【作者】 徐竞；

【导师】 刘良明；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 严重创伤/休克失代偿期常出现血管低反应性,主要表现为全身血管对缩血管物质和舒血管物质的反应降低或不反应,本实验室前期研究发现,除了以往认识的血管低反应性发生的血管平滑肌细胞(vascular smooth muscle cell,VSMC)的K+和Ca2+通道功能障碍、VSMC膜超极化之外,钙失敏(Ca2+ desensitization),即血管平滑肌细胞收缩蛋白对钙的敏感性(calcium sensitivity)降低,也就是力/钙比率(Force/Ca2+ ratio)和肌肉收缩效率的降低,也是休克血管低反应性发生的重要原因;蛋白激酶C (Protein kinase C,PKC)参与了休克后血管反应性和钙敏感性的调控。PKC是一组磷脂依赖性的钙激活的蛋白丝氨酸/苏氨酸激酶,按生化性质及结构可分为三大类:传统型PKC (classic PKC, cPKC):包括PKCα、βⅡ、βⅠ、γ;新型PKC (novel PKC, nPKC):包括PKCδ、ε、η、θ;非典型PKC (atypical PKC, aPKC):包括PKCζ、ι、λ。在大鼠主动脉和肠系膜上动脉发现,主要表达PKCα、PKCδ、PKCε和PKCζ四种收缩功能相关亚型。但是哪些亚型参与失血性休克后血管反应性和钙敏感性的调节,其机制如何?尚不清楚。据此,我们利用大鼠失血性休克模型和缺氧处理体外培养血管平滑肌细胞模型,研究了参与失血性休克后钙敏感性和血管反应性调节的PKC亚型,并从MLC20磷酸化的非钙依赖性调节途径入手研究其机制,以及缺血预适应和药物预处理对其的诱导作用。主要内容包括三部分:①明确参与失血性休克后血管反应性和钙敏感性调节的PKC亚型;②研究相关PKC亚型调节休克血管钙敏感性的MLC20磷酸化机制;③观察缺血预适应和吡那地尔预处理对PKC的诱导作用及其对失血性休克大鼠血管反应性和钙敏感性的保护作用。主要实验方法:第一部分明确参与失血性休克后血管反应性和钙敏感性调节的PKC亚型1.采用失血性休克大鼠肠系膜上动脉(superior mesenteric artery, SMA)的一级分支血管环,测定不同休克时间点(40 mmHg,即时、30min、1 h、2 h、4 h)血管环的血管反应性(用血管环对梯度浓度NE的收缩反应反映)和钙敏感性(用血管环对梯度浓度钙离子的收缩反应反映),观察变化规律。2.采用失血性休克大鼠(40 mmHg, 2 h)的肠系膜上动脉的一级分支血管环,观察各亚型PKC的激动剂和抑制剂对休克2h血管反应性、钙敏感性的影响。3.采用失血性休克大鼠肠系膜上动脉,测定休克不同时间点(40 mmHg, ,即时、30min、1 h、2 h、4 h)相关亚型PKC的mRNA表达、在胞浆和胞膜的蛋白表达,观察变化规律。第二部分相关亚型PKC调控失血性休克大鼠血管钙敏感性的MLC20磷酸化机制1.采用失血性休克大鼠(40 mmHg, 2 h)的肠系膜上动脉一级分支血管环,观察CPI-17、ILK和ZIPK的拮抗剂(褪膜后应用CPI-17、ILK和ZIPK的中和抗体)对相关亚型PKC激动剂调节休克血管敏感性作用的影响,明确相关亚型PKC调控休克血管钙敏感性是否与CPI-17、ILK和ZIPK有关。2.采用缺氧培养的肠系膜上动脉VSMC (缺氧处理2h),观察相关亚型PKC激动剂对缺氧后CPI-17磷酸化和表达、以及ILK、ZIPK的活性和表达的影响,明确相关亚型PKC究竟通过是影响CPI-17、ILK和ZIPK的活性还是表达来调控钙敏感性。3.采用缺氧培养的肠系膜上动脉VSMC (缺氧处理2h),应用免疫共沉淀和western blot技术观察缺氧2h后,以及相关亚型PKC激动剂处理后,CPI-17、ILK、ZIPK和相关亚型PKC之间的相互作用关系,以及CPI-17、ILK和ZIPK之间的相互作用关系。4.采用缺氧培养的肠系膜上动脉VSMC (缺氧处理2h),观察相关亚型PKC激动剂及CPI-17、ILK和ZIPK抑制剂对缺氧血管平滑肌细胞MLCP活性的影响;采用失血性休克大鼠(40 mmHg, 2 h)的肠系膜上动脉,观察相关亚型PKC激动剂及CPI-17、ILK和ZIPK抑制剂对休克后血管MLC20磷酸化的影响,分析CPI-17、ILK和IPK调节休克后血管钙敏感性与MLCP和MLC20的关系。第三部分观察缺血预适应和吡那地尔预处理对PKC的诱导作用及其对失血性休克大鼠血管反应性和钙敏感性的保护作用1.观察不同失血量(2.5%、5%、10%)预处理不同时间(30min、1h、2h、3h)后,以及不同剂量吡那地尔(12μg/kg体重、25μg/kg体重、50μg/kg体重)预处理不同时间(30min、1h、2h、3h)对失血性休克大鼠(40 mmHg,2 h)的NE (3μg/kg体重)的升压作用和缩血管作用的影响,以及对肠系膜上动脉一级分支血管环的血管反应性和钙敏感性的影响,确定可诱导对失血性休克后血管反应性保护作用的缺血预适应和吡那地尔预处理条件。2.观察相关亚型PKC拮抗剂对缺血预适应和吡那地尔预处理(5%失血量以及25μg/kg体重吡那地尔预处理30min)诱导的对失血性休克大鼠(40 mmHg,2 h)血管反应性和钙敏感性保护作用的影响。以及观察缺血预适应和吡那地尔预处理对失血性休克大鼠(40 mmHg, 2 h)肠系膜上动脉相关亚型PKC胞浆和胞膜蛋白表达的影响。主要结果:一、参与失血性休克后血管反应性和钙敏感性调节的PKC亚型1.失血性休克后大鼠肠系膜上动脉对NE和Ca2+的收缩反应性在休克早期增高(P<0.01),从休克30min开始降低(P<0.01),并随着休克时间延长逐渐降低(P<0.01)。2. PKCα激动剂thymelea toxin、PKCε激动剂carbachol和PKCδ的非特异性激动剂PMA可显著增高休克2h大鼠肠系膜上动脉对NE和Ca2+的收缩反应性(P <0.01),PKCα抑制剂G?-6976和PKCε假底物抑制肽可显著降低休克大鼠肠系膜上动脉对NE和Ca2+的收缩反应性(P <0.01),但PKCζ的激动剂和抑制剂不改变休克大鼠肠系膜上动脉对NE和Ca2+的收缩反应性,PKCδ的抑制剂不能抑制PMA的增高休克大鼠肠系膜上动脉对NE和Ca2+收缩反应性的作用。3. PKCα和PKCε的mRNA表达在失血性休克后逐渐增高(P<0.01)。失血性休克后,PKCα和PKCε在胞浆部分的蛋白表达降低(P<0.01),而在胞膜部分的蛋白表达增高(P<0.01),呈现由胞浆向胞膜的转位。二、相关亚型PKC调控失血性休克大鼠血管钙敏感性的MLC20磷酸化机制1. PKCα激动剂thymelea toxin和PKCε激动剂carbachol可部分恢复休克大鼠肠系膜上动脉的钙敏感性(P<0.01),CPI-17、ILK和ZIPK的中和抗体可消除PKCα和ε激动剂对休克血管钙敏感性的恢复作用(P<0.01)。2.缺氧2h的VSMC中CPI-17、ILK和ZIPK的蛋白表达、CPI-17磷酸化、ILK和ZIPK活性均降低(P<0.01),PKCα激动剂thymelea toxin和PKCε激动剂carbachol可增高其蛋白表达,增高CPI-17磷酸化、以及ILK和ZIPK活性(P<0.01)。3. PKCα的免疫沉淀中可杂交出ILK和ZIPK,尤其是ILK的蛋白条带,不能杂交出CPI-17的蛋白条带,PKCε的免疫沉淀中可杂交出ILK和ZIPK的蛋白条带,也不能杂交出CPI-17的蛋白条带,CPI-17的免疫沉淀中可杂交出ILK和ZIPK的蛋白条带,ILK的免疫沉淀中可杂交出ZIPK的蛋白条带。4.缺氧2h的VSMC中,MLCP活性显著增高(P<0.01),休克2h的大鼠肠系膜上动脉中MLC20磷酸化显著降低(P<0.01),PKCα和PKCε激动剂可降低缺氧后的MLCP活性(P<0.01),增高休克后的MLC20磷酸化(P<0.01),CPI-17、ILK和ZIPK的中和抗体可逆转PKCα和PKCε激动剂对缺氧后MLCP活性和MLC20磷酸化的作用(P<0.01)。三、缺血预适应和吡那地尔预处理对PKC的诱导作用及其对失血性休克大鼠血管反应性和钙敏感性的保护作用1.缺血预处理和吡那地尔预处理可改善失血性休克2h后NE诱导的升压效应和缩血管效应,改善SMA的血管反应性和钙敏感性,以5%失血量预适应30min和25μg/kg吡那地尔预处理30min效果最好(P<0.01)。2. PKCα抑制剂G?-6976和PKCε假底物抑制肽可显著抑制缺血预适应(5%失血量预适应30min)和吡那地尔预处理(25μg/kg吡那地尔预处理30min)诱导的对失血性休克2h后SMA一级分支血管反应性和钙敏感性的保护作用(P<0.01)。缺血预适应和吡那地尔预处理可诱导失血性休克后的PKCα和PKCε由胞浆向胞膜转位(P<0.01)。结论:1. PKCα和PKCε是参与大鼠失血性休克后血管反应性和钙敏感性调节的主要PKC亚型,也是失血性休克后血管反应性的重要内源性保护分子。2.失血性休克后,PKCα和PKCε可直接作用于ILK和ZIPK,也可通过ILK和ZIPK间接作用于CPI-17,改变CPI-17、ILK和ZIPK的蛋白表达和活性,调节MLCP活性,进而调节MLC20磷酸化,最终调节失血性休克大鼠的血管反应性和钙敏感性。3.缺血预适应和吡那地尔预处理可诱导大鼠肠系膜上动脉PKCα和PKCε由胞浆向胞膜的转位和活化,从而诱导对失血性休克后血管反应性和钙敏感性的保护效应。更多还原

【Abstract】 After severe trauma or shock, including hemorrhagic, endotoxic and septic shock, vascular reactivity to vasoconstrictors and vasodilators is greatly reduced. Our previous studies showed that the vascular hyporeactivity is not only caused by the functional disorder of the K+ and Ca2+ cannels in vascular smooth muscle cell (VSMC), the hyperpolarization of VSMC membrane, but also by the calcium desensitization (the decrease of force/Ca2+ ratio) in VSMC. Our previous study showed that calcium desensitization is one of the important mechanisms responsible for the occurrence of vascular hyporeactivity, PKC take part in the regulation of vascular reactivity and calcium sensitization following hemorrhagic shock (HS). Protein kinase C (PKC) is a widely distributed protein serine/threonine kinase with broad substrate specificity, it comprises of a family of at least 12 isozymes, which are classified into three main groups: conventional, novel and atypical PKC. Conventional PKC are the Ca2+-dependent isozymesα,β?,β??,γ. Novel PKC are the Ca2+-independent isoformsδ,ε,η,θ,μ. The atypical isozymes are PKCζ,ι,λ. Among these numerous isoforms, only PKCα,ε,δandζare identified as the contraction-related PKC isoforms in aorta and superior mesenteric artery of rats, While which isoform plays an important role in the regulation of calcium desensitivity and vascular hyporeactivity following hemorrhagic shock and what is the precise mechanism is still unknown. So with hemorrhagic shock model of rats and hypoxia-treated VSMCs, we investigated the PKC isoforms responsible for the regulation of the vascular reactivity and calcium sensitivity following hemorrhagic shock and its calcium-independent regulatory mechanism through the 20kDa myosin light chain (MLC20) phosphorylation signal transduction pathway, and the induction effects by ischemia precondition and pinacidil pretreatment. The experiments were conducted in three parts:①To identify the PKC isoforms responsible for the regulation of the vascular reactivity and calcium sensitivity following hemorrhagic shock.②To investigate the regulatory mechanism of the related PKC isoforms on vascular reactivity and calcium sensitivity.③To observe the induction effects of ischemia precondition and pinacidil pretreatment on the related PKC isoforms, and their protective effects on vascular reactivity and calcium sensitivity after hemorrhagic shock.Methods:Part I. To identify the PKC isoforms responsible for the regulation of the vascular reactivity and calcium sensitivity following hemorrhagic shock.1. The first class arborizations of superior mesenteric artery (SMA) from normal and hemorrhagic shock rats (40mmHg) at different time after shock (immediately, 30min, 1h, 2h and 4h) were adopted to assay the vascular reactivity and calcium sensitivity via observing the contraction initiated by norepinephrine (NE) and Ca2+ with isolated organ perfusion system.2. The first class arborizations of SMA from hemorrhagic shock rats (40mmHg, 2h) were adopted to observe the effects of agonists and antagonists of PKCα,ε,δandζon the vascular reactivity and calcium sensitivity following HS.3. The SMAs from normal and hemorrhagic shock rats (40mmHg) at different time after shock (immediately, 30min, 1h, 2h and 4h) were adopted to determine the mRNA, protein expression and distribution of the related PKC isoforms using RT-PCR and western blot technique.Part II. To investigate the regulatory mechanism of the related PKC isoforms on vascular reactivity and calcium sensitivity through the MLC20 phosphorylation signal transduction pathway.1. The first class arborizations of SMA from hemorrhagic shock rats (40mmHg, 2h) were adopted to observe the influence of inhibitors of PKC–potentiated phosphatase inhibitor of 17 kDa (CPI-17), integrin-linked kinase (ILK) and zipper-interacting protein kinase (ZIPK) (using their neutralizing antibodies after permeabilization) on the effects of the related PKC isoforms agonists on calcium sensitivity after shock.2. Two hours hypoxia-treated vascular smooth muscle cells (VSMCs) were adopted to measure the protein expression of CPI-17, ILK and ZIPK, the phosphorylation of CPI-17, and the activity of ILK and ZIPK after applying the agonists of the related PKC isoforms following hypoxia via western blot and substrate phosphorylation.3. Two hours hypoxia-treated VSMCs were adopted to observe the direct effect of CPI-17, ILK and ZIPK, and their relationship to PKC isoforms via co-immunoprecipitation and immunoblot analysis.4. Two hours hypoxia-treated VSMCs were adopted to determine the myosin light chain phosphatase (MLCP) activity, and SMA from hemorrhagic shock rats (40mmHg, 2h) were adopted to determine the MLC20 phosphorylation after applying the agonists of the related PKC isoforms and the inhibitors of CPI-17, ILK and ZIPK via substrate phosphorylation and western blot.Part III. To observe the induction effects of ischemia precondition and pinacidil pretreatment on the related PKC isoforms, and their protective effects on vascular reactivity and calcium sensitivity after hemorrhagic shock.1. The NE-induced pressor response and vasoconstriction response, the vascular reactivity and calcium sensitivity of the first class arborization of SMA after ischemia precondition with different hemorrhage (2.5%, 5%, 10%), and pinacidil pretreatment with different dosage (12μg/kg body weight, 25μg/kg body weight, 50μg/kg body weight) implemented at different time before hemorrhagic shock (30min, 1h, 2h and 3h) were measured to determine the optimal ischemia precondition and pinacidil pretreatment which induce the protection against vascular hyporeactivity and calcium desensitization after hemorrhagic shock2. The SMAs tissue and the first class arborization artery rings of SMA from hemorrhagic shock (40 mmHg, 2 h), the optimal ischemia preconditioned and pinacidil pretreatmented rats were used to observe the effects of the related PKC isoforms antagonists on the protection of vascular reactivity and calcium sensitivity after hemorrhagic shock, and the effects of ischemia precondition and pinacidil pretreatment on the protein translocation of the related PKC isoforms. Results:1. The PKC isoforms responsible for the regulation of the vascular reactivity and calcium sensitivity following HS(1) The contractile response of SMA to NE and Ca2+ were increased at the early stage of shock (P<0.01), and decreased since 30min after shock (P<0.01), and continued to decrease at the late stage of shock (P<0.01).(2) PKCαagonist thymelea toxin, PKCεagonist carbachol, and PKC nonselective agonist PMA significantly restored the contractile response to NE and Ca2+ as compared with shock group (P<0.01 or P<0.05), PKCαantagonist, G?-6976, and PKCεantagonist, PKCεpseudosubstrate inhibition peptide, further decreased the contractility to NE and Ca2+ as compared with shock group or permeabilized shock group (P<0.01 or P<0.05). While PKCζantagonist did not change the contractility to NE and Ca2+ after shock, and PKCδantagonist did not inhibit PMA induced increase of contractile response of SMA to NE and Ca2+.(3) The mRNA expression of PKCαand PKCεexhibited a time-dependent increase following HS as compared with normal control (P<0.01). The protein expression of PKCαand PKCεincreased in membrane fraction and decrease in cytosolic fraction following HS as compared with normal control (P<0.01), and exhibited a time-dependent translocation from cytoplasm to membrane fraction following HS as compared with normal control.2. The regulatory mechanism of the related PKC isoforms on vascular reactivity and calcium sensitivity through the MLC20 phosphorylation signal transduction pathway.(1) The contractile response of SMA to Ca2+ decreased after 2h shock was restored by PKCαagonist, thymelea toxin, and PKCεagonist, carbachol. The neutralizing antibodies of CPI-17, ILK and ZIPK abolished the increase of calcium sensitivity induced by the agonists of PKCαandε(P<0.01).(2) The protein expression of CPI-17, ILK and ZIPK after 2h hypoxia was decreased obviously as compared with normal control, and restored by PKCαagonist, thymelea toxin, and PKCεagonist, carbachol (P<0.01). The CPI-17 phosphorylation and the activity of ILK and ZIPK were also decreased in VSMC after 2h hypoxia as compared with normal control, and restored by PKCαagonist, thymelea toxin, and PKCεagonist, carbachol (P<0.01).(3) ILK and ZIPK, especially ILK was present in the immunoprecipitates of PKCα, while CPI-17 was not, ILK and ZIPK were both present in the immunoprecipitates of PKCε, while CPI-17 was not either. ILK and ZIPK were both presented in the immunoprecipitates of CPI-17, ZIPK was presented in the immunoprecipitates of ILK.(4) MLCP activity was significantly increased after 2h hypoxia as compared with the normal control (P<0.01), MLC20 phosphorylation was significantly decreased after 2h shock as compared with normal control (P<0.01). The agonists of PKCαand PKCεdecreased the MLCP activity and increased the MLC20 phosphorylation after 2h hypoxia or shock (P<0.01). The neutralizing antibodies of CPI-17, ILK and ZIPK obviously reversed the effects of the PKCαandεagonists on MLCP activity and MLC20 phosphorylation (P<0.01).3. The induction effects of ischemia precondition and pinacidil pretreatment on the related PKC isoforms, and their protective effects on vascular reactivity and calcium sensitivity after hemorrhagic shock.(1) Ischemia precondition, especially 5% hemorrhage precondition, and pinacidil pretreatment, especially 25μg/kg pinacidil pretreatment, implemented at 30min before shock, improved the decreased NE-induced pressor response and vasoconstriction response, and improved the decreased vascular reactivity and calcium sensitivity after hemorrhagic shock (P<0.01).(2) The inhibitor of PKCα, G?-6976, and the inhibitor of PKCε, PKCεpseudosubstrate inhibitory peptidee, could significantly suppress the protection effects of ischemia precondition (5% hemorrhage precondition implemented at 30min before shock) and pinacidil pretreatment (25μg/kg pinacidil pretreatment also implemented at 30min before shock) on the vascular reactivity and calcium sensitivity after hemorrhagic shock (P<0.01).The ischemia precondition and pinacidil pretreatment could further promote shock induced PKCαand PKCεtranslocation from cytosolic to membrane fraction (P<0.01). Conclusions:1. PKCαand PKCεmay be the main isoforms of PKC responsible for the regulation of the vascular reactivity and calcium sensitivity following hemorrhagic shock, and they may be the important endogenous protective molecule of vascular reactivity.2. The regulatory effects of PKCαand PKCεon vascular reactivity and calcium sensitivity may be through altering the protein expression and activity of CPI-17, ILK and ZIPK. PKCαandεmay directly act on ILK and ZIPK. ILK and ZIPK may directly alter or via CPI-17 to alter MLCP activity and MLC20 phosphorylation to regulate the vascular calcium sensitivity.3. Ischemia precondition and pinacidil pretreatment may induce the translocation and activation of PKCαand PKCε, leading to the protection against vascular hyporeactivity and calcium desensitization after hemorrhagic shock.更多还原