槲皮素通过PKC/MLCK信号通路缓解仔猪肠道机械屏障功能损伤的机制研究

The diarrhea and intestinal inflammation of piglets are common diseases that restrict the pig-breeding industry development, and impaired intestinal epithelial barrier function is one of the important factors that causing these diseases. In the trend of banning the use of medical feed additives, regulation of piglet intestinal health by nutritional strategies has become the topic issue in nutrition research of piglets. The current studies indicated that quercetin can maintain the integrity of intestinal mechanical barrier function by regulating the expression and distribution of tight junction proteins, but the underlying mechanism is still unclear. Therefore, through the establishment of intestinal epithelial barrier injure model induced by lipopolysaccharide in vivo and in vitro and the use of PKC and MLCK inhibitors, the present study aims to investigate the effects of quercetin on the growth performance and intestinal mechanical barrier function of piglets by immunofluorescence, transmission electron microscope, real-time PCR, and Western blot technology, together with the regulation mechanism of quercetin on the gene and protein expression of PKC/MLCK signaling pathway-associated effector proteins. These results will reveal the effect and regulation mechanism of quercetin on attenuating intestinal mechanical barrier function injury of piglets, which will provide nutritional regulation means for promoting intestinal healthy of piglets, and will provide a theoretical basis of the quercetin as a functional feed additive in pig production.

仔猪腹泻和肠道炎症是制约生猪产业发展的常见疾病，而肠上皮屏障功能受损是引发这些疾病的重要因素之一。在药物饲料添加剂将全面禁止趋势下，寻求通过营养调控策略保障仔猪肠道健康，已成为当前仔猪营养研究的重中之重。研究表明，槲皮素可通过调节紧密连接蛋白的表达和分布来维系肠道机械屏障功能的完整性，但其内在作用机制尚不清楚。因此，本项目拟通过脂多糖诱导仔猪肠道屏障功能损伤，采用免疫荧光染色、透射电镜扫描、real-time PCR、Western blot等技术研究槲皮素对其生长和肠道机械屏障功能的影响；并采用槲皮素、PKC和MLCK通路的抑制剂处理脂多糖诱导的IPEC-J2细胞，检测PKC/MLCK信号通路相关效应蛋白的基因和蛋白表达，旨在揭示槲皮素缓解仔猪肠道屏障功能受损的分子机制，为促进仔猪肠道健康提供营养调控手段，并为槲皮素作为绿色功能性饲料添加剂应用于养猪生产中提供理论依据。

断奶应激可能会引起肠道屏障功能损伤和肠道炎症，从而导致仔猪生长性能降低。因此，本试验采用脂多糖诱导的断奶仔猪免疫应激模型的体内试验和TNF-α诱导的猪空肠上皮细胞（IPEC-J2）免疫应激模型的体外试验，探讨了槲皮素缓解断奶仔猪肠道屏障免疫应激损伤的保护作用及其分子机制。选取胎次和体重相近（7.45 ± 0.09 kg）、健康的48头21日龄断奶的三元杂交仔猪（杜洛克×长白×大白）随机分为6个处理组，每个处理组8头猪。试验分组如下：对照组（Control，饲喂基础日粮并注射无菌生理盐水）；LPS模型组（LPS，饲喂基础日粮并注射LPS）；0.02%槲皮素组（饲喂基础日粮中添加0.02%的槲皮素并注射生理盐水）；LPS + 0.02%槲皮素组（饲喂基础日粮中添加0.02%的槲皮素并注射LPS）；0.04%槲皮素组（饲喂基础日粮中添加0.04%的槲皮素并注射生理盐水）；LPS + 0.04%槲皮素组（饲喂基础日粮中添加0.04%的槲皮素并注射LPS）。试验期19 d，在试验的第19天屠宰前4 h，在仔猪腹腔注射100 μg/kg‍ 体重（BW）LPS和相同剂量的生理盐水。选取IPEC-J2细胞作为研究对象，建立TNF-α诱导IPEC-J2细胞免疫应激损伤模型。利用特异性PKCα抑制剂和MLCK抑制剂靶向阻断PKCα和MLCK信号通路等方法，研究槲皮素缓解TNF-α对IPEC-J2细胞屏障免疫应激损伤的作用机制。研究表明，仔猪断奶应激，引起多种基因和蛋白的变化，从而肠道结构和功能的破坏。日粮添加槲皮素可改善免疫应激仔猪肠道形态结构、降低肠道通透性、增强肠道紧密连接蛋白的表达和分布来改善应激受损的仔猪肠道屏障功能，其潜在机制可能是通过调节PKCα/MLCK信号通路。通过体外细胞试验表明槲皮素可通过抑制PKCα/MLCK信号通路，调控IPEC-J2细胞紧密连接蛋白的表达和分布，从而缓解免疫应激导致的肠道屏障损伤。

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