h.The effect of AICAR on AMPK phosphorylation in corneal epithelial cells was detected by Western Blot to determine the best drug concentration and time.3.The experiment is divided into four groups:simple corneal epithelial cell group(Cell,C)(C group),corneal epithelial cell(Cell,C)+spore(Spore,S)(C+S group),corneal epithelial cell(Cell,C)+spore(Spore,S)+AICAR(A)(C+S+A group),corneal epithelial cell(Cell,C)+spore(Spore,S)+Compound C(CC)(C+S+CC group).The expression of AMPK and p-AMPK in corneal epithelial cells induced by fungal spores was detected by Western Blot,and ELISA was used to detect the expression of IL-6 in the culture supernatant of corneal epithelial cells.Results1.At 24h,36h and 48h,the cell index of AICAR 100 μM group,300 μM group,500 μM group and 1000 μM group was not significantly different from that of the control group(P>0.05).After 48h,the growth of corneal epithelial cells entered a plateau stage,and at 60 h and 72h,the cell index of AICAR 100 μM group,300 μM group,500 μ M group and 1000μ M group was not significantly different from that of the control group(P>0.05).2.There was no significant difference in the cell index of Compound C 10μM group and 12.5μM group compared with the control group at 24h,36h,48h,60h and 72h(P>0.05),and there was no significant difference between the Compound C 15μM group and the control group at 24h(P>0.05).At 36 h,48 h,60 h and 72h,the cell index of the,Compound C 15 μM group was significantly higher than that of the control group(P<0.05),and the growth of corneal epithelial cells entered the plateau phase after 48h.The cell index of Compound C 17.5 μM group and 20 μM group was significantly higher than that of the control group at 24h,36h,48h,60h and 72h(P<0.05).3.Compared with the control group,the level of AMPK phosphorylation in AICAR 50 μM group,500 μM group and 1000 μM group was statistically significant(P<0.05).In the selected concentration range,the level of AMPK phosphorylation in AICAR 1000 μM group was the highest.4.Compared with the control group,there was no significant difference in the level of AMPK phosphorylation at 1h and 2h,but at 4h,there was significant difference in the level of AMPK phosphorylation between the control group and the control group(P<0.05).5.Compared with group C,there was significant difference in the level of AMPK phosphorylation in group C+S(P<0.01),and the level of phosphorylation of AMPK in group C+S+A and C+S+CC was significant difference compared with group C+S(P<0.05).6.Compared with group C,there was significant difference in the expression of IL-6 in group C+S(P<0.01),and the expression of IL-6 in C+S+A group was not significantly higher than that in group C+S group(P>0.05),but there is an increasing trend.The expression of IL-6 in group C+S+CC was significant difference compared with group C+S(P<0.05).ConclusionsAMPK phosphorylation was expressed in corneal epithelial cells,and after fungal stimulation,the phosphorylation of AMPK in corneal epithelial cells increased,and the secretion of pro inflammatory cytokines IL-6 increased.AMPK phosphorylation and IL-6 may exist in the same signal pathway and participate in the pathogenesis of fungal keratitis.