芬普尼透過誘導粒線體凋亡、介白素-7 基因調控的失調及干擾 GABA 基因介導之免疫調節作用以擾亂免疫功能的恆定

Abstract

芬普尼是一種苯基吡唑類殺蟲劑，廣泛應用於農業、家庭害蟲防治與獸醫治療領域， 傳統上被認為對脊椎動物毒性較低。然而，越來越多研究指出，芬普尼暴露可能對肝臟、生 殖系統與神經系統造成預期之外的不良影響。儘管如此，其對免疫功能，特別是 T 淋巴細 胞反應的影響仍未被充分探討。本研究使用體內與體外兩種模型，系統性評估芬普尼對成熟 與發育中 T 淋巴細胞的免疫毒性。結果顯示，在免疫卵白蛋白小鼠中口服投予芬普尼後， 對卵白蛋白的抗原特異性免疫反應顯著增強，包括促進脾臟細胞代謝活性上升、增加細胞激 素 IL-2、IL-4 與 IFN-γ 分泌量，以及同時增加抗原專一性 OVA-IgG 1 與 OVA-IgG 2a 血清抗體 濃度。基因表現的分析顯示，GABA 調控相關的基因受到影響，其中 Gad67 的基因表現下 降，以及 GABA 受體次單元（β2 和 δ）的基因表現則上升。這些結果顯示，芬普尼可能 藉由干擾 GABA 路徑基因調控 T 淋巴細胞的免疫抑制作用，從而增強抗原特異性免疫反應 不正常的活化。

由於胸腺細胞的生成與 T 細胞的成熟分化是後天免疫系統發育的重要作用，我們進一 步評估芬普尼對胸腺發育的影響。結果發現，芬普尼會引起胸腺明顯萎縮、雙陽性胸腺細胞 比例與數量下降，T 細胞成熟受到抑制與 IL-7 與其受體表現下降密切相關。IL-7 是胸腺早期 發育中不可或缺的細胞激素，支持雙陰性階段後 T 淋巴細胞的的存活、增殖和分化。研究亦 發現芬普尼抑制 IL-7 軸相關基因和蛋白（包括 FOXN1、LYL1、SCF 和 c-KIT）的表現，進 一步破壞了細胞增生所需的胸腺微環境。除胸腺發育訊號受到抑制外，芬普尼也誘發胸腺細 胞的氧化壓力，包括細胞內活性氧含量上升、粒線體膜電位去極化、脂質過氧化、鈣離子與 穀胱甘肽濃度下降等現象。同時，BCL-2 家族基因表現失衡，抗凋亡基因抑制、促凋亡基因 如 Bim 與 Bnip3 表現異常，顯示芬普尼誘發粒線體凋亡機制，最終導致胸腺細胞死亡。綜合我們的研究結果所示，芬普尼具有兩種機制，其一是干擾 GABA 相關基因包含合成與傳 遞 GABA 的機制失調，進而誘發 Th1/Th2 細胞激素同時過度分泌，造成 T 細胞的過度活化； 另外是抑制 IL-7 訊號及維持胸腺微環境所需之轉錄因子和誘發氧化性傷害以破壞胸腺 T 淋 巴細胞發育，最終造成免疫細胞調控的失調。我們的研究成果突顯出暴露芬普尼對 T 細胞 功能與發育的潛在毒性作用，全面地重新評估芬普尼對於青春期脊椎動物的安全性為重要的 課題。

Fipronil (FPN) is a phenylpyrazole pesticide widely used in agriculture, household pest control, and veterinary medicine. It is traditionally considered to have low toxicity in vertebrates. However, growing evidence suggests that FPN exposure may lead to unexpected adverse effects on the liver, reproductive system, and nervous system. Despite these findings, its influence on immune function, particularly on T cell responses, remains poorly understood. In this study, I systematically investigated the immunotoxic effects of FPN on both mature and developing T cells using in vivo and ex vivo models. Oral administration of FPN in ovalbumin-sensitized mice enhanced antigen-specific immune responses, as indicated by increased splenocyte metabolic activity, elevated production of IL-2, IL-4, and IFN-γ, and higher serum levels of OVA-IgG 1 and OVA-IgG2a . Gene expression analysis revealed that GABAergic signaling was altered, with a significant decrease in Gad67 and an increase in GABA receptor subunits (β2 and δ). These findings suggest that FPN may interfere with the inhibitory role of GABAergic pathways in T cell regulation, thereby enhancing antigen-specific immune activation.

Because T cell lineage commitment and thymopoiesis are fundamental to functional adaptive immunity, I further examined the impact of FPN on thymic development. FPN exposure induced marked thymic atrophy, reduced the proportion of double-positive thymocytes, and impaired T cell maturation. These effects were closely linked to the suppression of IL-7 and IL-7 receptor expression. As a key cytokine in early thymocyte development, IL-7 supports survival, proliferation, and differentiation beyond the double-negative stage. Downregulation of IL-7 axis-related genes and proteins, including FOXN1, LYL1, SCF, and c-KIT, further disrupted the thymic microenvironment required for progenitor cell expansion. In addition to impairing developmental signaling, FPN exposure triggered oxidative stress in thymocytes. Elevated reactive oxygen species, mitochondrial membrane depolarization, lipid peroxidation, calcium depletion, and glutathione reduction were observed. Moreover, an imbalance in BCL-2 family gene expression was detected, with downregulation of anti-apoptotic genes and dysregulation of pro-apoptotic regulators such as Bim and Bnip3. These alterations activated mitochondrial apoptosis and led to thymocyte death. Taken together, our results demonstrate that FPN compromises immune system integrity through a dual mechanism involving enhanced peripheral T cell activity and disrupted thymic development via GABAergic imbalance, IL-7 signaling suppression, transcriptional factors for supporting thymic microenvironment, and oxidative damage. Our findings highlight the potential risk to immune system integrity from contaminant exposure to FPN and the need for a more comprehensive reassessment of the safety of fipronil in pubertal vertebrates.