Elsevier

Brain Research

Volume 1437, 9 February 2012, Pages 16-25
Brain Research

Research Report
Luteolin enhances cholinergic activities in PC12 cells through ERK1/2 and PI3K/Akt pathways

https://doi.org/10.1016/j.brainres.2011.12.019Get rights and content

Abstract

Luteolin, a 3′, 4′, 5, 7-tetrahydroxyflavone, is an active compound in Rosmarinus officinalis (Lamiacea), and has been reported to exert several benefits in neuronal cells. However cholinergic-induced activities of luteolin still remain unknown. Neuronal differentiation encompasses an elaborate developmental program which plays a key role in the development of the nervous system. The advent of several cell lines, like PC12 cells, able to differentiate in culture proved to be the turning point for gaining and understanding of molecular neuroscience. In this work, we investigated the ability of luteolin to induce PC12 cell differentiation and its effect on cholinergic activities. Our findings showed that luteolin treatment significantly induced neurite outgrowth extension, enhanced acetylcholinesterase (AChE) activity, known as neuronal differentiation marker, and increased the level of total choline and acetylcholine in PC12 cells. In addition, luteolin persistently, activated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt; while the addition of pharmacological MEK/ERK1/2 inhibitor (U0126) and PI3k/Akt inhibitor (LY294002) attenuated luteolin-induced AChE activity and neurite outgrowth in PC12 cells. The above findings suggest that luteolin induces neurite outgrowth and enhanced cholinergic activities, at least in part, through the activation of ERK1/2 and Akt signaling.

Highlights

► Luteolin induced NGF-like differentiation in PC12 cell. ► Luteolin promoted cholinergic activities in PC12 cell. ► Inhibition of ERK1/2 and PI3K-Akt suppressed luteolin induced activities. ► Luteolin treatment may promote neuronal differentiation and cholinergic activities.

Introduction

Flavonoids, a large group of natural compounds, exert beneficial effects in a multitude of disease states, including cancer, cardiovascular disease, and neurodegenerative disorders (Fiorani and Accorsi, 2005). They are commonly included in food additives and health food supplements and are also considered as the active ingredients in many herbal medicines (Coleta et al., 2009). Dietary intervention studies using flavonoid-rich plant or food extracts have indicated that flavonoids are capable to improve both memory and learning (Spencer, 2009). Previous study has shown that flavonoids have the ability to stimulate the differentiation of PC12 cells (Sagara et al., 2004). In our previous study, we found that Rosmarinus officinalis leave extracts promote PC12 cell differentiation, and after HPLC analysis several flavonoids like quercetin, apeginin, and luteolin were detected in cells (El Omri et al., 2010). Only, luteolin was able to induce clear morphological changes in PC12 cells.

Luteolin, a 3′, 4′, 5, 7-tetrahydroxyflavone, a naturally occurring flavonoid, is abundant in our daily dietary intake (Chowdhury et al., 2002). Pre-various studies showed that luteolin exhibits a wide spectrum of pharmacological properties. In fact, it has been demonstrated to possess a high DNA protective effect in the presence of H2O2, anti-inflammatory and phytoestrogen-like activities (Cheng et al., 2009). More recently, it has been demonstrated to protect PC12 cells against oxidative stress (Pavlica and Gebhardt, 2010) and serum-deprivation apoptosis (Lin et al., 2010).

In animal model, luteolin, was reported to have central nerve system (CNS) activity with anxiolytic-like effects through GABAergic mechanism (Coleta et al., 2009) and to have anti-amnesic and protective effect against the toxicity of amyloid-β25–35 (Liu et al., 2009). Luteolin, was also demonstrated to attenuate the deficits of passive avoidance performance induced by scopolamine hydrobromide through the activation of the central cholinergic neuronal system (Tsai et al., 2007).

It is well known that the cholinergic system is involved in the regulation of several CNS functions like cognition, memory, conscious arousal, attention and subsequently regulation of mood impairments such us depression and anxiety (Dagyté et al., 2010). The modulation of cholinergic activities in neuronal cells is presently of particular interest for the development of cognitive enhancers.

PC12 cells are well established model for the investigation of neuronal cell differentiation (Wang et al., 2006, Yang et al., 2008) and cholinergic activities (Isoda et al., 2002, Madziar et al., 2008). When treated with nerve growth factor (NGF), PC12 cells cease proliferation and take several phenotypic properties of cholinergic neurons with an increase of AChE activity as a marker of differentiation (Liu et al., 2006). NGF was reported as the most efficacious neurotrophic factor to induce neuronal differentiation and to prevent atrophy of cholinergic neurons in patient with Alzheimer's disease (AD) (Lin et al., 2010). However, this polypeptide cannot cross easily the blood brain barrier (BBB) and is metabolized by peptidases when administered peripherally (Hur et al., 2009). In this sense, discovery of phytochemicals with neurotrophic activities is a promising alternative for treatment of neurodegenerative disease (Ramassamy, 2006).

The above reports reveal that luteolin possesses neuroprotective effect. However, the detailed mechanism regarding its possible neurogenic action to promote neuronal differentiation and cholinergic activities remains limited. In the present study, we demonstrate that luteolin induces neuronal differentiation and enhanced cholinergic activities through the activation of ERK1/2 and Akt signal pathways in PC12 cells.

Section snippets

Luteolin promotes PC12 cell differentiation

In preliminary experiments using MTT assay, there was no significant difference in the proliferation or viability in luteolin-treated PC12 cells or pretreated with U0126 and LY294002, indicating that the current treatments are not cytotoxic to neuronal cells (data not shown).

The proportion of differentiated cells is used to describe cellular differentiation process but this single parameter could only give partial information (Blasina et al., 2009). Adding other morphological parameters like

Discussion

There is mounting evidence supporting the use of nonpeptidic neurotrophins against neurodegenerative disorders. Recently, attention has been focused on phytochemicals, such as polyphenolic compounds, that are able to induce neuronal differentiation and regulate neurotransmitter's functions in CNS. Luteolin is a well known reactive oxygen species (ROS) scavenger and serves as an antioxidant, anti-inflammatory, and anticancer agent (Lin et al., 2010). In animal experiments, luteolin has been

Reagents

Luteolin, NGF 7s, radioimmunoprecipitation assay (RIPA) buffer and p-ERK1/2 antibody were purchased from Sigma Aldrich Co., Ltd. (St Louis, USA), and acetylcholine iodide was from Wako (Japan). ERK1/2 antibody and goat anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, USA), and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126) was purchased from Promega (USA). Goat anti-mouse IgG-HRP was from Bethyl Laboratories Inc. (Montgomery, USA), and

Acknowledgments

This work was partially supported by Mitsui & Co., Ltd. Environment Fund.

References (38)

  • F.S. Tsai et al.

    Effects of luteolin on learning acquisition in rats: involvement of the central cholinergic system

    Life Sci.

    (2007)
  • Z.J. Wang et al.

    Panaxynol induces neurite outgrowth in PC12D cells via cAMP- and MAP kinase-dependent mechanisms

    Chem. Biol. Interact.

    (2006)
  • E.H. Wu et al.

    Activation of muscarinic M4 receptor augments NGF-induced pro-survival Akt signaling in PC12 cells

    Cell. Signal.

    (2006)
  • Y.J. Yang et al.

    Effect of scoparone on neurite outgrowth in PC12 cells

    Neurosci. Lett.

    (2008)
  • D.S. Auld et al.

    Neurotrophins differentially enhance acetylcholine release acetylcholine content and choline acetyltransferase activity in basal forebrain neurons

    J. Neurochem.

    (2001)
  • M.F. Blasina et al.

    Differentiation induced by Achyrocline satureioides (Lam) infusion in PC12 cells

    Phytother. Res.

    (2009)
  • A. Blokland

    Acetylcholine: a neurotransmitter for learning and memory

    Brain Res. Rev.

    (1996)
  • X. Castell et al.

    Exploring the regulation of the expression of ChAT and VAChT genes in NG108-15 cells: implication of PKA and PI3K signaling pathways

    Neurochem. Res.

    (2003)
  • Z. Chen et al.

    Liquiritin potentiate neurite outgrowth induced by nerve growth factor in PC12 cells

    Cytotechnology

    (2009)
  • Cited by (0)

    View full text