Fig 1: GDPD3 deficiency ameliorates CCI-induced neuropathic pain. GDPD3−/−, GDPD3, shNC, and shGDPD3 mice were subjected to the CCI model. Mechanical hypersensitivity trend was assessed by the percentage of paw withdrawal to 0.4 g von Frey filament (A, E) and 0.07 g von Frey filament (B, F), thermal hypersensitivity trend was assessed by paw withdrawal latency to Hargreaves test (C, G). (D) Fluorescence microscopy showed virus was injection in the DRG. The data were presented as mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001
Fig 2: The macrophage polarization and GDPD3 expression trends in DRG during neuropathic pain progressing. A Illustration of the Western blot results for macrophage polarization and GDPD3 expression trends in DRG. B Statistical trend results of M1 macrophage polarization marker (CD86) tested by Western blot in DRG. C Statistical trend results of the trend of M2 macrophage polarization marker (CD206) tested by Western blot in DRG. D Statistical GDPD3 relative protein level trends tested by Western blot in DRG. E Statistical GDPD3 relative mRNA expression trends tested by RT-QPCR analysis in DRG. The data were presented as mean ± SD, n = 3, **P < 0.01, ***P < 0.001.DRG, dorsal root ganglion
Fig 3: GDPD3 regulates neuropathic pain through PPARγ.A Immunohistochemical analysis of the expression levels of CD86 and CD206 in the DRG of various groups of mice. Mechanical hypersensitivity trend was assessed by the percentage of paw withdrawal to 0.4 g von Frey filament A and 0.07 g von Frey filament B thermal hypersensitivity trend was assessed by paw withdrawal latency to Hargreaves test C The data were presented as mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001
Fig 4: GDPD3 deficiency promotes the transformation of M1 macrophage polarization into M2 in vitro. A Gel electrophoresis assay and western blot assay to analyses the expression of GDPD3. B Western blot results of M1 macrophage polarization marker CD86 and M2 marker CD206 in GDPD3−/− and GDPD3+/− BMDMs treated with M1 stimulus (LPS) and M2 stimulus (IL-4). C, D Quantitation of relative band densities of CD86 and CD206 in GDPD3−/− and GDPD3+/− BMDMs treated with LPS and IL-4. E, F Relative mRNA levels of proinflammatory factor IL-1β and TNF-α in GDPD3−/− and GDPD3+/− BMDMs challenged with LPS and IL-4 tested by RT-QPCR analysis. G, H Relative M2 macrophage polarization gene IL-10 and Arg1 expression via RT-QPCR analysis in GDPD3−/− and GDPD3+/− BMDMs challenged with LPS and IL-4. The data were presented as mean ± SD, n = 3, **P < 0.01, ***P < 0.001
Fig 5: GDPD3 promotes M2 macrophage polarization through PPARγ. Volcano plot A and Hierarchical plot B show that the overexpressed genes of M2 macrophage polarization in the GDPD3−/− BMDMs were mainly related to the PPARγ-related pathway. PPARγ protein expression C and mRNA expression D in GDPD3−/− and GDPD3± BMDMs challenged with LPS. E FABP4 (downstream of PPARγ) mRNA expression in GDPD3-/- and GDPD3 ± BMDMs challenged with LPS. F, G PPARγ inhibitor GW9962 reversed the effect of GDPD3 on macrophage polarization-related gene CD206 (M2 polarization) and IL-1β (M1 polarization) expression via RT-QPCR analysis in BMDMs challenged with LPS. The data were presented as mean ± SD, n = 3, **P < 0.01, ***P < 0.001. PPARγ, peroxisome proliferator-activated receptor gamma; FABP4, fatty acid binding protein-4
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