The Synthetic Curcumin Derivative CNB-001 Attenuates Thrombin-Stimulated Microglial Inflammation by Inhibiting the ERK and p38 MAPK Pathways

Abstract

We have recently found that the synthetic curcumin derivative CNB-001 suppresses lipopolysaccharide (LPS)-induced nitric oxide (NO) production in cultured microglia, demonstrating that it exerts anti-neuroinflammatory effects by regulating microglial activation. To explore the molecular mechanisms underlying the anti-inflammatory effect of CNB-001, the present study investigated whether CNB-001 is also effective for microglial NO production induced by other stimulants than LPS. Treatment of primary cultured rat microglia with thrombin, a serine protease that has been proposed as a mediator of cerebrovascular injuries, caused the expression of inducible NO synthase (iNOS) and the production of NO. The thrombin-induced NO production was completely blocked by the presence of SCH-79797, a selective protease-activated receptor 1 (PAR-1) antagonist, suggesting that the effect of thrombin is mediated by PAR-1. CNB-001 (1–10 µM) attenuated the thrombin-induced iNOS expression and NO production without affecting the PAR-1 expression. In addition, thrombin treatment caused rapid phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). The changes in ERK and p38 MAPK were significantly suppressed by the presence of CNB-001. These results demonstrate that CNB-001 suppresses thrombin-stimulated microglial activation by inhibiting the ERK and p38 MAPK pathways.

INTRODUCTION

CNB-001, a novel hybrid molecule synthesized from curcumin and cyclohexyl bisphenol A, has neuroprotective effects against various types of toxic substances.¹⁾ Furthermore, it promotes hippocampal long-term potentiation and enhances memory in an object recognition test.²⁾ Therefore, CNB-001 is a promising drug candidate for the treatment of cerebrovascular and neurodegenerative diseases.

Microglia are major resident immune cells of the central nervous system (CNS) and play key roles in the host defense and tissue repair by producing pro- and anti-inflammatory cytokines.^3–6) However, hyperactivation of microglia may be the cause of progressive brain damage associated with cerebrovascular and neurodegenerative diseases.^4,7–9) We have recently found that CNB-001 suppresses lipopolysaccharide (LPS)-induced nitric oxide (NO) production in cultured microglia.¹⁰⁾ To further explore the mechanisms underlying the suppressive effect of CNB-001 on the microglial NO production, the present study focused on thrombin as an inducer of microglial inflammation.

Thrombin is a serine protease well-known as a blood coagulation factor, but also has been implicated in the pathology of brain ischemia, stroke, traumatic brain injury or neurogenerative diseases.^7,8,11–15) Although little is known about the role of thrombin in microglial activation, it has been reported that treatment of microglia with thrombin leads to increased expression of inducible NO synthase (iNOS), the major enzyme involved in the NO production during inflammation associated with neurodegenerative disorders.^16–19) Molecular mechanisms underlying thrombin-induced NO production in microglia are not fully understood. However, it is likely that thrombin and LPS differently activate their receptors, signaling pathways, and induce iNOS expression in microglia. Therefore, we investigated the effect of CNB-001 on thrombin-induced NO production in primary cultured rat microglial cells.

Protease-activated receptors (PARs) belong to a subfamily of G protein-coupled receptors involved in the diverse biological activities in many types of cells including microglia.^7,8,12–19) While thrombin activates the three members of this family (PAR-1, PAR-3 and PAR-4), trypsin and tryptase are considered to activate PAR-2.^20–22) However, it remains unclear which PARs are responsible for thrombin-induced NO production in microglia. Mitogen-activated protein kinases (MAPKs) belong to a large family of serine/threonine protein kinases involved in the inflammatory process.^{8,16,18,19,23)} The major MAPKs consist of the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK. In our previous study, CNB-001 inhibited LPS-stimulated activation of p38 MAPK, but not ERK and JNK.¹⁰⁾ To explore the molecular mechanisms underlying the anti-inflammatory effect of CNB-001 in microglia, we also investigated the roles of PARs and MAPKs in thrombin-induced NO production.

MATERIALS AND METHODS

Chemicals and Antibodies

CNB-001 was kindly given by Prof. David Schubert (The Salk Institute, San Diego, CA, U.S.A.). Procedures for preparation of CNB-001 were as in a previous paper.¹⁾ Since CNB-001 at 10 µM or less has been previously confirmed to show no toxic effect in cultured microglia,¹⁰⁾ the same concentration range (0.1–10 µM) of CNB-001 was tested in the present study. We have also confirmed that the vehicle (0.0002–0.02% dimethyl sulfoxide) had no effect in all experiments. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, U.S.A.). Thrombin was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Ser-Leu-Ile-Gly-Arg-Leu-NH₂ (SLIGRL-NH₂) and Ser-Phe-Asn-Gly-Gly-Pro-NH₂ (SFNGGP-NH₂) were purchased from Peptide Institute Inc. (Osaka, Japan). ML-354 was purchased from Cayman Chemical Inc. (Ann Arbor, MI, U.S.A.). SB203580 was obtained from Merck Millipore (Billerica, MA, U.S.A.). U0126 was from Promega (Madison, WI, U.S.A.). Mouse monoclonal anti-β-actin antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.). Rabbit polyclonal anti-iNOS antibody was from Abcam (Cambridge, U.K.). Rabbit polyclonal anti-PAR-1 antibody was purchased from Bioss Antibodies Inc. (Woburn, MA, U.S.A.). The following antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, U.S.A.): rabbit monoclonal anti-ERK1/2 (137F5) antibody, rabbit monoclonal anti-phospho ERK1/2 (Thr202/Tyr204) (D13.14.4E) antibody, rabbit monoclonal anti-p38 MAPK (D13E1) XP^® antibody, rabbit anti-phospho p38 MAPK (Thr180/Tyr182) (D3F9) XP^® antibody, rabbit polyclonal anti-JNK antibody, rabbit monoclonal anti-phospho JNK (Thr183/Tyr185) (81E11) antibody, horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) antibody, HRP conjugated anti-mouse IgG antibody. ECL prime Western blotting detection reagent was purchased from GE Healthcare (Buckinghamshire, U.K.). The other chemicals were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Microglia Culture

The experimental procedures conformed to the “Guiding Principles for the Care and Use of Laboratory Animals” approved by The Japanese Pharmacological Society, and were approved by the Institutional Animal Care and Use Committee of Musashino University (No. 17008, No. 07-A-2018). As described in our previous papers,^10,24) primary microglia cultures were prepared from the forebrains of Wistar rats (2-d-old neonates). The mixed glial cells were cultured in the DMEM containing 10% FBS and antibiotics. When the cells reached confluent (typically 11 d after dissociation), microglia were separated by the shaking procedure. Microglia were allowed to adhere for 1 h, and then washed to remove nonadherent cells. Microglia were incubated with serum-free DMEM for overnight prior to treatments.

Measurement of Nitrite Accumulation

The concentration of nitrite in the culture medium was determined by the Griess method.^10,24) The supernatant (50 µL) was mixed with an equal amount of Griess reagent and incubated for 10 min. The absorbance of the mixture at 570 nm was measured with a microplate reader (Model 680, Bio-Rad Laboratories, Hercules, CA, U.S.A.).

Western Blotting Analysis

The protein expression was analyzed by Western blotting as described in our previous paper.¹⁰⁾ After each treatment, the cells were lysed with RIPA buffer containing a protease and phosphatase inhibitor cocktail. In preliminary experiments, we have confirmed that all the treatments applied in the present study caused no significant change in total protein levels of cultured microglia. The samples were loaded onto a sodium dodecyl sulfate (SDS)-polyacrylamide gel, separated electrophoretically, and transferred to a polyvinylidine fluoride (PVDF) membrane filter. The filters were blocked with 2% bovine serum albumin (BSA) in Tris-buffered saline containing 1% Tween (TBS-T), and then incubated with rabbit anti-PAR-1 antibody (1 : 500), mouse anti-β actin antibody (1 : 1000), rabbit anti-iNOS antibody (1 : 250), rabbit anti ERK1/2 antibody (1 : 800), anti-phospho ERK1/2 antibody (1 : 1000), rabbit anti-p38 MAPK antibody (1 : 1000), rabbit anti-phospho p38 MAPK antibody (1 : 1000), rabbit anti-JNK antibody (1 : 500) or rabbit anti-phospho JNK antibody (1 : 500) for overnight at 4°C. After washing three times with TBS-T, the filters were incubated with HRP-conjugated anti-mouse IgG antibody (1 : 2500) or anti-rabbit IgG antibody (1 : 2500) for 1 h at room temperature. Membranes were visualized by ECL prime Western blotting detection reagent, and densitometric analysis was performed using Multi Gauge V3.0 software (FUJIFILM). Western blot images and quantitative data were presented as in our previous paper.¹⁰⁾

Statistical Analysis

All statistical methods used in the present study were the same as in our previous paper.¹⁰⁾ In experiments evaluating the effects of drugs on thrombin-induced events, the measured values were normalized by taking the value in the group treated with thrombin alone as 100%. The collected data were shown as the mean ± standard error of the mean (S.E.M.). Statistical significance of differences was evaluated using Dunnett’s test or Steel–Dwass’ test. When probability values were less than 5%, differences were considered significant.

RESULTS

We first checked the time course of thrombin-induced NO production in cultured microglia. As shown in Fig. 1A, the nitrite concentration in the culture medium began to increase more than 6 h after addition of 10 U/mL thrombin and continued to increase until 48 h. We also checked the concentration range of thrombin effective in inducing NO production in cultured microglia. As shown in Fig. 1B, more than 1 U/mL was required for thrombin to induce significant increases in NO production after both 24 and 48 h. According to these results, we chose 10 U/mL as the submaximal concentration of thrombin suitable for examining if test drugs can inhibit thrombin-induced events.

Fig. 1. Treatment of Cultured Rat Microglia with Thrombin Caused the Production of NO

(A) Time dependency of thrombin-induced NO production. Microglia were treated with none (○, n = 4) or 10 U/mL thrombin (●, n = 4) for the indicated time periods. (B) Concentration dependency of thrombin-induced NO production. Microglia were treated with none (n = 5) or 0.1–30 U/mL thrombin (n = 5) for 24 h (gray columns) or 48 h (black columns). Data are presented as mean ± S.E.M. * p < 0.05 vs. none.

To determine the role of PARs in thrombin-induced NO production in microglia, several PAR ligands were employed. When SCH-79797, a selective PAR-1 antagonist, was added simultaneously with thrombin, it almost completely blocked the thrombin-induced NO production in a concentration-dependent manner (Fig. 2A). When ML354, a selective PAR-4 antagonist, was added simultaneously with thrombin, it partly reduced thrombin-induced NO production only at a high concentration (10 µM; Fig. 2B). SCH-79797 (10 µM) or ML354 (10 µM) alone had no effect on the cell viability and nitrite concentration in the culture medium (data not shown). Since selective antagonists for PAR-2 and PAR-3 were not available, we tested the effects of selective agonists alone. As shown in Figs. 2C and D, the PAR-2 agonist SLIGRL-NH₂ (0.1–100 µM) or the PAR-3 agonist SFNGGP-NH₂ (0.1–100 µM) alone failed to elicit NO production. The expression of PAR-1 was confirmed by Western blotting analysis with anti-PAR-1 antibody. As shown in Fig. 3, treatment with CNB-001 (10 µM) did not significantly change the PAR-1 expression level (p = 0.343, Mann–Whitney Rank Sum Test).

Fig. 2. Effects of the Selective PAR-1 Antagonist SCH-79797 (A, n = 5), the PAR-4 Antagonist ML354 (B, n = 5), the PAR-2 Agonist SLIGRL-NH₂ (C, n = 3) or the PAR-3 Agonist SFNGGP-NH₂ (D, n = 3) on NO Production in Cultured Microglia

(A and B) Microglia were treated with 10 U/mL thrombin in the absence or presence of 0.1–10 µM each antagonist for 48 h. (C and D) Microglia were treated with 0.1–100 µM each agonist for 48 h. Data are presented as mean ± S.E.M. * p < 0.05 vs. none; #p < 0.05 vs. thrombin alone.

Fig. 3. CNB-001 Did Not Change the Expression of PAR-1 in Cultured Microglia

Microglia were treated with none (n = 4) or 10 µM CNB-001 (n = 4) for 24 h. The PAR-1 expression level was evaluated by Western blotting analysis. β-Actin was used as internal controls. Data are presented as mean ± S.E.M.

When CNB-001 was added simultaneously with thrombin (10 U/mL), the thrombin-induced NO production was significantly attenuated by the presence of 1 or 10 µM CNB-001 in a concentration-dependent manner (Fig. 4A). CNB-001 (1 or 10 µM) alone had no effect on the nitrite concentration in the culture medium (data not shown). Since CNB-001 is a compound designed to hybridize curcumin and cyclohexyl bisphenol A,¹⁾ we also examined the effects of curcumin and cyclohexyl bisphenol A on thrombin-induced NO production in microglia. Curcumin at 0.1–1 µM showed no significant effect, but partly suppressed the thrombin-induced NO production at 10 µM (Fig. 4B). Cyclohexyl bisphenol A showed no significant effect on the thrombin-induced NO production at concentrations of 0.1–10 µM (Fig. 4C). Since it is well known that iNOS is involved in inflammation-related NO production, we next examined the effects of thrombin and CNB-001 on the iNOS expression in microglia. As shown in Fig. 5, treatment of microglia with thrombin (10 U/mL) for 24 h caused remarkable increases in the iNOS expression. The thrombin-induced increase of iNOS expression was significantly inhibited by simultaneous treatment with 1–10 µM CNB-001 (Fig. 5). The effective concentrations of CNB-001 in suppressing NO production (Fig. 4A) and iNOS expression (Fig. 5) were well correlated.

Fig. 4. Effects of CNB-001 (A, n = 6), Curcumin (B, n = 5) and Cyclohexyl Bisphenol A (C, n = 5) on Thrombin-Induced NO Production in Cultured Microglia

Microglia were treated with 10 U/mL thrombin in the absence or presence of 0.1–10 µM each drug for 48 h. Data are presented as mean ± S.E.M. * p < 0.05 vs. none; #p < 0.05 vs. thrombin alone.

Fig. 5. CNB-001 Suppressed Thrombin-Induced iNOS Protein Expression in Cultured Microglia

Microglia were exposed to none (n = 5), 10 U/mL thrombin (n = 5) or 10 U/mL thrombin +0.1–10 µM CNB-001 (n = 5) for 24 h. The iNOS expression level was evaluated by Western blotting analysis. β-Actin was used as internal controls. Data are presented as mean ± S.E.M. * p < 0.05 vs. none; #p < 0.05 vs. thrombin alone.

We have previously found that CNB-001 suppresses LPS-induced NO production through inhibition of p38 MAPK, but not ERK or JNK.¹⁰⁾ To ask if the effect of CNB-001 on thrombin-induced NO production involves possible changes in the MAPKs, we checked thrombin-induced change in phosphorylation of MAPKs by Western blotting analysis. Treatment of microglia with thrombin (10 U/mL) for 0.5 h resulted in rapid increases in phosphorylation of ERK, JNK and p38 MAPK (Figs. 6A–C). When CNB-001 was added simultaneously with thrombin, it significantly suppressed the thrombin-induced phosphorylation of ERK (Fig. 6A) and p38 MAPK (Fig. 6B), but showed no effect on the thrombin-induced phosphorylation of JNK (Fig. 6C). The total ERK, JNK and p38 MAPK levels were not changed by any treatments tested here.

Fig. 6. CNB-001 Suppressed the Thrombin-Induced Phosphorylation of ERK (A, n = 5) and p38 MAPK (B, n = 5), but Not JNK (C, n = 5) in Cultured Microglia

Microglia were treated with 10 U/mL thrombin in the absence or presence of 0.1–10 µM CNB-001 for 30 min. The protein levels of phosphorylated MAPKs (p-ERK, p-p38, p-JNK) and total MAPKs (total ERK, total p38 MAPK, total JNK) were evaluated by Western blotting analysis. Total MAPKs were used as internal controls. Data are presented as mean ± S.E.M. * p < 0.05 vs. none; #p < 0.05 vs. thrombin alone.

To ask if the activation of ERK or p38 MAPK is required for NO production, we also checked the effects of specific inhibitors of these signaling pathways. When U0126 (0.1–10 µM), a specific inhibitor of MAPK/ERK kinases (MEK1 and MEK2), was added simultaneously with thrombin (10 U/mL), the thrombin-induced NO production was significantly attenuated by U0126 in a concentration-dependent manner (Fig. 7A). When the p38 inhibitor SB203580 (0.1–10 µM) was added simultaneously with thrombin (10 U/mL), the thrombin-induced NO production was significantly attenuated by SB203580 (Fig. 7B).

Fig. 7. The MEK Inhibitor U0126 (A, n = 5) or the p38 MAPK Inhibitor SB203580 (B, n = 5) Suppressed the Thrombin-Induced NO Production in Cultured Microglia

Microglia were treated with 10 U/mL thrombin in the absence or presence of 0.1–10 µM each inhibitor for 48 h. Data are presented as mean ± S.E.M. * p < 0.05 vs. none; #p < 0.05 vs. thrombin alone.

DISCUSSION

In the present study, we showed that the synthetic curcumin derivative CNB-001 at 1–10 µM suppressed thrombin-induced NO production in primary cultured rat microglia. Since this concentration range of CNB-001 had no effect on the cell viability of cultured microglia,¹⁰⁾ the effect is likely to be attributed to its anti-inflammatory action but not toxicity. Although thrombin is a well-known protease involved in blood coagulation and wound healing, accumulating evidence also shows that thrombin plays key roles in the pathogenesis of neurodegeneration such as cerebral ischemia, Alzheimer’s disease and Parkinson’s disease.^7,8,14,15) Disruption of the cerebral vasculature by brain ischemia, stroke, traumatic brain injury or other vascular insults results in thrombin extravasation into brain parenchyma.^7,14,15) Brain resident cells produce intrinsic thrombin in response to injury or degeneration.^7,8,11) Increased thrombin not only activates endothelial cells and induces leukocyte infiltration, but also activates microglia or astrocytes, leading to inflammation, reactive gliosis and neuronal cell death. It has been shown that thrombin induces the microglial iNOS expression, the major enzyme responsible for the NO production during inflammation, and causes degeneration of brain cortical, hippocampal or nigral dopaminergic neurons.^16,25–28) Furthermore, several lines of evidence indicate that orally administered CNB-001 is absorbed in the intestinal tract and pass the blood-brain barrier and reaches the CNS.^1,2) Liu et al.¹⁾ have shown that orally administered CNB-001 rapidly absorbed into the blood and quickly distributes the brain in mice. We have previously reported that oral administration of CNB-001 enhances memory in an object recognition test in rats.²⁾ Taken together, the present finding shows that CNB-001 has therapeutic potential for a variety of neurodegenerative disorders. CNB-001 is a compound designed as a hybrid of curcumin and cyclohexyl bisphenol A.¹⁾ In our previous¹⁰⁾ and present studies, curcumin was less effective than CNB-001 in suppressing LPS- and thrombin-induced NO production in microglia. Cyclohexyl bisphenol was virtually ineffective. These results demonstrated that CNB-001 has better anti-inflammatory activity than curcumin.

There is accumulating evidence that PARs play important roles in the regulation of microglial functions. For example, PAR-1 activation induces microglial proliferation, or potentiates microglial tumor necrosis factor-α (TNF-α) production by up-regulating the CD40 expression.¹⁹⁾ Stimulation of microglia with the PAR-2 agonist 2-Furoyl-LIGRLO-NH₂ induces the early transient release of brain-derived neurotrophic factor and the delayed release of inflammatory cytokines, such as TNF-α and interleukin-1β (IL-1β).²⁹⁾ Microglial PAR-4 activation also stimulates the TNF-α production.³⁰⁾ In addition, PAR-3 is found on microglia in the penumbra after transient or permanent focal ischemia.³¹⁾ In the present study, the PAR-1 expression in cultured microglia was confirmed by Western blot analysis, and the thrombin-induced NO production was completely blocked by the PAR-1 antagonist SCH-79797 at relatively low concentrations (0.1–10 µM). The PAR-2 agonist SLIGRL-NH₂ or the PAR-3 agonist SFNGGP-NH₂ alone failed to elicit NO production. In addition, the thrombin-induced NO production was partly reduced by 10 µM ML354, a putative PAR-4 antagonist (Fig. 2B). Since ML354 has been reported to have an IC₅₀ of 140 nM for PAR-4 and an IC₅₀ of 10 µM for PAR-1,³²⁾ the effect of ML354 observed in the present study may result from its blockade of PAR-1. Therefore, it is likely that the thrombin-induced NO production in our microglial cultures is mainly caused by stimulation of PAR-1.

The time course and concentration dependency of thrombin-induced microglial NO production observed in the present study were similar to those previously reported by Ryu et al.¹⁸⁾ However, our and their studies have a discrepancy regarding the role of PAR-1 in thrombin-induced microglial NO production. Ryu et al.¹⁸⁾ have demonstrated that thrombin-induced NO production in cultured microglia was not blocked by cathepsin G, a putative PAR-1 inhibitor. Considering that cathepsin G is also known to inhibit PAR-3 or activate PAR-1, PAR-2 and PAR-4,^33–35) their result does not rule out a role of PAR-1 in the thrombin-induced NO production in microglia. In contrast, we used the PAR-1 antagonist SCH-79797, which has better potency and selectivity for PAR-1,³⁶⁾ and successfully demonstrated the major role of PAR-1. In addition, Ryu et al.¹⁸⁾ have also reported that thrombin receptor agonist peptide, a PAR-1 agonist, failed to mimic the effect of thrombin. Therefore, it is possible that activation of PAR-1 is necessary but not sufficient for thrombin to produce iNOS expression and NO production in microglia. Further investigations are underway in our laboratory to elucidate the roles of PARs in thrombin-induced microglial activation.

Since CNB-001 did not affect the expression level of PAR-1 in cultured microglia, downregulation of PAR-1 cannot account for the suppressive effect of CNB-001 on thrombin-induced NO production. In addition, CNB-001 suppressed thrombin-induced iNOS expression in microglia, suggesting that its suppressive effect on thrombin-induced NO production was mediated by inhibition of the iNOS expression. Furthermore, CNB-001 suppressed thrombin-induced phosphorylation of ERK and p38, but not JNK. The inhibitory effect of CNB-001 on NO production was mimicked by blockade of the ERK and p38 MAPK pathways with U0126 and SB203580. Therefore, it is likely that that CNB-001 suppresses thrombin-stimulated microglial activation by inhibiting signaling processes upstream of ERK and p38 MAPK.

Although the direct molecular target of CNB-001 associated with its anti-inflammatory effect in microglia remains unclear, there are several possibilities that can be discussed by comparing the results in our previous and present studies. First, CNB-001 suppressed the activation of MAPKs induced by different two stimulants, LPS and thrombin, implying that degradation or inactivation of LPS or thrombin cannot solely account for the suppressive effects of CNB-001. Second, CNB-001 did not suppress the thrombin-induced or LPS-induced phosphorylation of JNK, ruling out possible interaction of CNB-001 with thrombin outside the cells, and indicating that CNB-001 does not act on the JNK signaling pathway. Third, CNB-001 suppressed the p38 MAPK activation induced by LPS and thrombin, suggesting that the molecular target of CNB-001 is the signaling molecule(s) by which LPS and thrombin commonly lead the p38 activation. Fourth, ERK was activated by LPS and thrombin, and CNB-001 suppressed the ERK activation induced by thrombin, but not by LPS. Therefore, it is likely that thrombin and LPS separately promote ERK phosphorylation through different signaling cascades, one of which is the target of CNB-001. These hypotheses are summarized as in Fig. 8.

Fig. 8. A Hypothesis That Can Explain the Effects of CNB-001 on LPS- and Thrombin-Induced NO Production in Microglia

In conclusion, we have found that CNB-001 suppresses the thrombin-induced NO production through the inhibition of ERK and p38 MAPK pathways in microglia. In our previous study, CNB-001 suppressed LPS-induced NO production through the inhibition of p38 MAPK, but not ERK. Therefore, additional studies on differences in signaling cascades by which LPS and thrombin promotes ERK phosphorylation will help to identify the molecular target of CNB-001 for exerting the anti-inflammatory effect.

Acknowledgments

This work was partly supported by a Grant-in-Aid for Young Scientists from the Japan Society for the Promotion of Science (KAKENHI 24790540, KAKENHI 26860358) and the Takeda Science Foundation awarded to T.A. We thank Dr. David Schubert, the Salk Institute, for critically reviewing the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

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