Cucurbitacin B Down-Regulates TNF Receptor 1 Expression and Inhibits the TNF-α-Dependent Nuclear Factor κB Signaling Pathway in Human Lung Adenocarcinoma A549 Cells

Pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), induce the expression of intracellular adhesion molecule-1 (ICAM-1) by activating the nuclear factor κB (NF-κB) signaling pathway. In the present study, we found that cucurbitacin B decreased the expression of ICAM-1 in human lung adenocarcinoma A549 cells stimulated with TNF-α or interleukin-1α. We further investigated the mechanisms by which cucurbitacin B down-regulates TNF-α-induced ICAM-1 expression. Cucurbitacin B inhibited the nuclear translocation of the NF-κB subunit RelA and the phosphorylation of IκBα in A549 cells stimulated with TNF-α. Cucurbitacin B selectively down-regulated the expression of TNF receptor 1 (TNF-R1) without affecting three adaptor proteins (i.e., TRADD, RIPK1, and TRAF2). The TNF-α-converting enzyme inhibitor suppressed the down-regulation of TNF-R1 expression by cucurbitacin B. Glutathione, N-acetyl-L-cysteine, and, to a lesser extent, L-cysteine attenuated the inhibitory effects of cucurbitacin B on the TNF-α-induced expression of ICAM-1, suggesting that an α,β-unsaturated carbonyl moiety is essential for anti-inflammatory activity. The present results revealed that cucurbitacin B down-regulated the expression of TNF-R1 at the initial step in the TNF-α-dependent NF-κB signaling pathway.


Introduction
Pro-inflammatory cytokines, such as those belonging to the tumor necrosis factor (TNF) family and interleukin-1 (IL-1) family, are mainly produced by macrophages [1]. In the intracellular signaling pathways, TNF-α and IL-1 primarily activate the transcription factor nuclear factor κB (NF-κB) [2,3]. NF-κB induces the transcriptional activation of many genes, some of which encode cell adhesion molecules, cytokines, and chemokines [4]. In vascular endothelial cells, cell adhesion molecules are up-regulated during inflammation and mediate the capture and migration of circulating leukocytes to local inflamed sites [5,6]. Intercellular adhesion molecule-1 (ICAM-1) is a cell adhesion molecule that regulates inflammatory and immune responses [7]. Its expression is mainly up-regulated at the transcriptional level by NF-κB subunits [8].
We identified a number of natural and synthetic compounds that exhibited antiinflammatory activity in evaluations of the down-regulation of ICAM-1 expression by pro-inflammatory cytokines [35]. In the present study, we found that cucurbitacin B reduced the level of ICAM-1 expression induced by TNF-α and IL-1α in human lung adenocarcinoma A549 cells. We further investigated the mechanisms by which cucurbitacin B down-regulates TNF-α-induced ICAM-1 expression. The results obtained revealed that cucurbitacin B down-regulated the expression of TNF-R1 at the initial step in the TNF-αinduced NF-κB signaling pathway.

Cucurbitacin B Down-Regulated TNF-α-Induced ICAM-1 Expression
A549 cells were treated with serial dilutions of cucurbitacin B for 1 h and were then incubated with or without TNF-α or IL-1α for 6 h. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cucurbitacin B at concentrations up to 30 µM did not markedly affect cell viability; however, a reduction in cell viability was observed at 100 µM ( Figure 1B; Figure S1). TNF-α-induced expression of ICAM-1 protein was reduced by cucurbitacin B in a dose-dependent manner ( Figure 1C). IL-1α-induced expression of the ICAM-1 protein was also decreased by cucurbitacin B ( Figure  S2). Flow cytometry confirmed that cucurbitacin B at 30 µM decreased the expression of cellsurface ICAM-1 induced by TNF-α and IL-1α ( Figure 1D,E; Figure S3). Based on cell-ELISA, cucurbitacin B appeared to preferentially reduce the TNF-α-induced expression of ICAM-1 protein over that induced by IL-1α ( Figure 1C; Figure S2). Therefore, we examined the mechanisms by which cucurbitacin B down-regulated TNF-α-induced ICAM-1 expression as well as the upstream intracellular process.

Cucurbitacin B Inhibited the TNF-α-Dependent NF-κB Signaling Pathway
Upon TNF-α stimulation, the NF-κB subunit RelA translocated from the cytosol to the nucleus and bound to the ICAM-1 promoter in A549 cells [36]. A549 cells were pretreated with cucurbitacin B for 1 h and were then stimulated with TNF-α for 30 min. Cell lysates were separated into nuclear and cytoplasmic fractions. The amount of RelA in the nuclear fraction was markedly increased by TNF-α stimulation but decreased by cucurbitacin B (Figure 3). The amount of RelA in the cytoplasmic fraction was not markedly affected by cucurbitacin B (Figure 3). (C) A549 cells were transfected with plasmid vectors encoding the ICAM-1 promoter-driven firefly luciferase reporter and cytomegalovirus (CMV) promoter-driven Renilla luciferase reporter for 17 h. Transfected A549 cells were pretreated with or without cucurbitacin B for 1 h and were then treated with (+) or without (−) TNF-α (2.5 ng/mL) for 3 h. ICAM-1 promoter-driven luciferase activity was normalized to CMV promoter-driven luciferase activity. Luciferase activity (fold) is shown as the mean ± S.E. of three independent experiments. *** p < 0.001. Upon TNF-α stimulation, the NF-κB subunit RelA translocated from the cytosol to the nucleus and bound to the ICAM-1 promoter in A549 cells [36]. A549 cells were pretreated with cucurbitacin B for 1 h and were then stimulated with TNF-α for 30 min. Cell lysates were separated into nuclear and cytoplasmic fractions. The amount of RelA in the nuclear fraction was markedly increased by TNF-α stimulation but decreased by cucurbitacin B (Figure 3). The amount of RelA in the cytoplasmic fraction was not markedly affected by cucurbitacin B (Figure 3).  In the TNF-α-induced NF-κB signaling pathway, a prerequisite to the nuclear translocation of RelA, we previously showed that IκBα was phosphorylated in A549 cells within 5 min and then degraded to undetectable levels after 15 min [37]. Cucurbitacin B inhibited the phosphorylation of IκBα in a dose-dependent manner ( Figure 4A,B), and this was accompanied by a slight increase in the IκBα protein ( Figure 4C,D). The degradation of IκBα 15 min after the TNF-α stimulation was inhibited by cucurbitacin B (Figure 4E,F). These results indicate that cucurbitacin B inhibited the TNF-α-dependent NF-κB signaling pathway. , x FOR PEER REVIEW 6 of 17

Cucurbitacin B Down-Regulated the Expression of TNF-R1
We previously showed that A549 cells express TNF-R1 but not TNF-R2 [38]. Upon a stimulation with TNF-α, TNF-R1 recruits TRADD, RIPK1, and TRAF2 as major adaptor proteins, which is essential for the activation of IκB kinase [3,9]. To clarify the mechanisms by which cucurbitacin B inhibited the TNF-α-induced NF-κB signaling pathway, we measured the expression levels of TNF-R1 and adaptor proteins. Western blotting using an antibody recognizing TNF-R1 CRD showed that cucurbitacin B down-regulated the expression of TNF-R1 ( Figure S4). Western blotting using another antibody recognizing an intracellular domain in TNF-R1 confirmed that cucurbitacin B down-regulated the expression of TNF-R1 ( Figure 5A,B). In contrast, cucurbitacin B at concentrations up to 30 µM did not markedly down-regulate the expression of TRADD, RIPK1, or TRAF2 ( Figure 5C). Cucurbitacin B did not reduce the expression of TNF-R1 mRNA; it actually increased its expression, particularly at 10 µM ( Figure 5D). To clarify whether cucurbitacin B affected exogenous human TNF-R1, A549 cells were transiently transfected with an expression vector encoding C-terminal FLAG-tagged TNF-R1, driven by the constitutive CMV promoter, and were then treated with cucurbitacin B for 1 h. Western blotting using the anti-FLAG-antibody showed that cucurbitacin B decreased the amount of FLAG-tagged TNF-R1 ( Figure 6A,B). These results indicate that cucurbitacin B selectively down-regulated the expression of TNF-R1 without affecting that of TRADD, RIPK1, or TRAF2. To clarify whether cucurbitacin B affected exogenous human TNF-R1, A549 cells were transiently transfected with an expression vector encoding C-terminal FLAG-tagged TNF-R1, driven by the constitutive CMV promoter, and were then treated with cucurbitacin B for 1 h. Western blotting using the anti-FLAG-antibody showed that cucurbitacin B decreased the amount of FLAG-tagged TNF-R1 ( Figure 6A,B). These results indicate that cucurbitacin B selectively down-regulated the expression of TNF-R1 without affecting that of TRADD, RIPK1, or TRAF2.
were transiently transfected with an expression vector encoding C-terminal FLAG-tagged TNF-R1, driven by the constitutive CMV promoter, and were then treated with cucurbitacin B for 1 h. Western blotting using the anti-FLAG-antibody showed that cucurbitacin B decreased the amount of FLAG-tagged TNF-R1 ( Figure 6A,B). These results indicate that cucurbitacin B selectively down-regulated the expression of TNF-R1 without affecting that of TRADD, RIPK1, or TRAF2.

Cucurbitacin B Promoted the TNF-α-Converting Enzyme (TACE)-Dependent Down-Regulation of TNF-R1
To investigate whether cucurbitacin B promoted TNF-R1 protein degradation, A549 cells were pretreated with the TACE inhibitor TAPI-2, the proteasome inhibitor MG-132, and the vacuolar type H + -ATPase inhibitor bafilomycin A 1 , which prevents lysosomal degradation, prior to the treatment with cucurbitacin B. TAPI-2 suppressed reductions in TNF-R1 protein levels in cucurbitacin B-treated A549 cells ( Figure 7A,B). In contrast, cucurbitacin B still decreased TNF-R1 protein levels in the presence of bafilomycin A 1 ( Figure 7A,B). Unexpectedly, MG-132 alone decreased TNF-R1 protein levels, and this was accompanied by the appearance of smaller TNF-R1 fragments ( Figure 7A,B). We previously demonstrated that MG-132 (20 µM) did not markedly affect the amount of RelA and IκBα in A549 cells during a 75-or 90-min incubation [36,[39][40][41]. Although further studies are needed to clarify the mode of action of MG-132 on the degradation of TNF-R1, these results showed that cucurbitacin B promoted the TACE-dependent down-regulation of TNF-R1.

Cucurbitacin B Promoted the TNF-α-Converting Enzyme (TACE)-Dependent Down-Regulation of TNF-R1
To investigate whether cucurbitacin B promoted TNF-R1 protein degradation, A549 cells were pretreated with the TACE inhibitor TAPI-2, the proteasome inhibitor MG-132, and the vacuolar type H + -ATPase inhibitor bafilomycin A1, which prevents lysosomal degradation, prior to the treatment with cucurbitacin B. TAPI-2 suppressed reductions in TNF-R1 protein levels in cucurbitacin B-treated A549 cells ( Figure 7A,B). In contrast, cucurbitacin B still decreased TNF-R1 protein levels in the presence of bafilomycin A1 (Figure 7A,B). Unexpectedly, MG-132 alone decreased TNF-R1 protein levels, and this was accompanied by the appearance of smaller TNF-R1 fragments ( Figure 7A,B). We previously demonstrated that MG-132 (20 µM) did not markedly affect the amount of RelA and IκBα in A549 cells during a 75-or 90-min incubation [36,[39][40][41]. Although further studies are needed to clarify the mode of action of MG-132 on the degradation of TNF-R1, these results showed that cucurbitacin B promoted the TACE-dependent down-regulation of TNF-R1. Cucurbitacin B possesses an α,β-unsaturated carbonyl moiety ( Figure 1A), which binds to thiol groups by the Michael reaction. To establish whether the α,β-unsaturated carbonyl moiety is essential for the inhibitory effects of cucurbitacin B, A549 cells were

An α,β-Unsaturated Carbonyl Moiety Was Necessary for the Inhibitory Effect of Cucurbitacin B
Cucurbitacin B possesses an α,β-unsaturated carbonyl moiety ( Figure 1A), which binds to thiol groups by the Michael reaction. To establish whether the α,β-unsaturated carbonyl moiety is essential for the inhibitory effects of cucurbitacin B, A549 cells were pretreated with thiol compounds and then with cucurbitacin B, followed by a TNF-α stimulation to induce ICAM-1 expression. Glutathione and N-acetyl-L-cysteine efficiently attenuated the inhibitory effects of cucurbitacin B on TNF-α-induced ICAM-1 expression ( Figure 8A,B). The inhibitory effects of cucurbitacin B were also reversed, but to a lesser extent, by L-cysteine ( Figure 8C). Glutathione and N-acetyl-L-cysteine are often used as thiol-type antioxidants, while ascorbic acid and Trolox are antioxidants that lack thiol groups. Ascorbic acid and Trolox at concentrations up to 10 mM did not attenuate the inhibitory effects of cucurbitacin B on TNF-α-induced ICAM-1 expression ( Figure S5). These results suggest that the α,β-unsatu- Glutathione and N-acetyl-L-cysteine are often used as thiol-type antioxidants, while ascorbic acid and Trolox are antioxidants that lack thiol groups. Ascorbic acid and Trolox at concentrations up to 10 mM did not attenuate the inhibitory effects of cucurbitacin B on TNF-α-induced ICAM-1 expression ( Figure S5). These results suggest that the α,βunsaturated carbonyl moiety was necessary for the inhibitory effects of cucurbitacin B.

Cytochalasin D and Jasplakinolide Only Partially Down-Regulated TNF-α-Induced ICAM-1 Expression
Cucurbitacins have been reported to induce the aggregation of actin, which decreases the globular actin (G-actin) pool, in Triton X-100-soluble fractions [42,43]. To clarify whether cucurbitacin B affected the actin cytoskeleton in A549 cells, the amount of γ1-actin in cytoplasmic fractions, which were collected as Triton X-100-soluble fractions, was analyzed by Western blotting. GAPDH, but not lamin A/C, was present in the cytoplasmic fraction, while lamin A/C was only detected in the nuclear fraction ( Figure 9). Cucurbitacin B decreased γ1-actin in the cytoplasmic fraction ( Figure 9). These results suggest that cucurbitacin B affected the actin cytoskeleton and decreased the G-actin pool in A549 cells.  We also investigated the effects of small-molecule ac α-induced ICAM-1 expression. Cytochalasin D has been s zation, while jasplakinolide stabilizes the actin cytoskeleto tochalasin D at concentrations higher than 3 µM only sli A549 cells (Figure 10A), whereas cytochalasin D at a narr regulated TNF-α-induced ICAM-1 expression by ~40% (F µM decreased cell viability by ~20% (Figure 10B), and d ICAM-1 expression by approximately 30% (Figure 10D). T We also investigated the effects of small-molecule actin-modulating agents on TNF-α-induced ICAM-1 expression. Cytochalasin D has been shown to inhibit actin polymerization, while jasplakinolide stabilizes the actin cytoskeleton [44]. In the present study, cytochalasin D at concentrations higher than 3 µM only slightly decreased the viability of A549 cells ( Figure 10A), whereas cytochalasin D at a narrow range of 0.3 or 1 µM down-regulated TNF-α-induced ICAM-1 expression by~40% ( Figure 10C). Jasplakinolide at 1 µM decreased cell viability by~20% (Figure 10B), and down-regulated TNF-α-induced ICAM-1 expression by approximately 30% (Figure 10D). These results indicate that cytochalasin D and jasplakinolide only partially down-regulated TNF-αinduced ICAM-1 expression. A549 cells (Figure 10A), whereas cytochalasin D at a narrow range of 0.3 or 1 µM downregulated TNF-α-induced ICAM-1 expression by ~40% ( Figure 10C). Jasplakinolide at 1 µM decreased cell viability by ~20% (Figure 10B), and down-regulated TNF-α-induced ICAM-1 expression by approximately 30% (Figure 10D). These results indicate that cytochalasin D and jasplakinolide only partially down-regulated TNF-α-induced ICAM-1 expression.
A previous study demonstrated that cucurbitacins B, E and I suppressed LPS-induced nuclear RelA translocation in mouse microglia [27]. Cucurbitacin E inhibited TNF-αinduced nuclear RelA translocation in human synoviocyte MH7A cells [26] and suppressed LPS-induced nuclear RelA translocation in mouse RAW264.7 cells without affecting the phosphorylation of IκBα [23]. In contrast to these findings, another study demonstrated that cucurbitacin B did not affect TNF-α-induced nuclear RelA translocation but inhibited its transcriptional activity in human cervical carcinoma HeLa cells [21]. Based on these findings, cucurbitacins appear to inhibit multiple steps in the NF-κB signaling pathway in a cell context-dependent manner. The present results showed that cucurbitacin B inhibited TNF-α-induced IκBα phosphorylation and selectively down-regulated the expression of TNF-R1 without affecting three adaptor proteins. Therefore, the inhibition of the TNF-αinduced NF-κB signaling pathway by cucurbitacin B appears to be at least partly attributed to the down-regulation of TNF-R1 in the initial step.
Glutathione, N-acetyl-L-cysteine, and, to a lesser extent, L-cysteine attenuated the inhibitory effects of cucurbitacin B. These results confirmed that the α,β-unsaturated carbonyl moiety of cucurbitacin B is critical, which is consistent with previous findings showing that N-acetyl-L-cysteine canceled the biological effects of cucurbitacin B in A549 cells [45]. Compounds possessing α,β-unsaturated carbonyl moieties have been reported to inhibit the NF-κB signaling pathway by targeting the Cys179 of IκB kinase β and Cys38 of the NF-κB subunit RelA [35,46]. We previously showed that eudesmane-type sesquiterpene lactones containing α,β-unsaturated carbonyl moieties, such as an α-methylene-γ-lactone or α-bromo ketone group, targeted multiple steps in the NF-κB signaling pathway induced by TNF-α and IL-1α [41]. In addition to the down-regulated expression of TNF-R1, IκB kinase β and the NF-κB subunit RelA are potential candidate targets for cucurbitacin B in the common NF-κB signaling pathway activated by TNF-α and IL-1α.
We previously reported that allantopyrone A, possessing two α,β-unsaturated carbonyl moieties, binds and crosslinks multiple proteins, including TNF-R1, which blocks the TNF-α-induced NF-κB pathway [47]. Allantopyrone A directly bound to TNF-R1 because it reduced the reactivity of the anti-TNF-R1 antibody to an epitope containing a cysteine residue in the extracellular CRD [47]. Western blotting using different antibodies recognizing TNF-R1 excluded the possibility that cucurbitacin B bound to TNF-R1 in a similar manner to allantopyrone A. We recently reported that isopanduratin A, a flavonoid possessing an α,β-unsaturated carbonyl moiety, down-regulated the expression of TNF-R1 by ectodomain shedding via the extracellular signal-regulated kinase (ERK)-dependent activation of TACE and the inhibition of de novo translation via eIF2α phosphorylation [48]. Translation inhibitors regulate the expression of TNF-R1 by reducing its de novo synthesis and by inducing its ectodomain shedding via the activation of ERK and/or p38 MAP kinase [49]. The TACE inhibitor TAPI-2 did not reverse the amount of TNF-R1 in isopanduratin A-treated A549 cells, which was the combined effect of enhanced ecodomain shedding and inhibited translation [48]. In contrast to isopanduration A, TAPI-2 suppressed reductions in TNF-R1 in cucurbitacin B-treated A549 cells. Based on the present results, we speculate that cucurbitacin B induces the TACE-dependent cleavage of TNF-R1 and then promotes the prompt degradation of intracellular TNF-R1 fragments. Further analyses are required to clarify the regulation of intracellular TNF-R1 levels by translation, ectodomain shedding, lysosomal degradation, and/or proteasomal degradation as well as the molecular mechanisms by which cucurbitacin B down-regulates TNF-R1.
Cucurbitacins have been reported to induce the aggregation of actin, which reduces the G-actin pool [42,43]. They have also been shown to covalently bind to cofilin1 via Cys39 and F-actin via Cys257 [50,51]. Cofilins cleave actin filaments and promote depolymerization, which is inactivated by phosphorylation [52]. Cucurbitacins were previously found to inhibit cofilin phosphorylation and promote actin aggregation, which was accompanied by a reduction in the G-actin pool [53,54]. Cucurbitacin B affected the actin cytoskeleton in A549 cells, as evidenced by a reduction in the G-actin pool. In contrast to cucurbitacin B, the actin-modulating agents cytochalasin D and jasplakinolide exerted only partial inhibitory effects on TNF-α-induced ICAM-1 expression in A549 cells. These results suggest that the effects of cucurbitacin B on the actin cytoskeleton are not the main mechanism by which it down-regulates TNF-α-induced ICAM-1 expression and inhibits the NF-κB signaling pathway.
In the present study, we demonstrated that cucurbitacin B down-regulated the expression of TNF-R1 and inhibited the common NF-κB signaling pathway induced by TNF-α and IL-1α. In addition, we showed that cucurbitacin B decreased γ1-actin in the cytoplasmic fraction. These results revealed that cucurbitacin B has multiple cellular targets. Consistent with this notion, cucurbitacin B has been shown to have many molecular targets in cancer signaling pathways [18]. The combined effects of cucurbitacin B targeting multiple steps in the NF-κB pathway may contribute to stronger anti-inflammatory activity. However, higher selectivity is preferred to reduce side effects and develop therapeutic drugs. Further studies are needed to identify the multiple cellular targets of cucurbitacin B in A549 cells and clarify their modes of action in the NF-κB signaling pathway induced by TNF-α and IL-1α.

Cell Viability Assay
Cell viability was evaluated by the MTT assay, which measured mitochondrial MTTreducing activity. A549 cells were incubated with MTT at a concentration of 500 µg/mL for the last 2 h. Formazan was extracted in the presence of 5% SDS overnight. Absorbance at 570 nm was measured by the iMark microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). Cell viability (%) was calculated as (experimental absorbance-background absorbance without cells)/(control absorbance-background absorbance without cells) × 100.

Flow Cytometry
Flow cytometry was performed as previously described [58]. A549 cells were stained either with the anti-ICAM-1 antibody (15.2) or an isotype control antibody (MOPC-21; BioLegend, San Diego, CA, USA) at a concentration of 1 µg/mL, followed by staining with a phycoerythrin-labeled anti-mouse IgG antibody (Jackson ImmunoResearch) at a concentration of 5 µg/mL. Fluorescent intensity was assessed by FACSCalibur (BD Biosciences). Histograms were analyzed using FlowJo software version 8.5.1 (Tomy Digital Biology, Tokyo, Japan).

Luciferase Reporter Assay
A549 cells were transfected with a pGL4.22[luc2CP/Puro] vector encoding an ICAM-1 promoter-driven firefly luciferase reporter and a pCR3 expression vector encoding a CMV promoter-driven Renilla luciferase reporter by HilyMax Transfection Reagent (Dojindo Laboratories, Kumamoto, Japan). Cell lysates were prepared and used for the luciferase assay as previously described [56]. Relative light units were measured by Lumitester C-110 (Kikkoman Biochemifa, Tokyo, Japan).

Western Blotting
Western blotting was performed as previously described [63]. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were then serially incubated with primary antibodies and peroxidase-conjugated secondary antibodies. Protein bands were acquired by Amersham Imager 680 (GE Healthcare Japan, Tokyo, Japan) and analyzed by ImageQuant TL software toolbox version 7.0 (GE Healthcare Japan).

Statistical Analysis
Statistical analyses were performed using a one-way ANOVA and Tukey's test by KaleidaGraph software version 4.5.1 (Hulinks, Tokyo, Japan).

Conclusions
The present results demonstrated that cucurbitacin B down-regulated the expression of TNF-R1 and inhibited the TNF-α-dependent NF-κB signaling pathway in A549 cells. TNF-α is produced by activated macrophages and other types of cells, while TNF-R1 is ubiquitously expressed in various cells. TNF-α-induced NF-κB-dependent gene expression plays an essential role in inflammatory and immune responses. The acute and chronic activation of the NF-κB signaling pathway has been implicated in the pathogenesis of inflammatory diseases. Natural products, such as cucurbitacins, that target the TNF-αinduced NF-κB pathway have potential as anti-inflammatory agents. Further studies are needed to elucidate the mechanisms of action of cucurbitacins on the NF-κB signaling pathway and develop more selective anti-inflammatory agents. While the present study focused on the cellular response of non-immune cells stimulated with inflammatory cytokines, macrophages are involved in detrimental inflammatory diseases by producing excess inflammatory cytokines, such as TNF-α. Future research is warranted to investigate the effects of cucurbitacin B on primary macrophages and a TNF-α-induced inflammation in vivo model.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in the present study are available upon request from the corresponding author.