Yuwen02f1 suppresses LPS-induced endotoxemia and adjuvant-induced arthritis primarily through blockade of ROS formation, NFkB and MAPK activation
Chun-Chieh Hsu a, Jin-Cherng Lien b,**, Chia-Wen Chang a, Chien-Hsin Chang a,
Sheng-Chu Kuo b, Tur-Fu Huang a,*
a Graduate Institute of Pharmacology, College of Medicine, National Taiwan
University, Taipei, Taiwan
b Graduate Institute of Pharmaceutical Chemistry, China Medical University,
Taichung, Taiwan
* Corresponding author at: Graduate Institute of Pharmacology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Rd, Taipei, Taiwan. Tel.: +886 2 23123456x88332; fax: +886 2 23214009.
** Corresponding author. Tel.: +886 4 22053366x5607. E-mail addresses: [email protected] (J.-C. Lien), [email protected] (T.-F. Huang).
Keywords: Diphenylpyrazole ROS NFkB MAPK Inflammatory animal
Phagocytes release inflammatory mediators to defense harmful stimuli upon bacterial invasion, however, excessive inflammatory reaction leads to tissue damage and manifestation of pathological states. Therefore, targeting on uncontrolled
inflammation seems feasible to control numerous inflammation-associated diseases. Under the drug screening process of synthetic diphenylpyrazole derivatives, we discovered compound yuwen02f1 possesses anti-inflammatory effects in decreasing the release of pro-inflammatory cytokines including TNFa and IL-6, nitric oxide, reactive oxygen species (ROS) as well as inhibiting migration of LPS-stimulated phagocytes. In addition, we observed that the molecular mechanism of yuwen02f1-mediated anti-inflammation is associated with decreasing phosphorylation of MAPK molecules including ERK1/2, JNK and p38, and attenuating translocation of p47phox and p67phox to the cell membrane. Yuwen02f1 also reverses IkBa degradation and attenuates the expression of NFkB-related downstream inducible enzymes like iNOS and COX-2. Furthermore, we found that yuwen02f1 attenuates some pathological syndromes of LPS-induced sepsis and adjuvant-induced arthritis in mice, as evidenced by decreasing the cytokine production, reversing thrombocyto- penic syndrome, protecting the mice from tissue injury in septic mice, and attenuating paw edema in arthritic mice as well. These results suggest that yuwen02f1 is a potential anti-inflammatory agent for alleviating syndromes of acute and chronic inflammatory diseases as evidenced by attenuating the generation of cytokines and down-regulating the expression of iNOS and COX-2 through the blockade of ROS generation and NADPH oxidase, NFkB and MAPK activation pathways in LPS-stimulated phagocytes.
1. Introduction
Inflammation is an adaptive response triggered by harmful stimuli and certain conditions, such as infection, toxin, tissue injury and irritation [1]. Appropriate inflammatory response is beneficial for the host to protect against pathogens and wound healing, but uncontrolled inflammation leads to extensive tissue damage and manifestation of pathological states [2,3]. On exposure to pro-inflammatory,
metabolic and immune stimuli, monocytes are recruited to the extravascular tissues and differentiate into macrophages. Once activated, macrophages are the main source of growth factors and cytokines, which profoundly affect endothelial, epithelial and mesenchymal cells in the local microenvironment and contribute to host defense, tissue remodel-ing and repair [4]. Macrophages have versatile roles. As scavenger cells, they get rid of cellular debris and pathogens. As secretory cells, they release over 100 different mediators, including pro-inflammatory and cytotoxic cytokines, growth factors, bioactive lipids, hydrolytic enzymes, reactive oxygen intermediates, and nitric oxide in biologic activity from induction of cell growth to cytotoxicity [5,6]. This complex defense network successfully restores normal homeostasis, but it is proved to be detrimental to the host if dysregulated and leads to disorders like endotoxemia, ischemia reperfusion injury, multiple organ failure, and acute respiratory distress syndrome.
Sepsis is a complex pathophysiological state and is still associated with a high degree of mortality. The initial symptoms of sepsis encompass those usually associated with acute inflammation, including fever or hypothermia, tachypnea, tachycardia, high white blood cell counts and pulmonary edema, and finally resulting in septic shock and a multiple organic failure system. Lipopolysaccharide (LPS) is an important structural component of the outer membrane of Gram-negative bacteria that
can trigger a variety of inflammatory reactions, including the release of
pro-inflammatory cytokines and other soluble factors. These mediators may trigger the systemic inflammation and even cause end-organ damage, sepsis, and death. Upon LPS recognition, LPS is transferred to CD14 by LPS binding protein (LBP) and recognized by TLR4-MD-2 complex on the cellular surface leading to the rapid and coordinated activation of various intracellular signaling pathways including activation of the protein kinase C, Src-related kinases, and the major MAP kinase cascades and translocation of NFkB [7,8]. These intracellular signaling pathways that control expression of genes of the inflammatory responses might become therapeutic targets in a wide range of diseases.
Here, we investigated the pharmacological effects of yuwen02f1, 1-(4-fluorobenzyl)-5-(5-ethoxycarbonyl-2-furyl)-3- phenylpyrazole (Fig. 1), which is a synthetic diphenylpyrazole (DPP) compound modified on the basis of herb extract. In 1979, DPPs were studied based on their anti-inflammatory, analgesic and antipyretic properties [9]. Recently, DPPs have been reported to be against prion infections [10] and HIV-1 nonnucleoside reverse transcriptase inhibitors [11]. YC-1 is most
investigated DPP, which was reported to possess anti-inflammatory activity, including lipoteichoic acid-induced iNOS expression in mouse macrophage cell line [12], LPS-induced cytokine release in peripheral blood mononuclear leukocytes and mouse models [13] and LPS-induced pro-inflammatory responses in microglia [14].
However, the full mechanism of how these derivates affect inflammatory process still remains uncertain.
In our study, we discovered that yuwen02f1 possesses anti-inflammatory effects of decreasing inflammatory cytokines, nitric oxide and ROS. We also identified the anti-inflammatory mechanisms of yuwen02f1 through modulating MAP kinases and
NF-kB signaling pathways. Furthermore, we used LPS- induced endotoxemic and adjuvant-induced arthritis animal models to investigate the effects of yuwen02f1 on acute and chronic inflammation.
2. Materials and methods 2.1. Materials
Yuwen02f1 was provided by Dr. Jin-Cherng Lien (China Medical College, Taichung, Taiwan). This synthetic compound yuwen02f1 is purified by
chromatography (silica gel/ethyl acetate: n-hexane = 1:1) after preparation and then crystallized from ethanol. The purity of yuwen02f1 is more than 95% by its spectral data (Fig. 1). The enzyme-linked immunosorbent assays (ELISA) kit for TNFa and IL-6 were from R&D Systems (Minneapolis, MN, USA). DMEM, FBS and all culture reagents were purchased from Gibco BRL (Life technologies, USA). Antibodies against phosphorylated p38, phosphorylated JNK, phosphorylated ERK, a-tubulin, COX-2, iNOS, p47phox, p67phox and p22phox were purchased from Santa Cruz (Biotechnology, Inc., USA). Lipopolysaccharide (LPS, from E. coli, 0127:B8), gelatin, dexamethasone, 2070-dichlorofluorescein-diacetate (DCF-DA) and other chemicals were purchased from Sigma Chemicals, Co. (St Louis, Mo, USA). Pam3CSK4 was from InvivoGen (San Diego, CA, USA).
2.2. Cell cultures
The RAW264.7 (mouse macrophage) and THP-1 (human monocyte) were obtained from the American Type Culture Collection and were cultured in DMEM and RPMI-1640 media with 10% FBS, respectively. The cells were maintained at 37 °C in an atmosphere containing 5% CO2.
2.3. Cytokine assays
After macrophages and monocytes were cultured with 100 ng/ ml or 1 mg/ml LPS or 1 mg/ml Pam3CSK4 alone or with varying concentrations of yuwen02f1 or dexamethasone (Dex) for 24 h, media were collected by centrifugation. Cytokines were measured by ELISA kit according to the manufacture’s instruction.
2.4. Migration assay
Migration assay of RAW264.7 macrophages were measured as previously described with modification [15]. In brief, Costar Transwells (5 mm pore size) were coated with 2% gelatin. RAW264.7 (1 _ 105 cells per well) treated with LPS (100 ng/ml) or not were incubated with different concentration of yuwen02f1 at 37 8C for 30 min, and then cells were seeded into the upper chamber. After incubation at 37 8C for 20 h, the macrophages that had transmigrated and bound to the membrane were fixed with 4% paraformadehyde and stained with 0.5% toluidine. Migration was quantified by counting the number of stained cells with light microscope (Nikon, Japan) in 3 random fields (200x). Results are expressed as a migration index, in which the number of control that migrated in the absence of LPS and yuwen02f1 was set at 1. Migration of THP-1 monocyte was conducted by similar method with different cell counts of 5 x 105 cells per well and LPS with a concentration of 1 mg/ml as inducer.
2.5. Cell viability assay
Cell viability was determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma, St Louis, MO, USA) reduction assay. In brief, RAW264.7 cells were pre-incubated overnight in 48-well plates at a density of 5 _ 104 cells per well, and were then treated with various concentrations of yuwen02f1 (3, 10, or 30 mM) or positive controls (Dexamethasone, Dex) coexisted with LPS
(100 ng/mL). After LPS stimulation for 24 h, the culture supernatants were replaced with MTT (0.5 mg/ml) at 37 °C with 5% CO2 for 30 min. The resulting dark blue
crystals were then dissolved with DMSO (200 ml/well) after the MTT were removed. Absorbance values were read at 550 nm with an automated SpectraMAX 340
(Molecular Devices, Sunnyvale, CA, USA). All determinations were obtained by replication in at least three independent experiments
2.6. LDH release assay
LDH (Lactate dehydrogenase) release assay was conducted by CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, CA, USA) kit. RAW264.7 cells were plated onto 48-well plates at a density of 4 x 104 cells per well, and then treated with
various concentrations of yuwen02f1 (3, 10, or 30 mM) at 37 °C for 24 h. During the last 30 min of experiment, the lysis buffer was added to perform the total lysis group. At the end of treatment, the cell supernatants were collected to mix with reconstituted substrate in 96-well plate and incubated at room temperature. After 30 min, the stop solution was added to stop the reaction and the absorbance values were read at 490 nm with an automated SpectraMAX 340 (Molecular Devices, Sunnyvale, CA, USA). The result was expressed as percentage of release relative to total lysis group (as 100% LDH release).
2.7. Nitric oxide assay
The nitrite accumulated in the culture medium was measured as an indicator of nitric oxide (NO) production based on the Griess 2.8. ROS measurement Production of intracellular ROS in RAW264.7 macrophage cells was evaluated by using the non-polar 2070-dichlorofluor- escein-diacetate (DCF-DA) dye [17,18]. Briefly, the cells were treated with various concentrations of yuwen02f1 coexisted with LPS (1
mg/mL) for 24 h. After washed with PBS, cells were incubated with 20 mM DCF-DA at 37 °C for 25 min and then analyzed by FACS Calibur (Becton Dickinson, USA) for relative florescence intensity.
2.9. Subcellular fractionation
For making whole cell lysates, the cells were lysed in radioimmune precipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail (Roche,
Mannheim, Germany). Membrane and cytoplasmic protein were obtained with Proteo JETTM membrane protein extraction kit (Thermo Fermentas, EU) according to manufacturer’s protocol.
2.10. Western blot analysis
RAW 264.7 cells (2–10 x 105 cells/ml) were plated onto 6-well plates and
pretreated with yuwen02f1 for 30 min or 1 h and then stimulated with the presence or absence of 100 ng/ml or 1 mg/ml LPS or 1 mg/ml Pam3CSK4 for 24 h (for iNOS), 30 min (for MAPKs, IkB and COX-2), or 15 min (for p22phox, p47phox and p67phox). Aliquots of total cell lysates or cell membrane fractions were analyzed by Western blotting. For quantification of the Western analysis, the density of each band was quantified by ImageQuant software.
2.11. Animals
Male ICR mice weighing 25–30 g were used in the animal model. All the experimental protocols regarding animal studying have been approved by the Laboratory Animal Use Committee of College of Medicine, National Taiwan
University. The animals were maintained on a 12 h light/dark cycle under controlled tempera-ture (20 ± 1 °C) and humidity (55 ± 5%). Animals were given continuous
access to food and water.
2.12. Acute inflammation model: LPS-induced endotoxemia
Mice were injected intraperitoneally (i.p.) with LPS at a dose of 30 mg/kg body weight, and then the mouse was intravenously injected with DMSO (as control, 20 ml) or yuwen02f1 (3.2 and 8 mg/g) through lateral tail vein.
2.13. Mice whole blood and serum collection
After injecting intraperitoneally (i.p.) with LPS and immediately followed by intravenous injection of yuwen02f1 for 2 h or 24 h, mice were anesthetized by i.p. injection of sodium pentobarbital (50 mg/g, i.p.) and whole blood was drawn by cardiac puncture or from the eyeholes and collected in citric acid-citrate dextrose (ACD; 9:1 blood vol/vol) at indicated time. The sera were obtained by centrifugation at 1000 _ g for 10 min.
2.14. Measurement of cytokine levels
The concentration of cytokines, TNFa and IL-6, in the sera was determined by ELISA kit according to the manufacture’s instruction.
2.15. Chronic inflammation model: adjuvant-induced arthritis
Arthritis progression is made in adjuvant-induced arthritis in the ICR mice. On day 0, the right hind paw of each ICR mice were injected intradermally with 0.04 ml of complete Freund’s adjuvant (CFA) which was heat-killed mycobacterium
butyricum suspended in mineral oil as an adjuvant (CFA, 1% suspension in olive oil; Difco) while the left one injected with 0.04 ml of normal saline as internal control.
Procedures were performed under anesthesia with sodium pentobarbital (50 mg/g, i.p.).
2.16. Yuwen02f1 treatment and evaluation of arthritis
The whole experimental period consists of 21 days. Yuwen02f1 (10 mg/g) was injected intraperitoneally (i.p.) into the mice every two days from day 0 (adjuvant-induction day) in whole period (day 1–21), and paw volumes were measured at the day 0, 3, 7, 11, 15, 19 and 21 by using digital plethysmometer. The magnitude of the arthritis was evaluated by measuring the volumes of both hind paws, and was
indicated by inflammation index that was a quotient by dividing the volume of adjuvant-injected paw by its contralateral internal control.
2.17. Histological examination
Lung, liver, kidney segments and the paws as well as ankle joints were cut off and fixed in 10% (v/v) phosphate-buffered formalin for 48–72 h and then embedded in paraffin. Next, the samples were sectioned (5 mm) using a microtome, stained with H&E, and examined with light microscopy at 400 x magnifications.
2.18. Statistical analysis
All values are presented as mean ± S.E.M. Differences between groups were assessed by one-way ANOVA and Newman-Keuls multiple comparison test where appropriate. Two groups were compared by unpaired Student’s t test. Odds ratio was calculated. P values less than 0.05 (p < 0.05) were considered as significant
difference.
3.1. Yuwen02f1 inhibits pro-inflammatory cytokines TNFa and IL-6 production in LPS-stimulated phagocytes
First of all, we performed cytokine release assay and cell viability assay to select compounds possessing anti-inflammatory effect with the least cytotoxicity. Under the investigation of a series of DPP-derivatives, we found several compounds exhibiting anti-inflammatory activity in suppressing the production of TNFa in LPS-stimulated RAW264.7 macrophages. Among them, yuwen02f1 showed the least effect on cell viability. Therefore, we focused on yuwen02f1 to further study its pharmacological effects.
Pro-inflammatory cytokines enhance inflammation at locally inflamed tissues by maintaining the inflammatory process at various steps [4]. To investigate the anti-inflammatory effect of yuwen02f1 on LPS-stimulated phagocytes, the production of pro-inflammatory cytokines in RAW 264.7 macrophages and THP-1 monocytes were evaluated by ELISA (Fig. 2). We observed that the production of inflammatory cytokines from these phagocytes, including TNFa and IL-6 increased after exposure to LPS. However, yuwen02f1 (3–30 mM) treatment led to marked attenuation of TNFa and IL-6 production in a concentration-dependent manner. On the other hand, dexamethasone (10 mM), a positive control, also showed inhibitory effect on LPS-induced cytokine release from these phagocytes used (Fig. 2).
To exclude the possibility that reductions in the levels of inflammatory cytokines occurred due to the direct toxicity of yuwen02f1, we conducted MTT assay to
determine the cell viability, and measured LDH release as an index of cell damage in RAW264.7 macrophage cells. The treatment of yuwen02f1 on macrophages had no apparent effect on cell viability and no cytotoxicity compared with control (Fig. 2G and H). Yuwen06f1 is devoid of cytotoxicity at 30 M.
We further used a TLR2-specific activator, Pam3CSK4, to test the effect of Yuwen06f1 on the production of pro-inflammatory cytokines in RAW 264.7 macrophages. Interestingly, yuwen02f1 (30 M) treatment also led to marked attenuation of TLR2-induced TNFa production (Fig. 2I).
3.2. Yuwen02f1 inhibits the migration of LPS-stimulated murine macrophages and human monocytes
During inflammation, monocytes transmigrate to extravascu- lar sites and differentiate into macrophages to clear pathogens, so we used gelatin-coated transwells to study the effect of yuwen02f1 on LPS-stimulated transmigration of murine macro- phage cells and human monocytes. Yuwen02f1 (3–30 M) exhibited a concentration-dependent inhibition on LPS-induced migration of RAW264.7
macrophge cells (Fig. 2C) and human THP- 1 monocytes (Fig. 2F).
3.3. Yuwen02f1 inhibits nitric oxide production and iNOS expression in LPS- stimulated RAW264.7 macrophages
Previously, it was reported that LPS induces TLR4 expression and increases nitric oxide (NO) production by increasing the expression of inducible nitric oxide synthase (iNOS) [19]. Yuwen02f1 treatment (30 M) significantly reduced LPS-induced NO production (Fig. 3A) and decreased LPS-stimulated iNOS expression (Fig. 3B). These data suggest that yuwen02f1 inhibits NO production at the transcriptional level of the iNOS gene in LPS-stimulated macrophages.
3.4. Yuwen02f1 inhibits COX-2 expression in LPS-stimulated RAW 264.7 cells COX-2 is an inducible enzyme that catalyzes the production of important biological mediators called prostaglandins that play an essential role in inflammation
and pain [16]. We next investigated the effects of yuwen02f1 on COX-2 expression in LPS-stimulated RAW264.7 cells. As shown in Fig. 3C, yuwen02f1 (3–30 M) significantly suppressed the LPS-induced COX-2 expression in a concentration-dependent manner.
3.5. Yuwen02f1 inhibits ROS production in LPS-stimulated RAW 264.7 macrophages Reactive oxygen species (ROS), which are synthesized by NADPH oxidase, serve as secondary messengers to activate multiple intracellular proteins and enzymes involved in physio-logical and pathological states. During inflammation, activated macrophages largely increase the oxygen uptake resulting in massive release of ROS called respiratory burst [20,21]. Overpro-duction of ROS is thought to be harmful in inflammatory diseases like sepsis, so we examined the anti-oxidant effects of
yuwen02f1 in LPS-stimulated macrophages. We observed that treatment of
yuwen02f1 (3–30 mM) during the 24 h stimulation with 1 mg/ml LPS resulted in a concentration-dependent decrease of measured fluorescence, thus demonstrating its inhibitory effect on intracel-lular ROS production (Fig. 3D and E).
3.6. Yuwen02f1 inhibits LPS-Induced translocation of the cytosolic subunits p47phox and p67phox to the cellular membrane
The activation of NADPH oxidase requires that the cytosolic component p47phox be phosphorylated, and subsequently trans-located along with the p67phox component to the plasma membrane, where they associate with the membrane-bound subunits, gp91phox and p22phox, to assemble into an active enzyme complex [22]. Because Yuwen02f1 showed potent effects on extracellular ROS production, we sought to determine whether Yuwen02f1 inhibits NADPH oxidase activation by preventing the translocation of the cytoplasmic subunits p47phox and p67phox from
cytosol to membrane following LPS stimulation. As shown in Fig. 4F, in the absence of LPS, weak basal p47phox and p67phox in membrane was detected, and an increase in the immunoreactivity of both p47phox and p67phox was observed 15 min
following LPS stimulation. LPS-induced translocation of p47phox and p67phox was significantly and concentration-dependently inhibited by Yuwen02f1 pretreatment for 30 min (Fig. 3F). Therefore, it is likely that Yuwen02f1-mediated inhibition of
superoxide produc-tion by LPS is primarily through the inhibition of p47phox and p67phox translocation to the cellular membrane.
3.7. Yuwen02f1 inhibits the phosphorylation of MAPK molecules in LPS-stimulated RAW 264.7 cells
MAPK molecules are among the important signaling pathways that control the synthesis and release of pro-inflammatory mediators like cytokines and NO by activated macrophages during the inflammatory response [23]. Therefore, the phosphorylation of three MAPK molecules, extracellular signal-related kinase 1/2 (ERK1/2), p38 MAP kinase (p38) and c-Jun NH2-terminal kinase (JNK) were examined by Western blot analysis. LPS stimulation rapidly induced the
phosphorylation of ERK1/2, p38 and JNK within 30 min in RAW 264.7 cells (Fig. 4A), and yuwen02f1 (3–30 mM) concentration-dependently suppressed the
expression of p-ERK, p- JNK and p-p38 in LPS-stimulated macrophages. These results indicate that signal transduction by MAPK molecules was blocked by yuwen02f1 in LPS-activated macrophages. We also investigate TLR2-specific
activation of MAPK, and found that yuwen06f1 (30 mM) suppressed the Pam3CSK4-induced phosphorylation of ERK1/2 in RAW 264.7 cells (Fig. 4C).
NFkB is a major transcription factor that has been shown to be essential for the expression of iNOS and COX-2, as well as for inflammatory cytokines [24]. A large variety of inflammatory conditions, including bacterial and viral infections, rapidly activate NFkB pathway. During the process, the degradation and phos-phorylation of IkBa are necessary to release NFkB from the cytoplasmic NFkB/IkBa complex and allow its subsequent translocation to the cell nucleus. We evaluated the effect of yuwen02f1 on LPS-induced degradation of IkBa in RAW 264.7 macrophages. As shown in Fig. 4B, we observed that yuwen02f1 (3–30 M) suppresses LPS-induced degradation of IkBa in a concentration-dependent manner, suggesting that yuwen02f1 inhibits LPS-induced NFkB activation through prevention of IkBa degradation and phosphorylation.
3.9. Yuwen02f1 inhibits pro-inflammatory cytokines production in vivo
We used the endotoxemic animal model to investigate the effects of yuwen02f1 on acute inflammation in vivo, and mice were injected intraperitoneally (i.p.) with LPS and immediately followed by intravenous injection of yuwen02f1 (3.2 and 8 mg/g) through lateral tail vein. After 2 h, the sera of mice were collected and the cytokine release was measured by ELISA kit. We observed that LPS elevated plasma TNFa and IL-6 levels while yuwen02f1 administration dose-dependently decreased the cytokine levels (Fig. 5A and B), indicating that yuwen02f1 efficiently reduced cytokine release in LPS-induced septic animal.
3.10. Yuwen02f1 reverses LPS-induced thrombocytopenia
Bacterial invasion disturbs the function of coagulation and fibrinolytic systems, causing systemic thrombocytopenic phenom-enon [25]. The number of platelets was reduced from 1114 ± 86/ml to 322 ± 30/ml in LPS-challenged mice after 24 h.
However, yuwen02f1 (3.2 and 8 mg/g) treatment increased the platelet number to 464 ± 36/ml and 504 ± 38/ml, respectively (Fig. 5C), indicating that yuwen02f1
significantly reversed LPS-induced thrombocytopenia.
In addition, we also monitored effect of yuwen02f1 on the mortality of mice with lethal endotoxemia, and found that 24 h survival rate for LPS-treated mice was 56% (9/16), but raised to 77% (17/22) and 78% (18/23), respectively, after yuwen02f1 treatment (3.2 and 8 mg/g; Odds ratio of survival: 2.64 and 2.80, respectively) (Fig. 5D). The tendency of reduced mortality indicated that yuwen06f1 could be beneficial to endotoxemia.
3.11. Effects of yuwen02f1 on tissue injury in endotoxemia examined by histochemistry
In comparison with non-induction mice (Fig. 6A), we found that liver section from LPS-induced mice exhibited leukocyte infiltration in liver parenchyma and showed peri-vascular infiltration (Fig. 6B). Histological examination of lung section from LPS-induced mice (Fig. 6E) was characterized by the presence of leukocytes in the lung interstitium and alveoli and thickening of the alveolar wall, while the kidney was also impaired by LPS induction with evidence of glomerular hypercellularity (Fig. 6H). However, treatment of yuwen02f1 protected the mice from these organ injuries caused by LPS induction (Fig. 6C, F and I).
3.12. Yuwen02f1 attenuates paw edema caused by adjuvant-induced arthritis In chronic inflammation model, we used complete Freund’s adjuvant-induced arthritis to investigate the anti-inflammatory effect of yuwen02f1. Under the investigation, the arthritic mice showed a soft tissue swelling that was noticeable around ankle joints and was believed due to edema of periarticular tissues such as
ligaments and joint capsules [26]. After induction, swelling and redness developed over a 24 h period in the foot injected with adjuvant followed by the continuous inflammation observed as increase in paw volumes (Fig. 7A). On the day 15, the inflammation reaction slightly subsided. In comparison, yuwen02f1 treatment markedly suppressed the primary inflam- mation (on day 7), and the effect was sustained to suppress secondary inflammation (on day 15).
3.13. Effects of yuwen02f1 on histopathology of adjuvant-induced arthritis Under histological examination, we observed that the synovial lining of
adjuvant-induction paw is hyperplastic, with multiple layers of cells (Fig. 7B, middle panel) compared with a normal lining of non-induction paw (Fig. 7B, upper panel). In addition, the adjuvant-induction paw revealed mixed inflammatory infiltration as well as pannus (membrane of granulation tissue, which is chronic and progressive and produce joint erosion) formation (*) as shown in Fig. 7B. However, adjuvant-induction paw of yuwen02f1- treated mice showed slight improvement in the pathophysiology of adjuvant-induced arthritis (Fig. 7B, lower panel) including the lesser intense infiltration and the thinner synovial lining.
4. Discussion
Upon exposure to bacterial infection, pro-inflammatory cyto-kines like TNFa and IL-6 are secreted mostly by human blood leukocytes such as
monocytes/macrophages to defense against bacterial infections. However,
overproduction of these cytokines leads to many inflammatory diseases. Under the drug screening process, we discovered that compound yuwen02f1, a synthetic phenylpyrazole derivative, effectively reduces LPS-stimulated TNFa and IL-6
cells and THP-1 monocytes.
During the process of inflammation, the activated monocytes/ macrophages attracted by chemokines migrate to site of inflam-mation and clear pathogens, so migration of leukocytes represents a good index of inflammation. We found that yuwen02f1 significantly inhibited LPS-induced migration of murine macro-phages and human monocytes (Fig. 2). However, yuwen02f1 did not inhibit LPS-induced adhesion of monocytes/macrophages to several kinds of matrix, including collagen, fibronectin, fibrinogen and vitronectin (data not shown).
LPS stimulates macrophages and activates the expression of genes responsible for the synthesis of inflammatory mediators, such as reactive oxygen and nitrogen species (NO, O2_, H2O2, and peroxynitrite). These free radicals are important mediators that provoke or sustain inflammatory processes to protect hosts from harmful stimuli, but high levels of them are cytotoxic in causing inflammatory diseases, including atherosclerosis, rheumatoid arthritis, diabetes, septic shock, transplant rejection, and multiple sclerosis [27,28]. In this report, we found that yuwen02f1 effectively decreased NO production via suppressing the protein expressions of iNOS in LPS-stimulated RAW264.7 cells. In addition, we demonstrated yuwen02f1’s capacity to decrease intracellular ROS production, inhibiting p47phox and p67phox translocation to the cell membrane, and subsequent NADPH oxidase activation in LPS-activated macrophages (Fig. 3F). These results suggest that yuwen02f1 possesses anti-oxidant effects in neutralizing free radicals and attenuates inflammation.
The detail mechanism of yuwen06f1 on TLRs-stimulated signaling pathway is still under study. From the preliminary data, it was shown that yuwen06f1 did not interrupt the recruitment of MyD88 and TRIF to the receptor-complex. Hmama et al.
purposed that LPS binding to CD14 switches on the small G-protein Rho leading to activation of PI3-kinase and leads to increased monocyte adherence [29]. In addition, yuwen06f1 does not inhibit LPS-induced macrophage adhesion. Accordingly,
yuwen06f1 seems to have no effect on TLR4 complex (LPS/CD14/TLR4), since yuwen06f1 did not interrupt the recruitment of MyD88 and TRIF to
receptor-complex. It is possible that the inhibitory effects of yuwen06f1 may primarily derive from NADPH oxidase-inhibition, though this needs to be further investigated.
PGE2 is another inflammatory mediator generated at inflammatory sites by COX-2, known as prostaglandin endoperoxide synthase. It is associated with many chronic inflammatory diseases, including cardiovascular diseases, arthritis,
inflammatory bowel disease, angiogenesis and chronic gastric ulcer [30–32]. Thus, the suppression of COX-2 upregulation induced by LPS (Fig. 3) is partially
responsible for its anti-inflammatory activity. To explore the possible underlying mechanism, two major kinase-mediated signaling pathway enzyme complexes activated by LPS stimulation: MAP Kinase and IkB kinase (IKK) were examined. Upon exposure to LPS, monocytes/macrophages acti-vate the MAPK pathway to induce migration and accumulation of leukocytes, production of cytokines and pro-inflammatory mediators. Treatment of yuwen02f1 significantly inhibited
LPS-stimulated phosphorylation of ERK1/2, p38 and JNK (Fig. 4A), which are responsible for compromising the over-reactive inflammatory responses. In addition, LPS is also a potent activator of NFkB pathways. When cell are activated, phosphorylation of IkB leads to ubiquitination and degradation. NFkB is free from complex and translocates to the nucleus, where it binds to DNA and induces activation of inflammatory response [24]. We showed that yuwen02f1 blocks LPS-induced degradation of IkBa in murine macrophages (Fig. 4B). Taken together, our data show that yuwen02f1 is an
effective anti-inflammatory agent that inhibits NFkB and MAPK pathway activation in a concentration-dependent manner. However, in comparison with TLR4 for Gram-negative bacterial component (lipopolysaccharide, LPS), the Gram-positive bacteria receptor TLR2 is still poorly defined. We found that yuwen06f1 significantly
inhibited production of TNFa and phosporylation of MAPK of Pam3CSK4-activated RAW 264.7 (Figs. 2 and 4I and C). These results suggest that the protective function of yuwen06f1 against both Gram-negative and -positive bacteria activated phagocytes may be attributed to its anti-TLRs activation caused by the complex microbial
patterns. After identifying its in vitro effects, we used LPS-induced septic model and adjuvant-induced arthritic model, respectively, to examine anti-inflammatory effects of yuwen02f1 on acute and chronic inflammation in vivo. In endotoxemic model, we observed that seral cytokines including TNFa and IL-6 were markedly reduced, and the thrombocytopenic condition was partially reversed in yuwen02f1-treated septic mice.
In addition, the tendency of reduced mortality and cytokine-producing assay indicated that yuwen06f1 could be beneficial to endotoxemia. Yuwen02f1 shows significant protective effects against the organ injuries, such as leukocyte infiltration in lung, liver and kidney caused by endotoxemia (Fig. 6). In arthritic model,
yuwen02f1- treated mice show attenuated inflammation with milder paw edema. From the histopathological examination, the arthritic mice showed redundant folds of the synovial lining, intense infiltration with inflammatory cells and the pannus
formation. These morphological changes were apparently attenuated in yuwen02f1-treated mice (Fig. 7).
In summary, yuwen02f1, a phenylpyrazole derivative with little cytotoxicity, possesses anti-inflammatory effects in sup-pressing release of
inflammation-associated mediators (TNFa, IL- 6, ROS), reducing inducible enzymes (iNOS and COX-2) over-expression, inhibiting cell migration in vitro and even attenuating acute and chronic inflammation of mice mainly through suppressing LPS-induced NADPH oxidase activation, ROS forma-tion, NF-kB and MAPK pathways in phagocytes (Fig. 8). Thus, yuwen02f1 may be a potential lead compound for developing
anti-inflammatory agents. Conflict interest
The authors declare that they have no competing interests.
Acknowledgment
This work was supported by grant from the National Science Council NSC100-2320-B-002-105-MY3, Taiwan.
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Fig. 1. The chemical structures of yuwen02f1. (A) Structure of yuwen02f1. Physical properties data of compound yuwen02mp: 97.8–97.9 8C. MS (m/z): 390 (M+). (B) 1H NMR (CDCl3-d1, 200 MHz)TM (ppm): 1.39 (3H, t, J = 7.1, –OCH2CH3), 4.38 (2H, q, J = 7.1, –OCH2CH3), 5.64 (2H, s, –CH2–), 6.53 (1H, d, J = 3.6 Hz, H-3000 ), 6.90 (1H, s, 4), 6.95 (2H, dd, J = 8.7, 8.7 Hz, 30,50 ), 7.12 (1H, d, J = 3.6 Hz, H-4000 ), 7.28–7.44 (5H, m, H-20 , 60 , 400 , 300 , 500 ), 7.84 (1H, d, J = 8.3 Hz, 200 , 600). (C) 13C NMR (CDCl3-d1, 50 MHz)TM (ppm): 14.37, 54.48, 61.16, 103.91, 110.01, 115.24, 115.66, 125.67(2C), 128.06, 128.69(2C), 129.08, 129.24, 132.69, 133.64, 144.35, 147.62, 151.19, 158.40, 159.88, 164.76.
Fig. 2. The effects of yuwen02f1 on LPS-induced cytokine release and cell migration in murine macrophages and human monocytes and cytotoxicity of yuwen02f1 on murine macrophages. (A–C) RAW264.7 macrophage cells and (D–F) THP-1 cells were pretreated with various concentrations of yuwen02f1 (3, 10, or 30 mM) or dexamethasone (Dex, 10 mM) for 30 min, and then activated with LPS (100 ng/ml) for RAW264.7 macrophages or LPS (1 mg/ml) for THP-1 monocytes or DMSO alone (control, CTL). Culture medium was collected by centrifugation and concentrations of cytokines were measured by ELISA after 24 h (A, B, D and E). Cell migrated through gelatin-coated transwells for 20 h. The migration index was calculated only as fold change of adhered cells normalized to that of unstimulated cells (C and F). CTL, DMSO alone as non-activated control. Data are expressed as the mean _ S.E.M. of three independent experiments, and *p < 0.05, **p < 0.01 and ***p < 0.001 as
compared with LPS-activated groups. (G) RAW264.7 macrophages were incubated in the presence or absence of yuwen02f1 (3, 10, or 30 mg/ml) with LPS (100 ng/ml) for 24 h. Cell viability was determined by MTT assay. S: starvation. Each value is a mean percentage _ S.E.M. compared with vehicle-treated control (100%) in three individual experiments. (H) LDH release assay was conducted. Each value is a mean percentage _ S.E.M. as compared with the release of LDH of total lysis group (100%). (I)
RAW264.7 cells were activated by treatment of LPS (1 mg/ml) or Pam3CSK4 (1 mg/ml) in the present of yuwen06f1 (30 mM) or not. After 24 h, the production of TNFa in the media was measured with ELISA kit. Data are expressed as the mean _ S.E.M. of three independent experiments, and ***p < 0.001 as compared with DMSO vehicle groups.
Fig. 3. The effects of yuwen02f1 on LPS-induced nitric oxide production, iNOS expression, COX-2 expression, ROS production and NADPH subunits expression in RAW264.7 macrophages. (A) The cells were treated with LPS (100 ng/ml) only or with various concentrations of yuwen02f1 or dexamethasone (Dex, 10 mM) and the nitric oxide production were measured after 24 h. Control (CTL) values were obtained in the absence of LPS. Data were obtained from three independent experiments and expressed as means _ S.E.M. **p < 0.01 compared with the LPS-activated only group. (B and C) RAW264.7 cells were treated with various concentration of yuwen02f1 or dexamethasone (Dex, 10 mM) and stimulated with LPS (100 ng/ml). Cells were harvested and iNOS (B) or COX-2 (C) were detected. The fold change was compared with LPS-activated group (as 1.0). (D) After incubation with 1 mg/ml LPS and yuwen02f1 for 24 h, RAW cells were harvested and intracellular reactive oxygen species were detected by flow cytometry using DCFDA dye. Dash line, no LPS
stimulated control. Bold line, LPS-stimulated cells. Thin line, LPS-stimulated cells with yuwen02f1. (E) Inhibitory effect of yuwen02f1 on LPS-stimulated ROS production (LPS alone as 100%). The relative fluorescence intensities of five
independent experiments were recorded. Each value is a mean percentage _ S.E.M. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with the LPS-stimulated ROS production (100%). (F) Effect of Yuwen02f1 on NADPH oxidase subunits expression in membrane subcellular fractions. RAW cells were preincubated with vehicle or Yuwen02f1 (10 or 30 mM) for 30 min followed by LPS (1 mg/ml) treatment or not for 15 min. Membrane protein was isolated to perform Western blot analysis. p22phox is as an internal membrane control. Each experiment has been performed three times.
Fig. 4. Effects of yuwen02f1 on the LPS-induced activation of MAP kinases and IkBa degradation in RAW 264.7 macrophages. RAW264.7 cells were treated with various concentration of yuwen02f1 or dexamethasone (Dex, 10 mM) and stimulated with LPS (100 ng/ml) or Pam3CSK4 (1 mg/ml) for 30 min. Cells were harvested and total cell extracts were prepared. (A and C) Phosphorylated-ERK, phosphorylated-JNK, phosphorylated-p38 or (B) IkBa were detected by Western blot analysis. a-tubulin was used as internal markers for loading variation. The fold change was expressed as relative intensity compared to LPS or Pam3CSK4-activated group (LPS or
Pam3CSK4 in the absence of inhibitor).
Fig. 5. The effects of yuwen02f1 on LPS-induced cytokines release, thrombocytopenia and survival rate. ICR mice (25–30 g) were treated with
B). After 2 h, mice were euthanized and blood was collected from eyeholes. The serum was obtained and the cytokines TNFa and IL-6 were measured by ELISA kit. Values are presented as mean _ S.E.M. (n = 11). **p < 0.01 and ***p < 0.001 as compared with the LPS-activated group. (C) After 24 h, mice were euthanized to collect blood by cardiac puncture and platelet numbers were counted. Each dot represents an individual mouse and horizontal bars represent the mean of each group, and *p < 0.05. (D) The 24 h survival rate was expressed as the percentage of the survival mice and the total number tested. CTL, mice receiving vehicle only.
Fig. 6. The effects of yuwen02f1 on tissue inflammation in LPS-induced endotoxemia in vivo. Histochemical changes in mouse liver (A–C), lung (D–F) and kidney (G–I) sections of ICR mice on LPS treatments (H&E staining, original magnification, 100_). (A, D and G), control mice; (B, E and H), LPS-induced endotoxemic mice; and (C, F and I), LPS-induced endotoxemic mice treated with yuwen02f1. Leukocyte infiltration and peri-vascular infiltration in liver and lung (arrowhead) and glomerular hypercellularity in kidney (arrow) were indicated.
Fig. 7. Effects of yuwen02f1 on hind paw in adjuvant-induced arthritis model. (A) Anti-inflammatory effect of yuwen02f1 on adjuvant-induced arthritis. The magnitude of inflammation was represented by inflammation index which was obtained by dividing the volume of adjuvant-injected paw by that of its contralateral internal control. Values are presented as mean _ S.E.M. (n = 4–5). Arthritis-induction mice without yuwen02f1-treatment, continuous line; Arthritis-induction mice with yuwen02f1-treatment, dash line. (B) Morphological changes in histological sections of paws and ankle joints of ICR mice on adjuvant-induced arthritis. Upper panel,
non-induction paw. Middle panel, non-induction paw. Lower panel, adjuvant-induction paw of yuwen02f1-treated mice. The arrow showed synovial cell and star (*) indicated pannus formation.
Fig. 8. The anti-inflammatory mechanisms of yuwen02f1 in phagocyte. After being stimulated with LPS, TLR4 activates the MyD88-dependent pathway through the MyD88-IRAK-TRAF6 complex, which in turn activates the downstream MAPKs and NFkB activation. In addition, ROS presumably produced by LPS-activated
membrane-bound NADPH-oxidase, triggers downstream MAPKs and NFkB activation [33]. Yuwen02f1 possesses anti-inflammatory effects mainly through attenuating LPS-induced NADPH oxidase assembling, ROS formation, NF-kB and MAPK pathways in phagocytes. Taken together, Yuwen02f1 suppresses release of inflammation-associated mediators (TNFa, IL-6, ROS), reduces inducible enzymes (iNOS and COX-2) over-expression, inhibits cell migration in vitro and even attenuates the acute and chronic inflammation of mice.
Fig. 4.
Fig. 6.
