Eupafolin reduces TNF-α-induced upregulation of ICAM-1 in A549 cells

A549 cells were incubated with various concentrations of TNF-α at different time intervals. ICAM-1 expression was significantly upregulated with 1 ng/mL, 3 ng/mL or 10 ng/mL of TNF-α treatment for 4 h, 8 h, 16 h, and 24 h or with 0.3 ng/mL of TNF-α for 24 h (Fig. 2). We next analyzed the effect of eupafolin on ICAM-1 expression under inflammation. A549 cells were pretreated for 24 h with 1, 3, 10, 30, or 50 μM eupafolin before incubation with 3 ng/mL TNF-α for 4 h, TNF-α-induced ICAM-1 expression was reduced (2.3±0.3, 2.1±0.2, 1.6±0.2, 0.9±0.2, 0.6±0.1 fold of control levels, respectively).

The reductions caused by the three highest concentrations were significant (P<0.05, Fig.

3A). The effect of eupafolin on ICAM-1 expression was also confirmed by immunofluorescent staining (Fig. 3B). Cells treated for 4 h with 3 ng/mL TNF-α showed strong ICAM-1 expression (T) and this effect was inhibited by pretreatment with eupafolin (50 μM, ET). According to these results, 3 ng/mL TNF-α and 50 μM eupafolin were used in all subsequent experiments to evaluate the anti-inflammatory effects and molecular mechanisms of eupafolin treatment.

3.2. The inhibition of ERK1/2 and JNK phosphorylation mediates eupafolin-increased reduction in TNF-α-induced ICAM-1 expression

Previous studies have reported that TNF-α-induced inflammation includes the production of inflammatory cytokines via the MAPK pathways (Lee et al., 2011; Lee et al., 2013). We next investigated whether TNF-α-induced ICAM-1 expression was mediated through MAPKs 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

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followed by a decline within 60 min (P<0.05, Fig. 4A). To determine the targets that were affected by eupafolin, the cells were preincubated with eupafolin for 24 h and then incubated with TNF-α for 5, 15, 30, 45, or 60 min. Eupafolin significantly inhibited TNF-α-induced ERK1/2, p38, and JNK phosphorylation (P<0.05). In addition, the PD98059 (50M, ERK1/2 inhibitor) or SP600125 (10M, 30M, 50M, JNK inhibitor) inhibited the TNF-α-induced ICAM-1 expression (PD: 40±13% of inhibition; SP:

26±10 % for 10M, 39±13% for 30M, and 43±15% for 50M, respectively, P<0.05).

In contrast, SB203580 (p38 inhibitor) had no effects (Fig. 4B). To further confirm the involvement of ERK1/2 and JNK in the TNF-α-induced ICAM-1 expression, we used siRNA transfection to knockdown the ERK1/2 or JNK expression in A549 cells. As shown in Fig. 5A, the expression levels of ERK1/2 or JNK were significantly

downregulated by siRNA transfection (P<0.05). Moreover, cells transfected with 1 nM ERK1/2- or with JNK-specific siRNA inhibited TNF-α-induced ICAM-1 expression (1.6±0.1 and 1.7±0.1 fold of control levels, respectively) (P<0.05, Fig. 5B). These results suggest that eupafolin inhibits TNF-α-induced ICAM-1 expression partly by inhibiting TNF-α-induced ERK1/2 and JNK phosphorylation.

3.3. The inhibition of AKT phosphorylation mediates eupafolin-increased reduction in TNF-α-induced ICAM-1 expression

The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is reported to be involved in adhesion molecule expression in TNF--treated various cells (Choi et al., 2012; Jang et al., 2012; Oh and Kwon, 2009). To investigate whether eupafolin affects TNF--induced PI3K/AKT activation, we examined the effect of eupafolin on the TNF-α-induced PI3K/AKT in A549 cells using Western blot analysis. The expression levels of phosphorylated PI3K and AKT were gradually increased after TNF-α

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stimulation, and pretreatment with eupafolin significantly attenuated the

phosphorylation of PI3K and AKT (P<0.05, Figs. 6A and 6B). Interestingly, eupafolin pretreatment completely blocked the increase of TNF-α-induced AKT phosphorylation.

Moreover, we determined whether the activation of PI3K/AKT was involved in TNF-α induced ICAM-1 expression. As shown in Fig. 6D, pretreatment with MK2206 (AKT inhibitor) caused a significant attenuation of ICAM-1 expression in TNF-α stimulated A549 cells (34±13% of inhibition, P<0.05). In contrast, pretreatment with LY294002 (PI3K inhibitor) did not reduce the ICAM-1 expression (P<0.05, Fig. 6C). These results suggest that eupafolin inhibits TNF-α-induced ICAM-1 expression partly by inhibiting TNF-α-induced AKT phosphorylation.

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

We investigated whether eupafolin reduced TNF-α-induced ICAM-1 expression via NF-κB signaling because the promoter of ICAM-1 gene contains consensus binding sites for the transcription factor (Rahman et al., 1999). At first, we examined the levels of phosphorylated NF-κB p65 in TNF--treated A549 cells by Western blotting and immunofluorescence staining. The phospho-p65 level was higher in TNF--treated A549 cells than in control cells and that eupafolin pretreatment significantly reduced the effect (P<0.05, Fig. 7A). The similar result was obtained for IκB phosphorylation (P<0.05, Fig. 7B), which is responsible for NF-κB activation (Choi et al., 2012). The results of immunofluorescent staining were consistent with the Western blot finding of NF-κB p65. Control A549 cells (C) or cells incubated only with eupafolin (E) showed no nuclear NF-κB p65 staining, but strong staining in the cytoplasm. In contrast, A549 cells stimulated with TNF- for 1 h showed strong NF-κB p65 staining in the nucleus

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(T), and this effect was significantly decreased by pretreatment for 24 h with eupafolin (Fig. 8). Furthermore, TNF--induced ICAM-1 expression by A549 cells was blocked by preincubation of the cells for 1 h with 5-50 M Bay11-7082, a NF-κB inhibitor (Fig.

9A). To further confirm the involvement of NF-B in the TNF-α-induced ICAM-1 expression, we used p65-specific siRNA transfection to knockdown the p65 expression in A549 cells. The expression level of NF-B was markedly downregulated by siRNA transfection (P<0.05, Fig. 9B). Moreover, the level of TNF-α-induced ICAM-1

expression was also attenuated in the NF-B p65-depleted A549 cells (76±3% of

inhibition, P<0.05, Fig. 9C). Furthermore, we use Western blot analysis to determine the expression levels of NF-B p65 in the nuclear portion of A549 cells. The expression level of p65 in the nuclear portion of TNF-α-treated A549 cells was reduced by eupafolin (P<0.05, Fig. 10A). Gel-shift assays were performed to determine the effect of eupafolin on NF-κB activation in TNF--treated A549 cells. As shown in Fig. 10B, low basal levels of NF-κB binding activity were detected in both untreated cells and cells treated only with eupafolin, but binding was increased by treatment with TNF-

for 1 h and further decreased by pretreatment with eupafolin. These results suggest that eupafolin-reduced ICAM-1 expression in TNF--treated A549 cells was mediated by the inhibition of NF-κB activation.

3.5. TNF-α-induced ICAM-1 expression was mediated by AKT/ERK1/2/JNK/NF-B signaling pathway

There are multiple cross-talk points between PI3K and MAPKs pathways, whose co-ordinated action determines the cell fate (Aksamitiene et al., 2012; Bölck et al., 2014). In addition, AKT activation has been shown to activate MAPK pathway (Binion et al., 2009). To further elucidate the detailed pathway, we examine the crosstalk among

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AKT, MAPK, IB, NF-B p65 in TNF--treated A549 cells. A549 cells pretreated with PD98059, SP600125 or MK2206 for 24h, and then stimulated with TNF- for 15min.

As shown in Figs. 11A-11D, PD98059 had no effect on the phosphorylation of p65 and AKT. In contrast, SP600125 reduced the phosphorylation of p65 (21±8% and 29±10%

of inhibition for 10 M and 50 M, respectively), but not AKT. In addition,

pretreatment with MK2206 significantly attenuated the phosphorylation of ERK1/2, JNK and p65 (70±3%, 48±1%, and 60±6% of inhibition, respectively, P<0.05, Figs.

11E-11G). Moreover, to confirm whether MAPK/AKT was linked to IB

phosphorylation, we performed that A549 cells pretreated with PD98059, SP600125 or MK2206 for 24h, and then stimulated with TNF- for 5min. PD98059 had no effect on the expression of IB phosphorylation. But both SP600125 and MK2206 reduced the IB phosphorylation (59±17% and 69±13% of inhibition, respectively, P<0.05, Figs.

12A-12C). These data suggest that TNF-α-induced ICAM-1 expression in A549 cells was mediated by AKT/IB/ERK1/2, JNK/NF-B signaling pathway.

3.6. Eupafolin suppressed the adhesion of monocytes to TNF-α-stimulated A549 cells

To evaluate the influence of eupafolin on the epithelial cell-leukocyte interaction, we investigated the adhesion of U937 cells to TNF-α-stimulated A549 cells (Fig. 13A).

A549 cells pretreated with 3ng/mL TNF-α for 4 h (T) for 4 h substantially increased monocyte adhesion than control cells (C). Pretreatment of A549 cells with eupafolin for 24 h (E/T) reduced the number of U937 cells adherent to TNF-α-treated A549 cells by 52±6% compared to TNF-α alone (P<0.05, Fig. 13B). As expected, A 549 cells pretreated with 1 or 2 μg/mL anti-ICAM-1 antibody decreased the adhesion of U937 cells to TNF-α-treated A549 cells. This result showed that ICAM-1 plays the important

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role in the adhesion of U937 cells to TNF-α-treated A549 cells. The adherence of U937 cells to TNF-α-treated A549 cells was also markedly inhibited by the introduction of PD98059, SB203580, or SP600125 (73±5%, 57±8%, and 71±4% of inhibition, respectively, P<0.05). Similarly, the adherence of U937 cells to TNF-α-treated A549 cells was also inhibited by Bay11-7082, MK2206, or LY294002 (69±7%, 67±6 %, and 45±12% of inhibition, respectively, P<0.05).

3.7. Eupafolin reduces ICAM-1 expression in lung tissues in TNF-α-treated mice

To detect the effect of eupafolin on ICAM-1 expression under inflammation in vivo, lung tissues of TNF--treated mice were examined by Western blotting and immunohistochemical staining. TNF- significantly induced the ICAM-1 expression in lung tissues and pretreatment with eupafolin could downregulate the ICAM-1 level by Western blotting (53±15%, P<0.05, Fig. 14A). Fig. 14B shows that no ICAM-1 staining was seen on the lung tissues in the control (C) and eupafolin-treated (E) groups,

whereas strong ICAM-1 staining was seen on the epithelial cells in the TNF-α-treated group (T) by the immunohistochemical staining. The stronger ICAM-1 expression was closely associated with type-II alveolar cells, which were identified by SP-D (Fig. 14C).

In contrast, preadministration of eupafolin showed weak ICAM-1 staining in TNF-α-treated mice (ET).

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第四章 討論

In the present study, our results showed that eupafolin significantly reduced ICAM-1 expression and monocytic cell line U937 adhesion in TNF-α-stimulated A549 cells in vitro. The influence was partly mediated through inhibition of

AKT/ERK1/2/JNK phosphorylation and NF-κB activation. Furthermore, eupafolin attenuates ICAM-1 expression in lung tissues of TNF-α-treated mice in vivo.

P. nodiflora, an important ingredient of herbal tea, has long been used in traditional medicine to treat inflammatory diseases (Lai et al., 2011). The bioactive compounds isolated from P. nodiflora include flavonoids (Tom´as-Barber´an et al., 1987), essential oils, resin (Elakovich and Stevens., 1985), quinol (Siddiqui et al., 2009), cyclohexenone (Ravikanth et al., 2000), and steroids (Wang and Huang., 2005), which are responsible for its antiseptic, antitussive, antipyretic, antiurolithiatic, antidiabetic, antinociceptive, and anti-inflammatory effects (Balakrishnan et al., 2010; Forestieri et al., 1996). Eupafolin, a flavonoid isolated from P. nodiflora, was chosen for using in the present research, which possesses the anti-inflammatory action (Lai et al., 2011; Maas et al., 2011). Eupafolin promoted iron release from ferritin and donated electrons to the stable free radical DPPH (Dabaghi-Barbosa et al., 2005). Eupafolin protected cultured neurons against glutamate-induced oxidative stress (Kim et al., 2002) and inhibited xanthine oxidase activity (Sanz et al., 1994). The recent study has showed that eupafolin inhibited pro-inflammatory iNOS and COX-2 protein expressions in LPS-stimulated RAW264.7 macrophages (Lai et al., 2011). In addition, eupafolin exhibited anti-tumor effects on MK-1 (human gastric adenocarcinoma), B16-F10 (murine melanoma), HeLa (human cervical adenocarcinoma) cells, and prostate cancer cells (Abe et al., 2002;

Chung et al., 2010; Ko et al., 2014; Liu et al., 2014). Eupafolin lessened virus-induced

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upregulation of IL-6 and RANTES in RD cells, derived from a human

rhabdomyosarcoma (Wang et al., 2013). Our previous study demonstrated that

eupafolin downregulated melanogenesis (Ko et al., 2014). The present study is the first to report that eupafolin strongly reduces the ICAM-1 expression in TNF-α-treated A549 cells and pulmonary tissues in TNF--treated mice. In addition, eupafolin treatment markedly inhibited leukocyte adhesion to these cells by the inhibiting ICAM-1 expression. The induced expression of adhesion molecules, especially ICAM-1, has been reported to be associated with airway inflammation and the migration and

recruitment of lymphocytes (Lee and Yang, 2013; Qureshi et al., 2003), indicating that an additional mechanism by which eupafolin treatment may be important in preventing the progression of airway inflammation.

MAPK pathways, such as phosphorylation of ERK, JNK, and p38 play the important role in the expressions of proinflammatory mediators, which lead to the initiation and progression of lung inflammation (Lee et al., 2013). The present study demonstrated that TNF-α caused strong activation of three MAPK subtypes in human alveolar epithelial A549 cells, as reported in previous studies (Lee et al., 2013; Jang et al., 2012; Oh and Kwon, 2009). However, the involvement of their activation in the protective mechanism of eupafolin has not been detected. Our results showed that eupafolin decreased TNF-α-induced ERK1/2, JNK and p38 phosphorylation. The increase in ICAM-1 expression induced by TNF-α was markedly suppressed in the presence of an ERK1/2 inhibitor or a JNK inhibitor, but not a p38 inhibitor. ICAM-1 expression was also inhibited by ERK1/2 or JNK-specific siRNA. Based on the results, we suggest one of the signals by which eupafolin attenuates TNF-α-induced ICAM-1 expression involves a reduction in ERK1/2 and JNK activation. Consistent with our results, eupafolin specifically reduced virus-induced upregulation of IL-6 and RANTES

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by inhibiting the ERK1/2 signaling pathway (Wang et al., 2013). Another study showed that nodiflora extract significantly inhibited the phosphorylation of ERK1/2 and JNK in LPS-treated RAW 264.7 macrophages (Balakrishnan et al., 2010). In contrast, eupafolin significantly induced the phosphorylation of ERK1/2 and p38 MAPK correlate well with the suppression of melanogenesis in B16F10 mouse melanoma cells (Ko et al., 2014). The differences between the above results in terms of the pathways involved may be related to differences in cell type, inducers, and cytokines.

The transcription factor NF-κB was served as the major activator in the regulation of inflammatory responses (Karin et al., 2002). MAPKs have been shown to

phosphorylate NF-κB transcriptional activity. Our results demonstrated that the

activation of NF-κB is necessary for TNF-α-induced ICAM-1 expression in A549 cells and that is in accordance with previous reports (Banerjee et al., 2002; Oh and Kwon, 2009; Wu et al., 2014). Our study further demonstrated that the eupafolin-decreased ICAM-1 expression in TNF--treated A549 cells was mediated through inactivation of NF-κB binding activity. The result is similar with a previous report that nodiflora extract inhibited LPS-induced TNF-, IL-1, and IL-6 production might be related the

reduction of NF-κB activation in RAW 264.7 macrophages (Balakrishnan et al., 2010).

NF-κB is activated by signals possibly involving of the 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 eupafolin-induced decrease in ICAM-1 expression was mediated through inhibition of IκB phosphorylation and p65 translocation.

PI3K and AKT pathway have been implicated that they played the crucial role in activation of inflammatory mediators, inflammatory cell recruitment and immune cell function (Koyasu et al., 2003). This notion is confirmed by our observation that TNF-

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activated the phosphorylation of PI3K and AKT. In the present study, the increase in ICAM-1 expression induced by TNF-α was markedly suppressed in the presence of an AKT inhibitor, but not a PI3K inhibitor. In addition, eupafolin decreased

TNF-α-induced PI3K and AKT phosphorylation. Eupafolin seems to have a more pronounced effect on AKT phosphorylation and subsequently reduced ICAM-1 expression. Thus, one of the mechanisms by which eupafolin reduces TNF-α-induced ICAM-1 expression involves a reduction in AKT activation. Moreover, it has been reported that there are multiple cross-talk points between PI3K and MAPKs pathways, whose co-ordinated action determines the cell fate (Aksamitiene et al., 2012; Bölck et al., 2014). In our study, the phosphylation of AKT was not affected by the inhibition of ERK1/2 and JNK, but the phosphylation of ERK1/2 and JNK was affected by the inhibition of AKT. These findings indicate that AKT is the upstream regulator of IB/ERK/JNK activation. Together these results suggest that eupafolin treatment inactivates TNF-α-induced AKT phosphorylation, which in turn reduces the phosphorylation of IB/ERK1/2/JNK MAPK cascades and NF-B pathways, and subsequently suppressed ICAM-1 expression, resulting in the 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 proinflammtory cytokines, the enhanced monocyte adhesion, and the accompanying inflmmatory signal (Lee and Yang, 2013), eupafolin may provide a new therapeutic approach for the prevention of

inflammation and lung diseases.

In summary, this study provides the first evidence that eupafolin reduces ICAM-1 expression under inflammatory conditions both in vitro and in vivo and also decreases leukocyte adhesion to alveolar epithelial cells. Our results show that the eupafolin inhibited ICAM-1 expression in A549 cells through blockade of AKT, ERK1/2, JNK,

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and NF-κB phosphorylation (Fig. 15). Eupafolin, an active component of P. nodiflora, exerts the anti-inflammatory effect on pulmonary epithelial cells in the present study.

Based on these findings, eupafolin should be considered as a novel therapeutic agent targeting epithelial activation in pulmonary inflammation.

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