Neutrophil peptide 1 accelerates the clearance of degenerative axons during Wallerian degeneration by activating macrophages after peripheral nerve crush injury

Abstract

JOURNAL/nrgr/04.03/01300535-202408000-00036/figure1/v/2023-12-16T180322Z/r/image-tiff Macrophages play an important role in peripheral nerve regeneration, but the specific mechanism of regeneration is still unclear. Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide closely involved in peripheral nerve regeneration. However, the mechanism by which neutrophil peptide 1 enhances nerve regeneration remains unclear. This study was designed to investigate the relationship between neutrophil peptide 1 and macrophages in vivo and in vitro in peripheral nerve crush injury. The functions of RAW 264.7 cells were elucidated by Cell Counting Kit-8 assay, flow cytometry, migration assays, phagocytosis assays, immunohistochemistry and enzyme-linked immunosorbent assay. Axonal debris phagocytosis was observed using the CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) optical clearing technique during Wallerian degeneration. Macrophage inflammatory factor expression in different polarization states was detected using a protein chip. The results showed that neutrophil peptide 1 promoted the proliferation, migration and phagocytosis of macrophages, and CD206 expression on the surface of macrophages, indicating M2 polarization. The axonal debris clearance rate during Wallerian degeneration was enhanced after neutrophil peptide 1 intervention. Neutrophil peptide 1 also downregulated inflammatory factors interleukin-1α, -6, -12, and tumor necrosis factor-α in vivo and in vitro. Thus, the results suggest that neutrophil peptide 1 activates macrophages and accelerates Wallerian degeneration, which may be one mechanism by which neutrophil peptide 1 enhances peripheral nerve regeneration.

Figure 1

RAW 264.7 cell proliferation situation. (A–D) Optical density (OD) values of the RAW 264.7 cells at 12 (A), 24 (B), 36 (C), or 72 hours (D) after neutrophil peptide 1 (NP-1) intervention. The results of 2 and 10 μg/mL were similar to adjacent concentrations, thus they were not shown. (E) Survival curves for each group at different time points. The cell survival rate of the 20 µg/mL group exceeded that of the NS group and was higher compared with the other groups. (F) Purple color shows the outcomes of the colony formation. Purple coloring was the RAW 264.7 cells. (G) Hematoxylin-eosin staining showed the proliferation of RAW 264.7 cells at 36 and 72 hours after 20 µg/mL NP-1 stimulation. The red arrow indicates the island-shape proliferation of RAW 264.7 cells. Original magnification, 100×. *P < 0.05, vs. 0 µg/mL group; #P < 0.05, vs. other groups. Data are expressed as the mean ± SD (n = 3; one way analysis of variance followed by the least significant difference post hoc test). NS: Normal saline.

Figure 2

Flow cytometry for RAW 264.7 cells cycle detection. (A, B) Cells were treated with blank-control (0 µg/mL; A) and neutrophil peptide 1 (NP-1 20 µg/mL; B) for 72 hours. (C–E) Cell-cycle distributions at different stages. No significant differences were observed in the number of cells in the G1 and S phases. Significantly more cells were found in the G2 phase in the NP-1 treatment group than in the blank-control group. *P < 0.05. Data are expressed as the mean ± SD (n = 3; one way analysis of variance followed by the least significant difference post hoc test).

Figure 3

Apoptosis analysis of RAW 264.7 cells cultured for 72 hours. (A, B) Flow cytometry showed cell apoptosis in the blank-control group and neutrophil peptide 1 group (20 µg/mL). (C, D) Statistical graphs of live cells and apoptotic cells. There was no significant difference in the number of live cells and apoptotic cells between the two groups (P > 0.05). Data are expressed as the mean ± SD (n = 3; one way analysis of variance followed by the least significant difference post hoc test).

Figure 4

Transwell cell migration assays in RAW 264.7 cells. (A, B) Representative images of crystal violet staining. Cells were treated with blank-control (0 µg/mL; A) and neutrophil peptide 1 (NP-1 20 µg/mL; B) for 24 hours. The irregular blue shape indicates that cells migrated onto the membrane. (C) The number of cell migration in NP-1 group was significantly higher than that in the blank-control group (*P < 0.05). Data are expressed as the mean ± SD (n = 3; one way analysis of variance followed by the least significant difference post hoc test). Original magnification, 100×.

Figure 5

Phagocytosis of fluorescent microspheres in RAW 264.7 cells. (A, B) Representative fluorescence images of cells were treated with blank-control (0 µg/mL; A) and NP-1 (20 µg/mL; B) for 72 hours. The orange color indicates fluorescent microspheres phagocytized by RAW 264.7 macrophage cells. Original magnification: 100×. (C) Significantly more fluorescent microspheres were phagocytosed in the neutrophil peptide 1 group than in the blank-control group. *P < 0.05. Data are expressed as the mean ± SD (n = 3; one way analysis of variance followed by the least significant difference post hoc test).

Figure 6

Neurodegeneration distance imaging using CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) optical clearing technique. (A, B) Neurodegeneration at 3 (A) and 5 (B) days post-operation. In each image, a fluorescence map, light micrograph, and composite map are shown (from right to left), with the scale indicating the distance from the stitch-mark point to the full-denaturation point. (C) Statistical plot of the complete neurodegeneration distance. The complete degeneration distance of the neutrophil peptide 1 (NP-1) group was significantly longer than that of the normal saline (NS) group. *P < 0.05. Data are expressed as the mean ± SD (n = 6; independent samples t-test).

Figure 7

Polarization of macrophages and secretion of inflammatory cytokines. (A) Representative immunohistochemistry images of F4/80, CD80 and CD206 in RAW 264.7 cells treated with blank-control (0 µg/mL) and NP-1 (20 µg/mL) for 24 hours. Brown cells indicated that RAW 264.7 cells expressed the protein. (B) Levels of inflammatory cytokines (integrated optical density) in cells after 1 day of NP-1 stimulation through enzyme-linked immunosorbent assay detection. (C) Levels of inflammatory cytokines (optical density) at 1, 3, 5, and 7 days after modeling. The trend of inflammatory factors over time was also shown in a single group. *P < 0.05. Data are expressed as the mean ± SD (n = 3; one way analysis of variance followed by the least significant difference post hoc test). Original magnification: 200×. NP-1: Neutrophil peptide 1.