基于LPS/TLR4信号通路研究牛磺酸对免疫应激仔猪腹泻预防作用及机制

The occurrence of diarrhea in piglets is closely related to the hyperactivation of inflammation.Our preliminary research shows that taurine can suppress TLR4 and IL - 6 mRNA expression in the ileum mucosa epithelial cells the to the immune stress piglets. It showed that taurine can inhibit the excessive activation of inflammation, but the concrete target is unclear.In this study, lipid A structure will be used to detect by ESI/MS and X-ray single crystal diffraction, collection rate of LPS and cell will be detected by the FCM , CD14 and MD2 expression levels will be detected by the q-PCR and WB to reveal the effect of taurine on the extracellular signal of TLR4 ; the level of total TLR4 , TLR4 on the cell membrane and the localization in the intracellular TLR4 will be detected by means of WB, flow cytometry, fluorescence triad combined with laser confocal technology to reveal the effects of taurine on TLR4 distribution; The key factor levels, channel negative regulatory factors level will be detected through the q - PCR and WB to clear the specific target of taurine on the intracellular signal transduction pathways of TLR4. Above all, the inhibitory mechanism of taurine on inflammatory hyperactivity will be revealed and to provide a theoretical basis for the application of taurine as a nutritional immune additive to prevent diarrhea in piglets.

仔猪腹泻的发生与炎症过度激活密切相关。本课题组前期研究表明，预防性添加牛磺酸能够抑制免疫应激仔猪回肠粘膜上皮细胞TLR4及IL-6mRNA表达，表明牛磺酸可以抑制炎症过度激活，但对作用的具体靶点尚不明确。本研究拟通过X单晶衍射结合电喷雾质谱方法（ESI/MS）检测类脂A结构、流式细胞术检测LPS与细胞结合率、q-PCR及WB检测CD14及MD2基因及蛋白表达水平，揭示牛磺酸对LPS与TLR4胞外信号作用靶点；通过WB、流式细胞术、荧光三标结合激光共聚焦显微技术分别检测细胞总TLR4及膜TLR4水平、胞内TLR4定位，探明牛磺酸对TLR4分布的影响；结合通路抑制剂及转染CSD干扰载体，通过q-PCR及WB检测TLR4信号转导通路关键因子、通路负调控因子基因及蛋白水平，明确牛磺酸对TLR4受体后作用靶点；揭示牛磺酸对炎症过度激活的抑制机制，为应用牛磺酸作为营养免疫添加剂预防仔猪腹泻提供理依据。

仔猪腹泻是最主要的仔猪疾病之一，也是引起仔猪死亡的重要原因。引起仔猪腹泻的原因错综复杂，其中，促炎因子的过度释放是引发仔猪免疫应激、腹泻发生的重要因素。由于仔猪耐受力差、耐药性等问题，仔猪腹泻的治疗效果并不理想。因此，采取有效措施对其进行预防要比发病后的治疗更有意义。本项目结合体内、体外试验，考察预防性添加牛磺酸对免疫应激仔猪TLR4 胞外信号、跨膜转导、通路负调控因子的作用，从而明确牛磺酸对 TLR4 信号通路作用具体靶点，揭示牛磺酸对仔猪腹泻预防作用及调控机制。研究结果表明：饲料中添加 0.3%牛磺酸可以降低料肉比，提高小肠绒毛长度、降低隐窝深度、提高绒/隐比值，调节结肠中微生物的组成成分和代谢物的含量，改善结肠紧密连接蛋白的表达，有效地缓解了 LPS造成的仔猪结肠屏障损伤；降低肠粘膜组织 TLR4 及 TLR4 前信号分子、TLR4 受体后信号分子、通路负调控因子基因及部分蛋白表达量，降低仔猪肠粘膜组织中炎性因子的基因表达量，降低 LPS 诱导仔猪腹泻率。体外培养IPEC-J2细胞，培养基中添加牛磺酸（20 mM）能够降低 LPS（10 µg/ml）与TLR4受体结合率，降低 TLR4 及受体前信号分子基因表达水平，降低 TLR4 受体后信号分子基因及蛋白表达量、减低LPS诱导的促炎因子水平。本研究结果将为应用牛磺酸作为营养免疫添加剂预防仔猪腹泻提供理论依据。