Nonphagocytic Activation of NOX2 Is Implicated in Progressive Nonalcoholic Steatohepatitis During Aging
Nonphagocytic Activation of NOX2 Is Implicated in Progressive Nonalcoholic Steatohepatitis During Aging
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DOI：
10.1002/hep.31118
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发表时间：
2020-10-09
期刊：
影响因子：
13.5
通讯作者：
Torok, Natalie J.
中科院分区：
文献类型：
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作者：
Jiang, Joy X.;Fish, Sarah R.;Torok, Natalie J.
背景和目的老年肥胖/II型糖尿病患者经常出现晚期NASH。这是否是由于在衰老过程中加速纤维化的特定分子途径尚不清楚。Src同源性2结构域的胶原蛋白相关（Shc）蛋白和氧化还原应激的激活已在衰老中得到认可;然而，尚未探索它们与NASH的联系。方法和结果通过真实的时间定量PCR（RT-qPCR）或蛋白质印迹法评估，患有NASH的老年患者的肝脏中Shc表达增加。在快餐饮食（FFD）的年轻/老年小鼠中研究了纤维化、Shc表达、衰老标志物和烟酰胺腺嘌呤二核苷酸磷酸、还原型氧化酶（NOX）。为了抑制老年小鼠中的Shc，在FFD期间使用慢病毒（LV）-短发夹Shc与对照-LV。对于肝细胞特异性效应，给FFD的floxed（fl/fl）Shc小鼠注射腺相关病毒8-甲状腺素结合球蛋白-Cre重组酶，与对照组相比。FFD时，老年小鼠的纤维化加速，老年小鼠LV的Shc抑制或肝细胞特异性缺失导致炎症显著改善，老年小鼠衰老标志物减少，脂质过氧化和纤维化。为了研究NOX 2活化，通过邻近连接和免疫共沉淀来评估p47（phox）（NOX 2调节亚基）和p52 Shc的相互作用。棕榈酸诱导p52 Shc与p47（phox）结合，激活NOX 2复合物，在老年人中更是如此。结合动力学进行了评估Src同源2域（SH 2）或磷酸酪氨酸结合（PTB）域缺失突变体的生物层干涉，揭示SH 2和PTB域的作用。最后，使用RosettaDock生成p52 Shc/p47（phox）相互作用的电子模型。结论p52 Shc/NOX 2参与了老年人肝纤维化的加速过程。我们显示了一种途径，通过p52 Shc结合并激活p47（phox）亚基，直接激活肝细胞中的吞噬性NOX 2，导致老年人氧化还原应激和加速纤维化。
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, anin silicomodel 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.