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The antioxidative potential of farrerol occurs via the activation of Nrf2 mediated HO-1 signaling in RAW 264.7 cells
Chemico-Biological Interactions 239 (2015) 192–199
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The antioxidative potential of farrerol occurs via the activation of Nrf2 mediated HO-1 signaling in RAW 264.7 cells Xinxin Ci a, Hongming Lv a,f, Lidong Wang a, Xiaosong Wang a, Liping Peng a, F. Xiao-Feng Qin a,b,c,d,⇑, Genhong Cheng a,b,c,e,⇑ a
Institute of Translational Medicine, The First Hospital, Jilin University, Changchun 130001, China Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China Suzhou Institute of Systems Medicine, Suzhou 215123, China d Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA e Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA f Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun 130061, China b c
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Article history: Received 6 January 2015 Received in revised form 4 May 2015 Accepted 22 June 2015 Available online 22 June 2015 Keywords: Farrerol Heme oxygenase-1 Nuclear translocation of NF-E2-related factor 2 Phosphatidylinositol 3-kinase Mitogen-activated protein kinase
a b s t r a c t Farrerol, (S)-2,3-dihydro-5,7-dihydroxy-2-(4-hydroxyphenyl)-6,8-dimethyl-4-benzopyrone, isolated from rhododendron, has been shown to have antioxidative potential, but the molecular mechanism underlying this activity remains unclear. The inducible expression of heme oxygenase-1 (HO-1), a potent antioxidative and cytoprotective enzyme, is known to play an important role in cytoprotection in a variety of pathological models. In this study, we evaluated the antioxidative potential of farrerol against oxidative damage and investigated its antioxidative mechanism in RAW 264.7 cells. The molecular mechanism underlying the cytoprotective function of farrerol was determined by analyzing intracellular signaling pathways, transcriptional activation and the inhibitory effect of HO-1 on ROS production. Farrerol induced antioxidant enzymes mRNA expression, HO-1 protein expression and nuclear translocation of NF-E2-related factor 2 in RAW 264.7 macrophage cells. Farrerol down-regulated the expression of the Keap1 protein and the thiol reducing agents attenuated farrerol-induced HO-1 expression. Further investigation utilizing Western blotting and specific inhibitors of Akt, p38, JNK and ERK demonstrated that Akt, p38, and ERK axis of signaling pathway mediates HO-1 expression. Moreover, tert-butyl hydroperoxide (t-BHP)-induced oxidative damage was ameliorated by farrerol treatment in a dose-dependent manner, which was abolished by Akt, p38, ERK and HO-1 inhibitors (Snpp). It is hence likely that farrerol inactivated KEAP-1 or activated the Akt, p38 and ERK to facilitate the release of Nrf2 from Keap1 and subsequent reduced the intracellular production of reactive oxygen species via the induction of HO-1 expression. These results support the central role of HO-1 in the cytoprotective effect of farrerol. Ó 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Oxidative stress is defined as an imbalance between the production of free radicals and reactive metabolites, so-called oxidants or reactive oxygen species (ROS), and their elimination by protective potential, referred to as antioxidants [1]. An overproduction of ROS can cause damage to lipids, proteins and DNA, resulting in a wide spectrum of diseases, including chronic inflammation and a wide variety of different cancers [2–6]. As mammalian cells have ⇑ Corresponding authors at: Institute of Translational Medicine, The First Hospital, Jilin University, Changchun 130001, China. E-mail addresses: [email protected] (F. Xiao-Feng Qin), [email protected]. edu (G. Cheng). http://dx.doi.org/10.1016/j.cbi.2015.06.032 0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
developed several protective mechanisms to prevent ROS formation or to detoxify ROS, the use of compounds with antioxidative properties may help to prevent or alleviate many diseases associated with ROS [7]. These mechanisms employ molecules called antioxidants as well as cytoprotective enzymes such as, NAD(P)H quinone oxidore ductase (NQO1), glutathione S-transferase (GST), heme oxygenase-1 (HO-1), and glutathione peroxidase (GSH-Px) [8,9]. Among these various cytoprotective enzymes, HO-1 has anti-inflammatory, antioxidative, and antiproliferative potential [10] and also performs critical roles in both biosynthetic and degradation pathways for heme. When HO-1 catalyzes heme to biliverdin and bilirubin, these degradation products have antioxidant properties. So HO-1 played a critical role in maintaining a
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cellular redox homeostasis against the oxidative stress [11]. The transcriptional regulation of the ho-1 gene is linked to the transcription factor NF-E2-related factor (Nrf2), which plays a crucial role in cellular defense [12]. Under normal conditions, Nrf2 is sequestered in the cytoplasm by binding to Keap 1, an actin-binding protein, which maintains Nrf2 in a constantly ubiquitylated state, leading to its degradation in the cytoplasm. Upon oxidative stress, Nrf2 is released from Keap1, translocates into the nucleus, forms a heterodimer with the small Maf protein and binds to anti oxidant-related elements (ARE) in the promoter region of phase II detoxifying enzymes, such as HO-1. Although the mechanism by which Nrf2 is liberated from the Keap1–Nrf2 complex remains to be established, recent studies have suggested that the phosphorylation of Nrf2 at serine and threonine residues by kinases such as phosphatidylinositol 3-kinase (PI3K), PKC, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) facilitates the release of Nrf2 from Keap1 and subsequent translocation, followed by the initiation of antioxidative cascades [13]. As growing evidence suggests that HO-1 provides cytoprotection, the pharmacological modulation of HO-1 expression may represent a novel target for therapeutic intervention [14,15]. In particular, the identification of a non-cytotoxic inducer of HO-1 may maximize the intrinsic antioxidative potential of cells. Many natural resources that scavenge free radicals have been identified and reported to induce HO-1 expression in a variety of cells, such as liver cells, retinal pigment epithelial cells and macrophage cells, and hence may function against oxidative stress [16–18]. Flavonoids, plant polyphenolic compounds that are abundant in fruits and vegetables, have been studied for their potent antioxidative capacity. Some flavonoids isolated from the roots of Rhododendron mucronulatum Turzaninov were reported to be potential anti-inflammatory agents based on the results of their dose-dependent inhibition of the expression of inflammatory mediators, NO and PGE2 [19]. Farrerol, a new type of 2,3-dihydro-flavonoid, has also been isolated from rhododendron, a traditional Chinese herbal medicine [20]. Accumulating evidence suggests that farrerol possesses many biological properties, including antitussive, antibacterial, anti-inflammatory effects, with also an inhibitory effect on vascular smooth muscle cells (VSMCs) proliferation [21,22]. Previous study showed that farrerol has significant cytoprotective potential against hydrogen peroxide (H2O2)-induced injury of human umbilical vein endothelial cells [23]. Accumulating evidence has also suggested that farrerol has a cytoprotective effect against oxidative stress of EA.hy926 cells, which is associated with the regulation of malondialdehyde (MDA), superoxide dismutase (SOD), ROS and GSH-Px [24]. Therefore, the present study aimed to evaluate the antioxidative potential of farrerol against oxidative damage of RAW 264.7 cells and to determine the underlying molecular mechanisms.
2. Materials and methods 2.1. Reagents Farrerol, ((S)-2,3-dihydro-5,7-dihydroxy-2-(4-hydroxypheny l)-6,8-dimethyl-4-benzopyrone, analytical grade, purity P 98%) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). 3-(4,5-Di methylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT), tert-butyl hydroperoxide (t-BHP), Dimethyl sulfoxide (DMSO), U0126, SB203580, SP600125 and LY294002 (specific inhibitors of the ERK1/2, p38, JNK1/2 and PI3K/Akt, respectively), DCFH-DA and Protoporphyrin IX zinc were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). Antibodies against Nrf2,
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HO-1, KEAP1, AKT, phosphor-AKT, phospho-extracellular signal-regulated kinase (ERK), ERK, phospho-c-Jun NH2-terminal kinase (JNK), JNK, phospho-p38, p38, as well as b-actin were purchased from Cell Signaling (Boston, MA, USA) or Abcam (Cambridge, MA, USA). The horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse IgG were purchased from proteintech (Boston, MA, USA). TRIzol reagent was from Invitrogen (Carlsbad, CA, USA). The control siRNA and Nrf-2 siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). X-treme GENE siRNA Transfection Reagent and Faststart Universal SYBR Green Master were purchased from Roche (Basel, Switzerland). Prime-Script RT-PCR kit was purchased from Takara (Dalian, China). 2.2. Cell culture and treatment The RAW 264.7 murine macrophage cell line was obtained from the China Cell Line Bank (Beijing, China). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 3 mM glutamine, antibiotics (100 U/mL penicillin and 100 U/mL streptomycin), and 10% heat-inactivated fetal bovine serum. The cells were maintained at 37 °C in a humidified incubator containing 5% CO2. In all experiments, cells were allowed to acclimate for 24 h before any treatments. 2.3. MTT assay for cell viability To measure cell viability, the MTT assay was performed. RAW 264.7 cells were mechanically scraped, seeded in 96-well plates at 4 105 cells/mL, 100 ll/well, and incubated in a 37 °C, 5% CO2 incubator overnight. Then the cells were treated with 100 lL different concentrations of farrerol (0–80 mg/L) for 20 h. Subsequently, 20 ll of 5 mg/mL MTT in FBS-free medium was added to each well and incubated for an additional 4 h. Cell-free supernatants were then removed and resolved cells with 150 lL/well DMSO. The optical density was measured at 570 nm on a microplate reader. To measure the antioxidative potential of farrerol against the t-BHP-induced oxidative damage in RAW 264.7 cell, the cells were treated with 50 lL different concentrations of farrerol (5, 10, 20 mg/L) for 12 h, followed by stimulation with 50 lL of t-BHP for 3 h. Subsequently, 20 lL of 5 mg/mL MTT in FBS-free medium was added to each well and incubated for an additional 4 h. Cell-free supernatants were then removed and resolved with 150 lL/well DMSO. The optical density was measured at 570 nm on a microplate reader. 2.4. Intracellular reactive oxygen species formation assay RAW 264.7 cells were grown in 24-well plates (1 105 cells/well) for 24 h incubation, and then the cells were pre-incubated with various concentrations of farrerol for 18 h. Next, the cells were stained with 10 lM of DCFH-DA for 2 h and subsequently incubated with t-BHP (0.5 mM) for 30 min to induce ROS generation. The fluorescence of the dye was measured using a multi-detection reader (Bio-Tek Instruments Inc.) at excitation and emission wave-lengths of 485 nm and 530 nm, respectively. 2.5. Isolation of total RNA from cells and qPCR Total RNA from cells was isolated using the Trizol reagent and the manufacturer’s instructions. After the concentration of RNA was determined by spectrophotometer, 1 lg of RNA was converted to cDNA using the Prime-Script RT-PCR kit, and a real-time PCR analysis was performed using the Applied Biosystems 7300 real-time PCR system and software (Applied Biosystems,
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Carlsbad, CA, USA). Real-time PCR was performed in 0.2 mL PCR tubes with forward and reverse primers and the SYBR green working solution using a custom PCR master mix with the following conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s. The relative gene expression was analyzed by normalization to b-actin mRNA expression.
2.6. Western blot analysis RAW 264.7 cell (1 106/well) were cultured in 6-well plates for 24 h and then treated for 3 h, 6 h and 24 h with various concentrations of farrerol. After each experiment, the cells were washed twice with cold PBS and then scraped from the plate with 500 lL of PBS. The cell homogenates were centrifuged at 3000 g for 5 min. Nuclear and cytoplasmic fractions of cell were prepared as previously described [25]. Whole cell lysates were lysed in 1% non-diet P-40 lysis buffer (1% NP-40, 150 mM Nacl, 50 mM Tris, pH 7.4) with freshly added protease and phosphatase inhibitors. After incubation on ice for 30 min, the lysates were centrifuged (12,000g at 4 °C) for 5 min to obtain the cytosolic fraction. Protein concentrations were determined using the Bradford assay before storage at 80 °C. The cell lysates were subjected to an immunoblotting analysis using the following antibodies: anti-pAKT, anti-pJNK, anti-pERK, anti-pp38, anti-AKT, anti-JNK, anti-ERK, anti-p38, anti-Nrf-2, anti-HO-1, and anti-b-actin. The membranes were further probed with HRP-conjugated secondary antibodies and detected using an ECL Western blot substrate. The band intensities were quantified using Image J gel analysis software. The fold increase in the level of protein expression was calculated by comparison with that of normal controls. The experiments were repeated three times for each experimental condition.
2.7. Nrf2-siRNA transient transfection For Nrf2-siRNA transfection, RAW 264.7 cells (4 105/well) were cultured in 6-well plates for at least 24 h until the confluence of cells reached approximately 50%. Then transfected with Nrf2-siRNA or Nrf2-negative control siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) using the X-treme GENE siRNA Transfection Reagent (Roche Applied Science), according to the manufacturer instructions for 24 h. The transfected cells were treated with farrerol for 24 h followed by lysis buffer for Western blot analysis.
2.8. Statistical analysis Two-tailed unpaired Student’s t tests were performed to determine p values. All the graphs represent the mean ± SEM of three independent experiments, and the asterisks represent p values: * p < 0.05, **p < 0.01.
3. Results
Fig. 1. Effect of farrerol on macrophage toxicity, the mRNA expression of antioxidative enzymes and the protein expression of HO-1. (A) Effect of farrerol on macrophage toxicity. Cell were cultured with farrerol (0–40 mg/L) for 24 h and cell viability was estimated by the MTT method; (B) effect of farrerol on the mRNA expression of antioxidative enzymes. RAW 264.7 cells were treated with 20 mg/L farrerol for 24 h and real-time PCR analyses was performed to assess HO-1, NQO1 and GCLC expression. (C) and (D) Effect of farrerol on the protein expression of HO1. Western blot analysis was conducted to confirm the (C) dose-dependent of HO-1 protein expression induction by farrerol for 24 h and (D) time-dependent of HO-1 protein expression induction by 20 mg/L farrerol. **p 6 0.01 vs. control group.
3.2. Effects of farrerol on the mRNA expression of antioxidant enzymes and the protein expression of HO-1
3.1. Cytotoxicity of farrerol in RAW 264.7 We first examined the effect of farrerol on the viability of RAW 264.7 cells. The maximum final concentration of farrerol was chosen with respect to its solubility in the culture medium. As shown in Fig. 1A, farrerol (0–20 mg/L) do not exert cytotoxicity in RAW 264.7 cells.
Real-time PCR analysis was performed to determine the effect of farrerol on phase II enzymes expression. Activation of the antioxidant response element (ARE) by the Nrf2 pathway causes the induction of phase II enzymes. mRNAs for HO-1, NQO1 and c-glutamate cysteine ligase catalytic subunit (GCLC) were increased in RAW 264.7 cells treated with farrerol (Fig. 1B). As
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HO-1 is an important component of the cellular defense against oxidative stress, we further assessed whether farrerol could induce HO-1 protein expression in a dose- or time- dependent manner. RAW 264.7 cells exposed to farrerol (0–20 mg/L) for 18 h showed a dose-dependent manner increase in HO-1 protein expression (Fig 1C), and the treatment of cells with 20 mg/L farrerol resulted in a time-dependent increase in HO-1 protein expression (Fig 1D). 3.3. Farrerol-induced expression of HO-1 was mediated by Nrf2 Nrf2 plays a key role in HO-1 expression and the role of Nrf2 in farrerol-induced HO-1 expression was confirmed using siRNA. RAW 264.7 cells were transiently transfected with control or Nrf2 siRNA, and Nrf2 protein expression was measured by Western blot analysis. Fig. 2A demonstrates that Nrf2 siRNA can inhibit Nrf2 protein expression. We further examined whether farrerol-induced up-regulation of HO-1 expression is mediated by Nrf2 by using Nrf2 siRNA to knockdown Nrf2. As shown in Fig. 2B, up-regulated HO-1 protein expression caused by farrerol in RAW 264.7 cells was abolished when Nrf2 expression was silenced with a specific siRNA. 3.4. Effects of farrerol on Nrf2 nuclear translocation and Keap1 expression Nrf2 is an important transcription factor that regulates the ARE-driven expression of phase II detoxifying and antioxidant enzymes such as HO-1. Treatment with farrerol increased Nrf2 translocation from the cytoplasm to the nucleus (Fig. 3A) in a dose-dependent manner. Moreover, farrerol down-regulated the expression of the Keap1 protein in a dose-dependent manner (Fig. 3B). 3.5. Cysteine residues of Keap1 may be putative targets of farrerol for its activation of Nrf2 and HO-1 induction Several investigations have shown that some phytochemicals directly interact with Keap1 by binding to its thiol group, facilitating the release of Nrf2 [26]. To explore this possibility, RAW 264.7 cells were incubated with farrerol with or without 100 lM DTT or 1.4 lM b-mercaptoethanol. Both disulfide reducing agents abrogated the farrerol-induced HO-1 expression. 3.6. Involvement of the PI3K/Akt and MAPK pathways in HO-1 expression and Nrf2 nuclear translocation by farrerol To further elucidate the upstream signaling pathway involved in farrerol-mediated Nrf2 activation and HO-1 induction, we examined the activation of PI3K/Akt and MAPKs in RAW 264.7 cells treated with farrerol. As shown in Fig. 4A and B, the induction of Akt and all three MAPKs was detected in farrerol-treated RAW 264.7 cells by Western blotting using specific antibodies. No change in the expression of total Akt and the three MAPK proteins was detected, whereas Akt, ERK and p38 phosphorylation was significantly increased in a dose-dependent manner. To address the role of the individual Akt and MAPK pathways in HO-1 expression induced by farrerol, we examined the effects of LY294002, U0126, SP600125, and SB203580, specific inhibitors for the PI3K/Akt, ERK, p38 and JNK pathways, on HO-1 expression. Although the JNK inhibitor had no effect on farrerol-induced HO-1 expression, the inhibitors of the Akt, ERK and p38 did significantly reduce farrerol-induced HO-1 expression (Fig 4C). Next, we examined whether the PI3K/Akt and MAPK pathways are involved in the process by which farrerol causes Nrf2 nuclear translocation. As shown in Fig. 4D, the inhibitors of the PI3K/Akt, p38 and ERK
Fig. 2. Abrogation of HO-1 expression induced by farrerol in RAW 264.7 transiently transfected with Nrf2-siRNA. (A) Cells were transfected with Nrf2-siRNA or Nrf2negative control siRNA for 24 h, after cells were harvested, proteins were detected by Western blot. (B) Nrf2 regulates farrerol-induced HO-1 protein expression. Cells were transfected with Nrf2-siRNA or Nrf2-negative control siRNA for 24 h. The transfected cells were treated with farrerol (20 mg/L) for 24 h and protein expression of HO-1 was measured by Western blot analysis. **p 6 0.01 vs. control siRNA + farrerol group.
pathways, but not the JNK pathway, blocked farrerol-induced Nrf2 nuclear accumulation. These results indicate a role for PI3K/Akt, p38 and ERK signaling in farrerol mediated HO-1 induction through the nuclear translocation of Nrf2.
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Fig. 4. Effect of thiol reducing agents on farrerol-induced HO-1 expression. (A) and (B) Abrogation of farrerol-induced HO-1 upregulation by the reducing agents DTT. RAW 264.7 cells were pretreated with 100 lM DTT for 1 h followed by 24 hincubation with 20 mg/L farrerol. (C) and (D) Abrogation of farrerol-induced HO-1 upregulation by the reducing agents b-mercaptoethanol (b-ME). RAW 264.7 cells were pretreated with 1.4 lM b-ME for 1 h followed by 24 h-incubation with 20 mg/ L farrerol. **p 6 0.01 vs. control group, ##p 6 0.01 vs. farrerol group.
3.8. Effect of farrerol on t-BHP-induced RAW 264.7 cell cytotoxicity Fig. 3. Effects of farrerol on the protein expression levels of Nrf2 and Keap1 in RAW 264.7 macrophages. (A) Nuclear and cytoplasmic extracts from RAW 264.7 cells were prepared after treatment with farrerol at 5, 10 and 20 mg/L for 24 h. (B) Total protein extracts from different dose of farrerol treatment were prepared for detecting KEAP1. **p 6 0.01 vs. control group.
3.7. Effect of farrerol on t-BHP-induced ROS generation in RAW 264.7 cells To identify the antioxidative potential of farrerol, a ROS scavenging assay was performed. The data shown in Fig. 5 indicate that farrerol significantly attenuated t-BHP-induced ROS formation in a dose-dependent manner in RAW 264.7 cells, which suggests that farrerol might be a potent antioxidant.
Free radicals trigger lipid peroxidation and subsequent cellular injury; therefore, increased free radical production could be a primary factor of cytotoxicity [27]. In this study, t-BHP, an organic hydroperoxide, was applied to induce oxidative damage in RAW 264.7 cells. Untreated and farrerol treated cells with or without inhibitors were exposed to 0.5 mM t-BHP for 3 h to induce cytotoxic damage. A sharp increase in cytotoxicity was found, as shown in Fig. 6, as a result of lipid peroxidation, which was significantly attenuated by farrerol treatment. In contrast, this cytoprotective potential against oxidative stress was not be found in LY294002, U0126, SP600125 and SnPP (Akt inhibitor, ERK inhibitors, p38 inhibitor and HO-1 inhibitors, respectively) treated cells due to the abolished HO-1 expression. The above results suggest that Nrf2 mediated HO-1 expression induced by farrerol strengthens the cellular antioxidative potential against t-BHP-induced oxidative
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Fig. 5. Induction of HO-1 and activation of Nrf2 by farrerol via phosphorylation of Akt, p38 and ERK. (A) and (B) Effect of farrerol on the phosphorylation of AKT and MAPKs. RAW 264.7 cell were treated with different dose of farrerol for 24 h and then immunoblotted with specific antibodies. (C) Effect of PI3K and MAPK inhibitors on farrerolinduced HO-1 expression. Cells were preincubated with 10 lM LY294002, 10 lM U0126, 20 lM SP600125, 10 lM SB203580 for 1 h and then incubated with 20 mg/L farrerol for 23 h. whole cell lysates were subjected to Western blotting analysis with anti-HO-1 and anti-b-actin antibodies. (D) Effect of PI3K and MAPK inhibitors on farrerol-induced Nrf2 translocation. Cells were preincubated with 10 lM LY294002, 10 lM U0126, 20 lM SP600125, 10 lM SB203580 for 1 h and then incubated with 20 mg/L farrerol for 23 h. Nuclear extracts were subjected to Western blotting analysis with anti-Nrf2 and anti-lamin B1 antibodies.
damage through the modulation of the PI3K/Akt and MAPK signaling pathways in RAW 264.7 cells (see Fig. 7). 4. Discussion
Fig. 6. Farrerol scavenged t-BHP-induced ROS generation in RAW 264.7 cells. RAW 264.7 cells were pre-incubated with farrerol (5, 10 and 20 mg/L) for 18 h and stained with 10 lM of DCFH-DA for 2 h. Then, the cells were and subsequently incubated with t-BHP (0.5 mM) for 30 min to induce the ROS generation. The data represent the mean ± standard deviation of triplicate experiments. **p 6 0.01 vs. tBHP group, #p 6 0.05 vs. t-BHP + farrerol (20 mg/L) group.
Farrerol, a new type of 2,3-dihydro-flavonoid, has been demonstrated that it is a highly interesting candidate for the use in antioxidative strategies in EA.hy926 cells. In our previous study, intraperitoneal injection of farrerol (40 mg/kg) for 5 constitutive days can alleviate the OVA-induced airway inflammation by inhibiting the PI3K and NF-jB pathway. In this study, we attempted to explore the possible molecular mechanisms underlying the chemopreventive/chemoprotective effect of farrerol-treated macrophages, with a special focus on its possible upregulation of HO-1, a cytoprotective enzyme with anti-inflammatory as well as antioxidative functions. The cytoprotective properties of antioxidants have been partially attributed to their ability to induce cytoprotective enzymes, such as NAD(P)H:quinine oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), c-glutamyl cysteine synthetase catalytic
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Fig. 7. Effect of farrerol on t-BHP-induced RAW 264.7 cell cytotoxcity. The cells were treated with indicated concentrations of farrerol (5, 10 and 20 mg/L) for 21 h in the presence or absence of each selective inhibitor. The untreated and farrerol treated cells with or without inhibitor were exposed to 0.5 mM t-BHP for 3 h. The data represent the mean ± standard deviation of triplicate experiments. **p 6 0.01 vs. t-BHP group, #p 6 0.01 vs. t-BHP + farrerol (20 mg/L) group.
subunit (GCLC) [28]. Among the various cytoprotective enzymes, HO-1 expression has been considered to be an adaptive and beneficial response to oxidative stress in a wide variety of cells, including macrophages. Macrophages defend the host cell against a variety of stimuli and stresses because of their phagocytosis ability, which can lead to cytoprotection. Furthermore, the stress-mediated induction of HO-1 expression in macrophages is involved in the protective repair responses [29]. In the present study, our study demonstrate that farrerol increases mRNA expression of HO-1, NQO1 and GCLC. Furthermore, farrerol-induced HO-1 protein expression occurred in a time and dose-dependent manner in RAW 264.7 cell which suggested that farrerol act as a antioxidative strategies may account for induction of HO-1 expression. Nrf2, a transcription factors, is known to play an important role in the induction of antioxidant response element (ARE)-dependent gene expression, including that of phase II enzymes. Our results suggested that farrerol enhanced the nuclear translocation of Nrf2, thereby stimulating the transcriptional activity of Nrf2. Silencing of Nrf2 by use of siRNA knock down abolished the upregulation of HO-1 expression induced by farrerol, confirming the role of Nrf2 as an essential regulator in the farrerol-induced HO-1 expression. A possible mechanism of Nrf2 activation involves cysteine residues of the Keap1 protein [30]. Indeed, Keap1 contains 25 cysteine residues, making Keap1 the protein an attractive target for thiol-reactive chemical species. Thus, Keap1 inactivation is suggested to be a mechanism for the dissociation of Nrf2-Keap1 complex, which leads to the nuclear translocation of the Nrf2 and the induction of HO-1 expression [31]. Our results demonstrates that treatment with farrerol reduced Keap1 protein expression in a dose-dependent manner. It is more likely that an electrophilic quinine formed as a consequence of oxidation of farrerol may directly interact with critical cysteine thiol of keap1, thereby facilitating the dissociation of Nrf2. Another possible mechanism of Nrf2 activation involves the posttranscriptional modification of Nrf2 by kinases. The PI3K/Akt and MAPK pathways have been reported to be involved in HO-1 expression and in Nrf2-dependent transcription in RAW 264.7 cells [32]. Phosphorylation is one of the key steps for activating the Nrf2 pathway, but the role of individual protein kinases and phosphatases in the Nrf2/ARE signal system largely depends on the cell type. The phosphorylation of Nrf2 at serine and threonine residues by kinases such as phosphatidylinositol 3-kinase (PI3K), c-Jun
N-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) is assumed to facilitate the release of Nrf2 from Keap1 and its subsequent translocation [33]. The current experiments were designed to determine a possible role for the PI3K/Akt and MAPK pathways in farrerol-induced HO-1 expression, and farrerol was indeed found to activate the PI3K/Akt, p38 and ERK1/2 pathways but with no effect on JNK1/2. Furthermore, the use of specific inhibitors of the PI3K/Akt, p38, JNK1/2 and ERK1/2 pathways confirmed the involvement of PI3K/Akt, p38 and ERK1/2, but not JNK1/2, in farrerol-induced HO-1 expression. The induction of HO-1 expression confers antioxidative potential and prevents oxidative stress-induced cell damage [34,35]. To identify the antioxidative potential of farrerol, a ROS scavenging assay was performed. Our results indicates that farrerol significantly attenuated ROS formation in a dose-dependent manner in RAW 264.7 cells, which suggests that farrerol might be a potent antioxidant. Moreover, we hypothesized that the induction of HO-1 by farrerol would be cytoprotective against oxidative stress. Free radicals trigger lipid peroxidation and subsequent cell injury; therefore, increased free radical production could be a primary factor of cytotoxicity. In this study, t-BHP, an organic hydroperoxide, was applied to induce oxidative damage in RAW 264.7 cells. The t-BHP is metabolized by cytochrome P450, which initiates lipid peroxidation, leading to cell death. In our study, untreated and farrerol-treated cells with or without inhibitors were exposed to 0.5 mM t-BHP for 3 h to induce cytotoxic damage and we found a sharp increase in cytotoxicity, which was significantly attenuated by farrerol treatment. However, this cytoprotective potential against oxidative stress was not be observed on SB202190, U0126 and SnPP (p38, ERK inhibitors and HO-1 inhibitor, respectively)-treated cells due to the abolished HO-1 expression. The above results suggest that the Nrf2-mediated HO-1 expression induced by farrerol strengthens the antioxidative potential against t-BHP-induced oxidative damage through the modulation of PI3K/AKT, ERK and JNK signaling pathways in RAW 264.7 cells. In conclusion, farrerol may have cytoprotective activity through the induction of HO-1 expression through activation of Nrf2. This is likely to be achieved by activating Akt, p38 and ERK signaling pathways or directly targeting Keap1, possibly through modification of cysteine thiols present in Keap1. All possibilities remain to be elucidated and is another subject of our current investigations. This study supports the potential therapeutic mechanism of farrerol in protecting against oxidative stress-related diseases. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.
Acknowledgement This work supported by a grant from the Natural Science Foundation of Jilin (No. 20150520050JH). References [1] M. Valko, D. Leibfritz, J. Moncol, et al., Free radicals and antioxidants in normal physiological functions and human disease, Int. J. Biochem. Cell Biol. 39 (2007) 44–84. [2] H. Bartsch, J. Nair, Chronic inflammation and oxidative stress in the genesis and perpetuation of cancer: role of lipid peroxidation, DNA damage, and repair, Langenbecks Arch. Surg. 391 (2006) 499–510.
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The antioxidative potential of farrerol occurs via the activation of Nrf2 mediated HO-1 signaling in RAW 264.7 cells Xinxin Ci a, Hongming Lv a,f, Lidong Wang a, Xiaosong Wang a, Liping Peng a, F. Xiao-Feng Qin a,b,c,d,⇑, Genhong Cheng a,b,c,e,⇑ a
Institute of Translational Medicine, The First Hospital, Jilin University, Changchun 130001, China Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China Suzhou Institute of Systems Medicine, Suzhou 215123, China d Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA e Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA f Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun 130061, China b c
a r t i c l e
i n f o
Article history: Received 6 January 2015 Received in revised form 4 May 2015 Accepted 22 June 2015 Available online 22 June 2015 Keywords: Farrerol Heme oxygenase-1 Nuclear translocation of NF-E2-related factor 2 Phosphatidylinositol 3-kinase Mitogen-activated protein kinase
a b s t r a c t Farrerol, (S)-2,3-dihydro-5,7-dihydroxy-2-(4-hydroxyphenyl)-6,8-dimethyl-4-benzopyrone, isolated from rhododendron, has been shown to have antioxidative potential, but the molecular mechanism underlying this activity remains unclear. The inducible expression of heme oxygenase-1 (HO-1), a potent antioxidative and cytoprotective enzyme, is known to play an important role in cytoprotection in a variety of pathological models. In this study, we evaluated the antioxidative potential of farrerol against oxidative damage and investigated its antioxidative mechanism in RAW 264.7 cells. The molecular mechanism underlying the cytoprotective function of farrerol was determined by analyzing intracellular signaling pathways, transcriptional activation and the inhibitory effect of HO-1 on ROS production. Farrerol induced antioxidant enzymes mRNA expression, HO-1 protein expression and nuclear translocation of NF-E2-related factor 2 in RAW 264.7 macrophage cells. Farrerol down-regulated the expression of the Keap1 protein and the thiol reducing agents attenuated farrerol-induced HO-1 expression. Further investigation utilizing Western blotting and specific inhibitors of Akt, p38, JNK and ERK demonstrated that Akt, p38, and ERK axis of signaling pathway mediates HO-1 expression. Moreover, tert-butyl hydroperoxide (t-BHP)-induced oxidative damage was ameliorated by farrerol treatment in a dose-dependent manner, which was abolished by Akt, p38, ERK and HO-1 inhibitors (Snpp). It is hence likely that farrerol inactivated KEAP-1 or activated the Akt, p38 and ERK to facilitate the release of Nrf2 from Keap1 and subsequent reduced the intracellular production of reactive oxygen species via the induction of HO-1 expression. These results support the central role of HO-1 in the cytoprotective effect of farrerol. Ó 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Oxidative stress is defined as an imbalance between the production of free radicals and reactive metabolites, so-called oxidants or reactive oxygen species (ROS), and their elimination by protective potential, referred to as antioxidants [1]. An overproduction of ROS can cause damage to lipids, proteins and DNA, resulting in a wide spectrum of diseases, including chronic inflammation and a wide variety of different cancers [2–6]. As mammalian cells have ⇑ Corresponding authors at: Institute of Translational Medicine, The First Hospital, Jilin University, Changchun 130001, China. E-mail addresses: [email protected] (F. Xiao-Feng Qin), [email protected]. edu (G. Cheng). http://dx.doi.org/10.1016/j.cbi.2015.06.032 0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
developed several protective mechanisms to prevent ROS formation or to detoxify ROS, the use of compounds with antioxidative properties may help to prevent or alleviate many diseases associated with ROS [7]. These mechanisms employ molecules called antioxidants as well as cytoprotective enzymes such as, NAD(P)H quinone oxidore ductase (NQO1), glutathione S-transferase (GST), heme oxygenase-1 (HO-1), and glutathione peroxidase (GSH-Px) [8,9]. Among these various cytoprotective enzymes, HO-1 has anti-inflammatory, antioxidative, and antiproliferative potential [10] and also performs critical roles in both biosynthetic and degradation pathways for heme. When HO-1 catalyzes heme to biliverdin and bilirubin, these degradation products have antioxidant properties. So HO-1 played a critical role in maintaining a
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cellular redox homeostasis against the oxidative stress [11]. The transcriptional regulation of the ho-1 gene is linked to the transcription factor NF-E2-related factor (Nrf2), which plays a crucial role in cellular defense [12]. Under normal conditions, Nrf2 is sequestered in the cytoplasm by binding to Keap 1, an actin-binding protein, which maintains Nrf2 in a constantly ubiquitylated state, leading to its degradation in the cytoplasm. Upon oxidative stress, Nrf2 is released from Keap1, translocates into the nucleus, forms a heterodimer with the small Maf protein and binds to anti oxidant-related elements (ARE) in the promoter region of phase II detoxifying enzymes, such as HO-1. Although the mechanism by which Nrf2 is liberated from the Keap1–Nrf2 complex remains to be established, recent studies have suggested that the phosphorylation of Nrf2 at serine and threonine residues by kinases such as phosphatidylinositol 3-kinase (PI3K), PKC, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) facilitates the release of Nrf2 from Keap1 and subsequent translocation, followed by the initiation of antioxidative cascades [13]. As growing evidence suggests that HO-1 provides cytoprotection, the pharmacological modulation of HO-1 expression may represent a novel target for therapeutic intervention [14,15]. In particular, the identification of a non-cytotoxic inducer of HO-1 may maximize the intrinsic antioxidative potential of cells. Many natural resources that scavenge free radicals have been identified and reported to induce HO-1 expression in a variety of cells, such as liver cells, retinal pigment epithelial cells and macrophage cells, and hence may function against oxidative stress [16–18]. Flavonoids, plant polyphenolic compounds that are abundant in fruits and vegetables, have been studied for their potent antioxidative capacity. Some flavonoids isolated from the roots of Rhododendron mucronulatum Turzaninov were reported to be potential anti-inflammatory agents based on the results of their dose-dependent inhibition of the expression of inflammatory mediators, NO and PGE2 [19]. Farrerol, a new type of 2,3-dihydro-flavonoid, has also been isolated from rhododendron, a traditional Chinese herbal medicine [20]. Accumulating evidence suggests that farrerol possesses many biological properties, including antitussive, antibacterial, anti-inflammatory effects, with also an inhibitory effect on vascular smooth muscle cells (VSMCs) proliferation [21,22]. Previous study showed that farrerol has significant cytoprotective potential against hydrogen peroxide (H2O2)-induced injury of human umbilical vein endothelial cells [23]. Accumulating evidence has also suggested that farrerol has a cytoprotective effect against oxidative stress of EA.hy926 cells, which is associated with the regulation of malondialdehyde (MDA), superoxide dismutase (SOD), ROS and GSH-Px [24]. Therefore, the present study aimed to evaluate the antioxidative potential of farrerol against oxidative damage of RAW 264.7 cells and to determine the underlying molecular mechanisms.
2. Materials and methods 2.1. Reagents Farrerol, ((S)-2,3-dihydro-5,7-dihydroxy-2-(4-hydroxypheny l)-6,8-dimethyl-4-benzopyrone, analytical grade, purity P 98%) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). 3-(4,5-Di methylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT), tert-butyl hydroperoxide (t-BHP), Dimethyl sulfoxide (DMSO), U0126, SB203580, SP600125 and LY294002 (specific inhibitors of the ERK1/2, p38, JNK1/2 and PI3K/Akt, respectively), DCFH-DA and Protoporphyrin IX zinc were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). Antibodies against Nrf2,
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HO-1, KEAP1, AKT, phosphor-AKT, phospho-extracellular signal-regulated kinase (ERK), ERK, phospho-c-Jun NH2-terminal kinase (JNK), JNK, phospho-p38, p38, as well as b-actin were purchased from Cell Signaling (Boston, MA, USA) or Abcam (Cambridge, MA, USA). The horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse IgG were purchased from proteintech (Boston, MA, USA). TRIzol reagent was from Invitrogen (Carlsbad, CA, USA). The control siRNA and Nrf-2 siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). X-treme GENE siRNA Transfection Reagent and Faststart Universal SYBR Green Master were purchased from Roche (Basel, Switzerland). Prime-Script RT-PCR kit was purchased from Takara (Dalian, China). 2.2. Cell culture and treatment The RAW 264.7 murine macrophage cell line was obtained from the China Cell Line Bank (Beijing, China). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 3 mM glutamine, antibiotics (100 U/mL penicillin and 100 U/mL streptomycin), and 10% heat-inactivated fetal bovine serum. The cells were maintained at 37 °C in a humidified incubator containing 5% CO2. In all experiments, cells were allowed to acclimate for 24 h before any treatments. 2.3. MTT assay for cell viability To measure cell viability, the MTT assay was performed. RAW 264.7 cells were mechanically scraped, seeded in 96-well plates at 4 105 cells/mL, 100 ll/well, and incubated in a 37 °C, 5% CO2 incubator overnight. Then the cells were treated with 100 lL different concentrations of farrerol (0–80 mg/L) for 20 h. Subsequently, 20 ll of 5 mg/mL MTT in FBS-free medium was added to each well and incubated for an additional 4 h. Cell-free supernatants were then removed and resolved cells with 150 lL/well DMSO. The optical density was measured at 570 nm on a microplate reader. To measure the antioxidative potential of farrerol against the t-BHP-induced oxidative damage in RAW 264.7 cell, the cells were treated with 50 lL different concentrations of farrerol (5, 10, 20 mg/L) for 12 h, followed by stimulation with 50 lL of t-BHP for 3 h. Subsequently, 20 lL of 5 mg/mL MTT in FBS-free medium was added to each well and incubated for an additional 4 h. Cell-free supernatants were then removed and resolved with 150 lL/well DMSO. The optical density was measured at 570 nm on a microplate reader. 2.4. Intracellular reactive oxygen species formation assay RAW 264.7 cells were grown in 24-well plates (1 105 cells/well) for 24 h incubation, and then the cells were pre-incubated with various concentrations of farrerol for 18 h. Next, the cells were stained with 10 lM of DCFH-DA for 2 h and subsequently incubated with t-BHP (0.5 mM) for 30 min to induce ROS generation. The fluorescence of the dye was measured using a multi-detection reader (Bio-Tek Instruments Inc.) at excitation and emission wave-lengths of 485 nm and 530 nm, respectively. 2.5. Isolation of total RNA from cells and qPCR Total RNA from cells was isolated using the Trizol reagent and the manufacturer’s instructions. After the concentration of RNA was determined by spectrophotometer, 1 lg of RNA was converted to cDNA using the Prime-Script RT-PCR kit, and a real-time PCR analysis was performed using the Applied Biosystems 7300 real-time PCR system and software (Applied Biosystems,
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Carlsbad, CA, USA). Real-time PCR was performed in 0.2 mL PCR tubes with forward and reverse primers and the SYBR green working solution using a custom PCR master mix with the following conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s. The relative gene expression was analyzed by normalization to b-actin mRNA expression.
2.6. Western blot analysis RAW 264.7 cell (1 106/well) were cultured in 6-well plates for 24 h and then treated for 3 h, 6 h and 24 h with various concentrations of farrerol. After each experiment, the cells were washed twice with cold PBS and then scraped from the plate with 500 lL of PBS. The cell homogenates were centrifuged at 3000 g for 5 min. Nuclear and cytoplasmic fractions of cell were prepared as previously described [25]. Whole cell lysates were lysed in 1% non-diet P-40 lysis buffer (1% NP-40, 150 mM Nacl, 50 mM Tris, pH 7.4) with freshly added protease and phosphatase inhibitors. After incubation on ice for 30 min, the lysates were centrifuged (12,000g at 4 °C) for 5 min to obtain the cytosolic fraction. Protein concentrations were determined using the Bradford assay before storage at 80 °C. The cell lysates were subjected to an immunoblotting analysis using the following antibodies: anti-pAKT, anti-pJNK, anti-pERK, anti-pp38, anti-AKT, anti-JNK, anti-ERK, anti-p38, anti-Nrf-2, anti-HO-1, and anti-b-actin. The membranes were further probed with HRP-conjugated secondary antibodies and detected using an ECL Western blot substrate. The band intensities were quantified using Image J gel analysis software. The fold increase in the level of protein expression was calculated by comparison with that of normal controls. The experiments were repeated three times for each experimental condition.
2.7. Nrf2-siRNA transient transfection For Nrf2-siRNA transfection, RAW 264.7 cells (4 105/well) were cultured in 6-well plates for at least 24 h until the confluence of cells reached approximately 50%. Then transfected with Nrf2-siRNA or Nrf2-negative control siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) using the X-treme GENE siRNA Transfection Reagent (Roche Applied Science), according to the manufacturer instructions for 24 h. The transfected cells were treated with farrerol for 24 h followed by lysis buffer for Western blot analysis.
2.8. Statistical analysis Two-tailed unpaired Student’s t tests were performed to determine p values. All the graphs represent the mean ± SEM of three independent experiments, and the asterisks represent p values: * p < 0.05, **p < 0.01.
3. Results
Fig. 1. Effect of farrerol on macrophage toxicity, the mRNA expression of antioxidative enzymes and the protein expression of HO-1. (A) Effect of farrerol on macrophage toxicity. Cell were cultured with farrerol (0–40 mg/L) for 24 h and cell viability was estimated by the MTT method; (B) effect of farrerol on the mRNA expression of antioxidative enzymes. RAW 264.7 cells were treated with 20 mg/L farrerol for 24 h and real-time PCR analyses was performed to assess HO-1, NQO1 and GCLC expression. (C) and (D) Effect of farrerol on the protein expression of HO1. Western blot analysis was conducted to confirm the (C) dose-dependent of HO-1 protein expression induction by farrerol for 24 h and (D) time-dependent of HO-1 protein expression induction by 20 mg/L farrerol. **p 6 0.01 vs. control group.
3.2. Effects of farrerol on the mRNA expression of antioxidant enzymes and the protein expression of HO-1
3.1. Cytotoxicity of farrerol in RAW 264.7 We first examined the effect of farrerol on the viability of RAW 264.7 cells. The maximum final concentration of farrerol was chosen with respect to its solubility in the culture medium. As shown in Fig. 1A, farrerol (0–20 mg/L) do not exert cytotoxicity in RAW 264.7 cells.
Real-time PCR analysis was performed to determine the effect of farrerol on phase II enzymes expression. Activation of the antioxidant response element (ARE) by the Nrf2 pathway causes the induction of phase II enzymes. mRNAs for HO-1, NQO1 and c-glutamate cysteine ligase catalytic subunit (GCLC) were increased in RAW 264.7 cells treated with farrerol (Fig. 1B). As
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HO-1 is an important component of the cellular defense against oxidative stress, we further assessed whether farrerol could induce HO-1 protein expression in a dose- or time- dependent manner. RAW 264.7 cells exposed to farrerol (0–20 mg/L) for 18 h showed a dose-dependent manner increase in HO-1 protein expression (Fig 1C), and the treatment of cells with 20 mg/L farrerol resulted in a time-dependent increase in HO-1 protein expression (Fig 1D). 3.3. Farrerol-induced expression of HO-1 was mediated by Nrf2 Nrf2 plays a key role in HO-1 expression and the role of Nrf2 in farrerol-induced HO-1 expression was confirmed using siRNA. RAW 264.7 cells were transiently transfected with control or Nrf2 siRNA, and Nrf2 protein expression was measured by Western blot analysis. Fig. 2A demonstrates that Nrf2 siRNA can inhibit Nrf2 protein expression. We further examined whether farrerol-induced up-regulation of HO-1 expression is mediated by Nrf2 by using Nrf2 siRNA to knockdown Nrf2. As shown in Fig. 2B, up-regulated HO-1 protein expression caused by farrerol in RAW 264.7 cells was abolished when Nrf2 expression was silenced with a specific siRNA. 3.4. Effects of farrerol on Nrf2 nuclear translocation and Keap1 expression Nrf2 is an important transcription factor that regulates the ARE-driven expression of phase II detoxifying and antioxidant enzymes such as HO-1. Treatment with farrerol increased Nrf2 translocation from the cytoplasm to the nucleus (Fig. 3A) in a dose-dependent manner. Moreover, farrerol down-regulated the expression of the Keap1 protein in a dose-dependent manner (Fig. 3B). 3.5. Cysteine residues of Keap1 may be putative targets of farrerol for its activation of Nrf2 and HO-1 induction Several investigations have shown that some phytochemicals directly interact with Keap1 by binding to its thiol group, facilitating the release of Nrf2 [26]. To explore this possibility, RAW 264.7 cells were incubated with farrerol with or without 100 lM DTT or 1.4 lM b-mercaptoethanol. Both disulfide reducing agents abrogated the farrerol-induced HO-1 expression. 3.6. Involvement of the PI3K/Akt and MAPK pathways in HO-1 expression and Nrf2 nuclear translocation by farrerol To further elucidate the upstream signaling pathway involved in farrerol-mediated Nrf2 activation and HO-1 induction, we examined the activation of PI3K/Akt and MAPKs in RAW 264.7 cells treated with farrerol. As shown in Fig. 4A and B, the induction of Akt and all three MAPKs was detected in farrerol-treated RAW 264.7 cells by Western blotting using specific antibodies. No change in the expression of total Akt and the three MAPK proteins was detected, whereas Akt, ERK and p38 phosphorylation was significantly increased in a dose-dependent manner. To address the role of the individual Akt and MAPK pathways in HO-1 expression induced by farrerol, we examined the effects of LY294002, U0126, SP600125, and SB203580, specific inhibitors for the PI3K/Akt, ERK, p38 and JNK pathways, on HO-1 expression. Although the JNK inhibitor had no effect on farrerol-induced HO-1 expression, the inhibitors of the Akt, ERK and p38 did significantly reduce farrerol-induced HO-1 expression (Fig 4C). Next, we examined whether the PI3K/Akt and MAPK pathways are involved in the process by which farrerol causes Nrf2 nuclear translocation. As shown in Fig. 4D, the inhibitors of the PI3K/Akt, p38 and ERK
Fig. 2. Abrogation of HO-1 expression induced by farrerol in RAW 264.7 transiently transfected with Nrf2-siRNA. (A) Cells were transfected with Nrf2-siRNA or Nrf2negative control siRNA for 24 h, after cells were harvested, proteins were detected by Western blot. (B) Nrf2 regulates farrerol-induced HO-1 protein expression. Cells were transfected with Nrf2-siRNA or Nrf2-negative control siRNA for 24 h. The transfected cells were treated with farrerol (20 mg/L) for 24 h and protein expression of HO-1 was measured by Western blot analysis. **p 6 0.01 vs. control siRNA + farrerol group.
pathways, but not the JNK pathway, blocked farrerol-induced Nrf2 nuclear accumulation. These results indicate a role for PI3K/Akt, p38 and ERK signaling in farrerol mediated HO-1 induction through the nuclear translocation of Nrf2.
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Fig. 4. Effect of thiol reducing agents on farrerol-induced HO-1 expression. (A) and (B) Abrogation of farrerol-induced HO-1 upregulation by the reducing agents DTT. RAW 264.7 cells were pretreated with 100 lM DTT for 1 h followed by 24 hincubation with 20 mg/L farrerol. (C) and (D) Abrogation of farrerol-induced HO-1 upregulation by the reducing agents b-mercaptoethanol (b-ME). RAW 264.7 cells were pretreated with 1.4 lM b-ME for 1 h followed by 24 h-incubation with 20 mg/ L farrerol. **p 6 0.01 vs. control group, ##p 6 0.01 vs. farrerol group.
3.8. Effect of farrerol on t-BHP-induced RAW 264.7 cell cytotoxicity Fig. 3. Effects of farrerol on the protein expression levels of Nrf2 and Keap1 in RAW 264.7 macrophages. (A) Nuclear and cytoplasmic extracts from RAW 264.7 cells were prepared after treatment with farrerol at 5, 10 and 20 mg/L for 24 h. (B) Total protein extracts from different dose of farrerol treatment were prepared for detecting KEAP1. **p 6 0.01 vs. control group.
3.7. Effect of farrerol on t-BHP-induced ROS generation in RAW 264.7 cells To identify the antioxidative potential of farrerol, a ROS scavenging assay was performed. The data shown in Fig. 5 indicate that farrerol significantly attenuated t-BHP-induced ROS formation in a dose-dependent manner in RAW 264.7 cells, which suggests that farrerol might be a potent antioxidant.
Free radicals trigger lipid peroxidation and subsequent cellular injury; therefore, increased free radical production could be a primary factor of cytotoxicity [27]. In this study, t-BHP, an organic hydroperoxide, was applied to induce oxidative damage in RAW 264.7 cells. Untreated and farrerol treated cells with or without inhibitors were exposed to 0.5 mM t-BHP for 3 h to induce cytotoxic damage. A sharp increase in cytotoxicity was found, as shown in Fig. 6, as a result of lipid peroxidation, which was significantly attenuated by farrerol treatment. In contrast, this cytoprotective potential against oxidative stress was not be found in LY294002, U0126, SP600125 and SnPP (Akt inhibitor, ERK inhibitors, p38 inhibitor and HO-1 inhibitors, respectively) treated cells due to the abolished HO-1 expression. The above results suggest that Nrf2 mediated HO-1 expression induced by farrerol strengthens the cellular antioxidative potential against t-BHP-induced oxidative
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Fig. 5. Induction of HO-1 and activation of Nrf2 by farrerol via phosphorylation of Akt, p38 and ERK. (A) and (B) Effect of farrerol on the phosphorylation of AKT and MAPKs. RAW 264.7 cell were treated with different dose of farrerol for 24 h and then immunoblotted with specific antibodies. (C) Effect of PI3K and MAPK inhibitors on farrerolinduced HO-1 expression. Cells were preincubated with 10 lM LY294002, 10 lM U0126, 20 lM SP600125, 10 lM SB203580 for 1 h and then incubated with 20 mg/L farrerol for 23 h. whole cell lysates were subjected to Western blotting analysis with anti-HO-1 and anti-b-actin antibodies. (D) Effect of PI3K and MAPK inhibitors on farrerol-induced Nrf2 translocation. Cells were preincubated with 10 lM LY294002, 10 lM U0126, 20 lM SP600125, 10 lM SB203580 for 1 h and then incubated with 20 mg/L farrerol for 23 h. Nuclear extracts were subjected to Western blotting analysis with anti-Nrf2 and anti-lamin B1 antibodies.
damage through the modulation of the PI3K/Akt and MAPK signaling pathways in RAW 264.7 cells (see Fig. 7). 4. Discussion
Fig. 6. Farrerol scavenged t-BHP-induced ROS generation in RAW 264.7 cells. RAW 264.7 cells were pre-incubated with farrerol (5, 10 and 20 mg/L) for 18 h and stained with 10 lM of DCFH-DA for 2 h. Then, the cells were and subsequently incubated with t-BHP (0.5 mM) for 30 min to induce the ROS generation. The data represent the mean ± standard deviation of triplicate experiments. **p 6 0.01 vs. tBHP group, #p 6 0.05 vs. t-BHP + farrerol (20 mg/L) group.
Farrerol, a new type of 2,3-dihydro-flavonoid, has been demonstrated that it is a highly interesting candidate for the use in antioxidative strategies in EA.hy926 cells. In our previous study, intraperitoneal injection of farrerol (40 mg/kg) for 5 constitutive days can alleviate the OVA-induced airway inflammation by inhibiting the PI3K and NF-jB pathway. In this study, we attempted to explore the possible molecular mechanisms underlying the chemopreventive/chemoprotective effect of farrerol-treated macrophages, with a special focus on its possible upregulation of HO-1, a cytoprotective enzyme with anti-inflammatory as well as antioxidative functions. The cytoprotective properties of antioxidants have been partially attributed to their ability to induce cytoprotective enzymes, such as NAD(P)H:quinine oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), c-glutamyl cysteine synthetase catalytic
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Fig. 7. Effect of farrerol on t-BHP-induced RAW 264.7 cell cytotoxcity. The cells were treated with indicated concentrations of farrerol (5, 10 and 20 mg/L) for 21 h in the presence or absence of each selective inhibitor. The untreated and farrerol treated cells with or without inhibitor were exposed to 0.5 mM t-BHP for 3 h. The data represent the mean ± standard deviation of triplicate experiments. **p 6 0.01 vs. t-BHP group, #p 6 0.01 vs. t-BHP + farrerol (20 mg/L) group.
subunit (GCLC) [28]. Among the various cytoprotective enzymes, HO-1 expression has been considered to be an adaptive and beneficial response to oxidative stress in a wide variety of cells, including macrophages. Macrophages defend the host cell against a variety of stimuli and stresses because of their phagocytosis ability, which can lead to cytoprotection. Furthermore, the stress-mediated induction of HO-1 expression in macrophages is involved in the protective repair responses [29]. In the present study, our study demonstrate that farrerol increases mRNA expression of HO-1, NQO1 and GCLC. Furthermore, farrerol-induced HO-1 protein expression occurred in a time and dose-dependent manner in RAW 264.7 cell which suggested that farrerol act as a antioxidative strategies may account for induction of HO-1 expression. Nrf2, a transcription factors, is known to play an important role in the induction of antioxidant response element (ARE)-dependent gene expression, including that of phase II enzymes. Our results suggested that farrerol enhanced the nuclear translocation of Nrf2, thereby stimulating the transcriptional activity of Nrf2. Silencing of Nrf2 by use of siRNA knock down abolished the upregulation of HO-1 expression induced by farrerol, confirming the role of Nrf2 as an essential regulator in the farrerol-induced HO-1 expression. A possible mechanism of Nrf2 activation involves cysteine residues of the Keap1 protein [30]. Indeed, Keap1 contains 25 cysteine residues, making Keap1 the protein an attractive target for thiol-reactive chemical species. Thus, Keap1 inactivation is suggested to be a mechanism for the dissociation of Nrf2-Keap1 complex, which leads to the nuclear translocation of the Nrf2 and the induction of HO-1 expression [31]. Our results demonstrates that treatment with farrerol reduced Keap1 protein expression in a dose-dependent manner. It is more likely that an electrophilic quinine formed as a consequence of oxidation of farrerol may directly interact with critical cysteine thiol of keap1, thereby facilitating the dissociation of Nrf2. Another possible mechanism of Nrf2 activation involves the posttranscriptional modification of Nrf2 by kinases. The PI3K/Akt and MAPK pathways have been reported to be involved in HO-1 expression and in Nrf2-dependent transcription in RAW 264.7 cells [32]. Phosphorylation is one of the key steps for activating the Nrf2 pathway, but the role of individual protein kinases and phosphatases in the Nrf2/ARE signal system largely depends on the cell type. The phosphorylation of Nrf2 at serine and threonine residues by kinases such as phosphatidylinositol 3-kinase (PI3K), c-Jun
N-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) is assumed to facilitate the release of Nrf2 from Keap1 and its subsequent translocation [33]. The current experiments were designed to determine a possible role for the PI3K/Akt and MAPK pathways in farrerol-induced HO-1 expression, and farrerol was indeed found to activate the PI3K/Akt, p38 and ERK1/2 pathways but with no effect on JNK1/2. Furthermore, the use of specific inhibitors of the PI3K/Akt, p38, JNK1/2 and ERK1/2 pathways confirmed the involvement of PI3K/Akt, p38 and ERK1/2, but not JNK1/2, in farrerol-induced HO-1 expression. The induction of HO-1 expression confers antioxidative potential and prevents oxidative stress-induced cell damage [34,35]. To identify the antioxidative potential of farrerol, a ROS scavenging assay was performed. Our results indicates that farrerol significantly attenuated ROS formation in a dose-dependent manner in RAW 264.7 cells, which suggests that farrerol might be a potent antioxidant. Moreover, we hypothesized that the induction of HO-1 by farrerol would be cytoprotective against oxidative stress. Free radicals trigger lipid peroxidation and subsequent cell injury; therefore, increased free radical production could be a primary factor of cytotoxicity. In this study, t-BHP, an organic hydroperoxide, was applied to induce oxidative damage in RAW 264.7 cells. The t-BHP is metabolized by cytochrome P450, which initiates lipid peroxidation, leading to cell death. In our study, untreated and farrerol-treated cells with or without inhibitors were exposed to 0.5 mM t-BHP for 3 h to induce cytotoxic damage and we found a sharp increase in cytotoxicity, which was significantly attenuated by farrerol treatment. However, this cytoprotective potential against oxidative stress was not be observed on SB202190, U0126 and SnPP (p38, ERK inhibitors and HO-1 inhibitor, respectively)-treated cells due to the abolished HO-1 expression. The above results suggest that the Nrf2-mediated HO-1 expression induced by farrerol strengthens the antioxidative potential against t-BHP-induced oxidative damage through the modulation of PI3K/AKT, ERK and JNK signaling pathways in RAW 264.7 cells. In conclusion, farrerol may have cytoprotective activity through the induction of HO-1 expression through activation of Nrf2. This is likely to be achieved by activating Akt, p38 and ERK signaling pathways or directly targeting Keap1, possibly through modification of cysteine thiols present in Keap1. All possibilities remain to be elucidated and is another subject of our current investigations. This study supports the potential therapeutic mechanism of farrerol in protecting against oxidative stress-related diseases. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.
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