Genistein and 17β-Estradiol Protect Hepatocytes from Fatty Degeneration by Mechanisms Involving Mitochondria, Inflammasome and Kinases Activation

Oxidative stress and mitochondria dysfunction could be involved in the onset of non-alcoholic fatty liver disease (NAFLD) and in its progression to non-alcoholic steatohepatitis (NASH). Estrogens/phytoestrogens could counteract liver fat deposition with beneficial effects against NAFLD by unclear mechanisms. We aimed to analyze the protective effects elicited by genistein/estradiol in hepatocytes cultured in NAFLD-like medium on cell viability, triglycerides accumulation, mitochondrial function and oxidative stress and the role of NLRP3 inflammasome, toll like receptors 4 (TLR4), Akt and 5' AMP-activated protein kinase (AMPK)α1/2.Human primary hepatocytes/hepatoma cell line (Huh7.5 cells) were incubated with a 2 mM mixture of oleate/palmitate in presence/absence of genistein/17β-estradiol. In some experiments, Huh7.5 cells were exposed to various inhibitors of the above pathways and estrogenic receptors (ERs) and G protein-coupled estrogen receptor (GPER) blockers, before genistein/17β-estradiol. Cell viability, mitochondrial membrane potential, reactive oxygen species and triglycerides content were examined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), 5,51,6,61-tetrachloro-1,11,3,31 tetraethylbenzimidazolyl carbocyanine iodide (JC-1), 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) and the Triglyceride Colorimetric Assay. The expression/activation of kinases was analyzed by means of Western blot.Genistein/17β-estradiol protected hepatocytes against NAFLD-like medium, by preventing the loss of cell viability and mitochondrial function, triglycerides accumulation and peroxidation. The blocking of kinases, ERs and GPER was able to reduce the above effects, which were potentiated by NLRP3 inflammasome.Our findings suggest novel mechanisms underlying the protective effects elicited by phytoestrogens/estrogens against NAFLD/NASH and open novel therapeutic perspectives in the management of NAFLD in postmenopausal women.