Li SS et al. (2023), A new peptide, VD11, promotes structural and fu...

XB-ART-59956

Neural Regen Res 2023 Oct 01;1810:2260-2267. doi: 10.4103/1673-5374.369119.

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A new peptide, VD11, promotes structural and functional recovery after spinal cord injury.

Li SS , Zhang BY , Yin SG , Wei ZQ , Liu NX , Li YL , Wang SY , Shi YH , Zhao J , Wang LJ , Zhang Y , Sun J , Wang Y , Yang XW .

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The regenerative capacity of the central nervous system is very limited and few effective treatments are currently available for spinal cord injury. It is therefore a priority to develop new drugs that can promote structural and functional recovery after spinal cord injury. Previous studies have shown that peptides can promote substantial repair and regeneration of injured tissue. While amphibians have a pronounced ability to regenerate the spinal cord, few studies have investigated the effect of amphibian spinal cord-derived peptides on spinal cord injury. Here we report for the first time the successful identification and isolation of a new polypeptide, VD11 (amino acid sequence: VDELWPPWLPC), from the spinal cord of an endemic Chinese amphibian (Odorrana schmackeri). In vitro experiments showed that VD11 promoted the secretion of nerve growth factor and brain-derived neurotrophic factor in BV2 cells stimulated with lipopolysaccharide, as well as the proliferation and synaptic elongation of PC12 cells subjected to hypoxia. In vivo experiments showed that intravertebral injection of VD11 markedly promoted recovery of motor function in rats with spinal cord injury, alleviated pathological damage, and promoted axonal regeneration. Furthermore, RNA sequencing and western blotting showed that VD11 may affect spinal cord injury through activation of the AMPK and AKT signaling pathways. In summary, we discovered a novel amphibian-derived peptide that promotes structural and functional recovery after spinal cord injury.

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Genes referenced: prkaa2
GO keywords: cellular response to oxidative stress [+]

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	Graphical Abstract
	Figure 1: VD11 cDNA sequence.(A) VD11 cDNA sequence. The VD11 precursor contains 58 amino acid residues encoded by a 221-bp mRNA, and the mature sequence is VDELWPPWLPC (underlined italics). (B) Sequence alignment of VD11 and Nigrocin-2N showed similar precursor sequences but no apparent similarity between the mature peptides. cDNA: Complementary DNA.
	Figure 2: VD11 increases NGF and BDNF secretion in vitro and in vivo.(A, B) VD11 significantly increased NGF (A) and BDNF (B) mRNA expression in BV2 cells, as detected by qPCR. NGF and BDNF expression levels were normalized to β-actin. (C, D) VD11 promoted NGF and BDNF secretion in a dose-dependent manner, as determined by enzyme-linked immunosorbent assay. Data were normalized to the control group. (E, F) Western blot assay showed that VD11 promoted BDNF (n = 4) and NGF (n = 3) secretion after SCI in rats. Data are presented as mean ± SD from three independent experiments performed in triplicate. P < 0.05, P < 0.01, P < 0.001, **P < 0.0001 (one-way analysis of variance with the Bonferroni’s post hoc test). BDNF: Brain-derived neurotrophic factor; NGF: nerve growth factor; SCI: spinal cord injury.
	Figure 3: VD11 promotes PC12 cell proliferation and axon extension.(A) Effect of VD11 on PC12 cell viability, normalized to the control group (n = 9). (B) Representative immunofluorescence images of the effects on OGD/R and VD11 on the cell survival and axon growth (detected by Tau expression, green-fluorescein isothiocyanate) in PC12 cells. Axon length decreased after OGD/R and increased after VD11 treatment. DAPI indicates nuclei (blue). Scale bars: 20 μm. (C, D) VD11 (100 nM) alleviated the decrease in cell number (C) and increased axon length (D) in PC12 cells. Three replicates were performed for each group, with each value representing mean cell number in three random visual fields. Data are presented as mean ± SD (n = 3). P < 0.05, P < 0.01, P < 0.001, **P < 0.0001 (one-way analysis of variance with the Bonferroni’s post hoc test). DAPI: 4′,6-Diamidino-2-phenylindole; OGD/R: oxygen glucose deprivation/reoxygenation.
	Figure 4: VD11 promotes hind limb functional recovery in rats after SCI, as determined by motor function assessment.(A) Experimental design and timeline. (B) Changes in BBB locomotor score. Data represent means of scores from three independent observers. (C) Animal weight 28 days after injury. Data are presented as mean ± SD (n = 9). P < 0.05, P < 0.01, P < 0.001, **P < 0.0001, vs. vehicle group; ##P < 0.01, vs. MPEP group (two-way (B) or one-way (C) analysis of variance with the Bonferroni’s post hoc test). BBB: Basso-Beattie-Bresnahan; MPED: methylprednisolone; SCI: spinal cord injury.
	Figure 5: VD11 reduces scar lengths in the spinal cord of rats after SCI.(A, B) Representative dorsal (A) and ventral (B) images of the spinal cord 30 days after SCI. Rats treated with MPED showed a reduction in spinal cord scarring compared with vehicle group, while rats treated with VD11 showed a more substantial reduction in scarring. Scar edge is denoted by red lines. (C, D) Spinal cord scar lengths (mm) in rats 30 days after surgery. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.01 (one-way analysis of variance with the Bonferroni’s post hoc test). MPED: Methylprednisolone; SCI: spinal cord injury.
	Figure 6: VD11 promotes tissue structure repair in SCI rats, as determined by H&E and Nissl staining.(A, B) Representative images of H&E (A) and Nissl (B) staining of spinal cord tissue from rats 30 days after SCI. The images in the second row of (B) are magnifications of the red boxes in the images in the first row. The images in the third row are high-magnification representative images of neurons with a diameter of 5 mm at the injury center. Some residual neurons could be seen in the VD11 group, but almost no neurons could be seen in the control and MPED groups. Red arrows indicate tissue cavities, and black arrows indicate neurons. (C, D) Statistical analysis of the cavity area in the spinal cord tissue stained with H&E (C) and the number of neurons stained with Nissl (D) in rats 30 days after surgery. Three replicates were performed for each group, with each value representing mean neuron number in three random visual fields. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.01 (one-way analysis of variance with the Bonferroni’s post hoc test). H&E: Hematoxylin and eosin; MPED: methylprednisolone; SCI: spinal cord injury.
	Figure 7: VD11 alleviates nerve injury and promotes nerve regeneration in SCI rats, as determined by immunofluorescence staining.(A) Representative immunofluorescence staining images of reactive astrocytes (marked with GFAP, green-fluorescein isothiocyanate) and neuronal axons (marked with Tau, red-Cy3) in the spinal cord 30 days after SCI. DAPI indicates nuclei (blue). Scale bars: 500 µm. (B, C) Statistical analysis of the GFAP+ (B) and Tau+ (C) areas. The area in the vehicle group was defined as 100%. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.01 (one-way analysis of variance with the Bonferroni’s post hoc test). DAPI: 4′,6-Diamidino-2-phenylindole; GFAP: glial fibrillary acidic protein; MPED: methylprednisolone; SCI: spinal cord injury.
	Figure 8: Mechanism underlying the effects of VD11 on neurological recovery.(A) DEGs as determined by RNA-seq analysis. (B) KEGG pathways enriched in DEGs based on RNA-seq analysis, arranged from top to bottom in terms of significance and presented as a bubble graph. (C, D) VD11 administration resulted in decreased AMPK (C) and AKT (D) phosphorylation compared with the OGD/R group. The value for the control group was set to 100%. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.01 (one-way analysis of variance with the Bonferroni’s post hoc test). AMPK: 5′-Monophosphate-activated protein kinase; DEG: differentially expressed gene; KEGG: Kyoto Encyclopedia of Genes and Genomes; OGD/R: oxygen-glucose deprivation/reoxygenation; P-Akt: phosphorylated Akt; P-AMPK: phosphorylated 5′-monophosphate-activated protein kinase; RNA-seq: RNA sequencing.
	Figure 1. VD11 cDNA sequence.(A) VD11 cDNA sequence. The VD11 precursor contains 58 amino acid residues encoded by a 221-bp mRNA, and the mature sequence is VDELWPPWLPC (underlined italics). (B) Sequence alignment of VD11 and Nigrocin-2N showed similar precursor sequences but no apparent similarity between the mature peptides. cDNA: Complementary DNA.
	Figure 2. VD11 increases NGF and BDNF secretion in vitro and in vivo.(A, B) VD11 significantly increased NGF (A) and BDNF (B) mRNA expression in BV2 cells, as detected by qPCR. NGF and BDNF expression levels were normalized to β-actin. (C, D) VD11 promoted NGF and BDNF secretion in a dose-dependent manner, as determined by enzyme-linked immunosorbent assay. Data were normalized to the control group. (E, F) Western blot assay showed that VD11 promoted BDNF (n = 4) and NGF (n = 3) secretion after SCI in rats. Data are presented as mean ± SD from three independent experiments performed in triplicate. P < 0.05, P < 0.01, P < 0.001, **P < 0.0001 (one-way analysis of variance with the Bonferroni’s post hoc test). BDNF: Brain-derived neurotrophic factor; NGF: nerve growth factor; SCI: spinal cord injury.
	Figure 3. VD11 promotes PC12 cell proliferation and axon extension.(A) Effect of VD11 on PC12 cell viability, normalized to the control group (n = 9). (B) Representative immunofluorescence images of the effects on OGD/R and VD11 on the cell survival and axon growth (detected by Tau expression, green-fluorescein isothiocyanate) in PC12 cells. Axon length decreased after OGD/R and increased after VD11 treatment. DAPI indicates nuclei (blue). Scale bars: 20 μm. (C, D) VD11 (100 nM) alleviated the decrease in cell number (C) and increased axon length (D) in PC12 cells. Three replicates were performed for each group, with each value representing mean cell number in three random visual fields. Data are presented as mean ± SD (n = 3). P < 0.05, P < 0.01, P < 0.001, **P < 0.0001 (one-way analysis of variance with the Bonferroni’s post hoc test). DAPI: 4′,6-Diamidino-2-phenylindole; OGD/R: oxygen glucose deprivation/reoxygenation.
	Figure 4. VD11 promotes hind limb functional recovery in rats after SCI, as determined by motor function assessment.(A) Experimental design and timeline. (B) Changes in BBB locomotor score. Data represent means of scores from three independent observers. (C) Animal weight 28 days after injury. Data are presented as mean ± SD (n = 9). P < 0.05, P < 0.01, P < 0.001, **P < 0.0001, vs. vehicle group; ##P < 0.01, vs. MPEP group (two-way (B) or one-way (C) analysis of variance with the Bonferroni’s post hoc test). BBB: Basso-Beattie-Bresnahan; MPED: methylprednisolone; SCI: spinal cord injury.
	Figure 5. VD11 reduces scar lengths in the spinal cord of rats after SCI.(A, B) Representative dorsal (A) and ventral (B) images of the spinal cord 30 days after SCI. Rats treated with MPED showed a reduction in spinal cord scarring compared with vehicle group, while rats treated with VD11 showed a more substantial reduction in scarring. Scar edge is denoted by red lines. (C, D) Spinal cord scar lengths (mm) in rats 30 days after surgery. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.01 (one-way analysis of variance with the Bonferroni’s post hoc test). MPED: Methylprednisolone; SCI: spinal cord injury.
	Figure 6. VD11 promotes tissue structure repair in SCI rats, as determined by H&E and Nissl staining.(A, B) Representative images of H&E (A) and Nissl (B) staining of spinal cord tissue from rats 30 days after SCI. The images in the second row of (B) are magnifications of the red boxes in the images in the first row. The images in the third row are high-magnification representative images of neurons with a diameter of 5 mm at the injury center. Some residual neurons could be seen in the VD11 group, but almost no neurons could be seen in the control and MPED groups. Red arrows indicate tissue cavities, and black arrows indicate neurons. (C, D) Statistical analysis of the cavity area in the spinal cord tissue stained with H&E (C) and the number of neurons stained with Nissl (D) in rats 30 days after surgery. Three replicates were performed for each group, with each value representing mean neuron number in three random visual fields. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.01 (one-way analysis of variance with the Bonferroni’s post hoc test). H&E: Hematoxylin and eosin; MPED: methylprednisolone; SCI: spinal cord injury.
	Figure 7. VD11 alleviates nerve injury and promotes nerve regeneration in SCI rats, as determined by immunofluorescence staining.(A) Representative immunofluorescence staining images of reactive astrocytes (marked with GFAP, green-fluorescein isothiocyanate) and neuronal axons (marked with Tau, red-Cy3) in the spinal cord 30 days after SCI. DAPI indicates nuclei (blue). Scale bars: 500 µm. (B, C) Statistical analysis of the GFAP+ (B) and Tau+ (C) areas. The area in the vehicle group was defined as 100%. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.01 (one-way analysis of variance with the Bonferroni’s post hoc test). DAPI: 4′,6-Diamidino-2-phenylindole; GFAP: glial fibrillary acidic protein; MPED: methylprednisolone; SCI: spinal cord injury.
	Figure 8. Mechanism underlying the effects of VD11 on neurological recovery.(A) DEGs as determined by RNA-seq analysis. (B) KEGG pathways enriched in DEGs based on RNA-seq analysis, arranged from top to bottom in terms of significance and presented as a bubble graph. (C, D) VD11 administration resulted in decreased AMPK (C) and AKT (D) phosphorylation compared with the OGD/R group. The value for the control group was set to 100%. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.01 (one-way analysis of variance with the Bonferroni’s post hoc test). AMPK: 5′-Monophosphate-activated protein kinase; DEG: differentially expressed gene; KEGG: Kyoto Encyclopedia of Genes and Genomes; OGD/R: oxygen-glucose deprivation/reoxygenation; P-Akt: phosphorylated Akt; P-AMPK: phosphorylated 5′-monophosphate-activated protein kinase; RNA-seq: RNA sequencing.

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	Graphical Abstract
	Figure 1: VD11 cDNA sequence.(A) VD11 cDNA sequence. The VD11 precursor contains 58 amino acid residues encoded by a 221-bp mRNA, and the mature sequence is VDELWPPWLPC (underlined italics). (B) Sequence alignment of VD11 and Nigrocin-2N showed similar precursor sequences but no apparent similarity between the mature peptides. cDNA: Complementary DNA.
	Figure 2: VD11 increases NGF and BDNF secretion in vitro and in vivo.(A, B) VD11 significantly increased NGF (A) and BDNF (B) mRNA expression in BV2 cells, as detected by qPCR. NGF and BDNF expression levels were normalized to β-actin. (C, D) VD11 promoted NGF and BDNF secretion in a dose-dependent manner, as determined by enzyme-linked immunosorbent assay. Data were normalized to the control group. (E, F) Western blot assay showed that VD11 promoted BDNF (n = 4) and NGF (n = 3) secretion after SCI in rats. Data are presented as mean ± SD from three independent experiments performed in triplicate. P < 0.05, P < 0.01, P < 0.001, **P < 0.0001 (one-way analysis of variance with the Bonferroni’s post hoc test). BDNF: Brain-derived neurotrophic factor; NGF: nerve growth factor; SCI: spinal cord injury.
	Figure 3: VD11 promotes PC12 cell proliferation and axon extension.(A) Effect of VD11 on PC12 cell viability, normalized to the control group (n = 9). (B) Representative immunofluorescence images of the effects on OGD/R and VD11 on the cell survival and axon growth (detected by Tau expression, green-fluorescein isothiocyanate) in PC12 cells. Axon length decreased after OGD/R and increased after VD11 treatment. DAPI indicates nuclei (blue). Scale bars: 20 μm. (C, D) VD11 (100 nM) alleviated the decrease in cell number (C) and increased axon length (D) in PC12 cells. Three replicates were performed for each group, with each value representing mean cell number in three random visual fields. Data are presented as mean ± SD (n = 3). P < 0.05, P < 0.01, P < 0.001, **P < 0.0001 (one-way analysis of variance with the Bonferroni’s post hoc test). DAPI: 4′,6-Diamidino-2-phenylindole; OGD/R: oxygen glucose deprivation/reoxygenation.
	Figure 4: VD11 promotes hind limb functional recovery in rats after SCI, as determined by motor function assessment.(A) Experimental design and timeline. (B) Changes in BBB locomotor score. Data represent means of scores from three independent observers. (C) Animal weight 28 days after injury. Data are presented as mean ± SD (n = 9). P < 0.05, P < 0.01, P < 0.001, **P < 0.0001, vs. vehicle group; ##P < 0.01, vs. MPEP group (two-way (B) or one-way (C) analysis of variance with the Bonferroni’s post hoc test). BBB: Basso-Beattie-Bresnahan; MPED: methylprednisolone; SCI: spinal cord injury.
	Figure 5: VD11 reduces scar lengths in the spinal cord of rats after SCI.(A, B) Representative dorsal (A) and ventral (B) images of the spinal cord 30 days after SCI. Rats treated with MPED showed a reduction in spinal cord scarring compared with vehicle group, while rats treated with VD11 showed a more substantial reduction in scarring. Scar edge is denoted by red lines. (C, D) Spinal cord scar lengths (mm) in rats 30 days after surgery. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.01 (one-way analysis of variance with the Bonferroni’s post hoc test). MPED: Methylprednisolone; SCI: spinal cord injury.
	Figure 6: VD11 promotes tissue structure repair in SCI rats, as determined by H&E and Nissl staining.(A, B) Representative images of H&E (A) and Nissl (B) staining of spinal cord tissue from rats 30 days after SCI. The images in the second row of (B) are magnifications of the red boxes in the images in the first row. The images in the third row are high-magnification representative images of neurons with a diameter of 5 mm at the injury center. Some residual neurons could be seen in the VD11 group, but almost no neurons could be seen in the control and MPED groups. Red arrows indicate tissue cavities, and black arrows indicate neurons. (C, D) Statistical analysis of the cavity area in the spinal cord tissue stained with H&E (C) and the number of neurons stained with Nissl (D) in rats 30 days after surgery. Three replicates were performed for each group, with each value representing mean neuron number in three random visual fields. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.01 (one-way analysis of variance with the Bonferroni’s post hoc test). H&E: Hematoxylin and eosin; MPED: methylprednisolone; SCI: spinal cord injury.
	Figure 7: VD11 alleviates nerve injury and promotes nerve regeneration in SCI rats, as determined by immunofluorescence staining.(A) Representative immunofluorescence staining images of reactive astrocytes (marked with GFAP, green-fluorescein isothiocyanate) and neuronal axons (marked with Tau, red-Cy3) in the spinal cord 30 days after SCI. DAPI indicates nuclei (blue). Scale bars: 500 µm. (B, C) Statistical analysis of the GFAP+ (B) and Tau+ (C) areas. The area in the vehicle group was defined as 100%. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.01 (one-way analysis of variance with the Bonferroni’s post hoc test). DAPI: 4′,6-Diamidino-2-phenylindole; GFAP: glial fibrillary acidic protein; MPED: methylprednisolone; SCI: spinal cord injury.
	Figure 8: Mechanism underlying the effects of VD11 on neurological recovery.(A) DEGs as determined by RNA-seq analysis. (B) KEGG pathways enriched in DEGs based on RNA-seq analysis, arranged from top to bottom in terms of significance and presented as a bubble graph. (C, D) VD11 administration resulted in decreased AMPK (C) and AKT (D) phosphorylation compared with the OGD/R group. The value for the control group was set to 100%. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.01 (one-way analysis of variance with the Bonferroni’s post hoc test). AMPK: 5′-Monophosphate-activated protein kinase; DEG: differentially expressed gene; KEGG: Kyoto Encyclopedia of Genes and Genomes; OGD/R: oxygen-glucose deprivation/reoxygenation; P-Akt: phosphorylated Akt; P-AMPK: phosphorylated 5′-monophosphate-activated protein kinase; RNA-seq: RNA sequencing.
	Figure 1. VD11 cDNA sequence.(A) VD11 cDNA sequence. The VD11 precursor contains 58 amino acid residues encoded by a 221-bp mRNA, and the mature sequence is VDELWPPWLPC (underlined italics). (B) Sequence alignment of VD11 and Nigrocin-2N showed similar precursor sequences but no apparent similarity between the mature peptides. cDNA: Complementary DNA.
	Figure 2. VD11 increases NGF and BDNF secretion in vitro and in vivo.(A, B) VD11 significantly increased NGF (A) and BDNF (B) mRNA expression in BV2 cells, as detected by qPCR. NGF and BDNF expression levels were normalized to β-actin. (C, D) VD11 promoted NGF and BDNF secretion in a dose-dependent manner, as determined by enzyme-linked immunosorbent assay. Data were normalized to the control group. (E, F) Western blot assay showed that VD11 promoted BDNF (n = 4) and NGF (n = 3) secretion after SCI in rats. Data are presented as mean ± SD from three independent experiments performed in triplicate. P < 0.05, P < 0.01, P < 0.001, **P < 0.0001 (one-way analysis of variance with the Bonferroni’s post hoc test). BDNF: Brain-derived neurotrophic factor; NGF: nerve growth factor; SCI: spinal cord injury.
	Figure 3. VD11 promotes PC12 cell proliferation and axon extension.(A) Effect of VD11 on PC12 cell viability, normalized to the control group (n = 9). (B) Representative immunofluorescence images of the effects on OGD/R and VD11 on the cell survival and axon growth (detected by Tau expression, green-fluorescein isothiocyanate) in PC12 cells. Axon length decreased after OGD/R and increased after VD11 treatment. DAPI indicates nuclei (blue). Scale bars: 20 μm. (C, D) VD11 (100 nM) alleviated the decrease in cell number (C) and increased axon length (D) in PC12 cells. Three replicates were performed for each group, with each value representing mean cell number in three random visual fields. Data are presented as mean ± SD (n = 3). P < 0.05, P < 0.01, P < 0.001, **P < 0.0001 (one-way analysis of variance with the Bonferroni’s post hoc test). DAPI: 4′,6-Diamidino-2-phenylindole; OGD/R: oxygen glucose deprivation/reoxygenation.
	Figure 4. VD11 promotes hind limb functional recovery in rats after SCI, as determined by motor function assessment.(A) Experimental design and timeline. (B) Changes in BBB locomotor score. Data represent means of scores from three independent observers. (C) Animal weight 28 days after injury. Data are presented as mean ± SD (n = 9). P < 0.05, P < 0.01, P < 0.001, **P < 0.0001, vs. vehicle group; ##P < 0.01, vs. MPEP group (two-way (B) or one-way (C) analysis of variance with the Bonferroni’s post hoc test). BBB: Basso-Beattie-Bresnahan; MPED: methylprednisolone; SCI: spinal cord injury.
	Figure 5. VD11 reduces scar lengths in the spinal cord of rats after SCI.(A, B) Representative dorsal (A) and ventral (B) images of the spinal cord 30 days after SCI. Rats treated with MPED showed a reduction in spinal cord scarring compared with vehicle group, while rats treated with VD11 showed a more substantial reduction in scarring. Scar edge is denoted by red lines. (C, D) Spinal cord scar lengths (mm) in rats 30 days after surgery. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.01 (one-way analysis of variance with the Bonferroni’s post hoc test). MPED: Methylprednisolone; SCI: spinal cord injury.
	Figure 6. VD11 promotes tissue structure repair in SCI rats, as determined by H&E and Nissl staining.(A, B) Representative images of H&E (A) and Nissl (B) staining of spinal cord tissue from rats 30 days after SCI. The images in the second row of (B) are magnifications of the red boxes in the images in the first row. The images in the third row are high-magnification representative images of neurons with a diameter of 5 mm at the injury center. Some residual neurons could be seen in the VD11 group, but almost no neurons could be seen in the control and MPED groups. Red arrows indicate tissue cavities, and black arrows indicate neurons. (C, D) Statistical analysis of the cavity area in the spinal cord tissue stained with H&E (C) and the number of neurons stained with Nissl (D) in rats 30 days after surgery. Three replicates were performed for each group, with each value representing mean neuron number in three random visual fields. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.01 (one-way analysis of variance with the Bonferroni’s post hoc test). H&E: Hematoxylin and eosin; MPED: methylprednisolone; SCI: spinal cord injury.
	Figure 7. VD11 alleviates nerve injury and promotes nerve regeneration in SCI rats, as determined by immunofluorescence staining.(A) Representative immunofluorescence staining images of reactive astrocytes (marked with GFAP, green-fluorescein isothiocyanate) and neuronal axons (marked with Tau, red-Cy3) in the spinal cord 30 days after SCI. DAPI indicates nuclei (blue). Scale bars: 500 µm. (B, C) Statistical analysis of the GFAP+ (B) and Tau+ (C) areas. The area in the vehicle group was defined as 100%. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.01 (one-way analysis of variance with the Bonferroni’s post hoc test). DAPI: 4′,6-Diamidino-2-phenylindole; GFAP: glial fibrillary acidic protein; MPED: methylprednisolone; SCI: spinal cord injury.
	Figure 8. Mechanism underlying the effects of VD11 on neurological recovery.(A) DEGs as determined by RNA-seq analysis. (B) KEGG pathways enriched in DEGs based on RNA-seq analysis, arranged from top to bottom in terms of significance and presented as a bubble graph. (C, D) VD11 administration resulted in decreased AMPK (C) and AKT (D) phosphorylation compared with the OGD/R group. The value for the control group was set to 100%. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.01 (one-way analysis of variance with the Bonferroni’s post hoc test). AMPK: 5′-Monophosphate-activated protein kinase; DEG: differentially expressed gene; KEGG: Kyoto Encyclopedia of Genes and Genomes; OGD/R: oxygen-glucose deprivation/reoxygenation; P-Akt: phosphorylated Akt; P-AMPK: phosphorylated 5′-monophosphate-activated protein kinase; RNA-seq: RNA sequencing.