RESUMO
Objective:To investigate the effects of long non-coding RNA (lncRNA) Gm13568 on the activation of A1 astrocytes and the progress of experimental autoimmune encephalomyelitis (EAE) in mice.Methods:A recombinant lentiviral vector (LV-Inhibit-Gm13568) carrying astrocyte-specific promoter of glial fibrillary acidic protein (GFAP) was established to inhibit the function of endogenous Gm13568. A control vector (LV-ctrl) was established as well. The recombinant vectors were packaged. C57BL/6 mice were injected with 1×10 7 transforming units of viral suspension via the tail vein and 7 d after the injection, myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55) was used to establish the mouse model of EAE. Four groups, PBS group, EAE group, LV-ctrl+ EAE group and LV-Inhibit-Gm13568+ EAE group, were included in this study. Clinical signs of the mice were monitored daily in a double-blinded manner. The mice were sacrificed 23 d after the EAE model was established and the spinal cord tissues were collected. The expression of Serping 1, C3, Srgn and H2-T23 at mRNA level was detected by real-time PCR. Changes in the expression of IL-6, TNF-α, macrophage chemotactic protein-1 (MCP-1) and interferon-inducible protein-10 (IP-10) were measured. Western blot was used to investigate the expression of GFAP and Notch1 in spinal cord tissues and the phosphorylation of signal transduction and transcription activator 3 (STAT3). The expression of Notch1 intracellular domain (NICD) and GFAP in spinal cord tissues was detected by immunofluorescence. Furthermore, the infiltration of inflammatory cells and the demyelination of spinal cord were observed using HE and Luxol fast blue (LFB) staining methods. Results:Compared with PBS group, A1 astrocytes were activated and Notch1 expression was significantly up-regulated in EAE group and LV-ctrl+ EAE group. The clinical score of mice in LV-Inhibit-Gm13568+ EAE group was decreased from an average score of 3.5 to less than 1 on 23 d after antigen induction and the clinical symptoms were alleviated as compared with the mice in LV-ctrl+ EAE group. Meanwhile, the activation of A1 astrocytes was down-regulated, and the production of inflammatory cytokines and chemokines was also reduced. The expression of Notch1, GFAP and NICD at protein level and the phosphorylation of STAT3 were significantly reduced. Moreover, the infiltration of inflammatory cells and demyelination of spinal cord tissues were alleviated significantly.Conclusions:LncRNA Gm13568 might regulate the activation of A1 astrocytes via the Notch1/STAT3 pathway, thus affecting the production of inflammatory cytokines and chemokines and participating in the process of EAE.
RESUMO
Objective:To investigate the regulatory effects of sphingosine kinase-2 (SphK2) on the function of activated astrocytes and the progression of experimental autoimmune encephalomyelitis (EAE) in mice.Methods:Primary mouse astrocytes were isolated from wild-type (WT) C57BL/6 mice and sphk2 gene knock-out ( sphk2 -/-) mice and stimulated in vitro with interleukin 17 (IL-17). Real-time PCR was used to measure the expression of inflammatory cytokines and chemokines at mRNA levels. Western blot and immunofluorescence were used to detect the expression of glial fibrillary acidic protein (GFAP) and the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3). An EAE mouse model was constructed using myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55) polypeptide. Western blot was used to detect the expression of GFAP and p-STAT3 at protein level and real-time PCR was used to detect the expression of inflammatory cytokines and chemokines at mRNA level in spinal cords. Hematoxylin-Eosin (HE) and Luxol fast blue (LFB) staining were used to observe the changes in inflammatory cell infiltration and demyelination in spinal cords. Results:Compared with the WT group, the phosphorylation of STAT3 was obviously reduced in in vitro activated mouse astrocytes of sphk2 -/- mice, and the expression of inflammatory cytokines and chemokines including monocyte chemotactic protein 1 (MCP-1), TNF-α and IL-6 at mRNA level was also significantly decreased. Compared with the WT EAE group, changes in the above-mentioned cytokines and relative proteins in sphk2 -/- EAE mice in vivo were similar to those in vitro. Moreover, inflammatory cell infiltration and demyelination were significantly reduced in spinal cords of sphk2 -/- EAE mice. However, no significant difference in in vitro or in vivo GFAP expression was observed between WT and sphk2 -/- mice. Conclusions:SphK2 might regulate the function of reactive astrocytes through STAT3 molecular pathway, thereby regulating the production of inflammatory cytokines and chemokines and participating in the pathological process of EAE.
