Pharmacological Research 144 (2019) 227–234

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Pharmacological Research
journal homepage: www.elsevier.com/locate/yphrs

Neuroprotective effects of andrographolide derivative CX-10 in transient T

focal ischemia in rat: Involvement of Nrf2/AE and TLR/NF-κB signaling
⁎,1
Ming-Yan Yanga, , Qing-Long Yub,1, Yao-Shi Huanga, Guo Yanga
a
Shandong Target Drug Research Co. Ltd., Yantai 264005, Shandong Province, China
b
Yantai Key Laboratory of Nanomedicine & Advanced Preparations, Yantai Institute of Materia Medica, Shandong, 264000, China

A R T I C LE I N FO A B S T R A C T

Keywords: Ischemic stroke is a major cause of mortality and disability worldwide. To date there is no ideal effective
Andrographolide treatment. 3, 14, 19-triacetyl andrographolide (CX-10) is a new molecule entity derived from andrographolide.
Inflammation The aim of the present study was to evaluate the neuroprotection of CX-10 against experimental cerebral
Ischemic stroke ischemia. The anti-inflammation of CX-10 was screened using LPS-induced inflammation in vitro and in vivo.
Nrf2 signaling
Rats were subjected to 1.5 h of middle cerebral occlusion (MCAO) and then reperfusion for 72 h. The infarct size
Oxidative stress
TLR4 signaling
was evaluated by TTC staining, and the behavioral disturbance was evaluated, and inflammatory cytokines and
anti-oxidant enzymes in brain tissues were examined. Western blot was used to analyze the expression of pro-
teins. The results showed that CX-10 exerted potent anti-inflammatory and anti-oxidation activities, which
significantly inhibited LPS-induced TNF-α and NO release, lowered TNF-α and IL-1β levels in the brain,
meanwhile increased activities of SOD, CAT and GSH-P × . The effect of CX-10 was equivalent to that of dex-
amethasone, and was obviously superior to that of andrographolide. CX-10 exhibited a neuroprotective effects,
manifested as reducing infarct size, improving neurological function and reducing motor impairments.
Furthermore, western blot analysis revealed that treatment with CX-10 down-regulated the expression of TLR4,
NF-κB, TNF-α and iNOS, induced Nrf2 and HO-1 expression. Overall, CX-10 has a favorable neuroprotection in
ischemic brain injury. The mechanism may involve inhibition of TLR4/NF-κB signaling pathway and upregu-
lation of Nrf2/ARE signaling pathway. All these indicated that CX-10 is likely to be a promising agent for
ischemic stroke.

1. Introduction Hence, development of effective and safe therapeutic strategies for

Stroke is a common and sudden cerebrovascular disease, including
hemorrhagic and ischemic strokes, in which ischemic stroke accounts
for approximately 87% of all strokes [1]. Ischemic stroke is a major
cause of morbidity, mortality and long-term disability worldwide. Ac-
cording to a report from the American Heart Association, the global
prevalence of cerebrovascular disease was 42.4 million people, in
which ischemic stroke was 24.9 million [1]. There were 6.3 million
stroke deaths worldwide in 2015, becoming the second-leading global
cause of death [1]. Such higher prevalence and mortality brought about
enormous economic and social burden. In the United States, the average
annual medical cost of stroke is $40.1 billion. The total cost of stroke in
2035 is predicted to be $94.3 billion [1]. In China, stroke is also the
leading cause of death. Moreover, the prevalence of stroke still in-
creases along with an increasing and rapidly ageing population [2,3].
stroke is an urgent and important task for modern medicine.
Up to now there is no ideal treatment for stroke. Current strategies
for treatment of stroke include re-establishing perfusion through
pharmacologic and mechanical thrombolysis and neuroprotection [4].
To date recombinant tissue plasminogen activator (rtPA), a thrombo-
lytic drug, is the only FDA-approved medical therapy for acute ischemic
stroke. However, clinical application of rtPA has some limitation due to
its narrow therapeutic time window and potential risk of hemorrhage.
Only ˜2% patients are eligible for treatment with rtPA [5,6]. There have
not yet effective neuroprotective agents for stroke patients in clinic,
except for rtPA [5,6]. Consequently, there is an urgent need to look for
novel therapeutics to attenuate nervous injury and promote recovery of
nervous function for ischemic stroke patients.
Andrographis paniculata (A. paniculata) is a versatile Chinese herbal
drug for the treatment of flu, upper respiratory tract infection and sore

⁎
Corresponding author at: Department of Pharmacology, Shandong Target Drug Research Co. Ltd., No. 1 Wanshoushan Road, 264005 Yantai, Shandong, China.
E-mail address: mingyan-123456@163.com (M.-Y. Yang).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.phrs.2019.04.023
Received 11 February 2019; Received in revised form 16 April 2019; Accepted 19 April 2019
Available online 24 April 2019
1043-6618/ © 2019 Elsevier Ltd. All rights reserved.
M.-Y. Yang, et al.
Fig. 1. Chemical structure of CX-10.
throat [7]. Andrographolide, the major active constituent isolated from
Andrographis paniculata (A. paniculata), exerts anti-viral, anti-throm-
botic, anti-inflammation properties [8]. In China, several products
based on andrographolide have been commercially available and
widely used in clinic [9]. Due to its extensive pharmacological activ-
ities, andrographolide is regarded as a good pharmacophore. Structure
modifications of andrographolide have been investigated for years,
such as esterification and epoxidation. Most of the modifications are
positive and have gained increasing biological activities [10]. The an-
drographolide derivative 3, 14, 19-triacetyl andrographolide, also
termed as CX-10, is an acetylated product with acetoxy-groups at C-3,
C-14, C-19 positions (Fig. 1) [11]. The 1H NMR spectroscopic data was
followed as below: 1H (400 MHz, CDCl3): δ = 7.01 (td, J = 6.84,
1.48 Hz, 1H, H-12), 5.92 (d, J = 5.96 Hz, 1H, H-14), 4.90 (bs, 1H, H-
17a), 4.60 (dd, J = 11.68, 4.26 Hz, 1H, H-15a), 4.55 (dd, J = 11.24,
6.08 Hz, 1H, H-15b), 4.53(bs, 1H, H-17b), 4.36(d, J = 11.80 Hz, 1H, H-
19a), 4.25 (dd, J = 11.24, 1.80 Hz, 1H, H-3), 4.12 (d, J = 11.80 Hz, 1H,
H-19b), 2.52˜2.37(m, 3 H), 2.12(s, 3 H), 2.05(s, 6 H), 2.00˜1.96(m,
1 H), 1.90˜1.86(m, 2 H), 1.80˜1.77(m, 2 H), 1.73˜1.66(m, 1 H),
1.57˜1.47(m, 1 H), 1.37˜1.31(m, 2 H), 1.03(s, 3 H), 0.76 (s, 3 H). MS
(ESI) m/z (%) = 499([M + Na]+, 100%), 477([M+H]+, 13%). Accu-
mulating evidence suggests that inflammation plays a crucial role in the
pathophysiology of acute ischemic stroke. Anti-inflammatory agents
could reduce ischemic brain tissue damage. Inflammation may serve as
a valid target for pharmacological intervention [12–15]. We herein
evaluated the anti-inflammation of CX-10 and explored its neuropro-
tective effects in transient focal ischemia in rat.
2. Materials and methods
2.1. Cell line
Mouse macrophage RAW264.7 cells were purchased from ATCC
(TIB-71) and cultured in RPMI1640 medium supplemented with 10%
heat-inactivated FBS, 100U/mL penicillin and 100 μg/mL streptomycin,
2 mM L-glutamine in a humidified atmosphere of 5% CO2 and 95% air
at 37℃.
2.2. Animals
Male BALB/c mice (7-week-old) were purchased from Jinan
Pengyue Experimental Animal Breeding Co. Ltd (certificate No.
0024579) and Sprague-Dawley (SD) rats (weight, 230–240 g) were
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Pharmacological Research 144 (2019) 227–234
purchased from Vital River Laboratory Animal Technology Co. Ltd
(certificated No. 11400700039133). All animals were acclimated for 1
week at a temperature of 24 ± 1℃ and humidity of 55% ± 5%. All
animals were housed in cages with food and tap water ad libitum. All
experiments were performed in accordance with relevant guidelines
and regulations approved by the Experimental Animal Research
Committee of Shandong Target Drug Research Co. Ltd.
2.3. Regents
CX-10, CX-10 micellar solution (for in vivo study) and andro-
grapholide (Andro) were provided by Shandong Target Drug Research
Co. Ltd. (Shandong, China). Dexamethasone sodium phosphate injec-
tion (Dex) was purchased from Guoyao Rongsheng Pharmaceutical Co.
Ltd. (Jiaozuo, China). Andrographolide Natrii Bisulfis Injection (Andro,
for in vivo study) was product of Shanhe Pharmaceutical Co. Ltd.
(Wuxi, China). Edaravone injection was product of Guorui
Pharmaceutical Co. Ltd. Lipopolysaccharide (LPS, Escherichia coli
O55:B5) and 2,3,5-triphenyltetrazolium chloride (TTC, T8877) were
products of Sigma. Antibodies used in this study were anti-TLR4 (sc-
293072, Santa Cruz), anti-NF-κB p65 (ab16502, Abcam), anti-TNF-α
(ab6671, Abcam), anti-iNOS antibody (ab15323, Abcam), anti-Nuclear
factor-E2-related factor 2 (Nrf2, sc-722, Santa Cruz) and anti-HO-1
(ab13248, Abcam). BCA protein assay kit, β-actin mouse monoclonal
antibody, histone H3 mouse monoclonal antibody, HRP-labeled goat
anti-mouse/rabbit IgG (H + L), superoxide dismutase (SOD) and
Glutathione peroxidase (GSH-Px) detection kits were obtained from
Beyotime Institute of Biotechnology (Jiangsu, China). Catalase (CAT)
assay kit was purchased from Nanjing Jiancheng Bioengineering
Institute (Nanjing, China). Mouse/rat TNF-α and IL-1β ELISA kits were
products of ExCell Bio (Jiangsu, China). All other chemicals were of
analytical grade.
2.4. Drug preparation
For in vitro study, CX-10 and Andro were dissolved in dimethyl
sulfoxide (DMSO) and diluted for cell treatment. DMSO used in our
study is suitable for cell culture or tissue culture grade DMSO. The final
DMSO concentration added into the culture medium was 0.1% (v/v),
which had no significant effect on the cell growth.
2.5. Measurement of TNF-α and NO production in vitro
Cell viability was tested by using MTT assay before conducting the
following tests. RAW264.7 cells were seeded in a 96-well plate at the
density of 2.5 × 105 cells/mL. After 1 h incubation, the cells were
treated with LPS (1 μg/mL) with or without CX-10 or Andro
(0.625–10 μM) for 24 h. The nitrite concentration in the supernatant
represented as the NO production was measured by the Griess method.
The TNF-α level was assayed by using a commercial enzyme-linked
immunosorbent assay (ELISA) kit according to the manufacturer’s in-
structions.
2.6. LPS-induced TNF-α release in mouse
BALB/c mice were randomly assigned into the following groups
(n = 6): Control, LPS alone (LPS), LPS plus Dex (10 mg/kg), LPS plus
Andro (50 mg/kg), LPS plus CX-10 (6.25, 12.5, 25, 50, 100 mg/kg). All
mice were intravenously administered once with above drugs/tested
materials. Mice in Control and LPS-only groups were injected with the
same volume of blank micellar solution. Thirty minutes after the
treatment, LPS (10 mg/kg) were given intravenously to mice to induce
inflammation. Mice in Control group were injected with normal saline
instead. Two hours after LPS injection, blood was collected from the
inner canthus of mice and anticoagulated with EDTA·Na2. Collected
blood was centrifuged at 5000 rpm for 15 min to prepared plasma. TNF-
M.-Y. Yang, et al. Pharmacological Research 144 (2019) 227–234

α level in plasma was measured by using a commercial ELISA kit ac-

cording to the manufacturer’s instructions.

2.7. Transient middle cerebral artery occlusion and treatment protocol

All animals were fasted 8–10 h before the study. A blinded manner
was performed in all animal procedures. In total, 45 male rats (weight,
270–290 g) were randomly assigned into the following five groups:
Sham-operated group, middle cerebral artery occlusion (MCAO),
edaravone 3 mg/kg, CX-10 (10, 50 mg/kg), Andro 50 mg/kg. Animals
were anaesthetized with 3% isoflurane and subsequently maintained
anesthetic status with 2% isoflurane delivered with a face mask. Each
rat was shaved and cleaned with a 75% alcohol swab. Rats were placed
in a prone position and subjected to left MCAO. The surgical procedure
was performed according to the intraluminal suture technique de-
scribed by Koizumi et al. [16]. Briefly, a midline incision was made at
the neck to expose the left carotid region. The common carotid arteries
(CCA), the external carotid arteries (ECA) and the internal carotid ar-
teries (ICA) were carefully separated from the surrounding tissue, in-
cluding the vagus nerve. A 2-cm length of a 4-0 monofilament nylon
suture with a silicon coated tip (diameter, 0.38 mm) was inserted
through the small incision in CCA and further pushed along the lumen
of ICA until resistance was felt, approximately 18 mm beyond the bi-
furcation of the CCA. After 90 min of occlusion, the filament was gently
removed and the incision in CCA was permanently ligated, and then the
midline neck wound was infiltrated with 1% lidocaine and was closed
with sutures. After disinfection of the surgical region, the animals were
placed in their normal habitat. Body temperature was maintained in the
normal rang (36.5℃ to 37.5℃) with a heating pad during the operation.
Temperature was monitored with a rectal probe. The sham-operated
animals were subjected to the same procedure without occlusion of
MCA.
CX-10, edaravone and Andro were administrated intravenously
5 min prior to reperfusion and again at 3 h after reperfusion, and then at
24 h and 48 h after reperfusion. The same volumes of blank micellar
solution were administered in the same manner to rats of the sham and
MCAO groups.

2.8. Neurological evaluation and infarct analysis

The neurological evaluation was performed at 24 h, 48 h and 72 h

after ischemia according to the method described by Yokoo et al. [17].
The neurological scoring system includes four different functions:
general status, simple motor deficit, complex motor deficit and sensory
deficit. The score was performed by an observer blinded to group as-
signment. The total score is 48, which represents worst function.
After the final neurological evaluation, animals were weighed, sa-
crificed with anaesthesia with isoflurane. The brains were removed,
immersed in normal saline to clean residual blood. Then the brains
were frozen at -20℃ for 15–20 min. Two-mm-thick coronal sections
were cut with the aid of a brain slicer matrix, and six coronal slices were
obtained. Brain slices were incubated with 2% TTC solution for 10 min
at 37℃, after which sections were fixed in 4% paraformaldehyde for
photography. Animals that developed hemorrhage were excluded from
further evaluation.
Brain areas were traced and measured using the Image-pro plus 6.0
software. The infarcted regions and the areas of both hemispheres were
calculated for each brain slice. The average area of infarction was ex-
pressed as the percentage of the whole coronal section.
Fig. 2. Effects of CX-10 on NO and TNF-α production in vitro. RAW264.7 cells
were treated with 1 μg/mL of LPS with or without CX-10 or Andro (0.625, 1.25,
2.5, 5 and 10 μM) for 24 h. The supernatant was taken out for the measurement
of NO production and TNF-α level using the Griess method and ELISA kit, re-
spectively. (A) Cell viability; (B) Inhibition on NO; (C) Inhibition on TNF-α.
was started. Rats were placed on the rotarod with the same accelerating
rotational speed. The length of time that each animal was able to stay
on the rod beginning at time 0 with switch to acceleration mode was
recorded as the latency to fall. Animals unable to grasp the rotating rod
were given a latency value of 0 s. Testing was repeated three times in
each daily session, with 5-min intervals.

2.9. Rotarod test

All animals were trained for three consecutive days on the rotarod
before MCAO procedure. The training consisted of three trials per day
at an accelerating rotational speed (4–40 rpm over a period of 5 min).
On the day of surgery and 24, 48 and 72 h after MCAO, rotarod testing
2.10. Determination of proinflammatory cytokines and antioxidant
enzymes activities in brain tissues
Seventy-two hours after MCAO, rats were sacrificed and the brains
were removed. The ischemic hemispheres were homogenized on ice in

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Fig. 3. Effects of CX-10 on LPS-induced TNF-α

release in mice. BALB/c mice were in-
travenously administered once with Dex
(10 mg/kg), Andro (50 mg/kg), CX-10 (6.25,
12.5, 25, 50, 100 mg/kg). Thirty minutes after
the treatment, LPS (10 mg/kg) were given in-
travenously to mice to induce inflammation.
Two hours after LPS injection, blood was col-
lected to prepared plasma. TNF-α level in
plasma was measured using ELISA kit. (A)
TNF-α levels of each group; (B) Dose-response
curve. Data are represented as mean ± S.D. of
6 animals of each group. ##P < 0.01 vs.
Control, **P < 0.01 vs. LPS group. & P <
0.05, && P < 0.01 vs. Andro.

Fig. 4. Neuroprotective of CX-10 in MCAO in rats. Rats were intravenously administered CX-10 (10 and 50 mg/kg), edaravone 3 mg/kg and Andro 50 mg/kg after
MCAO surgery. The frequency of administration was described in method section. (A) Infarct area of each group; (B) Representative coronal sections of each group;
(C) Neurological scores. Data are represented as mean ± S.D. of 8 animals of each group (n = 5 for Sham group). *P < 0.05 and **P < 0.01 vs. MCAO group.

PBS buffer to prepare a 10% homogenate. The homogenate was im- according to the manufacturers’ instructions. The final results were
mediately centrifuged at 12,000 rpm for 10 min, then the supernatant expressed as pg/g tissue for TNF-α and IL-1β and U/mg protein for
was collected for the determination of levels of TNF-α and IL-1β and SOD, GSH-Px and CAT activities.
activities of SOD, GSH-Px and CAT. The process of assay was performed

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Fig. 5. Effects of CX-10 on motor performance in rotarod test. Data are re-
presented as mean ± S.D. of 8 animals of each group. *P < 0.05 vs. MCAO
group.
2.11. Western blot analysis
Nuclear and cytoplasmic extracts were prepared using a nuclear and
cytoplasmic protein extraction kit (Beyotime, China). Briefly, brain
tissues (approximately 80 mg) were homogenized in cytoplasmic ex-
traction buffer. Tissue homogenates were rapidly lysed by vortexing.
The homogenate was centrifuged at 12,000 rpm for 10 min at 4 °C and
proteins were collected from the supernatant. The pellet was re-sus-
pended in 50 μL of nuclear protein extraction buffer, lysed by ultra-
sound in an ice water bath, and then centrifuged to yield the nuclear
fraction. Protein concentration was measured with a BCA protein assay
kit. Protein samples (80–100 μg) were separated using 8%–15%
SDS–PAGE gels and transferred onto PVDF membranes. The membranes
were blocked with 5% skim milk in Tris-buffered saline with Tween 20
for 2 h at room temperature and then incubated with primary anti-
bodies to anti-TLR4 (1:200 dilution), anti-NF-κB p65, anti-TNF-α, anti-
iNOS, anti-Nrf2 and anti-HO-1 (1:1000-1:5000 dilution) overnight at
4 °C. Then, the membranes were washed 3 times for 10 min each and
incubated with a horseradish peroxidase-conjugated anti-rabbit/mouse
IgG antibody. Protein bands were detected using an enhanced chemi-
luminescence detection kit (Beyotime Institute of Biotechnology) and
visualized by exposure to photographic film or detected using a BioRad
imaging system. Densitometric analysis of protein bands was performed
using Quantity One v4.4.0.36 analyzer software (BIO-RAD).
2.12. Statistical analysis
Statistical analysis was performed using SPSS 17.0 and
GraphPad_Prism software 7.0 for Windows. All results were expressed
as mean ± standard deviation (SD). Quantitative data were tested for
homogeneity of variance. If the variance was homogeneous, one-way
ANOVA followed by Dunnett test was used. Comparisons between
groups of nonparametric data were made using the Kruskal-Wallis test
followed by the Mann-Whitney U test. P < 0.05 was considered sig-
nificant.
3. Results
3.1. Effects of CX-10 on NO and TNF-α production in vitro
In our previous study, CX-10 did not show cytotoxicity against
RAW264.7 cells at the range of 0.01–10 μM (Fig. 2A). Therefore this
dose range was used in the following experiments. RAW264.7 cells
were treated with 1 μg/mL of LPS with or without CX-10 or Andro. As
shown in Fig. 2B and C, CX-10 and Andro suppressed the production of
NO and TNF-α in a concentration-dependent manner. The 50% in-
hibitory concentrations (IC50) were 2.6 and 2.1 μM for CX-10, and 4.3
and 3.9 μM for Andro, respectively.
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Pharmacological Research 144 (2019) 227–234
3.2. Effects of CX-10 on LPS-induced TNF-α release in mice
In order to explore the anti-inflammation of CX-10 in vivo. We used
LPS to induced systemic inflammatory response. As shown in Fig. 3, CX-
10 markedly decreased TNF-α production in a dose-dependent manner.
The median effective dose (ED50) was 20.7 mg/kg. The potency of CX-
10 is equivalent to that of positive control DEX, and superior to that of
Andro 50 mg/kg.
3.3. Neuroprotective of CX-10 in MCAO in rats
As shown in Fig. 4, the infarct size of MCAO group was
23.7 ± 4.9% at 72 h after ischemia injury. When treatment with
edaravone 3 mg/kg, the infarct size was 17.0 ± 4.7%, which was sig-
nificantly decreased as compared with MCAO group. Similarly, treat-
ment with CX-10 10 and 50 mg/kg inhibited the expansion of the in-
farct. The infarct size were 19.3 ± 5.5% and 14.4 ± 4.3%,
respectively. Treatment with CX-10 50 mg/kg caused approximately
40% reduction in infarct size, which was statistically significant. Al-
though the reduction in infarct size was not statistically significant
when administration of CX-10 10 mg/kg, infarct size tended to be
smaller than that of MCAO group. Representative sections of each
group are presented in Fig. 4B. In addition, CX-10 treated groups
showed reduced neurological scores, the effect was significant at dose
of 50 mg/kg. The effect of andrographolide at dose of 50 mg/kg was
less potent than that of CX-10 in reducing infarct size and neurological
scores.
3.4. Effects of CX-10 on motor performance in rats after MCAO
Animals in MCAO group showed gradually worse motor perfor-
mance in rotarod test from days 1 and 3 after brain injury. Compared
with MCAO group, animals treated with CX-10, edaravone and Andro
showed improved performance. CX-10 50 mg/kg showed significant
improvement of motor performance beginning at days 2 and 3. These
results suggested an increased rate of recovery of motor function fol-
lowing administration of CX-10 after ischemia-reperfusion injury
(Fig. 5).
3.5. Effects of CX-10 on proinflammatory cytokines and antioxidant
enzymes activities in brain tissues
As shown in Fig. 6, TNF-α and IL-1β markedly increased in brain
tissues after ischemia-reperfusion injury. Meanwhile, activities of an-
tioxidant enzymes SOD, CAT and GSH-Px significantly decreased. These
results confirmed that inflammatory and oxidative damage occurred in
ischemic brain injury. Treatment with CX-10 (10 and 50 mg/kg) de-
creased TNF-α and IL-1β levels and increased activities of SOD, CAT
and GSH-P × .
3.6. Effects of CX-10 on TLR4, NF-κB, TNF-α, iNOS, Nrf2 and HO-1
proteins expression
As shown in Fig. 7, TLR4, NF-κB, TNF-α, iNOS, Nrf2 and HO-1
protein levels were increased at 72 h after MCAO as compared with
Sham rats. Treatment with CX-10 down-regulated the expression of
TLR4, NF-κB, TNF-α and iNOS proteins, further induced Nrf2 and HO-1
expression.
4. Discussion
Due to the lack of effective and applicable therapeutic strategies for
the treatment of ischemic stroke, many pharmacological agents have
been investigated for years, though with limited clinical success. As we
known, ischemic stroke is a complex pathological process with multiple
mechanisms. Thus, it is appropriate to consider using pharmacological
M.-Y. Yang, et al. Pharmacological Research 144 (2019) 227–234

Fig. 6. Effects of CX-10 on proinflammatory cytokines and antioxidant enzymes activities in brain tissues. (A–B) TNF-α and IL-1β levels; (C–E) Activities of SOD, CAT
and GSH-P × . Data are represented as mean ± S.D. of 8 animals of each group. ##P < 0.01 vs. Sham; *P < 0.05, **P < 0.01 vs. MCAO group.

agents that affect multiple mechanisms simultaneously, either though activities, such as anti-oxidation, anti-inflammation, anti-apoptosis and
multi-target drugs or multidrug therapies consisting of drugs with dif- neurofunctional regulation. Therefore, looking for neuroprotective
ferent mechanisms of action. Many traditional herbal medicine or agents from natural products is a promising strategy for the develop-
natural products commonly possess a variety of pharmacological ment of novel drugs for the treatment of ischemic stroke. Previous

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M.-Y. Yang, et al.
Fig. 7. Effects of CX-10 on TLR4, NF-κB, TNF-α, iNOS, Nrf2 and HO-1 expression. (A–C) Protein bands; (D–E) Protein expression relative densities to Sham group.
Data are represented as mean ± S.D. of 3 animals of each group. ##P < 0.01 vs. Sham; *P < 0.05, **P < 0.01 vs. MCAO group.
studies have reported that many natural products or traditional medi-
cine, particularly traditional Chinese medicine (TCM), exert varying
degree of neuroprotection in experimental models of stroke [5,18–20].
In this study, we evaluated the neuroprotection of CX-10, a new
molecular entity derived from andrographolide, and preliminarily ex-
plored its underlying mechanisms. We firstly estimated the in vitro and
in vivo anti-inflammation of CX-10. TNF-α and NO are two common
inflammatory mediators. Elevated TNF-α and NO levels have been
shown in various experimental brain injury, and they involved in the
evolution of brain damage after an ischemia/reperfusion injury
[21,22]. In models of LPS-induced inflammatory response, CX-10 dose-
dependently inhibited LPS-induced TNF-α and NO release. The anti-
inflammatory effect of CX-10 appeared to be equivalent to that of
dexamethasone, and was obviously superior to that of parent compound
andrographolide.
The following study further demonstrated that systemic adminis-
tration of CX-10 post-reperfusion had significant neuroprotective
properties. CX-10 was effective in reducing infarct region, in improving
neurological function, in reducing motor impairments as measured with
a rotarod assay, and in decreasing levels of proinflammatory cytokines
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Pharmacological Research 144 (2019) 227–234
and enhancing anti-oxidant defense. These results confirmed that CX-
10 had a beneficial effect on ischemic stroke. In this study, andro-
grapholide showed less neuroprotective potency, which was incon-
sistent with a previously reported study. In that study, andrographolide
(0.1 mg/kg) exhibited favorable neuroprotective effects in a rat model
of permanent cerebral ischaemia [23]. The reason for the difference
may be that andrographolide exerts better effect at a lower dosage.
In order to explore the neuroprotective mechanism of CX-10, we
examined whether CX-10 could modulate some key molecules among
signaling pathways of inflammation and oxidative stress. TLR4 protein
is one of TLRs family members that function as key mediators of innate
immunity. TLR4 could induce the activation of NF-κB and the expres-
sion of NF-κB-regulated genes for inflammatory cytokines [24]. Evi-
dence confirmed that TLR4 knockout plays a neuroprotective role in
ischemic brain injury in mice [24,25]. Our data showed that treatment
with CX-10 effectively decreased the expression of TLR4, NF-κB, TNF-α
and iNOS proteins. iNOS expression is transcriptionally regulated by
NF-κB and oxidative radicals. NO derived from iNOS is deleterious in
cerebral ischemic injury [22]. Thus, inhibition of the activation of
TLR4/NF-κB signaling pathway may be the potential neuroprotective
M.-Y. Yang, et al.
mechanism of CX-10.
In addition to inflammation, oxidative stress also plays a key role in
the pathological process of stroke. During cerebral damage after stroke,
excessive of reactive oxygen species (ROS) are generated, with dys-
function of anti- and pro-oxidant enzymes systems [26]. Nrf2 is a well-
known regulator of endogenous antioxidant defense, which regulates
the expression of various antioxidant genes. Activation of Nrf2 could
alleviate ischemia/reperfusion injury by protecting against oxidative
stress [27,28]. Li et al. [29] previously reported that andrographolide
exerted protection effects against oxidative stress via activation of the
Keap1-Nrf2-Are pathway. As described in our results, treatment with
CX-10 effectively increased activities of several anti-oxidant enzymes in
brain tissues. Thus, we deeply explored the effect of CX-10 on Nrf2
signaling pathway. Our data showed that CX-10 could induced Nrf2
protein expression, as well as its target gene HO-1. These results sug-
gested that Nrf2/ARE signaling pathway may be one of the potential
mechanisms of neuroprotection of CX-10.
In conclusion, these results showed that CX-10 possessed potent
anti-inflammation and anti-oxidant activities. Systemic administration
of CX-10 effectively attenuated brain infarct region, improved neuro-
logical function and reduced motor impairments. The molecular me-
chanisms may involve inhibition of activation of TLR4/NF-κB signaling
pathway and upregulation of Nrf2/ARE signaling pathway. These novel
findings provide the pharmacological foundation for the further de-
velopment of CX-10 for the treatment of ischemic stroke.
Conflict of interest
All authors have no conflict of interest.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phrs.2019.04.023.
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