Paeoniflorin Protects against Ischemia-Induced Brain Damages in Rats via Inhibiting MAPKs/NF-κB-Mediated Inflammatory Responses
et al. (2012) Paeoniflorin Protects against Ischemia-Induced Brain Damages in Rats via Inhibiting MAPKs/NF-
kB-Mediated Inflammatory Responses. PLoS ONE 7(11): e49701. doi:10.1371/journal.pone.0049701
Paeoniflorin Protects against Ischemia-Induced Brain Damages in Rats via Inhibiting MAPKs/NF-kB-Mediated Inflammatory Responses
Ruo-Bing Guo 0
Guo-Feng Wang 0
An-Peng Zhao 0
Jun Gu 0
Xiu-Lan Sun 0
Gang Hu 0
Cesar V. Borlongan, University of South Florida, United States of America
0 1 Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University , Nanjing , China , 2 Department of Cadre Ward No. 3, the General Hospital of Jinan Military Area Command of PLA , Jinan , China
Paeoniflorin (PF), the principal component of Paeoniae Radix prescribed in traditional Chinese medicine, has been reported to exhibit many pharmacological effects including protection against ischemic injury. However, the mechanisms underlying the protective effects of PF on cerebral ischemia are still under investigation. The present study showed that PF treatment for 14 days could significantly inhibit transient middle cerebral artery occlusion (MCAO)-induced over-activation of astrocytes and microglia, and prevented up-regulations of pro-inflamamtory mediators (TNFa, IL-1b, iNOS, COX2 and 5-LOX) in plasma and brain. Further study demonstrated that chronic treatment with PF suppressed the activations of JNK and p38 MAPK, but enhanced ERK activation. And PF could reverse ischemia-induced activation of NF-kB signaling pathway. Moreover, our in vitro study revealed that PF treatment protected against TNFa-induced cell apoptosis and neuronal loss. Taken together, the present study demonstrates that PF produces a delayed protection in the ischemia-injured rats via inhibiting MAPKs/NF-kB mediated peripheral and cerebral inflammatory response. Our study reveals that PF might be a potential neuroprotective agent for stroke.
Funding: This study was supported by grants from the National Natural Science Foundation of China (No. 81273495), Major Porject of Jiangsu Provincial
Department of Education (No. 12KJA310002), National Key Basic Research Program of China (No. 2009CB521906) and Project Funded by the Priority Academic
Program Development of Jiangsu Higher Education Institutions (No. JX10131801042). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Stroke has a high incidence and is harmful to human health. It
is a leading cause of death and the third cause of disability. The
loss of neurological functions following stroke is caused by massive
loss of neurons resulting from hypoxic-ischemic insults. Up to now,
several hypotheses have been put forward, such as Ca2+ overload,
oxidative injury, excitotoxicity and apoptosis . A complex
interplay between different factors and signal cascades results in
neuronal cell degeneration after ischemia . Extensive loss of
neurons as well as glia over-activations in ischemic brain are the
characteristic pathological features of cerebral ischemia . The
acute neuronal damage is followed by a second round of neuronal
injury that occurs hours to days after brain ischemia in the
neighboring areas, which is called delayed neuronal death (DND)
. It is very important to study the mechanism underlying DND
for prolonging the timescale of clinical treatment .
Much progress has been made in developing novel therapeutics
to treat stroke, including glutamate receptor antagonists, calcium
channel blockers, radical scavengers, and anti-apoptotic agents
. Despite many promising neuroprotective agents have been
identified in extensive animal research, few has been translated
into clinically effective therapies . Tissue plasminogen activators
(tPAs) are still the only agents approved by the Food and Drug
Administration (FDA). tPAs has limited applicability and is
currently used in fewer than 5% of stroke victims , although
there is now some evidence that the window of opportunity for
tPA usage may be extended to 6 h after the event [11;12].
Therefore, arduous works should be directed at increasing efficacy
and extending the treatment window for stroke.
Given the complex pathophysiology of stroke, it may be
unrealistic to hope for a single magic bullet that will result in
neuroprotection and rescue of damaged but not-yet-destroyed
neurons . To date, no single agent has been shown to improve
stroke outcome in humans, and the current standard of care
remains primarily supportive. The herb Paeonia lactiflora pall,
which is known as Shao Yao in Chinese, has been used for more
than 1,000 years in traditional Chinese medicine to treat cramp,
pain, giddiness and congestion . Paeoniflorin (PF), a
monoterpene glucoside, is the principal bioactive component purified
and extracted from the root of Paeonia lactiflora pall. It has been
reported to exhibit many pharmacological effects such as
antiinflammation, anti-allergy, anti-hyperglycemia, analgesia ,
blocking neuromusculus  and enhancing cognition [17;18].
Previous studies indicated the protective effects of PF may be
related to its abilities to prevent apoptosis , scavenge free
radicals , adjust cerebral energy metabolism and nitric oxide
formation , prevent thrombosis , block Na+ channels,
activate adenosine A1 receptor [22;23] or facilitate the
translocation of protein kinase C and glucose transporter . These
studies suggest that multitargets may be involved in PF-mediated
protective effects. Regarding to the effects of PF in the cerebral
ischemia, it has been reported that treatment with PF attenuated
ischemia-induced pathological and behavioral changes as well as
cognitive impairments [18;23;2527]. However, the mechanisms
underlying the protective effects of PF on cerebral ischemia are still
under investigation. Therefore, the present study focused on
evaluating the delayed protective effects of PF in the transient
middle cerebral artery occlusion (MCAO) rat model, and
revealing the signaling pathways involved in the actions of PF.
Male SD rats weighing 220250 g were used in the present
study. Animals were allowed to acclimatize for at least 7 days prior
to experimentation. The animals were housed in individual cages
under light-controlled conditions and at room temperature. Food
and water were available ad libitum. All animals received care in
compliance with the Guide for the Care and Use of Laboratory
Animals published by the National Institutes of Health (NIH
publication 8023, revised 1996). The rats were randomly divided
into the following 3 groups (n = 2025 for each group): 1. MCAO
groups: MCAO(90 min)+saline (2 ml.kg21, i.p., twice per day for
14 days); 2. PF groups: PF (5 mg. kg21, i.p., twice per day) was
administrated for 14 days after MCAO (90 min) and reperfusion
24 h. PF (purity.98.5%) was purchased from Nanjing ZeLang
Medical Technology Co., LTD); 3. Sham group. Physiological
parameters of rats were monitored continuously, and the
temperature remains 3761uC throughout the intraoperative
2. Establishment of transient MCAO
The experimental MCAO rat model was conducted as
described previously (Takano et al., 1997), with minor
modifications. Briefly, the rats were anesthetized with choral hydrate
(300 mg. kg21, i.p.), the bifurcation of the left common carotid
artery was exposed. The right MCA was occluded for 90 min by
insertion of a monofilament nylon suture through the common
carotid artery as described previously. Then, the suture was
withdrawn allowing reperfusion.
3. Determination of neurological symptoms
The severity of neurological symptoms of the experimental
animals was graded on a scale of 0959 according to methods
described previously with slight modifications as follows: 0-no
neurological deficit; 19-retracts left forepaw when lifted by the tail;
29-circles to the left; 39-falls while walking; 49-does not walk
spontaneously; 59-dead. Neurological symptoms were evaluated
24 h after MCAO. The above behavioral observations were
carried out in a blinded manner.
4. Measurement of infarct size
Coronal sections of the brain were cut into 2 mm slices and
immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC;
Sigma, USA) at 37uC for 15 min, followed by 10% formaldehyde
solution. The image of each slice was captured by digital camera.
The infarct area and hemisphere area were traced and quantitated
by an image analysis system (Adobe ImageReady 7.0). Analysis of
cerebral ischemic damage included total (hemispheric), cortical
and subcortical (striatal) infarction.
For immunohistochemical analysis, rats were perfused with
saline followed by 4% paraformaldehyde and
immunohistochemistry was performed on 40 mm free-floating sections using
antiNeuN monoclonal antibody (1:100, Millipore), anti-GFAP rabbit
monoclonal antibody (1:400, Millipore), or CD11b monoclonal
antibody (1:400, Abcam). Sections were then incubated with
corresponding secondary antibodies and immunoreactivity was
visualized with 0.05% DAB as chromagen. Negative controls
received the same treatments omitting the primary antibodies and
showed no specific staining.
6. Western blot analysis
The isolated cortex, hippocampus and striatum were
homogenized in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1%
nonidet P-40, 0.5% sodium deoxycholate and protease inhibitor
cocktail), centrifuged at 3,000 g for 10 min. Protein concentration
was determined by BioRad protein assay. Samples were
electrophoresed in SDS/PAGE gels and transferred onto a PVDF
membrane. The membrane was blocked with 5% nonfat milk in
16TBS, 0.1% Tween-20 at 25uC for 1 h and subsequently
incubated overnight at 4uC with appropriate primary antibody
(anti-iNOS, anti-COX2, anti-5-LOX, anti-phospho-p38 MAPK,
anti-phospho-ERK, anti-phospho-JNK, anti-NF-kB p65,
antiIkBa, anti-cytochrome c, anti-Bcl-2 and anti-Bax were purchased
from Santa Cruz, Abcam or Cell Signaling) diluted in TBST
[TBS, 0.1% (v/v) Tween-20 and 5% (w/v) BSA]. After incubated
with horseradish peroxidase-conjugated secondary antibodies for
1 h, the blots were developed with chemiluminescence reagent.
7. Measurement of plasma TNFa and IL-1b levels
Plasma TNFa and IL-1b levels were measured using
enzymelinked immunosorbent assay (ELISA) kit (purchased from ExCell)
according to manufacturers recommendations.
8. Measurement of mRNA levels of TNFa and IL-1b in the
Total RNA was extracted using Trizol reagent (Invitrogen Life
technologies, USA) followed by treatment with RNase-free
DNaseI (Invitrogen Life technologies, USA). Reverse transcription
was performed with the One-Step RNA-PCR Kit (Takara),
according to the manufacturers protocol. PCR primers were as
follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
was used as housekeeping gene (forward 59-
CCTACCCCCAATGTATCCGTTGTG-39 and reverse 59-
GGAGGAATGGGAGTTGCTGTTGAA-39), TNF-a (forward
59CGAGTGACAAGCCCGTAG-39 and reverse 59-
GGATGAACACGCCAGTCG-39), IL1b (forward 59-
CCAGGATGAGGACCCAAGCA-39 and reverse 59-
TCCCGACCATTGCTGTTTCC-39). Quantitative real-time PCR was
performed on a 7300 Real-Time PCR System using the SYBR
Green PCR Master Mix. The samples were run in triplicate and
the experiments were repeated at least three times. The GAPDH
gene was used as an endogenous control to normalize for
differences in the amount of total RNA in each sample. All values
were expressed as fold increase or decrease relative to the
expression of GAPDH.
9. Hippocampal primary neuron cultures
Primary neuron cultures were prepared from the hippocampal
tissues of embryonic day 14/15 mice. Briefly cell were dissociated
by trypsinization [0.25% (w/v) Trypsin and 0.02% EDTA in Ca2+
and Mg2+ free Hanks balanced salt solution] at 37uC for 10 min,
followed by gentle triturating in plating medium (h-DMEM
supplemented with 10% fetal bovine serum and 10% horse serum,
Gibco-BRL). Cells were seeded onto poly-L-lysine (Sigma) -coated
24-well plates and 25-cm2 T-flask at a density of 2.56105 cells per
cm2 and incubated at 37uC in 5% CO2 atmosphere. After 18
24 h, after cells were adherenced, the medium was replaced by
Neurobasal medium supplemented with 2% B-27 (Gibco-BRL)
and 0.5 mM L-glutamine (Sigma) and treated with cytosine
arabinoside (1 mM) for 24 h to inhibit glial cell proliferation. Half
of the culture medium was replaced every 3.5 days. Cultures were
used after 7 days in vitro.
10. Analysis of cell viability
Cell viability was determined by use of an MTT assay. Cells
were seeded in 96-well plates at 16104 cells per well and grown to
70% confluence in culture medium. The medium was replaced by
medium containing TNFa (100 mg/L) or TNFa+PF(25,50,
100 mM) for 24 h. A total of 5 g/L MTT was added to each
well after 24 h, and the culture continued to incubate for another
4 h at 37uC. Then the medium was aspirated, dye crystals were
dissolved in DMSO and the absorbance was read on an ELISA
plate reader using a 490 nm filter.
11. Hoechst 33342 staining
After treatment with TNFa (100 mg/L) or TNFa+PF(50 mM)
for 24 h, the primary cultured neurons were fixed with 4%
paraformaldyhyde for 30 min at 25uC, then washed with
prechilled phosphate buffer saline (PBS) three times and exposed to
10 mg/L Hoechst 33342 at room temperature in the dark for
10 min. Samples were observed under a fluorescence microscopy
(Nikon Optical TE2000-S).
For statistical analyses, a standard software package (SPSS for
Windows 10.1) was used. All data were given as means 6 SD.
Differences between groups were compared by using a one-way
analysis of variance (ANOVA). P values,0.05 were considered
1. PF treatment improves neurological deficits and
decreases cerebral infarct size in rats
Rats treated with vehicle showed neurological deficits such as
retracting left forepaw when lifted by the tail, circling to the left or
falling while walking. PF treatment significantly improved the
neurological symptoms (Figure 1A). The neurological deficit score
and the infarct volume of rats were determined to evaluate the
impacts of PF treatment for 14 days after MCAO. TTC staining
analysis revealed a mean infarct volume of 250.1617.2% mm3 in
vehicle treated groups. Treatment with PF for 14 days significantly
reduced the magnitude of ischemic lesion to 106.0611.7 mm3
(P,0.01, Figure 1B and 1C). In addition, the neuroprotection of
PF treatment were not to be accounted for by the modification of
physiological variables, since the parameters (e.g. blood pH, pO2,
pCO2 and blood glucose) were kept within normal physiologic
limit (Table 1).
2. PF treatment prevents ischemia-induced loss of neuron, and inhibits activations of microglia and astrocytes in the brain
As shown in Figure 2A and 2B, there was a robust loss of
neurons in ipsilateral striatum and cortex after ischemia. However,
neuronal injury in striatum and cortex was significantly prevented
by PF treatment.
After ischemia, astrocytes displayed reactive changes in cortex
and striatum of vehicle treated groups which are characterized by
increasing the expression of glial fibrillary acidic protein (GFAP)
and the number of GFAP-positive cells. The number of astrocytes
of MCAO-treated groups was higher than that of sham groups. PF
treatment significantly inhibited the astrocytic activation in the
brain (Figure 2C and 2D).
Ischemia also induced significantly activation of microglia which
was characterized by changing the resting ramified types to the
fully amoeboid morphology. The number of activated microglia
and the soma volume of microglia were markedly increased
induced by ischemia, which were inhibited by treatment with PF
(Figure 2E and 2F).
15 minutes after reperfusion
15 minutes after reperfusion
15 minutes after reperfusion
15 minutes after reperfusion
Physiologic data obtained from control and drug-treated groups are presented as mean 6 SD. All animals were maintained at 3761uC. There were no statistically
differences within or between the groups at any time point.
3. PF treatment decreases the levels of TNFa and IL-1b in
plasma, down-regulates the protein expression levels of
iNOS and COX2 and 5-LOX in the brain
Ischemia and reperfusion resulted in 1.4-fold elevation in the
concentrations of plasma interleukin 1b (IL-1b) and 1.2-fold
elevation in the concentrations of plasma tumor necrosis factor a
(TNFa). Treatment with PF markedly decreased the IL-1b level to
39.7, and the TNFa level to 92.5, respectively (Figure 3).
Simultaneously, we determined the protein expressions of
inducible nitric oxide synthase (iNOS), cyclooxygenase 2
(COX2) and 5-lipoxidase (5-LOX) in the cortex, hippocampus
and striatum via western blotting. The results showed that the
protein levels of iNOS, COX2 and 5-LOX in the brain of
MCAOinjured groups were higher compared to those in the sham groups.
PF treatment for 14 days significantly inhibited the three protein
expressions in the brain (Figure 4).
4. PF treatment inhibited mRNA expressions of TNFaand IL-1b in the brain
We further observed the changes in mRNA levels of IL-1b and
TNFa in brain by real-time PCR analysis. Our results showed that
ischemia attack significantly up-regulated the mRNA levels of
TNFa to 145%, 158% and 352% in cortex, hippocampus and
striatum, respectively. The mRNA levels of IL-1b in the three
brain regions were also robustly increased by ischemic injury to
359%, 223% and 747% in cortex, hippocampus and striatum,
respectively. PF treatment reversed the increase of mRNA levels of
TNFa (Figure 5A), and remarkably decreased the mRNA levels of
IL-1b in cortex, hippocampus and striatum of rats (Figure 5B).
5. PF treatment regulates MAPK and NF-kB signalling
Since MAPK signaling pathway plays important roles in
inflammatory response, the effects of PF treatment on mitogen
activated protein kinases (MAPKs) signaling pathways including
extracellular signal-regulated kinase (ERK), Jun N-terminal kinase
(JNK) as well as p38 MAPK were explored. Ischemia and
reperfusion up-regulated the phosphorylations of JNK and p38
MAPK, but down-regulated ERK phosphorylation. Treatment
with PF significantly suppressed the activations of JNK(Figure 6D)
and p38 MAPK(Figure 6B), but enhanced ERK activation
We further observed the ischemic injury-induced changes in the
nuclear factor kappa B (NF-kB) signaling pathway. As shown in
figure 6E and 6F, the expression of p65 was increased but
ikappaB-alpha (I-kBa) was significantly decreased by ischemic
insult. These changes in pathway were reversed by PF treatment.
6. PF treatment down-regulates the expressions of cytochrome c and Bax, but up-reglulates the expressions of Bcl-2
The pro-apoptotic protein Bax and the anti-apoptotic protein
Bcl-2 are crucial determinants of the apoptotic response and also
control the release of cytochrome c. To address whether PF
treatment influences the expression of apoptosis-related proteins
participated in ischemia and reperfusion. Western blot analysis
revealed a significant increases in the protein levels of cytoplasmic
cytochrome c and Bax in the cortex, hippocampus and striatum,
whereas there was a marked decrease in Bcl-2 expression
(Figure 7), indicating higher apoptosis existed in the ischemic
brain. Chronic treatment with PF could prevent the increases of
Bax and cytochrome c (Figure 7B and 7D) and significantly
elevated the protein levels of Bcl-2 (Figure 7C).
7. PF treatment protects TNFa-induced cytotoxicity in
Many studies have been revealed that TNFa could activate both
MAPKs and NF-kB signal pathways. So we used TNFa to induce
inflammatory damage in hippocampal neurons, and to verify
whether MAPKs and NF-kB signal pathways were involved in the
neuroprotective effects of PF. After incubation with TNFa, the cell
viability was decreased to 72.1%. Treatment with PF (25, 50,
100 mM) decreased the cell death rate in a
concentrationdependent manner (cell survival ratio was 81.2%, 87.1% and
92.3%, respectively. Figure 8A).
The Hoechst 33342 staining that is sensitive to DNA was used
to assess changes in nuclear morphology following TNFa or
TNFa+PF treatment. The nuclei in normal cells were normal and
exhibited diffused staining of the chromatin. However, after
exposure to TNFa 100 mg/L for 24 h, neurons underwent typical
Figure 3. PF treatment decreases the levels of IL-1b(A) and TNFa(B) in plasma. Sham, rats received surgery without vessel occlusion; MCAO,
rats treated with saline for 14 days after transient MCAO; PF, PF (5 mg. kg21) was administered after MCAO for 14 days. n = 9. All data were expressed
as mean 6 SD. * P,0.05,** P,0.01 vs sham; #P,0.05, ##P,0.01 vs MCAO.
Figure 4. PF treatment inhibits the protein expressions of iNOS, COX-2 and 5-LOX in the brain. (A) represents the protein expression of
iNOS, COX-2 and 5-LOX in the cortex, hippocampus and striatum of rats. (BD) indicates the relative optical density of these proteins expression.
Sham, rats received surgery without vessel occlusion; MCAO, rats treated with saline for 14 days after transient MCAO; PF, PF (5 mg. kg21) was
administered for 14 days after MCAO. n = 4. All data were expressed as mean 6 SD. ** P,0.01 vs sham; ##P,0.01 vs MCAO.
Figure 5. PF treatment decreases the mRNA expressions of TNFa (A) and IL-1b (B) in the brain. Sham, rats received surgery without vessel
occlusion; MCAO, rats treated with saline for 14 days after transient MCAO; PF, PF (5 mg. kg21) was administered for 14 days after MCAO; n = 4. All
data were expressed as mean 6 SD. ** P,0.01 vs sham, #P,0.05; ##P,0.01 vs MCAO.
morphologic changes of apoptosis such as condensed chromatin
and shrunken nucleus. Treatment with PF (50 mM) almost
reversed TNFa-induced neuronal apoptosis (Figure 8B and 8C).
An important delayed mechanism beginning within hours from
the onset of ischemia is the robust inflammatory response in the
ischemic tissue [5;28;29]. There is increasing evidence showing a
detrimental effect of the post-ischemic inflammatory reaction.
Therefore, therapeutic strategies targeting the delayed
inflammatory response could inhibit the progression of the tissue damage
providing an extended therapeutic window for neuroprotection.
Within hours after the onset of focal cerebral ischemia,
peripheral leukocytes (granulocytes, monocytes/macrophages,
lymphocytes) adhere to the cerebral endothelium, cross the vessel
wall and invade the damaged parenchyma [28;30]. Activation and
accumulation of leukocytes results in further damage. Current
evidence suggests a detrimental role of iNOS and COX2 from
neutrophils and vascular cells in the ischemic brain . iNOS is
transcriptionally induced in the ischemic core generating toxic
levels of NO continuously . Inhibition of iNOS by
pharmacological and genetic approaches prevents ischemia-induced
neurodegeneration. For example, iNOS null mice were reported
to have smaller infarcts and better neurological outcomes after
focal ischemia . In the present study, ischemia elicited
significantly increased expressions of iNOS, COX2 and 5-LOX
in three brain regions, PF treatment inhibited the overexpressions
of the three proteins. Simultaneously, we found that levels of
TNFa and IL-1b in the plasma were also increased, PF treatment
significantly down-regulated the levels of the two inflammatory
In response to ischemic injury in brain, microglia and astrocytes
are activated . Microglia are the resident macrophages of the
brain. They are very sensitive to subtle alterations in their
neuronal microenvironment. The surrounding astrocytes are also
sensitive to the increased release of these immunomodulatory
peptides and therefore severe ischemia also compromises
astrocytic function [28;34]. In response to the ischemic injury, glial cells
quickly become activated and undergo morphological
transformations, and are accompanied by functional changes, such as
increasing expression of cytokines: interleukins (IL-1b, IL-4, IL-6,
IL-10), TNFa, interferons and chemokines [35;36]. The
accumulaiton of pro-inflammatory factors should further induce ischemic
damages [35;37;38]. In the present study, we found ischemic insult
resulted in over-activation of astrocytes and microglia, and thereby
robustly elevated the mRNA expressions of TNFa and IL-1b in
the brain. Chronic treatment with PF could inhibit glial
overactivations, and reduce the mRNA levels of these inflamamtory
factors. Moreover, our other study via comparative proteomics
analysis showed that ischemic injury substantially increased the
expressions of astrocyte marker proteins (S100b and GFAP), PF
treatment significantly reversed these changes (data not published).
Therefore, our results reveal that PF exerts the delay
neuroprotective effects via inhibiting peripheral and cerebral inflammatory
The secretion of inflammatory molecules in cerebral ischemia
triggers the activation of several transcription factors involved in
the inflammatory response . Among them, the activation of
NF-kB and subsequent degradation of I-kBa are the key events in
ischemia and reperfusion . The activated NF-kB further
induces the expression of genes encoding cell adhesion molecules
and cytokines, thereby triggering the vicious cycle and
exacerbating inflammatory injury . Our results demonstrated that
ischemia caused the overexpression of p65 and decreased
expression of its endogenous inhibitor I-kBa, while these changes
were reversed by PF treatment. Consequently, inhibiting NF-kB
activation by PF resulted in the down-regulations of IL-1b and
TNFa mRNA levels in the brain.
MAPKs are a family of key proteins which are involved in a
wide range of cell responses, including cell proliferation,
differentiation and apoptosis . MAPK signaling pathways also
positively regulate transcription of inflammatory genes, such as
those coding for TNFa, IL-1b, and COX2 . Both the JNK
pathway and pro-inflammatory mediators further potentiate the
brain tissue injury and lead to apoptotic and necrotic cell death of
the potential viable tissue within hours and days . Inhibition of
MAP kinases (MAPK), especially p38 and JNKs, could lead to a
reduction in pro-inflammatory molecule production by
inflammatory cells, especially microglia/macrophages in which the MAPK
cascades are highly activated after an ischemic injury [43;44]. In
contrast, activation of ERK signaling pathway was critical for
Figure 6. Effects of PF on MAPK and NF-kB signaling effectors expression. p-P38 MAPK, p-ERK, p-JNK, p65 and IkB protein expressions in
the cortex, hippocampus and striatum of rats were indicated in (A). The relative optical densities were indicated in (BF). Sham, rats received surgery
without vessel occlusion; MCAO, rats treated with saline for 14 days after transient MCAO; PF, PF (5 mg. kg21) was administered for 14 days after
MCAO. n = 4. All data were expressed as mean 6 SD. ** P,0.01 vs sham; ##P,0.01 vs MCAO.
delayed neuroprotection [41;45]. We therefore investigated the
effect of PF on the MAPK signaling pathways including JNK, p38
and ERK. We found that PF treatment potently restrained the
activation of p38 MAPK and JNK, which were reported
persistently activated during ischemia [43;44]. The expression of
phosphorylated ERK was inhibited by ischemia in our results,
Figure 7. PF treatment decreases expressions of cytochrome c and Bax, but increases the expressions of Bcl-2. Cytochrome c, Bax and
Bcl-2 protein expressions in the cortex, hippocampus and striatum of rats were indicated in (A). The relative optical densities were indicated in (BD).
Sham, rats received surgery without vessel occlusion; MCAO, rats treated with saline for 14 days after transient MCAO; PF, PF (5 mg. kg21) was
administered for 14 days after MCAO. n = 4. All data were expressed as mean 6 SD. ** P,0.01 vs sham; ##P,0.01 vs MCAO.
Figure 8. PF treatment protects TNFa-induced cytotoxicity in hippocampal neurons as assessed by MTT(A) and staining with
Hoechst33342 (B and C). A and C. TNFa stimulation increased cell apoptosis and cell death in hippocampal neurons. PF inhibited the apoptotic
ratio of neurons and promoted cell survival. B. Fluorescence photomicrographs of neurons with Hoechst 33242 staining. Bar = 50 mm. **P,0.01 vs.
control group, # P,0.05, ## P,0.01 vs. TNFa group. Data are means 6 S.E.M. n = 4.
whereas PF treatment promoted its activation. Therefore, MAPK
pathway is involved in the protective effects of PF.
Compelling evidence indicates that apoptosis is crucially
important in transient cerebral ischemia . A variety of cell
death signals in ischemia affect mitochondria, so the central role of
mitochondria in apoptosis has been widely accepted . The
Bcl2 proteins are a family of mitochondrial proteins involved in the
response to apoptosis. Some of these proteins (such as bcl-2 and
bcl-XL) are anti-apoptotic, while others (such as Bad, Bax or Bid)
are pro-apoptotic . Our results showed that ischemic insult
significantly increased Bax expression but decreased Bcl-2
expression. The sensitivity of cells to apoptotic stimuli depends
on the balance of pro- and anti-apoptotic bcl-2 proteins . The
pro- and antiapoptotic members of the Bcl-2 family control the
release of cytochrome c and other factors from mitochondria. The
release of cytochrome c from the mitochondria is particularly
important in the induction of apoptosis. Once cytochrome c has
been released into the cytosol, it is able to interact with Apaf-1 that
leads to the recruitment of pro-caspase 9 into a multi-protein
complex and results in the formation of apoptosome . In our
study, the increased levels of cytoplasmic cytochrome c were found
in the ischemic rat brain which indicated more severe apoptosis in
ischemia-injured rats. Importantly, we found that PF treatment for
14 days could efficaciously prevent the changes in these
apoptosisrelated proteins, thereby protecting against ischemia-induced
neuronal loss in the rats.
The pro-inflammatory cytokine TNF plays a key role in a wide
variety of physiological processes, including inflammation,
proliferation and programmed cell death . These pleiotropic
biological effects of TNF result from its ability to initiate different
intracellular signaling pathways. TNF binding to TNF receptor 1
(TNF-R1) leads to the recruitment of TNF-R associated death
domain (TRADD), TNF-R associated factor 2 (TRAF2), and
receptor interacting protein 1 (RIP1), forming complex I .
Signaling from complex I leads to NF-kB activation via activation
of the IKK complex . Signaling from complex I also activates
the p38, ERK and JNK MAP kinases . Whereas recruitment
of FADD and procaspase-8 results in the formation of the cytosolic
complex II, where caspase-8 is activated. Caspase-8 initiates the
mitochondrial pathway by cleaving Bid to tBid, which induces
mitochondrial permeabilization that results in the release of
cytochrome c. This initiates an amplification loop that results in
full-blown caspase activity and subsequent apoptosis [51;52].
Therefore, TNFa was used to activate MAPKs and NF-kB signal
pathways in our in vitro study. The results revealed that PF could
significantly protect against TNFa-induced hippocampal neuron
damage, suggestting that MAPKs and NF-kB signal pathways
were involved in PF-mediated neuroprotective effects.
In conclusion, our data demonstrate that PF produces a delayed
protective effect on ischemic injury in the rats via inhibiting
peripheral and cerebral inflammatory response. The MAPKs and
NF-kB signaling pathways are involved in the protective effects of
PF. The present study reveals that PF might be a potential
neuroprotective agent for stroke.
Conceived and designed the experiments: X-LS GH. Performed the
experiments: R-BG G-FW A-PZ JG. Analyzed the data: A-PZ JG X-LS.
Contributed reagents/materials/analysis tools: R-BG G-FW A-PZ JG.
Wrote the paper: X-LS GH.
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