Suppressive activities of KC1–3 on HMGB1-mediated septic responses

Article abstract of DOI:10.1016/j.bcp.2019.02.027

In the present study, several decursin analogues (KC1–3) were synthesized and evaluated in terms of their anti-septic activities on high mobility group box 1 (HMGB1)-mediated septic responses and survival rate in a mouse model of sepsis. KC1 and KC3, but not KC2, significantly reduced HMGB1 release in lipopolysaccharide (LPS)-activated human umbilical vein endothelial cells (HUVECs) and attenuated the cecal ligation and puncture (CLP)-induced release of HMGB1. Additionally, in vitro analyses revealed that KC1 and KC3 both alleviated HMGB1-mediated vascular disruptions and inhibited hyperpermeability in mice, and in vivo analyses revealed that KC1 and KC3 reduced sepsis-related mortality and tissue injury. Taken together, the present results suggest that KC1 and KC3 both reduced HMGB1 release and septic mortality and, thus, may be useful for the treatment of sepsis.

Full text of DOI:10.1016/j.bcp.2019.02.027

Biochemical Pharmacology 163 (2019) 260–268
Contents lists available at ScienceDirect
Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm
Suppressive activities of KC1–3 on HMGB1-mediated septic responses
Wonhwa Leea,1, O. Yuseok , Changhun Lee , So Yeon Jeong , Jee-Hyun Lee ,
b,1
c
c
d
e
b,d,⁎
, Jong-Sup Baec,⁎
Moon-Chang Baek , Gyu-Yong Song
Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea
College of Pharmacy, Chungnam National University, Daejon 34134, Republic of Korea
a
b
c
College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, BK21 Plus KNU Multi-Omics Based Creative Drug Research Team, Kyungpook National
University, Daegu 41566, Republic of Korea
d
AREZ Co. Ltd., 197 Songam-ro, Sejong 30066, Republic of Korea
Department of Molecular Medicine, CMRI, School of Medicine, Kyungpook National University, Daegu 700-422, Republic of Korea
e
A R T I C L E I N F O
A B S T R A C T
Keywords:
KC1-3
Endothelium
HMGB1
Sepsis
In the present study, several decursin analogues (KC1–3) were synthesized and evaluated in terms of their anti-
septic activities on high mobility group box 1 (HMGB1)-mediated septic responses and survival rate in a mouse
model of sepsis. KC1 and KC3, but not KC2, significantly reduced HMGB1 release in lipopolysaccharide (LPS)-
activated human umbilical vein endothelial cells (HUVECs) and attenuated the cecal ligation and puncture
(CLP)-induced release of HMGB1. Additionally, in vitro analyses revealed that KC1 and KC3 both alleviated
HMGB1-mediated vascular disruptions and inhibited hyperpermeability in mice, and in vivo analyses revealed
that KC1 and KC3 reduced sepsis-related mortality and tissue injury. Taken together, the present results suggest
that KC1 and KC3 both reduced HMGB1 release and septic mortality and, thus, may be useful for the treatment of
sepsis.
1. Introduction
hand, HMGB1-neutralizing antibodies confer protection against lethal
endotoxemia and sepsis even when administered 24 h after the onset of
Sepsis is an overwhelming systemic inflammatory response to se-
sepsis, which suggests that HMGB1 is a critically important late med-
iator of lethal sepsis [7–9]. Thus, therapeutic agents capable of in-
hibiting HMGB1 release might be potential candidates for the treatment
of lethal systemic inflammatory diseases.
vere infections that is a common cause of morbidity and mortality de-
spite recent advances in antibiotic therapies and intensive care [1].
Sepsis-associated mortality is highly related to the development of
multi-organ dysfunction syndrome (MODS) or multiple organ failure
2,3]. Although patients with sepsis may have profound life-threatening
hypoxemia, most die from MOF rather than oxygen deficiencies [4].
The lungs and kidney are the organs most commonly affected by sepsis
Angelica gigas Nakai, also known as Cham-Dang-Gui, is a Korean
traditional herbal medicine and one of the most popular herbal re-
medies in many Asian countries, including Korea, Japan, and China
(Angelica sinensis). Angelica gigas Nakai has been extensively studied and
used for the treatment of anemia, as a sedative, and as an anodyne or
tonic agent. The major bioactive components of Angelica gigas Nakai
include (+)-decursin and (+)-decursinol angelate [10], which have
been shown to exert various pharmacological activities, including anti-
cancer, anti-oxidant, wound healing, antibacterial, neuroprotective,
and anti-inflammatory effects [10–15]. Thus, considerable effort has
been directed towards the synthesis of several types of (+)-decursin
analogues with various pharmacological activities, including the in-
hibition of melanin formation, anti-asthma effects, and anti-tumor ac-
tivities [16–18]. Recently, ongoing research from our laboratory aimed
[
[
2], but acute sepsis-related kidney injury subsequently mediates a
systemic inflammatory response that causes remote damage to the
heart, lung, brain, spleen, liver, and gut [3]. Although the pathogenesis
of sepsis is rather complex, this process is partly mediated by en-
dotoxins that stimulate macrophages and/or monocytes to sequentially
release early (e.g., tumor necrosis factor [TNF]-α and interleukin [IL]-
1
β) and late (e.g., high mobility group box 1 [HMGB1]) proin-
flammatory mediators [5,6]. In animal models of endotoxemia and
sepsis, circulating HMGB1 levels plateau at 24–36 h, which differ-
entiates it from TNF-α and other early cytokines [7,8]. On the other
⁎
Corresponding authors at: College of Pharmacy, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejon 34134, Republic of Korea (G.-Y. Song).
College of Pharmacy, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 41566, Republic of Korea (J.-S. Bae).
E-mail addresses: gysong@cnu.ac.kr (G.-Y. Song), baejs@knu.ac.kr (J.-S. Bae).
1
First two authors contributed equally to this work.
https://doi.org/10.1016/j.bcp.2019.02.027
Received 2 January 2019; Accepted 25 February 2019
Available online 26 February 2019
0006-2952/ © 2019 Elsevier Inc. All rights reserved.

W. Lee, et al.
Biochemical Pharmacology 163 (2019) 260–268
at developing novel inhibitors of HMGB1 release from natural products
for sepsis treatment has identified novel synthetic (+)-decursin ana-
logues that were labeled KC1, KC2, and KC3. The present study de-
monstrated that KC1–3 decreased HMGB1 release from lipopoly-
saccharide (LPS)-activated human endothelial cells as well as increased
the survival of and reduced circulating HMGB1 levels in septic mice.
diethyl ether, the organic layer was dried with sodium sulfate and
concentrated in vacuo, and the residue was purified using flash silica
gel column chromatography to obtain (3-bromo-prophenyl)-benzene
(2d).
A solution of (+)-decursinol (0.41 mM) in N, N-dimethylformamide
anhydrous was cooled to −20 °C and added to (3-bromo-prophenyl)-
benzene (2d, 0.609 mM). After stirring the reaction mixture at −20 °C
for 24 h, it was quickly filtrated using silica gel short-column chroma-
tography with 5:1 n-hexane-ethyl acetate. After the solvent was re-
moved under reduced pressure, the residue was purified using silica gel
column chromatography (n-hexane:ethylacetate at a 8:1 to 4:1 gradient
elution) to obtain a decursin ether derivative (2, KC2) with the fol-
lowing characteristics: yield = 35.3%, white solid, mp: 143 °C,
2. Materials and methods
2.1. Method for the synthesis of (7S)-(+)-(E)-2-(pyridin-3-yl)ethenyl
carbamic acid, 8,8-dimethyl-2-oxo-6,7-dihydro-2H,8H-pyrano[3,2-g]
chromen-7-yl-ester (1, KC1)
2
5
To synthesize KC-1, triethylamine (TEA; 1.67 mM) and diphenyl
phosphoryl azide (DPPA; 1.67 mM) were added to an ice-cooled solu-
tion of trans-3-(3-pyridyl)acrylic acid (1a, 1.67 mM) in 5 mL of dry
benzene and the reaction mixture was stirred at room temperature for
R
f
= 0.39 (2:1 n-hexane–ethyl acetate); [α]D + 117.6 (c = 1, CHCl
3
);
1
H NMR(400 MHz, CDCl
3
): δ
H
7.56(d, J = 9.6 Hz, 1H), 7.38–7.23(m,
5H), 7.15(s, 1H), 6.76(s, 1H), 6.59(d, J = 16.0 Hz, 1H), 6.30–6.23(m,
1H), 6.20(d, J = 9.6 Hz, 1H), 4.34(dd, J = 6.0, 12.8 Hz, 1H), 4.21(dd,
J = 6.0, 12.4 Hz, 1H), 3.59(dd, J = 5.2, 7.6 Hz, 1H), 3.07(d, J = 4.8,
5
h. Then, the mixture was isolated by diluting the solution with cold
water and extracting it with ether. Next, the organic phase was dried
over anhydrous sodium sulfate and the solvent was removed under
reduced pressure to provide a crude product that contained aryl azide
16.0 Hz, 1H), 2.85(dd, J = 7.2, 16.4 Hz, 1H), 1.41(s CH
3
), 1.36(s, CH
3
);
13
C NMR (100 MHz, acetone‑d
132.9, 130.4, 129.6, 128.6, 127.5, 127.4, 118.3, 113.7, 113.6, 104.5,
6
) δ
C
161.2, 157.8, 155.3, 144.5, 137.9,
+
(
1b). The crude product was dissolved in dry benzene, heated at reflux
78.8, 76.4, 70.8, 27.8, 26.1, 22.2; ESI-MS: m/z = 363 [M+H] .
for 3 h; then, (+)-decursinol (1.11 mM), TEA (1.341 mM), and 4-(di-
methylamino)pyridine (4-DMAP; 0.447 mM) were added to the re-
actant, which contained isocyanate (1c). The mixture was stirred at
2.3. Method for the synthesis of (7S)-(+)-(E)-2-phenylethenyl carbamic
acid, 8,8-dimethyl-2-oxo-6,7-dihydro-2H,8H-pyrano[3,2-g]chromen-7-yl
–ester (3, KC3)
80 °C for 3 h, and cooled and concentrated under reduced pressure;
then, the residue was purified using a medium pressure liquid chro-
matography (MPLC) system (Yamazen, Osaka, Japan) with a silica gel
column (n-hexane:ethylacetate = 2:1–1:5 gradient elution) to obtain a
decursin carbamate derivative (1, KC1) with the following character-
To synthesize KC-3, TEA (3.37 mM) and DPPA (3.37 mM) were
added to an ice-cooled solution of trans-cinnamic acid (3a, 3.37 mM,
1 eq) in 5 mL of dry benzene and the reaction mixture was stirred at
room temperature for 5 h. Then, the mixture was isolated by diluting
the solution with cold water and extracting it with ether. Next, the
organic phase was dried over anhydrous sodium sulfate and the solvent
was removed under reduced pressure to provide a crude product that
contained aryl azide (3b). After the crude product was dissolved in dry
benzene and heated at reflux for 3 h, (+)-decursinol (2.25 mM), TEA
(2.70 mM), and 4-DMAP (0.90 mM) were added to reactant, which
contained isocyanate (3c), and the mixture was stirred at 80 °C for 3 h.
Then, the mixture was cooled and concentrated under reduced pressure
and the residue was purified using MPLC (Yamazen) with a silica gel
column (n-hexane:ethylacetate = 8:1–2:1 gradient elution) to obtain a
decursin carbamate derivative (3, KC3) with the following character-
istics: yield = 53.0%; ivory-white solid, mp: 125.9 °C, R = 0.12 (1:2 n-
f
2
5
1
hexane–ethyl acetate); [α] + 138.5667 (c = 3, CHCl
3
); H NMR
10.03(d, J = 10.4 Hz, NH), 8.44(s, 1H),
.30(dd, J = 1.2, 4.8 Hz, 1H), 7.92(d, J = 9.6 Hz, 1H), 7.75(d,
D
(
8
400 MHz, DMSO‑d
6
): δ
H
J = 7.6 Hz, 1H), 7.49(s, 1H), 7.250(m, 2H), 6.80(s, 1H), 6.26(d,
J = 9.6 Hz, 1H), 6.00(d, J = 14.8 Hz, 1H), 5.06(t, J = 3.6 Hz, 1H),
3
CH
1
1
.26(dd, J = 4.4, 18.0 Hz, 1H), 2.92(dd, J = 3.2, 17.6 Hz, 1H), 1.39(s,
1
3
3
), 1.32(s, CH
3
); C NMR(100 MHz, DMSO‑d
6
C
): δ 160.4, 156.0,
53.7, 153.3, 146.9, 146.8, 144.1, 132.5, 131.3, 129.7, 126.9, 123.7,
15.8, 112.8, 112.7, 106.6, 103.5, 76.9, 70.3, 27.2, 24.3, 23.6; IT-TOF/
+
MS 415.1281 [M+Na] .
2.2. Method for the synthesis of 3-(7S)-(+)-8,8-dimethyl-7-(3-phenyl-
allyloxy)-7,8-dihydro-6H-pyrano[3,2-g]chromen-2-one (2, KC2)
istics: yield = 13.8%; brown-white solid, mp: 112.4 °C, R
f
= 0.45 (1:1
); H NMR
25
1
n-hexane–ethyl acetate); [α] + 93.4467 (c = 3, CHCl
3
D
(
400 MHz, DMSO‑d
J = 10.1 Hz, 1H), 7.50(s, 1H), 7.25(m, 4H), 7.12(m, 2H), 6.80(s, 1H),
.26(d, J = 9.6 Hz, 1H), 6.01(d, J = 14.8 Hz, 1H), 5.05(s, 1H), 3.26(dd,
J = 3.6, 18.0 Hz, 1H), 2.92(d, J = 17.2 Hz, 1H), 1.39(s, CH3), 1.32(s,
6
):
δ
H
9.87(d, J = 10.4 Hz, –NH), 7.92(d,
To synthesize KC-2, a solution of trans-cinnamic acid (1a, 34.2 mM,
1
eq) and concentrated sulfuric acid (five drops) in MeOH (20 mL) was
6
warmed to reflux overnight. After cooling to room temperature, the
solvent was removed under reduced pressure and the residue was
purified using flash silica gel column chromatography to obtain 3-
phenyl-acrylic acid methyl ester (2b). A solution of 3-phenyl-acrylic
acid methyl ester (2b, 24.7 mM) in dichloromethane anhydrous was
cooled to −78 °C under nitrogen gas. Then, the mixture was slowly
added to a 1 M solution of diisobutylalluminum hydride (DIBAL-H) in
toluene (74 mM). After stirring the mixture at 0 °C for 1 h, it was added
to methanol (22 mL) and stirred at room temperature for 30 min. Then,
aqueous saturated Rochelle’s salt (88 mL) was added to the mixture and
it was vigorously stirred for 2 h. The reaction mixture was separated
with dichloromethane and dried sodium sulfate and the solvent was
removed under reduced pressure. The residue was purified by flash
silica gel column chromatography to obtain 3-phenyl-pro-2-pen-1-ol
1
3
CH
1
1
3
); C NMR(100 MHz, DMSO‑d
6
C
): δ 160.4, 156.0, 153.7, 153.4,
44.1, 136.6, 129.7, 128.7, 125.9 125.1, 124.9, 115.8, 112.8, 112.6,
10.4, 103.5, 76.9, 70.2, 27.3, 24.3, 23.6; IT-TOF/MS 390.1346 [M
+
+
Na] .
2.4. Cell culture and reagents
Primary human umbilical vein endothelial cells (HUVECs) were
obtained from Cambrex Bio Science (Charles City, IA) and maintained
using a previously described method [19,20]. In all experiments, the
HUVECs were used at cell culture passages 3–5. LPS (from Escherichia
coli), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(
2c, 7.45 mM), which was then dissolved in dichloromethane anhy-
(
(
MTT), Evans blue, crystal violet, 2-mercaptoethanol, the antibiotics
penicillin G and streptomycin), and dimethyl sulfoxide (DMSO) were
drous and added to a 1 M solution of boron tribromide in di-
chloromethane (2.61 mM) in an ice bath for 1 h. Next, the mixture was
added to ice water (50 mL) and stirred for 10 min. Subsequently, the
reaction mixture was separated using saturated sodium bicarbonate and
purchased from Sigma Chemical Co. (St. Louis, MO). Human re-
combinant HMGB1 was purchased from Abnova (Taipei City, Taiwan).
261

W. Lee, et al.
Biochemical Pharmacology 163 (2019) 260–268
2.5. Animals and the cecal ligation and puncture procedure
extracts, respectively.
For this study, male C57BL/6 mice (6–7 weeks old, 27 g) were
2.8. Cell viability assay
purchased from Orient Bio Co. (Sungnam, Republic of Korea) and
maintained as described previously [19]. The model of cecal ligation
and puncture (CLP)-induced sepsis was also prepared as described
previously [19]. Briefly, a 2-cm midline incision was made to expose
the cecum and adjoining intestine. The cecum was tightly ligated with a
An MTT assay was performed to determine cell viability, as de-
scribed previously [22]. The HUVECs were incubated with KC1–3 for
48 h.
3.0-silk suture approximately 5.0 mm from the cecal tip and then
2.9. Permeability assay
punctured once with a 22-gauge needle. Next, the cecum was gently
squeezed to extrude a small amount of feces from the perforation sites
and returned to the peritoneal cavity. The laparotomy site was stitched
with 4.0-silk in the absence of antibiotics or fluid resuscitation. In a
group of sham control animals, the cecum was exposed but not ligated
or punctured and then returned to the abdominal cavity. Next, a KC
compound was injected 24 h after the CLP procedure to evaluate
HMGB1 secretion (Fig. 2C), cell permeability (Fig. 3C), and leukocyte
migration (Fig. 4D). Alternatively, a KC compound was injected at 12
and 50 h after CLP to assess survival rate (Fig. 6). This protocol was
approved by the Animal Care Committee at Kyungpook National Uni-
versity prior to conducting the study (IRB No. KNU 2017-102).
For the spectrophotometric quantification of endothelial cell per-
meability in response to increasing concentrations of KC1–3 in vitro,
the flux of Evans blue-bound albumin across functional cell monolayers
was measured using a modified two-compartment chamber model, as
described previously [22]. For the in vivo experiment in the present
study, male mice were first treated with HMGB1 (2 μg/mouse, in-
travenous [i.v.]) for 16 h and then received i.v. injections of KC1–3
(0.15–0.58 mg/kg). Vascular permeability was expressed as μg of dye in
the peritoneal cavity per mouse and determined using a standard curve,
as described previously [22].
2.10. Cell-cell adhesion assay
2.6. Competitive enzyme-linked immunosorbent assay for HMGB1
The cellular adhesion of human neutrophils to HUVECs was de-
A competitive enzyme-linked immunosorbent assay (ELISA) was
termined as described previously [22,23]. Briefly, purified human
neutrophils (1.5 × 10 cells/mL, 200 μL/well) were labeled with Vy-
6
performed to determine HMGB1 concentrations in the cell culture
media or mouse serum as described previously [19]. The HUVEC
monolayers were first treated with LPS (100 ng/mL) for 16 h and then
incubated with KC1–3 for 16 h.
brant DiD dye and then added to washed and stimulated HUVECs.
HUVEC monolayers were treated with HMGB1 (1 μg/mL) for 16 h and
then incubated with KC1–3 for 6 h. The amounts of labeled cells added
were assessed by recording the fluorescence signal (total signal) using a
spectrometer equipped with a microplate reader (Tecan, Austria GmbH,
Grödig, Austria). After incubation at 37 °C for 60 min, nonadherent cells
were removed by washing them four times with PBS and then the
fluorescence signal was reassessed with the microplate reader (i.e.,
adherent signal). The percentage of adherent neutrophils was calcu-
lated using the following formula: % adherence = (adherent signal/
total signal) × 100.
2
.7. Preparation of cytoplasmic and nuclear extracts and Western blot
analyses
The cells were harvested rapidly by sedimentation and their nuclear
and cytoplasmic extracts were prepared on ice, as described previously
21]. Briefly, the cells were harvested and washed with 1 mL of buffer A
10 mM HEPES, pH 7.9, 1.5 mM MgCl , and 19 mM KCl) at 600×g for
min. Subsequently, the cells were resuspended in buffer A, cen-
[
(
5
2
trifuged at 600×g for 3 min, resuspended in 30 μL of buffer B (20 mM
HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl , and 0.2 mM
EDTA), rotated at 4 °C for 30 min, and then centrifuged at 13,000×g for
0 min. The supernatant was used as the nuclear extract. The nuclear
and cytosolic extracts were analyzed for protein content using the
Bradford assay. For the Western blot analyses, the cells were first rinsed
with ice-cold phosphate-buffered saline (PBS) and then treated with a
lysis buffer composed of 0.5% SDS, 1% NP-40, 1% sodium deox-
ycholate, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), and protease in-
hibitors.
2.11. In vitro migration assay
2
The migration of human neutrophils toward HUVECs was de-
termined as described previously [22,23]. Briefly, the migration assays
were performed in 6.5-mm Transwell plates that contained filters with a
2
4
pore size of 8 µm. The HUVECs (6 × 10 ) were cultured for 3 days to
obtain confluent endothelial monolayers. Prior to the addition of neu-
trophils to the upper compartment, the cell monolayers were treated
with HMGB1 (1 μg/mL) for 16 h and then incubated with KC1–3 for 6 h.
Subsequently, the Transwell plates were incubated at 37 °C for 2 h in
Equal sample volumes were mixed with 2 × loading dye and boiled
at 95 °C for 5 min. Samples of culture medium and whole cell lysate
were separated by electrophoresis in polyacrylamide gels of different
percentages depending on the size of the protein of interest. The gels
were transferred to polyvinylidene difluoride (PVDF) membranes via
semidry electrophoretic transfer at 20 mA for 2 h. Then, the PVDF
membranes were blocked at room temperature for 2 h in 5% bovine
serum albumin (BSA) and incubated with a primary antibody (1:500,
anti-IL-6, anti-TNF-α, anti-NF-κB p65, or anti-lamin B or -actin anti-
bodies; Santa Cruz Biotechnology Inc., Dallas, TX) in Tris-buffered
saline/Tween 20 (TBS-T) containing 5% BSA overnight at 4 °C.
Subsequently, they were incubated with a secondary antibody (1:5000)
in TBS-T containing 1% BSA at room temperature for 1 h. After in-
cubation, the membranes were washed three times with TBS-T, in-
cubated with Western blot enhanced chemiluminescence (ECL) detec-
tion reagents (Amersham; Piscataway, NJ), and exposed to Xomat AR
films (Eastman Kodak; Rochester, NY). The antibodies for actin and
lamin B were used as loading controls for the cytoplasmic and nuclear
2
5% CO . Then, the cells in the upper chamber were aspirated and the
non-migrating cells on top of the filter were removed using a cotton
swab. Neutrophils on the lower side of the filter were fixed with 8%
glutaraldehyde and stained with 0.25% crystal violet in 20% methanol
(w/v). Nine randomly selected high-power microscopic fields (200×)
were counted. All experiments were repeated twice per well on dupli-
cate wells and the results are presented as migration indices.
2.12. In vivo leukocyte migration assay
For the in vivo experiment in the present study, male mice were
anesthetized with 2% isoflurane (Forane; JW Pharmaceutical, Seoul,
South Korea) in oxygen via a small rodent gas anesthesia machine (RC2;
Vetequip, Pleasanton, CA) that was first administered in a breathing
chamber and then via facemask. The in vivo migration of leukocytes
was determined as described previously [22,23] and the mice were
allowed to breath spontaneously during the procedure. The mice were
treated with HMGB1 (2 μg/mouse, i.v.) for 16 h and then received an
262

W. Lee, et al.
Biochemical Pharmacology 163 (2019) 260–268
i.v. administration of KC (0.14–0.56 mg/kg). To assess leukocyte mi-
gration, the mice were sacrificed after 6 h and the peritoneal cavities
were washed with 5 mL of normal saline. The obtained samples of
peritoneal fluids (20 μL) were mixed with 0.38 mL of Turk’s solution
2.17. Purification of natural decursin
The natural compound decursin was purified using a previously
reported method [25]. Briefly, the dried powdered roots of Angelica
gigas (1 kg) were extracted with methanol (2 L) using an ultrasonic
apparatus. Upon removal of the solvent in vacuo, the methanolic ex-
(
0.01% crystal violet in 3% acetic acid) and the number of leukocytes
was counted under a light microscope.
tract yielded 75 g. This methanolic extract was then suspended in H
and partitioned successively with CH Cl . Silica gel column chroma-
tography of the CH Cl fraction (42 g) with a mixture of n-hexane-
CHCl -MeOH as an eluent afforded nine fractions. Fraction 3 (11.5 g)
was subjected to silica gel column chromatography with n-hex-
ane:ethylacetate (7:3) to obtain crude decursin. Next, the crude de-
cursin was recrystallized with EtOH to obtain pure decursin (3.2 g); the
purity of the decursin was determined using high-performance liquid
chromatography (HPLC; Fig. 7).
2
O
2
2
2
.13. Expression levels of cell adhesion molecules and HMGB1 receptors
2
2
3
The expression levels of vascular cell adhesion molecule (VCAM)-1,
intercellular CAM (ICAM)-1, and E-selectin were determined using
whole-cell ELISAs, as described previously [19,24]. Briefly, confluent
monolayers of HUVECs were treated with HMGB1 (1 μg/mL) for either
1
6 h (VCAM-1 and ICAM-1) or 22 h (E-selectin), and were then ad-
ministered KC1–3. Next, the HUVECs were fixed in 1% paraformalde-
hyde and washed three times; then, mouse anti-human monoclonal
antibodies (VCAM-1, ICAM-1, and E-selectin, 1:50 each; Chemicon
Temecula, CA) were added before the samples were incubated at 37 °C
2.18. Statistical analysis
2
for 1 h in 5% CO . Finally, the cells were washed, treated with perox-
idase-conjugated anti-mouse IgG antibody (Sigma) for 1 h, washed an
additional three times, and treated with o-phenylenediamine substrate
All data are expressed as mean ± standard deviation (SD) of three
independent experiments. A one-way analysis of variance (ANOVA) and
Tukey’s post-hoc tests were performed to make comparisons among the
different groups. P values < 0.05 were considered to indicate statis-
tical significance. The Kaplan-Meier method was used to compare dif-
ferences in survival following the CLP-induced sepsis outcomes.
(
Sigma). The same experimental procedures were used to monitor the
cell surface expression levels of Toll-like receptor (TLR)2, TLR4 and the
receptor for advanced glycation end products (RAGE) using specific
antibodies (A-9, H-80, and A-9, respectively; Santa Cruz Biotechnology
Inc.).
3. Results and discussion
2
.14. ELISAs for phosphorylated p38 mitogen-activated protein kinase, NF-
3
.1. KC reduced HMGB1 release in LPS-activated HUVECs and the CLP-
κB, TNF-α, extracellular signal-regulated kinase 1/2, IL-1β, and IL-6
induced sepsis mouse model
The activity of phosphorylated p38 mitogen-activated protein ki-
nase (MAPK) was quantified using a commercially available ELISA kit
Cell Signaling Technology, Danvers, MA) in accordance with the
manufacturer’s instructions. The activities of the total and phosphory-
lated forms of p65 (nuclear factor kappa B (NF-κB) (#7174, #7173; Cell
Signaling Technology) and extracellular signal-regulated kinase (ERK)
The HMGB1 protein secreted by activated immune cells and da-
maged cells functions as a sepsis mediator [8] and LPS has been used as
a research tool to assess severe vascular inflammation in animal and cell
studies [26,27]. Consistent with a previous report showing that HMGB1
release is induced by LPS [27], the present study found that LPS sig-
nificantly stimulated HMGB1 secretion in HUVECs. However, this LPS-
induced stimulation was inhibited by the independent administration of
KC1 and KC3, but not KC2 (Fig. 2B). To confirm the inhibitory effects of
KC1–3 on HMGB1 release in vivo, KC was i.v. injected 12 h after CLP
surgery and it was shown that KC1 and KC3 both significantly reduced
CLP-induced HMGB1 secretion (Fig. 2C). Next, the effects of the KC
compounds on the expression levels of HMGB1 receptors, including
TLR2, TLR4, and RAGE, were assessed. KC1 and KC3 (data not shown)
both diminished the HMGB1-induced expression levels of TLR2, TLR4,
and RAGE in HUVECs (Fig. 1D) whereas KC2 did not (data not shown).
To determine the toxicity of the KC compounds, a cellular viability
assay was performed by applying MTT to the HUVECs. The KC com-
pounds did not affect the viability of cells that were treated with con-
centrations up to 50 μM over 48 h (Fig. 2E). Taken together, these re-
sults indicate that KC1 and KC3 may be viable early interventions for
the prevention of HMGB1 release and progression to severe sepsis and
septic shock.
(
1
/2 (R&D Systems, Minneapolis, MN) in the nuclear lysates were de-
termined using ELISA kits (R&D Systems). The concentrations of IL-1β,
IL-6, and TNF-α in the cell culture supernatants were also determined
using ELISA kits.
2
.15. Hematoxylin & eosin staining and histopathological examinations
Male C57BL/6 mice (n = 5) were subjected to CLP and then re-
ceived i.v. administrations of KC1–3 (0.15–0.58 mg/kg) at 12 and 50 h
after CLP. At 96 h after CLP, the mice were euthanized. Light micro-
scopic analyses of lung specimens were performed via blinded ob-
servation to evaluate pulmonary architecture, tissue edema, and the
infiltration of inflammatory cells, as previously described [22]. The
results were classified into four grades: Grade 1 indicated normal his-
topathology; Grade 2 indicated minimal neutrophil leukocyte infiltra-
tion; Grade 3 indicated moderate neutrophil leukocyte infiltration,
perivascular edema formation, and partial destruction of pulmonary
architecture; and Grade 4 indicated dense neutrophil leukocyte in-
filtration, abscess formation, and the complete destruction of pul-
monary architecture.
2.16. Measurement of tissue injury markers
The plasma levels of aspartate transaminase (AST), alanine transa-
minase (ALT), blood urea nitrogen (BUN), creatinine, and lactate de-
hydrogenase (LDH) were measured using commercial assay kits (Pointe
Scientific, Lincoln Park, MI).
Fig. 1. Structure of (+)-decursin and decursinol angelate.
263

W. Lee, et al.
Biochemical Pharmacology 163 (2019) 260–268
Fig. 2. Structures of KC1–3 and the effects of
KC on HMGB1 release and the expression
levels of high mobility group box 1 (HMGB1)
receptors. (A) Chemical structures of KC1,
KC2, and KC3. (B) After stimulation with li-
popolysaccharide (LPS) (100 ng/mL, 16 h),
the human umbilical vein endothelial cells
(HUVECs) were treated with the indicated
concentrations of KC for 16 h and HMGB1
release was measured with an enzyme-linked
immunosorbent assay (ELISA). (C) At 12 h
after cecal ligation and puncture (CLP), male
C57BL/6 mice (n = 5) received intravenous
(i.v.) injections of the indicated amount of
KC and were then euthanized 24 h after CLP;
serum HMGB1 levels were measured with an
ELISA. (D) Confluent HUVECs were activated
with HMGB1 (1 μg/mL, 16 h) and then in-
cubated with KC1 for 6 h; the expression le-
vels of Toll-like receptor (TLR)2 (white bar),
TLR4 (gray bar), and receptor for advanced
glycation end products (RAGE) (black bar)
were determined with a cell-based ELISA. (E)
The effects of KC on cellular viability were
measured with a 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT)
assay; all data are shown as mean ±
standard deviation (SD) from three separate
experiments conducted in triplicate on dif-
ferent days. D = 0.2%, dimethyl sulfoxide
*
(
DMSO) was the vehicle control. p < 0.05
versus LPS alone (B) or CLP alone (C).
3
.2. KC suppressed HMGB1-mediated vascular barrier disruption
that the vascular destruction response caused by HMGB1 occurs via the
activation of p38 MAPK [29–31]. Thus, the independent effects of KC1
and KC3 on the activation of p38 were assessed and it was shown that
HMGB1 increased the activation of p38, but that this enhancement was
reduced by both KC1 and KC3 (Fig. 3D). The reductions in HMGB1-
mediated hyperpermeability and p38 activation indicate that KC1 and
KC3 have potential as anti-septic agents.
Because HMGB1 and LPS destroy the integrity of vascular barriers
[
28], vascular permeability was analyzed in the present study to eval-
uate the ability of KC to maintain barrier consistency in HUVECs.
HUVECs were activated with either LPS (100 ng/mL; Fig. 3A) or
HMGB1 (1 µg/mL; Fig. 3B) and then treated with KC for 16 h. LPS- and
HMGB1-mediated hyperpermeability were inhibited by both KC1 and
KC3 (Fig. 3A and B); the barrier-protective effects of KC1 and KC3 were
confirmed in vivo using mice (Fig. 3C). Previous reports have indicated
Fig. 3. Effects of KC on HMGB1-mediated
permeability in vitro and in vivo. (A, B)
Effects of treatment with different con-
centrations of KC for 16 h on barrier disrup-
tion caused by LPS (100 ng/mL, 4 h; A) and
HMGB1 (1 μg/mL, 16 h; B); changes were
assessed by monitoring the flux of Evans
blue-bound albumin across the HUVECs. (C)
At 12 h after CLP, male C57BL/6 mice
(n = 5) received i.v. injections of the in-
dicated amount of KC and were then eu-
thanized 24 h after CLP; the effects of KC on
CLP-induced vascular permeability were ex-
amined by measuring Evans blue dye in
peritoneal washings (expressed as μg/mouse,
n = 5). (D) HUVECs were activated with
HMGB1 (1 μg/mL, 16 h) and then treated
with different concentrations of KC for 16 h;
the effects of KC1–3 on the HMGB1-mediated
expression of phospho-p38 were determined
with an ELISA. All data are expressed as
mean ± SD of three separate experiments
*
on different days. p < 0.05 vs. LPS (A) or
HMGB1 (B, C, D).
264

W. Lee, et al.
Biochemical Pharmacology 163 (2019) 260–268
Fig. 4. Effects of KC on HMGB1-mediated
pro-inflammatory responses. (A–C) HUVECs
were stimulated with HMGB1 (1 μg/mL) for
1
6 h and then treated with KC for 6 h. The
HMGB1-mediated (A) expression levels of
VCAM-1 (white bar), ICAM-1 (gray bar), and
E-selectin (black bar) in HUVECs, (B, E) ad-
herence of human neutrophils to HUVEC
monolayers, and (C) migration of neutrophils
through HUVEC monolayers were analyzed.
(
(
D) At 12 h after CLP, male C57BL/6 mice
n = 5) received i.v. injections of the in-
dicated amount of KC and were then eu-
thanized 24 h after CLP; the effects of KC
treatment on CLP-induced leukocyte migra-
tion into the peritoneal cavities of mice were
analyzed. Representative images from three
separate experiments on different days with
similar results are shown. All data are ex-
pressed as mean ± SD of three separate ex-
*
periments on different days. p < 0.05 vs.
HMGB1.
Fig. 5. Effects of KC on the HMGB1-stimu-
lated production of interleukin (IL)-6, tumor
necrosis factor (TNF)-α, and IL-1β and the
activation of the nuclear factor kappa B/ex-
tracellular signal-regulated kinase (NF-κB/
ERK) pathways. (A–D) HUVECs were stimu-
lated with HMGB1 (1 μg/mL) for 16 h and
then treated with KC for 16 h. The HMGB1-
mediated production of TNF-α (A), IL-6 (B),
and IL-1 β (C) in the HUVECs was shown
with ELISAs (A–C) or Western blot analyses
(D). (E, F) Confluent HUVECs were activated
with HMGB1 (1 μg/mL, 16 h) and then in-
cubated with KC for 16 h; the HMGB1-
mediated activation of phospho-NF-κB p65
(white bar) and total NF-κB p65 (black bar)
and (E) phospho-ERK1/2 (white box) and
total ERK1/2 (black box) in HUVECs was
analyzed (F). (G) Expression levels of NF-κB
in nuclear and cytoplasmic extracts were
evaluated with Western blot analyses; actin
and lamin B were used as loading controls for
the cytoplasmic and nuclear extracts, re-
spectively. All data are expressed as
mean ± SD of three separate experiments
*
on different days. p < 0.05 vs. HMGB1.
265

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Biochemical Pharmacology 163 (2019) 260–268
Fig. 6. Effects of KC on lethality and tissue injury after the CLP procedure. (A) Male C57BL/6 mice (n = 20) received i.v. injections of KC at 0.58 mg/kg at 12 h and
0 h after CLP; animal survival was monitored every 12 h for 132 h after CLP. Control CLP mice (●) and sham-operated mice (○) were administered sterile saline
n = 20). A Kaplan-Meier survival analysis was conducted to determine the overall survival rates of the CLP-treated mice. (B) Male C57BL/6 mice (n = 20) received
5
(
i.v. injections of KC at 0.58 mg/kg at 12 h and 50 h after CLP; serum HMGB1 levels at 96 h after CLP were measured with an ELISA. (C) Male C57BL/6 mice were
subjected to CLP, received i.v. injections of KC at 12 h and 50 h after CLP (n = 5), and were then euthanized at 96 h after CLP; the histopathological scores of the
injured lung tissues were recorded as described in the Methods section. (D) Photomicrographs of lung tissues (H&E staining, 200×); the illustrations are re-
presentative images from three independent experiments conducted on different days with similar results, and the arrows indicate leukocyte infiltration. (E–H) Male
C57BL/6 mice (n = 20) received i.v. injections of KC at 0.58 mg/kg at 12 h and 50 h after CLP; the mice were bled and then euthanized to determine the plasma levels
of (E) aspartate transaminase (AST) and alanine transaminase (ALT), (F) creatinine, (G) blood urea nitrogen (BUN), and (H) lactate dehydrogenase (LDH). All data
are expressed as mean ± SD of three separate experiments conducted on different days with similar results. *p < 0.05 versus CLP.
reduced the expression levels of CAMs in a dose-dependent manner,
which suggests that the inhibitory effects of KC1 and KC3 on the ex-
pression levels of CAMs were mediated by attenuation of HMGB1 sig-
naling. In addition to decreasing CAM expression levels, KC1 and KC3
reduced the adherence of human neutrophils to HUVECs, as well as
their subsequent migration (Fig. 4B, C, and E). An in vivo experiment in
the present study corroborated these results by showing inhibition of
the HMGB1-induced migration of leukocytes in the peritoneal space
(
Fig. 4D). Thus, the present results suggest that KC1 and KC3 both in-
hibited the adhesion and migration of leukocytes to the inflamed en-
dothelium.
3.4. KC inhibited HMGB1-stimulated activation of NF-κB/ERK and the
production of IL-1β, IL-6, and TNF-α
Fig. 7. Purity (> 98%) of decursin as determined with an LC-20AT ultraviolet-
visible spectrometer (254 nm; Shimadzu, Kyoto, Japan). For the purity test, an
ACE C18 column (4.6 × 250 mm) was used at a column temperature of 40 °C.
The isocratic elution system consisted of n-hexane (70%) and isopropanol
HMGB1 exacerbates the pathological and physiological conditions
of sepsis by increasing the expression levels of inflammatory cytokines,
including TNF-α, IL-1β, and IL-6, through a variety of signaling path-
ways, such as ERK 1/2 and NF-κB [19,33,34]. Thus, the present study
evaluated the inhibitory effects of KC on the HMGB1-induced produc-
tion of TNF-α, IL-1β, and IL-6, and the activation of the NF-κB and ERK
(30%); the solvent flow rate was held constant at 0.5 mL/min, and the sample
injection volume was 5 µL.
1
/2 pathways. The production of inflammatory cytokines and activa-
3.3. KC suppressed the HMGB1-mediated expression of CAMs, human
tion of transcriptional factors were enhanced by HMGB1 in HUVECs,
but were independently inhibited by treatment with KC1 and KC3
neutrophil adhesion, and leukocyte migration
(
Fig. 5A–F); the effects of KC2 and KC3 on the activation of NF-κB and
HMGB1 increases the endothelial cell surface expression levels of
adhesion molecules, including ICAM-1, VCAM-1, and E-selectin, and
has also been implicated in diseases such as atherosclerosis [32]. These
adhesion molecules aid in the migration of leukocytes across the en-
dothelium to the site of inflammation. In the present study, KC1
ERK 1/2 are not shown. Additionally, HMGB1 increased the expression
of p65 NF-κB in the nucleus but this was inhibited by independent
treatment with KC1 and KC3 in HUVECs (Fig. 5G).
(
Fig. 4A) and KC3 (data not shown), but not KC2 (data not shown),
266

W. Lee, et al.
Biochemical Pharmacology 163 (2019) 260–268
Fig. 8. Effects of natural decursin, KC1, and KC3 on
HMGB1 release. (A) After stimulation with LPS
(
100 ng/mL, 16 h), the HUVECs were treated with
the indicated concentrations of the compounds for
6 h and HMGB1 release was measured with an
ELISA. (C) At 12 h after CLP, male C57BL/6 mice
n = 5) received i.v. injections of the indicated
1
(
amount of compound and were then euthanized 24 h
after CLP; serum HMGB1 levels were measured with
an ELISA. All data are expressed as mean ± SD
from three separate experiments conducted in tri-
plicate on different days. D = 0.2%, DMSO was the
vehicle control. *p < 0.05 vs. LPS alone (A) or CLP
alone (B).
Fig. 9. Effects of natural decursin, KC1, and KC3 on
HMGB1-mediated permeability in vitro and in vivo.
(A) Effects of treatment with different concentra-
tions of the compounds for 16 h on the barrier dis-
ruption caused by HMGB1 (1 μg/mL, 16 h); changes
were monitored by measuring the flux of Evans blue-
bound albumin across the HUVECs. (C) The effects
of the compounds on CLP-induced vascular perme-
ability in mice were examined by measuring Evans
blue dye in the peritoneal washings (expressed as
μg/mouse, n = 5). All data are expressed as
mean ± SD of three separate experiments on dif-
ferent days. *p < 0.05 vs. HMGB1 (A) or CLP (B).
Fig. 10. Reagents and conditions. (a) dry benzene, TEA, and DPPA, 5 h; (b) dry benzene, reflux, 3 h; (c) MeOH, cH
2 4
SO , 80 °C, 12 h; (d) DIBAL-H in toluene 1 M
solution, −78 °C, MeOH; (e) PBr , dry MC; (f) TEA, 4-DMAP, 80 °C, 3 h; and (g) NaH, DMF anhydrous, −20 °C, 4 h.
3
3
.5. KC administration increased the survival rate and reduced tissue injury
inhibit further secretion of HMGB1 and HMGB1-mediated in-
flammatory responses. These results indicate that both KC1 and KC3
may be useful for the control of sepsis and septic shock.
in CLP-induced septic mice
The present study assessed the protective effects of KC1–3 against
CLP-induced septic lethality by treating mice with KC following CLP
surgery. A single dose of KC (0.58 mg/kg, 12 h after CLP) did not pro-
tect against CLP-induced lethality (data not shown); therefore, KC was
subsequently administered twice at 12 and 50 h after CLP. This ad-
ministration regimen for KC1 and KC3 improved the survival rate of
mice dying from sepsis (p < 0.00001; Fig. 6A). These results indicate
that, although HMGB1 levels were significantly reduced by KC at 12 h
after CLP (Fig. 2C), a single treatment with KC could not inhibit the
further secretion of HMGB1. Therefore, KC was administered twice to
Next, the potential protective effects of KC1 and KC3 against CLP-
induced lung injury were assessed. CLP caused interstitial edema via
the large-scale infiltration of inflammatory cells into the alveolar space,
as well as severe impairment of lung tissue, but KC1 and KC3 both
ameliorated these changes (Fig. 6C and D). The systemic inflammation
that occurs during sepsis frequently causes MOF, with the liver and
kidney being the major target organs [35]. In the present study, CLP
significantly increased the plasma levels of ALT and AST (Fig. 6E),
which are markers of hepatic injury, and of creatinine and BUN (Fig. 6F
and G), which are markers of renal injury. However, these increases
267

W. Lee, et al.
Biochemical Pharmacology 163 (2019) 260–268
were mitigated by both KC1 and KC3. LDH, which is another important
marker of tissue injury, was also reduced by both KC1 and KC3 in CLP-
induced mice (Fig. 6H).
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[
[
This study aimed to evaluate the barrier-protective effects of KC
against the vascular barrier-disruptive responses induced by septic
conditions. Under normal pathophysiological conditions, the vascular
endothelium plays a pivotal role in maintaining vascular barrier in-
tegrity in response to the vascular endothelial extracellular environ-
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and important process involved in septic responses because it can result
in vascular hyperpermeability and body cavity edema in septic patients
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S)-(+)-decursin and its analogues as potent inhibitors of melanin formation in B16
(
In the present study, the natural compound (+)-decursin, which has
an ester group in the dihydropyranocoumarin ring, exhibited weak anti-
septic activity (Figs. 7–9). However, it is noteworthy that the synthetic
compounds KC1 and KC3, which have a carbamate moiety in the di-
hydropyranocoumarin ring, exhibited strong anti-septic activities. In
contrast, when the carbamate moiety was replaced with ether moiety in
the dihydropyranocoumarin ring, the resulting synthetic compound K2
exhibited a dramatic decrease in anti-septic activity, which suggests
that the carbamate moiety played key role in anti-septic activity.
The present results demonstrated that KC1 and KC3 (See Fig. 10)
both reduced HMGB1 release in LPS-activated HUVECs, suppressed the
CLP-mediated release of HMGB1, and alleviated HMGB1-mediated
barrier disruption by increasing barrier integrity. Furthermore, the
barrier-protective effects of KC1 and KC3 were confirmed using an in
vivo mouse model of sepsis, in which treatment with KC1 and KC3
reduced CLP-induced mortality and pulmonary injury. The present
findings indicate that KC1 and KC3 might be potential candidates for
the treatment of severe vascular inflammatory diseases, including sepsis
and septic shock.
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vative, (S)-(+)-3-(3,4-dihydroxy-phenyl)-acrylic acid 2,2-dimethyl-8-oxo-3,4-di-
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of (+)-decursin derivatives as inhibitors of the Wnt/beta-catenin pathway, Bioorg.
Med. Chem. Lett. 26 (2016) 3529–3532.
[
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19] B. Jung, H. Kang, W. Lee, H.J. Noh, Y.S. Kim, M.S. Han, et al., Anti-septic effects of
dabrafenib on HMGB1-mediated inflammatory responses, BMB Rep. 49 (2016)
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14–219.
[20] J. Kim, J.S. Bae, ROS homeostasis and metabolism: a critical liaison for cancer
therapy, Exp. Mol. Med. 48 (2016) e269.
[
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tional activation of the human tissue factor gene in THP-1 monocytic cells requires
both activator protein 1 and nuclear factor kappa B binding sites, J. Exp. Med. 174
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[
22] W. Lee, S.K. Ku, J.S. Bae, Zingerone reduces HMGB1-mediated septic responses and
improves survival in septic mice, Toxicol. Appl. Pharmacol. 329 (2017) 202–211.
[23] I.C. Lee, D.Y. Kim, J.S. Bae, Sulforaphane reduces HMGB1-mediated septic re-
sponses and improves survival rate in septic mice, Am. J. Chin. Med. 45 (2017)
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three diketopiperazines from marine-derived bacteria on TGFBIp-mediated septic
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Acknowledgements
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Angelica gigas inhibit acetylcholinesterase: structure-activity relationships, J. Nat.
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This research was supported the National Research Foundation of
Korea (NRF) grant funded by the Korea government (MSIP) (NRF-
2017R1A2B2005851 and 2017M3A9G8083382).
Conflict of interest statement
The authors have no conflicts of interest to declare.
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