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.引言：孕激素（progestin）常被用于口服避孕与早产预防，但其对大脑及胃肠（gastrointestinal, GI）功能存在多种脱靶副作用，具体分子机制尚不明确。本研究旨在探究孕激素对雌性小鼠胃肠功能与类焦虑行为的影响。 方法：本研究利用结肠干细胞，探究孕激素17-羟己酸孕酮（17-hydroxyprogesterone caproate, 17-OHPC）介导的紧密连接蛋白-1（claudin-1, CLDN1）抑制机制——CLDN1对上皮屏障完整性至关重要。通过染色质免疫共沉淀与荧光素酶活性实验，研究团队在CLDN1启动子区域鉴定出潜在的孕激素应答元件，并后续检测了氧化应激与促炎细胞因子的释放水平。通过调控维生素D受体（vitamin D receptor, VDR）或雌激素受体β（estrogen receptor β, ERβ）的表达，明确二者在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与类焦虑行为的影响。此外，研究团队还对杏仁核、下丘脑与海马体等多个脑区的基因表达进行了分析。 结果：17-OHPC处理可通过表观遗传修饰及VDR从CLDN1启动子区域解离，抑制CLDN1的表达。此外，17-OHPC可加剧氧化应激与促炎细胞因子的释放。VDR敲低可部分模拟17-OHPC的效应，而VDR或ERβ过表达则可部分逆转17-OHPC介导的生物学变化。肠道特异性VDR敲除可部分重现17-OHPC诱导的胃肠功能障碍，但对17-OHPC介导的类焦虑行为影响极小。 结论：17-OHPC通过VDR抑制CLDN1的表达，进而导致雌性小鼠胃肠功能障碍，这一通路与17-OHPC诱导的类焦虑行为机制不同。本研究揭示了孕激素暴露对胃肠道的潜在负面影响与全新分子机制，同时证实其可诱导雌性小鼠产生类焦虑行为。