基于LiP-MS探讨芍药苷减轻肠嗜铬细胞敏化的机制研究
| 作 者：李红燕, 唐大东, 闻永, 占煜 |
| 单 位：成都市中西医结合医院/成都市第一人民医院 |
| 基金项目：四川省中医药管理局科学技术研究专项课题；成都市卫健委-成都中医药大学2024年度委校联合科技创新基金 |
| 摘 要： |
| 背景：中药白芍的缓急止痛功效显著，临床常应用于肠易激综合征的治疗方剂中，可改善内脏高敏感状态。目的：探讨芍药苷对肠嗜铬细胞敏化的作用及其机制。方法：采用BON-1细胞株模拟肠嗜铬细胞，以异戊酸（ISV）构建BON-1细胞敏化模型。通过浓度梯度实验确定ISV与芍药苷的量效关系。以蛋白质印迹法检测色氨酸羟化酶1（TPH1）表达，ELISA法检测5-羟色胺（5-HT）水平，CCK-8法检测细胞活性。采用限制性蛋白酶解-质谱联用（LiP-MS）筛选芍药苷的潜在结合靶点，并通过分子对接、表面等离子体共振（SPR）和蛋白质印迹法验证芍药苷与目标蛋白的亲和力及其调控作用。结果：诱导BON-1细胞敏化的最佳ISV浓度为25 μmol/L，芍药苷（100~1 600 μmol/L）可剂量依赖性地降低敏化BON-1细胞中的TPH1表达和5-HT水平。LiP-MS共筛选出7个潜在结合靶点，分别为溶质载体家族25成员5（SLC25A5）、二氢硫辛酰胺脱氢酶（DLD）、20S蛋白酶体β4亚基（PSMB4）、X连锁核糖体蛋白S4（RPS4X）、角蛋白9（KRT9）、真核翻译起始因子5A（EIF5A）、微管蛋白α-1c链（TUBA1C）。分子对接显示，SLC25A5、PSMB4与芍药苷的结合活性最好，结合能分别为-9.2、-10.1 kcal/mol；SPR显示两者的亲和常数（KD）分别为20.2、13.7 μmol/L。蛋白质印迹法结果证实芍药苷可剂量依赖性地上调敏化BON-1细胞中的SLC25A5、PSMB4蛋白表达水平。结论：芍药苷可通过调控SLC25A5、PSMB4表达，减轻ISV诱导的BON-1细胞敏化。 |
| 关键词：芍药苷； 肠易激综合征； 内脏高敏感； 肠嗜铬细胞 |
Study on Mechanism of Paeoniflorin in Alleviating Sensitization of Enterochromaffin Cells Based on LiP-MS
| Author's Name: |
| Institution: |
| Abstract: |
| Background: Radix Paeoniae Alba, a traditional Chinese medicine, exerts a remarkable effect on relieving spasm and alleviating pain. It is frequently prescribed in formulas for treating irritable bowel syndrome by ameliorating visceral hypersensitivity. Aims: To explore the effect and its underlying mechanism of paeoniflorin on enterochromaffin cell sensitization. Methods: BON-1 cell line was used as an in vitro model of enterochromaffin cells, and isovaleric acid (ISV) was applied to establish the cell sensitization model. Dose-response relationships of ISV and paeoniflorin were determined through concentration gradient experiments. The expression of tryptophan hydroxylase 1 (TPH1) was detected by Western blotting, the level of 5-hydroxytryptamine (5-HT) by ELISA, and cell viability by CCK-8 assay. Limited proteolysis-mass spectrometry (LiP-MS) was used to screen potential binding targets of paeoniflorin. Molecular docking, surface plasmon resonance (SPR) and Western blotting were further performed to verify the binding affinity of paeoniflorin to target proteins and its regulatory effects. Results: The optimal concentration of ISV for inducing BON-1 cell sensitization was 25 μmol/L. Paeoniflorin at concentrations ranging from 100 to 1 600 μmol/L reduced TPH1 expression and 5-HT level in sensitized BON-1 cells in a dose-dependent manner. A total of seven potential binding targets were identified by LiP-MS, including solute carrier family 25 member 5 (SLC25A5), dihydrolipoyl dehydrogenase (DLD), proteasome 20S subunit beta 4 (PSMB4), ribosomal protein S4 X-linked (RPS4X), keratin 9 (KRT9), eukaryotic translation initiation factor 5A (EIF5A), and tubulin alpha-1C chain (TUBA1C). Molecular docking results revealed that SLC25A5 and PSMB4 exhibited the strongest binding activity with paeoniflorin, with binding energies of −9.2 kcal/mol and −10.1 kcal/mol, respectively. SPR assay showed that their affinity constant (KD) values were 20.2 μmol/L and 13.7 μmol/L, respectively. Western blotting confirmed that paeoniflorin upregulated the protein expressions of SLC25A5 and PSMB4 in sensitized BON-1 cells in a dose-dependent manner. Conclusions: Paeoniflorin alleviates ISV-induced BON-1 cell sensitization by regulating the expressions of SLC25A5 and PSMB4. |
| Keywords: Paeoniflorin; Irritable Bowel Syndrome; Visceral Hypersensitivity; Enterochromaffin Cells |
| 投稿时间：2025-10-22 |
|
|
|
查看pdf文件
|
|
