Role of Nrf2/ARE Pathway in Protective Effect of Electroacupuncture against Endotoxic Shock-Induced Acute Lung Injury in Rabbits
et al. (2014) Role of Nrf2/ARE Pathway in Protective Effect of Electroacupuncture against Endotoxic Shock-
Induced Acute Lung Injury in Rabbits. PLoS ONE 9(8): e104924. doi:10.1371/journal.pone.0104924
Role of Nrf2/ARE Pathway in Protective Effect of Electroacupuncture against Endotoxic Shock-Induced Acute Lung Injury in Rabbits
Jian-bo Yu 0 1
Jia Shi" 0 1
Li-rong Gong" 0 1
Shu-an Dong 0 1
Yan Xu 0 1
Yuan Zhang 0 1
Xin-shun Cao 0 1
Li-li Wu 0 1
John Calvert, Emory University, United States of America
0 Funding: This research was supported by grants No. 81372096 from the National Natural Science Foundation of China , Beijing, China , and grant 12ZCZDSY03300 from the Key Project of Tianjin Science and Technology Support , Tianjin , China. The funders had no role in study design, data collection and analysis, decision to publish,or preparation of the manuscript
1 Department of Anesthesiology, Tianjin Nankai Hospital, Tianjin Medical University , Tianjin , China
NF-E2 related factor 2 (Nrf2) is a major transcription factor and acts as a key regulator of antioxidant genes to exogenous stimulations. The aim of current study was to determine whether Nrf2/ARE pathway is involved in the protective effect of electroacupuncture on the injured lung in a rabbit model of endotoxic shock. A dose of lipopolysaccharide (LPS) 5 mg/kg was administered intravenously to replicate the model of acute lung injury induced by endotoxic shock. Electroacupuncture pretreatment was handled bilaterally at Zusanli and Feishu acupoints for five consecutive days while sham electroacupuncture punctured at non-acupoints. Fourty anesthetized New England male rabbits were randomized into normal control group (group C), LPS group (group L), electroacupuncture + LPS group (group EL) and sham electroacupuncture + LPS (group SEL). At 6 h after LPS administration, the animals were sacrificed and the blood samples were collected for biochemical measurements. The lungs were removed for calculation of wet-to-dry weight ratios (W/D), histopathologic examination, determination of heme oxygenase (HO)-1 protein and mRNA, Nrf2 total and nucleoprotein, as well as Nrf2 mRNA expression, and evaluation of the intracellular distribution of Nrf2 nucleoprotein. LPS caused extensive morphologic lung damage, which was lessened by electroacupuncture treatment. Besides, lung W/D ratios were significantly decreased, the level of malondialdehyde was inhibited, plasma levels of TNF-a and interleukin-6 were decreased, while the activities of superoxide dismutase, glutathione peroxidase and catalase were enhanced in the electroacupucnture treated animals. In addition, electroacupuncture stimulation distinctly increased the expressions of HO1 and Nrf2 protein including Nrf2 total protein and nucleoprotein as well as mRNA in lung tissue, while these effects were blunted in the sham electroacupuncture group. We concluded that electroacupuncture treatment at ST36 and BL13 effectively attenuates lung injury in a rabbit model of endotoxic shock through activation of Nrf2/ARE pathway and following up-regulation of HO-1 expression.
" JbY, JS and LrG are co-first authors on this work.
Pulmonary dysfunction is documented as a hallmark of sepsis,
and diffuse lung injury resulting in acute respiratory distress
syndrome has been put forward as the major characters of
pulmonary dysfunction after endotoxin administration .
Intravenous infusion of LPS induced acute lung injury and caused
alterations in lung physiologic processes, which was similar to
those in humans . However, the precise mechanism involved in
the formation of acute lung injury (ALI) or acute respiratory
distress syndrome (ARDS) induced by endotoxin is not, as yet,
fully understood. Our previous study showed that the increased
oxidative stress may be a major cause of organ failure and high
mortality during endotoxic shock . Oxidative stress is defined as
a condition of imbalance between reactive oxygen species (ROS)
formation and cellular antioxidant capacity owing to
overproduction of ROS or dysfunction of the antioxidant system .Thus,
repairing the imbalance status by scavenging ROS or enhancing
cellular antioxidant capacity may have implication for a wide
array of pathology and disease models.
Acupuncture as an integral part of a traditional Chinese medical
system for more than 2500 years. It is worthy of applying
acupuncture to specific acupoints to achieve favorable regional or
systemic effects . It was proven that electroacupuncture
pretreatment significantly inhibited systemic inflammatory
responses and improved survival rate in rats with lethal endotoxemia
. Feishu (BL13) acupoint is considered of choice to treat lung
diseases and regulate pulmonary functions, Pan et al. reported that
treatment with electroacupuncture on BL13 showed beneficial
effects on hypoxia-induced pulmonary hypertention in rats .
Traditionally, acupuncture at Zusanli (ST36) acupoints was
known as the modulation of immune functions and is often used
in clinical disorders of the immune system .
Nuclear factor erythroid-2 related factor-2 (Nrf2) as a Cap n
Collar basic leucine zipper transcription factor plays a crucial role
in regulating antioxidant and cytoprotective genes in response to
oxidative stress . An accumulation of ROS or electrophilic
compounds give rise to the disruption of Nrf2/Kelch-like
ECHassociated protein-1 (Keap1) complex and result in the
translocation of Nrf2 from the cytoplasm into the nucleus where it
dimerizes with antioxidant response element (ARE) DNA
sequence ultimately activates the expression of ARE-dependent
genes . Downstream targets of Nrf2 include direct antioxidant
proteins such as catalase (CAT) and glutathione peroxidase (GPx),
stress-response proteins such as HO-1 and phase II metabolizing
enzymes such as glutathione S-transferase (GST), and others .
Among these, CAT and GPx directly neutralize ROS and are
regarded as very important antioxidant enzymes . Heme
oxygenases (HO-1), and with the productions of heme catabolism
including biliverdin, bilirubin, carbon monoxide (CO) andiron,
shows anti-inflammatory and antioxidant properties . Our
preliminary studies have confirmed that up-regulation of HO-1
protein followed by CO increasing could lessen the mortality in
septic shock rats . Another previous study elucidated the
protective effects of electroacupuncture stimulation at ST36 and
BL13 acupoints against acute lung injury evoked by endotoxic
shock in rabbits were dependent on up-regulated HO-1 expression
However, it is not known whether electroacupuncture
stimulation play a protective role in impaired lung by activating Nrf2/
ARE pathway during endotoxic shock. Based on these previous
data, we hypothesized that electroacupuncture treatment at ST36
and BL13 acupoints protect against endotoxic shock-induced lung
injury via modulating Nrf2/ARE pathway.
Materials and Methods
The current study was conducted in accordance with the
Institutional Animal Use Guidelines and approved by the Animal
Care Committee of Tianjin Nankai Hospital (NKH-20120818,
Tianjin, China). Two-month-old male New England white rabbits
(1.5,2.0 kg) was provided by Laboratory Animal Center of
Nankai Clinical Institution of Tianjin Medical University. The
animals were housed at 18,22uC on a 12-h light-dark cycle.
Besides, food and water were supplied ad libitum for a 5-day
period prior to the experiment protocols.
Prior to the induction of anesthesia, the rabbits were fasted for
12 h but allowed free access to water. All the rabbits were
anesthetized with 20% urethane (5 ml/kg) via the marginal ear
vein and anesthesia was maintained with intravenous infusion of
ketamine at 3 mg/Kg/h throughout the experiment. Then, a
3.5 mm non-cuffed endotracheal tube was inserted and tied in
place through the tracheotomy. Mean arterial pressure was
continuously monitored with a PE-50 catheter inserted through
the right carotid artery by using Hellige monitor instruments
(Germany), while a 24-g catheter was inserted into internal jugular
vein for intravenous injections. As Nishina et al. described ,
the lungs of animals were mechanically ventilated with an infant
ventilator (IV100B, Sechrist, Anaheim, CA) at an inspired oxygen
concentration of 40%. Tidal volume was set to 10 ml/kg (peak
inspiratory pressure was 1113 cm H2O), as measured by
pneumotachograph, and 2 cm H2O of peak expiratory pressure
was added . Respiratory rate was controlled to produce initial
PaCO2 of 3540 mmHg while the inspiratory/expiratory time
ratio was set at 1:2 . The rabbits were placed on a heating pad
under a radiant heat lamp so as to keep the body temperature at
37.740.3uC. Lactated Ringers solution was administered
intravenously at a rate of 8 ml/kg/h.
All animals were lightly immobilized using hands to minimize
stress during acupuncture stimulation which was initiated for a
five-consecutive-day before the experiment. Besides,
electroacupunture stimulation was performed throughout the operating steps
for 6 h during the experimental day . The selected acupoints
in this study were Zusanli (ST36), located between the tibia and
fibular approximately 5 mm lateral to the anterior tubercle of the
tibial, and Feishu (BL13), located between T3 and T4 of the spine
approximately 1.5 cm lateral to the midline. A set of
nonacupoints located on 5 mm lateral to the ST36 or BL13 original
location as controls. Two pairs of stainless steel needles (diameter,
0.3 mm) were inserted bilaterally to a depth of 5 mm into the
acupoints and kept in place. The parameter of electroacupuncture
was applied for 15 minutes with a disperse-dense wave (ie,
alternating frequencies of 2 Hz and 15 Hz) once a day by an
electrical stimulation device (HANS G6805-1A, Huayi Co,
Shanghai, China) , and the intensity was adjusted to induce
moderate muscle contract of the hindlimb (#1 mA) .
Acupuncture points were identified by an experienced
Forty Rabbits were randomized into four different groups
(n = 10/group): group C, group L, group EL and group SEL.
Rabbits in group L, EL and SEL were treated with intravenous
injection of 0.5 ml (5 mg/kg) LPS (L2630, sigma, USA) to
replicate the experimental model of acute lung injury induced
by endotoxic shock, while group C received 0.5 ml normal saline
intravenously as a control. Electroacupuncture treatment at ST36
and BL13 bilaterally was conducted in group EL from the
preparation of the model until the end of the experiment for 6 h.
Meanwhile, group SEL was acupunctured at non-acupoints with
the same frequency and intensity described as above. There was
no electroacupuncture stimulation in group C. Mean arterial
blood pressure (MAP) was monitored continuously and recorded
at 30, 60, 90 and 120 min after the start of administration of LPS.
MAP did not decrease within 2 h or rabbits died within 6 h after
LPS administration were regarded as the exclusion criteria of the
Preparation and analysis of samples
The whole blood was withdrawn from the right carotid artery at
6 h after LPS or normal saline administration. Approximately
1 ml of the arterial blood samples were analyzed for the
calculation of oxygenation indexes by a blood gas analyzer
(Gem premier 3000, USA) before death. Meanwhile, the blood
specimen remained were centrifuged at 4uC (3000 rpm for
15 min). The plasma was removed and the aliquots of the
supernatant were separately frozen at 280uC for subsequent
analysis. At the end of the experiment, the rabbits were sacrificed
by exsanguination, and the lungs were removed and quickly
flushed with phosphate-buffered saline (PBS) to remove the blood.
Ultimately, sections of the left lung tissues were put in 10%
formaldehyde for histopathological analysis, and the remaining
tissues were snap frozen in liquid nitrogen and stored at 280uC for
The tissue homogenate was prepared for biochemical assays by
the upper lobe of the right lung. The levels of superoxide
dismutase (SOD) activities and malondialdehyde (MDA) contents
in the lung tissues were determined by spectrophotometry 
and measured by means of Loewenberg , respectively. And
both were expressed as per unit of protein determined by the
Lowry method . Moreover, the serum level of glutathione
peroxidase (GPx) and catalase (CAT) activities were measured
using commercial assay kits supplied by the Nanjing Jiancheng
Bioengineering Institute (Nanjing, China). Plasma tumor necrosis
factor-alpha (TNF-a) and interleukin-6 (IL-6) were assayed with a
commercial enzyme-linked immunosorbent assay kit (R&D
systems, USA). All the procedures were performed according to
Lung wet-to-dry (W/D) weight ratios
The W/D weight ratio of the lung was calculated to evaluate
the severity of pulmonary edema. The harvested tissue of left
upper lobe was rinsed with normal sodium to scour off the
superfluous water. After that, the lung tissue was weighed as wet
weight. Dry weight was recorded after the specimen was dried to a
constant weight at 70uC for 24 h in an electric air blast drier. The
W/D weight ratio was then calculated .
Preparation of BALF and Measurements
The Bronchoaleveolar lavage fuild (BALF) analysis was
measured to quantify the magnitude of the pulmonary edema.
40 ml saline with ethylendiamine tetraacetic acid (EDTA)-2Na at
4uC was slowly infused for 5 times through the right mainstem
bronchus and withdrawn. BALF was analyzed for cell
differentiation and cell counts by the Bu rker-Tu rk method . Lavage
samples were centrifuged at 250 g at 4uC for 10 min to remove the
cells. The cell-free supernatant was analyzed for albumin
determined by immune-nephelometry.
Immediately after the rabbits were killed (,5 min), the middle
lobe of right lung was fixed in 10% formaldehyde for 24 h, and
then dehydrated with graded alcohol followed by embedding in
paraffin at 60uC. A battery of microsections (4 mm) stained with
hematoxylin and eosin were examined under a light microscope
(6400) and ten different visual fields were observed for each slice.
The lung pathologic change was assessed by alveolar edema,
airway congestion, widening of the interstitium or hyaline
membrane formation and reactive cell infiltration or aggregation
. Each item was graded according to a 5-point scale described
as follows : 0 = minimal damage, 1+ = mild damage, 2+
= moderate damage, 3+ = severe damage, and 4+ = maximal
damage. The individual scores were added together to rack up a
final score ranging from 0 to 16. The lung injury assessment was
quantified by a blinded expert pathologist.
Western blot analysis
The expression of HO-1, Nrf2 total protein and Nrf2
nucleoprotein of the lung samples were analyzed by Western blot
technique. The tissues stored at 280uC were homogenized in
13.2 mmol/L Tris-HCl, 5.5%glycerol, 0.44%SDS and 10%
bmercaptoethanol. The proteins were extracted according to the
instructions of the total protein and nuclear protein extraction kit
(Thermo, USA), while the protein concentration was detected on
the basis of the BCA protein assay kit (Thermo, USA). Equal
amounts of soluble protein were fractionated by 12% SDS-PAGE
and were transferred to a PVDF membrane (Bio-Rad, USA). Blots
were washed triple for 5 min in TBS and then were incubated
overnight at 4uC with polyclonal rabbit antibodies against HO-1
(1:800, Abcam, UK) or Nrf2 (1:300, Biorbyt, UK). Primary
antibodies were diluted in blocking solution containing 1% nonfat
milk plus 0.5% BSA in TBS-0.05% Tween 20. After three washes
with TBS-0.05% Tween 20, blots was incubated at 37uC for 1 h
with the horseradish peroxidase (HRP)-conjugated goat anti-rabbit
IgG (1:3000 dilation, Biorbyt, UK). The blots were visualized with
the enhanced chemiluminesence (Bio-Rad, USA) according to the
manufacturers instruction , and the relative density of bands
was quantified by densitometry (Molecular Analyst Image-analysis
Software, Bio-Rad, USA).
RNA isolation and Real-time PCR
Total RNA was extracted from lung tissue using a Total Quick
RNA kit (TA200TQR, Talent, Italy). Tissue was lysed in the
provided buffer and RNA was eluted by RNAse-free water. Total
amount of RNA was determined by absorbance at 260 nm, while
the purity of RNA were measured by 260/280 nm absorbance
ratio, respectively. 1 mg total RNA was reversely transcribed into
cDNA with random hexamers using a Revert AidTM First Strand
cDNA Synthesis kit (MetaBiosInc, Canada). The final PCR
reaction of volume of 20 ml was established with SYBR Green
master mix. Predegeneration of the PCR mix was at 95uC for
10 min, and the thermal cycle profile was denaturing for 20 s at
94uC, annealing for 20 s at 59uC and extension for 20 s at 59uC. A
total of 40 PCR cycles were used. The primers were as followed:
bactin sense, 59-CGCGACATCAAGGAGAAGCTG-39 and
bactin antisense, 59-ATTGCCAATGGGTGATACCTG-39,
128 bp; HO-1sense, 59-TGCCGAGGGTTTTAAGCTGGT-39
and HO-1 antisense, 59-AGAAGGCCATGTCCAGCTCCA-39,
158 bp; Nrf2 sense, 59-CCCACAAGTTCGGCATCCAC-39 and
Nrf2 antisense, 59-TGGCGATTCCTCTGGCGTCT-39, 182 bp.
The comparative Ct (threshold cycle) method was applied for
quantitiation of target gene expression as described by Schmittgen
et al . The relative gene expression of HO-1 and Nrf2 mRNA
were normalized to that of b-actin.
The intracellular distribution of Nrf2 was displayed by
immunofluorescence technique. Paraffin-embedded tissue sections
(4 mm) were dewaxed in xylene and rehydrated in graded ethanol
solutions. The antigen retrieval method with citric acid solution
was proceeded for 5 min at 95uC, and then was washed in PBS.
Follwing that, the tissues were permeabilized with 0.5%Triton
X100 for 15 min and blocked in normal goat serum at room
temperature for 30 min . After incubation with the polyclonal
Nrf2 primary antibody conjugated to FITC (1:150, Biorbyt, UK)
at 4uC overnight, the sections was washed triple with PBS. At last,
the nuclei were counterstained with DAPI (Roche, Shanghai,
China) and visualized using a fluorescent microscope (Olympus
U25ND25, Tokyo, Japan). The green staining was showed in
cytoplasm and nucleus, while blue staining was in nucleus. The
overlay color was considered to be positive. Ultimately, the results
were evaluated by semi-quantitative analysis based on the
proportion of the Nrf2 nucleoprotein to the number of nuclei in
five fields of each slices at a 400 multiple signal magnification .
Values were expressed in mean6SD or median (range), except
for lung injury scores, for which the Kruskal-Wallis rank test was
used. Parametric data were analyzed by one-way analysis of
variance (ANOVA) and variations of different groups were
compared using the Tukey-Kramer post hoc test.
Repeatedmeasures data (eg. PaO2/FiO2) were determined by
repeatedmeasures ANOVA. The W/D ratios were analyzed by two-way
ANOVA followed by Bonferroni correction. Data of real-time
PCR were tested by a t test with two-tailed hypothesis testing.
SPSS19.0 statistical software was used for data analysis and P,
0.05 was deemed as statistically significant.
The death rate in group EL (1 rabbits) was lower than that in
group L (4 rabbits), and was higher than that in group C (zero
rabbits). There was no significant difference in death rate between
group SEL (3 rabbits) and group L. And, the animals were
supplemented according to the randomized crossover principles.
Hemodynamic and oxygenation indexes
MAP in rabbits were remained stable throughout the
experiment and the baseline MAP of each group were similar
(105,109 mmHg) (table 1). Sixty minutes after LPS injection,
MAP in group EL was distinctly lower than that in group C, and
higher than that in group L (P,0.05). There was no significant
difference between group L and SEL (P.0.05). Oxygenation
indexes were decreased to less than 300 mmHg in group L, EL
and SEL at the end of LPS administration. However,
electroacupuncture treatment, rather than sham electroacupuncture
stimulation could attenuate the reduction, which revealed that
oxygenation indexes in group EL was higher than group L (P,
Lung W/D weight ratio
The lung wet-to-dry weight ratio was calculated as an indicator
of pulmonary edema. W/D ratio was increased in the rabbits
received LPS (Group L, EL and SEL) compared to group C (P,
0.05) (Table 2). Electroacupuncture treatment attenuated the
increase of W/D weight ratio (attenuation 50.5%, P,0.05), while
group SEL did not show the protective effect (P.0.05).
MDA contents and SOD activities in the lung tissue
The comparisons of MDA contents and SOD activities were
showed in Table 2. Intravenous administration of LPS showed an
apparent increase of MDA contents and decrease of SOD
activities compared to group C (P,0.05). However,
electroacupuncture reduced the contents of MDA by 53.5% and enhanced
activities of SOD by 34.3% to counteract the effects induced by
LPS (P,0.05). No significant influence in above parameters were
discovered when compared group SEL with group L (P.0.05).
GPx and CAT activities in serum
Our data revealed that rabbits from group L, EL plus group
SEL possessed lower GPx and CAT activities than control group
(P,0.05) (Table 2). Moreover, the activities of GPx and CAT,
which were known as the ROS direct scavengers were enhanced
significantly in group EL (augment 30.6% for GPx and 50.4% for
CAT) compared with group L or group SEL (P,0.05). There
were no significant differences between group SEL and group L
Plasma levels of TNF-a and IL-6
As shown in Table 3, plasma levels of TNF-a and IL-6 in group
L, EL and SEL were significantly higher than in group C.
However, the EL group showed lower levels of TNF-a and IL-6
Table 2. Comparisons of W/D ratio, MDA contents and SOD activities in the lung tissue, as well as CAT and GPx activities in serum
among four groups.
than the L group (TNF-a, 19.6264.89 and 26.7967.65, P,0.05;
IL-6, 87.53616.23 and 112.32625.76, P,0.05). We did not find
a significant difference in plasma levels of TNF-a and IL-6
between group SEL and group L (P.0.05).
Analysis of BALF
The recovery percentages of BALF among the four groups were
83%,88%, indicating no differences in the groups. Compared
with group C, the number of leukocytes and albumin
concentrations in the supernatant of BALF were obviously higher in group
L, EL and SEL (Table 3). However, electroacupuncture treatment
mitigated the increase in leukocyte counts and albumin
concentrations in the BALF in rabbits receiving LPS. In contrast, there
was no significant difference in BALF concentrations between
group SEL and group L.
The expressions of HO-1 mRNA, Nrf2 mRNA, HO-1
protein, Nrf2 total protein and Nrf2 nucleoprotein in lung
The results of the experiment determined by Real-time PCR
and Western blot were shown in Figure 2 and Figure 3. Exposure
to LPS notably increased the mRNA expression of HO-1 and Nrf2
as well as the protein expression of HO-1 and Nrf2 containing
nucleoprotein and total protein compared with group C (P,0.05).
In addition, the expression of HO-1 m RNA and Nrf2 mRNA
plus the levels of HO-1 protein and Nrf2 total and nucleoprotein
were markedly up-regulated in group EL in contrast to group L
and group SEL (P,0.05). Nevertheless, there were no significant
differences between group SEL and group L in terms of the above
mentioned mRNA or protein expressions (P.0.05).
The distribution ratio of Nrf2 nucleoprotein expression
Immunofluorescence analysis of Nrf2 expression was
represented in Figure 4. Group C showed a negligible Nrf2 nucleoprotein
expression, while an enhanced expression with concomitant
increase in Nrf2 positive protein was apparent in group L (P,
0.05). Meanwhile, electroacupuncture stimulation at acupoints of
ST36 and BL13 resulted in a significant increase in the number of
Nrf2 nucleoprotein comparison with group L (augment 70.2%,
P,0.05). However, sham electroacupuncture treatment exhibited
the similar expression of Nrf2 to that of group L (P.0.05).
Data from the current study demonstrated that
electrostimulation at ST36 and BL13 acupoints dramatically mitigated
LPSinduced ALI in endotoxic shock rabbits. Furthermore, the
BALF Albumin (mg/dl)
Abbreviations: BALF, Bronchoalveolar lavage fluid; TNF-a, tumor necrosis factor-alpha; IL-6, interleukin-6. Values as means 6 SD (n = 10).
*P,0.05 versus control group;
+P,0.05 versus LPS group.
Acute lung injury scores
Values were expressed as medians (range). The lung injury was scored by a 5-point scale according to combined assessments of alveolar congestion, hemorrhage and
edema, infiltration or aggregation of neutrophils in the airspace or vessel wall and thickness of alveolar wall/hyaline membrane formation: Score of 0 = minimal (little)
damage; 1+ = mild damage; 2+ = moderate damage; 3+ = severe damage; and 4+ = maximal damage. Minimum and maximum possible lung injury scores are 0 and 16,
*P,0.05 versus Group C;
+P,0.05 versus Group L.
production of HO-1 mRNA and HO-1 protein in group EL were
notably higher than group L, which were consistent with the
expression of Nrf2 mRNA, Nrf2 total protein and nucleoprotein.
In addition, electroacupuncture treatment enhanced the activities
of SOD, GPx and CAT with the increase of Nrf2 and following
HO-1 expression. In brief, the present study for the first time
confirmed that electroacupuncture at bilaterally ST36 and BL13
produced powerful protection against lung injury through
activation of the Nrf2/ARE pathway and induction of the
following antioxidant enzymes.
Systemic LPS exposure was used for establishing the standard
model of endotoxic shock, which was invariably associated with
ALI or ARDS and even multiorgan dysfunction . ALI induced
by endotoxin was manifested with hypoxemia and pulmonary
edema depended on severe leukocytes infiltration, increased
microvascular permeability and endothelial barrier disruption.
The excess production of ROS by polymorphonuclear leucocytes
exceeded the antioxidant defense capacity of cells and extracellular
fluids, which leaded to oxidative damage in multiple organs .
MDA was a reliable marker of oxidative stress mediated lipid
peroxidation . Therefore, we applied it to reflect the degree of
cell damage caused by reactive oxygen metabolites. The first line
of defense against ROS mediated oxidative stress injury involved
endogenous antioxidant enzymes such as SOD, GPx and CAT
[4,33]. Moreover, plasma levels of TNF-a and IL-6 were
measured as indicators of systemic inflammatory responses
[34,35]. In our study, the activities of SOD, GPx and CAT were
significantly decreased in LPS induced groups, accompanied with
the increased MDA contents as well as the higher levels of TNF-a
and IL-6. Concordant with previous studies , the rabbit model
of injured lung induced by endotoxic shock in this research was
defined by the lowered MAP,75% of the baseline values and
oxygenation index (PaO2/FiO2) #300 mmHg.
HO-1 is highly inducible under conditions of ischemia/
reperfusion injury or inflammatory cytokines and serves as one
of the most prominent lines of defense of the cell against oxidative
stress . Previous research showed that hemin pretreatment
with ulinastatin in endotoxin treated rats resulted in an improved
response by upregulating HO-1 protein followed by increasing
CO and restraining increased oxidative stress . Furthermore,
Takaki et al. indicated , oxidative stress was closely related to
HO-1 expression, and the expression of HO-1 protein was
increased in critically ill patients, especially those with severe sepsis
or septic shock. To our knowledge, the parameters of
electroacupuncture treatment including the frequency of EA were critical
for producing prophylactic effects . Therefore, acupuncture
was performed with a disperse-dense wave with 2 Hz/15 Hz
lasted 15 min for 5 days consecutively before the experiment in the
current study [8,19]. Data from our research revealed that
electroacupuncture stimulation attenuated ALI induced by LPS in
rabbits through upregulation of HO-1 and reduction of MDA
content, W/D weight ratio and lung injury scores as well as
augment of SOD activities, which was compatible with our prior
Nrf2/ARE signaling pathway is essential for upregulating the
expression of numerous antioxidant genes in response to a wide
array of stimuli, and also protecting the cell against oxidative stress
and inflammation . Under physiological conditions, Keap1
promoted cytosolic Nrf2 degradation via the Cul3-dependent
ubiquitin proteasome pathway. When exposure to redox
modulators, the reactive cysteine residues of Keap1 was modified,
leading to Nrf2 translocation and accumulation in the nucleus.
Subsequently, Nrf2 dimerized with small Maf or Jun proteins that
binded to the ARE sequence in the promoter regions of phase II
detoxification enzymes and antioxidant proteins, which were
activated ultimately to protect cells from ROS generation . As
mentioned, SOD, GPx and CAT are directly involved in ROS
scavenge, thus, are deemed as very important antioxidant
enzymes. The SOD decomposes superoxide radicals and produces
H2O2. And, H2O2 is subsequent removed to water by CAT in the
peroxisomes or by GPx oxidizing GSH in the cytosol .
Consequently, the activities of above antioxidant enzymes are
proportional to the Nrf2 expression. Moreover, Nrf2 had been
clarified in Tsai PS et al. research to mediate upregulation of
HO1 by LPS in human monocytic cells . Judging from the present
study, upregulation of HO-1mRNA and protein by
electroacupuncture in endotoxic shock rabbits was displayed in the lung
tissue, which showed the same tendency with expression of Nrf2
mRNA as well as Nrf2 total and nucleoprotein, followed the
increase of SOD, GPx and CAT activities. Consistent with
Western blot and real-time PCR analyses, immunofluorescence
staining identified increments in Nrf2 protein accumulation in the
nucleus of cells subsequent to electroacupuncture stimulation.
Collectively, our research has several limitations. First of all, the
experimental model of injured lung induced by endotoxic shock
was established by intravenous LPS injection, which was extracted
from the cell wall of Gram-negative bacteria. However, the
infection of pathogenic bacteria was not the common cause in
clinical patients with endotoxic shock. As a result, it is not easily for
us to extrapolate our conclusions to the clinical setting. Secondly,
lung hyper-permeability causing pulmonary edema was deemed as
the main mechanism of ALI/ARDS . Therefore, the
determination of albumin content and leukocyte count in
bronchoalveolar lavage fluid should be added in further
exploration to more fully evaluate the effect of electroacupuncture on the
impaired lung. Finally, the expression levels of Nrf2 including total
protein and nucleoprotein in our present study were both
increased significantly. The findings were in coincidence with
the research of Chen et al. , which still need further
exploration for a suitable explanation.
In summary, treatment with electroacupuncture at ST36 and
BL13 acupoints alleviated ALI induced by LPS in rabbits, which
was counted on activation of Nrf2/ARE pathway and
upregulation of HO-1 expression. In addition, several kinases and
signaling pathways have been implicated in the activation of Nrf2/
ARE pathway. It has been reported that the MAPK cascade,
PI3K/AKT and PKC signaling pathways facilitate the
dissociation of Nrf2 from Keap1 and thereby influence the Nrf2/ARE
pathway . Nevertheless, the accurate mechanism by which
electroacupuncture activates Nrf2 pathway has not been fully
understood and requires further investigation. Above all, the
present findings provide the scientific foundation for the
development of electroacupuncture as a prophylactic treatment for acute
lung injury induced by endotoxic shock in clinic.
We thank Dr. Jian-bo Wang (Washington University School of Medicine)
for his technical support and valuable advice.
Conceived and designed the experiments: JbY LrG. Performed the
experiments: JS SaD YX YZ XsC LlW. Analyzed the data: JS LrG SaD.
Contributed reagents/materials/analysis tools: JbY SaD. Contributed to
the writing of the manuscript: JbY JS.
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