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Asiatic acid enhances Nrf2 signaling to protect HepG2 cells from oxidative damage through Akt and ERK activation
Biomedicine & Pharmacotherapy 88 (2017) 252–259
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Original article
Asiatic acid enhances Nrf2 signaling to protect HepG2 cells from oxidative damage through Akt and ERK activation Zhimin Qia,1, Xinxin Cia,1, Jingbo Huangc , Qinmei Liua , Qinlei Yua , Junfeng Zhoub,* , Xuming Denga,* a b c
Institute of Translational Medicine, The First Hospital of Jilin University, College of Veterinary Medicine, Jilin University, Changchun, 130061, China Department of Dermatology and Venereology, The First Hospital of Jilin University, Changchun, 130061, China Department of Traditional Chinese Medicine, The First Hospital of Jilin University, Changchun, 130061, China
A R T I C L E I N F O
Article history: Received 28 November 2016 Received in revised form 4 January 2017 Accepted 10 January 2017 Keywords: Asiatic acid Nrf2 Oxidative stress HepG2 cells
A B S T R A C T
Asiatic acid (AA), a natural triterpene isolated from the plant Centella asiatica, have antioxidative potential, but the molecular mechanism of AA against oxidative stress remains unclear. Our study was performed to investigate the antioxidative effect of AA against oxidative stress and the antioxidative mechanism in tert-butyl hydroperoxide (t-BHP) -stimulated the HepG2 cells. The results showed that AA suppressed t-BHP-induced cytotoxicity, apoptosis, and reactive oxygen species (ROS) generation. Additionally, AA activated the nuclear factor erythroid 2-related factor 2 (Nrf2) signal, which was closely related to induction Nrf2 nuclear translocation, reduction the expression of Keap1 and up-regulation the activity of the antioxidant response element (ARE). Meanwhile, activation of Nrf2 signal upregulated the protein expressions of antioxidant genes, including heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidase (NQO-1), and glutamyl cysteine ligase catalytic subunit (GCLC). Excitingly, Knockout of Nrf2 almost abolished AA-mediated antioxidant activity and cytoprotection against t-BHP. Further studies showed the mechanism underlying that AA induced Nrf2 activation in HepG2 cells via Akt and ERK signal activation. We found Akt and ERK inhibitors treatment attenuated AA-mediated Nrf2 nuclear translocation. Furthermore, treatment with either Akt or ERK inhibitor also decreased AA-mediated cytoprotection against t-BHP-induced cellular damage. Collectively, these results presented in this study indicate that AA has the protective effect against t-BHP-induced cellular damage and oxidative stress by modulating Nrf2 signaling through activating the signals of Akt and ERK. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Oxidative stress is generally associated with various disorders and diseases, such as various cancers, diabetes and aging [1–4]. Under oxidative stress conditions, excessive reactive oxygen species (ROS) cause lipid peroxidation and seriously damage to DNA and proteins [5]. In the oxidative stress, the cytoprotection mechanisms have been developed in mammalian cells, which is to counteract ROS generation via regulating the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling [6–8]. Nrf2, which is the key nuclear transcription factor regulating expression of antioxidative enzymes, plays a vital role against oxidative stress [9]. Under normal or unstressed conditions, the inactive form of
* Corresponding authors. E-mail addresses: [email protected] (J. Zhou), [email protected] (X. Deng). These authors contributed equally to this work.
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http://dx.doi.org/10.1016/j.biopha.2017.01.067 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
Nrf2 is bound to Kelch-like ECH associated protein 1 (Keap1) in the cytoplasm. Under specific conditions, including oxidative stress, Nrf2 is dissociated from Nrf2-Keap1 complex and transfered into the nucleus [9]. In the nucleus, Nrf2 combines with the antioxidant response element (ARE) at the upstream region of many antioxidative genes, subsequently transcription of antioxidative genes are initiated [10,11]. Activation of Nrf2 signal induces the expressions of antioxidant enzymes, such as heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidase (NQO-1), glutamyl cysteine ligase catalytic subunit (GCLC), glutamyl cysteine ligase modulatory subunit (GCLM). In these above-mentioned enzymes, they act as the multiple roles against oxidative stress. HO-1 is a cytoprotective enzyme that responses to oxidative stress. It is involved in maintaining the redox homeostasis against oxidative stress [12]. Glutamate cysteine ligase (GCL), the rate-limiting enzyme of glutathione (GSH) biosynthesis, is a heterodimer which consists of GCLC and GCLM. GSH and GSH synthetases play crucial roles which involve in eliminating ROS,
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maintaining redox status, and suppressing cell apoptosis [13]. NQO-1, which is among the most clearly induced-phase II enzymes, is well-known to be a relevant marker for the nuclear factor erythroid 2-related factor 2 (Nrf2) activation [14]. Nrf2 signal activation plays a crucial role against cellular oxidative stress, but the mechanism of Nrf2 dissociation from the Keap1-Nrf2 complex is still unclear. Recent studies have suggested that several signal transduction pathways, such as phosphatidylinositol 3-kinase (PI3K) pathways and mitogen-activated protein kinases (MAPKs), may involve in releasing Nrf2 from Keap1-Nrf2 complex and promote Nrf2-mediated translocation [15–17]. Triterpenes possess a variety of biological activities, and they are ubiquitous in the plant. Furthermore, the pharmaceutical activities have received extensive attention. Some triterpenoids derived from the plant have demonstrated potential effects as antiinflammatory agents [18,19]. Asiatic acid (Fig. 1) is a triterpenoid that has been isolated from the plant Centella asiatica, one of the traditional Chinese medicines (TCM). Increasing evidence showed that AA possesses biological effects, including antioxidant and anti-inflammation [20]. It could alleviate H2O2-induced cell death and suppressed free radical generation [21]. Furthermore, previous study suggested that the protect effect of asiatic acid against oxidative damage and inflammation is related to the reduction in the expressions of malondialdehyde (MDA), cyclooxygenase-2 (COX-2), and upregulated the expressions of catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver [20]. Metabolism of t-BHP by cytochrome P-450 (in hepatocytes), which can initiate lipid peroxidation, affect the integrity of cell membrane leading to the cell damage [22,23]. Thus as, the aim of the current study was to explore the cytoprotective effect of asiatic acid (AA) and the underlying mechanisms against oxidative stress by the t-BHP-induced model in HepG2 cells.
Asiatic acid was purchased from the Chengdu Herbpurify Co., Ltd. (Chengdu, China). DMEM and fetal bovine serum (FBS) were obtained from Life Technologies Inc. 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), tert-butyl hydroperoxide (t-BHP), puromycin, U0126 (ERK1/2 inhibitor) and LY294002 (Akt inhibitor) were obtained from Sigma-Aldrich Co (St. Louis, MO). Antibodies against Nrf2, Keap1, HO-1, NQO-1, GCLC, GCLM, phosphatidylinositol 3-kinase (PI3K), phospho-Akt, phospho-extracellular signalregulated kinase (ERK), ERK, phospho-c-Jun NH2-terminal kinase (JNK), JNK, phospho-p38, p38, HADC1, and b-actin were purchased from Cell Signaling (Boston, MA, USA). The horseradish peroxidase- (HRP-) conjugated anti-rabbit or anti-mouse IgG were purchased from Proteintech (Boston, MA, USA). RIPA Lysis Buffer was purchased from Cell Signaling Technology (Beverly, MA). BCA protein assay kit and NE-PER Nuclear and Cytoplasmic Extraction Reagent were purchased from Thermo scientific (Waltham, MA, USA). ViaFect transfection reagent and Dual-Glo luciferase assay kit were purchased from Promega (Madison, WI, USA).
2. Materials and methods
HepG2 cells were grown in 12-well plates (2 105 cells/well) for 24 h, and then were treated to various concentrations of AA for 6 h and subjected to t-BHP (1 mM) for 1 h. Then, cells were washed 3 times with PBS, collected and centrifuged at 1000 rpm/min for 5 min at 4 C. Subsequently, cells were stained Hoechst 33342 and Propidium Iodide (PI). The percentage of apoptosis and necrosis were analyzed by flow cytometry (LSR II Flow Cytometer; BD Biosciences).
2.1. Cell culture, plasmids and reagents Human liver hepatocellular carcinoma cell line HepG2 cells was obtained from the China Cell Line Bank (Beijing, China) and cultured in DMEM (Life Technologies Inc.) medium containing 10% fetal bovine serum (FBS), penicillin (100 IU/mL) and streptomycin (100 mg/mL) at 37 C with 5% CO2. ARE-driven reporter gene plasmid pGL4.37 was obtained from Promega (Madison, WI, USA). Plasmid pGL4.74 was kindly provided by Dr. Guangyun Tan (Jilin University). These vectors for knockout of Nrf2 gene were constructed using CRISPR/Cas9 system in this study by our lab.
2.2. Cell viability assay Cell viability was evaluated by MTT assay, it was performed as described previously [24]. HepG2 cells were grown in 96-well plates (1 104 cells/well) for 24 h incubation at 37 C with 5% CO2. Then the HepG2 cells were incubated with AA for 6 h at various concentration. Then, MTT (5 mg/mL) was added to each well and incubated for 4 h, the supernatant was discarded, and DMSO was added to lyse the cells. Then, the absorbance of MTT was measured at 570 nm by the microplate reader (Bio-Tek Instruments Inc.). 2.3. Quantification of apoptotic cells and necrotic cells
2.4. Detection of intracellular ROS levels HepG2 cells were cultured in 96-well plates (1 104 cells/well). After 24 h, and then the cells were treated with various concentrations of AA for 6 h. Then, the cells were stained with 50 mM of DCFH-DA for 40 min and treated with t-BHP (1 mM) for 15 min to generate ROS. The fluorescence intensities were measured by themicroplate reader with excitation at 485 nm and emission at 535 nm. 2.5. Immunoblotting analysis Immunoblotting was performed as previous description [24]. In short, cells were harvested and lysed in RIPA lysis buffer containing protease and phosphatase inhibitors. The protein concentrations were measured using a Pierce BCA protein assay kit (Thermo Scientific). Nuclear and cytoplasmic fractions of HepG2 cells were extracted as previously described [25]. The immunoblotting analysis used the following specific antibodies: anti-Nrf2, antiHO-1, anti-NQO-1, anti-GCLM, anti-GCLC, anti-p-PI3K, anti-Akt, anti-pAkt, anti-JNK, anti-pJNK, anti-ERK, anti-pERK, anti-pp38, anti-p38, anti-HADC1, and anti-b-actin.
Fig. 1. Chemical structure of the triterpene asiatic acid.
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2.6. CRISPR/Cas9 knockout of Nrf2 gene
3. Results
HepG2 cells were cultured in 12-well plates at the density of 3 105 cells/well for 24 h. The plasmids of expressing Cas9 with Nrf2-sgRNA and puromycin resistant gene were co-transfected into HepG2 cells using Viafect transfection reagent (Promega). At 48 h after transfection, cells were added puromycin at a concentration of 2 mg/ml and harvested for immunoblotting analysis with Nrf2 antibody. After 7 days, cells were cultured in a 96-well plate (1 cell/well). The efficiency of gene editing was evaluated by the western-blot and DNA sequencing was performed to confirm the edited gene of Nrf2.
3.1. AA protects HepG2 cells from t-BHP-induced cytotoxicity and reduces ROS production, apoptosis
2.7. ARE promoter activity assay HepG2 cells were cultured in 96-well plates (1 104 cells/well) for 24 h. Then plasmids pGL4.37 and pGL4.74 were transfected into HepG2 cells using Viafect transfection reagent. After AA treatment with various concentrations for 6 h, The ARE promoter activity was detected by Dual-Glo Luciferase Assay System. ARE activity was measured in the microplate reader. ARE promoter activity was analysed in relative light intensity, which a ratio of ARE-dependent firefly luciferase activity to ARE-independent Renilla luciferase activity. 2.8. Statistical analysis All experimental results were displayed as mean SEM of three independent experiments. The data was analyzed with one-way ANOVA, while multiple comparisons were made using the LSD (least significant difference) tests using SPSS 19.0 software. P < 0.05 was thought statistically significant.
We firstly assessed the potential effect of AA on cell viability by MTT assay. The result showed that AA at tested concentrations (0– 24 mM) was not toxic to HepG2 cells for 6 h (Fig. 2A). This model of oxidative stress (t-BHP treatment) was commonly used in biological systems. Thus, we examined the cytoprotection of AA against t-BHP-induced cell injury. HepG2 cells were pretreated with increasing concentrations of AA (6, 12, and 24 mM) for 6 h and treated with t-BHP (1 mM) for 2.5 h. The data shows that t-BHPinduced cytotoxicity was restrained by AA in a dose-dependent manner (Fig. 2B). Furthermore, our results indicated that AA inhibited intracellular ROS production, which was reduced by treatment with t-BHP (Fig. 2C). Previous reports suggested that increased ROS production are closely associated with apoptosis, and t-BHP treatment could result in cell death via inducing apoptosis. Our result showed that AA effectively decreased t-BHP-induced apoptosis and necrosis in HepG2 cells (Fig. 2D and E). 3.2. AA mediates Nrf2 nuclear translocation and ARE activity Nrf2 plays an essential role in preventing numerous diseases, which is an oxidative stress-mediated nuclear transcription factor that leads to the ARE-driven expression of phase II detoxifying enzymes and antioxidative proteins. We examined whether AA could induce Nrf2 activation. HepG2 cells were incubated with AA (3 h, 6 h, and 18 h) for 24 mM or AA (6, 12, and 24 mM) for 18 h. The result showed AA significantly activated the total protein
Fig. 2. Effects of AA on t-BHP-induced cell death, ROS generation and apoptosis. (A) HepG2 cells were treated with various concentrations of AA for 6 h. (B) HepG2 cells were pro-treated with AA for 6 h and incubated with t-BHP for 2.5 h. (A, B) MTT assay was used to determine the cell viability. (C) HepG2 cells were pro-incubated with AA for 6 h and stained with DCFH-DA (50 mM) for 40 min, cells were exposed to t-BHP (1 mM) for additional 15 min. (D, E) HepG2 cells were exposed to various concentrations of AA for 6 h and subsequently subjected to t-BHP (1 mM) for 1 h. The percentage of cell apoptosis and necrosis were determined using flow cytometry. All results were showed as mean SEM from three independent experiments. Columns marked by different letters differ significantly at P < 0.05 according one-way ANOVA followed by LSD tests.
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Fig. 3. Effects of AA on the activation of Nrf2 and the ARE activity. (A, B) HepG2 cells were treated with AA (24 mM) indicated time periods, or cells were treated with increasing doses of AA for 6 h. Immunoblotting tested the expressions of listed proteins. The levels of proteins were normalized to b-actin or HADC1,??-actin and HADC1 acted as internal controls. (C) The luciferase plasmids pGL4.37 and pGL4.74 were co-transfected into HepG2 cells for 24 h and subsequently exposed to AA. ARE activity was detected by a dual-luciferase reporter assay system. All results were showed as mean SEM from three independent experiments. Columns marked by different letters differ significantly at P < 0.05 according one-way ANOVA followed by LSD tests.
expression of Nrf2 and the degradation of Keap1 (Fig. 3A and B). Further, we confirmed that AA could result in a decrease in the cytoplasmic level and a concomitant increase in the nuclear level of Nrf2 in Fig. 3B. Meanwhile, increasing Nrf2 expression in the nucleus is required for ARE-driven transcription. ARE activity assay suggested that AA markedly increased ARE-driven luciferase activity in Fig. 3C. 3.3. Nrf2 mediates AA-induced antioxidant activities and cytoprotection in HepG2 cells Immunoblotting tested the protein expressions of antioxidative enzymes (HO-1, NQO-1, GCLC and GCLM etc.). The result showed that AA enhanced the protein expression of HO-1, NQO-1 GCLC but not GCLM (Fig. 4A). According to the above results we hypothesized Nrf2 signal plays a crucial role against oxidative stress and cytotoxicity. To confirm the hypothesis, Nrf2 was knocked out in HepG2 cells using the CRISPR/Cas9 gene editing system. So, we examined the protein expressions of antioxidative enzymes in HepG2 WT cells and Nrf2-/- cells. The excitingly result showed that AA-mediated antioxidant activity was almost abolished in Nrf2-/cells (Fig. 4B). In addition, we also tested the cytoprotection effect of AA against t-BHP-induced cell injury in HepG2 cells and Nrf2-/cells. The result showed that the cytoprotection effect of AA almost disappeared in Nrf2-/- cells (Fig. 4C). Based on these results, we suggested that Nrf2 mediated AA-induced antioxidant activities and HepG2 cytoprotection.
3.4. AA induces Nrf2 nuclear translocation via Akt and ERK activation Previous reports have suggested that the PI3K/Akt and MAPK pathways are related to the regulation of Nrf2 nuclear translocation. Therefore, we examined if AA induces the activation of PI3K/ Akt and MAPK pathways in HepG2 cells. After HepG2 cells were incubated with AA (6, 12, and 24 mM) for 6 h, immunoblotting result indicated that AA activated Akt and ERK phosphorylation but not phosphorylation of PI3K, p38, and JNK signals (Fig. 5A and B). To further determine the upstream signaling pathway (Akt, ERK) which were necessary for AA-mediated Nrf2 nuclear translocation, HepG2Cells were treated with either LY294002 (Akt inhibitor, 20 mM) or U0126 (ERK inhibitor, 10 mM) for 6 h and then exposed to AA (24 mM) for 6 h. The data showed that AAmediated Nrf2 nuclear translocation was abolished when Akt or ERK was blocked by respectively inhibitor (Fig. 6A and B). The protein levels of p-Akt was not completely blocked at the Akt inhibitor concentration of 20 mM. Because the increasing concentration of AKT inhibitor is toxic to the HepG2 cells by MTT assay, we selected the appropriate Akt inhibitor concentration to inhibit the protein level of p-Akt. Therefore, these results suggested that AA mediated Nrf2 nuclear translocation via the activation of Akt and ERK signals. 3.5. AA alleviated cellular injury by up-regulating Nrf2 via Akt and ERK activation in HepG2Cells Based on the above results, we hypothesized that the cytoprotection effect of AA against t-BHP-induced oxidative stress
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Fig. 4. Nrf2 mediates AA-induced antioxidant activity and cytoprotection. (A and B) HepG2 WT and Nrf2-/- cells were treated were pro-treated with AA for 6 h. The levels of proteins were examined by Western blot analysis. The levels of proteins were normalized to b-actin. (C) HepG2 WT and Nrf2-/- cells were pro-treated with AA for 6 h followed by exposed to t-BHP (1 mM) for 2.5 h. MTT assay was used to determine the cell viability. All results were showed as mean SEM from three independent experiments. Columns marked by different letters differ significantly at P < 0.05 according one-way ANOVA followed by LSD tests.
Fig. 5. Effects of AA on the activation of the PI3K/Akt and MAPK pathways in HepG2 cells. (A, B) HepG2 cells were treated with increasing doses of AA for 6 h. Immunoblotting tested the levels of listed proteins. The levels of proteins were normalized to b-actin. All results were showed as mean SEM from three independent experiments. Columns marked by different letters differ significantly at P < 0.05 according one-way ANOVA followed by LSD tests.
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Fig. 6. Effects of Akt and ERK activation on AA-induced Nrf2 nuclear translocation. (A, B) HepG2 cells were incubated with LY294002 (Akt inhibitor, 20 mM) or U0126 (ERK inhibitor, 10 mM) for 18 h followed by 6 h incubation with AA (24 mM). Immunoblotting tested the expressions of listed proteins. The levels of proteins were normalized to HADC1 and the unphosphorylated forms (Akt, ERK). All results were showed as mean SEM from three independent experiments. Columns marked by different letters differ significantly at P < 0.05 according one-way ANOVA followed by LSD tests.
was related to the Nrf2 nuclear translocation. Thus we measured the cell viability by MTT assay. HepG2 cells were treated with LY294002 (Akt inhibitor, 20 mM) and U0126 (ERK inhibitor, 10 mM) for 6 h respectively then incubated with AA (24 mM) for 6 h and t-BHP (1 mM) for 2.5 h. Our results showed that AA attenuated t-BHP-induced the reduction of cytoprotection (Fig. 7). 4. Discussion The molecular mechanisms underlying the known pharmacological effects of natural products are largely unclear, but a variety of studies suggest that several mechanisms might be associated with their abilities to serve as antioxidants, free radical scavengers, and modulators of gene expression [26,27]. Oxidative stress is an imbalance between the oxygen free radical generation and their elimination by antioxidants. Overproduction of ROS is associated with the mechanisms of various diseases. The previous research demonstrated that scavenging ROS can slow the progress of the disease such as the neurodegenerative diseases [28]. Thus as, it is probably effective therapeutic strategy that regulating oxidative stress in relevant liver diseases. Asiatic acid, a new type of triterpenoid acid, has been demonstrated that it is a potential agent for the use in antioxidative strategies in SH-SY5Y (human neuroblastoma cell line) cells [29]. In the current study, we are aimed at exploring the antioxidant potential and the mechanism underlying of the natural plant triterpene AA in HepG2 cells. This
Fig. 7. Effects of Akt and ERK activation on t-BHP-induced cytotoxicity. HepG2Cells were pre-incubated with LY294002 (20 mM), U0126 (10 mM), for 1 h and treated with AA for 6 h followed by exposed to t-BHP (1 mM) for 2.5 h. MTT assay was used to determine the cell viability. All results were showed as mean SEM from three independent experiments. Columns marked by different letters differ significantly at P < 0.05 according one-way ANOVA followed by LSD tests.
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study provides an example of this therapeutic strategy in hepatopathy research. t-BHP exposure leads to cell death via inducing apoptosis and result in oxidative stress via the overproduction of ROS. The metabolites of t-BHP destroy the cell membrane integrity by lipid peroxidation resulting in the cell injury [22]. In the present study, AA treatment could alleviate the cytotoxicity, ROS accumulation, and cell apoptosis. These protective effects can attribute to the exposure of AA, which protects HepG2 cells from the cell damage caused by t-BHP. One important finding of this study is that AA activates Nrf2 signal in HepG2 cells. It is well-known that Nrf2 promotes transcriptional activation of a variety of antioxidant genes through binding to ARE, such as HO-1, NQO-1, GCLC and GCLM [11]. The transcriptional activation of Nrf2 is dependent on the rate of nuclear translocation followed by the disaggregation of Nrf2 from Nrf2-Keap1 complex. Here, we discovered that AA induced the protein expressions of ARE-dependent genes (HO-1, NQO-1 and GCLC). Meanwhile, AA suppressed t-BHP-induced ROS generation and apoptosis in HepG2 cells. These results could explain the significant HepG2 cytoprotection and antioxidant. However, these effects of AA almost completely disappeared by the knockout of Nrf2 signal. So, the activation of Nrf2 could be the key signal of AAinduced cytoprotective and antioxidant effects in HepG2 cells. One possible mechanism of Nrf2 activation could relate to the modified cysteine residues of Keap1. Some compounds could change the conformation of Keap1 by the covalent modification, and lead to the release Nrf2 from Nrf2-Keap1 and Nrf2 nuclear translocation [30–32]. Phosphorylation of Nrf2 is probably another mechanism for the Nrf2 activation by the protein kinases. Previous reports have suggested that the PI3K/Akt and MAPK pathways play key roles in regulating Nrf2-dependent transcription [33,34]. One or more MAPK families (e.g., ERK, JNK, and p38) function to activate the downstream genes following stimulation [35–37]. ERK signaling plays an important role in regulating cell cycle, cell proliferation, and cell differentiation [38], and JNK and p38 signals are stimulated by a variety of environmental stresses, such as ultraviolet (UV) radiation, cytokines, inflammatory factors, and ROS [39]. In vivo and in vitro, MAPK and PI3K signaling is involved in the Nrf2-dependent transcription of ARE-related antioxidant genes, such as NQO-1 gene [40]. In this study, these kinases possibly promote the release of Nrf2 from Keap1 and its subsequent translocation by the phosphorylation of Nrf2 at serine and threonine residues. We found that AA treatment enhanced expression of Akt and ERK signals which might be required for subsequent Nrf2-dependent transcription, whereas PI3K, JNK and p38 signals did not be activated. Moreover, the treatment of Akt or ERK inhibitor significant decreased subsequent Nrf2 phosphorylation and nuclear translocation in HepG2 cell. AA-mediated cytoprotection against t-BHP was also almost abolished by these inhibitors treatment. The above results supported that the cytoprotection of AA is associated with Nrf2 activation through the modulation of Akt and ERK pathways. In conclusion, AA has cytoprotective effect against t-BHPinduced cell damage via suppressing cytotoxicity, ROS generation and apoptosis. Furthermore, AA enhanced the expressions of antioxidant enzymes by activation of Nrf2 signaling. This antioxidation mechanism of AA-mediated Nrf2 activation and nuclear translocation is attributed to Akt and ERK signals activation. Our study supports that AA has the potential therapeutic effect in the oxidative stress-induced diseases.
Conflict of interest The authors declare to have no conflict of interest. Acknowledgment This work was supported by the General Financial Grant from the China Postdoctoral Science Foundation (Grant No. 168847). References [1] D. Pitocco, M. Tesauro, R. Alessandro, G. Ghirlanda, C. Cardillo, Oxidative stress in diabetes: implications for vascular and other complications, Int. J. Mol. Sci. 14 (11) (2013) 21525–21550. [2] L.H. Breimer, Molecular mechanisms of oxygen radical carcinogenesis and mutagenesis: the role of DNA base damage, Mol. Carcinog. 3 (4) (1990) 188– 197. [3] A.E. Vendrov, K.C. Vendrov, A. Smith, J. Yuan, A. Sumida, J. Robidoux, M.S. Runge, N.R. Madamanchi, NOX4 NADPH oxidase-dependent mitochondrial oxidative stress in aging-associated cardiovascular disease, Antioxid. Redox Signal. 23 (18) (2015) 1389–1409. [4] V. Sosa, T. Moline, R. Somoza, R. Paciucci, H. Kondoh, M.E. Leonart, Oxidative stress and cancer: an overview, Ageing Res. Rev. 12 (1) (2013) 376–390. [5] H. Bartsch, J. Nair, Chronic inflammation and oxidative stress in the genesis and perpetuation of cancer: role of lipid peroxidation, DNA damage, and repair, Langenbeck’s Arch. Surg. Deutsche Gesellschaft fur Chirurgie 391 (5) (2006) 499–510. [6] H.E. de Vries, M. Witte, D. Hondius, A.J. Rozemuller, B. Drukarch, J. Hoozemans, J. van Horssen, Nrf2-induced antioxidant protection: a promising target to counteract ROS-mediated damage in neurodegenerative disease? Free Radic. Biol. Med. 45 (10) (2008) 1375–1383. [7] X.L. Chen, C. Kunsch, Induction of cytoprotective genes through Nrf2/ antioxidant response element pathway: a new therapeutic approach for the treatment of inflammatory diseases, Curr. Pharm. Des. 0 (8) (2004) 879–891. [8] S. Kovac, P.R. Angelova, K.M. Holmstrom, Y. Zhang, A.T. Dinkova-Kostova, A.Y. Abramov, Nrf2 regulates ROS production by mitochondria and NADPH oxidase, Biochim. Biophys. Acta 1850 (4) (2015) 794–801. [9] T.W. Kensler, N. Wakabayashi, S. Biswal, Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway, Annu. Rev. Pharmacol. Toxicol. 47 (2007) 89–116. [10] Q. Ma, Role of nrf2 in oxidative stress and toxicity, Annu. Rev. Pharmacol. Toxicol. 53 (2013) 401–426. [11] C.L. Liu, Y.T. Chiu, M.L. Hu, Fucoxanthin enhances HO-1 and NQO1 expression in murine hepatic BNL CL.2 cells through activation of the Nrf2/ARE system partially by its pro-oxidant activity, J. Agric. Food Chem. 59 (20) (2011) 11344– 11351. [12] C.D. Ferris, S.R. Jaffrey, A. Sawa, M. Takahashi, S.D. Brady, R.K. Barrow, S.A. Tysoe, H. Wolosker, D.E. Baranano, S. Dore, K.D. Poss, S.H. Snyder, Haem oxygenase-1 prevents cell death by regulating cellular iron, Nat. Cell Biol. 1 (3) (1999) 152–157. [13] T.P. Dalton, M.Z. Dieter, Y. Yang, H.G. Shertzer, D.W. Nebert, Knockout of the mouse glutamate cysteine ligase catalytic subunit (Gclc) gene: embryonic lethal when homozygous, and proposed model for moderate glutathione deficiency when heterozygous, Biochem. Biophys. Res. Commun. 279 (2) (2000) 324–329. [14] L. Marrot, C. Jones, P. Perez, J.R. Meunier, The significance of Nrf2 pathway in (photo)-oxidative stress response in melanocytes and keratinocytes of the human epidermis, Pigm. Cell Melanoma Res. 21 (1) (2008) 79–88. [15] Y.J. Lee, H.Y. Jeong, Y.B. Kim, Y.J. Lee, S.Y. Won, J.H. Shim, M.K. Cho, H.S. Nam, S.H. Lee, Reactive oxygen species and PI3K/Akt signaling play key roles in the induction of Nrf2-driven heme oxygenase-1 expression in sulforaphanetreated human mesothelioma MSTO-211H cells, Food Chem. Toxicol. 50 (2) (2012) 116–123. [16] R. Yu, C. Chen, Y.Y. Mo, V. Hebbar, E.D. Owuor, T.H. Tan, A.N. Kong, Activation of mitogen-activated protein kinase pathways induces antioxidant response element-mediated gene expression via a Nrf2-dependent mechanism, J. Biol. Chem. 275 (51) (2000) 39907–39913. [17] K. Nakaso, H. Yano, Y. Fukuhara, T. Takeshima, K. Wada-Isoe, K. Nakashima, PI3K is a key molecule in the Nrf2-mediated regulation of antioxidative proteins by hemin in human neuroblastoma cells, FEBS Lett. 546 (2–3) (2003) 181–184. [18] G.J. Huang, S.S. Huang, S.S. Lin, Y.Y. Shao, C.C. Chen, W.C. Hou, Y.H. Kuo, Analgesic effects and the mechanisms of anti-inflammation of ergostatrien3beta-ol from Antrodia camphorata submerged whole broth in mice, J. Agric. Food Chem. 58 (12) (2010) 7445–7452. [19] M. Saleem, Lupeol, a novel anti-inflammatory and anti-cancer dietary triterpene, Cancer Lett. 285 (2) (2009) 109–115. [20] S.S. Huang, C.S. Chiu, H.J. Chen, W.C. Hou, M.J. Sheu, Y.C. Lin, P.H. Shie, G.J. Huang, Antinociceptive activities and the mechanisms of anti-inflammation of asiatic Acid in mice, Evid. Based Complement. Altern. Med. eCAM 2011 (2011) 895857.
Z. Qi et al. / Biomedicine & Pharmacotherapy 88 (2017) 252–259 [21] I. Mook-Jung, J.E. Shin, S.H. Yun, K. Huh, J.Y. Koh, H.K. Park, S.S. Jew, M.W. Jung, Protective effects of asiaticoside derivatives against beta-amyloid neurotoxicity, J. Neurosci. Res. 58 (3) (1999) 417–425. [22] G.F. Rush, J.R. Gorski, M.G. Ripple, J. Sowinski, P. Bugelski, W.R. Hewitt, Organic hydroperoxide-induced lipid peroxidation and cell death in isolated hepatocytes, Toxicol. Appl. Pharmacol. 78 (3) (1985) 473–483. [23] N. Masaki, M.E. Kyle, J.L. Farber, Tert-butyl hydroperoxide kills cultured hepatocytes by peroxidizing membrane lipids, Arch. Biochem. Biophys. 269 (2) (1989) 390–399. [24] X. Ci, H. Lv, L. Wang, X. Wang, L. Peng, F.X. Qin, G. Cheng, The antioxidative potential of farrerol occurs via the activation of Nrf2 mediated HO-1 signaling in RAW 264.7 cells, Chemico-biol. Interact. 239 (2015) 192–199. [25] H. Lv, H. Ren, L. Wang, W. Chen, X. Ci, Lico A enhances Nrf2-mediated defense mechanisms against t-BHP-induced oxidative stress and cell death via Akt and ERK activation in RAW 264.7 cells, Oxid. Med. Cell. Longev. 2015 (2015) 709845. [26] R.J. Nijveldt, E. van Nood, D.E. van Hoorn, P.G. Boelens, K. van Norren, P.A. van Leeuwen, Flavonoids: a review of probable mechanisms of action and potential applications, Am. J. Clin. Nutr. 74 (4) (2001) 418–425. [27] M. Zhu, Q. Chang, L.K. Wong, F.S. Chong, R.C. Li, Triterpene antioxidants from ganoderma lucidum, Phytother. Res. PTR 13 (6) (1999) 529–531. [28] B. Uttara, A.V. Singh, P. Zamboni, R.T. Mahajan, Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options, Curr. Neuropharmacol. 7 (1) (2009) 65–74. [29] Y. Xiong, H. Ding, M. Xu, J. Gao, Protective effects of asiatic acid on rotenone- or H2O2-induced injury in SH-SY5Y cells, Neurochem. Res. 34 (4) (2009) 746– 754.
259
[30] S. Tanigawa, M. Fujii, D.X. Hou, Action of Nrf2 and Keap1 in ARE-mediated NQO1 expression by quercetin, Free Radic. Biol. Med. 42 (11) (2007) 1690–1703. [31] X. Chen, J. Liu, S.Y. Chen, Over-expression of Nrf2 diminishes ethanol-induced oxidative stress and apoptosis in neural crest cells by inducing an antioxidant response, Reprod. Toxicol. 42 (2013) 102–109. [32] H. Cao, X. Pan, C. Li, C. Zhou, F. Deng, T. Li, Density functional theory calculations for resveratrol, Bioorg. Med. Chem. Lett. 13 (11) (2003) 1869–1871. [33] J. Kim, Y.N. Cha, Y.J. Surh, A protective role of nuclear factor-erythroid 2-related factor-2 (Nrf2) in inflammatory disorders, Mutat. Res. 690 (1–2) (2010) 12–23. [34] M. Torres, H.J. Forman, Redox signaling and the MAP kinase pathways, BioFactors 17 (1–4) (2003) 287–296. [35] G.L. Johnson, R. Lapadat, Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases, Science 298 (5600) (2002) 1911–1912. [36] T. Obata, G.E. Brown, M.B. Yaffe, MAP kinase pathways activated by stress: the p38 MAPK pathway, Crit. Care Med. 28 (4 Suppl) (2000) N67–77. [37] B.B. Aggarwal, S. Shishodia, Molecular targets of dietary agents for prevention and therapy of cancer, Biochem. Pharmacol. 71 (10) (2006) 1397–1421. [38] A.N. Kong, R. Yu, V. Hebbar, C. Chen, E. Owuor, R. Hu, R. Ee, S. Mandlekar, Signal transduction events elicited by cancer prevention compounds, Mutat. Res. 480–481 (2001) 231–241. [39] K.M. Cheng, C.M. Chan, Y.L. Cheung, C.C. Liang, M.K. Lee, C.L. Leung, H.M. Chiu, C.H. Chan, Transvenous embolisation of spontaneous carotid-cavernous fistulas by sequential occlusion of the cavernous sinus, Intervent. Neuroradiol. 5 (3) (1999) 225–234. [40] A. Giudice, M. Montella, Activation of the Nrf2-ARE signaling pathway: a promising strategy in cancer prevention, BioEssays: news and reviews in molecular, Cell. Dev. Biol. 28 (2) (2006) 169–181.
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Original article
Asiatic acid enhances Nrf2 signaling to protect HepG2 cells from oxidative damage through Akt and ERK activation Zhimin Qia,1, Xinxin Cia,1, Jingbo Huangc , Qinmei Liua , Qinlei Yua , Junfeng Zhoub,* , Xuming Denga,* a b c
Institute of Translational Medicine, The First Hospital of Jilin University, College of Veterinary Medicine, Jilin University, Changchun, 130061, China Department of Dermatology and Venereology, The First Hospital of Jilin University, Changchun, 130061, China Department of Traditional Chinese Medicine, The First Hospital of Jilin University, Changchun, 130061, China
A R T I C L E I N F O
Article history: Received 28 November 2016 Received in revised form 4 January 2017 Accepted 10 January 2017 Keywords: Asiatic acid Nrf2 Oxidative stress HepG2 cells
A B S T R A C T
Asiatic acid (AA), a natural triterpene isolated from the plant Centella asiatica, have antioxidative potential, but the molecular mechanism of AA against oxidative stress remains unclear. Our study was performed to investigate the antioxidative effect of AA against oxidative stress and the antioxidative mechanism in tert-butyl hydroperoxide (t-BHP) -stimulated the HepG2 cells. The results showed that AA suppressed t-BHP-induced cytotoxicity, apoptosis, and reactive oxygen species (ROS) generation. Additionally, AA activated the nuclear factor erythroid 2-related factor 2 (Nrf2) signal, which was closely related to induction Nrf2 nuclear translocation, reduction the expression of Keap1 and up-regulation the activity of the antioxidant response element (ARE). Meanwhile, activation of Nrf2 signal upregulated the protein expressions of antioxidant genes, including heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidase (NQO-1), and glutamyl cysteine ligase catalytic subunit (GCLC). Excitingly, Knockout of Nrf2 almost abolished AA-mediated antioxidant activity and cytoprotection against t-BHP. Further studies showed the mechanism underlying that AA induced Nrf2 activation in HepG2 cells via Akt and ERK signal activation. We found Akt and ERK inhibitors treatment attenuated AA-mediated Nrf2 nuclear translocation. Furthermore, treatment with either Akt or ERK inhibitor also decreased AA-mediated cytoprotection against t-BHP-induced cellular damage. Collectively, these results presented in this study indicate that AA has the protective effect against t-BHP-induced cellular damage and oxidative stress by modulating Nrf2 signaling through activating the signals of Akt and ERK. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Oxidative stress is generally associated with various disorders and diseases, such as various cancers, diabetes and aging [1–4]. Under oxidative stress conditions, excessive reactive oxygen species (ROS) cause lipid peroxidation and seriously damage to DNA and proteins [5]. In the oxidative stress, the cytoprotection mechanisms have been developed in mammalian cells, which is to counteract ROS generation via regulating the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling [6–8]. Nrf2, which is the key nuclear transcription factor regulating expression of antioxidative enzymes, plays a vital role against oxidative stress [9]. Under normal or unstressed conditions, the inactive form of
* Corresponding authors. E-mail addresses: [email protected] (J. Zhou), [email protected] (X. Deng). These authors contributed equally to this work.
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http://dx.doi.org/10.1016/j.biopha.2017.01.067 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
Nrf2 is bound to Kelch-like ECH associated protein 1 (Keap1) in the cytoplasm. Under specific conditions, including oxidative stress, Nrf2 is dissociated from Nrf2-Keap1 complex and transfered into the nucleus [9]. In the nucleus, Nrf2 combines with the antioxidant response element (ARE) at the upstream region of many antioxidative genes, subsequently transcription of antioxidative genes are initiated [10,11]. Activation of Nrf2 signal induces the expressions of antioxidant enzymes, such as heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidase (NQO-1), glutamyl cysteine ligase catalytic subunit (GCLC), glutamyl cysteine ligase modulatory subunit (GCLM). In these above-mentioned enzymes, they act as the multiple roles against oxidative stress. HO-1 is a cytoprotective enzyme that responses to oxidative stress. It is involved in maintaining the redox homeostasis against oxidative stress [12]. Glutamate cysteine ligase (GCL), the rate-limiting enzyme of glutathione (GSH) biosynthesis, is a heterodimer which consists of GCLC and GCLM. GSH and GSH synthetases play crucial roles which involve in eliminating ROS,
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maintaining redox status, and suppressing cell apoptosis [13]. NQO-1, which is among the most clearly induced-phase II enzymes, is well-known to be a relevant marker for the nuclear factor erythroid 2-related factor 2 (Nrf2) activation [14]. Nrf2 signal activation plays a crucial role against cellular oxidative stress, but the mechanism of Nrf2 dissociation from the Keap1-Nrf2 complex is still unclear. Recent studies have suggested that several signal transduction pathways, such as phosphatidylinositol 3-kinase (PI3K) pathways and mitogen-activated protein kinases (MAPKs), may involve in releasing Nrf2 from Keap1-Nrf2 complex and promote Nrf2-mediated translocation [15–17]. Triterpenes possess a variety of biological activities, and they are ubiquitous in the plant. Furthermore, the pharmaceutical activities have received extensive attention. Some triterpenoids derived from the plant have demonstrated potential effects as antiinflammatory agents [18,19]. Asiatic acid (Fig. 1) is a triterpenoid that has been isolated from the plant Centella asiatica, one of the traditional Chinese medicines (TCM). Increasing evidence showed that AA possesses biological effects, including antioxidant and anti-inflammation [20]. It could alleviate H2O2-induced cell death and suppressed free radical generation [21]. Furthermore, previous study suggested that the protect effect of asiatic acid against oxidative damage and inflammation is related to the reduction in the expressions of malondialdehyde (MDA), cyclooxygenase-2 (COX-2), and upregulated the expressions of catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver [20]. Metabolism of t-BHP by cytochrome P-450 (in hepatocytes), which can initiate lipid peroxidation, affect the integrity of cell membrane leading to the cell damage [22,23]. Thus as, the aim of the current study was to explore the cytoprotective effect of asiatic acid (AA) and the underlying mechanisms against oxidative stress by the t-BHP-induced model in HepG2 cells.
Asiatic acid was purchased from the Chengdu Herbpurify Co., Ltd. (Chengdu, China). DMEM and fetal bovine serum (FBS) were obtained from Life Technologies Inc. 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), tert-butyl hydroperoxide (t-BHP), puromycin, U0126 (ERK1/2 inhibitor) and LY294002 (Akt inhibitor) were obtained from Sigma-Aldrich Co (St. Louis, MO). Antibodies against Nrf2, Keap1, HO-1, NQO-1, GCLC, GCLM, phosphatidylinositol 3-kinase (PI3K), phospho-Akt, phospho-extracellular signalregulated kinase (ERK), ERK, phospho-c-Jun NH2-terminal kinase (JNK), JNK, phospho-p38, p38, HADC1, and b-actin were purchased from Cell Signaling (Boston, MA, USA). The horseradish peroxidase- (HRP-) conjugated anti-rabbit or anti-mouse IgG were purchased from Proteintech (Boston, MA, USA). RIPA Lysis Buffer was purchased from Cell Signaling Technology (Beverly, MA). BCA protein assay kit and NE-PER Nuclear and Cytoplasmic Extraction Reagent were purchased from Thermo scientific (Waltham, MA, USA). ViaFect transfection reagent and Dual-Glo luciferase assay kit were purchased from Promega (Madison, WI, USA).
2. Materials and methods
HepG2 cells were grown in 12-well plates (2 105 cells/well) for 24 h, and then were treated to various concentrations of AA for 6 h and subjected to t-BHP (1 mM) for 1 h. Then, cells were washed 3 times with PBS, collected and centrifuged at 1000 rpm/min for 5 min at 4 C. Subsequently, cells were stained Hoechst 33342 and Propidium Iodide (PI). The percentage of apoptosis and necrosis were analyzed by flow cytometry (LSR II Flow Cytometer; BD Biosciences).
2.1. Cell culture, plasmids and reagents Human liver hepatocellular carcinoma cell line HepG2 cells was obtained from the China Cell Line Bank (Beijing, China) and cultured in DMEM (Life Technologies Inc.) medium containing 10% fetal bovine serum (FBS), penicillin (100 IU/mL) and streptomycin (100 mg/mL) at 37 C with 5% CO2. ARE-driven reporter gene plasmid pGL4.37 was obtained from Promega (Madison, WI, USA). Plasmid pGL4.74 was kindly provided by Dr. Guangyun Tan (Jilin University). These vectors for knockout of Nrf2 gene were constructed using CRISPR/Cas9 system in this study by our lab.
2.2. Cell viability assay Cell viability was evaluated by MTT assay, it was performed as described previously [24]. HepG2 cells were grown in 96-well plates (1 104 cells/well) for 24 h incubation at 37 C with 5% CO2. Then the HepG2 cells were incubated with AA for 6 h at various concentration. Then, MTT (5 mg/mL) was added to each well and incubated for 4 h, the supernatant was discarded, and DMSO was added to lyse the cells. Then, the absorbance of MTT was measured at 570 nm by the microplate reader (Bio-Tek Instruments Inc.). 2.3. Quantification of apoptotic cells and necrotic cells
2.4. Detection of intracellular ROS levels HepG2 cells were cultured in 96-well plates (1 104 cells/well). After 24 h, and then the cells were treated with various concentrations of AA for 6 h. Then, the cells were stained with 50 mM of DCFH-DA for 40 min and treated with t-BHP (1 mM) for 15 min to generate ROS. The fluorescence intensities were measured by themicroplate reader with excitation at 485 nm and emission at 535 nm. 2.5. Immunoblotting analysis Immunoblotting was performed as previous description [24]. In short, cells were harvested and lysed in RIPA lysis buffer containing protease and phosphatase inhibitors. The protein concentrations were measured using a Pierce BCA protein assay kit (Thermo Scientific). Nuclear and cytoplasmic fractions of HepG2 cells were extracted as previously described [25]. The immunoblotting analysis used the following specific antibodies: anti-Nrf2, antiHO-1, anti-NQO-1, anti-GCLM, anti-GCLC, anti-p-PI3K, anti-Akt, anti-pAkt, anti-JNK, anti-pJNK, anti-ERK, anti-pERK, anti-pp38, anti-p38, anti-HADC1, and anti-b-actin.
Fig. 1. Chemical structure of the triterpene asiatic acid.
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2.6. CRISPR/Cas9 knockout of Nrf2 gene
3. Results
HepG2 cells were cultured in 12-well plates at the density of 3 105 cells/well for 24 h. The plasmids of expressing Cas9 with Nrf2-sgRNA and puromycin resistant gene were co-transfected into HepG2 cells using Viafect transfection reagent (Promega). At 48 h after transfection, cells were added puromycin at a concentration of 2 mg/ml and harvested for immunoblotting analysis with Nrf2 antibody. After 7 days, cells were cultured in a 96-well plate (1 cell/well). The efficiency of gene editing was evaluated by the western-blot and DNA sequencing was performed to confirm the edited gene of Nrf2.
3.1. AA protects HepG2 cells from t-BHP-induced cytotoxicity and reduces ROS production, apoptosis
2.7. ARE promoter activity assay HepG2 cells were cultured in 96-well plates (1 104 cells/well) for 24 h. Then plasmids pGL4.37 and pGL4.74 were transfected into HepG2 cells using Viafect transfection reagent. After AA treatment with various concentrations for 6 h, The ARE promoter activity was detected by Dual-Glo Luciferase Assay System. ARE activity was measured in the microplate reader. ARE promoter activity was analysed in relative light intensity, which a ratio of ARE-dependent firefly luciferase activity to ARE-independent Renilla luciferase activity. 2.8. Statistical analysis All experimental results were displayed as mean SEM of three independent experiments. The data was analyzed with one-way ANOVA, while multiple comparisons were made using the LSD (least significant difference) tests using SPSS 19.0 software. P < 0.05 was thought statistically significant.
We firstly assessed the potential effect of AA on cell viability by MTT assay. The result showed that AA at tested concentrations (0– 24 mM) was not toxic to HepG2 cells for 6 h (Fig. 2A). This model of oxidative stress (t-BHP treatment) was commonly used in biological systems. Thus, we examined the cytoprotection of AA against t-BHP-induced cell injury. HepG2 cells were pretreated with increasing concentrations of AA (6, 12, and 24 mM) for 6 h and treated with t-BHP (1 mM) for 2.5 h. The data shows that t-BHPinduced cytotoxicity was restrained by AA in a dose-dependent manner (Fig. 2B). Furthermore, our results indicated that AA inhibited intracellular ROS production, which was reduced by treatment with t-BHP (Fig. 2C). Previous reports suggested that increased ROS production are closely associated with apoptosis, and t-BHP treatment could result in cell death via inducing apoptosis. Our result showed that AA effectively decreased t-BHP-induced apoptosis and necrosis in HepG2 cells (Fig. 2D and E). 3.2. AA mediates Nrf2 nuclear translocation and ARE activity Nrf2 plays an essential role in preventing numerous diseases, which is an oxidative stress-mediated nuclear transcription factor that leads to the ARE-driven expression of phase II detoxifying enzymes and antioxidative proteins. We examined whether AA could induce Nrf2 activation. HepG2 cells were incubated with AA (3 h, 6 h, and 18 h) for 24 mM or AA (6, 12, and 24 mM) for 18 h. The result showed AA significantly activated the total protein
Fig. 2. Effects of AA on t-BHP-induced cell death, ROS generation and apoptosis. (A) HepG2 cells were treated with various concentrations of AA for 6 h. (B) HepG2 cells were pro-treated with AA for 6 h and incubated with t-BHP for 2.5 h. (A, B) MTT assay was used to determine the cell viability. (C) HepG2 cells were pro-incubated with AA for 6 h and stained with DCFH-DA (50 mM) for 40 min, cells were exposed to t-BHP (1 mM) for additional 15 min. (D, E) HepG2 cells were exposed to various concentrations of AA for 6 h and subsequently subjected to t-BHP (1 mM) for 1 h. The percentage of cell apoptosis and necrosis were determined using flow cytometry. All results were showed as mean SEM from three independent experiments. Columns marked by different letters differ significantly at P < 0.05 according one-way ANOVA followed by LSD tests.
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Fig. 3. Effects of AA on the activation of Nrf2 and the ARE activity. (A, B) HepG2 cells were treated with AA (24 mM) indicated time periods, or cells were treated with increasing doses of AA for 6 h. Immunoblotting tested the expressions of listed proteins. The levels of proteins were normalized to b-actin or HADC1,??-actin and HADC1 acted as internal controls. (C) The luciferase plasmids pGL4.37 and pGL4.74 were co-transfected into HepG2 cells for 24 h and subsequently exposed to AA. ARE activity was detected by a dual-luciferase reporter assay system. All results were showed as mean SEM from three independent experiments. Columns marked by different letters differ significantly at P < 0.05 according one-way ANOVA followed by LSD tests.
expression of Nrf2 and the degradation of Keap1 (Fig. 3A and B). Further, we confirmed that AA could result in a decrease in the cytoplasmic level and a concomitant increase in the nuclear level of Nrf2 in Fig. 3B. Meanwhile, increasing Nrf2 expression in the nucleus is required for ARE-driven transcription. ARE activity assay suggested that AA markedly increased ARE-driven luciferase activity in Fig. 3C. 3.3. Nrf2 mediates AA-induced antioxidant activities and cytoprotection in HepG2 cells Immunoblotting tested the protein expressions of antioxidative enzymes (HO-1, NQO-1, GCLC and GCLM etc.). The result showed that AA enhanced the protein expression of HO-1, NQO-1 GCLC but not GCLM (Fig. 4A). According to the above results we hypothesized Nrf2 signal plays a crucial role against oxidative stress and cytotoxicity. To confirm the hypothesis, Nrf2 was knocked out in HepG2 cells using the CRISPR/Cas9 gene editing system. So, we examined the protein expressions of antioxidative enzymes in HepG2 WT cells and Nrf2-/- cells. The excitingly result showed that AA-mediated antioxidant activity was almost abolished in Nrf2-/cells (Fig. 4B). In addition, we also tested the cytoprotection effect of AA against t-BHP-induced cell injury in HepG2 cells and Nrf2-/cells. The result showed that the cytoprotection effect of AA almost disappeared in Nrf2-/- cells (Fig. 4C). Based on these results, we suggested that Nrf2 mediated AA-induced antioxidant activities and HepG2 cytoprotection.
3.4. AA induces Nrf2 nuclear translocation via Akt and ERK activation Previous reports have suggested that the PI3K/Akt and MAPK pathways are related to the regulation of Nrf2 nuclear translocation. Therefore, we examined if AA induces the activation of PI3K/ Akt and MAPK pathways in HepG2 cells. After HepG2 cells were incubated with AA (6, 12, and 24 mM) for 6 h, immunoblotting result indicated that AA activated Akt and ERK phosphorylation but not phosphorylation of PI3K, p38, and JNK signals (Fig. 5A and B). To further determine the upstream signaling pathway (Akt, ERK) which were necessary for AA-mediated Nrf2 nuclear translocation, HepG2Cells were treated with either LY294002 (Akt inhibitor, 20 mM) or U0126 (ERK inhibitor, 10 mM) for 6 h and then exposed to AA (24 mM) for 6 h. The data showed that AAmediated Nrf2 nuclear translocation was abolished when Akt or ERK was blocked by respectively inhibitor (Fig. 6A and B). The protein levels of p-Akt was not completely blocked at the Akt inhibitor concentration of 20 mM. Because the increasing concentration of AKT inhibitor is toxic to the HepG2 cells by MTT assay, we selected the appropriate Akt inhibitor concentration to inhibit the protein level of p-Akt. Therefore, these results suggested that AA mediated Nrf2 nuclear translocation via the activation of Akt and ERK signals. 3.5. AA alleviated cellular injury by up-regulating Nrf2 via Akt and ERK activation in HepG2Cells Based on the above results, we hypothesized that the cytoprotection effect of AA against t-BHP-induced oxidative stress
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Fig. 4. Nrf2 mediates AA-induced antioxidant activity and cytoprotection. (A and B) HepG2 WT and Nrf2-/- cells were treated were pro-treated with AA for 6 h. The levels of proteins were examined by Western blot analysis. The levels of proteins were normalized to b-actin. (C) HepG2 WT and Nrf2-/- cells were pro-treated with AA for 6 h followed by exposed to t-BHP (1 mM) for 2.5 h. MTT assay was used to determine the cell viability. All results were showed as mean SEM from three independent experiments. Columns marked by different letters differ significantly at P < 0.05 according one-way ANOVA followed by LSD tests.
Fig. 5. Effects of AA on the activation of the PI3K/Akt and MAPK pathways in HepG2 cells. (A, B) HepG2 cells were treated with increasing doses of AA for 6 h. Immunoblotting tested the levels of listed proteins. The levels of proteins were normalized to b-actin. All results were showed as mean SEM from three independent experiments. Columns marked by different letters differ significantly at P < 0.05 according one-way ANOVA followed by LSD tests.
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Fig. 6. Effects of Akt and ERK activation on AA-induced Nrf2 nuclear translocation. (A, B) HepG2 cells were incubated with LY294002 (Akt inhibitor, 20 mM) or U0126 (ERK inhibitor, 10 mM) for 18 h followed by 6 h incubation with AA (24 mM). Immunoblotting tested the expressions of listed proteins. The levels of proteins were normalized to HADC1 and the unphosphorylated forms (Akt, ERK). All results were showed as mean SEM from three independent experiments. Columns marked by different letters differ significantly at P < 0.05 according one-way ANOVA followed by LSD tests.
was related to the Nrf2 nuclear translocation. Thus we measured the cell viability by MTT assay. HepG2 cells were treated with LY294002 (Akt inhibitor, 20 mM) and U0126 (ERK inhibitor, 10 mM) for 6 h respectively then incubated with AA (24 mM) for 6 h and t-BHP (1 mM) for 2.5 h. Our results showed that AA attenuated t-BHP-induced the reduction of cytoprotection (Fig. 7). 4. Discussion The molecular mechanisms underlying the known pharmacological effects of natural products are largely unclear, but a variety of studies suggest that several mechanisms might be associated with their abilities to serve as antioxidants, free radical scavengers, and modulators of gene expression [26,27]. Oxidative stress is an imbalance between the oxygen free radical generation and their elimination by antioxidants. Overproduction of ROS is associated with the mechanisms of various diseases. The previous research demonstrated that scavenging ROS can slow the progress of the disease such as the neurodegenerative diseases [28]. Thus as, it is probably effective therapeutic strategy that regulating oxidative stress in relevant liver diseases. Asiatic acid, a new type of triterpenoid acid, has been demonstrated that it is a potential agent for the use in antioxidative strategies in SH-SY5Y (human neuroblastoma cell line) cells [29]. In the current study, we are aimed at exploring the antioxidant potential and the mechanism underlying of the natural plant triterpene AA in HepG2 cells. This
Fig. 7. Effects of Akt and ERK activation on t-BHP-induced cytotoxicity. HepG2Cells were pre-incubated with LY294002 (20 mM), U0126 (10 mM), for 1 h and treated with AA for 6 h followed by exposed to t-BHP (1 mM) for 2.5 h. MTT assay was used to determine the cell viability. All results were showed as mean SEM from three independent experiments. Columns marked by different letters differ significantly at P < 0.05 according one-way ANOVA followed by LSD tests.
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study provides an example of this therapeutic strategy in hepatopathy research. t-BHP exposure leads to cell death via inducing apoptosis and result in oxidative stress via the overproduction of ROS. The metabolites of t-BHP destroy the cell membrane integrity by lipid peroxidation resulting in the cell injury [22]. In the present study, AA treatment could alleviate the cytotoxicity, ROS accumulation, and cell apoptosis. These protective effects can attribute to the exposure of AA, which protects HepG2 cells from the cell damage caused by t-BHP. One important finding of this study is that AA activates Nrf2 signal in HepG2 cells. It is well-known that Nrf2 promotes transcriptional activation of a variety of antioxidant genes through binding to ARE, such as HO-1, NQO-1, GCLC and GCLM [11]. The transcriptional activation of Nrf2 is dependent on the rate of nuclear translocation followed by the disaggregation of Nrf2 from Nrf2-Keap1 complex. Here, we discovered that AA induced the protein expressions of ARE-dependent genes (HO-1, NQO-1 and GCLC). Meanwhile, AA suppressed t-BHP-induced ROS generation and apoptosis in HepG2 cells. These results could explain the significant HepG2 cytoprotection and antioxidant. However, these effects of AA almost completely disappeared by the knockout of Nrf2 signal. So, the activation of Nrf2 could be the key signal of AAinduced cytoprotective and antioxidant effects in HepG2 cells. One possible mechanism of Nrf2 activation could relate to the modified cysteine residues of Keap1. Some compounds could change the conformation of Keap1 by the covalent modification, and lead to the release Nrf2 from Nrf2-Keap1 and Nrf2 nuclear translocation [30–32]. Phosphorylation of Nrf2 is probably another mechanism for the Nrf2 activation by the protein kinases. Previous reports have suggested that the PI3K/Akt and MAPK pathways play key roles in regulating Nrf2-dependent transcription [33,34]. One or more MAPK families (e.g., ERK, JNK, and p38) function to activate the downstream genes following stimulation [35–37]. ERK signaling plays an important role in regulating cell cycle, cell proliferation, and cell differentiation [38], and JNK and p38 signals are stimulated by a variety of environmental stresses, such as ultraviolet (UV) radiation, cytokines, inflammatory factors, and ROS [39]. In vivo and in vitro, MAPK and PI3K signaling is involved in the Nrf2-dependent transcription of ARE-related antioxidant genes, such as NQO-1 gene [40]. In this study, these kinases possibly promote the release of Nrf2 from Keap1 and its subsequent translocation by the phosphorylation of Nrf2 at serine and threonine residues. We found that AA treatment enhanced expression of Akt and ERK signals which might be required for subsequent Nrf2-dependent transcription, whereas PI3K, JNK and p38 signals did not be activated. Moreover, the treatment of Akt or ERK inhibitor significant decreased subsequent Nrf2 phosphorylation and nuclear translocation in HepG2 cell. AA-mediated cytoprotection against t-BHP was also almost abolished by these inhibitors treatment. The above results supported that the cytoprotection of AA is associated with Nrf2 activation through the modulation of Akt and ERK pathways. In conclusion, AA has cytoprotective effect against t-BHPinduced cell damage via suppressing cytotoxicity, ROS generation and apoptosis. Furthermore, AA enhanced the expressions of antioxidant enzymes by activation of Nrf2 signaling. This antioxidation mechanism of AA-mediated Nrf2 activation and nuclear translocation is attributed to Akt and ERK signals activation. Our study supports that AA has the potential therapeutic effect in the oxidative stress-induced diseases.
Conflict of interest The authors declare to have no conflict of interest. Acknowledgment This work was supported by the General Financial Grant from the China Postdoctoral Science Foundation (Grant No. 168847). References [1] D. Pitocco, M. Tesauro, R. Alessandro, G. Ghirlanda, C. Cardillo, Oxidative stress in diabetes: implications for vascular and other complications, Int. J. Mol. Sci. 14 (11) (2013) 21525–21550. [2] L.H. Breimer, Molecular mechanisms of oxygen radical carcinogenesis and mutagenesis: the role of DNA base damage, Mol. Carcinog. 3 (4) (1990) 188– 197. [3] A.E. Vendrov, K.C. Vendrov, A. Smith, J. Yuan, A. Sumida, J. Robidoux, M.S. Runge, N.R. Madamanchi, NOX4 NADPH oxidase-dependent mitochondrial oxidative stress in aging-associated cardiovascular disease, Antioxid. Redox Signal. 23 (18) (2015) 1389–1409. [4] V. Sosa, T. Moline, R. Somoza, R. Paciucci, H. Kondoh, M.E. Leonart, Oxidative stress and cancer: an overview, Ageing Res. Rev. 12 (1) (2013) 376–390. [5] H. Bartsch, J. Nair, Chronic inflammation and oxidative stress in the genesis and perpetuation of cancer: role of lipid peroxidation, DNA damage, and repair, Langenbeck’s Arch. Surg. Deutsche Gesellschaft fur Chirurgie 391 (5) (2006) 499–510. [6] H.E. de Vries, M. Witte, D. Hondius, A.J. Rozemuller, B. Drukarch, J. Hoozemans, J. van Horssen, Nrf2-induced antioxidant protection: a promising target to counteract ROS-mediated damage in neurodegenerative disease? Free Radic. Biol. Med. 45 (10) (2008) 1375–1383. [7] X.L. Chen, C. Kunsch, Induction of cytoprotective genes through Nrf2/ antioxidant response element pathway: a new therapeutic approach for the treatment of inflammatory diseases, Curr. Pharm. Des. 0 (8) (2004) 879–891. [8] S. Kovac, P.R. Angelova, K.M. Holmstrom, Y. Zhang, A.T. Dinkova-Kostova, A.Y. Abramov, Nrf2 regulates ROS production by mitochondria and NADPH oxidase, Biochim. Biophys. Acta 1850 (4) (2015) 794–801. [9] T.W. Kensler, N. Wakabayashi, S. Biswal, Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway, Annu. Rev. Pharmacol. Toxicol. 47 (2007) 89–116. [10] Q. Ma, Role of nrf2 in oxidative stress and toxicity, Annu. Rev. Pharmacol. Toxicol. 53 (2013) 401–426. [11] C.L. Liu, Y.T. Chiu, M.L. Hu, Fucoxanthin enhances HO-1 and NQO1 expression in murine hepatic BNL CL.2 cells through activation of the Nrf2/ARE system partially by its pro-oxidant activity, J. Agric. Food Chem. 59 (20) (2011) 11344– 11351. [12] C.D. Ferris, S.R. Jaffrey, A. Sawa, M. Takahashi, S.D. Brady, R.K. Barrow, S.A. Tysoe, H. Wolosker, D.E. Baranano, S. Dore, K.D. Poss, S.H. Snyder, Haem oxygenase-1 prevents cell death by regulating cellular iron, Nat. Cell Biol. 1 (3) (1999) 152–157. [13] T.P. Dalton, M.Z. Dieter, Y. Yang, H.G. Shertzer, D.W. Nebert, Knockout of the mouse glutamate cysteine ligase catalytic subunit (Gclc) gene: embryonic lethal when homozygous, and proposed model for moderate glutathione deficiency when heterozygous, Biochem. Biophys. Res. Commun. 279 (2) (2000) 324–329. [14] L. Marrot, C. Jones, P. Perez, J.R. Meunier, The significance of Nrf2 pathway in (photo)-oxidative stress response in melanocytes and keratinocytes of the human epidermis, Pigm. Cell Melanoma Res. 21 (1) (2008) 79–88. [15] Y.J. Lee, H.Y. Jeong, Y.B. Kim, Y.J. Lee, S.Y. Won, J.H. Shim, M.K. Cho, H.S. Nam, S.H. Lee, Reactive oxygen species and PI3K/Akt signaling play key roles in the induction of Nrf2-driven heme oxygenase-1 expression in sulforaphanetreated human mesothelioma MSTO-211H cells, Food Chem. Toxicol. 50 (2) (2012) 116–123. [16] R. Yu, C. Chen, Y.Y. Mo, V. Hebbar, E.D. Owuor, T.H. Tan, A.N. Kong, Activation of mitogen-activated protein kinase pathways induces antioxidant response element-mediated gene expression via a Nrf2-dependent mechanism, J. Biol. Chem. 275 (51) (2000) 39907–39913. [17] K. Nakaso, H. Yano, Y. Fukuhara, T. Takeshima, K. Wada-Isoe, K. Nakashima, PI3K is a key molecule in the Nrf2-mediated regulation of antioxidative proteins by hemin in human neuroblastoma cells, FEBS Lett. 546 (2–3) (2003) 181–184. [18] G.J. Huang, S.S. Huang, S.S. Lin, Y.Y. Shao, C.C. Chen, W.C. Hou, Y.H. Kuo, Analgesic effects and the mechanisms of anti-inflammation of ergostatrien3beta-ol from Antrodia camphorata submerged whole broth in mice, J. Agric. Food Chem. 58 (12) (2010) 7445–7452. [19] M. Saleem, Lupeol, a novel anti-inflammatory and anti-cancer dietary triterpene, Cancer Lett. 285 (2) (2009) 109–115. [20] S.S. Huang, C.S. Chiu, H.J. Chen, W.C. Hou, M.J. Sheu, Y.C. Lin, P.H. Shie, G.J. Huang, Antinociceptive activities and the mechanisms of anti-inflammation of asiatic Acid in mice, Evid. Based Complement. Altern. Med. eCAM 2011 (2011) 895857.
Z. Qi et al. / Biomedicine & Pharmacotherapy 88 (2017) 252–259 [21] I. Mook-Jung, J.E. Shin, S.H. Yun, K. Huh, J.Y. Koh, H.K. Park, S.S. Jew, M.W. Jung, Protective effects of asiaticoside derivatives against beta-amyloid neurotoxicity, J. Neurosci. Res. 58 (3) (1999) 417–425. [22] G.F. Rush, J.R. Gorski, M.G. Ripple, J. Sowinski, P. Bugelski, W.R. Hewitt, Organic hydroperoxide-induced lipid peroxidation and cell death in isolated hepatocytes, Toxicol. Appl. Pharmacol. 78 (3) (1985) 473–483. [23] N. Masaki, M.E. Kyle, J.L. Farber, Tert-butyl hydroperoxide kills cultured hepatocytes by peroxidizing membrane lipids, Arch. Biochem. Biophys. 269 (2) (1989) 390–399. [24] X. Ci, H. Lv, L. Wang, X. Wang, L. Peng, F.X. Qin, G. Cheng, The antioxidative potential of farrerol occurs via the activation of Nrf2 mediated HO-1 signaling in RAW 264.7 cells, Chemico-biol. Interact. 239 (2015) 192–199. [25] H. Lv, H. Ren, L. Wang, W. Chen, X. Ci, Lico A enhances Nrf2-mediated defense mechanisms against t-BHP-induced oxidative stress and cell death via Akt and ERK activation in RAW 264.7 cells, Oxid. Med. Cell. Longev. 2015 (2015) 709845. [26] R.J. Nijveldt, E. van Nood, D.E. van Hoorn, P.G. Boelens, K. van Norren, P.A. van Leeuwen, Flavonoids: a review of probable mechanisms of action and potential applications, Am. J. Clin. Nutr. 74 (4) (2001) 418–425. [27] M. Zhu, Q. Chang, L.K. Wong, F.S. Chong, R.C. Li, Triterpene antioxidants from ganoderma lucidum, Phytother. Res. PTR 13 (6) (1999) 529–531. [28] B. Uttara, A.V. Singh, P. Zamboni, R.T. Mahajan, Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options, Curr. Neuropharmacol. 7 (1) (2009) 65–74. [29] Y. Xiong, H. Ding, M. Xu, J. Gao, Protective effects of asiatic acid on rotenone- or H2O2-induced injury in SH-SY5Y cells, Neurochem. Res. 34 (4) (2009) 746– 754.
259
[30] S. Tanigawa, M. Fujii, D.X. Hou, Action of Nrf2 and Keap1 in ARE-mediated NQO1 expression by quercetin, Free Radic. Biol. Med. 42 (11) (2007) 1690–1703. [31] X. Chen, J. Liu, S.Y. Chen, Over-expression of Nrf2 diminishes ethanol-induced oxidative stress and apoptosis in neural crest cells by inducing an antioxidant response, Reprod. Toxicol. 42 (2013) 102–109. [32] H. Cao, X. Pan, C. Li, C. Zhou, F. Deng, T. Li, Density functional theory calculations for resveratrol, Bioorg. Med. Chem. Lett. 13 (11) (2003) 1869–1871. [33] J. Kim, Y.N. Cha, Y.J. Surh, A protective role of nuclear factor-erythroid 2-related factor-2 (Nrf2) in inflammatory disorders, Mutat. Res. 690 (1–2) (2010) 12–23. [34] M. Torres, H.J. Forman, Redox signaling and the MAP kinase pathways, BioFactors 17 (1–4) (2003) 287–296. [35] G.L. Johnson, R. Lapadat, Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases, Science 298 (5600) (2002) 1911–1912. [36] T. Obata, G.E. Brown, M.B. Yaffe, MAP kinase pathways activated by stress: the p38 MAPK pathway, Crit. Care Med. 28 (4 Suppl) (2000) N67–77. [37] B.B. Aggarwal, S. Shishodia, Molecular targets of dietary agents for prevention and therapy of cancer, Biochem. Pharmacol. 71 (10) (2006) 1397–1421. [38] A.N. Kong, R. Yu, V. Hebbar, C. Chen, E. Owuor, R. Hu, R. Ee, S. Mandlekar, Signal transduction events elicited by cancer prevention compounds, Mutat. Res. 480–481 (2001) 231–241. [39] K.M. Cheng, C.M. Chan, Y.L. Cheung, C.C. Liang, M.K. Lee, C.L. Leung, H.M. Chiu, C.H. Chan, Transvenous embolisation of spontaneous carotid-cavernous fistulas by sequential occlusion of the cavernous sinus, Intervent. Neuroradiol. 5 (3) (1999) 225–234. [40] A. Giudice, M. Montella, Activation of the Nrf2-ARE signaling pathway: a promising strategy in cancer prevention, BioEssays: news and reviews in molecular, Cell. Dev. Biol. 28 (2) (2006) 169–181.