嘌呤核苷酸对模拟高原缺氧大鼠脑线粒体UCPs活性和表达的影响及其在呼吸氧耗与能量合成中的作用
发布时间：2019-05-19 18:06
【摘要】： 线粒体氧化磷酸化是由呼吸链在将电子传递给氧的过程中将质子从内膜基质转到内膜外,从而在线粒体内膜两侧形成质子梯度(△H+)和电位梯度(△Ψ),两者共同构成驱动ATP合成酶的跨膜质子电动势(△P ),催化ADP和无机磷合成ATP。质子还可通过内膜上的另一质子通道——脱偶联蛋白(Uncoupling Proteins,UCPS)漏回到基质,形成质子漏,降低△P,使氧化磷酸化脱偶联,减少ATP的生成,从而降低用氧效率,这部分氧耗为“无效氧耗”。高原缺氧时线粒体中脱偶联程度增加,线粒体膜电位降低,能量生成减少,导致机体功能和代谢障碍。脱偶联蛋白4(Uncoupling Proteins 4,UCP4)和脱偶联蛋白5(Uncoupling Proteins 5,UCP5)是特异存在于哺乳动物脑组织中的UCPs家族成员,占脑中UCPs的84%以上。嘌呤核苷酸(GDP)是UCPs的特异抑制剂。本实验利用体外线粒体呼吸氧耗测定体系,观察GDP对UCPs的活性和“含量”以及对高原缺氧大鼠脑线粒体呼吸氧耗和能量代谢的影响,以探讨UCPs在缺氧大鼠脑线粒体能量生成和氧利用效率中的作用。 方法 健康雄性SD大鼠暴露于模拟海拔5000米高原低压舱内,23小时/天,分别连续缺氧3天(急性缺氧组)和30天(慢性缺氧组),同时设立平原对照组。分别在平原和模拟高原低压舱断头处死,分离大鼠脑线粒体。Clark氧电极法测定线粒体氧化呼吸活性,寡霉素抑制法测定F0F1-ATP酶活性,罗丹明123法测定线粒体膜电位,高压液相色谱法分析脑组织线粒体内腺苷酸含量,[3H]-GTP结合法测定脑组织UCPs的活性,RT-PCR和Western blot分别测定UCP4、UCP5mRNA和蛋白表达。 结果 1.缺氧组UCPs活性明显升高,其中急性组升高程度最大,Kd值降低43.08%,Bmax升高1.7倍;同时脑组织中UCP4和UCP5的mRNA和蛋白质表达也明显升高,其中急性组UCP4和UCP5的mRNA量分别升高1.47倍和3.69倍,蛋白质表达升高 2.44倍和3.62倍;GDP能显著抑制UCPs活性,对急性缺氧组抑制率最大,达83.13%。但对各组UCP4和UCP5的mRNA和蛋白质表达改变无统计学意义。 2.缺氧组ST3、RCR、OPR、P/O和MMP明显降低,其中急性缺氧组分别降低16.96%、38.98%、23.55%、8%和18.04%;但ST4则明显升高36.12%。GDP可使各组呼吸氧耗显著降低, MMP显著升高,其中对急性缺氧组的影响最显著,ST3和ST4分别降低31.54%和60.91%,RCR、OPR和P/O分别升高75.12%、15.08%和24.24%, MMP提高39.73%。 3.缺氧组F0F1-ATP酶活性、ATP含量、ATP/ADP和ATP/总腺苷酸比值显著降低,以急性缺氧组降低最显著,F0F1-ATP酶活性降低39.06%,ATP含量、ATP/ADP以及ATP/总腺苷酸比值分别降低47.67%、51.14%和47.83%。GDP可显著升高F0F1-ATP酶活性、ATP含量、ATP/ADP和ATP/总腺苷酸比值以及能荷,其中急性组F0F1-ATP酶活性升高22.29%,ATP含量升高70.19%,ATP/ADP和ATP/总腺苷酸比值分别升高49.41%和70.85%,能荷升高30.16%。 结论 1.模拟高原缺氧暴露可显著升高大鼠脑线粒体UCPs活性,增强UCP4和UCP5的mRNA和蛋白表达,并且急性缺氧组更显著;GDP可抑制正常以及缺氧暴露的大鼠脑线粒体UCPs活性,尤以急性缺氧组更明显,但在体外对UCP4和UCP5mRNA和蛋白表达无影响。 2.模拟高原缺氧暴露可显著增加大鼠脑线粒体脱偶联呼吸氧耗,降低呼吸控制率和氧化磷酸化效率及膜电位;GDP具有减轻缺氧大鼠脑线粒体脱偶联效应,降低“无效氧耗”的作用,从而提高呼吸控制率和氧化磷酸化效率。 3.模拟高原缺氧暴露可降低大鼠脑线粒体中F0F1-ATP酶活性、ATP含量、ATP/ADP和ATP/总腺苷酸比值; GDP具有升高F0F1-ATP酶活性和ATP含量的作用。 综上所述 模拟高原缺氧暴露可使大鼠脑线粒体UCPs活性和含量增加,膜电位降低,从而使“无效氧耗”增加,氧化磷酸化效率降低,线粒体能量合成减少;GDP则具有抑制缺氧大鼠脑线粒体UCPs活性,从而提高膜电位,降低“无效氧耗”,增加氧化磷酸化效率和能量生成效率的作用,且作用程度与缺氧暴露时间有关。
[Abstract]:the mitochondrial oxidative phosphorylation is defined by the transfer of protons from the inner membrane matrix to the outer membrane during the transfer of the electron transfer to the oxygen by the respiratory chain, so as to form a proton gradient (hhh +) and a potential gradient (bph) on both sides of the mitochondrial inner membrane, The two together form a transmembrane proton-electromotive force (OZP) that drives the ATP synthase, which catalyzes the synthesis of ATP from ADP and inorganic phosphorus. The protons can also leak back to the matrix through the other proton channel _ decoupling protein (UCPS) on the inner membrane to form a proton leak, reduce the P, decoupling the oxidative phosphorylation, and reduce the generation of ATP, thereby reducing the oxygen efficiency, and the oxygen consumption is the "dead oxygen consumption". At the time of hypoxia, the degree of decoupling in the mitochondria is increased, the mitochondrial membrane potential is reduced, the energy generation is reduced, and the function and the metabolic disorder of the body are caused. The unconjugated protein 4 (UCP4) and the unconjugated protein 5 (UCP5) are members of the UCPs, which are specific to the brain of the mammal, and occupy more than 84% of the UCPs in the brain. The concentration of methotrexate (GDP) is a specific inhibitor of UCPs. In this experiment, the effects of GDP on the activity and "content" of UCPs and the effects of UCPs on the oxygen consumption and energy metabolism of the brain mitochondria in rats with hypoxia were studied by using the in vitro mitochondrial respiratory oxygen consumption measurement system. Methods Healthy male SD rats were exposed to the low-pressure cabin of the simulated altitude of 5000 meters,23 hours/ day, respectively for 3 days (acute hypoxia group) and 30 days (chronic shortage). The control group of the plain is set up at the same time. The low-pressure cabin in the plain and the simulated plateau is respectively established. The rat brain mitochondria were isolated by decapitation. The mitochondrial oxidative respiratory activity was determined by the Clark oxygen electrode method. The activity of F0F1-ATPase and the determination of the activity of F0F1-ATPase and the determination of the mitochondrial membrane potential by the rhodamine 123 were determined by the method of low-pressure liquid chromatography. The content of the adenoid acid in the mitochondria of the brain was analyzed by high-pressure liquid chromatography.[3H]-GTP binding The activities of UCPs in brain tissue were determined by RT-PCR and Western blot. ,U Results 1. The activity of UCPs in the hypoxic group increased significantly, in which the elevation of UCPs in the acute group was the largest, the Kd value decreased by 43.08%, and the Bmax increased by 1.7 times, and the expression of UCP4 and UCP5 in the brain tissue was also significantly increased, with the UCP in the acute group. 4 and UCP5 The mRNA levels increased by 1.47 and 3.69 times, respectively, and the protein expression was 2.44-fold and 3.62-fold. The activity of UCPs can be obviously inhibited by the GDP, and the inhibition rate of the acute hypoxia group is up to 83.13. 2. The expression of mRNA and protein of UCP4 and UCP5 in each group was not statistically significant.2. The levels of ST3, RCR, OPR, P/ O and MMP in the group were lower than those in the group of hypoxia group, and the acute hypoxia group was decreased by 16.96%, 38.98%, 23.55%,8% and 18.04%, respectively; however, ST4 increased significantly by 36.1. The effect of ST3 and ST4 was 31.54% and 60.91%, RCR, OP respectively. R and P/ O increased by 75.12%, 15.08% and 24.24%, respectively, and MMPs increased by 39.73%.3. The activity of F0F1-ATPase, ATP content, ATP/ ADP and ATP/ total adenoid acid in the hypoxia group were significantly reduced, the most significant in the acute hypoxia group, the activity of F0F1-ATPase was decreased by 39.06%, the ATP content, ATP/ ADP and the ratio of ATP/ total adenoid acid were decreased respectively. The activity of F0F1-ATPase, ATP content, ATP/ ADP and ATP/ total adenoid acid were decreased by 47.67%, 51.14% and 47.83%. The activity of F0F1-ATPase in acute group was 22.29% and the ATP content was increased by 70.19. %, The ratio of ATP/ ADP and ATP/ total adenoid acid increased by 49.41% and 70.85%, respectively, and it could be increased by 30.16%. P5 mRNA and protein expression, and acute hypoxia group is more significant; GDP The activity of UCPs in the brain of rats with normal and hypoxic exposure was inhibited, especially in the acute hypoxia group, but there was no effect on UCP4 and UCP5 mRNA and protein expression in vitro. Acidification efficiency and membrane potential; GDP has the effect of reducing oxygen deficiency 3. The effect of the decoupling of the mitochondria in the rat brain is reduced, and the effect of the "dead oxygen consumption" is reduced, so that the respiratory control rate and the oxidative phosphorylation efficiency are improved. exposure can be reduced F0F1-ATPase activity, ATP content, ATP/ ADP and ATP/ total adenoid acid ratio in low rat brain mitochondria, and the effect of increasing F0F1-ATPase activity and ATP content in the rat brain mitochondria. To sum up, the activity and content of the mitochondrial UCPs in the brain of rats can be increased by simulating the hypoxia exposure of the plateau. And the membrane potential is reduced, so that the "dead oxygen consumption" is increased, the oxidative phosphorylation efficiency is reduced, the synthesis of the mitochondrial energy is reduced, and the GDP has the effect of inhibiting the hypoxia rat.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363
本文编号：2480945
[Abstract]:the mitochondrial oxidative phosphorylation is defined by the transfer of protons from the inner membrane matrix to the outer membrane during the transfer of the electron transfer to the oxygen by the respiratory chain, so as to form a proton gradient (hhh +) and a potential gradient (bph) on both sides of the mitochondrial inner membrane, The two together form a transmembrane proton-electromotive force (OZP) that drives the ATP synthase, which catalyzes the synthesis of ATP from ADP and inorganic phosphorus. The protons can also leak back to the matrix through the other proton channel _ decoupling protein (UCPS) on the inner membrane to form a proton leak, reduce the P, decoupling the oxidative phosphorylation, and reduce the generation of ATP, thereby reducing the oxygen efficiency, and the oxygen consumption is the "dead oxygen consumption". At the time of hypoxia, the degree of decoupling in the mitochondria is increased, the mitochondrial membrane potential is reduced, the energy generation is reduced, and the function and the metabolic disorder of the body are caused. The unconjugated protein 4 (UCP4) and the unconjugated protein 5 (UCP5) are members of the UCPs, which are specific to the brain of the mammal, and occupy more than 84% of the UCPs in the brain. The concentration of methotrexate (GDP) is a specific inhibitor of UCPs. In this experiment, the effects of GDP on the activity and "content" of UCPs and the effects of UCPs on the oxygen consumption and energy metabolism of the brain mitochondria in rats with hypoxia were studied by using the in vitro mitochondrial respiratory oxygen consumption measurement system. Methods Healthy male SD rats were exposed to the low-pressure cabin of the simulated altitude of 5000 meters,23 hours/ day, respectively for 3 days (acute hypoxia group) and 30 days (chronic shortage). The control group of the plain is set up at the same time. The low-pressure cabin in the plain and the simulated plateau is respectively established. The rat brain mitochondria were isolated by decapitation. The mitochondrial oxidative respiratory activity was determined by the Clark oxygen electrode method. The activity of F0F1-ATPase and the determination of the activity of F0F1-ATPase and the determination of the mitochondrial membrane potential by the rhodamine 123 were determined by the method of low-pressure liquid chromatography. The content of the adenoid acid in the mitochondria of the brain was analyzed by high-pressure liquid chromatography.[3H]-GTP binding The activities of UCPs in brain tissue were determined by RT-PCR and Western blot. ,U Results 1. The activity of UCPs in the hypoxic group increased significantly, in which the elevation of UCPs in the acute group was the largest, the Kd value decreased by 43.08%, and the Bmax increased by 1.7 times, and the expression of UCP4 and UCP5 in the brain tissue was also significantly increased, with the UCP in the acute group. 4 and UCP5 The mRNA levels increased by 1.47 and 3.69 times, respectively, and the protein expression was 2.44-fold and 3.62-fold. The activity of UCPs can be obviously inhibited by the GDP, and the inhibition rate of the acute hypoxia group is up to 83.13. 2. The expression of mRNA and protein of UCP4 and UCP5 in each group was not statistically significant.2. The levels of ST3, RCR, OPR, P/ O and MMP in the group were lower than those in the group of hypoxia group, and the acute hypoxia group was decreased by 16.96%, 38.98%, 23.55%,8% and 18.04%, respectively; however, ST4 increased significantly by 36.1. The effect of ST3 and ST4 was 31.54% and 60.91%, RCR, OP respectively. R and P/ O increased by 75.12%, 15.08% and 24.24%, respectively, and MMPs increased by 39.73%.3. The activity of F0F1-ATPase, ATP content, ATP/ ADP and ATP/ total adenoid acid in the hypoxia group were significantly reduced, the most significant in the acute hypoxia group, the activity of F0F1-ATPase was decreased by 39.06%, the ATP content, ATP/ ADP and the ratio of ATP/ total adenoid acid were decreased respectively. The activity of F0F1-ATPase, ATP content, ATP/ ADP and ATP/ total adenoid acid were decreased by 47.67%, 51.14% and 47.83%. The activity of F0F1-ATPase in acute group was 22.29% and the ATP content was increased by 70.19. %, The ratio of ATP/ ADP and ATP/ total adenoid acid increased by 49.41% and 70.85%, respectively, and it could be increased by 30.16%. P5 mRNA and protein expression, and acute hypoxia group is more significant; GDP The activity of UCPs in the brain of rats with normal and hypoxic exposure was inhibited, especially in the acute hypoxia group, but there was no effect on UCP4 and UCP5 mRNA and protein expression in vitro. Acidification efficiency and membrane potential; GDP has the effect of reducing oxygen deficiency 3. The effect of the decoupling of the mitochondria in the rat brain is reduced, and the effect of the "dead oxygen consumption" is reduced, so that the respiratory control rate and the oxidative phosphorylation efficiency are improved. exposure can be reduced F0F1-ATPase activity, ATP content, ATP/ ADP and ATP/ total adenoid acid ratio in low rat brain mitochondria, and the effect of increasing F0F1-ATPase activity and ATP content in the rat brain mitochondria. To sum up, the activity and content of the mitochondrial UCPs in the brain of rats can be increased by simulating the hypoxia exposure of the plateau. And the membrane potential is reduced, so that the "dead oxygen consumption" is increased, the oxidative phosphorylation efficiency is reduced, the synthesis of the mitochondrial energy is reduced, and the GDP has the effect of inhibiting the hypoxia rat.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363
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