Advances in Bioscience and Biotechnology, 2012, 3, 731-739 ABB
http://dx.doi.org/10.4236/abb.2012.326094 Published Online October 2012 (http://www.SciRP.org/journal/abb/)
Pentoxifylline suppressed LPS-induced inflammatory and
apoptotic signaling in neuronal cells
Sameer K. Muchhala, Kenza E. Benzeroual*
Division of Pharmaceutical Sciences, Arnold & Marie Schwartz College of Pharmacy & Health Sciences, Long Island University,
Brooklyn, USA
Received 16 August 2012; revised 23 September 2012; accepted 3 October 2012
ABSTRACT
Several studies have reported a relation between in-
creased pro-inflammatory mediators such as TNF-α
and apoptosis in neurodegenerative diseases such as
Alzheimer’s disease (AD). It is known that lipopoly-
saccharide (LPS) treatment induces neuroinflamma-
tion and memory deterioration, and it has been re-
ported that LPS induces apoptosis mostly through the
production of TNF-α. Pentoxifylline (PTX), is a vas-
cular protective agent and a potent TNF-alpha in-
hibitor. However, the molecular neuroprotective me-
chanisms of PTX against LPS-induced neurotoxicity
have not been well studied. In this study, we inves-
tigated the direct protective effect of PTX against
LPS-induced toxicity in Rat pheochromocytoma (PC12)
cell line. The results showed that a pretreatment with
PTX prior exposure to LPS significantly decreased
LPS-induced cell death. Mechanisms study showed
that PTX has the potential to inhibit pro-inflamma-
tory and pro-apoptotic pathways via the suppression
of TNF-α and a caspase-dependent pathway in neu-
ronal PC12 cells. This is the first study to report the
anti-inflammatory and anti-apoptotic effects of PTX
via inhibition of TNF-α and a caspase-dependent
pathway in neuronal PC12 cells. Altogether, these
observations indicate that PTX is capable of promot-
ing neuroprotective effects, meanwhile also present
some insights into the potential signaling pathways
that are involved. Thus, these findings support the
potential for PTX to be investigated as a potential
agent fo r the treat ment of neurod egenerativ e diseases
such as AD.
Keywords: Pentoxifylline; Lipopolysaccharide (LPS);
Neuronal Cells; Apoptosis; Inflammatory Cytokines;
Neurodegeneration; TNF-Alpha; Alzheimer’s Disease
1. INTRODUCTION
Alzheimer’s disease (AD) is an irreversible and progres-
sive disorder affecting regions of the brain that control
memory and cognitive functions. The disease gradually
results in memory loss, and the ability to communicate
and carry out daily activities [1]. The three main struc-
tural changes in the AD brain include extracellular de-
posits of amyloid beta (Aβ) peptides known as senile
plaques, intracellular deposits of hyper-phosphorylated
tau proteins termed neurofibrillary tangles (NFT), and
diffuse neuronal loss [2]. In addition, inflammation has
been reported to occur in pathologically vulnerable re-
gions of the AD brain [3]. In line with this, pro-inflame-
matory mediators such as tumor necrosis factor-alpha
(TNF-α) has been reported to play an important role in
apoptosis and neuronal cell death [4-6], and apoptosis
has been documented in AD brains [2,7] and in models
of neuronal cell death utilizing various neurotoxins in-
cluding lipopolysaccharide (LPS) [8,9].
LPS is an endotoxin constituent of the outer mem-
brane of gram-negative bacteria, and is widely distrib-
uted in the environment including in house dust [10] and
various agricultural settings [11]. Studies have reported
that a direct injection of LPS to the brain leads to neu-
ronal death in the hippocampus [12]. This may be due to
the presence of inflammatory factors as LPS administra-
tion to the brain in rodents induces chronic neuroin-
flammation associated with impaired hippocampal-de-
pendent spatial cognitive function [13-15]. In line with
this, LPS administration to mice showed altered expres-
sions of genes associated with learning and memory [16].
Moreover, behavioral and biochemical studies have also
shown that spatial memory [17], object recognition and
long-term potentiation (LTP) [18,19], and neuronal net-
work activity [15] are all impaired following acute LPS
injections to models that mimic AD pathology. At the
cellular level, LPS binding to the toll-like receptor
(TLR4) on host cells can trigger a signaling cascade that
induces the expression of inflammatory cytokines such
*Corresponding author.

S. K. Muchhala, K. E. Benzeroual / Advances in Bioscience and Biotechnology 3 (2012) 731-739 732
as TNF-α, and other signaling pathways such as caspases
that subsequently contribute to apoptosis and neuronal
death [20]. In line with this, TNF-α may directly induce
neuronal apoptosis and injury and its elevated levels in
AD have been documented [21,22]. In the present study,
we used LPS-induced pheochromocytoma (PC12) cells,
as an in vitro model of neurodegeneration to screen for
neuroprotective agents.
Since the neuropathology of several neurodegenerative
diseases, especially AD, has been linked to mitochon-
drial dysfunctions, and inflammation-induced apoptosis
[21,22] there has been intense interest in screening for
neuroprotective agents with anti-inflammatory and anti-
apoptotic effects. Pentoxifylline (PTX), a methylated
xanthine derivative, is a competitive nonselective phos-
phodiesterase inhibitor used as a vascular-protecting
agent to improve blood blood circulation in the arms and
legs in the case of intermittent claudication, and possibly
to improve cerebral blood flow in the case of vascular
dementia [23]. In addition to its ability to improve
microcirculation, PTX has potent anti-TNF-α action [24]
which have prompted investigations into its potential
efficacy in disease states such as AD, where TNF-α lev-
els are elevated and may intimately be associated with
neuronal death [21,22]. Evidence from clinical practices
and experimental animal studies indicate that treatment
with PTX improves learning, memory and brain metabo-
lism [25]. Others reported that PTX inhibited kainic acid-
induced cognitive impairment in mice [26]. Only few
studies have shown that PTX inhibited apoptosis in non-
neuronal cells [27-29]. However, its anti-inflammatory
and anti-apoptotic mechanisms in LPS-induced PC12
cells, a model of phenotypic neuronal cells, have not
been studied. PTX, if able to protect against inflamma-
tory mediators and apoptosis, may play a protective role
in neurodegenerative diseases, especially vascular di-
mentia of AD.
Hence, in the present study we investigated the possi-
ble protective mechanisms of PTX in LPS-induced neu-
rotoxicity in PC12 cells. Our results showed that PTX
significantly and dose-dependently reduced cell death
caused by LPS. Furthermore, PTX significantly and
dose-dependently suppressed LPS-induced TNF-α ex-
pression, and prevented the apoptotic mechanisms. The
present result showed that PTX exerts its neuroprotection
probably via the inhibition of TNF-α expression and a
caspase-dependent apoptotic pathway.
2. MATERIAL AND METHODS
2.1. Materials
Rat pheochromocytoma (PC12) cells, Fetal Bovine Se-
rum, Horse Serum, Penicillin-Streptomycin, and Tryp-
sin-EDTA were obtained from American Type Culture
Collection (ATCC, USA). Mouse monoclonal primary
antibodies (TNF α, β-actin, Bad, and Bcl2) and Anti-
mouse IgG-HRP conjugated secondary antibody, were
purchased from Santa Cruz biotechnology. Pentoxifyl-
line, LPS, Dulbeccos’s Modified Eagle Medium (DMEM),
PBS, DMSO, SDS, Glycine, TBE Buffer, Tris base, β-
mercaptoethanol, ammonium persulfate and other chemi-
cals were obtained from Sigma-Aldrich (St. Louis, MO).
Caspase-3 colorimetric assay kit was obtained from Invi-
trogen.
2.2. Cell Culture and Treatments
Rat pheochromocytoma (PC12) cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM) (Sigma,
Aldrich), supplemented with 10% horse serum, 5% fetal
bovine serum and 1% Penicillin/Streptomycin in an in-
cubator with a constant supply of 5% CO2 and 95%
oxygen at 37˚C, and medium was changed every 2 days.
Cells were separated by trypsin-EDTA, when confluent.
In all experiments, cells were seeded in 12 or 96-well
plates and grown to a confluence of 75% - 85%.
PC12 cells were treated as follows:
For MTT assay, vehicle (control cells were treated
with 0.1% DMSO for 24 hr, 48 hr, and 72 hr), LPS (cells
were pretreated with 0.1% DMSO for 2 hr followed with
LPS treatment for 24 hr, 48 hr, and 72 hr), Pentoxifylline
(PTX) (cells were treated with 1.0 mM of PTX for 24 hr,
48 hr, and 72 hr). For all other conditions, cells were
pretreated with varying concentrations of PTX (0.1 mM,
0.5 mM, and 1 mM) for 2 hr prior explosure to LPS (4
g/ml) for 24 hr, 48 hr, and 72 hr.
For all other experiments, cells were treated with 0.1%
DMSO (vehicle for 48 hr) or PTX only (1 mM for 48
hrs). Pretreatment consisted of 0.1% DMSO (LPS condi-
tion) or PTX (at varying concentrations) for 2 hr, prior
exposure to LPS (4 g/ml) for 48 hr.
PTX was dissolved in DMSO at a final concentration
of 0.1%, and final DMSO concentration in the media was
below 0.1%.
2.3. MTT Assay and Cell Viability
Cell viability was determined using 3-(4,5-dimethylthi-
azol-2-yl)-2,5-diph enyltetrazolium bromide (MTT) re-
duction assay according to the following method. In 96
well plates, 1 × 105 PC12 cells/well were seeded and
treated as described previously. MTT solution (0.5 mg/
mL) was then added in each well, and the plates were
kept at 37˚C in a 5% CO2 and 95% air humidified envi-
ronment for 4 hours. The medium was discarded and 100
µL of Dimethyl sulphoxide (DMSO) was added to each
cell, and the plate was kept on a shaker for 15 min to
dissolve the formazan crystals formed in intact cells.
Micro plate reader (Bio-Rad) was used to measure the

S. K. Muchhala, K. E. Benzeroual / Advances in Bioscience and Biotechnology 3 (2012) 731-739 733
absorbance at 595. Results were expressed as the per-
centages of reduced MTT, assuming the absorbance of
control cells as 100%.
2.4. Western Blot Analysis
Proteins were extracted from PC12 cells using RIPA
lysis buffer after 48 hours of drug treatment. The protein
concentration was measured using BCA assay kit and
equal concentration of proteins was run using western
blot assembly. TNF-α, Bad, and Bcl2 were probed using
the respective primary antibodies on nitrocellulose
membranes. The membranes were then probed with HRP
conjugated anti-mouse secondary antibody. X-ray film
was used to reveal protein expression using chemilumi-
nescence kit. Beta-actin blot was run to assure equal
protein loading. Quantification of results was performed
by densitometric scan of films. Data analysis was done
by Image J. [30]
2.5. DNA Extraction and Agarose Gel
Electrophoresis
To determine whether cytotoxicity was due to apoptosis
and/or necrosis, DNA was extracted from PC12 cells
after 48 hours, using Pierce DNA extraction kit. Equiva-
lent amounts of DNA were loaded into wells of 1.2%
agarose gel and electrophoresed in 0.5 TAE buffer (40
mM Tris-acetate, 1 mM EDTA) at 54 volts for 240 min-
utes. DNA was visualized by ethidium bromide staining.
Gel pictures were taken by UV transillumination with a
Polaroid camera. Quantification of results was performed
by densitometric scan of films. Data analysis was done
by Image.J [30].
2.6. Caspase 3 Colorimetric Assay
To evaluate caspase-3 activity, cell lysates were prepared
after their respective treatment of LPS and/or PTX As-
says were performed in 96-well microtitre plates by in-
cubating cell lysates in 2× reaction buffer containing the
caspase-3 substrate DEVD-pNA, a pseudosubstrate used
to measure caspase-3 activity, at 37˚C for 2 h. p-Nitro-
analine (pNA) is released from the substrate DEVD-pNA
upon cleavage by DEVDase. Free pNA produces a yel-
low color after cleavage that is proportional to the
amount of DEVDase activity present in the sample. The
optical density (OD) was then read at 405 nm in a mi-
croplate reader. The increase in OD positively correlates
with the amount of caspase-3 activity.
2.7. Statistical Analysis
All experiments were performed in triplicate, and the
results are presented as mean ± standard deviation (S.D.).
Overall differences among all the conditions were deter-
mined by analysis of variance (ANOVA). Specific pair-
wise differences were determined using the tukey’s range
test. Differences were considered significant when P <
0.05.
3. RESULTS
3.1. Pentoxifylline Prevented LPS-Induced PC
12 Cell Death
MTT Cell Viability Assay was carried out to determine
the protective effect of PTX against LPS-induced cell
death. In the MTT assay (Figures 1 and 2), vehicle
(0.1% DMSO) was considered as 100%.
Figure 1 shows LPS time-response curve at 24 hr, 48
hr, and 72 hr. LPS dose-response curve shows a LD50 of
4 µg/ml in our experimental condition at 48 hr. The rest
of the experiments were then carried out with LPS 4
µg/ml.
Figure 2 shows no significant changes in viability of
cells treated with PTX (PTX only), as compared to vehi-
cle (0.1% DMSO). LPS treatment reduced cell viability
to 45.05%, whereas pretreatment with increasing doses
of PTX for 2 hr prior LPS stimulation caused a gradual
increase in the cell viability from 45.5% to 73%, 77%
and 84% at 0.1 mM, 0.5 mM, and 1 mM, respectively at
48 hr. These results showed that PTX tends to reverse the
toxicity induced by LPS and increase the cell viability to
about 84% at a concentration of 1 mM.
3.2. Pentoxifylline Suppressed the Expression of
TNF-α
PTX, known as an anti-inflammatory and protective
Figure 1. Effect of LPS on PC12 cell death. Cell viability was
assessed by the MTT assay. Cells were treated with vehicle
(0.1% DMSO), or LPS at varying concentrations for 24 hr, 48
hr, and 72 hr. For statistical evaluations two-way ANOVA
analysis, followed by tukey’s range test was performed. *p <
0.05 compared with vehicle. Data are expressed as percentage
of viable cells compared with vehicle, and mean ± S.D. of at
least 3 separate experiments.

S. K. Muchhala, K. E. Benzeroual / Advances in Bioscience and Biotechnology 3 (2012) 731-739 734
Figure 2. Effect of Pentoxifylline on cell viability. Cell viabil-
ity was assessed by the MTT assay. Cells were treated with
vehicle (0.1% DMSO), or PTX (1 mM). Pretreatment consisted
of 0.1% DMSO (LPS condition) or PTX at different concentra-
tions (0.1 mM, 0.5 mM, and 1 mM) for 2 hrs prior to stimula-
tion with LPS (4 g/ml) for 24 hr, 48 hr, and 72 hr. For sta-
tistical evaluations two-way ANOVA analysis, followed by
tukey’s range test was performed. *p < 0.05 compared with
vehicle; **p < 0.05 compared with LPS treated cells only. Data
are expressed as percentage viable cells compared with vehicle,
and mean ± S.D. of at least 3 separate experiments.
agent in a variety of cells, has been used in this study to
confirm this property in neuronal cells. To determine the
effect of PTX on LPS-induced cytokine expression,
western blot assay of TNF-α expression, as an inflame-
matory marker, was carried out as described under mate-
rial and methods. Cells treated with LPS showed 8.3-fold
increase in the expression of TNF-α as compared to ve-
hicle. PTX only treated cells did not show any significant
increase in the expression of TNF-α suggesting that no
toxicity was imparted by the drug itself on neuronal cells.
Pretreatment of cells with 0.1 mM, 0.5 mM, 1 mM of
PTX, significantly reduced the LPS-induced expression
of TNF-α to 4.8, 2.3, and 1.1 fold, respectively, as com-
pared to LPS-treated cells (Figure 3). Over expression of
TNF-α in the presence of LPS was reduced dose-depen-
dently, and was even suppressed at the highest concen-
tration (1 mM) of PTX. The lack of significant differ-
ences in the corresponding beta-actin immunonoreactiv-
ity among the groups suggests that differences in the
expression of TNF-α are not attributed to loading differ-
ent amount of proteins per lane.
3.3. Pentoxifylline Inhibited LPS-Induced DNA
Fragmentation
To examine whether apoptotic mechanism were involved
in LPS-induced TNF-α mediated cell death, DNA frag-
mentation method was performed as described under
Section 2.5. As depicted in Figure 4, LPS induced a lad-
der-like pattern on agarose gel electrophoresis, free of
β-actin
TNFα
PTX (mM) prior LPS stimulation
Vehicle LPS PTX 0.1 0.5 1
Figure 3. Effect of Pentoxifylline on the expression of TNF-α.
Cells were treated with vehicle, or PTX only for 48 hr. Pre-
treatment consisted of 0.1% DMSO (LPS condition) or PTX at
different concentrations (0.1 mM, 0.5 mM, and 1 mM) for 2 hr
prior to stimulation with LPS (4 g/ml) for 48 hrs. Quantitative
data for the expression of TNF-α in all groups was performed.
For each treatment the corresponding beta-actin immunoreac-
tivity is reported to show equal protein loading of the lanes. For
statistical evaluations two-way ANOVA analysis, followed by
tukey’s range test was performed. Data are expressed as rela-
tive intensity of protein expression as compare with vehicle,
and mean ± S.D. of at least 3 separate experiments.*p < 0.05
compared with vehicle; **p < 0.05 compared with LPS treated
cells only.
Figure 4. Effect of Pentoxifylline on DNA fragmentation.
Representative agarose gel electrophoresis showing DNA lad-
dering of cells treated as described in Figure 3 legends. MW
represents the 100-base pair DNA ladder marker.
smear like pattern. This result shows that neurotoxicity
induced by LPS is in the form of apoptosis. Vehicle and
PTX treated cells show intact DNA which rules out drug-

S. K. Muchhala, K. E. Benzeroual / Advances in Bioscience and Biotechnology 3 (2012) 731-739 735
induced apoptosis. PTX pretreatment at a concentration
of 0.1 and 0.5 mM show a decrease in DNA fragmenta-
tion while PTX at a concentration of 1 mM shows an
intact DNA as compared to vehicle, suggesting that the
drug may exerts its protective effect through the inhibit-
tion of apoptotic pathways.
highest dose used (1 mM), and increased Bcl2 levels
significantly (Figure 5(b)), as compared to LPS-treated
cells. These results show that pretreatment of PC 12 cells
with PTX inhibited LPS-induced expression of the pro-
apoptotic bad protein (Figure 5(a)) and increased the
expression of the anti-apoptotic Bcl-2 protein (Figure
5(b)). PTX only treated cells did not show any signifi-
cant increase in the expression of Bad Protein suggesting
that PTX by itself, did not induce apoptosis in neuronal
cells. Thus, PTX mediated its protective effect on PC 12
cells through its anti-apoptotic mechanism.
3.4. Pentoxifylline Reversed LPS-Induced
Apoptotic Pathways
Apoptosis, also known as programmed cell death, plays
an important role in neurodegeneration [4]. One pro-
posed mechanism is the upregulation of the pro-apoptotic
Bad protein. The over-expression of Bad protein affects
the permeability of mitochondrial membranes and leads
to the activation of caspases which proteolyses cellular
components and leads to cell death. In contrast, anti-
apoptotic genes such as Bcl-2 are known to down regu-
late Bad, and therefore, has the potency to inhibit the
intrinsic mitochondrial pathway of apoptosis [31]. As
shown in Figures 5(a) and (b), in the presence of LPS,
apoptosis went along with a significant increase of in the
expression of Bad protein as compared to vehicle, while
the expression of Bcl-2 protein was not changed signifi-
cantly. This confirms results reported by other studies
investigating LPS effects on PC12 cells [32]. However,
pretreatment of cells with increasing doses of PTX de-
creased Bad expression and even suppressed it at the
Caspases also play an important role in the induction,
and amplification of apoptotic mechanisms [9,33], and
previous studies reported that caspase inhibitors pre-
vented LPS-induced apoptosis and cell death in PC12
cells [34]. To further confirm the protective effect of
PTX on LPS-induced cell death, we measured caspase-3
levels by caspase-3 colorimetric assay. A fold increase in
the absorbance at 405 nm reflects an increase in the cas-
pase-3 activity that may indicate ongoing apoptosis. A
decrease would reflect inhibition of apoptosis and rever-
sal of cell death. Figure 6 shows that LPS treated cells
exhibited a significant increase of 11 fold in absorbance
as compared to vehicle and PTX only treated cells. This
suggests that LPS increased caspase-3 activity in cells
leading to ongoing apoptosis. Pretreatment of cells with
0.1 mM, 0.5 mM, and 1 mM of PTX dose-dependently
(a) (b)
Figure 5. Effect of Pentoxifylline on the expression of Bad (5A) and Bcl2 (5B) proteins. PC12 cells were pretreated with 0.1%
DMSO (LPS condition) or 0.1 mM, 0.5 mM, and 1 mM of PTX for 2 hr and then exposed to LPS for 48 hr. Proteins were separated
on SDS-PAGE, Western blotted, probed with anti-Bad and/or anti-Bcl-2 antibodies, and reprobed with anti-β-actin antibody (One
representative Western blot was shown; n = 3). The relative intensity of corresponding bands was measured and the median of three
ndependent experiments is shown. *p < 0.05 compared with vehicle; **p < 0.05 compared with LPS treated cells only. i

S. K. Muchhala, K. E. Benzeroual / Advances in Bioscience and Biotechnology 3 (2012) 731-739 736
Figure 6. Caspase-3 levels in PC12 cells pretreated with Pen-
toxifylline. Caspase levels were assessed by the caspase col-
orimetric assay. PC12 cells were pretreated with 0.1% DMSO
(LPS condition) or 0.1 mM, 0.5 mM, and 1 mM of PTX for 2
hrs and then exposed to LPS for 48 hr. *p < 0.05 compared with
vehicle; **p < 0.05 compared with LPS treated cells only. Data
are expressed as arbitrary unit of capsase-3 activity as com-
pared with vehicle, and mean ± S.D. of 3 separate experi-
ments.
and significantly reduced the effect of LPS on caspase-3
levels to 7-, 3.6-, and 2-fold, respectively as compared to
LPS-treated cells (11 fold), suggesting the ability of PTX
to inhibit caspase-3 activity and to suppress LPS-induced
apoptosis in PC12 cells.
4. DISCUSSION
Pro-inflammatory cytokines such as TNF-α have been
reported to contribute to neurodegenerative disorders
initiation and progression [35,36], and have been in-
volved as cellular stimulators of apoptotic pathways [37,
38]. In addition, increasing genetic and clinical evidence
supports that an excess of TNF-α plays a central role in
neurodegeneration [39,40]. In fact, studies reported that
patients with mild cognitive function that progress into
AD showed as an early event an increased TNF-α level
in cerebrospinal fluid, and this rise correlates well with
the progression of the disease [41,42]. Others reported
that in rodent models of chronic neuroinflammation the
expression of genes associated with learning and mem-
ory has been altered by LPS administration [16]. In line
with this, LPS is one of the most potent cytotoxic induc-
ers of inflammation, and of a cascade of intracellular
events involved in cell death [43]. Since there has been
considerable efforts to search for compounds capable of
inhibiting pro-inflammatory mediators and thus related
apoptotic mechanisms in neuronal degeneration [44], this
study focused on the prevention by PTX of pro-inflame-
matory and pro-apoptotic mediators induced by LPS in
PC12 cells.
PC 12 cells have been widely used as a model of neu-
ronal cells to investigate mechanisms of signal transduc-
tion and cell death [45,46], and exposure to LPS has
been reported to result in apoptosis [32,33]. Our data
confirmed that treatment of PC12 cells with LPS resulted
in cell death as reported by others [8,9], which was
greatly decreased in the presence of PTX. To investigate
PTX neuroprotective mechanisms, we first target TNF- α
expression. In fact, TNF-α is one of the major cytokines
implicated in the neuroinflammation and neurodegenera-
tion [36] and has been shown to regulate numerous cel-
lular processes such as inflammation and cell death [47].
The results showed an increase in the expression of
TNF-α in PC12 cells treated with LPS, confirming LPS-
induced pro-inflammatory mediators [48]. Moreover,
intracellular generation of the inflammatory mediator
TNF-α was significantly and dose-dependently decreased
by PTX. Our study suggests that PTX reversal of cyto-
toxicity induced by LPS is probably through the inhibit-
tion of TNF-α expression. Others reported that while
TNF-α was released from activated microglia, it only has
a minor neurotoxic effect in microglia-neuron cocultures
treated with LPS (100 g/ml) for 48 hr with or without
PTX (500 M) [49]. The discrepancy between this study
and ours reside in the study design and the dose of LPS
used. Our study used a higher LPS dose (4 g/ml), and
neuronal PC12 cells were directly exposed and pretreated
with PTX, meanwhile the study by Xie et al. (2002) used
a co-culture and a co-treatment where microglia were
directly exposed to LPS and PTX, but neurons were only
exposed to microglial culture media [49].
To investigate whether PTX cytoprotective mecha-
nisms was mediated through the inhibition of apoptosis
or necrosis, DNA fragmentation experiment was carried
out. Results showed that LPS treated cells showed a dis-
tinct DNA ladder consistent with ongoing apoptosis in
our experimental model. On pre-treatment with PTX,
there was a significant dose-dependent inhibition in the
DNA fragmentation. The present result, confirmed that
PTX exerts its protective effect through inhibition of
apoptosis. Several studies indicate that apoptosis con-
tributes to AD onset and progression [38]. To co-relate
the release of pro-inflammatory mediators to apoptosis
and neurodegeneration, we examined the effect of PTX
on apoptotic pathways. The balance between pro-apop-
totic and anti-apoptotic proteins of the Bcl-2 family plays
an important role in the induction of apoptosis [31]. The
pro-apoptotic members of the Bcl-2 family such as Bad
react with the mitochondrial membrane and induce the
release of cytochrome c and activation of caspase-3, re-
sulting in the degradation of DNA, and eventually apop-
totic cell death [41,50]. The effect of PTX on the expres-

S. K. Muchhala, K. E. Benzeroual / Advances in Bioscience and Biotechnology 3 (2012) 731-739 737
sion of pro-apoptotic protein Bad and anti-apoptotic pro-
tein Bcl2 were investigated in this study. Our results
showed an increase in the pro-apoptotic Bad expression
on LPS treatment, indicating the ongoing apoptosis, and
a significant suppression of Bad levels when treated with
the highest concentration of PTX. On the other hand,
PTX treated PC12 cells showed an increase in the anti-
apoptotic Bcl-2 protein expression, thereby supporting an
anti-apoptotic role of the drug. To confirm our anti-
apoptotic hypothesis, caspase-3 colorimetric assay showed
a significant increase in the caspase-3 activity in the
LPS-induced cells as reported by others in neuronal
PC12 cells [9], and a gradual decrease in PTX pretreated
cells confirming inhibition of apoptosis and hence a
neuroprotective mechanism of the drug. Considering that
caspase-3 in particular, promote apoptotic cell death, it is
possible that neuroprotection by PTX is achieved, par-
tially by a reduction in a caspase-dependent pathway.
These effects on cell death may also be partly due to the
attenuating effects of PTX on TNF-α expression. In ac-
cordance with our results, others have reported that PTX
may induce protection against ischemia injury in the
spinal cord in rabbits by preventing both necrosis and
apoptosis. The study also reported a significant decrease
in serum TNF-α and spinal cord caspase-3 activity in the
presence of PTX [52]. Thus, confirming a protective
mechanism of the drug.
PTX is a vascular protective drug and also exhibits
anti-inflammatory and potent anti-TNF-
 action [24]. In
addition, the association between hemorheological changes
and alterations of TNF-α has been reported and indicates
a potential immunorheologic mechanism associated with
cerebrovascular damages in dementia of AD, suggesting
the use of vascular protective drugs such as PTX to sup-
port the main pharmacological and non-pharmacological
therapy of AD [53]. While PTX have been reported to
produce a significant improvement in vascular dementia
[54] others reached the conclusions that PTX have
shown some, but partly limited benefits in vascular di-
mentia patients [55]. The study reported that the possible
causes of the negative results of many randomized clini-
cal trials in vascular dimentia include enrollment of pa-
tients with heterogeneous subtypes of vascular dimentia,
the small sample size, and the use of endpoints and cog-
nitive tests inadequate for vascular dimentia setting be-
cause derived from previous experience in the field of
AD [55]. Thus, PTX may need to be reevaluated for its
neuroprotective effects in vascular dimentia of AD.
In summary, our study reports a neuroprotective effect
of PTX against LPS-induced PC12 cell death. PTX has
been shown to exert a cytoprotecitve effect through the
suppression of the expression of the pro-inflammatory
mediator TNF-α, and the increase in cell viability. It also
inhibited apoptosis pathways induced by LPS, by abol-
ishing the pro-apoptotic Bad protein expression and in-
creasing the anti-apoptotic Bcl2 protein expression. To
the best of our knowledge, this is the first study to dem-
onstrate the direct anti-inflammatory mechanisms of
PTX in neuronal PC12 cells through inhibition of TNF-α
expression and a caspase-3-dependent apoptotic pathway,
suggesting potential protective effects against inflamma-
tion-mediated neurodegeneration. Future works along
this line will lead to a potential therapeutic use of the
compound for the treatment of neurodegenerative disor-
ders, especially AD.
5. ACKNOWLEDGEMENTS
This work was supported by Science Research Funds from Arnold and
Marie Schwartz College of Pharmacy of Long Island University.
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