ABSTRACT
The low intrinsic growth capacity of neurons and an injury-induced inhibitory milieu are major contributors to the failure of sensory and motor functional recovery following spinal cord injury. Heat shock transcription factor 1 (HSF1), a master regulator of the heat shock response, plays neurogenetic and neuroprotective roles in the damaged or diseased central nervous system. However, the underlying mechanism has not been fully elucidated. In the present study, we used a gecko model of spontaneous nerve regeneration to investigate the potential roles of gecko HSF1 (gHSF1) in the regulation of neurite outgrowth and inflammatory inhibition of macrophages following spinal cord injury. gHSF1 expression in neurons and microglia at the lesion site increased dramatically immediately after tail amputation. gHSF1 overexpression in gecko primary neurons significantly promoted axonal growth by suppressing the expression of suppressor of cytokine signaling-3, and facilitated neuronal survival via activation of the mitogen-activated extracellular signal-regulated kinase/extracellular regulated protein kinases and phosphatidylinositol 3-kinase/protein kinase B pathways. Furthermore, gHSF1 efficiently inhibited the macrophage-mediated inflammatory response by inactivating IkappaB-alpha/NF-kappaB signaling. Our findings show that HSF1 plays dual roles in promoting axonal regrowth and inhibiting leukocyte inflammation, and provide new avenues of investigation for promoting spinal cord injury repair in mammals.
ABSTRACT
In the search for a therapeutic schedule for spinal cord injury, it is necessary to understand key genes and their corresponding regulatory networks involved in the spinal cord injury process. However, ad hoc selection and analysis of one or two genes cannot fully reveal the complex molecular biological mechanisms of spinal cord injury. The emergence of second-generation sequencing technology (RNA sequencing) has provided a better method. In this study, RNA sequencing technology was used to analyze differentially expressed genes at different time points after spinal cord injury in rat models established by contusion of the eighth thoracic segment. The numbers of genes that changed significantly were 944, 1362 and 1421 at 1, 4 and 7 days after spinal cord injury respectively. After gene ontology analysis and temporal expression analysis of the differentially expressed genes, C5ar1, Socs3 and CCL6 genes were then selected and identified by real-time polymerase chain reaction and western blot assay. The mRNA expression trends of C5ar1, Socs3 and CCL6 genes were consistent with the RNA sequencing results. Further verification and analysis of C5ar1 indicate that the level of protein expression of C5ar1 was consistent with its nucleic acid level after spinal cord injury. C5ar1 was mainly expressed in neurons and astrocytes. Finally, the gene Itgb2, which may be related to C5ar1, was found by Chilibot database and literature search. Immunofluorescence histochemical results showed that the expression of Itgb2 was highly consistent with that of C5ar1. Itgb2 was expressed in astrocytes. RNA sequencing technology can screen differentially expressed genes at different time points after spinal cord injury. Through analysis and verification, genes strongly associated with spinal cord injury can be screened. This can provide experimental data for further determining the molecular mechanism of spinal cord injury, and also provide possible targets for the treatment of spinal cord injury. This study was approved ethically by the Laboratory Animal Ethics Committee of Jiangsu Province, China (approval No. 2018-0306-001) on March 6, 2018.
