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Available online at www.sciencedirect.com

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journal homepage: www.JournalofSurgicalResearch.com

Beneficial effect of cyclosporine A on traumatic

hemorrhagic shock
Yan Lei, MS,a,b Xiaoyong Peng, MS,b Liangming Liu, MD, PhD,b,*,1
Zhaojun Dong, MD,c and Tao Li, MDb,*,1
a

Department of General Medicine, Medical Service Training Base, Third Military Medical University,
Chongqing, China
b
State Key Laboratory of Trauma, Burns and Combined Injury, Second Department of Research Institute of Surgery,
Daping Hospital, Third Military Medical University, Chongqing, China
c
Department of Chemical Defense, Institute of Toxicology, Third Military Medical University, Chongqing, China

article info

abstract

Article history:

Background: Vascular hyporeactivity plays an important role in severe trauma and shock.

Received 27 October 2014

We investigated the beneficial effect of cyclosporine A (CsA) on traumatic shock and its

Received in revised form

relationship to vascular reactivity improvement and mitochondrial permeability transition

29 January 2015

pore (MPTP).

Accepted 4 February 2015

Materials and methods: Sodium pentobarbital-anesthetized rats were used to induce trau-

Available online 12 February 2015

matic hemorrhagic shock by left femur fracture and hemorrhage, the beneficial effects of
CsA (1, 5, and 10 mg/kg, intravenously) on animal survival, cardiovascular function, tissue

Keywords:

blood perfusion, and mitochondrial function of vital organs were observed. In addition,

Mitochondrial permeability

hypoxia-treated vascular smooth muscle cells from normal rats were used to investigate

transition pore

the relationship of this beneficial effect of CsA to Rho-associated serine/threonine kinase

Cyclosporine A

(ROCK) and protein kinase C.

Vascular hyporeactivity

Results: CsA prolonged the survival time and increased the 24-h survival rate of traumatic

Traumatic hemorrhagic shock

hemorrhagic shock (31%, 56%, and 56% in 1, 5, and 10 mg/kg CsA group versus 25% in

Rho-associated

lactated Ringer solution group). Five milligrams per kilogram of CsA had the best effect,

serine/threonine kinase

which stabilized and improved the hemodynamics, increased the tissue blood flow, and
improved the liver and kidney function including its mitochondrial function in shock rats.
CsA had no significant influences on the production of inflammatory mediators and cardiac output after traumatic hemorrhagic shock. Further results indicated that CsA significantly improved the vascular constriction and dilation reactivity of superior mesenteric
artery to norepinephrine and acetylcholine, which was antagonized by ROCK inhibitor,
Y27632, but not by protein kinase C inhibitor, staurosporine. Further studies showed that
CsA restored hypoxia-induced decrease of ROCK activity and inhibited the opening of
MPTP in hypoxia-treated vascular smooth muscle cells.
Conclusions: CsA is beneficial for the treatment of traumatic hemorrhagic shock. The
mechanism is mainly through improving the vascular reactivity, stabilizing the

* Corresponding authors. State Key Laboratory of Trauma, Burns and Combined Injury, Second Department of Research Institute of
Surgery, Daping Hospital, Third Military Medical University, Daping, Chongqing 400042, China. Tel.: 86 23 68757421; fax: 86
23 68813806.
E-mail addresses: liangmingliu@yahoo.com (L. Liu), lt200132@163.com (T. Li).
1
Equal contribution to this work.
0022-4804/$ e see front matter 2015 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jss.2015.02.005

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hemodynamics, and increasing tissue perfusion. This beneficial effect of CsA is related to
the inhibitory effect of CsA on MPTP opening. ROCK is an important regulator molecule in
this process.
2015 Elsevier Inc. All rights reserved.

1.

Introduction

Traumatic injury is a leading cause of death and disability

globally. There are approximately 5.8 million people who
die each year in the world from traumatic injuries [1].
About 50% death during the early stage after trauma accounts for hemorrhage or hemorrhagic shock [2,3]. Therefore, developing the optimal therapeutic strategies is very
important.
Vascular hyporesponsiveness plays an important role in
the incidence and development of shock. Our previous
studies and other teams have demonstrated that improving
the vascular hyporesponsiveness is beneficial to severe
trauma and shock [4e6]. However, the mechanisms for the
occurrence of vascular hyporesponsiveness are not
completely understood, and current measures based on the
present mechanisms can only restore the vascular responsiveness partly.
It has been well documented that the mitochondrial
permeability transition pore (MPTP) has a crucial role in
ischemia and reperfusion injury of the heart and brain [7,8].
Under pathologic conditions, MPTP will open and lead to cell
dysfunction or death [9,10]. Cyclosporine A (CsA), an inhibitor of MPTP opening, has been shown to fight against
myocardial and neuronal ischemiaereperfusion injury,
vascular hyperpermeability, and shock-induced hypotension [11e14]. Many studies showed that the opening of MPTP
resulted in the release of cytochrome c and subsequent
caspases activation and induced cell apoptosis [10,13].
Although MPTP-mediated apoptotic signaling might not
participate in the regulation of vascular hyporesponsiveness
after hemorrhagic shock, some studies indicated that
Rho-associated serine/threonine kinase (ROCK) and protein
kinase C (PKC) may participate in MPTP-associated cardioprotection and neuroprotection against ischemia and
reperfusion injury [15,16]. Our previous studies demonstrated that ROCK and PKC played a critical role in the
regulation of vascular reactivity after shock [4,17].
Therefore, we hypothesized that CsA may have beneficial effect on traumatic shock by improving shock-induced
vascular hyporeactivity and restoring the hemodynamics
and tissue perfusion. This effect may be related to its
MPTP inhibitory effect. To elucidate this hypothesis, with
traumatic hemorrhagic shock rats and hypoxia-treated
vascular smooth muscle cells (VSMCs), we investigated:
(1) the beneficial effects of CsA on animal survival, blood
gases, and organ function in traumatic hemorrhagic shock;
(2) if CsA elicits the antishock effects via restoring the
vascular hyporeactivity and subsequently improving the
hemodynamics and the tissue perfusion; (3) the relationship of CsA-regulating vascular reactivity to ROCK or PKC
signaling.

2.

Materials and methods

This study was approved by the Research Council and Animal

Care and Use Committee of Research Institute of Surgery,
Daping Hospital, Third Military Medical University. The
investigation conformed to the Guide for the Care and Use of
Laboratory Animals (Eighth Edition, 2011, Washington, D.C., National Academies Press, USA), and all efforts were made to
minimize the animal suffering.

2.1.

Animal preparation

SpragueeDawley (SD) rats (180e220 g), both male and female,

were used in the present study and housed in controlled
temperature and light conditions. On the day of the experiment, rats were anesthetized with sodium pentobarbital
(initial dosage was 30 mg/kg, intraperitoneally) and jingsongling (xylidinothiazole, initial dosage was 0.1 mg/kg, intramuscularly; then the two drugs were added until the rats had
no response to a needle stimulus). During the experiment
period, anesthesia was maintained by an intermittent injection of sodium pentobarbital/xylidinothiazole, and the body
temperature of the rats was maintained at 37 C with a heating
pad.
The right femoral artery and vein were catheterized for
monitoring the mean arterial pressure (MAP) and/or bleeding
and drug administration, respectively. The right carotid artery
and vein were catheterized for monitoring the hemodynamics
or cardiac output. After completion of the surgical procedure,
rats were allowed to stabilize for 10 min. A model of traumatic
hemorrhagic shock was induced by femur fracture and arterial bleeding as previously described by our research team [18].
Briefly, the left midshaft femur was fractured using a hemostatic clamp. Simultaneously, the rats were hemorrhaged via
the right femoral artery catheter, and the MAP was maintained at 40 mm Hg for 3 h. At the end of each experiment, all
animals were euthanatized with a pentobarbital-based
euthanasia solution (Sleepaway, 2 mL, intravenously; Fort
Dodge Laboratories, Fort Dodge, IA) through the femoral vein
catheter.

2.2.

VSMCs hypoxia

VSMCs were obtained from mesenteric arteries of normal rats

by an explant technique [17]. Briefly, the mesenteric arteries
from normal rats were cut into small pieces and placed in a
culture flask for culture for 5e7 d in Dulbecco modified Eagle
medium-F12 supplemented with 20% fetal bovine serum and
1% penicillin/streptomycin. After VSMCs outgrowth, the tissue pieces were removed and the thirdeto-sixth passage cells
were used. For hypoxia treatment, VSMCs were starved for

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24 h in serum-free medium and transferred into a hypoxia

culture compartment, which was continuously bubbled with
95% N2 and 5% CO2 at 10 L/min for 15 min, and then equilibrated for 10 min. This procedure was repeated five times
until the O2 concentration in the chamber was less than 0.2%
[19]. Under this hypoxic condition, VSMCs were maintained
for 3 h and then used for subsequent experiments.

2.3.

Experimental protocol

2.3.1.

Experimental phases and management

The in vivo experiments were defined as three phases. Phase I

was the traumatic hemorrhagic shock period (shock model
stage) in which femur was fractured, and the MAP was
maintained at 40 mm Hg for 3 h. Phase II was the treatment
period (30e40 min) in which rats received lactated Ringer (LR)
solution and CsA 1, 5, and 10 mg/kg administration. The
amount of LR was two volumes of blood loss. All fluids were
administered with an infusion pump (Model AS 50; B.Braun
Melsungen AG, Melsungen, Germany). Phase III was the
observation period (120 min) in which the effects of CsA were
observed. Anesthesia and analgesia were maintained
throughout the experiments as described previously (Fig. 1).

2.3.2. Part I: beneficial effects of CsA on traumatic

hemorrhagic shock in rats
The first part was aimed to investigate the effects of CsA on
animal survival, blood gases, vital organs function, and cytokines production after traumatic hemorrhagic shock in rats.

2.3.2.1. Animal survival time and 24-h survival rate. Ninetysix SD rats were randomly divided into six groups (n 16 per
group) as follows: normal control (sham-operated), shock
control, shock LR, and shock LR CsA 1, 5, or 10 mg/kg. At
the end of shock, rats in CsA groups received a continuous
infusion of CsA (1, 5, and 10 mg/kg, respectively) with two
volumes of LR. The LR group was just infused with two volumes of LR. The shock control group did not receive any

531

treatment. The sham-operated group just experienced the

same operation but without hemorrhage and fluid infusion.
After the treatment period, all catheters were removed and
the incisions were closed. Anesthesia was discontinued to
allow the animals to free access to food and water, whereas
the analgesics were applied every 6 h (xylidinothiazole,
0.2 mg/kg, intramuscularly). The survival time and 24-h survival rate were observed.

2.3.2.2. Blood gases, liver and/or kidney function, and their

mitochondrial function. Eighty SD rats were randomly divided
into ten groups (n 8 per group) as follows: sham-operated,
shock control, LR and CsA 1, 5, and 10 mg/kg. LR and CsA 1,
5, and 10 mg/kg groups were further divided into two time
points as follows: 1 h and 2 h after resuscitation. In this
experiment, the blood gases and the function of the liver and
kidney were observed by withdrawing blood at baseline, at the
end of phase I, and at 1 h and 2 h of phase III. After these
measurements, rats were then sacrificed to obtain liver, kidney, and small intestine for the measurement of their mitochondrial function.
Blood gases (including blood pH, lactate, bicarbonate
[HCO
3 ], base excess [BE], partial pressure of oxygen and carbon dioxide in arterial blood [PaO2, PaCO2], and oxygen saturation [SaO2]) were determined with a blood gas analyzer
(Phox plus L; Nova Biomedical, Waltham, MA). The variables
of liver and kidney function (including aspartate aminotransferase [AST], alanine aminotransferase [ALT], blood urea
nitrogen [BUN], and serum creatinine [CREA]) were measured
using a biochemical analyzer (Beckman, Fullerton, CA).
Mitochondrial function of the liver, kidney, and intestine
was measured by a mitochondrial function analyzer (MT 200;
Strathkelvin, Lanarkshire, Scotland) as described in our previous work [20]. Briefly, the mitochondria of the liver, kidney,
or intestine was extracted and resuspended with isolation
buffer. The concentration of mitochondrial protein was
measured by the Lowry method. Measurement buffer of
1.4 mL (Tris 0.2 mol/L, pH 7.6, KCl 15 mmol/L, KH2PO4

Fig. 1 e Timeline of the experimental phases. Phase I: The traumatic hemorrhagic shock (THS) model was induced by femur
fracture and bleeding to MAP maintained at 40 mmHg for 3 h. Phase II: Two volumes of blood loss of LR or CsA (1, 5, and
10 mg/kg) were administered after shock. Phase III: Observed the effects of these treatments. (Color version of figure is
available online.)

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15 mmol/L, Na2EDTA 1 mmol/L, MgCl2 5 mmol/L, and sucrose

0.25 mol/L) warmed to 30 C was added into the reaction
chamber and equilibrated for 2 min. Then, 0.2 mL of 3 mg/mL
of a mitochondrial mixture was put into the reaction chamber
and equilibrated for 20 s. Ten microliters of 0.5 mol/L sodium
malate and sodium glutamate and 5 mL of adenosine diphosphate (400 nmol/L) were added in order. The oxygen consumption rate was determined by a mitochondrial function
analyzer (MT 200; Strathkelvin). Mitochondrial function was
reflected by the respiration control rate (consumed oxygen
rate with and without adenosine diphosphate).

by NE to the 124-mmol/L K-induced reference contraction,

and the vasodilator reactivity was expressed as the percentage of the decreased tension induced by Ach to the increased
tension induced by NE.
Another forty-eight SD rats were randomly divided into six
groups (n 8 per group) as follows: sham-operated, shock
control, shock LR, shock LR CsA (1, 5, and 10 mg/kg)
groups. At baseline, at the end of phase I, and at 1 h and 2 h in
phase III, the cardiac output was assessed using a CardiomaxIII Thermo dilution Cardiac Output system (Columbus Instruments, Columbus, OH) [20].

2.3.2.3. Inflammatory mediators. Thirty-two SD rats were

randomly divided into four groups (n 8 per group) as follows:
sham-operated, shock, LR, CsA 5 mg/kg groups. At 2 h after
resuscitation, 2-mL blood were collected, and the concentration of tumor necrosis factor-a (TNF-a), interleukin-1b (IL), IL6, total nitric oxide synthase (tNOS), and inducible nitric oxide
synthase (iNOS) were detected with commercial assay kits
(NeoBioscience Technology Co, Shenzhen, China; Nanjing
Jiancheng Bioengineering Institute, Nanjing, China) according
to the manufacturers instructions.

2.3.4. Part III: relationship of CsA-regulating vascular

reactivity to ROCK, PKC, and the opening of MPTP

2.3.3. Part II: effects of CsA on vascular reactivity,

hemodynamics, cardiac output, and the tissue blood perfusion
of vital organs after traumatic hemorrhagic shock in rats
Eighty SD rats were also randomly divided into ten groups and
treated identically as described in section 2.3.2. At each time
point (baseline, at the end of phase I, and at 1 h and 2 h in
phase III), the MAP and hemodynamic parameters (including
left intraventricular systolic pressure [LVSP], maximal change
rate in left intraventricular pressure [dp/dtmax] and heart
rate [HR]) were determined with a polygraph physiological
recorder (SP844; AD Instruments, Castle Hill, Australia).
Thereafter, rats underwent a laparotomy, and the blood flow
in the liver and kidney were measured by a laser Doppler
blood flowmeter (Feriflux system 5000; Perimed, Stockholm,
Sweden), and the superior mesenteric arteries (SMAs) were
isolated for the measurement of vascular reactivity.
The vasoconstriction to norepinephrine (NE) and vasorelaxation to acetylcholine (Ach) were observed using an isolated organ perfusion system (Scientific Instruments,
Barcelona, Spain) [21]. Briefly, each SMA was cut into a 2e3mm long ring, which was suspended between a force transducer and a post attached to a micrometer, then immersed
into a 10-mL isolated organ chamber (Scientific Instruments)
containing KrebseHenseleit solution (K-H solution). After 2-h
equilibration, 124 mmol/L K K-H solution was added into the
organ chamber to elicit a reference contraction. Then the SMA
rings were washed twice with K-H solution and were allowed
a 30-min recovery period. Thereafter, we determined contractile responses by cumulative administration of NE (final
concentration: 109, 108, 107, 106, and 105 mol/L). When
the constriction of SMA rings to NE (105 mol/L) reached to
stable plateau, the relaxation reactivity of SMA to Ach (final
concentration: 109, 108, 107, 106, and 105 mol/L) was
tested. The tension of the artery rings was recorded by a
PowerLab System via a force transducer (AD Instruments).
The vascular constriction reactivity was expressed as a percentage of the increased vascular contractile tension induced

To further explore the molecular mechanisms for the protection of CsA on traumatic shock, we observed the relationship of CsA-regulating vascular reactivity to ROCK and PKC
and the effects of CsA on protein expression and phosphorylation of myosin phosphatase target subunit (MYPT1, the
target of ROCK, which was used to reflect the activity of ROCK)
and the opening of MPTP in VSMCs.

2.3.4.1. For vascular reactivity. Forty SD rats were randomly

divided into five groups (n 8) as follows: CsA (5 mg/kg) group,
CsA U46619 (ROCK agonist) group, CsA Y27632 (ROCK inhibitor) group, CsA PMA (phorbol-12-myristate-13-acetate,
PKC agonist) group, and CsA staurosporine (PKC inhibitor)
group. (These control groups including normal control group,
shock control group, and LR group were same as Part II). After
the model of traumatic hemorrhagic shock was established,
rats in CsA groups were treated with 5 mg/kg of CsA, rats in
CsA U46619 group, CsA Y27632 group, CsA PMA group,
and CsA staurosporine group were treated with U46619
(25 mg/kg), Y27632 (2 mg/kg), PMA (1 mg/kg), or staurosporine
(1 mg/kg), respectively, 5 min before CsA (5 mg/kg) treatment.
At 2 h after resuscitation, SMAs were isolated, vascular contractile reactivity to NE and relaxation reactivity to Ach were
determined as described previously.
2.3.4.2. For the expression and phosphorylation of MYPT1.
VSMCs were divided into control group, hypoxia 3-h group,
and hypoxia 3-h CsA group. After 3-h hypoxia, VSMCs in
hypoxia CsA group were incubated with CsA (1 mmol/L) for
2 h, then the cells were collected, the protein was extracted,
and the expression of MYPT1 and ROCK activity were performed as previously described [18]. The expression of MYPT1
was normalized to b-actin to correct for loading differences.
The activity of ROCK was reflected by the phosphorylation of
MYPT1. Antibodies were as follows: MYPT1 and phosphoMYPT1 (1:1000; Upstate Biotechnology, Lake Placid, NY) and
b-actin (1:5000; SigmaeAldrich, St. Louis, MO).

2.3.4.3. For MPTP opening. VSMCs were put on the confocal

dish (cover glass-bottom dish). They were also divided into
three groups as follows: control group, hypoxia 3-h group, and
hypoxia 3-h CsA group. After treatment with hypoxia and/or
CsA (1 mmol/L) as described previously, the opening of VSMC
MPTP was determined by the calcein-CoCl2 method using
laser scanning confocal microscopy [22]. Briefly, the treated
cells were incubated with 2 mmol/L calcein-acetoxymethyl

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ester and 100 nmol/L MitoTracker Deep Red (Sigma-Aldrich,

St. Louis, MO) for 30 min (protected from light). Cells were
subsequently washed twice with phosphate-buffered saline
and then exposed to 2 mmol/L CoCl2 for 15 min. After three
times of washing, the cells were excited at 488 nm (calcein) or
633 nm (MitoTracker) using a Leica TCS SP5 confocal system
(Leica Microsystems, Wetzlar, Germany). Images were
collected, and the mean intensity of calcein uorescence was
calculated using the Leica TCS software.

2.4.

Statistical analysis

Data are presented as means  standard errors of the mean.

Differences between experimental groups were analyzed by
repeated measures analysis of variance, followed by post hoc
Tukey tests. The analysis was performed using SPSS 13.0 software (SPSS Inc, Chicago, IL). P < 0.05 was considered significant.

533

with the baseline. LR infusion slightly decreased the blood

lactic acid. CsA (1, 5, and 10 mg/kg) significantly decreased the
blood level of lactic acid. The lactic acid level in 5 mg/kg of CsA
group was significantly lower than that in the LR group at 1 h
and 2 h after resuscitation (P < 0.01), whereas the lactic acid
level in 1 and 10 mg/kg CsA groups was lower than that in the
LR group only at 1 h after resuscitation (P < 0.05; Table 1).

3.1.3.

HCO
3 and BE

Both HCO
3 and BE at the end of shock were significantly
reduced as compared with baseline. LR infusion only slightly
increased blood HCO
3 and BE. Five milligrams per killogram of
CsA significantly increased the level of HCO
3 and BE, which
were markedly higher than those in the LR group (P < 0.01;
Table 1).

3.1.4.

Blood pH, PaO2, PaCO2, and SaO2

3.1.
Part I: beneficial effects of CsA on traumatic
hemorrhagic shock

The blood pH value significantly decreased after shock; LR and

three doses of CsA infusion all restored the pH to a normal
level. LR and CsA also improved the blood PaCO2, but there
were no significant differences among LR and three CsA dosage
groups. PaO2 and SaO2 did not show significant changes in all
groups during the entire experiment period (data not shown).

3.1.1.

3.1.5.

3.

Results

Animal survival

All rats in the sham-operated group survived over 24 h. All rats

in the shock control group (without any treatment at phase II)
died within 8 h after traumatic hemorrhagic shock, and the
mean survival time of rats in this group was about 3 h. LR
infusion slightly increased the survival time and 24-h survival
rate. CsA (5 and 10 mg/kg) infusion significantly prolonged the
survival time and increased the survival rate of shock rats as
compared with the LR alone group. The 24-h survival rates in
sham-operated, shock control, LR, and CsA 1, 5, and 10 mg/kg
groups were 16 of 16 (100%), 0 of 16 (0), 4 of 16 (25%), 5 of 16 (31%),
9 of 16 (56%), and 9 of 16 (56%), respectively (Fig. 2A and B).

3.1.2.

Lactic acid

At the end of the shock period, the blood lactic acid was
significantly increased, it reached to 8.7e9.3 mmol/L at the
end of the shock and increased about seven folds as compared

Liver and kidney function

The liver and kidney function parameters including AST, ALT,

BUN, and CREA were significantly increased after shock. LR or
CsA had no significant influences on blood AST and BUN (data
not shown). CsA (1 and 5 mg/kg) significantly decreased the
level of ALT and CREA as compared with the LR group (P < 0.05
or 0.01; Fig. 3A and B).

3.1.6.

Mitochondrial function

The respiratory control rate of mitochondria in the liver, kidney, and intestine was significantly reduced after shock. CsA
(5 mg/kg) infusion could increase the respiratory control rate
of mitochondria in these organs as compared with the LR
infusion (P < 0.01). The respiratory control rate of mitochondria in these organs in the CsA (5 mg/kg) group was recovered
to 92.8%, 96.1%, and 90.8% of sham-operated group, respectively, which were all close to the normal level (Fig. 3CeE).

Fig. 2 e Effects of CsA on the 24-h survival rate (A) and survival time (B) after traumatic hemorrhagic shock in rats. (n [ 16
per group). **P < 0.01 as compared with the sham-operated group; #P < 0.05 as compared with the LR group. (Color version
of figure is available online.)

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Table 1 e Effects of CsA on blood gases after traumatic hemorrhagic shock.

Group

Baseline

End of shock

After administration
1h

Lactic acid (mmol/L)

Sham-operated
Shock control
LR
CsA 1 mg/kg
CsA 5 mg/kg
CsA 10 mg/kg
HCO
3 (mmol/L)
Sham-operated
Shock control
LR
CsA 1 mg/kg
CsA 5 mg/kg
CsA 10 mg/kg
BE (mmol/L)
Sham-operated
Shock control
LR
CsA 1 mg/kg
CsA 5 mg/kg
CsA 10 mg/kg

2h

1.3 
1.4 
1.6 
1.2 
1.4 
1.0 

0.1
0.1
0.2
0.1
0.1
0.1

1.8
8.7
8.7
8.6
9.3
8.9

 0.2
 0.3**
 0.6
 0.2
 0.4
 0.5

2.3 
8.5 
8.4 
6.9 
6.0 
6.7 

0.2
0.4**
0.4
0.2#
0.4##
0.3#

2.1 
8.2 
7.0 
6.0 
4.5 
5.8 

0.2
0.4**
0.3
0.5
0.3##
0.4

25.8 
23.6 
23.2 
22.4 
25.2 
24.8 

0.7
1.0
0.7
0.8
0.9
0.8

25.6
13.2
14.5
12.2
12.6
10.3

 1.3
 0.8**
 0.4
 0.6
 0.8
 0.9

23.7 
11.8 
14.3 
15.3 
19.6 
14.1 

0.9
1.0**
0.7
0.8
1.1##
0.8

24.6 
12.1 
15.5 
17.8 
19.4 
15.8 

1.0
1.0**
0.5
1.0
1.2##
0.5

1.6 
0.5 
1.1 
2.3 
0.6 
0.1 

0.5
0.8
0.7
0.6
1.0
0.6

0.3
13.6
13.9
14.6
13.8
16.7

 0.8
 0.8**
 0.8
 0.7
 0.9
 1.1

1.0 
14.6 
12.3 
10.4 
8.4 
12.1 

1.9 
14.1 
11.4 
7.2 
6.3 
9.5 

0.6
1.3**
1.0
1.0
1.1##
0.9

0.7
1.3**
0.9
1.0
1.3
1.1

Data are presented as the mean  standard errors of the mean (n 8 per group).
**P < 0.01 as compared with the sham-operated group; #P < 0.05, ##P < 0.01 as compared with the LR group.

3.1.7.

Blood levels of TNF-a, IL-1b, IL-6, tNOS, and iNOS

The blood levels of TNF-a and IL-1b were significantly

increased after traumatic hemorrhagic shock. LR infusion
further increased the serum levels of TNF-a and IL-1b. CsA
(5 mg/kg) infusion only slightly decreased the TNF-a and IL-1b
levels, but there were no significant differences between the
CsA group and LR group. The levels of IL-6, tNOS, and iNOS did
not show significant changes after shock and treatment in all
groups (Table 2).

3.2.
Part II: effects of CsA on vascular reactivity,
hemodynamics, and tissue perfusion
3.2.1.

Vascular contractile reactivity

As compared with that of the sham-operated group, the

vascular constriction reactivity of SMAs to NE was significantly reduced after shock; the maximal contraction
decreased to 59.3% of the normal level. LR only slightly
increased the constriction reactivity. CsA markedly restored

Fig. 3 e Effects of CsA on liver and kidney function and their mitochondrial function after traumatic hemorrhagic shock
(showed 2-h data after resuscitation). Data represent the mean standard errors of the mean (n [ 8 per group). (A) ALT; (B)
serum CREA; (C), (D); and (E) mitochondrial function (respiratory control rate, RCR) of the liver, kidney, and small intestine.
**P < 0.01 as compared with the sham-operated group; #P < 0.05, ##P < 0.01 as compared with the LR group.

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Table 2 e Effects of CsA on inflammatory mediators after traumatic hemorrhagic shock.

Group

TNF-a (pg/mL)

Sham-operated
Shock control
LR
CsA 5 mg/kg

10.8 
61.1 
74.2 
69.9 

0.9
1.8**
5.3
2.8

IL-1b (pg/mL)
53.5
97.6
137.3
130.6






IL-6 (pg/mL)
22.2 
20.1 
21.9 
19.8 

3.4
3.6**
5.6
5.0

1.9
1.5
1.2
0.7

tNOS (U/mL)
28.3
25.7
27.2
25.4






1.0
1.6
1.3
1.4

iNOS (U/mL)
10.4 
8.5 
9.7 
9.0 

0.6
0.5
0.6
0.5

Data are presented as the mean  standard errors of the mean (n 8/group).
**P < 0.01 as compared with the sham-operated group; #P < 0.05, ##P < 0.01 as compared with the LR group.

the decreased constriction reactivity of SMAs to NE, which

restored to 80.5%, 93.4%, and 86.1% of the normal level in CsA
1, 5, and 10 mg/kg groups, respectively (Fig. 4A and B).

3.2.2.

Vascular relaxation reactivity

CsA also significantly restored the decreased vasodilator

reactivity of SMAs to Ach after shock. The maximal relaxation
reactivity of SMA rings to Ach was 92.8%, 68.9%, 71.6%, 77.6%,
84.6%, and 80.0% in the sham-operated group, shock control
group, LR group, and CsA 1, 5, and 10 mg/kg groups, respectively (Fig. 4C and D).

3.2.3.

Hemodynamics

All hemodynamic parameters including MAP, LVSP, dp/

dtmax, and HR were significantly decreased after traumatic
shock. Two volumes of LR infusion only slightly increased
these parameters. Five and 10 mg/kg of CsA significantly
increased the MAP, LVSP, and dp/dtmax, which were higher

than those in the LR alone group at 2 h after resuscitation

(P < 0.05). One milligram per kilogram of CsA only slightly
increased the MAP, LVSP, and dp/dtmax, and there were no
significant differences between the CsA 1 mg/kg group and LR
group (Fig. 5AeD). CsA infusion also restored the decreased HR
of shock animals, but there were no significant differences
between CsA groups and LR group (HR data not shown).

3.2.4.

Blood flow

At the end of the shock period, the blood flow in the liver and
kidney was significantly reduced, which was decreased to
57.9% and 22.3% as compared with the baseline level, respectively (P < 0.01). LR slightly increased the blood flow of the liver
and kidney, whereas CsA (5 and 10 mg/kg) significantly
increased them (P < 0.05 or 0.01). At 2 h after resuscitation, the
recovery rate of blood flow in the liver was 72.2%, 69.7%, and in
kidney was 74.4%, 63.5% as compared with the baseline level
in CsA 5 and 10 mg/kg groups, respectively (Fig. 5E and F).

Fig. 4 e Effects of CsA on the vascular contractile and relaxation reactivity of SMAs after traumatic hemorrhagic shock (2-h
data after resuscitation). Data represent the mean standard errors of the mean (n [ 8 per group). (A) dose-response curves
of SMA to NE; (B) the maximal contractile response of SMA to NE; (C) dose-response curves of SMA to Ach; and (D) the
maximal relaxation response of SMA to Ach. The vascular constriction response was reflected by a percentage of the
increased vascular contractile tension induced by NE to the 124-mM KD-induced reference contraction, and the relaxation
response was expressed by the percent of the decreased tension by Ach to the increased tension by NE. **P < 0.01 as
compared with the sham-operated group; #P < 0.05, ##P < 0.01 as compared with the LR group. (Color version of figure is
available online.)

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j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 5 ( 2 0 1 5 ) 5 2 9 e5 4 0

Fig. 5 e Effects of CsA on hemodynamics, cardiac output, and blood flow in the liver and kidney after traumatic hemorrhagic
shock. Data represent the mean standard errors of the mean (n [ 8 per group). (A) MAP; (B) LVSP; (C and D) maximal
change rate in left intraventricular pressure (dp/dtmax); (E and F) blood flow in the liver and kidney; and (G) cardiac output.
**P < 0.01 as compared with the sham-operated group; #P < 0.05, ##P < 0.01 as compared with the LR group. (Color version
of figure is available online.)

3.2.5.

Cardiac output

At the end of the shock period, the cardiac output was

significantly decreased as compared with baseline. Infusion of
LR significantly restored the decreased cardiac output of shock
animals, which was close to the baseline level at 1 h and 2 h
after resuscitation. CsA did not further increase the cardiac
output; there were no significant differences among three CsA
groups and the LR group at each time point (Fig. 5G).

3.3.
Part III: relationship of CsA-regulating vascular
reactivity to ROCK and PKC
3.3.1.

Role of ROCK and PKC agonists and inhibitors

ROCK inhibitor Y27632 significantly antagonized CsA-induced

increase in vascular constriction reactivity of SMAs to NE after
shock (P < 0.01). ROCK agonist U46619 further increased the
contractile response of SMA to NE, whereas the agonist and
inhibitor of PKC (PMA and staurosporine) had no significant
influences on CsA-regulating vascular reactivity after shock
(Fig. 6A and B).

3.3.2.

Expression and phosphorylation of MYPT1

After 3-h hypoxia, the protein expression of MYPT1 did not

show significant changes, whereas the phosphorylation of
MYPT1 (reflecting the activity of ROCK) was significantly
decreased (P < 0.01). CsA (1 mmol/L) reversed hypoxia-induced
decrease of MYPT1 phosphorylation (P < 0.01; Fig. 6C and D).

3.3.3.

The opening of MPTP

The mean intensity of calcein-related mitochondrial fluorescence in VSMCs from the hypoxia group was significantly
decreased as compared with the control group (P < 0.01). CsA
(1 mmol/L) inhibited the opening of MPTP after hypoxia; it
appeared that the intensity of calcein-related mitochondrial
fluorescence in VSMCs was increased (Fig. 7A and B).

4.

Discussion

Mitochondria are the key organelles, which play a critical role

in maintaining cell function and determining cell fate.
Therefore, the abnormal function of mitochondria has been

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 5 ( 2 0 1 5 ) 5 2 9 e5 4 0

537

Fig. 6 e Influences of ROCK and PKC on CsA regulation of vascular reactivity in SMAs and effects of CsA on the expression
and phosphorylation of MYPT1 (reflecting the activity of ROCK) in VSMC after hypoxia. Data represent the mean standard
errors of the mean. (A) The maximal contractile response of SMA to NE; (B) the maximal relaxation response of SMA to Ach
(n [ 8 per group); (C) the expression and phosphorylation of MYPT1, immunoblot analyses were repeated thrice; and (D) the
ratio of the optical density (OD) for MYPT1/b-actin and p-MYPT1/b-actin; U46619: ROCK agonist; Y27632: ROCK inhibitor;
PMA: PKC agonist; staurosporine, PKC inhibitor. **P < 0.01 as compared with the sham-operated group; #P < 0.05,
##P < 0.01 as compared with the shock group or hypoxia group; ^^P < 0.01 as compared with the CsA group. (Color version
of figure is available online.)
implicated in many diseases, including ischemiaereperfusion
injury, trauma, and Alzheimer disease, and so forth. The
permeability of the inner mitochondrial membrane is essential to maintain the normal function of mitochondria, which

was mainly regulated by the gating of MPTP [7e10].

Many studies have demonstrated that the MPTP remains
closed under normal physiological conditions but opens on
some pathologic conditions, such as ischemia or

Fig. 7 e Effects of CsA on the opening of MPTP in VSMC after hypoxia. Data represent the mean standard errors of the mean.
(A) Images acquired by confocal microscopy showing VSMCs loaded from calcein-acetoxymethyl ester D CoCl2 and stained
with MitoTracker Deep Red. Images show: left column: calcein uorescence inside mitochondria; middle column:
MitoTracker uorescence, MitoTracker is a mitochondrion-selective fluorescent dye; right column: merged image.
Experiments were repeated thrice; (B) quantitative data of the mean intensity of mitochondrial uorescence. ##P < 0.01 as
compared with the control group; ^^P < 0.01 as compared with the hypoxia group.

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j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 5 ( 2 0 1 5 ) 5 2 9 e5 4 0

ischemiaereperfusion [23]. To date, there are increasing evidences demonstrating that inhibition of MPTP opening plays
an important role in cardioprotection and neuroprotection
against ischemiaereperfusion injury and prevents from
vascular hyperpermeability and hypotension after hemorrhagic shock [11e14]. Our present study found that CsA, an
inhibitor of MPTP, was beneficial for traumatic hemorrhagic
shock in rats via improving vascular reactivity, stabilizing
hemodynamic, and increasing the tissue blood perfusion and
then improving the vital organ function and blood gases. Five
and 10 mg/kg of CsA significantly improved the animal survival of shock rats.
CsA is a potent immunosuppressant, which is widely used
in organ transplantation and various autoimmune diseases
[11]. The effects of CsA on inflammatory cytokines are well
known. Some studies have revealed that hemorrhagic shock
may elicit an inflammatory response characterized by inflammatory factors production [24]. To elucidate the relationship between the beneficial effects of CsA on traumatic
hemorrhagic shock and its inflammatory effects, we observed
the effects of CsA on some key inflammatory mediators
including TNF-a, IL-1b, IL-6, tNOS, and iNOS. We found that the
blood level of TNF-a and IL-1b was significantly increased after
shock, whereas CsA only slightly decreased their blood level.
The blood level of IL-6, tNOS, and iNOS did not show significant
changes after CsA administration. It is suggested that the
strong immunosuppressive effects (or anti-inflammatory effect) of CsA may be depended on a continuous administration
for a long period. In the present study, because a moderate
dose of CsA (5 mg/kg, the discussion of varying CsA doses as
seen in the following) and just a short-term infusion were
adopted, CsA did not show a significant inhibitory effect on
inflammatory cytokines after shock. These results suggest that
the beneficial effects of CsA on traumatic hemorrhagic shock
are mainly coming from the vascular function protection, not
mainly from the immunosuppressive effects.
It has been known that a long-term or high-dose application of CsA would result in a number of side effects, including
hepatotoxicity and nephrotoxicity. To obtain the optimal dose
of CsA for the treatment of traumatic hemorrhagic shock, we
compared three doses of CsA (1, 5, and 10 mg/kg). The results
showed that 5 mg/kg of CsA could protect the liver and kidney
function against the ischemia injury after shock, whereas
10 mg/kg of CsA induced hepatotoxicity and nephrotoxicity
and failed to improve the liver and kidney function. It suggests
that 5 mg/kg of CsA may be a safe and effective dose for the
treatment of traumatic shock. Similarly, 5 mg/kg of CsA
improved the mitochondrial function in the liver, kidney, and
intestine after shock, but 10 mg/kg of CsA did not. This finding
is supported by other studies. For example, Halestrap et al. [25]
and Niemann et al. [26] reported that CsA at low doses
inhibited the opening of MPTP to protect against reperfusion
injury, but CsA at high doses could inhibit mitochondrial
respiration and damages proteins and lipids in isolated
mitochondria. A number of studies showed that CsA exerted
the protection against ischemia and/or hypoxia and reperfusion injury in the heart, brain, spinal cord, lung, liver, kidney,
gut, and skeletal muscle [11,12,26e28]. Only few studies
explored the effects of CsA on the vascular function in hemorrhagic shock. Tharakan et al. [13] demonstrated that CsA

could prevent hemorrhagic shock-induced vascular hyperpermeability. Wang et al. [29] reported that polydatin and CsA
could protect the neuron damage in severe hemorrhagic
shock. Whether CsA can improve the vascular dysfunctions
such as vascular hyporesponsiveness after hemorrhagic
shock is not known. Our present study showed that CsA
(1e10 mg/kg) markedly restored the decreased vascular contractile and relaxation reactivity of SMAs after shock and
improved and stabilized the hemodynamics and tissue blood
flow, and via this contributed the beneficial effects for traumatic shock. Meanwhile, we also observed the effects of CsA
on the cardiac output. The results showed that CsA did not
increase the cardiac output of traumatic shock rats. It suggests that under this shock and/or resuscitation conditions,
CsA stabilized the hemodynamics mainly by improving the
vascular function after shock, whereas the effect of CsA on the
myocardial contractile function needed further elucidation.
Cardiovascular dysfunction is the major cause of death
after severe trauma and/or shock. Studies showed vascular
hyporeactivity occurred at 1 h after shock and need early
prevention and treatment [30]. Catecholamines (dopamine,
epinephrine, and NE, and so forth) are the most widely used
vasoactive agents for cardiovascular dysfunction during septic and hemorrhagic shock. Currently, catecholamines are
often used to increase and maintain the arterial pressure and
tissue perfusion. However, catecholamine resistance is a wellknown clinical dilemma in critical illness such as severe
trauma, shock, and sepsis, which will result in the cardiovascular dysfunction, unstable hemodynamics, and refractory hypotension. Progressively increasing catecholamine
application has poor effects and high mortality [31]. Therefore,
a new drug that can improve cardiovascular function (catecholamine resistance) and stabilize hemodynamics in the
refractory shock states would be of great benefit. In the present study, we investigated the beneficial effects of early
application of CsA on traumatic hemorrhagic shock. The results indicated that early application of CsA can increase the
cardiovascular function, especially for the vascular function.
It is suggested that CsA can be a proposed application at early
stage of shock. Of course, the most suitable time for initiation
of therapy with CsA still needs to be determined in further
studies and clinical trials.
ROCK is a downstream effector of small GTPase RhoA that
has been known to play an important role in smooth muscle
contraction [32]. PKC constitutes a family of protein-serine
and/or threonine kinases that serve as central signaling molecules and regulators of multiple cellular processes [33]. Our
previous studies have demonstrated that activation of ROCK
and PKC (such as using vasopressin and pinacidil) could increase the contractile response of isolated mesenteric arteries
after hemorrhagic shock and improved the hemodynamics
and the survival outcome in hemorrhagic shock, which suggested that ROCK and PKC played a critical role in the regulation of vascular reactivity after shock [4,17,34,35]. Our present
study showed that the highly selective ROCK inhibitor, Y27632,
significantly antagonized CsA-induced increase of vascular
contractile reactivity, and its agonist U46619 further increased
the effects of CsA, whereas the agonist and inhibitor of PKC
had no significant influences on the effects of CsA. Further
studies showed that CsA increased the activity of ROCK and

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 5 ( 2 0 1 5 ) 5 2 9 e5 4 0

inhibited the opening of MPTP in VSMCs after hypoxia. It is

suggested that ROCK, not PKC, participated in the regulatory
effect of CsA on vascular reactivity after shock; the inhibitory
effect of CsA on MPTP may be the main factor that CsA play
beneficial effect on traumatic shock.

5.

Conclusions

In conclusion, our results indicate that CsA is beneficial for the

treatment of traumatic hemorrhagic shock in rats, and this
beneficial effect of CsA may mainly comes from the inhibitory
effect of CsA on MPTP, not by anti-inflammation effect. CsA
elicits the antishock effects mainly by restoring the decreased
vascular reactivity after shock, which contributed to the improvements on the hemodynamics, tissue perfusion, and
organ function for shock animals. ROCK may participate in
the beneficial effect of CsA. However, the precise mechanism
responsible for the activation of ROCK by CsA needs further
elucidation.

Acknowledgment
This study was supported by the National Natural Science
Foundation of China (81270400 and 30901559), Natural Science
Foundation of Chongqing City (Cstc2013jcyjA10012), and
Foundation of State Key Laboratory of Trauma, Burns and
Combined Injury (SKLZZ201020, SKLZZ200912, SKLZZ201207).
Authors contributions: Y.L., L.L., and T.L. participated in
the design. Y.L. participated in the entire experiments, and the
preparation of the article. X.P. and Z.D. created the animal
model and carried out the measurements of the hemodynamics, the vascular reactivity, and protein expression. L.L.
and T.L. conceived the study, participated in the coordination,
and edited the article. All authors approved the final article.
The authors thank Dr Shen (a statistician in Chongqing
Center for Disease Control and Prevention) for his statistical
assistance in this article.

Disclosure
The authors report no proprietary or commercial interest in
any product mentioned or concept discussed in this article.

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