竹节香附素A对实验性自身免疫性脑脊髓炎小鼠炎症的影响及其调控机制研究

Effects of Raddeanin A on inflammation and the underlying mechanisms in EAE mice

摘要:

目的 探讨竹节香附素A（RA）对实验性自身免疫性脑脊髓炎（EAE）小鼠炎症的影响及其调控机制。方法 将60只C57BL/6小鼠随机分为对照组、EAE模型组、RA低剂量组、RA中剂量组、RA高剂量组，其中EAE模型组、RA低剂量组、RA中剂量组、RA高剂量组使用MOG35-55多肽复制EAE模型，RA低剂量组、RA中剂量组、RA高剂量组进行药物干预，持续至发病高峰期。观察各组小鼠的临床症状、神经功能缺损评分，HE染色检测脊髓组织炎症细胞浸润情况，Western blotting检测脊髓组织p-NF-κB蛋白表达，酶联免疫吸附试验检测脊髓组织白细胞介素-1β（IL-1β）、IL-17表达水平，实时荧光定量聚合酶链反应检测脊髓组织IL-1β和IL-17 mRNA表达。结果 高剂量组发病潜伏期较RA低剂量组、RA中剂量组、EAE模型组长（P <0.05），RA中剂量组较RA低剂量组、EAE模型组长（P <0.05），RA低剂量组较模型组长（P <0.05）。高剂量组疾病进展期较RA低剂量组、RA中剂量组、EAE模型组短（P <0.05），RA中剂量组较RA低剂量组、EAE模型组短（P <0.05），RA低剂量组较EAE模型组短（P <0.05）。高剂量组神经功能缺损评分较RA低剂量组、RA中剂量组、EAE模型组低（P <0.05），RA中剂量组较RA低剂量组、EAE模型组低（P <0.05），RA低剂量组较EAE模型组低（P <0.05）。RA高剂量组HE染色评分较RA低剂量组、RA中剂量组和EAE模型组低（P <0.05），RA中剂量组较RA低剂量组、EAE模型组低（P <0.05），RA低剂量组较EAE模型组低（P <0.05）。对照组脊髓组织IL-1β和IL-17 mRNA相对表达量低于RA低剂量组、RA中剂量组、RA高剂量、EAE模型组（P <0.05），RA高剂量组低于RA低剂量组、RA中剂量组、EAE模型组（P <0.05），RA中剂量组低于RA低剂量组、EAE模型组（P <0.05），RA低剂量组低于模型组（P <0.05）。对照组脊髓组织IL-1β、IL-17相对表达量低于RA低剂量组、RA中剂量组、RA高剂量、EAE模型组（P <0.05），RA高剂量组低于RA低剂量组、RA中剂量组、EAE模型组（P <0.05），RA中剂量组低于RA低剂量组、EAE模型组（P <0.05），RA低剂量组低于EAE模型组（P <0.05）。对照组脊髓组织NF-κB蛋白磷酸化水平低于RA低剂量组、RA中剂量组、RA高剂量组、EAE模型组（P <0.05），RA高剂量组低于RA低剂量组、RA中剂量组和EAE模型组（P <0.05），RA中剂量组低于RA低剂量组和EAE模型组（P <0.05）；RA低剂量组低于EAE模型组（P <0.05）。结论 RA能改善EAE小鼠发病情况及脊髓组织炎症，其机制可能与通过调节NF-κB，减少炎症因子分泌有关。

Abstract:

Objective To investigate the effects of Raddeanin A (RA) on inflammation in experimental autoimmune encephalomyelitis (EAE) mice and to explore the underlying mechanisms.Methods Sixty C57BL/6 mice were randomly divided into the control group, EAE model group and low-dose, medium-dose and high-dose RA groups. Among them, mice in the EAE model group and low-dose, medium-dose and high-dose RA groups were treated with MOG35-55 polypeptide to establish EAE models, while those in the low-dose, medium-dose and high-dose RA groups were continuously subject to drug intervention until the acme of the disease. The clinical symptoms, neurological severity scores, and inflammatory cell infiltration in the spinal cord detected via HE staining were observed. The protein expression of p-NF-κB in the spinal cord was detected via Western blotting, the levels of interleukin (IL)-1β and IL-17 in the spinal cord were detected via enzyme-linked immunosorbent assay (ELISA), and the mRNA expressions of IL-1β and IL-17 in the spinal cord were detected by quantitative real-time polymerase chain reaction (qRT-PCR).Results The onset latency in the high-dose RA group was longer than that in the low-dose and medium-dose RA groups and the EAE model group (P < 0.05). Compared with the low-dose RA group and the EAE model group, the onset latency in the medium-dose RA group was longer (P < 0.05). Besides, the onset latency in the low-dose RA group was longer than that in the EAE model group (P < 0.05). The duration of disease progression in the high-dose RA group was shorter than that in the low-dose and medium-dose RA groups and the EAE model group (P < 0.05). The tduration of disease progression in the medium-dose RA group was shorter than that in the low-dose RA group and the EAE model group (P < 0.05), while that in the low-dose RA group was even shorter than that in the EAE model group (P < 0.05). The neurological severity scores in the high-dose RA group were lower than those in the low-dose and medium-dose RA groups and the EAE model group (P < 0.05), those in the medium-dose RA group were lower than those in the low-dose RA group and the EAE model group (P < 0.05), while those in the low-dose RA group were even lower than those in the EAE model group (P < 0.05). The HE staining scores in the high-dose RA group were lower than those in the low-dose and medium-dose RA groups and the EAE model group (P < 0.05), those in the medium-dose RA group were lower than those in the low-dose RA group and the EAE model group (P < 0.05), while those in the low-dose RA group were even lower than those in the EAE model group (P < 0.05). The mRNA expressions of IL-1β and IL-17 in the spinal cord in the control group were lower than the other groups (P < 0.05), those in the high-dose RA group were lower than those in the low-dose and medium-dose RA groups and the EAE model group (P < 0.05), those in the medium-dose RA group were lower than those in the low-dose RA group and the EAE model group (P < 0.05), while those in the low-dose RA group were even lower than those in the EAE model group (P < 0.05). Consistently, the levels of IL-1β and IL-17 in the spinal cord as determined via ELISA exhibited parallel differences among the groups (P < 0.05). As for the level of p-NF-κB, that in the control group was lower than the other groups (P < 0.05), that in the high-dose RA group was lower than that in the low-dose and medium-dose RA groups and the EAE model group (P < 0.05), that in the medium-dose RA group was lower than that in the low-dose RA group and the EAE model group (P < 0.05), while that in the low-dose RA group was even lower than that in the EAE model group (P < 0.05).Conclusions Raddeanin A may slow the disease onset and progression and alleviate the inflammation in the spinal cord of EAE mice, which may be achieved via regulating NF-κ B to reduce the secretion of inflammatory factors.