The role of heat shock protein 70 in the protective effect of YC-1 on heat stroke rats

The role of heat shock protein 70 in the protective effect of YC-1 on heat stroke rats

Kwok-Keung Lam

, Pao-Yun Cheng

, Yen-Mei Lee

, Yu-Pei Liu

, James Cheng

Ding

, Won-Hsiung Liu

^, Mao-Hsiung Yen

a

Department of Pharmacology, Taipei Medical University, Taiwan;

Department of Anesthesiology, Catholic Mercy Hospital, Hsinchu, Taiwan;

Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung, Taiwan;

Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan;

Department of Pharmacology, Taipei Medical University, Taipei, Taiwan;

Center of Coronary Heart Disease, Fu Wai Hospital & Cardiovascular Institute, Chinese Academy of Medical Sciences &

Peking Union Medical College, China.

Department of Pediatrics, Chi Mei Medical Center, Tainan, Taiwan

Corresponding Author: Mao-Hsiung Yen, Department of Pharmacology,

National Defense Medical Center, No. 161, Sec 6, Min-Chuan East Road, Nei hu (114), Taipei, Taiwan, TEL & FAX: 886-2-87921704, E-

mail:mhyen@mail.ndmctsgh.edu.tw; or Won-Hsiung Liu, Department of Pediatrics, Chi Mei Medical Center, No.901, Zhonghua Rd., Yongkang Dist., Tainan City 710, Taiwan, TEL: 886-6-2812811, E-mail:x47937@yahoo.com.tw

# Mao-Hsiung Yen and Won-Hsiung Liu contributed equally to this work.

Abstract

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-(5’- Hydoxymethyl-2’-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-1+HS) 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-1+HS group were 21,

480, and 445 min, respectively. The expression of Hsp70 and HSF-1 in liver and

renal of YC-1+HS 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 (400mg/kg). Results indicate that YC-1 may improve survival due to induce Hsp70 overexpression.

Key words: YC-1; heat stroke; heat shock response; heat shock protein; heat

shock factor-1

1. Introduction

In 2003, Europe experienced 22000-45000 heat related deaths during a summer heat wave (Luterbacher et al., 2004; Schar and Jendritzky, 2004). Heat stroke is a life-threatening illness characterized by an elevated core body temperature that rises above 40 °C 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., 2007).

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 protective organ damage and lethality (Lee et al., 2006;

Wang et al., 2005; Chen et al., 2009).

3-(5’-Hydoxymethyl-2’-furyl)-1-benzyl-indazol (YC-1) was discovered that

have capacity to exert significant control over soluble guanylate cyclase (sGC)

and cyclic guanosine 3’,5’-cyclic 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-β1 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 23±1 °C, humidity of 55±5% 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

°C with a heating chamber at a room temperature of 24±1 °C, throughout the entire experiments. 2) Vehicle-treated heat stroke (HS) groups: the heat stroke experiment preparative as below. 3) 3-(5’-Hydoxymethyl-2’-furyl)-1-benzyl- indazol (YC-1) pretreatment with heat stroke (YC-1+HS) 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 (Q+YC-1+HS) 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 (Q+HS) 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 wee killed with an overdose of sodium

pentobarbital.

2.2 Induction of Heat Stroke

This study, an animal heat stroke model is modified by Niu (2007). The heat

stroke was induced by putting the animals in a heating chamber (42 °C) 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 °C. After the onset of heat stroke, the rats were removed from heating chamber

and the animals were allowed to recover at room temperature (24 °C). 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 °C for exactly 60 min and then allowed to recover at room temperature (24±0.1 °C). 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 x 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 Extraction

The liver and kidney tissue were obtained and frozen at -80

C 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 μg/ml of aprotinin, 1 mM

phenylmethylsulfonylfluoride and 10 μg/ml of leupeptin) and agitated at 4

C for 1 h to evaluate protein expression. After centrifugation for 30 min at 10,000 × g (4

o

C), 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

o

C 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-β-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

Image-Pro software (Media Cybemetrics, Inc.).

2.5 Statistical Analysis

Results are presented as mean±S.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 P < 0.05.

3. Results

3.1 YC-1 Attenuates Heat Stroke Induced Physiologic Dysfunction

Fig. 2 depicts the effects of heat exposure (42°C for 60 min) on several physiologic variables between different groups. In HS and YC-1+HS 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-1+HS group, the MAP and heart rate were all significantly higher at 15-120 min after the onset of heat stroke than HS, Q+HS and Q+YC-1+HS 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-1+HS 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 multi-

organ dysfunction.

3.3 YC-1 Improves Survival Time during Heat Stroke

Fig. 4 summarizes the effects of heat stroke (42 °C 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 21±3.8 min. The survival time of YC-1+HS group was significantly prolonged to 445±44.3 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. 5, A 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. 6, A 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-1+HS 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.

4. Discussion

This is a first study to demonstrated that YC-1 significantly attenuated the hypotension, tachycardia, hepatic and renal 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 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 stroke rats.

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., 2005). 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 lethality (Lee et al., 2006; Wang et al., 2005; 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-

1+HS 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-1+HS 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 needed to clarify the mechanism via which YC-1

regulates the expression of Hsp70 and HSF-1.

A high (above 40.6°C) 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 (Szelényi 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.

Acknowledgments

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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Figure legends

Fig. 1 Experimental protocol. NT, normothermal control; HS, heat stroke; YC- 1+HS, HS pretreated with YC-1(20 mg/kg); Q+YC-1+HS, HS pretreated with quercetin (400 mg/kg) and YC-1 (20 mg/kg); Q+HS, HS pretreated with quercetin (400 mg/kg).

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-1+HS), HS pretreated with quercetin (400 mg/kg) and YC-1 (20 mg/kg) (Q+YC-1+HS), and HS pretreated with quercetin (400 mg/kg)

(Q+HS) groups. Depicted are change Data are expressed as mean ± S.E.M. (n=5).

* P<0.05 compared with NT group, # P<0.05 compared with HS group, ***

P<0.05 compared with YC-1+HS group.

Fig. 3 Effects of YC-1 pretreatment on plasma value of GOT, GPT, blood urea nitrogen and creatinine in normothermal (NT), heat stroke (HS), HS pretreated with YC-1(20 mg/kg) (YC-1+HS), HS pretreated with quercetin (400 mg/kg) and YC-1 (20 mg/kg) (Q+YC-1+HS), and HS pretreated with quercetin (400 mg/kg) (Q+HS) groups. Depicted are change Data are expressed as mean ±S.E.M. (n=5).

* P<0.05 compared with NT group, # P<0.05 compared with HS group, **

P<0.05 compared with YC-1+HS group.

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-1+HS), HS pretreated with quercetin (400 mg/kg) and YC-1 (20 mg/kg) (Q+YC-1+HS), and HS pretreated with quercetin (400 mg/kg) (Q+HS) groups. Depicted are change Data are expressed as mean ±S.E.M. (n=5). * P<0.05 compared with NT group, # P<0.05 compared with HS group, ** P<0.05 compared with YC-1+HS group.

Fig. 5 Effects of YC-1 pretreatment on the expression of Hsp70 in liver (A) and kidney (B) in normothermal (NT), heat stroke (HS), HS pretreated with YC-1(20 mg/kg) (YC-1+HS), HS pretreated with quercetin (400 mg/kg) and YC-1 (20 mg/kg) (Q+YC-1+HS), and HS pretreated with quercetin (400 mg/kg) (Q+HS) groups. Depicted are change Data are expressed as mean ±S.E.M. (n=5). * P<0.05 compared with NT group, # P<0.05 compared with HS group, ** P<0.05

compared with YC-1+HS group.

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-1+HS), HS pretreated with quercetin (400 mg/kg) and YC-

1 (20 mg/kg) (Q+YC-1+HS), and HS pretreated with quercetin (400 mg/kg) (Q+HS) groups. Depicted are change Data are expressed as mean ±S.E.M. (n=5).

* P<0.05 compared with NT group, # P<0.05 compared with HS group, **

P<0.05 compared with YC-1+HS group.