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Introduction
Glutamate, the major excitatory neurotransmitters in the
mammalian central nervous system, plays an important role
in many physiological functions by acting on its receptors.
At the same time, abnormally intense exposure to glutamate
can be neurotoxic, primarily through the overactivation of
the N-methyl-D-aspartate (NMDA) subtype of glutamate
receptors, a phenomenon known as excitotoxicity, which has
been implicated in neuronal degeneration and loss in some
acute conditions and chronic neurodegenerative
diseases[1]. Recently, sufficient evidence has been demonstrated for the
presence of NMDA glutamate receptors in the lungs and
some other non-neuronal tissues[2]. Said
et al reported that the activation of NMDA receptors in perfused, ventilated rat
lungs by NMDA (1×10-3 mol/L), a synthetic agonist that
selectively activates NMDA receptors, triggered acute injury,
marked by high-permeability edema[2]. Our previous study
demonstrated that glutamate (0.5 g/kg, ip) in
vivo also provoked acute lung injury. Both injuries were attenuated by
the NMDA receptor antagonist, MK801 (dizocilpine maleate),
which suggests that the activation of NMDA receptors leads
to acute lung injury[2]. Ginsenoside Rg1 is an important
active component of ginseng and they share many
pharmacological effects. It was proved that ginsenoside Rg1 has a
partial neurotrophic and neuroprotective role against the toxic
effects of glutamate in cultured dopaminergic
cells[3], hippocampal
neurons[4], and the neurons in the spinal cord
culture[5]. We speculate that ginsenoside Rg1 also ameliorates
glutamate-induced lung injuries. In the current study, we
first investigated the in vivo effect of ginsenoside Rg1 on
acute lung injury induced by glutamate to afford a
theoretical basis of clinical administration of ginsenoside Rg1 for
the treatment of lung diseases associated with the
activation of the NMDA receptors.
Materials and methods
Materials Kunming male mice (clean grade), weighing
25±5 g, were obtained from the Experimental Animal Center
of Xiangya School of Medicine, Central South University
(Changsha, China). Ginsenoside Rg1 and glutamate were
obtained from the School of Pharmacy, Jilin University (Jilin,
China) and Sigma (St Louis, MO, USA), respectively. MS302
bioinstrumentation was from Guangzhou Long-fei-da
Technology Ltd Co (Guangzhou, China). Detection kits for nitric
oxide (NO), total nitric oxide synthetase (NOS), xanthine
oxidase (XOD), superoxide dismutase (SOD), catalase (CAT),
and malonaldehyde (MDA) were all from Nanjing Jiancheng
Bioengineering Institute (Nanjing, China). All other
chemicals were up to the analytical chemistry standard.
Experimental groups The animals were housed at room
temperature and kept on an unlimited supply of standard
diet and tap water. All studies were performed in accordance
with the National Institute of Health Criteria for the Care of
Laboratory Animals. The mice were randomly divided into 4
groups and were given normal saline alone (group NS),
glutamate (group Glu), ginsenoside Rg1 (group Gin), or both
ginsenoside Rg1 and glutamate (group Gin+Glu),
respec-tively. Ginsenoside Rg1 was given intraperitoneally at a
dose of 0.03 g/kg for 30 min before the injection of glutamate
(0.5 g/kg, ip).
All the mice were anesthetized 2 h after the insult of
glutamate; the heart rate (HR) and breathing rate (BR) were
recorded by MS302 bioinstrumentation. After that, the mice
were sacrificed immediately for determinations as below.
Lung wet weight/body weight (LW/BW), lung wet
weight/lung dry weight (W/D) After sacrificing each mouse by
femoral artery bleeding, both lungs were carefully taken out
and weighed immediately in a weighing bottle. Then the
lungs were dried in a microwave oven with progressively
increasing power[6], cooled in the bottle, and
weighed again until no further weight loss occurred. After that, the weight
deducted from the bottle weight was reported as dry weight.
Histological examination After sacrifice,
the lung samples were immersed in 10% buffered formalin.
Formalin-preserved specimens were then embedded in paraffin, cut into 5 µm
thick sections, and stained with hematoxylin and eosin for
histological examination. This experiment was repeated 5
times.
Analysis of lung homogenates
Preparation of lung homogenate samples In some
experiments, the left lung was removed, washed in 0.9% NaCl,
and homogenized (1/10, w/v) in a glass homogenizer in
ice-cold NaCl solution. The homogenates were centrifuged, and
the supernatant was stored at 4 °C and used for the
following determination. All biochemical parameters in the
homogenates were studied on the same day. Tissue
concentrations of the respective parameters were related to the
protein content in the samples. The protein concentration
was determined by the Lowry method[7]
.
NO and NOS activity Tissue concentrations of NO
were measured through its stable metabolites nitrate and
nitrite. Nitrate was first reduced by nitrate reductase to
nitrite and then nitrite was determined spectrophotometrically
at 550 nm by the Griess reaction using commercial kits. NOS
activities were measured spectrophotometrically at 530 nm
by enzymatic methods using commercial kits.
XOD activity determination XOD activity was assayed
spectrophotometrically at 37 °C with the
corresponding substrate using commercial kits, and the red-violet product of
the reaction was measured in the visible range at 530 nm.
Antioxidase activity measurement Tissue SOD
activities were determined by the inhibition of nitroblue
tetrazolium (NBT) reduction with xanthine/XOD used as a
superoxide generator using commercial kits. One unit of SOD was
defined as the amount of protein that inhibits the rate of
NBT reduction by 50%.
CAT activity was measured by the breakdown of
hydrogen peroxide catalysed by catalase enzymes using
commercial kits.
MDA determination MDA, an end product of fatty acid
peroxidation, was measured in lung homogenates by the
thiobarbituric acid reactivity assay using commercial kits.
Statistical analysis All statistical analyses were carried
out using SPSS statistical software (SPSS for Windows
version 11.0, SPSS Inc, Chicago, USA,). Data were analyzed by
ANOVA, and the Student Newman-Keuls method was used
to estimate the level of significance of
differences between means. The data are expressed as mean±SD. The criterion
for significance was P<0.05.
Results
Heart rate and breathing rate Glu (0.5 g/kg) treatment
elevated HR and BR (P<0.01). Gin (0.03 g/kg) pretreatment
abolished the enhancing effect of Glu on HR and BR
(P<0.01; Table 1).
LW/BW and W/D The addition of Gin (0.03 g/kg) 30 min
before Glu treatment prevented an increase of lung water
induced by Glu (0.5 g/kg). Both LW/BW and W/D of the Glu
group were elevated (P<0.05), while those of group Gin+Glu
decreased to the same level as group NS (P<0.01; Table 2).
Histological examination Significant lung injury was
present in the animals of the Glu group compared with the
NS group. There was edematous thickening of the alveolar
walls with occasional alveoli containing coagulated edema
fluid. The alveolar interstitium showed marked
sequestration of inflammatory cells, which were predominantly
neutrophils. Gin treatment reduced lung injurious
pathology change caused by the administration of Glu (Figure 1).
Activity of XOD, SOD, CAT, and levels of MDA in lung
tissues Treatment of Glu (0.5 g/kg) increased XOD activity
(P<0.01) and MDA (P<0.05) in lung tissues. Gin (0.03 g/kg)
abolished the activation of XOD and increased MDA levels
induced by Glu (Table 3).
The lung SOD and CAT activities in the Glu group were
lower than in the NS group (P<0.01), and SOD and CAT
activities in the Gin+Glu group increased to the same level as
group NS. No significant difference of tissue SOD and CAT
activities was observed between the Gin and NS groups
(Table 3).
NOS activity and NO production The injection of Glu
(0.5 g/kg) led to the activation of NOS, following the
elevation of NO content (P<0.01). With the pretreatment of Gin
(0.03 g/kg), NOS activity and the NO level decreased to the
control level (Table 4).
Discussion
Acute respiratory distress syndrome (ARDS) is a
common clinical syndrome. The pathogenesis of ARDS has been
undefined up to now. The neutrophils sequestration and
activation of neutrophils and alveolar macrophages play key
roles in the development of ARDS[8].
Neutrophils[9] and
macrophages[10] can release glutamate when stimulated. The
glutamate concentrations in the pulmonary veins of rat model
septicemia are higher than in the pulmonary
artery[11]. Our previous study demonstrated that the administration of
glutamate via intraperitoneal injection provoked acute lung
injury characterized by edema, neutrophil sequestration, and
increased alveolar-microvascular membrane permeability.
This injury was attenuated by MK801, a NMDA receptor
antagonist, which suggests that glutamate in
vivo causes lung injury through the activation of NMDA receptors. These
results raise the possibility of new therapeutic uses of
MK-801 which have potential protective effects against
glutamate-induced toxicity in related lung impairment.
Although there is promising therapeutic potential of
MK-801, the delivery remains a challenge due to its side
effects[12].
Ginseng is the best-known and most popular herbal
medicine. It serves as an important component of many
Chinese prescriptions for thousands of years and is now
popular in the world as a natural medicine. The molecular
components responsible for ginseng actions are ginsenosides,
which are also known as ginseng
saponins[5]. Ginsenoside Rg1 is an important active component of
ginseng and has been reported to protect neurons from excitotoxicity induced
by glutamate[3_5]. Several recent studies have reported that
ginsenoside Rg1 has some beneficial actions on lungs. It
inhibits the releases of histamine and leukotrienes during
the activation of guinea pig lung mast
cells[13]. Ginsenoside Rg1 can enhance the synthesis of pulmonary surfactant of
cultured rat lung explants[14]. Whether ginsenoside Rg1 has
protective actions on the glutamate-induced lung injury has
not been reported.
The simplest way to evaluate edema formation in lungs is
to use a gravimetric approach.
Because inbred strains have relatively uniform LW/BW ratios
between animals at any given age, one can potentially compare
LW/BW in treatment groups to that predicted based on body weight. The measure of
W/D is a more useful tool since it accounts
for changes in lung dry mass as well. This research shows that ginsenoside
Rg1 attenuated the glutamate-induced elevation of LW/BW,
W/D, HR, BR, and the histology changes of lung tissues.
This study first proved that ginsenoside Rg1 could
attenuate glutamate-induced lung injury in
vivo.
There is general agreement about the mechanism of
neuroexcitotoxicity induced by glutamate that it is
Ca2+-dependent. It is also generally accepted that the NMDA
receptors play a key role in mediating glutamate toxicity
owing to its high Ca2+
permeability[1]. Excessive
Ca2+ loading exceeding the capacity of
Ca2+-regulating mechanisms could activate several cell death-related genes and pathways.
These include the calcium-dependent activation of
XOD[15]. XOD is known to generate deleterious oxygen-free radicals
such as superoxide, and hydroxyl radicals such as
hypoxanthine are metablized to uric acid in the final steps of purine
degradation[16]. In this study, the impairment of SOD and
CAT was also noted in the lungs of of the mice in the Glu
group. SOD converts superoxide radical to
H2O2, which is in turn broken down to water and oxygen by
CAT[17]. Therefore XOD, SOD, and CAT play important roles in the balance
between the oxidation and antioxidation of the
organism[17]. The activation of XOD and the impairment of SOD and CAT
lead to overproduction of reactive oxygen species. Higher
doses of oxygen-derived free radicals destabilize cell
membranes, increase membrane permeability, oxidize
cyto-solic, and membrane-bound
proteins[18]. These alterations
of cellular function impact cellular defense mechanisms and
membrane integrity, contributing to pulmonary edema
accumulation. The augmentation of antioxidant defense
mechanisms attenuates lung injury. It has been reported
that ginsenoside Rg1 inhibits lipid peroxidation within the
liver and brain by upregulating
catalases[19] and suppresses oxidative stress in neurons by the activation
SOD[20]. Our data indicates that the administration of ginsenoside Rg1
reversed the activation of XOD and reduced the impairment
of SOD and CAT. As a result, cell injury mediated by free
radicals was relieved in the Gin+Glu group, which was
demonstrated by the drop in levels of MDA, an end product of
fatty acid peroxidation. Ginsenosides are reported for the
inhibition of Ca2+ over-influx into the mitochondria of the
surviving cells, thus lowering free radical production by
depolarized mitochondria and increasing energy production
crucial for cell survival[3]. Additionally, the modulation
effect of ginsenoside Rg1 on NMDA receptors binding in the
rat brain was also reported[21]. Whether ginsenoside Rg1
influences the binding of NMDA receptors or
Ca2+ over-influx in this model needs further research.
Evidence has been presented for a key mediator role for
NO in glutamate-stimulated
neurotoxicity[22]. Based on the research of the isolated perfused and ventilated rat lung,
Said et al reported that as in central neuronal glutamate
toxicity, lung injury caused by the NMDA in the perfusate
was NMDA receptor-mediated and NO dependent, and was
associated with the increased production of
NO[2]. The present study shows that glutamate
in vivo leads to an increase in NOS activity and NO generation in mouse lungs,
which suggests NO may contribute to lung injury caused by
glutamate in vivo through some mechanism. NO, in high
enough concentrations produced in pathological conditions,
is known to effectively compete with SOD for
superoxide[23]. When NO and superoxide
anion are present in large amounts,
peroxynitrite (ONOO_), a potent oxidant and nitrating reagent,
is produced[24]. However, glutamate-induced NO
generation by the upregulation of NOS activity may also promote
transcription and the translation of numerous inflammatory
cytokines, and as a result, more neutrophils and
macrophages are recruited and activated, which produce a burst of
free radicals[25_27]. Although these possible alterations could
explain cell injury and the dysfunction of the alveolar
capillary-epithelial permeability barrier, which lead to the leak of
plasma into the pulmonary interstitium and alveolar spaces
resulting in high-permeability pulmonary edema in the
present study, further evidence is still required. In this study,
pretreatment with ginsenoside Rg1 almost completely
inhibited an increase of NOS activity, and subsequently NO
generation and eventually relieved inflammatory responses and
free-radical-mediated lipid peroxidation. Three isoforms of
the enzyme responsible for NO production, NOS have been
described: neuronal (nNOS, NOS I), inducible (iNOS, NOS
II), and endothelial (eNOS, NOS III). All 3 isoforms of NOS
are expressed in lungs[28]. The NOS isoenzymes and
location involved in the protective effects of ginsenoside Rg1
against glutamate-induced lung injury require further
investigation in the future.
In summary, the present study indicates that ginsenoside
Rg1, an extract of ginseng, could attenuate
glutamate-induced lung injury by interrupting the generation of reactive
oxygen species and NO. Although other studies have
demonstrated beneficial effects of ginsenoside Rg1 on
neuro-toxicity, this is the first study to our knowledge that
examined the effect of ginsenoside Rg1 on the glutamate-induced
impairment of pulmonary tissue in vivo. Given that ginseng
has only minor side effects in animals and
humans[5], our research might offer a potential means to rescue or protect
lungs from some diseases associated with glutamate toxicity.
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