Free Radical Research, 2014; Early Online: 1–9 © 2014 Informa UK, Ltd. ISSN 1071-5762 print/ISSN 1029-2470 online DOI: 10.3109/10715762.2014.966705
ORIGINAL ARTICLE
iosgenin inhibits superoxide generation in FMLP-activated mouse neutrophils via D multiple pathways Y. Lin*, R. Jia*, Y. Liu, Y. Gao, X. Zeng, J. Kou & B. Yu
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
Jiangsu Provincial Key Laboratory for TCM Evaluation and Translational Research, China Pharmaceutical University, Nanjing, P. R. China Abstract Diosgenin possesses anti-inflammatory and anticancer properties. Activated neutrophils produce high concentrations of the superoxide anion which is involved in the pathophysiology of inflammation-related diseases and cancer. In the present study, the inhibitory effect and possible mechanisms of diosgenin on superoxide generation were investigated in mouse bone marrow neutrophils. Diosgenin potently and concentration-dependently inhibited the extracellular and intracellular superoxide anion generation in Formyl-Met-Leu-Phe (FMLP)activated neutrophils, with IC50 values of 0.50 0.08 mM and 0.66 0.13 mM, respectively. Such inhibition was not mediated by scavenging the superoxide anion or by a cytotoxic effect. Diosgenin inhibited the phosphorylation of p47phox and membrane translocation of p47phox and p67phox, and thus blocking the assembly of nicotinamide adenine dinucleotide phosphate oxidase. Moreover, cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) expression were also effectively increased by diosgenin. It attenuated FMLP-induced increase of phosphorylation of cytosolic phospholipase A (cPLA2), p21-activated kinase (PAK), Akt, p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK). Our data indicate that diosgenin exhibits inhibitory effects on superoxide anion production through the blockade of cAMP, PKA, cPLA2, PAK, Akt and MAPKs signaling pathways. The results may explain the clinical implications of diosgenin in the treatment of inflammation-related disorders. Keywords: diosgenin, mouse neutrophils, superoxide anion, NADPH oxidase, signaling pathways
Introduction Reactive oxygen species (ROS) overproduction is involved in the pathophysiology of cancer, inflammation, and cardiovascular diseases [1]. Neutrophils play a critical role in the host defense by activating superoxide anion production, which can be further processed to generate more ROS such as hydroxyl radical and hypochlorous acid [2]. The enzyme responsible for superoxide generation is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. NADPH oxidase activation coupled with ROS overproduction often leads to oxidative stress, contributing to development of inflammation diseases. Antioxidants are mostly ineffective in alleviating the burden of oxidative stress. Inhibition of ROS production through NADPH oxidase inhibitors offers an alternative approach to antioxidant therapies [3]. NADPH oxidase of neutrophils is a multiprotein complex that comprises a membrane-bound flavocytochrome b558 (gp91phox and p22phox) and cytosolic proteins (p40phox, p47phox, p67phox, and Rac2 guanosine triphosphate (GTPase)) in the resting state. Upon cell stimulation by microorganisms or inflammatory mediators, such as FMLP, the cytosolic components translocate to the membrane and associate with cytochrome b558 to form an activated NADPH oxidase complex [4]. NADPH oxidase assembly at the plasma membrane results
in the release of extracellular superoxide anion, whereas oxidase assembly on an intracellular membrane would result in intracellular superoxide anion which may be involved in signaling functions of gp91phox [5]. During neutrophils stimulation, p47phox is heavily phosphorylated. p47phox phosphorylation and subsequent translocation to flavocytochrome b558 are essential steps for the activation of NADPH oxidase [6]. The signaling mechanisms responsible for p47phox phosphorylation in neutrophils are complex. A number of kinases participate in p47phox phosphorylation events, including mitogenactivated protein kinases (MAPKs) [7], Akt [8], p21-activated kinase (PAK) [9], and protein kinase C (PKC). Furthermore, cyclic AMP (cAMP) and cytosolic phospholipase A (cPLA2) are two important second messengers involved in the activation of neutrophils and the assembled NADPH oxidase [10,11]. Therefore, modulating such signaling pathways or second messengers could produce significant effects on neutrophils activation. Diosgenin, a steroid sapogenin found in several plants including Dioscorea species, Trigonella foenum-graecum, and Costus speciosus, possesses a wide range of biological activities. It has potential chemopreventive capacity [12,13], anti-inflammatory [14,15] and anti-thrombosis properties [16], cytoprotective effect on vascular endothelial cells, etc. [17]. It can modulate antioxidant defense and decrease
*These authors contributed equally to this work. Correspondence: Dr. Junping Kou and Dr. Boyang Yu, Jiangsu Provincial Key Laboratory for TCM Evaluation and Translational Research, China Pharmaceutical University, 639 Longmian Avenue, Nanjing 211198, P. R. China. Tel: 86-25-86185158. E-mail address: junpingkou@ cpu.edu.cn; [email protected] (Received date: 16 June 2014; Accepted date: 13 September 2014; Published online: 16 October 2014)
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
2 Y. Lin et al. oxidative stress damage [18,19]. Despite that diosgenin affected various cells, such as cancer cells, endothelial cells, and monocytes [20], by multiple signaling pathways, there are few reports about its effect on activated neutrophils, and its molecular mechanisms on oxidative stress remain to be elucidated. Therefore, in the present study, we investigated the effects of diosgenin on the superoxide generation in FMLP-stimulated mouse bone marrow neutrophils and possible molecular mechanisms involved. This study would put some new insights on the potential beneficial use of diosgenin in the treatment of inflammationrelated diseases and cardiovascular diseases.
described [21,22]. Briefly, mouse bone marrow was flushed out from the rear femurs with Hank’s balanced salt buffer without Ca2 and Mg2 (HBSS¯), and centrifuged at 400 g for 10 min at 4°C. The pellet was resuspended in HBSS¯. Cells were layered on a 3-step Percoll gradient (72%, 64%, and 52%). Following centrifugation at 1000 g for 30 min, neutrophils were obtained from the 64%/72% interface and washed twice with HBSS¯. The neutrophils were resuspended in HBSS. The purity of the cells was greater than 90% and viability was greater than 95%, as determined by vital staining and microscopy.
Materials and methods
Superoxide production was measured using an isoluminol chemiluminescence assay [23,24]. Neutrophils (6 105 cells) were preincubated for 15 min with drugs prior to FMLP stimulation. One milliliter of reaction mixture containing neutrophils, HRP (20 U/mL), and isoluminol (50 mM) was equilibrated in the luminometer for 10 min at 37°C, after which 100 mL of FMLP (final concentration, 2 mM) was added to activate the reaction. The light emission was recorded continuously in a BPCL1-TGC luminometer apparatus (Academia Sinica Biophysics Institute, Beijing, China). Nonstimulated cells were recorded as background. Inhibition percentage was calculated according to (Mf - M) 100/(Mf - Mb), where M, Mb, and Mf represent maximum chemiluminescence value of drug FMLP sample, background sample, and FMLP sample, respectively.
Materials Diosgenin with purity higher than 98% was purchased from Shanghai Winherb Medical Technology Co. Ltd. (Shanghai, China). Superoxide dismutase (SOD) and FMLP were obtained from Sigma (MA, USA). Cytochalasin B (CB), horseradish peroxidase (HRP), and apocynin were obtained from Aladdin (Shanghai, China). Nitro blue tetrazolium (NBT) was purchased from Amresco (Solon, USA). Isoluminol was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). The cAMP assay kit was obtained from Jiancheng Bioengineering Institute (Nanjing, China). Antibodies against p47phox, p67phox, p38MAPK, phospho-p38MAPK (T180), extracellular signal-regulated kinase (ERK1/2), phospho-ERK1/2 (T202/Y204), cPLA2, phospho-cPLA2, PAK1/2, and protein kinase A (PKA) were purchased from Bioworld Technology (St. Paul, MN, USA). Antibodies against Akt (pan) and phospho-Akt (Ser473), c-Jun N-terminal kinase (JNK), phospho-JNK (Thr183/Tyr185), and phospho-PAK1 (Thr423)/PAK2 (Thr402) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-phosphoserine was purchased from Millipore (Bedford, MA, USA). When drugs were dissolved in dimethyl sulfoxide (DMSO), the final concentration of DMSO in the cell experiments did not exceed 0.4% and did not affect the parameters measured. Mice Male Institute of Cancer Research (ICR) mice were purchased from Nanjing Qinglong Experimental Animal Co. Ltd., China; 10–15-week-old mice were used throughout the study. All procedures and assessments were proved by Animal Ethics Committee of the School of Chinese Materia Medica, China Pharmaceutical University. These experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) revised in 1996.
Measurement of extracellular superoxide generation
Measurement of intracellular superoxide generation Quantitation of intracellular superoxide production was measured using a colorimetric NBT assay [25]. Neutrophils (6 105 cells) were incubated with diosgenin for 15 min, and then 100 mL of 2 mg/mL NBT solution was added. Cells were incubated with 2.5 mg/mL of cytochalasin B for 3 min, and then activated with FMLP (10 mM). As negative controls, cells were incubated in the NBT solution containing 100 U/mL of SOD along with stimulus. After incubation for 30 min at 37°C, cells were washed twice with PBS and once with methanol, and then airdried. The NBT deposited inside the cells was then dissolved by adding 120 mL of 2 M KOH and 140 mL of DMSO with vortex mixing for 2 min. The dissolved NBT solution was monitored at 620 nm. Superoxide scavenging activity The superoxide scavenging ability of diosgenin was determined by a kit using xanthine/xanthine oxidase assay in a cell-free system. The assay was performed according to the manufacturer’s instructions.
Isolation of mouse bone marrow neutrophils
Measurement of LDH release
Neutrophils were isolated from mouse bone marrow by isotonic Percoll gradient centrifugation, as previously
Cell membrane damage was measured by lactate dehydrogenase (LDH) release. Neutrophils (2 106 cells) were
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
Diosgenin inhibits mouse neutrophils activation 3
equilibrated at 37°C for 5 min and then incubated with diosgenin for 30 min. The cell-free supernatant fluids were collected. The activity of LDH released into the supernatant was measured using the test kit. Release of LDH in the supernatants was expressed as the percentage of total LDH that was present in an equivalent number of lysed neutrophils with 0.2% Triton X-100 at 37°C for 30 min and ultrasonication.
significance of differences among the groups. A p value of less than 0.05 was considered statistically significant.
Determination of cellular cAMP concentration
Production of both the extracellular and the intracellular superoxide in neutrophils was measured. The effects were compared with apocynin, a known inhibitor of NADPH oxidase. At the extracellular level, diosgenin (0.1–10 mM) inhibited superoxide production in a concentration-dependent manner in activated neutrophils, with an IC50 value of 0.50 0.08 mM (Figure 1A). Diosgenin (10 mM) showed strong inhibitory activity (about 96% inhibition). Apocynin (10 mM) significantly attenuated FMLP-induced superoxide generation (about 84% inhibition). Intracellular superoxide anion production was measured by the colorimetric NBT assay. Diosgenin (0.1–10 mM) prevented the increase in intracellular superoxide production in a dose-dependent manner, with an IC50 value of 0.66 0.13 mM (Figure 1B). Significant inhibition (p 0.05) was observed at diosgenin concentrations of greater than or equal to 1 mM. The inhibitory efficacy of diosgenin was significantly higher than that of apocynin. To determine whether diosgenin directly scavenges superoxide anion, experiments with xanthine/xanthine oxidase in a cell-free system were performed. Diosgenin (1–10 mM) was found to have no effect on superoxide scavenging (Figure 1C). The data suggest that the inhibitory effect of diosgenin on superoxide generation is independent of the superoxide scavenging activity. Neutrophils in the presence of diosgenin (0.1–10 mM) for 30 min did not damage the cell membranes, as assayed by LDH release (data not shown). These data indicate that inhibition of superoxide generation by diosgenin is not due to cytotoxicity.
The cAMP levels were determined as previously described [26]. Freshly isolated neutrophils (2.5 106 cells) were incubated with drugs for 15 min at 37°C before stimulation with or without 10 mM of FMLP for another 5 min, and the reaction was terminated by cooling to 4°C. Samples were then centrifuged at 800 g for 5 min at 4°C. The precipitate was treated with 0.1 M HCl for 20 min at room temperature, and then the cells were disrupted by sonication (5 cycles on ice, each for 5 s), followed by centrifugation at 12 000 g for 10 min. The supernatants were used as a source for the cAMP samples, which were assayed using an enzyme immunoassay kit. Western blot analysis To analyze their effects on PKA expression, and p47phox, cPLA2, PAK1/2, Akt, p38MAPK, ERK1/2, and JNK phosphorylation, neutrophils were pretreated with diosgenin (1, 3, and 10 mM) for 15 min at 37°C, and stimulated with 10 mM of FMLP. The reaction was stopped on ice, and the mixture was centrifuged at 800 g for 5 min at 4°C. The precipitate was washed twice with HBSS and lysed in 100 mL of RIPA buffer. The lysate was centrifuged at 13 000 g for 10 min at 4°C. Proteins (40 mg) were electrophoresed through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA). The blots were incubated with primary antibodies overnight at 4°C. A goat anti-rabbit antibody conjugated to HRP was used as the secondary antibody. Signal detection was determined using enhanced chemiluminescence followed by exposure to X-ray film. For the measurement of p47phox and p67phox translocation, the cytosolic and plasma membrane fractions were prepared using a membrane and cytosol protein extraction kit (Beyotime, Jiangsu, China). Protein concentrations were measured using the bicinchoninic acid (BCA) protein assay kit. Proteins were resolved by 12% SDS-PAGE and the blots were probed for either p47phox or p67phox using anti-p47phox or anti-p67 antibodies. Statistical analysis Results are expressed as means S.E.M. Data were analyzed with GraphPad Prism software (San Diego, CA, USA). Student’s t-test was used to determine the statistical significance of differences between two group means, and one-way analysis of variance was used to determine the
Results Effect of diosgenin on FMLP-induced superoxide production
Effect of diosgenin on NADPH oxidase activity The phosphorylation of cytosolic protein p47phox plays a key role in NADPH oxidase activation. This study examined whether diosgenin could modulate FMLP-induced p47phox phosphorylation of NADPH oxidase. As shown in Figure 1D, the level of p47phox phosphorylation was significantly increased in FMLP-stimulated neutrophils. Pretreatment of neutrophils with 10 mM diosgenin resulted in significant attenuation of phosphorylation of serine residues in p47phox. NADPH oxidase can produce superoxide after assembly of the plasma membrane flavocytochrome b558 and the cytosolic components. Furthermore, we investigated the effect of diosgenin on the translocation of p47phox and p67phox to the cell membrane in FMLP-stimulated neutrophils. As shown in Figure 1E and F, the band immunointensities of p47phox and p67phox were notably upregulated
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
4 Y. Lin et al.
Figure 1. Effects of diosgenin on superoxide generation and NADPH oxidase activation. Effects on extracellular (A) and intracellular (B) superoxide generation. Neutrophils were incubated with DMSO (as the control), diosgenin (0.1–10 mM), or apocynin (APO) for 15 min and then activated using FMLP (A) and FMLP/CB (B). The concentration of APO is 10 mM (A) and 100 mM (B). *p 0.05; **p 0.01; ***p 0.001 compared with the control. All data are expressed as the mean S.E.M. (n 426). (C) Effect on superoxide generation in a cell-free xanthine/xanthine oxidase system. All data are expressed as the mean S.E.M. (n 426). (D) Effect on p47phox phosphorylation in intact neutrophils. Cell lysates prepared from the cells treated as described above were immunoblotted with the specific antibody against p47phox and phosphoserine. Effects on p47phox (E) and p67phox (F) translocation. Representative immunoblots for the p47phox and p67phox proteins in the membrane and cytosolic fractions. The ratios between the quantified proteins in membrane and cytosolic fractions were used to quantify the FMLP-induced translocation degree. All data are expressed as mean S.E.M. (n 3). *p 0.05; **p 0.01 compared with FMLP. #p 0.05; ##p 0.01 compared with the control.
in the membrane fractions of FMLP-stimulated neutrophils. Diosgenin blocked the expression levels of subunits of p47phox and p67phox in the membrane. The data suggest that diosgenin may reduce the translocation of p47phox and p67phox from the cytosol to the membrane. The results coincided with the effect of diosgenin on superoxide generation in FMLP-stimulated neutrophils.
Effects of diosgenin on cellular cAMP levels and PKA activation cAMP is an important second messenger involved in a variety of physiological and pathophysiological processes. Increases in intracellular cAMP concentrations have been reported to inhibit FMLP-induced superoxide generation
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
Diosgenin inhibits mouse neutrophils activation 5
Figure 2. Effects of diosgenin on cAMP levels and PKA expression in FMLP-activated mouse bone marrow neutrophils. Neutrophils were incubated with DMSO (control) or diosgenin (1, 3, and 10 mM) for 15 min at 37°C before stimulation with or without FMLP (10 mM). (A) cAMP levels were measured using an enzyme immunoassay kit. (B) PKA protein expression was immunoblotted with the specific antibody against PKA. GAPDH was included as a reference for protein normalization. All data are expressed as the mean S.E.M. (n 3). *p 0.05; **p 0.01; ***p 0.001 compared with FMLP. #p 0.05; # # #p 0.001 compared with the control (1st lane).
[27]. PKA is a downstream mediator of the cAMP pathway [28]. We, therefore, examined whether diosgenin stimulated cAMP production and PKA expression in FMLP-induced neutrophils. Diosgenin (1, 3, and 10 mM) caused a slight increase in the cAMP concentration, and caused a synergistic increase in FMLP-induced cAMP levels in a concentration-dependent manner (Figure 2A). Furthermore, diosgenin (10 mM) significantly increased PKA protein expression in FMLP-stimulated neutrophils (Figure 2B). Effects of diosgenin on activations of cPLA2 and PAK cPLA2 serves as second messengers and regulates NADPH oxidase activity by modulating the assembly of the active
NADPH oxidase complex through controlling the translocation of both p47phox and p67phox [29]. PAK may participate in a variety of neutrophil responses, including rapid activation of several distinct MAPKs cascades [30,31]. Inhibition of PAK activity in neutrophils could reduce FMLP-stimulated superoxide generation. In the present study, phosphorylation of cPLA2 and PAK occurred in FMLP-activated neutrophils. Diosgenin (1, 3, and 10 mM) inhibited the phosphorylation of cPLA2 and PAK in a concentration-dependent manner (Figure 3A and B). The highest concentration of diosgenin (10 mM) reduced the phosphorylation of cPLA2 (45%) and PAK (61%), respectively. Thus, the blockade of the signaling pathways by diosgenin may account for the attenuation of neutrophils activation.
Figure 3. Effects of diosgenin on phosphorylation of cPLA2 and PAK1/2 in FMLP-activated mouse bone marrow neutrophils. Neutrophils were incubated with or without diosgenin (1, 3, and 10 mM) for 15 min before stimulation with FMLP (10 mM) for 3 min (cPLA2) or 30 s (PAK1/2) at 37°C. Phosphorylations of cPLA2 (A) and PAK1/2 (B) were analyzed by an immunoblot analysis using antibodies against the phosphorylated form and the total of each protein. All data are expressed as the mean S.E.M. (n 3). *p 0.05; **p 0.01 compared with FMLP. #p 0.05; # #p 0.01 compared with the control (1st lane).
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
6 Y. Lin et al. Effect of diosgenin on activation of MAPKs and Akt
Discussion
The MAPK proteins consist of the three major subfamilies of p38MAPK, ERK1/2, and JNK, which are known to be involved in FMLP-induced superoxide generation [32]. Akt is involved in the activation of MAPK cascades [33]. Here, we also investigated the effects of diosgenin on phosphorylation of p38MAPK, ERK1/2, JNK, and Akt in FMLP-stimulated neutrophils. Stimulation of neutrophils with FMLP resulted in the rapid phosphorylation of p38MAPK, ERK1/2, JNK, and Akt. Diosgenin (1–10 mM) reduced the phosphorylation of p38MAPK, ERK1/2, and Akt in FMLP-stimulated neutrophils in a concentration-dependent manner (Figure 4A, B, and D), whereas diosgenin (10 mM) slightly affected the phosphorylation of JNK in FMLP-stimulated neutrophils (Figure 4C).
Regulation of neutrophil activity is an important approach in avoiding inflammation. Diosgenin has been shown to have a variety of biological activities including antidiabetic activity [18], antioxidant activity, and anti-inflammatory activity. However, the action site of diosgenin on ROS generation and possible molecular mechanisms linking regulation of ROS production remain to be established. In the present study, we investigated the effects of diosgenin on superoxide generation in mouse bone marrow neutrophils to elucidate the signaling pathways responsible for the regulation of superoxide production. Diosgenin exhibited powerful activity in the inhibition of FMLP-induced extracellular and intracellular superoxide generation. Superoxide anion produced by NADPH oxidase is rapidly converted to hydrogen peroxide (H2O2)
Figure 4. Effects of diosgenin on phosphorylation of p38MAPK, ERK1/2, JNK, and Akt in FMLP-activated mouse bone marrow neutrophils. Neutrophils were incubated with DMSO (control) or diosgenin (1, 3, and 10 mM) for 15 min before stimulation with FMLP (10 mM) for 3 min (p38MAPK, ERK1/2, and JNK) or 1 min (Akt) at 37°C. Phosphorylations of p38MAPK (A), ERK1/2 (B), JNK (C), and Akt (D) were analyzed by an immunoblot analysis using antibodies against the phosphorylated form and the total of each protein. All data are expressed as mean S.E.M. (n 3). *p 0.05; **p 0.01; ***p 0.001 compared with FMLP. # #p 0.01; # # #p 0.001 compared with the control (1st lane).
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
by superoxide dismutase. Superoxide anion and H2O2 can be further processed to generate more reactive metabolites such as hydroxyl radical (OH·) and hypochlorous acid (HOCl). ROS play a very important physiological role as second messengers. They could activate diverse signaling molecules such as PKC, MAPKs, Akt, and nuclear factorkappa B (NF-kB), all of which induce inflammation in various cell types [34]. Thus, these signaling pathways and ROS may cooperate to generate amplified signaling responsible for pathological process. Our data showed that diosgenin suppressed ROS production by inhibiting MAPKs and Akt signaling pathways. The inhibition by diosgenin of ROS generation may be linked to the attenuation of signaling pathways activation. The phosphorylation of p47phox plays a key role in NADPH oxidase activation and further superoxide production in neutrophils. We then found that diosgenin also inhibited phosphorylation of p47phox and translocation of p47phox and p67phox, eventually blocking the assembly of NADPH oxidase complex (Figure 1D–F). These findings provide the first evidence of diosgenin inhibiting NADPH oxidase in activated neutrophils. As reported, NADPH oxidase is not only highly expressed in neutrophils, but also expressed in a variety of other cell types, including vascular smooth muscle cells (VSMC) and endothelial cells [35]. Previous studies have shown that diosgenin suppresses intracellular ROS production in mouse VSMC [15] and human umbilical vein endothelial cells [19], and elevates the glutathione (GSH) and restores the endothelial nitric oxide synthase (eNOS) mRNA expression level in chronic renal failure rats [36]. Our data indicated that diosgenin might inhibit NADPH oxidase in these cells and present some new explanation for these activities. On the other hand, a number of kinases have been proposed to participate in p47phox phosphorylation, including MAPKs, Akt, and PAK [37,7,9]. The p38MAPK inhibitor, SB203580, significantly reduced p47phox phosphorylation and diminished superoxide production in neutrophils. The specific inhibitors of the ERK kinase, PD98059 and U0126, inhibited p47phox phosphorylation in FMLP-stimulated human neutrophils [38]. Diosgenin concentration-dependently diminished the FMLP-induced phosphorylation of Akt, PAK1/2, p38MAPK, and ERK1/2, thus explaining the inhibitory effects of diosgenin on NADPH oxidase and ROS hyperproduction in inflammatory diseases. Previous studies have shown that pharmacological inhibition or genetic deletion of cPLA2 attenuates neutrophilmediated bacterial killing in vitro, and impairs arthritis [39], septic shock [40], and pulmonary inflammation [41] in vivo. The cPLA2 pathway is the major route of arachidonic acid production, which is implicated in the translocation, assembly, or activation of the NADPH oxidase in neutrophils [11]. The inhibition of cPLA2 has been proposed as a potential therapeutic strategy for the management of arthritis and pulmonary inflammation. Moreover, PAK can regulate leukocyte-dependent inflammatory responses in inflammatory diseases, which contributes to
Diosgenin inhibits mouse neutrophils activation 7
neutrophils adhesion and migration across the endothelium through effects on neutrophils and endothelial cells [42]. PAK may actually play a key role in the regulation of NADPH oxidase in intact cells via its involvement in p47phox phosphorylation [43]. The cAMP/PKA signaling pathways were involved in the regulation of ROS production and showed an inverse correlation between cAMP and ROS levels mediated by PKA [44]. PKA has been shown to suppress respiratory burst through inhibition of phosphoinositide 3-kinase (PI3-K) activation and translocation of both PDK1 and Akt [45,46,27]. However, the actions of diosgenin on cPLA2, PAK1/2, and cAMP/PKA pathways are less investigated in neutrophils. Our data showed that diosgenin inhibited the phosphorylation of cPLA2 and PAK1/2, and increased cAMP levels and PKA protein expression. The modulation of multiple pathways should be beneficial for diosgenin in inhibition of neutrophils activation. Neutrophils act as the first line of defense against invading bacterial. Neutrophils express a number of G-proteincoupled receptors (GPCR) that participate in host defense and inflammation. FMLP is used as a model chemoattractant due to its effective ability to activate neutrophils by binding to the GPCR on the membrane. Stimulation of the GPCR resulted in the rapid phosphorylation of several proteins via G-protein, including PKC, MAPKs, PI3-K, and PAK [47]. An increase in cAMP levels has also been demonstrated [27]. Activation of these signal transduction pathways is known to lead neutrophils activation. Based on the results obtained in the present study, we show that diosgenin appears to interfere with the multiple FMLPstimulated signaling pathways. But it remains to be defined whether diosgenin may inhibit the binding to FMLP recep-
Figure 5. Schematic diagram of the action site by diosgenin on superoxide generation in mouse bone marrow neutrophils. Upon formyl peptide binding, a number of signal transduction events are activated, which result in superoxide generation. Diosgenin can increase cAMP level and PKA expression in FMLP-activated neutrophils. Diosgenin regulates superoxide production by inhibition of cPLA2, PAK, Akt, and MAPKs activation.
8 Y. Lin et al.
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
tor to suppress the activation of these signal transduction pathways. Taken together, these results indicate that diosgenin may be an effective NADPH oxidase inhibitor. The suppressive effects of diosgenin are associated with inhibition of the cPLA2, PAK1/2, Akt, p38MAPK, ERK1/2, and JNK signaling pathways and mediated by a cAMP/PKA pathway in FMLP-activated neutrophils (Figure 5). Diosgenin does not show direct superoxide-scavenging property. It could serve as a new source of therapeutic alternatives for the treatment of cardiovascular and/or inflammatory diseases related to NADPH oxidase hyperactivity. Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. This work was financially supported by the National Natural Science Foundation of China (81073008) and National Found for College Students Innovation Project for the R&D of Novel Drugs (J1030830). References [1] Bonner MY, Arbiser JL. Targeting NADPH oxidases for the treatment of cancer and inflammation. Cell Mol Life Sci 2012;69:2435–2442. [2] Maghzal GJ, Krause KH, Stocker R, Jaquet V. Detection of reactive oxygen species derived from the family of NOX NADPH oxidases. Free Radic Biol Med 2012;53: 1903–1918. [3] Manea A. NADPH oxidase-derived reactive oxygen species: involvement in vascular physiology and pathology. Cell Tissue Res 2010;342:325–339. [4] Babior BM. NADPH Oxidase: an update. Blood 1999;93: 1464–1476. [5] Karlsson A, Dahlgren C. Assembly and activation of the neutrophil NADPH oxidase in granule membranes. Antioxid Redox Signal 2002;4:49–60. [6] El-Benna J, Dang PM, Gougerot-Pocidalo MA, Marie JC, Braut-Boucher F. p47phox, the phagocyte NADPH oxidase/ NOX2 organizer: structure, phosphorylation and implication in diseases. Exp Mol Med 2009;41:217–225. [7] El Benna J, Han J, Park JW, Schmid E, Ulevitch RJ, Babior BM. Activation of p38 in stimulated human neutrophils: phosphorylation of the oxidase component p47phox by p38 and ERK but not by JNK. Arch Biochem Biophys 1996;334:395–400. [8] Didichenko SA, Tilton B, Hemmings BA, Ballmer-Hofer K, Thelen M. Constitutive activation of protein kinase B and phosphorylation of p47phox by a membrane-targeted phosphoinositide 3-kinase. Curr Biol 1996;6:1271–1278. [9] Martyn KD, Kim MJ, Quinn MT, Dinauer MC, Knaus UG. p21-activated kinase (Pak) regulates NADPH oxidase activation in human neutrophils. Blood 2005;106:3962–3969. [10] Yu HP, Hsieh PW, Chang YJ, Chung PJ, Kuo LM, Hwang TL. DSM-RX78, a new phosphodiesterase inhibitor, suppresses superoxide anion production in activated human neutrophils and attenuates hemorrhagic shock-induced lung injury in rats. Biochem Pharmacol 2009;78:983–992.
[11] Dana R, Leto TL, Malech HL, Levy R. Essential requirement of cytosolic phospholipase A2 for activation of the phagocyte NADPH oxidase. J Biol Chem 1998;273:441–445. [12] Chiang CT, Way TD, Tsai SJ, Lin JK. Diosgenin, a naturally occurring steroid, suppresses fatty acid synthase expression in HER2-overexpressing breast cancer cells through modulating Akt, mTOR and JNK phosphorylation. FEBS Lett 2007;581: 5735–5742. [13] Rajalingam K, Sugunadevi G, Arokia Vijayaanand M, Kalaimathi J, Suresh K. Anti-tumour and anti-oxidative potential of diosgenin against 7, 12-dimethylbenz(a)anthracene induced experimental oral carcinogenesis. Pathol Oncol Res 2012;18:405–412. [14] Gao MY, Chen L, Yu HX, Sun Q, Kou JP, Yu BY. Diosgenin down-regulates NF-kB p65/p50 and p38MAPK pathways and attenuates acute lung injury induced by lipopolysaccharide in mice. Int Immunopharmacol 2013;15:240–245. [15] Choi KW, Park HJ, Jung DH, Kim TW, Park YM, Kim BO, et al. Inhibition of TNF-a-induced adhesion molecule expression by diosgenin in mouse vascular smooth muscle cells via downregulation of the MAPK, Akt and NF-kB signaling pathways. Vascul Pharmacol 2010;53:273–280. [16] Gong G, Qin Y, Huang W. Anti-thrombosis effect of diosgenin extract from Dioscorea zingiberensis C.H. Wright in vitro and in vivo. Phytomedicine 2011;18:458–463. [17] Song JX, Ma L, Kou JP, Yu BY. Diosgenin reduces leukocytes adhesion and migration linked with inhibition of intercellular adhesion molecule-1 expression and NF-kB p65 activation in endothelial cells. Chin J Nat Med 2012;10:142–149. [18] Pari L, Monisha P, Mohamed Jalaludeen A. Beneficial role of diosgenin on oxidative stress in aorta of streptozotocin induced diabetic rats. Eur J Pharmacol 2012;691:143–150. [19] Gong G, Qin Y, Huang W, Zhou S, Wu X, Yang X, et al. Protective effects of diosgenin in the hyperlipidemic rat model and in human vascular endothelial cells against hydrogen peroxideinduced apoptosis. Chem Biol Interact 2010;184:366–375. [20] Yang HP, Yue L, Jiang WW, Liu Q, Kou JP, Yu BY. Diosgenin inhibits tumor necrosis factor-induced tissue factor activity and expression in THP-1 cells via down-regulation of the NF-kB, Akt, and MAPK signaling pathways. Chin J Nat Med 2013;11:608–615. [21] Van der Hoeven D, Wan TC, Auchampach JA. Activation of the A(3) adenosine receptor suppresses superoxide production and chemotaxis of mouse bone marrow neutrophils. Mol Pharmacol 2008;74:685–696. [22] Itou T, Collins LV, Thorén FB, Dahlgren C, Karlsson A. Changes in activation states of murine polymorphonuclear leukocytes (PMN) during inflammation: a comparison of bone marrow and peritoneal exudate PMN. Clin Vaccine Immunol 2006;13:575–583. [23] Li S, Yamauchi A, Marchal CC, Molitoris JK, Quilliam LA, Dinauer MC. Chemoattractant-stimulated Rac activation in wild-type and Rac2-deficient murine neutrophils: preferential activation of Rac2 and Rac2 gene dosage effect on neutrophil functions. J Immunol 2002;169:5043–5051. [24] Dahlgren C, Karlsson A. Respiratory burst in human neutrophils. J Immunol Methods 1999;232:3–14. [25] Choi HS, Kim JW, Cha YN, Kim C. A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells. J Immunoassay Immunochem 2006;27:31–44. [26] Van der Hoeven D, Gizewski ET, Auchampach JA. Activation of the A(3) adenosine receptor inhibits FMLP-induced Rac activation in mouse bone marrow neutrophils. Biochem Pharmacol 2010;79:1667–1673. [27] Ahmed MU, Hazeki K, Hazeki O, Katada T, Ui M. Cyclic AMP-increasing agents interfere with chemoattractant-induced respiratory burst in neutrophils as a result of the inhibition of phosphatidylinositol 3-kinase rather than receptor-operated Ca2 influx. J Biol Chem 1995;270:23816–23822.
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
[28] Haskó G, Szabó C. Regulation of cytokine and chemokine production by transmitters and co-transmitters of the autonomic nervous system. Biochem Pharmacol 1998;56: 1079–1087. [29] Rane MJ, Carrithers SL, Arthur JM, Klein JB, McLeish KR. Formyl peptide receptors are coupled to multiple mitogen- activated protein kinase cascades by distinct signal transduction pathways: role in activation of reduced nicotinamide adenine dinucleotide oxidase. J Immunol 1997;159:5070–5078. [30] Dewas C, Fay M, Gougerot-Pocidalo MA, El-Benna J. The mitogen-activated protein kinase extracellular signal-regulated kinase ½ pathway is involved in formyl-methionyl-leucylphenylalanine-induced p47phox phosphorylation in human neutrophils. J Immunol 2000;165:5238–5244. [31] Zu YL, Qi J, Gilchrist A, Fernandez GA, Vazquez-Abad D, Kreutzer DL, et al. p38 mitogen-activated protein kinase activation is required for human neutrophil function triggered by TNF-alpha or FMLP stimulation. J Immunol 1998;160: 1982–1989. [32] Rane MJ, Carrithers SL, Arthur JM, Klein JB, McLeish KR. Formyl peptide receptors are coupled to multiple mitogenactivated protein kinase cascades by distinct signal transduction pathways: role in activation of reduced nicotinamide adenine dinucleotide oxidase. J Immunol 1997;159:5070–5078. [33] Ding J, Vlahos CJ, Liu R, Brown RF, Badwey JA. Antagonists of phosphatidylinositol 3-kinase block activation of several novel protein kinases in neutrophils. J Biol Chem 1995; 270:11684–11691. [34] Dworakowski R, Anilkumar N, Zhang M, Shah AM. Redox signaling involving NADPH oxidase-derived reactive oxygen species. Biochem Soc Trans 2006;34:960–964. [35] Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 2007;87:245–313. [36] Manivannan J, Balamurugan E, Silambarasan T, Raja B. Diosgenin improves vascular function by increasing aortic eNOS expression, normalize dyslipidemia and ACE activity in chronic renal failure rats. Mol Cell Biochem 2013;384: 113–120. [37] Chen Q, Powell DW, Rane MJ, Singh S, Butt W, Klein JB, McLeish KR. Akt phosphorylates p47phox and mediates respiratory burst activity in human neutrophils. J Immunol 2003;170:5302–5308.
Diosgenin inhibits mouse neutrophils activation 9 [38] Dewas C, Fay M, Gougerot-Pocidalo MA, El-Benna J. The mitogen-activated protein kinase extracellular signal-regulated kinase ½ pathway is involved in formyl-methionyl-leucylphenylalanine-induced p47phox phosphorylation in human neutrophils. J Immunol 2000;165:5238–5244. [39] Hegen M, Sun L, Uozumi N, Kume K, Goad ME, Nickerson-Nutter CL, et al. Cytosolic phospholipase A2 adeficient mice are resistant to collagen-induced arthritis. J Exp Med 2003;197:1297–1302. [40] Levy R, Dana R, Hazan I, Levy I, Weber G, Smoliakov R, et al. Elevated cytosolic phospholipase A(2) expression and activity in human neutrophils during sepsis. Blood 2000;95: 660–665. [41] Rubin BB, Downey GP, Koh A, Degousee N, Ghomashchi F, Nallan L, et al. Cytosolic phospholipase A2-a is necessary for platelet-activating factor biosynthesis, efficient neutrophilmediated bacterial killing, and the innate immune response to pulmonary infection: cPLA2-a does not regulate neutrophil NADPH oxidase activity. J Biol Chem 2005;280:7519–7529. [42] Reutershan J, Stockton R, Zarbock A, Sullivan GW, Chang D, Scott D, et al. Blocking p21-activated kinase reduces lipopolysaccharide-induced acute lung injury by preventing polymorphonuclear leukocyte infiltration. Am J Respir Crit Care Med 2007;175:1027–1035. [43] Shalom-Barak T, Knaus UG. A p21-activated kinasecontrolled metabolic switch up-regulates phagocyte NADPH oxidase. J Biol Chem 2002;277:40659–40665. [44] Ouedraogo R, Wu X, Xu SQ, Fuchsel L, Motoshima H, Mahadev K, et al. Adiponectin suppression of high-glucoseinduced reactive oxygen species in vascular endothelial cells: evidence for involvement of a cAMP signaling pathway. Diabetes 2006;55:1840–1846. [45] Burelout C, Naccache PH, Bourgoin SG. Dissociation between the translocation and the activation of Akt in fMLP-stimulated human neutrophils-effect of prostaglandin E2. J Leukoc Biol 2007;81:1523–1534. [46] Bengis-Garber C, Gruener N. Protein kinase A downregulates the phosphorylation of p47 phox in human neutrophils: a possible pathway for inhibition of the respiratory burst. Cell Signal 1996;8:291–296. [47] Futosi K, Fodor S, Mócsai A. Neutrophil cell surface receptors and their intracellular signal transduction pathways. Int Immunopharmacol 2013;17:638–650.
ORIGINAL ARTICLE
iosgenin inhibits superoxide generation in FMLP-activated mouse neutrophils via D multiple pathways Y. Lin*, R. Jia*, Y. Liu, Y. Gao, X. Zeng, J. Kou & B. Yu
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
Jiangsu Provincial Key Laboratory for TCM Evaluation and Translational Research, China Pharmaceutical University, Nanjing, P. R. China Abstract Diosgenin possesses anti-inflammatory and anticancer properties. Activated neutrophils produce high concentrations of the superoxide anion which is involved in the pathophysiology of inflammation-related diseases and cancer. In the present study, the inhibitory effect and possible mechanisms of diosgenin on superoxide generation were investigated in mouse bone marrow neutrophils. Diosgenin potently and concentration-dependently inhibited the extracellular and intracellular superoxide anion generation in Formyl-Met-Leu-Phe (FMLP)activated neutrophils, with IC50 values of 0.50 0.08 mM and 0.66 0.13 mM, respectively. Such inhibition was not mediated by scavenging the superoxide anion or by a cytotoxic effect. Diosgenin inhibited the phosphorylation of p47phox and membrane translocation of p47phox and p67phox, and thus blocking the assembly of nicotinamide adenine dinucleotide phosphate oxidase. Moreover, cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) expression were also effectively increased by diosgenin. It attenuated FMLP-induced increase of phosphorylation of cytosolic phospholipase A (cPLA2), p21-activated kinase (PAK), Akt, p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK). Our data indicate that diosgenin exhibits inhibitory effects on superoxide anion production through the blockade of cAMP, PKA, cPLA2, PAK, Akt and MAPKs signaling pathways. The results may explain the clinical implications of diosgenin in the treatment of inflammation-related disorders. Keywords: diosgenin, mouse neutrophils, superoxide anion, NADPH oxidase, signaling pathways
Introduction Reactive oxygen species (ROS) overproduction is involved in the pathophysiology of cancer, inflammation, and cardiovascular diseases [1]. Neutrophils play a critical role in the host defense by activating superoxide anion production, which can be further processed to generate more ROS such as hydroxyl radical and hypochlorous acid [2]. The enzyme responsible for superoxide generation is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. NADPH oxidase activation coupled with ROS overproduction often leads to oxidative stress, contributing to development of inflammation diseases. Antioxidants are mostly ineffective in alleviating the burden of oxidative stress. Inhibition of ROS production through NADPH oxidase inhibitors offers an alternative approach to antioxidant therapies [3]. NADPH oxidase of neutrophils is a multiprotein complex that comprises a membrane-bound flavocytochrome b558 (gp91phox and p22phox) and cytosolic proteins (p40phox, p47phox, p67phox, and Rac2 guanosine triphosphate (GTPase)) in the resting state. Upon cell stimulation by microorganisms or inflammatory mediators, such as FMLP, the cytosolic components translocate to the membrane and associate with cytochrome b558 to form an activated NADPH oxidase complex [4]. NADPH oxidase assembly at the plasma membrane results
in the release of extracellular superoxide anion, whereas oxidase assembly on an intracellular membrane would result in intracellular superoxide anion which may be involved in signaling functions of gp91phox [5]. During neutrophils stimulation, p47phox is heavily phosphorylated. p47phox phosphorylation and subsequent translocation to flavocytochrome b558 are essential steps for the activation of NADPH oxidase [6]. The signaling mechanisms responsible for p47phox phosphorylation in neutrophils are complex. A number of kinases participate in p47phox phosphorylation events, including mitogenactivated protein kinases (MAPKs) [7], Akt [8], p21-activated kinase (PAK) [9], and protein kinase C (PKC). Furthermore, cyclic AMP (cAMP) and cytosolic phospholipase A (cPLA2) are two important second messengers involved in the activation of neutrophils and the assembled NADPH oxidase [10,11]. Therefore, modulating such signaling pathways or second messengers could produce significant effects on neutrophils activation. Diosgenin, a steroid sapogenin found in several plants including Dioscorea species, Trigonella foenum-graecum, and Costus speciosus, possesses a wide range of biological activities. It has potential chemopreventive capacity [12,13], anti-inflammatory [14,15] and anti-thrombosis properties [16], cytoprotective effect on vascular endothelial cells, etc. [17]. It can modulate antioxidant defense and decrease
*These authors contributed equally to this work. Correspondence: Dr. Junping Kou and Dr. Boyang Yu, Jiangsu Provincial Key Laboratory for TCM Evaluation and Translational Research, China Pharmaceutical University, 639 Longmian Avenue, Nanjing 211198, P. R. China. Tel: 86-25-86185158. E-mail address: junpingkou@ cpu.edu.cn; [email protected] (Received date: 16 June 2014; Accepted date: 13 September 2014; Published online: 16 October 2014)
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
2 Y. Lin et al. oxidative stress damage [18,19]. Despite that diosgenin affected various cells, such as cancer cells, endothelial cells, and monocytes [20], by multiple signaling pathways, there are few reports about its effect on activated neutrophils, and its molecular mechanisms on oxidative stress remain to be elucidated. Therefore, in the present study, we investigated the effects of diosgenin on the superoxide generation in FMLP-stimulated mouse bone marrow neutrophils and possible molecular mechanisms involved. This study would put some new insights on the potential beneficial use of diosgenin in the treatment of inflammationrelated diseases and cardiovascular diseases.
described [21,22]. Briefly, mouse bone marrow was flushed out from the rear femurs with Hank’s balanced salt buffer without Ca2 and Mg2 (HBSS¯), and centrifuged at 400 g for 10 min at 4°C. The pellet was resuspended in HBSS¯. Cells were layered on a 3-step Percoll gradient (72%, 64%, and 52%). Following centrifugation at 1000 g for 30 min, neutrophils were obtained from the 64%/72% interface and washed twice with HBSS¯. The neutrophils were resuspended in HBSS. The purity of the cells was greater than 90% and viability was greater than 95%, as determined by vital staining and microscopy.
Materials and methods
Superoxide production was measured using an isoluminol chemiluminescence assay [23,24]. Neutrophils (6 105 cells) were preincubated for 15 min with drugs prior to FMLP stimulation. One milliliter of reaction mixture containing neutrophils, HRP (20 U/mL), and isoluminol (50 mM) was equilibrated in the luminometer for 10 min at 37°C, after which 100 mL of FMLP (final concentration, 2 mM) was added to activate the reaction. The light emission was recorded continuously in a BPCL1-TGC luminometer apparatus (Academia Sinica Biophysics Institute, Beijing, China). Nonstimulated cells were recorded as background. Inhibition percentage was calculated according to (Mf - M) 100/(Mf - Mb), where M, Mb, and Mf represent maximum chemiluminescence value of drug FMLP sample, background sample, and FMLP sample, respectively.
Materials Diosgenin with purity higher than 98% was purchased from Shanghai Winherb Medical Technology Co. Ltd. (Shanghai, China). Superoxide dismutase (SOD) and FMLP were obtained from Sigma (MA, USA). Cytochalasin B (CB), horseradish peroxidase (HRP), and apocynin were obtained from Aladdin (Shanghai, China). Nitro blue tetrazolium (NBT) was purchased from Amresco (Solon, USA). Isoluminol was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). The cAMP assay kit was obtained from Jiancheng Bioengineering Institute (Nanjing, China). Antibodies against p47phox, p67phox, p38MAPK, phospho-p38MAPK (T180), extracellular signal-regulated kinase (ERK1/2), phospho-ERK1/2 (T202/Y204), cPLA2, phospho-cPLA2, PAK1/2, and protein kinase A (PKA) were purchased from Bioworld Technology (St. Paul, MN, USA). Antibodies against Akt (pan) and phospho-Akt (Ser473), c-Jun N-terminal kinase (JNK), phospho-JNK (Thr183/Tyr185), and phospho-PAK1 (Thr423)/PAK2 (Thr402) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-phosphoserine was purchased from Millipore (Bedford, MA, USA). When drugs were dissolved in dimethyl sulfoxide (DMSO), the final concentration of DMSO in the cell experiments did not exceed 0.4% and did not affect the parameters measured. Mice Male Institute of Cancer Research (ICR) mice were purchased from Nanjing Qinglong Experimental Animal Co. Ltd., China; 10–15-week-old mice were used throughout the study. All procedures and assessments were proved by Animal Ethics Committee of the School of Chinese Materia Medica, China Pharmaceutical University. These experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) revised in 1996.
Measurement of extracellular superoxide generation
Measurement of intracellular superoxide generation Quantitation of intracellular superoxide production was measured using a colorimetric NBT assay [25]. Neutrophils (6 105 cells) were incubated with diosgenin for 15 min, and then 100 mL of 2 mg/mL NBT solution was added. Cells were incubated with 2.5 mg/mL of cytochalasin B for 3 min, and then activated with FMLP (10 mM). As negative controls, cells were incubated in the NBT solution containing 100 U/mL of SOD along with stimulus. After incubation for 30 min at 37°C, cells were washed twice with PBS and once with methanol, and then airdried. The NBT deposited inside the cells was then dissolved by adding 120 mL of 2 M KOH and 140 mL of DMSO with vortex mixing for 2 min. The dissolved NBT solution was monitored at 620 nm. Superoxide scavenging activity The superoxide scavenging ability of diosgenin was determined by a kit using xanthine/xanthine oxidase assay in a cell-free system. The assay was performed according to the manufacturer’s instructions.
Isolation of mouse bone marrow neutrophils
Measurement of LDH release
Neutrophils were isolated from mouse bone marrow by isotonic Percoll gradient centrifugation, as previously
Cell membrane damage was measured by lactate dehydrogenase (LDH) release. Neutrophils (2 106 cells) were
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
Diosgenin inhibits mouse neutrophils activation 3
equilibrated at 37°C for 5 min and then incubated with diosgenin for 30 min. The cell-free supernatant fluids were collected. The activity of LDH released into the supernatant was measured using the test kit. Release of LDH in the supernatants was expressed as the percentage of total LDH that was present in an equivalent number of lysed neutrophils with 0.2% Triton X-100 at 37°C for 30 min and ultrasonication.
significance of differences among the groups. A p value of less than 0.05 was considered statistically significant.
Determination of cellular cAMP concentration
Production of both the extracellular and the intracellular superoxide in neutrophils was measured. The effects were compared with apocynin, a known inhibitor of NADPH oxidase. At the extracellular level, diosgenin (0.1–10 mM) inhibited superoxide production in a concentration-dependent manner in activated neutrophils, with an IC50 value of 0.50 0.08 mM (Figure 1A). Diosgenin (10 mM) showed strong inhibitory activity (about 96% inhibition). Apocynin (10 mM) significantly attenuated FMLP-induced superoxide generation (about 84% inhibition). Intracellular superoxide anion production was measured by the colorimetric NBT assay. Diosgenin (0.1–10 mM) prevented the increase in intracellular superoxide production in a dose-dependent manner, with an IC50 value of 0.66 0.13 mM (Figure 1B). Significant inhibition (p 0.05) was observed at diosgenin concentrations of greater than or equal to 1 mM. The inhibitory efficacy of diosgenin was significantly higher than that of apocynin. To determine whether diosgenin directly scavenges superoxide anion, experiments with xanthine/xanthine oxidase in a cell-free system were performed. Diosgenin (1–10 mM) was found to have no effect on superoxide scavenging (Figure 1C). The data suggest that the inhibitory effect of diosgenin on superoxide generation is independent of the superoxide scavenging activity. Neutrophils in the presence of diosgenin (0.1–10 mM) for 30 min did not damage the cell membranes, as assayed by LDH release (data not shown). These data indicate that inhibition of superoxide generation by diosgenin is not due to cytotoxicity.
The cAMP levels were determined as previously described [26]. Freshly isolated neutrophils (2.5 106 cells) were incubated with drugs for 15 min at 37°C before stimulation with or without 10 mM of FMLP for another 5 min, and the reaction was terminated by cooling to 4°C. Samples were then centrifuged at 800 g for 5 min at 4°C. The precipitate was treated with 0.1 M HCl for 20 min at room temperature, and then the cells were disrupted by sonication (5 cycles on ice, each for 5 s), followed by centrifugation at 12 000 g for 10 min. The supernatants were used as a source for the cAMP samples, which were assayed using an enzyme immunoassay kit. Western blot analysis To analyze their effects on PKA expression, and p47phox, cPLA2, PAK1/2, Akt, p38MAPK, ERK1/2, and JNK phosphorylation, neutrophils were pretreated with diosgenin (1, 3, and 10 mM) for 15 min at 37°C, and stimulated with 10 mM of FMLP. The reaction was stopped on ice, and the mixture was centrifuged at 800 g for 5 min at 4°C. The precipitate was washed twice with HBSS and lysed in 100 mL of RIPA buffer. The lysate was centrifuged at 13 000 g for 10 min at 4°C. Proteins (40 mg) were electrophoresed through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA). The blots were incubated with primary antibodies overnight at 4°C. A goat anti-rabbit antibody conjugated to HRP was used as the secondary antibody. Signal detection was determined using enhanced chemiluminescence followed by exposure to X-ray film. For the measurement of p47phox and p67phox translocation, the cytosolic and plasma membrane fractions were prepared using a membrane and cytosol protein extraction kit (Beyotime, Jiangsu, China). Protein concentrations were measured using the bicinchoninic acid (BCA) protein assay kit. Proteins were resolved by 12% SDS-PAGE and the blots were probed for either p47phox or p67phox using anti-p47phox or anti-p67 antibodies. Statistical analysis Results are expressed as means S.E.M. Data were analyzed with GraphPad Prism software (San Diego, CA, USA). Student’s t-test was used to determine the statistical significance of differences between two group means, and one-way analysis of variance was used to determine the
Results Effect of diosgenin on FMLP-induced superoxide production
Effect of diosgenin on NADPH oxidase activity The phosphorylation of cytosolic protein p47phox plays a key role in NADPH oxidase activation. This study examined whether diosgenin could modulate FMLP-induced p47phox phosphorylation of NADPH oxidase. As shown in Figure 1D, the level of p47phox phosphorylation was significantly increased in FMLP-stimulated neutrophils. Pretreatment of neutrophils with 10 mM diosgenin resulted in significant attenuation of phosphorylation of serine residues in p47phox. NADPH oxidase can produce superoxide after assembly of the plasma membrane flavocytochrome b558 and the cytosolic components. Furthermore, we investigated the effect of diosgenin on the translocation of p47phox and p67phox to the cell membrane in FMLP-stimulated neutrophils. As shown in Figure 1E and F, the band immunointensities of p47phox and p67phox were notably upregulated
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
4 Y. Lin et al.
Figure 1. Effects of diosgenin on superoxide generation and NADPH oxidase activation. Effects on extracellular (A) and intracellular (B) superoxide generation. Neutrophils were incubated with DMSO (as the control), diosgenin (0.1–10 mM), or apocynin (APO) for 15 min and then activated using FMLP (A) and FMLP/CB (B). The concentration of APO is 10 mM (A) and 100 mM (B). *p 0.05; **p 0.01; ***p 0.001 compared with the control. All data are expressed as the mean S.E.M. (n 426). (C) Effect on superoxide generation in a cell-free xanthine/xanthine oxidase system. All data are expressed as the mean S.E.M. (n 426). (D) Effect on p47phox phosphorylation in intact neutrophils. Cell lysates prepared from the cells treated as described above were immunoblotted with the specific antibody against p47phox and phosphoserine. Effects on p47phox (E) and p67phox (F) translocation. Representative immunoblots for the p47phox and p67phox proteins in the membrane and cytosolic fractions. The ratios between the quantified proteins in membrane and cytosolic fractions were used to quantify the FMLP-induced translocation degree. All data are expressed as mean S.E.M. (n 3). *p 0.05; **p 0.01 compared with FMLP. #p 0.05; ##p 0.01 compared with the control.
in the membrane fractions of FMLP-stimulated neutrophils. Diosgenin blocked the expression levels of subunits of p47phox and p67phox in the membrane. The data suggest that diosgenin may reduce the translocation of p47phox and p67phox from the cytosol to the membrane. The results coincided with the effect of diosgenin on superoxide generation in FMLP-stimulated neutrophils.
Effects of diosgenin on cellular cAMP levels and PKA activation cAMP is an important second messenger involved in a variety of physiological and pathophysiological processes. Increases in intracellular cAMP concentrations have been reported to inhibit FMLP-induced superoxide generation
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
Diosgenin inhibits mouse neutrophils activation 5
Figure 2. Effects of diosgenin on cAMP levels and PKA expression in FMLP-activated mouse bone marrow neutrophils. Neutrophils were incubated with DMSO (control) or diosgenin (1, 3, and 10 mM) for 15 min at 37°C before stimulation with or without FMLP (10 mM). (A) cAMP levels were measured using an enzyme immunoassay kit. (B) PKA protein expression was immunoblotted with the specific antibody against PKA. GAPDH was included as a reference for protein normalization. All data are expressed as the mean S.E.M. (n 3). *p 0.05; **p 0.01; ***p 0.001 compared with FMLP. #p 0.05; # # #p 0.001 compared with the control (1st lane).
[27]. PKA is a downstream mediator of the cAMP pathway [28]. We, therefore, examined whether diosgenin stimulated cAMP production and PKA expression in FMLP-induced neutrophils. Diosgenin (1, 3, and 10 mM) caused a slight increase in the cAMP concentration, and caused a synergistic increase in FMLP-induced cAMP levels in a concentration-dependent manner (Figure 2A). Furthermore, diosgenin (10 mM) significantly increased PKA protein expression in FMLP-stimulated neutrophils (Figure 2B). Effects of diosgenin on activations of cPLA2 and PAK cPLA2 serves as second messengers and regulates NADPH oxidase activity by modulating the assembly of the active
NADPH oxidase complex through controlling the translocation of both p47phox and p67phox [29]. PAK may participate in a variety of neutrophil responses, including rapid activation of several distinct MAPKs cascades [30,31]. Inhibition of PAK activity in neutrophils could reduce FMLP-stimulated superoxide generation. In the present study, phosphorylation of cPLA2 and PAK occurred in FMLP-activated neutrophils. Diosgenin (1, 3, and 10 mM) inhibited the phosphorylation of cPLA2 and PAK in a concentration-dependent manner (Figure 3A and B). The highest concentration of diosgenin (10 mM) reduced the phosphorylation of cPLA2 (45%) and PAK (61%), respectively. Thus, the blockade of the signaling pathways by diosgenin may account for the attenuation of neutrophils activation.
Figure 3. Effects of diosgenin on phosphorylation of cPLA2 and PAK1/2 in FMLP-activated mouse bone marrow neutrophils. Neutrophils were incubated with or without diosgenin (1, 3, and 10 mM) for 15 min before stimulation with FMLP (10 mM) for 3 min (cPLA2) or 30 s (PAK1/2) at 37°C. Phosphorylations of cPLA2 (A) and PAK1/2 (B) were analyzed by an immunoblot analysis using antibodies against the phosphorylated form and the total of each protein. All data are expressed as the mean S.E.M. (n 3). *p 0.05; **p 0.01 compared with FMLP. #p 0.05; # #p 0.01 compared with the control (1st lane).
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
6 Y. Lin et al. Effect of diosgenin on activation of MAPKs and Akt
Discussion
The MAPK proteins consist of the three major subfamilies of p38MAPK, ERK1/2, and JNK, which are known to be involved in FMLP-induced superoxide generation [32]. Akt is involved in the activation of MAPK cascades [33]. Here, we also investigated the effects of diosgenin on phosphorylation of p38MAPK, ERK1/2, JNK, and Akt in FMLP-stimulated neutrophils. Stimulation of neutrophils with FMLP resulted in the rapid phosphorylation of p38MAPK, ERK1/2, JNK, and Akt. Diosgenin (1–10 mM) reduced the phosphorylation of p38MAPK, ERK1/2, and Akt in FMLP-stimulated neutrophils in a concentration-dependent manner (Figure 4A, B, and D), whereas diosgenin (10 mM) slightly affected the phosphorylation of JNK in FMLP-stimulated neutrophils (Figure 4C).
Regulation of neutrophil activity is an important approach in avoiding inflammation. Diosgenin has been shown to have a variety of biological activities including antidiabetic activity [18], antioxidant activity, and anti-inflammatory activity. However, the action site of diosgenin on ROS generation and possible molecular mechanisms linking regulation of ROS production remain to be established. In the present study, we investigated the effects of diosgenin on superoxide generation in mouse bone marrow neutrophils to elucidate the signaling pathways responsible for the regulation of superoxide production. Diosgenin exhibited powerful activity in the inhibition of FMLP-induced extracellular and intracellular superoxide generation. Superoxide anion produced by NADPH oxidase is rapidly converted to hydrogen peroxide (H2O2)
Figure 4. Effects of diosgenin on phosphorylation of p38MAPK, ERK1/2, JNK, and Akt in FMLP-activated mouse bone marrow neutrophils. Neutrophils were incubated with DMSO (control) or diosgenin (1, 3, and 10 mM) for 15 min before stimulation with FMLP (10 mM) for 3 min (p38MAPK, ERK1/2, and JNK) or 1 min (Akt) at 37°C. Phosphorylations of p38MAPK (A), ERK1/2 (B), JNK (C), and Akt (D) were analyzed by an immunoblot analysis using antibodies against the phosphorylated form and the total of each protein. All data are expressed as mean S.E.M. (n 3). *p 0.05; **p 0.01; ***p 0.001 compared with FMLP. # #p 0.01; # # #p 0.001 compared with the control (1st lane).
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
by superoxide dismutase. Superoxide anion and H2O2 can be further processed to generate more reactive metabolites such as hydroxyl radical (OH·) and hypochlorous acid (HOCl). ROS play a very important physiological role as second messengers. They could activate diverse signaling molecules such as PKC, MAPKs, Akt, and nuclear factorkappa B (NF-kB), all of which induce inflammation in various cell types [34]. Thus, these signaling pathways and ROS may cooperate to generate amplified signaling responsible for pathological process. Our data showed that diosgenin suppressed ROS production by inhibiting MAPKs and Akt signaling pathways. The inhibition by diosgenin of ROS generation may be linked to the attenuation of signaling pathways activation. The phosphorylation of p47phox plays a key role in NADPH oxidase activation and further superoxide production in neutrophils. We then found that diosgenin also inhibited phosphorylation of p47phox and translocation of p47phox and p67phox, eventually blocking the assembly of NADPH oxidase complex (Figure 1D–F). These findings provide the first evidence of diosgenin inhibiting NADPH oxidase in activated neutrophils. As reported, NADPH oxidase is not only highly expressed in neutrophils, but also expressed in a variety of other cell types, including vascular smooth muscle cells (VSMC) and endothelial cells [35]. Previous studies have shown that diosgenin suppresses intracellular ROS production in mouse VSMC [15] and human umbilical vein endothelial cells [19], and elevates the glutathione (GSH) and restores the endothelial nitric oxide synthase (eNOS) mRNA expression level in chronic renal failure rats [36]. Our data indicated that diosgenin might inhibit NADPH oxidase in these cells and present some new explanation for these activities. On the other hand, a number of kinases have been proposed to participate in p47phox phosphorylation, including MAPKs, Akt, and PAK [37,7,9]. The p38MAPK inhibitor, SB203580, significantly reduced p47phox phosphorylation and diminished superoxide production in neutrophils. The specific inhibitors of the ERK kinase, PD98059 and U0126, inhibited p47phox phosphorylation in FMLP-stimulated human neutrophils [38]. Diosgenin concentration-dependently diminished the FMLP-induced phosphorylation of Akt, PAK1/2, p38MAPK, and ERK1/2, thus explaining the inhibitory effects of diosgenin on NADPH oxidase and ROS hyperproduction in inflammatory diseases. Previous studies have shown that pharmacological inhibition or genetic deletion of cPLA2 attenuates neutrophilmediated bacterial killing in vitro, and impairs arthritis [39], septic shock [40], and pulmonary inflammation [41] in vivo. The cPLA2 pathway is the major route of arachidonic acid production, which is implicated in the translocation, assembly, or activation of the NADPH oxidase in neutrophils [11]. The inhibition of cPLA2 has been proposed as a potential therapeutic strategy for the management of arthritis and pulmonary inflammation. Moreover, PAK can regulate leukocyte-dependent inflammatory responses in inflammatory diseases, which contributes to
Diosgenin inhibits mouse neutrophils activation 7
neutrophils adhesion and migration across the endothelium through effects on neutrophils and endothelial cells [42]. PAK may actually play a key role in the regulation of NADPH oxidase in intact cells via its involvement in p47phox phosphorylation [43]. The cAMP/PKA signaling pathways were involved in the regulation of ROS production and showed an inverse correlation between cAMP and ROS levels mediated by PKA [44]. PKA has been shown to suppress respiratory burst through inhibition of phosphoinositide 3-kinase (PI3-K) activation and translocation of both PDK1 and Akt [45,46,27]. However, the actions of diosgenin on cPLA2, PAK1/2, and cAMP/PKA pathways are less investigated in neutrophils. Our data showed that diosgenin inhibited the phosphorylation of cPLA2 and PAK1/2, and increased cAMP levels and PKA protein expression. The modulation of multiple pathways should be beneficial for diosgenin in inhibition of neutrophils activation. Neutrophils act as the first line of defense against invading bacterial. Neutrophils express a number of G-proteincoupled receptors (GPCR) that participate in host defense and inflammation. FMLP is used as a model chemoattractant due to its effective ability to activate neutrophils by binding to the GPCR on the membrane. Stimulation of the GPCR resulted in the rapid phosphorylation of several proteins via G-protein, including PKC, MAPKs, PI3-K, and PAK [47]. An increase in cAMP levels has also been demonstrated [27]. Activation of these signal transduction pathways is known to lead neutrophils activation. Based on the results obtained in the present study, we show that diosgenin appears to interfere with the multiple FMLPstimulated signaling pathways. But it remains to be defined whether diosgenin may inhibit the binding to FMLP recep-
Figure 5. Schematic diagram of the action site by diosgenin on superoxide generation in mouse bone marrow neutrophils. Upon formyl peptide binding, a number of signal transduction events are activated, which result in superoxide generation. Diosgenin can increase cAMP level and PKA expression in FMLP-activated neutrophils. Diosgenin regulates superoxide production by inhibition of cPLA2, PAK, Akt, and MAPKs activation.
8 Y. Lin et al.
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
tor to suppress the activation of these signal transduction pathways. Taken together, these results indicate that diosgenin may be an effective NADPH oxidase inhibitor. The suppressive effects of diosgenin are associated with inhibition of the cPLA2, PAK1/2, Akt, p38MAPK, ERK1/2, and JNK signaling pathways and mediated by a cAMP/PKA pathway in FMLP-activated neutrophils (Figure 5). Diosgenin does not show direct superoxide-scavenging property. It could serve as a new source of therapeutic alternatives for the treatment of cardiovascular and/or inflammatory diseases related to NADPH oxidase hyperactivity. Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. This work was financially supported by the National Natural Science Foundation of China (81073008) and National Found for College Students Innovation Project for the R&D of Novel Drugs (J1030830). References [1] Bonner MY, Arbiser JL. Targeting NADPH oxidases for the treatment of cancer and inflammation. Cell Mol Life Sci 2012;69:2435–2442. [2] Maghzal GJ, Krause KH, Stocker R, Jaquet V. Detection of reactive oxygen species derived from the family of NOX NADPH oxidases. Free Radic Biol Med 2012;53: 1903–1918. [3] Manea A. NADPH oxidase-derived reactive oxygen species: involvement in vascular physiology and pathology. Cell Tissue Res 2010;342:325–339. [4] Babior BM. NADPH Oxidase: an update. Blood 1999;93: 1464–1476. [5] Karlsson A, Dahlgren C. Assembly and activation of the neutrophil NADPH oxidase in granule membranes. Antioxid Redox Signal 2002;4:49–60. [6] El-Benna J, Dang PM, Gougerot-Pocidalo MA, Marie JC, Braut-Boucher F. p47phox, the phagocyte NADPH oxidase/ NOX2 organizer: structure, phosphorylation and implication in diseases. Exp Mol Med 2009;41:217–225. [7] El Benna J, Han J, Park JW, Schmid E, Ulevitch RJ, Babior BM. Activation of p38 in stimulated human neutrophils: phosphorylation of the oxidase component p47phox by p38 and ERK but not by JNK. Arch Biochem Biophys 1996;334:395–400. [8] Didichenko SA, Tilton B, Hemmings BA, Ballmer-Hofer K, Thelen M. Constitutive activation of protein kinase B and phosphorylation of p47phox by a membrane-targeted phosphoinositide 3-kinase. Curr Biol 1996;6:1271–1278. [9] Martyn KD, Kim MJ, Quinn MT, Dinauer MC, Knaus UG. p21-activated kinase (Pak) regulates NADPH oxidase activation in human neutrophils. Blood 2005;106:3962–3969. [10] Yu HP, Hsieh PW, Chang YJ, Chung PJ, Kuo LM, Hwang TL. DSM-RX78, a new phosphodiesterase inhibitor, suppresses superoxide anion production in activated human neutrophils and attenuates hemorrhagic shock-induced lung injury in rats. Biochem Pharmacol 2009;78:983–992.
[11] Dana R, Leto TL, Malech HL, Levy R. Essential requirement of cytosolic phospholipase A2 for activation of the phagocyte NADPH oxidase. J Biol Chem 1998;273:441–445. [12] Chiang CT, Way TD, Tsai SJ, Lin JK. Diosgenin, a naturally occurring steroid, suppresses fatty acid synthase expression in HER2-overexpressing breast cancer cells through modulating Akt, mTOR and JNK phosphorylation. FEBS Lett 2007;581: 5735–5742. [13] Rajalingam K, Sugunadevi G, Arokia Vijayaanand M, Kalaimathi J, Suresh K. Anti-tumour and anti-oxidative potential of diosgenin against 7, 12-dimethylbenz(a)anthracene induced experimental oral carcinogenesis. Pathol Oncol Res 2012;18:405–412. [14] Gao MY, Chen L, Yu HX, Sun Q, Kou JP, Yu BY. Diosgenin down-regulates NF-kB p65/p50 and p38MAPK pathways and attenuates acute lung injury induced by lipopolysaccharide in mice. Int Immunopharmacol 2013;15:240–245. [15] Choi KW, Park HJ, Jung DH, Kim TW, Park YM, Kim BO, et al. Inhibition of TNF-a-induced adhesion molecule expression by diosgenin in mouse vascular smooth muscle cells via downregulation of the MAPK, Akt and NF-kB signaling pathways. Vascul Pharmacol 2010;53:273–280. [16] Gong G, Qin Y, Huang W. Anti-thrombosis effect of diosgenin extract from Dioscorea zingiberensis C.H. Wright in vitro and in vivo. Phytomedicine 2011;18:458–463. [17] Song JX, Ma L, Kou JP, Yu BY. Diosgenin reduces leukocytes adhesion and migration linked with inhibition of intercellular adhesion molecule-1 expression and NF-kB p65 activation in endothelial cells. Chin J Nat Med 2012;10:142–149. [18] Pari L, Monisha P, Mohamed Jalaludeen A. Beneficial role of diosgenin on oxidative stress in aorta of streptozotocin induced diabetic rats. Eur J Pharmacol 2012;691:143–150. [19] Gong G, Qin Y, Huang W, Zhou S, Wu X, Yang X, et al. Protective effects of diosgenin in the hyperlipidemic rat model and in human vascular endothelial cells against hydrogen peroxideinduced apoptosis. Chem Biol Interact 2010;184:366–375. [20] Yang HP, Yue L, Jiang WW, Liu Q, Kou JP, Yu BY. Diosgenin inhibits tumor necrosis factor-induced tissue factor activity and expression in THP-1 cells via down-regulation of the NF-kB, Akt, and MAPK signaling pathways. Chin J Nat Med 2013;11:608–615. [21] Van der Hoeven D, Wan TC, Auchampach JA. Activation of the A(3) adenosine receptor suppresses superoxide production and chemotaxis of mouse bone marrow neutrophils. Mol Pharmacol 2008;74:685–696. [22] Itou T, Collins LV, Thorén FB, Dahlgren C, Karlsson A. Changes in activation states of murine polymorphonuclear leukocytes (PMN) during inflammation: a comparison of bone marrow and peritoneal exudate PMN. Clin Vaccine Immunol 2006;13:575–583. [23] Li S, Yamauchi A, Marchal CC, Molitoris JK, Quilliam LA, Dinauer MC. Chemoattractant-stimulated Rac activation in wild-type and Rac2-deficient murine neutrophils: preferential activation of Rac2 and Rac2 gene dosage effect on neutrophil functions. J Immunol 2002;169:5043–5051. [24] Dahlgren C, Karlsson A. Respiratory burst in human neutrophils. J Immunol Methods 1999;232:3–14. [25] Choi HS, Kim JW, Cha YN, Kim C. A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells. J Immunoassay Immunochem 2006;27:31–44. [26] Van der Hoeven D, Gizewski ET, Auchampach JA. Activation of the A(3) adenosine receptor inhibits FMLP-induced Rac activation in mouse bone marrow neutrophils. Biochem Pharmacol 2010;79:1667–1673. [27] Ahmed MU, Hazeki K, Hazeki O, Katada T, Ui M. Cyclic AMP-increasing agents interfere with chemoattractant-induced respiratory burst in neutrophils as a result of the inhibition of phosphatidylinositol 3-kinase rather than receptor-operated Ca2 influx. J Biol Chem 1995;270:23816–23822.
Free Radic Res Downloaded from informahealthcare.com by University of Washington on 10/27/14 For personal use only.
[28] Haskó G, Szabó C. Regulation of cytokine and chemokine production by transmitters and co-transmitters of the autonomic nervous system. Biochem Pharmacol 1998;56: 1079–1087. [29] Rane MJ, Carrithers SL, Arthur JM, Klein JB, McLeish KR. Formyl peptide receptors are coupled to multiple mitogen- activated protein kinase cascades by distinct signal transduction pathways: role in activation of reduced nicotinamide adenine dinucleotide oxidase. J Immunol 1997;159:5070–5078. [30] Dewas C, Fay M, Gougerot-Pocidalo MA, El-Benna J. The mitogen-activated protein kinase extracellular signal-regulated kinase ½ pathway is involved in formyl-methionyl-leucylphenylalanine-induced p47phox phosphorylation in human neutrophils. J Immunol 2000;165:5238–5244. [31] Zu YL, Qi J, Gilchrist A, Fernandez GA, Vazquez-Abad D, Kreutzer DL, et al. p38 mitogen-activated protein kinase activation is required for human neutrophil function triggered by TNF-alpha or FMLP stimulation. J Immunol 1998;160: 1982–1989. [32] Rane MJ, Carrithers SL, Arthur JM, Klein JB, McLeish KR. Formyl peptide receptors are coupled to multiple mitogenactivated protein kinase cascades by distinct signal transduction pathways: role in activation of reduced nicotinamide adenine dinucleotide oxidase. J Immunol 1997;159:5070–5078. [33] Ding J, Vlahos CJ, Liu R, Brown RF, Badwey JA. Antagonists of phosphatidylinositol 3-kinase block activation of several novel protein kinases in neutrophils. J Biol Chem 1995; 270:11684–11691. [34] Dworakowski R, Anilkumar N, Zhang M, Shah AM. Redox signaling involving NADPH oxidase-derived reactive oxygen species. Biochem Soc Trans 2006;34:960–964. [35] Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 2007;87:245–313. [36] Manivannan J, Balamurugan E, Silambarasan T, Raja B. Diosgenin improves vascular function by increasing aortic eNOS expression, normalize dyslipidemia and ACE activity in chronic renal failure rats. Mol Cell Biochem 2013;384: 113–120. [37] Chen Q, Powell DW, Rane MJ, Singh S, Butt W, Klein JB, McLeish KR. Akt phosphorylates p47phox and mediates respiratory burst activity in human neutrophils. J Immunol 2003;170:5302–5308.
Diosgenin inhibits mouse neutrophils activation 9 [38] Dewas C, Fay M, Gougerot-Pocidalo MA, El-Benna J. The mitogen-activated protein kinase extracellular signal-regulated kinase ½ pathway is involved in formyl-methionyl-leucylphenylalanine-induced p47phox phosphorylation in human neutrophils. J Immunol 2000;165:5238–5244. [39] Hegen M, Sun L, Uozumi N, Kume K, Goad ME, Nickerson-Nutter CL, et al. Cytosolic phospholipase A2 adeficient mice are resistant to collagen-induced arthritis. J Exp Med 2003;197:1297–1302. [40] Levy R, Dana R, Hazan I, Levy I, Weber G, Smoliakov R, et al. Elevated cytosolic phospholipase A(2) expression and activity in human neutrophils during sepsis. Blood 2000;95: 660–665. [41] Rubin BB, Downey GP, Koh A, Degousee N, Ghomashchi F, Nallan L, et al. Cytosolic phospholipase A2-a is necessary for platelet-activating factor biosynthesis, efficient neutrophilmediated bacterial killing, and the innate immune response to pulmonary infection: cPLA2-a does not regulate neutrophil NADPH oxidase activity. J Biol Chem 2005;280:7519–7529. [42] Reutershan J, Stockton R, Zarbock A, Sullivan GW, Chang D, Scott D, et al. Blocking p21-activated kinase reduces lipopolysaccharide-induced acute lung injury by preventing polymorphonuclear leukocyte infiltration. Am J Respir Crit Care Med 2007;175:1027–1035. [43] Shalom-Barak T, Knaus UG. A p21-activated kinasecontrolled metabolic switch up-regulates phagocyte NADPH oxidase. J Biol Chem 2002;277:40659–40665. [44] Ouedraogo R, Wu X, Xu SQ, Fuchsel L, Motoshima H, Mahadev K, et al. Adiponectin suppression of high-glucoseinduced reactive oxygen species in vascular endothelial cells: evidence for involvement of a cAMP signaling pathway. Diabetes 2006;55:1840–1846. [45] Burelout C, Naccache PH, Bourgoin SG. Dissociation between the translocation and the activation of Akt in fMLP-stimulated human neutrophils-effect of prostaglandin E2. J Leukoc Biol 2007;81:1523–1534. [46] Bengis-Garber C, Gruener N. Protein kinase A downregulates the phosphorylation of p47 phox in human neutrophils: a possible pathway for inhibition of the respiratory burst. Cell Signal 1996;8:291–296. [47] Futosi K, Fodor S, Mócsai A. Neutrophil cell surface receptors and their intracellular signal transduction pathways. Int Immunopharmacol 2013;17:638–650.
