Butyric acid alleviates LPS-induced intestinal mucosal barrier damage by inhibiting the RhoA/ROCK2/MLCK signaling pathway in Caco2 cells

• Published in: PloS one 2024-12, Vol.19 (12), p.e0316362
• Main Authors: Liu, Luqiong, Chen, Tong, Xie, Zhenrong, Zhang, Yongjin, He, Chenglu, Huang, Yongkun
• Format: Article
• Language: English
• Subjects: Amides - pharmacology; Biology and Life Sciences; Butyric acid; Butyric Acid - pharmacology; Caco-2 Cells; Cell culture; Cholecystokinin; Dextran; Dextrans; Dietary fiber; Electrical resistivity; Enzymes; Epithelium; Ethylenediaminetetraacetic acid; Fluctuations; Fluorescence; Gene amplification; Gene expression; Homeostasis; Humans; Immunofluorescence; Intestinal Mucosa - drug effects; Intestinal Mucosa - metabolism; Intestinal Mucosa - pathology; Intestine; Kinases; Lipopolysaccharides; Localization; Medicine and Health Sciences; Membrane permeability; Metabolites; Mucosa; Myosin; Myosin-light-chain kinase; Myosin-Light-Chain Kinase - metabolism; Occludin - genetics; Occludin - metabolism; Permeability; Physical Sciences; Proteins; Pyridines - pharmacology; Research and Analysis Methods; Rho-associated kinase; rho-Associated Kinases - metabolism; rhoA GTP-Binding Protein - metabolism; RhoA protein; RNA; Roles; Saturated fatty acids; Signal transduction; Signal Transduction - drug effects; Zonula occludens-1 protein; Zonula Occludens-1 Protein - genetics; Zonula Occludens-1 Protein - metabolism
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0316362

Butyric acid (BA) can potentially enhance the function of the intestinal barrier. However, the mechanisms by which BA protects the intestinal mucosal barrier remain to be elucidated. Given that the Ras homolog gene family, member A (RhoA)/Rho-associated kinase 2 (ROCK2)/Myosin light chain kinase (MLCK) signaling pathway is crucial for maintaining the permeability of the intestinal epithelium, we further investigated whether BA exerts a protective effect on epithelial barrier function by inhibiting this pathway in LPS-induced Caco2 cells. First, we aimed to identify the optimal treatment time and concentration for BA and Lipopolysaccharide (LPS) through a CCK-8 assay. We subsequently measured Trans-epithelial electrical resistance (TEER), FITC-Dextran 4 kDa (FD-4) flux, and the mRNA expression of ZO-1, Occludin, RhoA, ROCK2, and MLCK, along their protein expression levels, and average fluorescence intensity following immunofluorescence staining. We then applied the ROCK2 inhibitor Y-27632 and reevaluated the TEER, FD-4 flux, and mRNA, and protein expression of ZO-1, Occludin, RhoA, ROCK2, and MLCK, as well as their distribution in Caco2 cells. The optimal treatment conditions were determined to be 0.2 mmol/L BA and 5 μg/mL LPS for 24 hours. Compared with LPS treatment alone, BA significantly mitigated the reduction in the TEER, decreased FD-4 flux permeability, increased the mRNA expression of ZO-1 and Occludin, and normalized the distribution of ZO-1 and Occludin in Caco2 cells. Furthermore, BA inhibited the expression of RhoA, ROCK2, and MLCK, and normalized their localization within Caco2 cells. Following treatment with Y-27632, the epithelial barrier function, along with the mRNA and protein expression and distribution of ZO-1 and Occludin were further normalized upon inhibition of the pathway. These findings contribute to a deeper understanding of the potential mechanisms through which BA attenuates LPS-induced impairment of the intestinal epithelial barrier.