保护性预热应激模型的创建及其信号转导通路机制的研究
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摘要
目的：探讨预热应激对大鼠中暑休克的保护作用及其信号转导机制
方法：实验分为两部分。第一部分：保护性预热应激模型的创建及信号转导通路作用的研究。此部分分为两个实验：1．预热应激(42℃，15min)对大鼠中暑休克的保护作用。30只SD雄性大鼠随机分为预热应激组(42℃，15min，HS组)、预热应激对照组(不给予预热应激处理，SC组)和正常体温对照组(NC组)，三组室温恢复20h。然后给予热暴露至其死亡，气象条件：干球温度(39.5±0.5)℃，湿球温度(34.0±0.5)℃，相对湿度(65±3)％。监测受热全程肛温等生理指标的变化及其生存时间、存活率，测定死亡时大脑皮层、心肌、肝脏的丙二醛(MDA)及热休克蛋白70(HSP70)的含量；2．预热应激后信号转导通路的研究。32只SD雄性大鼠随机分为HS组、SC组、NC组和蛋白激酶C抑制剂组(预热前10min注入蛋白激酶C抑制剂chelerythrine chloride，PKC组)，同样室温恢复20h。给予热暴露，气象条件同前，受试时间为73min。期间全程监测生理指标，测定实验终止时大脑皮层、心肌和血清中MDA、一氧化氮(NO)含量。第二部分：保护性预热应激模型建立过程中信号转导作用机制的研究。此部分亦分为两个实验：1．保护性预热应激后PKC介导的细胞内信号转导通路中肿瘤坏死因子(TNF)、NO的研究。66只SD雄性大鼠随机分为预热应激后0h、2h、4h、8h、12h、24h组以及各时间点的对照组。检测各时间点血清TNF、NO的含量；2．保护性预热应激模型建立过程中PKC介导的细胞内信号转导通路对HSP70表达的影响。24只SD雄性大鼠随机分为HS组，NC组和PKC组，室温恢复20h后测定大脑皮层、心肌HSP70的含量。
结果：第一部分：1．HS组热暴露后的生存时间、存活率明显高于SC组。中暑后，HS组血压、心率高于SC组，而肛温却明显低于SC组。死亡时，大脑皮层和肝脏MDA、HSP70含量较SC组明显增加；2．中暑
后，相对于SC组，HS组表现出更高的血压、心率值，而肛温却明显下
降，大脑皮层、心肌、血清中MDA、NO的含量亦显著降低。相对于
HS组，PKC组表现出更低的血压、心率值，肛温却明显升高，大脑皮
层、心肌和血清中MDA、NO含量均明显高于HS组。第二部分：1．预
热应激后，大鼠血清 WF 4h达到峰值，NO sh达到峰值，并均较受热前
明显升高；2．HS组心肌、大脑皮层HsP7二表达量较NC组显著增加，
而PKC组心肌、大脑皮层HSP72的含量明显低于HS组。
结论：1．预热应激（42 oC，15min）可以有效延缓热暴露大鼠中暑
休克的到来，提高大鼠热耐受能力，降低中暑死亡率，填补了国内研究
空白；2．预热应激增强了机体心脏储备，提高了心血管系统功能；3．预
热应激可有效减轻中暑后体温调节中枢的损害，从而减轻中暑后机体体
温调节能力的下降；4．预热应激可减轻中暑诱导的MDA、NO对机体的
损害，同时还可提高机体对MDA的耐受能力，此研究结果国内外尚无
报道：5．首次提出保护性热应激模型的概念：给予动物适当、短暂的热
应激，从而使机体在细胞、器官、整体水平增强对抗外界有害应激的能
力；6．首次观察到预热应激可在多器官组织水平激活PKC，并通过其诱
导多器官组织HSP72的大量表达，从而使机体热耐受能力提高；7提出
了“ TNF、NO可能是预热应激激活 PKC介导的信号转导通路的触发因
子”的新见解。8．总结归纳出预热应激保护作用可能的信号转导通路图。
Objective To investigate protective effects of heat stress preconditioning on the heatstroke in rats and the mechanism of signal transduction about heat stress preconditioning.
Methods The experiment consisted of two parts. The first part:the establishment of protective heat stress preconditioning model and investigation of effects of signal transduction pathway. This part included two experiments:1. Protective effects of heat stress preconditioning(42C,15min) on heatstroke in rats. 30 SD male rats were randomly divided into 3 groups:heat stress preconditioning group(42C,15min,HS group),sham heat stress control group(without heat stress preconditioning,SC group) and normal temperature control group(NC group). Three groups recovered for 20h at room temperature. Then all groups exposed to death in thermal environment(Td 39.5+0.5C,Tw 34.0+0.5C,relative humidity 65+3%). The existent time(interval between onset of heat stress and death),survival rate,the rectal temperatrue(Tr),heart rate(HR),mean arterial pressure(MAP) were measured continuously during heat exposure. The cerebral cortex,heart and liver were removed at the end of the experiment for detection of MDA and HSP70;2.1nvestigat
ion of signal transduction pathway after heat stress preconditioning. 32 SD male rats were randomly divided into 4 groups:HS group,SC group,NC group and protein kinase C inhibitor group(inject protein kinase C inhibitor-chelerythrine chloride into rats' abdominal cavity lOmin before heat stress preconditioning,PKC group). Four groups recovered for 20h at room temperature. Then all groups exposed in thermal environment(Td 39.5+0.5C,Tw 34.0+0.5C,relative humidity 65 + 3%) for 73 min. The physiological index were measured continuously. The cerebral cortex,serum and heart were removed at the end of the experiment for detection of MDA and NO. The second part:Investigation of the
mechanism of signal transduction during establishment of protective heat stress preconditioning model. This part included two experiments:1. Study of tumor necrosis factor(TNF) and NO in PKC signal transduction pathway after heat stress preconditioning. 66 SD male rats were randomly divided into Oh group,2h group,4h group,8h group,12h group,24h group and control groups of each time point. The serum were separated and the concentration of TNF and NO were measured between Oh and 24h after heat stress preconditioning respectively. 2. Influence of PKC signal transduction pathway on the expression of HSP72 during establishment of the model. 24 SD male rats were randomly divided into 3 groups:HS group,NC group and PKC group. All groups recovered for 20h at room temperature. Then HSP72 of the cerebral cortex and myocardium were detection.
Results The first part:1. Compared with SC group,existent time and survival rate increased significantly in HS group after heat exposure. MAP and HR were higher,but Tr was lower in HS group after heatstroke. At the end of experiments the content of MDA and HSP70 in the cerebral cortex and liver increased significantly vs SC group. 2. Compared with SC group,MAP,HR were higher,but Tr and MDA,NO in cerebral cortex,myocardium and serum was lower in HS group. Compared with HS group,the content of MDA and NO in the cerebral cortex,serum and myocardium increased significantly in PKC group,however MAP and HR was lower. The second part:1. The concentration of TNF reached the peak 4h after hat stress preconditioning. The concentration of NO amounted to peak 8h after hat stress preconditioning. 2. compared with NC group,the content of HSP72 in cerebral cortex and myocardium increased significantly in HS group. After application of chelerythrine chloride(a potent,selective PKC inhibitor),the content of HSP72 in cerebral cortex and myocardium decreased significantly compared with HS group.
Conclusion 1. Heat stress preconditioning(42C,15min) delayed the
heatstroke,enhanced thermotolerance and decreased death rate after heatstroke,which filled up the vacancy in China. 2. Heat stress preconditioning could enhance heart reserve and
方法：实验分为两部分。第一部分：保护性预热应激模型的创建及信号转导通路作用的研究。此部分分为两个实验：1．预热应激(42℃，15min)对大鼠中暑休克的保护作用。30只SD雄性大鼠随机分为预热应激组(42℃，15min，HS组)、预热应激对照组(不给予预热应激处理，SC组)和正常体温对照组(NC组)，三组室温恢复20h。然后给予热暴露至其死亡，气象条件：干球温度(39.5±0.5)℃，湿球温度(34.0±0.5)℃，相对湿度(65±3)％。监测受热全程肛温等生理指标的变化及其生存时间、存活率，测定死亡时大脑皮层、心肌、肝脏的丙二醛(MDA)及热休克蛋白70(HSP70)的含量；2．预热应激后信号转导通路的研究。32只SD雄性大鼠随机分为HS组、SC组、NC组和蛋白激酶C抑制剂组(预热前10min注入蛋白激酶C抑制剂chelerythrine chloride，PKC组)，同样室温恢复20h。给予热暴露，气象条件同前，受试时间为73min。期间全程监测生理指标，测定实验终止时大脑皮层、心肌和血清中MDA、一氧化氮(NO)含量。第二部分：保护性预热应激模型建立过程中信号转导作用机制的研究。此部分亦分为两个实验：1．保护性预热应激后PKC介导的细胞内信号转导通路中肿瘤坏死因子(TNF)、NO的研究。66只SD雄性大鼠随机分为预热应激后0h、2h、4h、8h、12h、24h组以及各时间点的对照组。检测各时间点血清TNF、NO的含量；2．保护性预热应激模型建立过程中PKC介导的细胞内信号转导通路对HSP70表达的影响。24只SD雄性大鼠随机分为HS组，NC组和PKC组，室温恢复20h后测定大脑皮层、心肌HSP70的含量。
结果：第一部分：1．HS组热暴露后的生存时间、存活率明显高于SC组。中暑后，HS组血压、心率高于SC组，而肛温却明显低于SC组。死亡时，大脑皮层和肝脏MDA、HSP70含量较SC组明显增加；2．中暑
后，相对于SC组，HS组表现出更高的血压、心率值，而肛温却明显下
降，大脑皮层、心肌、血清中MDA、NO的含量亦显著降低。相对于
HS组，PKC组表现出更低的血压、心率值，肛温却明显升高，大脑皮
层、心肌和血清中MDA、NO含量均明显高于HS组。第二部分：1．预
热应激后，大鼠血清 WF 4h达到峰值，NO sh达到峰值，并均较受热前
明显升高；2．HS组心肌、大脑皮层HsP7二表达量较NC组显著增加，
而PKC组心肌、大脑皮层HSP72的含量明显低于HS组。
结论：1．预热应激（42 oC，15min）可以有效延缓热暴露大鼠中暑
休克的到来，提高大鼠热耐受能力，降低中暑死亡率，填补了国内研究
空白；2．预热应激增强了机体心脏储备，提高了心血管系统功能；3．预
热应激可有效减轻中暑后体温调节中枢的损害，从而减轻中暑后机体体
温调节能力的下降；4．预热应激可减轻中暑诱导的MDA、NO对机体的
损害，同时还可提高机体对MDA的耐受能力，此研究结果国内外尚无
报道：5．首次提出保护性热应激模型的概念：给予动物适当、短暂的热
应激，从而使机体在细胞、器官、整体水平增强对抗外界有害应激的能
力；6．首次观察到预热应激可在多器官组织水平激活PKC，并通过其诱
导多器官组织HSP72的大量表达，从而使机体热耐受能力提高；7提出
了“ TNF、NO可能是预热应激激活 PKC介导的信号转导通路的触发因
子”的新见解。8．总结归纳出预热应激保护作用可能的信号转导通路图。
Objective To investigate protective effects of heat stress preconditioning on the heatstroke in rats and the mechanism of signal transduction about heat stress preconditioning.
Methods The experiment consisted of two parts. The first part:the establishment of protective heat stress preconditioning model and investigation of effects of signal transduction pathway. This part included two experiments:1. Protective effects of heat stress preconditioning(42C,15min) on heatstroke in rats. 30 SD male rats were randomly divided into 3 groups:heat stress preconditioning group(42C,15min,HS group),sham heat stress control group(without heat stress preconditioning,SC group) and normal temperature control group(NC group). Three groups recovered for 20h at room temperature. Then all groups exposed to death in thermal environment(Td 39.5+0.5C,Tw 34.0+0.5C,relative humidity 65+3%). The existent time(interval between onset of heat stress and death),survival rate,the rectal temperatrue(Tr),heart rate(HR),mean arterial pressure(MAP) were measured continuously during heat exposure. The cerebral cortex,heart and liver were removed at the end of the experiment for detection of MDA and HSP70;2.1nvestigat
ion of signal transduction pathway after heat stress preconditioning. 32 SD male rats were randomly divided into 4 groups:HS group,SC group,NC group and protein kinase C inhibitor group(inject protein kinase C inhibitor-chelerythrine chloride into rats' abdominal cavity lOmin before heat stress preconditioning,PKC group). Four groups recovered for 20h at room temperature. Then all groups exposed in thermal environment(Td 39.5+0.5C,Tw 34.0+0.5C,relative humidity 65 + 3%) for 73 min. The physiological index were measured continuously. The cerebral cortex,serum and heart were removed at the end of the experiment for detection of MDA and NO. The second part:Investigation of the
mechanism of signal transduction during establishment of protective heat stress preconditioning model. This part included two experiments:1. Study of tumor necrosis factor(TNF) and NO in PKC signal transduction pathway after heat stress preconditioning. 66 SD male rats were randomly divided into Oh group,2h group,4h group,8h group,12h group,24h group and control groups of each time point. The serum were separated and the concentration of TNF and NO were measured between Oh and 24h after heat stress preconditioning respectively. 2. Influence of PKC signal transduction pathway on the expression of HSP72 during establishment of the model. 24 SD male rats were randomly divided into 3 groups:HS group,NC group and PKC group. All groups recovered for 20h at room temperature. Then HSP72 of the cerebral cortex and myocardium were detection.
Results The first part:1. Compared with SC group,existent time and survival rate increased significantly in HS group after heat exposure. MAP and HR were higher,but Tr was lower in HS group after heatstroke. At the end of experiments the content of MDA and HSP70 in the cerebral cortex and liver increased significantly vs SC group. 2. Compared with SC group,MAP,HR were higher,but Tr and MDA,NO in cerebral cortex,myocardium and serum was lower in HS group. Compared with HS group,the content of MDA and NO in the cerebral cortex,serum and myocardium increased significantly in PKC group,however MAP and HR was lower. The second part:1. The concentration of TNF reached the peak 4h after hat stress preconditioning. The concentration of NO amounted to peak 8h after hat stress preconditioning. 2. compared with NC group,the content of HSP72 in cerebral cortex and myocardium increased significantly in HS group. After application of chelerythrine chloride(a potent,selective PKC inhibitor),the content of HSP72 in cerebral cortex and myocardium decreased significantly compared with HS group.
Conclusion 1. Heat stress preconditioning(42C,15min) delayed the
heatstroke,enhanced thermotolerance and decreased death rate after heatstroke,which filled up the vacancy in China. 2. Heat stress preconditioning could enhance heart reserve and
引文
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49 朱冠山，秦一中，万谟彬，等．TNF对小鼠急性肝坏死的双重作用．第二军医大学学报，1997，18(2)：173-6．
50 Malyshev IYu, Manukhina EB, MikoyanVD, et al. Nitric oxide is involved in heat-induced HSP70 accumulation. FEBS Lett, 1995, 370(3):159-62.
51 Membrillo-Hernandez HJ, Coopamah MD, Anjum MF, et al. The flavohemoglobin of Escherichia coli confers resistance to a nitrosating agent, a "Nitric oxide Releaser," and paraquat and is essential for transcriptional responses to oxidative stress. J Biol Chem, 1999,274(2):748-54.
52 Kitakaze M, Hori M, Takashima S, et al. Ischemic preconditioning increases adenosine release and 5'-nucleotidase activity during myocardial ischemia and reperfusion in dogs. Circulation, 1993, 87(1):208-15.
53 车玉英，贾国良，陈士良．缺血预处理和“腺苷预处理”对兔心肌梗塞面积的影响．中国危重病急救医学，1997，9(9)：515．
54 Maulik N, Engelman DT, Watanabe M, et al. Nitric oxide/carbon monoxide. A molecular switch for myocardial preservation during ischemia. Circulation, 1996, 94(9 Suppl): Ⅱ398-406.
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44 Munro S, Pelham HR. An hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell, 1986, 46(2): 291-300.
45 Martin J, Langer T, Boteva R, etal Hartl FU.Chaperonin-mediated protein folding at the surface of groEL through a 'molten globule'-like intermediate. Nature, 1991, 352(6330): 36-42.
46 Ellis R J, van der Vies SM.Molecular chaperones. Annu. Rev. Biochem, 1991 (60): 321-47.
47 Kone BC. Nitric oxide in renal health and disease. Am J Kidney Dis, 1997, 30(3): 311-3.
48 Nakano M, Knowlton AA, Dibbs Z, et al. Tumor necrosis factor-alpha confers resistance to hypoxic injury in the adult mammalian cardiac myocyte. Circulation, 1998, 97(14): 1392-1400.
49 朱冠山，秦一中，万谟彬，等．TNF对小鼠急性肝坏死的双重作用．第二军医大学学报，1997，18(2)：173-6．
50 Malyshev IYu, Manukhina EB, MikoyanVD, et al. Nitric oxide is involved in heat-induced HSP70 accumulation. FEBS Lett, 1995, 370(3):159-62.
51 Membrillo-Hernandez HJ, Coopamah MD, Anjum MF, et al. The flavohemoglobin of Escherichia coli confers resistance to a nitrosating agent, a "Nitric oxide Releaser," and paraquat and is essential for transcriptional responses to oxidative stress. J Biol Chem, 1999,274(2):748-54.
52 Kitakaze M, Hori M, Takashima S, et al. Ischemic preconditioning increases adenosine release and 5'-nucleotidase activity during myocardial ischemia and reperfusion in dogs. Circulation, 1993, 87(1):208-15.
53 车玉英，贾国良，陈士良．缺血预处理和“腺苷预处理”对兔心肌梗塞面积的影响．中国危重病急救医学，1997，9(9)：515．
54 Maulik N, Engelman DT, Watanabe M, et al. Nitric oxide/carbon monoxide. A molecular switch for myocardial preservation during ischemia. Circulation, 1996, 94(9 Suppl): Ⅱ398-406.