背景:双氢青蒿素(DHA),青蒿素的衍生物和活性代谢产物,具有各种免疫调节特性。然而,其在重症肌无力(MG)中的作用尚未明确探讨。这里,我们研究了DHA在实验性自身免疫性重症肌无力(EAMG)中的作用及其潜在机制。
方法:在Lewis大鼠中建立AChR97-116肽诱导的EAMG模型,并用DHA处理。流式细胞术用于评估Th细胞亚群和Treg细胞的释放,应用16SrRNA基因扩增子序列分析探讨DHA处理后肠道菌群变化的关系。此外,利用网络药理学和分子对接来探索DHA抗EAMG的潜在机制,通过免疫组织化学和RT-qPCR在大鼠模型中进一步验证。
结果:在这项研究中,我们证明口服DHA改善了EAMG大鼠模型的临床症状,降低Th1和Th17细胞的表达水平,并增加Treg细胞的表达水平。此外,16SrRNA基因扩增子序列分析表明,DHA通过降低Ruminococcus的丰度和增加梭菌的丰度来恢复EAMG大鼠的肠道菌群失调,双歧杆菌,和Allobaculum.使用网络药理学,确定了103个与MG相关的DHA潜在靶标,和京都基因和基因组百科全书(KEGG)分析表明,PI3K-AKT信号通路与DHA对EAMG的治疗有关。同时,分子对接验证了DHA对AKT1、CASP3、EGFR、IGF1免疫组化染色显示DHA处理显著抑制EAMG大鼠脾组织中AKT和PI3K的磷酸化表达。在EAMG大鼠中,RT-qPCR结果还显示DHA降低了PI3K和AKT1的mRNA表达水平。
结论:DHA通过抑制PI3K-AKT信号通路改善EAMG,调节CD4+T细胞和调节肠道菌群,为MG的治疗提供了一种新的治疗途径。
BACKGROUND: Dihydroartemisinin (DHA), a derivative and active metabolite of artemisinin, possesses various immunomodulatory properties. However, its role in myasthenia gravis (MG) has not been clearly explored. Here, we investigated the role of DHA in experimental autoimmune myasthenia gravis (EAMG) and its potential mechanisms.
METHODS: The AChR97-116 peptide-induced EAMG model was established in Lewis rats and treated with DHA. Flow cytometry was used to assess the release of Th cell subsets and Treg cells, and 16S rRNA gene amplicon sequence analysis was applied to explore the relationship between the changes in the intestinal flora after DHA treatment. In addition, network pharmacology and molecular docking were utilized to explore the potential mechanism of DHA against EAMG, which was further validated in the rat model by immunohistochemical and RT-qPCR for further validation.
RESULTS: In this study, we demonstrate that oral administration of DHA ameliorated clinical symptoms in rat models of EAMG, decreased the expression level of Th1 and Th17 cells, and increased the expression level of Treg cells. In addition, 16S rRNA gene amplicon sequence analysis showed that DHA restored gut microbiota dysbiosis in EAMG rats by decreasing Ruminococcus abundance and increasing the abundance of Clostridium, Bifidobacterium, and Allobaculum. Using network pharmacology, 103 potential targets of DHA related to MG were identified, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that PI3K-AKT signaling pathway was related to the treatment of DHA on EAMG. Meanwhile, molecular docking verified that DHA has good binding affinity to AKT1, CASP3, EGFR, and IGF1. Immunohistochemical staining showed that DHA treatment significantly inhibited the phosphorylated expression of AKT and PI3K in the spleen tissues of EAMG rats. In EAMG rats, RT-qPCR results also showed that DHA reduced the mRNA expression levels of PI3K and AKT1.
CONCLUSIONS: DHA ameliorated EAMG by inhibiting the PI3K-AKT signaling pathway, regulating CD4+ T cells and modulating gut microbiota, providing a novel therapeutic approach for the treatment of MG.