内质网CHOP信号介导热应激猪肠道屏障损伤的机制研究

Intestine is a target tissue susceptible to heat stress. Chronic heat stress causes thin and even absent mucous layer. The damage to protective layer facilitates the diffusion of virus, bacteria, etc. from the gastrointestinal lumen to the blood, leading to intestinal inflammation and diseases. Our previous studies have demonstrated that chronic heat exposure induces endoplasmic reticulum (ER) stress response of intestinal mucosa in pigs. However, the mechanisms by which it regulates intestine mucosa injure due to heat loads are not extensively elucidated. In this study, regulatory mechanisms will be studied that ER stress-mediated apoptosis mediate intestine injure because of heat exposure in vitro and in vivo. We will establish pig model of heat stress to investigate the effects of heat exposure on expression of ER stress sensor proteins, marker proteins, and apoptosis proteins as well as goblet cell number, mucous layer, mucin contents and intestinal permeability using real-time PCR, western blot, immunofluorescence and PAS assay, so as to analyze the relationship between ER stress-mediated apoptosis pathway CHOP and heat exposure-induced damage to intestine. Furthermore, we also use the CHOP siRNA porcine small intestinal epithelial cell line (IPEC-J2) and growing pigs with inhibitor to further clarify the mechanisms of intestine injury regulated by ER stress-mediated apoptosis pathway CHOP under heat stress. These findings will provide a new insight for developing future mitigating nutrition to reduce intestinal injury due to high ambient temperature.

肠道是动物热应激的靶器官，长期热应激造成猪肠道黏液层变薄甚至缺失，致使病毒、细菌等穿过肠道屏障进入血液，诱发各种疾病。前期研究发现，热应激导致猪肠道黏膜发生内质网应激。内质网应激调控黏膜屏障热损伤机制尚待探究。本项目通过体内动物试验和体外细胞试验，研究内质网CHOP信号介导热应激猪肠道屏障损伤的分子机制。通过构建生长猪热应激模型，应用Western blot、免疫荧光、PAS染色等方法研究热应激对小肠黏膜内质网应激感应蛋白、标志蛋白、凋亡蛋白的表达和分布，杯状细胞的数量、黏液层厚度、黏蛋白含量、黏膜通透性的影响，分析内质网应激性凋亡通路CHOP与黏膜屏障损伤的关系；进一步通过构建猪肠道上皮细胞和生长猪热应激模型，应用CHOP siRNA结合信号通路阻断抑制技术，解析内质网应激性凋亡通路CHOP介导热应激猪肠道黏膜损伤的作用机制，为利用营养素缓解肠道黏膜屏障热应激损伤提供新的思路。

本项目通过体内动物试验和体外细胞试验，研究内质网CHOP信号介导热应激猪肠道屏障损伤的分子机制。通过肥育猪热应激模型，应用气相色谱和质谱（GC-MS）技术，分析热应激猪血清代谢组，证实热应激导致猪肠道屏障损伤，导致肠腔微生物有害代谢产物进入血液。通过检测质内质网应激的标志蛋白GRP78、内质网促凋亡蛋白CHOP表达变化，发现长期热应激诱发内质网应激响应。构建了猪小肠上皮细胞（IPEC-J2）的热应激模型，通过转录组测序、内质网应激特异性抑制剂（4-PBA）处理和siRAN干扰技术分析首次发现短期热应激（43 ℃，2 h）诱导eif2α的磷酸化，激活p-eif2α-CHOP通路，导致内质网应激性凋亡。抑制内质网应激缓解了热应激诱导的肠道屏障损伤。本研究首次发现，长期热应（43 ℃，12 h）激特异性诱导内质网应激支路ATF4-CHOP，一方面调控促凋亡蛋白BAX重定位于线粒体膜，导致线粒体膜去极化， Cyt c渗漏到细胞质，启动线粒体凋亡途径；另一方面ATF4-CHOP调控下游谷胱甘肽降解酶CHAC1, 直接降解还原型谷胱甘肽，造成肠道黏膜屏障氧化还原失衡，导致氧化应激和细胞凋亡。这些新机制的解析提示我们抑制细胞内质网应激是缓解热应激的重要途径，这些新的发现将为靶向营养素干预热应激提供新的思路。