2. Academic Validation
  3. Fluoride disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated RhoA/ROCK signaling and myosin light chain kinase

Fluoride disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated RhoA/ROCK signaling and myosin light chain kinase

  • Ecotoxicol Environ Saf. 2023 Apr 24;257:114940. doi: 10.1016/j.ecoenv.2023.114940.
Lianxin Li 1,  Jinge Xin 1,  Hesong Wang 2,  Yadong Wang 3,  Weiqi Peng 2,  Ning Sun 1,  Haonan Huang 1,  Yanxi Zhou 1,  Xingmei Liu 1,  Yu Lin 3,  Jing Fang 1,  Bo Jing 1,  Kangcheng Pan 1,  Yan Zeng 1,  Dong Zeng 1,  Xiang Qin 4,  Yang Bai 5,  Xueqin Ni 6
Affiliations

Affiliations

  • 1 Animal Microecology Institute, College of Veterinary, Sichuan Agricultural University, Chengdu, Sichuan, China.
  • 2 Baiyun Branch, Nanfang Hospital, Southern Medical University, Guangzhou, China.
  • 3 Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Institute of Gastroenterology of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China.
  • 4 School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China. Electronic address: [email protected].
  • 5 Baiyun Branch, Nanfang Hospital, Southern Medical University, Guangzhou, China; Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Institute of Gastroenterology of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China. Electronic address: [email protected].
  • 6 Animal Microecology Institute, College of Veterinary, Sichuan Agricultural University, Chengdu, Sichuan, China. Electronic address: [email protected].
PMID: 37099960 DOI: 10.1016/j.ecoenv.2023.114940
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.

Keywords

Fluoride; Intestinal epithelial barrier; Intracellular calcium level; Myosin light chain kinase; Rho kinase; Tight junction.

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