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Introduction

Nitric oxide (NO) has been known to involve in the regulation of various physiological processes in mam-mals and the overproduction of NO is responsible for the pathological development of inflammation, cancer, and diabetes.(1,2) The production of NO depended on the activity of nitric oxide synthases (NOS) including induc-ible- (iNOS), neuronal- (nNOS) and endothelial- (eNOS) nitric oxide synthases. The iNOS is normally not present in resting cells but is expressed in several pathophysi-ological conditions, and produces large amounts of NO in response to inflammatory signals, such as cytokines and lipopolysaccharides (LPS).(3) Transcriptional nuclear factor-κB (NF-κB), a family of Rel domain-containing proteins, has been demonstrated to regulate the expres-sion of genes involving in cancer, arthritis, asthma,

inflammatory bowel disease and other inflammatory conditions.(4,5) In most cell types, this transcription factor seems to be an essential requirement for iNOS induction in response to inflammatory signals.(6,7) Therefore, the inhibition of NF-κB that could prevent the expression of iNOS and NO production become important target for the treatment of the related diseases.(8,9)

The natural Diels-Alder type products sanggenon C (SC) and sanggenon O (SO) are diastereomers (Figure 1) and present in various Moraceous plants. Previous stud-ies reported that SC showed strong cytotoxicity,(10) inhi-bition of hypoxia-inducible factor-1,(11) protein tyrosine phosphatase 1B,(12) COX-1 and -2,(13) and platelet cyclooxygenase.(14) SC also suppressed the adhesion of polymorphonuclear leukocyte to human synovial cells by targeting the NF-κB gene VCAM-1.(15) The biological

RESEARCH ARTICLE

Sanggenon C and O inhibit NO production, iNOS expression and NF-κB activation in LPS-induced RAW264.7 cells

Nguyen Tien Dat1, Phung Thi Xuan Binh1,2, Le Thi Phuong Quynh2, Hoang Thanh Huong2, and Chau Van Minh1

1Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, Hanoi, Vietnam, and 2Institute of Natural Product Chemistry, Vietnam Academy of Science and Technology, Hanoi, Vietnam

AbstractObjective: The NO production through the iNOS induction by activation of nuclear factor (NF-κB) is known to involve in various inflammatory conditions. Sanggenon C and O, two Diels-Alder type adducts isolated from Morus alba, a plant has been used for the anti-inflammatory purpose in the Oriental medicine, were investigated for their effect on the NO production, iNOS expression and NF-κB activity.

Methods: The inhibitory effects of sanggenon C and O on the NF-κB activity were investigated in LPS-stimulated RAW264.7 cells by SEAP reporter assay. The regulation of the iNOS expression and IκBα activation by two compounds was also evaluated by Western blot.

Results: Both compounds strongly inhibited NO production and NF-κB activation in a dose-dependent manner. The expression of the iNOS protein was also suppressed by treatment of the compounds (10 and 1 µM). Sanggenon O showed stronger inhibition than the diastereomer sanggenon C. Both compounds prevented the phosphorylation and degradation of IκBα protein.

Conclusion: We demonstrated that sanggenon C and O inhibited NO production and iNOS expression by suppressing NF-κB activity and IκBα activation.

Keywords: Sanggenon C, sanggenon O, Morus alba, nitric oxide, iNOS, NF-κB

Address for Correspondence: Nguyen Tien Dat, Department of Bioactive Products, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Hanoi, Vietnam. E-mail: ngtiend@hotmail.com

(Received 03 March 2011; revised 30 March 2011; accepted 11 April 2011)

Immunopharmacology and Immunotoxicology, 2012; 34(1): 84–88© 2012 Informa Healthcare USA, Inc.ISSN 0892-3973 print/ISSN 1532-2513 onlineDOI: 10.3109/08923973.2011.580755

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activity of SO, a diastereomer of SC, has not been well studied except for the hypoxia-inducible factor-1 and COX inhibitory effects.(11,13) In our continuing search for anti-inflammatory agents of natural origin, we found that SC and SO isolated from Morus alba strongly sup-pressed the production of NO in LPS-induced RAW264.7 cells. The present paper describes the effects of SC and SO on the NO production, iNOS expression and NF-κB inhibition in LPS-induced RAW264.7 cells.

Experimental

Reagents and cell cultureSC and SO were previously isolated from Morus alba and identified by spectroscopic methods.(16) Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and Griess reagent were purchased from Invitrogen, Grand Island, NY. Rabbit polyclonal anti-iNOS, anti-IκBα, anti-phosphor-IκBα and β-actin antibodies were purchased from Santa Cruz Technology, Santa Cruz, CA . Murine macrophage RAW264.7 cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM supplemented with 10% (v/v) FBS, penicillin, and streptomycin and maintained in a humidified 5% CO

2 atmosphere at 37°C.

Measurement of NO productionRAW264.7 cells were seeded in 96-well plate at 2 × 105 cells/well and incubated for 3 h. The plates were pre-treated with various concentrations of compounds for 30 min and then incubated for another 24 h with or with-out 1 μg/mL LPS. As a parameter of NO synthesis, nitrite concentration in the culture supernatant was measured by the Griess method.(17) The culture supernatant of 100 μL was transferred to other 96-well plate and 100 μL of Griess reagent was added. The absorbance of the reac-tion solution was read at 570 nm with a microplate reader

(Molecular Devices Co., Menlo Park, CA). The remain-ing cell solutions in cultured 96-well plate were used to evaluate cell viability by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.

NF-κB reporter assayRAW264.7 cells were transiently transfected with a plas-mid containing eight copies of κB elements linked to the SEAP (secreted alkaline phosphatase) gene, then the SEAP assay was performed as previously described.(18) Briefly, transfected cells were seeded in a 96-well plate at a density of 5 × 104 cells/well. After 3 h incubation at 37°C, cells were treated with samples and stimulated with 1 µg/mL LPS for 24 h. Each culture supernatants of 100 μL was transferred to 96-well plate and heated at 65°C for 10 min followed by adding 100 μL of 2× SEAP buffer to each well and incubated for another 10 min. The reaction was initiated by adding 20 μL of 31.6 mg/mL p-nitrophenyl phosphate dissolved in 1× SEAP buffer and incubated at 37°C for 4 h. The optical density values of solutions were measured at 405 nm with a microplate reader (Molecular Devices Co., Menlo Park, CA). Deguelin, a potent NF-κB inhibitor was used for positive control.

Protein extraction for Western blot analysisRAW264.7 cells were cultured in 6 cm dishes at 37°C overnight. Various concentrations of SC and SO were added and incubated for 30 min, and then further incu-bated with 1 µg/mL LPS for 24 h (for iNOS detection) or 15 min (for IκBα and phospho-IκBα detection). At the end of incubation, the cells were harvested by scraping, followed by centrifugation at 1000 g for 5 min at 4°C, and washed twice with ice-cold phosphate-buffered saline. Total cells lysates (for iNOS detection) were obtained using Amresco’ total protein lysis buffer (Amresco, Solon, OH ). Cytosolic extracts (for IκBα and phospho-IκBα detection) were prepared using NE-PER Nuclear

Figure 1. Structure of sanggenon C and sanggenon O.

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and Cytoplasmic Extraction Reagents (Perbio, Germany) supplemented with protease inhibitor cocktail.

Western blot analysisCell lysates were prepared using a lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin, 150 mM NaCl). Lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to Hybond-C membrane (Amersham Biosciences, Piscataway, NJ). The membrane

was blocked with 5% nonfat milk in phosphate-buffered saline containing 0.1% Tween-20 (PBST) at room tem-perature for 1 h and then incubated overnight at 4°C with primary antibodies of iNOS (1:500 dilution), IκBα and phospho-IκBα (1:1000 dilution). The secondary anti-body (HRP-conjugated anti-rabbit IgG, 1:5000 dilution) was then added for 1 h incubation at room temperature, and the antigen-antibody complexes were visualized by enhanced chemiluminescence according to the instruc-tions of the manufacturer (Amersham Bioscience, Piscataway, NJ).

Figure 2. Effect of sanggenon C and sanggenon O on the NO production and NF-κB activity in RAW264.7 cells. (A) Cells were pretreated with compounds for 30 min, then stimulated with 1 μg/mL LPS for 24 h. NO concentrations were determined by Griess assay. (B) Cells transfected with NF-κB plasmid linked to SEAP gene were pretreated with compounds for 30 min, then stimulated with 1 μg/mL LPS for 24 h. SEAP activity of the culture media was measured as described in Experimental section . Bars represent standard deviations from three experiments. Asterisks indicate a significance of p < 0.05 (*) and p < 0.01 (**) when compared to the untreated control.

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Statistical analysisEach experiment was performed at least three times, and representative data are shown. Means were checked for statistical differences by using the Student’s t-test with error probabilities of p < 0.05.

Results and discussion

SC and SO inhibited NO production in LPS-stimulated RAW264.7 cellsThe inhibitory effects of SC and SO on the NO produc-tion from LPS-stimulated RAW264.7 cells were evalu-ated by Griess method as described above. The cells were treated with compounds and stimulated by 1 µg/mL LPS for 24 h and the NO levels in the culture media were measured as nitrite concentration. The result showed that SC and SO strongly inhibited NO produc-tion in a dose-dependent manner with IC

50 of 2.82

and 1.15 μM, respectively (Figure 2A). The MTT assay showed that the two compounds had no significant toxicity at their effective doses for the NO inhibition (data not shown).

SC and SO inhibited NF-κB activityNF-κB is considered as one of the major factors that regulate the expression of iNOS. Therefore, we examined whether the iNOS inhibition of SC and SO is through the inhibition of NF-κB. The effects of SC and SO on the NF-κB activation were evaluated in a NF-κB-dependent reporter gene assay in RAW264.7 cells stimulated by 1 μg/mL LPS. The result showed that SC and SO strongly inhibited NF-κB activation at concentrations compa-rable to the NO inhibition with IC

50 of 3.38 and 1.29 μM,

respectively (Figure 2B). The activity of SC and SO was significant in comparison with deguelin, a natural com-pound that strongly inhibits NO production and NF-κB signaling.(18)

SC and SO inhibited iNOS expressionThe induced expression of iNOS is primary responsible for the NO production. Since SC and SO strongly down regulated NO production, we examined the effect of these compounds on the iNOS expression in LPS-stimulated RAW264.7 cells. The cells pretreated with SC and SO were stimulated by 1 μg/mL LPS for 24 h and the iNOS level in the total cells lysates was analyzed by Western blot. Figure 3 showed that SC and SO at 1 and 10 µM strongly suppressed LPS-induced iNOS expression.

SC and SO prevented IκBα degradation and phosphorylationSince the NF-κB activation by various stimuli is preceded by the phosphorylation, ubiquitination and proteolytic degradation of the IκBα protein,(4) we examined the effect of compounds on the LPS-induced phosphory-lation and degradation of IκBα in RAW264.7 cells. As shown in Figure 3, the LPS induced the degradation and phosphorylation of IκBα (lanes 1 and 2). In the presence of SC and SO, the degradation and phosphorylation were significantly blocked (lanes 3–6). Thus the NF-κB inhibi-tory effect of SC and SO could be due to the inactivation of IκBα protein.

Sanggenon C and O are belonging to the phenolic constituents found in Morus alba and other Moraceous plants. It has reported that SC prevented the adhesion of polymorphonuclear leukocyte to human synovial cells through the inhibition of ICAM-1 and VCAM-1 expres-sions by targeting NF-κB/DNA binding activity.(15) In the present study, we showed that SC inhibited the produc-tion of NO and iNOS expression by suppressing NF-κB activity. It is noted that SC and SO are diasteromers at C-2 and C-3 configurations. Interestingly, the NF-κB and NO inhibitory effects of SO were stronger than those of SC suggesting that the stereo configurations at C-2 and C-3 could be important for the inhibition of NO production

Figure 3. Effect of sanggenon C and sanggenon O on the iNOS expression and IκBα phosphorylation and degradation. RAW264.7 cells were pretreated with compounds for 30 min, then the cells were stimulated by 1 μg/mL LPS for indicated times. iNOS, IκBα and phosphor-IκBα proteins in cell lysates were examined by Western blot. β-actin was detected for protein loading control.

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and NF-κB activity. It has been reported that the extracts as well as the compounds isolated from Morus alba showed strong anti-inflammatory effects by suppres-sion of NO production and iNOS expression via NF-κB pathway.(19–23) Our result also indicated that the Diels-Alder type constituents of M. alba, SC and SO, strongly prevented NO production though the inhibition of NF-κB activity. These results may explain the effective usage of M. alba in the prevention and treatment of inflammation in the Oriental medicine.(24)

Conclusion

In summary, we demonstrated that SC and SO inhibited the production of NO, iNOS expression and NF-κB activ-ity in LPS-stimulated macrophages. It also suggested that the stereo orientation of the chemical substituents at C-2 and C-3 in the SC and SO structures could be important for the inhibition of NO production and NF-κB activity. Therefore, SC and SO might be potential leads for the inflammatory agents.

Declaration of interest

This work is supported by a grant from the Vietnam’s National Foundation for Science and Technology Development (NAFOSTED-104.01.41.09) to one of the authors (N.T.D.).

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