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Safrole-induced expression of proinflammatory responses is associated with phosphorylation of mitogen-activated protein kinase family and the nuclear factor-κB/inhibitor of κB pathway in macrophages

Objective: Safrole, also called shikimol and Sassafras, is the carcinogenic and phenylpropanoid compound extracted from Sassafras tree and anise, betel, and camphor. Moreover, a high concentration of safrole can be occur in the saliva because of betel nut or areca quid chewing which a common habit observed in Southern and Southeastern Asia. Notably, macrophages are crucial phagocytic cells of the immune system. Nonetheless, to date, no evidence has been reported regarding safrole-induced proinflammatory response and the corresponding mechanism in macrophages. Materials and Methods: In the present study, the cytokines expression, NO generation, protein phosphorylation, and expression were assessed by enzyme-linked immunosorbent assay, Griess reagent, and Western blot assay, respectively. Results: In this study, we determined that safrole induces the generation of nitric oxide and proinflammatory cytokines, including tumor necrosis factor-α, interleukin-1β, and IL-6 in RAW264.7 macrophages in a concentration-dependent manner. Furthermore, inhibitor of κB (IκB) degradation was caused by safrole in a concentration-dependent manner. In addition, the phosphorylation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) family, including p38 MAPK, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase, was induced by safrole began to increase at 10 μM and attained a plateau at 100 μM. Conclusion: These results indicated that safrole induces the expression of proinflammatory responses in macrophages through the NF-κB/IκB pathway and its upstream factor, MAPK family phosphorylation.


Introduction
S afrole, also called shikimol and Sassafras, is a natural phenylpropanoid compound. Safrole is a colorless oily liquid that can be obtained through natural extraction from Sassafras trees (Sassafras albidum) and anise (Illicium anisatum), betel (Piper betle L.), and camphor (Ocotea pretiosa) [1][2][3]. Safrole and its metabolites, including 1-hydroxysafrole, isosafrole, and dihydrosafrole, are carcinogenic and contribute to the development of several cancers, such as oral, esophageal, and hepatocellular cancers [4][5][6][7]. Notably, chewing betel nut or areca quid is a common habit observed in Southern and Southeastern Asian countries, including Taiwan, Vietnam, Malaysia, Thailand, and the Philippines [8]. P. betle inflorescence is the common component of betel nut or areca quid and contains safrole at a high concentration of approximately 15 mg/g [9]. A previous study determined that chewing betel nut or areca quid with and metastasis in the tumor microenvironment [13]. The mitogen-activated protein kinase (MAPK) family, including p38 MAPK, extracellular signal-regulated protein kinase (ERK)-1/2, and c-Jun N-terminal kinase (JNK), mediates crucial signaling responses to generate proinflammatory mediators via the proinflammatory transcription factor and nuclear factor (NF)-κB pathways [13]. Notably, a previous study demonstrated macrophage phagocytosis was induced by safrole in mice and cell models [14,15]. In the recent study, we have purposed toxic effects, which including cytotoxicity, genotoxicity, and apoptosis, induced by safrole via intracellular reactive oxygen species generation and Akt phosphorylation in macrophages [16]. However, there is no evidence to purpose that the mechanism of the proinflammatory effects induced by safrole in macrophages. The present study attempted to assess the potency of safrole as a macrophage stimulator and explored the possible involvement of the MAPK family and NF-κB pathway in proinflammatory responses.

Cell culture
The RAW264.7 mouse macrophage cell line (Bioresource Collection and Research Center, Number: 60001) was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). RAW264.7 cells were cultured in DMEM supplemented with 10% FBS, 1% antibiotic-antimycotic solution, 25 mM HEPES, 1 mM sodium pyruvate, and 0.2% NaHCO 3 maintained at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . After 1 day of culture, the medium was changed to serum-free DMEM for further experiments [17].

Measurement of proinflammatory cytokines
The protein concentrations of TNF-α, IL-1 β, and IL-6 were measured using ELISA assay kits. Briefly, 5 × 10 4 cells were incubated with or without safrole at the indicated concentration for 12 h. The cytokine level in the culture medium was measured using ELISA kits according to the manufacturer's instructions [12].

Determination of NO generation
The NO content in the culture medium was determined through a method previously described [12]. Briefly, RAW264.7 cells were treated with or without safrole at the indicated concentration for 12 h. An equal volume of Griess reagent was added to the culture medium at room temperature for 30 min. The optical density was measured at 540 nm using a microplate reader.

Western blotting
After treatment, the RAW264.7 cells were harvested and lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% deoxycholate, 0.2% aprotinin, and 1 mM phenylmethylsulfonyl fluoride at 4°C for 30 min. The protein content in the supernatant was measured using the Bradford assay after centrifugation. Equal protein samples were separated using SDS gel electrophoresis and electrotransferred onto polyvinylidene difluoride membranes in a wet electrophoretic transfer cell. After blocking with skim milk, the membranes were incubated with antibodies, including P-p65, P-p38 MAPK, P-ERK, P-JNK, p65, p38 MAPK, ERK, JNK, iNOS, IκB, and β-actin. The membranes were washed and reprobed with appropriate horseradish peroxidase-conjugated secondary antibodies. The membranes were visualized using enhanced chemiluminescence Western blotting reagents and developed using the Fusion Solo (Vilber, Paris, France) [18].

Statistical analysis
The data from threat least three independent experiments were expressed as means ± standard deviation. Statistical analysis was performed using a one-way analysis of variance followed by Bonferroni's test for multigroup comparisons, and statistical significance (P < 0.05) was evaluated using SPSS 11.0.

Effects of safrole on nitric oxide generation and inducible nitric oxide synthase expression
Generation of NO plays a crucial role in macrophage activation via the upregulation of iNOS expression. The level of NO was estimated using the Griess reaction. The level of NO generated from 3.4 ± 0.6 (control) to 5.9 ± 0.7, 7.9 ± 0.8, and 8.3 ± 1.2 µM by 10, 100, and 300 µM safrole, respectively. Safrole significantly induced NO generation in a concentration-dependent manner, with the significant induction starting at 10 µM [P < 0.05, Figure 2]. The expression of iNOS was estimated using the Western blot assay. The expression of iNOS induced from 1.0 ± 0.0 (control) to 1.4 ± 0.2, 2.0 ± 0.5, and 3.2 ± 0.7 µM by 10, 100, and 300 µM safrole, respectively. Notably, safrole significantly induced iNOS expression in a concentration-dependent manner, with the significant induction starting at 10 µM [P < 0.05, Figure 2].

Effects of safrole on nuclear factor-κB p65 phosphorylation
The secretion of proinflammatory cytokines and NO is regulated by NF-κB p65 activation and phosphorylation.

Effects of safrole on phosphorylation of the mitogen-activated protein kinase family
Degradation of IκB and phosphorylation of NF-κB p65 are stimulated by activation and phosphorylation of the MAPK family, including p38 MAPK, ERK, and JNK. Phosphorylation of the MAPK family was measured  Figure 5].

Discussion
Macrophages are the predominant cells of the innate immune system in human beings and provide a nonspecific, first-line defense system [11]. After RAW264.7 cells treated with safrole at the concentration of 10 µM for  Results are expressed as means ± standard deviation (n = 3). *P < 0.05 considers significant as compared with control group 24 h significantly induce cytotoxicity, DNA damage, and apoptosis [16]. There were no cytotoxic effects on RAW264.7 cells treated with safrole at 300 µM for 12 h (the survival rate of the control group and 300 µM safrrole were 100.0 ± 0.0 and 99.9 ± 1.8%, respectively). For avoidance of the toxic effects, the proinflammatory effect of RAW264.7 cells incubated with safrole at the indicated concentration was performed all experiments for 12 h in the present study. Invading pathogens activate macrophages, which then secrete proinflammatory cytokines, including TNFα, IL-1β, and IL-6, that assist in pathogen defense [12]. In addition, the expression of proinflammatory cytokines results in inflammatory  -1 β (b), and interleukin-6 (c) were induced by safrole in RAW264.7 macrophages. The cells incubated with SB203580 at 30 µM, U0126 at 1 µM, SP600125 at 30 µM, and JSH23 at 30 µM for 1 h, and then the cells treated with safrole at 300 µM for 12 h. The nitric oxide generation was measured by Griess reaction. The secretion of tumor necrosis factor-α, interleukin-1 β, and interleukin-6 were detected by by Enzyme-linked immunosorbent assay kits. Results are expressed as means ± standard deviation (n = 3). *P < 0.05 considers significant as compared with control group. # P < 0.05 considers significant as compared with safrole group d c b a responses that precede tumor development [19]. The primary producers of TNFα, IL-1β, and IL-6 are macrophages, which are highly sensitive to them. Therefore, these proinflammatory cytokines play the role of master regulator of inflammatory cytokine production and are implicated in several inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, juvenile chronic arthritis, atherosclerosis, and sepsis [20][21][22]. Furthermore, macrophage functions, such as adhesion, phagocytosis, proliferation, and production of bactericidal substances, are promoted by these proinflammatory cytokines [20][21][22]. In addition, the generation of NO is a characteristic feature of macrophage activation and participation in the defense against foreign pathogens [11,12]. The amount of NO produced by iNOS after stimulation has been implicated in host-defense immunology and inflammatory responses [11]. Therefore, NO is a crucial bactericidal and proinflammatory mediator in macrophage activation [11]. To the best of our knowledge, the present study was the first to determine that safrole induces the secretion of proinflammatory cytokines and mediators in a concentration-dependent manner in macrophages. These results suggest that macrophage overexpression of proinflammatory cytokines and mediators induced by safrole could result in excessive inflammation and peripheral tissue destruction.
The proinflammatory transcription factor, NF-κB, modulates multiple signaling mechanism pathways involved in the generation of TNFα, IL-1β, and IL-6 and expression of iNOS. NF-κB, the inducible and proinflammatory transcription factor family, is composed of five proteins, namely p65 or RelA, RelB, c-Rel, p50 and its precursor p105 (also called NF-κB1), and p52 and its precursor p100 (also called NF-κB2) [23]. The most abundant subunit of NF-κB is p65, which regulates various cell functions through the highly potent transcriptional activation domain on C-terminal ends in the nucleus [23]. In a resting condition, NF-κB p65 is in the inactive form as a latent complex binding to IκB within the cytoplasm [24]. After activation, NF-κB p65 dissociates because of the degradation of IκB and translocates from the cytoplasm to the nucleus [25]. Notably, NF-κB expression is induced by safrole in human buccal mucosa fibroblasts and human umbilical vein endothelial cells [26,27]. According to our data, phosphorylation of NF-κB p65 and degradation of IκB were significantly increased by safrole at the concentration of 10 µM. We also found significantly increased generation of proinflammatory cytokines and mediators by safrole at the concentration of 10 µM. These findings indicate that the generation of proinflammatory cytokines and mediators was induced by safrole via the NF-κB pathway, including NF-κB phosphorylation and IκB degradation.
Notably, the degradation of IκB is triggered by the phosphorylation of the MAPK family [28]. In mammals, the three subclasses of the MAPK family include ERK, JNK, and p38 MAPK. Activation of the MAPK family is induced by phosphorylation on the threonine and tyrosine residues within a conserved Thr-X-Tyr motif in the activation loop. The MAPK family is involved in transmitting signals from various extracellular ligands and converting them into a wide range of intracellular responses [29]. The MAPK family plays a crucial role in proinflammatory responses [30]. After human buccal mucosal fibroblasts were treated with PD98059, the inhibitor of ERK activation reduced the NF-κB expression induced by safrole. These results indicate that safrole-induced NF-κB expression was mediated by ERK activation in fibroblasts [27]. Herein, the results indicated that phosphorylation of ERK, JNK, and p38 MAPK was markedly induced by safrole at the concentration of 10 µM. The level of MPAK phosphorylation induced by safrole at the concentration of 100 and 300 µM was higher than 10 µM. However, there are similar effects of MAPK phosphorylation induced by safrole at the concentration of 100 and 300 µM. These results suggested that the phosphorylation level of MAPK began to increase at 10 µM and attained a plateau at 100 µM. This reactive trend would be also found in phosphorylation of NF-κB p65. These results suggest that NF-κB phosphorylation was induced by safrole through phosphorylation of ERK, JNK, and p38 MAPK.
In the previous study, we have demonstrated that induction of cytotoxicity, DNA damage, and apoptosis in macrophage treatment with safrole for 24 h was through the generation of ROS and inhibition of antioxidative enzymes possibly via Akt phosphorylation. To the best of our knowledge, this study is the first systemic attempt to evaluate the proinflammatory responses and mechanisms of safrole on RAW264.7 macrophages. We demonstrated for the first time that proinflammatory cytokines, including TNFα, IL-1β, IL-6, and NO, were induced by safrole. Furthermore, we determined that safrole induced NF-κB phosphorylation and IκB degradation, leading to the generation of proinflammatory cytokines and NO. In addition, we observed that phosphorylation of ERK, JNK, and p38 MAPK induced by safrole exhibited similar concentration trends as NF-κB phosphorylation and IκB degradation did. These results suggested that the expression of proinflammatory cytokines and NO in macrophages treated with safrole occurs via the NF-κB/IκB pathway and its upstream factor, MAPK family phosphorylation [ Figure 7].