Department of Postgraduate, Jiangxi University of Chinese Medicine, Nanchang 330004, Jiangxi Province, China.
Laboratory Animal Research Center for Science and Technology, Jiangxi University of Chinese Medicine, Nanchang 330004, Jiangxi Province, China.
Phytomedicine. 2024 Dec;135:156035. doi: 10.1016/j.phymed.2024.156035. Epub 2024 Sep 19.
Ulcerative colitis (UC) is an autoimmune disease that is highly susceptible to recurrence, which is still a lack of effective drugs with minor side effects in clinic. Intervention of inflammatory differentiation of dendritic cells (DCs) might be an effective strategy to treat UC. Sishen Pills (SSP) is a classic Chinese herbal formula which has been demonstrated the protective effect of UC, but the mechanism remains unclear.
To elucidate the protective effects of SSP against UC in mice and reveal its regulatory mechanism of DCs and the key active ingredients for the UC treatment based on transcriptomics, network pharmacology and experiments validation in vivo and vitro.
The key active ingredients of SSP were detected and screened integrating LC-MS/MS and network pharmacology. A mouse UC model was induced with 3% sodium dextran sulfate and treated with SSP for 14 days to evaluate the efficacy. ELISA was used to detect the levels of IL-6, IL-1β and TNF-α in the colon; flow cytometry was used to detect the expression levels of DCs and their subpopulations; whole transcriptomic sequencing of differential RNAs in the colon and RT-PCR to detect key miRNAs to verify the sequencing results. Mouse bone marrow-derived dendritic cells (BMDCs) were isolated, an inflammatory model was constructed using 100 ng/ml LPS, and the effects of SSP on DC proliferation and apoptosis and their surface co-stimulatory molecule expression were examined; IL-6, IL-1β, TNF-α levels were measured by ELISA; RT-PCR and WB were performed to detect miR-505-3p, CDH1, E-cadherin expression. BMDCs with low expression of miR-505-3p were constructed by lentiviral transfection for further validation. The potential key ingredient was re-validated in vivo and vitro experiment.
Animal experiments showed that SSP alleviated DSS-induced UC symptoms and colonic pathological injury in mice, and inhibited IL-6, IL-1β, TNF-α secretion and inflammatory DC proliferation and activation maturation. Network pharmacology predicted that evodiamine, isobavachalcone, curcumin, and engenol may play a key role in SSP. RNA sequencing revealed that miR-505-3p, as the differential miRNA, shared a large number of transcription factors with E-cadherin, and was involved in inflammatory differentiation regulation. In vivo experiments confirmed that SSP accelerated apoptosis, slowed down proliferation, inhibited inflammatory differentiation and IL-6, IL-1β, and TNF-α secretion in BMDCs, and decreased miR-505-3p, CDH1, and E-cadherin levels. After knocking down miR-505-3p, SSP could not regulate the inflammatory differentiation and IL-6, IL-1β, TNF-α level in BMDCs. Additionally, evodiamine was found and verified to be the key active ingredient of SSP in preventing the inflammatory differatiation of DCs.
SSP prevented the inflammatory differentiation of DCs by downregulating the expression of miR-505-3p, in which Evodiamine may played a key role.
溃疡性结肠炎（UC）是一种易复发的自身免疫性疾病，临床上仍缺乏副作用小的有效药物。树突状细胞（DC）炎症分化的干预可能是治疗 UC 的有效策略。四神丸（SSP）是一种经典的中药方剂，已被证明对 UC 具有保护作用，但机制尚不清楚。
阐明 SSP 对 UC 小鼠的保护作用，并通过转录组学、网络药理学和体内外实验验证，揭示其对 DCs 的调节机制及 UC 治疗的关键活性成分。
采用 LC-MS/MS 结合网络药理学检测 SSP 的关键活性成分，并进行筛选。采用 3%葡聚糖硫酸钠诱导 UC 模型，并用 SSP 治疗 14 天，评价疗效。采用 ELISA 法检测结肠中 IL-6、IL-1β 和 TNF-α 的水平；采用流式细胞术检测 DCs 及其亚群的表达水平；对结肠差异 RNA 进行全转录组测序，并通过 RT-PCR 检测关键 miRNA，以验证测序结果。分离小鼠骨髓源性树突状细胞（BMDCs），用 100ng/ml LPS 构建炎症模型，观察 SSP 对 DC 增殖、凋亡及其表面共刺激分子表达的影响；采用 ELISA 法检测 IL-6、IL-1β、TNF-α 水平；采用 RT-PCR 和 WB 法检测 miR-505-3p、CDH1、E-cadherin 表达。通过慢病毒转染构建低表达 miR-505-3p 的 BMDCs，进一步验证。对潜在的关键成分进行体内和体外实验的重新验证。
动物实验表明，SSP 可缓解 DSS 诱导的 UC 症状和小鼠结肠病理损伤，抑制 IL-6、IL-1β、TNF-α 的分泌和炎症性 DC 增殖及活化成熟。网络药理学预测，吴茱萸碱、异甘草素、姜黄素和莪术醇可能是 SSP 的关键成分。RNA 测序显示，miR-505-3p 作为差异 miRNA，与 E-cadherin 共享大量转录因子，参与炎症分化调节。体内实验证实，SSP 可加速 BMDCs 的凋亡，减缓增殖，抑制炎症分化及 IL-6、IL-1β、TNF-α 的分泌，降低 miR-505-3p、CDH1 和 E-cadherin 水平。敲低 miR-505-3p 后，SSP 不能调节 BMDCs 的炎症分化和 IL-6、IL-1β、TNF-α 水平。此外，发现并验证吴茱萸碱是 SSP 预防 DC 炎症分化的关键活性成分。
SSP 通过下调 miR-505-3p 的表达来防止 DC 的炎症分化，其中吴茱萸碱可能发挥关键作用。
