Many enteral and systematic diseases lead to intestinal epithelial barrier damage. The pathogenesis and potential therapies for intestinal epithelial barrier damage have been widely researched16. However, appropriate techniques through food supplement with no adverse effect for reversing intestinal epithelial barrier damage to improve patient outcomes and prognosis have not yet definitely been established. Therefore, finding a new regimen with therapeutic value for intestinal epithelial barrier damage is of great significance for basic and clinical research. BA not only provides energy for colon cells, but also is important to maintain intestinal epithelial integrity17. Correspondingly, many studies have found that LPS induces intestinal barrier damage and has been a common molding methods10. Therefore, in this study, LPS was selected as a stimulating factor to create an in vitro intestinal barrier damage model. At the same time, BA as a protective factor of Caco2 cells to study the role of BA in LPS-induced intestinal barrier damage and possible related mechanisms. In this present study, it was found that BA could improve cell viability, improve the growth status of Caco2 cells, maintain the integrity of intestinal epithelial barrier, up-regulate ZO-1 and Occludin expression, maintain their subcellular distribution, inhibit RhoA/ROCK2/MLCK signaling pathway, reversed intracellular distribution of RhoA, ROCK2 and MLCK, leading to alleviate the LPS-caused damage of intestinal barrier. After addition of Y-27632, BA could further improve the barrier function.
The intestinal mucosal barrier, composed of intestinal epithelial cells and mucus in the intestinal cavity, forms the first defensive barrier against pathogens18. Its function is closely related to the tight junctions and gap junctions between epithelial cells, especially the tight junctions at the apical side of epithelial cells. Tight junctions are formed by the polymerization of several proteins, including transmembrane proteins junction adhesion molecules and triglycerides. Transmembrane proteins include claudins, occludins, and so on. Perimembrane proteins are mainly ZOs, including ZO-1, ZO-2 and ZO-3, which bridge membrane proteins to actin skeleton structure and signal transmitting proteins. Tight junction regulates the transcellular movement of substances including ions and water and closely related to transcellular permeability19.
BA is a short-chain fatty acid that provides 60–70% of the energy required for intestinal mucosal epithelial cells. Studies have confirmed that BA enhances the intestinal barrier integrity by up-regulating the expression of Claudin 2 and decreasing intestinal permeability through an IL-10 receptor-dependent mechanism20. In the presence of hypoxia-inducable-factor-1, as its stabilizer, BA activates genes encoding tight junction protein, and up-regulates the expression of tight junction proteins through transcription factor recombinant protein, to improve intestinal epithelial barrier function21. In this experiment, low final concentration (0-0.5mM) improved cell viability, while high concentration showed the opposite effect. A final concentration of 0.2mM BA treatment Caco2 cells for 24 h could significantly improve cell viability, confirming that low concentration of BA plays a protective role in maintaining intestinal epithelial barrier22 .
In the present study, different concentration of LPS was administrated to Caco2 cells for 18, 24 and 30 h, respectively. Through CCK-8 test, it was found that IC50 of LPS fluctuated in the range of 2–50 ug/ml, therefore, 2–50 ug/ml LPS were added to Caco2 cells for 24 h, the results indicated that BA played a role in protecting epithelial barrier when the concentration of LPS was 2–10 ug/ml. Finally, in vitro model was created by using 0.2 mM BA and 5 ug/ml LPS for 24 h. LPS decreased TEER, and increased FD-4 flux, deteriorated the growth status of cells, down-regulated the expression of ZO-1 and Occludin, changed their intracellular distribution. The data were similar to the previous researches23,24, indicating LPS induced intestine epithelial barrier dysfunction. When BA was administrated, these effects were mitigated significantly, inhibiting BA could reverse the epithelial barrier injury. These results related to BA improving barrier junction were consistent with previous studies9.
RhoA plays a vital role in junction polymerization and function, which leads to abnormal tight junction function of epithelial cells25. Studies have shown that EphB2-Exos has been proved to protect the activity of Caco2 by inhibiting RhoA/ROCK pathway, and promote cell proliferation and migration26. Extracellular enzymes C3, Y-27632 and selective RhoA, ROCK1 and ROCK2 siRNAs significantly inhibited Caco2 epithelial cell migration27. LPS and TNF-α induced ROCK activation in Caco2 cells, which resulted in the relocation of ZO-1 and caludin-2, and ultimately down-regulated TEER and up-regulated FD-4 flux28. In brain capillary endothelial cells, the viability of cells decreased, TEER decreased, and fluorescence yellow permeability increased after LPS treatment. Transmission electron microscopy showed that cell tight junction were damaged after LPS exposure, and the expressions of claudin-5 and ZO-1 decreased, meanwhile, the mRNA expression of claudin-5 and Occludin decreased. By inhibiting RhoA/ROCK2 pathway, catalpol reversed the expression of proteins such as ZO-1 and Occludin, and reversed the increased blood-brain barrier permeability caused by LPS29. In addition, MLCK is also important in the physiological and pathophysiological regulation of epithelial tight junction protein. In vitro experiments showed that ZO-1 remodeling induced by MLCK was similar to Occludin and F-actin. Inflammatory stimulation, such as TNF-α, promoted MLCK activation, and Occludin bridging onto MLCK-dependent actin loops, resulting in endocytosis of Occludin, sequentially increasing paracellular permeability30. After LPS was given to Caco2 cells, the distribution of ZO-1 and claudin-1 changed and activated the MLCK/MLC pathway, resulting in high permeability31. Sodium fluoride activated the calcium-dependent RhoA/ROCK pathway and MLCK, promoted the fracture of ZO-1 in Caco2 cells, changed the intracellular rearrangement of ZO-1 and F-actin, and induced intestinal mucosal damage, while Y-27632 reversed these effects induced by sodium fluoride32. In the study, LPS increased the mRNA expression of RhoA33, ROCK2 34 and MLCK 35 and affected the distribution of these proteins in Caco2 cells. In the control group, RhoA was mainly distributed in the cell membrane and cytoplasm36, ROCK2 and MLCK mainly located around cytoplasm and nucleus37. The expression of RhoA was significantly increased, and moved to the nucleus, the expression of ROCK2 around cytoplasm and nucleus was significantly increased, while the expression level of MLCK around the cell membrane was significantly increased after LPS stimulation38. After BA treatment, the mRNA expression levels of these proteins decreased and their distribution in cells also reversed39. When Y-27632 was added to Caco2 cells for 24 h, the viability and growth status of Caco2 cells, integrality of barrier, the expression and location of ZO-1 and Occludin, as well as the activated status of RhoA/ROCK2/MLCK pathway were further improved40. In summary, Y-27632 played a synergistic role with BA, which further proved that RhoA/ROCK/MLCK pathway participated in the protection of BA from epithelial barrier damage induced by LPS.
There are several limitations to the present investigation. In the present study, we did not evaluate the role of inhibitor of RhoA and MLCK41, nor did we conduct related researches using agonists of RhoA, ROCK2 and MLCK, further research is required to include agonists and other protein inhibitors except Y-27632 to further clarify the function of RhoA/ROCK2/MLCK signaling pathway. Furthermore, we only investigate one signaling pathway, it is still necessary to further expand experiments which relate to mechanisms. In addition, this experiment was only conducted on Caco2 cells, but there are many intestinal epithelial cell lines, which need to be further researched. Moreover, precise and versatile molecular researches should be adopted to better explore the direct target of BA on the RhoA/ROCK2/MLCK signaling pathway.