摘要
目的探讨甘露特钠对实验性自身免疫性脑脊髓炎(EAE)小鼠发病的治疗作用及对肠道菌群、小胶质细胞极化的影响。方法将50只雌性C57BL/6小鼠用随机数字表法分为对照组、EAE模型组及甘露特钠低、中、高剂量组,每组各10只。EAE模型组、甘露特钠各剂量组采用MOG35-55多肽皮下多点注射制备EAE模型。甘露特钠低、中、高剂量组从造模次日起分别用甘露特钠40、80、160 mg/kg,2次/d,灌胃进行干预,持续至小鼠处死。观察各组小鼠发病情况、脊髓组织炎症细胞浸润及脱髓鞘情况。收集小鼠粪便,通过16S rRNA测序进行肠道菌群检测,免疫荧光染色检测脊髓组织中小胶质细胞激活指标Iba-1蛋白表达情况,Western印迹法和免疫荧光染色法检测脊髓组织中小胶质细胞M1型标志物iNOS、CD16和M2型标志物Arg1、CD206的蛋白表达水平。结果对照组小鼠均未发病,其余各组小鼠均有不同程度发病,表现为尾部下垂、步态不稳、后肢无力等。与EAE模型组比较,甘露特钠各剂量组小鼠发病潜伏期延长、高峰期延迟、高峰期神经功能障碍评分降低[(3.6±0.6)分比(3.0±0.6)分、(2.8±0.5)分、(1.8±0.6)分,均P<0.05],且干预剂量越大症状越轻,组间两两比较差异均有统计学意义(均P<0.05)。对照组小鼠脊髓组织无炎性细胞浸润及脱髓鞘变化;EAE模型组发病高峰期脊髓组织炎性细胞浸润及脱髓鞘变化明显;与EAE模型组比较,甘露特钠各剂量组发病高峰期脊髓组织炎症细胞浸润及脱髓鞘变化减轻。与对照组比较,EAE模型组拟杆菌门相对丰度较低,厚壁菌门相对丰度较高;与EAE模型组比较,甘露特钠各剂量组拟杆菌门相对丰度较高,厚壁菌门相对丰度较低,拟杆菌门与厚壁菌门比值增加(0.20±0.05比0.37±0.02、0.61±0.03、0.91±0.08,均P<0.01),且干预剂量越大变化越明显,组间两两比较差异均有统计学意义(P<0.01)。与对照组比较,EAE模型组Iba-1、CD16、iNOS水平增加,Arg-1、CD206水平降低;与EAE模型组比较,甘露特钠各剂量组Iba-1、CD16、iNOS水平降低,Arg-1、CD206水平增加(均P<0.01),且剂量越大变化越明显,组间两两比较各指标差异均有统计学意义(均P<0.01)。结论甘露特钠对EAE具有治疗作用,且呈剂量依赖性;其作用机制可能与改善肠道微生态、调控小胶质细胞极化有关。
Objective To investigate the therapeutic effect of sodium oligomannate on experimental autoimmune encephalomyelitis(EAE)mice and its effect on intestinal flora and microglia polarization.Methods Fifty female C57BL/6 mice were randomly divided by the random number table method into the control group,EAE model group and low-dose,medium-dose and high-dose group of sodium oligomannate with 10 mice each.The EAE model group and each dose group of sodium oligomannate were induced by subcutaneous multi-point injection of MOG35-55 peptide for the EAE model.Mice in the low-dose,medium-dose and high-dose group of sodium oligomannate were gavaged sodium oligomannate 40,80,and 160 mg/kg twice a day,respectively,starting from the day after modeling.The intervention continued until the mice were euthanized.Observe the incidence of disease,infiltration of inflammatory cells in spinal cord tissue,and demyelination in each group of mice..The mice feces were collected and tested for intestinal flora by 16S rRNA sequencing.Immunofluorescence staining was used to observe the expression of Iba-1 protein,an activation indicator of microglia,in spinal cord tissue.The protein levels of M1 type markers iNOS,CD16,and M2 type markers Arg1 and CD206 were tsested in the spinal cord by Western blotting and immunofluorescence staining.Results None of the mice in the control group developed any disease,while the mice in other groups showed varying degrees of disease,including tail sag,unstable walking,and hind limb weakness.Compared with the EAE model group,the incubation period was prolonged,the peak was delayed and the peak neurological dysfunction score was reduced(3.6±0.6 vs 3.0±0.6,2.8±0.5,1.8±0.6,P<0.05)in all sodium oligomannate groups,with milder symptoms at higher doses.The differences in pairwise comparisons between the groups were all statistically significant(all P<0.05).In the control group,no inflammatory cell infiltration or demyelinating changes were observed in spinal cord tissue.In the EAE model group,inflammatory cell infiltration and demyelination changes were evident in the spinal cord tissues at the onset peak.Compared with the EAE model group,inflammatory cell infiltration and demyelination were ameliorated in all sodium oligomannate groups.Compared with the control group,the relative abundance of Bacteroidota decreased and that of Firmicutes increased in the EAE model group.Compared with the EAE model group,the relative abundance of Bacteroidota increased and that of Firmicutes decreased,the ratio of Bacteroidetes to Firmicutes increased(0.20±0.05 vs 0.37±0.02,0.61±0.03,0.91±0.08,P<0.01)in the respective dose groups.The difference in pairwise comparison between groups was statistically significant(P<0.01),with greater changes at higher doses.Compared with the control group,the levels of Iba-1、CD16 and iNOS increased,while the levels of Arg-1 and CD206 decreased in the EAE model group.Compared with the EAE model group,the levels of Iba-1、CD16 and iNOS decreased,while the levels of Arg-1 and CD206 increased in all sodium oligomannate groups(P<0.01),with greater changes at higher doses.The difference between groups was statistically significant(P<0.01).Conclusions Sodium oligomannate has a therapeutic effect on EAE and is dose-dependent.Its mechanism of action may be related toimproving intestinal microecology and the modulation of microglial polarization.
作者
赵怡琳
李作孝
Zhao Yilin;Li Zuoxiao(Department of Neurology,the Affiliated Hospital of Southwest Medical University,Luzhou 646000,China)
出处
《中华医学杂志》
CAS
CSCD
北大核心
2024年第17期1521-1528,共8页
National Medical Journal of China
基金
泸州市人民政府-西南医科大学科技战略合作基金(2018LZXNYD-ZK17)
作者简介
通信作者:李作孝,Email:lzx3235@sina.com
