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

Toxicol Appl Pharmacol. 2014 Jan 15;274(2):293-301. doi: 10.1016/j.taap.2013.10.026. Epub 2013 Nov 7.


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.

Keywords: 5′ AMP-activated protein kinase; AMPK; ARE; Cytoprotection; ERK1/2; GCS; HO-1; Isorhamnetin; Methylated flavonoid; NAD(P)H:quinone reductase; NF-E2-related factor-2; NQO1; Nrf2; PI3K; PKC; ROS; Sesn2; antioxidant response element; extracellular signal-regulated kinase; hemeoxygenase 1; phosphoinositide 3-kinase; protein kinase C; reactive oxygen species; sestrin2; t-BHQ; tert-butylhydroquinone; γ-glutamylcysteine synthetase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases / genetics
  • AMP-Activated Protein Kinases / metabolism
  • Anti-Inflammatory Agents / pharmacology*
  • Antioxidant Response Elements / drug effects
  • Antioxidants / pharmacology
  • Cell Proliferation / drug effects
  • Cell Survival / drug effects
  • Glutamate-Cysteine Ligase / genetics
  • Glutamate-Cysteine Ligase / metabolism
  • Glutathione / metabolism
  • Heme Oxygenase-1 / genetics
  • Heme Oxygenase-1 / metabolism
  • Hep G2 Cells
  • Hepatocytes / drug effects
  • Hepatocytes / metabolism
  • Humans
  • MAP Kinase Signaling System / drug effects
  • NF-E2-Related Factor 2 / genetics
  • NF-E2-Related Factor 2 / metabolism*
  • Oxidative Stress / drug effects*
  • Phosphorylation
  • Protein Kinase C-delta / genetics
  • Protein Kinase C-delta / metabolism
  • Quercetin / analogs & derivatives*
  • Quercetin / pharmacology
  • tert-Butylhydroperoxide / toxicity


  • Anti-Inflammatory Agents
  • Antioxidants
  • NF-E2-Related Factor 2
  • NFE2L2 protein, human
  • 3-methylquercetin
  • tert-Butylhydroperoxide
  • Quercetin
  • HMOX1 protein, human
  • Heme Oxygenase-1
  • Protein Kinase C-delta
  • AMP-Activated Protein Kinases
  • GCLM protein, human
  • Glutamate-Cysteine Ligase
  • Glutathione