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Differential effects of Radix Paeoniae Rubra (Chishao) on cytokine and chemokine expression inducible by mycobacteria

  • 1,
  • 1,
  • 2,
  • 4,
  • 4,
  • 1, 2 and
  • 1, 2, 3Email author
Chinese Medicine20116:14

  • Received: 2 April 2010
  • Accepted: 30 March 2011
  • Published:



Upon initial infection with mycobacteria, macrophages secrete multiple cytokines and chemokines, including interleukin-6 (IL-6), IL-8 and tumor necrosis factor-α (TNF-α), to mediate host immune responses against the pathogen. Mycobacteria also induce the production of IL-10 via PKR activation in primary human monocytes and macrophages. As an anti-inflammatory cytokine, over-expression of IL-10 may contribute to mycobacterial evasion of the host immunity. Radix Paeoniae Rubra (RPR, Chishao), a Chinese medicinal herb with potentials of anti-inflammatory, hepatoprotective and neuroprotective effects, is used to treat tuberculosis. This study investigates the immunoregulatory effects of RPR on primary human blood macrophages (PBMac) during mycobacterial infection.


The interaction of Bacillus Calmette-Guerin (BCG) with PBMac was used as an experimental model. A series of procedures involving solvent extraction and fractionation were used to isolate bioactive constituents in RPR. RPR-EA-S1, a fraction with potent immunoregulatory effects was obtained with a bioactivity guided fractionation scheme. PBMac were treated with crude RPR extracts or RPR-EA-S1 before BCG stimulation. The expression levels of IL-6, IL-8, IL-10 and TNF-α were measured by qPCR and ELISA. Western blotting was used to determine the effects of RPR-EA-S1 on signaling kinases and transcriptional factors in the BCG-activated PBMac.


In BCG-stimulated macrophages, crude RPR extracts and fraction RPR-EA-S1 specifically inhibited IL-10 production while enhanced IL-8 expression at both mRNA and protein levels without affecting the expressions of IL-6 and TNF-α. Inhibition of BCG-induced IL-10 expression by RPR-EA-S1 occurred in a dose- and time-dependent manner. RPR-EA-S1 did not affect the phosphorylation of cellular protein kinases including MAPK, Akt and GSK3β. Instead, it suppressed the degradation of IκBα in the cytoplasm and inhibited the translocation of transcription factor NF-κB1 p50 to the nucleus.


RPR crude extracts and its fraction RPR-EA-S1 inhibited anti-inflammatory cytokine IL-10 and enhanced pro-inflammatory chemokine IL-8 expression in BCG-activated PBMac. The inhibitory effects of RPR-EA-S1 on IL-10 expression in BCG-activated PBMac may be due to the reduced nuclear translocation of NF-κB1 p50.


  • High Performance Liquid Chromatography
  • Chemokine Expression
  • Immunoregulatory Effect
  • Autologous Plasma
  • Invigorate Blood Circulation


Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide as a result of Mycobacterium tuberculosis (Mtb) infection. In 2008, an estimated 9.4 million new cases of TB and 1.3 million deaths were recorded globally [1]. Although anti-TB drugs and vaccines have been used for decades, the emergence of multidrug-resistant TB (MDR-TB) and the co-infection of Mtb and related mycobacteria with human immunodeficiency virus (HIV) pose new challenges to the treatment of TB [1, 2].

Upon Mtb infection, multiple immune cells including macrophages/monocytes [3], dendritic cells (DCs) [4], neutrophils [5], natural killer cells [6] and T cells [6] are activated to mediate host defense against the pathogen. Among these cell types, macrophages are the main immunocytes in initiating innate immunity against Mtb. Macrophages are professional antigen presenting cells that are important in bridging innate and adaptive immunity [7]. Macrophages exert their direct anti-microbial effects through phagocytosis [8], generation of reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs) [9], autophagy [10], activation of vitamin D pathway [11], apoptosis [12] and secretion of cytokines and chemokines [13].

Specific cytokines and chemokines produced by macrophages during Mtb infection play important roles in regulating the host innate and adaptive immune responses [13]. For example, tumor necrosis factor-α (TNF-α) enhances the activity of macrophage to kill replicating Mtb in synergy with interferon-γ (IFN-γ) [14] and contributes to the formation of granuloma and prevention of mycobacterial dissemination through decreased cell migration [15]. Another key cytokine is interleukin-6 (IL-6), which is a pro-inflammatory cytokine that regulates B and T cell activities and contributes to the initial innate responses to Mtb [16].

IL-8, another important chemokine against Mtb, is primarily secreted by monocytes and macrophages but also produced by fibroblasts, keratinocytes, and lymphocytes [17]. The induction of IL-8 can be stimulated by multiple factors including pathogens, toxins and cytokines [18]. Previous studies have demonstrated the connection between IL-8 production and Mtb infection. In clinical studies, remarkably elevated levels of IL-8 were found in tuberculous pleural exudate, bronchoalveolar lavage fluid, cerebrospinal fluid and tuberculous granulomas [19]. In contrast, decreased IL-8 secretion was observed in HIV-infected patients with miliary TB, suggesting that the absence of IL-8 induction is detrimental to restricting Mtb dissemination [20]. Such important role of IL-8 was illustrated in a previous study that upon Mtb infection IL-8 attracted neutrophils and T lymphocytes to the infection sites at an early stage, resulting in the formation of granuloma [21].

Unlike the pro-inflammatory cytokines IL-6 and IL-8, interleukin-10 (IL-10) is a key anti-inflammatory cytokine produced by macrophages, Th1 cells [22], Th2 cells [22], regulatory CD4+ T cells [23], CD8+ T cells [23], DCs and B cells [24]. IL-10 mainly plays a negative role in the regulation of immunity to prevent uncontrolled responses to pathogens [25, 26]. It inhibits the production of pro-inflammatory cytokines, such as IFN-γ, IL-12, IL-18, IL-1 and TNF-α, by macrophages and DCs [27]. Together with reduced induction of inducible nitric oxide synthases (iNOS) and ROIs, IL-10 expression results in reduced killing of intracellular pathogens by macrophages [28]. IL-10 is also an inhibitor of antigen presentation by macrophages and DCs through down-regulation of MHCII expression [2931]. In the event of TB progression, over-expression of IL-10 results in a higher mycobacterial burden and reactivation of pulmonary TB [32]. Macrophages from IL-10 deficient mice, when challenged with BCG, produce higher levels of TNF-α with concomitant accelerated clearance of mycobacteria [33]. These observations were further validated by clinical studies, showing higher levels of IL-10 production in patients with active pulmonary TB [34, 35]. Moreover, overproduction of IL-10 by T cells was shown to be related to suppressed immunity and increased susceptibility to mycobacteria infection [36].

Radix Paeoniae Rubra (RPR, Chishao), the dried root of Paeonia lactiflora Pallas or Paeonia veitchii Lunch [37], has been widely used by Chinese medicine practitioners to treat cardiovascular, inflammation and female reproductive diseases [38]. Based on the principle of Chinese medicine, historical literatures described RPR with the functions of tonifying blood, cooling blood, cleansing heat, and invigorating blood circulation. It is often used as an "Assistant" herb to counteract or ameliorate the undesirable side effects of the "King" herbs [37]. In contemporary literatures, RPR is used for the treatment of the following diseases: seasonal febrile diseases with eruptions, bleeding, menstrual disorders, trauma, skin infection and conjunctivitis as well as pain over the chest, hypochondrium and abdomen [39]. RPR is also used in some herbal formulae to treat TB patients [40]. RPR has protective effects on lung injury through the induction of heme oxygenase-1 (HO-1) and suppression of nitric oxide (NO) in rats [41, 42]. In a mouse model, the extracts obtained from RPR and Astragalus membranaceus protected the liver from BCG/endotoxin-induced injury. These protective effects were associated with downregulation of pro-inflammatory cytokines [43]. While some studies on the biological effects and chemical constituents of RPR have been reported [38], the immunoregulatory effects of RPR and their detailed mechanisms of action at cellular signaling levels are yet to be investigated, especially the interaction of BCG with human blood macrophages.

In the present study, an active fraction, namely RPR-EA-S1, was isolated from the crude extracts of RPR using a bioactivity-guided fractionation scheme. The chemical profile of RPR-EA-S1 was analyzed by high performance liquid chromatography (HPLC) and gas chromatography mass spectrometry (GC-MS). The regulatory effects of crude RPR extract and its fraction RPR-EA-S1 on BCG-induced cytokine and chemokine expression in human PBMac were examined. The underlying mechanisms of the inhibitory effects of RPR-EA-S1 on BCG-induced IL-10 expression were delineated.


Plant material

Radix Paeoniae Rubra was obtained from PuraPharm International Ltd. (Hong Kong, China). The raw material of the herb was authenticated by PuraPharm Corporation (Guangxi, China) according to the method in the Pharmacopoeia of the People's Republic of China 2005 [44] which states that the RPR sample should not contain less than 1.8% of paeoniflorin, calculated with reference to the dried substance. The Radix Paeoniae Rubra used in our study contained more than 4% of paeoniflorin.

Preparation of the crude extract from Radix Paeoniae Rubra

The procedures for raw herb extraction and fractionation were shown in Figure 1. Briefly, the herbs (300 g) were ground into powder and extracted with eight folds (volume) of Milli Q water (Sartorius, Germany) under reflux for one hour. The extraction was repeated for three times. The resulting extracts were combined and concentrated at 70°C under reduced pressure until the density reached 1.28 g/mL. Then the paste collected was precipitated with five folds (volume) of ethanol (EtOH). After filtration, the supernatant was evaporated to dryness in an evaporator (Rotavapor R-200, BUCHI, Switzerland), yielding crude RPR extract (81 g). The extract was then dissolved in methanol (MeOH) and partitioned with an equal volume of hexane (n-C6H14) for three times. The MeOH extract was dried and re-dissolved in water and then partitioned sequentially with an equal volume of ethyl acetate (EtOAc) and n-butanol (n-BuOH) for four times. Four fractions were obtained, namely RPR-H, RPR-EA (10 g), RPR-Bu and RPR-W. According to a bioassay guided scheme, the fraction RPR-EA with the most potent activity on IL-10 suppression was selected and further separated by silica gel column chromatography (Merck, Germany). After loading the sample, the column was washed sequentially with a series of solvents: 50% n-C6H14 in EtOAc, 100% EtOAc, EtOAc combined with 10%, 30%, 50% or 70% of MeOH and finally 100% MeOH. These serial washings yielded seven fractions: RPR-EA-S1 (0.78 g), RPR-EA-S2, RPR-EA-S3, RPR-EA-S4, RPR-EA-S5, RPR-EA-S6 and RPR-EA-S7.
Figure 1
Figure 1

Extraction and separation scheme of RPR-EA-S1 from RPR. RPR powder was extracted by Milli Q water under reflux and the extract was precipitated with EtOH. The resulting supernatant was concentrated and partitioned sequentially with n-C6H14, EtOAc and n-BuOH. The extracts were tested for their inhibitory effects on IL-10 production in primary human blood macrophages stimulated by BCG. The bioactive RPR-EA was further separated by silica gel column chromatography. RPR-EA-S1 fraction was selected by bioactivity assay and analyzed by HPLC and GC-MS.

Chemicals and antibodies

All the chemical solvents such as EtOH, MeOH, n-BuOH, EtOAc and n-C6H14 were purchased from Merck (Germany). Antibodies against NF-κB1 p50, actin, IκBα and Lamin B were purchased from Santa Cruz Biotechnology (USA). Antibodies against phospho-ERK1/2, phospho-p38, phospho-Akt and phospho-GSK3β as well as antibodies against total ERK1/2, p38, Akt and GSK3β were purchased from Cell Signaling Technology (USA). Anti-rabbit IgG HRP-conjugated secondary antibodies were purchased from BD Biosciences (USA). Anti-goat IgG HRP-conjugated secondary antibodies were purchased from ZYMED Laboratory (USA).

Bacillus Calmette-Guerin

Bacillus Calmette-Guerin (BCG) is a live freeze-dried vaccine made from an attenuated strain of Mycobacterium bovis, Danish strain 1331. The vaccine, which contains no virulent Mtb, was purchased from Statens Serum Institut (Denmark).

Isolation of primary human peripheral blood macrophages

Using Ficoll-Paque (GE Healthcare, USA) density gradient centrifugation as previously described [3, 29], we isolated primary human peripheral blood macrophages (PBMac) from buffy coats of healthy blood donors obtained from the Hong Kong Red Cross Blood Transfusion Service. Briefly, the fresh blood samples were centrifuged at 3000 rpm (1811g, Eppendorf AG, Germany) for 20 minutes and separated into plasma and blood cell layers. The plasma layer was carefully removed and heat-inactivated for 30 minutes at 56°C in water bath, followed by chilling on ice for ten minutes. It was centrifuged at 4000 rpm (3220g, Eppendorf AG, Germany) for ten minutes to remove the precipitates. The clear supernatant was then filtered through 0.45 μm membranes and later used as the autologous plasma added to culture macrophages. The blood cell layer was diluted with an equal volume of phosphate buffered saline (PBS) at a ratio of 1:1 and then slowly overlaid over the Ficoll and centrifuged for 20 minutes at 2300 rpm (1065g, Eppendorf AG, Germany). The white blood cell layer was removed and washed with RPMI 1640 (Invitrogen, USA) for three times and the supernatant was clear after washes. The cell pellet was re-suspended with RPMI medium containing 5% autologous plasma and 1% penicillin/streptomycin and plated onto a tissue culture Petri dish. The dish was incubated at 37°C in a humidified atmosphere with 5% CO2 for one hour to allow monocytes to adhere. The unattached cells were washed away by warm RPMI medium and the adherent monocytes were incubated at 37°C overnight. Subsequently, cold RPMI medium with 5 mM ethylenediaminetetraacetic acid (EDTA) was used to wash and detach the monocytes, which were finally seeded onto the tissue culture plates in RPMI medium supplemented with 5% autologous plasma and 1% penicillin/streptomycin. After 14 days of culturing, with replenishment of medium every three to four days, the monocytes derived macrophages were ready for treatment.

Total RNA extraction and reverse transcription

Total RNA was extracted with TRIzol Reagent (Invitrogen, USA). SuperScript II transcriptase (Invitrogen, USA) was used to synthesize cDNA from the total RNA according to the manufacturer's instructions. The cDNA samples were kept at -20°C until use.

Quantitative real-time PCR

The expression levels of IL-6, IL-8, IL-10 and TNF-α mRNA were determined with quantitative real-time PCR (q-PCR) on an ABI 7500 system using TaqMan (Applied Biosystems, USA). The relative quantification method was used to calculate the mRNA expression levels [45]. Briefly, we applied a 20 μL reaction system containing the sample cDNA, TaqMan 2× Master Mix, TaqMan probes for target genes (gene-specific Assays-on-Demand reagent kits, Applied Biosystems, USA) and TaqMan probe for 18 s rRNA as the internal control. Each sample was run in duplicates. The threshold cycle number (Ct) of the target gene was normalized to the Ct of 18 S rRNA for each sample (-ΔCt). The -ΔCt value of the mock-treated sample was subtracted from the -ΔCT value of other samples, yielding -ΔΔCt. And the value of 2-ΔΔCt refers to the fold change of target gene's mRNA expression level compared to that of the corresponding mock-treated sample.

Enzyme-linked immunosorbent assay (ELISA)

To study the RPR's differential effects on cytokines inducible by BCG, we applied ELISA to measure the amounts of IL-6, IL-8, IL-10 and TNF-α in the cell culture supernatants. Briefly, macrophages (PBMac) derived from human primary monocytes were seeded onto a 24-well plate at a concentration of 106/mL. After pretreatment of PBMac with the RPR extract or its designated partially purified fractions for 16 hours, BCG (MOI = 1) was added to the cell cultures. After 24 hours of incubation, the supernatants were collected and the concentrations of cytokines were measured with ELISA according to the manufacturer's instructions (R&D Systems, USA).

Western blot analysis

Cytoplasmic proteins and nuclear proteins were extracted separately with buffer A and buffer C as previously described [46] with modifications. Cells were washed twice with cold PBS and lysed on ice with buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 2 μg/mL aprotinin, 1 mM sodium orthovanadate, 2 μg/mL pepstatin, 2 μg/mL leupeptin and 50 mM sodium fluoride) for 15 minutes. Then 10% NP-40 was added to the cells at a final concentration of 0.625%, followed by several seconds of vortexing. The cells were then removed with a clean scraper. The lysate was then centrifuged at 15000g for 30 seconds. The supernatant containing cytoplasmic proteins was stored at -70°C until use. The pellet was washed gently with buffer A and centrifuged again (15000g, Eppendorf AG, Germany) to obtain a pellet by discarding the supernatant. The nuclear proteins were extracted by re-suspending the pellet in buffer C (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1 μg/mL aprotinin, 1 mM sodium orthovanadate, 2 μg/mL pepstatin, 2 μg/mL leupeptin and 50 mM sodium fluoride). The sample was lysed on ice for 15 minutes with vortexing every five minutes. The nuclear extract was centrifuged (15000g, Eppendorf AG, Germany) at 4°C for five minutes and the supernatant was stored at -70°C until use.

Protein concentrations were quantified with the Bio-Rad protein assay kit (USA). Then an equal amount of proteins (30 μg of cytoplasmic or 4 μg of nuclear proteins) was separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. After three hours of incubation with 1% BSA in tris-buffered saline Tween-20 (TBST), the membrane was incubated with a specific antibody (1:1000 dilution) to the protein of interest for overnight at 4°C. Then the membrane was washed with TBST and incubated with the corresponding secondary antibody (1:4000 dilution) for one hour at room temperature. After five times of washes, the bands in the membrane were detected with a GE Healthcare Enhanced Chemiluminescence System (USA) according to the manufacturer's instructions. ImageJ (National Institutes of Health, USA) was used to quantify the levels of proteins captured in the laser densitometry results.

MTT assay

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were used to test the cytotoxicity of the RPR extract and its partially purified fractions on the cells. PBMac were seeded at a concentration of 106/mL in a 24-well plate. After 48 hours of treatment with the RPR extract or its designated fractions, the cells were incubated with RPMI 1640 medium containing 0.5 mg/mL MTT (Invitrogen, USA) for one hour. Then the cell culture medium was removed and isopropanol (IPP) was added to the wells and incubated at room temperature for ten minutes. Absorbance values of the culture medium at 570 nm were measured with a microplate reader (Bio-Rad, USA).

High performance liquid chromatography (HPLC) analysis of the extracts

The RPR extract and its fractions were dissolved in HPLC grade MeOH (Merck, Germany) before analysis. HPLC was performed with an Agilent 1200 liquid chromatography system (Agilent, USA) equipped with a reverse-phase HPLC column (Lichrospher 100, RP C18 EC 5 μm, 250 × 4.6 mm ID) described in our previous reports [47, 48]. A gradient elution from 10% acetonitrile (CH3CN) to 90% CH3CN at a flow rate of 1 mL/min was used to separate the peaks which were detected at 210 nm with an Agilent 1200 series (Agilent, USA), a fast scanning photodiode array detector.

Gas chromatography mass spectrometry

Before GC-MS analysis, the RPR extracts first underwent silylation. Briefly, 100 μL RPR-EA-S1 in CH3CN was mixed with 50 μL of pyridine and 50 μL of the derivatizing agent BSTFA [N, O-bis(trimethylsilyl)trifloroacetamide] in a 1 mL reaction vial (Alltech, USA). After incubation for two hours at 70°C, bis-trimethyl silyl trifluoroacetamide (BSTFA) replaced the labile hydrogen atom with a -Si(CH3)3 group on a wide range of polar compounds. Then the resulting mixture was analyzed by GC-MS (GC: 7890A, MS: 5975C, Agilent, USA) equipped with a HP-5MS column (Agilent, USA) (30 m × 250 μ m × 0.25 μ m). The sample (1 μl) was injected to the column. Helium was used as the carrier gas at a flow rate of 1 mL/min. The oven temperature was started at 70°C for one minute, and then increased to 180°C at a rate of 10°C/min; after two minutes' holding, increased to 280°C at a rate of 10°C/min and held for three minutes. The interface temperature was 250°C, ion source temperature was 230°C, and electron impact ionization (EI) was at 200 eV. Mass spectra were analyzed in the range of 50-700 atom mass units (amu) for a run time of 22 minutes. The G1701EA Chemstation (Agilent, USA) was used to perform the MS data analysis. The peaks of the eluants with more than 90% similarity to the compounds listed in the NIST GC-MS library were selected.

Statistical analysis

All data were presented as the mean ± standard deviation (SD). Data were analyzed with the SPSS statistical package (version 11.5.0, SPSS Inc, USA). The statistical differences in the respective protein levels and mRNA levels among treatments were tested by one-way ANOVA followed by an Honestly Significant Difference (HSD) post-hoc Tukey test. P value of less than 0.05 is considered statistically significant.


Extraction and bioassay guided fractionation of RPR

Using solvent partitioning extraction, we obtained RPR-H, RPR-EA, RPR-Bu and RPR-W. MTT assays showed that all four fractions were not toxic to PBMac at 50 μg/mL (Additional file 1). Therefore, each fraction at 50 μg/mL was tested for their inhibitory effects on BCG (MOI = 1, MOI: multiplicity of infection) induced IL-10 production in PBMac. We reported previously that 24 hours of BCG stimulation was an optimal time point for studying BCG-induced IL-10 expression [30]. The present study used the same time point. The ELISA assays found that PBMac pretreated with RPR-EA showed 68% reduction in IL-10 production induced by BCG while RPR-H, RPR-Bu and RPR-W did not show any inhibitory effect (Figure 2A). Using silica gel column chromatography, we further separated RPR-EA, the most potent fraction, into seven sub-fractions, namely RPR-EA-S1 to RPR-EA-S7 (Figure 1). Additional MTT assays demonstrated that none of them was toxic to PBMac at 50 μg/mL (Additional file 2). The ELISA results found that pretreatment of PBMac with fractions RPR-EA-S1, RPR-EA-S2 and RPR-EA-S6 (20 μg/mL) showed 66%, 40% and 62% reduction of BCG-induced IL-10 production respectively whereas the remaining fractions did not have inhibitory effects (Figure 2B). RPR-EA-S1, the most biologically active fraction, was selected for further analysis and investigation. The chemical profiles of the crude RPR extract as well as RPR-EA and RPR-EA-S1 were analyzed with reverse phase HPLC (Additional file 3).
Figure 2
Figure 2

Bioassay guided fractionation of RPR. PBMac (5 × 105) were pretreated with 0.05% DMSO or the indicated RPR fractions (A) or RPR-EA subfractions (B) overnight and then stimulated with BCG (MOI = 1). Culture supernatants were collected after 24 hours. The levels of IL-10 in the supernatants were measured by ELISA. The IL-10 concentration in the supernatant of DMSO + BCG sample was set as 100%, and the rest were compared to it to obtain a percentage value. Results are shown as mean ± SD from independent experiments on PBMac obtained from three different healthy donors. *P < 0.05, **P < 0.001 compared to the DMSO + BCG sample (one-way ANOVA, Tukey's test).

Differential effects of RPR extracts on BCG-induced cytokine and chemokine expression

Since cytokines and chemokines secreted by macrophages play a critical role in the host immune responses against mycobacteria, we examined the RPR's regulatory effects on BCG-induced IL-6, IL-8, IL-10 and TNF-α expression in PBMac. Pretreatment of the immune cells with RPR extract had no effect on BCG-induced IL-6 or TNF-α expression; however, it significantly reduced the production of IL-10 and increased that of IL-8 at both mRNA and protein levels (Figure 3). The differential effects of RPR on BCG-induced cytokine and chemokine expression were further confirmed with three batches of RPR extracts (Additional file 4).
Figure 3
Figure 3

Effects of RPR crude extract on the expression levels of cytokines in BCG-stimulated PBMac. PBMac (5 × 105) were pretreated with 0.1% DMSO or 100 μg/mL RPR extract overnight and then stimulated with BCG (MOI = 1). (A) Total mRNA was extracted after 3 hours for cDNA synthesis. The levels of mRNA were determined by q-PCR. The mRNA level of the DMSO + BCG sample was set as 100%, and the rest were compared to the DMSO + BCG sample to obtain a percentage value. (B) Culture supernatants were collected after 24 hours. The levels of IL-6, IL-8, IL-10 and TNF-α in the supernatants were measured by ELISA. The individual cytokine level in the supernatant of the DMSO + BCG sample was set as 100%, and the rest were compared to it to obtain a percentage value. Results are shown as mean ± SD from independent experiments on PBMac obtained from three (A) or six (B) different healthy donors. *P < 0.05, **P < 0.001 compared to the DMSO + BCG sample (one-way ANOVA, Tukey's test).

Differential effects of RPR-EA-S1 on BCG-induced cytokine and chemokine expression

To investigate whether the partially purified fraction RPR-EA-S1 contained the immuno-regulatory activity derived from the crude RPR extract, we performed ELISA on IL-6, IL-8, IL-10 and TNF-α. RPR-EA-S1's regulatory patterns on the four cytokines or chemokines were similar to those of RPR crude extracts (Figure 3 and 4). RPR-EA-S1 inhibited IL-10 and enhanced IL-8 expression, while had no effect on IL-6 and TNF-α expression in the BCG-induced PBMac. When compared to the crude RPR extract (Figure 3B), RPR-EA-S1 was more effective in terms of the levels of IL-10 inhibition and IL-8 induction in BCG-stimulated PBMac (Figure 4).
Figure 4
Figure 4

Effects of RPR-EA-S1 on the production of different cytokines or chemokines in BCG-stimulated PBMac. PBMac (5 × 105) were pretreated with 0.01% DMSO or 20 μg/mL of RPR-EA-S1 overnight and then stimulated with BCG (MOI = 1). Supernatants were collected after 24 hours. The levels of IL-6, IL-8, IL-10 and TNF-α in the supernatants were measured by ELISA. The individual cytokine level in the supernatant of DMSO + BCG sample was set as 100%, and the rest were compared to it to obtain a percentage value. Results are shown as mean ± SD from independent experiments on PBMac obtained from four different healthy donors. **P < 0.001 compared to the DMSO + BCG sample (one-way ANOVA, Tukey's test).

Moreover, the inhibitory effect of RPR-EA-S1 on BCG-induced IL-10 production was in a dose-dependent manner between 5 μg/mL and 50 μg/mL both at the protein and mRNA levels (Figure 5A and 5B). The regulatory effects were significant even at a low concentration of 5 μg/mL.
Figure 5
Figure 5

Dose-dependent and time course effects of RPR-EA-S1 on the production of IL-10 in BCG-stimulated PBMac. PBMac (5 × 105) were pretreated with 0.05% DMSO or different dose of RPR-EA-S1 overnight and then stimulated with BCG (MOI = 1). (A) Supernatants were collected after 24 hours. The levels of IL-10 in the supernatants were measured by ELISA. (B) Total mRNA was extracted after 3 hours for cDNA synthesis. The levels of mRNA were determined by q-PCR. The mRNA or protein expression levels of DMSO + BCG sample were set as 100%, and the rest were compared to DMSO + BCG to obtain a percentage value. (C) PBMac (5 × 105) were pretreated with 0.01% DMSO or 20 μg/mL of RPR-EA-S1 overnight and then stimulated with BCG (MOI = 1) for 0, 1, 3, 6, 12 and 24 hours. Total mRNA was used to synthesize cDNA. The levels of IL-10 mRNA were determined by q-PCR. Results are shown as mean ± SD from independent experiments on PBMac obtained from three different healthy donors. *P < 0.05, **P < 0.0001 compared to the DMSO + BCG sample (one-way ANOVA, Tukey's test for Figures 5A and B, t-test for Figure 5C).

The IL-10 mRNA expression levels were studied over a time course of 24 hours. Figure 5C shows that the BCG-induced IL-10 expression reached the peak level at three hours before declining to the basal level at 24 hours; however, pretreatment of the cells with RPR-EA-S1 inhibited BCG-induced IL-10 mRNA expression throughout the time course.

RPR-EA-S1, a fraction of RPR, demonstrated differential effects on cytokine and chemokine expression in BCG-stimulated PBMac, enhanced BCG-induced IL-8 production and inhibited IL-10 production in a dose and time-dependent manner.

Mechanisms underlying RPR-EA-S1 inhibition of IL-10 expression in BCG-stimulated blood macrophages

After confirming the inhibitory effect of RPR-EA-S1 on BCG-induced IL-10 expression, we investigated the mechanisms underlying the decrease of IL-10 expression. Mitogen-activated protein kinases (MAPK) are involved in IL-10 regulation [3, 25]. BCG increases the phosphorylation of ERK1/2 and p38 [3]. This study showed that pretreatment with RPR-EA-S1 did not affect the phosphorylation levels of ERK1/2 and p38 induced by BCG stimulation (Figure 6A and 6B). Since Akt/GSK3β is a key pathway involved in BCG-induced IL-10 expression [30], we examined the effect of RPR-EA-S1 on this pathway. The study found that the phosphorylation of Akt or GSK3β, inducible by BCG, was not affected by pretreatment of the cells with RPR-EA-S1 (Figure 6A and 6B). Neither MAPK kinases nor the Akt/GSK3β pathway was involved in the inhibitory effects of RPR on BCG-induced IL-10 production.
Figure 6
Figure 6

Effects of RPR-EA-S1 on signaling kinases and NF-κB expression in BCG-stimulated PBMac. PBMac (2 × 106) were pretreated with 0.01% DMSO or 20 μg/mL of RPR-EA-S1 overnight and then stimulated with BCG (MOI = 1) for 30 minutes. Cytoplasmic proteins and nuclear proteins were extracted separately. (A) and (C): The levels of phospho-MAPK, phospho-GSK and phospho-Akt, as well as NF-κB1 p50 and IκBα were analyzed with Western blot. The figures showed one set of representative results from independent experiments on PBMac obtained from three different healthy donors. (B) and (D): Quantification of the Western blot was measured by laser densitometry. Intensities of phospho-MAPK, phospho-GSK, phospho-Akt, NF-κB1 p50 and IκBα were normalized to the corresponding MAPK, GSK, Akt, Lamin B and Actin, respectively. Results are shown as mean ± SD from independent experiments on PBMac obtained from three different healthy donors. *P < 0.05 compared to the DMSO + BCG sample (one-way ANOVA, Tukey's test).

Downstream of these kinases, specific transcriptional factors may also affect IL-10 expression. Our previous study demonstrated a correlation between the translocation of NF-κB1 p50 into the nucleus and the induction of IL-10 by BCG [30]. In the present study, Western blotting was performed to examine the translocation of NF-κB1 p50 to the nucleus. The results confirmed the induced translocation of NF-κB1 p50 by BCG while RPR-EA-S1 pretreatment of the cells reduced such effects (Figure 6C and 6D). Similarly, the IκBα was degraded after BCG stimulation while pretreatment of the cells with RPR-EA-S1 significantly inhibited this degradation (Figure 6C and 6D).

Suppression of BCG-induced IL-10 by RPR-EA-S1 were achieved through suppression of IκBα degradation and inhibition of NF-κB1 p50 translocation to the nucleus (Figure 7).
Figure 7
Figure 7

Mechanisms underlying the immunomodulation effects of RPR-EA-S1. In BCG-stimulated macrophages, RPR-EA-S1 can specifically inhibit IL-10 production and yet enhance IL-8 expression. RPR-EA-S1 did not affect the phosphorylation of cellular protein kinases including ERK, p38, Akt and GSK3β. Its inhibitory effects in BCG-activated IL-10 expression may be, at least in part, through the suppression of the degradation of IκBα in the cytoplasm and thus inhibited the translocation of transcription factor NF-κB1 p50 to the nucleus.

GC-MS analysis of the chemical constituents of RPR-EA-S1

To further investigate the chemical components of RPR-EA-S1, we performed GC-MS analysis (Additional files 5 and 6). The mass spectra generated from the extracts were compared to the compounds in NIST GC-MS library. Two major groups of compounds were found in RPR-EA-S1, namely phenols (including hydroquinone, 4-hydroxybenzoic acid and ferulic acid) and fatty acids (including unsaturated and saturated fatty acids).


Recent resurgence of mycobacterial infection is in part due to the spread of AIDS in developing countries. Combined administration of specific anti-mycobacterial drugs is the current TB treatment recommended by the World Health Organization [49]. With increases in multidrug-resistant-TB (MDR-TB) and extensively drug-resistant (XDR)-TB strains, the need to develop new anti-Mtb drugs is urgent and immuno-modulatory agents have been considered to enhance TB treatment [50].

We demonstrated that the crude RPR extract and its fraction RPR-EA-S1 augmented BCG-induced IL-8 expression (Figure 3 and 4). Decreased IL-8 secretion was observed in HIV-infected patients with miliary TB [20]. In vivo studies showed that anti-IL-8 antibody inhibited the formation of granuloma in rabbits [51], suggesting a significant role for IL-8 in controlling Mtb infection by the host immunity. Recent studies showed that IL-8 attracted neutrophils and T lymphocytes to the infection sites for the formation of granuloma and augmentation of cell mediated immunity [52]. IL-8 also activated neutrophils to exhibit bactericidal responses [53]. Therefore, the enhanced IL-8 induction by RPR may be beneficial to the initiation of host immune responses against Mtb and the subsequent formation of the granuloma in human [51].

We also showed that crude RPR extract and its fraction RPR-EA-S1 can inhibit BCG-induced IL-10 expression (Figure 3, 4, 5). IL-10 is a well known anti-inflammatory cytokine produced by macrophages and T cells during Mtb infection [3]. The relationship between IL-10 production and TB progression was well observed in both animal and human studies [32, 33, 36]. Recent studies revealed that Mtb utilized IL-10 to evade the host immunity. Firstly, induction of IL-10 inhibited the production of bactericidal molecules including NO and RNIs by macrophages [54]. Secondly, IL-10 suppressed pro-inflammatory cytokines such as IL-12, TNF-α and IFN-γ, resulting in reduced Th1 cell immunity, CD4+ and CD8+ lytic activity, as well as delayed macrophage activation [31]. Thirdly, IL-10 suppressed antigen presentation by down-regulating MHC and other co-stimulatory molecules on the cell surface of monocytes/macrophages [31]. We recently showed that this effect was due to IL-10 activation of STAT3 to suppress cathepsin S expression [29]. Fourthly, IL-10 inhibited TNF-α activity by down-regulating TNF-α expression and inducing soluble TNF receptor 2, leading to reduced apoptosis and increased pathogen survival [55]. Fifthly, macrophage-derived IL-10 triggered alternative macrophage activation in Mtb infection, in which arginase-1 gene expression was strikingly enhanced. In turn, arginase-1 negatively regulated microbicidal mechanisms in macrophages and promotes Mtb recrudescence [56]. The inhibitory effects of RPR on BCG-induced IL-10 production may have a beneficial effect on the host for the reversal of the immunosuppressive effects of IL-10 thereby speeding up the killing of Mtb.

Multiple pathways are involved in the regulation of BCG-induced IL-10 expression, including ERK1/2, p38 and PI3K/GSK3 [3, 30]. In the present study, RPR showed no effect on either of these pathways (Figure 6A and 6B); however, RPR inhibited NF-κB1 p50 translocation to the nucleus (Figure 6C and 6D). NF-κB plays a role in promoting the production of cytokines [57]. NF-κB is a family of transcriptional factors, including RelA (p65), NF-κB1 (p50 and p105), NF-κB2 (p52 and p100), c-Rel and RelB, bound with an inhibitory protein named IκB in the cytoplasm. After stimulation, IκB undergoes phosphorylation and degradation, resulting in the release of NF-κB for translocation to the nucleus, leading to transcription of their regulated target genes [58]. Among members of the NF-κB family, NF-κB1 p50/p50 homodimer was involved in IL-10 transcription [59]. In murine macrophages, NF-κB1 p50/p50 homodimer activated transcription of IL-10 together with the CREB protein [59]. Macrophages from NF-κB1 p50 knock-out mice expressed less IL-10 compared to normal mice upon lipopolysaccharide challenge [59]. The present study shows that RPR-EA-S1 significantly reduced the degradation of IκBα in the cytoplasm and inhibited the translocation of NF-κB1 p50 to the nucleus in BCG-induced human blood macrophages (Figure 6). Therefore, the inhibition of IL-10 by RPR-EA-S1 may be mediated by the effects of its constituents on NF-κB1 translocation (Figure 7).

While NF-κB pathway also regulates IL-6, IL-8 and TNF-α, previous studies indicated that the transcriptions of IL-6, IL-8 and TNF-α were not mainly regulated by NF-κB1 p50 homodimer [5961]. Instead, silencing of NF-κB p65 rather than p50 gene was effective in down-regulating TNF-α induced IL-6 and IL-8 mRNA synthesis [61]. In luciferase activity assays of the TNF-α promoter, p50 plays little role in driving the transcription, while c-Rel and p65 plays dominant roles in inducing the transcription [60]. Therefore, the inhibition of NF-κB1 p50 translocation to the nucleus cannot affect BCG-induced IL-6, IL-8 and TNF-α expression. Moreover, the transcriptional regulation of cytokines involved multiple transcriptional factors and was inducer-dependent [6264]. For example, respiratory syncytial virus (RSV) may increase IL-8 production in the airway epithelium partly via the activation of transcription factors including NF-κB and NF-IL-6 [65] whereas AP-1 and NF-κB are essential transcription factors for IL-1β-induced IL-8 gene expression [66]. In BCG stimulated macrophages, it is possible that in addition to NF-κB, multiple transcriptional factors may contribute to the expression of IL-6, IL-8 and TNF-α. The inhibition of NF-κB pathway may be compensated by other transcriptional factors. This may also explain why the BCG-induced IL-6, IL-8 and TNF-α expression were not inhibited by RPR-EA-S1.

The major groups of compounds in the RPR-EA-S1 fraction were phenols and fatty acids. Both phenols and fatty acids are biologically active compounds commonly present in the genus of Paeonia[6769]. For example, ferulic acid, one of the phenols isolated from Paeonia, scavenges free reactive radicals as determined by DPPH (1, 1-Diphenyl-2-picrylhydrazyl) assay and inhibits lipid peroxidation and protects DNA from oxidative damage [69]. Phenylacetic acid, plant hormone that had been used to treat type II hyperammonemia [70], was also detected in RPR-EA-S1. Our future studies will isolate the compounds responsible for the immuno-regulatory effects of RPR-EA-S1.


RPR crude extracts and its fraction RPR-EA-S1 specifically inhibited anti-inflammatory cytokine IL-10 and enhanced pro-inflammatory chemokine IL-8 expression in BCG-activated PBMac. The inhibitory effects of RPR-EA-S1 on IL-10 expression in BCG-activated PBMac may be due to the reduced nuclear translocation of NF-κB1 p50.



atom mass units


Bacillus Calmette-Guerin


bis-trimethyl silyl trifluoroacetamide




threshold cycle number


dendritic cells


ethylenediaminetetraacetic acid


electron impact ionization


enzyme-linked immunosorbent assay


ethyl acetate




gas chromatography mass spectrometry


glycogen synthase kinase-3β


human immunodeficiency virus


heme oxygenase-1


high performance liquid chromatography


Honestly Significant Difference










inducible nitric oxide synthases




mitogen-activated protein kinases


multidrug-resistant TB




multiplicity of infection


Mycobacterium tuberculosis


3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

n-BuOH n




NF-κB1 p50: 

nuclear factor-κB1 p50


nitric oxide


peripheral blood monocytes-derived macrophages


phosphate buffered saline


quantitative real-time PCR


reactive nitrogen intermediates


reactive oxygen intermediates


Radix Paeoniae Rubra


respiratory syncytial virus


standard deviation




tris-buffered saline Tween-20


tumor necrosis factor alpha


extensively drug-resistant TB



LW is a recipient of the HKU postgraduate research studentship. This study was funded by grants to ASL from Prof. Francis SK Lau and Mr William Au Research Fund. The authors wish to thank PuraPharm International for the provision and authentication of medicinal herbs as well as collaborations in the extraction and purification processes.

Authors’ Affiliations

Molecular Chinese Medicine Laboratory, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, PR, China
Cytokine Biology Group, Department of Paediatrics and Adolescent Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, PR, China
Department of Pharmacology and Pharmacy, The University of Hong Kong, Pokfulam, Hong Kong SAR, PR, China
Purapharm International Hong Kong, Pokfulam, Hong Kong SAR, PR, China


  1. Global Tuberculosis Control: a Short Update to the 2009 Report. []
  2. Ahmad S, Mokaddas E: Recent advances in the diagnosis and treatment of multidrug-resistant tuberculosis. Respir Med. 2009, 103: 1777-1790. 10.1016/j.rmed.2009.07.010.View ArticlePubMedGoogle Scholar
  3. Cheung BKW, Lee DCW, Li JCB, Lau YL, Lau ASY: A role for double-stranded RNA-activated protein kinase PKR in Mycobacterium-induced cytokine expression. J Immunol. 2005, 175: 7218-7225.View ArticlePubMedGoogle Scholar
  4. Mellman I, Steinman RM: Dendritic cells: specialized and regulated antigen processing machines. Cell. 2001, 106: 255-258. 10.1016/S0092-8674(01)00449-4.View ArticlePubMedGoogle Scholar
  5. Brown AE, Holzer TJ, Andersen BR: Capacity of human-neutrophils to kill Mycobacterium-tuberculosis. J Infect Dis. 1987, 156: 985-989. 10.1093/infdis/156.6.985.View ArticlePubMedGoogle Scholar
  6. Yoneda T, Ellner JJ: CD4(+) T cell and natural killer cell-dependent killing of Mycobacterium tuberculosis by human monocytes. Am J Respir Crit Care Med. 1998, 158: 395-403.View ArticlePubMedGoogle Scholar
  7. Boom WH, Canaday DH, Fulton SA, Gehring AJ, Rojas RE, Torres M: Human immunity to M-tuberculosis: T cell subsets and antigen processing. Tuberculosis. 2003, 83: 98-106. 10.1016/S1472-9792(02)00054-9.View ArticlePubMedGoogle Scholar
  8. Desjardins M, Huber LA, Parton RG, Griffiths G: Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus. J Cell Biol. 1994, 124: 677-688. 10.1083/jcb.124.5.677.View ArticlePubMedGoogle Scholar
  9. Fang FC: Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat Rev Microbiol. 2004, 2: 820-832. 10.1038/nrmicro1004.View ArticlePubMedGoogle Scholar
  10. Gutierrez MG, Master SS, Singh SB, Taylor GA, Colombo MI, Deretic V: Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell. 2004, 119: 753-766. 10.1016/j.cell.2004.11.038.View ArticlePubMedGoogle Scholar
  11. Liu PT, Stenger S, Li HY, Wenzel L, Tan BH, Krutzik SR, Ochoa MT, Schauber J, Wu K, Meinken C, Kamen DL, Wagner M, Bals R, Steinmeyer A, Zugel U, Gallo RL, Eisenberg D, Hewison M, Hollis BW, Adams JS, Bloom BR, Modlin RL: Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science. 2006, 311: 1770-1773. 10.1126/science.1123933.View ArticlePubMedGoogle Scholar
  12. Molloy A, Laochumroonvorapong P, Kaplan G: Apoptosis, but not necrosis, of infected monocytes is coupled with killing of intracellular bacillus-calmette-guerin. J Exp Med. 1994, 180: 1499-1509. 10.1084/jem.180.4.1499.View ArticlePubMedGoogle Scholar
  13. Wang J, Wakeham J, Harkness R, Xing Z: Macrophages are a significant source of type 1 cytokines during mycobacterial infection. J Clin Invest. 1999, 103: 1023-1029. 10.1172/JCI6224.PubMed CentralView ArticlePubMedGoogle Scholar
  14. Flesch IEA, Kaufmann SHE: Activation of tuberculostatic macrophage functions by gamma-interferon, interleukin-4, and tumor-necrosis-factor. Infect Immun. 1990, 58: 2675-2677.PubMed CentralPubMedGoogle Scholar
  15. Flynn JL, Goldstein MM, Chan J, Triebold KJ, Pfeffer K, Lowenstein CJ, Schreiber R, Mak TW, Bloom BR: Tumor-necrosis-factor-alpha is required in the protective immune-response against Mycobacterium-tuberculosis in mice. Immunity. 1995, 2: 561-572. 10.1016/1074-7613(95)90001-2.View ArticlePubMedGoogle Scholar
  16. Saunders BM, Frank AA, Orme IM, Cooper AM: Interleukin-6 induces early gamma interferon production in the infected lung but is not required for generation of specific immunity to Mycobacterium tuberculosis infection. Infect Immun. 2000, 68: 3322-3326. 10.1128/IAI.68.6.3322-3326.2000.PubMed CentralView ArticlePubMedGoogle Scholar
  17. Munk ME, Emoto M: Functions of T-cell subsets and cytokines in Mycobacterial infections. Eur Respir J. 1995, 8: S668-S675.Google Scholar
  18. Hoffmann E, Dittrich-Breiholz O, Holtmann H, Kracht M: Multiple control of interleukin-8 gene expression. J Leukoc Biol. 2002, 72: 847-855.PubMedGoogle Scholar
  19. Dlugovitzky D, Rateni L, TorresMorales A, RuizSilva J, Pinesky R, Canosa B, Molteni O, Bottasso O: Levels of interleukin-8 in tuberculous pleurisy and the profile of immunocompetent cells in pleural and peripheral compartments. Immunol Lett. 1997, 55: 35-39. 10.1016/S0165-2478(96)02649-1.View ArticlePubMedGoogle Scholar
  20. Law KF, Jagirdar J, Weiden MD, Bodkin M, Rom WN: Tuberculosis in HIV-positive patients: Cellular response and immune activation in the lung. Am J Respir Crit Care Med. 1996, 153: 1377-1384.View ArticlePubMedGoogle Scholar
  21. Pace E, Gjomarkaj M, Melis M, Profita M, Spatafora M, Vignola AM, Bonsignore G, Mody CH: Interleukin-8 induces lymphocyte chemotaxis into the pleural space - role of pleural macrophages. Am J Respir Crit Care Med. 1999, 159: 1592-1599.View ArticlePubMedGoogle Scholar
  22. Mosser DM, Zhang X: Interleukin-10: new perspectives on an old cytokine. Immunol Rev. 2008, 226: 205-218. 10.1111/j.1600-065X.2008.00706.x.PubMed CentralView ArticlePubMedGoogle Scholar
  23. O'Garra A, Vieira P: Regulatory T cells and mechanisms of immune system control. Nat Med. 2004, 10: 801-805.View ArticlePubMedGoogle Scholar
  24. Fillatreau S, Gray D, Anderton SM: Not always the bad guys: B cells as regulators of autoimmune pathology. Nat Rev Immunol. 2008, 8: 391-397. 10.1038/nri2315.View ArticlePubMedGoogle Scholar
  25. Li JCB, Lau ASY: A role for mitogen-activated protein kinase and Ets-1 in the induction of interleukin-10 transcription by human immunodeficiency virus-1 Tat. Immunology. 2007, 121: 337-348. 10.1111/j.1365-2567.2007.02580.x.PubMed CentralView ArticlePubMedGoogle Scholar
  26. Li JCB, Lee DCW, Cheung BKW, Lau ASY: Mechanisms for HIV Tat upregulation of IL-10 and other cytokine expression: kinase signaling and PKR-mediated immune response. FEBS Lett. 2005, 579: 3055-3062. 10.1016/j.febslet.2005.04.060.View ArticlePubMedGoogle Scholar
  27. Bogdan C, Vodovotz Y, Nathan C: Macrophage Deactivation by Interleukin-10. J Exp Med. 1991, 174: 1549-1555. 10.1084/jem.174.6.1549.View ArticlePubMedGoogle Scholar
  28. Flesch IEA, Kaufmann SHE: Role of cytokines in tuberculosis. Immunobiology. 1993, 189: 316-339.View ArticlePubMedGoogle Scholar
  29. Chan LLY, Cheung BKW, Li JCB, Lau ASY: A role for STAT3 and cathepsin S in IL-10 downregulation of IFN-γ-induced MHC class II molecule on primary human blood macrophages. J Leukoc Biol. 2010, 88: 303-311. 10.1189/jlb.1009659.View ArticlePubMedGoogle Scholar
  30. Chan MMP, Cheung BKW, Li JCB, Chan LLY, Lau ASY: A role for glycogen synthase kinase-3 in antagonizing mycobacterial immune evasion by negatively regulating IL-10 induction. J Leukoc Biol. 2009, 86: 283-291. 10.1189/jlb.0708442.View ArticlePubMedGoogle Scholar
  31. Moore KW, Malefyt RD, Coffman RL, O'Garra A: Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol. 2001, 19: 683-765. 10.1146/annurev.immunol.19.1.683.View ArticlePubMedGoogle Scholar
  32. Turner J, Gonzalez-Juarrero M, Ellis DL, Basaraba RJ, Kipnis A, Orme IM, Cooper AM: In vivo IL-10 production reactivates chronic pulmonary tuberculosis in C57BL/6 mice. J Immunol. 2002, 169: 6343-6351.View ArticlePubMedGoogle Scholar
  33. Jacobs M, Brown N, Allie N, Gulert R, Ryffel B: Increased resistance to mycobacterial infection in the absence of interleukin-10. Immunology. 2000, 100: 494-501. 10.1046/j.1365-2567.2000.00053.x.PubMed CentralView ArticlePubMedGoogle Scholar
  34. Lee JS, Song CH, Kim CH, Kong SJ, Shon MH, Kim HJ, Park JK, Paik TH, Jo EK: Profiles of IFN-gamma and its regulatory cytokines (IL-12, IL-18 and IL-10) in peripheral blood mononuclear cells from patients with multidrug-resistant tuberculosis. Clin Exp Immunol. 2002, 128: 516-524. 10.1046/j.1365-2249.2002.01858.x.PubMed CentralView ArticlePubMedGoogle Scholar
  35. Torres M, Herrera T, Villareal H, Rich EA, Sada E: Cytokine profiles for peripheral blood lymphocytes from patients with active pulmonary tuberculosis and healthy household contacts in response to the 30-kilodalton antigen of Mycobacterium tuberculosis. Infect Immun. 1998, 66: 176-180.PubMed CentralPubMedGoogle Scholar
  36. Boussiotis VA, Tsai EY, Yunis EJ, Thim S, Delgado JC, Dascher CC, Berezovskaya A, Rousset D, Reynes JM, Goldfeld AE: IL-10-producing T cells suppress immune responses in anergic tuberculosis patients. J Clin Invest. 2000, 105: 1317-1324. 10.1172/JCI9918.PubMed CentralView ArticlePubMedGoogle Scholar
  37. Zhu YP: Chinese Materia Medica: Chemistry, Pharmacology, and Applications. 1998, Amsterdam: OPA; Harwood AcademicGoogle Scholar
  38. Wu SH, Wu DG, Chen YW: Chemical constituents and bioactivities of plants from the Genus Paeonia. Chem Biodivers. 2010, 7: 90-104. 10.1002/cbdv.200800148.View ArticlePubMedGoogle Scholar
  39. Zhao Z: An Illustrated Chinese Materia Medica in Hong Kong. 2004, Hong Kong: School of Chinese Medicine, Hong Kong Baptist UniversityGoogle Scholar
  40. Guo S: Tuberculose pleural effusion treated by xiongmoyan decoction plus Western drugs. Shanxi J Tradit Chin Med. 2007, 28: 1580-1581.Google Scholar
  41. Chen C, Zhang F, Xia ZY, Lin H, Mo AS: Protective effects of pretreatment with Radix Paeoniae Rubra on acute lung injury induced by intestinal ischemia/reperfusion in rats. Chin J Traumatol. 2008, 11: 37-41. 10.1016/S1008-1275(08)60008-5.View ArticlePubMedGoogle Scholar
  42. Zhan LY, Xia ZY, Chen C, Wang XY: Effect of Radix Paeoniae Rubra on the expression of HO-1 and iNOS in rats with endotoxin-induced acute lung injury. Chin J Traumatol. 2006, 9: 181-186.PubMedGoogle Scholar
  43. Sun WY, Wei W, Gui SY, Wu L, Wang H: Protective effect of extract from Paeonia lactiflora and Astragalus membranaceus against liver injury induced by Bacillus Calmette-Guerin and lipopolysaccharide in mice. Basic Clin Pharmacol Toxicol. 2008, 103: 143-149. 10.1111/j.1742-7843.2008.00243.x.View ArticlePubMedGoogle Scholar
  44. The State Pharmacopoeia Commission of the People's Repubilic of China: Pharmacopoeia of the People's Republic of China (Chinese edition). 2005, Beijing: Chemical Industry PressGoogle Scholar
  45. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262.View ArticlePubMedGoogle Scholar
  46. Schreiber E, Matthias P, Muller MM, Schaffner W: Rapid detection of octamer binding-proteins with mini-extracts, prepared from a small number of cells. Nucleic Acids Res. 1989, 17: 6419-6419. 10.1093/nar/17.15.6419.PubMed CentralView ArticlePubMedGoogle Scholar
  47. Yang CLH, Chik SCC, Li JCB, Cheung BKW, Lau ASY: Identification of the bioactive constituent and its mechanisms of action in mediating the anti-inflammatory effects of black cohosh and related Cimicifuga species on human primary blood macrophages. J Med Chem. 2009, 52: 6707-6715. 10.1021/jm9006164.View ArticlePubMedGoogle Scholar
  48. Lee DCW, Yang CLH, Chik SCC, Li JCB, Rong JH, Chan GCF, Lau ASY: Bioactivity-guided identification and cell signaling technology to delineate the immunomodulatory effects of Panax ginseng on human promonocytic U937 cells. J Transl Med. 2009, 7: 34-10.1186/1479-5876-7-34.PubMed CentralView ArticlePubMedGoogle Scholar
  49. Ducati RG, Ruffino-Netto A, Basso LA, Santos DS: The resumption of consumption - A review on tuberculosis. Mem Inst Oswaldo Cruz. 2006, 101: 697-714. 10.1590/S0074-02762006000700001.View ArticlePubMedGoogle Scholar
  50. Iseman MD: Tuberculosis therapy: past, present and future. Eur Respir J. 2002, 20: 87s-94s. 10.1183/09031936.02.00309102.View ArticleGoogle Scholar
  51. Larsen CG, Thomsen MK, Gesser B, Thomsen PD, Deleuran BW, Nowak J, Skodt V, Thomsen HK, Deleuran M, Thestrup-Pedersen K: The delayed-type hypersensitivity reaction is dependent on Il-8 - inhibition of a tuberculin skin reaction by an anti-Il-8 monoclonal-antibody. J Immunol. 1995, 155: 2151-2157.PubMedGoogle Scholar
  52. Mukaida N: Pathophysiological roles of interleukin-8/CXCL8 in pulmonary diseases. Am J Physiol Lung Cell Mol Physiol. 2003, 284: L566-L577.View ArticlePubMedGoogle Scholar
  53. Godaly G, Young DB: Mycobacterium bovis bacille Calmette Guerin infection of human neutrophils induces CXCL8 secretion by MyD88-dependent TLR2 and TLR4 activation. Cell Microbiol. 2005, 7: 591-601. 10.1111/j.1462-5822.2004.00489.x.View ArticlePubMedGoogle Scholar
  54. Cunha FQ, Moncada S, Liew FY: Interleukin-10 (Il-10) inhibits the induction of nitric-oxide synthase by interferon-gamma in murine macrophages. Biochem Biophys Res Commun. 1992, 182: 1155-1159. 10.1016/0006-291X(92)91852-H.View ArticlePubMedGoogle Scholar
  55. Balcewicz-Sablinska MK, Keane J, Kornfeld H, Remold HG: Pathogenic Mycobacterium tuberculosis evades apoptosis of host macrophages by release of TNF-R2, resulting in inactivation of TNF-alpha. J Immunol. 1998, 161: 2636-2641.PubMedGoogle Scholar
  56. Schreiber T, Ehlers S, Heitmann L, Rausch A, Mages J, Murray PJ, Lang R, Holscher C: Autocrine IL-10 induces hallmarks of alternative activation in macrophages and suppresses antituberculosis effector mechanisms without compromising T cell immunity. J Immunol. 2009, 183: 1301-1312. 10.4049/jimmunol.0803567.PubMed CentralView ArticlePubMedGoogle Scholar
  57. Ghosh S, Hayden MS: New regulators of NF-kappa B in inflammation. Nat Rev Immunol. 2008, 8: 837-848. 10.1038/nri2423.View ArticlePubMedGoogle Scholar
  58. Ghosh S, May MJ, Kopp EB: NF-kappa B and rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol. 1998, 16: 225-260. 10.1146/annurev.immunol.16.1.225.View ArticlePubMedGoogle Scholar
  59. Cao SJ, Zhang X, Edwards JP, Mosser DM: NF-kappa B1 (p50) homodimers differentially regulate pro- and anti-inflammatory cytokines in macrophages. J Biol Chem. 2006, 281: 26041-26050. 10.1074/jbc.M602222200.PubMed CentralView ArticlePubMedGoogle Scholar
  60. Lee UJ, Choung SR, Prakash KVB, Lee EJ, Lee MY, Kim YJ, Han CW, Choi YC: Dual knockdown of p65 and p50 subunits of NF-kappaB by siRNA inhibits the induction of inflammatory cytokines and significantly enhance apoptosis in human primary synoviocytes treated with tumor necrosis factor-alpha. Mol Biol Rep. 2008, 35: 291-298. 10.1007/s11033-007-9084-4.View ArticlePubMedGoogle Scholar
  61. Kunsch C, Rosen CA: Nf-Kappa-B subunit-specific regulation of the interleukin-8 promoter. Mol Cell Biol. 1993, 13: 6137-6146.PubMed CentralView ArticlePubMedGoogle Scholar
  62. Gray JS, Pestka JJ: Transcriptional regulation of deoxynivalenol-induced IL-8 expression in human monocytes. Toxicol Sci. 2007, 99: 502-511. 10.1093/toxsci/kfm182.View ArticlePubMedGoogle Scholar
  63. Tang XR, Fenton MJ, Amar S: Identification and functional characterization of a novel binding site on TNF-alpha promoter. Proc Natl Acad Sci USA. 2003, 100: 4096-4101. 10.1073/pnas.0630562100.PubMed CentralView ArticlePubMedGoogle Scholar
  64. Hershko DD, Robb BW, Luo GJ, Hasselgren PO: Multiple transcription factors regulating the IL-6 gene are activated by cAMP in cultured Caco-2 cells. Am J Physiol Regul Integr Comp Physiol. 2002, 283: R1140-R1148.View ArticlePubMedGoogle Scholar
  65. Mastronarde JG, He B, Monick MM, Mukaida N, Matsushima K, Hunninghake GW: Induction of interleukin (IL)-8 gene expression by respiratory syncytial virus involves activation of nuclear factor (NF)-kappa B and NF-IL-6. J Infect Dis. 1996, 174: 262-267. 10.1093/infdis/174.2.262.View ArticlePubMedGoogle Scholar
  66. Jung YD, Fan F, McConkey DJ, Jean ME, Liu WB, Reinmuth N, Stoeltzing O, Ahmad SA, Parikh AA, Mukaida N, Ellis LM: Role of P38 MAPK, AP-1, and NF-kappa B in interleukin-1 beta-induced IL-8 expression in human vascular smooth muscle cells. Cytokine. 2002, 18: 206-213. 10.1006/cyto.2002.1034.View ArticlePubMedGoogle Scholar
  67. Galli C, Calder PC: Effects of fat and fatty acid intake on inflammatory and immune responses: a critical review. Ann Nutr Metab. 2009, 55: 123-139. 10.1159/000228999.View ArticlePubMedGoogle Scholar
  68. Chae HS, Kang OH, Lee YS, Choi JG, Oh YC, Jang HJ, Kim MS, Kim JH, Jeong SI, Kwon DY: Inhibition of LPS-induced iNOS, COX-2 and inflammatory mediator expression by paeonol through the MAPKs inactivation in RAW 264.7 cells. Am J Chin Med. 2009, 37: 181-194. 10.1142/S0192415X0900676X.View ArticlePubMedGoogle Scholar
  69. Lee SC, Kwon YS, Son KH, Kim HP, Heo MY: Antioxidative constituents from Paeonia lactiflora. Arch Pharm Res. 2005, 28: 775-783. 10.1007/BF02977342.View ArticlePubMedGoogle Scholar
  70. Scaglia F, Carter S, O'Brien WE, Lee B: Effect of alternative pathway therapy on branched chain amino acid metabolism in urea cycle disorder patients. Mol Genet Metab. 2004, 81: S79-S85. 10.1016/j.ymgme.2003.11.017.View ArticlePubMedGoogle Scholar


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