Original ContributionEssential role of Nrf2 in the protective effect of lipoic acid against lipoapoptosis in hepatocytes
Graphical abstract
Section snippets
Reagents and antibodies
Fetal bovine serum (FBS) and culture media were obtained from Invitrogen (Carlsbad, CA, USA). PA and LA were from Sigma–Aldrich (St. Louis, MO, USA). Anti-cleaved (Asp175) caspase 3 (9661), anti-phospho-JNK (Thr183/Tyr185) (4668), anti-phospho-p38 MAPK (Thr180/Tyr182) (9211), anti-phospho-PERK (Thr980) (3179), anti-phospho-eIF2α (Ser51) (9721), anti-phospho-p53 (Ser15) (9286), anti-Bax (2772), anti-Puma (7467), and anti-Bim (2919) antibodies were purchased from Cell Signaling Technology
LA prevents the effects of PA in the activation of stress kinases, ER stress, and lipoapoptosis and increases the expression of Nrf2-related proteins in primary human hepatocytes
We first analyzed the effects of LA on PA-induced lipoapoptosis in primary human hepatocytes treated with PA (800 μM) in the absence or presence of LA (250 μM). Quantification of TUNEL-positive hepatocytes revealed a significant increase after PA treatment for 16 h, and this effect was not observed in cells cotreated with LA (Fig. 1A). At the molecular level, phosphorylation of stress kinases such as JNK and p38 MAPK, as well as the ER stress kinase eIF2α, was observed at 8 h in PA-treated
Discussion
Nonalcoholic fatty liver disease may result in progressive liver disease with risks for cirrhosis and HCC [53]. Intracellular lipid accumulation in hepatocytes (first hit) followed by enhancement of oxidative stress (second hit) is required for the progression from simple fatty liver to NASH [54], [55], [56], [57]. Moreover, several lines of evidence suggest that toxic ROS can cause ER stress responses such as the UPR [58]. Among therapies suggested for slowing disease progression, we have
Conclusion
In conclusion and as summarized in the graphical abstract, our results strongly suggest that in hepatocytes, Nrf2 is an essential early player in the rescue of oxidative stress by LA leading to protection against PA-mediated lipoapoptosis. Importantly, we have demonstrated for the first time by in vitro and in vivo approaches that an intervention protocol based on the administration of LA after PA overload is able to substantially decrease hepatocyte lipoapoptosis.
Acknowledgments
We acknowledge the following grant support: SAF2012-33283 (Ministry of Economy and Competitiveness, MINECO, Spain), Comunidad de Madrid S2010/BMD-2423, EFSD and Amylin Paul Langerhans Grant, and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (ISCIII, Spain) to A.M.V.; SAF2010-17822 (MINECO, Spain) and Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (ISCIII, Spain) to A.C.; PI09/0185 (ISCIII, Spain) and Centro de
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