Strigolactones: A plant phytohormone as novel anti-inflammatory agents

Article abstract of DOI:10.1039/c7md00461c

Strigolactones (SLs) are a novel class of plant hormones with enormous potential for the prevention and treatment of inflammation. To further investigate the anti-inflammatory activities of SLs, a representative SL, GR24, and the reductive products of its D-ring were synthesized and their anti-inflammatory activities were fully evaluated on both in vitro and in vivo models. Among these compounds, the two most active optical isomers (2a and 6a) demonstrated strong inhibitory activity on the release of inflammatory cytokines, including nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) by blocking the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways; they also greatly inhibited the migration of neutrophils and macrophages in fluorescent protein labeled zebrafish larvae. These results identified the promising anti-inflammatory effects of SLs, and suggested that both the absolute configuration of SL and the α,β-unsaturated D-ring structure are essential for the observed anti-inflammatory activity.

Full text of DOI:10.1039/c7md00461c

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ARTICLE
Strigolactones: A plant phytohormone as novel anti-inflammatory
agents
Jun-Xia Zhenga‡, Yu-Shui Hanc‡, Jin-Cai Wangc‡, Hui Yangc, Hao Kongc, Kang-Jia Liuc, Si-Yu
Chenc, Yi-Rui Chenb, Yi-Qun Changc, Wei-Min Chenc, Jia-Liang Guob,*, Ping-Hua Sunc,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Strigolactones (SLs) are a novel class of plant hormones with enormous potential in the prevention and treatment of
inflammation. To further investigate the anti-inflammatory activities of SLs, a representative SL, GR24 and its reductive
products of D-ring were synthesized and their anti-inflammatory activities were fully evaluated both in vitro and in vivo
models. Among these compounds, two most active optical isomers (2a and 6a) demonstrated strong inhibitory activity on
the release of inflammatory cytokines including nitric oxide (NO), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-
6) by blocking the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, as well
as greatly inhibited the migration of neutrophils and macrophages in fluorescent protein labeled zebrafish larvae model.
These results identified the promising anti-inflammatory effects of SLs, and suggested that both the absolute configuration
of SL and the α, β-unsaturated D-ring structure are essential for anti-inflammatory activity.
etc. However, no study related to SLs’ promising anti-
inflammatory effects have been reported.
Introduction
To date, more than 15 natural SLs have been isolated from
Multifaceted biological functions in mammals were recognized
in massive phytohormones, abscisic acids (ABA)1, methyl
jasmonates (MJ)2, and cytokinins (CTK)3, 4, for instance, which
indicates highly potential in the treatment of various clinical
diseases. It seems those phytohormones are likely to be an
excellent source for the discovery of potential drugs from food
and plants. The strigolactones (SLs) are a novel family of plant
hormones produced in potential host plants such as wheat,
rice and corn, which control shoot branching architecture by
inhibiting growth and self renewal of axillary meristem cells5.
As far, most initial studies were focus on the bioactivity of SLs
of their action as a germination stimulant of parasitic seed
weeds6-9. Interestingly, Yarden et al. recently reported that SL
and its analogues are potent inhibitors of mammosphere
formation and cancer stem-like cell survival, which opens up
the possibility to discover promising anticancer agents from
SLs10-12. As we known, inflammation is an immune response
initiated by pathogen invasion or tissue injury, and increasing
studies have realized that it is always accompanied by various
plant root extracts or exudates19. Based on the structure-
activity relationships, a number of synthetic SL analogues, so-
called GR compounds, were designed and synthesized. Among
them, GR24,
a synthetic analogue of SLs with simpler
structure, is the most active and widely applicable as a
reference compound for research into parasitic weeds, such as
Striga and Orobanche in bioassays20-23. Since its inhibitory
effects toward breast cancer cell lines growth and survival10,
GR24 become an interesting leading compound and research
hotspot in pharmaceutical studies.
Therefore, in the present work, the (±)-GR24 and their
reductive products of D-ring were synthesized and the anti-
inflammatory effects were evaluated on both in vitro and in
vivo models, followed by our in-depth study on their
mechanism by western-blot analysis. Toxicity on cells were
also
evaluated
using
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. The results showed
the promising anti-inflammatory activity of GR24, especially
two most active optical isomers (2a and 6a), which greatly
inhibited the release of TNF-α and NO in LPS-activated murine
macrophages by affecting MAPK and NF-κB pathways. These
findings provided supports for the potential future
pharmaceutical application of SLs as a novel anti-inflammatory
leading compound.
diseases, such as cancer13, 14, atherosclerosis15, 16, diabetes17, 18
,
a. School of Chemical Engineering and Light Industry, Guangdong University of
Technology, Guangzhou, PR China.
b. School of Stomatology and Medicine, Foshan University, Foshan, PR China.
c. College of Pharmacy, Jinan University, Guangzhou, PR China.
*Corresponding author, Tel.:+86 2085224497, E-mail address: janalguo@126.com.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
‡ These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 20xx
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Table 1. The inhibitory effects of the synthesized compounds on NO production in
LPS-stimulated RAW264.7 cells.
Results and discussion
Compounds
Dexb
IC50 (µM)a
Compounds
1b
IC50 (µM)a
Preparation of optically active synthetic GR24 derivatives
42.05±5.77
2.66±0.50
The synthetic routes of GR24 followed previously reported
methods, with some improvements to increase the yield
32.27±4.23
60.45±6.54
44.48±4.72
64.59±4.32
1a
2a
3a
4a
5a
6a
4.06±0.47
3.14±0.24
2b
3b
4b
5b
6b
(shown in Scheme S1). The synthetic of GR24 enantiomers (2a
,
6.34±0.76
4.32±0.39
6.62±0.51
3.51±0.22
3a, 5a and 6a) are shown in Scheme 1, in order to investigate
the effect of double bond in D-ring, reduction product GR24
derivatives (1b and 4b) were obtained by a chemical reduction
method from 1a and 4a, respectively. The optically active
35.44±2.83
a Results were showed as means ± SD (n=4) of at least three independent experiments.
b Dex: dexamethasone.
reduction GR24 derivatives (2b, 3b, 5b and 6b) were separated
by chiral column chromatography. All the GR24 enantiomers
were confirmed by 1H NMR, 13C NMR and MS (show in
D-ring, are important chemical pharmacophore. Besides,
supplementary information). The structures of 2a, 6a and
compounds 3a, 3b, 5a and 5b showed weak bioactivity in vitro
positive control dexamethasone were shown in Figure 1.
because of the steric-hindrance.
Inhibition of NO production in (LPS)-stimulated RAW264.7
Cytotoxicity in RAW264.7 cells
cells
To determine whether the NO inhibitory activities of 2a and 6a
were related to cell viabilities, a further investigation was
demonstrated on the effect of compounds upon RAW264.7
cells growth, by adopting the method of MTT assay26. As can
be seen from Table 2, the relative cell viabilities of RAW264.7
were all above 99% which indicated no obvious cytotoxicity of
Nitric oxide (NO) acts as
a significant pro-inflammatory
mediator for acute or chronic inflammation. It is generally
reported that NO inhibitors may offer potential opportunities
to develop new therapeutic methods for inflammatory
diseases24, 25. In this study, the inhibitory activity against NO
release of synthesized strigolactones compounds were firstly
investigated in lipopolysaccharide (LPS)-stimulated RAW264.7
cells. As depicted in Table 1, it was found that a novel class of
GR24 derivatives (1a-6a) displayed improving NO inhibitory
the tested agents (dexamethasone, 2a, 6a and LPS) were
shown at the detected concentrations. A further indication of
this study also showed that NO inhibitory effects of 2a and 6a
were likely to be attributed to the structure of these two
compounds. These non-toxic concentrations were also used in
the following experiments.
activities compared to dexamethasone. In particular, series
1a-6a) exhibited better activities than series 1b-6b). Among
which, compounds 2a 2b 6a and 6b showed better activities
than corresponding compounds 3a 3b 5a and 5b
a
(
b (
,
,
,
,
.
Inhibition of TNF-α and IL-6 production in RAW264.7 cells
This work confirmed that the unreduced D-ring of
strigolactones plays an essential role in anti-inflammation
TNF-α and IL-6 play an important role in many biological
processes and has been proposed as the main contributing
factors to induce inflammatory diseases27, 28. The inhibitory
effect of 2a and 6a on the production of pro-inflammatory
cytokines is one of the major and effective methods for
treatment of inflammation. So the expression of TNF-α and
IL-6 cytokines in RAW264.7 cells was investigated and
measured by ELISA29. Dexamethasone was also used as a
positive reference drug. The effects of these three compounds
bioactivity. Compounds 2a
and (8bR, 2’R)-configuration, respectively. In contrast,
compounds 6a 6b and 5a 5b have the configuration of (8bR,
, 2b and 3a, 3b have the (8bS, 2’R)
,
,
2’S) and (8bS, 2’S), respectively. So we can speculate that the
α,β-unsaturated furanones, including strigolactones with their
(
2a, 6a and dexamethasone) were evaluated using the
concentrations of 10 μM, 5 μM, 2.5 μM and 1.25 μM in a time-
dependent manner.
Table 2. Effects of compounds on the viability of RAW264.7 cells
Compounds
Blankb
Dexc
2a
Concentrations
Cell viability (%)a
100.0±3.5
100.2±2.1
100.2±1.6
99.2±2.1
Scheme 1. Synthesis of GR24 derivatives: (1) H2/Pd, 1 h; (2) Optically active
10 μM
column.
10 μM
6a
10 μM
LPS
500 ng/mL
100 ng/mL
99.8±2.0
LPS
101.5±4.2
a Results were expressed as means ± SD (n=5) of three independent experiments.
b Blank: the cells cultured with fresh medium only.
c Dex: dexamethasone.
Figure 1. Chemical structures of dexamethasone, 2a and 6a.
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Table 3. The inhibitory effects of dexamethasone, 2a and 6a on LPS-induced TNF-α production in RAW264.7 cells.
TNF-α (pg/mL)a
Compounds
Blank
6 h
12 h
24 h
75.9±6.5
107.2±10.4
204.1±23.8
LPS
4377.1±318.6##
2655.0±106.2**
3219.4±208.8**
3671.8±97.6**
4056.3±231.3
3862.3±300.1*
4140.7±427.4
4276.9±415.4
4388.3±425.2
3927.4±222.8*
4209.3±316.3
4307.9±271.9
4367.3±264.7
5313.7±420.9##
3393.5±280.9**
4063.6±326.8**
4455.7±446.9*
4810.1±462.8
4685.5±348.6*
5036.1±413.2
5186.9±386.2
5303.7±405.9
4721.3±286.7*
5079.1±376.0
5212.4±404.2
5319.6±354.3
9635.7±377.3##
6458.3±362.9**
7533.7±260.2**
8187.9±184.9**
8748.1±370.5**
8660.7±363.2**
9085.5±327.1*
9397.7±483.9
9565.4±450.4
8796.7±342.8**
9318.5±348.4
9444.5±260.6
9582.9±362.0
10 μM
5 μM
LPS+Dexb
2.5 μM
1.25 μM
10 μM
5 μM
LPS+2a
2.5 μM
1.25 μM
10 μM
5 μM
LPS+6a
2.5 μM
1.25 μM
##P < 0.01, #P < 0.05 versus the blank (cultured with fresh medium only) group; **P < 0.01, *P < 0.05 versus the LPS (treated with LPS only) group.
a Results were showed as means ± SD (n=3) of three independent experiments.
b Dex: dexamethasone.
Table 4. The inhibitory effects of dexamethasone, 2a and 6a on LPS-induced IL-6 production in RAW264.7 cells.
IL-6 (pg/mL)a
Compounds
Blank
6 h
12 h
24 h
2.3±0.5
6.8±0.7
12.4±1.9
LPS
472.3±25.8##
269.5±38.9**
332.8±32.8**
393.4±30.9**
422.7±23.0*
302.3±21.8**
387.1±15.0**
433.6±14.2*
461.2±27.3
340.6±37.8**
410.3±26.2**
447.9±19.7
470.1±25.7
1394.0±62.6##
791.0±82.9**
927.7±57.4**
1120.3±60.7**
1240.5±42.8**
869.0±47.2**
1090.7±30.2**
1267.7±40.5**
1351.9±57.7
998.2±84.1**
1177.0±21.7**
1296.0±42.8*
1374.3±56.2
1966.6±102.3##
924.1±84.1**
1336.5±117.4**
1594.9±95.4**
1770.4±107.2*
1002.2±87.5**
1499.2±43.3**
1729.3±11.3**
1892.3±47.2
1061.3±82.1**
1561.1±162.1**
1787.6±65.3*
1936.5±137.1
10 μM
5 μM
LPS+Dexb
2.5 μM
1.25 μM
10 μM
5 μM
LPS+2a
2.5 μM
1.25 μM
10 μM
5 μM
LPS+6a
2.5 μM
1.25 μM
##P < 0.01, #P < 0.05 versus the blank (cultured with fresh medium only) group; **P < 0.01, *P < 0.05 versus the LPS (treated with LPS only) group.
a Results were showed as means ± SD (n=3) of three independent experiments.
b Dex: dexamethasone.
According to the results (Table 3, Table 4, Figure 2 and agents which showed obvious anti-inflammation effect. Next,
Figure 3), we found that the levels of TNF-α and IL-6 in LPS- in order to further examine the regulatory effect of 2a and 6a
stimulated RAW264.7 cells were significantly decreased by 2a on inflammatory response, numerous efforts were performed
and 6a treatment in concentration- and time-dependent to explore the possible anti-inflammatory mechanism of 2a
manners as compared with that in RAW264.7 control cells, and and 6a
.
their inhibitory effects were slightly weaker than that of the
Effects of GR24 on migration of neutrophils and primitive
macrophages in the injuries of transgenic zebrafish larvae
positive control drug treatment on both TNF-α and IL-6
production at all three (6 h, 12 h and 24 h) detection time. For
example, compounds 2a, 6a, and dexamethasone at 10 μM
In order to further evaluate anti-inflammatory activity of GR24
derivatives, the injury transgenic zebrafish larvae was used as
in vivo model for the next step. To our best knowledge,
neutrophils and macrophages are two most prominent types
of inflammatory cells in living body. Once inflammation occurs
in vivo, higher quantities of them were produced and were
could significantly reduce the production of LPS-induced
inflammation cytokine IL-6 with the inhibition index of
1002.2±87.5 pg/mL, 1061.3±82.1 pg/mL, and 924.1±84.1
pg/mL, respectively. So the above results found in our
laboratory suggested that 6a, especially 2a, were excellent
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Figure 3. Effects of dexamethasone, 2a and 6a on the LPS-induced production of IL-6 in
RAW264.7 cells. RAW264.7 cells were treated with dexamethasone, 2a and 6a (at the
concentrations of 10 µM, 5 µM, 2.5 µM and 1.25 µM) and LPS (100ng/mL) for 6 h, 12 h
and 24 h. Data were presented as means ± SD (n=3). ##P < 0.01, #P < 0.05 versus the
blank (cultured with fresh medium only) group; **P < 0.01, *P < 0.05 versus the LPS
(treated with LPS only) group.
Figure 2. Effects of dexamethasone, 2a and 6a on the LPS-induced production of TNF-
α in RAW264.7 cells. RAW264.7 cells were treated with dexamethasone, 2a and 6a (at
the concentrations of 10 µM, 5 µM, 2.5 µM and 1.25 µM) and LPS (100ng/mL) for 6 h,
12 h and 24 h. Data were presented as means ± SD (n=3). ##P < 0.01, #P < 0.05 versus
the blank (cultured with fresh medium only) group; **P < 0.01, *P < 0.05 versus the LPS
(treated with LPS only) group.
slightly reduced after treatment with 5 μM of 2a and 6a, but
obviously reduced after treatment with 10 and 20 μM dosages
during the evaluation time, which is similar to positive control
group. This is also consistent with the behavior described in
our early reports30. In summary, all these results illustrate the
in vivo anti-inflammation activity of GR24 derivatives 2a and
6a. Therefore, further efforts were essential to explore their
possible anti-inflammatory mechanism.
being transported to the inflammation sites. In this study, the
anti-inflammatory effects of 2a and 6a were investigated using
a double-transgenic line (Coronilla-eGFP/Lyc-dsRed), in which
neutrophils were double labeled by eGFP and dsRed,
presenting yellow color; while primitive macrophages were
labeled by eGFP only, presenting green color.
Western Blot for interpreting possible mechanism
The generation of injury inflammation was induced by tail-
cutting; dexamethasone was used as the control drug. As
shown in Figure 4, the blank group without tail cutting, which
was the healthy larvae, always generated a clear image. But
after tail cutting for 3 h and 6 h, the control group generated a
fluorescence image, in which visualize these inflammatory cells
neutrophils and primitive macrophages obviously accumulated
around the wound. Compared to the control group, the level
of neutrophils and primitive macrophages around the wound
It has been reported that NF-κB and MAPK pathways were
quite paramount and significant in inflammation by
interplaying with various inflammatory diseases because of
their crucial roles in the regulation of the intracellular and
extracellular production of inflammatory cytokines in activated
macrophages, such as NO, TNF-α, IL-6, and other inflammatory
mediators31-33. Accumulating literatures indicated that the
inflammatory mediators induced by LPS could activate NF-κB
and MAPK signaling pathways34, 35
.
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Figure 5. Effects of compounds 2a and 6a on the LPS-induced phosphorylation of NF-
κB p65, IκBα, ERK1/2 and p38 MAPK in RAW264.7 cells. RAW264.7 cells were treated
with 2a (10 μM, 5 μM, 2.5 μM, 1.25 μM), 6a (10 μM, 5 μM, 2.5 μM, 1.25 μM), and LPS
(500 ng/mL) for 4 h. The levels of NF-κB p65, IκBα, ERK1/2, and p38 MAPK proteins,
and their phosphorylated forms were analyzed using western blotting. Data were
presented as means ± SD (n=3). ##P < 0.01, #P < 0.05 versus the blank (cultured with
fresh medium only) group; **P < 0.01, *P < 0.05 versus the LPS (treated with LPS only)
group.
Figure 4. Effects of 2a and 6a on migration of neutrophils and macrophage in injury
transgenic zebrafish larvae. After tail cutting for half hour, 4 dpf transgenic zebrafish
larvae were treated by 2a and 6a (5, 10 and 20 µM). Dexamethasone (Dex) was used
as control drug. The image of neutrophil (yellow) and macrophage (green) in 3 h and 6
h were recorded by fluorescent microscope. The blank were healthy larvae. The
control were cut-tail larvae but without compounds treatment. 6-8 larvae in each
group were recorded. Scale bar = 400 μm.
was performed. 62 proteins of NF-κB were obtained from PDB
website, and its protein target structure database was built
(Table S1). The molecular docking software AutoDock Vina was
used to dock 2a with the protein targets. According to the
docking scores and the docking conformational interaction,
the potential targets were selected. 3 target proteins including
PARP1, CK2, and AKT were found to interact with GR24 with
high affinity. By comparison of their docking conformational
interaction (Figures S1~S3), PARP1 is superior to CK2 and AKT.
Anti-inflammation of GR24 relates to its function of
competitive inhibition of the 3 target proteins, which provides
an important reference for the study of the anti-inflammatory
mechanism of strigolactone and its analogues. However, it also
needs to be confirmed further.
Therefore, in order to investigate whether the anti-
inflammatory effects of 2a and 6a were associated with the
activation of NF-κB and MAPK, western blot analysis were
employed in this study. Herein, the proteins we detected
including two NF-κB pathway proteins: NF-κB p65 and inhibitor
kappa B alpha (IκBα) kinase36-38, as well as two MAPK cascades:
extracellular regulated protein kinases (ERK1/2, or p42/44) and
p38 MAPK39-41. As shown in Figure 5, the levels of
phosphorylated NF-κB p65, IκBα, ERK1/2 and p38 MAPK was
significantly increased by treatment of LPS, and decreased in
varying degrees in RAW264.7 cells by 2a and 6a treatment.
Furthermore, we observed that the phosphorylations of NF-
κBp65, IκBα, ERK1/2 and p38 MAPK were obviously inhibited
after treatment with 2a and 6a in a concentration dependent
manner when compared with LPS-induced macrophages, while
they had slightly antagonized effect on phosphorylation of
IκBα. Taken together, it can be concluded that anti-
inflammation effects of 2a and 6a were due in part, to their
ability to suppress the activation of NF-κB and MAPK cascades,
resulting in decreased NO, TNF-α and IL-6 levels.
Conclusions
In conclusion, a novel class of synthetic GR24 derivatives was
synthesized and their bioactivities were evaluated. To our
knowledge, the anti-inflammatory activity of SLs and
derivatives was studied for the first time. Among these SLs,
two promising compounds (2a and 6a) for the treatment of
inflammatory diseases were identified. In respect of the in
vitro anti-inflammatory experiment, a similar or even better
inhibitory effect was demonstrated on the release of
inflammatory mediator and cytokines (NO, TNF-α, and IL-6)
comparing to dexamethasone. Moreover, the migration of
To further investigate the anti-inflammatory mechanism of
GR24, reverse docking of 2a in the NF-κB signaling pathway
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neutrophils and primitive macrophage were also measured in protein assay kit (KGPBCA) was purchased from KeyGEN
a in vivo zebrafish model, the results also revealed a strongly Biotechnology (Nanjing, China). The enhanced
inhibition on 2a and 6a. It was demonstrated that anti- chemiluminescent (ECL) Detection Kit (WBKLS0050) was
inflammation activity of 2a and 6a was due at least in part, to purchased from Merck Millipore Corporation (Merck KGaA,
their ability to suppress the activation of NF-κB and MAPK Darmstadt, Germany).
cascades, resulting in decreased NO, TNF-α and IL-6 levels.
Chemicals
These results further confirm that compounds with the
unreduced D-ring generally exhibit improved anti-
inflammation activity, which suggests that reserved D-ring
would be an effective strategy to develop preferable anti-
inflammatory agents. Further work will be undertaken in our
group in order to evaluate the possible anti-inflammatory
action target of these SLs.
(±)-GR24 and its derivatives were synthesized from
commercially available material indan-1-one using previously
reported methods42-44 as an epimer mixtures of 1a and 4a (ca.
1:1) at C-2’, which were then separated by chromatography on
silica gel with hexane/EtOAc (v/v, 7:3) as the elution. Then, a
solution of 1a/4a (50 mg) in ethyl acetate (5mL) was added
Pd/C (5mg). The mixture was stirred under hydrogen
atmosphere for 0.5 h at room temperature before being
filtered, and the filtrate was concentrated in vacuo to provide
the crude product which was purified by flash chromatography
Experimental
Apparatus
1H and 13C NMR spectra were taken in CDCl3 with a Bruker 300
spectrometer (Karlsruhe, Germany), using tetramethylsilane
(TMS) as an internal standard. Chemical shifts are shown in δ
(ppm). Mass spectra were recorded with a Thermo Finnigan
LCQ Advantage MAX mass spectrometer (Applied Biosystems,
4000 Q TRAP). Optical rotation was obtained on a SEPA-300
(Horiba, Tokyo, Japan). Melting points were determined with a
Reichert Thermopan microscope and are uncorrected.
Kieselgel 60 F254 (Merck, Germany) was used for column
chromatography, and pre-coated silica gel 60 F254 plates were
used to analyze thin layer chromatography (TLC). Spots were
visualized using a UV lamp. The fluorescence photos of larvae
on a silica gel column to provide the diastereomer 1b/4b. The
optical isomers of 2a and 3a were achieved by an optically
active column from compound 1a, as reported by Ueno45.
Similarly, the isomers of 5a and 6a were also prepared from
compound 4a. Additionally, a series of optical isomers of 2b,
3b, 5b and 6b were obtained in the same way, respectively
(Scheme 1). All products in stock including dexamethasone
were dissolved in dimethyl sulfoxide (DMSO) to
a
concentration of 50 mM and the amount of DMSO was fixed at
0.1% (v/v) when added to cells.
Cell culture
were recorded by using a fluorescence microscope (Olympus, RAW264.7 murine macrophages cells were routinely cultured
Japan).
in DMEM with 10% fatal calf serum (FCS), 100 units/mL
penicillin, and 100 μg/mL streptomycin at 37 °C in a 5% CO2
humidified atmosphere.
Materials and reagents
RAW264.7 murine macrophages was purchased from
Assay for NO production
American Type Culture Collection (ATCC) and cultured in
DMEM containing 10% FBS. Fetal bovine serum (FBS) and RAW264.7 cells,
Dulbecco's modified Eagle's medium (DMEM) were purchased inoculated at 5×104 cells per well in 96-well plate and left to
from Gibco (Invitrogen, California, USA). The culture for 18 h before any treatment. The concentrations of
a murine macrophage cell line, were
lipopolysaccharide (LPS, Escherichia coli 0127:B8) and MTT each compound including the positive drug (dexamethasone)
were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). were designed to 100 µM, 50 µM, 20 µM, 10 µM, 5 µM, 2 µM
Dexamethasone was purchased from national institutes for and 1 µM. The cells were then pre-treated with different
food and drug control (Beijing, China). N-(1-naphthyl) concentrations of compounds for 2 h before stimulation with
ethylenediamine dihydrochloride and sulfanilamide were LPS (100 ng/mL), while the cells in control group were treated
purchased from Sinopharm chemical reagent Co. Ltd with LPS only. After stimulated for 24 h by LPS, the NO
(Shanghai, China). TNF-α enzyme immunoassay kits (EK2822) produced in the culture medium was quantified by Greiss
and IL-6 enzyme immune assay kits (EK2062) were purchased reagent, in brief, transferred 100 μL cell culture supernatant to
from Multi Sciences Biotechnology (Hangzhou, China). another 96-well plate, added 100 μL of Griess reagent and
Phorbol-12-myristate-13-acetate (PMA, S1819), IP cell lysis incubated for 10 min at room temperature. Then the
buffer (P0013) and phenylmethanesulfonyl fluoride (PMSF, absorbance of samples was measured at 540 nm (OD540) in a
ST506) were purchased from Beyotime Biotechnology microplate reader (Bio-Rad Laboratories, CA, USA).
(Shanghai, China). Anti-beta actin monoclonal antibody Dexamethasone was used as the positive control. IC50, the half
(E021020-01) was purchased from EarthOx, LLC, CA. Primary maximal inhibitory
concentration,
represented
the
anti-rabbit inducible NO synthase (iNOS, #2982), NF-κB concentration of a compound that is required for inhibition of
pathway sampler Kit (#9936S), p-MAPK family antibody 50% NO production in RAW264.7 cells and was determined
sampler kit (#9910S) and MAPK family antibody sampler kit constructing a dose-response curve in vitro.
(#9926) were purchased from Cell Signaling Technology. BCA
6 | J. Name., 2012, 00, 1-3
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concentration was determined using the BCA protein assay kit
(Thermo Scientific, 23227). Furthermore, each protein sample
was mixed with a quarter volume of 5X SDS-PAGE sample
loading buffer and the mixture was boiled for 10 min. Equal
amounts of total cellular protein were separated in a 12.5%
SDS-PAGE gel and then transferred to polyvinylidenedifluoride
membranes (Bio-Rad). After wet transfer, the membranes
were blocked for 4 h at room temperature in 5% non-fat dry
milk in TBS containing 0.1% Tween 20 (TBST), washed 3 times
in TBST, incubated with the primary antibody (anti-
phosphorylation of ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-
phosphorylation of p38 (Thr180/Tyr182), anti-p38, anti-
phosphorylation of IκBα (Ser32/36), anti-IκBα, anti-
phosphorylation of NF-κB p65, and anti-NF-κB p65) at 4 °C
overnight (all the primary antibodies were purchased from Cell
Signaling Technology and diluted in TBST at the ratio of
1:1000), washed 3 times in TBST, incubated with anti-rabbit or
anti-mouse secondary antibody (1:1000 in TBST, Cell Signaling
Technology) for 1.5 h at room temperature, washed in TBST
and exposed to ECL reagents.
Cell cytotoxicity
Cell cytotoxicity of RAW264.7 cells was measured by MTT
assay. Briefly, cells were seeded at 4×103 cells per well in a 96-
well plate. After cultured overnight, the cells were treated with
different compounds for 48 h. Then to each well, 20 μL of 0.5
mg/mL MTT solution was added. Cells were incubated for 4 h
at 37 °C. After four hours, cell culture supernatant was
removed and then followed by solubilization with 150 μL
dimethyl sulfoxide (DMSO) solutions. The optical density was
measured at 570 nm (OD570) using a Bio-Radmicroplate reader.
Each experiment was repeated thrice, and the data was
calculated from three measurement results. The density of
formazan formed in blank group was set as 100% of viability.
Cell viability (%) = [Compound (OD570) - Blank (OD570)] /
[Control (OD570) - Blank (OD570)] × 100%
Blank: cultured with fresh medium only
Control: the cells cultured with fresh medium only
Compound: the cells treated with different compounds
Measurement of TNF-α and IL-6
Statistical analysis
Results are presented as mean ± standard error (SD). Statistical
analysis were performed using Student’s t test (two-tailed,
paired) versus vehicle controls and regarded as being
significant when p < 0.05.
The levels of TNF-α and IL-6 were measured with the mouse
ELISA kit (TNF-α: MultiSciences, EK2822; IL-6: MultiSciences,
EK2062) which is operated according to the manufacturer’s
instructions. All samples were obtained as follows: RAW264.7 Acknowledgements
cells (5×105 cells/well) were cultured in 24-well plate and
We express our gratitude to Key Laboratory of Zebrafish
pretreated with 10 μM, 5 μM, 2.5 μM, 1.25 μM of compounds
for 2 h, and then LPS (100 ng/mL) was added. The production
of TNF-α and IL-6 was incubated for 6 h, 12 h and 24 h.
Modeling and Drug Screening for Human Diseases of
Guangdong Higher Education Institutes, Southern Medical
University, for supplements of zebrafish larvae. This research
was supported financially by the National Natural Science
Foundation of China (Grant: 81503031), the Science and
Technology Project of Guangdong Province (Grant:
2016A020226004), and the Initial Scientific Research Fund of
Doctor in Foshan University (Grant: gg040952).
Zebrafish larvae live image analysis
The neutrophil/macrophage double transgenic zebrafish
(Coroninla-eGFP / Lyc-dsRed) of 2a and 6a was performed
according to our previously study30, 46-48. The preparing of four
days post fertilization zebrafish larvae, tail-cutting surgery,
compound treatment and image analysis were all followed this
reported methods with a minor modification. Dexamethasone Conflict of Interest
was used as the control drug. All experiments were performed
in compliance with the relevant laws and institutional
The authors declare no competing interests.
guidelines, and the Zebrafish Research Centre of Southern
Medical University in China has approved the experiments.
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Table of Contents Graphic
Strigolactones (SLs)，A representative SL, GR24 and its reductive products of D-ring were synthesized and their
anti-inflammatory activities were fully evaluated on both in vitro and in vivo models.