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Okanin, a chalcone found in the genus Bidens, and 3-penten-2-one inhibit inducible nitric oxide synthase expression via heme oxygenase-1 induction in RAW264.7 macrophages activated with lipopolysaccharide

Okanin, a chalcone found in the genus Bidens, and 3-penten-2-one inhibit inducible nitric oxide... Original Article JC J 0912 1880 the Society Ky j 10.3164/j Original Article c ournal of bn BN ot 11- o, - -0009 5086 Jap 30 cb Clinica afor n n.11-30 Free Ra l Biochem dical Res istryearch Ja and Nupan trition Okanin, a chalcone found in the genus Bidens, and 3penten2one inhibit inducible nitric oxide synthase expression via heme oxygenase1 induction in RAW264.7 macrophages activated with lipopolysaccharide 1 2 2 3 4 5 5 JinSang Kil, Young Son, YongKwan Cheong, NamHo Kim, Hee Jong Jeong, JiWung Kwon, EohJin Lee, 5 6 7, TaeOh Kwon, HunTaeg Chung and HyunOck Pae * 1 2 4 5 Department of Neurosurgery, Department of Anesthesiology and Pain Medicine, Department of Urology, College of Life Science and Natural Resources and Department of Microbiology and Immunology, Wonkwang University School of Medicine, 3442 Shinyongdong, Iksan 570749, Republic of Korea Department of Cardiovascular Medicine, Wonkwang University Hospital, Iksan 570711, Republic of Korea Department of Biological Science, University of Ulsan, Ulsan 680749, Republic of Korea (Received 21 February, 2011; Accepted 28 March, 2011; Published online 15 December, 2011) ?? Excess production of nitric oxide by activated macrophages via Creativ stricted vided the or C 200? This is opy e Com right © u sae, distribution, and reproduc nig op inal m 200? en acce ons wo Attribution L J rk C ss is B a N properly rticle dis cited. tiributed under the term cense tio,n whic in any h m peerm diuits unre- m s of , pro- the inflammatory cytokines and the prolonged expression of inducible inducible nitric oxide synthase leads to the development of pro-inflammatory enzymes, such as inducible nitric oxide synthase (11,12) various inflammatory diseases. Heme oxygenase1 expression via (iNOS). The inflammatory enzyme iNOS, once expressed in activation of nuclear factorerythroid 2related factor 2 inhibits activated macrophages, can generate a large amount of nitric oxide (11,12) nitric oxide production and inducible nitric oxide synthase expres (NO) for a long period. The free radical NO has been sion in activated macrophages. Okanin is one of the most abundant implicated as an important inflammatory mediator in the process (12) chalcones found in the genus Bidens (Asteraceae) that is used as of macrophage-mediated inflammation. However, uncontrolled/ various folk medications in Korea and China for treating inflam excess NO production by activated macrophages leads to the (12,13) mation. Here, we found that okanin (possessing the αβ unsatu development of various inflammatory diseases. Hence, rated carbonyl group) induced heme oxygenase1 expression via pharmacological inhibition of NO production and/or iNOS nuclear factorerythroid 2related factor 2 activation in RAW264.7 expression is a promising strategy for reducing the potentially (13) macrophages. 3Penten2one, of which structure, as in okanin, harmful pro-inflammatory activity of macrophages. possesses the αβ unsaturated carbonyl group, also induced Heme oxygenase-1 (HO-1) is an inducible enzyme that nuclear factorerythroid 2related factor 2dependent heme catalyzes the rate-limiting step in the conversion of free heme into oxygenase1 expression, while both 2pentanone (lacking a carbon monoxide, free iron, and biliverdin, which is subsequently (14) double bond) and 2pentene (lacking a carbonyl group) were catabolized into bilirubin by biliverdin reductase. In addition virtually inactive. In lipopolysaccharideactivated RAW264.7 macro to its primary role in heme degradation, HO-1 has been also phages, both okanin and 3penten2one inhibited nitric oxide recognized to play other important roles in resolution of inflam- production and inducible nitric oxide synthase expression via mation, which has been demonstrated in HO-1 knockout mice and (15,16) heme oxygenase1 expression. Collectively, our findings suggest a human case of genetic HO-1 deficiency. Particularly, HO-1 that by virtue of its αβ unsaturated carbonyl functional group, and its enzymatic metabolites are the critical regulators of okanin can inhibit nitric oxide production and inducible nitric inflammation, with activated macrophages acting as the critical (14–16) oxide synthase expression via nuclear factorerythroid 2related targets. Nuclear factor-erythroid 2-related factor 2 (Nrf 2) is a factor 2dependent heme oxygenase1 expression in lipopolysac redox sensitive transcription factor that is critical for induction of (17) charideactivated macrophages. the gene encoding HO-1. Under normal conditions, Nrf2 is sequestered in the cytoplasm by forming a complex with the Key Words: okanin, heme oxygenase1, inducible nitric oxide negative regulator of Nrf 2, Klech-like ECH-associated protein 1 (17) synthase, nuclear factorerythroid 2related factor 2, (Keap1). This complex is disrupted by several naturally occurring αβ unsaturated carbonyl group compounds, and Nrf 2 is liberated and translocated to the nucleus where it binds to antioxidant response element (ARE) sequences (18,19) Int Okanin (chem roduction ical structure shown in Fig. 1) is one of the most in the ho-1 gene promoter. Oabundant chalcone [1,3-diaryl-2-propen-1-one] compounds Recently, a series of naturally occurring compounds from found in the genus Bidens (Asteraceae) that has been used as medicinal plants have been reported to induce HO-1 expression in various folk medications in Korea and China for treating inflam- different cell types, and in some of these studies, HO-1 has been mation, malaria, hypertension, diabetes, peptic ulcer, snake bite shown to mediate their anti-inflammatory properties through (1–3) (20–24) and smallpox. Although ethnopharmacological studies carried inhibition of NO production and iNOS expression. In the out with plants of the genus Bidens have demonstrated anti- present study, we have also reported that okanin inhibited NO (4–10) inflammatory activity, whether okanin would also have production and iNOS expression through Nrf 2-dependent HO-1 anti-inflammatory activity is not yet investigated. Activated macrophages play a pivotal role in a variety of *To whom correspondence should be addressed. inflammatory diseases via the excess production of pro- Email: hopae@wonkwang.ac.kr doi: 10.3164/jcbn.1130 J. Clin. Biochem. Nutr. | January 2012 | vol. 50 | no. 1 | 53–58 ©2012 JCBN expression in RAW264.7 macrophages activated with the endo- or β-actin (1:1000 dilution) at 4°C overnight. The membrane was toxin lipopolysaccharide (LPS). then washed four times with PBS-Tween 20 buffer and further incubated with secondary antibodies for 1 h at room temperature. Materials and Methods Specific bands were detected using enhanced chemiluminescence detection system (Amersham Biosciences), and the membrane Chemicals and reagents. Previously, we had isolated okanin was exposed to X-ray film. (25) from the ethanol extract of the flowers of Bidens bipinnata L. Transfection of siRNA. RAW264.7 macrophages were and this compound was used in this study. 3-Penten-2-one, 2- grown in 6- or 12-well plates and transiently transfected with Nrf 2 pentanone, 2-pentene, Dulbecco’s modified Eagle’s medium siRNA or HO-1 siRNA mixed with siRNA transfection reagent (DMEM), hemin, LPS (Escherichia coli 055:B5), 3-(4,5-dimethyl- (Santa Cruz Biotechnology) according to the manufacturer’s 2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), N-acetyl-L- instructions. After incubation at 37°C and 5% CO2 for 30 h, cells cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, were treated with reagents. The samples were then prepared for MO). Antibodies against iNOS, HO-1, Nrf 2, lamin B and β-actin Western blot analysis. and small interfering RNA (siRNA) products against Nrf2 and Nrf2ARE binding assay. The amount of Nrf2 available in HO-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, the nucleus to bind to AREs was determined using the ELISA- TM CA). All other reagents used were of analytical grade. based TransAM Nrf 2 Kit (Active Motif, Carlsbad, CA). Briefly, Cell culture. RAW264.7 macrophages were obtained from nuclear extracts were added to wells containing the immobilized the American Type Culture Collection (Manassas, VA). The cells consensus ARE oligonucleotide. A primary antibody against Nrf 2 were cultured in DMEM supplemented with 2 mM glutamine, was added to each well. Then a secondary antibody conjugated to antibiotics (100 U/ml of penicillin A and 100 U/ml of strepto- horseradish peroxidase that binds to the primary antibody was mycin) and 10% heat-inactivated fetal bovine serum (Gibco/BRL, added to each well. The signal was detected at 450 nm, and Nrf 2- Rockville, MD) and maintained at 37°C in a humidified incubator ARE binding was reported as optical density (OD) units at containing 5% CO2. 450 nm. Cell viability assay. Cell viability was determined by a Statistical analysis. Data are expressed as means ± SE. One- modified MTT reduction assay. MTT is a pale yellow substance way analysis of variance procedures were used to assess signifi- that is reduced by living cells to yield a dark blue formazan cant differences among treatment groups. For each treatment product. This process requires active mitochondria, and even fresh showing a statistically significant effect, the Newman-Keuls test dead cells do not reduce significant amounts of MTT. RAW264.7 was used for comparisons of multiple group means. The criterion macrophages were cultured in a 96-well flat-bottom plate at for statistical significance was set at p<0.05 or 0.01. concentration of 5 × 10 cells/ml. After 12 h of preconditioning, the cells were treated with various concentrations of okanin for Results 24 h. Thereafter, culture medium was aspirated and 100 μl of MTT dye (1 mg/ml in phosphate-buffered saline) was added; the Okanin and 3penten2one, but not 2pentanone and 2 cultures were incubated for 4 h at 37°C. The formazan crystals pentene, can induce HO1 expression. The chemical struc- produced through dye reduction by viable cells were dissolved tures of okanin and other three synthetic compounds tested in this using acidified isopropanol (0.1 N HCl). Index of cell viability study are shown in Fig. 1. Both okanin and 3-penten-2-one contain was calculated by measuring the optical density of color produced an α-β unsaturated carbonyl group in the central five-carbon by MTT dye reduction at 570 nm. chain. In comparison with 3-penten-2-one, 2-pentanone and 2- Nitrite assay. The nitrite concentration in the medium was pentene lack a double bond and a carbonyl group, respectively. 3- measured as an indicator of NO production according to the Griess penten-2-one, 2-pentanone and 2-pentene were used to explore a reaction. One hundred microliters of each supernatant was mixed possible mechanism of action of okanin. In RAW264.7 macro- with the same volume of Griess reagent (1% sulfanilamide in 5% phages, okanin and 3-penten-2-one, but not 2-pentanone and 2- phosphoric acid and 0.1% naphthylethylenediamine dihydro- pentene, exhibited cytotoxicity at more than 40 μM (data not chloride in water); absorbance of the mixture at 550 nm was shown). determined with an ELISA plate reader. Treatment of RAW264.7 macrophages with non-cytotoxic Western blot analysis. RAW264.7 macrophages were incu- concentrations of okanin (0.5–10 μM) for 6 h resulted in a bated with or without reagents. They were harvested, washed ice- cold phosphate-buffered saline (PBS) and kept on ice for 1 min. The suspension was mixed with buffer A (10 mM HEPES, pH 7.5, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 5 μg/ml aprotinin, 5 μ/ml pepstatin, and 10 μg/ml leupeptin) and lysed by three freeze-thaw cycles. Cytosolic frac- tion was obtained by centrifugation at 12,000 × g for 20 min at 4°C. The pellets were re-suspended in buffer C (20 mM HEPES, pH 7.5, 0.4 M NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 5 μg/ml aprotinin, 5 μg/ml pepstatin, and 10 μg/ml leupeptin) on ice for 40 min and centrifuged at 14,000 × g for 20 min at 4°C. The resulting supernatant was used as soluble nuclear fraction. Protein content was determined with BCA pro- tein assay reagent (Pierce, Rockford, IL). Total cellular or nuclear fractions were separated on 10% SDS-polyacrylamide gels, and transferred to the nitrocellulose membranes (Amersham Bio- sciences, Inc., Piscataway, NJ). The membrane was then blocked in blocking buffer containing 20 mM sodium phosphate buffer, pH 7.6, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk Fig. 1. Chemical structures of okanin, 3penten2one, 2pentanone for 1 h at room temperature. Thereafter, the membrane was and 2pentene. In comparison with 3penten2one, 2pentanone and incubated with antibodies against HO-1 (1:1000 dilution), Nrf 2 2pentene lack a double bond and a carbonyl group, respectively. The (1:500 dilution), iNOS (1:1000 dilution), lamin B (1:1000 dilution) αβ unsaturated carbonyl group is marked with dotted circles. 54 doi: 10.3164/jcbn.1130 ©2012 JCBN Fig. 2. Effects of okanin, 3penten2one, 2pentanone and 2pentene on HO1 expression. (A) RAW264.7 macrophages were incubated for 6 h without or with 10 μM of hemin, a wellknown inducer of HO1, or indicated concentrations of okanin. (B) RAW264.7 macrophages were incubated without or with 10 μM of okanin for indicated time periods. (C) RAW264.7 macrophages were incubated for 6 h without or with 10 μM of okanin, 3penten2one, 2pentanone, or 2pentene. Western blot analysis for HO1 expression was performed as described under Materials and Methods. Blots shown are representative of three independent experiments. Fig. 3. Effects of okanin and 3penten2one on Nrf2 activation. (A) RAW264.7 macrophages were incubated without or with 10 μM of okanin for indicated time periods. Cytosolic and nuclear extracts were isolated, and the levels of Nrf2 protein were determined by Western blot analysis. (B) RAW264.7 macrophages were incubated for 2 h concentration-dependent increase in HO-1 protein levels, as without or with 10 μM of okanin, 3penten2one, 2pentanone, or 2 confirmed by Western blot analysis (Fig. 2A). Induction of HO-1 pentene. Nrf2 activation was assessed in nuclear extracts by analysis of expression by okanin was robust at 6 h and slightly decreased over Nrf2 binding to ARE. Data are expressed as mean ± SE from 3 to 4 time (Fig. 2B). Similarly, treatment with 3-penten-2-one for 6 h experiments. *p<0.05 with respect to untreated control group. (C) RAW264.7 macrophages were transiently transfected with Nrf2 siRNA, resulted in a marked increase in HO-1 protein levels (Fig. 2C). In and then exposed to 10 μM of okanin or 3penten2one for 6 h. contrast, neither 2-pentanone nor 2-pentene induced HO-1 expres- Western blot analysis for HO1 expression was performed as described sion (Fig. 2C). under Materials and Methods. Blots shown are representative of three Okanin and 3penten2one can induce Nrf2 activation independent experiments. that mediates HO1 expression. A time-dependent increase in Nrf 2 protein in the nucleus, along with a significant decrease in Nrf2 protein in the cytoplasm, was observed when RAW264.7 macrophages were incubated with 10 μM okanin (Fig. 3A). Similarly, 3-penten-2-one also increased levels of nucleus Nrf 2 inhibited. HO-1 expression by okanin and 3-penten-2-one was protein (not shown). To study Nrf 2 activation, the nuclear extracts prevented by siRNA against Nrf 2 (Fig. 3C). of RAW264.7 macrophages were isolated and the binding of Nrf 2 Nrf2 activation and HO-1 expression in RAW264.7 macro- TM to ARE was quantified by using a TransAM assay. Both okanin phages observed with okanin and 3-penten-2-one may be attribut- and 3-penten-2-one increased Nrf2 activation, whereas 2-pentanone able to changes in the production of reactive oxygen species and and 2-pentene had no significant effect on Nrf2 activation the redox environment, and/or to direct thiol modification of (26) (Fig. 3B). We next conducted the experiments of Nrf 2 knockdown Keap1 and other proteins. To better characterize Nrf 2 activation by using siRNA to investigate whether HO-1 expression would be and HO-1 expression, we treated normal RAW264.7 macrophages J.S. Kil et al. J. Clin. Biochem. Nutr. | January 2012 | vol. 50 | no. 1 | 55 ©2012 JCBN Fig. 4. Effects of NAC on Nrf2 activation and HO1 expression by okanin and 3penten2one. (A) RAW264.7 macrophages were treated with 10 μM of okanin or 3penten2one together with 1 mM NAC for 2 h. Nrf2 activation was assessed in nuclear extracts by analysis of Nrf2 binding to ARE. Data are expressed as mean ± SE from 3 to 4 experi ments. *p<0.05 with respect to untreated control group. p<0.05. (A) RAW264.7 macrophages were treated with 10 μM of okanin or 3 penten2one together with 1 mM NAC for 6 h. Western blot analysis for HO1 expression was performed as described under Materials and Methods. Blots shown are representative of three independent experi ments. with high concentrations of NAC, a thiol donor, along with okanin and 3-penten-2-one. The NAC treatment partially inhibited Nrf 2 activation (Fig. 4A) and HO-1 expression (Fig. 4B) by okanin and 3-penten-2-one. Okanin and 3penten2one inhibit LPSinduced NO Fig. 5. Effects of okanin and 3penten2one on NO production and production and iNOS expression via HO1 expression. iNOS expression. RAW264.7 macrophages were preincubated for 6 h RAW264.7 macrophages were pre-incubated for 6 h with okanin without or with indicated concentrations of okanin or 3penten2one, and activated with LPS. Okanin inhibited LPS-induced NO and then exposed to 1 μg/ml of LPS for 6 h (B, D) or 18 h (A, B). Nitrite production (Fig. 5A) and iNOS expression (Fig. 5B) in a dose- assay for NO production (A, C) and Western blot analysis for iNOS dependent manner. Similarly, 3-penten-2-one also inhibited LPS- expression (B, D) were performed as described under Materials and Methods. Blots shown are representative of three independent experi induced NO production (Fig. 5C) and iNOS expression (Fig. 5D). ments. Data are expressed as mean ± SE from 3 to 4 experiments. Unlike 3-penten-2-one, 2-pentanone and 2-pentene had no effect *p<0.05. on LPS-induced NO production and iNOS expression (not shown). To explore a potential involvement of HO-1 expression in the observed inhibitory effects of okanin and 3-penten-2-one on LPS-induced NO production and iNOS expression, siRNA against HO-1 was used to inhibit cellular synthesis of HO-1 protein. As behind the HO-1 expression by okanin, and demonstrated that shown in Fig. 6, inhibition of HO-1 expression by siRNA signifi- HO-1 expression was dependent on Nrf2 activation. cantly reversed the inhibitory effects of okanin and 3-penten-2- From a chemical viewpoint, okanin consists of two hydroxy- one on LPS-induced NO production and iNOS expression. phenyl rings linked by a three-carbon unit forming an α-β unsaturated carbonyl moiety (see Fig. 1). The α-β unsaturated Discussion carbonyl group (also known as classical Michael acceptor) has been shown to be a crucial structure of okanin, because both 2- The present study demonstrates that okanin, one of the most pentanone (lacking a double bond) and 2-pentene (lacking a abundant chalcones found in the genus Bidens (Asteraceae), carbonyl group) were virtually inactive in inducing HO-1 expres- induced the expression of the anti-inflammatory HO-1 in sion; on the contrary, 3-penten-2-one (possessing an α-β unsatu- RAW264.7 macrophages and that HO-1 expression by okanin was rated carbonyl group) had an ability to induce HO-1 expression associated with its inhibition of LPS-induced NO production and (Fig. 2C). In fact, some phytochemicals carrying this reactive (27) (28) (29) iNOS expression. We have also studied a possible mechanism functional group, such as avicins, curcumin, costunolide, 56 doi: 10.3164/jcbn.1130 ©2012 JCBN many experimental studies showing that α-β unsaturated carbonyl- containing compounds are capable of inducing HO-1 expression (27,29,30,32) through Nrf2 nuclear translocation. Thus, the present study examined whether okanin would also activate Nrf 2 nuclear translocation in RAW264.7 macrophages. Indeed, both okanin and its core component (i.e., 3-penten-2-one) induced Nrf2 nuclear translocation, and in turn increased Nrf2-ARE biding activity. Moreover, Nrf 2 activation by okanin was associated with HO-1 expression, because RAW264.7 macrophages in which the cellular synthesis of Nrf2 was suppressed by siRNA did not display any change in HO-1 expression following exposure to okanin (Fig. 3C). Our results, therefore, suggest that HO-1 expres- sion by okanin may occur through the Nrf 2-dependent pathway. HO-1 represents a major protective factor because of its anti- (14–16) oxidant and anti-inflammatory properties. It has been shown that HO-1 expression has anti-inflammatory effects that are presumably mediated by its enzymatic metabolites, such as carbon (35,36) monoxide and biliverdin/bilirubin. In activated macrophages, HO-1 expression has been shown to inhibit the release of pro- inflammatory cytokines and the expression of pro-inflammatory (36) enzymes, including iNOS. Our data shows that knocking down HO-1 largely blocked the inhibition of okanin and its core component (i.e., 3-penten-2-one) on NO production and iNOS expression in LPS-activated RAW264.7 macrophages (Fig. 6), suggesting that the inhibitory effect of okanin on LPS-induced NO production and iNOS expression may be, at least in part, mediated through HO-1 expression. Our data are in agreement with other studies demonstrating that HO-1 expression in activated macro- (37–40) phages has anti-inflammatory effects and, therefore, provide one of possible mechanisms that could explain the anti- Fig. 6. Effect of HO1 siRNA on the inhibitory actions of okanin and 3 (1–10) penten2one in NO production and iNOS expression. RAW264.7 macro inflammatory effects of medicinal plants of the genus Bidens. phages transiently transfected with HO1 siRNA were preincubated for In summary, the results of the present study demonstrate that: 6 h without or with 10 μM of okanin or 3penten2one, and then (i) okanin, one of the most abundant chalcones found in medicinal activated for 6 h (B) or 18 h (A) with 1 μg/ml of LPS. Nitrite assay for NO plants of the genus Bidens, induces HO-1 expression through production (A) and Western blot analysis for iNOS expression (B) were Nrf 2-dependent pathway in RAW264.7 macrophages; (ii) the α-β performed as described under Materials and Methods. Blots shown are unsaturated carbonyl functional group of okanin is crucial for representative of three independent experiments. Data are expressed as mean ± SE from 3 to 4 experiments. *p<0.05. Nrf2-dependent HO-1 expression; and (iii) okanin and its core component (i.e., 3-penten-2-one) inhibit LPS-induced NO produc- tion and iNOS expression by inducing HO-1 expression. (30) and dehydrocostus lactone, have been reported to induce HO-1 Conflicts of Interests expression. Moreover, 2'-hydroxychalcone, of which chemical structure is similar to that of okanin with the exception of four This paper was supported by Wonkwang University in 2010. hydroxyl substituents on two aromatic rings, has been previously reported to induce HO-1 expression in RAW264.7 macro- Abbreviations (31) phages, implying that okanin would have a chemical property resembling that of 2'-hydroxychalcone. Thus, we speculate that ARE antioxidant response element okanin may be effective in inducing HO-1 expression, at least in DMEM Dulbecco’s modified Eagle’s medium part, because it bears the α-β unsaturated carbonyl group. HO-1 heme oxygenase-1 At present, the mechanism(s) by which the α-β unsaturated iNOS inducible nitric oxide synthase carbonyl group of okanin is capable of inducing HO-1 expression Keap1 Klech-like ECH-associated protein 1 is not clear. It has been hypothesized that compounds containing LPS lipopolysaccharide the α-β unsaturated carbonyl group may cause the disruption of MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium the Keap1-Nrf 2 complex, most likely through interaction with the bromide (32–34) thiols present on Keap1, thereby resulting in Nrf 2 activation. NAC N-acetyl-L-cysteine This hypothesis is further supported by our observation that Nrf 2 NO nitric oxide activation by okanin was partially abrogated by high concentra- Nrf 2 nuclear factor-erythroid 2-related factor 2 tions of NAC (Fig. 4A). Presumably, the reaction of okanin with a OD optical density large excess of NAC may result in reversible formation of the PBS phosphate-buffered saline thiol-okanin product, allowing for a limited amount of free okanin SE standard error to be available for interaction with Keap1. Additionally, there are siRNA small interfering RNA References 1 Yuan LP, Chen FH, Ling L, et al. 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Mol Nutr 202: 579–590. 58 doi: 10.3164/jcbn.1130 ©2012 JCBN http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Clinical Biochemistry and Nutrition Pubmed Central

Okanin, a chalcone found in the genus Bidens, and 3-penten-2-one inhibit inducible nitric oxide synthase expression via heme oxygenase-1 induction in RAW264.7 macrophages activated with lipopolysaccharide

Journal of Clinical Biochemistry and Nutrition , Volume 50 (1) – Dec 15, 2011

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Abstract

Original Article JC J 0912 1880 the Society Ky j 10.3164/j Original Article c ournal of bn BN ot 11- o, - -0009 5086 Jap 30 cb Clinica afor n n.11-30 Free Ra l Biochem dical Res istryearch Ja and Nupan trition Okanin, a chalcone found in the genus Bidens, and 3penten2one inhibit inducible nitric oxide synthase expression via heme oxygenase1 induction in RAW264.7 macrophages activated with lipopolysaccharide 1 2 2 3 4 5 5 JinSang Kil, Young Son, YongKwan Cheong, NamHo Kim, Hee Jong Jeong, JiWung Kwon, EohJin Lee, 5 6 7, TaeOh Kwon, HunTaeg Chung and HyunOck Pae * 1 2 4 5 Department of Neurosurgery, Department of Anesthesiology and Pain Medicine, Department of Urology, College of Life Science and Natural Resources and Department of Microbiology and Immunology, Wonkwang University School of Medicine, 3442 Shinyongdong, Iksan 570749, Republic of Korea Department of Cardiovascular Medicine, Wonkwang University Hospital, Iksan 570711, Republic of Korea Department of Biological Science, University of Ulsan, Ulsan 680749, Republic of Korea (Received 21 February, 2011; Accepted 28 March, 2011; Published online 15 December, 2011) ?? Excess production of nitric oxide by activated macrophages via Creativ stricted vided the or C 200? This is opy e Com right © u sae, distribution, and reproduc nig op inal m 200? en acce ons wo Attribution L J rk C ss is B a N properly rticle dis cited. tiributed under the term cense tio,n whic in any h m peerm diuits unre- m s of , pro- the inflammatory cytokines and the prolonged expression of inducible inducible nitric oxide synthase leads to the development of pro-inflammatory enzymes, such as inducible nitric oxide synthase (11,12) various inflammatory diseases. Heme oxygenase1 expression via (iNOS). The inflammatory enzyme iNOS, once expressed in activation of nuclear factorerythroid 2related factor 2 inhibits activated macrophages, can generate a large amount of nitric oxide (11,12) nitric oxide production and inducible nitric oxide synthase expres (NO) for a long period. The free radical NO has been sion in activated macrophages. Okanin is one of the most abundant implicated as an important inflammatory mediator in the process (12) chalcones found in the genus Bidens (Asteraceae) that is used as of macrophage-mediated inflammation. However, uncontrolled/ various folk medications in Korea and China for treating inflam excess NO production by activated macrophages leads to the (12,13) mation. Here, we found that okanin (possessing the αβ unsatu development of various inflammatory diseases. Hence, rated carbonyl group) induced heme oxygenase1 expression via pharmacological inhibition of NO production and/or iNOS nuclear factorerythroid 2related factor 2 activation in RAW264.7 expression is a promising strategy for reducing the potentially (13) macrophages. 3Penten2one, of which structure, as in okanin, harmful pro-inflammatory activity of macrophages. possesses the αβ unsaturated carbonyl group, also induced Heme oxygenase-1 (HO-1) is an inducible enzyme that nuclear factorerythroid 2related factor 2dependent heme catalyzes the rate-limiting step in the conversion of free heme into oxygenase1 expression, while both 2pentanone (lacking a carbon monoxide, free iron, and biliverdin, which is subsequently (14) double bond) and 2pentene (lacking a carbonyl group) were catabolized into bilirubin by biliverdin reductase. In addition virtually inactive. In lipopolysaccharideactivated RAW264.7 macro to its primary role in heme degradation, HO-1 has been also phages, both okanin and 3penten2one inhibited nitric oxide recognized to play other important roles in resolution of inflam- production and inducible nitric oxide synthase expression via mation, which has been demonstrated in HO-1 knockout mice and (15,16) heme oxygenase1 expression. Collectively, our findings suggest a human case of genetic HO-1 deficiency. Particularly, HO-1 that by virtue of its αβ unsaturated carbonyl functional group, and its enzymatic metabolites are the critical regulators of okanin can inhibit nitric oxide production and inducible nitric inflammation, with activated macrophages acting as the critical (14–16) oxide synthase expression via nuclear factorerythroid 2related targets. Nuclear factor-erythroid 2-related factor 2 (Nrf 2) is a factor 2dependent heme oxygenase1 expression in lipopolysac redox sensitive transcription factor that is critical for induction of (17) charideactivated macrophages. the gene encoding HO-1. Under normal conditions, Nrf2 is sequestered in the cytoplasm by forming a complex with the Key Words: okanin, heme oxygenase1, inducible nitric oxide negative regulator of Nrf 2, Klech-like ECH-associated protein 1 (17) synthase, nuclear factorerythroid 2related factor 2, (Keap1). This complex is disrupted by several naturally occurring αβ unsaturated carbonyl group compounds, and Nrf 2 is liberated and translocated to the nucleus where it binds to antioxidant response element (ARE) sequences (18,19) Int Okanin (chem roduction ical structure shown in Fig. 1) is one of the most in the ho-1 gene promoter. Oabundant chalcone [1,3-diaryl-2-propen-1-one] compounds Recently, a series of naturally occurring compounds from found in the genus Bidens (Asteraceae) that has been used as medicinal plants have been reported to induce HO-1 expression in various folk medications in Korea and China for treating inflam- different cell types, and in some of these studies, HO-1 has been mation, malaria, hypertension, diabetes, peptic ulcer, snake bite shown to mediate their anti-inflammatory properties through (1–3) (20–24) and smallpox. Although ethnopharmacological studies carried inhibition of NO production and iNOS expression. In the out with plants of the genus Bidens have demonstrated anti- present study, we have also reported that okanin inhibited NO (4–10) inflammatory activity, whether okanin would also have production and iNOS expression through Nrf 2-dependent HO-1 anti-inflammatory activity is not yet investigated. Activated macrophages play a pivotal role in a variety of *To whom correspondence should be addressed. inflammatory diseases via the excess production of pro- Email: hopae@wonkwang.ac.kr doi: 10.3164/jcbn.1130 J. Clin. Biochem. Nutr. | January 2012 | vol. 50 | no. 1 | 53–58 ©2012 JCBN expression in RAW264.7 macrophages activated with the endo- or β-actin (1:1000 dilution) at 4°C overnight. The membrane was toxin lipopolysaccharide (LPS). then washed four times with PBS-Tween 20 buffer and further incubated with secondary antibodies for 1 h at room temperature. Materials and Methods Specific bands were detected using enhanced chemiluminescence detection system (Amersham Biosciences), and the membrane Chemicals and reagents. Previously, we had isolated okanin was exposed to X-ray film. (25) from the ethanol extract of the flowers of Bidens bipinnata L. Transfection of siRNA. RAW264.7 macrophages were and this compound was used in this study. 3-Penten-2-one, 2- grown in 6- or 12-well plates and transiently transfected with Nrf 2 pentanone, 2-pentene, Dulbecco’s modified Eagle’s medium siRNA or HO-1 siRNA mixed with siRNA transfection reagent (DMEM), hemin, LPS (Escherichia coli 055:B5), 3-(4,5-dimethyl- (Santa Cruz Biotechnology) according to the manufacturer’s 2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), N-acetyl-L- instructions. After incubation at 37°C and 5% CO2 for 30 h, cells cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, were treated with reagents. The samples were then prepared for MO). Antibodies against iNOS, HO-1, Nrf 2, lamin B and β-actin Western blot analysis. and small interfering RNA (siRNA) products against Nrf2 and Nrf2ARE binding assay. The amount of Nrf2 available in HO-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, the nucleus to bind to AREs was determined using the ELISA- TM CA). All other reagents used were of analytical grade. based TransAM Nrf 2 Kit (Active Motif, Carlsbad, CA). Briefly, Cell culture. RAW264.7 macrophages were obtained from nuclear extracts were added to wells containing the immobilized the American Type Culture Collection (Manassas, VA). The cells consensus ARE oligonucleotide. A primary antibody against Nrf 2 were cultured in DMEM supplemented with 2 mM glutamine, was added to each well. Then a secondary antibody conjugated to antibiotics (100 U/ml of penicillin A and 100 U/ml of strepto- horseradish peroxidase that binds to the primary antibody was mycin) and 10% heat-inactivated fetal bovine serum (Gibco/BRL, added to each well. The signal was detected at 450 nm, and Nrf 2- Rockville, MD) and maintained at 37°C in a humidified incubator ARE binding was reported as optical density (OD) units at containing 5% CO2. 450 nm. Cell viability assay. Cell viability was determined by a Statistical analysis. Data are expressed as means ± SE. One- modified MTT reduction assay. MTT is a pale yellow substance way analysis of variance procedures were used to assess signifi- that is reduced by living cells to yield a dark blue formazan cant differences among treatment groups. For each treatment product. This process requires active mitochondria, and even fresh showing a statistically significant effect, the Newman-Keuls test dead cells do not reduce significant amounts of MTT. RAW264.7 was used for comparisons of multiple group means. The criterion macrophages were cultured in a 96-well flat-bottom plate at for statistical significance was set at p<0.05 or 0.01. concentration of 5 × 10 cells/ml. After 12 h of preconditioning, the cells were treated with various concentrations of okanin for Results 24 h. Thereafter, culture medium was aspirated and 100 μl of MTT dye (1 mg/ml in phosphate-buffered saline) was added; the Okanin and 3penten2one, but not 2pentanone and 2 cultures were incubated for 4 h at 37°C. The formazan crystals pentene, can induce HO1 expression. The chemical struc- produced through dye reduction by viable cells were dissolved tures of okanin and other three synthetic compounds tested in this using acidified isopropanol (0.1 N HCl). Index of cell viability study are shown in Fig. 1. Both okanin and 3-penten-2-one contain was calculated by measuring the optical density of color produced an α-β unsaturated carbonyl group in the central five-carbon by MTT dye reduction at 570 nm. chain. In comparison with 3-penten-2-one, 2-pentanone and 2- Nitrite assay. The nitrite concentration in the medium was pentene lack a double bond and a carbonyl group, respectively. 3- measured as an indicator of NO production according to the Griess penten-2-one, 2-pentanone and 2-pentene were used to explore a reaction. One hundred microliters of each supernatant was mixed possible mechanism of action of okanin. In RAW264.7 macro- with the same volume of Griess reagent (1% sulfanilamide in 5% phages, okanin and 3-penten-2-one, but not 2-pentanone and 2- phosphoric acid and 0.1% naphthylethylenediamine dihydro- pentene, exhibited cytotoxicity at more than 40 μM (data not chloride in water); absorbance of the mixture at 550 nm was shown). determined with an ELISA plate reader. Treatment of RAW264.7 macrophages with non-cytotoxic Western blot analysis. RAW264.7 macrophages were incu- concentrations of okanin (0.5–10 μM) for 6 h resulted in a bated with or without reagents. They were harvested, washed ice- cold phosphate-buffered saline (PBS) and kept on ice for 1 min. The suspension was mixed with buffer A (10 mM HEPES, pH 7.5, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 5 μg/ml aprotinin, 5 μ/ml pepstatin, and 10 μg/ml leupeptin) and lysed by three freeze-thaw cycles. Cytosolic frac- tion was obtained by centrifugation at 12,000 × g for 20 min at 4°C. The pellets were re-suspended in buffer C (20 mM HEPES, pH 7.5, 0.4 M NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 5 μg/ml aprotinin, 5 μg/ml pepstatin, and 10 μg/ml leupeptin) on ice for 40 min and centrifuged at 14,000 × g for 20 min at 4°C. The resulting supernatant was used as soluble nuclear fraction. Protein content was determined with BCA pro- tein assay reagent (Pierce, Rockford, IL). Total cellular or nuclear fractions were separated on 10% SDS-polyacrylamide gels, and transferred to the nitrocellulose membranes (Amersham Bio- sciences, Inc., Piscataway, NJ). The membrane was then blocked in blocking buffer containing 20 mM sodium phosphate buffer, pH 7.6, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk Fig. 1. Chemical structures of okanin, 3penten2one, 2pentanone for 1 h at room temperature. Thereafter, the membrane was and 2pentene. In comparison with 3penten2one, 2pentanone and incubated with antibodies against HO-1 (1:1000 dilution), Nrf 2 2pentene lack a double bond and a carbonyl group, respectively. The (1:500 dilution), iNOS (1:1000 dilution), lamin B (1:1000 dilution) αβ unsaturated carbonyl group is marked with dotted circles. 54 doi: 10.3164/jcbn.1130 ©2012 JCBN Fig. 2. Effects of okanin, 3penten2one, 2pentanone and 2pentene on HO1 expression. (A) RAW264.7 macrophages were incubated for 6 h without or with 10 μM of hemin, a wellknown inducer of HO1, or indicated concentrations of okanin. (B) RAW264.7 macrophages were incubated without or with 10 μM of okanin for indicated time periods. (C) RAW264.7 macrophages were incubated for 6 h without or with 10 μM of okanin, 3penten2one, 2pentanone, or 2pentene. Western blot analysis for HO1 expression was performed as described under Materials and Methods. Blots shown are representative of three independent experiments. Fig. 3. Effects of okanin and 3penten2one on Nrf2 activation. (A) RAW264.7 macrophages were incubated without or with 10 μM of okanin for indicated time periods. Cytosolic and nuclear extracts were isolated, and the levels of Nrf2 protein were determined by Western blot analysis. (B) RAW264.7 macrophages were incubated for 2 h concentration-dependent increase in HO-1 protein levels, as without or with 10 μM of okanin, 3penten2one, 2pentanone, or 2 confirmed by Western blot analysis (Fig. 2A). Induction of HO-1 pentene. Nrf2 activation was assessed in nuclear extracts by analysis of expression by okanin was robust at 6 h and slightly decreased over Nrf2 binding to ARE. Data are expressed as mean ± SE from 3 to 4 time (Fig. 2B). Similarly, treatment with 3-penten-2-one for 6 h experiments. *p<0.05 with respect to untreated control group. (C) RAW264.7 macrophages were transiently transfected with Nrf2 siRNA, resulted in a marked increase in HO-1 protein levels (Fig. 2C). In and then exposed to 10 μM of okanin or 3penten2one for 6 h. contrast, neither 2-pentanone nor 2-pentene induced HO-1 expres- Western blot analysis for HO1 expression was performed as described sion (Fig. 2C). under Materials and Methods. Blots shown are representative of three Okanin and 3penten2one can induce Nrf2 activation independent experiments. that mediates HO1 expression. A time-dependent increase in Nrf 2 protein in the nucleus, along with a significant decrease in Nrf2 protein in the cytoplasm, was observed when RAW264.7 macrophages were incubated with 10 μM okanin (Fig. 3A). Similarly, 3-penten-2-one also increased levels of nucleus Nrf 2 inhibited. HO-1 expression by okanin and 3-penten-2-one was protein (not shown). To study Nrf 2 activation, the nuclear extracts prevented by siRNA against Nrf 2 (Fig. 3C). of RAW264.7 macrophages were isolated and the binding of Nrf 2 Nrf2 activation and HO-1 expression in RAW264.7 macro- TM to ARE was quantified by using a TransAM assay. Both okanin phages observed with okanin and 3-penten-2-one may be attribut- and 3-penten-2-one increased Nrf2 activation, whereas 2-pentanone able to changes in the production of reactive oxygen species and and 2-pentene had no significant effect on Nrf2 activation the redox environment, and/or to direct thiol modification of (26) (Fig. 3B). We next conducted the experiments of Nrf 2 knockdown Keap1 and other proteins. To better characterize Nrf 2 activation by using siRNA to investigate whether HO-1 expression would be and HO-1 expression, we treated normal RAW264.7 macrophages J.S. Kil et al. J. Clin. Biochem. Nutr. | January 2012 | vol. 50 | no. 1 | 55 ©2012 JCBN Fig. 4. Effects of NAC on Nrf2 activation and HO1 expression by okanin and 3penten2one. (A) RAW264.7 macrophages were treated with 10 μM of okanin or 3penten2one together with 1 mM NAC for 2 h. Nrf2 activation was assessed in nuclear extracts by analysis of Nrf2 binding to ARE. Data are expressed as mean ± SE from 3 to 4 experi ments. *p<0.05 with respect to untreated control group. p<0.05. (A) RAW264.7 macrophages were treated with 10 μM of okanin or 3 penten2one together with 1 mM NAC for 6 h. Western blot analysis for HO1 expression was performed as described under Materials and Methods. Blots shown are representative of three independent experi ments. with high concentrations of NAC, a thiol donor, along with okanin and 3-penten-2-one. The NAC treatment partially inhibited Nrf 2 activation (Fig. 4A) and HO-1 expression (Fig. 4B) by okanin and 3-penten-2-one. Okanin and 3penten2one inhibit LPSinduced NO Fig. 5. Effects of okanin and 3penten2one on NO production and production and iNOS expression via HO1 expression. iNOS expression. RAW264.7 macrophages were preincubated for 6 h RAW264.7 macrophages were pre-incubated for 6 h with okanin without or with indicated concentrations of okanin or 3penten2one, and activated with LPS. Okanin inhibited LPS-induced NO and then exposed to 1 μg/ml of LPS for 6 h (B, D) or 18 h (A, B). Nitrite production (Fig. 5A) and iNOS expression (Fig. 5B) in a dose- assay for NO production (A, C) and Western blot analysis for iNOS dependent manner. Similarly, 3-penten-2-one also inhibited LPS- expression (B, D) were performed as described under Materials and Methods. Blots shown are representative of three independent experi induced NO production (Fig. 5C) and iNOS expression (Fig. 5D). ments. Data are expressed as mean ± SE from 3 to 4 experiments. Unlike 3-penten-2-one, 2-pentanone and 2-pentene had no effect *p<0.05. on LPS-induced NO production and iNOS expression (not shown). To explore a potential involvement of HO-1 expression in the observed inhibitory effects of okanin and 3-penten-2-one on LPS-induced NO production and iNOS expression, siRNA against HO-1 was used to inhibit cellular synthesis of HO-1 protein. As behind the HO-1 expression by okanin, and demonstrated that shown in Fig. 6, inhibition of HO-1 expression by siRNA signifi- HO-1 expression was dependent on Nrf2 activation. cantly reversed the inhibitory effects of okanin and 3-penten-2- From a chemical viewpoint, okanin consists of two hydroxy- one on LPS-induced NO production and iNOS expression. phenyl rings linked by a three-carbon unit forming an α-β unsaturated carbonyl moiety (see Fig. 1). The α-β unsaturated Discussion carbonyl group (also known as classical Michael acceptor) has been shown to be a crucial structure of okanin, because both 2- The present study demonstrates that okanin, one of the most pentanone (lacking a double bond) and 2-pentene (lacking a abundant chalcones found in the genus Bidens (Asteraceae), carbonyl group) were virtually inactive in inducing HO-1 expres- induced the expression of the anti-inflammatory HO-1 in sion; on the contrary, 3-penten-2-one (possessing an α-β unsatu- RAW264.7 macrophages and that HO-1 expression by okanin was rated carbonyl group) had an ability to induce HO-1 expression associated with its inhibition of LPS-induced NO production and (Fig. 2C). In fact, some phytochemicals carrying this reactive (27) (28) (29) iNOS expression. We have also studied a possible mechanism functional group, such as avicins, curcumin, costunolide, 56 doi: 10.3164/jcbn.1130 ©2012 JCBN many experimental studies showing that α-β unsaturated carbonyl- containing compounds are capable of inducing HO-1 expression (27,29,30,32) through Nrf2 nuclear translocation. Thus, the present study examined whether okanin would also activate Nrf 2 nuclear translocation in RAW264.7 macrophages. Indeed, both okanin and its core component (i.e., 3-penten-2-one) induced Nrf2 nuclear translocation, and in turn increased Nrf2-ARE biding activity. Moreover, Nrf 2 activation by okanin was associated with HO-1 expression, because RAW264.7 macrophages in which the cellular synthesis of Nrf2 was suppressed by siRNA did not display any change in HO-1 expression following exposure to okanin (Fig. 3C). Our results, therefore, suggest that HO-1 expres- sion by okanin may occur through the Nrf 2-dependent pathway. HO-1 represents a major protective factor because of its anti- (14–16) oxidant and anti-inflammatory properties. It has been shown that HO-1 expression has anti-inflammatory effects that are presumably mediated by its enzymatic metabolites, such as carbon (35,36) monoxide and biliverdin/bilirubin. In activated macrophages, HO-1 expression has been shown to inhibit the release of pro- inflammatory cytokines and the expression of pro-inflammatory (36) enzymes, including iNOS. Our data shows that knocking down HO-1 largely blocked the inhibition of okanin and its core component (i.e., 3-penten-2-one) on NO production and iNOS expression in LPS-activated RAW264.7 macrophages (Fig. 6), suggesting that the inhibitory effect of okanin on LPS-induced NO production and iNOS expression may be, at least in part, mediated through HO-1 expression. Our data are in agreement with other studies demonstrating that HO-1 expression in activated macro- (37–40) phages has anti-inflammatory effects and, therefore, provide one of possible mechanisms that could explain the anti- Fig. 6. Effect of HO1 siRNA on the inhibitory actions of okanin and 3 (1–10) penten2one in NO production and iNOS expression. RAW264.7 macro inflammatory effects of medicinal plants of the genus Bidens. phages transiently transfected with HO1 siRNA were preincubated for In summary, the results of the present study demonstrate that: 6 h without or with 10 μM of okanin or 3penten2one, and then (i) okanin, one of the most abundant chalcones found in medicinal activated for 6 h (B) or 18 h (A) with 1 μg/ml of LPS. Nitrite assay for NO plants of the genus Bidens, induces HO-1 expression through production (A) and Western blot analysis for iNOS expression (B) were Nrf 2-dependent pathway in RAW264.7 macrophages; (ii) the α-β performed as described under Materials and Methods. Blots shown are unsaturated carbonyl functional group of okanin is crucial for representative of three independent experiments. Data are expressed as mean ± SE from 3 to 4 experiments. *p<0.05. Nrf2-dependent HO-1 expression; and (iii) okanin and its core component (i.e., 3-penten-2-one) inhibit LPS-induced NO produc- tion and iNOS expression by inducing HO-1 expression. (30) and dehydrocostus lactone, have been reported to induce HO-1 Conflicts of Interests expression. Moreover, 2'-hydroxychalcone, of which chemical structure is similar to that of okanin with the exception of four This paper was supported by Wonkwang University in 2010. hydroxyl substituents on two aromatic rings, has been previously reported to induce HO-1 expression in RAW264.7 macro- Abbreviations (31) phages, implying that okanin would have a chemical property resembling that of 2'-hydroxychalcone. Thus, we speculate that ARE antioxidant response element okanin may be effective in inducing HO-1 expression, at least in DMEM Dulbecco’s modified Eagle’s medium part, because it bears the α-β unsaturated carbonyl group. HO-1 heme oxygenase-1 At present, the mechanism(s) by which the α-β unsaturated iNOS inducible nitric oxide synthase carbonyl group of okanin is capable of inducing HO-1 expression Keap1 Klech-like ECH-associated protein 1 is not clear. It has been hypothesized that compounds containing LPS lipopolysaccharide the α-β unsaturated carbonyl group may cause the disruption of MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium the Keap1-Nrf 2 complex, most likely through interaction with the bromide (32–34) thiols present on Keap1, thereby resulting in Nrf 2 activation. NAC N-acetyl-L-cysteine This hypothesis is further supported by our observation that Nrf 2 NO nitric oxide activation by okanin was partially abrogated by high concentra- Nrf 2 nuclear factor-erythroid 2-related factor 2 tions of NAC (Fig. 4A). Presumably, the reaction of okanin with a OD optical density large excess of NAC may result in reversible formation of the PBS phosphate-buffered saline thiol-okanin product, allowing for a limited amount of free okanin SE standard error to be available for interaction with Keap1. Additionally, there are siRNA small interfering RNA References 1 Yuan LP, Chen FH, Ling L, et al. 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Journal of Clinical Biochemistry and NutritionPubmed Central

Published: Dec 15, 2011

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