急性缺氧对氯霉素处理大鼠脑线粒体氧化呼吸功能及细胞色素氧化酶亚基表达的影响及其机制
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摘要
缺氧是常见而又重要的病理生理过程，由其导致的中枢神经系统功能障碍与线粒体能量生成不足密切相关。脑组织对缺氧最敏感，急性缺氧时脑线粒体氧化磷酸化功能改变及细胞核与线粒体两基因组之间相互调控的机制还不清楚。线粒体结构和功能的完整相当程度上依赖于呼吸链上酶功能的正常发挥。由线粒体DNA(mtDNA)和核DNA(nDNA) 编码的13个蛋白亚基组成的细胞色素氧化酶(COX)是呼吸链上的关键酶，其亚基的正确匹配对酶功能的正常发挥极其重要，也是线粒体能量合成的基础。氯霉素是线粒体蛋白翻译特异抑制剂，利用氯霉素处理大鼠建立实验模型，观察急性缺氧时脑线粒体氧化呼吸功能、COX活性及其亚基表达及相关调控因子mtTFA和NRF-1的表达，探讨急性缺氧时脑能量代谢障碍的机制及线粒体氧化呼吸功能改变在其中的作用，进而探讨线粒体自身遗传系统在维持其氧化呼吸功能中的作用及其与细胞核基因组之间存在的协调调控关系。Wistar系大鼠随机分为四组：平原对照组、药物处理组、急性缺氧组和药物处理+急性缺氧组。药物处理为0.5%氯霉素，腹腔注射， 50mg/kg体重，1次/12小时，连续注射7天后低压舱内取材；动物缺氧条件为：低压舱模拟海拔5000米高原，大气压为405.35mmHg，连续减压 24h。平原对照组与药物处理组于舱外同时喂养，并于实验前7天开始腹腔注射等量的生理盐水。采用本室建立的方法提取脑皮质线粒体，Clark电极法测定线粒体呼吸活性，极谱法测量COX活性，Western Blot分析COXⅠ、COXⅣ和NRF-1蛋白表达，RT-PCR检测COXⅠ、COXⅣ、12s rRNA、mtTFA和NRF-1 mRNA稳态量（state level）。
主要结果
1、急性缺氧暴露24小时，大鼠脑线粒体ST3和RCR显著降低，ST4显著升高，与正常对照组相比，差异非常显著；而氯霉素处理大鼠经急性缺氧暴露后，脑线粒体ST3较正常对照组显著降低，与单纯缺氧组相比无显著差异， ST4比单纯缺氧组显著降低，而且也显著低于正常对照组， RCR比单纯缺氧组显著升高，但仍低于正常组。
2、急性缺氧暴露24小时，大鼠脑线粒体COX活性显著降低，而氯霉素处理大鼠急性缺氧后COX活性显著回升，由65.7%回升到86.5%，但仍低于正常组。
3、急性缺氧暴露24小时，大鼠脑线粒体COXⅠ蛋白表达比正常组显著降低，致使COXⅣ/COXⅠ比值显著升高，COXⅣ和脑组织NRF-1的表达则无改变；急性缺氧与氯霉素处理两因素对COXⅠ蛋白表达、COXⅣ/COXⅠ比值有显著交互影响，与正常组比无差异，脑组织NRF-1蛋白的表达未受影响。
4、急性缺氧暴露24小时，大鼠脑皮质COXⅣ、mtTFA、NRF-1 mRNA均显著降低，COXⅠ、COXⅣ/COXⅠ mRNA比值及12s rRNA未受影响；而氯霉素处理大鼠经急性缺氧暴露后，COXⅠ、COXⅣ、COXⅣ/COXⅠ比值及mtTFA mRNA较单纯缺氧时均显著升高，与正常组比较无显著差异， NRF-1 mRMA与单纯缺氧组比较无显著差异，但显著低于正常组。
结 论
1. 急性缺氧暴露可造成大鼠脑线粒体氧化呼吸功能障碍，但经线粒体蛋白合成特异抑制剂氯霉素处理的大鼠再经急性缺氧暴露时可以改善单纯缺氧造成的线粒体功能障碍，提高氧化磷酸化效率。而COX活性的变化与线粒体氧化呼吸改变的一致，提示COX在缺氧和氯霉素处理的大鼠脑线粒体呼吸链氧化磷酸化功能改变中的重要作用。
2. 急性缺氧以及氯霉素处理后大鼠脑线粒体COXⅠ蛋白量的改变与COX活性相一致，提示COXⅠ在全酶功能发挥中起着主导作用，同时表明缺氧对COX活性的影响至少在一定程度上受到酶蛋白亚基表达的定量调节。
3. 通过对COXⅠ、COX Ⅳ和mtTFA、NRF-1的mRNA和蛋白水平表达的观察发现，缺氧以及氯霉素处理对COX亚基表达的调控表现在细胞核与线粒体两个基因组的转录与翻译两个水平上，而转录后或翻译水平上存在微细调整。
It is known that brain is the most sensitive organ to hypoxia and mitochondrial dyfunction resulted from acute hypoxia is a key factor for the disorder in brain energy metabolism. Objective To understand the changing aspects of mitochondrial oxidative phosphorylation function and cytochrome c oxidase activity during acute hypoxia exposure and their mechanism regulated by gene expression encoded by mtDNA and nDNA. An animal model set by chloramphenicol(CAP) administration ,CAP is a specific inhibitor for mitochondrial protein synthesis,was used. Methods Adult male Wistar rats were divided into four groups.They were hypoxia exposure group(H group), medication group(M group), medication plus hypoxia exposure group(MH group) and control group(C group). Medication was administrated by giving CAP(50mg/kg, intraperitoneal injection) every 12 hours for 7 days. Hypoxia exposure was administrasted by exposing rats to a hypobaric chamber simulated 5000m high altitude for 24 hours. C group received only equal amount of normal saline by intraperitoneal injection every 12 hours for 7 days. All animals were sacrificed by decapitation under normoxic(C and M) and hypoxic(H and MH)conditions respectively at 12 hours after the last injection. The rat cerebral cortex was removed and the mitochondria was isolated by centrifugation programme. Mitochondrial respiratory function and COX activity were measured by Clark oxygen electrode. The protein content of COX subunit I and IV in mitochondria and NRF-1 in cerebral tissues was detected by Western blot analysis. And mRNA state levels of COXⅠ,COXⅣ,12s rRNA,mtTFA and NRF-1 in tissues were determined by RT-PCR. Results 1) Compared with C group, H group showed elevated state 4 respiration(ST4) and decreased state 3 respiration(ST3) and respiratory control rate(RCR) in mitochondrial respiration during acute hypoxic exposure significantly; ST3 in MH group was significantly lower than that in C group but not decrease than in H group,while ST4 in MH group is lower significantly than that in H group as well as C group. RCR in MH group was higher than that in H group but lower than that in C group; 2) COX
activity in H group decreased significantly than in C group. In MH group, COX activity increased and was higher than that in H group, but which was still lower than in C group, restored from 65.7% to 86.5% of the control(C group) level; 3) A decreased protein content of COX subunit I and an elevated ratio of subunit IV/ I were observed in H group than in C group. There was no significant difference of the protein content of COXI and the ratio of subunit IV/ I between C and MH groups. But a mutual effect by two factors(medication and acute hypoxia) was observed in MH group.The protein content of COXIV and NRF-1 remained similar among all groups; 4) Compared with C group, H group showed significant decreased COXIV, mtTFA and NRF-1 mRNA state level, while 12s rRNA, COXI and the ratio of subunit IV/ I mRNA had no significant change among groups; In MH group, mtTFA,COXI , COXIV mRNA and the ratio of subunit IV/ I mRNA showed significant higher than those in H group but not than in C group. The state level of NRF-1 mRNA decreased significantly in MH group than in C group, but not than in H group;12s rRNA state level had no significant changes among all groups.
Conclusion：1) Acute hypoxic exposure could lead to mitochondrial respiratory dyfunction, bur CAP admistration might be beneficial to the recovering of rat respiratory function and oxidative phosphorylation efficiency during acute hypoxic exposure. The change of COX activity is consistent with that of mitochondrial respiratory function during acute hypoxic exposure and CAP-admistration, which indicated that COX played an important role in oxidative phosphorylation function of motochondria from cerebral cortex of hypoxic and CAP-administrated rats; 2) The alteration in content of COX subunit I protein in mitochondria from cerebral cortex of rats exposed to acute hypoxia and CAP-administrated, is consistent with that of COX activity
主要结果
1、急性缺氧暴露24小时，大鼠脑线粒体ST3和RCR显著降低，ST4显著升高，与正常对照组相比，差异非常显著；而氯霉素处理大鼠经急性缺氧暴露后，脑线粒体ST3较正常对照组显著降低，与单纯缺氧组相比无显著差异， ST4比单纯缺氧组显著降低，而且也显著低于正常对照组， RCR比单纯缺氧组显著升高，但仍低于正常组。
2、急性缺氧暴露24小时，大鼠脑线粒体COX活性显著降低，而氯霉素处理大鼠急性缺氧后COX活性显著回升，由65.7%回升到86.5%，但仍低于正常组。
3、急性缺氧暴露24小时，大鼠脑线粒体COXⅠ蛋白表达比正常组显著降低，致使COXⅣ/COXⅠ比值显著升高，COXⅣ和脑组织NRF-1的表达则无改变；急性缺氧与氯霉素处理两因素对COXⅠ蛋白表达、COXⅣ/COXⅠ比值有显著交互影响，与正常组比无差异，脑组织NRF-1蛋白的表达未受影响。
4、急性缺氧暴露24小时，大鼠脑皮质COXⅣ、mtTFA、NRF-1 mRNA均显著降低，COXⅠ、COXⅣ/COXⅠ mRNA比值及12s rRNA未受影响；而氯霉素处理大鼠经急性缺氧暴露后，COXⅠ、COXⅣ、COXⅣ/COXⅠ比值及mtTFA mRNA较单纯缺氧时均显著升高，与正常组比较无显著差异， NRF-1 mRMA与单纯缺氧组比较无显著差异，但显著低于正常组。
结 论
1. 急性缺氧暴露可造成大鼠脑线粒体氧化呼吸功能障碍，但经线粒体蛋白合成特异抑制剂氯霉素处理的大鼠再经急性缺氧暴露时可以改善单纯缺氧造成的线粒体功能障碍，提高氧化磷酸化效率。而COX活性的变化与线粒体氧化呼吸改变的一致，提示COX在缺氧和氯霉素处理的大鼠脑线粒体呼吸链氧化磷酸化功能改变中的重要作用。
2. 急性缺氧以及氯霉素处理后大鼠脑线粒体COXⅠ蛋白量的改变与COX活性相一致，提示COXⅠ在全酶功能发挥中起着主导作用，同时表明缺氧对COX活性的影响至少在一定程度上受到酶蛋白亚基表达的定量调节。
3. 通过对COXⅠ、COX Ⅳ和mtTFA、NRF-1的mRNA和蛋白水平表达的观察发现，缺氧以及氯霉素处理对COX亚基表达的调控表现在细胞核与线粒体两个基因组的转录与翻译两个水平上，而转录后或翻译水平上存在微细调整。
It is known that brain is the most sensitive organ to hypoxia and mitochondrial dyfunction resulted from acute hypoxia is a key factor for the disorder in brain energy metabolism. Objective To understand the changing aspects of mitochondrial oxidative phosphorylation function and cytochrome c oxidase activity during acute hypoxia exposure and their mechanism regulated by gene expression encoded by mtDNA and nDNA. An animal model set by chloramphenicol(CAP) administration ,CAP is a specific inhibitor for mitochondrial protein synthesis,was used. Methods Adult male Wistar rats were divided into four groups.They were hypoxia exposure group(H group), medication group(M group), medication plus hypoxia exposure group(MH group) and control group(C group). Medication was administrated by giving CAP(50mg/kg, intraperitoneal injection) every 12 hours for 7 days. Hypoxia exposure was administrasted by exposing rats to a hypobaric chamber simulated 5000m high altitude for 24 hours. C group received only equal amount of normal saline by intraperitoneal injection every 12 hours for 7 days. All animals were sacrificed by decapitation under normoxic(C and M) and hypoxic(H and MH)conditions respectively at 12 hours after the last injection. The rat cerebral cortex was removed and the mitochondria was isolated by centrifugation programme. Mitochondrial respiratory function and COX activity were measured by Clark oxygen electrode. The protein content of COX subunit I and IV in mitochondria and NRF-1 in cerebral tissues was detected by Western blot analysis. And mRNA state levels of COXⅠ,COXⅣ,12s rRNA,mtTFA and NRF-1 in tissues were determined by RT-PCR. Results 1) Compared with C group, H group showed elevated state 4 respiration(ST4) and decreased state 3 respiration(ST3) and respiratory control rate(RCR) in mitochondrial respiration during acute hypoxic exposure significantly; ST3 in MH group was significantly lower than that in C group but not decrease than in H group,while ST4 in MH group is lower significantly than that in H group as well as C group. RCR in MH group was higher than that in H group but lower than that in C group; 2) COX
activity in H group decreased significantly than in C group. In MH group, COX activity increased and was higher than that in H group, but which was still lower than in C group, restored from 65.7% to 86.5% of the control(C group) level; 3) A decreased protein content of COX subunit I and an elevated ratio of subunit IV/ I were observed in H group than in C group. There was no significant difference of the protein content of COXI and the ratio of subunit IV/ I between C and MH groups. But a mutual effect by two factors(medication and acute hypoxia) was observed in MH group.The protein content of COXIV and NRF-1 remained similar among all groups; 4) Compared with C group, H group showed significant decreased COXIV, mtTFA and NRF-1 mRNA state level, while 12s rRNA, COXI and the ratio of subunit IV/ I mRNA had no significant change among groups; In MH group, mtTFA,COXI , COXIV mRNA and the ratio of subunit IV/ I mRNA showed significant higher than those in H group but not than in C group. The state level of NRF-1 mRNA decreased significantly in MH group than in C group, but not than in H group;12s rRNA state level had no significant changes among all groups.
Conclusion：1) Acute hypoxic exposure could lead to mitochondrial respiratory dyfunction, bur CAP admistration might be beneficial to the recovering of rat respiratory function and oxidative phosphorylation efficiency during acute hypoxic exposure. The change of COX activity is consistent with that of mitochondrial respiratory function during acute hypoxic exposure and CAP-admistration, which indicated that COX played an important role in oxidative phosphorylation function of motochondria from cerebral cortex of hypoxic and CAP-administrated rats; 2) The alteration in content of COX subunit I protein in mitochondria from cerebral cortex of rats exposed to acute hypoxia and CAP-administrated, is consistent with that of COX activity
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