The role of heat shock protein 70 in the protective effect
of YC-1 on heat stroke rats
Kwok-Keung Lam a,b
, Pao-Yun Cheng c
, Yen-Mei Lee d,e
, Yu-Pei Liu d
, Cheng Ding f
Won-Hsiung Liu g,****,1
, Mao-Hsiung Yen d,*,1
a Department of Pharmacology, Taipei Medical University, Taipei 114, Taiwan
b Department of Anesthesiology, Catholic Mercy Hospital, Hsinchu, Taiwan
c Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung, Taiwan
d Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan
e Department of Pharmacology, Taipei Medical University, Taipei, Taiwan
f Center of Coronary Heart Disease, Fu Wai Hospital & Cardiovascular Institute, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China g Department of Pediatrics, Chi Mei Medical Center, Tainan, Taiwan
Received 16 July 2012
Received in revised form
22 November 2012
Accepted 23 November 2012
Available online 5 December 2012
Heat shock response
Heat shock protein
Heat shock factor-1
Heat stroke is a life-threatening illness characterized by an elevated core body temperature. Despite
adequate lowering of the body temperature and support treatment of multiple organ-system function,
heat stroke is often fatal. 3-(50
-furyl)-1-benzyl-indazol (YC-1) been identified as an
activator of soluble guanylate cyclase. To evaluate whether YC-1 protects multiple organ dysfunctions
and improves survival during heat stroke and its mechanism. Male Sprague-Dawley rats untreated or
treated with either YC-1 or quercetin (heat shock protein (Hsp) 70 inhibitor) were exposures to heat as
a model of heat stroke. The mean arterial pressure (MAP), heart rate, rectal temperature (Tco), survival
time, and plasma biochemical data, intracellular Hsp70 and heat shock factor-1 expression were
measured. The value of MAP, heart rate and Tco of untreated heat stroke (HS) group were all
significantly lower than that of normothermal (NT) group. Biochemical markers evidenced that liver
and kidney injuries of HS group were significantly higher than that of NT groups. YC-1 (20 mg/kg)
pretreatment with heat stroke (YC-1þHS) group, the MAP and heart rate were return to normal, and
the biochemical markers were all significantly recovered to normal. The survival time of HS group, NT
group and YC-1þHS group were 21, 480, and 445 min, respectively. The expression of Hsp70 and HSF-1
in liver and renal of YC-1þHS group was significantly higher than that of HS group. All of the protective
effects of YC-1 were all significantly suppressed when pretreated with quercetin (400 mg/kg). Results
indicate that YC-1 may improve survival due to induce Hsp70 overexpression.
& 2012 Elsevier B.V. All rights reserved.
In 2003, Europe experienced 22,000–45,000 heat related deaths
during a summer heat wave (Luterbacher et al., 2004a, 2004b).
Heat stroke is a life-threatening illness characterized by an
elevated core body temperature that rises above 40 1C and induced
that multi-organ system failure (such as circulatory shock, central
nervous system dysfunction, acute renal failure and liver failure)
was due to the combined effects of heat cytotoxicity, coagulopathies, and a systemic inflammatory response syndrome (Bouchama
and Knochel, 2002; Pease et al., 2009; Remick, 2003). The mechanisms of multiple organ system failure are not fully understood, in
spite of optimal cooling and supportive treatment in intensive care,
the overall mortality can exceed 60%, because as yet, there is no
specific treatment available (Misset et al., 2006; Argaud et al.,
Heat shock response (coordinated activation of heat shock proteins expression) is a universal mechanism of protection against
adverse environment conditions (Shamovsky and Nudler, 2008). The
heat shock proteins (Hsp) are subdivided into multi-member families
based on the molecular weights of the proteins encoded (the Hsp90,
Hsp70, Hsp60, and the small Hsp familities), of which Hsp70 is one of
the most extensively studied in mammalian cells. Hsp can function as
molecular chaperones in normal physiological conditions, facilitating
protein folding, preventing protein aggregation, or targeting improperly folded proteins to specific degradative pathways (Freeman and
Morimoto, 1996). In response to cellular stress, such as hyperthermia,
oxidative damage, physical injury or chemical stressors the expression of Hsp increases dramatically (Lindquist, 1986). Several studies
reported that overexpression of Hsp72 in response to heat stress can
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European Journal of Pharmacology
0014-2999/$ – see front matter & 2012 Elsevier B.V. All rights reserved.
* Corresponding author. Tel./fax: þ886 2 87921704. **** Corresponding author. Tel.: þ886 6 2812811.
E-mail addresses: [email protected] (W.-H. Liu),
[email protected] (M.-H. Yen). 1 Mao-Hsiung Yen and Won-Hsiung Liu contributed equally to this work.
European Journal of Pharmacology 699 (2013) 67–73
protective organ damage and lethality (Lee et al., 2006; Wang et al.,
2005a, 2005b; Chen et al., 2009).
-furyl)-1-benzyl-indazol (YC-1) was
discovered that have capacity to exert significant control over
soluble guanylate cyclase (sGC) and cyclic guanosine 30
monophosphate (cGMP) signaling in the cardiovascular system
(Tulis, 2008). YC-1 first discovered by Teng and colleagues in 1994
as NO-independent activator of platelet sGC and cGMP synthesis
in rabbits (Ko et al., 1994; Wu et al., 1995). Several studies have
shown that YC-1 provided protection against vascular injuries.
YC-1 reduces vascular smooth muscle growth through inhibiting
the proliferative factor TCF-b1 and via reducing focal adhesion
kinase and through alteration of matrix balance by suppression of
matrix metalloproteinase biology (Wu et al., 2004; Liu et al.,
2006). YC-1 also induces Hsp70 expression and prevents oxidized
LDL-mediated apoptosis (Liu et al., 2008). For the reason that
there were no specific drugs to improve survival rate of heat
stroke, we tried to investigate whether YC-1 can enhance Hsp70
production to protect heat stroke-induced multiple organ injury.
2. Materials and methods
2.1. Experimental animal preparation
Male Sprague-Dawley rats (300–350 g) were obtained from the
National Laboratory Animal Breeding and Research Center of the
National Science Council, Taiwan. Handling of the animals was in
accordance with the Guide for the Care and Use of Laboratory
Animals published by the US National Institutes of Health (NIH
Publication No. 85-23, revised 1996). All animals were housed at an
ambient temperature of 2371 1C, humidity of 5575% and maintained on 12 h light/12 h dark schedule. This study was approved by
the National Defense Medical Center Institutional Animal Care and
Use Committee, Taiwan. The rats were anesthetized by intraperitoneal injections of urethane (1.4 g/kg). The right femoral artery was
cannulated with a polyethylene-50 catheter and connected to a
pressure transducer (P231D, Statham, Oxnard, CA, USA) for the
measurement of blood pressure, mean arterial pressure (MAP) and
heart rate, which were displayed on a Gould model TA5000 polygraph recorder (Gould, Valley View, OH, USA). The right femoral vein
was cannulated for the administration of drugs and for the collection
of blood sample. Core temperature (Tco) was monitored continuously
by a thermocouple inserted into the rectum. After the completion of
surgery, all cardiovascular parameters were allowed to stabilize for
30–60 min. Rats under anesthesia were randomized into five major
groups, as described in Fig. 1: (1) Normothermic control (NT) group:
the Tco was maintained at about 36 1C with a heating chamber at a
room temperature of 2471 1C, throughout the entire experiments.
(2) Vehicle-treated heat stroke (HS) groups: the heat stroke experiment preparative as below. (3) 3-(50
-furyl)-1-benzyl-indazol (YC-1) pretreatment with heat stroke (YC-1þHS) group:
the rats received YC-1 20 mg/kg for 3 h before heat stress.
(4) Quercetin (Hsp inhibitor) and YC-1 pretreatment with heat stroke
(QþYC-1þHS) group: the rats received quercentin 400 mg/kg for 6 h
and YC-1 20 mg/kg for 3 h before heat stress. (5) Quercetin pretreatment with heat stroke (QþHS) group: the rats received quercentin
400 mg/kg for 6 h before heat stress. At the end of the experiments,
control rats and any rats that had survived heat stroke were killed
with an overdose of sodium pentobarbital.
2.2. Induction of heat stroke
This study, an animal heat stroke model is modified by Niu
et al. (2007). The heat stroke was induced by putting the animals
in a heating chamber (42 1C) and was remained about 60 min.
The onset of heat stroke was taken as the time at which MAP fell
to about 25 mmHg from the peak level and Tco was elevated to
about 42 1C. After the onset of heat stroke, the rats were removed
from heating chamber and the animals were allowed to recover at
room temperature (24 1C). This pilot study showed that the
latency for onset of heat stroke in vehicle-treated rats was about
60 min. Therefore, in the following experiments, all heat-stressed
animals were exposed to 42 1C for exactly 60 min and then
allowed to recover at room temperature (2470.1 1C). Use of
higher temperature or longer period of hyperthermia would
reduce both latency for onset of heat stroke and survival time
(interval between the onset of heat stroke and death).
2.3. Biochemical analysis
Whole blood (0.5 ml) was collected into sodium citrate tubes
and centrifuged (10,000 g for 3 min) to prepare plasma. The three
different time points of obtained blood sample were the following:
(1) 0 min before the start of heat stress, (2) 60 min after the start of
heat stress, and (3) 75 min after start of heat stress. The plasma
levels of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen and creatinine were
determined by spectrophotometry (Fiji DRI-CHEM 303, Japan).
2.4. West blot analysis of Hsp70 and HSF-1 and nuclear protein
The liver and kidney tissue were obtained and frozen at 80 1C
before assay. The tissue was ground in a mortar containing liquid
nitrogen. The powdered tissue was then suspended in 1 ml of lysis
buffer (50 mM HEPES, 5 mM EDTA, 50 mM NaCl, pH 7.5) containing
protease inhibitors (10 mg/ml of aprotinin, 1 mM phenylmethylsulfonylfluoride and 10 mg/ml of leupeptin) and agitated at 4 1C for 1 h
to evaluate protein expression. After centrifugation for 30 min at
10,000 g (4 1C), the protein concentration was determined using a
BCA protein assay kit (Pierce, Rockford, IL, USA). Nuclear and
cytosolic extracts were prepared using a nuclear/cytosol fractionation kit (BioVision, USA) according to the manufacturer’s protocol.
Protein concentrations adjusted to 1 mg/ml.
Samples containing equal amounts of protein were loaded
onto 10% sodium dodecyl sulfate-polyacrylamide gels, subjected
to electrophoresis, and subsequently blotted onto nitrocellulose
membrane (Millipore, Bedford, USA). Membranes were blocked
with Tris-buffered saline buffer (TBS), pH 7.4, containing 0.1%
Tween-20 and 5% skim milk, and then incubated overnight at 4 1C
with various primary antibodies in TBS containing 0.1% Tween-20.
The antibodies included mouse polyclonal anti-Hsp70 antibody
(1:1000 dilution, Stressgen Biotechnologies Co., Victoria, BC,
Canada), anti-heat shock factor-1(HSF-1) antibody (1:1000 dilution, Santa Cruze, sc9144), mouse anti-b-actin (1:2000 dilution,
Sigma-Aldrich, St. Louis, MO, USA). The membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary
antibodies (1:1000 dilutions, Cell Signaling). The blots were
detected with an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA), and the membrane exposed to X-ray film (Kodak,
Rochester, NY, USA) for 5 min. The density of the respective bands
was quantified by densitometric scanning of the blots using ImagePro software (Media Cybemetrics, Inc.).
2.5. Statistical analysis
Results are presented as mean7S.E.M. and were evaluated
statistically by one-way analysis of variance (ANOVA) with
Newman–Keuls multiple comparisons test for the post hoc
determination of significant differences. Differences were considered significant at Po0.05.
68 K.-K. Lam et al. / European Journal of Pharmacology 699 (2013) 67–73
3.1. YC-1 attenuates heat stroke induced physiologic dysfunction
Fig. 2 depicts the effects of heat exposure (42 1C for 60 min) on
several physiologic variables between different groups. In HS and YC-
1þHS group, the MAP, heart rate and Tco were all significantly higher
at 30–60 min after the start of heat stress than they were for NT
group. In YC-1þHS group, the MAP and heart rate were all significantly higher at 15–120 min after the onset of heat stroke than HS,
QþHS and QþYC-1þHS groups. These results indicated that pretreatment with YC-1 for 3 h before heat stress significantly attenuated the heat stroke induced arterial hypotension and tachycardia. In
contrast, pretreatment with quercetin for 6 h before heat stress
significantly enhanced the heat stroke induced circulatory shock.
3.2. YC-1 attenuates heat stroke induced liver and kidney injuries
Fig. 3 summarizes the plasma levels of GOT, GPT, blood urea
nitrogen and creatinine among different groups at 0, 60, 75 min
after the start of heat stress. The plasma levels of these parameters in HS group were all significantly higher at 75 min after
the start of heat stress than NT group and YC-1þHS group.
Pretreatment with YC-1 for 3 h before onset of heat stress
significantly attenuated the heat stroke induced increment of
plasma levels of all these parameters. These results indicated that
pretreatment with YC-1 attenuate heat stroke induced multiorgan dysfunction.
3.3. YC-1 improves survival time during heat stroke
Fig. 4 summarizes the effects of heat stroke (42 1C for 60 min)
on survival time in different groups. The survival time of NT group
were 480 min, the survival time of HS group were only
2173.8 min. The survival time of YC-1þHS group was signifi-
cantly prolonged to 445744.3 min. These results indicated that
the administration of YC-1 to the rats in a prophylactic manner
resulted in a significant reduction in the mortality rate.
3.4. YC-1 induces heat shock protein 70 and nuclear heat shock
factor-1 expression of liver and kidney during heat stroke
Fig. 5A and B showed that a vast increase Hsp70 expression was
detected after heat stroke in rat livers and kidneys. Nevertheless, this
increase was boosted when the pretreatment with YC-1 was
achieved, but this increase was suppressed by the pretreatment with
quercetin (400 mg/kg). Fig. 6A and B showed that increments of HSF-
1 expression were detected after heat stroke in livers and kidneys of
rats. Compared with the NT and HS group, YC-1þHS group had
higher levels of HSF-1 in livers and kidneys. These results indicated
that pretreatment with YC-1 increase hepatic and renal Hsp70
expression by up-regulated HSF-1 production during heat stroke.
This is a first study to demonstrated that YC-1 significantly attenuated the hypotension, tachycardia, hepatic and renal
0’ 60’ 75’
Blood sample Blood sample Blood sample
Fig. 1. Experimental protocol. NT, normothermal control; HS, heat stroke; YC-1þHS, HS pretreated with YC-1(20 mg/kg); QþYC-1þHS, HS pretreated with quercetin
(400 mg/kg) and YC-1 (20 mg/kg); QþHS, HS pretreated with quercetin (400 mg/kg).
K.-K. Lam et al. / European Journal of Pharmacology 699 (2013) 67–73 69
dysfunction induced by heat stroke in rats. It also prolonged the
survival time during heat stroke. In addition, the expression of
Hsp70 protein in hepatic and renal tissues was significantly
increased in heat stroke rats, and more enhanced the expression
of Hsp70 protein in YC-1 pretreated heat stroke rats. Treatment
with quercetin, an Hsp70 inhibitor, produced a significantly
Fig. 2. Effects of YC-1 pretreatment on mean arterial pressure, heart rate, and rectal temperature in normothermal (NT), heat stroke (HS), HS pretreated with YC-1(20 mg/
kg) (YC-1þHS), HS pretreated with quercetin (400 mg/kg) and YC-1 (20 mg/kg) (QþYC-1þHS), and HS pretreated with quercetin (400 mg/kg) (QþHS) groups. Depicted
are change data are expressed as mean7S.E.M. (n¼5). *
Po0.05 compared with NT group, #Po0.05 compared with HS group, ***Po0.05 compared with YC-1þHS group.
70 K.-K. Lam et al. / European Journal of Pharmacology 699 (2013) 67–73
suppressed the expression of Hsp70 protein in hepatic and renal
tissues and the protective effect of YC-1 in heat stroke rats.
Results suggest that increased Hsp70 protein expression may
play an important role in the protective effect of YC-1 in heat
An epidemiological study of military exertional heat stroke
patients showed 40% increased mortality risk from cardiovascular,
kidney, and liver failure within 30 years of hospitalization compared
with individuals treated for a non-heat related illness (Pease et al.,
2009; Giercksky et al., 1999; Wallace et al., 2007). The hepatic and
renal failure may be related to tissue inflammatory, hypoxia and
ischemia (due to circulatory shock) and thermal injury (Garcin et al.,
2008; Wang et al., 2005a, 2005b). In a rat experimental model for
heat stroke, as demonstrated in the present and previous results,
elevating core temperature and renal (e.g., an increased plasma
levels of blood urea nitrogen and creatinine), hepatic dysfunctions
(e.g., an increased plasma levels of GOT, GPT) occurred during heat
stroke (Lee et al., 2006; Chang et al., 2006; Chen et al., 2006).
However, pretreatment with YC-1 significantly improved hepatic
and renal injuries induced by heat stroke (Fig. 3). Several lines of
evidence have showed that administration of YC-1 pretreatment, a
sGC activator, which has an anti-platelet aggregation (Ko et al.,
1994), anti-inflammatory activation of LPS treated-animal model
(Lu et al., 2007) and inhibit choroidal neovascularization of rat (Song
et al., 2008). The present study shown that the heat stroke responses
(hypotension, tachycardia, hyperthermia, hepatic and renal dysfunction and mortality) were all ameliorated when pretreated with YC-1
(20 mg/kg) for 3 h before the start of heat stress in heat stroke rats.
This result suggests that YC-1 has a great potential as a new
protective agent for heat stroke.
Previous studies have established that the sublethal heat
stress-induced accumulation of inducible Hsp70 is necessary for
acquired thermotolerance, which is defined as the ability of a cell
or organism to become resistant to heat stress (Lee et al., 2006;
Moseley, 1997). Several studies reported that overexpression of
Hsp72 in response to heat stress can protective organ damage and
Fig. 4. Effects of YC-1 pretreatment on survival time in normothermal (NT), heat
stroke (HS), HS pretreated with YC-1(20 mg/kg) (YC-1þHS), HS pretreated with
quercetin (400 mg/kg) and YC-1 (20 mg/kg) (QþYC-1þHS), and HS pretreated
with quercetin (400 mg/kg) (QþHS) groups. Depicted are change data are
expressed as mean 7S.E.M. (n¼5). *
Po0.05 compared with NT group, #Po0.05
compared with HS group, **Po0.05 compared with YC-1þHS group.
K.-K. Lam et al. / European Journal of Pharmacology 699 (2013) 67–73 71
lethality (Lee et al., 2006; Wang et al., 2005a, 2005b; Chen et al.,
2006). The current results demonstrated that pretreatment with
YC-1 (20 mg/kg) for 3 h before the start of heat stress significantly
increases the expression of Hsp70 protein in liver and kidney;
even without heat stress, and that this effect is exacerbated under
heat stroke. Moreover, the protective effects of YC-1 were all
attenuated when the heat stroke rats pretreated with quercetin,
an Hsp70 inhibitor. These results indicate that pretreatment with
YC-1 may improve survival by ameliorating multi-organ injuries
during heat stroke due to induce Hsp70 overexpression.
The production of Hsp70 is mainly regulated by HSF-1 in
mammals (Christians et al., 2002; Dai et al., 2007; Sarge et al.,
1993). Under physiological conditions, HSF-1 remains as a monomer in cytosol. During heat stress or other stresses, HSF-1 is
rapidly converted to its active form. The activation event is
associated with the transition of the monomer to a trimer and
translocates into the nucleus (Sarge et al., 1993; Westwood and
Wu, 1993), where it binds to the heat shock element present in
the promoter of heat shock genes and initiates transcription and
synthesis of Hsp after activation (Morimoto, 1998). In this study,
the expression of nuclear HSF-1 in livers and kidneys in the rats of
YC-1 and YC-1þHS groups were significantly greater than in the
HS group, while the expression of Hsp70 in liver and kidney in the
rats of YC-1 and YC-1þHS groups were significantly greater than
in the HS group. These results further suggest that the YC-1
induced overexpression of Hsp70 by increment of nuclear HSF-1
may be involved in the improvement of multi-organ dysfunction
in the heat stroke rats. Although we have no direct evidence of the
modulation of YC-1 on the expression of Hsp70 via HSF-1, it is
plausible that (1) YC-1 may affect hyperphosphorylation of HSF-1
(Yamanaka et al., 2003), (2) YC-1 may induce HSF-1 release and
translocate to the nucleus through inhibiting of Hsp90 (Whitesell
et al., 2003), (3) YC-1 may directly activate nuclear HSF-1
production (Sun et al., 2000; Xu et al., 1997). Further studies are
Fig. 6. Effects of YC-1 pretreatment on the expression of nuclear HSF-1 in liver (A) and kidney (B) in normothermal (NT), heat stroke (HS), HS pretreated with YC-1 (20 mg/
kg) (YC-1þHS), HS pretreated with quercetin (400 mg/kg) and YC-1 (20 mg/kg) (QþYC-1þHS), and HS pretreated with quercetin (400 mg/kg) (QþHS) groups. Depicted
are change data are expressed as mean 7S.E.M. (n¼5). *
Po0.05 compared with NT group, #Po0.05 compared with HS group, **Po0.05 compared with YC-1þHS group.
72 K.-K. Lam et al. / European Journal of Pharmacology 699 (2013) 67–73
needed to clarify the mechanism via which YC-1 regulates the
expression of Hsp70 and HSF-1.
A high (above 40.6 1C) body temperature is widely viewed as
the crucial symptom of heat stroke; however, this criterion should
not be considered absolute, because many patients with severe
exertional heat stroke have a lower body temperature, presumably because of the time elapsed after the actual heat overload
(Romanovsky and Blatteis, 2000). In several animal species, both
whole-body heat exposure and intraperitoneal heating have been
shown to result in hypothermia that occurring after heating
ceases (‘‘hyperthermia-induced hypothermia’’) (Romanovsky
and Blatteis, 1996, 2000). A priori, the thermoregulatory mechanism of this phenomenon could involve either the inhibition of
metabolism or the enhancement of heat loss (e.g., generalized
peripheral vasodilation). The latter possibility is unlikely because
the post-intraperitoneal heating hypothermia was accompanied
by marked skin vasoconstriction (Romanovsky and Blatteis, 1996,
2000). In addition, several study have demonstrated that
hyperthermia-induced hypothermia involves the transient
depression of cold defenses, i.e., a pronounced but reversible
decrease in the threshold body temperature for activation
of metabolic heat production (Szele´nyi et al., 1996). In the
present study, the ‘‘hyperthermia-induced hypothermia’’ did not
observed, but the heat stroke-induced hyperthermia was followed
by a return of body temperature to its pre-heat stroke level
(Fig. 2C). This pattern may be result from a shorter duration of
observation. Further studies are needed to clarify the relationship
between YC-1 and thermoregulation.
In conclusion, the current results showed that YC-1 pretreatment increases Hsp70 and HSF-1 expression attenuates circulatory shock, liver and kidney injuries under heat stroke, which
results improved survival. These findings suggest that YC-1 seems
to be a pharmacological inducer of Hsp70, and it would be a good
candidate as a protector against heat stroke.
This work was supported in part by a research grant from the
National Science Council (NSC 98-2320-B-016-002), National
Defense Medical Research (D101-41, Mao-Hsiung Yen) and the
Chi-Mei Medical Center (CMNDMC9907), Taiwan.
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