Induction of heme oxygenase-1 by Macleaya cordata extract and its constituent sanguinarine in RAW264.7 cells

Fitoterapia 83 (2012) 329–335

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Induction of heme oxygenase-1 by Macleaya cordata extract and its constituent sanguinarine in RAW264.7 cells Jiri Vrba a,⁎, Eva Orolinova a, b, Jitka Ulrichova a, b a b

Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 77515 Olomouc, Czech Republic Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 77515 Olomouc, Czech Republic

a r t i c l e

i n f o

Article history: Received 27 October 2011 Accepted in revised form 16 November 2011 Available online 4 December 2011 Keywords: Sanguinarine Antioxidant Heme oxygenase-1 Thioredoxin 1 Nrf2 Human hepatocytes

a b s t r a c t Macleaya cordata (plume poppy) is used in traditional Chinese medicine for its antiinflammatory and antibacterial activities. In this study, we examined whether M. cordata extract and/or its major alkaloid constituents, protopine, allocryptopine, sanguinarine and chelerythrine activate the Nrf2 signalling pathway which regulates the expression of cytoprotective enzymes including heme oxygenase-1 (HO-1) and thioredoxin 1. In murine macrophage RAW264.7 cells, M. cordata extract increased both mRNA and protein levels of HO-1. Of the alkaloids examined, only sanguinarine appeared to be responsible for these effects. At the concentration of 2 μM, sanguinarine induced nuclear accumulation of Nrf2, increased the expression of Hmox1 gene encoding HO-1 and elevated HO-1 protein levels. Sanguinarine-induced Hmox1 mRNA expression was suppressed by SB203580, a pharmacologic inhibitor of p38 mitogen-activated protein kinases (p38 MAPKs). The upregulation of HO-1 in RAW264.7 cells by sanguinarine was, however, accompanied by decrease in cell viability. Nonetheless, sanguinarine at micromolar, non-cytotoxic concentrations elevated protein levels of HO-1 and thioredoxin 1 in primary cultures of human hepatocytes. We conclude that sanguinarine may, under appropriate conditions, increase the capacity of the enzymatic antioxidant defence system via activation of the p38 MAPK/Nrf2 pathway. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Macleaya cordata (Willd.) R. Br., a plant of the Papaveraceae family, is used in traditional Chinese medicine for its anti-inflammatory and antibacterial activities. The aerial part is also used as an active component in the natural antibacterial feed additive, Sangrovit. The biological effects of M. cordata are assumed due to the bioactivity of its alkaloid constituents. It contains a number of isoquinoline alkaloids among which protopine, allocryptopine, sanguinarine and chelerythrine (Fig. 1) are the most abundant [1]. The major alkaloids from M. cordata have been the focus of a large number of biological studies [2,3]. For instance, both protopine and allocryptopine exhibited no cytotoxicity in primary cultures of human hepatocytes [4]. Protopine was also reported ⁎ Corresponding author. Tel.: + 420 585632310; fax: + 420 585632302. E-mail address: [email protected] (J. Vrba). 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.11.022

to be cytoprotective against oxidative stress-induced cell death [5] and to diminish hepatotoxicity of D-galactosamine in rats [6]. In contrast, toxicological studies of sanguinarine and chelerythrine provide controversial results. Both alkaloids were found to be cytotoxic in a variety of cell models [7–9]. Sanguinarine toxicity has been demonstrated in fish [10] and following ip administration in mice [11,12]. This aside, it was also reported that sanguinarine could have teratogenic effects in mice [13]. No toxic response was, however, found following oral administration of sanguinarine and chelerythrine in rats [14] and pigs [15]. The chemoprotective activity of natural substances is linked, ceteris paribus, with the ability to upregulate enzymes involved in antioxidant defence and phase II xenobiotic metabolism such as heme oxygenase-1, thioredoxin 1, glutathione peroxidase and glutathione S-transferase [16]. Both the basal and inducible expression of genes coding for these cytoprotective enzymes is controlled by nuclear factor

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O

O

O

O +

+

N

N

CH3

H3CO

CH3

O

OCH3

O Sanguinarine

Chelerythrine

O

O

O

O OH

OH

+

+

N O O

CH3 Protopine

N H3CO

CH3

OCH3

Allocryptopine

Fig. 1. Cationic forms of the tested isoquinoline alkaloids.

erythroid 2-related factor 2 (Nrf2). Nrf2 is a constitutively active transcription factor, albeit its activity is negatively regulated by the repressor Kelch-like ECH-associated protein 1 (Keap1). Keap1 binds to Nrf2 and hence it produces sequestration of Nrf2 in the cytoplasm and proteasomal Nrf2 degradation. Conversely, dissociation of the Nrf2–Keap1 complex leads to stabilization and nuclear accumulation of Nrf2 followed by activation of Nrf2-dependent transcription [17]. The interaction between Nrf2 and Keap1 may be disrupted by two mechanisms. Under oxidative or electrophilic stress, Nrf2 stabilization is mediated by oxidation or covalent modification of critical cystein residues of Keap1 which in turn undergoes proteasomal degradation. Alternatively, Nrf2 may be released from the Nrf2–Keap1 complex via phosphorylation of Nrf2 by upstream kinases such as extracellular signal-regulated kinases (ERKs), c-Jun NH2-terminal kinases (JNKs), p38 mitogen-activated protein kinases (p38 MAPKs) and protein kinases C. To date, a number of phytochemicals such as flavonoids and organosulfur compounds have been recognized to induce the expression of Nrf2 target genes [16]. The aim of the present study was to examine whether the Nrf2 signalling pathway could be activated by M. cordata extract and/or its major alkaloid constituents.

and procedures were performed at the Department of Medical Chemistry and Biochemistry (Palacky University, Olomouc, Czech Republic), and specimens of the preparations are deposited in the alkaloid collection of the Department. Allocryptopine (S450987), quercetin (Q4951), PD98059 (P215), SB203580 (S8307), SP600125 (S5567) and dimethyl sulfoxide were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2.2. RAW264.7 cell culture and treatment The murine macrophage cell line RAW264.7 (No. 91062702, ECACC, Salisbury, UK) was cultured at 37 °C in Dulbecco's modified Eagle's medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) in a humidified atmosphere containing 5% CO2. The cells were grown in 75 cm 2 flasks and sub-cultured before confluence. For all experiments, RAW264.7 cells (passages 5 to 25) were seeded in the complete culture medium at a density of 1 × 10 5 cells/cm 2. After 8 h stabilization, the culture medium was exchanged for the serum-free medium. Following overnight incubation, cells were treated in the serum-free medium with the tested compounds. Negative controls were treated with 0.1% (v/v) DMSO only.

2. Materials and methods 2.3. Primary cultures of human hepatocytes 2.1. Chemicals Macleaya cordata extract, containing 667 g of protopine, 146 g of allocryptopine, 14 g of sanguinarine and 8 g of chelerythrine per kg, was supplied by the Resources and the Development of TCM (College of Horticulture and Landscape Architecture, Hunan Agricultural University, Changsha, China). Sanguinarine chloride (99% purity) and chelerythrine chloride (95% purity) were isolated from sanguiritrine, an extract from Macleaya species, purchased from Camas Technologies (Broomfield, CO, USA). Protopine (98.5% purity) was isolated from Fumaria schrammii. All the separations

Human tissue samples were obtained from multi-organ donors according to the protocols approved by the local ethics committee of the University Hospital, Olomouc, Czech Republic. Hepatocytes were isolated as described previously [18] and seeded on collagen-coated culture dishes at a density of 1.3 × 10 5 cells/cm 2 in culture medium [19] containing 5% bovine serum (Invitrogen). On the following day, the medium was exchanged for serum-free medium. After 24 h of stabilization, hepatocytes were treated in fresh serum-free medium with 0.1% (v/v) DMSO (control), sanguinarine or 100 μM quercetin (positive control). Hepatocyte

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cultures used in this study were prepared from liver samples of three donors: a 58-year-old woman (culture LH30), a 28year-old man (culture LH31) and a 70-year-old man (LH32). All donors had normal liver histology.

Student t-tests. A p value of less than 0.05 was considered as statistically significant.

2.4. Cell viability assay

3.1. Cytotoxicity of Macleaya cordata extract and its constituents in RAW264.7 cells

2.5. Reverse transcription and quantitative real-time PCR After treatment, total RNA was extracted using the TRI Reagent Solution (Applied Biosystems, Foster City, CA, USA) and the concentration of RNA was determined by spectrophotometry at 260 nm. RNA samples (2 μg) were reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's recommendations. Real-time PCR was performed on a LightCycler 480 II system (Roche Diagnostics, Mannheim, Germany) using the TaqMan Universal PCR Master Mix and the TaqMan Gene Expression Assays consisting of specific primers and FAM dye-labelled TaqMan minor groove binder probes (Applied Biosystems). The assay ID for Hmox1 was Mm00516005_m1 and for Gapdh Mm99999915_g1. Amplification conditions were 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Crossing point values, equivalent to CT, were determined automatically using the second derivative maximum analysis. Relative changes in gene expression were calculated by the comparative CT method using the 2 -ΔΔCT equation with results normalized to Gapdh mRNA levels. 2.6. Western blot analyses After treatment, total cellular extracts [4] or nuclear fractions [20] were prepared as described previously. Proteins in all samples were quantified by the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Aliquots containing equal amount of protein were subjected to electrophoresis through 10% or 12.5% SDS-polyacrylamide gel and proteins were transferred to polyvinylidene difluoride membrane by electroblotting. Nrf2, heme oxygenase-1, actin and thioredoxin 1 were detected with rabbit polyclonal Nrf2 (C-20), rabbit polyclonal heme oxygenase-1 (H-105), goat polyclonal actin (I-19) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit monoclonal thioredoxin 1 (C63C6) antibodies (Cell Signaling Technology, Danvers, MA, USA), respectively. Primary antibodies were detected with anti-goat immunoglobulin G-horseradish peroxidase (IgG-HRP) (Santa Cruz) or anti-rabbit IgG-HRP (Cell Signaling Technology) conjugated secondary antibodies using the chemiluminescent reaction. 2.7. Statistical analysis Results were expressed as means ± standard deviation (SD). The differences in mean values were analysed by

We first examined the effect of M. cordata extract and its alkaloid constituents, protopine, allocryptopine, sanguinarine and chelerythrine on the viability of RAW264.7 cells. The maximum final concentration of M. cordata extract was chosen with respect to its solubility in the culture medium. As shown by the MTT reduction assay, 3 or 6 h cell exposure to M. cordata extract at concentration equivalent to 50 μM protopine, 10.6 μM allocryptopine, 1.02 μM sanguinarine and 0.56 μM chelerythrine decreased the cell viability to 69% and 57%, respectively (Fig. 2). After the cell treatment with individual alkaloids, only sanguinarine displayed significant cytotoxic effects with viability reduced to 68% and 47% at 2 μM concentration and 3 or 6 h exposure, respectively. A small, statistically non-significant decrease in cell viability was also found after 6 h treatment with 2 μM chelerythrine (Fig. 2). Protopine and allocryptopine, in contrast, showed no cytotoxicity in RAW264.7 cells after treatment with up to 50 μM alkaloids (data not shown). 3.2. M. cordata extract and sanguinarine increase mRNA and protein levels of heme oxygenase-1 in RAW264.7 cells To examine whether M. cordata extract and/or its constituents affect the expression of genes regulated by the transcription factor Nrf2, we analysed the expression of mouse Hmox1 gene coding for heme oxygenase-1 (HO-1). As determined by quantitative real-time PCR, 6 h exposure of RAW264.7 cells to 50 μM quercetin, used as a positive control [21], resulted in 4.6-fold induction of Hmox1 mRNA expression when normalized to Gapdh mRNA levels (Fig. 3). After the cell treatment with M. cordata extract equivalent to 50 μM protopine, the level of Hmox1 mRNA significantly increased to 6.8-fold of the control value. Cell exposure to the major extract constituents showed that sanguinarine induced 6h

3h

Viability (% of control)

After treatment of RAW264.7 cells or human hepatocytes with 0.1% DMSO (control), tested compounds or 1.5% (v/v) Triton X-100 (positive control), the cell viability was assessed by MTT reduction assay as described previously [4].

3. Results

140 120 100 80

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Fig. 2. Cytotoxicity of Macleaya cordata extract, sanguinarine and chelerythrine in RAW264.7 cells. Cells were treated for 3 or 6 h with 0.1% DMSO (control), M. cordata extract equivalent to 10 or 50 μM protopine or with indicated concentrations of sanguinarine (SAN) or chelerythrine (CHE). The cell viability was determined by the MTT reduction assay. Data are means ± SD of three experiments. * p b 0.05; *** p b 0.001, significantly decreased versus control.

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Hmox1 mRNA (fold induction)

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10

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Fig. 3. Relative changes in Hmox1 gene expression by Macleaya cordata extract and its alkaloid constituents in RAW264.7 cells. Cells were treated for 6 h with 0.1% DMSO (control), 50 μM quercetin (QUE; positive control), M. cordata extract equivalent to 10 or 50 μM protopine or with indicated concentrations of sanguinarine (SAN), chelerythrine (CHE), protopine (PRO) or allocryptopine (AL). The levels of Hmox1 mRNA were determined by quantitative real-time PCR with results normalized to Gapdh mRNA. Data are means ± SD of three experiments. * p b 0.05, significantly increased versus control.

a dose-dependent increase in Hmox1 mRNA levels. At the concentrations of 1 and 2 μM, sanguinarine significantly increased the levels of Hmox1 mRNA to 1.6-fold and 5.8-fold of the control value, respectively. In contrast, chelerythrine, protopine or allocryptopine at concentrations similar to those during M. cordata extract treatment induced negligible changes in Hmox1 mRNA levels (Fig. 3). The western blot analysis showed that the induction of Hmox1 mRNA expression by M. cordata extract or sanguinarine was accompanied by an increase in HO-1 protein levels (Fig. 4). Upregulation of HO-1 was found after 6 h treatment of RAW264.7 cells with M. cordata extract equivalent to 50 μM protopine or with 2 μM sanguinarine. In contrast, the levels of HO-1 remained unaffected following cell exposure to chelerythrine, protopine or allocryptopine (Fig. 4). 3.3. Sanguinarine induces nuclear accumulation of Nrf2 in RAW264.7 cells The inducible expression of Nrf2 target genes is associated with translocation of Nrf2 from the cytosol to the nucleus [17]. For this reason, we examined whether sanguinarine af-

HO-1 Actin DMSO

1 2 SAN

2 1 CHE Extract

fected nuclear levels of Nrf2 in RAW264.7 cells. Western blot analysis of nuclear fractions showed that 50 μM quercetin, a positive control [22], increased nuclear levels of Nrf2 after 3 h treatment (Fig. 5). At 3 h exposure, the nuclear accumulation of Nrf2 was also induced by 2 μM sanguinarine. In contrast, the structurally related alkaloid chelerythrine was found not to increase the nuclear levels of Nrf2 under the same conditions (Fig. 5).

3.4. Induction of Hmox1 gene expression by sanguinarine involves p38 MAPK activity We further investigated possible involvement of MAPK cascades in sanguinarine-induced Hmox1 gene expression. For this purpose, we used pharmacologic inhibitors of MAPKs including PD98059 for blocking ERKs, SB203580 for blocking p38 MAPKs and SP600125 to inhibit JNKs [21]. Our results showed that sanguinarine-induced Hmox1 mRNA expression in RAW264.7 cells was significantly reduced by p38 MAPK inhibitor SB203580. A 30 min cell pre-treatment with 15 μM SB203580 decreased the induction of Hmox1 mRNA levels by 2 μM sanguinarine at 6 h exposure by 75% (Fig. 6). In contrast, the inhibition of ERKs or JNKs by pharmacologically efficient doses of respective inhibitors [23] had no substantial effects on sanguinarine-induced Hmox1 mRNA levels (Fig. 6).

Nrf2

HO-1

Actin

Actin 10 DMSO

50 AL

10

2

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PRO

Fig. 4. Effect of Macleaya cordata extract and its constituents on heme oxygenase-1 (HO-1) protein levels in RAW264.7 cells. Cells were treated for 6 h with 0.1% DMSO (control), sanguinarine (SAN), chelerythrine (CHE), protopine (PRO), allocryptopine (AL) or M. cordata extract equivalent to 50 μM protopine. After treatment, proteins in the whole cell lysates (30 μg/lane) were analysed by western blotting and HO-1 and actin were visualized by chemiluminescent detection. The figures are representative of three experiments.

DMSO

SAN

1

2 CHE

1 QUE

Fig. 5. Effect of sanguinarine and chelerythrine on nuclear levels of Nrf2 in RAW264.7 cells. Cells were treated for 3 h with 0.1% DMSO (control), sanguinarine (SAN), chelerythrine (CHE) or 50 μM quercetin (QUE; positive control). After treatment, cells were fractionated and proteins in nuclear fractions (30 μg/lane) were analysed by western blotting. Nrf2 and actin were visualized by chemiluminescent detection. The figures are representative of three experiments.

Hmox1 mRNA (% of SAN-induced expression)

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Culture LH30, 6h 150

Trx1 100

HO-1 50

**

Actin 1

0

DMSO

PD

SB

SP

QUE

SAN 2 µM Fig. 6. Effect of MAPK inhibitors on sanguinarine-induced Hmox1 gene expression in RAW264.7 cells. Cells were pre-treated for 30 min with 0.1% DMSO (control), 15 μM PD98059 (PD), 15 μM SB203580 (SB) or 30 μM SP600125 (SP) and then incubated in the absence or presence of 2 μM sanguinarine (SAN) for additional 6 h. The levels of Hmox1 mRNA were determined by quantitative real-time PCR and normalized to Gapdh mRNA. Results are expressed as the percentage of sanguinarine-induced Hmox1 mRNA expression. Data are means ± SD of three experiments. ** p b 0.01, significantly different from cells treated with sanguinarine in the absence of MAPK inhibitors.

5 SAN

DMSO

Culture LH31, 12h Trx1 HO-1 Actin

3.5. Sanguinarine upregulates HO-1 and thioredoxin 1 levels in primary cultures of human hepatocytes We also tested whether sanguinarine could stimulate the Nrf2 pathway in normal human hepatocytes which metabolize sanguinarine to the less bioactive dihydrosanguinarine [24,25]. Hepatocyte cultures LH30, LH31 and LH32 were treated for 6 or 12 h with up to 5 μM concentrations of sanguinarine, which were previously found to be non-cytotoxic to human hepatocytes [26], and the levels of HO-1 and thioredoxin 1 were analysed by western blotting. Using the MTT assay, we confirmed that 1 to 5 μM sanguinarine caused no decrease in the viability of human hepatocytes after up to 24 h of incubation (data not shown). Yao et al. [23] found that quercetin (positive control) upregulated HO-1 in human hepatocytes at the concentration of 100 μM. In our study, 100 μM quercetin produced a slight increase in the level of HO-1 only in culture LH32. In contrast, the levels of thioredoxin 1 were elevated by 100 μM quercetin in all three hepatocyte cultures (Fig. 7). Sanguinarine was found to upregulate both HO-1 and thioredoxin 1. At the concentration of 5 μM, sanguinarine induced an apparent increase in HO-1 levels in all hepatocyte cultures. In culture LH30, the level of HO-1 was elevated by as low as 1 μM sanguinarine as well. This aside, 1 to 5 μM sanguinarine caused a dosedependent increase in the levels of thioredoxin 1 in hepatocyte cultures LH30 and LH31. In culture LH32, the increased level of thioredoxin 1 was only found in cells exposed to 1 μM sanguinarine (Fig. 7). 4. Discussion This study was designed to investigate whether Macleaya cordata extract and/or its major alkaloid constituents activate the Nrf2 signalling pathway which regulates the expression of enzymes involved in the antioxidant defence or the elimination of xenobiotics such as heme oxygenase-1 (HO-1), thioredoxin 1 and glutathione S-transferase [16]. Traditional Chinese medicine employs M. cordata and its extracts for

1 DMSO

QUE

5 SAN

Culture LH32, 6h Trx1 HO-1 Actin 1 DMSO QUE

2

5

SAN

Fig. 7. Effect of sanguinarine on the protein levels of heme oxygenase-1 (HO1) and thioredoxin 1 (Trx1) in primary cultures of human hepatocytes. Three different cultures of human hepatocytes were treated for 6 or 12 h (as indicated) with 0.1% DMSO (control), 100 μM quercetin (QUE; positive control) or sanguinarine (SAN). After treatment, proteins in the whole cell lysates (40 μg/lane) were analysed by western blotting and HO-1, Trx1 and actin were visualized by chemiluminescent detection.

the treatment of skin diseases and inflammation. This aside, M. cordata is also used in agriculture and veterinary medicine [27]. Sangrovit, the feed additive from M. cordata, attenuates dextran sulfate sodium-induced colitis in rats [28]. The main alkaloids found in M. cordata are protopine, allocryptopine, sanguinarine and chelerythrine [1]. Research on their antiinflammatory activity has been primarily focused on possible interactions with the nuclear factor-κB (NF-κB) pathway which plays an important role in regulating the expression of cyclooxygenase-2 and pro-inflammatory cytokines [29]. In cell models, sanguinarine was found to inhibit NF-κB activation [30] and chelerythrine suppressed inducible expression of cyclooxygenase-2 [31]. The anti-inflammatory action of sanguinarine and chelerythrine could also be associated with their ability to inhibit formation of superoxide radical by phagocyte NADPH oxidase [32]. In contrast, the relevant

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molecular targets of protopine and allocryptopine have not been identified to date. In this study, we examined the effect of M. cordata extract and its major constituents, protopine, allocryptopine, sanguinarine and chelerythrine on the expression of HO-1 in murine macrophage RAW264.7 cells. HO-1 is an inducible form of heme oxygenase, a cytoprotective enzyme that cleaves heme to produce antioxidant biliverdin, iron and carbon monoxide [21,33]. We found that the tested extract of M. cordata increased both Hmox1 mRNA and HO-1 protein levels. Of the alkaloids examined, only sanguinarine showed significant induction of HO-1 expression in RAW264.7 cells. M. cordata extract increased HO-1 protein levels at a concentration equivalent to 1 μM sanguinarine, while the upregulation of HO-1 by sanguinarine alone required higher doses of the alkaloid. These results suggest that the effect of sanguinarine on HO-1 expression might be potentiated by other extract constituents. We also found that HO-1 induction by sanguinarine involved p38 MAPK activity and nuclear accumulation of Nrf2. In contrast, the activities of ERKs and JNKs were found to play a mild or negligible role. SB203580, a pharmacologic inhibitor of p38 MAPKs, decreased sanguinarineinduced Hmox1 mRNA levels by 75%. Our results correspond with those of Aburai et al. [34] who found that sanguinarine at micromolar concentrations produced phosphorylation of p38 MAPKs in human leukemia HL-60 cells. This effect may be associated with sanguinarine's ability to inhibit protein phosphatase 2C [34] and MAPK phosphatase-1 [35]. Inhibition of these enzymes leads to phosphorylation and activation of p38 MAPKs [34,35] which in turn phosphorylate Nrf2 and hence induce Nrf2 nuclear accumulation [16]. It has been shown that MAPK phosphatase-1 is inhibited with similar efficiencies by both sanguinarine and chelerythrine [35]. On the other hand, efficient inhibition of protein phosphatase 2C requires approximately fourfold higher concentrations of chelerythrine than those of sanguinarine [34]. Considering the inability of chelerythrine to upregulate HO1 in RAW264.7 cells, we may presume that inhibition of protein phosphatase 2C could play a major role in sanguinarine-induced HO-1 expression. It should be mentioned that sanguinarine could also activate Nrf2 through oxidation or covalent modification of cystein residues within the repressor Keap1, since sanguinarine is known to induce generation of reactive oxygen species at cytotoxic concentrations [36] and to alkylate thiol groups in proteins [37]. Nonetheless, we found that sanguinarine at non-cytotoxic concentrations [26] upregulated HO-1 in primary cultures of human hepatocytes. This aside, as low as 1 μM sanguinarine also increased levels of thioredoxin 1 in human hepatocytes. Thioredoxin 1 is a cytosolic form of thioredoxin, an enzyme which reduces oxidized cystein groups on proteins, thus playing a vital role in the maintenance of a reduced intracellular redox state [38]. In summary, we have shown that M. cordata extract induces the expression of HO-1 in RAW264.7 cells and that the isoquinoline alkaloid sanguinarine is primarily responsible for this effect. The mechanism of sanguinarine-induced HO-1 expression involves activation of the p38 MAPK/Nrf2 pathway. Since sanguinarine at micromolar concentrations also upregulates HO-1 and thioredoxin 1 in human hepatocytes, we conclude that sanguinarine may, under appropriate

conditions, increase the capacity of the enzymatic antioxidant defence system. The ability of sanguinarine to activate the Nrf2 signalling pathway in vivo remains to be investigated.

Conflict of interest The authors declare that there are no conflicts of interest.

Acknowledgements We thank Dr. Alexander Oulton for providing language help. This work was supported by grants from the Ministry of Education, Youth and Sports of the Czech Republic (ME 10071, MSM 6198959216 and CZ.1.05/2.1.00/01.0030).

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