The Protective Effects And Molecular Mechanisms Of Ferulic Acid Against Heat Stress-induced Intestinal Mucosa Barrier Dysfunction In Rat

Intestinal mucosal barrier is an important protective barrier, when livestock exposed to heat stress, it causes the intestinal mucosal barrier dysfunction, which increasing the permeability of intestinal mucosal for bacteria and toxins, further triggering inflammatory bowel disease, sepsis and multiple organ failure syndrome. Therefore, it is essential to study the molecular mechanism of heat stress-induced intestinal mucosal barrier disruption, and develop Chinese medicine to against intestinal heat stress. In this study, in order to investigate the molecular mechanism of heat stress-induced intestinal mucosal barrier dysfunction, and to discuss the protective effect of FA against heat stress-induced intestinal mucosal barrier disruption, high-throughput proteomic technology was employed to analyze the effect of heat stress on global protein in rat jejunum.Experiment 1In this study, the rat heat stress model was duplicated to investigate the effects of heat stress on intestinal mucosal barrier function. The results showed that heat stress significantly increase the rectal temperature, serum corticosterone, HSP27/70/90 mRNA expression. Moreover, heat stress significantly increased the permeability to FD4 and pro-inflammatory cytokines TNF-α、IFN-γ、IL-1β mRNA expression. Histological examination and electron microscopy observed that heat stress induced damage to the rat small intestine, and destroyed the tight junction and microvilli structure. These findings revealed that heat stress damage the rat intestinal mucosa barrier function.Experiment 2In order to investigate the molecular mechanism of heat stress-induced intestinal mucosal barrier dysfunction, iTRAQ method was employed to analyze heat stress-induced changes in the intestinal proteome. The results showed that a total of 1689 proteins were identified in rat intestine tissue, of which 41 showed significantly altered expression between the heat-stressed and control groups. And these significant alterations proteins were involved in biological processes such as cellular assembly and organization, cell morphology, cell death and survival. Top canonical pathway analysis showed that Actin Cytoskeleton, MAPK, Tight junction were related to heat stress. We found that members of the MAPK and NF-κB signaling pathways act as hub proteins in the network interaction analysis. Furthermore, western blot analysis verified that the MAPK and NF-κB signaling pathways were activated by heat stress as expected, and heat stress significantly decreased the Occludin, ZO-1, and E-cadherin proteins expression. These findings suggested that heat stress induce the activation of the MAPK and NF-κB signaling pathways, and decrease of the tight junction protein expression, which may contribute ultimately to intestinal mucosal barrier dysfunction.Experiment 3In the present study, we investigated the protective effect and the possible molecular mechanisms of ferulic acid against heat stress-induced intestinal mucosal barrier dysfunction. The results showed that FA could significantly reduce the increase of intestinal permeability induced by heat stress and improve the heat stress-induced intestinal mucosal damage and destruction of the tight junction structure. Additionally, FA significantly attenuated the decrease in occludin, ZO-1 and E-cadherin expression observed with heat stress. Furthermore, we found that the expression of TNF-a, IFN-y and IL-lβ could be effectively inhibited by FA, which mainly regulated by MAPKs and NF-κB signaling pathway, thus significantly weakened heat stress-induced rat intestinal mucosal barrier dysfunction.Experiment 4In this study, the IEC-6 cell heat stress model was established to investigate the effects of heat stress on IEC-6 cell barrier function. The results demonstrated that heat stress caused decreased transepithelial electrical resistance (TER) and increased permeability to 4-kDa fluorescein isothiocyanate (FITC)-dextran (FD4). Both effects were inhibited by FA (5、10、20 μM) in a dose-dependent manner. FA significantly attenuated the decrease in occludin, ZO-1 and E-cadherin expression observed with heat stress. The distortion and redistribution of occludin, ZO-1 and E-cadherin proteins were also effectively prevented by FA pretreatment. Moreover, heat stress diminished electron-dense material detected in tight junctions (TJs), an effect also alleviated by FA in a dose-dependent manner. These findings approved that FA could effectively alleviate the heat stress-induced IEC-6 cell barrier dysfunction.In conclusion, heat stress damaged the rat intestinal mucosa barrier function. FA could inhibite heat stress induced MAPK and NF-κB signaling pathway activation, the release of pro-inflammatory cytokines, and attenuated the decrease in occludin, ZO-1 and E-cadherin expression, which significantly weakened heat stress-induced rat intestinal mucosal barrier dysfunction. Furthermore, FA could effectively alleviate the heat stress-induced IEC-6 cell barrier dysfunction.