burns 38 (2012) 743–750

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Effects of Ligustrazine on pulmonary damage in rats

following scald injury

Chengjin Gao *, Yuhao Liu, Lijie Ma, Xiangyu Zhang, Sheng Wang
Emergency Department and Surgical Intensive Care Unit, Shanghai 10th People’s Hospital, Tongji University School of Medicine, China

article info abstract

Article history: Organ protection is a routine therapy in severe burn/scald injuries, and damage mecha-
Accepted 21 December 2011 nisms following early scald injury was not been fully elucidated. Our aim was to verify the
beneficial effects of Ligustrazine on pulmonary damage associated with scald injury.
Keywords: Lewis rats were subjected to 30% total body surface area (TBSA) scald injury, and were
Ligustrazine randomly divided into a burn control (S group) and an Ligustrazine-treated group (L group).
Burn Lung malondialdehyde (MDA) and superoxide dismutase (SOD) levels were determined and
Lipid peroxidation the lungs were examined histologically with immunohistochemistry (IHC) as well for the MHC
class I chain-related antigen A (MICA) and Bcl-2 at 24, 48 and 72 h after the injury. The
expression of spleen HLA-DR was detected by immunohistochemistry analysis. Selectins and
adhesion molecules in lungs and serum as well as pulmonary interleukins were also detected.
The lung injury degree was represented as wet/dry (W/D) values and alveolar thickness.
Ligustrazine decreased MDA levels and ameliorated the down-regulation of SOD activity.
MICA was up-regulated after the scald, and this up-regulation was greatly diminished by
Ligustrazine. Bcl-2 was up-regulated after the scald, especially in the L group. The spleen
HLA-DR expression demonstrated the immunoregulatory effects of Ligustrazine, which
effectively protected pulmonary tissues from scald-induced injury.
Our results demonstrated that pulmonay damage associated with autoimmunity and
oxidant attack occurred after severe scald. Ligustrazine exhibits significant protective
effects on these effects.
# 2012 Elsevier Ltd and ISBI. All rights reserved.

Ligustrazine on pulmonary injury has not been reported.

1. Introduction
Ligustrazine, a purified and chemical identified component of
a Chinese herbal remedy, has strong effects on scavenging
cytotoxic oxygen free radicals, promoting blood flow, anti-
platelet aggregation and radical scavenging action [1]. Fur-
thermore, our previous study has also demonstrated that
Ligustrazine could reduce renal dysfunction associated with
I/R of the kidney by attenuating lipid peroxidation (LPO),
apoptosis and ICAM-1 expression [2]. We also found out the
protective effects of Ligustrazine on early renal and pancreatic
injury on scalded rats. However, the protective effects of
Visceral damage is a key factor affecting survival in severe
burns, even after timely resuscitation. The pathogenesis of
such damage is not clear. Acute lung injury (ALI) is unavoid-
able in severely burned patients, and the application of
Ligustrazine on the burned as far as early to protect organ
function might be feasible and effective.
Human major histocompatibility complex (MHC) class I
chain-related antigen A, B (MICA, B) are polymorphic stress
regulated molecules deemed as ‘‘cell stress sensors,’’ which
are absent on normal adult tissue, but expressed on epithelial,
endothelial cells, and activated CD4+ and CD8+ T cells under
stringent state, showing histological evidence of cellular

* Corresponding author at: 301 Yanchang Road, Shanghai 200072, China. Tel.: +86 21 66307174; fax: +86 21 66307174.
E-mail address: chengjingao2003@yahoo.com.cn (C. Gao).
0305-4179/$36.00 # 2012 Elsevier Ltd and ISBI. All rights reserved.
doi:10.1016/j.burns.2011.12.026
744 burns 38 (2012) 743–750
injury [3,4]. Ectopic expression of MIC rendered MHC class I-
bearing targets susceptible to natural killer (NK) cell attack.
Reactive oxygen species (ROS) play a critical role in organ
injuries associated with severe burn injury [5]. ROS per se have
also been shown to compromise organ function and induce
apoptosis in epithelial cells. Antioxidants can attenuate
apoptosis and improve organ function. The involvement
of ROS in severe burn injury includes increased lipid
peroxidation (LPO). Levels of malondialdehyde (MDA) in
kidneys or pancreas may be determined as indicators of
LPO [1]. In this study, we designed experiments to investigate
the effects of Ligustrazine on scald-induced pulmonary injury
of severely scalded rats to elucidate the action mechanism
underlying its protection in pulmonary injury after severe
thermal trauma.
2. Materials and methods
2.1. Animals
Male Lewis rats (230  30 g, Grade II, Certificate no. 220010014)
were provided from Experimental Animals Center, Fudan
University, Shanghai, China. All rats were provided with
standard laboratory food stuff and water and housed in
accordance with institutional animal care policies. This
investigation conforms to the regulations stipulated by Fudan
University Animal Care Committee which follows the protocol
outlined in The Guide for the Care and Use of Laboratory
Animals published by the US National Institute of Health (NIH
publication no. 85-23, revised 1996).
2.2. Burn procedure
One day before receiving burns, a catheter was placed in rat
abdominal cavities for administration of fluids in each rat
under general anaesthesia (pentobarbital 40 mg/kg i.p.). Rats
dorsal surfaces were shaved closely and rats were secured in a
constructed template device. The surface area of the skin
exposed through the template device was immersed in 98 8C
water for 12 s on the dorsal surface. All were quickly dried after
each exposure to avoid additional injury. With the use of this
technique, full-thickness dermal burns comprising 30% TBSA
(total body surface area) were obtained [6].
2.3. Experimental design
All animals were randomly divided into two groups as follows
(n = 60each group): Scalded group (S group, control) and
Ligustrazine treated group (L group). All rats in these two
groups received a 30%TBSA full thickness scald injury and
were resuscitated with lactated Ringer’s solution by a
continuous infusion pump (Zhejiang Medical Apparatus and
Instruments Factory, Zhejiang, China) at a rate of 4 ml/kg/h.
Ligustrazine was administered intraperitoneally at a dose of
40 mg/kg/d in animals of L group. The harvesting of eight
lungs/speens was carried out at 24, 48 and 72 h after the scald
injury, respectively. Eight normal rats acted as controls. These
rats were executed by exsanguination from abdominal aorta
at each timepoint under anaesthesia.
2.4. Quantification of soluble endothelial-adhesion
molecules and interleukins
Plasma and lung homogenates were assayed to evaluate the
level of VCAM-1, E-selectin, P-selectin and IL-1b, IL-6 and IL-8
using Fluorokine1 MultiAnalyte Profiling kits and a Luminex1
Bioanalyzer (R&D Systems, Oxford, UK) according to the
manufacturers’ instructions.
2.5. Pulmonary function assessment by measurement of
lung wet to dry ratios and measurement of alveolar wall
thickness
At the time-points of 24 h, 48 h and 72 h post-scald injury, the
mice were exanguinated from the abdominal aorta. The two
lungs harvested from each animal were separated. The right
was deposited for the subsequent tests (immunohistochem-
istry staining, et al.). The left was homogenized, and the
homogenate was weighed. The homogenate was centrifuged
(14,000  g, 10 min), was dessicated in an oven (70 8C for 24 h)
for determination of dry weight. Lung wet to dry weight ratio
(W/D) was computed from lung wet and dry weights.
HE staining was performed on every slice section. Five
portions of every section were randomly selected and
analyzed by Spot Advanced Computer Photo Analysis Micro-
soft System (Silicon Graphic Inc, USA) to measure the
thickness of the alveolar wall. An average value and its
standard deviation were calculated.
2.6. Immunohistochemistry
Immunohistochemistry staining was performed on sections
using the avidin–biotin peroxidase complex method and
DAKO Liquid DAB+ Substrate-Chromogen System (DAKO Co,
Carpinteria U.S.A.), which led to the development of a brown
reaction product at sites of HLA-DR protein expression. The in
situ expression of HLA-DR protein in spleens, BCL-2 and MICA
in lungs were stained with primary monoclonal anti-mouse
HLA-DR, BCL-2 and MICA antibodies, and calculated by scoring
25 sequential fields using a semiquantitative scale. Each score
primarily reflects changes in the intensity of staining and
depends on the percentage of the area showing positive
staining: 0:0% to 5% stained; 1: >5% to 25%; 2: >25% to 50%; 3:
>50 to 75%; 4: >75%. Data was expressed as mean score
S.E.M. per field. The mean expressions were found by
measuring 8 different fields in each histopathological prepa-
ration prepared separately for each subject, and then by
calculating the means of these values for each group.
2.7. Determination of MDA levels and total superoxide
dismutase (SOD) activity
Trichloroacetic acid was added to pulmonary homogenates.
Then, tetrabromobisphenol A (TBA)-water solution was added
to the supernatant and boiled for 60 min. After cooling, the
optical density (OD) at 532 nm was measured. The total SOD
activity was determined by monitoring the rate of reduction of
nitroblue tetrazolium using a xanthine–xanthine oxidase
system as the source of O2. One enzyme unit of SOD was
defined as the amount that produces 50% inhibition of the
 burns 38 (2012) 743–750 745

Fig. 1 – HE staining of lung tissues. A significant increase in the thickeness of alveolar wall was observed after scald injury,
while lifustrazine alleviated the increase.

formation of formazan [1]. Data are expressed as mean values recruiting the leukocytes to the site of inflammation. Adhesion
S.E.M. (n = 8 per group). molecules play an important role in the leukocyte endothelial
interactions and resulting leukocyte migration into the site of
2.8. Statistical analysis injury or infection. ELISA assay was performed to analyze the
lung and serum levels of adhesion molecules such as E- and P-
All results are presented as mean value S.E.M. Statistical selectin, and VCAM-1. Table 1 represents E-selectin levels in
evaluation of the continuous data was performed using the serum. Animals with scald injury had a significantly
Bonferroni-corrected t-tests. The significance level was con- higher level of E-selectin in the serum at the time-points of
sidered to be P < 0.05. 24hcompared with the level in the L group.
Table 2 represents E-selectin levels in lungs. Animals of S
group had a significantly higher level of E-selectin in lungs at
3. Results the time-points of 48 h and 72 h compared with the level in the
L group.
3.1. The effect of Ligustrazine on the alveolar thickness Table 3 represents P-selectin levels in the serum. There is
no difference of serum P-selectin between the two groups.
Scald injury induced the thickening of alveolar wall (Fig. 1), and Table 4 represents P-selectin levels in lungs. Animals of S
at the timepoints of 24 h, 48 h, 72 h postinjection, the mean group had a significantly higher level of P-selectin in lungs at
alveolar wall thickness were 3.58  0.76 mm, 4.73  1.05 mm, and
12.68  2.57 mm respectively, while with Ligustrazine treat-
ment, the mean alveolar wall thickness were 1.12  0.18 mm,
1.47  0.28 mm, 1.41 0.24 mm at the same timepoints. The
mean alveolar wall thickness was 0.97  0.08 mm in normal
mices. A statistically significant difference was found between S
group and L group (P < 0.01), and significant difference between
S group and normal rats (P < 0.01). There was no difference of
alveolar wall thickness between Ligustrazine treated and the
normal animals.
Fig. 2 showed the W/D values, which were enhanced in the
scalded rats lungs, and were kept at low levels after
Ligustrazine administration.

3.2. Selectins and adhesion molecules in lungs and serum Fig. 2 – W/D values of lung homogenates. Differences are
shown between S treated group and L group. #P < 0.05
Selectins are a major class of adhesion molecules known to versus the same timepiont of L group. ##P < 0.05 versus the
play an important role in early inflammation stages in same timepiont of L group.
746 burns 38 (2012) 743–750

Table 1 – The expression of E-selectin in serum. Table 6 – The expression of VCAM-1 in lung tissues.
Time (h) S Group (pg/mL) L Group (pg/mL) Time (h) S Group (pg/mL) L Group (pg/mL)
0 25.62  2.58 25.62  2.58 0 8.54  1.24 8.54  1.24
24 41.67  5.05 37.83  5.27 24 23.32  4.36 15.47  3.06##
48 67.28  9.68 46.32  8.43# 48 19.47  5.07 17.83  2.97
72 43.59  6.85 45.13  7.65 72 26.42  4.38 14.84  2.71##
Differences are shown between the two groups. Values are means Differences are shown between the two groups. Values are means
for 5 rats. for 5 rats.
# ##
P < 0.05 versus the same timepiont of S Group. P < 0.01 versus the same timepoint of S Group.

Table 6 represents VCAM-1 levels in lungs. Animals of S

Table 2 – The expression of E-selectin in lung tissues. group had a significantly higher level of VCAM-1 levels in lungs
Time (h) S Group (pg/mL) L Group (pg/mL) at the time-points of 24 h and 72 h compared with the level in L
group.
0 123.24  22.35 123.24  22.35
24 183.75  39.35 169.57  31.25
48 241.37  41.64 173.46  28.66# 3.3. Interleukins in lungs
72 235.26  46.75 158.77  25.38##
Differences are shown between the two groups. Values are means Table 7 represents IL-1b levels in lungs. Animals of S group had
for 5 rats. a significantly higher level of IL-1b in lungs at the time-points
#
P < 0.05 versus the same timepoint of S Group. of 24 h and 48 h compared with the level in the L group.
##
P < 0.01 versus the same timepoint of S Group. Table 8 represents IL-6 levels in lungs. Animals of S group
had a significantly higher level of IL-6 in lungs at the time-points
of 24 h, 48 h and 72 h compared with the level in the L group.
Table 3 – The expression of P-selectin in serum. Table 9 represents IL-8 levels in lungs. Animals of S group
Time (h) S Group (pg/mL) L Group (pg/mL) had a significantly higher level of IL-8 in lungs at the time-

0 51.62  7.87 51.62  7.87

24 75.04  11.34 68.86  9.33
48 65.33  8.45 63.37  8.82 Table 7 – The expression of IL-1b in lung tissues.
72 63.92  7.82 59.73  7.71
Time (h) S Group (pg/mL) L Group (pg/mL)
There is no difference of serum P-selectin between the two groups.
0 13.46  2.72 13.46  2.72
24 57.38  8.77 21.63  4.32###
Table 4 – The expression of P-selectin in lung tissues. 48 68.73  11.54 32.33  6.28###
72 46.37  7.89 38.55  6.36
Time (h) S Group (pg/mL) L Group (pg/mL)
Differences are shown between the two groups. Values are means
0 81.27  10.51 81.27  10.51 for 5 rats.
24 96.82  8.39 93.71  10.33 ###
P < 0.001 versus the same timepoint of S Group.
48 132.47  16.12 87.46  12.25#
72 145.67  23.93 107.26  14.52#
Differences are shown between the two groups. Values are means Table 8 – The expression of IL-6 in lung tissues.
for 5 rats. Time (h) S Group (pg/mL) L Group (pg/mL)
#
P < 0.05 versus the same timepoint of S Group.
0 4.43  0.73 4.43  0.73
24 26.32  5.31 9.36  2.02###
48 21.38  4.45 12.37  2.68##
the time-point 48 h and 72 h compared with the level in the L 72 31.36  7.33 18.52  4.36##
group. Differences are shown between the two groups. Values are means
Table 5 represents VCAM-1 levels in serum. Animals of S for 5 rats.
##
group had a significantly higher level of VCAM-1 in the serum P < 0.01 versus the same timepoint of S Group.
###
at the time-point 72 h compared with the level in the L group. P < 0.001 versus the same timepoint of S Group.

Table 9 – The expression of IL-8 in lung tissues.

Table 5 – The expression of VCAM-1 in serum.
Time (h) S Group (pg/mL) L Group (pg/mL)
Time (h) S Group (pg/mL) L Group (pg/mL) 0 17.23  4.73 17.23  4.73
0 5.36  1.03 5.36  1.03 24 98.36  21.30 29.54  5.23###
24 6.92  0.97 6.48  1.14 48 87.55  18.32 36.38  6.76###
48 7.67  1.38 7.52  0.97 72 66.57  10.46 41.58  7.44##
72 12.56  2.12 7.53  1.26##
Differences are shown between the two groups. Values are means
Differences are shown between the two groups. Values are means for 5 rats.
##
for 5 rats. P < 0.01 versus the same timepoint of S Group.
## ###
P < 0.01 versus the same timepoint of S Group. P < 0.001 versus the same timepoint of S Group.
 burns 38 (2012) 743–750
Fig. 3 – MDA level of lung tissues. The values represent the
mean W SEM. Significance by #P < 0.05 with respect to L
group. Significance by ##P < 0.05 with respect to L group.
points of 24 h, 48 h and 72 h compared with the level in the L
group.
3.4. Effects of Ligustrazine on the pulmonary MDA level
and SOD activity
As shown in Fig. 3, from 24 to 72 h after injury, the pulmonary
MDA levels were significantly higher in the S group as
compared to the L group at the same time point (P < 0.01
and P < 0.05, respectively). The data in Fig. 4 demonstrate that
the pulmonary SOD levels in the S group when compared with
that of the L group were much lower, from 24 to 72 h at the
same time point (P < 0.05, respectively).
3.5. Expressions of MICA and BCL-2 in lungs
The data in Figs. 5 and 6 show the MICA scores of each group.
From 24 to 72 h post-scald, expression of MICA of lungs in the S
Fig. 5 – Immunohistochemistry detection of MICA in lung tissues (original magnification 400T).
747
Fig. 4 – SOD level of lung tissues. The values represent the
mean W SEM. Significance by #P < 0.05 with respect to L
group.
group was higher than that of rats in the L group at the same
time point. Conversely, data on the scores of BCL-2 are shown
in Figs. 7 and 8, demonstrating that expression of BCL-2 in the
S group was lower than that of the L group at the same time
point.
3.6. Expression of HLA-DR in spleen
Fig. 9 shows the histological examination of HLA-DR in spleens
by immunohistochemistry staining. At the timepoints of 24 h,
48 h, 72 h post scald injury, the mean scores were 2.98  0.57,
3.13  0.49, and 3.36  0.41 respectively, while in U group, the
mean scores were 0.93  0.17, 0.86  0.10, and 0.86  0.14 at
the same timepoints. The mean score was 0.45  0.07 of
normal spleens. A statistically significant difference was
found between S group and L group 24 h, 48 h and 72 h
postinjection (P < 0.01).
748 burns 38 (2012) 743–750
Fig. 6 – Expression of MICA on lung tissues. The values
represent the mean WSEM. Significance by ###P < 0.001
with respect to L group.
4. Discussion
Ligusztrazine is the main active compound isolated from
Chuanxiong, which has a variety of pharmacological function
with little toxicity and side effects, and consequently, it has
been widely used clinically.
In this study, some useful insights into the protective
effects of Ligustrazine against pulmonary injury associated
with severe scald injury were detected. MICA and MICB, the
human MHC class I-related products of polymorphic MHC
genes, are expressed early during embryonic development,
but are rare in healthy adult tissues [7]. It was also
demonstrated that MIC is absent in many normal tissues,
but is expressed in the epithelial cells under a severe stress,
such as that in ischaemia–reperfusion (I/R) injury, which show
histological evidence of cellular injury [8]. In this study, severe
scald injury produced a significant increase of MICA expres-
sion on pulmonary epithelial cells, while Ligustrazine dimin-
Fig. 7 – Immunohistochemistry detection of BCL-2 in lung tissues (original magnification 400T).
Fig. 8 – Expression of BCL-2 on lung tissues. The values
represent the mean WSEM. Significance by ###P < 0.001
with respect to L group.
ished the increase of the pulmonary expression of MICA,
which showed a protective effect against autoimmune injury.
There has been increasing evidence that free radicals
generated by stress contribute to organ functional injury [9].
Free radicals attack a variety of critical biological molecules,
including membrane lipids, essential cellular proteins and
DNA [10,11]. Free radicals can also be generated after scalding,
and free radicals including superoxide and hydroxyl radicals
are involved in the damage induced by scald injury [12].
Tissues with high levels of polyunsaturated fatty acids that
constitute the cell membrane and mitochondrial membrane
were particularly sensitive to oxygen radical-mediated injury
due to their low concentrations of oxygen radical scavenging
enzymes [13]. The degree of injury initiated by LPO, which is
initiated by free radicals, could be measured in terms of MDA
levels. In the present study, scald injury enhanced the MDA
levels, indicating elevation of free radicals and LPO in lungs.
Ligustrazine reversed the increase in MDA levels to a
 burns 38 (2012) 743–750
Fig. 9 – Immunohistochemistry detection of HLA-DR in spleen tissues. Ligustrazine could suppress the expression of HLA-
DR protein of the lung tissues (original magnification 400T).
considerable extent, thereby confirming its antioxidant role.
The SOD is the first line of defence against free-radical
generation and is generally referred to as one of the primary
antioxidants; furthermore, we showed that SOD levels
increased following Ligustrazine treatment. The elevated
SOD levels induced by Ligustrazine may contribute towards
reducing free radicals following severe scald injury. Elevated
SOD in the lungs confirmed the protective effects of
Ligustrazine, and elevated bcl-2 and decreased MICA in lungs
also supported the above-mentioned conclusion.
Another principal finding of this work is that Ligustrazine
increased bcl-2 expression in lungs of scalded rats. The results
showed that the bcl-2 level was degraded after scald. After
treatment with Ligustrazine, the expression of bcl-2 protein
was enhanced, suggesting that Ligustrazine exhibits an
inhibitory effect on organ dysfunction through modulation
of bcl-2. The major mechanism can also probably be attributed
to the interference of Ligustrazine with free-radical release.
Free radicals induce apoptosis by causing DNA damage,
oxidation of lipid membranes and activation of the proteins
responsible for apoptosis. Among these apoptosis-regulatory
proteins, the bcl-2 acts as a cell-death preventer, which can
prevent the apoptosis induced by free radicals and LPO [14].
Bcl-2 has the antioxidative characteristics in cells through its
participation in the reduction and inhibition of the formation
of active oxygen.
The spleen is the largest peripheral immune organ and it
plays an important role in both innate and adaptive immune
responses. DCs are located in marginal zones of the spleen
white pulp. HLA-DR is a known indicator of NK cell activation
in vitro. HLA-DR positive NK cells have been noted in several
pathologic states and their presence in tissue during infection
and their association with autoimmunity imply that they may
749
have physiologic importance in vivo [15]. Our IHC results
showed higher level of HLA-DR was detected in spleens from
scalded rats, indicating a more mature state of HLA-DR+ NK
cells. A hallmark of NK cells function is their ability to enlarge
the innate immune reaction. As a cell-stress sensor, MICA can
initiate the innate immunity of the cell by engagement with
NKG2D, which is expressed by all NK cells and other
immunocytes [7]. Higher expression of MICA found in lung
tissues triggered the innate-immunity injury to lungs can
accelerate apoptosis or necrosis of pulmonary epithelial cells.
Ligustrazine downregulated the expression of spleen HLA-DR,
which even exhibited an immunosuppressive function and a
pulmonary protective effects.
Innate immunity is the first line of defence against
microbial invasion. Interleukins, selectins and VCAM-1 medi-
ate neutrophil interactions, neutrophil migration, and the
infiltration of neutrophils into the inflammatory areas,
enlarging the regional and even systemic injuries [16].
Expression of endothelial adhesion molecules such as E-
selectin, P-selectin and VCAM-1, are a consistent feature of
sepsis. Higher levels serumal E- and P-selectin, and pulmonary
IL-1b, IL-6, IL-8 and VCAM-1 in scalded rats were found,
showing a more severe pulmonary injury. The productions of
these cytokines were decreased by the Ligustrazine treatment.
The thickness of alveolar wall and W/D values of lung tissues
could well reflect the pulmonary microvascular permeability
and the pulmonary injury degree associated with scald injury,
while Ligustrazine could effectively protect the pulmonary
tissues from scalded injury.
In summary, the experimental data showed that Ligus-
trazine reduced scald-induced pulmonary injury. The mecha-
nism of its action might involve the immunoloregulation and
the mitigation of excessive inflammatory reaction.
750 burns 38 (2012) 743–750
Funding
This study was funded by National Natural Science Founda-
tion of China (no. 81000024) and Shanghai Science and
Technology Committee Foundation (no. 11QA1405100).
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