College of Basic Medicine, Zhejiang Chinese Medical University, Hangzhou 310053, China
背景：T细胞异常增殖在系统性红斑狼疮（SLE）的发病机制中起重要作用。白花蛇舌草(HDW)治疗SLE的药物活性已经被证实。然而，其中的生物医学机制仍不清楚。目的：本研究旨在评估HDW乙酸乙酯部位(EAHDW)对狼疮小鼠的治疗效果，并探讨其潜在的治疗机制。方法：EAHDW由80%乙醇回流提取后经过分级萃取得到，并用HPLC和UPLC-Q/TOP-MS进行了成分分析。利用网络药理学预测了其潜在靶点和STAT3亲和调节分子。研究中采用MRL/lpr小鼠模型评价了EAHDW的药物活性，通过ELISA 方法定量分析细胞因子和自身抗体浓度。肾小球的病理损伤和肾脏中STAT3的表达分别通过组织化学和免疫组化技术检测。细胞周期的特征用流式细胞术分析。蛋白质印迹和双荧光素酶报告基因检测分别被用于评估STAT3的翻译和转录活性。结果：采用本研究的方法制备EAHDW的提取率为2.7±1%，鉴定出了其中19种活性成分。网络药理学预测EAHDW可以改善SLE发病时T细胞的异常增殖。EAHDW有益于改善MRL/lpr狼疮小鼠肾小球的病理变化和STAT3表达，以及血清中相关细胞因子和自身抗体的水平。在细胞学研究中发现，EAHDW处理削弱了STAT3的转录和磷酸化，通过延长细胞周期S期抑制了T细胞增殖。EAHDW中的5个化合物均对STAT3的DNA结合位点表现出很强的对接亲和力。结论：EAHDW可以减轻炎症反应，并通过STAT3信号通路抑制T 细胞增殖对SLE起到了治疗作用。
Background: Abnormal proliferation of T cells plays an essential role in the pathogenesis of Systemic lupus erythematosus (SLE). The pharmaceutical effect of Hedyotis Diffusa Willd (HDW) on SLE has been investigated previously. Nevertheless, the biomedical mechanism is still left unclear. Objective: This study has been arranged to evaluate the therapeutic effect of the ethyl acetate fraction of HDW (EAHDW) on lupus mice and explore the potential therapeutic mechanism.Methods: EAHDW was prepared with 80% ethanol reflex extraction followed by successive extraction, and analyzed with HPLC and UPLC-Q/TOP-MS. The potential targets and STAT3 affinity regulators were predicted with network pharmacology. The pharmaceutic effect of EAHDW was studied with MRL/lpr mice. Cytokines and autoantibodies were quantified with ELISA assays. The pathological damage of glomerulus and STAT3 expression in the kidney was detected with histochemical and immunohistochemical techniques. The cell cycle properties in cell proliferation were identified with the flow cytometry. The western blot and dual-Luciferase reporter assay were applied to evaluate translational and transcriptional activity of STAT3,respectively. Results: In this study, the extraction ratio of EAHDW was 2.7±1%, in which 19 ingredients were identified.Network pharmacological analysis showed that the target genes of EAHDW were highly focused on influencing the abnormal T cell proliferation in SLE. EAHDW showed the beneficial effects on pathological changes and STAT3 expression in the glomerulus of lupus mice, and the levels of cytokines and autoantibodies in serum. In cytological study, EAHDW treatment attenuated the transcription and phosphorylation of STAT3, which inhibited T cell proliferation by prolonged S-phase of the cell cycle. A total of 5 compounds in EAHDW exhibited high docking affinity to the DNA-binding site of STAT3. Conclusion: EAHDW could reduce the inflammatory response and inhibit the proliferation of T cells by interfering with the STAT3 signaling pathway, thereby playing a therapeutic effect on SLE.