Metformin induces M2 polarization via AMPK/PGC-1α/PPAR-γ pathway to improve peripheral nerve regeneration

Abstract

Objectives: Investigating the effect of metformin on peripheral nerve regeneration and the molecular mechanism.

Methods: In this study, a rat model of sciatic nerve injury and an inflammatory bone marrow-derived macrophage (BMDM) cell model were established. We assessed the sensory and motor function of the hind limbs four weeks after sciatic nerve injury, immunofluorescence was used to detect axonal regeneration and myelin formation, as well as local macrophage subtypes. We investigated the polarizing effect of metformin on inflammatory macrophages, and western blotting was applied to detect the molecular mechanisms behind it.

Results: Metformin treatment accelerated functional recovery, axon regeneration and remyelination, and promoted M2 macrophage polarization. In vivo, metformin transformed pro-inflammatory macrophages into pro-regeneration M2 macrophages. Protein expression levels of phosphorylated AMP-activated protein kinase (p-AMPK), proliferator-activated receptor-γ co-activator 1α (PGC-1α), and peroxisome proliferator-activated receptor-γ (PPAR-γ) increased upon metformin treatment. Moreover, inhibition of AMPK abolished the effects of metformin treatment on M2 polarization.

Conclusion: Metformin promoted M2 macrophage polarization by activating the AMPK/PGC-1α/PPAR-γ signaling axis, thereby promoting peripheral nerve regeneration.

Keywords: AMPK/PGC-1α/PPAR-γ signaling axis; Metformin; macrophage polarization; peripheral nerve regeneration.

Figure 1

Metformin treatment improved functional recovery after peripheral nerve injury. A, B. Sciatic functional index (SFI) and Basso, Beattie, and Bresnahan (BBB) scores during functional motor recovery after sciatic nerve injury. C. Ankle angles at 4 weeks post-operation. D. The gastrocnemius muscle index (GMI) was used to evaluate the degree of muscle atrophy. E. Chemosensitivity analysis. F. Von-Frey test for functional sensory recovery at 4 weeks post-operation. G. Electrophysiology recordings at 2 weeks and 4 weeks post-operation. H. Quantitative electrophysiology data for compound motor action potential (CMAP) at 2 and 4 weeks post-operation. I. Retrograde labeling with Dil in dorsal root ganglia (DRG) at 4 weeks post-operation (original magnification × 200). Scale bars are 25 μm. J. Quantitative analysis of retrograde labeling with Dil in DRG. Data are presented as mean ± SD, n = 6, *P < 0.05, **P < 0.01.

Figure 2

Immunofluorescence staining of neural tissue showed that metformin promoted axon regeneration and remyelination and accelerated M2 macrophage polarization after peripheral nerve injury. A. At 4 weeks post-operation, immunofluorescence staining with anti-neurofilament-200 (NF-200, red) was used to evaluate axon regeneration and immunofluorescence staining with anti-myelin basic protein (MBP, green) was used to evaluate remyelination. Tissues were counterstained with DAPI to mark the nuclei (blue) (original magnification × 200). Scale bars are 25 μm. B, C. Quantitation of NF200 and MBP signals in the metformin and control groups. D. Immunofluorescence staining revealed different subpopulations of macrophages. CD68-positive (green) and CD206-positive (red) cells were defined as M2 macrophages (original magnification × 400). Scale bars are 25 μm. E. Quantification of CD68⁺CD206⁺ cells. Data are presented as mean ± SD, n = 6, *P < 0.05, **P < 0.01.

Figure 3

Metformin treatment accelerated M2 macrophage polarization in vitro. A, B. Cell viability of BMDMs upon treatment with various concentrations of metformin was evaluated by CCK-8 assay. C, E. Flow cytometry was used to detect surface markers on bone-marrow-derived macrophages. F4/80 was used as the pan-macrophage marker. CD86 was used as the M1 marker and CD206 was used as the M2 marker. D, F. Quantification of M1 and M2 macrophages in the metformin and control groups. Data are presented as mean ± SD, n = 3, *P < 0.05, **P < 0.01.

Figure 4

mRNA expression in M1 and M2 macrophages. A-C. The relative mRNA expression levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β (markers of M1 macrophages) were lower in the metformin treatment group than in the control group. D-F. The relative mRNA expression levels of arginase (Arg)-1, IL10, and chitinase 3-like 3 (Ym-1) (markers of M2 macrophages) were higher in the metformin treatment group than in the control group. Relative mRNA expression was measured by qPCR. Data are presented as mean ± SD, n = 3, *P < 0.05, **P < 0.01.

Figure 5

Metformin-induced M2 macrophage polarization via the AMPK/PGC-1α/PPAR-γ signaling pathway. A. Protein expression levels of AMP-activated protein kinase (AMPK), phosphorylated (p)-AMPK, proliferator-activated receptor-γ co-activator 1α (PGC-1α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and GAPDH were evaluated by Western blot. B. The quantified p-AMPK/AMPK ratio is a measure of AMPK activation. C, D. Quantification of relative expression of PGC-1α and PPAR-γ. Abbreviations: MET, metformin; Cpd C, Compound C. Data are presented as mean ± SD, n = 3, **P < 0.01.

Figure 6

Inhibition of AMP-activated protein kinase activity by compound C reversed the regulatory function of metformin in vitro. A, C. Flow cytometry detection of surface markers on bone-marrow-derived macrophages. F4/80 was used as the pan-macrophage marker. CD86 was used as the M1 macrophage marker and CD206 was used as the M2 macrophage marker. B, D. Quantification of the proportion of M1 and M2 macrophages. Abbreviations: MET, metformin; Cpd C, Compound C. Data are presented as mean ± SD, n = 3, *P < 0.05, **P < 0.01.