Rutaecarpine inhibits hypoxia/reoxygenation-induced apoptosis in rat hippocampal neurons.
ABSTRACT Our previous studies showed that rutaecarpine (Rut) protected against myocardial ischemia/reperfusion (I/R) injury, which was associated with activation of transient receptor potential vanilloid subtype 1 (TRPV1). Recently, TRPV1 activation was also reported to exert neuroprotective effects. The present study was to investigate the effect of Rut on hypoxia/reoxygenation (H/R)-induced apoptosis in primary rat hippocampal neurons. Three-hour hypoxia (1% O2) and consequent 24-h reoxygenation significantly increased the apoptotic death of hippocampal neurons as evidenced by increases in both TUNEL-positive cell number and caspase-3 activity. However, pretreatment with Rut (1-10microM) or caspase-3 specific inhibitor DEVD-CHO could markedly attenuate H/R-induced apoptosis in neurons. Rut markedly induced the phosphorylation of Akt and PI3K inhibitor LY294002 prevented the survival effect of Rut on neurons. Intracellular oxidative stress was significantly induced after H/R, which was inhibited by Rut and LY294002 as well as antioxidant PDTC. TRPV1 antagonist capsazepine or intracellular Ca2+ chelator BAPTA/AM could abolish these effects of Rut mentioned above. In summary, the present data suggest that Rut inhibits H/R-induced apoptosis of hippocampal neurons via TRPV1-[Ca2+]i-dependent and PI3K/Akt signaling pathway, which is related to inhibiting oxidative stress and caspase-3 activation.
- SourceAvailable from: Nicola B Mercuri[show abstract] [hide abstract]
ABSTRACT: Growing evidence regarding the function of vanilloid receptor-1 (VR1) in the brain suggests potential central roles of this receptor, previously described to occur primarily in peripheral sensory neurons. In the present study, we used electrophysiological and biochemical techniques to investigate the function and the endogenous stimulation of VR1 in dopaminergic neurons of the substantia nigra pars compacta (SNc). The VR1 agonist capsaicin increased the frequency of both TTX-sensitive and -insensitive spontaneous EPSCs (sEPSCs) without affecting their amplitude, suggesting a presynaptic site of action. In contrast, no effect was detected with regard to GABAergic transmission. No increase in sEPSC frequency was observed in the presence of cadmium chloride, while the voltage-dependent calcium channel antagonist omega-conotoxin MVIIC did not prevent capsaicin action. The VR1 antagonists capsazepine and iodoresiniferatoxin (IRTX) blocked the effects of capsaicin. Importantly, IRTX per se reduced sEPSC frequency, suggesting a tonic activity of VR1. The endogenous VR1 agonist anandamide (AEA) produced an IRTX-sensitive increase in the frequency of sEPSCs on dopaminergic neurons that was more pronounced when protein kinase A had been activated. Furthermore, mass spectrometric analyses and binding experiments revealed high levels of endogenous AEA and specific binding of AEA to VR1 receptors in the SNc. These data suggest a tonic facilitation of glutamate release exerted by VR1 in the SNc through a stimulation of VR1 by endovanilloids, including anandamide. The increase in sEPSC frequency by VR1 onto midbrain dopaminergic neurons suggests the involvement of these receptors in motor and cognitive functions involving the dopaminergic system.Journal of Neuroscience 05/2003; 23(8):3136-44. · 6.91 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The fruits of Evodia rutaecarpa Benth (Rutaceae) has long been used for inflammatory disorders and some anti-inflammatory actions of its constituents such as dehydroevodiamine, evodiamine and rutaecarpine were previously reported. Since the pharmacological data is not sufficient to clearly establish the scientific rationale of anti-inflammatory medicinal use of this plant material and the search for its active principles is limited so far, three major constituents (evodiamine, rutaecarpine, goshuyuamide II) were evaluated for their anti-inflammatory cellular action mechanisms in the present study. From the results, evodiamine and rutaecarpine were found to strongly inhibit prostaglandin E2 synthesis from lipopolysaccharide-treated RAW 264.7 cells at 1-10 microM. Evodiamine inhibited cyclooxygenase-2 induction and NF-kappaB activation, while rutaecarpine did not. On the other hand, goshuyuamide II inhibited 5-lipoxygenase from RBL-1 cells (IC50 = 6.6 microM), resulting in the reduced synthesis of leukotrienes. However, these three compounds were not inhibitory against inducible nitric oxide synthase-mediated nitric oxide production from RAW cells up to 50 micorM. These pharmacological properties may provide the additional scientific rationale for anti-inflammatory use of the fruits of E. rutaecarpa.Archives of Pharmacal Research 05/2006; 29(4):293-7. · 1.54 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: A signaling pathway was delineated by which insulin-like growth factor 1 (IGF-1) promotes the survival of cerebellar neurons. IGF-1 activation of phosphoinositide 3-kinase (PI3-K) triggered the activation of two protein kinases, the serine-threonine kinase Akt and the p70 ribosomal protein S6 kinase (p70(S6K)). Experiments with pharmacological inhibitors, as well as expression of wild-type and dominant-inhibitory forms of Akt, demonstrated that Akt but not p70(S6K) mediates PI3-K-dependent survival. These findings suggest that in the developing nervous system, Akt is a critical mediator of growth factor-induced neuronal survival.Science 02/1997; 275(5300):661-5. · 31.03 Impact Factor
Rutaecarpine inhibits hypoxia/reoxygenation-induced apoptosis
in rat hippocampal neurons
Zhong Daia,b, Jian Xiaoa, Si-Yu Liua, Liao Cuib, Gao-Yun Huc, De-Jian Jianga,*
aDepartment of Pharmacology, School of Pharmaceutical Sciences, Central South University, No. 110 Xiang-Ya Road, Changsha 410078, China
bDepartment of Pharmacology, Guangdong Medical College, Dongguan, China
cDepartment of Medicinal Chemistry, School of Pharmaceutical Sciences, Central South University, Changsha, China
a r t i c l e i n f o
Received 31 October 2007
Received in revised form 20 June 2008
Accepted 14 August 2008
Potential vanilloid subtype 1
a b s t r a c t
Our previous studies showed that rutaecarpine (Rut) protected against myocardial ischemia/reperfusion
(I/R) injury, which was associated with activation of transient receptor potential vanilloid subtype 1
(TRPV1). Recently, TRPV1 activation was also reported to exert neuroprotective effects. The present study
was to investigate the effect of Rut on hypoxia/reoxygenation (H/R)-induced apoptosis in primary rat
hippocampal neurons. Three-hour hypoxia (1% O2) and consequent 24-h reoxygenation significantly
increased the apoptotic death of hippocampal neurons as evidenced by increases in both TUNEL-positive
cell number and caspase-3 activity. However, pretreatment with Rut (1–10 mM) or caspase-3 specific
inhibitor DEVD-CHO could markedly attenuate H/R-induced apoptosis in neurons. Rut markedly induced
the phosphorylation of Akt and PI3K inhibitor LY294002 prevented the survival effect of Rut on neurons.
Intracellular oxidative stress was significantly induced after H/R, which was inhibited by Rut and
LY294002 as well as antioxidant PDTC. TRPV1 antagonist capsazepine or intracellular Ca2þchelator
BAPTA/AM could abolish these effects of Rut mentioned above. In summary, the present data suggest that
Rut inhibits H/R-induced apoptosis of hippocampal neurons via TRPV1-[Ca2þ]i-dependent and PI3K/Akt
signaling pathway, which is related to inhibiting oxidative stress and caspase-3 activation.
? 2008 Published by Elsevier Ltd.
Ischemic cerebral stroke is one of the leading causes of human
death and disability across the world. Global brain ischemia fol-
lowed by reperfusion results in cell death in hippocampal Cornus
Ammon’s field 1 (CA1) neurons in patients and in animal models.
Apoptosis is active process of cell suicide, morphologically and
biochemically different from necrosis. Some neuronal cells dis-
played the histological and biochemical markers characteristic of
apoptosis in cerebral tissues following ischemia/reperfusion (I/R)
event and in cultured neurons suffering hypoxia/reoxygenation
(H/R) process, suggesting that neuronal apoptosis is associated
with brain damages elicited by I/R (Tagami et al.,1999; Yermolaieva
et al., 2004; Zhou et al., 2004).
Transient receptor potential vanilloid subtype 1 (TRPV1) is
a ligand-gated nonselective cation channel, which is highly
expressed in the brain tissues including hippocampus. The patho-
logical changes in brain temperature or pH after a severe stroke
may influence TRPV1 (Tominaga et al., 1998), suggesting an
important role of TRPV1 in events occurring after I/R. TRPV1 can be
activated by endogenously generated compounds such as ananda-
mide and lipoxygenase products which accumulate during brain
injury (Marinelli et al., 2003). The involvement of TRPV1 has been
also reported in the protective effects of some exogenous drugs,
such as rimonabant and capsaicin, in different ischemic animal
models (Veldhuis et al., 2003; Pegorini et al., 2005, 2006).
Rutaecarpine (Rut), a quinazolinocarboline alkaloidal compound,
exerts extensive biological and pharmacological activities, such as
inhibiting inflammatory responses, lowering blood tension and
protecting against I/R-induced myocardial damages (Choi et al.,
blocked by capsazepine, a TRPV1 antagonist, suggesting the key role
of TRPV1 in these protective effects of Rut. Based on these data
mentioned above, we hypothesized that Rut might have neuro-
protective effects due to activation of TRPV1. Thus, the present study
was to observe the effect of Rut on H/R-induced apoptotic death of
primary rat hippocampal neurons.
2. Material and methods
Rut was synthesized by the following scheme (Scheme 1). Trifluoroacetic
anhydride was added to the isatoic anhydride in pyridine to afford a reactive
* Corresponding author. Tel./fax: þ86 731 2355078.
E-mail address: firstname.lastname@example.org (D.-J. Jiang).
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/neuropharm
0028-3908/$ – see front matter ? 2008 Published by Elsevier Ltd.
Neuropharmacology 55 (2008) 1307–1312
intermediate. After refluxed with tryptamine, the product was treated with the
mixture of acetic acid and hydrochloric acid. Then stirring with a hot solution of KOH
in ethanol and water and some necessary treatments, rutaecarpine crystals were
obtained. The purity of Rut (>99%) was determined by HPLC and its structure was
confirmed by MS,1H- and13C-NMR spectroscopy. Rut was initially dissolved in
dimethyl sulfoxide (DMSO) and further diluted in conditioned medium to proper
final concentration. The final concentration of DMSO in solution did not exceed 0.1%.
Capsazepine, LY294002 and pyrrolidine dithiocarbamate (PDTC) were obtained
from Sigma. Fura-2/AM, BAPTA/AM and H2DCF-DA were purchased from Molecular
Probes. Acetyl-DEVD-CHO was purchased from Calbiochem. Antibodies to Akt and
phosphor-Akt were obtained from Santa Cruz.
2.2. Cell culture and treatment
Primary cultures of rat hippocampal neurons were prepared from day 18
Sprague–Dawley rat embryos as described previously (Tamatani et al.,1999). Female
SD rats were obtained from Laboratory Animal Center, Central South University.
Animals received humane care in compliance with the ‘‘Guide for the Care and Use
of Laboratory Animals’’ published by the National Institutes of Health (NIH publi-
cation No. 8023, revised 1978). All experiments were performed on cells cultured for
8–10 days. As described previously (Tamatani et al., 1999), hypoxic treatment was
performed using an incubator set to 1% O2, with 5% CO2and 94% N2. Cultures
exposed to hypoxia were then returned to the ambient atmosphere (reoxygenation)
and the conditioned medium was rapidly exchanged with fresh medium. To observe
the effect of Rut on H/R-induced apoptosis, hippocampal neurons were pretreated
with different concentrations of Rut (1–10 mM) for 1 h before 3-h hypoxia and 24-h
reoxygenation. For other experiments, cells were co-treated with various tool drugs
in the presence of Rut (10 mM).
2.3. In situ TUNEL assay
The cultured hippocampal neurons were fixed in phosphate-buffered saline
(PBS) containing 4% paraformaldehyde. After washed in PBS, cells were per-
meabilized with 0.1% Triton X-100. In situ cell apoptosis was detected by using
terminal deoxynucleotidyltransferase dUTP nick end-labeling (TUNEL) assay kit
according to the manufacturer’s protocol (Roche). Then cells were washed and
double stained with 1 mg/ml propidium iodide (PI) at room temperature for another
20 min. The labelled cells were observed with a confocal microscope (LSM510,
Zeiss). The nuclei of all cells were stained with red, whereas cells with green nuclei
are TUNEL-positive indicating apoptosis. Five hundreds cells were counted in
randomly chosen fields for each sample, and the numbers of apoptotic cells are
expressed as a percentage of the total cells counted.
2.4. Measurement of caspase-3 activity
The activity of caspase-3-like protease in the lysate was measured using
a colorimetric caspase-3 assay kit (Sigma) according to the manufacturer’s protocol
(Jiang et al., 2006a). Caspase-3 activity was expressed by value of OD405.
2.5. Determination of ROS generation
Intracellular ROS levels were determined by H2DCF-DA, an oxidant-sensitive
fluorescent indicator, according to our previous description (Jiang et al., 2006a). The
fluorescence was monitored using a fluorescence microscope equipped with a FITC
filter and the average intensity values were measured at 200 ?magnification in five
randomlychosen fields of each nine replicates from threeindependent experiments.
2.6. Western blot analysis
Cells were lysed for 1 h at 4?C in a lysis buffer and total protein concentration
was determined by Lowry assay. Samples containing 60 mg of total cellular protein
were loaded and separated on 8% SDS-PAGE gel electrophoresis. The gel was then
transferred onto a PVDF membrane, which was then incubated at room temperature
for 2 h with a primary antibody. The polyclonal antibodies to phosphorylated and
total Akt were used at 1:1000 dilution. After washes the membranewas incubated at
room temperature for 1 h with corresponding HRP-conjugated second antibodies.
The washes were repeated before the membrane was developed using ECL reagent.
Band intensities were analyzed densitometrically using the Scion image software
and results were expressed as ratio of phosphorylated Akt to total Akt, which of
control was set to 1.
2.7. Measurements of intracellular Ca2þ
[Ca2þ]i was determined using the fluorescent Ca2þindicator Fura-2/AM as
described in our previous publication (Jiang et al., 2006b). The excitationwavelength
alternated between 340 and 380 nm. Emitted light was detected at 510 nm. Changes
of [Ca2þ]i were analyzed and data were expressed as ratio values of Fura-2
fluorescence at 340 and 380 nm.
3.1. Rut inhibited H/R-induced apoptosis and caspase-3 activation
in hippocampal neurons
At 8th dayof invitroculture, embryonic rat hippocampus grown
was identified as 95.2?2.0% microtubule-associated protein 2-
positive cells, suggesting these cells contained mainly neurons.
Expectedly, 3-h hypoxia and consequent 24-h reoxygenation
greatly increased the number of TUNEL-positive cells and the
activity of caspase-3. As shown in Figs.1 and 2A, pretreatment with
Rut (1–10 mM) could significantly attenuate H/R-induced increases
in apoptotic ratio of hippocampal neurons and caspase-3 activity in
a concentration-dependent manner. The vehicle (DMSO, concen-
tration: 0.1%) had no effect on these changes induced by H/R (data
not shown). Furthermore, pretreatment with caspase-3 specific
inhibitor DEVD-CHO (100 mM) markedly prevented H/R-induced
apoptotic death in rat hippocampal neurons (Fig. 1). However, Rut
(10 mM) itself had no effect on cellular apoptosis (data not shown).
3.2. The anti-apoptotic effect of Rut was related to decreasing
intracellular ROS production
H/R could markedly increase intracellular DCF fluorescence
intensity, reflecting elevation of intracellular ROS level, which was
concentration-dependently attenuated by Rut (Fig. 2B). Moreover,
treatment with intracellular antioxidant PDTC (50 mM) significantly
Scheme 1. The synthesis of rutaecarpine (Rut). Reagent and condition: (i) (CF3CO)2O, pyridine, 25?C, 15 min,115?C, 5 min; (ii) 115?C, 30 min; (iii) HCl/HOAc, reflux; (iv) KOH/H2O,
Z. Dai et al. / Neuropharmacology 55 (2008) 1307–13121308
attenuated H/R-induced caspase-3 activation in hippocampal
neurons (OD405nm: 0.082?0.009 vs H/R 0.268?0.028, P<0.01).
However, Rut (10 mM) or PDTC (50 mM) itself had no significant
effect on intracellular ROS level (data not shown).
3.3. PI3K/Akt signaling pathway was involved in the anti-apoptotic
effect of Rut
As shown in Fig. 3A, the level of phospho-Akt was low in
hippocampal neurons under unchallenged condition and H/R had
no significant effect on phosphorylation of Akt. However, upregu-
lated levels of phospho-Akt were detected in the presence of Rut at
the concentration of 3 and 10 mM. The phosphoinositide 3 kinase
(PI3K) inhibitor LY294002 (10 mM) thoroughly abolished Rut-
induced phosphorylation of Akt, indicating that Rut activates Akt
likely through PI3-kinase (Fig. 3A). Interestingly, the inhibitory
effects of Rut on intracellular ROS production and apoptosis
induced by H/R were mostly abolished in the presence of LY294002
(10 mM) (Fig. 3B and C).
3.4. The anti-apoptotic effect of Rut was dependent on
TRPV1 and [Ca2þ]i
Rut (10 mM) was found to induce a transient increase in [Ca2þ]i
in hippocampal neurons. TRPV1 antagonist capsazepine (1 mM)
could completely block Rut-induced transient increase in [Ca2þ]i
(Fig. 4A). As shown in Fig. 4B and C, pretreatment with capsazepine
or intracellular Ca2þchelator BAPTA/AM (5 mM) could abolish Rut-
induced Akt phosphorylation and anti-apoptotic effects. However,
capsazepine (1 mM) or BAPTA/AM (5 mM) itself had no effect on
Ca2þinflux and Akt phosphorylation as well as neuronal apoptosis
There is increasing evidence that neuronal apoptotic death
contributes to I/R-induced brain damage. Similar to neurons in the
brains of living animals exposed to I/R, primary cultures of neurons
from embryonic animals suffering H/R events have been shown to
display manyof same metabolic markers of apoptosis (Tagami et al.,
1999; Yermolaieva et al., 2004; Zhou et al., 2004). As a natural
TRPV1 agonist, Rut exerts extensive pharmacological actions,
including lowering blood tension and protecting against myocar-
dial I/R damage (Deng et al., 2004; Hu et al., 2002). The present
result showed that Rut could significantly attenuate H/R-induced
apoptosis in primary cultured rat hippocampal neurons. The find-
ings were supported by several previous reports that capsaicin and
anandamide, well known as exogenous and endogenous TRPV1
agonist, protected neuronal cells against hypoxia-induced damages
in different animal models (Veldhuis et al., 2003; Pegorini et al.,
Fig. 1. Effects of rutaecarpine (Rut) on hypoxia/reoxygenation (H/R)-induced apoptosis of rat hippocampal neurons. (A) The representative images and (B) the statistic graph of
TUNEL assay. All cells nuclei were stained by propidium iodide (red) and TUNEL-positive cells were stained with green. Scale bar, 50 mm. Mean ? SEM from three independent
experiments.þþP<0.01 vs control; *P<0.05, **P<0.01 vs H/R group. (For interpretation of colour in this figure, the reader is referred to the web version of this article.)
Fig. 2. Effects of rutaecarpine (Rut) on hypoxia/reoxygenation (H/R)-induced increases in (A) intracellular ROS level reflected by DCF fluorescence intensity and (B) caspase-3-like
activity indicated by OD405nmvalues. Mean ?SEM from three independent experiments.þþP<0.01 vs control; *P<0.05, **P<0.01 vs H/R group.
Z. Dai et al. / Neuropharmacology 55 (2008) 1307–1312 1309
ROS has been thought as an intracellular signal molecular, which
plays an important role in causing apoptotic cell death. I/R and H/R
can increase intracellular ROS level and induce apoptosis in
neuronal cells, which were greatly suppressed by antioxidants
(Fujimura et al., 2005; Rayner et al., 2006). The present study
showed that H/R significantly increased intracellular oxidant
production as shown by DCF fluorescence and induced the
apoptosis of hippocampal neurons, which were abolished by
pretreatment with antioxidant PDTC or Rut, suggesting that inhi-
bition of intracellular ROS production might be involved in Rut-
induced anti-apoptotic effect in hippocampal neurons.
In neuronal cell, H/R-induced apoptosis is associated with acti-
vation of caspase-3, whereas inhibition of caspase activity has been
shown to attenuate apoptosis, indicating that caspase-3 represents
a potential therapeutic target for suppression of neuronal cells
apoptosis induced byH/R (Lievreet al., 2000; Tamatani et al., 2000).
We identified a significant increased caspase-3 activity in the
extracts from H/R-treated hippocampal neurons, which was
H/R-induced neuronal apoptosis could be inhibited by DEVD-CHO,
the caspase-3 specific inhibitor. Our current data suggest that the
anti-apoptotic effect of Rut may be related to inhibition of caspase-
3 activity via decreasing H/R-induced intracellular ROS production.
PI3K and its downstream effectors serine/threonine kinase Akt
is a well-known signaling pathway involved in cell protection
under various stresses, including oxidative stress (Dudek et al.,
1997). It was recently reported that the PI3K/Akt pathway delivers
an anti-apoptotic signal in human glioblastoma against oxidative
Rut andPDTC. Moreover,
stress (Sonoda et al., 1999). In cardiomyocytes, the PI3K/Akt
pathway was also demonstrated to promote cell survival against
oxidative stress-induced apoptosis (Hong et al., 2001). In the
dependently induced phosphorylation of Akt in the presence of
H/R. In addition, LY294002, the specific inhibitor of PI3K could
markedly reverse Rut-induced phosphorylation of Akt and cell
rescue from H/R. Moreover, LY294002 also abolished Rut-induced
decrease in intracellular ROS level, further certified the opinion that
PI3K/Akt-mediated neuroprotection is related to inhibiting oxida-
tive stress (Di Segni et al., 2006). Thus, these results indicate that
PI3K/Akt survival pathway may be involved in Rut-mediated rescue
from H/R-induced neuron apoptosis.
It is well known that activation of TRPV1 results in a transient
Ca2þinflux and the latteras second messengercan activate series of
signaling proteins and exert biological effects. Our present results
showed that Rut could cause a transient [Ca2þ]iincrease, which
abolished by TRPV1 antagonist capsazepine. Moreover, both cap-
sazepine and intracellular Ca2þchelator BAPTA/AM could block the
protective effect of Rut against H/R-induced neuronal apoptosis.
Similarly, previous studies showed that TRPV1 and [Ca2þ]imedi-
ated the neuroprotective effects of some molecules such as xenon
and rimonabant (Di Segni et al., 2006; Pegorini et al., 2006). Thus,
these data suggest that TRPV1-Ca2þ-dependent pathway is
involved in these effects of Rut.
Though our present results showed the TRPV1-mediated neu-
roprotective effects of Rut, it is very complicated about TRPV1 and
neuronal survive or apoptosis. Contrary to its neuroprotection
that Rut concentration-
Fig. 3. PI3K/Akt signaling pathway mediated the anti-apoptotic effect of rutaecarpine (Rut) in rat hippocampal neurons. (A) Rut-induced Akt phosphorylation. Representative result
and quantification of western blots are shown. LY294002 attenuated the inhibitory effects of Rut on the increases in intracellular ROS level (B) and cell apoptosis (C) induced by H/R.
Mean ?SEM from three independent experiments.þþP<0.01 vs control; *P<0.05, **P<0.01 vs H/R group;##P <0.01 vs Rut-treated group.
Z. Dai et al. / Neuropharmacology 55 (2008) 1307–13121310
against in vivo excitotoxicity (Veldhuis et al., 2003), anandamide,
the endogenous cannabinoid which can activate TRPV1, has been
reported to induce neuronal apoptosis (Movsesyan et al., 2004). It
has been also reported that capsazepine alone exerts the neuro-
protective action (Veldhuis et al., 2003). Thus, further study will
need to investigate the effect of Rut on endocannabinoid system
and its possible mechanisms independent on TRPV1 activation in
In summary, the present data show that Rut protects against
H/R-induced apoptosis of hippocampal neurons via TRPV1-
[Ca2þ]i-dependent and PI3K/Akt-mediated signaling pathway. Our
results suggest that Rut may be useful for treatment of ischemia-
induced brain damage.
This work was supported by the National Postdoctoral Science
Foundation (20060390262) and the Provincial Natural Science
Foundation of Hunan (06jj3012), China.
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Fig. 4. The anti-apoptotic effect of rutaecarpine (Rut) was dependent on TRPV1 and [Ca2þ]i. (A) Effect of Rut on transient Ca2þinflux. [Ca2þ]iwas expressed as ratio of fluorescence
intensity at 340–380 nm. Either capsazepine or BAPTA/AM abolished Rut-induced Akt phosphorylation (B) and anti-apoptotic effect (C). Mean ?SEM from three independent
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Z. Dai et al. / Neuropharmacology 55 (2008) 1307–13121311