The Molecular Mechanism Of TLR4/NF-?B Signaling Pathway Contributing To Heat Stress-induced Porcine Inflammatory Bowel Disease

Heat stress is one of the most harmful stress factors to farm animals in southern China.It can induce mucosal damage and inflammation,typical characteristics of inflammatory bowel disease(IBD).TLR4/NF-?B signaling pathway plays an important role in anti-infective immunity and inflammatory processes.However the mechanism of TLR4/NF-?B signaling pathway mediated heat stress-induced porcine IBD is still unclear.This study was conducted to demonstrate the role of TLR4/NF-?B signaling pathway in heat-stress induced porcine IBD,and develop feasible technologies for the prevention and control of this disease.Forty eight pigs(Controls and test groups were 24 and 24 pigs,respectively.)weighed 15 ± 2 kg were used in the study.The control animals were exposed to 21? and 75%-85% humidity.The test animals were subjected to heat stress a higher temperature(34 ± 1?)but similar humidity(75%-85% humidity).The pigs were sacrificed on days 1,7,14,and 21.The extent of inflammation and severity of histopathological lesions in the duodenal,cecal and colonic mucosa were analyzed.The pathological features of heat stress-induced porcine IBD were examined.Western Blot method was used to analyze the response of TLR4,My D88,TRAF6,p65 in heat stress-induced porcine IBD.It is well established that TAK-242 inhibits the activity of TLR4 in the heat shock macrophage model.This model was used to study the mechanism of TLR4/NF-?B pathway and its contribution to heat stress-induced porcine IBD in vitro.Results showed that heat stress induced diarrhea in pigs.Villus length,crypt depth / width,and goblet cell number were significantly decreased in heat stressed pigs.Chronic heat stress increased the number of intestinal intra-epithelial immune cells,expression levels of IL-6,IL-8,and IL-17,and promoted intestinal cellular apoptosis.The ultrastructural pathology showed that the number of mitochondria were reduced and the length of microvilli was decreased in the epithelial cells.In pigs subjected to chronic heat stress the expression of TLR4 and TRAF6 in the duodenum,cecum and colon of the pigs were significantly up-regulated,and p65 activity was enhanced.TLR4 and TRAF6 positive cells were goblet cells,mucosal basal layer immune cells,vascular endothelial cells,and near columnar epithelial cells in the duodenum,cecum and colon.The relative expression of My D88 was also increased but the results were not statistically significant.TLR4/NF-?B signaling pathway was activated by macrophages after heat shock treatment.Following inhibition of the TLR4 activity,the relative expression of its downstream proteins TRAF6 and nuclear import p65 were significantly decreased.After LTR4 activity was inhibited,the relative expression of TRAF6 and nuclear import p65 were significantly higher than that of the controls,but still significantly lower than that of the heat shock treated group.The immunofluorescence results were consistent with the western blot results.Conclusion:(1)This study successfully developed a porcine heat stress-induced IBD model.(2)Heat stress activated TLR4/NF-?B signaling pathway and up-regulated IL-6,IL-8,IL-17 expression in vivo.(3)TLR4/NF-?B signaling pathway mediates the expression of inflammatory factors in heat shock macrophages model in vitro.