Saha Sarmistha, Capozzi Antonella, Profumo Elisabetta, Alessandri Cristiano, Sorice Maurizio, Saso Luciano, Buttari Brigitta
Department of Cardiovascular, Endocrine-Metabolic Diseases and Aging, Italian National Institute of Health, 00161 Rome, Italy.
Department of Biotechnology, Institute of Applied Sciences & Humanities, GLA University, Mathura 281406, India.
Int J Mol Sci. 2025 Jul 25;26(15):7179. doi: 10.3390/ijms26157179.
Oxysterols such as 7-ketocholesterol (7KCh) contribute to the pathogenesis of autoimmune and chronic inflammatory diseases by inducing oxidative stress and promoting pro-inflammatory immune cell activation. Dendritic cells (DCs) play a central role in maintaining immune tolerance, and their dysregulation is a key driver of autoimmunity. Targeting DCs by using natural compounds offers a promising strategy to restore redox balance and suppress aberrant immune responses. This study investigated the immunomodulatory and antioxidant properties of Lupeol, a natural triterpenoid, in human monocyte-derived DCs exposed to 7KCh. Flow cytometry and cytokine profiling demonstrated that Lupeol preserved the immature, tolerogenic phenotype of DCs by promoting a dose-dependent increase in the anti-inflammatory cytokine IL-10. Lupeol also inhibited the 7KCh-induced upregulation of maturation markers (CD83, CD86) and suppressed the release of pro-inflammatory cytokines IL-1β and IL-12p70. Functionally, Lupeol-treated DCs directed T cell polarization toward an anti-inflammatory and regulatory profile while dampening the inflammatory responses triggered by 7KCh. This immunoregulatory effect was further supported by the decreased secretion of the pro-inflammatory cytokines IL-1β and IL-12p70 in DC culture supernatants. Mechanistic analyses using immunofluorescence showed that Lupeol alone significantly increased nuclear NRF2 levels and upregulated HO-1 expression. Western blot analysis further confirmed Lupeol's ability to activate the KEAP1-NRF2 signaling pathway, as evidenced by increased expression of NRF2 and its downstream target, NQO1. The use of ML385, a selective NRF2 inhibitor, in ROS and cytokine assays supported the involvement of NRF2 in mediating the Lupeol antioxidant and anti-inflammatory effects in DCs. Notably, the oxidative burden induced by 7KCh limited the full activation of NRF2 signaling triggered by Lupeol. Furthermore, docking and MM/PBSA analyses revealed the specific interactions of Lupeol with the kelch domain of KEAP1. These findings suggest that Lupeol may serve as a promising orally available immunomodulatory agent capable of promoting tolerogenic DCs, offering potential applications in autoimmune and other chronic inflammatory diseases.
诸如7-酮胆固醇（7KCh）之类的氧化甾醇通过诱导氧化应激和促进促炎免疫细胞活化，在自身免疫性疾病和慢性炎症性疾病的发病机制中发挥作用。树突状细胞（DCs）在维持免疫耐受中起核心作用，其失调是自身免疫的关键驱动因素。利用天然化合物靶向DCs提供了一种恢复氧化还原平衡和抑制异常免疫反应的有前景的策略。本研究调查了天然三萜类化合物羽扇豆醇在暴露于7KCh的人单核细胞衍生DCs中的免疫调节和抗氧化特性。流式细胞术和细胞因子分析表明，羽扇豆醇通过促进抗炎细胞因子IL-10剂量依赖性增加，保留了DCs的未成熟、耐受性表型。羽扇豆醇还抑制了7KCh诱导的成熟标志物（CD83、CD86）上调，并抑制了促炎细胞因子IL-1β和IL-12p70的释放。在功能上，经羽扇豆醇处理的DCs引导T细胞极化朝向抗炎和调节性表型，同时抑制由7KCh触发的炎症反应。DC培养上清液中促炎细胞因子IL-1β和IL-12p70分泌减少进一步支持了这种免疫调节作用。使用免疫荧光进行的机制分析表明，单独的羽扇豆醇显著增加核NRF2水平并上调HO-1表达。蛋白质印迹分析进一步证实了羽扇豆醇激活KEAP1-NRF2信号通路的能力，NRF2及其下游靶点NQO1表达增加证明了这一点。在ROS和细胞因子测定中使用选择性NRF2抑制剂ML385支持了NRF2参与介导羽扇豆醇在DCs中的抗氧化和抗炎作用。值得注意的是，7KCh诱导的氧化负担限制了羽扇豆醇触发的NRF2信号通路的完全激活。此外，对接和MM/PBSA分析揭示了羽扇豆醇与KEAP1的kelch结构域的特异性相互作用。这些发现表明，羽扇豆醇可能作为一种有前景的口服免疫调节剂，能够促进耐受性DCs，在自身免疫性疾病和其他慢性炎症性疾病中具有潜在应用。
