The aim of this study was to investigate the role of necroptosis in deoxynivalenol (DON)-induced liver injury and inflammation in weaned piglets. In Exp. 1, 12 weaned piglets were divided into 2 groups including pigs fed basal diet and pigs fed diet contaminated with 4 mg/kg DON for 21 d. In Exp. 2, 12 weaned piglets were divided into 2 groups including control piglets and piglets given a gavage of 2 mg/kg body weight (BW) DON. In Exp. 3, 24 weaned piglets were used in a 2 × 2 factorial design and the main factors including necrostatin-1 (Nec-1) (DMSO or 0.5 mg/kg BW Nec-1) and DON challenge (saline or 2 mg/kg BW DON gavage). On 21 d in Exp. 1, or at 6 h post DON gavage in Exp. 2 and 3, pigs were killed for blood samples and liver tissues. Liver histology, blood biochemical indicators, and liver inflammation and necroptosis signals were tested. Dietary or oral gavage with DON caused liver morphological damage in piglets. Dietary DON led to hepatocyte damage indicated by increased aspartate transaminase (AST) activity and AST/alanine aminotransferase (ALT) ratio, and DON gavage also caused hepatocyte damage and cholestasis indicated by increased AST and alkaline phosphatase (AKP) activities. Dietary DON caused liver necroptosis indicated by increased protein abundance of total receptor interacting protein kinase 3 (t-RIP3) and total mixed lineage kinase domain-like protein (t-MLKL). Moreover, DON gavage increased mRNA expression of interleukin (IL)-6 and IL-1β in liver. DON gavage also induced liver necroptosis demonstrated by increased protein abundance of t-RIP3, phosphorylated-RIP3 (p-RIP3), t-MLKL and p-MLKL. However, pretreatment with Nec-1, a specific inhibitor of necroptosis, inhibited liver necroptosis indicated by decreased protein expression of t-RIP3, p-RIP3, t-MLKL and p-MLKL. Nec-1 pretreatment reduced liver morphological damage after DON gavage. Pretreatment with Nec-1 also attenuated liver damage induced by DON indicated by decreased activities of AST and AKP. Furthermore, Nec-1 pretreatment inhibited liver mRNA expression of IL-6 and IL-1β after DON challenge. Our data demonstrate for the first time that necroptosis contributes to DON-induced liver injury and inflammation in piglets.

中文翻译：

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坏死性凋亡导致脱氧雪腐镰刀菌烯醇诱导的断奶仔猪肝损伤和炎症


本研究的目的是探讨坏死性凋亡在脱氧雪腐镰刀菌烯醇 （DON） 诱导的断奶仔猪肝损伤和炎症中的作用。在实验 1 中，将 12 头断奶仔猪分为 2 组，包括饲喂基础日粮的仔猪和饲喂被 4 mg/kg DON 污染的日粮的仔猪 21 天。在实验 2 中，将 12 头断奶仔猪分为 2 组，包括对照仔猪和饲喂 2 mg/kg 体重 （BW） DON 的仔猪。在实验 3 中，24 头断奶仔猪采用 2 × 2 析因设计，主要因素包括坏死他汀-1 （Nec-1）（DMSO 或 0.5 mg/kg BW Nec-1）和 DON 攻击（生理盐水或 2 mg/kg BW DON 强饲法）。在实验 1 的 21 天，或在实验 2 和 3 的 DON 强饲法后 6 小时，杀死猪以获取血液样本和肝组织。检测肝脏组织学、血液生化指标、肝脏炎症和坏死性凋亡信号。含 DON 的日粮或口服强饲法导致仔猪肝脏形态损伤。膳食 DON 导致肝细胞损伤，表现为天冬氨酸转氨酶 （AST） 活性和 AST/丙氨酸氨基转移酶 （ALT） 比值增加，DON 管饲法也导致肝细胞损伤和胆汁淤积，表现为 AST 和碱性磷酸酶 （AKP） 活性增加。膳食 DON 引起的肝坏死性凋亡表现为总受体相互作用蛋白激酶 3 （t-RIP3） 和总混合谱系激酶结构域样蛋白 （t-MLKL） 的蛋白质丰度增加。此外，DON 强饲法增加了肝脏中白细胞介素 （IL）-6 和 IL-1β 的 mRNA 表达。DON 管饲法还诱导肝脏坏死性凋亡，表现为 t-RIP3 、 磷酸化 RIP3 （p-RIP3） 、 t-MLKL 和 p-MLKL 的蛋白质丰度增加。 然而，用坏死性凋亡的特异性抑制剂 Nec-1 预处理可抑制 t-RIP3、p-RIP3、t-MLKL 和 p-MLKL 蛋白表达降低的肝脏坏死性凋亡。Nec-1 预处理减少了 DON 灌胃后肝脏形态学损伤。用 Nec-1 预处理也减轻了 DON 诱导的肝损伤，表现为 AST 和 AKP 活性降低。此外，NEC-1 预处理抑制了 DON 攻击后肝脏 IL-6 和 IL-1β mRNA 的表达。我们的数据首次表明坏死性凋亡会导致 DON 诱导的仔猪肝损伤和炎症。