Original Contribution
Neuroprotective effects of PEP-1-carbonyl reductase 1 against oxidative-stress-induced ischemic neuronal cell damage

https://doi.org/10.1016/j.freeradbiomed.2014.01.006Get rights and content

Highlights

  • PEP-1–CBR1 protein was transduced into HT-22 cells in a time- and dose-dependent manner.

  • PEP-1–CBR1 protein significantly protects against H2O2-induced neuronal cell death dose-dependently via inhibition of ROS generation and cellular apoptosis.

  • PEP-1–CBR1 protein protects against neuronal cell death in an in vivo ischemic animal model by inhibiting lipid peroxidation.

  • PEP-1–CBR1 protein shows potential for therapeutic treatment of oxidative-stress diseases including ischemic injury.

Abstract

Human carbonyl reductase 1 (CBR1) is a member of the NADPH-dependent short-chain dehydrogenase/reductase superfamily that is known to play an important role in neuronal cell survival via its antioxidant function. Oxidative stress is one of the major causes of degenerative disorders including ischemia. However, the role CBR1 plays with regard to ischemic injury is as yet poorly understood. Protein transduction domains such as PEP-1 are well known and now commonly used to deliver therapeutic proteins into cells. In this study, we prepared PEP-1–CBR1 protein and examined whether it protects against oxidative-stress-induced neuronal cell damage. PEP-1–CBR1 protein was efficiently transduced into hippocampal neuronal HT-22 cells and protected against hydrogen peroxide (H2O2)-induced neuronal cell death. Transduced PEP-1–CBR1 protein drastically inhibited H2O2-induced reactive oxygen species production, the oxidation of intracellular macromolecules, and the activation of mitogen-activated protein kinases, as well as cellular apoptosis. Furthermore, we demonstrated that transduced PEP-1–CBR1 protein markedly protected against neuronal cell death in the CA1 region of the hippocampus resulting from ischemic injury in an animal model. In addition, PEP-1–CBR1 protein drastically reduced activation of glial cells and lipid peroxidation in an animal model. These results indicate that PEP-1–CBR1 protein significantly protects against oxidative-stress-induced neuronal cell death in vitro and in vivo. Therefore, we suggest that PEP-1–CBR1 protein may be a therapeutic agent for the treatment of ischemic injuries as well as oxidative-stress-induced cell damage and death.

Section snippets

Materials

Ni2+–nitrilotriacetic acid Sepharose Superflow was purchased from Qiagen (Valencia, CA, USA). The indicated primary antibodies and actin were obtained from Cell Signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). 4-Hydroxy-2-nonenal (4-HNE) and 8-hydroxy-2-deoxyguanosine (8-OHdG) antibodies were purchased from Santa Cruz Biotechnology. CBR1 inhibitor (hydroxyl-PP-Me) was obtained from Sigma–Aldrich (St. Louis, MO, USA). The polymerase chain reaction (PCR)

Purification and transduction of PEP-1–CBR1 protein into HT-22 cell lines

Supplementary Fig. 1 shows the PEP-1–CBR1 protein expression vector that is based on a pET-15b vector that has a PEP-1 sequence, six histidines, and a CBR1 gene. PEP-1–CBR1 proteins were overexpressed by adding IPTG and purified from the overexpressed whole extract of E. coli using a Ni2+–nitrilotriacetic acid Sepharose affinity column and PD-10 column chromatography. As shown in Figs. 1A and B, purified PEP-1–CBR1 protein was confirmed through SDS–PAGE and Western blotting using a

Discussion

Human CBR1 is known to be a ubiquitous NADPH-dependent enzyme belonging to the short-chain dehydrogenase/reductase superfamily, which catalyzes the reduction of carbonyl compounds. So far, the known kinds of carbonyl reductases are CBR1, CBR3, and CBR4 [43]. These enzymes are known to have a crucial role in detoxification in cancer, diabetes, and other neuronal diseases [10], [11], [12], [13], [14], [15], [43], [44], [45], [46], [47], [48]. CBR1 plays a critical role in biological processes as

Acknowledgments

This work was supported by a Priority Research Centers Program grant (NRF-2009-0093812) and a Mid-career Researcher Program grant (NRF-2012R1A2A2A06043084) through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning in the Republic Korea.

References (65)

  • M.A. Bradford

    A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding

    Anal. Biochem

    (1976)
  • I.K. Hwang et al.

    Changes in the expression of mitochondrial peroxiredoxin and thioredoxin in neurons and glia and their protective effects in experimental cerebral ischemic damage

    Free Radic. Biol. Med.

    (2010)
  • Q. Wang et al.

    Apocynin protects against global cerebral ischemia–reperfusion-induced oxidative stress and injury in the gerbil hippocampus

    Brain Res.

    (2006)
  • R.L. Bateman et al.

    Human carbonyl reductase 1 is an S-nitrosoglutathione reductase

    J. Biol. Chem.

    (2008)
  • E. Tak et al.

    Human carbonyl reductase 1 upregulated by hypoxia renders resistance to apoptosis in hepatocellular carcinoma cells

    J. Hepatol.

    (2011)
  • A. Murakami et al.

    Decreased carbonyl reductase 1 expression promotes malignant behaviours by induction of epithelial mesenchymal transition and its clinical significance

    Cancer Lett.

    (2012)
  • B. Wermuth

    Purification and properties of an NADPH-dependent carbonyl reductase from human brain: relationship to prostaglandin 9-ketoreductase and xenobiotic ketone reductase

    J. Biol. Chem

    (1981)
  • S.H. Kwon et al.

    The neuroprotective effects of Lonicera japonica Thunb. against hydrogen peroxide-induced apoptosis via phosphorylation of MAPKs and PI3K/Akt in SH-SY5Y cells

    Food Chem. Toxicol.

    (2011)
  • H.A. Kim et al.

    PTEN/MAPK pathways play a key role in platelet-activating factor-induced experimental pulmonary tumor metastasis

    FEBS Lett.

    (2012)
  • A. Contreras-Paredes et al.

    E6 variants of human papillomavirus 18 differentially modulate the protein kinase B/phosphatidylinositol 3-kinase (Akt/PI3K) signaling pathway

    Virology

    (2009)
  • H.Y. Shim et al.

    Acacetin-induced apoptosis of human breast cancer MCF-7 cells involves caspase cascade, mitochondria-mediated death signaling and SAPK/JNK1/2-c-Jun activation

    Mol. Cells

    (2007)
  • Y. Numagami et al.

    Attenuation of rat ischemic brain damage by aged garlic extracts: a possible protecting mechanism as antioxidants

    Neurochem. Int.

    (1996)
  • I.K. Hwang et al.

    Copper chaperone for Cu,Zn-SOD supplement potentiates the Cu,Zn-SOD function of neuroprotective effects against ischemic neuronal damage in the gerbil hippocampus

    Free Radic. Biol. Med.

    (2005)
  • E.H. Lo et al.

    Mechanisms, challenges and opportunities in stroke

    Nat. Rev. Neurosci.

    (2003)
  • C.A. Piantadosi et al.

    Mitochondrial generation of reactive oxygen species after brain ischemia in the rat

    Stroke

    (1996)
  • L.M. Sayre et al.

    Chemistry and biochemistry of oxidative stress in neurodegenerative disease

    Curr. Med. Chem.

    (2001)
  • R.A. Floyd

    Role of oxygen free radicals in carcinogenesis and brain ischemia

    FASEB J

    (1990)
  • T.M. Dawson et al.

    Molecular pathways of neurodegeneration in Parkinson’s disease

    Science

    (2003)
  • H. Pradeep et al.

    Oxidative stress—assassin behind the ischemic stroke

    Folia Neuropathol.

    (2012)
  • K.P. Loh et al.

    Oxidative stress: apoptosis in neuronal injury

    Curr. Alzheimer Res.

    (2006)
  • P.H. Chan

    Reactive oxygen radicals in signaling and damage in the ischemic brain

    J. Cereb. Blood Flow Metab

    (2001)
  • E. Ismail et al.

    Carbonyl reductase: a novel metastasis-modulating function

    Cancer Res.

    (2000)
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    These authors contributed equally to this work.

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