Geraniin exerts cytoprotective effect against cellular oxidative stress by upregulation of Nrf2-mediated antioxidant enzyme expression via PI3K/AKT and ERK1/2 pathway

doi:10.1016/j.bbagen.2015.04.010

Biochimica et Biophysica Acta (BBA) - General Subjects

Volume 1850, Issue 9, September 2015, Pages 1751-1761

https://doi.org/10.1016/j.bbagen.2015.04.010 Get rights and content

Highlights

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Geraniin suppressed H₂O₂-induced cytotoxicity in HepG2 cells.
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Geraniin induced the expression of antioxidant genes and intracellular GSH level.
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Geraniin induced the nuclear translocation of Nrf2.
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Geraniin activates PI3K/AKT and ERK 1/2 signaling pathways.

Abstract

Background

Geraniin, an active compound with remarkable antioxidant activity, was isolated from Geranium sibiricum. The present study aimed to investigate whether geraniin has the ability to activate Nrf2, induce antioxidant enzyme expression and protect cells from oxidative damage.

Methods

The cells were pretreated with geraniin for 24 h and exposed to hydrogen peroxide (H₂O₂) for 4 h. Intracellular reactive oxygen species (ROS) levels, mitochondrial membrane potential and apoptosis were measured. We also investigated intracellular glutathione (GSH) levels and changes in nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated signaling cascade in cells treated with geraniin.

Results

We investigated the protective effects of geraniin against H₂O₂-induced apoptosis in HepG2 cells. Geraniin significantly reduced H₂O₂-induced oxidative damage in a dose dependent manner. Further, geraniin induced the expression of heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase-1 (NQO1) and level of glutathione (GSH) in a concentration- and time-dependent manner, and increased Nrf2 nuclear translocation. The Nrf2-related cytoprotective effects of geraniin were PI3K/AKT and extracellular signal-regulated protein kinase1/2 (ERK1/2) pathway-dependent. However, inhibitors of PI3K/AKT and ERK1/2 (LY294002 or U0126) not only suppressed geraniin-induced nuclear translocation of Nrf2 but also abolished the expression of HO-1, NQO1 and GSH.

Conclusions

These results demonstrated that geraniin induced Nrf2-mediated expression of antioxidant enzymes HO-1 and NQO1, presumably via PI3K/AKT and ERK1/2 signaling pathways, thereby protecting cells from H₂O₂-induced oxidative cell death.

General significance

Geraniin, at least in part, offers an antioxidant defense capacity to protect cells from the oxidative stress-related diseases.

Graphical abstract

Introduction

The excessive generation of reactive oxygen species is associated with cell death and involved in various chronic diseases including atherosclerosis [1], cancer and aging [2]. If the generation and elimination of ROS fail to keep cellular homeostasis, excessive ROS can cause damage to the lipids, proteins and DNA [3]. Therefore, it is necessary for cells to effectively counteract ROS generation by triggering their own defensive mechanisms with the help of antioxidants.

Under normal conditions, nuclear factor-erythroid 2-related factor 2 (Nrf2), a member of the cap “n” collar subfamily of bZIP transcription factors [4], is sequestered in the cytoplasm binding with Kelch-like ECH-associated protein-1 (Keap1), which regulates proteasomal degradation of Nrf2 via the Cullin3-based E3-ligase ubiquitination complex [5]. In response to intracellular oxidative stress, activated Nrf2 is released from Keap1, and translocates to the nucleus where it forms a heterodimer with the small Maf protein. Then, it binds to antioxidant-related elements (ARE) in the DNA and activates the transcription of a series of phase II detoxifying antioxidant genes [6], such as hemeoxygenase 1 (HO-1), NAD(P)H: quinone oxidoreductase1 (NQO1) and γ-glutamate cysteine ligase (GCL). The latter one is the rate-limiting enzyme in GSH synthesis. These findings have suggested the importance of the Nrf2–ARE pathway for enhancing antioxidative defenses and survival of cells from oxidative damage. Moreover, some studies have identified a possible link between both mitogen-activated protein kinases (MAPKs), phosphoinositole 3-kinase (PI3K/AKT) and Nrf2 activation [7], [8]. Indeed, MAPKs and PI3K/AKT play an important role in regulating cell proliferation, survival and apoptosis associated with Nrf2 activation.

So far, many natural resources have been proposed as therapeutic agents to counteract the oxidative stress. It is known that polyphenolic compounds possess a multitude of biological activities, such as antioxidant [9], antihypertensive [10] and antibacterial [11] properties. In human's everyday life, regular consumption of vegetables and fruits can effectively reduce the risk of illness, because they are rich in polyphenols, which are strong antioxidants for free radicals scavenging in the body [12]. Geranium sibiricum is commonly consumed as additive in distilled spirit for drink and for foods [13]. Among the polyphenolic compounds in G. sibiricum, geraniin is a major polyphenolic compound with strong antioxidant activity due to 2,4-hexahydroxydiphenoyl (HHDP)-bearing glucopyranose within 3,6-bridges. The human hepatoma cell line HepG2 represents a suitable model to study xenobiotic metabolism and antioxidant activity, because it is similar with normal hepatocytes and keeps many liver-specific characteristics, especially the activity of phase I, phase II and antioxidant enzymes [14]. Therefore, we have chosen HepG2 cells for our experiments.

In our previous study, geraniin showed efficient radical scavenging activities, including superoxide radical scavenging activity, DPPH radical scavenging activity and the reducing power [15]. To the best of our knowledge, the molecular mechanisms of geraniin-mediated antioxidant effects have not been elucidated yet. Therefore, the present study aimed to investigate whether geraniin conferred an antioxidant defense capacity via activation of Nrf2 and induction of its downstream target genes in hepatocytes. Nrf2 activation by geraniin was found to be due to the phosphorylations of PI3K/AKT and MAPK signaling pathways. Furthermore, geraniin had a cytoprotective effect against oxidative damage in hepatocytes.

Section snippets

Reagents and chemicals

Geraniin (purity ≥ 98%) was purchased from Delta Co. Ltd. (Anhui province, China). A 10 mM stock solution of geraniin was prepared in dimethyl sulfoxide (DMSO) and stored at − 80 °C.

MTT (3-[4,5-dimehyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) and dimethylsulfoximine (DMSO) were purchased from Sigma (St. Louis, MO). Antibodies against ERK, phospho-ERK1/2 (Thr158 + Tyr187 + Thr202 + Tyr204), Akt, phospho-Akt (Ser473), phospho-p38MAPK, p38 MAPK, JNK, phospho-JNK (Thr183/Tyr185), Bcl-2, Bax,

Cell proliferation assay

First of all, the effect of geraniin on HepG2 cell proliferation was examined. HepG2 cells were treated with 0 to 50 μM geraniin for 24 h and cell proliferation was analyzed using the MTT assay. Compared with untreated control cell, we found that geraniin did not inhibit the proliferation of HepG2 cells (Fig. 1B).

Geraniin significantly inhibited H₂O₂-induced cell cytotoxicity

To examine the cytotoxic potential of H₂O₂-induced cell damage, cells were treated with increasing concentrations of H₂O₂ (0–1000 μM) for 4 h, the cell viability was significantly

Discussion

Oxidative stress has been involved in the etiology of numerous diseases and symptoms, Moreover, cardiovascular development is highly sensitive to excessive free radical molecules [19]. Cells possess a plenty of cytoprotective enzymes, which protect living cells from oxidative stress-induced damage. However, exogenous administration of antioxidants is also an important strategy to protect cells against ROS. Numerous polyphenols are involved in the balance between the cellular production of free

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

The authors gratefully acknowledge the financial supports by the Application Technology Research and Development Program of Harbin (2013AA3BS014), Special Fund of the National Natural Science Foundation of China (31270618) and Key Laboratory of Myocardial Ischemia, Harbin Medical University, Chinese Ministry of Education (KF201416).

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¹: These authors contributed equally to the work.

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Geraniin exerts cytoprotective effect against cellular oxidative stress by upregulation of Nrf2-mediated antioxidant enzyme expression via PI3K/AKT and ERK1/2 pathway

Highlights

Abstract

Background

Methods

Results

Conclusions

General significance

Graphical abstract

Introduction

Section snippets

Reagents and chemicals

Cell proliferation assay

Geraniin significantly inhibited H2O2-induced cell cytotoxicity

Discussion

Conflict of interest statement

Acknowledgments

Atherosclerosis

Methods Enzymol.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

J. Biol. Chem.

Food Chem. Toxicol.

Food Bioprod. Process.

Toxicology

Toxicol. Lett.

Food Chem.

Int. J. Biochem. Cell Biol.

Mol. Asp. Med.

Cancer Lett.

Free Radic. Biol. Med.

J. Biol. Chem.

Cancer Lett.

Arch. Biochem. Biophys.

J. Biol. Chem.

Ageing Res. Rev.

Geraniin significantly inhibited H₂O₂-induced cell cytotoxicity