Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Mar;112(5):1316-26.
doi: 10.1111/j.1471-4159.2009.06552.x. Epub 2009 Dec 17.

Plumbagin, a novel Nrf2/ARE activator, protects against cerebral ischemia

Tae Gen Son  1 , Simonetta Camandola, Thiruma V Arumugam, Roy G Cutler, Richard S Telljohann, Mohamed R Mughal, Tyson A Moore, Weiming Luo, Qian-Sheng Yu, Delinda A Johnson, Jeffrey A Johnson, Nigel H Greig, Mark P Mattson
Affiliations

Plumbagin, a novel Nrf2/ARE activator, protects against cerebral ischemia

Tae Gen Son et al. J Neurochem. 2010 Mar.

Abstract

Many phytochemicals function as noxious agents that protect plants against insects and other damaging organisms. However, at subtoxic doses, the same phytochemicals may activate adaptive cellular stress response pathways that can protect cells against a variety of adverse conditions. We screened a panel of botanical pesticides using cultured human and rodent neuronal cell models, and identified plumbagin as a novel potent activator of the nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. In vitro, plumbagin increases nuclear localization and transcriptional activity of Nrf2, and induces the expression of the Nrf2/ARE-dependent genes, such as heme oxygenase 1 in human neuroblastoma cells. Plumbagin specifically activates the Nrf2/ARE pathway in primary mixed cultures from ARE-human placental alkaline phosphatase reporter mice. Exposure of neuroblastoma cells and primary cortical neurons to plumbagin provides protection against subsequent oxidative and metabolic insults. The neuroprotective effects of plumbagin are abolished by RNA interference-mediated knockdown of Nrf2 expression. In vivo, administration of plumbagin significantly reduces the amount of brain damage and ameliorates-associated neurological deficits in a mouse model of focal ischemic stroke. Our findings establish precedence for the identification and characterization of neuroprotective phytochemicals based upon their ability to activate adaptive cellular stress response pathways.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Plumbagin activates ARE- driven transcription at subtoxic concentrations. SH-SY5Y cells were cultured on 96 well plates and incubated with the indicated concentrations of plumbagin for 24 hours. Viability was assessed by alamar blue (A) and lactate dehydrogenase activity (LDH) (B) assays. Data are presented as mean and S.D. (n=3). ***, p< 0.001 compared with DMSO (−) treated cells. SH-SY5Y cells were transfected with ARE- (C), AP-1- (D), NF-κB- (E) or Foxo- (F) firefly luciferase reporter and pRL-TK control vector, and treated with the indicated concentrations of plumbagin or, respectively, 5 μM sulforaphane (SF), 100 nM phorbol-12-myristate-13-acetate (PMA), 2 μg pRelA (RelA), or 2 μg pECE-Foxo3A (Foxo) as positive controls for 24 hours. The relative light intensities were normalized, and signal averages were determined. Data are mean and S.D. (n=3–6).***, p< 0.001 compared with DMSO (−) treated cells. (G) Structure of sulforaphane, plumbagin and its analogs. (H) Effect of plumbagin and its analogs on ARE-driven luciferase activity. SH-SY5Y were transfected with ARE-luciferase reporter and treated with the indicated concentrations of phytochemicals. Luciferase activity was assessed after 24 hours. Data are normalized values compared to the activity of DMSO treated cells set equal to 1. Data are mean and S.D. of triplicate samples from 2–3 separate experiments. **, p< 0.05 compared with control group; ***, p< 0.001 compared with control group.
Fig. 2
Fig. 2
Effect of plumbagin on Nrf2 endogenous genes. (A) SH-SY5Y cells were treated with 1 μM plumbagin for 6 hrs. Total RNA was extracted, retrotranscribed, and the transcript levels of the indicated Nrf2-dependent genes were analyzed by semi quantitative RT-PCR. Data are mean and S.D. from three independent experiments. *, p<0.01 compared with vehicle treated cells; **, p<0.001 compared with vehicle treated cells. Protein steady state levels of HO-1 were measured in cell extracts from SH-SY5Y treated either for 20 hrs with increasing concentrations of plumbagin (B), or with 1 μM plumbagin for the indicated time points (C). Autoradiograms are representative of two-three independent experiments. Right panels show densitometric quantitation as mean and S.D. of 3–4 samples.*p<0.05 compared control set equal to 1.
Fig. 3
Fig. 3
Plumbagin affords cell protection trough the Nrf2/ARE pathway. (A) SH-SY5Y were treated with either vehicle (Vh) or 1 μM plumbagin (PL) for 6 hrs, and subsequently exposed to the indicated concentrations of tert-butyl-hydroperoxide (BHP). Cell survival was assessed by blinded random counting of Hoescht stained cells. Data are mean and S.E.M. of three independent experiments. *, p<0.0001 compared to the corresponding BHP concentration in Vh treated cells. SH-SY5Y were treated for 48 hrs with control (siCnt) or Nrf2 siRNA (siNrf2), than subjected to plumbagin (PL) for 6 hrs. (B) Nuclear extracts were assessed for Nrf2 levels, using tubulin and hnRNP as internal controls for subcellular fractionation purity. HO-1 representative immunostaining (C) and quantification (D) following PL treatment in control and Nrf2 siRNA treated cells. Data are mean and S.D. of three independent experiments (90–100 cells per treatment). ***, p<0.001 compared to control; #, p<0.001 compared to PL. (E) Representative images and (F) survival quantification following challenge with BHP 200 μM (BHP2) and 500 μM (BHP5) in cells pretreated with control (siCnt) or Nrf2 siRNA (siNrf2) for 48 hrs, then vehicle or 1 μM PL for 6 hrs.; ***, p<0.0001 compared to control siRNA BHP2 or BHP5; ##, p<0.0001 compared to control siRNA PL BHP2 or control siRNA PL BHP5.
Fig. 4
Fig. 4
Plumbagin activates the PI3K/AKT and ERK pathways. (A) SH-SY5Y cells were loaded with 25 μM DCFDA for 45 min at 37 °C, washed with PBS and exposed to 400 μM hydrogen peroxide (H2O2) or the indicated concentrations of plumbagin (PL). The relative levels of DCF fluorescence were quantified at the indicated time points. (B) Levels of the lipid peroxidation end product 4-hydroxy-2,3-nonenal (4HNE) were assessed in SH-SY5Y cells treated for 24 hrs with increasing concentrations of plumbagin. Total lipids extracts were analyzed by mass spectrometry. Data are mean and S.E.M of triplicate samples. ***, p<0.001 versus DMSO (−) treated cells. (C) SH-SY5Y cells were treated with 1 μM plumbagin for the indicated time points, and cell lysates were analyzed by western blotting using anti- pAKT, AKT, pERK and ERK antibodies. Right panels show densitometric quantitation as mean and S.D. (n=3).*p<0.05 compared to time zero. (D) Cells were pretreated with 1 μM wortmannin (W) (PI3K inhibitor), or 10 μM PD98059 (PD) (MEK1 inhibitor) for 1 h, were washed with PBS and treated with 1 μM plumbagin for 6 hrs. Cell lysates were blotted with anti-HO-1 and NQO1 antibodies. Right panels show quantification as mean and S.D. (n=3).*, p=0.026 compared to control (−); #, p=0.024 compared to PL; **, p=0.0007 compared to control; ##, p=0.0023 compared to PL; ###. P=0.0005 compared to PL. (E) Cell survival quantification and (F) representative images of cells pretreated as above mentioned than subjected to either 200 μM tert-butyl hydroperoxide (BHP2) or 500 μM (BHP5) for 20 hrs. Values are the mean ± S.E.M. of three experiments. *, p<0.0001 compared to control; ***, p<0.0001 compared to BHP2; **, p<0.0001 compared to BHP5; #, p<0.0001 compared to BHP2+PL; ##, p<0.0001 compared to BHP5+PL.
Fig. 5
Fig. 5
Plumbagin protects cultured primary neurons against OGD, reduces brain damage and improves functional outcome in a mouse stroke model. Neuronal cultures transfected with pEF (Vector) or dominant negative Nrf2(DN-Nrf2) were treated with either vehicle (Control) or 0.5 μM plumbagin (PL) then subjected to glucose deprivation (GD) for 24 hrs. Cell survival was assessed by random blinded cell counting and MTT assays. Representative images (A) and quantification of apoptotic neurons (B). Data are mean and S.D. of three independent experiments (~300–600 neurons per experiment). ***, p<0.0001 compared to GD; ##, p<0.0001 compared to vector transfected cells. (C) MTT data are mean and S.E.M. (n=3). *, p=0.003 compared to GD; #, p=0.02 compared to vector transfected cells. (D) Immunoblot showing levels of the pro-apoptotic marker cleaved caspase 3 (csp3) in neurons pretreated with vehicle or plumbagin (PL) and subjected to oxygen and glucose deprivation (OGD) for the indicated time points. (E) Levels of the Nrf-2-target gene HO-1 in cortex and striatum from mice injected with either vehicle or 3 mg/kg of plumbagin for the indicated time points. (F) Neurological scores of mice sham operated or subjected to 1 hr ischemia followed by reperfusion (I/R) for the indicated time points. Mice were intravenously injected with either vehicle (control) (n=11) or 3 mg/kg of plumbagin (PL) 6 hrs (n=9) and 24 hrs (n=9) before surgery, or 1 hr post ischemia (n=7). *p<0.001 compared to control mice. (G) Ischemic infarct size 72 hrs post ischemia/reperfusion in vehicle-treated (n=9), 6 hrs PL-pretreated (n=10), 24 hrs PL-pretreated (n=8), or 1 hr PL post treated (n=9) mice. ***p,0.001 compared to sham operated mice. ++ p<0.001 compared to vehicle- treated mice.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alam J, Stewart D, Touchard C, Boinapally S, Choi AM, Cook JL. Nrf2, a cap ‘n’ Collar transcription factor, regulates induction of the heme oxygenase-1 gene. J Biol Chem. 1999;274:26071–26078. - PubMed
    1. Arumugam TV, Tang SC, Lathia JD, et al. Intravenous immunoglobulin (IVIG) protects the brain against experimental stroke by preventing complement-mediated neuronal cell death. Proc Natl Acad Sci USA. 2007;104:14101–14109. - PMC - PubMed
    1. Calabrese EJ, Bachmann KA, Bailer AJ, et al. Biological stress response terminology: Integrating the concepts of adaptive response and preconditioning stress within a hormetic dose-response framework. Toxicol Appl Pharmacol. 2007;222:122–128. - PubMed
    1. Calixto JB, Kassuya CA, Andre E, Ferreira J. Contribution of natural products to the discovery of the transient receptor potential (TRP) channels family and their functions. Pharmacol Ther. 2005;106:179–208. - PubMed
    1. Camandola S, Cutler RG, Gary DS, Milhavet O, Mattson MP. Suppression of calcium release from IP3-sensitive stores mediates the anti-apoptostic function of NF-κB. J Biol Chem. 2005;280:22287–22296. - PubMed

Publication types

MeSH terms