Purpose To determine whether Activated 5'AMP-activated protein kinase (AMPK) activation
enhances Fusarium solani (F. solani) fungicidal capacity of neutrophils. Methods The
expression of AMPK and phosphorylated-AMPK (p-AMPK) proteins was tested using Western
Blot. Plate counting studied the effects of the fungicidal capacity of neutrophils
enhanced by AMPK activation. Phagocytized spores by neutrophils were assessed by immunostaining
and inhibited hyphal growth images were captured by JULI Stage real-time cell history
recorder. Flow cytometry assay tested Reactive Oxygen Species (ROS) production and
the percentage of apoptosis neutrophils. ROS Assay Kit also tested ROS production
at different time points. The F. solani keratitis murine model was established, and
slit-lamp microscopy captured corneal photographs. Results Our experiments were divided
into the following groups. Neutrophils (N), neutrophils + spores (N + S), neutrophils
+ spores+ 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) (N + S + A), neutrophils
+ spores + Compound C (N + S + C). AMPK activator AICAR significantly increased the
expression of p-AMPK in neutrophils. The plate counting experiment showed that the
number of colonies in the N + S + A was significantly less than in the N + S group.
Immunostaining results showed phagocytized spores were significantly increased in
the N + S + A group compared with the N + S group. Captured photographs by a real-time
cell history recorder camera showed F. solani hyphal growth in the N + S + A group
was significantly inhibited than in the N + S group. ROS release in the N + S + A
group was significantly higher in the N + S + A group than in other groups. The percentage
of apoptosis neutrophils in the N + S + A group was decreased than in the N + S group.
Captured photographs by slit-lamp showed that AICAR eye drop treatment alleviated
the severity and decreased clinical score at 12 and 24 hours post-infection (h.p.i)
Conclusion AMPK activation enhances the efficacy of neutrophils in killing F. solani
in vitro and in vivo.
