银莲花素A对实验性自身免疫性脑脊髓炎小鼠自噬的调控作用及机制研究

Research about regulation and mechanism of Raddeanin A on autophagy in experimental autoimmune encephalitis mice

目的:银莲花素A(RA)对实验性自身免疫性脑脊髓炎(EAE)小鼠自噬及p38MAPK通路的影响。方法:选取60只C57BL/6小鼠随机分为空白对照组、EAE模型组及RA低、中、高剂量组,每组12只,后4组建立EAE模型。RA低、中、高剂量组分别腹腔注射RA 0.05、0.1、0.2 mg/(kg·d),连续10 d,空白对照组及EAE模型组给予等量生理盐水。观察小鼠发病、脊髓组织脱髓鞘以及LC3、p-p38MAPK、p-mTOR、p-p70S6K蛋白表达情况。结果:空白对照组小鼠未发病,EAE模型组及RA各剂量组小鼠出现不同程度发病;与EAE模型组相比,RA各剂量组小鼠发病潜伏期延长、进展期缩短、神经功能评分降低,且剂量越大作用越明显(P<0.05或P<0.01)。空白对照组小鼠脊髓组织未见明显脱髓鞘,EAE模型组髓鞘脱失最明显,RA药物干预组脱髓鞘情况较轻,且随着剂量的增加,脱髓鞘的情况逐渐减轻。与空白组相比,EAE模型组LC3表达、LC3Ⅱ/LC3Ⅰ比值下降,p-p38MAPK、p-mTOR、p-p70S6K表达上升(P<0.05或P<0.01);与EAE模型组对比,RA各剂量组LC3表达、LC3Ⅱ/LC3Ⅰ比值上升,p-p38MAPK、p-mTOR、p-p70S6K表达下降,且剂量越大作用越明显(P<0.05或P<0.01)。结论:银莲花素A能改善EAE小鼠发病情况及脱髓鞘情况,且呈剂量依赖关系。其作用机制可能是通过调控p38MAPK通路,抑制mTOR磷酸化,上调LC3Ⅱ/LC3Ⅰ比值,诱导EAE小鼠细胞自噬有关。

Objective:To investigate the effects of Raddeanin A(RA)on autophagy and p38MAPK pathway in experimental autoimmune encephalomyelitis(EAE)mice.Methods:Sixty C57BL/6 mice were randomly divided into blank control group,EAE model group and RA low,medium and high dose group(12 mice per group),and the latter 4 groups were established EAE model.Low,medium and high doses of RA were intraperitoneally injected with RA 0.05,0.1,0.2 mg/(kg·d)for 10 days.The blank control group and the EAE model group were given the same amount of normal saline.Disease condition,demyelinating,LC3,p-p38MAPK,p-mTOR,and p-p70S6K protein expression in spinal cord tissue were measured and recorded.Results:The mice in the blank control group did not suffer from the disease.The mice in EAE model group and RA dose groups showed different degrees of the disease.Compared with the EAE model group,the incubation period was prolonged,the progressive stage was shortened and the neurological function score was decreased in each dose group of RA,and the greater the dose,the more obvious the effect(P<0.05 or P<0.01).There was no obvious demyelination in the spinal cord tissue of the blank control group.The demyelination of the EAE model group was the most obvious.The demyelination of the RA drug intervention group was mild,and the demyelination was gradually reduced with the increase of the dose.Compared with the blank group,the expression of LC3 and LC3Ⅱ/LC3Ⅰin EAE model group decreased,while the expression of p-p38MAPK,p-mTOR and p-p70S6K increased(P<0.05 or P<0.01).Compared with the EAE model group,LC3 expression,LC3Ⅱ/LC3Ⅰratio increased,p-p38MAPK,p-mTOR,p-p70S6K expression decreased in each dose group,and the greater the dose,the more obvious the effect(P<0.05 or P<0.01).Conclusion:Raddeanin A can improve the incident and demyelination of EAE mice in a dose-dependent manner.Its mechanism may be related to the inhibition of mTOR phosphorylation,the up-regulation of LC3Ⅱ/LC3Ⅰratio and the induction of autophagy in EAE mice by regulating p38MAPK pathway.