Notopterygium forbesii Boiss Extract and Its Active Constituents

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Notopterygium forbesii Boiss Extract and Its Active Constituents Increase Reactive Species and Heme Oxygenase-1 in Human Fetal Hepatocytes: Mechanisms of Action Soon Yew Tang, Huansong Wang, Wenxia Zhang, and Barry Halliwell* Department of Biochemistry, Centre for Life Sciences, Yong Loo Lin School of Medicine, National UniVersity of Singapore, 28 Medical DriVe, Singapore 117456 ReceiVed August 9, 2008

Notopterygium forbesii Boiss (NF) has been used as a traditional Chinese medicine for the treatment of common cold and rheumatism. However, there has been limited research on the biological properties of NF, and the mechanisms of action remain unknown. Here, we aimed to study the mechanism of NF-induced heme oxygenase-1 (HO-1) in human fetal hepatocytes (HFHs) and to identify the constituents responsible. Exposure of HFHs to NF causes oxidative stress with the accumulation of reactive species, which in turn leads to the phosphorylation of p38 MAPK and nuclear accumulation of Nrf2 transcription factor, and eventually increased levels of HO-1 mRNA and protein. The increases in reactive species and HO-1 protein are inhibited by agonists of glucocorticoid receptors (GR), such as RU28362, prednisolone, and dexamethasone, as well as by N-acetyl-L-cysteine and SB203580 (a p38 inhibitor), suggesting a role of GR in NF-induced increases in reactive species and HO-1. Assay-guided fractionation of NF led to three active compounds, phenethyl ferulate, bergaptol, and isoimperatorin, that were found to increase oxidative stress and HO-1 protein levels in HFHs. The induction of HO-1 protein in response to moderate oxidative stress may explain some of the beneficial pharmacological effects of NF. Introduction Rhizoma seu Radix Notopterygii, which is known as Qianghuo in Chinese, has been studied as part of our ongoing search for biologically active agents from medicinal plants. According to the Pharmacopoeia of the People’s Republic of China, both Notopterygium incisum Ting ex. H.T and Notopterygium forbesii Boiss (NF)1 are named as Qianghuo (1). Rhizoma seu Radix Notopterygii is a plant belonging to the Umbelliferae family. The rhizomes and roots of Rhizoma seu Radix Notopterygii have been used as a traditional Chinese medicine as crude drugs or in at least 13 prescriptions for the treatment of common cold, rheumatism, and headache (2). Moreover, this plant has been reported to possess anti-inflammatory (3), diaphoretic, and analgesic (4) properties. The chemical constituents of Rhizoma seu Radix Notopterygii have been reported (5, 6). Recently, Jiang and colleagues (7) have developed an accurate high-performance liquid chromatography-diode array detector (HPLC-DAD) fingerprint method for the quality control of Qianghuo. Many cells respond to moderate oxidative stress by increasing the defense system, such as the synthesis of heat shock proteins (Hsp) (8). Among the various Hsp, heme oxygenase-1 (Hsp32/ HO-1) has attracted most interest as it is inducible by a large number of structurally diverse chemicals, including stress conditions and heavy metals, and generates products that might * To whom correspondence should be addressed. Tel: +65 6516 3247. Fax: +65 6775 2207. E-mail: [email protected]. 1 Abbreviations: BGT, bergaptol; DCF, 2,7-dichlorofluorescein; DAD, diode array detector; DEM, dexamethasone; H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; HFHs, human fetal hepatocytes; HO-1, heme oxygenase-1; IMP, isoimperatorin; MAPKs, mitogen-activated protein kinases; MTT, 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide; NAC, N-acetyl-L-cysteine; NF, Notopterygium forbesii Boiss; Nrf2, nuclear factor erythroid 2-related factor 2; PF, phenethyl ferulate; PNS, prednisolone; RS, reactive species; RU28362, (11β,17β)-11,17-dihydroxy-6-methyl-17-(1propynyl)-androsta-1,4,6-trien-3-one.

have important biological activities (9). For example, HO-1 converts hemin, a potent pro-oxidant, into other bioactive metabolites, such as carbon monoxide and bilirubin. HO-1 is regulated by the nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH associating protein 1 (Keap1) transcription factor system (10, 11). In the course of our in vitro screening of medicinal plants for NAD(P)H:quinone oxidoreductase-1 (NQO1) activity in mouse hepatomas (Hepa1c1c7), undertaken because up-regulation of endogenous defense systems may contribute to the protective effects of medicinal plants, NF extract was found to increase NQO1 activity by 6-fold as compared to untreated control (unpublished data). However, the extract showed no effects on NQO1 activity and its protein levels when tested on normal human fetal hepatocytes (HFHs). We next sought to determine whether the extract up-regulates other phase II enzymes and found that the extract induced HO-1 protein expression but not HO-2 in HFHs. Thus, we aimed to study the mechanisms of NF in HO-1 induction and identify the constituents responsible. We report herein that the mechanism of HO-1 induction due to NF-induced oxidative stress in HFHs involves generation of reactive species (RS) and the subsequent activation of the p38 MAPK pathway and Nrf2 transcription factor. On the basis of HO-1 protein induction in HFHs, we have purified and identified phenethyl ferulate (PF), bergaptol (BGT), and isoimperatorin (IMP) from NF as components contributing to this effect.

Experimental Procedures Materials. All reagents used were purchased from SigmaAldrich (St. Louis, MO) unless otherwise stated. 2′-Amino-3′methoxyflavone (PD98059), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), 1,4-

10.1021/tx800301f CCC: $40.75  2008 American Chemical Society Published on Web 11/04/2008

NF Increases ReactiVe Species and HO-1

diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene(U0126), and 1,9-pyrazoloanthrone (SP600125) were obtained from Calbiochem (San Diego, CA). 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) was purchased from Molecular Probes Inc. (Eugene, OR). Antibodies against HO-1 and Nrf2 (C-20) and siRNA of A and Nrf2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). MAPK antibodies were from Cell Signaling Technology (Danvers, MA). Active-MAPK antibodies were from Promega Corp. (Madison, WI). Fetal bovine serum (FBS) was from Hyclone (Logan, UT). Cell Culture. The HFHs (12), a generous gift from Professor Sit Kim Ping (Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore), were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% (v/v) penicillin and streptomycin (PS) and 10% (v/v) FBS and grown in a humidified incubator with 5% CO2 at 37 °C. The cells were maintained in the logarithmic growth phase by routine passage every 3-4 days. 3-(4,5-Dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Assay. Cell viability was measured by the MTT method. Cells in medium were seeded overnight at a density of 1.0 × 104 cells per well in 96 well plates. After exposure to 0-60 µg/mL of NF for 6, 12, and 24 h, 200 µL of MTT (0.5 mg/mL final concentration) dissolved in Earle’s balanced salt solution (EBSS) was added. Cells were incubated at 37 °C in the dark for 1 h, and then, MTT was removed. Two hundred microliters of DMSO was added to solubilize the formazan formed. The absorbance at 570 nm was determined using a SpectraMax190 microplate reader (Molecular Devices, Sunnyvale, CA) after shaking in the dark for 15 min. Cell Cycle Analysis Using Flow Cytometry. Cytotoxicity of NF on HFHs was also assessed by staining with propidium iodide (PI) as described previously (13). Briefly, HFHs were exposed to 40 µg/mL NF for 3, 6, 12, and 24 h. Treated cells were harvested and washed twice with phosphate-buffered solution (PBS), followed by fixation with 2 mL of fixing solution consisting of PBS (200 µL) and 70% (v/v) ice-cold ethanol (1800 µL) overnight at 4 °C. The fixing solution was removed by centrifugation (300g, 5 min), and cells were stained with 0.5 mL of PI solution consisting of 1% (v/v) Triton X-100 in PBS (1 mL), 10 mg/mL RNase A (0.2 mL), 2.5 mg/mL PI solution (0.08 mL), and PBS (8.72 mL). The percentages of cells in subG1 were calculated using WinMDI 2.8 software (Scripps Institute, La Jolla, CA). Kinetic Study of Rise in RS with H2DCFDA. RS were determined with H2DCFDA fluorescent dye (Molecular Probes, Eugene, OR) using a Gemini Fluorescence microplate reader (Molecular Devices, Sunnyvale, CA). Cells in medium were seeded overnight at a density of 4.0 × 104 cells per well in 24 well plates for attachment. Cells at 80% confluency were washed twice with PBS and preincubated with H2DCFDA fluorescent dye dissolved in EBSS (final concentration 2.5 µM, 0.5 mL) for 30 min at 37 °C. Excess H2DCFDA solution was subsequently removed by washing twice with PBS. For treatment, 0-40 µg/mL of NF dissolved in EBSS was added to cells, and fluorescence measurement was started immediately at Ex/Em 488/535 nm with an interval of 2.5 min for 30 min at 37 °C. RS Measurement with H2DCFDA by Flow Cytometry. HFHs treated with 40 µg/mL of NF for 30 and 60 min were harvested by centrifugation (500g, 5 min) and washed twice with EBSS followed by staining with 2.5 µM H2DCFDA dissolved in EBSS for 30 min. Excess H2DCFDA fluorescent dye was removed by washing with PBS, and cells were

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resuspended in EBSS and kept on ice until flow cytometry analysis (Coulter Altra, Hialeah, FL). Control samples were analyzed again at the end to ensure that there were no significant changes to the distribution of intracellular fluorescent dye. NF was added prior to the addition of H2DCFDA because it was previously found that in the reverse procedure the measurement of DCF fluorescence was interfered with by medicinal plant extracts present at higher concentrations. The effects of (11β,17β)11,17-dihydroxy-6-methyl-17-(1-propynyl)-androsta-1,4,6-trien3-one (RU28362), prednisolone (PNS), dexamethasone (DEM), N-acetyl-L-cysteine (NAC), and SB203580 on NF-induced RS were also examined using the flow cytometer. Nrf2 Silencing by siRNA. Cells were seeded on six well plates at a density of 2.0 × 105 cells/well in 2 mL of antibioticfree DMEM supplemented with 10% FBS. Cells were allowed to grow to 80% confluency before transfection with siRNA (small interfering RNAs). Solution A (6 µL; 60 pmol of siRNA duplex in 100 µL of siRNA transfection medium) was mixed with solution B (6 µL of transfection reagent in 100 µL of transfection medium) and incubated at room temperature for 30 min. Following this incubation, HFHs were washed with transfection medium once. For each transfection, 800 µL of transfection medium was added to 200 µL of siRNA duplex/transfection reagent mix (solution A + B), and the entire volume was added gently onto the cells. After incubation at 37 °C for 6 h, transfection solution was aspirated, and 1 mL of normal growth medium was added to each well. Incubation was continued for 12 h, and thereafter, medium was replaced with fresh growth medium containing 40 µg/mL of NF, and cells were incubated for another 6 h. Cells were harvested by trypsinization and subjected to immunoblot analysis for evaluation of transfection effects on HO-1 protein induction in the presence of NF. A scrambled siRNA sequence (A siRNA) was used as a negative control. RNA Extraction and Reverse Transcription-Polymerase Chain Reaction Analysis. For reverse transcription-polymerase chain reaction (RT-PCR) analysis of mRNA levels, total RNA of HFHs was isolated at 3, 6, 12, and 24 h after exposure to NF at 40 µg/mL using TRIZOL Reagent (Invitrogen Corp., Carlsbad, CA). Experimental RNA (1 µg) samples were converted to single-stranded cDNA by the ImProm-II Reverse Transcription System (Promega, Madison, WI) according to the manufacture’s instruction, and the resulting cDNA was amplified by the GoTagGreen Master Mix (Promega). PCR conditions comprised an initial denaturation step at 94 °C for 1.5 min, followed by 30 cycles of denaturation (94 °C for 1.5 min), annealing (60-65 °C for 1 min), and extension (72 °C for 1.30 min) steps. The final extension step was at 72 °C for 2 min. The forward and reverse primers used for amplifying HO-1 and GAPDH were 5′-AGCTCTTTGAGGAGTTGCAGGA-3′ and 5′-TTGCACTTTGTTGCTGGCC-3′ and 5′-CATCACCATCTTCCAGGAGC-3′ and 5′-CATGAGTCCTTCCACGATACC3′, respectively. GAPDH was used as an internal control. At the completion of PCR, 5-10 µL of PCR products was electrophoresed in 2% (w/v) agarose gel in the presence of 0.5 µg/mL of ethidium bromide. The amplified DNA fragments were visualized with a Kodak Image Station 2000R (Kodak, Rochester, NY). Western Blot Analysis. Cells (0.5 × 106 cells per well in six well plates) stimulated with 0-40 µg/mL of NF for 3, 6, 12, and 24 h were harvested and lysed with cell lysis buffer (Cell Signaling Technology, Danvers, MA). Cells were centrifuged at 20000g at 4 °C for 15 min using a desktop centrifuge (Centrifuge 5417C, Eppendorf) to remove unbroken cells, nuclei,

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Figure 1. NF-induced loss of cell viability by MTT assay and cell cycle analysis. (A) HFHs exposed to 0-60 µg/mL of NF for 6, 12, and 24 h as assessed by the MTT assay as described in the Experimental Procedures. Data points are means ( SDs of at least three independent experiments performed in triplicate. (B) HFHs treated with 40 µg/mL of NF for 3, 6, 12, and 24 h were determined after staining the cells and were fixed overnight with PI solution as outlined in the Experimental Procedures. At least 104 cells were collected for cell cycle analysis. The percentage of nonviable cells is represented by SubG1. Data are means ( SDs of three separate experiments performed in duplicate. **p < 0.01 vs 0 µg/mL NF or control.

mitochondria, and other organelles after incubation on ice for 15 min. The protein concentration of the supernatant was determined with the DC Protein Assay Kit (Bio-Rad, Hercules, CA), and 40 µg of protein in loading dye was heated at 95 °C for 5 min before loading into a 10% SDS-PAGE gel for immunoblotting unless otherwise stated. The separated proteins were transferred to nitrocellulose membranes (Bio-Rad, 0.2 µM) and probed with antibodies against HO-1, Nrf2 (C-20), phosphorylated and total p38, ERK, and JNK, followed by the appropriate HRP-conjugated secondary antibodies. Detection was performed by enhanced chemiluminescence (Pierce, Rockfold, IL) using a Kodak Image Station 2000R (Kodak, Rochester, NY). Statistical Analysis. Differences between means were evaluated using an unpaired two-sided Student’s t test (*p < 0.05 and **p < 0.01 considered as significant).

Results Cytotoxicity of NF on HFHs by MTT Assay and Cell Cycle Analysis. The principle of the tetrazolium salt assay is based on the ability of viable cells to convert MTT to the purplecolored formazan crystals, serving as an indirect measurement of cell proliferation and loss of viability. As shown in Figure 1A, viability of HFHs decreased modestly in a dose- and timedependent manner at 12 h after incubation with NF. Interestingly, NF at concentrations of 20, 40, and 60 µg/ml showed a trend of increased MTT reduction ability by HFHs at 6 h. Because NF was removed prior to the addition of MTT, this is not an effect of NF on MTT itself. The number of cells treated with 20, 40, and 60 µg/ml of NF for 6 h as determined with trypan blue dye exclusion assay did not show a significant increase in cell population as compared to control (data not shown). Therefore, the increased MTT reduction could be due

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Figure 2. NF-induced rise in intracellular RS as determined by H2DCFDA using a Gemini Fluorescence microplate reader and flow cytometer. (A) HFHs were preincubated with 2.5 µM H2DCFDA for 30 min, and measurement started immediately with 0, 5, 10, and 20 µg/mL of NF after excess H2DCFDA fluorescent dye was removed as described in the Experimental Procedures. Kinetic studies were stopped after 30 min due to cell detachment. (B) HFHs were exposed to 20 and 40 µg/mL of NF for 30 and 60 min prior to the incubation with 2.5 µM H2DCFDA for 30 min. At least 104 cells were collected for flow cytometry analysis. Data are means ( SDs of three separate experiments performed in duplicate. *p < 0.05 and **p < 0.01 vs control.

to the activation of dehydrogenases by treatment with NF. Viability of cells after 24 h of incubation at 40 and 60 µg/mL of NF was reduced to 91 ( 3.6 and 81 ( 3.1%, respectively. A 40 µg/mL amount of NF, unless otherwise stated, was used throughout this study. We also studied HFHs cell cycle by PI staining using the flow cytometer after NF treatment to confirm the observed loss of cell viability by MTT assay. Nonviable cells were detected on PI histogram as a hypodiploid peak (subG1). As shown in Figure 1B, exposure of HFHs to 40 µg/mL of NF increased the percentage of nonviable cells in a time-dependent manner. The percentage of nonviable cells after 12 and 24 h of incubation was 10.2 ( 0.7 and 9.8 ( 1.8% (p < 0.01), respectively. The untreated cells showed 3.4 ( 0.6% of nonviable cells. These findings are similar to the results that we obtained by the MTT assay. NF Induces Rapid Rises in Intracellular RS. The generation of intracellular RS was monitored by the fluorescence emission of H2DCFDA. H2DCFDA is hydrolyzed by intracellular esterases to a nonfluorescent product that is readily oxidized to fluorescent 2,7-dichlorofluorescein (DCF) by a range of RS (14). As shown in Figure 2A, NF induced rapid generation of RS as determined by the increase in DCF fluorescence. The increased DCF fluorescence at 5 and 10 µg/mL of NF was not significant, but at 20 µg/mL, the DCF fluorescence detected was significantly higher (p < 0.05) at 10 min as compared to control and continued to rise (p < 0.01). However, at a higher concentration of 40 µg/mL, NF was found to interfere with the measurement


of DCF fluorescence (data not shown). The measurement was stopped after 30 min due to detachment of cells as observed under the microscope. It is not uncommon for medicinal plant extracts to interfere with the measurement of DCF fluorescence due to their complex nature. We have previously encountered a similar problem with Psoralea coryliforia extract (15). This problem can be overcome by measuring the generation of RS with H2DCFDA flow cytometrically in a reverse procedure; that is, cells treated with NF are washed with PBS prior to the addition of H2DCFDA as described in the Experimental Procedures. Figure 2B shows a dose- and time-dependent increase in DCF fluorescence positive cells, confirming that NF-induced generation of RS was detectable by DCF. For 30 min treatment with 40 µg/mL of NF, DCF fluorescence positive cells increased by 3-fold as compared to control (p < 0.01). To confirm that the rise in DCF fluorescence was not an artifact contributed by fluorescent compounds in NF after excitation at the wavelength of the DCF fluorescent dye, we measured cells treated with NF in the absence of H2DCFDA. No increase in fluorescence positive cells was observed in the absence of the fluorescent dye (data not shown). Some phenolic compounds have been reported to react with the ingredients in cell culture media to generate H2O2, which can have several effects on cells (16). In our system, the measurement of H2O2 by Fox 2 assay (17) showed that only a very low concentration of H2O2 (11.0 ( 3 µM, n ) 4) was detected in the media (DMEM + 1% PS + 10% FBS) after 24 h of incubation with 40 µg/mL of NF at 37 °C (data not shown). In the presence of cells, the levels of H2O2 generated in the media 1, 3, 6, 12, and 24 h after incubation with 40 µg/ mL of NF were below 10 µM as determined by the O2-electrode method (16) (data not shown). Moreover, HFHs treated with 12.5 and 25 µM H2O2 for 3, 6, 12, and 24 h did not show HO-1 protein up-regulation by Western blotting (data not shown). Hence, the effect of NF is not an artifact of pro-oxidant events occurring in the cell culture media. NF Increases HO-1 mRNA and Protein Levels. Many chemical and environmental stimuli are known to induce HO-1 by means of oxidative stress (18). Exposure of HFHs to NF resulted in a time-dependent increase in HO-1 mRNA levels (Figure 3A); the increase continued up to 24 h. This correlated with an increase in HO-1 protein expression in a time-dependent manner as shown in Figure 3B, which was maximal at 12 h and remained until 24 h. Figure 3C shows the dose-dependent increase in HO-1 protein expression. The lowest concentration examined at 2.5 µg/mL of NF also increased HO-1 protein modestly after 24 h of incubation at 37 °C. DMSO, the vehicle in which NF was solubilized (0.04%, final concentration), did not produce significant changes in HO-1 protein expression. NF Stimulates Nrf2 Nuclear Translocation and Nrf2 siRNA Blocks NF-Induced HO-1 Protein Expression. HO-1 is one of the phase II enzymes, also referred to as a Hsp32, which is widely distributed in mammalian tissues and regulated by the Nrf2/Keap1 transcription factor system (10, 11). Thus, we also determined Nrf2 proteins in the cytosolic and nuclear fractions of HFHs after exposure to NF. The nuclear fractions from NF-treated HFHs exhibited a dose- and time-dependent increase in Nrf2 proteins. As shown in Figure 4A, two faint bands of phosphorylated Nrf2 (118 and 98 kDa) were detected at the lower doses of 2.5 and 5 µg/mL of NF after 24 h of incubation. The phosphorylated Nrf2 proteins increased markedly at 10 µg/mL and higher of NF. Pi and colleagues have demonstrated that human Nrf2 is a casein kinase 2 (CK2)


Figure 3. HO-1 mRNA and protein levels of HFHs stimulated with NF. (A) HFHs were stimulated with 40 µg/mL of NF, and total RNA was extracted at the times indicated. Semiquantitative RT-PCRs, as described in the Experimental Procedures, were performed to determine HO-1 gene expression. GAPDH was applied as a loading control. (B and C) Western blots of HO-1 protein expression in HFHs. Total cellular proteins were isolated from cells treated with NF at the times (B) and doses (C) indicated, and Western blot analyses were performed using specific antibody for HO-1. β-Actin was used to control protein loading. Results are representative of three or more independent experiments.

substrate and that CK2 mediates two steps of Nrf2 phosphorylation, resulting in two forms of Nrf2 (19). With time, the accumulation of phosphorylated Nrf2 proteins was detected at as early as 3 h and continued to increase until 12 h and started to decline at 24 h (118 kDa band only) (Figure 4B). Cytosolic human Nrf2 proteins with a predicted molecular mass of 66 kDa were found to decrease modestly in a dose- and timedependent manner in HFHs after exposure to NF. Western blotting of the cytosolic fractions against histone 1 antibody showed satisfactory separation of the nuclei from the cytoplasm of the cells (data not shown). The role of Nrf2 in regulating NF-induced HO-1 expression was confirmed using siRNA of Nrf2. Nrf2 proteins decreased after silencing with Nrf2 siRNA as compared to untreated and negative controls (A siRNA) in total cell lysates (Figure 4C). With 60 pmol of Nrf2 siRNA, the rise in HO-1 protein was completely abolished, while A siRNA has no effect on HO-1 protein induced by NF (Figure 4D). These results indicate a key role for Nrf2 in regulating NF-induced HO-1 protein expression in HFHs and agree well with the established roles of Nrf2. P38 MAPK Pathway Is Important for NF-Induced HO-1 Protein Expression. Mitogen-activated protein kinases (MAPKs) are serine/threonine protein kinases that have been shown to be activated by a wide range of signaling molecules including RS. To determine the role, if any, of MAPKs in NFinduced HO-1 expression, we examined phosphorylated and total MAPKs (p38, ERK, and JNK) proteins by Western blotting. As shown in Figure 5A, both phosphorylated p38 and ERK can be detected but not JNK (data not shown). Phosphorylated p38 increased with incubation times, while ERK remained relatively constant up to 24 h. The increase in the levels of phosphorylated p38 was not due to a concomitant elevation in the amount of p38 since the level of p38 remained the same, suggesting that p38 MAPK pathway was activated in HFHs treated with NF.

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Figure 5. NF induces HO-1 involving phosphorylation of p38 MAPKs but not ERK and JNK. (A) Time-course activation of p38 by phosphorylation. HFHs were exposed to NF for 3, 6, 12, and 24 h. Total cellular proteins were prepared from cells for Western blot analyses using specific antibodies for p-p38, p38, p-ERK, and ERK. Results for p-JNK and JNK were not shown. Total MAPKs were used as loading control. (B) SB203580, an inhibitor of p38, decreases NFinduced HO-1 protein levels. HFHs were treated with 1, 2, and 5 µM SB203580 for 30 min prior to the addition of NF for 12 h. Total cellular proteins were prepared for Western blot analyses using specific antibody for HO-1. β-Actin was used to control protein loading. Results are representative of two or more independent experiments.

Figure 4. NF-induced HO-1 requires Nrf2 nuclear translocation. Dose (A)- and time (B)-dependent accumulation of Nrf2 in the nuclear and cytosolic fractions of HFHs stimulated with NF. Cells exposed to various doses of NF for various time points as indicated were collected for nuclear and cytosolic separation as described in the Supporting Information. (C) Reduction of Nrf2 protein in total cell lysate after Nrf2 silencing with 60 pmol of Nrf2 siRNA for 6 h. A scrambled sequence that will not lead to the specific degradation of any known cellular mRNA (A siRNA, 60 pmol) was included as a negative control. (D) Nrf2 siRNA abrogates NF-induced HO-1 protein up-regulation. Nrf2 in HFHs were silenced with 60 pmol of siRNA for 6 h. Twelve hours after the siRNA was removed, cells were exposed to NF for 6 h before total cellular proteins were collected for Western blot analyses for HO-1. β-Actin was used to control protein loading. Results are representative of three or more independent experiments.

To further address the role of individual MAPK pathways in HO-1 protein expression, we studied the effects of MAPK pathway inhibitors (PD98059: 5, 10, and 15 µM; U0126: 1, 2, and 5 µM), p38 inhibitor (SB203580: 1, 2, and 5 µM), and JNK inhibitor (SP600125: 1, 2, and 5 µM). Preincubation with SB203580 at various concentrations for 30 min prior to the addition of NF for 12 h attenuated the expression of HO-1 protein in a dose-dependent manner (Figure 5B). This suggests that the p38 MAPK pathway may be important for NF-induced HO-1 in HFHs. The other inhibitors of the MAPK pathways tested did not affect the increases in HO-1 protein, suggesting that the ERK and JNK MAPKs are not involved in our cell system (data not shown). Effects of Antioxidant and Antioxidant Defense Enzymes on NF-Induced HO-1 Protein Levels. Antioxidants have been used in many studies to counteract the damage caused by RS. Preincubation of HFHs with 2.5, 5, and 7.5 mM NAC for 30

Figure 6. N-Acetyl-L-cysteine (NAC) attenuates NF-induced HO-1 in HFHs. HFHs were exposed to NF for 12 h after preincubation with 2.5, 5, and 7.5 mM NAC for 30 min. Total cellular proteins were collected from cells for Western blot analyses for HO-1. β-Actin was used to control protein loading. Results are representative of three or more independent experiments.

min prior to the addition of 40 µg/mL of NF for 12 h dose dependently attenuated HO-1 protein expression (Figure 6). Because NF-induced HO-1 protein up-regulation did not deplete intracellular glutathione levels (data not shown), the effect of NF on HO-1 may act by mechanisms other than changing glutathione levels. Unlike NAC, preincubation of HFHs with 2.5 and 5 µM ebselen (a synthetic seleno-organic compound showing glutathione peroxidase-like activity (20)), 5 µM R-tocopherol, or 500 and 1000 U/mL each of catalase (EC 1.11.1.6) and superoxide dismutase (EC 1.15.1.1) did not affect HO-1 protein induction by NF (data not shown). Furthermore, these reagents also did not abolish NF-induced rises in intracellular RS as determined with H2DCFDA using the flow cytometer (data not shown). RU28362, PNS, and DEM Attenuate NF-Induced HO-1 Protein Levels. CYP3A7 is the major cytochrome P450 (CYP) isoform detected in the human embryonic, fetal, and newborn liver, although its levels are much lower than those of CYP3A4 in the adult liver (21). Moreover, expression of CYP3A genes is known to be controlled by pregnane X receptor (PXR) and constitutive androstane receptor (CAR), which are partly under the regulation of glucocorticoid receptor (GR) (22). Therefore, we examined the effect of GR agonists such as RU28362, PNS, and DEM on NF-induced HO-1 protein expression. As shown in Figure 7, preincubation with GR agonists for 30 min prior


Figure 7. Agonists of glucocorticoid receptor (GR) attenuate NFinduced HO-1 in HFHs. HFHs were preincubated with 0.5, 1, and 2 µM RU28362 and 2.5, 5, and 10 µM PNS or DEM for 30 min prior to the addition of NF for 12 h. For Western blot analyses, total cellular proteins were collected from cells for HO-1. β-Actin was used to control protein loading. Results are representative of at least three independent experiments.

to the addition of 40 µg/mL of NF for 12 h dose dependently attenuated the induced HO-1 protein levels. The protective effect of RU28362 was particularly strong; at 0.5 µM, the level of HO-1 protein induced by NF was reduced to control level. Both PNS and DEM at 2.5 µM also have modest protection against NF-induced HO-1 protein expression (Figure 7). Preliminary results showed that exposure of HFHs to NF resulted in a timedependent decline in CYP3A5 and CYP3A7 mRNA and protein levels (data not shown), which are known to be regulated indirectly by GR. These data suggest that NF may inhibit the functions of GR, and GR agonists can therefore protect HFHs from the effect of NF. RU28362, PNS, DEM, NAC, and SB203580 Suppress NF-Induced Generation of RS but Not with Nrf2 siRNA. To confirm that the induction of HO-1 was indeed due to the accumulation of RS generated by NF, we examined the effects of RU28362, PNS, DEM, NAC, and SB203580, agents that attenuated HO-1 protein expression, on RS generation by H2DCFDA using the flow cytometer. Preincubation of HFHs with 1 µM RU28362 and 5 µM each of PNS, DEM, NAC, and SB203580 for 30 min prior to stimulation with 20 µg/mL of NF for 12 h reduced the elevated RS detectable with DCF, suggesting that RS indeed were responsible for HO-1 induction [Figure 8A, p < 0.01 vs NF (20 µg/mL)]. The other inhibitors of the MAPK pathways, such as PD98059, U0126, and SP600125, did not reduce NF-induced increase in RS (data not shown). Although Nrf2 siRNA strongly attenuated HO-1 protein expression, it did not suppress the rise in RS, suggesting that RS generation was an upstream event to the activation of Nrf2 (Figure 8B). RS Are Not Generated from the Mitochondria and Other Oxidase Systems. Mitochondria are often a target for toxic agents that generate RS, such as clofibrate (23). However, preincubation of HFHs with 0.2 and 0.5 µM rotenone (a specific mitochondrial complex I inhibitor) or 0.25 µM CCCP (carbonyl cyanide m-chlorophenyl hydrazone, a mitochondrial uncoupling agent) has no effect on the rise of RS and HO-1 protein


Figure 8. Effects of RU28362, PNS, DEM, NAC, and SB203580 and Nrf2 siRNA on NF-induced intracellular RS as determined by H2DCFDA using the flow cytometer. (A) HFHs were stimulated with 20 µg/mL of NF for 12 h after preincubation with 1 µM RU28362 and 5 µM each of PNS, DEM, NAC, or SB203580. (B) Nrf2 silencing with siRNA has no effect on the rise in intracellular RS induced by NF. Nrf2-silenced cells were treated with 20 µg/mL of NF for 6 h before H2DCFDA loading. For both A and B, cells were loaded with 2.5 µM H2DCFDA for 30 min and immediately analyzed with the flow cytometer after washing off excess fluorescent dye. At least 104 cells were collected for analysis, and data were calculated using WinMDI 2.8 software. Data are means ( SDs from at least two independent experiments performed in triplicate. **p < 0.01 vs control, #p < 0.05 vs NF (20 µg/mL), and ##p < 0.01 vs NF (20 µg/mL).

expression, suggesting that NF did not target the mitochondria (data not shown). Furthermore, the source of RS is not likely to arise from the cyclooxygenase, NADPH oxidases, and the plasma membrane redox system because inhibitors of these systems [indomethacin (2.5 and 5 µM), diphenylene iodonium (0.5 and 1 µM), and potassium ferricyanide (extracellular electron acceptor, 0.5 mM), respectively] have no effect on RS and HO-1 protein induced by NF (data not shown). Furthermore, inhibitors of the caspases [Z-VAD-FMK (general), Z-DEVDFMK (caspase 3), Z-IETD-FMK (caspase 8), and Z-LEHDFMK (caspase 9)] and calpains (calpain I and II and calpeptin) at 6.25 and 12.5 µM failed to block HO-1 induction by NF in HFHs (data not shown). This suggests that NF-induced HO-1 protein expression was independent of the activation of caspases and calpains in HFHs. Isolation and Characterization of PF, BGT, and IMP. Successive liquid-liquid extraction yielded n-hexane and chloroform fractions with strong inductive effects on HO-1 protein expression in HFHs. These fractions were further fractionated by preparative and analytical HPLC to obtain active compounds with high purity. The active compounds were characterized based on MS and NMR spectra. The EI-MS data showed that the molecular ions at m/z 298, 202, and 270 were in agreement with the molecular formulae of PF, BGT, and IMP, respectively (data not shown). The chemical structures of PF, BGT, and IMP are shown in Figure 9A, and their yields were estimated to be about 0.92, 0.10, and 1.12% of the crude extract (w/w), respectively. The EI-MS spectrum of BGT agreed with that of the authentic compound. 1H-1H COSY, HMQC, and

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Figure 9. Characterization of PF, BGT, and IMP isolated from NF. (A) Chemical structures of PF, BGT, and IMP. (B) Effects of PF, BGT, and IMP on HO-1 induction in HFHs. HFHs were treated with various concentrations of PF, BGT, and IMP for 24 h. Total cellular proteins were prepared for Western blot analyses using specific antibody for HO-1. β-Actin was used to control protein loading. Cell lysates treated with 40 µg/mL of NF for 24 h were included as positive controls. (C) RS determination by H2DCFDA fluorescent dye using the flow cytometer. HFHs were stimulated with 2.5 and 5 µg/mL of PF, BGT, and IMP for 12 h prior to the incubation with 2.5 µM H2DCFDA for 30 min. At least 104 cells were collected for flow cytometry analysis. Data are means ( SDs from at least two independent experiments performed in triplicate. **p < 0.01 vs control.

HMBC were also employed to help assign the chemical shifts of PF and IMP. Our data are consistent with the published data (5, 24). These compounds increased HO-1 protein levels in HFHs at various concentrations (Figure 9B). The data from the densitometric analysis of Western blots showed that PF, BGT, and IMP contributed approximately 8.5, 0.8, and 5.1%, respectively, to NF-induced up-regulation of HO-1 protein levels. This correlated with the increase in DCF fluorescence positive cells as determined using the flow cytometer, suggesting that these compounds induce some form of RS (Figure 9C). However, only for IMP was the increase in RS and HO-1 protein levels dose-dependent (Figure 9C). We used NF but not its isolated compounds to study the mechanism of HO-1 induction in HFHs because these three compounds only account for a small part of the effect of NF.

Discussion Animal studies with NF have shown some beneficial pharmacological effects. For examples, Yang and colleagues reported that subchronic pretreatment with oral doses of NF extract significantly suppressed CCl4-induced lipid peroxidation products in mice (3). A close species of NF, Notopterygium incisum Ting ex H.T (NI), was demonstrated to possess analgesic activity

in mice in the acetic acid-induced writhing test and antiinflammatory activity in the vascular permeability test (4). However, the mechanisms of actions in the above-mentioned studies remained to be determined. In the present study, we have validated that the plant that we used was NF but not NI. Recently, Jiang and colleagues have shown that peak 3 was the highest peak in NI, while peak 4 (ferulic acid) was the highest in NF chromatogram (7). On the basis of this fingerprint, we confirmed that the medicinal plant used in this study belongs to NF (data not shown). The yield of NF was estimated to be about 320 mg per gram of dried raw material according to our laboratory extraction protocol. Moreover, on the basis of induction of HO-1 protein in HFHs, we have isolated and identified PF, BGT, and IMP that are partly responsible for the effect of NF. These active compounds were characterized based on MS and NMR spectra. The NF extract contains approximately 0.92, 0.10, and 1.12% (w/w) of PF, BGT, and IMP, respectively. Moreover, they account for about 8.5, 0.8, and 5.1%, respectively, of the NF inducing ability on HO-1 protein in HFHs. Ferulic acid was also identified in the NF extract, but it did not induce HO-1 protein in HFHs (data not shown). However, ferulic acid ethyl ester (FAEE), an ester derivative of ferulic acid, was reported to protect rat astrocytes


Figure 10. Schematic diagram of the interplay of GR, RS, p38 MAPK, Nrf2, and HO-1 in HFHs after NF treatment. The inhibition of GR or its functions with NF are followed by a rapid rise in intracellular RS. The accumulation of RS and intense oxidative stress in turn activate p38 MAPK and lead to the accumulation of Nrf2 transcription factor in the nucleus that up-regulates HO-1 protein expression. The effects of RU28362, PNS, DEM, NAC, and SB203580 on NF-mediated rise in RS and HO-1 protein levels suggest a signaling cascade involving the GR, RS, p38, Nrf2, and HO-1. Solid and dotted lines indicate activation and inhibition processes, respectively.

from glucose oxidase-mediated oxidative damage through HO-1 induction (25). FAEE was also found to reduce AAPH-, Fe2+/ H2O2- and amyloid-β peptide (1-42) induced oxidative stress and cytotoxicity in synaptosomal systems by radical scavenging effects and inducing Hsps (26, 27). In this study, PF isolated from NF was another ester derivative of ferulic acid that was shown to induce HO-1 protein in HFHs. Both BGT and IMP are furanocoumarin derivatives that have been reported to inhibit the CYP enzymes in vitro (2, 28). The effects of BGT and IMP on HO-1 protein induction in HFHs have not before been reported. Efficient detoxification of harmful xenobiotics is essential to the survival of living organisms. It is well-established that human fetal tissues metabolize many foreign compounds and endogenous substrates (21). The HFHs used in this study have been demonstrated to show complete sulfate conjugating activity when supplied with the necessary precursors (29) and UDPglucuronyl transferase activity when stimulated with mercuric chloride (12). Therefore, this cell line could be a suitable model for evaluating the potential cytotoxicity of xenobiotics, which would otherwise be difficult to examine due to the scarcity of human fetal live tissue samples. We also tested the effect of NF and the isolated compounds (PF, BGT, and IMP) on HepG2 cells as normal human adult hepatocytes are not easily accessible. We found that NF and the three compounds induced HO-1 protein levels after 12 h of incubation, and the protein levels were reduced in the presence of GR agonists such as RU28362, DEM, and PNS (unpublished results). Therefore, we believe that the observations that we obtained from the studies of NF and the isolated compounds on HFHs may be relevant to normal human adult hepatocytes. The metabolism of xenobiotics by CYP3As is regulated by the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) that are orphan nuclear receptors that crosstalk with glucocorticoid receptor (GR); thus, a signal transmission cascade GR-(PXR/CAR)-xenobiotic metabolizing and transporter systems was proposed (22). Studies with glucocorticoid receptor agonists such as RU28362, PNS, and DEM at


various concentrations inhibited the rise in intracellular RS and HO-1 protein up-regulation in the presence of NF, suggesting that NF may inhibit the GR or its functions. However, the exact roles of GR in NF-induced rise in RS and HO-1 protein upregulation require further study. We are currently investigating the expression of genes known to be controlled in part by GR such as tyrosine aminotransferase (TAT), PXR, CAR, CYP2C9, and UGT1A1 and/or indirectly by PXR and CAR, including CYP3A4, CYP3A5, and CYP3A7 in HFHs after NF treatment. Many chemical and environmental stimuli are known to induce HO-1 by means of generating oxidative and nitrosative stress (30-32). HO-1 is also referred to as Hsp32, which is widely distributed in mammalian tissues and regulated by the Nrf2/Keap1 transcription factor system (11). Phenolic compounds such as curcumin, resveratrol, and epigallocatechin-3gallate are some of the examples that have been shown to induce HO-1 involving Nrf2 in various cell types (33-35). In the cytoplasm, Nrf2 is sequestered by Keap1 from nuclear localization. Upon oxidation or modification of Keap1 by electrophiles, Nrf2 translocates into the nucleus where it binds to the antioxidant response element (ARE), which is present in the promoter regions of phase II genes such as HO-1. Different cytosolic kinases, including protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), ER-localized pancreatic endoplasmic reticulum kinase (PERK), and MAPK, have been shown to modify Nrf2 (36-38). Our data on the accumulation of Nrf2 in the nucleus in a dose- and time-dependent manner and the effect of Nrf2 siRNA on HO-1 support the notion that HO-1 is closely associated with Nrf2 transcription factor. The MAPKs are serine/threonine protein kinases that have been shown to be activated by inducers of oxidative stress (32, 39). Our results suggest that p38 mediates the MAPK induction of HO-1 and that ERK and JNK are unlikely to be involved. This conclusion is supported not only by studies with kinase inhibitors but could also be predicted from the MAPK activation profiles in Western blot analyses. The activation of the p38 MAPK pathway is likely attributable to the rapid rise in intracellular RS, as determined with H2DCFDA fluorescent dye, which caused intense oxidative stress. The isolated compounds of PF, BGT, and IMP from NF were also shown to increase intracellular RS. Although multiple MAPK pathways may be needed for an optimal response, the p38 cascade is most frequently activated for HO-1 induction, whereas the JNK is least stimulated (18). Because MAPKs are components of signaling cascades that can directly phosphorylate and activate transcription factors, it is possible for activated p38 to phosphorylate Nrf2 leading to nuclear translocation that modulates gene expression (18). In this study, we did not examine whether the activation of p38 acts up stream of Nrf2 translocation. However, Alam and colleagues (40) have demonstrated that SB203580 (p38 inhibitor) inhibited Nrf2 activity in cadmiuminduced HO-1 induction in MCF-7 cells, suggesting that nuclear Nrf2 translocation could be a down stream event of the activation of MAPKs. Hence, direct phosphorylation of Nrf2 by MAPKs has yet to be demonstrated. Up-regulation of HO-1 has been demonstrated to show protective effects against oxidative stress-induced damage both in vitro and in vivo. For example, curcumin reduced glucose oxidase-mediated loss of cell viability in bovine aortic endothelial cells (41), and quercetin exerted a dose-dependent protective effect against ethanol-induced elevated LDH and AST in human hepatocytes (42). Triphlorethol-A isolated from the brown alga Ecklonia caVa was reported to reduce H2O2- and γ-irradiation-induced loss of cell viability in Chinese hamster

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lung fibroblast cells (43). Attempts to determine the protective effect of NF from ethanol-, H2O2-, cumene hydroperoxide-, and 3-morpholinosynonimine hydrochloride-induced loss of cell viability in HFHs were not successful (unpublished data). However, NF inhibited lipopolysaccharide-stimulated iNOS and COX-2 expression and reactive nitrogen species production in RAW 264.7 murine macrophages (unpublished data). These data suggest that the protective effect of various HO-1 inducers may be selective against the types of RS inducers. Taking the data all together, we propose a model for NF action (Figure 10). We believe that NF inhibits the GR or its signaling cascades in HFHs, followed by a rapid rise in intracellular RS detectable by DCF fluorescence. The accumulation of RS and intense oxidative stress in turn activates p38 MAPK and leads to the accumulation of nuclear Nrf2 transcription factor that up-regulates HO-1 protein expression. Studies with GR agonists such as RU28362, PNS, and DEM, NAC, SB203580, and siRNA technology provide evidence for a possible involvement of GR in NF-induced RS and HO-1 protein. The possible role of GR in NF-induced rise in intracellular RS and HO-1 protein is currently being investigated in our laboratory. Acknowledgment. We thank Prof. Sit Kim Ping for her generous gift of the HFHs. We are grateful to the Biomedical Research Council of Singapore for their research support (Grant R-183-000-101-305). Supporting Information Available: Extraction of NF, validation of NF, purification and identification of active compounds from NF, preparation of cytosolic and nuclear extracts for Nrf2 immunoblot analysis, and NMR data of PF, BGT, and IMP. This material is available free of charge via the Internet at http://pubs.acs.org.

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TX800301F

Notopterygium forbesii Boiss Extract and Its Active Constituents

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