Qiu Wenyue, Zhang Xinting, Pang Xiaoyue, Huang Jianjia, Zhou Shuilian, Wu Ruixia, Wang Rongmei, Tang Zhaoxin, Su Rongsheng
College of Veterinary Medicine, South China of Agricultural University, Guangzhou, China.
Yingdong College of Biology and Agriculture, Shaoguan University, Shaoguan, China.
J Anim Physiol Anim Nutr (Berl). 2024 Jan;108(1):194-205. doi: 10.1111/jpn.13878. Epub 2023 Sep 7.
Inflammatory response induced by biological stress usually occurs in weaning piglets, it reduces the production performance of piglets and even causes death. Tert-butylhydroquinone (TBHQ) is a food additive that has the effect of anti-inflammation and anti-oxidation. However, there are few reports related to the protective mechanisms of TBHQ on lipopolysaccharide (LPS) induced injury in intestinal porcine epithelial (IPEC-J2) cells. Quantitative real-time polymerase chain reaction and western blot analysis, respectively, detected the mRNA levels and protein expressions related to pyroptosis, tight junction (TJ) protein and high-mobility group box 1/toll-like receptor 4/nuclear factor kappa-B (HMGB1/TLR4/NF-κB) axis. Localisation and expression of NOD-like receptor pyrin domain containing 3 (NLRP3), HMGB1 and P-NF-κB proteins detected by immunofluorescence. The results showed that TBHQ (12.5 and 25 μM) can increase cell activity and reduce intracellular lactate dehydrogenase (LDH) levels in a dose-dependent manner. LPS significantly decreases cell viability and increases the LDH level. However, pretreatment with TBHQ evidently increases cell viability and decreases the LDH level of IPEC-J2 cells. In addition, treatment with LPS decreased the mRNA level and protein expression of zonula occludens-1, occludin and claudin-1, and increased the mRNA level and protein expression of pyroptosis and HMGB1/TLR4/NF-κB axis. Interestingly, pretreatment with TBHQ increased the TJ protein expressions as well as decreased the mRNA level and protein expressions of pyroptosis and HMGB1/TLR4/NF-κB axis. Moreover, the results of immunofluorescence showed that TBHQ significantly reduced the expression of NLRP3, HMGB1 and P-NF-κB in LPS-induced injury of IPEC-J2 cells. Therefore, we come to the conclusion that TBHQ attenuates LPS-induced pyroptosis in IPEC-J2 cells through downregulation of the HMGB1/TLR4/NF-κB axis, TBHQ may become a potential feed additive for preventing inflammatory diarrhoea in piglets.
生物应激诱导的炎症反应通常发生在断奶仔猪中，它会降低仔猪的生产性能，甚至导致死亡。叔丁基对苯二酚（TBHQ）是一种具有抗炎和抗氧化作用的食品添加剂。然而，关于TBHQ对脂多糖（LPS）诱导的猪肠上皮（IPEC-J2）细胞损伤的保护机制的相关报道较少。分别采用定量实时聚合酶链反应和蛋白质免疫印迹分析检测与细胞焦亡、紧密连接（TJ）蛋白以及高迁移率族蛋白B1/ Toll样受体4/核因子κB（HMGB1/TLR4/NF-κB）轴相关的mRNA水平和蛋白表达。通过免疫荧光检测含NOD样受体吡咯结构域蛋白3（NLRP3）、HMGB1和磷酸化核因子κB（P-NF-κB）蛋白的定位和表达。结果表明，TBHQ（12.5和25μM）能以剂量依赖的方式增加细胞活性并降低细胞内乳酸脱氢酶（LDH）水平。LPS显著降低细胞活力并增加LDH水平。然而，用TBHQ预处理明显增加了IPEC-J2细胞的活力并降低了LDH水平。此外，LPS处理降低了闭合蛋白-1、闭锁蛋白和Claudin-1的mRNA水平和蛋白表达，并增加了细胞焦亡以及HMGB1/TLR4/NF-κB轴的mRNA水平和蛋白表达。有趣的是，用TBHQ预处理增加了TJ蛋白表达，同时降低了细胞焦亡以及HMGB1/TLR4/NF-κB轴的mRNA水平和蛋白表达。此外，免疫荧光结果显示，TBHQ显著降低了LPS诱导的IPEC-J2细胞损伤中NLRP3、HMGB1和P-NF-κB的表达。因此，我们得出结论，TBHQ通过下调HMGB1/TLR4/NF-κB轴减轻LPS诱导的IPEC-J2细胞焦亡，TBHQ可能成为预防仔猪炎症性腹泻的潜在饲料添加剂。
