Free fatty acids impair autophagic activity and activate nuclear factor kappa B signaling and NLR family pyrin domain containing 3 inflammasome in calf hepatocytes.
Free fatty acids impair autophagic activity and activate nuclear factor kappa B signaling and NLR family pyrin domain containing 3 inflammasome in calf hepatocytes.
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游离脂肪酸损害小牛肝细胞中的自噬活性并激活核因子κ B信号传导和含3个炎性体的NLR家族pyrin结构域。
DOI：
10.3168/jds.2021-20273
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发表时间：
2021-08
影响因子：
3.5
通讯作者：
Taiyu Shen;Xinwei Li;Bo Jin;J. Loor;A. Aboragah;Lingxue Ju;Zhiyuan Fang;Hao Yu;Meng Chen-Meng-Che
中科院分区：
文献类型：
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作者：
Taiyu Shen;Xinwei Li;Bo Jin;J. Loor;A. Aboragah;Lingxue Ju;Zhiyuan Fang;Hao Yu;Meng Chen-Meng-Che
游离脂肪酸（FFA）诱导的肝脏炎症加重了酮病或脂肪肝奶牛的肝损伤和代谢功能障碍。在应激条件下，自噬通常被认为是一种细胞保护机制，但FFA诱导的肝细胞炎症和应激效应是否涉及自噬反应尚不清楚。因此，本研究的目的是探讨游离脂肪酸对小牛肝细胞自噬的影响以及自噬在核因子-κB（nuclear factor kappa B）信号转导和NLRP 3（NLR family pyrin domain containing 3）炎性体激活中的作用。从3头健康荷斯坦雌性新生小牛（1日龄，30-40 kg）中分离小牛肝细胞，并在用或不用自噬抑制剂氯喹（CQ）或自噬激活剂雷帕霉素处理后暴露于各种浓度的FFA（0、0.3、0.6或1.2 mM）。自噬标志物LC 3（微管相关蛋白1轻链3）和p62（隔离体1）、NF-κB信号传导和NLRP 3炎性小体相关分子的表达通过蛋白质印迹和定量实时PCR进行分析。结果显示，0.6和1.2 mM FFA激活NF-κB信号传导和NLRP 3炎性体，如p-NF-κB/NF-κB比率升高、NLRP 3和CASP 1（半胱天冬酶1）的蛋白丰度、CASP 1活性以及IL 1 B和IL 18的mRNA丰度所示。此外，用0.6和1.2 mM FFA或自噬抑制剂CQ处理的肝细胞显示p62和LC 3-II的蛋白丰度增加。此外，在用1.2 mM FFA和1.2 mM FFA加CQ处理的小牛肝细胞之间，p62和LC 3-II的蛋白丰度没有差异，表明FFA抑制小牛肝细胞中的自噬活性。CQ处理导致NF-κB信号和NLRP 3炎性体过度活化。此外，CQ加1.2 mM FFA加重FFA诱导的炎症。相比之下，雷帕霉素诱导的自噬改善了FFA激活的NF-κB信号传导和NLRP 3炎性体，如通过较低的p-NF-κB/NF-κB比率、NLRP 3和CASP 1的蛋白丰度、CASP 1的活性以及IL 1 B和IL 18的mRNA丰度所证明的。总的来说，抑制自噬加剧，而诱导自噬减轻，FFA诱导的小牛肝细胞的炎症过程，这表明自噬损伤可能是部分负责肝脏炎症和随后的肝损伤的奶牛酮病或脂肪肝。因此，调节自噬可能是控制体内明显炎症反应的有效治疗策略。
Free fatty acids (FFA)-induced hepatic inflammation agravates liver injury and metabolic dysfunction in dairy cows with ketosis or fatty liver. Under stressful conditions, autophagy is generally considered as a cell protection mechanism, but whether the FFA-induced inflammatory and stress effect on hepatocytes involves an autophagy response is not well known. Thus, the objective of this study was to investigate the effects of FFA on autophagy and the role of autophagy in the activation of NF-κB (nuclear factor kappa B) signaling and NLRP3 (NLR family pyrin domain containing 3) inflammasome in calf hepatocytes. Calf hepatocytes were isolated from 3 healthy Holstein female new-born calves (1 d of age, 30-40 kg) and exposed to various concentrations of FFA (0, 0.3, 0.6, or 1.2 mM) after treatment with or without the autophagy inhibitor chloroquine (CQ) or the autophagy activator rapamycin. Expression of autophagy markers, LC3 (microtubule-associated protein 1 light chain 3) and p62 (sequestosome 1), NF-κB signaling, and NLRP3 inflammasome-related molecules were analyzed via western blot and quantitative real-time PCR. Results revealed that 0.6 and 1.2 mM FFA activated NF-κB signaling and NLRP3 inflammasome as indicated by an elevated ratio of p-NF-κB/NF-κB, protein abundance of NLRP3 and CASP1 (caspase 1), activity of CASP1, and mRNA abundance of IL1B and IL18. In addition, hepatocyte treated with 0.6 and 1.2 mM FFA or autophagy inhibitor CQ displayed increased protein abundance of p62 and LC3-II. Moreover, there was no difference in protein abundance of p62 and LC3-II between calf hepatocytes treated with 1.2 mM FFA and 1.2 mM FFA plus CQ, indicating that FFA inhibits autophagic activity in calf hepatocytes. Treatment with CQ led to overactivation of NF-κB signaling and NLRP3 inflammasome. Furthermore, CQ plus 1.2 mM FFA aggravated FFA-induced inflammation. In contrast, induction of autophagy by rapamycin ameliorated the FFA-activated NF-κB signaling and NLRP3 inflammasome as demonstrated by a lower ratio of p-NF-κB/NF-κB, protein abundance of NLRP3 and CASP1, activity of CASP1, and mRNA abundance of IL1B and IL18. Overall, inhibition of autophagy exacerbated, whereas induction of autophagy alleviated, FFA-induced inflammatory processes in calf hepatocytes, suggesting that impairment of autophagy might be partly responsible for hepatic inflammation and subsequent liver injury in dairy cows with ketosis or fatty liver. As such, regulation of autophagy may be an effective therapeutic strategy for controlling overt inflammatory responses in vivo.