Zearalenone-Induced Mechanical Damage of Intestinal Barrier via the RhoA/ROCK Signaling Pathway in IPEC-J2 Cells
Abstract
:1. Introduction
2. Results
2.1. Measurement of Transepithelial Electrical Resistance (TEER)
2.2. Effects of ZEN on Permeability Barrier Function and Intestinal Integrity
2.2.1. Effect of ZEN on Intestinal Epithelial Cell Viability
2.2.2. Alkaline Phosphatase Activity and The Permeation of FITC-Dextran
2.3. Analysis of Transepithelial Electrical Resistance (TEER)
2.4. Effect of ZEN on IPEC-J2 Intracellular ATP
2.5. Effects of ZEN on IPEC-J2 Cytoskeletal Microfilament (F-Actin)
2.6. Effects of ZEN on the Distribution and Expression of TJ Proteins
2.6.1. The Expression mRNA of ROCK1 and Tight Junction Proteins
2.6.2. Immunofluorescence
2.7. Western Blotting
3. Discussion
4. Materials and Methods
4.1. Chemicals and Reagents
4.2. Cell Culture Conditions
4.3. Transepithelial Electrical Resistance (TEER)
4.4. Cell Activity Assay
4.5. Alkaline Phosphatase Activity
4.6. Measurements of Paracellular Permeability
4.7. Design of RhoA/ROCK Signaling Pathway Validation Experiment
4.8. Quantitative Real-Time PCR
4.9. Immunofluorescence Staining
4.10. Intracellular ATP Determination
4.11. Filamentous Actin (F-actin) Staining Filaments of Actin of F-Actin Analysis
4.12. Western Blot Analysis
4.13. Statistics
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
References
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Genes | Orientation | Sequences (5′-3′) | Fragments Size (bp) | Accession Number |
---|---|---|---|---|
GAPDH | Forward | GATGGTGAAGGTCGGAGTGAAC | 153 | NM_001206359.1 |
Reversed | TGGGTGGAATCATACTGGAACA | |||
Occludin | Forward | ACGAGCAGCAAAGGGATTCTTC | 152 | NM_001163647.2 |
Reversed | TCACACCCAGGATAGCACTCATT | |||
Claudin-1 | Forward | TGCCTCAGTGGAAGATTTACTCC | 147 | NM_001244539.1 |
Reversed | TGGTGTTCAGATTCAGCAAGGA | |||
ZO-1 | Forward | AGTTTGATAGTGGCGTTGACAC | 106 | XM_005659811.1 |
Reversed | GCTGAAGGACTCACAGGAACA | |||
Claudin-3 | Forward | GTCCATGGGCCTGGAGAT | 130 | NM_001160075.1 |
Reversed | GATCTGCGCTGTGATAATGC | |||
ROCK1 | Forward | TTGTGCCTTCCTTACTGACAGG | 125 | XM_005653400.3 |
Reversed | CTGGTGCCACAGTGTCTCG |
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Huang, B.; Wang, J.; Gu, A.; Wang, T.; Li, J.; Shan, A. Zearalenone-Induced Mechanical Damage of Intestinal Barrier via the RhoA/ROCK Signaling Pathway in IPEC-J2 Cells. Int. J. Mol. Sci. 2022, 23, 12550. https://doi.org/10.3390/ijms232012550
Huang B, Wang J, Gu A, Wang T, Li J, Shan A. Zearalenone-Induced Mechanical Damage of Intestinal Barrier via the RhoA/ROCK Signaling Pathway in IPEC-J2 Cells. International Journal of Molecular Sciences. 2022; 23(20):12550. https://doi.org/10.3390/ijms232012550
Chicago/Turabian StyleHuang, Biying, Jingjing Wang, Aixin Gu, Tianhu Wang, Jianping Li, and Anshan Shan. 2022. "Zearalenone-Induced Mechanical Damage of Intestinal Barrier via the RhoA/ROCK Signaling Pathway in IPEC-J2 Cells" International Journal of Molecular Sciences 23, no. 20: 12550. https://doi.org/10.3390/ijms232012550