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Suppressive effects of Indigofera suffruticosa Mill extracts on lipopolysaccharide-induced inflammatory responses in murine raw 264. 7 macrophages


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Suppressive effects of Indigofera suffruticosa Mill extracts on lipopolysaccharide-induced inflammatory responses in murine RAW 264.7 macrophages

Tzy-Yen Chena,b,1, Hai-Lun Sun b,c,1, Hsien-Tsung Yaod, Chong-Kuei Liid, Haw-Wen Chend, Pei-Yin Chene, Chien-Chun Lie,f** and Kai-Li Liue,f*


aDivision of Gastroenterology, Chung Shan Medical University Hospital, Taichung, Taiwan

bSchool of Medicine, Chung Shan Medical University, Taichung, Taiwan

cDivision of Allergy, Asthma and Rheumatology, Department of Pediatrics, Chung Shan Medical University Hospital, Taichung, Taiwan

dDepartment of Nutrition, China Medical University, Taichung, Taiwan

eDepartment of Nutrition, Chung Shan Medical University, NO. 110, Sec. 1, Chien-Kuo N. Rd., Taichung 40203, Taiwan

fDepartment of Dietitian, Chung Shan Medical University Hospital, Taichung, Taiwan

1Tzy-Yen Chen and Hai-Lun Sun contributed equally to this work

**Corresponding authors. Tel.: +886 4-24730022.

E-mail address: licc@csmu.edu.tw (C.C. Li).

*Corresponding author. Tel.: 8864-2473-0022.

E-mail address: kaililiu@csmu.edu.tw (K.L. Liu)

Abbreviations: ARE, antioxidant response element; CO, carbon monoxide; DMSO, dimethyl sulfoxide; EE-IDS, ethanol extracts of Indigofera suffruticosa Mill, EMSA, electrophoretic mobility shift assay; ERK1/2, extracellular signal-regulated kinase 1/2; FE, formononetin equivalent; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HO-1, heme oxygenase-1; HPLC/MS, High performance liquid chromatography /mass spectrometry; IL-1, interleukin-1; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; NF-B, nuclear factor-kappa B; NO, nitric oxide; Nrf2, nuclear factor E2-related factor 2; NTC, non-targeting control; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; SE, syringaldehyde equivalent; SEAP, secretory alkaline phosphatase; siRNA, small interfering RNA; Syr, Syringaldehyde; TNF-, tumor necrosis factor-; WE-IDS, water extracts of Indigofera suffruticosa Mill.
1. Introduction

Inflammation is an important body defense against tissue injury and microbial invasion which involve the recruitment and activation of circulating leukocytes, along with the generation of large amount of pro-inflammatory mediators such as nitric oxide (NO), tumor necrosis factor- (TNF-), and interleukin-1 (IL-1) at the inflammatory site (MacMicking et al., 1997; Stylianou and Saklatvala, 1998; Kofler et al., 2005). Overproduction of the pro-inflammatory mediators resulting from inappropriate activation of macrophages is implicated in the pathologies of many chronic inflammatory diseases of modern society, such as rheumatoid arthritis, atherosclerosis, diabetes, and cancer (Manzi and Wasko, 2000; King, 2008; Kundu and Surh, 2008). Therefore, the inhibition of pro-inflammatory mediator production and the suppression of mechanisms responsible for the activation of inflammatory responses are regarded as clinical strategies for the treatment of chronic inflammation.

NO from oxidation of L-arginine to citrulline by nitric oxide synthase (NOS) has a profound influence on diverse physiological and pathological events (Moncada et al., 1992). In macrophages and invading immune cells, the high amount of NO produced by inducible NOS (iNOS) in response to lipopolysaccharide (LPS) and/or inflammatory cytokines plays a crucial role in inflammation and cytotoxicity (MacMicking et al., 1997). Although there are several binding sites for transcription factors in the promoter region of iNOS, nuclear factor-kappa B (NF-B) is critical to LPS-induced iNOS gene expression in mouse macrophages (Xie et al., 1994). The transcription factor NF-B exists in the cytoplasm of most eukaryotes by forming homodimers or heterodimers with proteins of the NF-B family, including p65 (RelA), p50/p105 (NF-B1), p52/p100 (NF-B2), RelB, and c-Rel. In unstimulated cells, an inhibitor protein termed IB (,  or ) noncovalently binds to NF-B sequestered in the cytoplasm. In response to inflammatory cytokines, oxidative stress, ultraviolet irradiation, or bacterial endotoxins, the inhibitor protein IB is phosphorylated, ubiquitinated, and degraded, which leads to NF-B activation. Then, the activated NF-B is translocated into the nucleus and induces transcriptional expression of its target genes, most of which are involved in pro-inflammatory responses, such as iNOS, TNF-, and IL-1 (Chen et al., 1999). Growing experimental evidence has indicated that regulation and control of NF-B activation may be a key molecular target for the anti-inflammatory therapy (Chen et al., 1999; Makarov et al., 2000).

There are three isoforms of heme oxygenase (HO) that catalyze the oxidative degradation of heme to carbon monoxide (CO), biliverdin, and ferrous iron in mammalian cells (Maines, 1988). In contrast to the constitutive expression of HO-2 and HO-3, the expression of HO-1 is inducible in response to a variety of stimuli, such as cytokines, oxidative stress, and endotoxin. HO-1 has been considered as a cytoprotective enzyme due to the antioxidant activity of biliverdin and its metabolite, bilirubin, as well as the anti-inflammatory ability of CO (Ryter et al., 2006). Induction of HO-1 expression can down-regulate the inflammatory responses of TNF--activated endothelial cells and Pseudomonas aeruginosa-stimulated lung tissue (Tsuburai et al., 2004; Seldon et al., 2007). Data from various studies have suggested that the increased HO-1 expression plays a vital role in the inhibitory modulation of iNOS expression and, subsequently, NO production in LPS-activated macrophages (Wang et al., 2004; Lee et al., 2005; Hu et al., 2009). Together, the above findings suggest that the activation of HO-1 gene expression may represent a new potential for chronic inflammation-related diseases.

A species belonging to family Fabaceae, Indigofera suffruticosa Mill is a wild herb growing in tropical and subtropical areas. While it is used as a natural indigo dye in the textile industry, in vivo and in vitro studies have investigated the therapeutic effect of I. suffruticosa on anti-inflammation (Leite et al, 2003), anti-epilepsy (Wong et al., 1999), anti-cancer (Vieira et al., 2007) as well as anti-mycobacterial, anti-bacterial and anti-fungal activity (Leite et al., 2006; de A Carli et al., 2010). Although the alkaloidal fraction of I. suffruticosa methanol extracts could induce NO and TNF-a production in mouse peritoneal macrophages(Lopes et al., 2011), the aqueous extract of I. suffruticosa leaves similar to the positive control acetylsalicylic acid can decrease carrageenan-induced paw edema in Swiss albino mice (Leite et al., 2003). However, the molecular mechanisms underlying the anti-inflammatory effects of I. suffruticosa are not completely understood. The present study aimed to assess the regulatory efficacy of I. suffruticosa on the LPS-induced inflammatory responses in RAW 264.7 macrophages and to explore the possible molecular mechanisms behind these activities.

2. Materials and methods

2.1. Materials

The mouse macrophage-like cell line RAW 264.7 was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan), and fetal bovine serum was from HyClone Laboratories (Losan, UT), RPMI 1640 medium and media supplements for cell culture were obtained from Invitrogen Corporation (Carlsbad, CA). LPS, syringaldehyde (98% purity), formononetin (99% purity) and Folin-Ciocalteu phenol reagent were from Sigma Chemical Company (St. Louis, MO). The specific antibodies for iNOS, pro-IL-1, HO-1 and -actin were obtained form Santa Cruz Biotechnology (Santa Cruz, CA), CytoLab Ltd. (Rehovot, Israel), Calbiochem (La Jolla,Ca) and Sigma Chemical Company, respectively. The antibodies against extracellular signal-regulated kinase 1/2 (ERK1/2) as well as phosphorylated ERK1/2 and IB- were from Cell Signaling Technology Inc. (Beverly, MA). Nucleotides and RNase inhibitor and M-MMLV reverse transcriptase were got form Promega Co. (Madison, WI). Real-time quantitative polymerase chain reaction (PCR) primers and TaqMan® Universal PCR Master Mix were from Applied Biosystems (Foster City, CA). The biotin-labeled and unlabeled double-stranded NF-B and antioxidant response element (ARE) consensus oligonucleotides and a mutant double-stranded NF-B oligonucleotide for electrophoretic mobility shift assay (EMSA) were synthesized by MDBio Inc. (Taipei, Taiwan). 



2.2. Preparation of extractions

Commercially available, air-dried I. suffruticosa were purchased from Tainan, Taiwan and were identified by Dr. Yi-Ching Li (Department of Pharmacology, Chung Shan Medical University). Voucher specimen was kept in our laboratory, at the Department of Nutrition, Chung Shan Medical University, Taichung, Taiwan, for further reference.

The powdered air-dried I. suffruticosa were extracted with 95% ethanolic (plant material: solvent, 1: 13.3, w/v) for 2 h at 40oC with continuous stirring. After filtration through a 0.22 m pore size membrane, the ethanolic was removed at 37oC under reduced pressure. The ethanolic extract of I. suffruticosa was weighted to measure the extraction yield and then dissolved in dimethyl sulfoxide (DMSO) for cell treatments.

The air-dried I. suffruticosa were boiled in Milli-Q water (5 ml/g plant material) for 1 h and then were filtered through Whatman #1 filter paper. The resulting solution was freeze-dried to yield the water extract. The water extract of I. suffruticosa was weighted to measure the extraction yield and was stored at -20oC.



2.3. Determination of total phenolics and flavonoids

The amount of total phenolics in the water and ethanolic extracts of I. suffruticosa was measured according to a modification of the Folin-Ciocalteu method (Nurmi et al., 1996). A 300-L aliquot of each extract or standard solution was mixed with 300 L of Folin-Ciocalteu reagent. The mixture was allowed to stand for 5 min followed by the addition of 600 L of 20% Na2CO3. After a 10-min incubation at room temperature, the reaction mixture’s absorbance was measured at 730 nm and total phenolics were calibrated by using a standard curve of syringaldehyde. The total phenolic contents of the I. suffruticosa extracts were expressed as the in mg/g dry material.

A 250-L aliquot of each extract or standard solution was mixed with 1.25 ml of distilled water and 75 l of 5% NaNO2 solution. After 6 min, 150 l of 10% AlCl3-H2O solution was added. After 5 min, 0.5 ml of 1 M NaOH solution was added and the total volume was brought up to 2.5 ml with dd H2O. The reaction mixture’s absorbance was measured at 510 nm and total flavonoids were calibrated by using a standard curve of formononetin. The total flavonoid contents of the I. suffruticosa extracts were expressed as the formononetin equivalent (FE) in mg/g dry material (Jia et al., 1999).

2.4. Cell culture

The RAW 264.7 macrophages of passages 10 to 15 were maintained in RPMI-1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 10% heat-inactivated fetal bovine serum at 37C in a humidified atmosphere of 5% CO2.

Cells were treated with the water and ethanolic extracts of I. suffruticosa in the presence of 0.01 g/ml LPS for indicated time periods or pre-treated with syringaldehyde or I. suffruticosa extracts 12 h prior to addition of LPS (0.01 g/ml). Cells treated with phosphate buffered saline (PBS) and DMSO alone were used as vehicle controls of water and ethanolic extracts of I. suffruticosa, respectively.

2.5. Cell viability assay

The mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan was used to measure cell respiration as an indicator of cell viability (Denizot and Lang, 1986). Cells were incubated in RPMI medium containing 0.5 mg/ ml MTT for an additional 3 h and isopropanol was added to dissolve the formazan. After centrifugation at 5000  g for 5 min, the absorbance of supernatant was read at 570 nm in a VersaMaxTM Tunable Microplate Reader (Molecular Devices Corporation, Sunnyvale, CA).



2.6. Nitrite determination

The nitrite in the medium was measured by Griess assay and used as an indicator of NO synthesis in cells (Green et al., 1982). Culture supernatants were mixed with Griess solution [1:1 mixture of 1% sulfanilamide and 0.1% N-(naphthyl) ethyl-enediamine dihydrochloride in 5% H3PO4] and stood at room temperature for 10 min. Absorbance was measured at 550 nm and nitrite concentration was determined by using a standard curve of sodium nitrite prepared in the culture medium.



2.7. Western blot analysis

Cells were harvested in 150 L lysis buffer containing 10 mM Tris-HCl, 5 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 20 g/ml aprotinin, pH 7.4. The protein content in each sample was quantified by use of the Coomassie Plus Protein Assay Reagent Kit (Pierce Chemical Co., Rockford, IL). Equal amounts of proteins were denatured and separated on SDS-polyacrylamide gels and were then transferred to polyvinylidene difluoride membranes (NewTM Life Science Product, Inc., Boston, MA). Nonspecific binding sites on the membranes were blocked with 5% nonfat dry milk in a buffer containing 10 mM Tris-HCl and 100 mM NaCl, pH 7.5, at 4C overnight. The blots were then incubated sequentially with primary antibody and horseradish peroxidase-conjugated anti-goat or anti-rabbit IgG (Bio-Rad, Hercules, CA). Immunoreactive protein bands were developed by enhanced chemiluminescence kits (Amersham Life Sciences, Arlington Heights, IL) and then were quantified through densitometric analysis by Zero-Dscan (Scanalytics Inc., Fairfax, VA).



2.8. RNA Isolation and real-time quantitative reverse transcriptase-PCR

Total RNA was isolated from cells by using Tri-Reagent TM (Molecular Research Center Inc., Cincinnati, OH) as described by the manufacturer. RNA were reverse transcribed with M-MMLV reverse transcriptase for synthesis of complementary DNA. Complementary DNA was amplified with TaqMan® Universal PCR Master Mix primers and probes and the reactions were measured in ABI 7000 Real Time PCR System (Applied Biosystems). The primers and probes were order from Applied Biosystems: iNOS (Mm00440502_m1), HO-1 (Mm00516005_m1), TNF- (Mm00434770_m1), IL-1 (Mm01336189_m1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Mm00484668_m1). GAPDH was used as an internal standard gene and the threshold cycles (Ct) of a test sample to a control sample (Ct method) was used for relative quantification of target gene expressions (Livak and Schmittgen, 2001).



2.9. HO-1 siRNA transfection

Predesigned small interfering RNA (siRNA) against mouse HO-1 and non-targeting control (NTC) siRNA were purchased from Dharmacon Inc. (Lafayette, CO). RAW 264.7 macrophages were transfected in Opti-MEM (Invitrogen) using lipofectamine (Invitrogen) with HO-1 siRNA SMARTpool® or NTC siRNA according to the manufacturer’s instructions. Then cells were treated with I. suffruticosa extracts 12 h prior to addition of LPS for 18 h.



2.10. Preparation of nuclear protein and EMSA

At the time of harvest, cells were scraped with cold PBS and centrifuged. The pellets were resuspended in the hypotonic extraction buffer (10 mM HEPES, 10 mM KCl, 1 mM MgCl2, 1 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF, 4 g/ml leupeptin, 20 g/ml aprotinin, and 0.5% NP-40) for 15 min on ice and were then centrifuged at 6000  g for 15 min. The pelleted nuclei were resuspended in 50 L hypertonic extraction buffer (10 mM HEPES, 400 mM KCl, 1 mM MgCl2, 1 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF, 4 g/ml leupeptin, 20 g/ml aprotinin, and 10% glycerol), were constantly shaken at 4C for 30 min, and were then centrifuged at 10,000  g for 15 min. The resultant supernatants containing nuclear proteins were collected and stored at –70C until the EMSA was performed.

EMSA was performed according to our previous study (Liu et al., 2006). The LightShiftTM Chemiluminescent EMSA Kit from Pierce Chemical Co. and synthetic biotin-labeled double-stranded consensus oligonucleotides of ARE (5’-TGGGGAACCTGTGCTGAGTCACTGGAG-3’) and NF-B (5’-AGTTGAGGGGACTTTCCCAGGC-3’) were used to measure the effect of the I. suffruticosa extracts on nuclear factor E2-related factor 2 (Nrf2) and NF-B nuclear protein-DNA binding activity, respectively. Nuclear proteins (2 g), poly (dI-dC), and biotin-labeled double-stranded oligonucleotides of ARE and NF-B were mixed with the binding buffer and were incubated at room temperature for 30 min. In addition, the excess amount of unlabeled double-stranded oligonucleotides of ARE and NF-B as well as a mutant double-stranded NF-B oligonucleotide (5’-AGTTGAGGCGACTTTCCCAGGC-3’) were used for the competition assay to confirm specificity of binding. The nuclear protein-DNA complex was separated by electrophoresis on a 6% Tris/Boric acid/EDTA-polyacrylamide gel and was then electrotransferred to a nylon membrane (HybondTM-N+, Amersham Pharmacia Biotech Inc, Piscataway, NJ). The membrane was treated with streptavidin-horseradish peroxidase, and the nuclear protein-DNA bands were developed by using a SuperSignal West Pico kit (Pierce Chemical Co.).

2.11. Reporter Gene Assay

The pNF-B-SEAP reporter plasmid that contains four tandem copies of the NF-B consensus sequence, and permit expression of secretory alkaline phosphatase (SEAP) in response to NF-B activity was a gift form Dr. Jaw-Ji Yang (Chung Shan Medical University, Taiwan). When RAW264.7 cells reached confluence, transiently transfection of reporter plasmids and pSV--galactosidase control vector was performed using LipofectamineTM transfection reagent for 6 h. Cells were then changed into fresh culture media for 18 h before treating with vehicle control or LPS plus syringaldehyde or I. suffruticosa extracts for 48 h. Cell culture media were used to measure the fluorescence from the product of SEAP by Great EscAPe™ SEAP Chemiluminescence Detection Kits (C1ontech Laboratories, Inc., Mountain View, CA) and the supernatants of cell lysates were applied to measure the -galactosidase activity by -Galactosidase Enzyme Assay System with Reporter Lysis Buffer (Promega Corp).



2.12. High performance liquid chromatography /mass spectrometry (HPLC/MS) analysis

The phenolic compounds of I. suffruticosa extracts were determined by HPLC/MS method (Yao et al., 2011). The phenolic compounds were identified by their retention times, compared to those of the reference standards in HPLC systems and by the mass of the selected ions. The phenolic compounds in our in-house library were used as the reference standards. In the HPLC/MS system, an Agilent Zorbax Eclipse XDB-C18 column (5 m, 2503.0 mm i.d.) was used. The mobile phase consisted of solvents A (10 mM ammonia acetate containing 0.5% formic acid) and B (acetonitrile containing 0.5% formic acid). The gradient system was 10-90% B (0-45 min), 90-10% B (45-50 min), and 10% B (50-60 min). The flow rate was 0.6 ml/min. Data acquisition was via selected ion monitoring. Ions representing negative species of the compounds were selected, and peak areas were measured. The calibration curves of authentic standards were linear over the concentration range of 0.005-40 g/ml with correlation coefficients of 0.99 (Supplementary Fig. S1).



2.13. Determination of Plasma salicylic acid Concentrations

Six male 5-week-old C57BL/6JNarl mice weight 19-21g were fasted overnight before being administrated the water and ethanolic of I. suffruticosa (2.4 g/kg body weight by intragastric gavage). At 1 h after dosing, mice were sacrificed, and blood was collected. Plasma was separated from the blood by centrifugation (1750  g) at 4oC for 20 min. For determining phenolic compounds concentration in plasma, plasma (100 l) was mixed with 200 l of acetonitrile (1:2, v/v), vortexed, and then centrifuged at 10000  g for 20 min. The supernatant was used to determine the salicylic acid. Calibration standards of salicylic acid were prepared by serial dilution of a stock salicylic acid solution with blank plasma. The plasma salicylic acid concentration was determined by HPLC/MS as described above. The mice were treated in obedience to the Guide for the Care and Use of Laboratory Animals (NIH publication #85-23, 1985)



2.14. Statistical Analysis

Data are expressed as the meansSD from at least three independent experiments. Differences among treatments or time periods were analyzed by one-way analysis of variance and Tukey’s multiple-range test by using the Statistical Analysis System (Cary, NC). A value of p < 0.05 was considered statistically significant.



3. Results

3.1. Extraction yields and phytochemical constituents of I. suffruticosa extracts

The extraction yields of water and ethanolic extracts of I. suffruticosa were 1.580.16% and 1.670.16%, respectively. In addition, the phenolic contents, expressed as SE, of water and ethanolic extracts of I. suffruticosa were 55.222.71 and 53.353.70 mg/g dry extract, respectively. The amount of flavonoids, expressed as FE, of the ethanolic extract of I. suffruticosa was 2.060.37 mg/g dry extract, which was 8.24 times higher than that of the water extract.

In the I. suffruticosa extracts, eight phenolic compounds were identified and quantified by HPLC/MS, and salicylic acid (661.5 g/g) and syringic acid (861.3 g/g) had the highest concentration in the water and ethanolic extracts of I. suffruticosa, respectively (Table 1).

3.2. Effects of I. suffruticosa extracts on cell viability and LPS-induced nitrite production as well as iNOS and HO-1 expression in RAW264.7 macrophages

As shown in Table 2, nitrite production was substantially higher in cells treated with LPS than in those treated with the vehicle control (LPS treatment: 50.672.19 M vs. control: 0.970.43 M). With water and ethanolic extracts of I. suffruticosa treatments, LPS-induced nitrite production was significantly inhibited in a dose-dependent manner with a half maximal inhibitory concentration (IC50) of 879.2 g/ml and 151.9 g/ml, respectively (p < 0.05). Addition of 1000 g/ml I. suffruticosa water extract and of 200 g/ml I. suffruticosa ethanolic extract caused a 26% and 72% reduction in LPS-induced nitrite production, respectively.

The immunoblot assay showed that iNOS protein was hardly detectable in the resting RAW 264.7 macrophages, but was highly induced in the presence of LPS. The addition of exogenous water and ethanolic extracts of I. suffruticosa significantly reduced LPS-induced iNOS protein expression (p < 0.05, Fig. 1A). Real-time quantitative reverse transcriptase-polymerase chain reaction further showed that LPS-induced iNOS mRNA expression was significantly decreased by I. suffruticosa extracts (Table 2). Compared with the control, LPS induced a moderate increase of HO-1 expression, and this response was augmented by I. suffruticosa extracts, with an approximately 5.30.2 and 4.60.1-fold increase in HO-1 protein expression and 12.81.0 and 11.92.7-fold increase in HO-1 mRNA expression with 1000 g/ml water extract and 200 g/ml ethanolic extract of I. suffruticosa, respectively (Table 2, Fig. 1B). Notably, the inhibitory effects of I. suffruticosa extracts on LPS-induced iNOS expression were coincidentally correlated with I. suffruticosa extract-enhanced HO-1 expression.

Compared with the DMSO vehicle control, LPS slightly influenced cell viability as evaluated by use of the MTT assay (cell viability was 96.07.8% of the control). In the presence of LPS, both extracts of I. suffruticosa did not adversely affect the cell viability of RAW 264.7 macrophages at the test concentrations for 18 h (Table 2).



3.3. Effects of I. suffruticosa extracts and syringaldehyde on induction of HO-1 expression as well as ERK and Nrf2 activation in RAW 264.7 macrophages

The NO levels in cells treated with 60 g/ml of formononetin, quercetin, syringic acid, vanillin, and syringaldehyde were 87±5%, 5±1%, 94±3%, 97±2%, and 488%, respectively, of that of cells treated with LPS alone. Although quercetin was more potent to decrease LPS-induced NO production than syringaldehyde, the amount of quercetin in I. suffruticosa extract was much less than syringaldehyde. Consequently, we used syringaldehyde as a reference active compound of I. suffruticosa extracts.

In the RAW 264.7 macrophages treated with the water extract (1000 g/ml), the ethanolic extract (200 g/ml), and syringaldehyde (60 g/ml), HO-1 protein expression was induced in a time-dependent manner, and the maximum expression was at 12 h among the selected time points of treatment (Supplementary Fig.S2A). A significant induction of HO-1 mRNA expression after 12 h of treatment was exhibited in the macrophages treated with the I. suffruticosa extracts but not in those treated with syringaldehyde (Supplementary Fig.S2B).

We further examined the possible involvement of mitogen-activated protein kinase (MAPK) cascade activation in I. suffruticosa-induced HO-1 expression. Among the treatment groups, the water extract of I. suffruticosa had the highest induction of phosphorylation of ERK1/2 proteins (Supplementary Fig.S2C). The water extract of I. suffruticosa induced the maximum activation of ERK1/2 protein at 30 min, and this activation was sustained until 120 min.

We then examined whether I. suffruticosa extracts could induce Nrf2 activation, an important transcription factor in HO-1 expression (Alam et al., 1999). Syringaldehyde and both extracts, especially the water extract of I. suffruticosa, noticeably increased Nrf2-ARE binding affinity (Supplementary Fig.S2D). The specificity of Nrf2 binding was shown by competition assays with excess amounts of the unlabeled ARE probe and the mutant biotin-labeled NF-B probe.

3.4. Effect of I. suffruticosa extract-induced HO-1 expression on LPS-induced inflammatory responses

We subsequently examined whether HO-1 induction was crucial to the anti-inflammatory action of I. suffruticosa. Pretreatment of cells with I. suffruticosa extracts and syringaldehyde for 12 h significantly reduced LPS-induced NO production and the mRNA levels of iNOS, TNF-, and IL-1 (Supplementary Table S1) as well as protein expression of iNOS and IL-1 (Supplementary Fig.S3). An HO-1 si RNA SMARTpool system was applied to knock down HO-1 expression induced by I. suffruticosa extracts. HO-1 siRNA diminished the inhibitory activity of I. suffruticosa extracts on LPS-induced NO production and iNOS protein expression (Fig. 2). These results suggest the importance of HO-1 in the inhibitory effects of I. suffruticosa extracts on LPS-induced inflammatory responses.



3.5. Effects of I. suffruticosa extracts and syringaldehyde on LPS-induced NF-B activation

Upon LPS treatment, the phosphorylated IB protein expression tremendously increased when compared with the control. LPS-induced phosphorylated IB- expression was abolished by both extracts but not by syringaldehyde (p < 0.05, Fig 3A).

EMSA experiments were used to evaluate the effect of I. suffruticosa extracts on the activation of NF-B (Fig. 3B). After treatment with LPS, the DNA-binding activity of the NF-B nuclear protein was markedly higher than that of the vehicle control. LPS-induced NF-B nuclear protein-DNA binding activity was noticeably reduced in cultures pretreated with water and ethanolic extracts of I. suffruticosa. The specificity of NF-B binding was shown by competition assays with excess amounts of the unlabeled probe and the mutant biotin-labeled probe of NF-B.

To investigate the transcriptional activity of NF-B, the expression of reporter genes in cells transfected transiently with pNF-B-SEAP and the internal control pSV--galactosidase was analyzed. Consistent with the above results, the expression of LPS-induced NF-B-SEAP activity was significantly inhibited in cultures treated with I. suffruticosa extracts but not in those treated with syringaldehyde (Fig. 3C, p < 0.05).



3.6. Plasma salicylic acid concentrations in mice

About 1 h after the oral administration of a single dose (2.4 g/kg body weight) of water and ethanolic extracts of I. suffruticosa to C57BL/6JNarl mice, salicylic acid could be detected in the plasma. The concentrations of salicylic acid in the plasma were 1574.2349.4 ng/ml and 786.3113.6 ng/ml, respectively, after administered with water and ethanolic extracts of I. suffruticosa. Other selected phenolic compounds in I. suffruticosa extracts were not detectable in the plasma.



4. Discussion

I. suffruticosa is grown in the northeast countryside of Brazil and is a popular herbal medicine with anti-inflammation and anti-epilepsy effects (Leite et al., 2003; Wong et al., 1999). Since Chinese people most often use water or ethanol to decoct herbal medicines, we investigate the anti-inflammatory properties of water and ethanolic extracts of I. suffruticosa and analyze the possible molecular mechanism involved in this anti-inflammatory action. Both the water and ethanolic extracts of I. suffruticosa significantly decreased NO production as well as expression of iNOS, IL-1, and TNF- in LPS-induced RAW 264.7 macrophages. The anti-inflammatory property of I. suffruticosa is likely associated with its ability to induce HO-1 expression and diminish LPS-induced NF-B activation.

Through down-regulation of inflammatory cytokine expression, HO-1 induction exerts therapeutic effects in a variety of diseases, such as cardiovascular diseases and neurodegenerative diseases (Chan et al., 2011; Jazwa and Cuadrado, 2010). Our data demonstrated that I. suffruticosa extracts are transcriptional inducers of HO-1 and that this effect may be related to the I. suffruticosa-induced activation of Nrf2, a key transcription factor in HO-1 expression (Alam et al., 1999). A blockade of I. suffruticosa-induced HO-1 expression with siRNA diminished the efficacy of I. suffruticosa in suppressing iNOS expression and NO production in LPS-stimulated macrophages. These data suggest the involvement of increased HO-1 expression in the inhibitory effects of I. suffruticosa extracts on LPS-induced inflammatory responses. A number of upstream signaling pathways, including MAPK, have been shown to convey the extracellular signals to activation of the Nrf2/ARE pathway for transcriptional induction of HO-1 in response to various HO-1 inducers (Alam et al., 2007). Our data also demonstrated that the activation of ERK1/2 was significantly induced by the water extract but not the ethanolic extract of I. suffruticosa. These data were consistent with our finding regarding HO-1 expression, which showed that the water extract was more potent than the ethanolic extract in the activation of Nrf2 and the induction of HO-1 expression. These data indicated that, besides Nrf2/HO-1 pathway, the other mechanism is behind the anti-inflammatory activity of ethanolic extract of I. suffruticosa.

In macrophages, activation of NF-B is considered crucial in LPS-induced inflammation due to its ability to induce pro-inflammatory gene expression (Tak et al., 2001). Our data clearly showed that I. suffruticosa extracts effectively inhibited the NF-B pathway by blocking LPS-induced IB- phosphorylation and subsequent DNA binding affinity and transcriptional activity of NF-B. These data suggested that direct modification of NF-B activation by I. suffruticosa extracts may account for the inhibitory effect of I. suffruticosa on the expression of pro-inflammatory mediators, including iNOS, TNF-, and IL-1. Given that the blocking of NF-B activation in macrophages is a clinical approach to the treatment of inflammation (Chen et al., 1999; Makarov, 2000), the therapeutic role of I. suffruticosa in chronic inflammatory diseases are worth investigating.

Our data demonstrated that, among the test compounds, 60 μg/ml syringaldehyde showed the most potent inhibitory effect on inflammatory responses in LPS-stimulated macrophages. The test concentrations (20~60 μg/ml) do not exist in the composition of I. suffruticosa extracts. Notably, salicylic acid, an anti-inflammatory phenolic compound (Fürst et al., 2006), was found in the mouse plasma 1 h after oral administration with I. suffruticosa extracts. The different absorption rate of phenolic compounds in I. suffruticosa extracts could result in that only salicylic acid was found in the plasma of mice after 1h administration of I. suffruticosa extracts. Moreover, the diverse reactions of metabolization and biotransformation among the phenolic compounds of I. suffruticosa extracts could cause the discrepancy of phenolic compound profiles between plasma and original dietary source. In vivo data presented here suggest that salicylic acid may be an active compound in I. suffruticosa extracts, as an anti-inflammatory agent. While we cannot exclude that all phenolic compounds in the I. suffruticosa extract together contribute to its anti-inflammatory properties. Our data showed that the total phenolic contents of both extracts of I. suffruticosa were similar, but the amount of flavonoids in the water extract was far less than that in the ethanolic extract. Whether different profiles of phenolic compounds between water and ethanolic extracts of I. suffruticosa account for the diverse effects of I. suffruticosa extracts on ERK/Nrf2/HO-1 pathway and healthy benefits is worth further studying.

In conclusion, we demonstrated that both water and ethanolic extracts of I. suffruticosa modulate LPS-induced inflammatory events in RAW 264.7 macrophages. Apart from decreased NF-B activation, the induction of HO-1 expression mediated by activation of the ERK-Nrf2 signal pathway is involved in the reduction of LPS-induced iNOS expression and, subsequent NO production. Our experimental results revealed that the water and ethanolic extracts of I. suffruticosa may be considered as candidates for the treatment and prevention of chronic inflammation-related diseases. Further studies are required to assess the anti-inflammatory potency of I. suffruticosa in an in vivo model.
Conflict of Interest

The authors declare that there are no conflicts of interest.



Acknowledgements

This research was funded by the National Science Council, Republic of China, under Grant 99-2320-B-040 -005 -MY3 and by Chung Shan Medical University Hospital, under Grant CSH-2010-C-006.

ABI 7000 Real Time PCR System was performed in the Instrument Center of Chung Shan Medical University.

Figure captions

Fig. 1. Effects of water extracts (WE) and ethanol extracts (EE) of Indigofera suffruticosa Mill (IDS) on LPS-induced iNO and HO-1 protein expressions in RAW 264.7 macrophages. Cells were treated with or without LPS (0.01 g/ ml) plus vehicle control and various concentrations of WE and EE of IDS for 18 h. Cells were lysed and Western blot analysis was used to measure the protein content of iNOS and HO-1. Data are the mean  SD of at least four separate experiments. Values are expressed as the percentage of the culture treated with LPS alone (iNOS) and as the fold induction relative to the control treatment (HO-1). Values not sharing the same letter are significantly different (p < 0.05).

Fig. 2. Effect of HO-1 siRNA on the inhibitory activity of WE and EE of IDS on LPS-induced NO production and iNOS expression in RAW 264.7 macrophages. Cells were transfected in Opti-MEM (Invitrogen) using lipofectamine (Invitrogen) with HO-1 siRNA SMARTpool® or NTC siRNA according to the manufacturer’s instructions. Cells were then treated with IDS extracts 12 h prior to LPS addition (0.01 g/ml) for 18 h. Griess assay and Western blot analysis were used to measure the nitrite production (A) as well as iNOS and HO-1 protein expressions (B), respectively. Data are the mean  SD of at least three separate experiments and are expressed as the percentage of the culture transfected with NTC si RNA and treated with LPS alone. Within the same siRNA treatment, values not sharing the same letter are significantly different (p < 0.05). An asterisk (*) indicates a significant difference between the mean of cells transfected with NTC siRNA and that of cells transfected with HO-1 siRNA within the same treatment (p < 0.05).

Fig. 3. Effects of WE and EE of IDS on LPS-induced the activation of NF-B. Cells. were preincubated with WE and EE of IDS for 12 h and then treated with either vehicle control or LPS (0.01 g/ml) for 2 h. Western blot analysis was used to measure the protein content of phosphorylated IB- in the cytosolic factions (A). EMSA experiments were carried out by using the LightShift Chemiluminescent EMSA Kit from Pierce Chemical Co. The unlabeled double-stranded oligonucleotide of NF-B and the unlabeled double-stranded mutant NF-B oligonucleotide were added for the competition assay and specificity assay, respectively (B). Cells were transiently transfected with pSV--galactosidase and pNF-B-SEAP reporter gene for 6 h. After washing with PBS, cells were treated were preincubated with WE and EE of IDS for 12 h and then treated with either DMSO vehicle control or LPS (0.01 g/ml) for 48 h. The fluorescence from the product of SEAP was measured by using a fluorometer (VersaFluorTM fluorometer, Bio-Rad Laboratories, Hercules, CA) and the -galactosidase activity was measured by -Galactosidase Enzyme Assay System with Reporter Lysis Buffer from Promaga Corp (C). Data are the mean  SD of at least three separate experiments and are expressed as the percentage of the culture treated with LPS alone. Values not sharing the same letter are significantly different (p < 0.05).

Table 1. Concentration of phenolic compounds in water extract (WE) and ethanolic extract (EE) of Indigofera suffruticosa Mill. (IDS, g/g dry weight)

Component

Retention time (min)

Amount (g/g dry weight)*

WE-IDS

EE-IDS

Syringic acid

7.6

530.9

861.3

p-coumaric acid

9.8

16.9

38.7

Vanillin

10.0

166.2

276.2

Syringaldehyde

10.2

49.5

131.6

Salicylic acid

15.3

661.5

750.9

Quercetin

15.8

0.27

6.4

Isoliquiritigenin

20.3

0.06

81.5

Formononetin

20.9

0.03

6.2

*Values are the mean of three determinations

Table 2. Effects of water extract (WE) and ethanolic extract (EE) of I. suffruticosa (IDS) on MTT assay and LPS-induced nitrite production as well as iNOS and HO-1 mRNA expression in RAW 264.7 macrophages



Treatment*

MTT#

Nitrite#

iNOS#

HO-1$

Control

104.78.4a

13.12.7f

2.51.3d

1.00.0e

LPS

100.00.0b

100.00.0a

100.00.0a

2.70.1e

LPS+ WE-IDS 250 g/ml

101.40.2b

83.54.3b

74.013.2b

7.40.4cd

LPS+ WE-IDS 500 g/ml

101.35.2b

63.66.0c

68.96.5b

9.70.0bc

LPS+ WE-IDS 1000 g/ml

108.52.5ab

47.23.0d

63.82.7cb

12.81.0a

LPS+ EE-IDS 50 mg/ml

103.5±1.8b

79.6±2.7b

69.7±7.6b

6.4±0.5d

LPS+ EE-IDS 100 mg/ml

98.8±2.5b

63.8±2.7c

69.4±2.0b

9.2±0.6bcd

LPS+ EE-IDS 200 mg/ml

99.9±5.2b

37.9±3.0e

49.1±5.1c

11.9±2.7ab

*RAW 264.7 macrophages were treated with or without LPS (0.01 g/ml) plus vehicle control, WE-IDS and EE-IDS for 18 h (MTT assay and nitrite production) or for 8 h (iNOS and HO-1 mRNA expression).

#Data are the mean  SD of at least three separate experiments and are expressed as the percentage of the culture treated with LPS alone. Values in the same column with different letters are significantly different (P < 0.05).

$Data are the mean  SD of at least three separate experiments and are expressed as the fold induction relative to the control treatment. Values in the same column with different letters are significantly different (P < 0.05).

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