Abstract: BACKGROUND: In acute lung injury, the activation and tissue infiltration of neutrophils are key contributing factors. Inhibiting poly(ADP-ribose) polymerase 1 activity can reduce the number of infiltrating neutrophils in lung tissue following lipopolysaccharide treatment and alleviate lung injury. However, the specific mechanism by which poly(ADP-ribose) polymerase 1 regulates neutrophil activation and function has not been fully elucidated.
OBJECTIVE: To investigate the effect and mechanism of poly(ADP-ribose) polymerase 1 in regulating neutrophil activation on acute lung injury in mice.
METHODS: (1) Using the human promyelocytic leukemia cell line HL-60 as a basis, cells were induced to differentiate into neutrophil-like cells (dHL-60) with dimethyl sulfoxide. Lipopolysaccharide was used to stimulate dHL-60 to obtain activated neutrophils, and Western blot was used to detect poly(ADP-ribose) polymerase 1 expression in activated neutrophils. (2) A neutrophil cell line with low poly(ADP-ribose) polymerase 1 expression was prepared using shRNA targeting human poly(ADP-ribose) polymerase 1, and the knockdown efficiency of poly(ADP-ribose) polymerase 1 was verified by western blot. (3) Western blot and immunofluorescence assays were used to detect the effect of low poly(ADP-ribose) polymerase 1 expression on neutrophil polarization; cell migration assays were used to detect the effect on neutrophil migration; real-time quantitative PCR and flow cytometry were used to detect the effect on chemokine receptor expression; and reactive oxygen species, interleukin 1β, and neutrophil extracellular traps were used to assess the effect on neutrophil activity. (4) Primary mouse neutrophils with low poly(ADP-ribose) polymerase 1 expression were prepared using shRNA targeting mouse poly(ADP-ribose) polymerase 1. In a mouse neutrophil adoptive transfer model, the effect of low poly(ADP-ribose) polymerase 1 expression on lipopolysaccharide-induced acute lung injury was verified by assessing pulmonary edema, myeloperoxidase activity, and hematoxylin-eosin staining. (5) A mouse sepsis survival experiment was performed to evaluate the effect of the poly(ADP-ribose) polymerase 1 inhibitor AG14361 on acute lung injury and survival time in mice.
RESULTS AND CONCLUSION: (1) Poly(ADP-ribose) polymerase 1 expression was significantly upregulated in activated neutrophils. (2) A neutrophil cell line with low poly(ADP-ribose) polymerase 1 expression was successfully constructed. (3) Compared with the control group, low poly(ADP-ribose) polymerase 1 expression did not affect neutrophil polarization but significantly inhibited neutrophil migration, activation, and inflammatory factor expression. The expression of C-X-C chemokine receptor 2 and C-X-C chemokine receptor 4 on the neutrophil surface was significantly downregulated. (4) Neutrophil adoptive transfer experiments confirmed that specifically knocking down poly(ADP-ribose) polymerase 1 expression in neutrophils reduced neutrophil infiltration in lung tissue and alleviated lipopolysaccharide-induced acute lung injury. (5) The poly(ADP-ribose) polymerase 1 inhibitor AG14361 alleviated lipopolysaccharide-induced acute lung injury and prolonged the survival time of septic mice. Overall, these findings indicate that poly(ADP-ribose) polymerase 1 affects neutrophil migration and inflammatory effector functions by regulating the expression of C-X-C chemokine receptor 2 and C-X-C chemokine receptor 4. Targeted inhibition of poly(ADP-ribose) polymerase 1 can reduce neutrophil infiltration in lung tissue, thereby alleviating lung injury and prolonging survival in mice.

Key words: acute lung injury, neutrophil, inflammatory response, poly(ADP-ribose) polymerase 1, chemokine receptor