Synthesis and biological evaluation of myricetin-pentadienone hybrids as potential anti-inflammatory agents in vitro and in vivo

Article abstract of DOI:10.1016/j.bioorg.2020.103597

Some important pro-inflammatory cytokines such as interleukin-6, tumor necrosis factor-α and nitric oxide are thought to play key roles in the destruction of cartilage and bone tissue in joints affected by rheumatoid arthritis. In the present study, a series of new myricetin-pentadienone hybrids were designed and synthesized. Majority of them effectively inhibited the expressions liposaccharide-induced secretion of IL-6, TNF-α and NO in RAW264.7. The most prominent compound 5o could significantly decrease production of above inflammatory factors with IC₅₀ values of 5.22 μM, 8.22 μM and 9.31 μM, respectively. Preliminary mechanism studies indicated that it could inhibit the expression of thioredoxin reductase, resulting in inhibiting of cell signaling pathway nuclear factor (N-κB) and mitogen-activated protein kinases. Significantly, compound 5o was found to effectively inhibit Freund's complete adjuvant induced rat adjuvant arthritis in vivo.

Full text of DOI:10.1016/j.bioorg.2020.103597

BioorganicChemistry96(2020)103597
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and biological evaluation of myricetin-pentadienone hybrids as
potential anti-inflammatory agents in vitro and in vivo
⁎
⁎
Chao Liub, Xu Hana, Pei Jing Yua, Liu Zeng Chena, Wei Xuec, , Xin Hua Liua,
a School of Pharmacy, Anhui Medical University, Hefei 230032, PR China
b School of Chemistry and Chemical Engineering, Suzhou University, Suzhou 234000, PR China
c State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Guizhou University, Guiyang 550025, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Some important pro-inflammatory cytokines such as interleukin-6, tumor necrosis factor-α and nitric oxide are
thought to play key roles in the destruction of cartilage and bone tissue in joints affected by rheumatoid arthritis.
In the present study, a series of new myricetin-pentadienone hybrids were designed and synthesized. Majority of
them effectively inhibited the expressions liposaccharide-induced secretion of IL-6, TNF-α and NO in RAW264.7.
The most prominent compound 5o could significantly decrease production of above inflammatory factors with
IC50 values of 5.22 µM, 8.22 µM and 9.31 µM, respectively. Preliminary mechanism studies indicated that it
could inhibit the expression of thioredoxin reductase, resulting in inhibiting of cell signaling pathway nuclear
factor (N-κB) and mitogen-activated protein kinases. Significantly, compound 5o was found to effectively inhibit
Freund’s complete adjuvant induced rat adjuvant arthritis in vivo.
Myricetin-pentadienone
Design
Synthesis
Anti-inflammatory
1. Introduction
In nucleus, reduced TRX-1 potentiates NF-κB binding to DNA, and
consequently, enhances transcription of inflammatory mediators [16].
Rheumatoid arthritis (RA) is defined as the chronic inflammation of
one or more joints, with presence of pain, erythema, swelling, an in-
crease in the temperature in the affected areas [1,2]. RA is a complex
process involving numerous inflammatory mediators [3]. Although RA
changes have been extensively investigated, there is still a still lack of
effective drugs. During RA, there are many pro-inflammatory cytokines
such as IL-1, IL-6, TNF-α and chemokines release to the synovial space
[4]. Therefore, it is urgent to find effective drugs for RA by inhibiting
the production and release of pro-inflammatory cytokines.
On the other hand, when H2O2 stimulates macrophage, oxidized Trx
can activate mitogen-activated protein kinases (MAPK) which enter the
nucleus to induce cell apoptosis [17].
Curcumin has attracted much attention regarding its anti-in-
flammatory activity [18]. But, due to low bioavailability, the potential
utility is limited by its instability and poor solubility. Thus, on basis of
improving stability and solubility, a number of analogs/derivatives
have been designed and synthesized. Among those compounds, dia-
rylpentadienone derivatives with good biological profile have been
discovered [19,20].
The thioredoxin system, which consists of thioredoxin (Trx), thior-
edoxin reductase (TrxR) and nicotinamide adenine dinucleotide phos-
phate (NADPH), plays an essential role in oxidative stress, nitrosative
stress, redox regulation and regulating intracellular signaling pathways
[5–7]. Trx, a small redox active protein, is one of the defense proteins
induced in response to various oxidative stress conditions [8,9]. The
reduction of oxidized Trx by NADPH is catalyzed by TrxR [10,11]. In
addition to its effective antioxidant effect, Trx system also has anti-
inflammatory properties [12,13], mainly because it can suppress neu-
trophil chemotaxis to inflammatory sites and inhibit the expression and
activation of macrophage migration-inhibitory factors [14]. On the one
hand, stimulated by LPS, then activation of nuclear factor (NF-κB) en-
ters nucleus to induce the release of pro-inflammatory cytokines [15].
Myricetin, a polyphenolic flavonoid, is present in a wide variety of
fruits with anti-cancer activity, its derivatives inhibiting of intestinal
tumor igenesis through the NF-κB/IL-6 pathway has been reported
[21,22]. However, substantial limitation of availability, due to low
stability in body has been observed [23,24]. It is presumed that the
polyphenolic moiety may affect this property. To overcome this lim-
itation, methylated-myricetin derivatives should be avoid it.
Based on the above-mentioned, we have introduced methylated-
myricetin moiety into the diarylpentadienone skeleton, a series of
myricetin-pentadienone hybrids were synthesized at first. The pre-
liminary activity results showed some compounds could inhibit ex-
pression of TrxR. It is becoming increasingly clear that TrxR plays a key
⁎ Corresponding authors.
E-mail addresses: wxue@gzu.edu.cn (W. Xue), xhliuhx@163.com (X.H. Liu).
https://doi.org/10.1016/j.bioorg.2020.103597
Received 17 September 2019; Received in revised form 16 January 2020; Accepted 19 January 2020
Availableonline29January2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

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Fig. 1. Idea of synthesis.
2.3. Anti-inflammatory screening
role in inflammation, and accumulating evidences indicate that TrxR
may be a promising anti-inflammatory target. Herein, in continuation
to extend our research of inflammation, more compounds with me-
thylated-myricetin-pentadienone moieties (Fig. 1) were designed and
screened for their anti-inflammatory activities in this study.
IL-6, TNF-α and NO have been shown contribute to the initiation
and progression of an inflammation-cause RA [26]. We use an enzyme-
linked immunosorbent assay (ELISA) and Griess Reagent assay to screen
for changes in LPS-induced IL-6, TNF-α and NO release by the syn-
thesized compounds in RAW264.7 cells. Macrophages were pre-treated
with 20 μM compounds for 1 h and then treated with LPS (0.5 µg/mL)
for 24 h. The cell conditioned medium was collected, the IL-6, TNF-α
and NO in the media was detected by ELISA and Griess Reagent assay.
The screening results indicated that most of the title compounds re-
duced the LPS-induced IL-6, TNF-α and NO secretion at a dosage of
20 µM (Fig. 4). It is noteworthy that compounds 5o, 5p and 5q ex-
hibited stronger inhibiting of IL-6, TNF-α and NO production compared
with the referecce Indomethacin. Therefore, these compounds were
used for further evaluation.
2. Results and discussion
2.1. Chemistry
Acetone and benzaldehyde in presence of 5% NaOH through Claisen-
Schmidt condensation, α, β-unsaturated ketone (compound 1) with good
yield was obtained. The key intermediate pentadienone (compound 2) was
synthesized with the reaction of aldehyde ketone condensation. During the
above reaction, the pH was adjusted to 5 by 5% HCl. Myricetin used as the
raw material, 3-hydroxy-4H-chromen-4-one (compound 3) was synthesized
by removing glycosides. Then, compound 3 and 1,3 (or 4) dibrombutane
with K2CO3 dissolved in DMF, 3-(3-bromopropoxy/butoxy)−5,7-di-
methoxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (compound 4) was
prepared. Compounds 5a–5w (Fig. 2) have been fully characterized as
Supporting information.
2.4. Antioxidant screening
H2O2 (100 μM) reduced cell viability in these experiments to
45–60% of that in the vehicle control group[27,28]. In order to eval-
uate the antioxidant activity of synthetic compounds, RAW 264.7 cells
were pre-incubated with compounds 5a–5w (10, 1, 0.1 µM) for 1 h and
treated with H2O2 (100 μM) for 24 h. MTT was used to detect cyto-
toxicity. The screening results indicated that most of the compounds
reduced the H2O2-induced cytotoxicity at a dosage of 10 µM (Fig. 5). Of
these, compounds 5o, 5p and 5q showed potential activity.
2.2. Assessment of toxicity
In order to ensure the safety of the title compounds and avoid their
false positive anti-inflammatory activity, MTT was used to detect cy-
totoxicity against RAW264.7 cells. As shown in Fig. 3, most of the
compounds showed low toxicity at 20 µM. Among then, compounds 5o,
5p and 5q showed lower toxicity. Therefore, these compounds are
valuable for further evaluation. Though the interference of drugs over
the mitochondrial activity can be interpreted as loss of viability. Still,
there is an extensive use of this assay as a viability assay [25].
2.5. Inhibiting of LPS-induced cytokine release and H2O2-induced oxidation
reaction
Our next objective was to determine whether compounds 5o, 5p
and 5q inhibitory effect on inflammatory cytokine release with dose-
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Fig. 2. Structures of 5a–5w.
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Fig. 2. (continued)
dependent pattern. We pretreated RAW264.7 cells with compounds 5o,
5p, 5q at concentrations of 20, 10, 5 and 2.5 μM for 1 h, and subse-
quently incubated the cells with 0.5 μg/mL LPS for 24 h. The release of
inflammatory cytokines into the culture medium was measured by
ELISA and Griess Reagent assay. Compounds 5o, 5p and 5q showed
well-defined a dose-dependent inhibitory activity on IL-6, TNF-α and
NO releases (Fig. 6A–C). When the concentration is 2.5–20 μM, the IC50
values of IL-6, TNF-α and NO were observed for compound 5o
(5.22 μM, 8.22 μM, 9.31 μM, respectively), compound 5p (7.68 μM,
11.88 μM, 14.88 μM, respectively), compound 5q (10.53 μM, 15.57 μM,
11.04 μM, respectively). Moreover, the IC50 values of these tested
compounds could be very useful for our next study. On the other hand,
RAW 264.7 cells were pre-incubated with all compounds (10, 1, 0.1,
0.01, 001 µM) for 1 h and treated with H2O2 (150 μM) for 24 h. MTT
results showed that when the concentration of the compound was
10 nM, it still had protective effect compared with the H2O2-induced
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Fig. 3. The cytotoxic evaluation of compounds 5a–5w against RAW264.7 cells.a (aThe cell viability was evaluated by MTT assay. *p
***p < 0.001 compare with control group.)
< 0.05, **p < 0.01,
Group (Fig. 6D). It's not hard to find out that compound 5o is the best
one. Therefore, we choose it as the title compound for further me-
chanism study.
2.6.4. Inhibition LPS-induced TrxR/NF-κB signaling pathways activation
The thioredoxin system contains Trx, TrxR, NAPDH. Trx and TrxR
are over expressed in various cancer and inflammation tissues [34–36].
Trx is a 12 KD protein responsible for cell redox homeostasis [37]. In the
nucleus, Trx in the reduced state has the ability to enhance the binding
of transcription factors to DNA [38]. The subunits p50 and p65 of NF-κB
are usually isolated in the cytoplasm by tight association with IκB-α
protein [39]. After LPS stimuli cell activation of the toll-like-receptor 4
(TLR4), IκB-α phosphorylation and proteasomal-degradation allows the
transcription factor NF-κB subunits to translate to the nucleus [40]. In
the nucleus, transcription factor NF-κB undergo several post-transla-
tional modifications, which leads to their binding to DNA and tran-
scription of several pro-inflammation proteins and inflammation fac-
tors, such as inflammatory mediators and TLRs. TrxR is the only
enzyme in the cell which reduces oxidized Trx [41]. So, the expression
of TrxR is essential for the regulation of inflammation. As shown in
Fig. 10, the LPS (0.5 µg/mL) stimulation could markedly augment TrxR
expression. However, compound 5o concentration dependently sup-
pressed LPS-induced TrxR expression (Fig. 10A). Further experiments,
showed compound 5o could inhibit the activation of NF-κB pathway by
effects of IκB and p65 phosphorylation and degradation (Fig. 10B and
C). This results demonstrated that compound 5o prevented LPS-induced
inflammatory response in macrophages.
2.6. Mechanism exploration of compound 5o
2.6.1. Morphological changes of the cells
RAW264.7 cells were seeded in 6 cm culture dishes overnight and
pretreated with compound 5o, at concentrations of 20, 10, 5, 2.5 μM for
1 h before with LPS (0.5 μg/mL) for 24 h. RAW264.7 cells were round
and bright in normal. When LPS stimulates cells, a large number of cell
pseudopods appear, and a large number of vacuoles appear on the cell
membrane. Title compound 5o ameliorated the degree of cell de-
formation compared with the LPS group. Morphologically, the in-
flammatory state of cells was reversed (Fig. 7).
2.6.2. Inhibits TrxR activity and intracellular ROS
The Trx system plays an important role in the balance of redox in
cells to prevent excessive ROS accumulation [29]. The inhibiting ex-
pression of TrxR and Trx may disturb the redox balance, leading to
intracellular ROS unbalance [30–32]. Previously, we reported that
curcumin analogue hybrids could exhibit anticancer activity and was
found to have efficacy in potential cytokines inhibitory activity through
inhibiting of TrxR. Compared with the control group, TrxR activity in
the LPS-treated group decreased. We found an interesting phenomenon
as shown in Fig. 8, title compound 5o did not increase the activity of
TrxR, and there was no statistical difference between the different
concentrations (p > 0.05). The results showed that compound 5o had
no effect on TrxR activity (Fig. 8A). Further research findings, the LPS-
treated group cells excessive ROS accumulation, while compound 5o
reduce ROS in a dose-dependent manner (Fig. 8B). Therefore, these
results deserve further study.
2.6.5. Inhibition H2O2-induced TrxR/MAPK signaling pathways activation
Reductive Trx binds with ASK and inhibits its activity, which also
makes ASK1 ubiquitin degradation. While oxidized Trx cannot bind to
ASK1, it directly or indirectly activates ASK1 mediated apoptotic
pathway [42]. Reactive oxygen radicals can dissociate Trx from ASK1
and activate ASK1. TrxR is the only enzyme that can reduce oxidation
state Trx in cells [43]. The TrxR expression is necessary for cell anti-
oxidant stress and apoptosis inhibition. TrxR activity was inhibited by
H2O2 stimulation, resulting in high expression of Trx oxidation. These
changes led to dissociation of ASK1 from Trx (reduced)-ASK1 com-
plexes, resulting in activation of the MAPK pathway, and the tran-
scription factor AP-1 enters the nucleus, causing them to bind to DNA
and transcription [44]. From Fig. 11, we found compound 5o could
inhibit the TrxR, P38 and JNK phosphorylation.
2.6.3. Suppresses LPS-induced inflammatory response
COX-2 and iNOS are important pro-inflammatory proteins, which
are associated with a variety of inflammatory diseases [33]. Because
COX-2 and iNOS proteins are at the downstream of cellular signaling
pathways, when LPS stimulates cells, activation of the cellular pathway
results in high expression of COX-2 and iNOS proteins. Therefore, the
detection of COX-2 and iNOS expressions is necessary. As shown in
Fig. 9, the expressions of COX-2 and iNOS increased significantly when
LPS (0.5 μg/mL) stimulated for 24 h. Title compound 5o could reduce
the expressions of COX-2 and iNOS in a concentration-dependent
manner. These results demonstrated that compound 5o could inhibit
COX-2 and iNOS in macrophages and LPS-induced inflammation.
2.6.6. Effect on histopathological changes of AA-induced rat ankle joints
Oxidative stress is closely related to rheumatoid arthritis. The ef-
fects on pathological changes of AA-induced rat ankle joints was de-
tected to confirm the activity of compound 5o in vivo.
Photomicrographs of knee joints sections stained with H&E showed AA
group synovial hyperplasia, serious cartilage destruction and acute in-
flammatory cells infiltration compared with the normal group. H&E
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Fig. 4. Inhibiting of IL-6, TNF-α and NO productions by compounds 5a–5w. (A) Effects of all compounds on IL-6 secretion. (B) Effects of all compounds on TNF-α
secretion. (C) Effects of all compounds on NO secretion. Ind: positive Indomethacin ***p < 0.001, **p < 0.01, *p < 0.05 vs LPS group. ###p < 0.001 vs control
group. &&&p < 0.001, &&p < 0.01, &p < 0.05 vs Ind group.
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Fig. 5. Compounds 5a–5w inhibited H2O2-induced oxidant response.a (aThe cell viability was evaluated by MTT assay. ###p < 0.001 vs control group. *p < 0.05,
**p < 0.01, ***p < 0.001 compare with H2O2 group.)
showed AA rats treated with Aspirin (50 mg/kg) exhibited moder-
atesynovial hyperplasia and serious inflammatory cells infiltration. AA
rats treated with compound 5o (45 mg/kg) exhibited moderatesynovial
hyperplasia and serious cartilage damage, but compound 5o (90 mg/
kg) only mild synovial hyperplasia with intact cartilage although slight
inflammatory cells infiltration remained. In conclusion, compound 5o
could significantly inhibit synovial hyperplasia and cartilage destruc-
tion in a dose-dependent manner and had a certain therapeutic effect on
adjuvant arthritis in rats (Fig. 12).
4. Experimental section
4.1. Chemistry
The reactions were monitored by thin layer chromatography (TLC).
Melting points were determined on a XT4MP apparatus (Taike Corp.,
Beijing, China), and are uncorrected. 1H and 13C NMR spectra were
recorded on a Brucker AM-500 (500 MHz) spectrometer with the sol-
vent of DMSO‑d6 and CDCl3. All reagents were purchased from standard
commercial suppliers.
3. Conclusions
4.2. General procedure for compounds 5a–5w
In summary, to better treat rheumatoid arthritis, based on rational
design, a series of myricetin-pentadienone hybrids were designed and
synthesized. Compared with previous studies, myricetin derivatives
were usually used as anti-tumor activity, most of which had strong cell
cytotoxicity. In this study, new myricetin derivatives were designed and
discovered, presenting low cell toxicity and high anti-inflammatory
activity. Preliminary inflammatory screening showed that most of the
compounds had good IL-6, TNF-α and NO inhibitory activity. Among
them, some compounds (5o, 5p and 5q), presenting low toxicity were
selected for further assessment of their IL-6, TNF-α and NO inhibitory
activities. Specifically, title compound 5o could inhibit IL-6 (5.22 μM),
TNF-α (8.22 μM) and NO (9.31 μM) secretion in a dose-dependent
manner. The preliminary mechanism indicated that title compound
could target TrxR proteins, reduce cellular inflammation and increase
cellular antioxidant stress. One side, this compound inhibited the high
expression of TrxR and inhibited Trx binding to NF-κB. On the other
hand, it could regulate expression of TrxR and reduction of TXR in the
oxidized state to the reduced state. The further study showed that this
compound effectively reduced AA-induced rat ankle joints in vivo
(Fig. 13).
To a N, N-dimethylformamide (30 mL) solution of 3-(3-bromopro-
poxy)-5,7-dimethoxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
(3.0 mmol), (1E,4E)-1-(3,4-dimethoxyphenyl)-5-(4-hydroxyphenyl)
penta-1,4-dien-3-one (3.0 mmol) and potassium carbonate (3.0 mmol)
was added, stirred for 20 min, then refluxed for 8 h. Until the reaction
was complete, 200 mL ice water was added, twenty hours later, the
crude residue was purified by chromatography on SiO2 (chloroform:
ethanol = 4:1, v/v) to give the title compound 5a as yellow solids.
Compounds 5b ~ 5w were synthesized by the same method. Synthesis
of them as Supporting information.
4.3. Cell culture
Mouse peritoneal macrophages were purchased from BeNa Culture
Collection Company. RAW264.7 cells were cultured in DMEM medium
(Hyclone, USA) supplemented with 10% fetal bovine serum (Biological
Industries, Israel), 100 μg/mL streptomycin and100 U/mL penicillin
(Beyotime). Cells were cultured at 37 °C with 5% CO2 in a humidified
atmosphere.
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Fig. 6. Compounds 5o–5p inhibiting of LPS-induced cytokine release and H2O2-induced oxidant in a dose-dependent manner. (A) IL-6. (B) TNF-α. (C) NO. (D)
antioxidant. ###p < 0.001 vs control group. *p < 0.05, **p < 0.01, ***p < 0.001 compare with LPS group or H2O2 group.
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Fig. 7. Morphology of RAW264.7 cells using inverted phase-contrast microscope (magnification × 10). RAWW264.7 cells treated with title compound 5o (10, 5, 2.5
and 1.25 μM) for 24 h. The concentration of LPS is 0.5 μg/mL.
Fig. 8. Physiological significance of targeting TrxR by compound 5o. (A) Inhibition of TrxR activity in RAW264.7 cells by compound 5o; (B) Treatment with LPS
induced the accumulation of toxic ROS in RAW264.7 cells. The compound 5o concentration is 5 μM, 10 μM, 20 μM. ###p < 0.001 vs control group. *p < 0.05,
**p < 0.01, ***p < 0.001 compared with LPS group.
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Fig. 9. Compound 5o inhibited LPS-induced iNOS
and COX-2 proteins expression in RAW 264.7 cells.
Pretreatment with compound 5o (2.5–10 µM) 1 h
before cells were stimulated with LPS (0.5 µg/mL)
for 24 h. COX-2, iNOS and β-actin were detected by
Western blot. Bay11-7082 (5 µM) is the NF-κB in-
hibitor. ###p
< 0.001 compared with control
cells, ***p < 0.001 compared with LPS-stimulated
cells; Three separate experiments were shown by
blots.
4.4. Detection of toxicity
1 h before with LPS (0.5 μg/mL) for 24 h. Morphological changes were
observed in these cells under the microscope (OLYMPUS, Japan).
RAW264.7 cells were seeded into 96-well plates at a density of
1 × 104 cells/well and permitted to continue to grow about 24 h. All
compounds (20 μM) pretreatment RAW264.7 cells for 24 h. Then 20 μL
MTT (5 mg/mL) was added into each well and were incubated for 4 h at
37 °C. After 4 h of incubation, the culture media were removed, 150 μL
DMSO were added to each well and shaking the 96-well in a horizontal
shaker about 15 min. At last, the absorbance at 492 nm was measured
by a microplate reader (MQX200, Bio-Tek, USA).
4.8. Detection of intracellular ROS
Detection of intracellular reactive oxygen species (ROS) with DCFH-
DA fluorescent probe. RAW264.7 cells were seeded into 6-well plates at
a density of 1.5 × 106 cells/well grow about 24 h. cells were pre-in-
cubated with all compounds (20, 10, 5 µM) for 1 h and treated with LPS
(0.5 μg/mL) for 6 h. The cells were treated with DCFH-DA (BestBio,
China) for 30 min at 37 °C in the dark. According to the manufacturer's
instructions, use flow cytometry detecting the intracellular reactive
oxygen species and analyze by flowjo software.
4.5. Determination of IL-6, TNF-α and NO
RAW264.7 cells were seeded into 48-well plates at a density of
6 × 104 cells/well and permitted to continue to grow about 24 h.
RAW264.7 cells were pretreated with compounds (20 μM) for 1 h, in-
cubated with LPS (0.5 μg/mL Sigma, USA) for 24 h. Collect cell su-
pernatant for detection. The levels of TNF-α and IL-6 in the culture
medium were measured by ELISA kit (eBioScience, San Diego, CA). NO
production was measured using Griess Reagent assay (Beyotime,
China), according to the manufacturer's instructions.
4.9. Determination of TrxR activity in RAW264.7 cells lysates
TrxR activity was measured using the TrxR Assay kit (BestBio,
China). Firstly, we should add 150 μL cell lysate to the prepared cells.
Collect supernatant for detection. Secondly, mixed all additives rapidly
with supernatant according to the protocol. Finally, we measured the
absorbance at 412 nm at 10 s (a1) and 310 s (a2) respectively. BCA
assay kit (Beyotime, China) was used determine the concentration of
proteins (Cpr). The activity of TrxR was calculated by formula: TrxR
(U/mg prot) = 147 × (ΔA sample − ΔA blank) ÷ Cpr.
4.6. Oxidative stress screening
RAW264.7 cells were seeded into 96-well plates at a density of
2 × 104 cells/well and permitted to continue to grow about 24 h·H2O2
(150 μM) reduced cell viability in these experiments to 45–60% of that
in the vehicle control group. RAW 264.7 cells were pre-incubated with
all compounds (10, 1, 0.1 µM) for 1 h and treated with H2O2 (150 μM)
for 24 h. MTT was used to detect cytotoxicity.
4.10. Western blotting
Cells were seeded into 6 cm culture dishes with 3 × 106 cells and
maintained overnight. RAW264.7 cells were pretreated with compound
5o (10, 5, 2.5 μM) for 1 h before were stimulated LPS (0.5 μg/mL) for
0.5 h. Add 300 μL RIPA cell lysis buffer (Beyotime, China) to the pre-
pared cells and collect supernatant for detection. Total proteins were
separated by 12% SDS-PAGE. The resulting blots were blocked with 5%
milk (about 2 h) and incubated with anti-p65 antibody (1:1000; abcam,
USA), anti-p-p65 antibody (1:1000; abcam, USA), COX-2 (1:1000;
4.7. Morphological changes of the cells
RAW264.7 cells were seeded in 6 cm culture dishes overnight and
pretreated with compound 5o, at concentrations of 20, 10, 5, 2.5 μM for
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Fig. 10. Compound 5o inhibited LPS-induced TrxR/NF-κB active in RAW 264.7 cells by Western blot analysis. (A) Detection of TrxR level by Western blotting; (B, C)
NF-κB protein expression level in RAW264.7 cells were measured. RAW 264.7 cells were treatment with compound 5o (2.5–10 µM) 1 h and were stimulated with LPS
(0.5 µg/mL) for 24 h. TrxR, p-IκB, IκB, p-P65, P65 and β-actin were detected by Western blot. Bay11-7082 (5 µM) is the NF-κB inhibitor. ###p < 0.001 compared
with Control cells, ***p < 0.001 compared with LPS-stimulated cells; Three separate experiments were shown by blots.
abcam, USA), iNOS (1:1000; abcam, USA), p-IκB (1:1000; CST, USA),
IκB (1:1000; CST, USA), P38 (1:1000; proteintech, China), p-P38
(1:500; proteintech, China), JNK (1:500; proteintech, China), p-JNK
(1:500; proteintech, China), TrxR (1:500; proteintech, China) overnight
at 4 °C. At last, the blots were detected with the chemiluminescence
(ECL) system.
obtained from Animal Department of Anhui Medical University. About one
week adaptive environment, SD rats were randomly divided into five
groups (7 rats per group): normal, AA (adjuvant arthritis), aspirin (50 mg/
kg) and compound 5o (45, 90 mg/kg). All experiments and animal care
procedures were approved by the Animal Resource Center of Anhui Medical
University. Compound 5o and aspirin in 0.5% carboxymethyl cellulose
sodium (CMC-Na) solution at concentrations of 9, 18 mg/mL and 10 mg/
mL, were administered intragastrically one milliliter per mouse, starting
from day 14–28, once a day. Animals were anaesthetized and executed
28 days later. The right ankle joint was fixed with 4% polyformaldehyde
4.11. In vivo experiments
Thirty-five male Sprague-Dawley (SD) rats weighing 160–180 g were
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Fig. 11. Compound 5o inhibit H2O2-induced TrxR/MAPK active in RAW 264.7 cells by Western blot. (A) Detection of TrxR level by Western blotting; (B, C) MAPK
protein expression level in RAW264.7 cells were measured. After pretreatment with compound 5o (2.5–10 µM) 1 h, RAW 264.7 cells were stimulated with H2O2
(150 µM) for 24 h. P38, p-P38, JNK, p-JNK and β-actin were detected by Western blot. ###p < 0.001 compared with control cells, ***p < 0.001 compared with
LPS-stimulated cells; Three separate experiments were shown by blots.
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BioorganicChemistry96(2020)103597
Fig. 12. Effect of compound 5o on pathological changes of AA-induced rat ankle joints by HE staining (magnification 100×). (A) Normal; (B) AA; (C) Aspirin 50 mg/
kg; (D) Compound 5o 45 mg/kg; (E) Compound 5o 90 mg/kg. AA, adjuvant arthritis; Aspirin used as the positive control.
Fig. 13. The possible molecular mechanisms involved of compound 5o against TrxR protein.
buffer and decalcified with dilute nitric acid. The ankle joint tissue was
paraffin embedded and sliced for histopathological analysis.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
4.12. Statistical analysis
All data are expressed as mean (SD). Analysis of variance (ANOVA)
was used to determine statistical data. The value of p < 0.05 was
considered to be statistically significant. Graphpad Prism 5 soft and
SPSS 17.0 were used for mapping and data analysis. Repeat all the
experimental data at least three times.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103597.
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