A low toxicity synthetic cinnamaldehyde derivative ameliorates renal inflammation in mice by inhibiting NLRP3 inflammasome and its related signaling pathways

Article abstract of DOI:10.1016/j.freeradbiomed.2015.12.003

Uncontrolled inflammation is a leading cause of various chronic diseases. Cinnamaldehyde (CA) is a major bioactive compound isolated from the essential oil of the leaves of Cinnamomum osmophloeum kaneh that exhibits anti-inflammatory activity; however, th

Full text of DOI:10.1016/j.freeradbiomed.2015.12.003

Free Radical Biology and Medicine 91 (2016) 10–24
Contents lists available at ScienceDirect
Free Radical Biology and Medicine
journal homepage: www.elsevier.com/locate/freeradbiomed
Original Contribution
A low toxicity synthetic cinnamaldehyde derivative ameliorates renal
inflammation in mice by inhibiting NLRP3 inflammasome and its
related signaling pathways
a
b
c
d
e
Shuk-Man Ka , Louis Kuoping Chao , Jung-Chen Lin , Shui-Tein Chen , Wen-Tai Li ,
f
f
c
c
c,f,
n
Chien-Nan Lin , Jen-Che Cheng , Huei-Ling Jheng , Ann Chen , Kuo-Feng Hua
Graduate Institute of Aerospace and Undersea Medicine, National Defense Medical Center, Taipei, Taiwan
Department of Cosmeceutics, China Medical University, Taichung, Taiwan
Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
National Research institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan
Department of Biotechnology and Animal Science, National Ilan University, Ilan, Taiwan
a
b
c
d
e
f
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 July 2015
Received in revised form
Uncontrolled inflammation is a leading cause of various chronic diseases. Cinnamaldehyde (CA) is a
major bioactive compound isolated from the essential oil of the leaves of Cinnamomum osmophloeum
kaneh that exhibits anti-inflammatory activity; however, the use of CA is limited by its cytotoxicity. Here,
we synthesized three CA derivatives and identified 4-hydroxycinnamaldehyde-galactosamine (HCAG) as
a low toxicity anti-inflammatory compound in vitro (HCAG IC50c1600 mM; CA IC50¼40 mM) and in vivo.
HCAG reduced pro-inflammatory mediator expression in LPS-activated macrophages by inhibiting MAPK
14 November 2015
Accepted 4 December 2015
Available online 7 December 2015
Keywords:
and PKC-
reduced NLRP3 inflammasome-derived IL-1
of AKT and PKC- . In a mouse model of LPS-induced renal inflammation, we observed reduced al-
α
/
δ
phosphorylation, decreasing ROS generation and reducing NF-
κ
B activation. HCAG also
Cinnamaldehyde
NLRP3 inflammasome
Renal inflammation
β
secretion by inhibiting the ATP-mediated phosphorylation
α/δ
buminuria and a mild degree of glomerular proliferation, glomerular sclerosis and periglomerular in-
flammation in the HCAG-treated mice compared with the vehicle-treated mice. The underlying me-
chanisms for these renoprotective effects involved: (1) inhibited NLRP3 inflammasome activation;
(
2) decreased superoxide anion levels and apoptosis; and (3) suppressed activation of NF-
κB and related
downstream inflammatory mediators.
&
2015 Elsevier Inc. All rights reserved.
1
. Introduction
recent study reported that peripheral blood mononuclear cells
from chronic kidney disease patients showed higher expression of
NLRP3 inflammasome components (NLRP3, caspase-1, and ASC)
Inflammation is one of the most important host defense me-
chanisms in mammals and is also required for wound healing;
however, uncontrolled inflammation is harmful to health. Many
studies have shown that inflammation is a risk factor for metabolic
diseases [1]. Recently, a caspase-1-containing multi-protein com-
plex called the NLRP3 inflammasome was identified that controls
and products (IL-1
[11]. These results suggest that the NLRP3/IL-1
potential therapeutic target for renal disease in humans.
β
and IL-18) compared with healthy subjects
pathway may be a
β
Corticosteroids, some cytotoxic agents, and cyclosporine A are
commonly used in the clinic to treat chronic kidney disease [12];
however, the prevention of the progression of renal lesions is ac-
companied by unsatisfactory side effects in the patients, which
remains a major concern [13]. Thus, the development of new
agents with sufficient therapeutic effects and negligible side ef-
fects is clinically important. Some small molecules or bioactive
ingredients isolated from natural products ameliorated renal da-
mage by inhibiting the NLRP3 inflammasome [14–16]. Recently,
we isolated several small molecules from natural products and
demonstrated their renal protective functions in various mouse
the release of IL-1
2]. A strong link between the NLRP3 inflammasome and the de-
velopment of metabolic diseases is becoming increasingly evident
3,4]. For example, the NLRP3 inflammasome promotes renal in-
β
and IL-18 during the inflammatory processes
[
[
flammation and contributes to chronic kidney disease [5–10]. A
n
Correspondence to: No. 1, Sec. 1, Shen-Lung Road, Ilan, Taiwan.
Tel.: þ886 3 935 7400x7626; fax: þ886 3 931 1526.
E-mail address: kuofenghua@gmail.com (K.-F. Hua).
http://dx.doi.org/10.1016/j.freeradbiomed.2015.12.003
0891-5849/& 2015 Elsevier Inc. All rights reserved.

S.-M. Ka et al. / Free Radical Biology and Medicine 91 (2016) 10–24
11
chronic kidney disease models, including IgA nephropathy, focal
segmental glomerulosclerosis, and lupus nephritis [17–22].
Cinnamaldehyde (CA) is a major bioactive compound isolated
from the essential oil of leaves of Cinnamomum osmophloeum ka-
neh [23]. CA exhibits immune modulation properties in bacteria-
infected zebrafish by enhancing the host’s defenses against pa-
thogen infection [24], in rat cerebral microvascular endothelial
temperature. The reaction mixture was stirred at ꢀ20 °C for 4 h
and monitored by TLC. Saturated NaHCO aqueous solution
3
(150 ml) was added to the mixture. The aqueous layer was sepa-
rated and extracted with CH Cl (2 ꢁ 150 ml). The organic layers
2
2
were combined, dried over MgSO4 and concentrated. Purification
by silica gel column chromatography yielded 2 (3.8 g, 42%) as a
white solid.
cells by decreasing IL-1
duction [25], and in TNF-
the adhesion of monocytes to the endothelial cells [26]. Our pre-
vious study showed that CA inhibited cytokine secretion from li-
popolysaccharide (LPS)-activated macrophages but was cytotoxic
β
-induced COX-2 activity and PGE
2
pro-
α-treated endothelial cells by inhibiting
2.4. Preparation of 4-(2,3,4,6-Tetra-O-acetyl-
D-glucopyranosyloxy)-
(E)-2-styryl-1,3-dioxolane (4)
4-(2,3,4,6-Tetra-O-acetyl-D-glucopyranosyloxy)benzaldehyde
at concentrations Z40
μ
M [23]. Additionally, CA induced caspase-
2
(3.4 g, 7.5 mmol) was dissolved in THF (75 ml, potassium tert-
3
-dependent apoptosis of human hepatoma cells by enhancing
butoxide, 0.84 g, 7.5 mmol); then, (1,3-dioxolan-2-ylmethyl)tri-
phenylphosphonium bromide 3 (3.95 g, 9.2 mmol) was added
portionwise. The reaction mixture was heated to reflux for 10 h.
After completion of the reaction, the mixture was added to water
ROS generation and disrupting mitochondrial function [27]. No-
tably, one study showed that food consumption and the body
weights of rats and mice fed CA were reduced, suggesting the
possibility of a side effect associated with CA [28]. Another study
indicated that CA induced eryptosis of erythrocytes and hemolysis
and extracted with CH
combined, dried over MgSO
purified by silica gel column chromatography to yield 4 (2.5 g,
2
Cl
2
(2 ꢁ 100 ml). The organic layers were
4
and concentrated. The residue was
[29]. These results indicate that safety should be a concern when
CA is used as an anti-inflammatory agent. Here, we report the
development of a nontoxic synthetic CA derivative (4-hydro-
xycinnamaldehyde-galactosamine, HCAG) that inhibits the LPS-
induced inflammation in macrophages and reduces the LPS-in-
duced renal inflammation in mice.
6
4%) as a white powder.
2
.5. Preparation of 4-( -glucopyranosyloxy)-(E)-3-(4-methox-
D
yphenyl)acrylaldehyde (5)
Compound 4 (1.8 g, 3.4 mmol) was dissolved in dry MeOH
(40 ml). Then, NaOMe in MeOH was added at 0 °C. The solution
was stirred at room temperature for 3 h. The progress of this re-
action was monitored by TLC. The reaction mixture was con-
centrated in a vacuum and purified by Sephadex LH-20 column
chromatography to yield a pale yellow powder. The pale yellow
2
. Materials and methods
2.1. Ethics statement
All animal experiments were performed after approval by the
Institutional Animal Care and Use Committee of The National Ilan
University, Taiwan (Permit number: NIU 102-7) and were consistent
with the NIH Guide for the Care and Use of Laboratory Animals.
powder (1.1 g) in CH
HOAc (10 ml) at 0 °C. Then, the mixture was warmed to room
temperature and stirred for 10 h. Saturated aqueous NaHCO so-
3 2
OH (20 ml) was added to H O (10 ml) and
3
lution was added, and the aqueous layer was extracted with
2
.2. Chemistries used in compound preparation
CH Cl (2 ꢁ 50 ml). The organic layers were combined, dried over
2
2
4
MgSO and evaporated to give a yellow powder 5 (0.8 g, 77% in
1
The goal of this study is to develop the nontoxic CA derivatives
two steps); H NMR (DMSO-d ): 9.61 (d, J¼7.8 Hz, 1 H), 7.70 (d,
6
that can be used in ameliorating renal inflammation by inhibiting
inflammation and NLRP3 inflammasome. To synthesize the CA
derivatives, all reactions were conducted in dried glassware
overnight in a 120 °C oven. All reagents were used as received
from commercial suppliers unless otherwise stated. Di-
chloromethane, chloroform, and methanol were distilled over
J¼8.4 Hz, 2 H), 7.67 (d, J¼15.6 Hz, 1 H), 7.09 (d, J¼8.4 Hz, 2 H), 5.18
(d, J¼5.4 Hz, 1 H), 4.92 (d, J¼7.8 Hz, 1 H), 4.87 (d, J¼6.0 Hz, 1 H),
4
3
.65 (t, J¼5.4 Hz, 1 H), 4.51 (d, J¼4.8 Hz, 1 H), 3.71–3.69 (m, 1 H),
13
.62–3.56 (m, 5 H). C NMR (DMSO-d
6
): 194.2, 159.8, 153.0, 130.5,
127.7, 126.7, 116.6, 100.5, 75.6, 73.2, 70.1, 68.1, 60.3.
2 6
CaH under nitrogen. Spectrograde chloroform-d and DMSO-d
2
.6. Cell cultures
were used as solvents. All NMR chemical shifts were reported as
values in parts per million (ppm), and coupling constants (J) were
given in hertz (Hz). The splitting pattern abbreviations are as fol-
lows: s, singlet; d, doublet; t, triplet; m, unresolved multiplet due
to the field strength of the instrument; and dd, doublet of doublet.
The purification was performed using preparative separations in
flash column chromatography (Merck silica gel 60, particle size
The murine macrophage cell lines RAW 264.7 and J774A.1 were
obtained from the American Type Culture Collection (Rockville,
MD, USA). RAW 264.7 macrophages stably transfected with the
NF-
InvivoGen (San Diego, CA, USA). All cells were cultured in RPMI
640 medium supplemented with 10% heat-inactivated fetal bo-
vine serum and 2 mM -glutamine (all from Life Technologies,
Carlsbad, CA, USA) at 37 °C in a 5% CO incubator. In the case of the
g/ml of zeocin was added to the medium.
κB reporter gene (RAW-Blue™ cells) were purchased from
1
2
30–400 mesh). Analytical TLC was performed on precoated plates
L
(
Merck silica gel 60, F254). Compounds analyzed on the TLC plates
2
were visualized using UV light, I
acid in ethanol with heating.
2
vapor, or 2.5% phosphomolybdic
RAW-Blue™ cells, 100
μ
s
2.7. AlamarBlue assay for cell viability
2.3. Preparation of 4-(2,3,4,6-Tetra-O-acetyl-
D
-glucopyranosyloxy)
benzaldehyde (2)
To evaluate the possible toxicity of CA derivatives toward
3
2
,3,4,6-Tetra-O-acetyl-
α
-
D
-galactopyranosyl
(9.85 g, 20 mmol) and 4-hydroxybenzaldehyde
Cl (100 ml); then, the
2,2,2-trichloroa-
macrophages, RAW 264.7 cells (5 ꢁ 10 cells in 0.1 ml medium)
cetimidate
3.66 g, 30 mmol) were dissolved in CH
1
were incubated with or without samples for 24 h. The Alamar-
s
(
2
2
Blue assay was used to determine the cytotoxicity of the samples
reaction mixture was cooled to ꢀ20 °C. Boron trifluoride diethyl
according to the protocol described in the manufacturer’s in-
structions (AbD Serotec, Oxford, UK).
etherate (46%, 16 ml, 60 mmol) was added dropwise at this

12
S.-M. Ka et al. / Free Radical Biology and Medicine 91 (2016) 10–24
2.8. Western blotting
2.12. ROS detection
To detect the levels of protein expression and protein phos-
phorylation, Western blotting was adapted. Briefly, the samples
were harvested, resuspended in sample buffer, and subjected to
2–15% SDS-PAGE, followed by transfer to polyvinylidene di-
fluoride membranes (EMD Millipore, Darmstadt, Germany). The
membranes were blotted with primary antibodies and secondary
HRP-conjugated antibodies, followed by ECL (EMD Millipore).
ROS is an important signaling molecule mediating in-
flammatory response and NLRP3 inflammasome activation. We
asked whether HCAG inhibits inflammatory response and NLRP3
inflammasome through reducing ROS level. Intracellular ROS
production was measured by detecting the fluorescence intensity
of 2′,7′-dichlorofluorescein (the oxidation product of 2′,7′-di-
chlorofluorescein diacetate, Molecular Probes, Eugene, OR, USA).
For LPS-induced ROS generation (as shown in Fig. 4A), RAW 264.7
1
3
2
.9. Inflammatory mediator expression
macrophages (5 ꢁ 10 cells in 0.1 ml medium) were incubated
with or without 50 mM HCAG, 10 mM N-acetyl cysteine (NAC), or
DMSO (vehicle) for 30 min, with 2 mM 2′,7′-dichlorofluorescein
diacetate for 30 min, and then with or without 0.1 mg/ml LPS for
0–40 min. For ATP-induced general ROS generation (shown in
To evaluate the anti-inflammatory activity of HCAG, the levels
of inflammatory mediator generated by LPS-activated macro-
phages were measured. For the NO generation and IL-6 and TNF-
α
5
3
secretion assay, RAW 264.7 macrophages (2 ꢁ 10 cells in 0.25 ml
medium) were incubated with or without the indicated con-
centrations of HCAG for 30 min and then treated with or without
Fig. 4F), J774A.1 macrophages (5 ꢁ 10 cells in 0.1 ml medium)
were incubated with 0.1 mg/ml LPS for 5.5 h, with or without
5
0 mM HCAG, 10 mM NAC, or DMSO for 30 min, with 2 mM 2′,7′-
dichlorofluorescein diacetate for 30 min, and then with or without
mM ATP for 0–60 min. The fluorescence intensity of 2′,7′-di-
chlorofluorescein was detected at an excitation wavelength of
85 nm and an emission wavelength of 530 nm on a microplate
the addition of 0.1
culture medium was measured by the Griess reaction and ELISA,
respectively. For the proIL-1 and NLRP3 protein expression assay,
μ
g/ml of LPS for 24 h. NO, IL-6, and TNF- in the
α
5
β
6
J774A.1 macrophages (2 ꢁ 10 cells in 2 ml medium) were in-
4
cubated with or without the indicated concentrations of HCAG for
absorbance reader (Bio-Tek, VT, USA). In each time point, the
fluorescent intensity fold change of each group was calculated by
comparison to control group (DMSO/PBS), as control group have a
fold change of “one”.
3
0 min and then treated with or without the addition of 0.1
of LPS for 6 h. Protein expression levels of iNOS, proIL-1
NLRP3 in the cells were measured by Western blotting.
μ
g/ml
and
β
2.10. IL-1β secretion and caspase-1 activation
2.13. NF-κB reporter assay
To evaluate the inhibitory effect of HCAG on NLRP3 inflamma-
some, IL-1 secretion and caspase-1 activation, the hallmarks of
NLRP3 inflammasome activation, were measured. For the IL-1
secretion and caspase-1 activation assays depicted in Fig. 3B,
J774A.1 macrophages (2ꢁ 10 cells in 0.25 ml medium) were in-
cubated with or without 0.1 g/ml LPS for 5.5 h, followed by
treatment with or without HCAG for 30 min. The cells were treated
with or without 5 mM ATP for 0.5 h. IL-1 in the culture medium
NF-
ulating the inflammatory gene expression. We asked whether
HCAG inhibits NF- B activation in LPS-activated macrophages.
κ
B is one of the most important transcription factors reg-
β
β
κ
TM
5
RAW-Blue
cubated with or without HCAG for 30 min and then with or
without 0.1 g/ml LPS for 24 h. The medium (20 l) from treated
cells (2 ꢁ 10 cells in 0.25 ml medium) were in-
5
μ
μ
μ
TM
TM
RAW-Blue
cells was mixed with 200 l of QUANTI-Blue
μ
β
medium (Invitrogen, Carlsbad, CA, USA) in 96-well plates and in-
cubated at 37 °C for 15 min. The secreted embryonic alkaline
phosphatase activity was assessed by measuring the optical den-
sity at 655 nm using a microplate absorbance reader (Bio-Tek, VT,
USA).
was measured by ELISA, and activated caspase-1 (p10) and pro-
caspase-1 (p45) in the cells were measured by Western blotting. For
IL-1
2ꢁ 10 cells in 0.25 ml medium) were incubated for 5.5 h with or
without 0.1 g/ml LPS, followed by treatment with or without
β
secretion (as shown in Fig. 3C and D), J774A.1 macrophages
5
(
μ
HCAG for 30 min. Then, the cells were treated with or without ni-
gericin (10 mM, 0.5 h), monosodium urate (MSU) (500 mg/ml, 24 h),
calcium pyrophosphate dehydrate (CPPD) crystals (100 mg/ml, 24 h),
2.14. Induction of LPS-induced renal inflammation
Experiments were performed on 8-week-old female C57/B6
nanoparticles of silicon dioxide (nano SiO
crystals (500 mg/ml, 24 h), flagellin from S. typhimurium (FLA-ST)
100 g/ml, 24 h), or muramyl dipeptide (MDP) (100 ng/ml, 24 h).
IL-1 in the culture medium was measured by ELISA.
2
) (200 mg/ml, 24 h), alum
mice (22–25 g body weights) obtained from the National Labora-
tory Animal Center, Taipei, Taiwan. LPS-induced renal inflamma-
tion was induced by injection of E coli LPS as described previously
with mild modifications [30]. Briefly, LPS was administered in-
traperitoneally at a dose of 1 mg/kg body weight for 8 consecutive
weeks. Two days prior to the first dose LPS injection, a daily dose
of HCAG (100 mg/kg body weigh) or corn oil (vehicle) was ad-
ministered via the intraperitoneal route until the sacrifice of the
mice at week 8. The dose of HCAG used for the mice was extra-
polated using an equation according to the dose employed
for in vitro experiments as described in our previous study [31].
The equation is as the follows:
(
μ
β
2.11. Phosphorylation levels of MAPKs, IKK-α, IκB-α, PKC, and AKT
To investigate which signaling pathway is affected by HCAG,
the phosphorylation levels of MAPKs, IKK- , I B- , PKC- , PKC-
and AKT in LPS- or ATP-activated macrophages were measured.
For the phosphorylation levels of IKK- and I B- (Fig. 4B), MAPKs
(Fig. 4E), RAW 264.7 macrophages
2 ꢁ 10 cells in 2 ml medium) were incubated for 30 min with or
g/ml
α
κ
α
α
δ
,
α
κ α
(
(
Fig. 4D), PKC-
α
and PKC-
δ
6
without HCAG and then for 0–60 min with or without 0.1
LPS. The phosphorylation levels of these proteins were analyzed by
μ
Dose of HCAG (mg/kg/day) = 40 × (V × P) × O/W
Western blotting. For the phosphorylation levels of AKT (Fig. 4G)
V is the volume of blood of a mouse on average (ꢂ3 ml), P is the
percentage of whole body water content of a mouse (ꢂ70%), O is
the optimal dose of HCAG used in cell models (100 mM; 0.031 mg/
ml), W is the mouse body weight on average (ꢂ25 g).Weekly urine
samples were collected in metabolic cages. The concentration of
urine albumin was determined by ELISA (Exocell, Philadelphia, PA,
6
and PKC-
α
(Fig. 4H) in Fig. 5, J774A.1 macrophages (2 ꢁ 10 cells in
2
ml medium) were incubated for 5.5 h with 0.1
μ
g/ml LPS, then
for 30 min with or without HCAG, and then for 0–60 min with or
without 5 mM ATP. The phosphorylation levels of these proteins
were analyzed by Western blotting.

S.-M. Ka et al. / Free Radical Biology and Medicine 91 (2016) 10–24
13
USA); urine albumin levels were expressed relative to urine
2.21. Real-time polymerase chain reaction analysis
creatinine (Cr) levels measured using a kit from Wako Pure Che-
mical Industries (Osaka, Japan) as described previously [32]. Renal
cortical tissues and blood samples were collected at week 8 and
stored appropriately for further analysis. Serum levels of blood
urea nitrogen (BUN) and Cr were measured using a BUN kit or Cr
kit (both from Fuji Dry-Chem Slide, Fuji Film Medical, Tokyo,
Japan) as described previously [33].
Renal cortex RNA was extracted using the TRIzol reagent and
subjected to cDNA synthesis (Invitrogen, Carlsbad, CA, USA). The
real-time PCR reaction was performed in the ABI Prism 7700 Se-
quence Detection System (Perkin Elmer Applied Systems, Foster
City, CA, USA) using the SYBR Green I PCR kit (Perkin Elmer Applied
Systems) to measure NLRP3, Caspase-1, IL-1
ceptor 4 (TLR4) and MyD88 gene expression as described previously
35]. The following primers were used: NLRP3 (5′-CCCTTGGAGA-
CACAGGACTC-3′ and 5′-GAGGCTGCAGTTGTCTAATTCC-3′); Caspase-
(5′-TCCGCGGTTGAATCCTTTTCAGA-3′ and 5′-ACCACAATTGCT-
GTGTGTGCGCA-3′); IL-1 (5′-TGTAATGAAAGACGGCACACC-3′ and
′-TCTTCTTTGGGTATTGCTTGG-3′); IL-18 (5′-CTGTACAACCGG-
AGTAATACGG-3′ and 5′-TCCATCTTGTTGTGTCCTGG-3′); and TLR4
5′-ACCAGGAAGCTTGAATCCCT-3′ and 5′-TCCAGCCACTGAAGTTCT-
GA-3′).
β, IL-18, toll-like re-
[
2.15. Renal histopathology
1
The tissues were fixed in 10% buffered formalin and embedded
β
in paraffin. Sections (4 m) were stained with hematoxylin and
μ
5
eosin. A nephropathologist (A Chen) was assigned to examine one
hundred glomeruli in a blinded way by light microscopy at the
magnification of 400 with at least two renal tissue sections each.
Scoring of the severity of individual renal lesions was performed as
described previously [34]. The percentage (proportion) was de-
termined for the following three major components: glomerular
proliferation, glomerular sclerosis, or periglomerular inflammation.
(
2.22. Flow cytometry
Splenocytes were double-stained with FITC-conjugated antibodies
against mouse CD3, CD4, or CD19 (B cell marker) and phycoerythrin
PE)-conjugated anti-mouse CD69 antibodies (H1.2F3; marker of ac-
2.16. Immunohistochemistry
(
tivated T and B cells) (BD Biosciences, CA, USA) and analyzed on a
FACSCalibur (BD Biosciences) as described previously [34].
Formalin-fixed and paraffin-embedded tissue sections were
incubated overnight at 4 °C with antibodies against CD3 (pan-T
cells, BD Biosciences, CA, USA), F4/80 (monocytes/macrophages;
2
.23. Statistical analysis
Serotec, NC, USA), phospho-NF-
κB p65 (pNF-
κB p65; Cell Signal-
ing, MA, USA), or IL-1 (Serotec) diluted in DAKO antibody dilution
β
All values are reported as the mean7standard deviation (SD).
buffer (DAKO, Denmark), followed by the incubation with horse-
radish peroxidase-conjugated second antibodies (DAKO) in the
same buffer as described previously [20]. Scoring of the IHC results
was performed using the Pax-it quantitative image analysis soft-
ware (Paxcam, IL, USA) as described previously [20].
The data were analyzed using a one-way ANOVA followed by a
Scheffé test.
3. Results
2
.17. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP
3.1. Synthesis of (E)-3-phenyl-2-propenoyl-
(EPPG)
β-D-galactosamine
nick end labeling (TUNEL)
Apoptosis staining was performed using ApopTag Plus Perox-
idase in Situ Apoptosis Detection kit (Chemiconm Temecula, CA)
according to the manufacturer’s instructions. Scoring of the TUNEL
results was performed using Pax-it quantitative image analysis
software (Paxcam, IL, USA) as described previously [34].
EPPG can be readily made in three steps from cinnamic anhy-
dride; the synthetic strategy is demonstrated in Scheme 1. Cin-
namic anhydride was prepared by treating the mixture of cin-
namic acid and thionyl chloride under basic conditions according
to known methods [36]. Subsequently, N-linked glycosylation was
conducted using galactosamine and cinnamic anhydride at room
temperature to produce a moderate yield of EPPG.
2.18. Renal ROS detection
3.2. Synthesis of N-cinnamyl-
β-D-galactosamine (NCAG)
An in situ staining procedure with the fluorescent dye dihy-
droethidium (DHE) was performed in renal tissues and quantified by
counting the percentage of total positive nuclei per kidney cross sec-
tion using the Pax-it software (Paxcam) as described previously [20].
The direct substitution of galactosamine to the allyl position of
cinnamyl bromide is readily accomplished at room temperature
using sodium methoxide in methanol to obtain NCAG (Scheme 2).
2.19. Renal NF-κB p65 activity assay
3.3. Synthesis of 4-(D-glucopyranosyloxy)-(E)-3-(4-methoxyphenyl)
acrylaldehyde (5) (4-hydroxycinnamaldehyde-galactosamine, HCAG)
Renal NF- B p65 activity was measured with the TransAM
κ
ELISA assay kit (Active Motif, CA, USA) according to the manu-
facturer’s instructions using a microplate absorbance reader (Bio-
Tek, VT, USA).
The strategy for the synthesis of 4-hydroxyphenylacrylaldehyde
glycoside 5 is shown in Scheme 3; the strategy involves a three-step
synthetic route from trichloroacetimidate derivative 1. Tri-
chloroacetimidate derivative 1 was prepared from 2,3,4,6-tetra-O-
2.20. Renal MCP-1 and IL-6 assay
acetyl-D-glucopyranose according to Schmidt’s trichloroacetimidate
procedure [37]. BF
3
-promoted glycosylation of 1 using 4-hydro-
Renal cortical samples were homogenized in protease in-
hibitors (Roche Applied Science, USA) using a sterile razor blade.
Both MCP-1 and IL-6 were measured using commercial ELISA kits
eBioscience, CA, USA) according to the manufacturer’s instruc-
tions and a microplate absorbance reader (Bio-Tek).
xybenzaldehyde afforded compound 2 with a 42% yield. Subse-
quently, compound 2 was treated with triphenyl-1,3-dioxolan-2-yl-
methylphosphonium bromide 3 and potassium tert-butoxide in THF
to produce a strong predominance of the E isomer of styryl-1,3-di-
oxolane glycoside 4. Then, the acetyl groups were removed from
(

14
S.-M. Ka et al. / Free Radical Biology and Medicine 91 (2016) 10–24
Scheme 1. Synthesis of EPPG.
Scheme 2. Synthesis of NCAG.
Scheme 3. Synthesis of HCAG.
styryl-1,3-dioxolane glycoside 4 using sodium methoxide in metha-
nol, followed by neutralization of the reaction mixture with a mild
acid to obtain HCAG with a 77% yield.
mouse macrophage cell line RAW 264.7 after 24 h of treatment. CA
exhibited high cytotoxicity against RAW 264.7 macrophages with
an IC50 of 40 mM. However, the cytotoxicity of HCAG was much
lower than CA because the cell viability was not significantly re-
duced by HCAG at a concentration of 1600 M (Fig. 1B). The cy-
totoxicity of EPPG and NCAG was also lower than CA because the
μ
3.4. Effect of CA derivatives on cell viability
CA and the synthesized CA derivatives EPPG, NCAG, and HCAG
cell viability was not significantly reduced by EPPG and NCAG at a
(Fig. 1A) were evaluated to determine their cytotoxicity against the
concentration of 100 M (data not shown).
μ

S.-M. Ka et al. / Free Radical Biology and Medicine 91 (2016) 10–24
15
3.5. Effect of HCAG on LPS-induced inflammatory response
The anti-inflammatory activity of the CA derivatives was in-
vestigated in LPS-activated RAW 264.7 macrophages. NO levels
were significantly reduced by HCAG in a dose dependent manner
(
Fig. 2A), while NO levels were slightly reduced by EPPG and were
not affected by NCAG (data not shown). HCAG also decreased iNOS
expression (Fig. 2B) and TNF- (Fig. 2C) and IL-6 (Fig. 2D) secretion
α
by LPS-activated macrophages. These results indicate that HCAG is
a nontoxic cinnamaldehyde derivative that is able to inhibit in-
flammatory response in LPS-activated macrophages.
3.6. HCAG reduces nlrp3 inflammasome activation
Full activation of the NLRP3 inflammasome requires both a
priming signal from LPS and an activation signal from a second
stimulus (e.g., ATP); the former controls the expression of NLRP3
and proIL-1
We found that proIL-1
macrophages was reduced compared with the control cells,
whereas NLRP3 expression was not affected (Fig. 3A). Additionally,
β
and the latter controls caspase-1 activation [38,39].
expression in HCAG-treated J774A.1
β
ATP-induced IL-1 secretion and caspase-1 activation (Fig. 3B) in
β
LPS-primed J774A.1 macrophages were reduced by HCAG in a
dose-dependent fashion. The inhibitory effect of HCAG on NLRP3
inflammasome was confirmed as HCAG reduced the induction of
IL-1
ing nigericin, monosodium urate crystal (MSU), calcium pyr-
ophosphate dihydrate (CPPD), SiO nanoparticles, and alum crys-
tals (Fig. 3C). Next, we determined whether the effect of HCAG on
β
secretion by other NLRP3 inflammasome activators, includ-
Fig. 1. Chemical structure of CA and its derivatives and their effect on cell viability.
A) Chemical structure of CA and its derivative (B) Cell viability. The data are ex-
pressed as the means7SD of three separate experiments.
(
2
Fig. 2. Effect of HCAG on LPS-induced inflammatory mediator expression. (A) NO production. (B) iNOS expression. (C) TNF-α secretion. (D) IL-6 secretion. The data are
expressed as the means7SD of three separate experiments. The Western blotting results are representative of results obtained in three separate experiments, and the
histograms represent the change in the ratio relative to actin compared with the control group. * and *** indicate a significant difference at the level of po0.05 and po0.001,
respectively, compared with LPS-treated group.

16
S.-M. Ka et al. / Free Radical Biology and Medicine 91 (2016) 10–24
Fig. 3. Effect of HCAG on NLRP3 inflammasome activation. (A) Expression levels of NLRP3 and proIL-1β. (B) IL-1β secretion and caspase-1 activation induced by ATP. (C) IL-1β
secretion induced by Nigericin, MSU, CPPD, nano SiO , and Alum. (D) IL-1β secretion induced by FLA-ST and MDP. The data are expressed as the means7SD of three separate
2
experiments. The Western blotting results are representative of results obtained in three separate experiments, and the histograms represent the change in the ratio relative
to p45 compared with the control group. * and *** indicate a significant difference at the level of po0.05 and po0.001, respectively, compared with the LPS-treated group
(
A) or LPSþinducer group (B–D).
IL-1
flammasome. HCAG was not selective for NLRP3-dependent in-
flammasome stimuli because HCAG also reduced IL-1 secretion
triggered by FLA-ST (flagellin from S. typhimurium) and muramyl
dipeptide (MDP) (Fig. 3D); these stimuli were dependent on the
NLRC4 and NLRP1 inflammasomes, respectively [2], and func-
tioned independent of NLRP3.
β
secretion was selective for the NLRP3-dependent in-
HCAG/LPS were not significantly different, indicating that HCAG is
not only able to reduce ROS levels induced by LPS, but is also able
β
to reduced endogenous ROS levels in untreated cells. NF-
vation leads to the production of proIL-1 and pro-inflammatory
mediators [40]. Accordingly, we found that LPS-induced IKK- and
B- phosphorylation (Fig. 4B) and NF- B transcriptional activity
κ
B acti-
β
α
I
κ
α
κ
(Fig. 4C) were reduced by HCAG. Additionally, mitogen-activated
protein kinase (MAPK) and PKC regulate the expression of proIL-
1
β
and pro-inflammatory mediators [41,42]. We found that the
phosphorylation levels of MAPK members p38, JNK1/2, and ERK1/
2 (Fig. 4D) as well as PKC- and PKC- (Fig. 4E) were reduced by
3
.7. Effect of HCAG on inflammatory signaling
ROS is one of the important signaling molecules mediating
α
δ
inflammatory signaling cascades in LPS-activated macrophages
23,39]. We asked whether HCAG is able to inhibit ROS generation
HCAG. These results indicated that HCAG inhibited pro-in-
flammatory mediator expression and the priming of the NLRP3
inflammasome; these effects were associated with reduced ROS
[
in activated macrophages. We found that LPS-induced ROS gen-
eration in the HCAG-treated RAW 264.7 macrophages was reduced
compared with the control cells (Fig. 4A). NAC (n-acetyl-cysteine),
an anti-oxidant, was used as a positive control in inhibiting ROS
generation. In addition, the fluorescent intensity of HCAG/PBS and
generation, NF-
κB activation, and MAPK and PKC-
α
/δ
phosphor-
ylation. Furthermore, ROS not only controls the priming step but
also controls the NLRP3 inflammasome activation step [43]. ATP
stimulates the phosphatidylinositol 3-kinase pathway and

S.-M. Ka et al. / Free Radical Biology and Medicine 91 (2016) 10–24
17
Fig. 4. Effect of HCAG on inflammatory signaling. (A) ROS generation induced by LPS. (B) Phosphorylation levels of IKK-α and IκB-α induced by LPS. (C) Activation levels of
NF-κB induced by LPS. (D) Phosphorylation levels of MAPKs induced by LPS. (E) Phosphorylation levels of PKC-α/δ induced by LPS. (F) ROS generation induced by ATP.
(
G) Phosphorylation levels of AKT induced by ATP. (H) Phosphorylation levels of PKC-α induced by ATP. The data are expressed as the means7SD of three separate
experiments. The Western blotting results are representative of results obtained in three separate experiments, and the histograms represent the change in the ratio relative
to actin or corresponding total protein compared with the control group. * and *** indicate a significant difference at the level of po0.05 and po0.001 compared with the
LPS group (B–E) or the ATP group (G and H).
subsequent AKT activation in a ROS-dependent manner, which is
required for caspase-1 activation and IL-1 secretion [44]. There-
fore, we investigated whether HCAG regulated ROS generation and
AKT activation during the NLRP3 inflammasome activation step.
We found that ATP-induced ROS generation was significantly re-
duced by NAC, but not affected by HCAG (Fig. 4F). Additionally,
of LPS-induced renal inflammation (LPRI) in mice that received daily
administration of HCAG for eight consecutive weeks. First, mice ad-
ministered HCAG showed a stable increase in their body weight
compared with the normal control group (data not shown), sug-
gesting that the appetite and activity of the mice were not affected by
the drug administration. Clinical assessment showed a significant
reduction in proteinuria at weeks 2, 4, 6 and 8 in the HCAG-treated
LPRI (HCAGþLPRI) mice compared with the vehicle-treated LPRI
β
phosphorylation levels of AKT (Fig. 4G) and PKC-
activated J774A.1 macrophages were reduced by HCAG.
α
(Fig. 4H) in ATP-
(
vehicleþLPRI) disease control mice (Fig. 5A). Significantly lower BUN
3.8. HCAG improves clinical conditions and renal histopathology and
serum levels were observed in the HCAGþLPRI mice compared with
the vehicleþLPRI mice, although there was no difference in the ser-
um Cr levels (Fig. 5B) at week 8. Although the vehicleþLPRI mice
exhibited glomerular proliferation and sclerosis and peri-glomerular
inflammation at week 8, this effect was greatly inhibited in the
HCAGþLPRI mice (Fig. 5C and D). In addition, significantly reduced
reduces renal ROS production
LPS from bacteria can induce inflammatory cell infiltration in the
kidney in a mouse model of accelerated lupus nephritis [45]. We
evaluated the effects of HCAG on the prevention of the development

18
S.-M. Ka et al. / Free Radical Biology and Medicine 91 (2016) 10–24
Fig. 5. Effect of HCAG on proteinuria, renal function, renal histopathology, renal ROS production, and renal apoptosis in the LPRI mouse model. (A) Proteinuria. (B) Serum
levels of BUN and creatinine. (C) Histopathology of the kidney. H&E stain. The arrow indicates periglomerular mononuclear leukocyte infiltration. (D) Scoring of renal
pathology. (E) Apoptosis detected by TUNEL assay. (F) Scoring for TUNEL assay. (G) ROS production detected by in situ DHE staining. (H) Scoring for DHE staining. Original
magnification, 400 ꢁ . The data are the mean7SEM for seven mice per group. *po0.05, **po0.01, ***po0.001. #: not detectable.
renal levels of apoptosis was observed in HCAGþLPRI mice, com-
pared to those of vehicleþLPRI mice (Fig. 5E and F). Next, we mea-
sured renal ROS levels. As shown in Fig. 5G and H, significantly in-
creased ROS production was observed in the renal tissues from the
vehicleþLPRI mice compared with the normal control mice, but this
effect was greatly suppressed in the HCAGþLPRI mice as demon-
strated by in situ DHE staining.
3.9. HCAG decreases NLRP3 inflammasome activation and TLR4/
MyD88 expression in the kidney
We evaluated the effects of HCAG on: (1) the activation of the
renal NLRP3 inflammasome, which has been implicated in the
development and progression of renal inflammation [5,6], and
(2) renal TLR4/MyD88 expression, which has been reported to

S.-M. Ka et al. / Free Radical Biology and Medicine 91 (2016) 10–24
19
Fig. 6. Effect of HCAG on renal NLRP3 inflammasome activation and TLR4/MyD88 expression in the LPRI mouse model. (A) mRNA expression levels of NLRP3, caspase-1,
IL-1β, IL-18, TLR4, and MyD88. (B) IHC staining of IL-1β. (C) Scoring for IL-1β staining. Positively stained cells in the glomeruli were mainly podocytes and mesangial cells.
Original magnification, 400 ꢁ . The data are the mean7SEM for seven mice per group. *po0.05, **po0.01, ***po0.001.
promote renal inflammation and may represent a novel ther-
factor for various types of glomerulonephritis [46,47]. As shown in
apeutic target for the prevention of renal injury [6]. Although the
Fig. 7A, vehicleþLPRI mice showed significantly increased renal
mRNA expression levels of NLRP3, caspase-1, IL-1
β
, IL-18, TRL4,
phosphorylation levels of NF- B p65 by IHC, but this effect was
κ
and MyD88 in renal tissues were all significantly increased in the
vehicleþLPRI mice compared with the normal controls, these ef-
fects were inhibited in the HCAGþLPRI mice (Fig. 6A). Ad-
ditionally, IHC demonstrated that the vehicleþLPRI mice had
significantly decreased in the HCAGþLPRI mice. This effect of
HCAG was confirmed by the observation of significantly elevated
levels of activated pNF- B p65 by ELISA (Fig. 7B); again, this effect
κ
was greatly decreased in the HCAGþLPRI mice. Significantly
higher renal expression levels of MCP-1 and IL-6 were also ob-
served in the vehicleþLPRI mice compared with the normal
control mice, but this effect was inhibited in the HCAGþLPRI mice
significantly higher renal IL-1 levels compared with the normal
β
control mice; this effect was also significantly inhibited in the
HCAGþLPRI mice (Fig. 6B and C).
(Fig. 7C).
3.10. HCAG decreases renal NF-
κ
B-mediated production of MCP-1
3.11. HCAG mitigates renal infiltration of macrophages and T cells
and IL-6
Infiltration of macrophages and T cells locally in the kidney
plays a pivotal pathogenic role in the development of glomerular
crescents of glomerulonephritis [21,48]. As shown in Fig. 8A and B,
Renal production of NF-
kines such as MCP-1 and IL-6 has been shown to be a pathogenic
κ
B-mediated pro-inflammatory cyto-

20
S.-M. Ka et al. / Free Radical Biology and Medicine 91 (2016) 10–24
a mechanism of action involved in the beneficial effects of HCAG in
the HCAGþLPRI mice. As shown in Fig. 9, increased activation of
þ
CD4 T cells in the spleen was observed in the vehicleþLPRI mice
compared with the normal control mice; this effect was sig-
nificantly suppressed in the HCAGþLPRI mice, as demonstrated by
flow cytometry. Similarly, the HCAGþLPRI mice exhibited sup-
pressed activation of CD3þT cells in the spleen, although the
difference did not reach significance (p¼0.07). Although the ve-
þ
hicleþLPRI mice showed higher levels of activation of CD19
B
cells in the spleen compared with the normal control mice, there
was no significant difference between the vehicleþLPRI mice and
HCAGþLPRI mice.
4. Discussion
LPS-induced TLR4 activation in macrophages triggers ROS re-
lease, which leads to the activation of NF-
κ
B and induces a rapid
cytokine storm, including spikes in TNF , IL-1
α
β, and IL-6 [51–53].
These cytokines, in turn, activate a large number of immune cells
that produce more ROS and inflammatory factors and damage the
surrounding tissue [54,55]. LPS not only activates macrophages but
also promotes the activation of intrinsic renal cells, such as me-
sangial and tubular epithelial cells, and these contribute to the
renal diseases [56,57]. LPS-induced VCAM-1 expression in me-
sangial cells was partially mediated through a TLR4/ROS-depen-
dent pathway associated with recruitment of monocyte adhesion
to kidney leading to the inflammatory responses in renal diseases
[58]. In this study, we showed that HCAG could ameliorate renal
lesions associated with glomerular proliferation, glomerular
sclerosis, periglomerular inflammation, and renal levels of apop-
tosis in a mouse model of LPS-induced renal inflammation. Our
data suggested that the beneficial effects of HCAG on LPS-induced
renal inflammation were mainly due to the inhibition of renal ROS
generation and NF- B activation, as well as reduced inflammatory
κ
cytokine expression and NLRP3 inflammasome activation in the
kidney (Fig. 10). We also demonstrated that HCAG was able to
inhibit LPS-induced inflammatory response in macrophages;
however, we have no solid evidences showing the direct effect of
HCAG on the renal cells yet.
In addition, our previous studies demonstrated that renal ROS
was increased in mice with renal disease, including IgA nephro-
pathy [17,22,48], lupus nephritis [19], focal segmental glomer-
ulosclerosis [20], and diabetic nephropathy [59]. Consistently, in-
hibition of renal ROS generation ameliorated the renal inflamma-
tion and injury in these mouse models of renal inflammation.
Moreover, HCAG administration significantly inhibited the in-
crease in ROS levels in renal tissues observed in the vehicleþLPRI
mice (Fig. 5G and H). This result was consistent with our in vitro
cell model finding that HCAG markedly inhibited ROS generation
in LPS-activated macrophages (Fig. 4A). ROS is one of the key
signaling molecules mediating the pro-inflammatory signaling
cascades downstream of TLR4 [48,60,61]. Thus, the inhibition of
ROS may be partially responsible for the reduced inflammation
induced by HCAG. ROS is also involved in the activation process of
the NLRP3 inflammasome [43]. The priming step of the NLRP3
inflammasome is regulated by ROS, which is required for NLRP3
expression [38]. HCAG did not affect NLRP3 expression in LPS-
activated macrophages (Fig. 3A), although the antioxidant activity
of HCAG in LPS-activated macrophages was observed (Fig. 4A).
NLRP3 expression in LPS-activated macrophages is also regulated
Fig. 7. Effect of HCAG on renal NF-κB activation and MCP-1 and IL-6 expression
levels in the LPRI mouse model. (A) Scoring of pNF-κB p65 staining. (B) Renal pNF-
κB p65 activity measured by ELISA. (C) Renal MCP-1 and IL-6 protein levels mea-
sured by ELISA. The data are the mean7SEM for seven mice per group. *po0.05,
**po0.01, ***po0.001.
the vehicleþLPRI mice showed an intense infiltration of macro-
phages and CD3þT cells in the periglomerular region of the kid-
ney, but few or no infiltrating macrophages and T cells were ob-
served in the HCAGþLPRI mice.
3.12. HCAG inhibits T cell activation in the spleen
by NF-
κ
B [62] and the MAPK pathways [39]. We showed that NF-
κB (Fig. 4B and C) and MAPK (Fig. 4D) were inhibited and that the
Systemic activation of T and B cells plays a crucial role in the
pathogenesis of renal inflammation and fibrosis [49,50]. We ex-
amined whether inhibition of the activation of T or B cells may be
NLRP3 inflammasome activation was suppressed (Fig. 3B and C) by
HCAG administration, although the expression level of the NLRP3
protein was not affected (Fig. 3A).

S.-M. Ka et al. / Free Radical Biology and Medicine 91 (2016) 10–24
21
Fig. 8. Effect of HCAG on the renal infiltration of macrophages and T cells in the LPRI mouse model. (A) Staining of renal tissues for F4/80þ macrophages and CD3þ T cells.
The arrows indicate positively stained cells. (B) Scoring of positively stained cells. Original magnification, 400 ꢁ . The data are the mean7SEM for seven mice per group.
**po0.01, ***po0.001.

22
S.-M. Ka et al. / Free Radical Biology and Medicine 91 (2016) 10–24
þ
þ
þ
þ
þ
þ
Fig. 9. Effect of HCAG on splenic T cell activation in the LPRI mouse model. Percentage of CD3 CD69 cells in CD3 splenocytes, CD4 CD69 cells in CD4 splenocytes and
þ
þ
þ
CD19 CD69 cells in CD19 splenocytes. The data are the mean7SEM for seven mice per group. **po0.01, ***po0.001.
Fig. 10. A diagram of HCAG-mediated anti-inflammatory and renal protective effects.
The full activation of the NLRP3 inflammasome requires both a
priming signal from LPS-stimulated TLR4 and an activation signal
from ATP; the former controls the expression of NLRP3 and proIL-
mitochondrial functions [64].
Mice with diabetic nephropathy exhibited significantly in-
creased expression of TLR4 and its associated protein MyD88 in
the kidney [65]. Interestingly, genetic deficiency of TLR4 amelio-
rated renal inflammation, fibrosis and podocytopathy [65,66].
TLR4-knockout mice were also protected from renal injury after
LPS exposure [67], suggesting that TLR4 signaling may represent a
potential therapeutic target for the prevention of renal injury [6].
In this study, we found that HCAG decreased renal TLR4 and
MyD88 expression. This reduced expression may be responsible
for the reduced renal injury induced by LPS. In our previous report,
we demonstrated that CA inhibited TLR4-mediated signaling [23];
these findings were supported by another report [68].
Hit to lead is a stage in early drug discovery where small mo-
lecule hits undergo limited optimization through the synthesis of
analogs to identify promising lead compounds with improved ef-
ficacy or reduced toxicity [69]. CA and its derivatives have been
reported to possess anti-tumor activity [70], anti-bacterial activity
1 and the latter controls caspase-1 activation. The up-regulation
β
of ROS levels in ATP-treated macrophages resulted in the activa-
tion of the PI3K/AKT pathway, thereby promoting caspase-1 acti-
vation and IL-1
phosphorylation may be responsible for the reduced caspase-1
activation and IL-1 secretion induced by HCAG (Fig. 4G); how-
β
secretion [44]. The inhibition of ATP-induced AKT
β
ever, HCAG did not inhibit ATP-induced ROS generation (Fig. 4F).
These results suggest that HCAG acts upstream of AKT and
downstream of ROS. Overproduction of mitochondrial ROS from
damaged mitochondria promotes a mitochondrial permeability
transition and facilitates the cytosolic release of mitochondrial
DNA, which in turn stimulates the activation of the NLRP3 in-
flammasome [17,63]. Although HCAG inhibits NLRP3 inflamma-
some activation, the effect of HCAG on mitochondrial function
requires further investigation. Interestingly, cinnamaldehyde in-
duces apoptosis of cancer cells by inducing derangement of
[71], anti-tyrosinase activity [72], and anti-AP-1 and -NF- B
κ

S.-M. Ka et al. / Free Radical Biology and Medicine 91 (2016) 10–24
23
activity [73,74]. In this study, we report for the first time that the
glycosylated CA derivative possesses anti-inflammatory activity,
and especially inhibits NLRP3 inflammasome activation. Im-
portantly, the toxicity of HCAG was reduced more than one hun-
dred-fold compared with CA (Fig. 1B). The reduced toxicity results
in a huge increase in the HCAG therapeutic window and suggests
that this molecule can treat disease effectively while staying
within the safety range. Furthermore, our data suggest that HCAG
may be helpful in ameliorating renal inflammation by targeting
the interlinked inflammatory pathway.
J.J. Huang, S.S. Yang, A. Chen, Epigallocatechin-3-gallate prevents lupus ne-
phritis development in mice via enhancing the Nrf2 antioxidant pathway and
inhibiting NLRP3 inflammasome activation, Free Radic. Biol. Med. 51 (2011)
744–754.
[20] S.M. Yang, Y.L. Chan, K.F. Hua, J.M. Chang, H.L. Chen, Y.J. Tsai, Y.J. Hsu, L.K. Chao,
Y. Feng-Ling, Y.L. Tsai, S.H. Wu, Y.F. Wang, C.L. Tsai, A. Chen, S.M. Ka, Osthole
improves an accelerated focal segmental glomerulosclerosis model in the
early stage by activating the Nrf2 antioxidant pathway and subsequently in-
hibiting NF-kappaB-mediated COX-2 expression and apoptosis, Free Radic.
Biol. Med. 73 (2014) 260–269.
21] S.M. Yang, K.F. Hua, Y.C. Lin, A. Chen, J.M. Chang, L. Kuoping Chao, C.L. Ho, S.
M. Ka, Citral is renoprotective for focal segmental glomerulosclerosis by in-
hibiting oxidative stress and apoptosis and activating Nrf2 pathway in mice,
Plos One 8 (2013) e74871.
[
[
22] S.M. Yang, S.M. Ka, K.F. Hua, T.H. Wu, Y.P. Chuang, Y.W. Lin, F.L. Yang, S.H. Wu,
S.S. Yang, S.H. Lin, J.M. Chang, A. Chen, Antroquinonol mitigates an accelerated
and progressive IgA nephropathy model in mice by activating the Nrf2
pathway and inhibiting T cells and NLRP3 inflammasome, Free Radic. Biol.
Med. 61 (2013) 285–297.
Acknowledgments
This study was supported by grants 102-2628-B-197-001-MY3,
03-2923-B-197-001-MY3, MOST 103-2321-B-016-002 and NSC
02-2320-B-016-006-MY3 from the Ministry of Science and
Technology, and MAB104-016 from National Defense Medical
Center, Taipei, Taiwan, ROC.
[
[
23] L.K. Chao, K.F. Hua, H.Y. Hsu, S.S. Cheng, I.F. Lin, C.J. Chen, S.T. Chen, S.T. Chang,
Cinnamaldehyde inhibits pro-inflammatory cytokines secretion from mono-
cytes/macrophages through suppression of intracellular signaling, Food Chem.
Toxicol. 46 (2008) 220–231.
24] E.N. Faikoh, Y.H. Hong, S.Y. Hu, Liposome-encapsulated cinnamaldehyde en-
hances zebrafish (Danio rerio) immunity and survival when challenged with
Vibrio vulnificus and Streptococcus agalactiae, Fish. Shellfish. Immunol. 38
1
1
(2014) 15–24.
[
25] J.Y. Guo, H.R. Huo, B.S. Zhao, H.B. Liu, L.F. Li, Y.Y. Ma, S.Y. Guo, T.L. Jiang, Cin-
namaldehyde reduces IL-1beta-induced cyclooxygenase-2 activity in rat cer-
ebral microvascular endothelial cells, Eur. J. Pharmacol. 537 (2006) 174–180.
26] B.C. Liao, C.W. Hsieh, Y.C. Liu, T.T. Tzeng, Y.W. Sun, B.S. Wung, Cinnamaldehyde
inhibits the tumor necrosis factor-alpha-induced expression of cell adhesion
molecules in endothelial cells by suppressing NF-kappaB activation: effects
upon IkappaB and Nrf2, Toxicol. Appl. Pharmacol. 229 (2008) 161–171.
27] L.T. Lin, C.J. Tai, S.P. Chang, J.L. Chen, S.J. Wu, C.C. Lin, Cinnamaldehyde-induced
apoptosis in human hepatoma PLC/PRF/5 cells involves the mitochondrial
death pathway and is sensitive to inhibition by cyclosporin A and z-VAD-fmk,
Anticancer Agents Med. Chem. 13 (2013) 1565–1574.
References
[
[
[
[
[
[
[
[
1] R.S. Goldszmid, G. Trinchieri, The price of immunity, Nat. Immunol. 13 (2012)
32–938.
2] E. Latz, T.S. Xiao, A. Stutz, Activation and regulation of the inflammasomes,
Nat. Rev. Immunol. 13 (2013) 397–411.
3] D. De Nardo, E. Latz, NLRP3 inflammasomes link inflammation and metabolic
disease, Trends Immunol. 32 (2011) 373–379.
4] H. Wen, J.P. Ting, L.A. O’Neill, A role for the NLRP3 inflammasome in metabolic
diseases – did Warburg miss inflammation? Nat. Immunol. 13 (2012) 352–357.
5] H.J. Anders, D.A. Muruve, The inflammasomes in kidney disease, J. Am. Soc.
Nephrol. 22 (2011) 1007–1018.
6] J.C. Leemans, L. Kors, H.J. Anders, S. Florquin, Pattern recognition receptors and
the inflammasome in kidney disease, Nat. Rev. Nephrol. 10 (2014) 398–414.
7] S.R. Mulay, O.P. Kulkarni, K.V. Rupanagudi, A. Migliorini, M.N. Darisipudi,
A. Vilaysane, D. Muruve, Y. Shi, F. Munro, H. Liapis, H.J. Anders, Calcium oxalate
crystals induce renal inflammation by NLRP3-mediated IL-1beta secretion, J.
Clin. Invest. 123 (2013) 236–246.
9
[
[
28] M.J. Hooth, R.C. Sills, L.T. Burka, J.K. Haseman, K.L. Witt, D.P. Orzech, A.
F. Fuciarelli, S.W. Graves, J.D. Johnson, J.R. Bucher, Toxicology and carcino-
genesis studies of microencapsulated trans-cinnamaldehyde in rats and mice,
Food Chem. Toxicol. 42 (2004) 1757–1768.
[
[
29] M. Theurer, N. Shaik, F. Lang, Stimulation of suicidal erythrocyte death by
trans-cinnamaldehyde, Phytomedicine 20 (2013) 1119–1123.
30] J.X. Zhai, Z.X. Zhang, Y.J. Feng, S.S. Ding, X.H. Wang, L.W. Zou, D.Q. Ye, PDTC
attenuate LPS-induced kidney injury in systemic lupus erythematosus-prone
MRL/lpr mice, Mol. Biol. Rep. 39 (2012) 6763–6771.
[
[
8] A. Vilaysane, J. Chun, M.E. Seamone, W. Wang, R. Chin, S. Hirota, Y. Li, S.
A. Clark, J. Tschopp, K. Trpkov, B.R. Hemmelgarn, P.L. Beck, D.A. Muruve, The
NLRP3 inflammasome promotes renal inflammation and contributes to CKD, J.
Am. Soc. Nephrol. 21 (2010) 1732–1744.
9] J. Zhao, H. Wang, C. Dai, H. Wang, H. Zhang, Y. Huang, S. Wang, F. Gaskin,
N. Yang, S.M. Fu, P2X7 blockade attenuates murine lupus nephritis by in-
hibiting activation of the NLRP3/ASC/caspase 1 pathway, Arthritis Rheum. 65
[31] S.M. Ka, Y.C. Kuo, P.J. Ho, P.Y. Tsai, Y.J. Hsu, W.J. Tsai, Y.L. Lin, C.C. Shen, A. Chen,
(S)-armepavine from Chinese medicine improves experimental autoimmune
crescentic glomerulonephritis, Rheumatology (Oxford) 49 (2010) 1840–1851.
32] S.M. Ka, T.T. Hsieh, S.H. Lin, S.S. Yang, C.C. Wu, H.K. Sytwu, A. Chen, Decoy
receptor 3 inhibits renal mononuclear leukocyte infiltration and apoptosis and
prevents progression of IgA nephropathy in mice, Am. J. Physiol. Ren. Physiol.
[
301 (2011) F1218–F1230.
(
2013) 3176–3185.
[
33] S.M. Ka, Y.C. Yeh, X.R. Huang, T.K. Chao, Y.J. Hung, C.P. Yu, T.J. Lin, C.C. Wu, H.
Y. Lan, A. Chen, Kidney-targeting Smad7 gene transfer inhibits renal TGF-beta/
MAD homologue (SMAD) and nuclear factor kappaB (NF-kappaB) signalling
pathways, and improves diabetic nephropathy in mice, Diabetologia 55 (2012)
509–519.
[
10] J. Zhao, H. Wang, Y. Huang, H. Zhang, S. Wang, F. Gaskin, N. Yang, S.M. Fu,
Lupus nephritis: glycogen synthase kinase 3beta promotion of renal damage
through activation of the NLRP3 inflammasome in lupus-prone mice, Arthritis
Rheumatol. 67 (2015) 1036–1044.
[
11] S. Granata, V. Masola, E. Zoratti, M.T. Scupoli, A. Baruzzi, M. Messa, F. Sallustio,
L. Gesualdo, A. Lupo, G. Zaza, NLRP3 inflammasome activation in dialyzed
chronic kidney disease patients, Plos One 10 (2015) e0122272.
12] T.M. Chan, Treatment of severe lupus nephritis: the new horizon, Nat. Rev.
Nephrol. 11 (2015) 46–61.
[34] S.M. Ka, H.K. Sytwu, D.M. Chang, S.L. Hsieh, P.Y. Tsai, A. Chen, Decoy receptor
3 ameliorates an autoimmune crescentic glomerulonephritis model in mice, J.
Am. Soc. Nephrol. 18 (2007) 2473–2485.
[35] S.M. Ka, C.W. Cheng, H.A. Shui, W.M. Wu, D.M. Chang, Y.C. Lin, A. Chen, Me-
sangial cells of lupus-prone mice are sensitive to chemokine production, Ar-
thritis Res. Ther. 9 (2007) R67.
[
[
13] A.T. Borchers, N. Leibushor, S.M. Naguwa, G.S. Cheema, Y. Shoenfeld, M.
E. Gershwin, Lupus nephritis: a critical review, Autoimmun. Rev. 12 (2012)
[36] F.H. Carpenter, The anhydride of benzylpenicillin, J. Am. Chem. Soc. 70 (1948)
174–194.
2964–2966.
[
14] Q.H. Hu, X. Zhang, Y. Pan, Y.C. Li, L.D. Kong, Allopurinol, quercetin and rutin
ameliorate renal NLRP3 inflammasome activation and lipid accumulation in
fructose-fed rats, Biochem. Pharmacol. 84 (2012) 113–125.
[37] J. Kerekgyarto, Z. Szurmai, A. Liptak, Synthesis of p-trifluoroacetamidophenyl
6-deoxy-2-O-(3-O-[2-O-methyl-3-O-(2-O-methyl-alpha- -rhamnopyranosyl)-
D
alpha- -fucopyranosyl]-alpha- -rhamnopyranosyl)-alpha-L-talopyranoside: a
L
L
[
15] Q. Shan, Y. Zheng, J. Lu, Z. Zhang, D. Wu, S. Fan, B. Hu, X. Cai, H. Cai, P. Liu, F. Liu,
Purple sweet potato color ameliorates kidney damage via inhibiting oxidative
stress mediated NLRP3 inflammasome activation in high fat diet mice, Food
Chem. Toxicol. 69 (2014) 339–346.
spacer armed tetrasaccharide glycopeptidolipid antigen of Mycobacterium
avium serovar 20, Carbohydr. Res. 245 (1993) 65–80.
[38] F. Bauernfeind, E. Bartok, A. Rieger, L. Franchi, G. Nunez, V. Hornung, Cutting
edge: reactive oxygen species inhibitors block priming, but not activation, of
the NLRP3 inflammasome, J. Immunol. 187 (2011) 613–617.
[39] P.C. Liao, L.K. Chao, J.C. Chou, W.C. Dong, C.N. Lin, C.Y. Lin, A. Chen, S.M. Ka, C.
L. Ho, K.F. Hua, Lipopolysaccharide/adenosine triphosphate-mediated signal
transduction in the regulation of NLRP3 protein expression and caspase-1-
mediated interleukin-1beta secretion, Inflamm. Res. 62 (2013) 89–96.
[40] K. Takeda, T. Kaisho, S. Akira, Toll-like receptors, Annu. Rev. Immunol. 21
(2003) 335–376.
[
16] C. Wang, Y. Pan, Q.Y. Zhang, F.M. Wang, L.D. Kong, Quercetin and allopurinol
ameliorate kidney injury in STZ-treated rats with regulation of renal NLRP3
inflammasome activation and lipid accumulation, Plos. One 7 (2012) e38285.
17] Y.P. Chang, S.M. Ka, W.H. Hsu, A. Chen, L.K. Chao, C.C. Lin, C.C. Hsieh, M.C. Chen,
H.W. Chiu, C.L. Ho, Y.C. Chiu, M.L. Liu, K.F. Hua, Resveratrol inhibits NLRP3
inflammasome activation by preserving mitochondrial integrity and aug-
menting autophagy, J. Cell. Physiol. 230 (2015) 1567–1579.
[
[
18] K.F. Hua, S.M. Yang, T.Y. Kao, J.M. Chang, H.L. Chen, Y.J. Tsai, A. Chen, S.S. Yang,
L.K. Chao, S.M. Ka, Osthole mitigates progressive IgA nephropathy by in-
hibiting reactive oxygen species generation and NF-kappaB/NLRP3 pathway,
Plos One 8 (2013) e77794.
[41] K.F. Hua, S.H. Wang, W.C. Dong, C.Y. Lin, C.L. Ho, T.H. Wu, High glucose in-
creases nitric oxide generation in lipopolysaccharide-activated macrophages
by enhancing activity of protein kinase C-alpha/delta and NF-kappaB, In-
flamm. Res. 61 (2012) 1107–1116.
[19] P.Y. Tsai, S.M. Ka, J.M. Chang, H.C. Chen, H.A. Shui, C.Y. Li, K.F. Hua, W.L. Chang,
[42] S.C. Su, K.F. Hua, H. Lee, L.K. Chao, S.K. Tan, H. Lee, S.F. Yang, H.Y. Hsu, LTA and

24
S.-M. Ka et al. / Free Radical Biology and Medicine 91 (2016) 10–24
LPS mediated activation of protein kinases in the regulation of inflammatory
cytokines expression in macrophages, Clin. Chim. Acta 374 (2006) 106–115.
43] J. Tschopp, K. Schroder, NLRP3 inflammasome activation: The convergence of
anti-NF-kappaB/NLRP3 inflammasome-mediated inflammation, enhancement
of NRF2 antioxidant activity and inhibition of apoptosis, Diabetologia 57
(2014) 424–434.
[
[
multiple signalling pathways on ROS production? Nat. Rev. Immunol. 10
[60] C.L. Ho, C.Y. Lin, S.M. Ka, A. Chen, Y.L. Tasi, M.L. Liu, Y.C. Chiu, K.F. Hua, Bamboo
vinegar decreases inflammatory mediator expression and NLRP3 inflamma-
some activation by inhibiting reactive oxygen species generation and protein
kinase C-alpha/delta activation, Plos One 8 (2013) e75738.
[61] P.C. Liao, S.C. Chien, C.L. Ho, E.I. Wang, S.C. Lee, Y.H. Kuo, N. Jeyashoke, J. Chen,
W.C. Dong, L.K. Chao, K.F. Hua, Osthole regulates inflammatory mediator ex-
pression through modulating NF-kappaB, mitogen-activated protein kinases,
protein kinase C, and reactive oxygen species, J. Agric. Food Chem. 58 (2010)
(
2010) 210–215.
44] C.M. Cruz, A. Rinna, H.J. Forman, A.L. Ventura, P.M. Persechini, D.M. Ojcius, ATP
activates a reactive oxygen species-dependent oxidative stress response and
secretion of proinflammatory cytokines in macrophages, J. Biol. Chem. 282
(
2007) 2871–2879.
[
[
[
[
45] P.Y. Tsai, S.M. Ka, J.M. Chang, J.H. Lai, M.S. Dai, H.L. Jheng, M.T. Kuo, P. Chen,
A. Chen, Antroquinonol differentially modulates T cell activity and reduces
interleukin-18 production, but enhances Nrf2 activation, in murine ac-
celerated severe lupus nephritis, Arthritis Rheum. 64 (2012) 232–242.
46] I. Batal, S.A. De Serres, B.G. Mfarrej, M. Grafals, G.S. Pinkus, A. Kalra, A. Weins,
V. Bijol, H.G. Rennke, I. Guleria, R. Abdi, A. Chandraker, N. Najafian, Glomerular
inflammation correlates with endothelial injury and with IL-6 and IL-1beta
secretion in the peripheral blood, Transplantation 97 (2014) 1034–1042.
47] H.R. Vianna, C.M. Soares, K.D. Silveira, G.S. Elmiro, P.M. Mendes, M. de Sousa
Tavares, M.M. Teixeira, D.M. Miranda, E.S.A.C. Simoes, Cytokines in chronic
kidney disease: potential link of MCP-1 and dyslipidemia in glomerular dis-
eases, Pediatr. Nephrol. 28 (2013) 463–469.
48] K.F. Hua, J.C. Chou, Y. Lam, Y.L. Tasi, A. Chen, S.M. Ka, Z. Fang, M.L. Liu, F.L. Yang,
Y.L. Yang, Y.C. Chiu, S.H. Wu, Polyenylpyrrole derivatives inhibit NLRP3 in-
flammasome activation and inflammatory mediator expression by reducing
reactive oxygen species production and mitogen-activated protein kinase ac-
tivation, Plos. One 8 (2013) e76754.
10445–10451.
[
62] F.G. Bauernfeind, G. Horvath, A. Stutz, E.S. Alnemri, K. MacDonald, D. Speert,
T. Fernandes-Alnemri, J. Wu, B.G. Monks, K.A. Fitzgerald, V. Hornung, E. Latz,
Cutting edge: NF-kappaB activating pattern recognition and cytokine re-
ceptors license NLRP3 inflammasome activation by regulating NLRP3 ex-
pression, J. Immunol. 183 (2009) 787–791.
[63] R. Zhou, A.S. Yazdi, P. Menu, J. Tschopp, A role for mitochondria in NLRP3
inflammasome activation, Nature 469 (2011) 221–225.
[
64] H. Ka, H.J. Park, H.J. Jung, J.W. Choi, K.S. Cho, J. Ha, K.T. Lee, Cinnamaldehyde
induces apoptosis by ROS-mediated mitochondrial permeability transition in
human promyelocytic leukemia HL-60 cells, Cancer Lett. 196 (2003) 143–152.
65] I. Jialal, A.M. Major, S. Devaraj, Global toll-like receptor 4 knockout results in
decreased renal inflammation, fibrosis and podocytopathy, J. Diabetes Com-
plicat. 28 (2014) 755–761.
66] J. Ma, S.J. Chadban, C.Y. Zhao, X. Chen, T. Kwan, U. Panchapakesan, C.A. Pollock,
H. Wu, TLR4 activation promotes podocyte injury and interstitial fibrosis in
diabetic nephropathy, Plos One 9 (2014) e97985.
67] J.A. Smith, L.J. Stallons, J.B. Collier, K.D. Chavin, R.G. Schnellmann, Suppression
of mitochondrial biogenesis through toll-like receptor 4-dependent mitogen-
activated protein kinase kinase/extracellular signal-regulated kinase signaling
in endotoxin-induced acute kidney injury, J. Pharmacol. Exp. Ther. 352 (2015)
[
[
[
[49] M.R. Clatworthy, K.G. Smith, B cells in glomerulonephritis: focus on lupus
nephritis, Semin. Immunopathol. 29 (2007) 337–353.
[
50] R.A. Cohen, G. Bayliss, J.C. Crispin, G.F. Kane-Wanger, C.A. Van Beek, V.
C. Kyttaris, I. Avalos, C.Y. Yu, G.C. Tsokos, I.E. Stillman, T cells and in situ
cryoglobulin deposition in the pathogenesis of lupus nephritis, Clin. Immunol.
128 (2008) 1–7.
[
51] T.M. El-Achkar, M. Hosein, P.C. Dagher, Pathways of renal injury in systemic
gram-negative sepsis, Eur. J. Clin. Invest. 38 (Suppl. 2) (2008) S39–S44.
52] L. Verstrepen, T. Bekaert, T.L. Chau, J. Tavernier, A. Chariot, R. Beyaert, TLR-4,
IL-1R and TNF-R signaling to NF-kappaB: variations on a common theme, Cell.
Mol. Life Sci. 65 (2008) 2964–2978.
346–357.
[
68] H.S. Youn, J.K. Lee, Y.J. Choi, S.I. Saitoh, K. Miyake, D.H. Hwang, J.Y. Lee, Cin-
namaldehyde suppresses toll-like receptor 4 activation mediated through the
inhibition of receptor oligomerization, Biochem. Pharmacol. 75 (2008)
[
494–502.
[
53] L.K. Chao, C.H. Lin, H.W. Chiu, W.T. Wong, H.W. Chiu, Y.L. Tasi, Y.H. Kuo, Y.C.
Chiu, M.L. Liu, C.L. Ho, K.F. Hua, Peroxyauraptenol inhibits inflammation and
NLRP3 inflammasome activation by inhibiting reactive oxygen species gen-
eration and preserving mitochondrial integrity, J. Agric. Food Chem. (2015).
54] S. Hong, Y. Lu, Omega-3 fatty acid-derived resolvins and protectins in in-
flammation resolution and leukocyte functions: targeting novel lipid mediator
pathways in mitigation of acute kidney injury, Front. Immunol. 4 (2013) 13.
55] P.R. Mayeux, L.A. MacMillan-Crow, Pharmacological targets in the renal peri-
tubular microenvironment: implications for therapy for sepsis-induced acute
kidney injury, Pharmacol. Ther. 134 (2012) 139–155.
[
[
69] G.M. Keseru, G.M. Makara, Hit discovery and hit-to-lead approaches, Drug.
Discov. today 11 (2006) 741–748.
70] E.H. Chew, A.A. Nagle, Y. Zhang, S. Scarmagnani, P. Palaniappan, T.D. Bradshaw,
A. Holmgren, A.D. Westwell, Cinnamaldehydes inhibit thioredoxin reductase
and induce Nrf2: potential candidates for cancer therapy and chemopreven-
tion, Free. Radic. Biol. Med. 48 (2010) 98–111.
[
[
[
71] X. Li, J. Sheng, G. Huang, R. Ma, F. Yin, D. Song, C. Zhao, S. Ma, Design, synthesis
and antibacterial activity of cinnamaldehyde derivatives as inhibitors of the
bacterial cell division protein FtsZ, Eur. J. Med. Chem. 97 (2015) 32–41.
[
[
[
56] H.J. Anders, B. Banas, D. Schlondorff, Signaling danger: toll-like receptors and
their potential roles in kidney disease, J. Am. Soc. Nephrol. 15 (2004) 854–867.
57] M.G. Robson, Toll-like receptors and renal disease, Nephron. Exp. Nephrol. 113
[72] Y. Cui, G. Liang, Y.H. Hu, Y. Shi, Y.X. Cai, H.J. Gao, Q.X. Chen, Q. Wang, Alpha-
substituted derivatives of cinnamaldehyde as tyrosinase inhibitors: inhibitory
mechanism and molecular analysis, J. Agric. Food Chem. 63 (2015) 716–722.
[73] J.Y. Kwon, S.H. Hong, S.D. Park, S.G. Ahn, J.H. Yoon, B.M. Kwon, S.A. Kim, 2′-
Benzoyloxycinnamaldehyde inhibits nitric oxide production in lipopoly-
saccharide-stimulated RAW 264.7 cells via regulation of AP-1 pathway, Eur. J.
Pharmacol. 696 (2012) 179–186.
[74] A.M. Reddy, J.H. Seo, S.Y. Ryu, Y.S. Kim, Y.S. Kim, K.R. Min, Y. Kim, Cinna-
maldehyde and 2-methoxycinnamaldehyde as NF-kappaB inhibitors from
Cinnamomum cassia, Planta Med. 70 (2004) 823–827.
(
2009) e1–e7.
58] I.T. Lee, R.H. Shih, C.C. Lin, J.T. Chen, C.M. Yang, Role of TLR4/NADPH oxidase/
ROS-activated p38 MAPK in VCAM-1 expression induced by lipopolysacchar-
ide in human renal mesangial cells, Cell. Commun. Signal.: CCS 10 (2012) 33.
59] S.M. Yang, S.M. Ka, H.L. Wu, Y.C. Yeh, C.H. Kuo, K.F. Hua, G.Y. Shi, Y.J. Hung, F.
C. Hsiao, S.S. Yang, Y.S. Shieh, S.H. Lin, C.W. Wei, J.S. Lee, C.Y. Yang, A. Chen,
Thrombomodulin domain 1 ameliorates diabetic nephropathy in mice via
[