苦参素靶向调节IRF7/IFN-β信号通路抑制中枢神经自体免疫性脱髓鞘疾病的机制研究

The pathogenic mechanism of central nervous system (CNS) autoimmune demyelinating diseases, including multiple sclerosis and optic neuritis, is not well addressed, and their treatments, such as IFN-β, are highly costly, thus limiting their applications. We have recently found that matrine suppresses experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, and inhibits central and peripheral inflammation, which promoted high production of IFN-β and enhanced phosphorylation of interferon-regulated factor-7 (IRF-7), a key regulatory molecule for IFN-β induction. Based on previous results, we hypothesize that matrine suppresses CNS autoimmunity via inducing IFN-β induction. To verify this hypothesis, we will establish and treat chronic EAE mouse models and experimental autoimmune optic neuritis (EON), and test whether the effect of matrine on these models can be blocked by neutralizing antibodies against IFN-β and/or its receptors. We will also cultivate macrophages/microglia, the major IFN-β-producing cells, using techniques such as Western blot, immunohistochemical, flow cytometry and electron microscopy, to define matrine effect and mechanism of its action, focusing on the IRF-7/IFN-β pathway, as well their downstream molecules IL-27 in macrophages/microglia and IL-10 in regulatory T cells. We will further compare/contrast the clinical effects of matrine and IFN-β on multiple EAE and EON models. Together, these studies will define the effect, target spots, and the mechanism of matrine therapy in CNS autoimmunity, and provide a novel, safe, low-cost, and effective therapy to replace IFN-β for CNS inflammatory demyelinating diseases.

中枢神经系统自体免疫性脱髓鞘疾病，包括多发性硬化(MS)、视神经炎等发病机制不清，治疗此类疾病的一线药物如β干扰素(IFN-β)价格高昂，很难推广应用。我们发现苦参素能降低MS动物模型EAE小鼠病情程度，抑制中枢和外周炎症，但其机制还不明确。我们新发现苦参素能促进IFN-β的产生及其上游分子IRF7的磷酸化，推测苦参素通过靶向IRF7诱导内源性IFN-β，实现免疫调节功能。本项目拟结合体内外实验明确苦参素对小胶质细胞/巨噬细胞的免疫调节、IFN-β诱导及抗原呈递功能的调控作用；利用IRF7-siRNA等模型，明确苦参素依赖IRF7促进IFN-β/IL-27产生，介导免疫调节性T细胞及IL-10产生的调节通路；系统评估苦参素和IFN-β对多个EAE模型及自体免疫性视神经炎(EON)的治疗效果的一致性。将阐明苦参素对免疫调节过程的调控机制和分子基础，并提供新的安全、经济的IFN-β替代药物。

多发性硬化症(MS)是一种慢性神经炎性脱髓鞘疾病，由免疫细胞渗入中枢神经系统(CNS)引起，主要的病理特征是血脑屏障破坏、少突胶质细胞丢失、髓鞘损伤、星形胶质细胞胶质增生和轴突变性。我们诱导Wistar大鼠和C57BL/6小鼠建立实验性自身免疫性（EAE）模型，根据处理条件不同对实验动物进行分组，在疾病高峰期取其脑组织和脊髓，分别用HE染色、LFB染色，检测并评估组织炎症和脱髓鞘情况。免疫荧光单标染色和免疫荧光双染色检测神经细胞、少突胶质细胞和星形胶质细胞中相关基因的表达变化，例如Cx32，Cx43，Cyt c，Beclin1等；Western blot、qRT-PCR检测脑和脊髓组织中的蛋白质和mRNA的表达变化，例如caspase-3、HSPB5、IL-1β、SPHK1和SPHK2 mRNA等；比色法检测EAE大鼠脑中MDA含量和GSH-Px活性。结果显示，与对照组比较，MAT治疗组小鼠/大鼠临床症状减轻，体重减轻较少，MAT能显著抑制EAE小鼠/大鼠脊髓的炎性浸润及CNS的脱髓鞘。脑组织中星形胶质细胞增生情况的western blot与免疫荧光染色结果显示，经过苦参素处理的动物模型脑内GFAP蛋白表达量降低且荧光强度与未处理组相比显著降低，证明了苦参素可以有效的调节星形胶质细胞的激活，抑制星形胶质细胞的异常增生。对小胶质细胞和巨噬细胞进行免疫荧光双标分析得出，CD11b+细胞是干扰素-β的主要产生来源，我们以前的研究表明MAT诱导外周和中枢神经系统产生干扰素-β，因此我们推断MAT可以诱导CD11b+细胞产生干扰素-β。IL-10是一种可以抑制T细胞增殖和Th1/Th17细胞分化的抗炎细胞因子，IL-27在感染和自身免疫中都有很强的抗炎作用，MAT处理的小鼠体内IL-β、IL-27和IL-10水平有明显升高，证明MAT通过干扰素-β/IL-27/IL-10途径在EAE中发挥治疗作用。综上，数据从多方面提示了苦参素CNS炎症中的治疗机制，因此，MAT治疗为多发性硬化症提供了一种新的治疗方法，有希望提供新的、安全、经济的干扰素-β替代药物。