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
  • 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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