Protective effect of rutaecarpine against t-BHP-induced hepatotoxicity by upregulating antioxidant enzymes via the CaMKII-Akt and Nrf2/ ARE pathways
A B S T R A C T
Rutaecarpine, an indolopyridoquinazolinone alkaloid isolated from the unripe fruit of Evodia rutaecarpa, has been shown to have cytoprotective potential, but the molecular mechanism underlying this activity remains unclear. Our study was designed to investigate the cytoprotective effect of rutaecarpine against tert-butyl hydroperoxide (t-BHP) and to elucidate its action mechanism of action of rutaecarpine in a cultured HepG2 cell line and in mouse liver. Rutaecarpine decreased t-BHPeinduced reactive oxygen species (ROS) production, cytotoxicity, and apoptosis in HepG2 cells. Pretreatment with rutaecarpine prior to the injection of t-BHP significantly prevented the increase in serum levels of AST, ALT, and lipid peroxidation in mice liver. It increased the transcriptional activity of NF-E2erelated factor 2 (Nrf2) as well as the products of the Nrf2 target genes hemeoxygenase-1 (HO-1), NAD(P)H:quinone oxidoreduc- tase 1 (NQO1), and glutamate cysteine ligase (GCL). Moreover, rutaecarpine also enhanced the phos- phorylation of Akt and Ca2þ/calmodulin-dependent protein kinase-II (CaMKII). The pharmaceutical inhibitors, such as KN-93 (CaMKII inhibitor) and LY294002 (Akt inhibitor) suppressed rutaecarpine- induced HO-1 expression and cytoprotection. Our findings identify the CaMKII-PI3K/Akt-Nrf2 cascade as an antioxidant pathway mediating rutaecarpine signaling and leading to HO-1 expression in hepatocytes.
1.Introduction
Reactive oxygen species (ROS) are involved in the homeostasis of cells and various diseases (Han et al., 2015). High levels of ROS damage cells and are related to a variety of cellular damage mechanism, such as inflammation, apoptosis, cancer, and metabolic diseases (El Assar et al., 2013; Han et al., 2015). Interestingly, intracellular ROS produced normally are scavenged by protective enzymes and endogenous antioxidants that are abundant in mammalian cells (Chen and Kunsch, 2004; Hwang et al., 2009). Numerous studies have demonstrated that the transcription factor NF-E2erelated factor 2 (Nrf2) is responsible for regulating the antioxidant response element (ARE)-driven expression of antioxidant enzymes, such as hemeoxygenase-1 (HO-1), NAD(P) H:quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase (GCL) (Itoh et al., 2010; Nguyen et al., 2009; Thimmulappa et al., 2002).
Among the various antioxidant enzymes, HO-1 has gained attention recently owing to its cytoprotective properties. HO-1 and its enzymatic metabolites provide a host defense system that can protect the body against oxidative injury (Hwang et al., 2011; Takahashi et al., 2004). In several studies, HO-1 has been reported to be a novel enzyme with potent antioxidant (Maines, 1998), anti- inflammatory (Takahashi et al., 2004), and antiproliferative effects (Tulis et al., 2001).Previous reports indicate that Nrf2 regulation depends on the activation of the Ca2þ/calmodulin-dependent protein kinase II (CaMKII), PI3K/Akt, c-Jun N-terminal kinase1/2 (JNK1/2), extracel- lular signal-regulated kinase1/2 (ERK1/2), and p38 MAPK pathways (Kong et al., 2001; Kim et al., 2010). Therefore, pharmacological approaches to reduce ROS and to activate the Akt/Nrf2/HO-1 pathway would be an effective strategy for preventing oxidative injury.The induction of antioxidant enzymes by natural compounds possesses marked therapeutic potential against diseases associated with inflammation and oxidative stress (Kim et al., 2010). Evodia rutaecarpa (E. rutaecarpa), a traditional oriental medicine, is used in the treatment of headache, hypertension, and gastrointestinal disorders (Jia and Hu, 2010). Rutaecarpine, a major bioactive com- ponents of E. rutaecarpa, has shown various biological activities such as antithrombotic activities and anti-inflammatory effects (Chen et al., 2013; Dai et al., 2008). Recently, Lee et al. (2012) re- ported that rutaecarpine inhibited hydrogen peroxide-induced oxidative injury in Hepa-1c1c7 cells. However, currently, there is no research on whether the antioxidative effect of rutaecarpine is related to HO-1 activity. Herein, we demonstrate that rutaecarpine protects against oxidative stress-induced cell injury in a cultured HepG2 cell line and in mouse liver, through the CaMKII/Akt/Nrf2/ HO-1 and calcium-signaling pathways.
2.Materials and methods
Chemicals and cell culture materials were obtained from the following sources: rutaecarpine (purity, > 98%), tert-butyl hydro- peroxide (t-BHP), dimethyl sulfoxide (DMSO), 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), 2,7-dichlorofluorescein diacetate (H2DCFDA), and zinc protopor- phyrin (ZnPP) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS), and Dulbecco’s modified Ea- gle’s medium (DMEM), and sodium pyruvate were obtained from Gibco-BRL (Grand Island, NY, USA). Antibodies against p-Akt, Akt, p-ERK1/2, ERK1/2, p-JNK1/2, JNK1/2, p-p38, and p38 were supplied by Cell Signaling Technology (Beverly, MA, USA). Antibodies against HO-1, Nrf2, p-CaMKIIa, CaMKIIa, b-actin, and Lamin B were pur- chased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). SB366791, BAPTA/AM, W7, LY294002, KN-93, SP600125, PD98059,
and SB203580 were supplied by Calbiochem (La Jolla, CA, USA). Luciferase reporter gene (HO-1-ARE) was kindly provided by Dr. J. Alam (University of Colorado Denver, Health Sciences Center, CO, USA).HepG2 cells were purchased from the ATCC (Manassas, VA, USA). HepG2 cells were maintained at 37 ◦C in an incubator with a humidified atmosphere of 5% CO2 and cultured in DMEM containing 10% heat-inactivated FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml). Rutaecarpine was dissolved in DMSO and the stock solutions were added directly to the culture media. The final concentration of DMSO was always <0.1%.
Cell viability was analyzed by an MTT reduction assay and lactate dehydrogenase (LDH) leakage assay. To measure cell viability, human HepG2 cells were plated at a density of 1 105 cells per well in 48-well plates and treated with 1 mM of t- BHP for 12 h either after, or without, pretreatment with rutae- carpine (1e10 mM, for 1 h). After incubation, cells were treated with MTT solution (final concentration, 0.5 mg/ml) for 1 h. The level of LDH release was measured in the media using an LDH kit (Cayman, Ann. Arbor, MI, USA).For the measurement of ROS, HepG2 cells were treated with 1 mM of t-BHP or a vehicle for 6 h, with or without of rutaecarpine, and incubated for 6 h. The cells were washed with PBS, and then incubated with 25 mM of H2DCFDA for 20 min. The cells were then washed twice with PBS and the fluorescence was detected on a fluorescence-detecting microplate reader (Varioskan, Thermo Electron Co., Vantaa, Finland) with excitation/emission wave- lengths of 485/530 nm.Caspase-3 assay was performed as described by previously published methods (Datta et al., 1997).A viability/cytotoxicity assay (Life Technologies, Seoul, Korea) was performed according to the manufacturer's instructions. Briefly, HepG2 cells were pretreated with rutaecarpine (1e10 mM) for 1 h, and then treated with 1 mM of t-BHP for an additional 12 h. The cells were stained with a 2 mM of calcein AM and 4 mM of ethidium homodimer, and incubated at 37 ◦C for 30 min. The cells were then visualized under a fluorescence microscope (Axiovert- 200M; Carl Zeiss, Jena, Germany).Western blot analysis was performed as described by previously published methods (Hwang et al., 2011). The PVDF membranes were probed with the appropriate primary antibodies such as anti- p-CaMKIIa, -CaMKIIa, -p-Akt, -Akt, -p-ERK1/2, -ERK1/2, -p-JNK1/2, -JNK1/2, -p-p38, and -p38, -NQO1, -GCL, -Nrf2, -HO-1, and -b-actin antibodies, followed by incubation with HRP-conjugated secondary antibodies. Immunoblot signals were visualized using an enhanced chemiluminescence (ECL) detection kit (Pierce Biotechnology, Rockford, IL, USA).A ChIP assay was performed using the EZChIP kit (Millipore, Billerica, MA,USA) according to the manufacturer's protocol. An anti-Nrf2 antibody was added to aliquots of precleared chromatin and incubated overnight. Input samples were incubated with the negative-control IgG. The immune complexes were captured by incubation with protein G agarose for 1 h at 4 ◦C. After the cross- links were reversed, DNA samples from the immunoprecipitates
Rutaecarpine prevents t-BHP-induced cellular damage. Cells were treated with rutaecarpine for 1 h, and then incubated with t-BHP (1 mM) for a further 12 h (A and B) Cell viability was measured by an MTT assay and LDH leakage assay. (C) Effects of rutaecarpine on t-BHP-induced cellular ROS production. Cells were pretreated with rutaecarpine or a vehicle for 1 h. Following removal of the medium, HepG2 cells were exposed to t-BHP for 6 h and fluorescence was then measured. (D) Effect of rutaecarpine on caspase-3 activation in HepG2 cells. (E) Effect of rutaecarpine on t-BHPeinduced death in HepG2 cells measured using a live/dead cell viability assay. Results are presented as means ± S.D. of three independent experiments. #p < 0.01, significantly different from the control; *p < 0.01, significantly different from t-BHP treated cells.Effect of rutaecarpine on Nrf2 transcriptional activity. (A and B) The cells were treated with rutaecarpine at the indicated doses (1e10 mM) or time (0e6 h). Nuclear lysates were analyzed for nuclear levels of Nrf2 and b-actin by western blotting. (C) HepG2 cells cultured on coverslips were treated with or without rutaecarpine for 3 h. Cells were fixed in 2% paraformaldehyde, permeabilized, and immunostained for Nrf2 (Alexa488; green). The nuclei were stained with DAPI (blue). The merger of DAPI and Alexa488 is shown in the right panel. The nuclear translocation of Nrf2 in cells was analyzed by fluorescence microscopy. (D) ChIP analysis of Nrf2 binding to the ARE-binding sequence in the HO-1 promoter in the presence of rutaecarpine. Cells were incubated with 10 mM of rutaecarpine for 12 h. The cells were then cross-linked with formaldehyde, and the associations of Nrf2 with DNA sequences were determined by a ChIP assay. (E) HO-1-ARE luciferase activity was analyzed in HepG2 cells transfected with the HO-1-ARE luciferase construct treated to rutaecarpine for 3 h. (F) Cells were treated with 10 mM of rutaecarpine for 6 h, and then incubated with t-BHP (1 mM) for a further 1 h. Nuclear lysates were analyzed for nuclear levels of Nrf2 and Lamin B by western blotting. Results are presented as means ± S.D. of three independent experiments. *p < 0.01, significantly different from the control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)Biotechnology). The transfected cells were incubated with rutae- carpine for 12 h followed by a lysis buffer (Cell Signaling Technol- ogy) for western blot analysis.Luciferase assay was performed as described previously pub- lished methods (Hwang et al., 2009). HepG2 cells were plated in 24- well plates overnight and transiently co-transfected with promoter-luciferase construct (HO-1-ARE) and pRL-SV plasmid using Lipofectamine. ARE-promoter-driven firefly luciferase activity was normalized to control Renilla luciferase expression.6e8 week male ICR mice with a body weight of 25 ± 1 g were purchased from Central Lab. Animal (Seoul, Korea), and allowed free access to a standard diet and tap water. Mice were maintained
in a controlled environment at 21 ± 2 ◦C and 50 ± 5% relative hu- midity with a 12 h dark/light cycle and acclimatized for at least one week prior to use. All animal handling was performed according to the instructions of the Committee for Ethical Usage of Experimental
The effect of rutaecarpine on the nuclear levels of Nrf2 target genes. (A and B) Cells were treated with rutaecarpine at the indicated doses (1e10 mM) or time (0e24 h), and then cell lysates were prepared for immunoblotting. (C) Cells were exposed to various concentrations of rutaecarpine for 12 h and then total RNA was extracted. HO-1, GCL, and NQO1 mRNA levels were analyzed by RT-qPCR. (D) The nuclear levels of Nrf2 target genes were assessed in HepG2 cells treated with t-BHP (1 mM) for 12 h after 12 h pre-incubation with vehicle or rutaecarpine (10 mM). Results are presented as means ± S.D. of three independent experiments. *p < 0.01, significantly different from the control.Animals in Chungnam National University. Twenty four healthy male mice were randomly assigned to four experimental groups of six mice each. To study its ability to protect against t-BHP-induced hepatotoxicity, rutaecarpine 5 mg/kg in corn oil was administered orally administrated for 3 consecutive days before t-BHP injection. Three hours after the final administration, animals were treated intraperitoneally with t-BHP (2 mmol/kg, 100:l dissolved in saline). Twenty-four hours after t-BHP administration, mice were anes- thetized with CO2, blood was removed by cardiac puncture to determine serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, and animals were euthanized by cervical dislocation. To assess hepatotoxicity, we measured the serum activity of ALT and AST using spectrophotometric diagnostic kits according to the manufacturer's recommendations (Sigma Chemical Co., St. Louis, MO, USA). After bleeding, livers were frozen quickly in dry ice/methanol and stored at 70 ◦C to determine lipid peroxidation levels.Hepatic lipid peroxidation level was determined by measuring thiobarbituric acid reactive substances (TBARS) (Lila, 2004). Briefly, samples were mixed with TBA reagent consisting of 0.375% TBA and 15% trichloroacetic acid in 0.25 M HCl. The reaction mixture was boiled in a water bath for 30 min and centrifuged at 2000 rpm for
10 min at 4 ◦C. Then, the TBARS concentration was determined based on the absorbance at 532 nm measured with a spectropho- tometer (Varioskan, Thermo Electron Co., Finland). Control tests were performed to ensure that rutaecarpine did not interfere with the lipid peroxidation assays. Protein
concentrations were determined using the Bradford method with bovine serum albumin (BSA) as the standard (Bradford, 1976).Values represent the means from three independent experi- ments, each performed in triplicate. The data were expressed as means ± S.D. Statistical significance was determined by ANOVA followed by the Tukey-Kramer test, using p < 0.01 or p < 0.05 as the level of significance.
3.Results
We first examined the cytotoxicity of rutaecarpine in human liver-derived HepG2 cells using MTT and LDH assays. Our results shows that rutaecarpine, at the tested concentrations (1e10 mM), did not cause significant cytotoxicity (data not shown). Thus, the cells were treated with rutaecarpine at a concentration of 1e10 mM in our subsequent experiments. Next, we examined the protective effects of rutaecarpine on t-BHP-induced HepG2 cell death. Cells exposed to 1 mM of t-BHP showed a 61% reduction in viability compared to the control. However, rutaecarpine (1e10 mM) pre- treatment reversed the effects of t-BHP in HepG2 cells by increasing viability in a concentration-dependent manner (Fig. 1A). Rupture of the plasma membrane during necrosis allows the release of cytosolic proteins such as LDH into the culture medium. LDH release due to t-BHP treatment significantly decreased in cells
4. Nrf2 regulates rutaecarpine-induced HO-1 expression. HepG2 cells were transiently transfected with Nrf2-siRNA or a non-specific control siRNA for 48 h. The transfected cells were treated with rutaecarpine (10 mM) for 12 h and the expression of HO-1 was measured by western blot analysis (A). (B) HO-1-ARE luciferase activity was measured by a luminometer. (C and D) After transfection with Nrf2-siRNA or non-specific control siRNA, cells were pretreated with rutaecarpine (10 mM) for 1 h, and then incubated with t-BHP (1 mM) for a further 12 h (C) or 6 h (D). (C) Cell viability was determined by an MTT assay. (D) The fluorescence was measured using an FL600 fluorescence spectrophotometer. Results are presented as means ± S.D. of three independent experiments. #p < 0.01, significantly different from the control; *p < 0.01, significantly different from t-BHP-treated cell pretreated with rutaecarpine (Fig. 1B). Oxidative stress is man- ifested by excessive generation of ROS and is involved in cellular damage and mitochondrial dysfunction (Loguercio and Federico, 2003). t-BHP induces oxidative stress, which may cause a signifi- cant increase in ROS formation and caspase-3 activity, which are associated with cell damage and apoptosis (Cuello et al., 2007).
Therefore, we determined whether rutaecarpine blocked the pro- duction of ROS and activation of caspase-3 activity by t-BHP treatment. The production of intracellular ROS in HepG2 cells was assayed using DCFH-DA. As expected, t-BHP significantly induced DCFH-DA oxidation, which was attenuated by rutaecarpine treat- ment in a concentration-dependent manner (Fig. 1C). To examine whether rutaecarpine prevented caspase-3 activity, cells were pretreated with rutaecarpine for 1 h followed by 12 h of t-BHP exposure, and then the DEVDase activity was determined. The level of t-BHP-induced DEVDase activity was significantly reduced by rutaecarpine pretreatment in HepG2 cells (Fig. 1D). To investigate the effects of rutaecarpine on t-BHPeinduced cell death, we used a cell viability live/dead assay. HepG2 cells were pretreated with rutaecarpine (1e10 mM) for 1 h, and then treated with 1 mM of t- BHP for an additional 12 h. The results showed that rutaecarpine decreased t-BHPeinduced apoptosis in HepG2 cells (Fig. 1E). These results indicated that the inhibition of ROS production and caspase-3 activity by rutaecarpine could protect HepG2 cells against t- BHPeinduced cell death. To evaluate the effect of rutaecarpine pretreatment on t-BHP-induced liver damage, we monitored serum AST and ALT activities and lipid peroxidation (Table 1). A single dose of t-BHP (2 mmol/kg) was hepatotoxic in mice, as demonstrated byelevated serum ALT and AST levels. However, rutaecarpine pre- treatment reduced serum ALT and AST levels in a dose-dependent manner after t-BHP administration (Table 1). Furthermore, rutae- carpine pretreatment significantly reduced t-BHP-induced lipid peroxidation in liver (Table 1).
Nuclear translocation of activated Nrf2 is an important up- stream regulator for ARE-driven expression of antioxidant en- zymes, such as HO-1, NQO1, and GCL (Itoh et al., 2010; Nguyen et al., 2009; Thimmulappa et al., 2002). To explore whether rutaecarpine could activate Nrf2, HepG2 cells were incubated with rutaecarpine for the indicated concentrations (1e10 mM) and times (0e6 h) and then the nuclear fractions were extracted for immunoblotting analysis. Treatment with rutaecarpine increased nuclear Nrf2 levels in a dose- and time-dependent manner. Consistent with this result, rutaecarpine stimulated translocation of Nrf2 into the nucleus (Fig. 2C). A ChIP assay was performed with HO-1-promoter-specific primers that contained a putative ARE binding sequence. Rutae- carpine induced the binding of Nrf2 to an ARE binding sequence that exists on the HO-1 promoter construct (Fig. 2D). Next, we determined the HO-1-ARE transcriptional activity in rutaecarpine-treated HepG2 cells. HO-1eARE luciferase constructs that contain 3-tandem repeats of ARE in the 50-upstream region of HO-1 were Induction of HO-1 and activation of Nrf2 by rutaecarpine via Akt phosphorylation. (A) Cells were treated with rutaecarpine (1e10 mM) for of 1 h and then whole cell extracts were prepared for western blotting. (B) HepG2 cells were preincubated with LY294002 (LY; 10 mM), PD98059 (PD; 20 mM), SB203580 (SB; 10 mM), and SP600125 (SP; 10 mM) for 30 min and then incubated with rutaecarpine (10 mM) for 12 h. Total cell extracts were subjected to immunoblotting. (C) Luciferase activity was measured using a luminometer. Results are presented as means ± S.D. of three independent experiments. #p < 0.01, significantly different from the control; *p < 0.01, significantly different from rutaecarpine- treated cells transfected into HepG2 cells to examine transactivation by rutae- carpine. As shown in Fig. 2E, rutaecarpine significantly increased the expression of the HO-1-ARE-Luc reporter gene in a concen- tration dependent manner, resulting in a 2.5-fold induction at 10 mM. These results demonstrate that rutaecarpine induced HO-1 transcriptional activity, stimulating the binding of Nrf2 to the HO-1 promoter and increasing HO-1 expression. To test whether t-BHP could affect Nrf2 translocation, HepG2 cells were incubated with
10 mM of rutaecarpine for 6 h and then incubated with t-BHP (1 mM) for a further 1 h. Treatment with t-BHP decreased nuclear Nrf2 levels but the effect was suppressed by rutaecarpine (Fig. 2F). Next, we determined the effect of rutaecarpine on Nrf2 target gene induction such as HO-1, NQO1, and GCL. Our results showed that the protein expression of HO-1, NQO1, and GCL was induced by rutaecarpine treatment (Fig. 3A and B). Furthermore, rutaecarpine (10 mM) significantly up-regulated HO-1, NQO1, and GCL mRNA expression by about 3.7-, 3.6- and 3.4-fold, respectively (Fig. 3C). Furthermore, t-BHP treatment reduced the protein expression of HO-1, NQO1, and GCL but pretreatment with rutaecarpine restored the Nrf2 target proteins (Fig. 3D), consistent with nuclear Nrf2 levels (Fig. 2F).
In recent studies, HO-1 was shown to exert cytoprotective ef- fects against oxidative stress-induced cell damage in many cell lines, including HepG2 cells. To test whether Nrf2 are required for HO-1 expression and cytoprotection by rutaecarpine, we used Nrf2-knockout (KO) HepG2 cells. As shown in Fig. 4A and B, rutaecarpine-induced up-regulation of HO-1 and ARE promoter activity in HepG2 cells was attenuated by Nrf2-siRNA, whereas
The CaMKII-PI3K/Akt pathway is involved in rutaecarpine-induced HO-1 expression and activation of the HO-1-ARE-Luc reporter. (A) HepG2 cells were stimulated with rutaecarpine for 1 h and then immunoblotted with activation-specific antibodies that recognize p-CaMKIIa and b-actin. (B) HepG2 cells were preincubated with KN-93 (10 mM) and BAPTA/AM (10 mM) for 30 min and then incubated with rutaecarpine (10 mM) for 12 h. Total cell extracts were subjected to immunoblotting using anti-HO-1 and anti-b-actin antibodies. (C) HepG2 cells were transfected with the HO-1-ARE-luciferase plasmid construct. After transfection for 24 h, cells were preincubated with SB366791 (10 mM), BAPTA/ AM (10 mM), W7 (10 mM), and KN-93 (10 mM) for 30 min and then treated with rutaecarpine for 12 h. Luciferase activity was measured using a luminometer. (D) Cells were preincubated with SB366791, BAPTA/AM, W7, and KN-93 for 30 min and then treated with rutaecarpine for 1 h. Total cell lysates were analyzed for phospho-CaMKIIa, phospho-Akt, and b-actin by immunoblot analysis. (E) HepG2 cells were preincubated with KN-93 and LY294002 for 30 min and then incubated with rutaecarpine for 1 h. Total cell lysates were analyzed for phospho-CaMKIIa and b-actin by immunoblot analysis. Results are presented as means ± S.D. of three independent experiments. #p < 0.01, significantly different from the control; *p < 0.01, significantly different from rutaecarpine-treated cells transfection of the cells with the same amount of control-siRNA was not effective. In addition, the rutaecarpine-induced cytopro- tection against t-BHP was suppressed by Nrf2-siRNA (Fig. 4C). Moreover, transfection of Nrf2-siRNA significantly attenuated the ROS depletion by rutaecarpine in t-BHPetreated HepG2 cells (Fig. 4D). Taken together, these observations suggest that the cytoprotective effect of rutaecarpine requires Nrf2 activation.
4.Discussion
In the present study, we examined the cytoprotective effect of rutaecarpine against t-BHP to elucidate the mechanism of action of rutaecarpine in hepatocytes and mice liver. Our data show that rutaecarpine inhibited t-BHP-induced cytotoxicity and apoptosis by inhibiting ROS production in HepG2 cells. The inhibitory effect of rutaecarpine was reinforced by inducing CaMKII-PI3K/Akt-Nrf2- mediated HO-1 expression. Rutaecarpine is one of the major components of E. rutaecarpa, which has long been used in tradi- tional oriental medicine for the treatment of many disorders such as hypertension, gastrointestinal disorders, and headache (Jia and Hu, 2010). Rutaecarpine has been reported to have a variety of biological properties, including anticancer, anti-inflammatory, antithrombotic, and antioxidative activities (Chen et al., 2013; Dai et al., 2008; Lee et al., 2012). A recent study has demonstrated that rutaecarpine inhibited hydrogen peroxide-induced cellular damage mechanisms in murine Hepa-1c1c7 cells (Lee et al., 2012). However, the effects of rutaecarpine on oxidative stress-induced injury in hepatocytes have not yet been reported. Oxidative stress is manifested by excessive generation of ROS, such as hydroxyl radicals and superoxide anion radicals, and is involved in several cellular damage such as apoptosis, cancer, and metabolic diseases (Loguercio and Federico, 2003; Han et al., 2015). Therefore, the use of natural compounds with antioxidant proper- ties may assist in preventing diseases associated with oxidative damage. In this study, we established the ability of rutaecarpine to improve t-BHP-induced ROS production and its related toxicity in human HepG2 cells. The present study showed that rutaecarpine decreased t-BHP-induced ROS production and oxidative stress in HepG2 cells. Moreover, the present study showed that rutaecarpine significantly lowered the t-BHP-induced serum levels of ALT and AST and reduced hepatic oxidative stress as determined by lipid peroxidation assays.
A previous study showed that phytochemicals protect against oxidative stress-induced toxicity (Dai et al., 2008; Hwang et al., 2011). The transcription factor Nrf2 plays a central role in the protection of many organs, including the liver against electrophilic and oxidative stress (Han et al., 2015; Ma and He, 2012). Recent evidence suggests that Nrf2 positively regulates the ARE-mediated expression of antioxidant enzyme genes, including GCL, NQO1, and HO-1 (Itoh et al., 2010; Nguyen et al., 2009; Thimmulappa et al., 2002). Therefore, it is generally accepted that the activation of Nrf2 is an attractive therapeutic target for the prevention and treatment of liver diseases (Aleksunes and Manautou, 2007; Han et al., 2015). Our previous study also showed that phytochemi- cals, such as puerarin, and anthocyanins, exhibit cytoprotective effects in cases of t-BHP-induced oxidative stress via the Nrf2- dependent expression of antioxidant enzymes (Hwang and Jeong, 2008; Hwang et al., 2011). In the present study, we showed that rutaecarpine significantly increased nuclear translocation of Nrf2 in HepG2 cells. In addition, rutaecarpine-induced upregulation of antioxidant enzymes and HO-1-ARE-luciferase activity in HepG2 cells was suppressed by Nrf2-siRNA expression, whereas trans- fection of the cells with the negative control siRNA was not effec- tive. These data suggest that rutaecarpine-induced expression of HO-1, NQO1, and GCL may occur via Nrf2/ARE signaling pathway in human hepatocytes.Many kinase signaling pathways, including CaMKII (Kim et al.,2010), PI3K/Akt (Kang et al., 2002), and MAPKs (Kong et al., 2001), may regulate Nrf2 activation and facilitate its accumula- tion in the nucleus for the induction of HO-1 gene expression (Kim et al., 2010). In this study, we found that rutaecarpine activated PI3K/Akt and its upstream activators, CaMKII. However, our result shows that specific inhibitors of MAPKs, such as PD98059, SB203580, and SP600125, had no inhibitory effect on rutaecarpine- induced HO-1 expression. In addition, rutaecarpine-induced HO-1 expression was inhibited by specific inhibitors of TRPV1, Ca2þ, calmodulin, and CaMKII, which confirmed the roles of these kinases in the activation of HO-1 by rutaecarpine. Furthermore, ZnPP, KN- 93, LY294002, and SB366791 alleviated the protective effect of rutaecarpine against t-BHP-induced oxidative stress. These results demonstrate that the activation of the CaMKII-PI3K/Akt signaling pathways was required for rutaecarpine-mediated up-regulation of HO-1 expression in HepG2 cells.
In conclusion, our findings clearly show that rutaecarpine in- creases the activation of the Nrf2/ARE signaling pathway via the phosphorylation of CaMKII-PI3K/Akt in hepatocytes. Furthermore, rutaecarpine inhibited t-BHP-induced ROS production and cell death. Taken together, rutaecarpine augments cellular antioxidant defense capacities through CaMKII-PI3K/Akt-dependent HO-1 induction via the Nrf2/ARE signaling pathway, thereby protecting cells from oxidative damage (Fig. 8). These findings are KN-93 anticipated to help develop E. rutaecarpa-based phytomedicine for the man- agement of oxidative injury.