Supplementary Material for: Exposure to Progestin 17-OHPC Induces Gastrointestinal Dysfunction through Claudin 1 Suppression in Female Mice with Increased Anxiety-Like Behaviors

Introduction: Progestin, commonly used in oral contraception and preventing preterm birth, elicits various off-target side effects on brain and gastrointestinal (GI) functions, yet the precise mechanisms remain elusive. This study aims to probe progestin's impact on GI function and anxiety-like behaviors in female mice. Methods: Colon stem cells were utilized to explore the mechanism underlying progestin 17-hydroxyprogesterone caproate (17-OHPC)-mediated suppression of claudin-1 (CLDN1), crucial for epithelial integrity. Chromatin immunoprecipitation and luciferase assays identified potential progestin-response elements on the CLDN1 promoter, with subsequent assessment of oxidative stress and pro-inflammatory cytokine release. Manipulation of vitamin D receptor (VDR) or estrogen receptor β (ERβ) expression elucidated their roles in 17-OHPC-mediated effects. Intestine-specific VDR deficient mice were generated to evaluate 17-OHPC's impact on GI dysfunction and anxiety-like behaviors in female mice. Additionally, gene expression was analyzed in various brain regions, including the amygdala, hypothalamus, and hippocampus. Results: Exposure to 17-OHPC suppressed CLDN1 expression via epigenetic modifications and VDR dissociation from the CLDN1 promoter. Furthermore, 17-OHPC intensified oxidative stress and proinflammatory cytokine release. VDR knockdown partly mimicked, while overexpression of either VDR or ERβ partly restored 17-OHPC-mediated effects. Intestinal VDR deficiency partly mirrored 17-OHPC-induced GI dysfunction, with minimal impact on 17-OHPC-mediated anxiety-like behaviors. Conclusions: 17-OHPC suppresses CLDN1 expression through VDR, contributing to GI dysfunction in female mice, distinct from 17-OHPC-induced anxiety-like behaviors. This study reveals a new mechanism and potential negative impact of progestin exposure on the gastrointestinal tract, alongside inducing anxiety-like behaviors in female mice.

前言：孕激素 (progestin) 常被应用于口服避孕与早产预防，但其对大脑及胃肠 (GI) 功能存在多种脱靶副作用，具体机制仍未明确。本研究旨在探究孕激素对雌性小鼠胃肠功能与焦虑样行为的影响。
方法：本研究采用结肠干细胞，探究17-羟孕酮己酸酯 (17-hydroxyprogesterone caproate, 17-OHPC) 介导的紧密连接蛋白-1 (claudin-1, CLDN1) 表达抑制的分子机制，CLDN1对上皮完整性至关重要。通过染色质免疫沉淀与荧光素酶实验，鉴定CLDN1启动子上的潜在孕激素反应元件，并后续评估氧化应激与促炎细胞因子释放情况。通过调控维生素D受体 (VDR) 或雌激素受体β (ERβ) 的表达，阐明二者在17-OHPC介导的生物学效应中的作用。构建肠道特异性VDR敲除小鼠，以评估17-OHPC对雌性小鼠胃肠功能障碍及焦虑样行为的影响。此外，本研究还分析了杏仁核、下丘脑与海马等多个脑区的基因表达水平。
结果：17-OHPC通过表观遗传修饰及VDR从CLDN1启动子上解离，抑制CLDN1的表达。此外，17-OHPC可加剧氧化应激与促炎细胞因子释放。VDR敲低可部分模拟17-OHPC的效应，而过表达VDR或ERβ则可部分逆转17-OHPC介导的上述作用。肠道特异性VDR敲除可部分模拟17-OHPC诱导的胃肠功能障碍，但对17-OHPC介导的焦虑样行为影响极小。
结论：17-OHPC通过VDR抑制CLDN1表达，进而导致雌性小鼠胃肠功能障碍，该通路与17-OHPC诱导的焦虑样行为无关。本研究揭示了孕激素暴露对胃肠道的全新作用机制与潜在负面影响，同时证实其可诱导雌性小鼠产生焦虑样行为。