Fig 1: FGF5 overexpression activates AMPK through cAMP/PKA pathway. (A, B) Mice were intraspinally injected with 2 μL lentivirus carrying FGF5 to overexpress FGF5 in the spinal cord, and cAMP level and PKA activity were detected 2 weeks post‐injection. (C) To inhibit PKA, mice with or without FGF5 overexpression were intraperitoneally injected with H89 every 2 days 1 week pre‐SCI. Western blot images and quantification of AMPK phosphorylation in the spinal cord were detected. (D) ROS level detected by DCFH‐DA probe. (E) The levels of MDA, 3‐NT and 8‐OHdG. (F) IL‐6 and TNF‐α levels in the spinal cord were detected. (G) BMS score, from 0 (no ankle movement) to 9 (normal gait), was determined at indicating times. (H, I) Sensitivities to mechanical and thermal stimulation were determined 28 days after SCI. (J) Extravasation of Evans blue dye was determined 28 days after SCI. n = 6 for each groups. Data are expressed as the mean ± standard deviation and p < 0.05 was considered statistically significant. *p < 0.05.
Fig 2: FGF5 overexpression reduces inflammation and oxidative stress in SCI mice. (A) Mice were overexpressed with FGF5 using lentiviral vectors, and then were exposed to SCI or sham surgery 2 weeks post‐injection. IL‐6 and TNF‐α levels in the spinal cord were detected. (B) MPO activity in the spinal cord. (C) Western blot images and quantification of p65 phosphorylation. (D) Relative NF‐κB transcription activity. (E, F) Relative levels of NRF2 protein and transcription activity. (G) ROS level detected by DCFH‐DA probe. (H) Quantification of hydrogen peroxide and superoxide anion in spinal cord. (I) The levels of MDA, 3‐NT and 8‐OHdG. n = 6 for each groups. Data are expressed as the mean ± standard deviation and p < 0.05 was considered statistically significant. *p < 0.05.
Fig 3: FGF5 overexpression reduces inflammation, oxidative stress and SCI through activating AMPK. (A) Mice were overexpressed with FGF5 using lentiviral vectors, and then were exposed to SCI or sham surgery 2 weeks post‐injection. Western blot images and quantification of AMPK phosphorylation in the spinal cord were detected. (B) To inhibit AMPK, mice with or without FGF5 overexpression were intraperitoneally injected with CC every 2 days 1 week pre‐SCI. IL‐6 and TNF‐α levels in the spinal cord were detected. (C) ROS level detected by DCFH‐DA probe. (D) The levels of MDA, 3‐NT and 8‐OHdG. (E) BMS score, from 0 (no ankle movement) to 9 (normal gait), was determined at indicating times. (F, G) Sensitivities to mechanical and thermal stimulation were determined 28 days after SCI. (H) Extravasation of Evans blue dye was determined 28 days after SCI. n = 6 for each groups. Data are expressed as the mean ± standard deviation and p < 0.05 was considered statistically significant. *p < 0.05.
Fig 4: FGF5 knockdown elevates inflammation and oxidative stress in SCI mice. (A) Mice were injected with shFGF5, and then were exposed to SCI or sham surgery 2 weeks post‐injection. IL‐6 and TNF‐α levels in the spinal cord were detected. (B) MPO activity in the spinal cord. (C) Western blot images and quantification of p65 phosphorylation. (D) Relative NF‐κB transcription activity. (E, F) Relative levels of NRF2 protein and transcription activity. (G) ROS level detected by DCFH‐DA probe. (H) Quantification of hydrogen peroxide and superoxide anion in spinal cord. (I) The levels of MDA, 3‐NT and 8‐OHdG. n = 6 for each groups. Data are expressed as the mean ± standard deviation and p < 0.05 was considered statistically significant. *p < 0.05.
Fig 5: FGF5 overexpression prevents SCI in mice. (A) Mice were exposed to Ctrl or SCI surgery, and FGF5 mRNA level in the spinal cord was detected at indicating times. (B) FGF5 level was detected using an ELISA kit. (C, D) Mice were intraspinally injected with 2 μL lentivirus carrying FGF5 to overexpress FGF5 in the spinal cord, and FGF5 mRNA and protein levels were detected 2 weeks post‐injection. (E) Mice were overexpressed with FGF5 using lentiviral vectors and then were exposed to SCI or sham surgery 2 weeks post‐injection. BMS score, from 0 (no ankle movement) to 9 (normal gait), was determined at indicating times. (F, G) Sensitivities to mechanical and thermal stimulation were determined 28 days after SCI. (H) Extravasation of Evans blue dye was determined 28 days after SCI. n = 6 for each groups. Data are expressed as the mean ± standard deviation and p < 0.05 was considered statistically significant. *p < 0.05.
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