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Effects of calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin on cell viability in LPS- and SNP-induced
macrophages

The cell viability of calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’,4’-dihydroseselin was evaluated in LPS- and SNP-stimulated
RAW264.7 cells (Fig. 2). No cytotoxic effect was observed in LPS-stimulated macrophages until treatment
with a 30-?M concentration of the derivatives. Consequently, non-toxic concentrations
were used for the following experiments.

Fig. 2. Effects of calipteryxin and (3’S,4’S)-3’,4’-disenecioyloxy-3’,4’-dihydroseselin on cell viability and NO production in
LPS- (a) and SNP- (b) stimulated-RAW 264.7 cells as described in the “Methods”. The data were derived
from three independent experiments and are expressed as the means?±?S.D. (***) p 0.001 indicates a significant difference from the LPS-challenged group. (
^###
) p 0.001 indicates a significant difference from the unstimulated control group. Control (vehicle),
LPS; (LPS?+?vehicle)-treated cells alone

Inhibitory effects of calipteryxin and (3’S,4’S)-3’,4’-disenecioyloxy-3’,4’-dihydroseselin on NO production in LPS- and SNP-induced
RAW 264.7 macrophages

To determine NO production, we measured the amount of nitrite released into the culture
medium. RAW 264.7 cells were treated with various concentrations of calipteryxin and
(3’S,4’S)-3’,4’-disenecioyloxy-3’,4’-dihydroseselin (2.5?~?30 ?M). Incubation with LPS alone
markedly increased NO production from these cells, compared with the NO production
generated under the control conditions (Fig. 2). However, pre-treatment with calipteryxin and (3’S,4’S)-3’,4’-disenecioyloxy-3’,4’-dihydroseselin prevented this increased level of NO production
in LPS-stimulated RAW 264.7 cells in a concentration-dependent manner (Fig. 2a)., However, SNP-induced NO production was slightly reduced after treatment with calipteryxin
and (3’S,4’S)-3’,4’-disenecioyloxy-3’,4’-dihydroseselin (Fig. 2b). By comparison, calipteryxin and (3’S,4’S)-3’,4’-disenecioyloxy-3’ ,4’-dihydroseselin
inhibition was more remarkable in LPS-stimulated macrophages than in SNP-induced macrophages.
Therefore, the LPS-stimulated RAW264.7 cell model was used for subsequent in vitro
experiments.

Effects of calipteryxin and (3’S,4’S)-3’,4’-disenecioyloxy-3’,4’-dihydroseselin on LPS-induced iNOS and COX-2 protein
and mRNA expression levels

As calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin inhibit NO production, we examined the
relationship between the protein and mRNA expression levels of iNOS and COX-2 (Fig. 3). The inhibitory effects of calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin on the protein and mRNA expression levels
of iNOS and COX-2 were determined through Western blotting and qRT-PCR analyses, respectively.
The iNOS and COX-2 protein and mRNA expression levels were markedly up-regulated after
LPS treatment, and calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin significantly attenuated iNOS and COX-2
mRNA expression in LPS-stimulated macrophages in a concentration-dependent manner
(Fig. 3).

Fig. 3. Effects of calipteryxin and (3’S,4’S)-3’,4’-disenecioyloxy-3’,4’-dihydroseselin on the LPS-induced iNOS protein (a), and COX-2 protein (b), iNOS mRNA (c), COX-2 mRNA (d) expression levels in RAW 264.7 macrophages using Western blotting as described in
“Methods”. The data were derived from three independent experiments and are expressed
as the means?±?S.D. (*) p 0.05, (**) p 0.01 and (***) p 0.001 indicate significant differences from the LPS-challenged group. (
^###
) p 0.001 indicates a significant difference from the unstimulated control group. Control (vehicle),
LPS; (LPS?+?vehicle)-treated cells alone; TPCK 30 ?M, N–p-tosyl-L-phenylalanyl chloromethyl ketone was used as a positive control

Fig. 4. Effects of calipteryxin and (3’S,4’S)-3’,4’-disenecioyloxy-3’,4’-dihydroseselin on various inhibitors induced by LPS in
RAW 264.7 macrophages as described in “Methods.” The data were derived from three
independent experiments and are expressed as the means?±?S.D. (*) p 0.05, (**) p 0.01 and (***) p 0.001 indicate significant differences from the LPS-challenged group. (
^###
) p 0.001 indicates a significant difference from the unstimulated control group. Control (vehicle),
LPS; (LPS?+?vehicle)-treated cells alone, M; only cells in the media

Effects of calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin on LPS-induced NO production using various
inhibitors in macrophages

To investigate the inflammatory signaling pathway involved in the inhibitory effects
of calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin on LPS-induced inflammatory mediators,
specific inhibitors of the NF-?B (TPCK, 20 ?M), MAPKs (SB202190, p38 MAPK inhibitor;
SP600125, JNK inhibitor; U0126, ERK inhibitor) and Akt (LY294002) were used (Fig. 4). The pretreatment of RAW 264.7 cells with TPCK, SB202190, SP600125, and LY294002
significantly inhibited LPS-induced nitrite production in the media, while U0126 showed
no effects at 20 ?M (Fig. 4). The combination of calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin with TPCK and specific inhibitors against
p38, JNK, ERK and Akt significantly inhibited LPS-induced NO production (Fig. 4). Overall, these results suggest that p38, JNK, ERK and Akt, in conjunction with
NF-?B inflammatory signaling, might contribute to the inhibitory effects of calipteryxin
and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin on inflammatory mediators.

Inhibitory effects of calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin on NF-?B signaling

Because the results indicated that calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin affect iNOS and COX-2 induction, we
focused on two transcription factors critical in iNOS and COX-2 induction, i.e., NF-?B
and AP-1 5], 12]. Initially, the inhibitory effects of calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin on NF-?B in the medium were evaluated.
As shown in Fig. 5, both compounds exhibited remarkable inhibitory effects on NF-?B in the culture media.

Fig. 5. Effects of calipteryxin and (3’S,4’S)-3’,4’-disenecioyloxy-3’,4’-dihydroseselin on LPS-induced and NF-?B-dependent alkaline
phosphatase (SEAP) expression in transfected-RAW 264.7 macrophages as described in
“Methods.” The data were derived from three independent experiments and are expressed
as the means?±?S.D. (***) p 0.001 indicates a significant difference from the LPS-challenged group. (
^###
) p 0.001 indicates a significant difference from the unstimulated control group. Control (vehicle),
LPS; (LPS?+?vehicle)-treated cells alone; TPCK 30 ?M, N–p-tosyl-L-phenylalanyl chloromethyl ketone was used as a positive control

To analyze whether calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin suppressed the phosphorylation and degradation
of I?B? and NF-?B nuclear translocation, time course experiments were performed in
LPS-stimulated macrophages using Western blot analysis (Fig. 6). After treatment with LPS alone for 1 h, I?B? activation levels were markedly increased,
and calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin) significantly blocked LPS-induced I?B?
phosphorylation after 60 min of LPS stimulation (Fig. 6). However, I?B? degradation through calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin was inhibited after 15 min of LPS (Fig. 6). Complete inhibition was observed after 30 min of LPS stimulation (Fig. 6).

Fig. 6. Effects of calipteryxin and (3’S,4’S)-3’,4’-disenecioyloxy-3’,4’-dihydroseselin on
p-IKK?/? (a), p-I?B? (b), and I?B? (c) protein expression levels in time course experiments

In an attempt to explore the effects of calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin on the inhibition of I?B kinase (IKK)
activity in RAW264.7 cells, Western blot analysis was performed to measure the phosphorylation
of IKK?/? after treatment with calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin (Fig. 6). Calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin significantly inhibited LPS-induced
activation of IKK?/? (Fig. 6).

Additionally, we examined the DNA-binding affinity of these transcription factors
using EMSA (Fig. 7a). Both Calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin attenuated the LPS-induced DNA binding
activity of both NF-?B (Fig. 7a). The specificity of the bands was confirmed after adding a 50-fold excess of unlabeled
NF-?B oligonucleotide to the binding reaction (Fig. 7a). Additionally, the nuclear translocation of p65, a component of the NF-?B heterodimer,
was further evaluated (Fig. 8b). LPS induced the translocation of p65 from the cytoplasm to the nucleus after treatment
for 1 h, and calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin markedly prevented the nuclear translocation
of p65 (Fig. 7).

Fig. 7. Effects of calipteryxin and (3’S,4’S)-3’,4’-disenecioyloxy-3’,4’-dihydroseselin on NF-?B-DNA binding activity (a) and p65 (b) protein expression

Fig. 8. Effects of calipteryxin and (3’S,4’S)-3’,4’-disenecioyloxy-3’,4’-dihydroseselin on the protein expression levels of the
MAPKs p-JNK1, p-p38, p-ERK and p-Akt in time course experiments

Effects of calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin on LPS-induced MAPKs and Akt activations
in macrophages

MAP kinases are important for the expression of both iNOS and COX-2 13], 14]. Thus, MAPKs act as specific targets in inflammatory responses. Because MAPK activation
plays an important role in NF-?B stimulation, we examined whether MAPK’s activity
is inhibited through treatment with calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin. Herein, we assessed the phosphorylation
levels of MAP kinases, including JNK1, p38 and ERK 1/2. When RAW 264.7 cells were
stimulated with LPS, in the presence of calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin, the levels of phosphorylated JNK1,
p38 and ERK 1/2 MAPK were observed to significantly start decreasing after 15 min
of LPS stimulation (Fig. 8). Additionally, Akt activation was significantly inhibited after treatment with calipteryxin
and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin after 60 min of LPS stimulation (Fig. 8). These results suggest that the MAPK and Akt pathways are relevant during the LPS-mediated
expression of iNOS and COX-2.

Effects of calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin on LPS-induced AP-1-DNA binding and
c-Jun expression in macrophages

AP-1 is another transcription factor involved in the regulation of inflammatory processes
13]. To evaluate the effects of calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin, we performed EMSA. The results clearly
demonstrated that calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin remarkably inhibited AP-1-DNA binding
activity (Fig. 9), while LPS-stimulated cells showed significantly high DNA-binding affinity (Fig. 9). In addition, the effects of calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin on c-jun were considerably promising
(Fig. 9).

Fig. 9. Effects of calipteryxin and (3’S,4’S)-3’,4’-disenecioyloxy-3’,4’-dihydroseselin on Ap-1-DNA binding activity (a) and c-Jun (b) protein expression

Effects of calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin on pro-inflammatory cytokines in LPS-stimulated
macrophages

As calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin inhibit the activation of the two pro-inflammatory
transcription factors NF-?B and AP-1, we examined the effects of calipteryxin and
(3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin on the expression of pro-inflammatory
cytokines using qRT-PCR analysis. TNF-? and IL-1? are predominantly regulated at the
transcriptional level, whereby the transcription factors NF-?B and AP-1 play crucial
roles 15]. Indeed, the treatment of LPS-activated cells with calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin significantly reduced the secretion
of TNF-? and IL-1? in RAW 264.7 cells (Fig. 10).

Fig. 10. Suppression effects of calipteryxin and (3’S,4’S)-3’,4’-disenecioyloxy-3’,4’-dihydroseselin on the mRNA expression of the pro-inflammatory
cytokines TNF-? (a) and IL-1? (b). Total RNA was isolated, and the expression of TNF-? and IL-1? was determined through
qRT-PCR, as described in the “Methods.” Con (vehicle), LPS; (LPS?+?vehicle)-treated
cells alone and TPCK (30 ?M) served as a positive control. (***) p 0.001 indicates significant differences from the LPS-treated group. (
^###
) p 0.001 indicates a significant difference from the unstimulated control group

Binding model analysis

To further elucidate the binding of compounds, we performed a docking analysis. Docking
was simulated using Glide XP (Schrödinger 2013) to examine the interactions between
calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin in the NIK
active site (Fig. 11). Calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin form two
hydrogen bonds with the LYS517 and SER476 residues. The secondary structure of this
protein is shown as a solid ribbon (gray). Key residues are displayed in line style
(blue), calipteryxin and (3’S,4’S)-3’ ,4’-disenecioyloxy-3’ ,4’-dihydroseselin are
displayed in stick style (carbon atoms in cyan), and hydrogen bonds are represented
as green dotted lines.

Fig. 11. Molecular docking study of calipteryxin (a) and (3’S,4’S)-3’,4’-disenecioyloxy-3’,4’-dihydroseselin (b)