Butyric acid alleviates LPS-induced intestinal mucosal barrier damage by inhibiting RhoA/ROCK2/MLCK signaling pathway
Authors: Liu Luqiong , Chen Tong , Xie Zhenrong , Zhang Yongjin , He Chenglu , Huang Yongkun 
Published: October 2023

Abstract
Bacground and Aim Butyric acid (BA), as a short-chain fatty acid, can improve intestinal barrier function, meanwhile intestinal mucosal epithelial injury is a common clinical phenomenon, especially in children. Moreover, RhoA/ROCK2/MLCK signaling pathway has been confirmed to play a vital role in the maintenance of intestinal epithelial permeability. However, the specific mechanism by which BA protects intestine mucosal barrier still needs to be clarified. This study intended to investigate the effect of BA in LPS-induced Caco2 cells, and determine whether BA protected epithelial barrier by inhibiting the RhoA/ROCK2/MLCK signaling pathway. Methods The optimal concentration and intervention time of the protective effect of BA on Caco2 were investigated by CCK-8 assay. On this basis of the above results, the damaged effect of LPS to Caco2 cells near the optimal time of BA protection was explored, then the optimal time and concentration were explored when BA and LPS were simultaneously administrated to Caco2 cells, the data were used to conduct further study. Then, cell growth status was observed, TEER and FD-4 permeability of monolayer barrier of Caco2 cells were detected, the mRNA expression of ZO-1 and Occludin, RhoA, ROCK2 and MLCK was detected by RT-qPCR. Moreover, immunofluorescence staning was adopted to observe the expression and distribution of ZO-1, Occludin, as well as RhoA, ROCK2 and MLCK in Caco2. After that, RhoA/ROCK2/MLCK pathway inhibitor Y-27632 was added to Caco2 cells, the cell growth status, TEER and FD-4 permeability of monolayers barrier, the expression level and distribution of ZO-1 and Occludin, RhoA, ROCK2 and MLCK in Caco2 cells were detected. Results The final concentration of 0.2mM BA action for 24 h had the greatest effect on the viability of Caco2 cells. After Caco2 cells exposure to LPS for 24 h, the final concentration of 5ug/ml LPS significantly decreased the viability of Caco2 cells. When combined with BA and LPS, compared with LPS alone, BA improved the growth state of Caco2 cells, restored the declined TEER, and reduced FD-4 permeability, as well as improved the mRNA expression of ZO-1, Occludin and inhibited the mRNA expression of RhoA, ROCK2 and MLCK, the expression and distribution of ZO-1, Occludin, RhoA, ROCK2 and MLCK were reversed in Caco2 cells. After treatment of Y-276432, the cell growth state and mucosal barrier function were further improved, the mRNA expression of ZO-1 and Occludin was further increased, the mRNA expression of RhoA, ROCK2 and MLCK was further decreased, and the expression and distribution of these proteins in Caco2 cells were further reversed. Conclusion This study provided complementary data for BA as a potential target for attenuating intestinal barrier injury induced by LPS through inhibiting the RhoA/ROCK2/MLCK signaling pathway, supporting a further research on BA protection intestinal barrier from damage and as a new therapeutic method.