【作者】 郭会彩；

【导师】 王永利；

【作者基本信息】 河北医科大学 ， 药理学， 2009， 博士

【摘要】 间歇性低氧是指一定时间暴露于低氧环境,而其余时间处于常氧环境。间歇性低氧是机体某种生理和病理状态下的低氧形式。研究表明:慢性间歇性低压低氧(chronic intermittent hypobaric hypoxia,CIHH),类似缺血预适应(ischemic preconditioning,IPC),具有明显的心脏保护作用,表现为增强心肌对缺血/再灌注损伤的耐受性、限制心肌梗死面积和形态学改变、抗细胞凋亡、促进缺血/再灌注心脏舒缩功能的恢复,以及抗心律失常。尽管CIHH对心脏的保护作用不容质疑,但其作用机制远未阐明。众所周知,氧自由基是缺血/再灌注和低氧/复氧诱发的心肌细胞膜损伤的一个重要原因。内源性抗氧化系统在CIHH心肌保护中的作用报道不一致。抗氧化是否参与了CIHH的心肌保护作用需要更进一步的研究。Na+,K+-ATP酶即钠钾泵是存在于细胞膜上的膜蛋白,主要由α亚基和β亚基组成,其功能主要是维持细胞内外钠钾离子的浓度梯度。当心肌缺血时,导致细胞内钠离子浓度升高,细胞内钙离子浓度增加造成钙超载,钙超载是心肌缺血引起损伤的重要因素之一。钠钾泵可以排出细胞内多余的钠离子,从而使细胞内钠离子降低,使钠钙交换体的功能增强排出细胞内多余的钙离子。有研究报道,心肌缺血或缺血/再灌注会抑制钠钾泵的活性。CIHH可减弱心肌缺血时钙离子的增加,这可能有钠钾泵的参与。是否CIHH对钠钾泵有影响尚无研究。有研究表明,活性氧簇(ROS)与钠钾泵关系密切,可显著抑制钠钾泵。在心肌缺血或缺血/再灌注时会有ROS的大量生成,这可能是心肌缺血或缺血/再灌注引起钠钾泵抑制的原因之一。本实验应用离体心脏灌流技术、细胞动缘系统、全细胞膜片钳技术及分子生物学方法,探讨了CIHH对豚鼠心脏缺血再灌注损伤的保护作用及抗氧化酶和Na,K-ATPase在CIHH对豚鼠心脏保护中所起的作用。研究分为三部分(1)CIHH对豚鼠缺血/再灌注心脏的保护作用:利用Langendorff离体心脏灌注系统,建立离体豚鼠心脏缺血/再灌注模型,观察CIHH对豚鼠心脏缺血/再灌注损伤的影响。(2)抗氧化酶在CIHH心脏保护中的作用:在豚鼠离体灌流心脏,通过药理学及分子生物学方法探讨抗氧化酶在CIHH心脏保护中的作用。(3)钠钾泵在CIHH心脏保护中的作用:利用细胞动缘及膜片钳技术观察CIHH对钠钾泵的作用及钠钾泵在CIHH心脏保护中的作用。第一部分CIHH对豚鼠心肌缺血再灌损伤的保护作用目的:观察CIHH对豚鼠心肌缺血再灌损伤是否有保护作用。方法:132只雄性成年豚鼠(250±20g)随机分为两组:非间歇性低氧组(non-CIHH)与间歇性低氧组(CIHH)。其中CIHH又分为CIHH处理14天组(CIHH14)、CIHH处理28天组(CIHH28)和CIHH处理42天组(CIHH42),而non-CIHH又相应分为non-CIHH14、non-CIHH28和non-CIHH42。CIHH处理组动物于低压氧舱分别接受14,28天、42天模拟5000米海拔高度(PB=404 mmHg, PO2=84 mmHg)的低压低氧处理,每天6小时。non-CIHH组动物除了不接受低氧处理外,其它处理均与CIHH组动物相同。离体心脏利用Langendorff灌流给予缺血(30min)/再灌注(60min)处理,观察心脏功能的变化。TTC染色方法测定心肌梗死面积。结果:1 CIHH14, 28, 42组豚鼠的体重(BW),全心/体重(THW/BW)、右心室/左心室重量(RVW/LVW、右心室/体重(RVW/BW)与相应non-CIHH组无显著差异。2基础状态下,除CIHH28和CIHH42组的CF较相应non-CIHH组增加外,其余心功能与non-CIHH组动物均无显著性差异。心脏停灌缺血30 min后再灌注60min, CIHH28和CIHH 42组的心功能参数,如LVDP,LVEDP,±dp/dtmax恢复都较相应non-CIHH组明显好转。3 CIHH28和CIHH 42天能明显降低缺血/再灌注后心肌梗死面积。小结:CIHH28和CIHH 42天增强成年豚鼠心脏对缺血/再灌注损伤的抵抗能力。第二部分抗氧化酶在CIHH心脏保护中的作用目的:探讨抗氧化酶在CIHH心脏保护中的作用。方法:142只雄性成年豚鼠(250±20g)随机分为两组:非间歇性低氧组(non-CIHH)与间歇性低氧组(CIHH)。CIHH处理组动物于低压氧舱分别接受28天、5000米海拔高度(PB=404 mmHg, PO2=84 mmHg)的低压低氧处理,每天6小时。non-CIHH组动物除了不接受低氧处理外,其它处理均与CIHH组动物相同。离体心脏利用Langendorff灌流给予缺血(30min)/再灌注(60min)处理,观察心脏功能的变化。生化方法测定心肌SOD、CAT和GPX的活性及MDA的含量。免疫印迹方法测定心肌SOD及CAT的蛋白含量。结果:1基础状态下, CIHH组心肌MDA含量与non-CIHH组相比无显著性差异。心脏停灌缺血30min后再灌注60min,各组MDA含量均明显升高,CIHH组MDA含量升高明显低于相应non-CIHH组(P<0.01)。2基础状态下, CIHH组心肌总SOD、SOD-2和CAT活性较non-CIHH组相比均明显增加,SOD-1和GPX活性与non-CIHH组相比无显著性差异。心脏停灌缺血30min后再灌注60min ,各组总SOD、SOD-1、SOD-2和CAT活性MDA含量均明显下降,但CIHH组总SOD、SOD-2和CAT活性仍明显高于相应non-CIHH组(P<0.01)。3预先应用CAT不可逆性阻断剂,可完全消除CIHH对缺血/再灌注心脏的保护作用;应用抗氧化酶复合物SOD+CAT可模拟CIHH的心肌保护作用。4 CIHH可对抗外源性H2O2引起的心功能降低及氧化应激。小结:CIHH通过上调抗氧化酶而发挥心肌保护作用。第三部分钠钾泵在CIHH心脏保护中的作用目的:探讨钠钾泵在CIHH心脏保护中的作用。方法:雄性成年豚鼠(250±20g)随机分为两组:非间歇性低氧组(non-CIHH)与间歇性低氧组(CIHH)。CIHH处理组动物于低压氧舱分别接受14,21,28,42天, 5000米海拔高度(PB=404 mmHg, PO2=84 mmHg)的低压低氧处理,每天6小时。non-CIHH组动物除了不接受低氧处理外,其它处理均与CIHH组动物相同。利用膜片钳技术记录心肌细胞钠钾泵电流。利用细胞动缘探测系统测定心肌细胞的长度及收缩力的大小。结果:1模拟缺血20分钟再灌30分钟可明显缩短细胞的长度,但CIHH组细胞缩短的长度明显小于相应non-CIHH组的细胞。Oua可取消CIHH的作用。2模拟缺血20分钟再灌30分钟可明显降低细胞的收缩幅度、细胞的最大收缩速率及最大舒张速率,但CIHH组细胞收缩的幅度、细胞的最大收缩速率及最大舒张速率恢复明显优于相应对照组。Oua可取消CIHH对细胞的收缩幅度,细胞的最大收缩速率的作用。3与相应non-CIHH组相比,CIHH处理21,28,42天明显增加心肌细胞钠钾泵电流。4 non-CIHH组△Ip-[Oua]关系曲线,从10-10到10-3 mol/LOua所对应的△Ip分别是0.088±0.03、0.150±0.03、0.060±0.01、-0.145±0.02、-0.391±0.06、-0.670±0.02、-0.98±0.01和-1.000±0.00。采用双亚基三位点结合模型可进行最优拟合,其解离常数(kd)包括高亲和力兴奋性位点Kd值(K+2)、高亲和力抑制性位点Kd值(K-2)和低亲和力抑制性位点Kd值(K1),分别为8.5 x 10-11M、5.2 x 10-8M和1.1 x 10-5M,高、低两种亲和力泵所占比例分别为f2 = 0.31和f1 = 0.68. CIHH组△Ip-[Oua]关系曲线,从10-10到10-3 mol/LOua所对应的△Ip分别是0.069±0.02、-0.036±0.03、-0.181±0.02、-0.202±0.05、-0.459±0.03、-0.770±0.02、-0.978±0.01和-1.000±0.00。采用双亚基三位点结合模型可进行最优拟合,其解离常数K+2、K-2和K1分别为2.4 x 10-10M、4.2 x 10-8M和2.8 x 10-6M, f2 = 0.20和f1 = 0.80。说明经CIHH处理后钠钾泵低亲和力泵即α1亚基的相对比例增加。5 CIHH处理28天使心肌细胞钠钾泵电流明显增强,当给予H2O2 (1mM)灌流5分钟时钠钾泵电流在non-CIHH组与CIHH组均明显降低。但CIHH组钠钾泵电流仍明显高于相应non-CIHH组(P<0.01)。6 0.1mM H2O2在non-CIHH组可明显抑制钠钾泵电流,而对CIHH组与CAT组钠钾泵电流无明显影响。1mM H2O2对各组钠钾泵电流均有明显抑制,但CIHH组及CAT组钠钾泵电流仍明显高于相应non-CIHH组(P<0.01)。1mM H2O2对各组的钠钾泵电流抑制基本已达到最大。但CIHH的最大抑制率明显低于non-CIHH组,CAT组的最大抑制率与non-CIHH组相比无明显差别。即CAT使H2O2与钠钾泵电流关系曲线平行右移,CIHH使H2O2与钠钾泵电流关系曲线右移,说明经CIHH处理后钠钾泵本身对H2O2的敏感性发生变化。小结:钠钾泵在CIHH心肌保护中发挥了重要作用。结论1 CIHH可明显改善豚鼠心肌缺血/再灌注损伤2 CIHH通过上调抗氧化酶的含量及活性而提高机体的抗氧化能力,这可能是其改善心肌缺血/再灌注损伤的机制之一。3 CIHH可增强钠钾泵电流及降低钠钾泵对氧化应激的敏感性,钠钾泵在CIHH的心肌保护作用中发挥重要作用。更多还原

【Abstract】 Intermittent hypoxia, or periodic exposure to hypoxia interrupted by return to normoxia or less hypoxic conditions, is encountered more frequently in life than sustained hypoxia. Many studies showed that chronic intermittent hypobaric hypoxia (CIHH) adaptation had the cardioprotective effects similar to those observed in ischemic preconditioning (IPC). However, the cardioprotective effect of CIHH lasts longer than that of IPC and it is easy to manipulate the CIHH model, thus it has significance in the study of CIHH. A number of studies have attempted to define the mechanisms of this phenomenon and several potential factors have been proposed to be involved in the protective mechanism afforded by CIHH, however, the precise mechanisms underlying the cardioprotective effects of CIHH are far from clear. It is well known that oxidative stress and oxygen-derived free radicals (mainly ROS) contribute to I/R injury. There are different reports on the role of antioxidation in cardioprotection of CIHH. Whether antioxidation contributed to the cardioprotection of CIHH and the detail mechanism of antioxidation in CIHH need further study.Na+,K+-ATPase (sodium pump) is a heterodimer protein composed ofα- andβ-subunits that plays a key role in regulating membrane potential and cation transport in the myocardium. When myocardial ischemia, Ca2+ and Na+ influx into myocardium, and Ca2+ overload is regarded as a crucial factor in the development of ischemic myocardial damage. Na+,K+-ATPase facilitates transportation of Na+ from the intracellular space. The decrease in the intracellular Na+ concentration enhances removal of Ca+ from the intracellular space by facilitating Na+/Ca2+ exchange mechanism. Some researches demonstrated that ischemia and I/R injury can depress the activity of the cardiac Na+,K+-ATPase. Therefore, prevention of Ca2+ overload by CIHH may involve changes in Na+,K+-ATPase activity. Whether CIHH treatment can affect cardiac Na+,K+-ATPase have not been studied. Some reports demonstrated that ROS and Na+,K+-ATPase have a crosstalk relationship. ROS can inhibit Na+,K+-ATPase activity. As oxidative stress has been shown to occur during development of I/R injury, it is likely that the depression of Na+,K+-ATPase activity in I/R hearts may be due to oxidative stress. The aim of the study is to investigate whether CIHH has a protective effect on guinea pig heart, and to explore the underlying mechanism. Our study consists of three parts: (1) Protective effect of CIHH against ischemia/reperfusion injury inguinea pig heart. (2) The role of antioxidant enzymes in the cardioprotection of CIHH. (3) The role of the sodium pump in the cardioprotection of CIHH.Part 1 Protective effect of CIHH against ischemia/reperfusion injury in guinea pig heartObjective: The aim of this study was to investigate the effect of CIHH on myocardial ischemia/reperfusion injury in guinea pigMethods: Adult male guinea pigs (n=132) were divided randomly into six groups: non-CIHH 14 days group (non-CIHH14), non-CIHH 28 days group (non-CIHH28), non-CIHH 42 days group (non-CIHH42), CIHH 14 days group (CIHH14), CIHH 28 days group (CIHH28), and CIHH 42 days group (CIHH42). In CIHH groups, guinea pigs were exposed to CIHH mimicking 5000m high altitude (PB=404 mmHg, PO2=84 mmHg) in a hypobaric chamber lasting 6 hrs/day for 14 days, 28 days and 42 days respectively. The animals in non-CIHH groups were kept in the same environment as the CIHH guinea pigs except for hypoxic exposure. Langendorff-perfused isolated guinea pig hearts were used to measure variables of left ventricular function during baseline perfusion, ischemia, and reperfusion period. The parameters of cardiac function including left ventricular developing pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), maximal differentials of LVDP (±LVdp/dtmax) and coronary flow (CF) were measured. Myocardium was stained by TTC to measure infarct size. Results: 1 The body weight of guinea pigs in CIHH group had no significant change compared with non-CIHH animals. No differences in the ratio of heart weight to body weight, ratio of right ventricular weight to left ventricular plus inter-ventricular septum weight, ratio of right ventricular weight to body weight were observed between CIHH and non-CIHH groups.2 The basic CF in CIHH28 and CIHH42 guinea pigs was significant higher than that in corresponding non-CIHH guinea pigs, while other parameters of cardiac function didn’t change. During the whole period of reperfusion, the recoveries of LVDP, LVEDP,±dp/dtmax, and CF in guinea pigs after 28 and 42 d of CIHH exposure were much better than those in the corresponding non-CIHH guinea pigs.3 The myocardial infarct size induced by ischemia and reperfusion was markedly reduced in CIHH28 and CIHH42 groups compared with those in corresponding non-CIHH guinea pigsConclusion: The results suggest that CIHH has a protective effect against ischemia/reperfusion injury on guinea pig heart.Part 2 The role of antioxidant enzymes in the cardioprotection of CIHHObjective: The aim of the present study was to evaluate: whether antioxidation was involved in the cardiacprotection afforded by CIHH.Methods: Adult male guinea pigs were exposed to CIHH mimicking 5000m high altitude (PB=404 mmHg, PO2=84 mmHg) in a hypobaric chamber lasting 6 hrs/day for 28 days. Langendorff-perfused isolated guinea pig hearts were used to measure variables of left ventricular function during baseline perfusion, ischemia, and reperfusion period. The activities and protein expressions of antioxidant enzymes in left ventricle were evaluated using biochemical methods and Western blotting, respectively. Intracellular reactive oxygen species (ROS) were assessed using ROS- sensitive fluorescence.Results:1 There was no significant difference in MDA contents between non-CIHH and CIHH group before ischemia/reperfusion. After reperfusion, MDA content increased significantly in both non-CIHH and CIHH hearts (p < 0.01), whereas MDA content in CIHH hearts was still lower than that in non-CIHH hearts (p < 0.01).2 The baseline activities of total SOD, SOD-2, and CAT in CIHH hearts were higher than those in non-CIHH hearts (p < 0.01), but SOD-1 and GPX activity did not significantly change. After reperfusion, SOD-2 and CAT activities decreased in both non-CIHH and CIHH hearts (p < 0.01), whereas the activities of SOD-2 and CAT in CIHH hearts were still higher than those in non-CIHH hearts (p < 0.01).3 Western blot analysis demonstrated that the baseline expressions of SOD-2 and CAT protein in CIHH hearts were higher than those in non-CIHH hearts; however, the expression of SOD-1 protein did not change. The protein expressions of SOD-2 and CAT were not significantly changed after reperfusion in both non-CIHH and CIHH hearts, whereas the expressions of SOD-2 and CAT in CIHH hearts were still higher than those in non-CIHH hearts (p < 0.01).4 Treatment with CAT inhibitor ATZ (1.0 g/kg) completely eliminated the protective effect of CIHH on cardiac function (p < 0.01), whereas it had no effect on non-CIHH hearts during reperfusion. A similar improvement in these cardiac function parameters was also observed in hearts treated with the antioxidant mixture containing SOD + CAT.5 Cardiac contractile dysfunction and oxidative stress induced by exogenous hydrogen peroxide (H2O2) were attenuated by CIHH and CAT. Conclusion: These data suggest that CIHH can protect heart against I/R injury through upregulation of antioxidant enzymes in guinea pig.Part 3 The role of sodium pump in the cardioprotection of CIHHObjective: whether Na+,K+-ATPase was involved the cardiacprotection afforded by CIHH.Methods: Adult male guinea pigs were exposed to CIHH mimicking 5000m high altitude (PB=404 mmHg, PO2=84 mmHg) in a hypobaric chamber lasting 6 hrs/day for 14, 21, 28, and 42days. The left ventricular myocytes were enzymatically isolated. The sodium pump current was recorded using whole cell patch clamp technique. The cell length and contraction were assessed by a video-based, motion-edge detection system.Results:1 After 20 min of ischemia followed by 30 min of reperfusion, cell length shortened in each group. CIHH significantly improved the recovery of cell length compared to that of non-CIHH myocytes (96.3±0.9% vs. non-CIHH 86.8±2.9%, P < 0.01). While Oua administered at 5 min before the ischemia completely abolished this beneficial effect in CIHH myocytes.2 Ischemia–reperfusion injury resulted in a marked decrease in the amplitude of contraction in each group. CIHH adaptation improved the recovery of contraction amplitude. When CIHH myocytes were treated with Oua at 5 min before the ischemia, all the beneficial effects were completely eliminated.3 The sodium pump currents in CIHH21, CIHH28, and CIHH42 guinea pigs were significant higher than those in corresponding non-CIHH guinea pigs.4 In the△Ip-[Oua] relation curve of non-CIHH, the△Ip values produced by each concentration of Oua from 10-10 to 10-3 mol/L were 0.088±0.03, 0.150±0.03, 0.060±0.01, -0.145±0.02, -0.391±0.06, -0.670±0.02, -0.98±0.01, and -1.000±0.00, respectively. K+2, K-2 and K1 were 8.5 x 10-11M, 5.2 x 10-8M, and 1.1 x 10-5M, repectively. f2 = 0.31, f1 = 0.68. In the△Ip-[Oua] relation curve of CIHH (Fig.5F), the△Ip values produced by each concentration of Oua from 10-10 to 10-3 mol/L were 0.069±0.02, -0.036±0.03, -0.181±0.02, -0.202±0.05, -0.459±0.03, -0.770±0.02, -0.978±0.01, and -1.000±0.00, respectively. K+2 , K-2 and K1 were 2.4 x 10-10M, 4.2 x 10-8M, 2.8 x 10-6M, f2 = 0.20, f1 = 0.80.5 Ip in cardiac myocytes after 28 CIHH was much higher than that in the corresponding non-CIHH (p < 0.01). After H2O2 (1mM) perfusion for 5 min, Ip decreased significantly in both non-CIHH and CIHH myocytes (p < 0.01 or p < 0.05), whereas the Ip in CIHH myocytes were still higher than those in non-CIHH myocytes (p < 0.01). 6 0.1mM H2O2 can inhibit Ip significantly in non-CIHH myocytes, but have no significantly effect on Ip in CIHH and CAT myocytes. 1mM H2O2 can inhibit Ip significantly in all myocytes, whereas the Ip in CIHH or CAT myocytes were still higher than those in non-CIHH myocytes. 10mM H2O2 can maximally inhibit Ip in all myocytes, whereas the Ip in CIHH myocytes was still higher than those in non-CIHH. CAT make the curve of concentration-dependence of H2O2 -induced inhibition of Ip move to the right parallelly. CIHH make the curve of concentration-dependence of H2O2 -induced inhibition of Ip move to the right, whereas maximal inhibition induced by H2O2 was smaller than those in non-CIHH or CAT myocytes.Conclusion: Sodium pump may play an important role in the cardioprotection of CIHH against I/R injury in guinea pig.SUMMARY1 CIHH has a protective effect against ischemia/reperfusion injury on guinea pig heart.2 CIHH upregulates the activity and protein expressions of antioxidant enzymes leading to an increase in antioxidant capacity, which may play an important role in the cardiac protection of CIHH against I/R injury in guinea pig.3 CIHH increase the sodium pump current and resistance to oxidative stress in cardiac myocytes. Sodium pump was involved in the cardiacprotection afforded by CIHH and may be an intermedium in the anti-oxidative cardiac protection mechanism of CIHH.更多还原