Discovering safe and effective drugs that promote neuron regeneration is an essential strategy for the recovery of central nervous system injuries. In this study, we found that L-leucine, an essential amino acid obtained from both supplements and food sources, could dramatically boost axonal outgrowth and regeneration. First, the effects of L-leucine on neurons were evaluated by cell apoptosis, survival, and death assays, and the results showed no changes in these processes after treatment. By live cell imaging, L-leucine was found to remarkably increase axonal length and growth velocity after axotomy. We also verified that L-leucine enhanced p-mTOR/p-S6K activation in neurons by testing with an mTOR inhibitor, rapamycin. Thereafter, we investigated the effects of L-leucine on the spinal cord injury in vivo. A mouse model of spinal cord hemi-section was established, and L-leucine was administered by tail intravenous injection. Basso mouse scale values revealed that L-leucine could improve functional recovery after injury. It was also notable that L-leucine treatment promoted axon growth across chondroitin sulfate proteoglycan (CSPG) areas.Furthermore, we used CSPGs as inhibitory environmental cues and clarified that L-leucine significantly enhanced axonal outgrowth and regeneration by promoting p-mTOR and p-S6K activation. Therefore, our study is the first to report that L-leucine promotes axonal regeneration in vitro and in vivo and could be candidate drug for axonal re-growth and nervous functional recovery.
Aims
This study aimed to evaluate the effects of the depletion of SAM and SH3 domain‐containing protein 1 (SASH1) on functional recovery after spinal cord injury (SCI) and to investigate the possible mechanism of SASH1 knockdown in astrocytes facilitating axonal growth.
Methods
SCI model was established in adult rats. SASH1 small interfering RNA (siSASH1) was used to investigate its function. Hindlimb motor function was evaluated by the Basso‐Bresnahan‐Beattie (BBB) assay. The gene expressions were evaluated by the methods of qRT‐PCR, Western‐blotting, ELISA, and immunohistochemistry.
Results
SASH1 knockdown improved the BBB scores after SCI and significantly reduced GFAP expression. In cultured spinal astrocytes, siSASH1 treatment decreased interferon‐γ release and increased brain‐derived neurotrophic factor (BDNF) release. When cocultured with SASH1‐knockdown astrocytes, axonal growth increased. The neuronal tropomyosin receptor kinase B (BDNF receptor) expression increased, especially in the axonal tips. SASH1 expression increased while NSCs differentiated into glial cells, instead of neurons. After SASH1 depletion, differentiated NSCs maintained a higher level of Nestin protein and an increase in BDNF release.
Conclusions
These results indicate that SASH1 acts as an astrocytic differentiation‐maintaining protein, and SASH1 downregulation limits glial activation and contributes toward functional recovery after SCI.
Antioxidant of bamboo leaves (AOB) was certified to be a natural antioxidant by the Chinese Ministry of Health in 2003. However, the effects of AOB on animal reproductive and developmental functions remain unclear. The present study aimed to investigate the effects of different concentrations of AOB on mouse embryonic fibroblast (MEF) cells, and to examine the underlying molecular mechanism through which AOB affects the proliferation and apoptosis of MEFs. MEFs prepared from individual embryos were treated with various dosages of AOB. Cell viability and apoptosis were detected by MTT and flow cytometry assays, respectively. Reverse transcription-quantitative polymerase chain reaction and western blot analyses were used for the detection of mRNA and protein expression. Functional annotation of differentially-expressed genes was performed according to the Gene Ontology database and Kyoto Encyclopedia of Genes and Genomes pathway analysis. Compared with the control group, ~50% of MEF cells were inhibited following treatment with a 400 µg/ml concentration of AOB. Treatment with 400 µg/ml AOB for 72 h significantly increased the apoptotic rate of MEF cells compared with the control group. Following treatment with AOB, dehydrogenase/reductase 9, phospholipase A2 group IVE and platelet derived growth factor B were downregulated, while 17 other genes were upregulated in MEF cells. Treatment with AOB markedly increased the expression of phosphorylated extracellular signal-regulated kinase (ERK), β-catenin, transcription factor SOX-17, calcium-binding tyrosine phosphorylation-regulated protein, and cholesterol side chain cleavage enzyme mitochondrial (P<0.01). Additionally, the ERK pathway inhibitor U0126 and Wnt pathway inhibitor dickkopf-related protein 1 markedly suppressed the expression of the above genes (P<0.01). AOB may impact the expression of proteins associated with embryonic fibroblast reproduction and embryonic development through activation of the ERK and Wnt signaling pathways, thus influencing cellular processes.
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