UCP4和BMCP-1在大鼠缺氧时脑组织线粒体能量合成与呼吸氧耗改变中的作用
发布时间：2019-06-14 14:27
【摘要】： 初上高原的人常常不能很快的适应高原低氧环境,从而产生一系列的病理生理过程和临床综合征,出现高原反应,严重的发展为高原脑水肿和高原肺水肿,危及生命。其根本原因是由于外界氧分压降低,导致低张性缺氧。线粒体是细胞的“能量工厂”,其通过消耗氧的氧化磷酸化过程为机体提供95%的能量。解偶联蛋白家族(Uncouplingprotein,UCPs)是线粒体内膜质子转运蛋白,作为质子漏通道,使氧化磷酸化形成的质子梯度降低,但不伴有ATP的生成,产生“无效氧耗”。近年来对UCPs的研究多涉及营养、代谢等领域,但在高原缺氧时UCPs在机体对于高原习服和适应中的作用未见报道。UCP4和BMCP-1是近年来才发现的特异在脑组织中丰富表达的UCPs家族成员,而缺氧时UCP4和BMCP-1在脑组织中的表达、异构体分布、功能特异性以及线粒体氧耗和ATP生成的关系尚无人问津。因此研究缺氧时UCPs活性和含量的变化及其对线粒体能量代谢和呼吸氧耗的影响,对提高氧的利用和能量的生成有重要的意义。 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;各缺氧组与对照组分别在模拟高原低压舱内和平原断头处死,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F_0-F_1ATP酶活性,罗丹明123法测定线粒体膜电位,HPLC分析脑组织线粒体内腺苷酸含量,[~3H]-GTP结合法同时测定脑组织UCPs的活性与含量,RT-PCR检测UCP4、BMCP-1mRNA表达量,Western blot测定UCP4、BMCP-1蛋白表达。 结果 1.急性缺氧暴露可显著降低大鼠脑线粒体ST3、RCR、OPR,而ST4则显著增高;慢性缺氧脑线粒体ST3、OPR较对照显著下降,而与急性组比较,ST4显著降低,RCR则显著升高。 2.急、慢性缺氧组大鼠脑线粒体ATP含量均显著下降,分别为对照的58.4%和71.4%。而慢性组则有所回升,显著高于急性组;急、慢性组AMP含量均较对照组显著增高;急性组ADP含量显著增高;急、慢性组ATP/ADP均显著降低,其中慢性组显著高于急性组。急、慢性缺氧组能荷均显著下降,相较于对照组分别降低20.1%、22.3%。 3.急、慢性组脑线粒体UCPs与[~3H]-GTP结合的解离常数Kd均显著下降,各为对照的58.1%、75.8%;而慢性组则高于急性组。急性组线粒体UCPs结合[~3H]-GTP的最大量Bmax显著增高,为对照组的2.9倍;慢性组则显著低于急性组,而与对照组无统计学意义差别。 急性组UCP4和BMCP-1 mRNA和蛋白的表达均显著高于对照组及慢性组,其中UCP4、BMCP-1 mRNA表达分别为对照组的2.34倍和4.08倍。慢性组BMCP-1 mRNA和蛋白表达较对照组显著增高,而UCP4 mRNA和蛋白表达则恢复至对照水平。 4.急、慢性缺氧组脑组织线粒体膜电位(MMP)和F_0-F_1ATP酶活性显著下降,其中急性组分别降为对照组的80.5%和55.4%,而慢性组则分别降为对照组的87.4%和77.5%,比急性组均有回升。 结论 1、缺氧可显著增加大鼠脑线粒体UCPs活性。急性缺氧暴露可诱导大鼠脑线粒体UCP4、BMCP-1 mRNA和蛋白的表达,增加脑组织中UCPs的含量,慢性缺氧时则有所回降,而急、慢性缺氧均可增加脑线粒体UCPs活性,表明UCPs(UCP4、BMCP-1)表达的增加是活性增高的主要因素。 2、缺氧时UCPs活性增强,驱散线粒体内膜质子梯度,同时增强线粒体ST4呼吸,使无效氧耗增强,从而使ATP合成减少,能量生成效率降低。 3、慢性缺氧暴露可在一定程度上使大鼠脑组织线粒体UCPs活性回降,增强氧化磷酸化功能,部分恢复ATP合成酶活性和线粒体内ATP含量。提示经长期缺氧习服-适应后脑组织UCPs活性的下降,使无效氧耗减少,能量生成效率提高,从而保持能量平衡,这无疑是慢性高原缺氧习服-适应的重要机制之一。
[Abstract]:The people of the first upper plateau often do not adapt to the high altitude hypoxia environment very quickly, so as to produce a series of pathophysiological processes and clinical syndromes, and the high altitude reaction occurs, and the serious development is high altitude cerebral edema and high altitude pulmonary edema, which is life-threatening. The root cause is due to the lower external oxygen partial pressure, resulting in low-tension oxygen deficiency. The mitochondria are the "energy plant" of the cells that provide 95% energy to the body by the oxidative phosphorylation of oxygen. Uncodingprotein (UCPs) is the mitochondrial membrane proton transport protein, which is used as the proton leakage channel, so that the proton gradient formed by the oxidative phosphorylation is reduced, but without the generation of ATP, the "dead oxygen consumption" is generated. In recent years, the research of UCPs in the field of nutrition, metabolism and so on, but the role of UCPs in the acclimatization and adaptation of the high altitude is not reported. UCP4 and BMCP-1 are members of the UCPs which are found only in the brain tissue in recent years, and the expression of UCP4 and BMCP-1 in the brain tissue, the distribution of the isomers, the function specificity and the relationship between the mitochondrial oxygen consumption and the ATP production are not yet known. Therefore, it is of great significance to study the changes of the activity and content of UCPs and its effect on the energy metabolism and the oxygen consumption of the mitochondria in the absence of hypoxia, and it is of great significance to improve the utilization of oxygen and the generation of energy. Methods Healthy male SD rats were exposed to the low-pressure cabin of the simulated altitude of 5000 m,23 hours/ day, respectively for 3 days (acute hypoxia group) and 30 days (chronic hypoxia group). in that control group of the plain, the hypoxia group and the control group were sacrificed at the low-pressure cabin and the plain end of the simulated plateau, respectively, the mitochondria of the brain of the rat were isolated, the activity of the mitochondrial oxidative respiration was determined by the Clark oxygen electrode method, the activity of the F _ 0-F _ 1 ATPase was determined by the oligomycin inhibition method, and the determination of the line by the rhodamine 123 method The activity and contents of UCP4 and BMCP-1 were determined by RT-PCR, and the expression of UCP4 and BMCP-1 was determined by Western blot. 1 egg Results:1. Acute hypoxia exposure could significantly lower the mitochondrial ST3, RCR and OPR of the rat brain, while the ST4 increased significantly; the chronic hypoxic-brain mitochondria ST3 and OPR decreased significantly compared with the acute group, and ST4 was significantly higher than that in the acute group. 2. The content of ATP in the brain of the rats in the acute and chronic hypoxia group decreased significantly, respectively. The control group was 58.4% and 71.4%, while the chronic group recovered, significantly higher than that of the acute group, and the content of AMP in the acute and chronic group was significantly higher than that in the control group; the content of ADP in the acute group was significantly increased; and the ATP/ ADP in the acute and chronic groups decreased significantly. The chronic group was significantly higher in the acute and chronic hypoxia group than in the control group. The dissociation constant (Kd) of the mitochondrial UCPs and[~ 3H]-GTP in the acute and chronic group decreased significantly, each of which was 58.1% of the control group. The most significant Bmax of the acute group of UCPs binding to[~ 3H]-GTP was significantly higher in the acute group than in the control group, and the chronic group was significantly lower than that in the control group. The expression of UCP4 and BMCP-1 mRNA and protein in the acute group was significantly higher than that in the control group and the chronic group, of which the UCP4 and BMCP-1 mRNA were significantly higher in the acute group than in the control group and the chronic group. The expression of BMCP-1 mRNA and protein in the chronic group was significantly higher than that of the control group, while the expression of BMCP-1 mRNA and protein in the chronic group was significantly higher than that of the control group. In the acute and chronic hypoxia group, the mitochondrial membrane potential (MMP) and F _ 0-F _ 1 ATPase activity of the brain tissue of the acute and chronic hypoxia group decreased significantly, and the acute group decreased to 80.5% and 55.4% in the control group, respectively, while the chronic group decreased to the control group, respectively. irradiation group 87.4% and 77.5% in the acute group. Conclusion 1. Hypoxia can significantly increase the activity of UCPs in the brain of rats. Acute hypoxia exposure can induce the expression of UCP4, BMCP-1 mRNA and protein in the brain of rats, increase the content of UCPs in the brain, and decrease the activity of UCPs in chronic hypoxia. The increase of the expression of s (UCP4, BMCP-1) is the main factor of the increase of activity. T4 respiration, which increases the effective oxygen consumption, decreases the synthesis of ATP and decreases the energy generation efficiency.3. Chronic hypoxia exposure can increase the activity of the mitochondrial UCPs in the brain of rats to a certain extent. In order to decrease the activity of UCPs in the brain tissue after long-term hypoxic acclimatization, the activity of UCPs in brain tissue can be reduced, and the effective oxygen consumption is reduced and the energy is effective.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R363
本文编号：2499456
[Abstract]:The people of the first upper plateau often do not adapt to the high altitude hypoxia environment very quickly, so as to produce a series of pathophysiological processes and clinical syndromes, and the high altitude reaction occurs, and the serious development is high altitude cerebral edema and high altitude pulmonary edema, which is life-threatening. The root cause is due to the lower external oxygen partial pressure, resulting in low-tension oxygen deficiency. The mitochondria are the "energy plant" of the cells that provide 95% energy to the body by the oxidative phosphorylation of oxygen. Uncodingprotein (UCPs) is the mitochondrial membrane proton transport protein, which is used as the proton leakage channel, so that the proton gradient formed by the oxidative phosphorylation is reduced, but without the generation of ATP, the "dead oxygen consumption" is generated. In recent years, the research of UCPs in the field of nutrition, metabolism and so on, but the role of UCPs in the acclimatization and adaptation of the high altitude is not reported. UCP4 and BMCP-1 are members of the UCPs which are found only in the brain tissue in recent years, and the expression of UCP4 and BMCP-1 in the brain tissue, the distribution of the isomers, the function specificity and the relationship between the mitochondrial oxygen consumption and the ATP production are not yet known. Therefore, it is of great significance to study the changes of the activity and content of UCPs and its effect on the energy metabolism and the oxygen consumption of the mitochondria in the absence of hypoxia, and it is of great significance to improve the utilization of oxygen and the generation of energy. Methods Healthy male SD rats were exposed to the low-pressure cabin of the simulated altitude of 5000 m,23 hours/ day, respectively for 3 days (acute hypoxia group) and 30 days (chronic hypoxia group). in that control group of the plain, the hypoxia group and the control group were sacrificed at the low-pressure cabin and the plain end of the simulated plateau, respectively, the mitochondria of the brain of the rat were isolated, the activity of the mitochondrial oxidative respiration was determined by the Clark oxygen electrode method, the activity of the F _ 0-F _ 1 ATPase was determined by the oligomycin inhibition method, and the determination of the line by the rhodamine 123 method The activity and contents of UCP4 and BMCP-1 were determined by RT-PCR, and the expression of UCP4 and BMCP-1 was determined by Western blot. 1 egg Results:1. Acute hypoxia exposure could significantly lower the mitochondrial ST3, RCR and OPR of the rat brain, while the ST4 increased significantly; the chronic hypoxic-brain mitochondria ST3 and OPR decreased significantly compared with the acute group, and ST4 was significantly higher than that in the acute group. 2. The content of ATP in the brain of the rats in the acute and chronic hypoxia group decreased significantly, respectively. The control group was 58.4% and 71.4%, while the chronic group recovered, significantly higher than that of the acute group, and the content of AMP in the acute and chronic group was significantly higher than that in the control group; the content of ADP in the acute group was significantly increased; and the ATP/ ADP in the acute and chronic groups decreased significantly. The chronic group was significantly higher in the acute and chronic hypoxia group than in the control group. The dissociation constant (Kd) of the mitochondrial UCPs and[~ 3H]-GTP in the acute and chronic group decreased significantly, each of which was 58.1% of the control group. The most significant Bmax of the acute group of UCPs binding to[~ 3H]-GTP was significantly higher in the acute group than in the control group, and the chronic group was significantly lower than that in the control group. The expression of UCP4 and BMCP-1 mRNA and protein in the acute group was significantly higher than that in the control group and the chronic group, of which the UCP4 and BMCP-1 mRNA were significantly higher in the acute group than in the control group and the chronic group. The expression of BMCP-1 mRNA and protein in the chronic group was significantly higher than that of the control group, while the expression of BMCP-1 mRNA and protein in the chronic group was significantly higher than that of the control group. In the acute and chronic hypoxia group, the mitochondrial membrane potential (MMP) and F _ 0-F _ 1 ATPase activity of the brain tissue of the acute and chronic hypoxia group decreased significantly, and the acute group decreased to 80.5% and 55.4% in the control group, respectively, while the chronic group decreased to the control group, respectively. irradiation group 87.4% and 77.5% in the acute group. Conclusion 1. Hypoxia can significantly increase the activity of UCPs in the brain of rats. Acute hypoxia exposure can induce the expression of UCP4, BMCP-1 mRNA and protein in the brain of rats, increase the content of UCPs in the brain, and decrease the activity of UCPs in chronic hypoxia. The increase of the expression of s (UCP4, BMCP-1) is the main factor of the increase of activity. T4 respiration, which increases the effective oxygen consumption, decreases the synthesis of ATP and decreases the energy generation efficiency.3. Chronic hypoxia exposure can increase the activity of the mitochondrial UCPs in the brain of rats to a certain extent. In order to decrease the activity of UCPs in the brain tissue after long-term hypoxic acclimatization, the activity of UCPs in brain tissue can be reduced, and the effective oxygen consumption is reduced and the energy is effective.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R363
【引证文献】
相关博士学位论文 前1条
1 徐丁洁;妇科实寒症与虚寒症代谢组学及证候形成过程中相关网络调控的比较研究[D];河北医科大学;2012年
，本文编号：2499456
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