黄芩素对实验性自身免疫性脑脊髓炎小鼠的 影响及机制研究

目的 探讨黄芩素（BAI）对实验性自身免疫性脑脊髓炎（EAE）小鼠的防治作用及其对中枢神经系统小胶 质细胞（MG）极化和炎性因子的影响。方法 将50只雌性C57BL/6小鼠按照随机数字表法分为空白对照组，EAE模 型组及BAI低、中、高剂量组，每组10只。空白对照组不做处理，其余各组建立EAE模型。BAI低、中、高剂量组每日 分别给予BAI 75、150、300 mg/kg灌胃，空白对照组及EAE模型组给予等体积生理盐水灌胃，连续14 d。观察小鼠的 神经功能缺损和脊髓组织炎性脱髓鞘情况。采用免疫荧光双重染色法检测小鼠脊髓组织离子钙接头蛋白1（Iba-1） 阳性MG中M1型MG标志物诱导型一氧化氮合酶（iNOS）、M2型MG标志物精氨酸酶1（Arg1）的表达情况及分歧指数 （RI）变化，实时荧光定量PCR法检测小鼠脊髓组织中iNOS、Arg1及炎性因子肿瘤坏死因子-α（TNF-α）、白细胞介 素-10（IL-10）mRNA的表达情况。结果 空白对照组小鼠未见发病，其余各组均不同程度发病。与EAE模型组比 较，BAI各剂量组小鼠发病潜伏期及达高峰期时间延长，神经功能障碍评分降低（均P＜0.05），脊髓组织脱髓鞘程度 减轻（均P＜0.05），Iba-1阳性MG中iNOS表达降低，Arg1表达升高，RI值减小（均P＜0.05），MG的形态倾向于M2型 极化，脊髓组织中iNOS、TNF-α mRNA表达降低，Arg1、IL-10 mRNA表达升高（均P＜0.05），且BAI剂量越大，变化越 明显。结论 BAI对EAE小鼠发病具有防治作用，且呈剂量依赖性，其机制可能与纠正M1/M2型MG失衡及调节炎 性因子TNF-α、IL-10表达有关。

The effect and mechanism of baicalein on experimental autoimmune encephalomyelitis in mice

Objective To investigate the preventive and therapeutic effects of baicalein (BAI) on experimental autoimmune encephalomyelitis (EAE) in mice and its effects on polarization and inflammatory factors of microglia in central nervous system. Methods Fifty female C57BL/6 mice were randomly divided into the blank control group, the EAE model group and the BAI low, medium and high dose groups, with 10 mice in each group. The blank control group was not treated, and EAE model was established in the other groups. BAI low, medium and high dose groups were given BAI (75, 150 and 300 mg·kg-1·d -1) by gavage respectively, while the blank control group and the EAE model group were given the same volume of normal saline by gavage for 14 consecutive days. The neurological deficit and inflammatory demyelination of spinal cord were observed. The immunofluorescence double staining was used to detect the expression of inducible nitric oxide synthase (iNOS) in M1 type microglia, arginase 1 (Arg1) in M2 type microglia and divergence index (RI) in Iba-1 positive microglia of spinal cord of mice. The real time fluorescent quantitative RT-PCR was used to detect iNOS, Arg1 and mRNA of TNF-α, IL-10 expression of spinal cord of mice. Results No disease was found in the blank control group, but the other groups all had the disease in varying degrees. Compared with the EAE model group, the onset latency and peak time were prolonged (P＜0.05), the peak neurological dysfunction score was decreased (P＜0.05), the demyelination degree of spinal cord was reduced, the expression level of iNOS in Iba-1 positive microglia was decreased (P＜0.05), the expression of Arg1 was increased (P＜0.05) and the RI value was decreased in the BAI intervention groups (P＜0.05). The morphology of microglia tended to be M2 polarization. The mRNA expressions of iNOS and TNF-α of spinal cord decreased (P＜0.05), but the mRNA expression of Arg1 and IL-10 increased (P＜0.05). The higher the dose of BAI, the more obvious the effect. Conclusion BAI can prevent and treat EAE in a dose-dependent manner in mice. Its mechanism may be related to the correction of M1/M2 microglia cell imbalance and regulation of the inflammatory factors TNF-α and IL-10 expression.