A new bisepoxylignan dendranlignan A isolated from Chrysanthemum Flower inhibits the production of inflammatory mediators via the TLR4 pathway in LPS-induced H9c2 cardiomyocytes

Article abstract of DOI:10.1016/j.abb.2020.108506

A new bisepoxylignan dendranlignan A (A1) and the known compound lantibeside D (D2) was isolated from Chrysanthemum Flower, the dried capitulum of Dendranthema morifolium (Ramat.) kitam. Their structures were determined on the basis of extensive spectroscopic methods, including 1D-NMR, 2D-NMR and MS data. Additionally, A1 and D2 were evaluated for their effects on the production of inflammatory mediators in H9c2 cardiomyocytes stimulated with lipopolysaccharide (LPS). Results demonstrated that A1 and D2 decreased LPS-induced production of inflammatory cytokines TNF-α, IL-2 and IFN-γ in H9c2 cells. Both compounds also decreased the nuclear localization of c-JUN, p-P65 and p-IRF3, but did not affect the level of TLR4. Molecular docking indicated that A1 and D2 occupied the ligand binding sites of TLR4-MD2. In the present study, we for the first time discovered a new bisepoxylignan compound A1, and found that this compound has a potential to inhibit inflammation by inhibiting TLR4 signaling.

Full text of DOI:10.1016/j.abb.2020.108506

Archives of Biochemistry and Biophysics 690 (2020) 108506
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
A new bisepoxylignan dendranlignan A isolated from Chrysanthemum
Flower inhibits the production of inflammatory mediators via the TLR4
pathway in LPS-induced H9c2 cardiomyocytes
Mengnan Zenga,b,1, Meng Lia,b,1, Yingjie Chen , Jingke Zhanga,b, Yangang Caoa,b, Beibei Zhanga,b
Weisheng Feng , Xiaoke Zheng
c
,
a,b
a,b,∗
c,∗∗
, Zhiling Yu
a
Henan University of Chinese Medicine, Zhengzhou, 450046, China
b
c
The Engineering and Technology Center for Chinese Medicine Development of Henan Province, Zhengzhou, 450046, China
Centre for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new bisepoxylignan dendranlignan A (A1) and the known compound lantibeside D (D2) was isolated from
Chrysanthemum Flower, the dried capitulum of Dendranthema morifolium (Ramat.) kitam. Their structures were
determined on the basis of extensive spectroscopic methods, including 1D-NMR, 2D-NMR and MS data.
Additionally, A1 and D2 were evaluated for their effects on the production of inflammatory mediators in H9c2
cardiomyocytes stimulated with lipopolysaccharide (LPS). Results demonstrated that A1 and D2 decreased LPS-
induced production of inflammatory cytokines TNF-α, IL-2 and IFN-γ in H9c2 cells. Both compounds also de-
creased the nuclear localization of c-JUN, p-P65 and p-IRF3, but did not affect the level of TLR4. Molecular
docking indicated that A1 and D2 occupied the ligand binding sites of TLR4-MD2. In the present study, we for
the first time discovered a new bisepoxylignan compound A1, and found that this compound has a potential to
inhibit inflammation by inhibiting TLR4 signaling.
Dendranlignan A
Bisepoxylignan
Structural elucidation
TLR4
Cardiomyocyte
1. Introduction
characteristics [10]. Flos chrysanthemum is recorded in the Shen
Nong's herbal classic and mainly contains flavonoids, triterpenoids,
Sepsis is a systemic inflammatory reactive disease. Its progress is
glycosides, and alkaloids [11,12]. Huai Flos Chrysanthemi, one of four
major Huai medicines, also one of Flos chrysanthemum, is mainly
produced in Jiaozuo of Henan province [13]. In our previous study,
four new sesquiterpenoids viz. chrysanthguaianolactones and four
known sesquiterpenoids were isolated from the acetone extract of Huai
Flos chrysanthemum, and the anti-inflammatory activity was in-
vestigated using the LPS induced H9c2 cardiomyocytes [14,15]. Since,
cardiomyocyte injury is the leading cause of morbidity and mortality in
cardiomyopathy patients, such as sepsis [16], diabetes [17], and hy-
pertension [18], the search for effective drugs to protect the cardio-
myocyte injury will ultimately cure many diseases.
In continuation of our study on the Huai Flos chrysanthemum,
further phytochemical studies were undertaken, which led to the iso-
lation of a new bisepoxylignan Dendranlignan A (A1) and a known
compounds lantibeside D (D2). The structures of the isolated com-
poundswere confirmed on the basis of spectroscopic analysis, including
rapid and life-threatening, thereby contributing largelytothe high sepsis
mortality [1]. Recent studies have shown that heart failure is a common
complication of sepsis [2,3]. Myocardial dysfunction caused by sepsis is
also the main cause of death in patients with sepsis [4]. However, the
causes of myocardial dysfunction caused by sepsis have not yet received
much attention. It was found that the proinflammatory factorsproduced
by cardiomyocytes, as well as the apoptosis of cardiomyocytes play a
key role in the pathogenesis of the myocardial dysfunction [5,6]. Li-
popolysaccharide (LPS) has been widely applied to induce apoptosis
and myocardial cells [7]. Therefore, the application of LPS-induced
apoptosis of the cardiomyocytes could be an effective model for
studying myocardial dysfunction caused by sepsis.
Dendranthema morifolium (Ramat.) (Flos chrysanthemum) is one of
the well-known daily beverage in China, and is known to exhibit anti-
bacterial [8], antioxidant [9], anti-inflammatory, and cardioprotective
∗
Corresponding author. Henan University of Chinese Medicine, Zhengzhou, 450046, China.
Corresponding author.
∗∗
E-mail addresses: zhengxk.2006@163.com (X. Zheng), zlyu@hkbu.edu.hk (Z. Yu).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.abb.2020.108506
Received 11 May 2020; Received in revised form 1 July 2020; Accepted 10 July 2020
Available online 14 July 2020
0003-9861/ © 2020 Elsevier Inc. All rights reserved.

M. Zeng, et al.
Archives of Biochemistry and Biophysics 690 (2020) 108506
UV, IR, 1D and 2D NMR, and HR-ESI-MS. The absolute configurations
of A1 and D2 were established by the comparison of the experimental
and calculated electronic circular dichroism (ECD) spectra. In addition,
we also investigated the protective effects of A1 and D2 from Huai Flos
chrysanthemum on the LPS-induced injury in H9c2 cardiomyocytes, in
order to evaluate its effectiveness to protect the cardiomyocyte injury.
Table 1
NMR spectral data of compounds 1 (A1) and 2 (D2).
No.
δ
H
δ
C
A1
D2
A1
D2
1
2
3
4
5
6
7
8
9
135.6
104.8
154.4
139.5
154.4
104.8
87.3
136.8
107.5
149.4
148.6
109.0
120.6
87.0
6.70 (1H, s)
6.87 (1H, d, 2.0)
6.77 (1H, d, 8.0)
6.83 (1H, dd, 2.0, 8.0)
4.70 (1H, d, 5.0)
3.10 (1H, m)
2. Materials and methods
6.70 (1H, s)
4.70 (1H, d, 5.0)
3.10 (1H, m)
4.25 (1H, m)
55.7
72.9
55.5
72.7
2.1. General experimental procedures
4.23 (1H, m)
3
.86 (1H, m)
3.86 (1H, m)
NMR spectra (including 1D and 2D) were recorded at room tem-
1′
2′
3′
4′
136.5
107.5
149.4
148.6
109.0
120.6
87.2
137.7
111.6
150.1
147.7
118.1
119.8
87.3
perature (approximately 20–25 °C) on a Bruker Avance III 500 MHz
6.87 (1H, d, 2.0)
7.01 (1H, d, 2.0)
1
spectrometer using TMS as a standard (500 MHz for H NMR and
1
3
1
25 MHz for C NMR). ECD spectra were obtained on a Chirascanq CD
5
6
′
′
6.77 (1H, d, 8.0)
6.84 (1H, dd, 2.0, 8.0)
4.75 (1H, d, 5.0)
3.10 (1H, m)
4.25 (1H, m)
3
4.80 (1H, d, 8.0)
3.46 (1H, m)
3.40 (1H, m)
3.40 (1H, m)
3.18 (1H, m)
7.13 (1H, d, 8.0)
6.90 (1H, dd, 2.0, 8.0)
4.74 (1H, d, 5.0)
3.10 (1H, m)
4.23 (1H, m)
3.86 (1H, m)
4.87 (1H, d, 7.0)
3.47 (1H, m)
3.38 (1H, m)
3.38 (1H, m)
3.38 (1H, m)
spectrometer (Applied Photophysics) at room temperature. Optical ro-
tation was analyzed using the APIV (Rudolph Research Analytical). IR
spectrometry was performed using Nicolet iS10 Microscope
Spectrometer (Thermo Fisher Scientific). HR-ESI-MS spectra were ob-
tained on a Brukerma Xis HD mass spectrometer. UV spectra were ob-
tained on a Shimadzu UV-2401PC apparatus. Preparative HPLC was
conducted using a Saipuruisi LC-50 instrument with a UV200 detector
and YMC-Pack ODS-A column (250 × 20 mm, 5 μm and 250 × 10 mm,
μm). Column chromatography was performed using a Diaion HP-20
Mitsubishi Chemical Corporation), Toyopearl HW-40, MCI gel CHP-20
TOSOH Corporation), Sephadex LH-20 (Amersham Pharmacia Biotech
AB), LiChroprep RP-18 gel (Merck, Darmstadt) and silica gel (Marine
Chemical Industry). TLC was performed using self-made silica gel G
plates (Qingdao Marine Chemical Industry). All chemical reagents were
supplied by Beijing Chemical Plant, China and Tianjin NO. 3 Reagent
Plant, China.
7′
8
9
′
′
55.5
72.8
55.6
72.7
.86 (1H, m)
1″
105.3
75.7
78.3
71.3
77.8
62.6
102.8
74.9
78.2
71.3
77.8
62.6
2″
3″
4″
5″
6″
5
(
(
3.80 (1H, dd, 2.0, 12.0)
3
3.85 (6H, s)
5.92 (2H, s)
3.80 (1H, dd, 2.0, 12.0)
3.65 (1H, dd, 7.0, 12.0)
3.86 (3H, s)
.65 (1H, dd, 7.0, 12.0)
2 × OCH
OCH
3
57.1
102.4
55.5
102.4
-
2
-
5.92 (2H, s)
2
.4. Acid hydrolysis
Compound 1 (1 mg) was treated with 2 mol/L aqueous HCl (2 mL)
sealed flask, heating in water bath, 80 °C, 3 h). Then the acidic aqueous
(
mixture was dried, water (2 mL) was added, and the mixture was ex-
tracted with EtOAc (3 × 2 mL). The aqueous layer was subjected to the
chiral-phase HPLC analysis under the following conditions: The car-
bohydrate products of the hydrolysis of 1 was separated and detected
using a CHIRALPAK AD-H column (250 × 4.6 mm) using n-hexane:
EtOH: TFA (750:250:0.25) as the mobile phase (0.5 mL min ) using an
evaporative light scattering detector (ELSD). D-Glucose was detected in
the acid hydrolysates of compound 1 by comparing the retention times
2
.2. Plant materials
Dendranthema morifolium (Ramat.) S. Kitam was collected from the
Jiaozuo, Henan Province, China, in 2015, and identified by Prof. Sui-
qing Chen (Henan University of Chinese Medicine). A voucher spe-
cimen (No. 20150715A) was deposited in the Research Department of
Natural Medicinal Chemistry, School of Pharmacy, Henan University of
Chinese Medicine.
−
1
of their derivatives with those of known D-glucose derivatives (t
R
,
18.3 min) prepared in the same manner. Glucose samples for compar-
ison were procured from Shanghai Yuanye Biotechnology Co., Ltd.,
China.
2.3. Extraction and isolation
The plant Chrysanthemum morifolium Ramat (11.2 Kg) was extracted
2
.5. Cell culture and treatment
using 50% acetone (25 L × 3) thrice in a flash extractor at room
temperature (25 °C). The filtered solution was concentrated under re-
duced pressure to give an extract (2.5 kg). The extract was concentrated
further, under reduced pressure to give brown syrup, which was added
with water (9 L) and extracted successively by partitioning with pet-
roleum ether (5 × 3 L), EtOAc (5 × 104 L), and n-BuOH (5 × 4 L); the
extracts were kept separately. The EtOAc-soluble fraction (352.0 g) was
passed through a silica gel (1.0 Kg) open column (30 cm × 16 cm i.d.)
The H9c2 cardiomyocytes were obtained from the American Type
Culture Collection (Manassas, VA, USA). The cells were cultured in
DMEM at 37 °C under a humidified atmosphere of 5% CO2 in air. Heat
inactivated FBS (10%) and antibiotics penicillin/streptomycin (1%)
were included in the culture. H9c2 cells were seeded on 96-well plates
(
5000 cells/well, 100 μL) and allowed to adhere overnight. Then, the
cells were divided into the control group, model group (LPS 20 μg/mL,
Sigma, USA), compound 1 group (10 μM, LPS 20 μg/mL) and compound
2
eluted with CH
:1, 30L) to provide fractions A–D on the basis of TLC analysis. Fraction
C (31.0 g) was separated on silica gel CC again, eluting with CH Cl
MeOH (80:1, 60:1, 40:1, 20:1, 10:1, and 5:1), to obtain fractions C1-C6.
Fraction C4 was separated by MCI using gradient elution (100% H O to
0% MeOH) to give seven fractions (Fr.C4a−Fr.C4g) based on TLC
analysis. Fr.C4f was subjected to Sephadex LH-20 CC by eluting with
MeOH: H O (70:30) to afford Fr.C4f1 and Fr.C4f2. Fr.C4f2 was further
purified by RP-C18 HPLC [YMC-Pack ODS-AA, CH CN-H O (23:77, v/
v) containing 0.2% TFA] to afford compound 1 (t 62.0 min, 2.19 mg)
and 2 (t 57.3 min, 2.80 mg).
2 2
Cl /MeOH (v/v, 50:1, 30L→ 30:1, 30L →20:1, 30L →
5
group (10 μM, LPS 20 μg/mL). Twenty-four hours later, the cell
2
2
-
morphology was observed and the cell vitality was detected by the MTT
assay (Amresco, Seattle, United States). Real Time Cellular Analysis
2
(
[
RTCA, Acea Biosciences, Inc.) was used to calculate EC50 of A1 and D2
19].
4
2
3
2
2.6. Cytometric bead array (CBA) assay
R
R
The cell processing was performed in a similar manner as mentioned
2

M. Zeng, et al.
Archives of Biochemistry and Biophysics 690 (2020) 108506
Fig. 1. Structures of compounds 1 (A1) and 2 (D2).
Fig. 2. HMBC and NOESY configuretion of compounds 1 (A1).
in section 2.5. Cell supernatant levels of TNF-α, IL-6, IFN-γ were
measured by CBA mice Inflammation Kit (BD Biosciences, Heidelberg,
Germany). 50 μl of cell supernatant was incubated with 50 μl of anti-
TNF-α (558299, BD), anti-IL-6 (558301, BD), anti–IFN–γ (558296, BD)
antibody (which was conjugated to beads) and 50 μl of the PE-labeled
anti-TNF-α, anti-IL-2, anti–IFN–γ and anti-IL-10 in the dark at room
temperature for 2 h with sufficient shaking. After washing with CBA
wash buffer, the samples were resuspended in 500 μl of CBA wash
buffer and analyzed immediately using a flow cytometry (FACS Aria III,
BD Biosciences, USA) and the Cell Quest software (BD).
Ltd, China). Cells in each group cultured on 6-well plates (same as 2.7.)
were trypsinized and collected by centrifugation. DCFH-DA (1 μM) was
added to the H9c2 cells and incubated at 37 °C for 20 min.
Subsequently, H9c2 cells were washed thrice with PBS, and thee DCF
fluorescence was measured by flow cytometry [20].
2.9. Cellular immunofluorescence
Assay for cellular immunofluorescence was performed in 96-well
plates (E190236X, PerkinElmer, USA). After treatment (same as section
.5.), the cells were fixed with 4% paraformaldehyde for 20 min at
2
2
.7. Apoptosis assay
room temperature. The cell monolayers were blocked for 90 min and
then incubated with primary antibodies (TLR4 ab22048, p-cJUN
ab30620, p-P65 ab86299, p-IRF3 SAB4503926, cleaved caspase-3
ab214430) diluted in blocking buffer (1:200, 50 μl) overnight at 4 °C.
After washing with PBS with Tween- 20 (PBST) buffer, the cell layers
were stained with the anti-mouse IgG or anti-rabbit IgG (1918277 or
1981155, 1:500, 50 μl; ThermoFisher Scientific, USA) for 1 h at room
temperature, rinsed, and scanned using an High-content imaging
system (Opera Phenix, PerkinElmer, USA). The relative protein ex-
pression level was normalized against the Harmony 4.8.
H9c2 cells were seeded on 6-well plates (5 × 105 cells/well) and
allowed to adhere overnight. Anti-apoptotic effects of compound 1 and
compound 2 (10 μM) on H9c2 cells were evaluated by Annexin V/PI
double staining according to the manufacturer's instructions (BD
Biosciences 556547, United States). Briefly, cells were harvested after
2
were then incubated in 100 μL labeling solution (5 μL of Annexin V-
FITC, 5 μL of PI, 10 μL of 10X binding buffer, and 80 μL of binding
buffer) in darkness at room temperature for 15 min. After that, 400 μL
of 1X binding buffer was added to stop the staining reaction. Flow cy-
tometric analyses were performed on a FACS AriaIII (BD Biosciences,
USA) utilizing 10,000 events. Three independent experiments were
performed.
5
4 h treatment (LPS 20 μg/mL, compound 10 μM), and 1 × 10 cells
2.10. Western blot
H9c2 cells were seeded and treated as described in Section 2.9. Cell
extracts were prepared, electrophoresed under denaturing conditions.
The proteins were transferred onto polyvinyldifluoridine membranes.
The membranes were washed in PBST and incubated over night at 4 °C
with corresponding primary antibodies (p-P65 ab86299, p-c-JUN
ab32385, SAB4503906 Sigma). The membranes were then incubated
with secondary antibodies (goat anti-rabbit 925-68071, goat-mouse
925-32210, Li-COR, MO, USA) and the intensity of the proteins was
2.8. Determination of ROS levels
The production of ROS in the H9C2 cells was fluorometrically
monitored using the nonfluorescent probe, 2,7-dichlorofuorescein dia-
cetate (DCFH-DA) (CA1410; Beijing Solarbio Science & Technology Co.,
3

M. Zeng, et al.
Archives of Biochemistry and Biophysics 690 (2020) 108506
Fig. 3. Effects of A1 and D2 on the production of cytokines in LPS-stimulated H9c2 cells using flow cytometry. The cells were incubated with the indicated
concentrations of A1 and D2 (10 μM) for 1 h and then stimulated with LPS (20 μg/mL) for 24 h. Cytokines production was quantified by CBA using fowcytometry. A:
Flow diagram. B: TNF-α. C: IL-6. D: IFN-γ. x ± SD, n = 4, *p < 0.05, **p < 0.01 vs. model group.
and ether groups (1202, 1123 and 1036 cm 1). The 1H- and 13C NMR
−
quantified using Odyssey (Clx, Li-COR Biosciences, USA).
spectra indicated that it was an unsymmetrically substituted fur-
1
ofurano-lignan. The H NMR signals of an ABX system at δ 6.87 (1H, d,
2
.11. Molecular docking
J = 2.0 Hz, H-2′), 6.84 (1H, d, J = 8.0 Hz, H-5′) and 6.77 (1H, dd,
J = 2.0, 8.0 Hz, H-6′), a 1,3,4,5-substituted aromatic ring at 6.70 (2H,
s, H-2, 6), a methylenedioxy group at δ
anomeric proton of the β-glucopyranose moieties resonated at δ
1H, d, J = 8.0 Hz, H-1″) and two methoxygroups at δ 3.85 (6H, s,
× OCH
). The 13C NMR spectroscopic data of 1 was similar to those
of (1R,2S,5R,6R)-5′-O-methylpluviatilol [22], except for the additional
signals associated with a glucopyranosyl unit concluded the signals
δ105.3, 75.7, 78.3, 71.3, 77.8, 62.6. On acid hydrolysis, compound 1
gave D-(+)-glucose, which was identified by HPLC with standard D-
The TLR4-MD-2 complex was used as the receptor (PDB: 5IJD),
5.92 (2H, s, -OCH
2
-), an
which was downloaded from the ZINC database (ID: ZINC01280591).
Since TLR4 needs to bind to the MD-2 protein for the combined TLR4-
MD-2 complex to recognize LPS and further induce the activation of the
TLR4 protein, the cavity in MD-2 of the TLR4-MD-2 complex was
treated as a docking site. Molecular docking was performed using the
AutoDock 4.2.6 software.
H
H
4.80
(
H
2
3
2.12. Statistical analysis
(
+)-glucose. The cross-peak between H-1″ of glucose and C-4 of the
aglycon HMBC spectrum indicated that the glucose unit was connected
to C-4 of the compound. The coupling constant between H-7 and H-8
was measured to be 5.0 Hz, which resulted in an erythro configuration
at H-7 and H-8. Also, the NOE cross peaks observed between H-7 and H-
Data were analyzed using SPSS 20.0 (IBM, NY, USA). Statistical
significance was assessed in comparison with the respective control for
each experiment using one-way analysis of variance (ANOVA). P values
less than 0.05 were accepted as significant.
8
indicated the relative configuration to be cis. The relative config-
uration of H-7′ and H-8′ was also indicated to be cis through the above
methods. In addition, the absolute configuration of 1 was defined by CD
spectrum. The CD spectrum of 1 showed similar Cotton effects (nega-
tive at 199 nm and positive at 212, 239, 278 nm) to that of
3
. Results
3.1. Identification
(
1R,2S,5R,6S) -(−)-methylpiperitol [23,24]. Therefore, the asymmetric
Compound 1 was obtained as a pale yellow amorphous powder,
centers of 1 were 7S,7′S,8R,8′R-configuration. From these data, the
structure of 1 was established and named as Dendranlignan A. In ad-
dition, the HMBC and NOESY configuretion of compounds 1 (A1) were
shown in Fig. 2.
The NMR spectroscopic data of compound 2 (Table 1) was similar to
that of compound 1, except for the substitution types of benzene rings.
with the molecular formula of C27
spectral data and the molecular ion peak at m/z = 571.1771 [M+Na]
calcd.: 571.1791) in HR-ESI-MS. Compound 1 displayed a similar UV
absorption spectrum to that of furofurano ligands at 206 (log ε 2.42),
28 (log ε 0.59) and 284 (log ε 0.23) nm [21]. The IR spectrum showed
H
32
O
12 deduced from the NM+R
(
2
−
1
−1
absorptions for hydroxyl (3445 cm ), methoxygroup (2925 cm
)
4

M. Zeng, et al.
Archives of Biochemistry and Biophysics 690 (2020) 108506
Fig. 4. Effects of A1 and D2 on ROS and apoptosis in LPS-induced H9c2 cells using flow cytometry. The cells were incubated with the indicated concentrations of A1
and D2 (10 μM) for 1 h and then stimulated with LPS (20 μg/mL) for 24 h. ROS and apoptosis were quantified by fowcytometry. A: ROS. B: apoptosis. C: Quantization
of ROS. D: Quantization of Apoptosis. E: Cleaved caspase-3. F: EC50 of A1 and D2. MD2-TLR4-IN-1, positive control compound, inhibitors for TLR4. x ± SD, n = 4,
*
p < 0.05, **p < 0.01 vs. model group.
The 1H NMR data of 2 (Table 1) indicated the presence of two ABX
patterns [δ 6.87 (1H, d, J = 2.0 Hz, H-2), 6.77 (1H, d, J = 8.0 Hz, H-
), 6.83 (1H, dd, J = 2.0, 8.0, H-6) and 7.01 (1H, d, J = 2.0, H-2′), 7.13
1H, d, J = 8.0, H-5′), 6.90 (1H, dd, J = 2.0, 8.0, H-6′)]. According to
determined to lantibeside D compared with the literature [21], and the
structure was shown in Fig. 1.
H
5
(
1
3
analyzing the C NMR, HSQC and HMBC spectrum, compound 2 was
5

M. Zeng, et al.
Archives of Biochemistry and Biophysics 690 (2020) 108506
Fig. 5. Effects of A1 and D2 on the levels of TLR4, p-cJUN, p-P65, p-IRF3 in LPS-stimulated H9c2 cells. Cells were treated with A1 and D2 at the indicated
concentrations (10 μM) for 1h and then stimulated with LPS for 60 min. Total and phosphorylated forms of corresponding proteins were detected by cellular
immunofluorescence. A: Cell immunofluorescence. B: p-P65/P65. C: p-c-JUN/c-JUN. D: p-IRF3/IRF3. x ± SD, n = 4, *p < 0.05, **p < 0.01 vs. model group.
3
.2. A1 and D2 decreased the production of inflammatory cytokines in LPS-
markedly increased after 1 h of LPS stimulation. A1 and D2 treatment
significantly reduced their nuclear localization and the levels of phos-
phorylated(Fig. 5), suggesting that A1 and D2 inhibits TLR4 signaling.
However, we did not observe any significant influence of A1 and D2 on
the protein expressions of TLR4 (Fig. 5), MyD88 and TRIF (data not
shown).
induced H9c2 cells
To assess the effect of A1 and D2 on the proinfammatory cytokine
production, the levels of TNF-α, IL-6, IFN-γ were evaluated by CBA
using fowcytometry. As shown in Fig. 3, LPS significantly elevated the
levels of inflammatory cytokines TNFα, IL-6, IFN-γ while A1 and D2
attenuated LPS-induced changes in the levels of inflammatory cytokines
to different degrees in the LPS-induced H9c2 cells.
3.5. Interaction between A1, D2 and MD-2 active-site residues
From the molecular docking analysis, it was revealed that the hy-
3
cells
.3. Effects of A1 and D2 on ROS and apoptosis in LPS-stimulated H9c2
drophobic cavity in MD-2 that recognized LPS was large enough to
occupy A1 or D2, and formed strong hydrophobic interactions with the
ligand binding site (A1: LEU78, ARG90, GLU437, ARG434, SER413. D2:
GLU92, ARG90, PHE126, PHE438, SER413, ARG434). After stable
binding in the cavity, A1 and D2 occupied the LPS binding site of TLR4-
MD-2 (Fig. 6).
The results of flow cytometry illustrate that the levels of ROS in LPS-
induced H9c2 cells was significantly increased, while A1 and D2 pre-
treatment significantly reduced this increase (Fig. 4A, C). Moreover,
flow cytometry analyses show that the rate of LPS-induced H9c2 cell
apoptosis was significantly higher than that in the control group
4. Discussion
(
p < 0.05). A1 and D2 significantly reduced the apoptosis of LPS-in-
duced H9c2 cells (Fig. 4B, D). In addition, A1 and D2 significantly in-
creased the cell viability of LPS-induced H9c2, results of EC50 indicated
that A1 (1.97 μM) and D2 (2.36 μM) were likely to be inhibitors of TLR4
Dendranthema morifolium (Ramat.) (Flos chrysanthemum) is
a
commonly used traditional Chinese herb medicinal for treating in-
flammatory disorders such as acute lung injury [25] and colitis [26].
Although, the literature shows that Huai Flos chrysanthemum has its
use in various inflammatory conditions, there is still a lack of research
on the anti-inflammatory effect and its mechanism of action. Our la-
boratory has been engaged in the research on the chemical components
and pharmacological effects. In our earlier report, it has been clear that
8 compounds isolated from Huai Flos chrysanthemum have protective
effects on LPS-induced cardiomyocytes. Based on the previous research,
(
Fig. 4F).
3.4. Effects of A1 and D2 on the expression of TLR4, p-cJUN, p-P65, p-
IRF3 in LPS-induced H9c2 cell
The nuclear protein levels of IRF3, NF-kB heterodimer components
(
p65) and one of the AP-1 components (c-Jun) was found to be
6

M. Zeng, et al.
Archives of Biochemistry and Biophysics 690 (2020) 108506
Fig. 6. The binding mode of A1 and D2 with TLR4-MD2 based on molecular simulation. A1 and D2 can bind to the active pocket of MD2. A: 3D stereogram. B: 2D
stereogram.
this study further separated the chemical constituents of HuaiFlos
chrysanthemum and defined its medicinal substance basis in the pro-
tection of the cardiomyocytes.
P65 and AP-1) and MyD88-independent (TRIF-dependent, resulting in
the phosphorylation and nuclear localization of IRF3) pathways
[28–30]. The MyD88-dependent pathway is responsible for the proin-
flammatory cytokine expression, while the MyD88-independent
pathway mediates the induction of Type I interferons and interferon-
inducible genes. In this study, the nuclear protein levels of IRF3, NF-kB
heterodimer components (p65), and one of the AP-1 components (c-
Jun) markedly increased after 1 h of LPS stimulation. On the other
hand, compound A1 and D2 pre-treatment significantly reduced their
nuclear localization, suggesting that A1 and D2 inhibited the TLR4
signaling.
Moreover, we observed that A1 and D2 inhibitory effects on c-JUN,
P65 and IRF3 phosphorylation were reduced by LPS application.
However, we did not observe significant influence of A1 and D2 on the
protein expressions of TLR4 (Fig. 3), MyD88 and TRIF (data not
shown). The possibility that compounds A1 and D2 affected the LPS
binding to TLR4 and/or led to the dimerization of TLR4, needs to be
further investigated. By molecular docking analysis, it was revealed
that the hydrophobic cavity in MD-2 that recognized LPS was large
enough to occupy A1 and D2 form strong hydrophobic interactions with
the active site residues (A1: LEU78, ARG90, GLU437, ARG434, SER413,
D2: GLU92, ARG90, PHE126, PHE438, SER413, ARG434). After stable
binding in the cavity, A1 and D2 occupied the LPS binding site of TLR4-
In this study, a new bisepoxylignan (A1) and a known compounds
lantibeside (D2) was isolated from the flowers of Dendranthema mor-
ifolium (Ramat.) kitam, determined on the basis of extensive spectro-
scopic methods, including 1D-, 2D-NMR and MS data. Additionally, A1
and D2 attenuated the LPS-induced changes in the levels of in-
flammatory cytokines (TNF-α, IL-2, IFN-γ, IL-17A), ROS, and apoptosis
(
reduced caspase-3 activation) to different degrees in H9c2 cells, sug-
gesting that A1 and D2 have protective effects on LPS-induced cardio-
myocytes.
TLR is an important member of innate immunity. It plays an im-
portant role in identifying pathogens and harmful factors in the body's
immune system, and is associated with inflammatory reactions and
autoimmune diseases. TLR4, a key member of the TLR family, has a
highest expression in the cardiomyocytes, when compared with other
TLR members, and is involved in the pathophysiological processes of
many diseases, including cardiovascular diseases [27]. TLR4 is a class I
transmembrane receptor expressed on the cell surface in response to
ligands of many different structural families, activated by ligand in-
duced dimerization. TLR4 signaling has been divided into MyD88-de-
pendent (resulting in the phosphorylation and nuclear localization of
7

M. Zeng, et al.
Archives of Biochemistry and Biophysics 690 (2020) 108506
MD-2.
Food 13 (2010) 306–311.
[
[
10] C.K. Lii, Y.P. Lei, H.T. Yao, Y.S. Hsieh, et al., Chrysanthemum morifolium Ramat.
reduces the oxidized LDL-induced expression of intercellular adhesion molecule-1
and E-selectin in human umbilical vein endothelial cells, Ethnopharmacol 128
(2010) 213–220.
11] J. Yuan, J. Huang, G. Wu, et al., Multiple responses optimization of ultrasonic-
assisted extraction by response surface methodology (RSM) for rapid analysis of
bioactive compounds in the flower head of Chrysanthemum morifolium Ramat, Ind.
Crop. Prod. 74 (2015) (2015) 192–199.
In conclusion, we for the first time discovered a new bisepoxylignan
compound A1, and found that this compound has a potential to inhibit
inflammation by inhibiting TLR4 signaling.
Authorship contribution statement
Mengnan Zeng, Xiaoke Zheng and Meng Li designed and performed
the experiments and analyzed the raw data. Yangang Cao, Yingjie Chen,
Jingke Zhang, Beibei Zhang assisted with the experiments. Zhiling Yu
and Weisheng Feng supervised the project.
[12] L. Qu, J.Y. Ruan, L.Y. Jin, et al., Xanthine oxidase inhibitory effects of the con-
stituents of Chrysanthemum morifolium stems, Phytochem. Lett. 19 (2017) 39–45.
[
13] Chinese Pharmacopoeia Committee, “Pharmacopoeia of the People's Republic of
China,” Part 1, China Medical Science Press, Beijing, 2015, pp. 310–311.
[14] W.J. Chen, M.N. Zeng, M. Li, et al., Four new sesquiterpenoids from Dendranthema
morifolium (Ramat.) kitam flowers, Phytochem. Lett. 23 (2018) 52–56.
[
15] B.B. Zhang, M.N. Zeng, M. Li, et al., Guaiane-type sesquiterpenoids from
Dendranthema morifolium (Ramat.) S. Kitam flowers protect H9c2 cardiomyocyte
from LPS-induced injury, Nat. Prod. Commun. 14 (2019), https://doi.org/10.1177/
Declaration of competing interest
1
934578X19864179.
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.
[
16] Y. Alhamdi, S.T. Abrams, Z. Cheng, et al., Circulating histones are major mediators
of cardiac injury in patients with sepsis, Crit. Care Med. 43 (2015) 2094–2103.
17] P.C. Trivedi, J.J. Bartlett, L.J. Perez, et al., Glucolipotoxicity diminishes cardio-
myocyte TFEB and inhibits lysosomal autophagy during obesity and diabetes,
Biochim. Biophys. Acta 1861 (2016) 1893–1910.
[
Acknowledgments
[18] A.N. Velic, D. Laturnus, J. Chhoun, et al., Diabetic basement membrane thickening
does not occur in myocardial capillaries of transgenic mice when metallothionein is
overexpressed in cardiac myocytes, Anat. Rec. 296 (2013) 480–487.
This study was supported by the National Key Research and
Development Project (2019YFC1708802, 2017YFC1702800) and
Henan province high-level personnel special support “ZhongYuan One
Thousand People Plan” -Zhongyuan Leading Talent (ZYQR201810080)
and the Central Government Guide Local Science and Technology
Development Funds ([2016]149).
[
19] Y.G. Cao, Y.L. Zhang, M.N. Zeng, et al., Renoprotective mono- and triterpenoids
from the fruit of Gardenia jasminoides, J. Nat. Prod. 83 (2020) 1118–1130.
20] M.N. Zeng, M. Qi, Y. Wang, et al., 5-O-methyldihydroquercetin and cilicicone B
isolated from Spina Gleditsiae ameliorate lipopolysaccharide-induced acute kidney
injury in mice by inhibiting inflammation and oxidative stress via the TLR4/
MyD88/TRIF/NLRP3 signaling pathway, Int. Immunopharm. 80 (2020) 106194.
[
[21] T. Li, X.J. Hao, Q.Q. Gu, W.M. Zhu, Minor furofurano lignans from the Tibetan herb,
Lancea tibetica, Planta Med. 74 (2008) 1391–1396.
[
22] T.H. Quang, N.T. Ngan, C.V. Minh, P.V. Kiem, et al., Anti-inflammatory and PPAR
transactivational effects of secondary metabolites from the roots of Asarum sie-
boldii, Bioorg. Med. Chem. Lett 22 (2012) 2527–2533.
References
[
[
[
23] H. Wu, Z. Li, Q. Zhang, Y. Pei, Application of CD in study of the absolute config-
uration of lignans, J. Shenyang Pharm. Univ. 27 (2010) 587–594.
[
1] J.D. Sammon, D.E. Klett, A. Sood, et al., Sepsis after major cancer surgery, J. Surg.
Res. 193 (2015) 788–794.
24] O. Hofer, R. Schoim, Stereochemistry of tetrahydrofurofuran derivatives-circular
dichroism and absolute conformation, Tetrahedron 37 (1981) 1181–1186.
25] X.H. Zhang, Study on the Changes of Chemical Composition, Anti-inflammatory
Efficacy and Mechanism of Different Cultivars of Fios Chrysanthemi, Nanjing
University of Chinese Medicine, Nanjing, 2014.
26] J.H. Tao, J.A. Duan, W. Zhang, et al., Polysaccharides from Chrysan-themum
morifolium Ramat ameliorate colitis rats via regulation of the metabolic profiling
and NF-kB/TLR4 and IL-6/JAK2/STAT3 signaling pathways, Front. Pharmacol. 9
[
[
2] M. Merx, C. Weber, Sepsis and the heart, Circulation 116 (2007) 793–802.
3] P. Yang, Y. Han, L. Gui, et al., Gastrodin attenuation of the inflammatory response
in H9C2 cardiomyocytes involves inhibition of NF⁃κB and MAPKs activation via the
phos⁃phatidylinositol 3⁃kinase signaling, Biochem. Pharmacol. 85 (2013)
1
124–1133.
[
[
4] M. Maeder, T. Fehr, H. Rickli, et al., Sepsis⁃associated myocardial dysfunction⁃-
diagnostic and prognostic impact of cardiac troponins and natriuretic peptides,
Chest 129 (2006) 1349–1366.
(2018) 746.
[
[
[
5] S.L. Zanotti, S.M. Hollenberg, Cardiac dysfunction in severe sepsis and septic shock,
[
[
27] Y.E. Wang, A.M. Abarbanell, J.L. Herrmann, et al., Toll-like receptor signaling
pathways and the evidence linking toll-like receptor signaling to cardiac ischemia/
reperfusion injury, Shock 34 (2010) 548–557.
Curr. Opin. Crit. Care 15 (2009) 392–397.
6] A. Ayala, M. Perl, F. Venet, et al., Apoptosis in sepsis:mechanisms,clinical impact
and potential therapeutic targets, Curr. Pharmaceut. Des. 14 (2008) 1853–1859.
7] H. Zhou, Y. Yuan, Y. Liu, et al., Icariin protects H9c2 cardiomyocytes from lipo-
polysaccharide⁃induced injury via inhibition of the reactive oxygen species⁃de-
pendent c-Jun N-terminal kinases/nuclear factor ⁃ kappa B pathway, Mol. Med. Rep.
28] X. Cheng, Y.L. Yang, H. Yang, et al., Kaempferol alleviates LPS-induced neuroin-
flammation and BBB dysfunction in mice via inhibiting HMGB1 release and down-
regulating TLR4/Myd88 pathway, Int. Immunopharm. 56 (2018) 29–35.
29] N.J. Gay, M.F. Symmons, M.A. Gangloff, Assembly and locali-zation of Toll-like
receptor signalling complexes, Nat. Rev. Immunol. 14 (2014) 546–558.
30] M. Sasai, M. Tatematsu, H. Oshiumi, et al., Direct binding of TRAF2 and TRAF6 to
TICAM-1/TRIF adaptor participates in activation of the Toll-like receptor3/4
pathway, Mol. Immunol. 47 (2010) 1283–1291.
[
[
1
1 (2015) 4327–4332.
[
8] Y. He, Z. Du, H. Lv, Q. Jia, Z. Tang, et al., Green synthesis of silver nanoparticles by
Chrysanthemum morifolium Ramat. Extract and their application in clinical ultra-
sound gel, Int. J. Nanomed. 8 (2013) 1809–1815.
[9] G.H. Lin, L. Lin, H.W. Liang, X. Ma, et al., Antioxidant action of a Chrysanthemum
morifolium extract protects rat brain against ischemia and reperfusion injury, Med.
8