WBGE decreased the TNF-α-induced ICAM-1 expression in A549 cells

A549 cells treated with 10-100 μg/mL WBGE or with 10-100 μg/mL charantin for

24 h did not cause cytotoxicity in the MTT assay (Fig. 2A). To analyze the effects of WBGE and charantin on ICAM-1 expression under inflammation, A549 cells were preincubated with WBGE for 24 h with 10, 30, 50, or 80 μg/mL or charantin with 0.5, 1, 3, 5 μg/mL and then followed by stimulation with 3 ng/mL TNF-α for 4 h. TNF-α

treatment significantly increased ICAM-1 expression in A549 cells (Fig. 2B). This effect was inhibited by pretreatment with WBGE or charantin. WBGE significantly reduced ICAM-1 expression in a concentration-dependent manner. Consistently, fluorescence microscopy images showed that ICAM-1 was strongly present in the cytosol of TNFα-treated A549 cells (T) (Fig. 3). In contrast, ICAM-1 expression was weaker in TNF-α-stimulated A549 cells pretreated with WBGE (WBGE/T) or charantin

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(CH/T). Luciferase activity of ICAM-1 promoter was significantly induced with TNF-α and was significantly reduced by WBGE (Fig. 4). Based on these results, 80 μg/mL WBGE was used in the present study to evaluate the anti-inflammatory effects and molecular mechanisms of WBGE treatment.

Since cell adhesion molecules are critical for monocyte adhesion, the next step was to examine the effects of WBGE on monocyte adhesion. As shown in Fig. 5, minimal binding of monocytes to confluent control cells was observed, but adhesion was substantially increased when the cells were treated with TNF-α. In contrast, WBGE-pretreated cells significantly reduced the binding of monocytes to

TNF-α-treated cells. In addition, pretreatment of the A549 cells with a neutralizing antibody against ICAM-1 led the number of monocytes binding to TNF-α-treated cells to be significantly reduced. The present study demonstrated that ICAM-1 played an important role in monocyte adhesion to TNF-α-treated A549 cells.

3.3 The inhibition of PI3K/AKT phosphorylation mediates the reduction in ICAM-1 expression by WBGE in TNF-α-treated A549 cells

Previous studies have demonstrated that MAPKs (Lee et al., 2013) and the

PI3K/AKT signaling pathway (Choi et al., 2012; Oh and Kwon, 2009) were involved in TNF-α-induced inflammation. We next investigated whether TNF-α-induced ICAM-1 expression was mediated through MAPKs or PI3K/AKT phosphorylation. The

phosphorylation of ERK1/2, p38, and JNK in A549 cells showed a significant increase at 15-30 min of TNF-α treatment and was followed by a decline at 60 min (Fig. 6). To determine the effects of WBGE, the cells were pretreated with WBGE for 24 h and then stimulated with TNF-α for 5, 15, 30, 45, or 60 min. WBGE had no effect on

TNF-α-induced ERK1/2, p38, and JNK phosphorylation (Fig. 6). The expression levels

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of phosphorylated PI3K and AKT were significantly increased after 15 min of TNF-α stimulation, and pretreatment with WBGE significantly attenuated the phosphorylation of PI3K and AKT (Fig. 7A). Additionally, we determined whether the activation of PI3K/AKT was involved in the suppression of WBGE on TNF-α-induced ICAM-1 expression. As shown in Fig. 7B, pretreatment with MK2206 (AKT inhibitor) and LY294002 (PI3K inhibitor) caused significant attenuation of ICAM-1 expression in TNF-α-treated A549 cells. In addition, pretreatment with WBGE and inhibitors enhanced the decrease in ICAM-1 expression. According to these results, WBGE suppressed TNF-α-induced ICAM-1 expression through PI3K/AKT phosphorylation and an undetermined pathway. In addition, inhibitors specific for PI3K and AKT

blocked TNF-α-induced monocyte adhesion (Fig. 8), which further supported the notion that sequential activation of the PI3K/AKT pathway resulted in upregulation of ICAM-1 and subsequent monocyte adhesion.

3.4 The inhibition of NF-κB p65 activation and translocation mediates WBGE-reduced ICAM-1 expression in TNF-α-treated A549 cells

We investigated whether WBGE reduced TNF-α-induced ICAM-1 expression via

NF-κB signaling because the promoter of the ICAM-1 gene contains consensus binding sites for the transcription factor (Rahman et al., 1999). First, we examined the levels of phosphorylated NF-κB p65 in TNF--treated A549 cells through Western blotting. The phospho-p65 level was higher in TNF--treated A549 cells than in control cells, and WBGE pretreatment significantly reduced that effect (Fig. 9A). A similar result was obtained for IκB phosphorylation (Fig. 9B), which is responsible for NF-κB activation (Choi et al., 2012). The results of immunofluorescent staining were consistent with the Western blot findings of NF-κB p65. Control A549 cells or cells incubated only with

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WBGE showed no nuclear NF-κB p65 staining, but there was strong staining in the cytoplasm. In contrast, A549 cells were stimulated with TNF- for 1 h and showed strong NF-κB p65 staining in the nucleus, and this effect was significantly decreased by pretreatment for 24 h with WBGE (Fig. 10A). A similar result was obtained through Western blotting (Fig. 10B). The binding of NF-κB/p65 to the ICAM-1 promoter was further examined by ChIP-PCR. TNF-α significantly increased the binding of p65 to the ICAM-1 promoter, whereas WBGE reduced the binding activity (Fig. 11A).

Furthermore, TNF--induced ICAM-1 expression in A549 cells was blocked by

preincubation of the cells for 1 h with 30 M Bay11-7082, which is an NF-κB inhibitor (Fig. 11B). Moreover, pretreatment of cells with Bay 11-7082 blocked TNF--induced monocyte adhesion (Fig. 12), which further supported the notion that sequential activation of the NF-kB pathway leads to upregulation of ICAM-1 and subsequent monocyte adhesion to TNF--treated A549 cells. These results suggested that WBGE-reduced ICAM-1 expression in TNF--treated A549 cells was mediated by inhibition of NF-κB activation.

3.5 WBGE reductions in endogenous ICAM-1 expression in A549 cells involves miR-222 upregulation

Recent studies suggest that ICAM-1 is a target of miR-221/-222, and both miRNAs

regulate ICAM-1 expression (Duan et al., 2013). miR-221 and miR-222 are encoded in tandem on X chromosomes and are conserved miRNAs with significant homology between them. According to TargetScan.org (http://www.targetscan.org), miR-221 and miR-222 are complementary to the ICAM-1 3’UTR and extend between 413 and 419 (Fig. 13A). Furthermore, we examined whether posttranscriptional regulation by

miR-221/-222 was critical for the ICAM-1 expression in A549 cells treated with TNF-

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and WBGE. There were no significant changes in the levels of both miR-221/-222 expression in A549 cells following TNF- stimulation for 4 h (Fig. 13B). To test the role of miR-221/-222 in TNF--mediated induction of ICAM-1, A549 cells were transfected with miR-221/-222 precursors for 44 h, followed by exposure to TNF- for 4 h with subsequent assessment of ICAM-1 protein expression via Western blot.

Transfection of cells with a precursor miR-221 or miR-222 sequence, however, did not affect TNF--induced ICAM-1 (Fig. 14A), p-p65, p-PI3K and p-IκB expression (Fig.

14B). We also found that miR-221 or miR-222 precursors had no effect on adhesion of U937 cells to TNF-treated A549 cells (Fig. 15).

Interestingly, we found that WBGE treatment significantly increased miR-221/-222 expression (Fig. 13B). In addition, WBGE treatment significantly reduced endogenous ICAM-1 expression (Fig. 2B). Additionally, the phosphorylation of PI3K, AKT, p65, and IB decreased after WBGE treatment compared to the control group (Figs. 7A, 9A, 9B). Therefore, we investigated whether miR-221/-222 was involved in the effects of WBGE on endogenous ICAM-1 expression in A549 cells. As shown in Fig. 16, the precursor of miR-222 significantly attenuated endogenous

ICAM-1 expression in A549 cells. An increase in the phosphorylation of AKT, p65, and IκB was detected in A549 cells, which was significantly ameliorated in cells transfected with a miR-222 precursor (Fig. 16). To determine the functional relevance of

miR-221/-222-regulated expression of ICAM-1 in A549 cells, monocyte adhesion assays were performed in the presence of A549 cells transfected with miR-221 or miR-222 precursors. As shown in Fig. 17, there was a significant reduction of monocyte adhesion in cells transfected with either of the precursors. We also found that ICAM-1 expression was downregulated by AKT, PI3K and p-65 inhibitors (Fig. 18). These data further corroborate the fact that WBGE-mediated upregulation of miR-222 is involved

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in endogenous expression of ICAM-1 in A549 cells and influences monocyte adhesion.

3.6 WBGE reduces ICAM-1 expression in lung tissues of TNF-α-treated WT mice

To determine the effects of WBGE on ICAM-1 expression in vivo, WT mice and

miR-221/-222 KO mice were fed WBGE for 5 days followed by an either an injection of TNF-α for 1 day or no injection; then, RT-PCR, Western blot and

immunohistochemical staining were performed to detect the expression of

miRNA-221/-222, ICAM-1 protein, and ICAM-1 in sections of lung tissues. As shown in Fig. 19, TNF-α treatment significantly reduced the levels of miRNA-222 expression.

WBGE significantly increased miRNA-221/-222 expression in lung tissues with or without TNF-α treatment. The levels of miRNA-221/-222 were very low for lung tissues of four groups in miRNA-221/-222 KO mice. As shown in Fig. 20A, TNF-α treatment significantly induced ICAM-1 expression, whereas WBGE decreased ICAM-1 expression in lung tissues of WT mice. Furthermore, TNF-α induced higher ICAM-1 expression in miRNA-221/-222 KO mice compared to wild-type mice. WBGE had little effect on ICAM-1 expression. As shown in Fig. 20B, in the control and

WBGE-treated groups, weak ICAM-1 staining was seen in the lung tissues, whereas in the TNF-α-treated group, strong ICAM-1 staining was seen in the lung tissues after immunohistochemical staining. In contrast, preadministration of WBGE resulted in weak ICAM-1 staining in the TNF-α-treated animals (WBGE /T). ICAM-1 expression was stronger in TNF-α-treated miR-221/-222 KO mice compared with the

TNF-α-treated WT animals. Additionally, WBGE mildly decrease ICAM-1 expression in miR-221/-222 KO mice. These beneficial effects of WBGE were lost in

miR-221/-222 KO mice.

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第四章討論

In this study, we demonstrated that WBGE treatment significantly decreased ICAM-1 expression and adhesion of monocyte U937 to TNF-α-stimulated A549 cells.

The influence was partially mediated through inhibition of the phosphorylation of PI3K/AKT/NF-κB/IB. Moreover, WBGE treatment significantly reduced endogenous ICAM-1 expression in A549 cells without TNF-α treatment. The effect was mediated through upregulation of miR-222 expression as well as decreased phosphorylation of PI3K/AKT/p65/IB. Furthermore, WBGE attenuated ICAM-1 expression and

upregulated miR-221/-222 expression in the lung tissues of mice with or without TNF-α treatment but had little effect in miR-221/-222 KO mice. The protection of lung cells by WBGE is due to the inhibition of PI3K/AKT/p65/IB and ICAM-1 by miR-221/-222.

Inflammation is a central feature of respiratory disorders. Substantial evidence has demonstrated that WBG is not only a functional food but also has anti-diabetic,

anti-inflammatory, and anti-tumor activities(Chao et al., 2014; Bai et al., 2016; Wu et al., 2009). The ethanol extract of WBG reduced iNOS and IL-1β expression in

LPS-treated RAW264.7 macrophages (Lii et al., 2009). Addition of WBG lyophilized powder reduced the inflammation biochemical markers IL-1, IL-6 and TNF-α in LPS-treated mice (Chao et al., 2014). A triterpenoid isolated from WBG exhibited an antiproliferative effect on breast cancer cells (Bai et al., 2016). The triglycerides and dicarboxylic acids in bitter gourd inhibited PGE2 production, which is a

proinflammatory mediator, in LPS-treated RAW264.7 macrophages (Wu et al., 2009).

This study is the first to report that WBGE reduced ICAM-1 expression in

TNF-α-stimulated A549 cells and lung tissues in mice. In addition, WBGE inhibited leukocyte adhesion to TNF-α-stimulated A549 cells. We also showed that the

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phytoconstituent from WBGE contained charantin, which is one of the major active compounds in WBGE. Charantin reduced ICAM-1 expression in TNF-α-stimulated A549 cells. These data indicated that the downregulation of ICAM-1 expression by WBGE may be partially attributed to the rich fraction of charantin, which is a natural steroidal glycoside. Because ICAM-1 has been reported as an important modulator of inflammation in epithelial cells of the respiratory airway and leukocyte adhesion (Lee et al., 2013), our experiments provide evidence that WBGE has the capability to reduce ICAM-1 expression and monocyte adhesion and may serve as a therapeutic reagent in lung inflammation.

MAPK and PI3K/AKT pathways played an important role in the expression of proinflammatory mediators and inflammatory cell recruitment, which led to the initiation and progression of lung inflammation (Lee et al., 2013). The present study demonstrated that TNF-α caused strong phosphorylation of three MAPK subtypes and PI3K/AKT in A549 cells, as reported in previous studies (Lee et al., 2013; Oh and Kwon, 2009). However, the involvement of their activation in the protective mechanism of WBGE has yet to be determined. Our results showed that WBGE significantly

decreased TNF-α-induced PI3K and AKT phosphorylation, whereas WBGE had no effects on TNF-α-induced MAPK phosphorylation. The increase in ICAM-1 expression induced by TNF-α was markedly suppressed in the presence of PI3K inhibitor or AKT inhibitor. Thus, one of the mechanisms through which WBGE reduces TNF-α-induced ICAM-1 expression involves a reduction in PI3K/AKT activation. Consistent with our results, bitter melon juice was effective at decreasing AKT/PI3K phosphorylation and viability of pancreatic cancer cells (Somasagara et al., 2015). A triterpenoid isolated from WBG induced breast cancer cell apoptosis through the downregulation of AKT signaling (Bai et al., 2016). However, our findings do not agree with several reports.

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The butanol fraction of bitter gourd suppressed phosphorylation of p38, JNK, and ERK in LPS-treated RAW 264.7 cells (Kobori et al., 2008). The preventive effects of bitter melon against insulin resistance and diabetes are associated with the inhibition of JNK pathway in the livers of rats fed a high-fat diet (Yang et al., 2015 ). The differences between these results in terms of the pathways involved may be related to differences in the cell type, inducers, and cytokines.

PI3K/AKT has been shown to phosphorylate NF-κB transcription activity, which is the major activator in the regulation of inflammatory responses (Lee and Yang, 2013). Our results demonstrated that activation of NF-κB is necessary for TNF-α-induced ICAM-1 expression in A549 cells and that this activation is similar to results from previous reports (Oh and Kwon, 2009). Our study further demonstrated that the

WBGE-decreased ICAM-1 expression in TNF--treated A549 cells was mediated through inactivation of NF-κB binding activity. The result was similar to the previous reports. Bitter melon preventive effects against insulin resistance and diabetes are associated with the modulation of NF-B pathway in high-fat-fed rats (Yang et al., 2015). Momordica charantia fruit extract protects cardiomyocytes from TNF--induced apoptosis partially through inhibition of the NF-B pathway (Hu et al., 2016). Wild bitter gourd lyophilized powder significantly inhibited the expression levels of NF-B, iNOS, and COX-2 in LPS-treated mice (Chao et al., 2014). Furthermore, inducible NF-κB activation is controlled by signals involved in IκB phosphorylation and its dissociation from the inactive cytoplasmic complex, followed by translocation of the active p50/p65 dimer to the nucleus and induced gene expression (Choi et al., 2012).

We demonstrated that the WBGE-induced decrease in ICAM-1 expression was mediated through inhibition of IκB phosphorylation and p65 translocation. Together these results suggest that WBGE treatment inactivates TNF-α-induced PI3K/AKT

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phosphorylation and NF-B /IB pathways as well as subsequently suppresses ICAM-1 expression, which results in decreased binding of leukocytes. Because the inflammation is involved in many kinds of chronic and acute lung tissues and it is characterized by the production of proinflammatory cytokines, the enhanced monocyte adhesion as well as the accompanying inflammatory signal (Lee and Yang, 2013), WBGE may provide a new natural therapeutic approach for the prevention of inflammation and lung diseases.

MicroRNAs have been reported to play a key role in mediating cellular responses under various stress conditions in lung diseases (Alipoor et al., 2016). COPD patients have lower miRNA-222 expression (Ikari et al., 2015). Recent reports indicated that ICAM-1 expression was controlled by a number of miRNA species, such as miR-221 and miR-222(Hu et al., 2010; Duan et al., 2013). These miRNAs are complementary to the ICAM-1 3’UTR region and modulate ICAM-1 expression at the posttranscriptional level by binding to the untranslated region(Marques-Rocha, 2015). The increased ICAM-1 expression in human immunodeficiency virus-1 (HIV-1) Tat-treated

endothelial cells was concomitant with a reduction in miR-221/-222 expression(Duan et al., 2013). IFN-γ suppressed miR-221 and resulted in increased ICAM-1 expression in cholangiocytes (Hu et al, 2010). Another study demonstrated that the upregulation of ICAM-1 protein in human intrahepatic biliary epithelial cells following the parasite infection involves downregulation of miRNA-221 (Gong et al., 2011). Endothelial microparticles promote an anti-inflammatory effect by reducing endothelial ICAM-1 expression through higher amounts of miR-222 (Jansen et al., 2015). In the present study, we showed that A549 cells treated with TNF- or with the TNF-+WBGE treatment did not alter the miRNA-221/-222 levels. Interestingly, only WBGE treatment can increase miRNA-221/-222 levels and decrease endogenous ICAM-1 expression in A549 cells. Furthermore, the overexpressed miRNA-222 levels significantly reduced

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the phosphorylation of PI3K/AKT and IB /NFB-p65 as well as the monocyte

adhesion. Furthermore, the in vivo study demonstrated that TNF- treatment decreased the miRNA-221/-222 levels and increased the ICAM-1 expression, whereas WBGE pretreatment increased the miRNA-221/-222 levels and attenuated the ICAM-1 expression in lung tissues. In contrast, WBGE treatment showed little effect on ICAM expression in lung tissues of miR-221/-222 KO mice with or without TNF- treatment.

These different results between in vivo and in vitro studies suggest that the effects of WBGE and miR-221/-222 expression probably did not depend on only one cell type.

The result in the in vivo study may be due to the component cells of lung tissues, including type I cells, endothelial cell, smooth muscle cells, and macrophages. Further investigations are needed to better understand the role of WBGE plays in the prevention and treatment of inflammatory lung diseases.

In summary, this study provides the first evidence that WBGE reduces ICAM-1 expression under inflammatory conditions both in vitro and in vivo and decreases leukocyte adhesion to alveolar epithelial cells. We also showed that WBGE inhibited endogenous ICAM-1 expression in A549 cells through blockade of

PI3K/AKT/NF-κB/IκB phosphorylation and regulation of miRNA-222 expression.

Based on these findings, WBGE should be considered a novel therapeutic agent for targeting epithelial activation in pulmonary inflammation. Taken together, the

pleiotropic mechanism against inflammation highlights the usefulness of WBGE as a healthy food.

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