miR­222-3p reduces neuronal cell apoptosis and alleviates spinal cord injury by inhibiting Bbc3 and Bim.

Spinal cord injury (SCI) is a severe traumatic event, but without any established effective treatment because of the irreversible neuronal death. Here, we investigated the role of miR-222-3p in neuronal apoptosis following SCI. Rat SCI models and neuron hypoxia models were accordingly established. The Bbc3, Bim, Bcl-2, Bax, cleaved-caspase 3, cleaved-caspase 9, Cytochrome c, and miR-222-3p expression levels were examined by Western blotting and real-time reverse transcription polymerase chain reaction (RT-qPCR). The possible association between miR-222-3p and Bbc3/Bim was analyzed by dual-luciferase assay. The neuron viability was assessed by Cell Counting Kit-8 assay and Nissl's staining. Live cell staining was performed to detect the mitochondrial membrane potential and neuronal apoptosis. Rat locomotor function was assessed using the Basso-Beattie-Bresnahan scores. Cytochrome c was outflowed from the mitochondria after SCI or hypoxia treatment, and Bbc3, Bim, Bax, cleaved-caspase 9, and cleaved-caspase 3 were significantly upregulated, while Bcl-2 and miR-222-3p were decreased remarkably. Meanwhile, neuronal cell viability was significantly inhibited. Treatment of miR-222-3p significantly suppressed the Cytochrome c efflux and neuronal apoptosis and improved neuronal cell viability and motor function in SCI rats. Moreover, we found that Bbc3 and Bim were the direct targets of miR-222-3p. Overall, our data suggest that miR-222-3p could alleviate the mitochondrial pathway-mediated apoptosis and motor dysfunction in rats after SCI by targeting Bbc3 and Bim.

Inhibition of ERK1/2 phosphorylation attenuates spinal cord injury induced astrocyte activation and inflammation through negatively regulating aquaporin-4 in rats.

The extracellular signal-regulated kinase (ERK) pathway has been reported to play a pivotal role in mediating spinal cord injury (SCI) progression. The present study aimed to investigate the effects of phosphorylated ERK1/2 (p-ERK1/2) inhibition on SCI-induced astrocyte activation and inflammation and its possible mechanism in rats. Here, female Sprague-Dawley rats were randomly assigned to four groups: (1) Sham group, (2) SCI group, (3) TGN-020 group (aquaporin-4, AQP4, blocking agent), (4) PD98059 group (ERK blocking agent). A well SCI model was established by compressing the thoracic vertebra 10 level (weight 35 g, time 5 min) in rats. Western blotting and immunofluorescence staining were used to measure the expression of associated proteins after SCI. HE staining and Nissl staining were performed to detect the morphological changes of spinal cords and the number of surviving neurons following SCI, respectively. The Basso-Beattie-Bresnahan open-field rating scale was used to evaluate functional locomotor recovery following SCI in rats. Our results demonstrated that SCI significantly induced the upregulation of aquaporin-4, p-ERK1/2, glial fibrillary acidic protein, proliferating cell nuclear antigen, and proinflammatory cytokines (tumor necrosis factor-α, interleukin-6 and interleukin-1ß). However, treatment with TGN-020 or PD98059 could effectively inhibit astrocyte proliferation and proinflammatory cytokine release, preserve the number of surviving ventral horn neurons, and subsequently improve the locomotor function of rats after SCI. Interestingly, the SCI-induced elevation of AQP4 expression was downregulated by p-ERK1/2 inhibition, suggesting that blocking ERK1/2 phosphorylation could attenuate astrocyte activation and inflammatory processes through negative regulation of AQP4. Therefore, p-ERK1/2 blockade may be employed as a therapeutic target for SCI.

Epidural Spinal Cord Stimulation Promotes Motor Functional Recovery by Enhancing Oligodendrocyte Survival and Differentiation and by Protecting Myelin after Spinal Cord Injury in Rats.

Epidural spinal cord stimulation (ESCS) markedly improves motor and sensory function after spinal cord injury (SCI), but the underlying mechanisms are unclear. Here, we investigated whether ESCS affects oligodendrocyte differentiation and its cellular and molecular mechanisms in rats with SCI. ESCS improved hindlimb motor function at 7 days, 14 days, 21 days, and 28 days after SCI. ESCS also significantly increased the myelinated area at 28 days, and reduced the number of apoptotic cells in the spinal white matter at 7 days. SCI decreased the expression of 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase, an oligodendrocyte marker) at 7 days and that of myelin basic protein at 28 days. ESCS significantly upregulated these markers and increased the percentage of Sox2/CNPase/DAPI-positive cells (newly differentiated oligodendrocytes) at 7 days. Recombinant human bone morphogenetic protein 4 (rhBMP4) markedly downregulated these factors after ESCS. Furthermore, ESCS significantly decreased BMP4 and p-Smad1/5/9 expression after SCI, and rhBMP4 reduced this effect of ESCS. These findings indicate that ESCS enhances the survival and differentiation of oligodendrocytes, protects myelin, and promotes motor functional recovery by inhibiting the BMP4-Smad1/5/9 signaling pathway after SCI.