Elsevier

NeuroToxicology

Volume 85, July 2021, Pages 186-200
NeuroToxicology

Formononetin attenuates H2O2-induced cell death through decreasing ROS level by PI3K/Akt-Nrf2-activated antioxidant gene expression and suppressing MAPK-regulated apoptosis in neuronal SH-SY5Y cells

https://doi.org/10.1016/j.neuro.2021.05.014Get rights and content

Highlights

  • Formononetin lowered H2O2-induced neurotoxicity in SH-SY5Y cells.

  • Formononetin decreased intracellular ROS level.

  • Formononetin increased the antioxidant gene expression through PI3K/Akt-Nrf2 signaling.

  • Formononetin suppressed apoptosis through inhibition of phosphorylation of MAPK.

Abstract

Formononetin is an isoflavone, found in herbs like Trifolium pratense, which executes a variety of physiological activities including anti-neurodegenerative effect. However, the molecular mechanism of formononetin-mediated neuroprotection remains unclear. In this study, we investigated the protective effect of formononetin on hydrogen peroxide (H2O2)-induced death of human neuroblastoma SH-SY5Y cells and its underlying molecular mechanism. Formononetin suppressed H2O2-induced cytotoxicity. H2O2-induced increase in the intracellular reactive oxygen species (ROS) levels was decreased by formononetin, together with the enhanced expression of the antioxidant genes. H2O2-induced elevation of the Bax/Bcl-2 ratio and cleaved caspase-3 and caspase-7 levels were lowered by formononetin treatment. Moreover, formononetin repressed H2O2-induced phosphorylation of mitogen-activated protein kinases (MAPKs). Nuclear factor erythroid 2-related factor 2 (Nrf2) siRNA decreased antioxidant gene expression and elevated the H2O2-induced ROS level in the formononetin-treated cells. Furthermore, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling is involved in the activation of the nuclear translocation of Nrf2. These results indicate that the neuroprotective effect of formononetin against H2O2-induced cell death is due to a decrease in the ROS level with the enhanced expression of the antioxidant genes through activation of the PI3K/Akt-Nrf2 signaling. In addition, formononetin suppressed apoptosis through inhibition of phosphorylation of MAPKs in SH-SY5Y cells. Thus, formononetin is a potential therapeutic agent for the treatment of neurodegenerative diseases.

Introduction

Oxidative stress triggered by reactive oxygen species (ROS) induces the cascade leading to damage in neuronal cells in various neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) (Barnham et al., 2004; Galli et al., 2005; Jenner, 2003; Singh et al., 2019). ROS include oxygen radicals such as superoxide and hydroxyl radicals, and non-free radicals such as hydrogen peroxide (H2O2). ROS are generated in a variety of redox processes (Galli et al., 2005), and can damage various biomolecules and organelles such as proteins, DNAs, lipids, and mitochondria (Schieber and Chandel, 2014). H2O2 is associated with the production of reactive hydroxyl radicals through Fenton’s reaction and can promote apoptosis (Singh et al., 2007). H2O2-induced apoptosis lowers the expression of anti-apoptotic proteins such as B-cell lymphoma 2 (Bcl-2), but increases pro-apoptotic proteins such as BCL2-associated X, apoptosis regulator (Bax) (Kale et al., 2018). Moreover, H2O2 activates the caspase cascade, including caspase-3 that is a key executor of apoptosis (Redza-Dutordoir and Averill-Bates, 2016). Thus, reduction of ROS is crucial in preventing various diseases including neurodegenerative disorders (Singh et al., 2019).

Nuclear factor erythroid 2-related factor 2 (Nrf2) is the major regulator of cytoprotective responses to oxidative stress (Li and Kong, 2009). Nrf2 is a basic region-leucine zipper-type transcription factor that regulates the expression of antioxidant response element (ARE)-containing genes such as heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), glutamate-cysteine ligase modifier subunit (GCLM), and thioredoxin reductase 1 (TXNRD1). Nrf2 is phosphorylated through two different mechanisms: kelch-like ECH-associated protein 1 (Keap1)-dependent and -independent mechanisms (Motohashi and Yamamoto, 2004). In the Keap1-dependent mechanism, Nrf2 is dissociated from Keap1 and moves into the nucleus. In the Keap1-independent mechanism, Nrf2 is phosphorylated by several kinases including mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), protein kinase C, and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathways (Espinosa-Diez et al., 2015; Zhang et al., 2016). Phosphorylated Nrf2 is translocated into the nucleus (Li and Kong, 2009).

Several antioxidant compounds prevent the development of oxidative stress-mediated disorders through scavenging ROS (Tabatabaei-Malazy et al., 2015). Plants are potential sources of natural antioxidants as they are enriched with compounds such as flavonoids, phenolic acids, and phenolic diterpenes (Heim et al., 2002). Numerous studies have reported that plant constituents possess capabilities to protect against ROS-mediated cell damage via their antioxidative effects (Heim et al., 2002; Kumar et al., 2014). Flavonoids are polyphenolic substances and are rich in several plant species. They exhibit antiviral, antimicrobial, anti-inflammatory, anti-cancer, and cytoprotective effects in a variety of cells (Nijveldt et al., 2001). Formononetin [7-hydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one] is an isoflavonoid abundantly found in medicinal herbs such as Trifolium pratense and Astragalus membranaceus (Kulling et al., 2002; Tay et al., 2019). Formononetin exerts various biological effects such as antimicrobial, anti-cancer, and antioxidative activities (Mu et al., 2009; Vishnuvathan et al., 2016). Moreover, formononetin shows protective actions against N-methyl-d-aspartate (NMDA)-triggered apoptosis in primary cultured cortical neuronal cells (Tian et al., 2013) and against l-glutamate-induced damage in neuronal cells (Yu et al., 2005). However, the molecular mechanism of neuroprotection by formononetin is still unclear. In this study, we investigated the protective effect of formononetin against H2O2-induced cell death and elucidated its underlying molecular mechanism in human neuroblastoma cells.

Section snippets

Materials

H2O2, wortmannin, SP600125, PD98059, and SB203580 were obtained from FUJIFILM Wako Pure Chemical (Osaka, Japan). 7'-Dichlorofluorescin diacetate (DCFH-DA) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Hoechst 33342 was from Nacalai Tesque (Kyoto, Japan). Anti-Bax (#2772), anti-Bcl-2 (#3498), anti-cleaved caspase-3 (#9661), anti-cleaved caspase-7 (#9491), anti-phospho-JNK (#9251), anti-JNK (#9252), anti-phospho-p44/42 MAPK (P-ERK; #9101), anti-p44/42 MAPK (ERK; #9102),

Extraction and purification of formononetin

Formononetin (Fig. 1A) was extracted from licorice, and purified by UHPLC. Analysis of the product showed high purity of over 99 % (Fig. 1B).

Prevention of H2O2-induced cell death by formononetin

Initially, the cell toxicity of formononetin was examined in SH-SY5Y cells. No apparent cytotoxic effect was observed up to a concentration of 50 μM formononetin (Fig. 2A). When SH-SY5Y cells were treated for 6 h with various concentrations of H2O2 (0−100 μM), cell viability was decreased in an H2O2-concentration-dependent manner (Fig. 2B). The total cell

Discussion

In the present study, we showed that formononetin can suppress H2O2-induced death of human neuroblastoma SH-SY5Y cells. The intracellular ROS level was reduced together with enhanced expression of the antioxidant genes, by formononetin treatment in H2O2-treated cells. Moreover, formononetin lowered the phosphorylation of MAPKs such as ERK, JNK, and p38, which activate the caspase cascade, resulting in suppression of apoptosis. The proposed neuroprotective mechanism of formononetin was shown in

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CRediT authorship contribution statement

Mayuko Sugimoto: Investigation, Formal analysis. Risa Ko: Investigation, Formal analysis. Hiromi Goshima: Investigation, Formal analysis. Atsushi Koike: Investigation, Validation, Formal analysis, Writing - review & editing. Makio Shibano: Investigation, Formal analysis, Validation, Resources, Writing - review & editing. Ko Fujimori: Conceptualization, Supervision, Investigation, Formal analysis, Writing - original draft, Writing - review & editing.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgments

We thank Drs. Minoru Sakaguchi and Satoshi Tanaka (Osaka Medical and Pharmaceutical University) for valuable suggestion.

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