The Role Of MTOR Pathway In Intestinal Mucosal Immune Stress Of Broilers Induced By LPS

The intestinal mucosal immune system plays an important role in the immune response, which is essential for maintaining the organism health. Based on the inflammatory response induced by LPS, we establish the chicken model in vivo and in the intestinal tissue cultured in vitro, to analyze whether mTOR/p70S6 K signaling pathway involved in the mechanism of inflammatory response activated by LPS. At the same time, we study the regulating function in the perspective of diet nutrition factors and discuss preliminarily the mechanism of mTOR/p70S6 K involving in nutritional regulation. 1. The effect of RAPA on the development of intestinal mucosaThree groups of 4-day-old male chicks were randomly subjected intraperitoneally to one of the following treatments for 6 days: high-dose RAPA(RAPA, a specific inhibitor of mTOR; 1.0 mg/kg body weight, once daily at 09:00 h), low-dose RAPA(0.4 mg/kg body weight), and controls(injected with RAPA vehicle). The results showed that RAPA treatments decreased body weight gain(P < 0.05), incressed the mortality rate(P < 0.05) and caused morphological damages in small intestinal, such as shorter, atrophied villi(P < 0.05) and significantly reduced the Clauding1 protein expression(P < 0.001). Meanwhile, immunohistochemistry and western blot results showed that the expression of IgA was significantly decreased(P < 0.001), which indicated the mTOR signaling involved in the development of the intestinal mucosa and SIgA protein synthesis. 2. The mechanism of intestinal mucosal immune induced by LPS orally treatment4-day-old AA broilers were respectively administrated orally by LPS at 0.25 mg/kg BW and 0.50 mg/kg BW, to study the influence of LPS on the intestinal mucosal immune mechanism at 4h-24 h time points. The results of SIgA concentration showed that there is no influence in the duodenum and jejunum and significant influence in ileum after LPS treatment, especially the LPS in high dose- 500 μg/kg BW, which presents a time-dependent increase. Meanwhile, phosphorylation-level of NF-κB p65 and p38 MAPK protein increased significantly at 4-6h(P < 0.05), and the difference is smaller and smaller with the time extension, and then, to 18 h, 21 h the protein expression significantly reduced(P < 0.05). The results demonstrated that the LPS orally treatment can cause the immune stress in the intestinal mucosal, and the effect on the intestine showed the specificity at different intestinal segments and different time points. 3. The culture of intestinal tissue in vitro and establishment of the immune stress model induced by LPSWe establish the model of intestinal tissue culture in vitro with the E19 SPF chicken embryos, which were treated by LPS at 0, 0.5, 1, 2 μg/m L to observe the effects on the intestinal immune at 2h and 4h after LPS treatment. According to the results, we found SIgA concentration increased gradually with the increase of LPS doses for 2h after LPS treatment, and lead to the significant difference at 2 μg/mL LPS(P < 0.05). And LPS increased the mRNA level of inflammatory cytokines and phosphorylation level of NF-κB and MAPK protein expression, respectively, especially in the group with 2 μg/ml LPS for 2h(P < 0.05). All of the results indicated that LPS can induce the immune stress in the intestinal tissue in vitro and activate the TLR- NF-κB and MAPK- inflammatory cytokines- SIgA signaling pathways.4. The mTOR signaling involved in mechanism of inflammatory response induced by LPSAt the experiment 3, the mTOR signaling molecules which maybe connect with the SIgA protein synthesis were detected and the results showed LPS significantly decreased the phosphorylation levels of mTOR/p70S6 K protein expression(P < 0.05), which suggested the mTOR signaling pathway may involve in the mechanism of the immune response induced by LPS. Leu at 10 mM, 20 mM and 40 mM was added to the intestinal tissue model by 2 μg/mL LPS for 2h to restore the expression of mTOR signaling. And the results showed that 40 mM Leu can restore the phosphorylation level of the mTOR/p70S6 K protein decreased by LPS, and inhibited the inflammatory cytokines, SIgA concentration and phosphorylation level of the NF-κB p65, p38, JNK MAPK protein activated by LPS(P < 0.05), which indicated that mTOR/p70S6 K signaling pathway involved the inflammatory response induced by LPS and regulated the immune response of downstream signaling molecules 5. The mTOR signaling involved in mechanism of nutritional factors regulating immunological stressThe study discussed the effect of LPS treatment with or without 5-HTP and VA diet on the intestinal mucosal immune.80 broilers at 7-day-old with similar weight were selected to given the 0.2% 5- HTP diet and basic diet for 4w, and then were injected intraperitoneally 1 mg/kg BW LPS. The results showed that LPS treatment with the basic diet can decreased significantly the phosphorylation level of mTOR downstream p70S6 K inhibited by LPS(P < 0.05), activated the inflammatory cytokines and the expression of NF-κB and/or MAPK signaling molecules(P < 0.05). However, the mRNA level of cytokines remarkably was decreased and NF-κB/MAPKcytokine signaling pathways were inhibited by LPS with the 5-HTP diet, suggesting that 5-HTP alleviate the inflammatory response induced by LPS. Meanwhile, LPS with 5-HTP diet restored phosphorylation level of mTOR downstream p70S6 K inhibited by LPS, and increased the SIgA concentration in the duodenum solution(P < 0.05).80 broilers with similar weight were selected to given the 1500IU/kg and 6000IU/kg VA diet for 10 d, and then were sprayed treatment with 1 mg/kg BW LPS for 2h which induced the inflammatory response in the respiratory tract. The results showed that LPS with the basic diet activated the expression of inflammatory cytokines and p38 MAPK signaling pathways(P < 0.05), while LPS with VA diet didn’t show the similiar changes, but it increased the expression of the TJs and the phosphorylation level of p70S6 K was higher than it in the basic diet, which reflect VA could protect the body from the external stress damage and enhance immunity. The reults indicated that the mTOR/p70S6 K signaling pathway may be involved in the mechanism of nutrition factors regulating the immune stress on intestinal mucosal immunity.In conclusion, mTOR signaling pathway involved in the development of intestinal mucosa and synthesis of SIgA. And the mTOR signaling pathways involved in mechanism of mucosal immune regulation in the model of intestinal tissue culture in vitro and in vivo induced by LPS in broilers. At the same time, 5- HTP and VA nutritional factors can relieve the inflammatory response induced by LPS, and also preliminarily discussed that the mTOR signaling pathway involved in mechanism of nutritional factors regulation on the mucosal immune, which has important significance for the research of the prevention of poultry’s stress and regulating mechanism of nutritional factors.