Free fatty acids impair autophagic activity and activate nuclear factor kappa B signaling and NLR family pyrin domain containing 3 inflammasome in calf hepatocytes

Abstract

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.