Nonphagocytic Activation of NOX2 Is Implicated in Progressive Nonalcoholic Steatohepatitis During Aging

Abstract

BaCKgRoUND aND aIMS: Older patients with obesity/ type II diabetes mellitus frequently present with advanced NASH. Whether this is due to specific molecular pathways that accelerate fibrosis during aging is unknown. Activation of the Src homology 2 domain-containing collagen-related (Shc) proteins and redox stress have been recognized in aging; however, their link to NASH has not been explored. appRoaCH aND ReSUltS: Shc expression increased in livers of older patients with NASH, as assessed by real time quantitative PCR (RT-qPCR) or western blots. Fibrosis, Shc expression, markers of senescence, and nicotinamide adenine dinucleotide phosphate, reduced form oxidases (NOXs) were studied in young/old mice on fast food diet (FFD). To inhibit Shc in old mice, lentiviral (LV)-short hairpin Shc versus control-LV were used during FFD. For hepatocyte-specific effects, floxed (fl/fl) Shc mice on FFD were injected with adeno-associated virus 8-thyroxine-binding globulin-Cre-recombinase versus control. Fibrosis was accelerated in older mice on FFD, and Shc inhibition by LV in older mice or hepatocyte-specific deletion resulted in significantly improved inflammation, reduction in senescence markers in older mice, lipid peroxidation, and fibrosis. To study NOX2 activation, the interaction of p47 phox (NOX2 regulatory subunit) and p52Shc was evaluated by proximity ligation and coimmunoprecipitations. Palmitate-induced p52Shc binding to p47 phox , activating the NOX2 complex, more so at an older age. Kinetics of binding were assessed in Src homology 2 domain (SH2) or phosphotyrosine-binding (PTB) domain deletion mutants by biolayer interferometry, revealing the role of SH2 and the PTB domains. Lastly, an in silico model of p52Shc/p47 phox interaction using RosettaDock was generated. CoNClUSIoNS: Accelerated fibrosis in the aged is modulated by p52Shc/NOX2. We show a pathway for direct activation of the phagocytic NOX2 in hepatocytes by p52Shc binding and activating the p47 phox subunit that results in redox stress and accelerated fibrosis in the aged. (Hepatology 2020;72:1204-1218). N ASH and associated cirrhosis are the leading causes of liver-related mortality in the United States and worldwide. (1) The incidence of NASH rises strikingly with age, contributing significantly to elder mortality. (2) Older patients are often excluded from liver transplantation because of comorbidities and the age limit for transplantation; therefore, a significant increase in NASH-related mortality is expected because NASH has no approved medical treatment. Most of the preclinical data in NASH/fibrosis research is derived from experiments employing young mice. In clinical trials, however, the target population is usually middle-aged or elderly. To

Serum Malondialdehyde is Associated with Non-Alcoholic Fatty Liver and Related Liver Damage Differentially in Men and Women

“…Age-related fibrosis in NASH patients was also connected to changes in redox cellular status. Fibrosis was demonstrated to be modulated by p52Shc/NOX2 that results in redox stress, and accelerated fibrosis in the aged due to increased production of hydrogen peroxides and superoxides that can promote lipid peroxidation as was demonstrated in old mice [ 41 ].…”

Aging and Chronic Liver Disease

“…In the MASH scenario, a recent study developed in aged mice receiving fast food diet demonstrated that direct activation of the phagocytic NOX2 by p52Shc binding in hepatocytes results in redox stress and accelerated fibrosis. 84 Old livers also present higher levels of macrophages and CD4þ cells recruitment after chronic injury. 79 It was described that the cytokine and chemokine expression profile of these inflammatory cells is completely different in aging.…”

The TGF-β/NADPH Oxidases Axis in the Regulation of Liver Cell Biology in Health and Disease

“…Furthermore, recent results indicate that accelerated fibrosis in the aged is modulated by p52Shc/NOX2. A direct activation of NOX2 in hepatocytes by p52Shc binding and activating the p47phox subunit results in redox stress and accelerated fibrosis in the aged [ 125 ] ( Figure 4 ). Finally, a role for Nox5 (which is not expressed in mice) has been proposed, mediating the proliferation and activation of human HSC in response to TGF-β, via p38 MAPK [ 126 ].…”