Abstract：
Fluoride is a common contaminant of groundwater and agricultural commodity, which poses challenges to animal and human health. A wealth of research has demonstrated its detrimental effects on intestinal mucosal integrity; however, the underlying mechanisms remain obscure. This study aimed to investigate the role of the cytoskeleton in fluoride-induced barrier dysfunction. After sodium fluoride (NaF) treatment of the cultured Caco-2 cells, both cytotoxicity and cytomorphological changes (internal vacuoles or massive ablation) were observed. NaF lowered transepithelial electrical resistance (TEER) and enhanced paracellular permeation of fluorescein isothiocyanate dextran 4 (FD-4), indicating Caco-2 monolayers hyperpermeability. In the meantime, NaF treatment altered both the expression and distribution of the tight junction protein ZO-1. Fluoride exposure increased myosin light chain II (MLC2) phosphorylation and triggered actin filament (F-actin) remodeling. While inhibition of myosin II by Blebbistatin blocked NaF-induced barrier failure and ZO-1 discontinuity, the corresponding agonist Ionomycin had effects comparable to those of fluoride, suggesting that MLC2 serves as an effector. Given the mechanisms upstream of p-MLC2 regulation, further studies demonstrated that NaF activated RhoA/ROCK signaling pathway and myosin light chain kinase (MLCK), strikingly increasing the expression of both. Pharmacological inhibitors (Rhosin, Y-27632 and ML-7) reversed NaF-induced barrier breakdown and stress fiber formation. The role of intracellular calcium ions ([Ca2+]i) in NaF effects on Rho/ROCK pathway and MLCK was investigated. We found that NaF elevated [Ca2+]i, whereas chelator BAPTA-AM attenuated increased RhoA and MLCK expression as well as ZO-1 rupture, thus, restoring barrier function. Collectively, abovementioned results suggest that NaF induces barrier impairment via Ca2+-dependent RhoA/ROCK pathway and MLCK, which in turn triggers MLC2 phosphorylation and rearrangement of ZO-1 and F-actin. These results provide potential therapeutic targets for fluoride-induced intestinal injury.
摘要：
氟化物是地下水和农产品的常见污染物，这对动物和人类健康构成了挑战。大量的研究已经证明了其对肠粘膜完整性的有害影响；然而，潜在的机制仍然模糊。本研究旨在探讨细胞骨架在氟化物诱导的屏障功能障碍中的作用。氟化钠（NaF）处理培养的Caco-2细胞后，观察到细胞毒性和细胞形态学变化(内部空泡或大量消融).NaF降低了跨上皮电阻（TEER）并增强了异硫氰酸荧光素葡聚糖4（FD-4）的细胞旁渗透，表明Caco-2单层高渗透性。同时,NaF处理改变了紧密连接蛋白ZO-1的表达和分布。氟化物暴露会增加肌球蛋白轻链II（MLC2）的磷酸化并触发肌动蛋白丝（F-肌动蛋白）的重塑。当Blebistatin对肌球蛋白II的抑制作用阻止NaF诱导的屏障破坏和ZO-1不连续性时，相应的激动剂离子霉素具有与氟化物相当的作用，表明MLC2是效应子。鉴于p-MLC2调节的上游机制，进一步研究表明,NaF激活了RhoA/ROCK信号通路和肌球蛋白轻链激酶(MLCK),显著增加两者的表达。药理学抑制剂(Rhosin,Y-27632和ML-7）逆转了NaF诱导的屏障击穿和应力纤维形成。研究了细胞内钙离子（[Ca2]i）在NaF对Rho/ROCK途径和MLCK的影响中的作用。我们发现NaF升高[Ca2+]i,而螯合剂BAPTA-AM减弱增加RhoA和MLCK表达以及ZO-1破裂，因此，恢复屏障功能。总的来说，上述结果表明，NaF通过Ca2+依赖的RhoA/ROCK通路和MLCK诱导屏障损伤，进而触发MLC2磷酸化和ZO-1和F-肌动蛋白的重排。这些结果为氟化物诱导的肠损伤提供了潜在的治疗靶点。
