Supplemental Materials and Methods:
Detailed Experimental protocols:
1. Effects of different doses of NS1619 pretreatment on survival rate and survival time
following hemorrhagic shock in rats
Fifty Sprague-Dawley (SD) rats were randomized into five groups (n=10/group): shock
control group and BkCa opener, NS1619 pretreatment groups (group I: NS1619 0.5mg/kg; group II:
NS1619 1mg/kg; group III: NS1619 2mg/kg, Ⅳ: NS1619 4 mg/kg). According to experimental
design, NS1619 in group I, II, III, Ⅳ were given intraperitoneally (i.p.) 30 min before hemorrhage.
30 minutes after NS1619 administration, the rats were rapidly hemorrhaged (within 10 min) from
the right femoral arterial catheter to MAP at 40 mmHg and maintained at this level for 3 hours by
withdrawal or infusion blood. After the completion of hemorrhagic shock model, the shed blood
and Lactated Ringer's Solution of one volume of shed blood were re-infused within 1h. The
femoral artery and vein catheters were then removed and ligated, and the wound was sutured. The
survival time and 72 h survival rate were observed.
2.Effects of NS1619 pretreatment on vascular reactivity and calcium sensitivity of SMA
after hemorrhagic shock in rats
Eighty-eight SD rats were randomly divided into three groups: sham operated group (n = 8),
hemorrhagic shock group (n = 40) and NS1619 pretreatment group (n = 40). Hemorrhagic shock
groups and NS1619 pretreatment groups were further divided into five subgroups (n
=8/group):10min, 0.5h, 1h, 2h, and 3h group after shock. Based on the results of the first
experiments, 2 mg/kg of NS1619 was given to the rats via i.p injection 30min before hemorrhage.
Rats in sham-operated group were subjected to identical operations as hemorrhagic shock control
group, but without hemorrhage. Rats were killed by decapitation at each time point, a laparotomy
was performed and SMA were obtained for the measurement of the vascular reactivity and
calcium sensitivity as described in our laboratory 1-4
Measurement of Vascular Reactivity
SMA rings were mounted on wire and suspended
between a force transducer and a post attached to a micrometer.
They were then immersed into a
10-mL isolated organ chamber (Scientific Instruments, Barcelona, Spain) containing
Krebs–Henseleit (K-H) solution (in millimoles per liter: 118 NaCl, 4.7 KCl, 25 NaHCO3, 1.03
KH2PO4, 0.45MgSO4·7H2O, 2.5CaCl2, and 11.1 glucose at pH 7.4), which was continuously
bubbled with 95% O2 /5% CO2 and the temperature maintained at 37 ℃. Preload was given 0.5 g
and K-H solution replaced every 15 minutes. The tension of SMA rings was determined by a
Power Lab System via a force transducer (Power Lab, AD Instruments, Castle Hill,
Australia). After a 2-hour equilibration when the tension curve of the SMA rings was stable, the
vascular reactivity of SMA to NE (10-9, 10-8, 10-7, 10-6, 10-5 mol/L) was determined. The maximal
contraction (Emax) and pD2 [-log (50% effective concentration)] were obtained from the
concentration–response curves and used to compare vascular reactivity. Emax reflects the
maximal contractive response of blood vessel to vasoactive (norepinephrine), which
directly reflects the vascular reactivity. The greater the Emax, the higher the vascular
reactivity. pD2 reflects the contractile sensitivity of blood vessel to vasoactive
(norepinephrine), which indirectly reflects the vascular reactivity. The greater the pD2,
the higher the sensitivity of blood vessel to vasoactives. 1-4
Measurement of Calcium Sensitivity SMA rings were incubated and equilibrated in K-H
solution for 2 hours as described above. The solution was then replaced with a depolarizing
solution containing (in millimoles per liter): 2.7 NaCl, 120 KCl, 25 NaHCO3 , 1.03 KH2 PO4,
0.45MgSO4 ·7H2O, and 11.1 glucose at pH 7.4. After 10–20minutes of equilibration, the
contractile responses of SMA rings to Ca2+ (3×10-5,1×10-4, 3×10-4,1×10-3, 2×10-3 , 6×10- 3, 1×10-2,
3×10- 2mol/L) were determined. The maximal contraction and pD2 were obtained from the
concentration–response curves and used to compare calcium sensitivity. 3, 4
3. Effects of NS1619 pretreatment on cardiac output (CO), oxygen delivery (DO2) and
oxygen consumption (VO2) after hemorrhagic shock in rats
Sixteen SD rats were randomly divided into shock control (n=8) and NS1619 pretreatment
group (n=8). In this experiment, all rats received catheterization of the left ventricle of heart
and right external jugular vein for measurement of cardiac output. The other procedures
(including NS1619 pretreatment, hemorrhage shock model preparation) were the same as
described in experiment 1. The cardiac output was measured by a Cardiomax-III machine
(Columbus Instruments, Columbus, OH, USA) before hemorrhage shock and 3 hours after
hemorrhage shock. At the same time, blood was sampled from the femoral artery and femoral
vein to analyze arterial and venous blood gases, respectively, using a blood gas analyzer
((Phox plus L, Nova Biomedical, Waltham, MA). The values of DO2 and VO2 were calculated
using the following equations:
DO2 = CI × 13.4 × Hb × SaO2
VO2 = CI × 13.4 × Hb × (SaO2 –SvO2)
where DO2 is oxygen delivery, VO2 is oxygen consumption, CI is the cardiac index, Hb is the
hemoglobin concentration, SaO2 is oxygen saturation of the arterial blood, and SvO2 is
oxygen saturation of the venous blood.
4.Effects of Rho-kinase antagonist and RhoA inhibitor on the protective effect of NS1619
pretreatment in vascular reactivity and calcium sensitivity
Thirty two SD rats were divided into shock control (n=8) and NS1619 pretreatment group
(n=21). NS1619 pretreatment group were further divided into NS1619 group, NS1619 +Y-27632
(Rho-kinase inhibitor) group, NS1619 + C3 Transferase (RhoA inhibitor) group (n=8/group).
NS1619 (2 mg/kg) was given 30 min before hemorrhage via i.p. injection. 3 hours after
hemorrhagic shock, rats were killed, SMA rings were obtained as described above for the
measurement of vascular reactivity and calcium sensitivity. Before the measurement, SMA rings
in NS1619+Y-27632 group were incubated with Y-27632(1×10−5mol/L) for 10 min, and SMA
rings in NS1619 + C3 Transferase group were treated with 5 μg/ml of cell permeable C3
Transferase (Cytoskeleton) in the sterile blood serum culture-medium for 6 h at 37°C.
5.Effect of NS1619 pretreatment on the activity of Rho-kinase following hemorrhagic shock
and the relationship to RhoA
Twelve Sprague-Dawley rats were randomly divided into four groups (n = 3/gourp): sham
operated group, shock control group, NS1619 pretreatment group, and NS1619 pretreatment + C3
Transferase group. Shock model preparation and NS1619 administration were as the same as
described above. SMA was obtained at 3h after shock for measurement of the activity of
Rho-kinase. Before the measurement, SMA tissues in NS1619 + C3 Transferase group were
treated with 5 μg/ml of cell permeable C3 Transferase (Cytoskeleton) in the sterile blood serum
culture-medium for 6 h at 37°C. Then, their proteins were isolated and used to determine the
activity of Rho-kinase.5
Measurment of the activity of Rho-kinase SMA tissues were homogenized in ice-cold
lysing buffer [50 mM Tris-base (pH 6.5), 150 mM NaCl, 1% Triton X-100, 1 mM ethylene glycol
tetraacetic acid, 1 mM ethylenediami-netetraacetic acid (EDTA), 1 mM Na3VO4, 10 mM NaF,
0.1%β-mercaptoethanol] with a protease inhibitor. The lysate was centrifuged at 10,000 g for 30
minutes and the supernatant collected. The protein concentration was determined using a Protein
Assay Kit (Pierce, Rockford, IL). 40μg of protein from each sample was used for sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (8% Tris–HCl gels; Bio-Rad, Hercules, CA) and
transferred to polyvinyli-dene difluoride membranes. The membranes were blocked with 5%
nonfat milk and 0.05% Tween 20 (Bio-Rad) in tris buffered saline buffer containing 10 mM
Tris–HCl and 50 Mm NaCl (pH 7.5) for 1 hour at room temperature. The membranes were then
incubated overnight at 4°C with polyclonal phosphorylated myosin light-chain phosphatase
target subunit 1 at site Thr850 (anti-pMYPT1 Thr850 ); 1:800; Upstate Bio-technology, Lake
Placid, NY) and anti-MYPT1 (1:1000;Upstate Biotechnology). The activity of Rho-kinase was
presented as the ratio of p-MYPT1 and MPYT1 (substrate of Rho-kinase). 5
6.Effects of NS1619 pretreatment on the activity of RhoA and Rho GEF in SMA
following hemorrhagic shock in rats
Nine Sprague-Dawley rats were randomly divided into 3 groups (n = 3/gourp): sham
operated group, hemorrhagic shock group and NS1619 pretreatment group. Shock model
preparation and NS1619 administration were as the same as described above. SMA tissues were
obtained at 3h after shock for measurement of the activity of RhoA and Rho GEF.
Measurment of the activity of RhoA The activity of RhoA was determined by G-LISA
RhoA Activation Assay Biochem Kit (Cytoskeleton Inc, Denver, CO, USA). Briefly, SMA tissues
were homogenized in lysis buffer (in 150mM NaCl, 50mM Tris pH 8, 10mM EDTA, 1% Triton
X-100, 0,05% sodium deoxycholate supplemented with protein inhibitors(Roche) and phosphatase
inhibitors (Sigma)), and were centrifuged at 14000 rpm for 2 min at 4°C. The supernatant,
containing 50μg of proteins, was transferred into a plate and equal volumes of ice-cold binding
buffer mixtures were added into each well. The plate was shaken on a cold orbital microplate
shaker (200 rpm) for 30 min at 4°C, flicked out of the solution from wells, and incubated with
diluted anti-RhoA primary antibodies(1:250) followed by secondary antibodies(1:250) on a
microplate shaker (250 rpm) at room temperature for 45 min each. The plate was incubated with
an HRP detection reagent for 5 min at 37°C, and after addition of an HRP stop buffer, the
absorbance was immediately recorded at 490 nm. RhoA activity is expressed as relative
luminescence unit (RLU).6
Measurement of the activity of Rho GEF The activity of Rho GEF including p115-Rho
GEF, PDZ-Rho GEF and LARG were determined via measurement of the exchange activity on
RhoA by fluorophore-based Rho GEF exchange activity assay kit (Cytoskeleton, Denver, USA).
Briefly, p115-Rho GEF, PDZ-Rho GEF and LARG immunoprecipitates were respectively
suspended in 100μl of exchange buffer containing 2μM GDP-loaded RhoA-His protein and
0.75μM N-methyl-anthraniloyl (mant) provided in the kits. Relative fluorescence intensity was
spectrometrically monitored every 30 s over 30 min, and Rho GEF exchange activity is expressed
as the exchange rate，which was calculated by the manufacturer’s formula. Exchange rate =
[Vmax(AFU/sec)] /0.75[Basal mant-GTP AFU×2](Vmax is obtained form exchange curve. ARU:
relative fluorescence units.)7
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