School of Traditional Chinese Medicine, Capital Medical University, Beijing, 100069, China.
School of Traditional Chinese Medicine, Capital Medical University, Beijing, 100069, China.
J Ethnopharmacol. 2023 Oct 28;315:116681. doi: 10.1016/j.jep.2023.116681. Epub 2023 May 23.
Rhizomes of Panax japonicus (RPJ), a traditional herbal medicine, was used for treating arthritis and physical weakness in China from the Ming dynasty. Triterpene saponins are the main bioactive components of RPJ. In this work, for the first time, we evaluate the therapeutic effect of the total saponin from RPJ (TSPJ) on experimental autoimmune encephalomyelitis (EAE) mice induced by myelin oligodendrocyte glycoprotein (MOG) , a commonly used animal model of Multiple sclerosis (MS).
To evaluate the therapeutic effect of TSPJ on EAE and explored its possible underlying mechanisms.
EAE was induced by MOG . Mice were administrated with TSPJ (36.5 mg/kg, 73 mg/kg) and prednisone acetate (positive control) orally once daily up to 28 days postimmunization, and their neurological deficit was scored. Hematoxylin and Eosin (HE), Luxol Fast Blue (LFB), and transmission electron microscopy (TEM) were carried out to evaluate the EAE-induced pathological changes in the brain and spinal cord. IL-17a and Foxp3 levels in central nervous system (CNS)were evaluated by immunohistochemical staining. The changes in IL-1β, IL-6, and TNF-α levels in serum and CNS were measured with ELISA. Quantitative reverse transcription PCR (qRT-PCR) was used to access mRNA expression in CNS of the above indices. The percentages of Th1, Th2, Th17and Treg cells in spleen were determined by Flow Cytometry (FCM). Furthermore, 16S rDNA sequencing was used to detect the intestinal flora of mice in each group. In vitro studies, lipopolysaccharides (LPS)-induced BV2 microglia cells were used and the expression of TLR4, MyD88, p65, and p-p65 in cells was detected by Western blot.
TSPJ treatment significantly alleviated neurological impairment caused by EAE. Histological examination confirmed the protective effects of TSPJ on myelin sheath and the reduction of inflammatory cell infiltration in the brain and spinal cord of EAE mice. TSPJ notably downregulated the ratio of IL-17a/Foxp3 at protein and mRNA levels in CNS, as well as Th17/Treg and Th1/Th2 cell ratios in the spleen of EAE mice. The levels of TNF-α, IL-6, and IL-1β in CNS and peripheral serum also decreased post-TSPJ treatment. In vitro, TSPJ suppressed LPS-induced production of inflammatory factors in BV2 cells via TLR4-MyD88-NF-κB signaling pathway. More importantly, TSPJ interventions altered the composition of gut microbiota and restored the ratio of Firmicutes to Bacteroidetes in EAE mice. Furthermore, Spearman's correlation analysis revealed that a relationship existed between statistically significantly altered genera and CNS inflammatory indices.
Our results demonstrated TSPJ had therapeutic effects on EAE. Its anti-neuroinflammation property in EAE was related to modulating gut microbiota and inhibiting TLR4-MyD88-NF-κB signaling pathway. Our study indicated that TSPJ may be a potential candidate for the treatment of MS.
人参（RPJ）的根茎是一种传统草药，自明朝以来，在中国一直被用于治疗关节炎和身体虚弱。三萜皂苷是 RPJ 的主要生物活性成分。在这项工作中，我们首次评估了 RPJ 总皂苷（TSPJ）对髓鞘少突胶质细胞糖蛋白（MOG）诱导的实验性自身免疫性脑脊髓炎（EAE）小鼠的治疗作用，MOG 是多发性硬化症（MS）的常用动物模型。
评估 TSPJ 对 EAE 的治疗效果，并探讨其可能的潜在机制。
通过 MOG 诱导 EAE。用 TSPJ（36.5mg/kg、73mg/kg）和醋酸泼尼松龙（阳性对照）每天口服一次，共 28 天，直至免疫后，对其神经功能缺损进行评分。用苏木精和伊红（HE）、卢索快速蓝（LFB）和透射电镜（TEM）评估大脑和脊髓中 EAE 引起的病理变化。用免疫组织化学染色评估中枢神经系统（CNS）中 IL-17a 和 Foxp3 水平。用 ELISA 测量血清和 CNS 中 IL-1β、IL-6 和 TNF-α 水平的变化。用定量逆转录 PCR（qRT-PCR）检测 CNS 中上述指标的 mRNA 表达。用流式细胞术（FCM）测定脾脏中 Th1、Th2、Th17 和 Treg 细胞的百分比。此外，用 16S rDNA 测序检测各组小鼠的肠道菌群。在体外研究中，用脂多糖（LPS）诱导 BV2 小胶质细胞，用 Western blot 检测细胞中 TLR4、MyD88、p65 和 p-p65 的表达。
TSPJ 治疗显著减轻了 EAE 引起的神经损伤。组织学检查证实 TSPJ 对 EAE 小鼠的髓鞘有保护作用，并减少了大脑和脊髓中炎性细胞的浸润。TSPJ 显著下调了 CNS 中 IL-17a/Foxp3 的蛋白和 mRNA 水平，以及 EAE 小鼠脾脏中 Th17/Treg 和 Th1/Th2 细胞的比值。TSPJ 治疗后，CNS 和外周血清中的 TNF-α、IL-6 和 IL-1β 水平也降低。在体外，TSPJ 通过 TLR4-MyD88-NF-κB 信号通路抑制 LPS 诱导的 BV2 细胞炎症因子的产生。更重要的是，TSPJ 干预改变了肠道微生物群的组成，并恢复了 EAE 小鼠中厚壁菌门与拟杆菌门的比例。此外，Spearman 相关性分析显示，统计上显著改变的属与 CNS 炎症指标之间存在相关性。
我们的结果表明 TSPJ 对 EAE 具有治疗作用。其在 EAE 中的抗炎作用与调节肠道微生物群和抑制 TLR4-MyD88-NF-κB 信号通路有关。我们的研究表明，TSPJ 可能是治疗 MS 的潜在候选药物。
