异槲皮苷通过调控ASIC3/RhoA/β-连环蛋白正反馈信号通路抑制喉咽反流病相关咽部上皮-间质转化

文献信息

中文摘要

背景： 喉咽反流病（LPRD）缺乏有效的靶向治疗，因为常规质子泵抑制剂无法缓解胆汁酸诱导的黏膜损伤和上皮-间质转化（EMT）。本研究旨在探讨酸性胆汁介导的LPRD进展机制以及异槲皮苷的治疗潜力。方法：建立酸性胆汁刺激的HHPC细胞模型和幽门结扎诱导的大鼠LPRD模型。采用高通量筛选鉴定最佳活性单体。进行多种分子生物学实验、分子对接、微量热泳动和荧光素酶报告基因实验，以验证蛋白质结合和信号调控。应用梯度剂量异槲皮苷治疗进行体外和体内验证。结果：在病理学上，酸性胆汁（pH 4.0）破坏咽上皮屏障完整性，降低E-钙黏蛋白表达，并通过激活ASIC3/RhoA/β-连环蛋白正反馈环路诱导EMT，其中β-连环蛋白/TCF/LEF进一步转录上调ASIC3。高通量筛选证实异槲皮苷（IC50 = 34.2 μM）是最佳抗EMT单体。在机制上，异槲皮苷直接结合β-连环蛋白的N426/S425位点，抑制β-连环蛋白核转位，从而阻断正反馈级联，恢复上皮屏障稳态，并逆转胆汁诱导的EMT。一致地，体内LPRD模型验证了异槲皮苷剂量依赖性地减轻炎症浸润，挽救咽组织结构性损伤，下调ASIC3，并恢复E-钙黏蛋白表达。结论：总之，异槲皮苷通过靶向ASIC3/RhoA/β-连环蛋白反馈通路抑制EMT，从而改善LPRD。这些发现表明异槲皮苷是LPRD的一种新型治疗候选药物。

英文摘要

Laryngopharyngeal reflux disease (LPRD) lacks effective targeted treatments, as conventional proton pump inhibitors cannot alleviate bile acid-induced mucosal damage and epithelial-mesenchymal transition (EMT). This study aimed to explore the mechanism of acidic bile-mediated LPRD progression and the therapeutic potential of isoquercitrin. An acidic bile-stimulated HHPC cell model and a pyloric ligation-induced rat LPRD model were established. High-throughput screening was used to identify optimal active monomers. Multiple molecular biological assays, molecular docking, microscale thermophoresis, and luciferase reporter assays were performed to verify protein binding and signaling regulation. Gradient-dose isoquercitrin treatment was applied for in vitro and in vivo validation. Pathologically, acidic bile (pH 4.0) disrupted pharyngeal epithelial barrier integrity, decreased E-cadherin expression, and induced EMT by activating the ASIC3/RhoA/β-catenin positive feedback loop, in which β-catenin/TCF/LEF further transcriptionally upregulated ASIC3. High-throughput screening confirmed isoquercitrin (IC50 = 34.2 μM) as the optimal anti-EMT monomer. Mechanistically, isoquercitrin directly bound the N426/S425 sites of β-catenin, restrained β-catenin nuclear translocation, and thereby blocked the positive feedback cascade, restored epithelial barrier homeostasis, and reversed bile-induced EMT. Consistently, in vivo LPRD models verified that isoquercitrin dose-dependently attenuated inflammatory infiltration, rescued pharyngeal tissue structural damage, downregulated ASIC3, and recovered E-cadherin expression. In conclusion, isoquercitrin ameliorates LPRD by inhibiting EMT via targeting the ASIC3/RhoA/β-catenin feedback pathway. These findings identify isoquercitrin as a novel therapeutic candidate for LPRD.