免疫细胞糖酵解基因表达及相关免疫细胞在实验性自身免疫性神经炎小鼠中的变化

Expression of glycolytic genes in immune cells and changes of related immune cells in experimental autoimmune neuritis

目的探讨免疫细胞糖酵解基因表达及相关免疫细胞在实验性自身免疫性神经炎(EAN)小鼠的变化,深化EAN发病机制研究。方法将24只雄性C57BL/6小鼠(6~8周龄,18~20 g)按随机数字表法分为四组:对照组(造模过程中用PBS替换P0_(180-199)肽段,第16天处死)、EAN小鼠造模结束后第8天处死组(EAN 8 d)、EAN小鼠造模结束后第16天处死组(EAN 16 d)、EAN小鼠药物干预并予造模结束后第16天处死组[初次免疫当天开始每日腹腔注射2脱氧-D-葡萄糖(2-DG),550 mg/kg,EAN 16 d+2-DG],每组6只。对各组小鼠每日观察并记录临床症状,临床评分。观察结束时采用水合氯醛麻醉处死,收取各组小鼠血清、脾脏、坐骨神经等组织备用。坐骨神经HE染色及LFB染色观察炎性细胞浸润及脱髓鞘程度。流式细胞术检测M1型巨噬细胞比例,Th17细胞比例,Tregs细胞比例。RT-PCR检测糖酵解相关基因mTORC1、HIF1α、GLUT1、LDHA的mRNA表达水平。Western印迹检测巨噬细胞及坐骨神经组织的泛-赖氨酸乳酸化水平。结果与对照组、EAN 8 d组及EAN 16 d+2-DG组相比,EAN 16 d组M1型巨噬细胞及坐骨神经糖酵解相关基因(mTORC1、HIF1α、GLUT1、LDHA)表达明显上调(均P<0.05);与对照组、EAN 8 d组及EAN 16 d+2-DG组相比,EAN 16 d组M1型巨噬细胞(1.25±0.02)及坐骨神经乳酸化修饰蛋白pankla相对水平(1.23±0.26)表达明显增加(M1型巨噬细胞:0.12±0.10、1.07±0.12、0.42±0.07,均P<0.05;坐骨神经:0.10±0.12、0.87±0.20、0.36±0.05,均P<0.05)。四组小鼠脾脏CD4+T细胞糖酵解基因(mTORC1、HIF1α、GLUT1、LDHA)表达比较差异均无统计学意义(均P>0.05)。对照组、EAN 8 d组、EAN 16 d组及EAN 16 d+2-DG组脾脏M1型巨噬细胞比例分别为4.28±0.13、7.54±0.25、13.16±0.33、4.13±0.38,EAN 16 d组明显高于其他组(均P<0.05)。四组小鼠脾脏Th17细胞比例分别为3.78±0.03、8.24±0.55、12.30±1.34、4.83±0.01,EAN 16 d组明显高于其他组(均P<0.05)。四组小鼠脾脏Tregs细胞比例分别为:10.01±1.05、7.54±0.70、3.82±0.47、8.22±1.21,EAN 16 d组明显低于其他组(均P<0.05)。结论EAN小鼠发病过程中脾脏巨噬细胞糖酵解基因表达明显增强,CD4+T细胞糖酵解基因表达未见明显增强。脾脏M1型巨噬细胞、Th17细胞比例逐渐增加,Tregs细胞比例逐渐降低。

Objective To investigate the expression of glycolytic genes in immune cells and the changes of related immune cells in experimental autoimmune neuritis(EAN),and deepen the understanding of pathogenesis of EAN.Methods Twenty-four male C57BL/6 mice(6-8 weeks old,18-20 g)were divided into four groups according to the random number table method:control group(P0_(180-199)was replaced by PBS during modeling and mice were sacrificed on the 16th day),EAN mice were sacrificed on the 8th day after the end of modeling(EAN 8 d),EAN mice were sacrificed on the 16th day after the end of modeling(EAN 16 d),and EAN mice received drug intervention and were sacrificed on the 16th day after the end of modeling(2-DG was intraperitoneally injected since the day of the first immunization,550 mg/kg;EAN 16 d+2-DG),with 6 rats in each group.The clinical symptoms and clinical scores were observed and recorded daily.At the end of the experiment,the mice were sacrificed under chloral hydrate anesthesia,and the serum,spleen,sciatic nerve and other tissues of each group were collected.The degree of inflammatory cell infiltration and demyelination of sciatic nerve were observed by hematoxylin and eosin(HE)staining and luxol fast blue(LFB)staining.Flow cytometry was used to detect the proportion of M1 macrophages,Th17 cells and Tregs cells.The mRNA expression levels of glycolysis-related genes(mTORC1,HIF1α,GLUT1 and LDHA)were detected by RT-PCR.Western blotting was used to detect the level of pan-lysine lactate in macrophages and sciatic nerve tissue.Results The expression of glycolysis-related genes(mTORC1,HIF1α,GLUT1 and LDHA)in spleen M1 macrophages and sciatic nerve was significantly up-regulated in EAN 16 d group,compared with control,EAN 8 d and EAN 16 d+2-DG groups(all P<0.05).The relative pan-lysine lactate(pankla)expression level of spleen M1 macrophages(1.25±0.02)and sciatic nerve tissue(1.23±0.26)significantly increased in EAN 16 d group,compared with control,EAN 8 d and EAN 16 d+2-DG groups(M1 macrophages:0.12±0.10,1.07±0.12 and 0.42±0.07;sciatic nerve:0.10±0.12,0.87±0.20 and 0.36±0.05)(all P<0.05).The expression of glycolytic genes in splenic CD4+T cells showed an increasing trend,but there were no statistically significant differences among the groups,and the expression of glycolytic genes did not decrease significantly after 2-DG treatment(all P>0.05).The proportion of spleen M1 macrophages in the control group,EAN 8 d group,EAN 16 d group and EAN 16 d+2-DG group was 4.28±0.13,7.54±0.25,13.16±0.33 and 4.13±0.38 respectively,which was significantly higher in the EAN 16 d group(all P<0.05).The proportion of spleen Th17 cells in the four groups was 3.78±0.03,8.24±0.55,12.30±1.34 and 4.83±0.01,respectively,which was significantly higher in the EAN 16 d group(all P<0.05).The proportion of spleen Tregs cells in the four groups was 10.01±1.05,7.54±0.70,3.82±0.47 and 8.22±1.21,respectively,which was significantly lower in the EAN 16 d group(all P<0.05).Conclusions The expression of glycolytic genes in splenic macrophages significantly increases during EAN,but not in CD4+T cells.The proportion of M1 macrophages and Th17 cells in spleen gradually increases,while the proportion of Tregs cells gradually decreases.