Tamarixetin Exhibits Anti-inflammatory Activity and Prevents Bacterial

May 31, 2018 - Department of Immunology, Lab of Dendritic Cell Differentiation & Regulation, School of Medicine, Konkuk ... Phone: +82-2-2049-6330...
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Article Cite This: J. Nat. Prod. 2018, 81, 1435−1443

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Tamarixetin Exhibits Anti-inflammatory Activity and Prevents Bacterial Sepsis by Increasing IL-10 Production Hee Jo Park,†,∥ Seung Jun Lee,†,∥ Joon Cho,‡,∥ Amal Gharbi,† Hee Dong Han,† Tae Heung Kang,† Yangmee Kim,§ Yeongjoon Lee,§ Won Sun Park,⊥ In Duk Jung,*,† and Yeong-Min Park*,†

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Department of Immunology, Lab of Dendritic Cell Differentiation & Regulation, School of Medicine, Konkuk University, Chungju, 380-701, South Korea ‡ Department of Neurosurgery, Konkuk University Hospital, Seoul, 05030, South Korea § Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, South Korea ⊥ Institute of Medical Sciences, Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 200-701, South Korea S Supporting Information *

ABSTRACT: Sepsis is a systemic inflammatory response to pathogenic infection that currently has no specific pharmaceutical interventions. Instead, antibiotics administration is considered the best available option, despite increasing drug resistance. Alternative strategies are therefore urgently required to prevent sepsis and strengthen the host immune system. One such option is tamarixetin (4′-Omethylquercetin), a naturally occurring flavonoid derivative of quercetin that protects against inflammation. The purpose of this study was to determine whether the anti-inflammatory effects of tamarixetin protect against the specific inflammatory conditions induced in lipopolysaccharide (LPS) or Escherichia coli K1 models of sepsis. Our study showed that tamarixetin reduced the secretion of various inflammatory cytokines by dendritic cells after activation with LPS. It also promoted the secretion of the anti-inflammatory cytokine interleukin (IL)-10 and specifically increased the population of IL-10-secreting immune cells in LPSactivated splenocytes. Tamarixetin showed general anti-inflammatory effects in mouse models of bacterial sepsis and decreased bacteria abundance and endotoxin levels. We therefore conclude that tamarixetin has superior anti-inflammatory properties than quercetin during bacterial sepsis. This effect is associated with an increased population of IL-10-secreting immune cells and suggests that tamarixetin could serve as a specific pharmaceutical option to prevent bacterial sepsis.

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factor 88 (MyD88), leading to the activation of several transcription factors. These include nuclear factor-κB (NFκB), a master regulator of inflammation that controls the transcription of many pro-inflammatory cytokines involved in sepsis.12 These pro-inflammatory cytokines cause excessive inflammatory responses throughout the body and promote systemic coagulation, leading to hypotension and multiple organ failure.13 Various therapeutic candidates have been investigated to overcome sepsis, with the efficacies of some evaluated in preclinical and clinical trials. Although several have shown promising preclinical results, candidates with satisfactory efficacies have not yet been identified in clinical trials.14 Multiple strategies have been employed to affect sepsis, including the use of monoclonal anti-TNF antibodies,15,16 IL1R inhibitors,17−19 TLR4 antagonists,20−23 and monoclonal anti-LPS antibodies.24−26 Although these therapies have shown

arious inflammatory responses are necessary for the host immune response that removes pathogens and prevents organ injury after infection. 1 However, an inadequate inflammatory reaction can lead to sepsis, a serious disease with high morbidity (exceeding 30% in some case).2 Sepsis is caused by an aggressive host inflammatory response that is often triggered by microbial infection.3,4 During sepsis, these inflammatory responses are exacerbated by a “cytokine storm” that can lead to multiple organ failure and even death.5 While sepsis is caused by a wide variety of pathogens, the majority of cases are associated with bacterial infections.6,7 Of these, Gramnegative bacteria are the most commonly reported sepsisrelated agent found in intensive care units.8,9 Therefore, controlling Gram-negative bacterial infection is crucial for successfully treating or preventing sepsis.7−10 Lipopolysaccharide (LPS) is a common endotoxin found in the outer membrane of Gram-negative bacteria and is a major pathogen-associated molecular pattern molecule that is associated with septic shock.11 After stimulation by LPS, Tolllike receptor (TLR) 4 interacts with myeloid differentiation © 2018 American Chemical Society and American Society of Pharmacognosy

Received: February 19, 2018 Published: May 31, 2018 1435

DOI: 10.1021/acs.jnatprod.8b00155 J. Nat. Prod. 2018, 81, 1435−1443

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or without 30 min of tamarixetin pretreatment. Tamarixetin pretreatment significantly reduced the secretion of the proinflammatory cytokines IL-6, TNF-α, and IL-12p70. Conversely, pretreatment increased levels of the anti-inflammatory cytokine IL-10 (Figure 1C). To identify the time point at which tamarixetin exhibits its inhibitory effects, BMDCs were simultaneously treated with LPS and tamarixetin at 30 min intervals for up to 120 min. After measuring cytokine levels along the time course, the inhibitory effects of tamarixetin were found to be the strongest 30 min post-treatment and gradually disappeared (Figure 1D). Taken together, these results show that tamarixetin elicited an inhibitory effect on the LPS-induced BMDC immune response. Tamarixetin Suppresses TLR4-Related Signaling Molecules in LPS-Activated BMDCs. TLR4 activates a signaling pathway common to other TLRs that recruits various downstream molecules, such as MAPKs, Akt, COX-2, and NF-κB.35 We used Western blotting to determine whether tamarixetin could control the activation of these TLR4mediated downstream factors in BMDCs stimulated by LPS. These data show that LPS increased the phosphorylation of MAPKs and Akt and the expression of COX-2 and promoted the degradation of IκBα in a time-dependent manner (Figure 2A). Tamarixetin inhibited the phosphorylation of JNK1, p38, and Akt, the expression of COX-2, and the degradation of IκBα by BMDCs stimulated with LPS, although it failed to decrease the phosphorylation of ERK (Figure 2A). These data show that tamarixetin strongly inhibits LPS-induced phosphorylation of JNK1. We next estimated the binding constant between tamarixetin and JNK1 using fluorescence quenching. As shown in Figure 2B, tryptophan fluorescence of JNK1 significantly decreased in the presence of tamarixetin, and the compound was found to tightly bind to JNK1 with a binding constant of 4.9 × 105 M−1. A docking modeling study suggested that tamarixetin binds to the ATP binding site of JNK1 (Figure 2C). At this site, the carboxyl oxygen side chain of the Asp169 residue forms a hydrogen bond with the 3′-hydroxy group of the tamarixetin Bring. The backbone amide proton and carbonyl oxygen of the Met111 residue also form hydrogen bonds with the carbonyl oxygen at the 4-position of the C-ring and 5-hydroxy group of the tamarixetin A-ring. The A- and C-rings form additional hydrophobic interactions with residue Ile32 in JNK1, and residue V158 also provides additional hydrophobic interactions with the C-ring. Finally, the B-ring of tamarixetin forms a hydrophobic interaction between the Val40 and Leu168 residues. This docking study provides insight into how tamarixetin inhibits JNK1 and suggests that the compound suppresses LPS immune cell activation by interfering with the activation of various TLR4-mediated signaling molecules, including JNK1. Tamarixetin Increases the Levels of Several microRNAs That Affect Cytokine Expression. It has previously been shown that IL-10 can also inhibit the translation of TNFα, IL-6, and IL-12 by destabilizing mRNA transcription.36 This can occur through microRNAs binding to the 3′ untranslated regions of TLR signaling genes.37 For example, microRNA-187 inhibits TNF-α, IL-6, and IL-12 expression by affecting mRNA stability.38 Our results showed that tamarixetin could increase the levels of miRNA187, miRNA 146a, and miRNA 155 in LPS-treated DCs (Figure S2). Taken together, we hypothesize that tamarixetin-increased IL-10 production may prevent a cytokine storm during sepsis by inhibiting pro-inflammatory

some effectiveness in animal models, they have unfortunately proven unsuccessful in human clinical trials. Activated drotrecogin alfa (Xigris) is so far the only drug to be approved by the U.S. FDA to treat some of the effects of sepsis. It was developed in 2001 and functions by inhibiting the blood coagulation that often occurs during sepsis. Despite Xigris being used in clinical practice to treat sepsis for more than a decade, the drug was withdrawn in 2011 after further investigation demonstrated its low therapeutic efficacy, alongside numerous side effects, such as an increased risk of bleeding.27 As a result, there are currently no specific drugs for sepsis, and the use of antibiotics remains the best treatment. In order to identify a specific treatment for LPS-induced endotoxemia and bacterial sepsis, we examined the effects of the immune-modulating phytochemical tamarixetin in an endotoxemia model. These phytochemicals are physiological compounds that are abundant in many foods, including fruits, vegetables, and nuts.28 Tamarixetin itself is a natural flavonoid derivative of quercetin that is methylated at the 4′ carbon position. Small amounts of quercetin in the body can be converted by methyltransferase into tamarixetin via methylation.29−31 If not methylated, quercetin has low stability and low cell permeability, making it difficult to enter the circulatory system. Moreover, the reactive oxidation product that is formed out of quercetin is known to channel the reactivity toward thiol groups, threatening vital cellular compounds. On the contrary, tamarixetin oxidation product prefers to pass the reactivity to the antioxidant network, specially ascorbate, leading to less thiol toxicity.32 Thus, methylated flavonoids, such as tamarixetin, may serve as a way to increase hepatic metabolic stability and intestinal absorption of flavonoids with less thiol toxicity and would be better suited for intracellular introduction.32,33 Previously, it has been demonstrated using an endotoxemia model that quercetin can inhibit LPS-induced anti-inflammatory responses.34 However, whether the more stable tamarixetin also has inhibitory effects on LPS- and bacteria-induced inflammation is unknown and was the focus of the study.

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RESULTS AND DISCUSSION Tamarixetin Attenuates LPS-Induced Immune Responses in Bone Marrow Dendritic Cells. Using H2O2 as a positive control, cell viability tests with bone marrow dendritic cells (BMDCs) showed that that tamarixetin is nontoxic at concentrations up to 100 μΜ (Figure 1B). To further investigate the effects of tamarixetin on the LPSmediated immune responses of BMDCs, the levels of cytokines produced by LPS-induced BMDCs were measured, either with 1436

DOI: 10.1021/acs.jnatprod.8b00155 J. Nat. Prod. 2018, 81, 1435−1443

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Figure 1. Structure, lack of toxicity, and potency of tamarixetin to inhibit the release of pro-inflammatory cytokines and promote the release of the anti-inflammatory cytokine IL-10 from cultured BMDCs exposed to LPS. (A) Chemical structures of tamarixetin and quercetin. (B) The cytotoxicity of tamarixetin to DCs was analyzed using a luminescent cell viability kit. DCs were treated with tamarixetin, DMSO, or H2O2 for 24 h. Data are presented as means ± SEM. ***, P < 0.001 compared to controls (CON). (C) DCs were pretreated with the indicated concentrations of tamarixetin for 30 min before being stimulated with LPS (50 ng·mL−1) for 6 h. (D) Time intervals at which tamarixetin (25 μM) and LPS stimulation (50 ng· mL−1) were given to DCs were −30 min, DCs were pretreated with tamarixetin for 30 min before LPS stimulation for 6 h; 0 min, DCs were cotreated with tamarixetin and LPS for 6 h; +30, +60, +90, or +120 min, after LPS stimulation for 6 h, DCs were treated with tamarixetin for 30, 60, 90, or 120 min, respectively. Supernatants were collected, and the levels of TNF-α, IL-6, IL-10, and IL-12p70 were determined by ELISA. Data are presented as means ± SEM. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared to DCs treated with LPS. n.s., no significance.

The amounts of TNF-α and IL-6 in the endotoxemic mouse sera were also significantly lower after treatment with tamarixetin. Conversely, secretion of IL-10 after induction by LPS was found to be increased (Figure 3B). Furthermore, the production of TNF-α and IL-6 in the lungs (Figure 3C) and infiltration of immune cells (Figure 3D) were both markedly decreased by tamarixetin treatment. Finally, LPS induced increases in various physiological parameters, including AST, ALT, and BUN (measures of organ damage), but these were significantly lower after tamarixetin injection (Figure 3E). These data confirmed that tamarixetin has a potential inhibitory effect on LPS-induced inflammatory responses in the endotoxemia mouse model. Tamarixetin Attenuates Escherichia coli-Induced Immune Response in BMDCs. Two strains of E. coli were used to determine whether tamarixetin affects the immune response induced by Gram-negative bacteria, a nonpathogenic DH5α E. coli strain and a pathogenic K1 E. coli strain. Dendritic cells (DCs) were pretreated for 30 min with tamarixetin and then inoculated with E. coli. Cytokine production was then measured at 30 min intervals for a total of 120 min. Similar to the LPS data (Figure 1D), an inhibitory effect was observed 30 min after tamarixetin treatment. As before, this effect gradually disappeared. In addition, levels of the anti-inflammatory cytokine IL-10 increased (Figure 4A and B). Taken together,

cytokines through increasing the expression of miRNA 187, miRNA 146a, and miRNA 155. Tamarixetin Has Anti-inflammatory Effects in an LPSInduced Endotoxemia Mouse Model. In order to assess the potential in vivo anti-inflammatory effects of tamarixetin in an LPS-induced endotoxemia mouse model, we assessed survival rates after treatment with tamarixetin (1 mg·kg−1, intraperitoneal [i.p.] injection) in mice pretreated 1 h before with LPS (25 mg·kg−1, i.p.) relative to untreated controls. This showed that tamarixetin pretreatment increased the survival rate to approximately 80% (Figure 3A). IL-10 is a well-described anti-inflammatory cytokine and is considered a key regulator of many infection-related diseases, including sepsis.39 Previous reports have shown that IL-10 reduces the secretion of pro-inflammatory cytokines and also lowers neutrophil infiltration induced by extracellular bacterial infections. It also inhibits DC maturation, leading to a reduction in their antigen presenting activities, affecting T cell activation. This subsequently improves the clearance of bacteria and related debris.40 In addition, IL-10 can be secreted by both innate and adaptive immune cells, including DCs, macrophages, T cells, and B cells.41 This leads to IL-10 acting as an antiinflammatory cytokine during sepsis. As such, it potentially suppresses the excessive inflammatory response that can occur during sepsis and prevent organ dysfunction. 1437

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Figure 2. Effects of tamarixetin on the TLR4-mediated signaling pathway in dendritic cells (DCs) activated by LPS. (A) DCs were pretreated with 25 μM tamarixetin for 30 min before being stimulated with LPS (50 ng·mL−1) for the indicated time points. The levels of p-ERK, ERK, p-JNK, JNK, p-p38, p38, p-Akt, Akt, COX-2, IκBα, and a-tubulin in cell lysates were detected by Western blot analysis. Results are representative of three independent experiments. (B) Fluorescence spectra of JNK1 in the presence of tamarixetin (0, 10, 20, 40, 60, or 100 μM) at pH 8.0. The sample was excited at 290 nm. Emission spectra were recorded for light scattering effects at 290 to 600 nm. (C) Binding model for the JNK1 and tamarixetin complex. Red dashed lines denote hydrogen bonds between tamarixetin and JNK1.

can prevent E. coli K1-induced sepsis by contributing to bacterial clearance and endotoxin elimination. Quercetin Has No Anti-inflammatory Effect on Bacterial Sepsis. Consistent with previous reports,34 our study also confirmed that quercetin inhibited the secretion of IL-10 induced by LPS, in contrast to tamarixetin (Figure 6A). However, no study has reported the anti-inflammatory effects of quercetin during bacterial sepsis. We therefore examined the antiseptic effects of quercetin relative to tamarixetin in the E. coli K1-induced bacterial sepsis model. The survival rate of mice treated with tamarixetin was 80% at a concentration of 1 mg· kg−1, while a similar concentration of quercetin did not improve survival rate at all (Figure 6B). Quercetin also did not inhibit bacterial growth in the lungs or kidneys after E. coli K1 infection (Figure 6C) and did not affect levels of endotoxin in the serum (Figure 6D). These results suggest that quercetin has no in vivo anti-inflammatory effects against bacterial sepsis. These data further demonstrate that tamarixetin has more effective therapeutic properties at lower doses than quercetin, which suggests that it has superior anti-inflammatory effects in a bacteria-induced response. It has also been reported that tamarixetin has higher hydrophobicity (consisting of a higher log P value of 1.86 and a lower polar surface area of 116.45) than quercetin (1.63 and 127.45, respectively).42 As hydrophobicity is an important determinant of membrane permeability, the higher values for tamarixetin than those for quercetin may mean it is more effective at penetrating cell membranes or tissues in vivo. This would lead to an increased

these results confirm that tamarixetin can inhibit the specific immune responses induced by Gram-negative E. coli. Tamarixetin Has an Antiseptic Effect in an E. coli K1Inoculated Sepsis Mouse Model. As tamarixetin had shown in vitro anti-inflammatory effects using DCs activated by E. coli (Figure 4), we next assessed whether it also had an antiinflammatory effect in vivo using an E. coli K1-induced sepsis mouse model. The 20 h survival rate of mice co-injected with tamarixetin in the model increased from 0% to 60% (Figure 5A). In addition, tamarixetin suppressed serum TNF-α and IL6 levels and increased IL-10 levels in the E. coli K1-induced sepsis mouse model (Figure 5B and C). Infiltration of immune cells into the lungs also increased (Figure 5D), and serum levels of AST, ALT, and BUN induced by E. coli K1 were lower after tamarixetin treatment (Figure 5E). To determine if tamarixetin also affected bacterial clearance in the organs of E. coli K1induced sepsis mice, we assessed the numbers of bacteria remaining in the major organs. The treatment with tamarixetin led to expansion in the IL10-producing DC, monocyte/ macrophage, Tr1, and B10 populations (Figure S1). The increase in these cell types induced by tamarixetin had subsequent effects on the levels of IL-10, leading to increased bacterial clearance and a reduction in bacterial counts (colony forming units [CFUs]) in the liver, lungs, and kidneys after E. coli K1 infection (Figure 5F).This was associated with a reduction in the amount of endotoxin present in the mouse sera (Figure 5G). These results demonstrate that tamarixetin 1438

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Figure 3. Effects of tamarixetin on an LPS-induced endotoxemia mouse model. (A) Mice were intraperitoneally (i.p.) injected with tamarixetin (1 mg·kg−1) 1 h before i.p. injection of LPS (20 mg·kg−1). The survival rate was observed over the next 72 h. (B) Mice were i.p. injected with tamarixetin (1 mg·kg−1) 1 h before i.p. injection of LPS (10 mg·kg−1) and left for 2 h. Serum cytokine levels (TNF-α, IL-6, and IL-10) were measured by ELISA. (C−E) Mice were i.p. injected with tamarixetin (1 mg·kg−1) 1 h before i.p. injection with LPS (10 mg·kg−1) and left for 12 h. (C) Cytokine levels (TNF-α and IL-6) in lung lysates measured by ELISA. (D) Lungs were perfused and fixed with formalin and sections stained with H&E. Results shown are representative of three independent experiments. (E) Serum levels of AST, ALT, and BUN were measured by ELISA. Data are presented as means ± SEM. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared to the LPS-injected group. 82), APC anti-mouse CD19 antibody (17-0193-82), APC anti-mouse CD11c antibody (17-0114-82), PE-cy7 anti-mouse CD11b antibody (25-0041-82), PE-cy7 anti-mouse CD4 antibody (25-0041-82), and monensin (00-4505-51) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cytokine ELISA kits specific for murine TNF-α, IL-6, IL-10, and IL-12p70 were purchased from eBioscience (San Diego, CA, USA). Anti-p-Akt, Akt, IκBα, p-ERK, ERK, p-JNK, JNK, p-p38, and p38 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-COX-2 and αtubulin antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). The anti-TLR4 antibody was purchased from Novus Biologicals (Littleton, CO, USA). LPS from either E. coli O111:B4 for the in vitro study or O127:B8 for the in vivo study was purchased from SigmaAldrich (St. Louis, MO, USA). A Limulus amebocyte lysate (LAL) assay kit was purchased from Lonza (Allendale, NJ, USA). LPS-Induced Endotoxemia and E. coli K1-Induced Sepsis Mouse Models. An LPS-induced endotoxemia mouse model was generated by intraperitoneal (i.p.) injection of LPS (either 10 and 20 mg·kg−1) dissolved in phosphate-buffered saline (PBS; n = 10). The E. coli K1-induced sepsis mouse model was generated using i.p. injection of E. coli K1 (1 × 107 CFU·mouse−1) diluted in LB broth (n = 10). Generation and Culture of BMDCs. Tibias and femurs from 6week-old female C57BL/6 mice were extracted, and the bone marrow was flushed out. This preparation was depleted of red blood cells (RBCs) by treatment with an RBC-lysing buffer (Sigma-Aldrich). BMDCs were then seeded onto six-well culture plates (1 × 106 cells· mL−1; 2 mL·well−1) in RPMI-1640 supplemented with 10% heatinactivated fetal bovine serum, 100 U·mL−1 penicillin, 100 mg·mL−1 streptomycin, and 10 ng·mL−1 rmGM-CSF at 37 °C under an atmosphere containing 5% CO2. On days 3 and 5, nonadherent cells were carefully removed, and fresh medium was added. On day 6, all

ability to effect its anti-inflammatory properties in response to bacteria. Concluding Remarks. Data presented in this study suggest that tamarixetin has a protective effect on sepsis induced by bacteria or LPS by increasing IL-10 secretion from innate and adaptive immune cells. Therefore, tamarixetin could serve as a potential health supplement or preventive agent to combat bacterial sepsis.

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EXPERIMENTAL SECTION

Animals. DCs were isolated from 6-week-old female C57BL/6 mice. BALB/c mice were used in both the LPS-induced endotoxemia model and the E. coli K1-induced sepsis model. All mice used in this study were purchased from Orient (Daejeon, Korea) and were housed under specific pathogen-free conditions in a temperature- and humidity-controlled environment for 1 week prior to the experiments. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Konkuk University, South Korea (IACUC number: KU17044). Bacterial Strains. E. coli DH5α (ATCC PTA-4750) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). E. coli K1 strain RS218 (O18:K1:H7) was kindly provided by Dr. Jang-Won Yoon of Kangwon National University (Gangwon-do, Korea). Reagents and Antibodies. RmGM-CSF was purchased from JW Creagene (Gyeonggi-do, Korea). Tamarixetin (purity ≥99% by HPLC; catalog number: 021140S) and quercetin (purity ≥99% by HPLC; catalog number: 021135S) were purchased from Indofine Chemical Company (Hillsborough, NJ, USA). FITC anti-mouse F40/ 80 antibody (11-4801-82), PE anti-mouse IL-10 antibody (12-71011439

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Figure 4. Effects of tamarixetin on dendritic cell (DC) activation when inoculated with E. coli. Time intervals at which tamarixetin (25 μM) and (A) E. coli DH5α (3 × 106 CFU·mL−1) or (B) E. coli K1 (1 × 1013 CFU·mL−1) cells were added to DCs were −30 min, DCs were pretreated with tamarixetin for 30 min before E. coli infection for 6 h; 0 min, DCs were cotreated with tamarixetin and E. coli for 6 h; +30, +60, +90, and +120 min, DCs were treated with tamarixetin for 30, 60, 90, and 120 min, respectively, and E. coli infection for 6 h. Supernatants were collected, and the levels of TNF-α, IL-6, IL-10, and IL-12p70 were determined by ELISA. Experiments were performed at least three times in triplicate. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared to DCs inoculated with E. coli. n.s., no significance. nonadherent cells, loosely adherent, and proliferating DC aggregates were harvested for analysis or LPS stimulation. On day 7, we found that 90% or more of nonadherent cells expressed CD11c (data not shown). Cytokine Enzyme-Linked Immunosorbent Assays (ELISAs). The quantities of TNF-α, IL-6, IL-10, and IL-12p70 in the culture supernatants and mouse sera were determined using sandwich ELISA kits (eBioscience), according to the manufacturer’s instructions. Detection of AST, ALT, and BUN in Mouse Serum. Serum levels of AST (aspartate aminotransferase), ALT (alanine aminotransferase), and BUN (blood urea nitrogen) were analyzed using total laboratory automation (Hitachi, Japan) and TBA-200FR NEO (Toshiba, Japan) systems at the Konkuk University Medical Center. Cell Viability Assays. BMDCs were seeded onto a 96-well white microplate at a density of 1 × 104 cells·well−1 and incubated with various concentrations of tamarixetin (6.25, 25, 50, 100, and 200 μM) for 24 h. A luminescent cell viability kit (Promega, Madison, WI, USA)

was used to assess the amount of ATP present, an indicator of metabolically active cells. The ATP present in each sample was quantified using a luminometer (Turner Biosystems, Inc., Sunnyvale, CA, USA). Hematoxylin and Eosin (H&E) Staining. Lungs were extracted from mice, fixed in 4% paraformaldehyde (PFA) solution, and dehydrated to prepare paraffin blocks. The paraffin blocks were then cut to a thickness of 5 μm to prepare tissue slides. Any remaining paraffin was removed with xylene. Tissue slides were then hydrated, stained with H&E, and dehydrated. After mounting the tissue slides, images were captured under a microscope. Western Blotting. Proteins (a total of 25 μg per sample) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. After blocking the membranes with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T), they were incubated with the indicated antibodies. After washing with TBS1440

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Figure 5. Effects of tamarixetin on a bacterial sepsis mouse model. (A) Mice were intraperitoneally (i.p.) injected with tamarixetin (1 mg·kg−1) 1 h before i.p. injection of E. coli K1 (1 × 107 CFU·mice−1). The survival rate was observed over the next 72 h. (B) Mice were i.p. injected with tamarixetin (1 mg·kg−1) 1 h before i.p. injection of E. coli K1 (1 × 106 CFU·mice−1) and left for 2 h. Serum cytokine levels (TNF-α, IL-6, and IL-10) were measured by ELISA. (C−G) Mice were i.p. injected with tamarixetin (1 mg·kg−1) 1 h before i.p. injection of E. coli K1 (1 × 106 CFU·mice−1) and left for 12 h. (C) Cytokine levels (TNF-α and IL-6) in lung lysates were measured by ELISA. (D) Lungs were perfused and fixed with formalin, and sections were stained with H&E. Results shown are representative of three independent experiments. (E) Serum levels of AST, ALT, and BUN were measured by ELISA. (F) Bacterial counts (CFUs) in the liver, lungs, and kidney were assessed after plating lysates onto LB agar plates. (G) Serum endotoxin levels were analyzed using an LAL endotoxin assay kit. Data are presented as means ± SEM. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared to the E. coli K1-injected group. T, the membranes were incubated with a secondary antibody conjugated to horseradish peroxidase and visualized using an enhanced chemiluminescence kit (Merck, Darmstadt, Germany). Determination of Bacterial Counts in Organ Tissues. At the time of sacrifice, the lungs, liver, and kidneys were removed aseptically and placed separately in 1 mL of sterile PBS. The tissues were then homogenized on ice using a tissue homogenizer under a vented hood. The lung, liver, and kidney homogenates were diluted with PBS to 1:1000. After plating 10 μL of each diluted sample onto LB agar, the plates were then incubated at 37 °C for 24 h. The numbers of colonies were then counted and used to assess the relative abundances of bacteria. Detection of Endotoxin in Mouse Serum. The levels of endotoxin in mouse sera were measured using an LAL chromogenic end point assay (Lonza Group Ltd., Allendale, NJ, USA), according to the manufacturer’s recommendations. Mouse sera were diluted 10-fold in endotoxin-free PBS before the assay. After subtracting background levels, the results were calculated relative to an E. coli endotoxin standard provided with the assay kit. The relative amounts of endotoxin in each sample are expressed as EU·mL−1. Fluorescence Quenching Experiments. We expressed and purified human JNK1 using a C-terminally His6-tag, as previously described.43 Tamarixetin was titrated with 10 μM JNK1 in a 50 mM sodium phosphate buffer containing 100 mM NaCl at pH 8.0. The final protein:inhibitor ratio was 1:10. The samples were then placed in 2 mL cuvettes with excitation and emission path lengths of 10 nm. Tryptophan emission was measured to determine the fluorescence quantum yields of JNK1 in the presence of tamarixetin at 25 °C using an RF-5301PC spectrofluorophotometer (Shimadzu, Kyoto, Japan), as described previously.44 Docking Study. Tamarixetin was docked to the ATP-binding site of JNK1 determined by the crystal structure (PDB ID: 3V3V) using CDOCKER, a CHARMm-based molecular dynamics method in

Discovery Studio (Accelrys Inc., San Diego, CA, USA). The CDOCKER algorithm uses the center of a sphere for the initial ligand placement. The final minimization steps optimize the docking pose of the ligand with the 50 steepest descent steps, followed by 200 conjugated-gradient steps, and using an energy tolerance of 0.001 kcal· mol−1, as described previously.42 Flow Cytometry. Mouse splenocytes were isolated from the spleens of each animal. To enrich white blood cells (WBCs), red blood cells (RBCs) were lysed using an RBC lysing buffer (Sigma-Aldrich) at room temperature at 25 °C for 15 min. The WBCs were washed with cold PBS, counted using a hemocytometer, and seeded onto a 24-well cell culture plate at a density of 3 × 106 cells·well−1. The splenocytes were stimulated with phorbol 12-myristate 13-acetate (50 ng), ionomycin (0.5 μg), monensin, and LPS (10 μg) at 37 °C under an atmosphere containing 5% CO2 for 5 h. Concurrently, surface marker antibodies (ms anti-F40/80-FITC, ms anti-CD19 APC, ms antiCD11c APC, ms anti-CD11b PE-cy7, ms anti-CD4 PE-cy7) and the intracellular marker antibody (ms anti-IL-10 PE) were prepared in PBS (1% ms anti-IL-10 PE, 99% PBS). The splenocyte WBCs were spun down at 1500 rpm for 5 min in a V-bottom plate, washed with cold PBS, and incubated with surface antibodies for 30 min on ice. Afterward, the cells were again spun down and washed with cold PBS. The WBCs were then fixed with 4% PFA, permeabilized using permeabilization buffer, and incubated with the intracellular antibody for 20 min. The proportions of immune cells expressing the indicated markers were then measured using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). miRNA Analysis. Total RNA was extracted from DCs using TRIzol reagent and reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Amplification of cDNA was performed using a LightCycler 480II (Roche, Basel, Switzerland) and a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific), according to the 1441

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Figure 6. Effects of tamarixetin and quercetin on bacterial sepsis. (A) Dendritic cells (DCs) were pretreated for 30 min with tamarixetin (25 μM) or quercetin (25 μM) for 30 min before stimulation with LPS (50 ng·mL−1) for 6 h. Supernatants were collected, and the levels of IL-10 were determined by ELISA. Data are presented as means ± SEM. ***, P < 0.001 compared to DCs treated with LPS only. n.s., no significance. (B) Mice were intraperitoneally (i.p.) injected with tamarixetin (1 mg·kg−1) or quercetin (50 mg·kg−1) 1 h before i.p. injection of E. coli K1 (1 × 107 CFU· mice−1). Survival rates were observed over the next 72 h. (C and D) Mice were i.p. injected with tamarixetin (1 mg·kg−1) or quercetin (50 mg·kg−1) 1 h before i.p. injection of E. coli K1 (1× 106 CFU·mice−1) and left for 12 h. (C) Bacterial counts (CFUs) in the lungs and kidney were measured after plating lysates onto LB agar plates. (D) Serum endotoxin levels were analyzed using an LAL endotoxin assay kit. Data are presented as means ± SEM. ***, P < 0.001 compared to the E. coli K1-injected group.

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manufacturer’s recommended conditions. The expression levels of miR-146a, miR-155, and miR-187 mRNA were quantified by real-time quantitative PCR using gene-specific primers (available upon request) and the cDNA as a template. Statistical Analysis. All experiments were repeated at least three times with consistent results. Unless otherwise stated, data are expressed as means ± SEM. Student’s t tests were performed to compare experimental groups and controls. A Tukey’s multiple comparison test (Prism 3.0; GraphPad Software Inc., La Jolla, CA, USA) was performed to compare multiple groups. Kaplan−Meier survival curves were analyzed using log rank tests. Comparisons were considered statistically significant at P < 0.05.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail (I. D. Jung): [email protected]. Phone: +82-2-20496330. Fax: +82-2-2049-6192. *E-mail (Y.-M. Park): [email protected]. Phone: +82-22049-6330. Fax: +82-2-2049-6192. ORCID

Yangmee Kim: 0000-0001-6438-4718 In Duk Jung: 0000-0002-0578-1817 Author Contributions ∥

H. J. Park, S. J. Lee, and J. Cho contributed equally to this work. Notes

ASSOCIATED CONTENT

The authors declare no competing financial interest.

S Supporting Information *

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00155.

ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and

Additional information (PDF) 1442

DOI: 10.1021/acs.jnatprod.8b00155 J. Nat. Prod. 2018, 81, 1435−1443

Journal of Natural Products

Article

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Technology (grants: 2013R1A4A1069575, 2016R1D1A3B03934384, and 2016R1A5A2012284).

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DOI: 10.1021/acs.jnatprod.8b00155 J. Nat. Prod. 2018, 81, 1435−1443