Bisperoxovanadium protects against spinal cord injury by regulating autophagy via activation of ERK1/2 signaling

Abstract

Background: Spinal cord injury (SCI) is a disease of the central nervous system with few restorative treatments. Autophagy has been regarded as a promising therapeutic target for SCI. The inhibitor of phosphatase and tensin homolog deleted on chromosome ten (PTEN) bisperoxovanadium (bpV[pic]) had been claimed to provide a neuroprotective effect on SCI; but the underlying mechanism is still not fully understood.

Materials and methods: Acute SCI model were generated with SD Rats and were treated with control, acellular spinal cord scaffolds (ASC) obtained from normal rats, bpV(pic), and combined material of ASC and bpV(pic). We used BBB score to assess the motor function of the rats and the motor neurons were stained with Nissl staining. The expressions of the main autophagy markers LC3B, Beclin1 and P62, expressions of apoptosis makers Bax, Bcl2, PARP and Caspase 3 were detected with IF or Western Blot analysis.

Results: The bpV(pic) showed significant improvement in functional recovery by activating autophagy and accompanied by decreased neuronal apoptosis; combined ASC with bpV(pic) enhanced these effects. In addition, after treatment with ERK1/2 inhibitor SCH772984, we revealed that bpV(pic) promotes autophagy and inhibits apoptosis through activating ERK1/2 signaling after SCI.

Conclusion: These results illustrated that the bpV(pic) protects against SCI by regulating autophagy via activation of ERK1/2 signaling.

Keywords: ERK1/2 signaling; apoptosis; autophagy; bisperoxovanadium; spinal cord injury.

Figure 1

ASC combined with bpV(pic) reverses tissue structure damage and improves functional recovery after acute SCI. Notes: (A) Plot of BBB locomotor scores of the different transplantation groups at different times (from 1 to 6 weeks) post-SCI. n=5. (B) Nissl staining of motor neurons in the large anterior horn in different transplantation groups 4 weeks after SCI. The arrows are pointing to the cell bodies of neurons. Scale bar =50 µm. (C) Number of motor neuron cell accounts in the anterior horns. n=5 (D) Representative immunofluorescence images of NeuN (green) and β-III-tubulin (red) staining in spinal cords from SCI rats with no graft, ASC-only graft, bpV(pic)-only graft, or ASC scaffold with bpV(pic) graft 4 weeks postsurgery. Scale bar =50 µm. (E) Quantification of the number of NeuN (green)-positive cells in (D). n=5. ^*In comparison with control group, P<0.05. The red ^* in (C) and (E) means P<0.05 between bpV(pic) and ASC combined with bpV(pic) group. ^#In comparison with ASC group, P<0.05. ns = not significant. All data are shown as the mean ± SD. Abbreviations: ASC, acellular spinal cord; BBB, Basso, Beattie, and Bresnahan; bpV(pic), bisperoxovanadium; SCI, spinal cord injury.

Figure 2

ASC combined with bpV(pic) promotes autophagy and attenuates apoptosis in rat acute SCI. Notes: (A) Representative immunofluorescence images of NeuN (green) and LC3B (red) staining in spinal cords from SCI rats from the different groups. The fields in the frame were enlarged on the right side. Scale bar =50 µm. (B) Quantification of the number of NeuN (green)-positive cells in (A), n=5. (C) Representative immunofluorescence images of Bcl2 (green) and Bax (red) staining in spinal cords from SCI rats from the different groups. Scale bar =50 µm. (D) Quantification of the Bax/Bcl2 ratio in (C), n=5. (E) Immunoblot analysis of p62, LC3B, Beclin1, Bax, Bcl2, and GAPDH levels in spinal cords from SCI rats with different treatments. n=3 independent experiments.^*In comparison with control group, P<0.05. The red ^* in (B) means P<0.05 between bpV(pic) and ASC combined with bpV(pic) group. ^#In comparison with ASC group, P<0.05. ns = not significant. All data are shown as mean ± SD. Abbreviations: ASC, acellular spinal cord; bpV(pic), bisperoxovanadium; LC3B, light chain 3B; SCI, spinal cord injury.

Figure 3

Treatment with bpV(pic) or ASC scaffolds combined with bpV(pic) promotes autophagy and attenuates apoptosis in cultured rat neurons. Notes: (A) Top, representative images of cocultured RNSCs with vehicle (Ctrl), ASC only, bpV(pic), or ASC combined with bpV(pic). Below, immunofluorescence analysis of LC3B expression (red) in RNSCs from different groups. Scale bar =50 µm. (B) RNSCs were cocultured with different materials for 24 hours, and then cell survival was analyzed by CCK-8 assay. n=3 independent experiments. (C) Quantification of the number of LC3B (red)-positive cells in (A), n=5. (D) Immunoblot analysis of p-PTEN, p62, Beclin1, LC3B, and GAPDH levels in RNSCs with different treatments. n=3 independent experiments. (E) Immunoblot analysis of cleaved-PARP, caspase 3, Bax, Bcl2, and GAPDH levels in RNSCs with different treatments. n=3 independent experiments. ^*In comparison with control group, P<0.05. The red ^* in (C) means P<0.05 between bpV(pic) and ASC combined with bpV(pic) groups. ^#In comparison with ASC group, P<0.05. ns = not significant. All data are shown as the mean ± SD. Abbreviations: ASC, acellular spinal cord; bpV(pic), bisperoxovanadium; CCK-8, cell counting kit-8; LC3B, light chain 3B; RNSCs, rat neuron stem cells; SCI, spinal cord injury.

Figure 4

bpV(pic) promotes autophagy and attenuates apoptosis through activation of ERK1/2 signaling. Notes: (A) Immunoblot analysis of pS6, S6, p-ERK1/2, ERK1/2, and β-actin levels in RNSCs with different treatments. n=3 independent experiments. (B) Representative immunofluorescence images of p-ERK1/2 in spinal cords from SCI rats from the different groups. Scale bar =50 µm. (C) Quantification of the p-ERK1/2-positive cells in (B). n=5. (D) Immunoblot analysis of p-ERK1/2, ERK1/2, Beclin1, LC3B, and β-actin levels with vehicle (Ctrl), ASC only, bpV(pic), ASC combined with bpV(pic), bpV(pic) plus SCH772984 or ASC combined with bpV(pic) plus SCH772984. n=3 independent experiments. ^*In comparison with control group, P<0.05. ^#In comparison with ASC group, P<0.05. ns = not significant. All data are shown as the mean ± SD. Abbreviations: ASC, acellular spinal cord; bpV(pic), bisperoxovanadium; LC3B, light chain 3B; RNSCs, rat neuron stem cells; SCI, spinal cord injury.