Magnesium lithospermate B mediates anti-inflammation targeting activator protein-1 and nuclear factor-kappa B signaling pathways in human peripheral T lymphocytes

International Immunopharmacology 13 (2012) 354–361

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Magnesium lithospermate B mediates anti-inflammation targeting activator protein-1 and nuclear factor-kappa B signaling pathways in human peripheral T lymphocytes Cheng-Chung Cheng a, Shih-Ping Yang a, Wei-Shiang Lin a, Ling-Jun Ho c, Jenn-Haung Lai b, Shu-Meng Cheng a,⁎, Wen-Yu Lin a a b c

Division of Cardiology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC Division of Rheumatology/Immunology and Allergy, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan, ROC

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Article history: Received 7 March 2012 Received in revised form 18 April 2012 Accepted 19 April 2012 Available online 6 May 2012 Keywords: Magnesium lithospermate B T lymphocytes c-Jun N-terminal kinase Activator protein-1 Nuclear factor kappa B

a b s t r a c t The activation of T lymphocytes contributes to the inflammatory processes of atherosclerotic diseases. Danshen is a traditional Chinese medicine and has shown therapeutic effects in patients with cardiovascular and cerebrovascular diseases. We investigated the effects of aqueous extract of Danshen (magnesium lithospermate B (MLB)) on phorbol 12-myristate acetate + ionomycin and anti-CD3 + anti-CD28 monoclonal antibody-activated T cells. We showed that MLB inhibited interleukin (IL)-2, IL-4, tumor necrosis factor-alpha and interferon-gamma production from activated T cells. The expressions of T cell activation markers CD 25 and CD 69 were effectively reduced. EMSA analysis indicated that MLB down-regulated activator protein-1 (AP-1), nuclear factor kappa B (NF-κB) and octamer binding transcription factor (Oct-1) DNA-binding activity. In addition, MLB inhibited c-jun N-terminal kinase (JNK) but not extracellular signal regulated protein kinase activity. MLB also inhibited IκBα degradation, nuclear translocation of p65 and p50 as well as decreased IκBα kinase (IKK) activity. Through suppressing JNK–AP-1, IKK–IκBα–NF-κB and Oct-1 signaling pathways by MLB in activated T cells, our results provide support for efficacy of MLB in inflammatory diseases and raise its therapeutic potential in activated T cell-mediated pathologies. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

1. Introduction Atherosclerotic diseases are an increasing threat to human health worldwide [1,2]. Their clinical presentations are diverse and vary with involved organs. Atherosclerosis is well known as an inflammatory disease which involves intense immunologic activity between vascular endothelial, smooth-muscle and immune cells [3,4]. Many immune cells exhibit signs of activation and produce many inflammatory cytokines [5,6]. Those markers indicating changes of systemic inflammation have been found to be directly associated with the risk of atherosclerosis, supporting the major role of inflammation in the pathogenesis of atherosclerosis [7,8]. T cells have been known to occur in human atherosclerotic plaques for decades [9] and they can be attracted into the atherosclerotic lesions at an early stage [10]. Subsequently, T cells can recognize antigens presented by inflammatory cells to transform atherosclerotic

⁎ Corresponding author at: Division of Cardiology, Department of Medicine, TriService General Hospital, National Defense Medical Center, No. 325, Section 2, ChengKung Road, Neihu 114, Taipei, Taiwan, ROC. Tel.: + 886 2 8792 7160; fax: + 886 2 66012656. E-mail address: [email protected] (S-M. Cheng).

lesions into unstable plaques. These activated T cells produce inflammatory cytokines, including interferon-gamma, tumor necrosis factor, interleukin-1, 2, 4, 12, 15 and 18, to modulate the functions of other cells in the atherosclerotic plaques [11–14]. In macrophages and vascular cells, these cytokines induce the production of large amounts of molecules downstream in the cytokine cascades, such as activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB) pathways. Acting synergistically, they can destroy the physiologically defensive mechanisms of vessel walls [15]. All these actions tend to promote atherosclerosis. Danshen, the dried root of Salvia miltiorrhiza of the family Labiatae, is one of the most versatile Chinese herbal medicines [16]. The most frequent clinical application of Danshen is for coronary artery disease [17–19]. In addition, Danshen has shown therapeutic effects in patients with hypertension, arrhythmia, stroke, peripheral vascular disease, and renal disease [20–24]. There are lipophilic and hydrophilic constituents in Danshen extracts. To date, at least 50 components have been identified from aqueous extracts of Danshen [25]. Among them, magnesium lithospermate B (MLB) is the major aqueous ingredient in Danshen, and has been demonstrated to possess anti-oxidative, free radical scavenging and antifibrotic effects [26–28]. In addition, MLB showed renoprotection, decreased neointimal formation, myocardial salvage

1567-5769/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2012.04.011

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and neuroprotective effects in previous reports [29–32]. All of these medicinal benefits seem to retard the progression of atherosclerosis. However, the effect of MLB on T cell immunologic activity is not well known. In the present study, we investigated whether MLB could effectively suppress the production of cytokines and their associated signaling pathways in activated human T cells by variable stimuli.

2. Materials and methods 2.1. Preparation of MLB Dried roots of S. miltiorrhiza plants cultivated at a local farm were prepared by Herbal Source Biotechnology Company (Tainan County, Taiwan). MLB was purified according to the protocol described by Tanaka et al. [33]. The dried roots (8.8 kg) were extracted with 50 L methanol under reflux for 8 h and concentrated to a brown syrup. The syrup was suspended in H2O and partitioned with chloroform. MLB was harvested after repeated column chromatography of the H2O extract using Sephadex LH-20 and H2O as an eluent.

2.2. Preparation of human peripheral blood T cells Human peripheral blood T cells were negatively selected from whole blood according to our previous report [34]. Briefly, the buffy coat was mixed with Ficoll-Hypaque, and after centrifugation the layer of mononuclear cells was collected. After lysis of the red blood cells, the peripheral blood mononuclear cells were placed on Petri dishes to remove adherent cells, and then incubated with antibodies including L243 (anti-DR; American Type Culture Collection (ATCC)), OKM1 (anti-CD 11b; ATCC), and LM2 (anti-Mac1; ATCC) for 30 min at 4 °C. The cells were then washed with a medium containing 0.1% fetal bovine serum and incubated with magnetic beads conjugated with goat anti-mouse IgG (Invitrogen). The antibody-stained cells were then removed with a magnet. Following a repeat of the above procedures, the T cells were obtained with a purity of more than 98% as determined by the percentage of CD3+ cells in flow cytometry (Becton Dickinson).

2.3. Cell stimulation and cytokine ELISA For cell activation, the following stimuli and concentrations were used: phorbol 12-myristate 13-acetate (PMA, Sigma) at 5 ng/ml (for cytokine measurements and transfection assays) or 20 ng/ml (for the rest of the studies); ionomycin (Sigma) at 1 μM; immobilized anti-CD3 monoclonal antibodies (mAbs, OKT3, ATCC) at 10 μg/ml and soluble anti-CD28 mAbs (clone 9.3) at 1 μg/ml concentrations. The cells were incubated with a series of stimuli for variable time points and the cell pellets or supernatants were collected for further analysis. The determination of cytokine concentrations was performed as described previously [35].

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2.5. Nuclear extract preparation Nuclear extracts were prepared as described previously [36]. Briefly, the treated cells were left at 4 °C in 70 μl of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol (DTT), 1 mM PMSF, and 3.3 μ/ml aprotinin) for 15 min with occasional gentle vortexing. The swollen cells were centrifuged at 25,000 g for 3 min after removal of the supernatants (cytoplasmic extracts), and the pelleted nuclei were washed with 70 μl buffer A and subsequently resuspended in 30 μl buffer C (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 1 mM DTT, 0.5 mM PMSF, and 3.3 μg/ml aprotinin) and incubated at 4 °C for 30 min with occasional vigorous vortexing. The mixtures were then centrifuged at 14,000 rpm for 20 min and the supernatants were used as nuclear extracts. 2.6. Electrophoresis mobility shift assay (EMSA) EMSAs were performed as described previously [36]. The oligonucleotides containing nuclear factor kappa B (NF-κB)-binding site (5′AGT TGA GGG GAC TTT CCC AGG C-3′) and AP-1-binding site (5′-CGC TTG ATG AGT CAG CCG GAA-3′) were purchased and used as DNA probes (Promega). The DNA probes were radiolabeled with [γ- 32P] ATP using T4 kinase according to the manufacturer's instructions (Promega). For the binding reaction, the radiolabeled NF-κB or AP-1 probe was incubated with 5 μg of nuclear extracts. The binding buffer contained 10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM MgCl2, 5% glycerol, and 2 μg poly (dI-dC). The reaction mixture was left at room temperature for the binding reaction to proceed for 20 min. If unradiolabeled competitive oligonucleotides were added, they were used as 100-fold molar excess and preincubated with nuclear extracts for 10 min before the addition of the radiolabeled probes. The final reaction mixture was analyzed in a 6.6% nondenaturing polyacrylamide gel with 0.5× Tris-borate/EDTA (TBE) as an electrophoresis buffer. 2.7. Western blotting ECL Western blotting (Amersham) was performed as described previously [34]. Briefly, after extensive washing, the treated cells were pelleted and resuspended in lysis buffer. After measurement of protein concentrations, equal amounts of whole cellular extracts were analyzed on 10% SDS-PAGE and transferred to the nitrocellulose filter. For immunoblotting, the nitrocellulose filter was incubated with TBS-T containing 5% nonfat milk (milk buffer) for 1 h, and then blotted with primary antibody against ERK, p38, JNK, p65, p50, IκBα (Santa Cruz Biotechnology), and phosphorylated IκBα kinase (IKK) (recognizes both phosphorylated IKKα and IKKβ), phosphorylated ERK, p38, or JNK (Cell Signaling) overnight at 4 °C. After washing with milk buffer twice, the filter was incubated with secondary antibody conjugated to horseradish peroxidase at a concentration of 1:5000 for 1 h. The filter was then incubated with the substrate and exposed to X-ray film.

2.4. Measurement of cell surface molecule expression

2.8. Transfection assays

Human peripheral blood T cells at a concentration of 1 × 10 6/ml were preincubated with various concentrations of MLB for 2 h. After adding stimuli for another 24 h, the cells were collected and washed with PBS. After washing, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-IL-2 receptor alpha (anti-IL2Rα or anti-CD25), anti-CD69 or FITC-conjugated isotype-matched mAbs (PharMingen) and the expression of cell surface molecules was determined with a flow cytometer (Becton Dickinson). The percentages of each cell surface molecule expression were used to evaluate the drug effects.

The transfection assays were performed according to our previous work [36] with some modifications. In order to reduce cell damage, we used the transfection reagent TransFast™ (Promega) to transfect plasmids into cells instead of using electroporation. In brief, 1 × 10 6 human leukemic Jurkat T cell line were mixed with 2 μg of reporter plasmid pNF-κB-Luc or pAP-1-Luc (Stratagene, La Jolla, CA, U.S.A.) and TransFast™ transfectant (6 μL) in triplicate. Forty-eight hours after transfection, the cells were equally distributed and pretreated or not with various dosages of MLB and then stimulated with or without PMA (5 ng/ml) and ionomycin (1 μM) for 24 h. Subsequently, the

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cell pellets were collected, the total cell lysates prepared and the luciferase activities determined according to the manufacturer's instructions (Promega).

studies showed that the MLB concentrations (up to 100 μg/ml) used in the experiments showed no apparent cytotoxicity (Fig. S1). 3.2. MLB effects on activation marker expressions

2.9. Measurement of nonspecific cytotoxicity LDH detection assays were performed according to the manufacturer's instructions (Roche, Indianapolis, IN) to evaluate drug toxicity in human peripheral T cells. 2.10. Statistics The results are expressed as means ± SD. A one-way ANOVA test was used to determine the significance of differences, and a p value less than 0.05 was considered statistically significant. 3. Results 3.1. MLB effects on cytokine production Because cytokines play important roles in atherosclerosis, we examined the effects of MLB on the cytokine production from PMA + ionomycin activated T cells as suggested in a previous report [37]. In the presence of MLB, the production of several cytokines, including IL2, IL-4, tumor necrosis factor-alpha, (TNF-α) and interferon-gamma (INF-γ) induced by PMA+ ionomycin stimulation was greatly inhibited in a dose-dependent manner from 10 to 100 μg/ml (Fig. 1). Meanwhile, consistent with previous studies indicating no cytotoxicity during incubation of hepatic stellate cells and MLB (greater than 74 μg/ml) for up to 48 h [38], the LDH assays performed on human T lymphocytes in our

Since the expression of activation markers has been commonly shown in atherosclerotic lesions, producing proatherogenic mediators and contributing to lesion growth and disease aggravation [39,40], we next determined whether MLB also affected the expression of T cell activation markers. As shown in Fig. 2, MLB effectively reduced expression of both CD 25 and CD 69, T cell activation markers, in a dosedependent manner. Suppression of T cell activation by MLB supports its potential role in the treatment of atherosclerosis. 3.3. MLB down-regulated AP-1, NF-κB and oct-1 DNA-binding activity We next checked the effects of MLB on the activation of NF-κB, a family of ubiquitously expressed transcription factors that play crucial roles in most inflammatory responses [41]. Peripheral blood T cells were preincubated with various concentrations of MLB and then stimulated with or without PMA + ionomycin. The cells were collected and the nuclear extracts prepared and analyzed with EMSA. As shown in Fig. 3B, MLB decreased PMA + ionomycin-induced NF-κB DNA-binding activity significantly at concentration of 100 μg/ml. We then examined another group of potential transcriptional factors, AP-1, that also bind to the enhancer/promoter regions of a variety of cytokine genes in activated T cells [42]. As shown in Fig. 3A, in the presence of MLB, the PMA + ionomycin-induced AP-1 DNA-binding activity was effectively suppressed. In addition, recent reports have suggested that octamer binding transcription factor (Oct-1) plays an important role in regulating atherosclerotic processes. Oxidized low density

Fig. 1. Effects of MLB on PMA + ionomycin-induced cytokines production from T cells. Human peripheral blood T cells at 1 × 106/ml were pretreated with various concentrations of MLB for 2 h and then stimulated with PMA + ionomycin for another 24 h. The supernatants were collected for IL-2 (A), TNF-α (B), IL-4 (C), and IFN-γ (D) measurements. The representative data of at least three independent experiments are shown. An asterisk (*) denotes statistical significance (p b 0.05) versus PMA + ionomycin treated only by one-way ANOVA test.

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Fig. 2. MLB inhibited the expression of CD25 and CD69 on activated T cells. Human peripheral blood T cells at 1 × 106/ml were treated with 100 μg/ml (B, D) or various concentrations (A, C) of MLB for 2 h and then stimulated with PMA + ionomycin for another 24 h. The expressions of CD25 and CD69 were measured by a flow cytometer. The representative data (A, C) and means ± SD (B, D) of at least six different donor cells are shown. An asterisk (*) denotes statistical significance (p b 0.05) as compared to the stimulated T cell in the absence of drug treatment.

lipoprotein (LDL) can induce oxidative DNA damage and activation of Oct-1 that results in metabolic dysfunction of endothelial cells [43,44]. As shown in Fig. 3C, MLB effectively suppressed the PMA + ionomycin-induced Oct-1 DNA-binding activity at a concentration of 100 μg/ml. For a more clinical approach, we used antiCD3 + anti-CD28 mAbs to costimulate T cells. By EMSA analysis, we found that MLB effectively down-regulated AP-1 and NF-κB DNAbinding activity (Fig. 3D and E). The nature of AP-1, NF-κB and Oct-1 complexes was confirmed by competition assays, which showed that the induced nuclear protein complexes bound to radiolabeled AP-1, NF-κB and Oct oligonucleotides could be blocked by wild-types but not mutant types (Fig. 3C, F and G). 3.4. MLB inhibited AP-1 and NF-κB transcription activity To further investigate whether the decrease of DNA-binding activity will also result in reduction of transcriptional activation, we transiently transfected AP-1-luciferase or NF-κB-luciferase reporter plasmids into human leukemic T cell line Jurkat. Forty-eight hours after transfection, the cells were treated with MLB at various dosages and then stimulated with PMA + ionomycin to induce NF-κB or AP-1 transcriptional activity. As shown in Fig. 4A and B, MLB at concentrations of 10–100 μg/ml significantly inhibited the transcriptional activity of AP-1 and NF-κB. The results of EMSA analysis of the Jurkat T cells were also consistent with those performed in human peripheral blood T cells (Fig. 4C and D). 3.5. MLB blocked JNK but not ERK and p38 activity Since mitogen-activated protein kinases (MAPKs) are the upstream signaling regulators of AP-1 activation, we evaluated the effects of

MLB on PMA + ionomycin-stimulated MAPK cascades, including the activities of c-jun N-terminal kinase (JNK), extracellular signal regulated protein kinase (ERK) and p38. The results showed that MLB inhibited the activation of p-JNK (Fig. 5A), but had no effects on ERK and p38 activities (Fig. 5B and C). Western blotting showed that the inhibition was not due to its effects on total JNK protein levels (Fig. 5A).

3.6. MLB inhibited IKK–IκBα–NF-κB signaling pathway To determine whether the decreased PMA + ionomycin-induced DNA-binding activity of NF-κB by MLB was due to a decreased accumulation of p65 and p50 subunits in the nucleus, we performed Western blotting to measure the levels of these two transcriptional factors in the nuclei of the cells. As shown in Fig. 6, levels of p65 and p50 in the nuclei were increased after PMA + ionomycin stimulation, and decreased after MLB treatment in a dose-dependent manner. The inhibition of nuclear translocation of NF-κB subunits by MLB suggests that this drug may have effects on the NF-κB-associated inhibitory protein, IκBα, which retains NF-κB in an inactive status in the cytosol. We treated T cells with PMA + ionomycin and the cytosolic IκBα was determined by Western blot. PMA + ionomycin stimulation induced IκBα degradation after treatment. In the presence of MLB, the PMA + ionomycin-induced degradation of IκBα was prevented, resulting in partial significant recovery of the cytosolic levels of IκBα. These indicated that MLB at concentrations of 10–100 μg/ml inhibited IκBα–NF-κB signaling pathway efficiently. Moreover, we further determine whether MLB has any effect on IKK activities. As shown in Fig. 6C, the PMA + ionomycin-induced IKKα/β phosphorylation was greatly reduced by MLB treatment at

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Fig. 3. MLB blocked Ap-1, NF-κB and Oct-1 DNA-binding activity. Human peripheral blood T cells at 1 × 107/ml were pretreated with various concentrations of MLB for 2 h and then stimulated with PMA + ionomycin (A, B, C, F and G) for 60 min or anti-CD3 + anti-CD28 (D and E) for 14 h. The nuclear extracts containing 5 μg of total protein from treated cells were analyzed by EMSA. The 32P-labeled oligonucleotides containing the AP-1 (A, D and F), NF-κB (B, E and G) or Oct-1 (C) sites were used as probes. The whole reaction mixtures were incubated for 20 min and then analyzed on a 6.6% native polyacrylamide gel. A competition study was done with 100-fold molar excess of unradiolabeled wild-type (wild) or mutant-type (mut) AP-1 (F), NF-κB (G) and Oct-1 (C) oligonucleotides. The competitors were preincubated with nuclear extracts for 10 min before the addition of the radiolabeled AP-1, NF-κB and Oct-1 probes. The representative results from three independent experiments with similar results are shown. 3 + 28 stands for anti-CD3+ anti-CD28 mAbs stimulation.

the concentration of 100 μg/ml. The total levels of IKKα were not affected by MLB treatment. 4. Discussion The present study was undertaken to examine and identify the potential MLB inhibitory effects on activated human peripheral blood T cells that play a key role in inflammatory responses in atherosclerotic cardiovascular diseases. We demonstrated that MLB efficiently blocked production of several cytokines, including IL-2, IL-4, TNF-α, and IFN-γ, from activated T cells (Fig. 1). Furthermore, MLB inhibited PMA + ionomycin-induced DNA-binding activities, and this inhibition was likely mediated by the down-regulation of JNK–AP1, IκBα-NF-κB and OCT-1 signaling pathways. In addition to the results observed, MLB inhibited the transcriptional activation of both AP-1 and NF-κB-dependent reporter genes (Fig. 4A and B) in human leukemic Jurkat T cell line. The broad spectrum of inhibitory abilities for different signaling pathways in T cells suggests that MLB has strong and efficient anti-inflammatory effects. In humans, atherosclerotic plaques, from fatty streaks to ruptured plaques, contain blood-borne inflammatory and immune cells, most of which are macrophages and T cells [3]. Many of these cells are activated and produce inflammatory cytokines causing cardiovascular damage. Therefore, inhibition of cytokine production by MLB is likely to be clinically relevant. Serum levels of IL-2 have been shown to be associated with carotid artery intima media thickness [45]. Levels of IL-2 and sIL-2 receptors are significantly elevated in patients with stable angina [46]. IFN-γ activates macrophages and increases their

production of pro-inflammatory cytokines, and pro-thrombotic and vasoactive mediators. In addition, IFN-γ also decreases cell and collagen content of the fibrous cap, reducing the stability of atherosclerotic plaques and promoting cardiovascular events [47]. TNF-α induces the production of reactive oxygen, proteolytic enzymes and prothrombotic tissue factors by endothelial cells [48]. TNF-α also has abundant metabolic effects, including suppression of lipoprotein lipase, which has been associated with heart disease in clinical studies [49,50]. Although IL-4 is traditionally considered to be an anti-inflammatory cytokine, recent in vitro and in vivo studies have provided robust evidence that IL-4 exerts pro-inflammatory effects on vascular endothelium and may play a critical role in the development of atherosclerosis [51]. During percutaneous coronary intervention, the ratio of IL-4 levels before and after procedure was higher in patients with multivessel coronary artery disease than those with single-vessel coronary artery disease [52]. Whereas the combined effects of several cytokines may give synergistically detrimental results, the broad spectrum inhibition of cytokine production from T cells by MLB provides additional therapeutic benefits. AP-1 is a transcription factor composed of Jun and Fos that mediates diverse signaling events [53,54]. The complex AP-1 activity is regulated by several upstream kinases, including MAPKs and phosphoinositide 3-kinase (PI3K). In the present study, we demonstrated that MLB down-regulated JNK but had no effects on ERK and p38 activities. Although belonging to the same group of MAPK signaling pathways, there are some differences between JNK, ERK and p38. ERK 1/2 regulates cell proliferation, differentiation and cell migration. It is activated by MEK 1/2 which is in turn activated by Raf. JNK and

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Fig. 4. MLB suppressed the transcriptional activity of AP-1 and NF-κB in Jurkat T cells. Human leukemic Jurkat T cells at 1 × 106/ml were mixed together with pAP-1-Luc (A, labeled as AP-1) or pNF-κB-Luc (B, labeled as NF-κB) reporter plasmids and the transfection reagent TransFast™. Forty-eight hours after transfection, the cells were pretreated with MLB at various dosages for 2 h. After stimulation with PMA (5 ng/ml) + ionomycin (1 μM) for another 24 h, cells were collected and the total cell lysates were analyzed for luciferase activities. Values are expressed as fold induction of luciferase activity when compared with unstimulated cells. The representative data (A and B) out of three independent experiments are shown. An asterisk (*) denotes statistical significance (p b 0.05) as compared with cells stimulated in the absence of MLB treatment. The EMSA analysis in Jurkat T cells was performed exactly as described in Fig. 3. The representative data (C and D) from three independent experiments with similar results are shown.

p38 are associated with cell proliferation, differentiation, apoptosis and inflammatory responses. The pathways consist of a MAP3K such as ASK1, MEKK1, or MLK3, as well as a MAP2K (such as MKK3 or MKK6 for the p38 pathway, or MKK4 or MKK7 for the JNK pathway) [55]. Because only the MAP kinase level was examined in the current study, it is possible that MLB may act on more upstream targets. Interestingly, MLB was shown to have discordant effects on these AP-1

Fig. 5. MLB blocked JNK but not ERK and p38 activity. Human T cells at 1× 106/ml were pretreated or not with MLB at various dosages for 2 h and then stimulated with PMA+ ionomycin for 60 min. MLB pretreatment suppressed the activities of p-JNK (A) but did not affect the activity of p-ERK and p-38 (B and C). Under these conditions, MLB had no effect on total JNK level (A). Data shown are representative of three independent experiments with similar results.

Fig. 6. MLB inhibited IκBα-NF-κB signaling pathway. Human peripheral blood T cells at 1 × 106/ml were pretreated with various concentrations of MLB for 2 h and then stimulated with PMA + ionomycin for 60 min. The cells were collected and nuclear extracts were probed for the expressions of p65 and p50 by Western blot (A). N: nucleus. In (B), human peripheral blood T cells were pretreated with MLB at various dosages for 2 h and then stimulated or not with PMA + ionomycin for 60 min. Cytoplasmic extracts were probed for the expression of IκBα by Western blot. In (C), total cell lysates were collected and Western blot analysis was performed using antibodies recognizing the phosphorylated form of both IKKα and IKKβ or using antibodies recognizing total IKKα or β-actin. Data shown are representative of three independent experiments with similar results.

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signaling pathways in different cells and different stimuli. In beta cells, pretreatment with MLB before IFN-γ and IL-1β exposure significantly reduced phosphorylation of p38, JNK and cleaved caspase-3 [56]. MLB inhibited platelet-derived growth factor (PDGF)-BB-induced phosphorylation of PI3K/Akt and ERK pathways by scavenging reactive oxygen species in vascular smooth muscle cells [32]. NF-κB, as well as AP-1, is a key transcriptional factor that orchestrates the expression of many genes involved in inflammation, oncogenesis and apoptosis [57]. In many cases, JNK and NF-κB are activated by the same signaling pathways, and the cross talk between these two pathways has been shown [58]. When NF-κB and AP-1 are simultaneously activated, the increased level of the NF-κB downstream target gene elk-1 can be further activated by ERK, p38 and JNK for more induction of fos expression and AP-1 activation [59]. Even though MLB can only suppress JNK in MAPK signaling pathways in T cells, it eliminates the synergic effects of the activation of AP-1 and NF-κB transcriptional factors by significantly reducing NF-κB activity as shown in our present study. The inhibitory effect of MLB on PMA plus ionomycin activated AP-1 and NF-κB signaling in Jurkat cells appeared to be more robust than in human peripheral T cells. Reasons for these different results may be due to different cells used in the studies. Moreover, PMA plus ionomycin activation of cytokines is dose-dependently inhibited by MLB, while activation of AP-1 and NF-kB by these agents could be only greatly reduced at 100 μg/ml of MLB in human peripheral T cells. Additional transcriptional factors, such as signal transducers and activators of transcription (STAT), cAMP response element-binding (CREB), nuclear factor of activated T cells (NAFT), might be involved in the MLB-inhibited human T cells inflammatory response [60–62]. Further studies are needed to investigate the detailed mechanisms. Although rarely explored in T cells, both the antioxidative and antiapoptotic effects of MLB shown in reports of other tissue cells activated by different stimuli may be related to the observed mechanisms demonstrated in this study [56,63]. It has been also reported that a methyl ester derivative of lithospermate B exhibits moderate inhibition of whole blood oxidative burst through unknown mechanisms without decrease of cytokine expression in LPS (bacterial lipopolysaccharide) and PMA-activated THP-1 (human monocytic leukemia cells), indicating the potential antioxidant effect of MLB [64,65]. Oxidative stress is well known to be activated by JNK–AP-1 and NF-κB signaling pathways [57,66,67]. More recently, the homeodomain transcription factor Oct-1 was added to the growing list of transcriptional factors regulated by DNA damage [68]. In coronary arterial endothelium, oxidized low density lipoprotein induces oxidative DNA damage and activates Oct-1, resulting in metabolic dysfunction [43]. Thus, the blockage of JNK–AP-1, NF-κB and Oct-1 signaling by MLB may explain in part its antioxidative effects. In addition, the induction of cellular apoptosis also requires the activation of JNK and NF-κB, and their blockage suppresses apoptotic effects [69–71]. Therefore, the inhibition of both JNK and NF-κB activation by MLB implicates its potential antiapoptotic effects. To delineate these interesting relationships, further studies are needed to clarify the underlying mechanisms. There is limitation to this study. We only showed in vitro MLB effects on human peripheral blood T cells isolated from healthy donors, even though we found that MLB could also down-regulate AP-1 and NF-κB transcriptional activity in Jurkat T cells. The investigation of T cells in MLB-treated disease models should provide stronger support for our conclusions. Nevertheless, this study is the first to examine the effects of MLB on human T cell immunity and the underlying signaling pathways. 5. Conclusion Our study indicates that an aqueous extract from Danshen, MLB, can reduce PMA + ionomycin-induced production of cytokines, activation of JNK–AP1, IKK–IκBα–NF-κB and Oct-1 signaling pathways in human peripheral T cells. The present study therefore reveals

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