The Mechanism Of Interleukin-17-Related Inflammatory Cytokines With A20 And NF-κB Regulatory Proteins On Astrocytes Of Spianl Cord In Mice

In the central nervous system, astrocytes have important roles of maintaining the nervous system functions and information-delivery; meanwhile, during the process of neurologic immunoreaction and pathophysiology, astrocytes play a vital role of participating in the development and regeneration of nervous tissue repair. At present, the spinal cord injury (SCI) is still a difficulty in the central nervous injury treatment. On the one hand, the tissue regeneration and repair ability of central nervous system is weak and it is very difficult to get back to normal after the injury; on the other hand, some pathological changes will promote and increase the related inflammatory reaction, which may ultimately affect the functional recovery of the spinal cord.Studies showed that after SCI, a series of secondary inflammatory reaction induced by inflammatory cytokines would aggravate the degree of SCI, including edema, inflammation, ischemia, re-perfusion injury and abnormal changes of peroxide group. And this would seriously damage the normal physiological function of the spinal cord. In the normal spinal cord, astrocytes supply metabolic supports for neurons. However, after the central nervous system injury, astrocytes would be activated to reactive astrocytes and could secrete a variety of inflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL) and interferon (IFN), which could in turn promote the activation of astrocytes and the formation of glial scars. Therefore, it is of great significance to investigate the mechanism of inflammatory factors affecting astrocytes which can provide theoretical basis for clinical treatment of SCI.In order to investigate the mechanism of IL-17-related inflammatory cytokines and regulatory proteins, the study are carried out two parts in the following.Section 1:Expressions of interleukin-17-related inflammatory factors of astrocytes in mice spinal cordObjective:To investigate the expressions of inflammatory cytokines stimulated with IL-17.Method:Astrocytes of spinal cord in mice were cultured in vitro. After stimulating astrocytes with different concentrations of IL-17, the expression of IL-6, TNF-a, MCP-1/5 and MIP-2 were determined using qRT-PCR and ELISA kits at different time points (3 h,6 h,12 h,24 h and 48 h).Results:Compared with the control group, the mRNA and protein expression levels of IL-6, TNF-a, MCP-1/5 and MIP-2 increased significantly at 6h and 12h respectively (p<0.05).Conclusion:IL-17 could promote the expressions of inflammatory cytokines of astrocytes in mice spinal cord.Section 2:The effects of interleukin-17-related A20 and NF-κB proteins on astrocytes in mice spinal cordObjective:To investigate the protein levels of A20 and NF-κB proteins stimulated with IL-17.Method:Astrocytes of spinal cord in mice were cultured in vitro. After stimulating astrocytes with IL-17 (50ng/ml), the expression of A20 and NF-κB were determined using western blotting (WB) and qRT-PCR at different time points (3 h,6 h,12 h,24 h and 48 h).Results:Compared with the control group, the mRNA and protein expression levels of NF-κB increased significantly at 6h and 12h respectively (p<0.05), which had a same tendency of IL-6, TNF-a, MCP-1/5 and MIP-2; the protein expression level of A20 significantly decreased at 12h (p<0.05) which showed an opposite response to p-NF-κB, while the mRNA expression level of A20 was decreased.Conclusion:After stimulating astrocytes with IL-17, NF-κB may have a relationship with inflammatory cytokines which would promote the expression of IL-6, TNF-a, MCP-1/5 and MIP-2, while A20 showed an inhibitory effect on these inflammatory cytokines.