| ObjectThe purpose of this study was to study the expression of AMPK phosphorylation in neutrophils in vitro,and the effects of AMPK phosphorylation on the anti-fungal abilities,mode and mechanism of neutrophils against Fusarium solani,and to verify the effect of AMPK signal pathway activation on the signs of fungal keratitis(FK)in mice,so as to provide a new idea for the development of new anti-fungal drugs.MethodsTwo parts are divided in this study: in vitro experiment and in vivo experiment.In vitro study: neutrophils were purified from human peripheral vein whole blood.500μmol/L AICAR and 10μmol/L Compound C were co-cultured with extracted fungal spores.Neutrophil(group N),neutrophil + spore(group N+S),neutrophil +spore + AICAR group(group N+S+A)and neutrophil + spore + Compound C(group N+S+C).The expression of AMPK and phosphorylated AMPK(p-AMPK)in neutrophils was detected by Western Blot.The experiment was divided into S group,N+S group,N+S+A group and N+S+C group by counting colonies.After in vitro co-culture for 4 h,6 h and 8 h,the mixture of each group was diluted and inoculated on potato glucose solid medium to grow for 36～48 h,and the colony growth of each group was photographed and observed.The phagocytosis of neutrophils was randomly selected by inverted fluorescence microscope and photographed.The experiment was divided into S group,N+S group,N+S+A group and N+S+C group.The phagocytic index of neutrophils was calculated at 4 h.The visual field was randomly selected and photographed every 30 minutes by real-time cell recorder.The experiment was divided into S group,N+S group,N+S+A group and N+S+C group.The hyphal length was calculated at 4 h,6 h,8 h,10 h and 12 h.The cells were stained with reactive oxygen species(ROS)kit,and the experiment was divided into N group,N+S group,N+S+A group and N+S+C group.The fluorescence intensity was measured every other 30 min for 5 h.The fluorescence intensity of each group was counted at 1 h,2 h,3 h,4 h and 5 h,and the fluorescence intensity of 4 h was verified by flow cytometry.The cells were stained with apoptosis kit,and the experiment was divided into N group,N+S group,N+S+A group and N+S+C group.The apoptosis rate of each group was detected by flow cytometry.In vivo study: the mouse model of FK was established,and the FK mice were randomly divided into normal saline(NS)group,AICAR group and Compound C group.The mice were treated with saline,500μmol/L AICAR and 10μmol/L Compound C at 5μL every 4 h.The corneal turbidity of 12 h,24 h,48 h and 72 h was observed under slit lamp microscope,and the corneal clinical score was calculated.ResultsIn vitro experiments,the level of AMPK phosphorylation in N+S+A group was significantly higher than that in N group(P = 0.0324),and the level of AMPK phosphorylation in N+S+C group was lower than that in N group(P = 0.0350;P =0.0409).The colony count of group S was the highest at each time point,and the colony count of N+S group was significantly lower than that of group S at 4 h,6 h and 8 h(P = 0.053;P = 0.0046;P < 0.001),and that of N+S+A group at 6 h and 8 h was significantly lower than that of group S at 6 h and 8 h(P = 0.0022;P = 0.0411),and the colony count of N+S+C group was significantly higher than that of N+S group at 4 h,6 h and 8 h(P = 0.0127;P = 0.0196;P = 0.0320).The phagocytic spore index of neutrophils in N+S+A group was significantly higher than that in N+S group at 4 h(P = 0.01),and the neutrophil phagocytic spore index in N+S+C group was significantly lower than that in N+S group(P < 0.001).The hyphal length of each group increased with the extension of time.The hyphal length of group S was the highest,the length of hypha in N+S group was lower than that in S group(P = 0.015)and has significantly differences at 8 h,10 h and 12 h(P = 0.0083;P = 0.0126;P =0.0348).There was no significant difference in hyphal length between group N+S+C and group S at each time point(P > 0.05).The hyphal length of N+S+A group was the lowest,and the hyphal length of N+S+A group was lower than that of N+S group(P < 0.001),and there were significant differences at 6 h,8 h,10 h and 12 h(P <0.001;P < 0.001;P < 0.001;P = 0.003).The hyphal length rate of each group was compared,and the rate of N+S group was significantly lower than that of S group(P = 0.038).The rate of N+S+A group was significantly lower than that of S group(P= 0.006),and the hyphal length rate of N+S+C group was significantly higher than that of S group(P = 0.042).The results of enzyme labeling instrument showed that the release of 1～5h ROS increased with time in all groups except group N.Compared with N group,the release of ROS in N+S+A group was significantly higher than that in N group at 3 h,4 h and 5 h(P < 0.001),and there was significant difference at 3 h,4 h and 5 h(P = 0.001;P < 0.001;P < 0.001).The release of ROS in N+S+C group was significantly lower than that in N group(P = 0.001),and the difference was statistically significant at 3 h,4 h and 5 h(P = 0.001;P = 0.05;P = 0.03).The flow cytometry showed that at 4 h,the release of ROS in N group was higher than that in N group(P = 0.0009),the release of ROS in N+S+A group was higher than that in N group(P = 0.0034),and the release of ROS in N+S+C group was lower than that in N group(P = 0.0208).At 4h,the apoptosis rate of neutrophils in N+S group was higher than that in N group(P = 0.016),the apoptosis rate of neutrophils in N+S+A group was decreased than that in N+S group(P < 0.01)and the apoptosis rate of neutrophils in N+S+C group was higher than that in N+S group(P < 0.001).In the in vivo experiment,there was no significant difference in the clinical score of in AICAR group at 72 h,but there was a tendency to decrease in.The clinical socre of Compound C group at 48 h and 72 h was significantly higher than NS group(P <0.01).ConclusionsThe increased expression of AMPK phosphorylation in neutrophils induced by fungal spores.The activation of AMPK signaling pathway enhances the phagocytosis and inhibition of hyphal length of neutrophils by enhancing the ROS releasing and inhibiting apoptosis of neutrophils,and enhances the anti-fungal ability of neutrophils.Meanwhile,our in vivo FK murine model proved that AMPK activation can improve the symptoms of FK.It shows that AMPK phosphorylation plays an important role in neutrophils during fungal killing process,which provides a new target for the treatment of FK. |
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