积雪草苷通过靶向STAT3恢复Th17/Treg稳态来减轻肾纤维化

摘要(中文译文)

肾纤维化（RF）是由慢性炎症和Th17/Treg失衡驱动的进行性病理过程。积雪草苷（AS），一种来自积雪草的三萜化合物。，具有抗炎和抗纤维化活性，但其分子机制尚不清楚。本研究旨在探讨AS是否通过“结合失稳”机制靶向信号转导子和转录激活子3（STAT3）来恢复Th17/Treg稳态，从而缓解RF。采用了一种结合网络药理学、转录组学和多模式实验验证的综合方法。UUO小鼠用AS（10、50、100mg/kg/d）治疗10天。进行了组织病理学、RNA-seq、流式细胞术、免疫荧光、Luminex、qPCR、DARTS-LC-MS/MS、分子对接/动力学模拟和SPR。包括使用Stattic（STAT3抑制剂）和Colivelin（STAT3激动剂）的药物干预，以在功能上验证STAT3的作用。网络药理学确定STAT3为核心靶点，Th17分化为关键途径。AS治疗显著减弱了UUO小鼠的RF，改善了肾功能，并重新平衡了Th17/Treg比值，同时伴有IL-17A降低和IL-10升高。转录组学分析显示，通过qPCR验证，Th17细胞分化基因富集。DARTS-LC-MS/MS证实AS与STAT3直接结合，并鉴定出一种衍生自SH2结构域的肽（残基582-602），表明构象不稳定。SPR显示出与人和小鼠STAT3的高亲和力结合。分子对接和动力学模拟表明，SH2结构域内存在“局部锚定变构效应”模式。qPCR分析显示，AS显著抑制UUO小鼠肾组织中IL-17A和总STAT3的mRNA表达。免疫荧光显示肾脏中STAT3和p-STAT3表达降低。STAT3抑制剂Stattic模拟了AS的抗纤维化和Th17抑制作用，而激动剂Colivelin加剧了纤维化，并被AS部分挽救。AS通过一种直接靶向STAT3 SH2结构域的新型“结合失稳”变构降解机制来缓解RF。这种相互作用诱导构象不稳定，抑制STAT3激活和转录活性，恢复Th17/Treg稳态，最终减轻肾脏炎症和纤维化。总的来说，这些发现为STAT3驱动的纤维化疾病建立了一种新的治疗策略。

Abstract

Renal fibrosis (RF) is a progressive pathological process driven by chronic inflammation and Th17/Treg imbalance. Asiaticoside (AS), a triterpenoid compound from Centella asiatica (L.) Urb., exhibits anti-inflammatory and antifibrotic activities, though its molecular mechanism remains unclear. This study aimed to investigate whether AS alleviates RF by targeting Signal transducer and activator of transcription 3 (STAT3) through a "bind to destabilize" mechanism to restore Th17/Treg homeostasis. An integrated approach combining network pharmacology, transcriptomics, and multimodal experimental validation was applied. UUO mice were treated with AS (10, 50, 100mg/kg/d) for 10 days. Histopathology, RNA‑seq, flow cytometry, immunofluorescence, Luminex, qPCR, DARTS‑LC‑MS/MS, molecular docking/dynamics simulations and SPR were performed. Pharmacological interventions using Stattic (STAT3 inhibitor) and Colivelin (STAT3 agonist) were included to functionally validate the role of STAT3. Network pharmacology identified STAT3 as the core target, with Th17 differentiation as the key pathway. AS treatment significantly attenuated RF, improved renal function, and rebalanced Th17/Treg ratios in UUO mice, accompanied by reduced IL-17A and elevated IL-10. Transcriptomic analysis revealed enriched Th17 cell differentiation genes, validated by qPCR. DARTS-LC-MS/MS confirmed direct binding of AS to STAT3 and identified a peptide derived from the SH2 domain (residues 582-602), indicating conformational destabilization. SPR showed high affinity binding to both human and murine STAT3. Molecular docking and dynamics simulations demonstrated a "local anchoring-allosteric effect" mode within the SH2 domain. qPCR analysis showed that AS significantly inhibited the mRNA expression of both IL-17A and total STAT3 in the renal tissues of UUO mice. Immunofluorescence revealed reduced STAT3 and p-STAT3 expression in kidneys. STAT3 inhibitor Stattic mimicked AS's antifibrotic and Th17 suppressive effects, whereas agonist Colivelin exacerbated fibrosis and was partially rescued by AS. AS alleviates RF via a novel "bind to destabilize" allosteric degradation mechanism that directly targets the STAT3 SH2 domain. This interaction induces conformational instability, suppresses STAT3 activation and transcriptional activity, restores Th17/Treg homeostasis, and ultimately mitigates renal inflammation and fibrosis. Collectively, these findings establish a new therapeutic strategy for STAT3-driven fibrotic diseases.

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