Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes.

Isorhamentin is a 3'-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes.

Yang JH ，Shin BY ，Han JY ，Kim MG ，Wi JE ，Kim YW ，Cho IJ ，Kim SC ，Shin SM ，Ki SH ... -  《-》

The chalcone compound isosalipurposide (ISPP) exerts a cytoprotective effect against oxidative injury via Nrf2 activation.

The chalcone compound isosalipurposide (ISPP) has been successfully isolated from the native Korean plant species Corylopsis coreana Uyeki (Korean winter hazel). However, the therapeutic efficacy of ISPP remains poorly understood. This study investigated whether ISPP has the capacity to activate NF-E2-related factor (Nrf2)-antioxidant response element (ARE) signaling and induce its target gene expression, and to determined the protective role of ISPP against oxidative injury of hepatocytes. In HepG2 cells, nuclear translocation of Nrf2 is augmented by ISPP treatment. Consistently, ISPP increased ARE reporter gene activity and the protein levels of glutamate cysteine ligase (GCL) and hemeoxygenase (HO-1), resulting in increased intracellular glutathione levels. Cells pretreated with ISPP were rescued from tert-butylhydroperoxide-induced reactive oxygen species (ROS) production and glutathione depletion and consequently, apoptotic cell death. Moreover, ISPP ameliorated the mitochondrial dysfunction and apoptosis induced by rotenone which is an inhibitor of complex 1 of the mitochondrial respiratory chain. The specific role of Nrf2 activation by ISPP was demonstrated using an ARE-deletion mutant plasmid and Nrf2-knockout cells. Finally, we observed that extracellular signal-regulated kinase (ERK) and AMP-activated protein kinase (AMPK), but not protein kinase C (PKC)-δ or other mitogen-activated protein kinases (MAPKs), are involved in the activation of Nrf2 by ISPP. Taken together, our results demonstrate that ISPP has a cytoprotective effect against oxidative damage mediated through Nrf2 activation and induction of its target gene expression in hepatocytes.

Han JY ，Cho SS ，Yang JH ，Kim KM ，Jang CH ，Park DE ，Bang JS ，Jung YS ，Ki SH ... -  《-》

Methyleugenol protects against t-BHP-triggered oxidative injury by induction of Nrf2 dependent on AMPK/GSK3β and ERK activation.

Methyleugenol (Mlg), a natural ingredient of many herbs and used as a flavoring substance in dietary products, inhibits inflammation and oxidative stress. The aim of the study is to explore the antioxidative potential of Mlg against tert-butyl hydroperoxide (t-BHP)-triggered oxidative injury and the involvement of antioxidative mechanisms. Our findings indicated that Mlg exposure significantly alleviated t-BHP-stimulated cytotoxicity, suppressed reactive oxygen species (ROS) generation, and increased superoxide dismutase (SOD) and glutathione (GSH) levels, which were related to the induction of the glutamate-cysteine ligase catalytic/modifier (GCLC/GCLM) subunit, heme oxygenase-1 (HO-1), and NAD (P) H: quinone oxidoreductase (NQO1) largely dependent upon upregulating the nuclear factor-erythroid 2-related factor 2 (Nrf2) induction, inhibiting the Keap1 protein expression, and heightening the antioxidant response element (ARE) activity. Additionally, Mlg exposure obviously induced AMP-activated protein kinase (AMPK), glycogen synthase kinase 3β (GSK3β) and extracellular signal-regulated kinase (ERK) phosphorylation, but AMPK and ERK inhibitors treatment exhibited effectively reduced levels of Mlg-enhanced Nrf2 nuclear translocation, respectively. Furthermore, Mlg exposure significantly lessened t-BHP-induced cytotoxicity and ROS production which were evidently abolished by treatment with AMPK and ERK inhibitors and Nrf2 siRNA. Accordingly, Mlg might exhibit a protective role against t-BHP-triggered cytotoxicity via the activation of the AMPK/GSK3β- and ERK-Nrf2 signaling pathways.

Zhou J ，Ma X ，Cui Y ，Song Y ，Yao L ，Liu Y ，Li S ... -  《-》

5-Caffeoylquinic acid ameliorates oxidative stress-mediated cell death via Nrf2 activation in hepatocytes.

Chen X ，Yang JH ，Cho SS ，Kim JH ，Xu J ，Seo K ，Ki SH ... -  《-》

Hepatocyte-protective effect of nectandrin B, a nutmeg lignan, against oxidative stress: Role of Nrf2 activation through ERK phosphorylation and AMPK-dependent inhibition of GSK-3β.

Oxidative stress can contribute to the development and progression of liver diseases, such as drug-induced or alcoholic liver injury, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis. Nectandrin B is a bioactive lignan isolated from nutmeg extract. To date, little information is available about its pharmacological activities in the liver. This study investigated the hepatocyte-protective effect of nectandrin B against tert-butylhydroperoxide-induced oxidative injury and the underlying molecular mechanism. The cell viability assay revealed that nectandrin B prevents apoptosis stimulated by tert-butylhydroperoxide in both HepG2 cells and primary mouse hepatocytes. Nectandrin B also attenuated ROS production and restored the depleted glutathione level. Real-time PCR and immunoblot analyses showed that the expression of glutamate-cysteine ligase, an enzyme responsible for the glutathione biosynthesis, was induced by nectandrin B, indicating its indirect antioxidative effect. The NF-E2-related factor-2 (Nrf2) regulates gene expression of an array of antioxidant enzymes in hepatocytes. Nectandrin B stimulated Nrf2 activation as evidenced by its enhanced nuclear accumulation and increased antioxidant response element (ARE)-luciferase activity. Intriguingly, the hepatocyte-protective effect of nectandrin B against oxidative damage was completely abrogated by Nrf2 knockdown using Nrf2 specific siRNA. Nectandrin B promoted ERK activation, but inactivated GSK-3β through the AMPK-mediated inhibitory phosphorylation. The enforced overexpression of dominant-negative mutant of MEK1 or AMPKα, or wild-type GSK-3β inhibited the increase in the NQO1-ARE-luciferase activity stimulated by nectandrin B, suggesting that both ERK and AMPK-GSK-3β signalings are involved in the activation of Nrf2/ARE pathway by nectandrin B. Consistent with this, cytoprotection and restoration of glutathione level by nectandrin B was also blocked by the overexpression of dominant-negative MEK1 or wild-type GSK-3β. Finally, our data demonstrate that nectandrin B has the ability to protect hepatocytes against oxidative injury through the activation of Nrf2/ARE pathway mediated by ERK phosphorylation and AMPK-dependent inactivation of GSK-3β.

Song JS ，Kim EK ，Choi YW ，Oh WK ，Kim YM ... -  《-》