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The role of endogenous opioids in the protective effects of local sublethal hyperthermia against the progression of burn injury
ARTICLE IN PRESS Journal of Thermal Biology 34 (2009) 286–289
Contents lists available at ScienceDirect
Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio
The role of endogenous opioids in the protective effects of local sublethal hyperthermia against the progression of burn injury Neda Karimi a, Zuhair Muhammad Hassan b, Morteza Behnam Rasuli a, Behrouz Ilkhanizadeh c, Shaker Salarilak d, Shahram Shahabi e, a
Department of Biology, Faculty of Sciences, Ferdowsi University of Mashad, Mashad, Iran Department of Immunology, School of Medical Sciences, Tarbiat Modarres University, Tehran, Iran c Department of Pathology, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran d Department of Health and Community Medicine, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran e Department of Microbiology, Immunology and Genetics, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran b
a r t i c l e in f o
a b s t r a c t
Article history: Received 14 January 2009 Accepted 31 March 2009
The aim of the present study was to evaluate the role of endogenous opioids in local sublethal hyperthermia-induced protection against burn injury. Second-degree burn wounds were induced on the back of Balb/c mice. Progression of burn injury and expression of heat shock protein (HSP)-70 were evaluated after 24 h. Both inhibition of HSP synthesis and blocking opioid receptors before applying local sublethal hyperthermia decreased the protective effects of sublethal hyperthermia against the progression of burn injury. Blocking opioid receptors attenuated induction of HSP-70 by sublethal hyperthermia. & 2009 Elsevier Ltd. All rights reserved.
Keywords: Sublethal hyperthermia Thermotolerance Burn Endogenous opioids HSP-70
1. Introduction Many studies have shown that if the environmental temperature rises severely, cells grown at normal temperature will die rapidly. This phenomenon is called ‘heat shock.’ If these cells are exposed to a sublethal temperature before being exposed to a lethal temperature, their resistance against heat shock will be increased due to the induction of the heat shock proteins (HSPs) at the sublethal temperature. This phenomenon is regarded as ‘heat shock tolerance’ (thermo tolerance; Lindquist, 1986; Ellis et al., 1993). Sublethal levels of other stresses can also induce stress tolerance when cells are exposed before being exposed to lethal stresses (Ellis et al., 1993). In addition to cells, tissues can also develop stress tolerance if they are exposed to a sublethal stress. Topping et al. (2001) have shown that if the skin is mildly warmed before being exposed to a high temperature, the thermal injury to the skin will be decreased as a result of the induction of HSPs. Also, if the myocardium undergoes a sublethal ischemia before undertaking a lethal ischemia, its resistance to the lethal ischemia will be increased. (Sommerschild and Kirkeboen, 2002).
Corresponding author. Tel.: +984413449548; fax: +984412780801.
E-mail addresses: [email protected], [email protected] (S. Shahabi). 0306-4565/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2009.03.007
It has been shown that in addition to HSPs, opioids and opioid receptors also have a significant role in the stress tolerance of cardiac tissue induced by ischemia and hyperthermia (Patel et al., 2002; Sommerschild and Kirkeboen, 2002; Sonneborn et al., 2004). Opioids are peptides that are mainly produced in the nervous system and are produced in lower quantities by cells of the immune system. Their receptors are on both nerve cells and immune cells (Wybran et al., 1979; Stein et al., 1990; Way et al., 2001). Skin burn can be regarded as a lethal heat shock to the skin. Cutaneous thermal injury initiates a pathophysiological response with a significant inflammatory component that involves several classes of chemical mediators. These mediators interact in a complex manner to cause the pain and secondary tissue damage associated with burn injury (Shakespeare, 2001). Hair follicles are epidermal appendages that have an important role in the healing process after a burn injury (Dyer and Roberts, 1990). Tissue necrosis (including necrosis of hair follicles) progresses after burning, and it is not limited to the time when the burn occurs (Topping et al., 2001; Shahabi et al., 2006). If a treatment can prevent progression of necrosis after the burn injury, it can prevent the rise in the number of necrotic hair follicles after burning. Thus, the count of hair follicles 24 h after the burn injury is used to assess the skin injury (Topping et al., 2001; Shahabi et al., 2006). We have previously shown that the endogenous opioid response plays an important role in the protective effects of
ARTICLE IN PRESS N. Karimi et al. / Journal of Thermal Biology 34 (2009) 286–289
post-burn local hyperthermia against the progression of seconddegree skin burn injury (Shahabi et al., 2006). The aims of the present study are to evaluate the role of the endogenous opioid response in the protective effects of pre-burn local sublethal hyperthermia against second-degree skin burn injury, and to test the hypothesis that the opioids play this role via induction of HSP70.
287
Table 1 Grouping of the mice. Treatment groups
Quercetin
NLX
Sublethal hyperthermia
Burn
Healthy Burn Pre NPre QPre
+
+
+ + +
+ + + +
2. Material and methods 2.1. Animals
2.6. Histological analysis
Forty male BALB/c mice (average weight of 20 g) aged 6 weeks were obtained from the Razi Institute (Karaj, Iran). The animals were depilated using a standard depilatory cream on their backs. All procedures on animals were conducted with approval from the research committee of the Faculty of Sciences, Ferdowsi University, Mashad, Iran.
Twenty-four hours after the burn injury, the central area of the wound together with 5 mm of the marginal tissue was harvested. The samples were fixed in a 10% buffered formalin solution and embedded in paraffin, and then 4 mm-thick sections were cut and stained with haematoxylin and eosin (H & E). The sections were assessed for the number of hair follicles. The assessor was blind to the allocation of the sections. Evaluation of the number of hair follicles was done by the following method: hair follicles in 10 fields ( 10 power) were counted, and the result was divided by 10.
2.2. Drugs Naloxone (NLX; Opioid Receptor Antagonist) was purchased from Sigma (USA). Mouse monoclonal anti-mouse HSP70 antibody was obtained from R&D systems (USA). Horseradish peroxidase-conjugated anti-mouse antibody, quercetin (HSP synthesis inhibitor) and diaminobenzidine (DAB) were purchased from Sigma (USA).
2.7. Western blotting for HSP-70 expression
The mice were anaesthetized by intra-peritoneal injection of ketamine/xylazine (100/10 mg/kg). Then local sublethal hyperthermia was induced by immersing the backs of the mice in 44 1C water for 15 min using the following procedure. The anesthetized mice were placed individually in a small wire-mesh basket in supine position. The basket was immersed in 44 1C water so deep that the backs of the mice were immersed in the water but the faces of the mice were out of the water.
HSP-70 expression was investigated using western blotting analysis. Equal amounts of the proteins (1.3 mg/ml) determined by Bradford assay from the lysate of burned skin were fractionated on 10% SDS-PAGE gels and transferred into a polyvinylidene difluoride membrane. After washing, the membrane was blocked with 10% bovine serum albumin at room temperature for 1 h and incubated with mouse monoclonal anti-HSP-70 (1:1000, R&D systems). After washing, the membrane was incubated with horseradish peroxidase-conjugated anti-mouse antibody (1:1000, Sigma) at room temperature for 1 h. After washing, the immunoreactive bands were detected by 0.5 mg/ml DAB and 0.1% H2O2 in PBS. After inverting the blots by NIH Image J software, the relative signal intensity was quantified by this software on a personal computer.
2.4. Induction of burn injury
2.8. Statistical analysis
Two hours after local sublethal hyperthermia, although the mice had not recovered completely from the first anesthesia but since they had become a little conscious, they were again anaesthetized by intra-peritoneal injection of ketamine/xylazine (100/10 mg/kg). No lethality had occurred due to anesthesia. The mice were securely placed into a burn template so that the dorsum was centered on the 2 1 cm2 window. The shaved skin on the back was immersed in 80 1C water for 4 s to achieve a second-degree burn.
Statistical analysis was performed using the Kruskal–Wallis test followed by the one-way ANOVA post-hoc LSD test. A value of po0.05 was considered to be statistically significant. All statistical analyses were conducted with SPSS 11.5 software.
2.3. Local sublethal hyperthermia
2.5. Grouping of the mice Five groups of mice were used, each of them consisting of eight animals. Burn wounds were induced for all except eight mice (group Healthy). Mice of the group Burn received no drugs or treatments before or after induction of the burn injury. Local sublethal hyperthermia was applied for 24 mice 2 h before induction of burn injury. Eight of these mice received no drugs before or after applying local sublethal hyperthermia (group Pre), whereas 16 of these mice received 5 mg/kg NLX i.p. 40 min before applying sublethal local hyperthermia (group NPre) or 100 mg/kg quercetin 6 h before applying sublethal local hyperthermia (group QPre). The grouping of the mice is shown in Table 1.
3. Results 3.1. Number of hair follicles To investigate the progression of burn injuries, the healthy hair follicles of burn wounds were counted after 24 h (Fig. 1). The numbers of hair follicles of the studied groups are shown in Fig. 2. Group Pre had significantly more hair follicles in comparison with group Burn (po.001). The number of hair follicles of group NPre was significantly lower than that of group Pre (po0.001). The difference in the number of hair follicles between group NPre and group Burn was not significant. The number of hair follicles of group QPre was significantly less than that of group Pre (po0.001), but it was not significantly different from those of groups NPre and Burn. The number of hair follicles of group Healthy was significantly more than those of all the other groups (po0.001).
ARTICLE IN PRESS 288
N. Karimi et al. / Journal of Thermal Biology 34 (2009) 286–289
Fig. 1. Histological sections of the central area of the burned wounds 24 h after burn injury. (A) Group Burn (burn without any treatment, H & E, 20). (B) Group Pre (sublethal hyperthermia+burn, H & E, 20). (C) Group Healthy (without burn, H & E, 20). (D) Group NPre (NLX+sublethal hyperthermia+burn, H & E, 20). (E) Group QPre (quercetin+sublethal hyperthermia+burn, H & E, 20). All groups, except group Healthy (C), received a second-degree burn injury. Note the intact epidermal layer and hair follicles in group Healthy (C). The number of hair follicles in group Pre (B) was more than those of groups Burn (A), NPre (D) and QPre (E).
70
Hair Follicles
60 50
was expressed as a fraction of the mean expression of HSP-70 in group Healthy. The relative expression of HSP-70 in the studied groups 24 h after burn injury is shown in Fig. 3. The relative expression of HSP-70 in group Pre was significantly higher than those observed in groups Burn, NPre, QPre and Healthy (po0.001, po0.05, po0.001 and po0.001, respectively). The relative expression of HSP-70 of group NPre was significantly higher than those of groups Burn, QPre and Healthy (po0.05, po0.05 and po0.01, respectively). There was no significant difference between the relative expression of HSP-70 of groups Burn and QPre. The relative expressions of HSP-70 of groups Burn and QPre were higher than that of group Healthy, but these differences were not significant.
***vs. NPre ***vs. Burn ***vs. Pre ***vs. QPre ***vs. NPre ***vs. Burn ***vs. QPre
40 30 20 10 0
Healthy
QPre
Pre
Burn
NPre
Fig. 2. Means of the number of hair follicles in the burn wounds of the five groups 24 h after burn injury. Group Pre (sublethal hyperthermia+burn) had significantly more hair follicles in comparison with groups Burn (received only burn), NPre (NLX+sublethal hyperthermia+burn) and QPre (quercetin+sublethal hyperthermia+burn). The difference in the number of hair follicles between group NPre and group Burn was not significant. The number of hair follicles of group QPre was not significantly different from those of groups NPre and Burn. The number of hair follicles of group Healthy, which did not receive burns, was significantly more than those of any of the other groups. Data are means7SD; ***po0.001. One-way ANOVA test, n ¼ 3.
3.2. Measurement of the relative HSP-70 expression by western blotting analysis The mean expression of HSP-70 in group Healthy was normalized to 1, and relative expression of HSP-70 in other mice
4. Discussion According to above mentioned results, applying sublethal local hyperthermia before burn injury could prevent progression of injury. This conclusion is in agreement with results obtained by Topping et al. (2001), who showed the same effect of sublethal local hyperthermia before thermal injury. Our results indicate that blocking opioid receptors by administration of NLX inhibits protective effects of sublethal hyperthermia against progression of burn injury. This result is in concordance with previous studies that showed that in addition to HSPs, opioids and opioid receptors also have a significant role in the stress tolerance of cardiac tissue against ischemia. (Sommerschild and Kirkeboen, 2002; Sonneborn et al., 2004). Also this result is in line with the findings of Patel et al. (2002), who indicated that whole-body hyperthermia-induced
ARTICLE IN PRESS N. Karimi et al. / Journal of Thermal Biology 34 (2009) 286–289
Healthy
QPre
Pre
Burn
NPre
30
Relative Density
25 **vs. NPre ***vs. Burn ***vs. QPre ***vs. Healthy
20 15
**vs. Burn *vs. QPre ***vs. Healthy
10 5 0
Healthy
Qpre
Pre
Burn
NPre
Fig. 3. Effect of sublethal local hyperthermia on the expression of HSP-70. Seconddegree burn wounds were induced on the back of Balb/c mice. Expression of HSP70 was evaluated after 24 h using the western blot method. (A) Representative western blot. (B) Blots were quantified by Image J software. The mean expression of HSP-70 in group Healthy, which did not receive burns, was normalized to 1 and the relative expression of HSP-70 in other groups was expressed as a fraction of the mean expression of HSP-70 in group Healthy. The relative expression of HSP-70 of group Pre (sublethal hyperthermia+burn) was significantly more than those of groups Burn (received only burn), NPre (NLX+sublethal hyperthermia+burn), QPre (quercetin+sublethal hyperthermia+burn) and Healthy. The relative expression of HSP-70 of groups NPre was significantly more than those of groups Burn, QPre and Healthy. Data are means7SD; *po0.05, **po0.01, ***po0.001. One-way ANOVA test, n ¼ 8.
289
hyperthermia before thermal injury. However, to our knowledge, our study is the first to use an inhibitor of HSP synthesis to show the role of induction of HSP-70 in the protective effect of sublethal local hyperthermia against progression of burn injury. There was no significant difference between inhibition of the protective effects of sublethal hyperthermia through the blockade of opioid receptors or by blockade of HSP synthesis. Blocking either opioid receptors or HSP synthesis nearly completely inhibited the protective effects of sublethal hyperthermia. However, inhibition of HSP synthesis was significantly greater when HSP synthesis was directly blocked by the inhibitor of HSP synthesis than when opioid receptors were blocked. Expression of HSP-70 due to local sublethal hyperthermia was nearly completely inhibited by blocking HSP synthesis, whereas it was only partially inhibited when opioid receptors were blocked (Fig. 3). This finding suggests that the role of the endogenous opioid response in the protective effect of sublethal local hyperthermia, in addition to HSP synthesis, involves other mechanism(s). In conclusion, this study showed that sublethal hyperthermia before skin burn attenuates the progression of burn injury. This effect is mediated by both HSP synthesis and the endogenous opioid response. Our results suggest that the role of the endogenous opioid response in the protective effects of local sublethal hyperthermia involves HSP synthesis, but also involves other mechanisms. To our knowledge, this study is the first to show that the endogenous opioid response activated by sublethal hyperthermia plays an important role in the induction of HSPs; therefore, follow-up studies are needed to confirm these results. References
cardioprotection can be attenuated by NLX. According to our results there was no significant difference between inhibition of HSP-70 synthesis and blockade of opioid receptors in reduction of protective effects of sublethal hyperthermia on burn injury (Fig. 2). Therefore, it may be concluded that the role of the endogenous opioid response is as important as that of HSP synthesis in protective effects of sublethal hyperthermia on burn injury. The results show that blocking opioid receptors by administration of NLX attenuates induction of HSP-70 by sublethal hyperthermia. This finding suggests that stimulation of endogenous opioid receptors by local sublethal hyperthermia plays an important role in the induction of HSPs by local sublethal hyperthermia. This finding is consistent with previous studies showing that HSP-70 mediated the delayed cardioprotection of prior stimulation of the k opioid receptor (Zhou et al., 2001; Liu et al., 2004). However, this finding was not in line with the findings of Patel et al. (2002), which indicated that pretreatment with NLX before whole-body hyperthermia did not affect the expression of HSP-70. This disagreement between our results and the findings of Patel et al. may be due to the difference in the kind of hyperthermia used by Patel et al., (whole-body hyperthermia), and that used in our study (local hyperthermia). Another cause may be the difference between the tissue studied by Patel et al. (heart) and the tissue type used in our study, i.e., skin. Our results show that blocking HSP-70 synthesis with an HSP synthesis inhibitor inhibits the protective effects of sublethal hyperthermia against progression of burn injury and the expression of HSP-70 induced by sublethal hyperthermia. This conclusion is in agreement with results obtained by Topping et al. (Patel et al., 2002), who showed the same effect of sublethal local
Dyer, C., Roberts, D., 1990. Thermal trauma. Nurs. Clin. North Am. 25, 85–117. Ellis, R.J., Laskey, R.A., Lorimer, G.H. (Eds.), 1993. Molecular Chaperones. Chapman & Hall, London. Lindquist, S., 1986. The heat-shock response. Annu. Rev. Biochem. 55, 1151–1191. Liu, J., Kam, K.W., Zhou, J.J., Yan, W.Y., Chen, M., Wu, S., Wong, T.M., 2004. Effects of heat shock protein 70 activation by metabolic inhibition preconditioning or kappa-opioid receptor stimulation on Ca2+ homeostasis in rat ventricular myocytes subjected to ischemic insults. J. Pharmacol. Exp. Ther. 310, 606–613. Patel, H.H., Hsu, A., Gross, G.J., 2002. Attenuation of heat shock-induced cardioprotection by treatment with the opiate receptor antagonist naloxone. Am. J. Physiol. Heart Circ. Physiol. 282, H2011–H2017. Shahabi, S., Hashemi, M., Hassan, Z.M., Javan, M., Bathaie, S.Z., Toraihi, T., Zakeri, Z., Ilkhanizadeh, B., Jazani, N.H., 2006. The effect of post-burn local hyperthermia on the reducing burn injury: the possible role of opioids. Int. J. Hyperthermia 22, 421–431. Shakespeare, P., 2001. Burn wound healing and skin substitutes. Burns 27, 517–522. Sommerschild, H.T., Kirkeboen, K.A., 2002. Preconditioning-endogenous defence mechanisms of the heart. Acta Anaesthesiol. Scand. 46, 123–137. Sonneborn, J.S., Gottsch, H., Cubin, E., Oeltgen, P., Thomas, P., 2004. Alternative strategy for stress tolerance: opioids. J. Gerontol. A. Biol. Sci. Med. Sci. 59, 433–440. Stein, C., Hassan, A.H., Przewlocki, R., Gramsch, C., Peter, K., Herz, A., 1990. Opioids from immunocytes interact with receptors on sensory nerves to inhibit nociception in inflammation. Proc. Natl. Acad. Sci. USA 87, 5935–5939. Topping, A., Gault, D., Grobbelaar, A., Green, C., Sanders, R., Sibbons, P., Linge, C., 2001. Successful reduction in skin damage resulting from exposure to the normal-mode ruby laser in an animal model. Br. J. Plast. Surg. 54, 144–150. Way, W.L., Fields, H.L., Schumacher, M.A., 2001. Opioid analgesics and antagonists. In: Katzurg, B. (Ed.), Basic and Clinical Pharmacology. McGraw-Hill, New York, pp. 512–548. Wybran, J., Appelboom, T., Famaey, J.P., Govaerts, A., 1979. Suggestive evidence for receptors for morphine and methionine-enkephalin on normal human blood T lymphocytes. J. Immunol. 123, 1068–1070. Zhou, J.J., Pei, J.M., Wang, G.Y., Wu, S., Wang, W.P., Cho, C.H., Wong, T.M., 2001. Inducible HSP70 mediates delayed cardioprotection via U-50488H pretreatment in rat ventricular myocytes. Am. J. Physiol. Heart Circ. Physiol. 281, H40–H47.
Contents lists available at ScienceDirect
Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio
The role of endogenous opioids in the protective effects of local sublethal hyperthermia against the progression of burn injury Neda Karimi a, Zuhair Muhammad Hassan b, Morteza Behnam Rasuli a, Behrouz Ilkhanizadeh c, Shaker Salarilak d, Shahram Shahabi e, a
Department of Biology, Faculty of Sciences, Ferdowsi University of Mashad, Mashad, Iran Department of Immunology, School of Medical Sciences, Tarbiat Modarres University, Tehran, Iran c Department of Pathology, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran d Department of Health and Community Medicine, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran e Department of Microbiology, Immunology and Genetics, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran b
a r t i c l e in f o
a b s t r a c t
Article history: Received 14 January 2009 Accepted 31 March 2009
The aim of the present study was to evaluate the role of endogenous opioids in local sublethal hyperthermia-induced protection against burn injury. Second-degree burn wounds were induced on the back of Balb/c mice. Progression of burn injury and expression of heat shock protein (HSP)-70 were evaluated after 24 h. Both inhibition of HSP synthesis and blocking opioid receptors before applying local sublethal hyperthermia decreased the protective effects of sublethal hyperthermia against the progression of burn injury. Blocking opioid receptors attenuated induction of HSP-70 by sublethal hyperthermia. & 2009 Elsevier Ltd. All rights reserved.
Keywords: Sublethal hyperthermia Thermotolerance Burn Endogenous opioids HSP-70
1. Introduction Many studies have shown that if the environmental temperature rises severely, cells grown at normal temperature will die rapidly. This phenomenon is called ‘heat shock.’ If these cells are exposed to a sublethal temperature before being exposed to a lethal temperature, their resistance against heat shock will be increased due to the induction of the heat shock proteins (HSPs) at the sublethal temperature. This phenomenon is regarded as ‘heat shock tolerance’ (thermo tolerance; Lindquist, 1986; Ellis et al., 1993). Sublethal levels of other stresses can also induce stress tolerance when cells are exposed before being exposed to lethal stresses (Ellis et al., 1993). In addition to cells, tissues can also develop stress tolerance if they are exposed to a sublethal stress. Topping et al. (2001) have shown that if the skin is mildly warmed before being exposed to a high temperature, the thermal injury to the skin will be decreased as a result of the induction of HSPs. Also, if the myocardium undergoes a sublethal ischemia before undertaking a lethal ischemia, its resistance to the lethal ischemia will be increased. (Sommerschild and Kirkeboen, 2002).
Corresponding author. Tel.: +984413449548; fax: +984412780801.
E-mail addresses: [email protected], [email protected] (S. Shahabi). 0306-4565/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2009.03.007
It has been shown that in addition to HSPs, opioids and opioid receptors also have a significant role in the stress tolerance of cardiac tissue induced by ischemia and hyperthermia (Patel et al., 2002; Sommerschild and Kirkeboen, 2002; Sonneborn et al., 2004). Opioids are peptides that are mainly produced in the nervous system and are produced in lower quantities by cells of the immune system. Their receptors are on both nerve cells and immune cells (Wybran et al., 1979; Stein et al., 1990; Way et al., 2001). Skin burn can be regarded as a lethal heat shock to the skin. Cutaneous thermal injury initiates a pathophysiological response with a significant inflammatory component that involves several classes of chemical mediators. These mediators interact in a complex manner to cause the pain and secondary tissue damage associated with burn injury (Shakespeare, 2001). Hair follicles are epidermal appendages that have an important role in the healing process after a burn injury (Dyer and Roberts, 1990). Tissue necrosis (including necrosis of hair follicles) progresses after burning, and it is not limited to the time when the burn occurs (Topping et al., 2001; Shahabi et al., 2006). If a treatment can prevent progression of necrosis after the burn injury, it can prevent the rise in the number of necrotic hair follicles after burning. Thus, the count of hair follicles 24 h after the burn injury is used to assess the skin injury (Topping et al., 2001; Shahabi et al., 2006). We have previously shown that the endogenous opioid response plays an important role in the protective effects of
ARTICLE IN PRESS N. Karimi et al. / Journal of Thermal Biology 34 (2009) 286–289
post-burn local hyperthermia against the progression of seconddegree skin burn injury (Shahabi et al., 2006). The aims of the present study are to evaluate the role of the endogenous opioid response in the protective effects of pre-burn local sublethal hyperthermia against second-degree skin burn injury, and to test the hypothesis that the opioids play this role via induction of HSP70.
287
Table 1 Grouping of the mice. Treatment groups
Quercetin
NLX
Sublethal hyperthermia
Burn
Healthy Burn Pre NPre QPre
+
+
+ + +
+ + + +
2. Material and methods 2.1. Animals
2.6. Histological analysis
Forty male BALB/c mice (average weight of 20 g) aged 6 weeks were obtained from the Razi Institute (Karaj, Iran). The animals were depilated using a standard depilatory cream on their backs. All procedures on animals were conducted with approval from the research committee of the Faculty of Sciences, Ferdowsi University, Mashad, Iran.
Twenty-four hours after the burn injury, the central area of the wound together with 5 mm of the marginal tissue was harvested. The samples were fixed in a 10% buffered formalin solution and embedded in paraffin, and then 4 mm-thick sections were cut and stained with haematoxylin and eosin (H & E). The sections were assessed for the number of hair follicles. The assessor was blind to the allocation of the sections. Evaluation of the number of hair follicles was done by the following method: hair follicles in 10 fields ( 10 power) were counted, and the result was divided by 10.
2.2. Drugs Naloxone (NLX; Opioid Receptor Antagonist) was purchased from Sigma (USA). Mouse monoclonal anti-mouse HSP70 antibody was obtained from R&D systems (USA). Horseradish peroxidase-conjugated anti-mouse antibody, quercetin (HSP synthesis inhibitor) and diaminobenzidine (DAB) were purchased from Sigma (USA).
2.7. Western blotting for HSP-70 expression
The mice were anaesthetized by intra-peritoneal injection of ketamine/xylazine (100/10 mg/kg). Then local sublethal hyperthermia was induced by immersing the backs of the mice in 44 1C water for 15 min using the following procedure. The anesthetized mice were placed individually in a small wire-mesh basket in supine position. The basket was immersed in 44 1C water so deep that the backs of the mice were immersed in the water but the faces of the mice were out of the water.
HSP-70 expression was investigated using western blotting analysis. Equal amounts of the proteins (1.3 mg/ml) determined by Bradford assay from the lysate of burned skin were fractionated on 10% SDS-PAGE gels and transferred into a polyvinylidene difluoride membrane. After washing, the membrane was blocked with 10% bovine serum albumin at room temperature for 1 h and incubated with mouse monoclonal anti-HSP-70 (1:1000, R&D systems). After washing, the membrane was incubated with horseradish peroxidase-conjugated anti-mouse antibody (1:1000, Sigma) at room temperature for 1 h. After washing, the immunoreactive bands were detected by 0.5 mg/ml DAB and 0.1% H2O2 in PBS. After inverting the blots by NIH Image J software, the relative signal intensity was quantified by this software on a personal computer.
2.4. Induction of burn injury
2.8. Statistical analysis
Two hours after local sublethal hyperthermia, although the mice had not recovered completely from the first anesthesia but since they had become a little conscious, they were again anaesthetized by intra-peritoneal injection of ketamine/xylazine (100/10 mg/kg). No lethality had occurred due to anesthesia. The mice were securely placed into a burn template so that the dorsum was centered on the 2 1 cm2 window. The shaved skin on the back was immersed in 80 1C water for 4 s to achieve a second-degree burn.
Statistical analysis was performed using the Kruskal–Wallis test followed by the one-way ANOVA post-hoc LSD test. A value of po0.05 was considered to be statistically significant. All statistical analyses were conducted with SPSS 11.5 software.
2.3. Local sublethal hyperthermia
2.5. Grouping of the mice Five groups of mice were used, each of them consisting of eight animals. Burn wounds were induced for all except eight mice (group Healthy). Mice of the group Burn received no drugs or treatments before or after induction of the burn injury. Local sublethal hyperthermia was applied for 24 mice 2 h before induction of burn injury. Eight of these mice received no drugs before or after applying local sublethal hyperthermia (group Pre), whereas 16 of these mice received 5 mg/kg NLX i.p. 40 min before applying sublethal local hyperthermia (group NPre) or 100 mg/kg quercetin 6 h before applying sublethal local hyperthermia (group QPre). The grouping of the mice is shown in Table 1.
3. Results 3.1. Number of hair follicles To investigate the progression of burn injuries, the healthy hair follicles of burn wounds were counted after 24 h (Fig. 1). The numbers of hair follicles of the studied groups are shown in Fig. 2. Group Pre had significantly more hair follicles in comparison with group Burn (po.001). The number of hair follicles of group NPre was significantly lower than that of group Pre (po0.001). The difference in the number of hair follicles between group NPre and group Burn was not significant. The number of hair follicles of group QPre was significantly less than that of group Pre (po0.001), but it was not significantly different from those of groups NPre and Burn. The number of hair follicles of group Healthy was significantly more than those of all the other groups (po0.001).
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Fig. 1. Histological sections of the central area of the burned wounds 24 h after burn injury. (A) Group Burn (burn without any treatment, H & E, 20). (B) Group Pre (sublethal hyperthermia+burn, H & E, 20). (C) Group Healthy (without burn, H & E, 20). (D) Group NPre (NLX+sublethal hyperthermia+burn, H & E, 20). (E) Group QPre (quercetin+sublethal hyperthermia+burn, H & E, 20). All groups, except group Healthy (C), received a second-degree burn injury. Note the intact epidermal layer and hair follicles in group Healthy (C). The number of hair follicles in group Pre (B) was more than those of groups Burn (A), NPre (D) and QPre (E).
70
Hair Follicles
60 50
was expressed as a fraction of the mean expression of HSP-70 in group Healthy. The relative expression of HSP-70 in the studied groups 24 h after burn injury is shown in Fig. 3. The relative expression of HSP-70 in group Pre was significantly higher than those observed in groups Burn, NPre, QPre and Healthy (po0.001, po0.05, po0.001 and po0.001, respectively). The relative expression of HSP-70 of group NPre was significantly higher than those of groups Burn, QPre and Healthy (po0.05, po0.05 and po0.01, respectively). There was no significant difference between the relative expression of HSP-70 of groups Burn and QPre. The relative expressions of HSP-70 of groups Burn and QPre were higher than that of group Healthy, but these differences were not significant.
***vs. NPre ***vs. Burn ***vs. Pre ***vs. QPre ***vs. NPre ***vs. Burn ***vs. QPre
40 30 20 10 0
Healthy
QPre
Pre
Burn
NPre
Fig. 2. Means of the number of hair follicles in the burn wounds of the five groups 24 h after burn injury. Group Pre (sublethal hyperthermia+burn) had significantly more hair follicles in comparison with groups Burn (received only burn), NPre (NLX+sublethal hyperthermia+burn) and QPre (quercetin+sublethal hyperthermia+burn). The difference in the number of hair follicles between group NPre and group Burn was not significant. The number of hair follicles of group QPre was not significantly different from those of groups NPre and Burn. The number of hair follicles of group Healthy, which did not receive burns, was significantly more than those of any of the other groups. Data are means7SD; ***po0.001. One-way ANOVA test, n ¼ 3.
3.2. Measurement of the relative HSP-70 expression by western blotting analysis The mean expression of HSP-70 in group Healthy was normalized to 1, and relative expression of HSP-70 in other mice
4. Discussion According to above mentioned results, applying sublethal local hyperthermia before burn injury could prevent progression of injury. This conclusion is in agreement with results obtained by Topping et al. (2001), who showed the same effect of sublethal local hyperthermia before thermal injury. Our results indicate that blocking opioid receptors by administration of NLX inhibits protective effects of sublethal hyperthermia against progression of burn injury. This result is in concordance with previous studies that showed that in addition to HSPs, opioids and opioid receptors also have a significant role in the stress tolerance of cardiac tissue against ischemia. (Sommerschild and Kirkeboen, 2002; Sonneborn et al., 2004). Also this result is in line with the findings of Patel et al. (2002), who indicated that whole-body hyperthermia-induced
ARTICLE IN PRESS N. Karimi et al. / Journal of Thermal Biology 34 (2009) 286–289
Healthy
QPre
Pre
Burn
NPre
30
Relative Density
25 **vs. NPre ***vs. Burn ***vs. QPre ***vs. Healthy
20 15
**vs. Burn *vs. QPre ***vs. Healthy
10 5 0
Healthy
Qpre
Pre
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Fig. 3. Effect of sublethal local hyperthermia on the expression of HSP-70. Seconddegree burn wounds were induced on the back of Balb/c mice. Expression of HSP70 was evaluated after 24 h using the western blot method. (A) Representative western blot. (B) Blots were quantified by Image J software. The mean expression of HSP-70 in group Healthy, which did not receive burns, was normalized to 1 and the relative expression of HSP-70 in other groups was expressed as a fraction of the mean expression of HSP-70 in group Healthy. The relative expression of HSP-70 of group Pre (sublethal hyperthermia+burn) was significantly more than those of groups Burn (received only burn), NPre (NLX+sublethal hyperthermia+burn), QPre (quercetin+sublethal hyperthermia+burn) and Healthy. The relative expression of HSP-70 of groups NPre was significantly more than those of groups Burn, QPre and Healthy. Data are means7SD; *po0.05, **po0.01, ***po0.001. One-way ANOVA test, n ¼ 8.
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hyperthermia before thermal injury. However, to our knowledge, our study is the first to use an inhibitor of HSP synthesis to show the role of induction of HSP-70 in the protective effect of sublethal local hyperthermia against progression of burn injury. There was no significant difference between inhibition of the protective effects of sublethal hyperthermia through the blockade of opioid receptors or by blockade of HSP synthesis. Blocking either opioid receptors or HSP synthesis nearly completely inhibited the protective effects of sublethal hyperthermia. However, inhibition of HSP synthesis was significantly greater when HSP synthesis was directly blocked by the inhibitor of HSP synthesis than when opioid receptors were blocked. Expression of HSP-70 due to local sublethal hyperthermia was nearly completely inhibited by blocking HSP synthesis, whereas it was only partially inhibited when opioid receptors were blocked (Fig. 3). This finding suggests that the role of the endogenous opioid response in the protective effect of sublethal local hyperthermia, in addition to HSP synthesis, involves other mechanism(s). In conclusion, this study showed that sublethal hyperthermia before skin burn attenuates the progression of burn injury. This effect is mediated by both HSP synthesis and the endogenous opioid response. Our results suggest that the role of the endogenous opioid response in the protective effects of local sublethal hyperthermia involves HSP synthesis, but also involves other mechanisms. To our knowledge, this study is the first to show that the endogenous opioid response activated by sublethal hyperthermia plays an important role in the induction of HSPs; therefore, follow-up studies are needed to confirm these results. References
cardioprotection can be attenuated by NLX. According to our results there was no significant difference between inhibition of HSP-70 synthesis and blockade of opioid receptors in reduction of protective effects of sublethal hyperthermia on burn injury (Fig. 2). Therefore, it may be concluded that the role of the endogenous opioid response is as important as that of HSP synthesis in protective effects of sublethal hyperthermia on burn injury. The results show that blocking opioid receptors by administration of NLX attenuates induction of HSP-70 by sublethal hyperthermia. This finding suggests that stimulation of endogenous opioid receptors by local sublethal hyperthermia plays an important role in the induction of HSPs by local sublethal hyperthermia. This finding is consistent with previous studies showing that HSP-70 mediated the delayed cardioprotection of prior stimulation of the k opioid receptor (Zhou et al., 2001; Liu et al., 2004). However, this finding was not in line with the findings of Patel et al. (2002), which indicated that pretreatment with NLX before whole-body hyperthermia did not affect the expression of HSP-70. This disagreement between our results and the findings of Patel et al. may be due to the difference in the kind of hyperthermia used by Patel et al., (whole-body hyperthermia), and that used in our study (local hyperthermia). Another cause may be the difference between the tissue studied by Patel et al. (heart) and the tissue type used in our study, i.e., skin. Our results show that blocking HSP-70 synthesis with an HSP synthesis inhibitor inhibits the protective effects of sublethal hyperthermia against progression of burn injury and the expression of HSP-70 induced by sublethal hyperthermia. This conclusion is in agreement with results obtained by Topping et al. (Patel et al., 2002), who showed the same effect of sublethal local
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