LncRNA Gm13568调控A1型星形胶质细胞活化及在EAE小鼠病变中的作用

LncRNA Gm13568 regulates the activation of A1 astrocytes and affects the EAE process in mice

目的探讨长链非编码RNA(long non-coding RNAs,lncRNA)Gm13568对A1型星形胶质细胞活化及实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)小鼠疾病进程的影响与分子机制。方法构建特异性靶向星形胶质细胞的lncRNA Gm13568沉默与对照的重组慢病毒载体,分别命名为LV-Inhibit-Gm13568和LV-ctrl,并包装病毒。1×107 TU病毒悬液静脉注射入C57BL/6小鼠体内7 d后,髓鞘少突胶质细胞糖蛋白35-55(myelin oligodendrocyte glycoprotein 35-55,MOG35-55)多肽诱导EAE模型。实验分为PBS组、EAE组、LV-ctrl+EAE组和LV-Inhibit-Gm13568+EAE组,采用双盲法每日对小鼠进行神经功能评分。EAE小鼠诱导后23 d,取各组小鼠脊髓组织,利用real-time PCR测定A1型星形胶质细胞活化指标Serping 1、C3、Srgn和H2-T23的mRNA转录水平,同时测定IL-6、TNF-α、巨噬细胞趋化蛋白-1(macrophage chemotactic protein-1,MCP-1)和干扰素诱导蛋白-10(interferon-inducible protein-10,IP-10)的生成变化;Western blot检测脊髓组织胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)和Notch1的表达及信号转导与转录激活子3(signal transducer and activator of transcription 3,STAT3)的磷酸化水平(phosphorylated STAT3,p-STAT3);免疫荧光法检测各组脊髓组织中Notch1活化后的胞内段蛋白(Notch1 intracellular domain,NICD)与GFAP的共表达;HE和劳克坚牢蓝染色(Luxol fast blue,LFB)分别观察脊髓组织内炎症细胞浸润及髓鞘脱失变化。结果与PBS组比较,EAE组和LV-ctrl+EAE组小鼠A1型星形胶质细胞活化增生,Notch1的表达显著上调。沉默内源性lncRNA Gm13568后,与LV-ctrl+EAE组比较,小鼠的神经功能评分在抗原诱导后23 d从平均3.5分下降至1分以下,临床症状缓解,A1型星形胶质细胞活化降低,同时炎性因子及趋化因子生成减少;Notch1、GFAP、NICD的蛋白质表达水平和STAT3的磷酸化水平显著降低;小鼠脊髓组织炎性细胞浸润与髓鞘脱失显著减轻。结论LncRNA Gm13568可能通过调控Notch1/STAT3信号调控A1型星形胶质细胞的活化,从而调节炎性因子及趋化因子的产生,参与EAE的病变进程。

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 1×107 transforming units of viral suspension via the tail vein and 7 d after the injection,myelin oligodendrocyte glycoprotein 35-55(MOG35-55)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.