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目的探讨β-榄香烯对实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)小鼠早期炎性损伤的治疗作用及可能的免疫学机制。方法 6～8周龄健康雌性野生型C57BL/6小鼠(SPF15)共64只,均分为对照组、模型组和β-榄香烯治疗组。采用髓鞘少突胶质细胞糖蛋白(myelinoligodendrocyte glycoprotein peptide35-55,MOG35-55)多肽作为抗原诱发EAE小鼠模型。于免疫后15日剥离脊髓组织进行病理学研究,MTT实验检测EAE小鼠MOG抗原特异性增殖反应及β-榄香烯(β-elemene)对MOG抗原特异性增殖反应的影响,定量PCR技术分别检测各组小鼠脊髓及淋巴结细胞白细胞介素17(interleukin-17,IL-17)、转录因子孤儿核受体(retinoid acid-related orphan receptors-γt,RORγt)、白细胞介素6(interleukin-6,IL-6)、白细胞介素23(interleukin-23,IL-23)、转化生长因子β(transforming growth factor-β,TGF-β)以及叉头蛋白3(Foxp3)的mRNA的表达。结果模型组的神经功能评分高于治疗组(<0.05),治疗组小鼠脊髓炎症及轴索损伤较模型组减轻。免疫后11d,与模型组比较,治疗组小鼠脊髓IL-17、RORγt、IL-6、IL-23的mRNA的表达减少(<0.05),Foxp3mRNA的表达增加(<0.05),并持续至免疫后15d。体外研究发现模型组T淋巴细胞增殖较对照组更明显(<0.05),治疗组β-榄香烯对T淋巴细胞增殖抑制呈现明显的量效关系(<0.05);与模型组比较,治疗组体外培养的淋巴细胞IL-17、RORγt、IL-6、IL-23mRNA表达水平增加,Foxp3mRNA表达水平减少(<0.05)。结论β-榄香烯减轻EAE小鼠早期炎性损伤,可能与其抑制髓鞘少突胶质细胞糖蛋白多肽反应性T细胞的激活以及维持Th17细胞/调节T细胞亚群的平衡密切相关。
Objective To investigate the therapeutic effect and possible immunological mechanism of β-elemene on early inflammatory injury in experimental autoimmune encephalomyelitis (EAE) mice. Methods Sixty-four healthy female wild-type C57BL / 6 mice (SPF15) aged 6-8 weeks were divided into control group, model group and β-elemene treatment group. EAE mouse model was induced by using myelinoligodendrocyte glycoprotein peptide35-55 (MOG35-55) polypeptide as antigen. Spinal cord tissue was dissected 15 days after immunization for pathological study. MTT assay was used to detect MOG antigen-specific proliferative response and β-elemene-specific MOG antigen-specific proliferative response in EAE mice. Quantitative PCR The levels of interleukin-17 (IL-17), RORγt, interleukin-6 (IL-6) , Interleukin-23 (IL-23), transforming growth factor-β (TGF-β) and forkp-3 (Foxp3) were detected by ELISA. Results The neurological score of the model group was higher than that of the treatment group (<0.05). The inflammation and axonal injury of the spinal cord in the treatment group were relieved compared with the model group. On the 11th day after immunization, the mRNA expression of IL-17, RORγt, IL-6 and IL-23 decreased (P <0.05) and the expression of Foxp3mRNA increased (<0.05) After 15d. In vitro, it was found that T-lymphocyte proliferation in model group was more obvious than that in control group (<0.05), while β-elemene in treatment group showed a significant dose-response relationship with inhibition of T lymphocyte proliferation (P <0.05) The mRNA expression of IL-17, RORγt, IL-6 and IL-23 in lymphocytes cultured in vitro increased and the expression of Foxp3 mRNA decreased (P <0.05). Conclusion β-elemene attenuates the early inflammatory injury in EAE mice, which may be related to the inhibition of the activation of reactive myeloid oligodendrocyte glycoprotein reactive T cells and the maintenance of Th17 / regulatory T cell subsets.