Ischemic stroke leads to high potentiality of
mortality and disability.The current
treatment for
ischemic stroke is mainly focused on intravenous
thrombolytic therapy.However,
ischemia/
reperfusion induces neuronal damage,which significantly influences the outcome of
patients with
ischemic stroke,and the exact mechanism implicated in
ischemia/
reperfusion injury remains unclear,although evidence shows that
oxidative stress is likely to be involved.
Betulinic acid is mainly known for its anti-
tumor and anti-inflammatory activities.Our previous study showed that
betulinic acid could decrease the
reactive oxygen species (ROS)
production by regulating the expression of
NADPH oxidase.Thus,we hypothesized that
betulinic acid may protect against
brain ischemic
injury in the
animal model of
stroke.Focal
cerebral ischemia was achieved by using the standard intraluminal occlusion
method and
reperfusion enabled after 2 h
ischemia.Neurological deficits were scored.
Infarct size was determined with 2,3,5-triphenyltetrazolium
chloride monohydrate (TTC)
staining and the
mRNA expression of
NADPH oxidase 4 (NOX4) was determined by RT-PCR in
infarct tissue.ROS generation and
apoptosis in ischemic
tissue were analyzed by measuring the oxidative conversion of
cell permeable 2',7'-dichloro-
fluorescein diacetate (DCF-DA) to fluorescent dichlorofluorescein (DCF) in
fluorescence microplate reader and
TUNEL assay,respectively.In Kunming
mice,2 h of
middle cerebral artery (MCA) occlusion followed by 24 or 72 h of
reperfusion led to an enhanced NOX4 expression in the ischemic hemisphere.This was associated with elevated levels of ROS generation and neuronal
apoptosis.Pre-
treatment with
betulinic acid (50 mg/kg/day for 7 days via gavage) prior to MCA occlusion prevented the
ischemia/
reperfusion-induced
up-regulation of NOX4 and ROS
production.In addition,
treatment with
betulinic acid could markedly blunt the
ischemia/
reperfusion-induced neuronal
apoptosis.Finally,
betulinic acid reduced
infarct volume and ameliorated the neurological deficit in this
stroke mouse model.Our results suggest that
betulinic acid protects against
cerebral ischemia/
reperfusion injury in
mice and the
down-regulation of NOX4 may represent a mechanism contributing to this effect.