TLR4/核因子κB介导热应激猪肠Treg/Th17失衡机制及在炎性肠病发生中的地位

Pig's inflammatory bowel disease(IBD) is widespread in China recently years.Heat stress, as well as a lot kinds of pathogenic bacterias,induce the take place of IBD in pigs. We found that the imbalance of Treg/Th17 in intestinal gut of pigs was correlated with the up-regulation of TLR4 in previous studies, speculated that TLR4 may mediated the imbalance of Treg/Th17 and play an important role in IBD induced by heat stress in pigs. In order to confirm our hypothesis, the following experiments will be conducted: (1) The expression of Foxp3 and RORγt will be detected by qRT-PCR and western blot as well the number of Treg/Th17 by flow cytometer in intestinal gut of heat stressed pigs;(2) Microstructure and ultrastructure of intestinal gut will be detected by light microscope and electron microscope as well the expression of proinflammatory cytokine by Elisa;(3) The expression of TLR4 and the activity of NF-κB will be detected by western-blot and EMSA,respectively. Elucidating the relationship between Treg/Th17 imbalance and the TLR4/NF-κB signal pathway of intestinal DC, which may be a upstream regulators of CD4+ T lymphocyte differention. (4) Investigating the role of TLR4/NF-κB pathway in the Treg/Th17 balance and intestinal inflammatory, the TLR4 high- expressing and gene silence plasmid will be constructed and transformed to DC, then co-cultured with intestinal cells or the supernatant be added to na?ve CD4+ T lymphocyte after heat shocked, and the number of Treg/Th17,the expression of RORγt/Foxp3 and the proinflammatory cytokine will be detected. (5) Investigating the role of TLR4/NF-κB in the formation of IBD induced by heat stress,the in vitro culture model of intestinal segment will be constructed, and the histologic structure and cytokines will be detected after the antagonist of TLR4/NF-κB pathway added and followed by heat stress. Consequently, the present study not only to elucidate the mechanism of TLR4/nuclear factor-κB mediates the imbalance of Treg/Th17 and it's positon in intestinal inflammation development in pigs under heat stress,but will constructe a base for completely discovery of pathology of IBD in pigs.

以腹泻为主要特征的猪炎性肠病（IBD）近年流行广泛，应激是诱因之一。前期研究发现TLR4上调与热应激猪肠Treg/Th17失衡高度相关，推测其介导促分化因子改变是失衡和炎症的重要原因。拟采用荧光定量、免疫印迹和基因沉默等技术，检测热应激猪：肠Treg/Th17数量、Foxp3、RORγt及促分化因子表达；肠结构和炎性因子表达；肠DC中TLR4表达和NF-κB活性，分析TLR4/NF-κB与失衡和炎症的相关性。分离培养DC，分别转染TLR4表达载体和RNAi载体，再热休克，检测NF-κB活性，并用上清处理幼稚CD4+T细胞，分析TLR4对NF-κB和Treg/Th17分化的影响；体外培养肠囊，拮抗物阻断TLR4/NF-κB通路，再热休克，分析对炎症的影响。体内外试验相互印证，阐述TLR4/NF-κB介导热应激猪肠Treg/Th17失衡机制及在IBD中的地位，为全面揭示IBD发病机制奠定基础。

以腹泻为主要特征的猪炎性肠病（IBD）近年流行广泛，应激是诱因之一，阐述其诱发IBD的分子机理可为该病的综合防控奠定基础。本项目采用荧光定量、免疫印迹和基因沉默等技术，对TLR4/NF-κB信号通路介导热应激猪肠Treg/Th17失衡的分子机制、Treg/Th17失衡在IBD发生中的地位及可能的防控策略进行了系统研究，发现：① 在温度35℃、湿度85%的饲养环境下，受试猪第7-14天出现以肠绒毛变短、隐窝变浅、炎性细胞浸润、粘膜下水肿及腹泻为主要特征的炎性肠病；② 热应激以激活MyD88非依赖性TLR4/NF-κB信号通路而上调炎性因子IL-6、IL-8和IL-17的表达，致肠CD4+T细胞由Treg向Th17飘移，从而驱动肠道的炎症过程是炎性肠病发生的主要原因；③ 构建了猪GM-CSF和IL-4的真核表达载体，转染IPEC-J2细胞后成功建立了GM-CSF和IL-4稳传细胞系。以该细胞系的培养上清为诱导剂，可刺激猪骨髓细胞分化成骨髓源DC细胞；④ HSP70过表达能显著增强肠上皮细胞和DC细胞NF-κB活性。但高表达TLR4，仅上调了DC细胞中NF-κB活性，肠上皮细胞未见明显改变。同时过表达HSP70和TLR4后，NF-κB活性显著增强，可见HSP70和TLR4能协同激活NF-κB信号通路；⑤ 在转录组水平上发现热应激处理的第7-21天TLR4/NF-κB信号通路关键分子显著上调，其下游炎性因子IL-6、IL-8、IL-17和IL-22表达水平亦见明显增高，再次印证了TLR4/NF-κB信号通路激活是热应激诱发炎性肠病重要机制的事实；⑥ 热应激猪肠道紧密连接蛋白Claudin-4下调，而磷酸化P38和ERK上调。体外培养的肠上皮细胞热休克处理5小时后紧密连接蛋白ZO-1、Claudin-4下调,而磷酸化P38和ERK仍上调，抑制P38和ERK后，上述现象消失。可见热应激经MAPK信号通路调控紧密连接蛋白表达致肠粘膜损伤是致病的另一重要机制；⑦ 海洋多糖（壳聚糖）能以TLR4信号通路为作用靶点，经抑制TLR4/NF-κB和TLR4/P38信号通路的激活而下调致炎因子IL-6和IL-8的表达，发挥抗炎和粘膜修复作用，是炎性肠病防控的潜在天然海洋药物。研究结果将为全面揭示猪应激性炎性肠病的分子机制奠定基础，为基于天然海洋药物的防控技术开发提供依据。