嘌呤核苷酸對(duì)模擬高原缺氧大鼠腦線粒體UCPs活性和表達(dá)的影響及其在呼吸氧耗與能量合成中的作用
發(fā)布時(shí)間：2019-05-19 18:06
【摘要】： 線粒體氧化磷酸化是由呼吸鏈在將電子傳遞給氧的過(guò)程中將質(zhì)子從內(nèi)膜基質(zhì)轉(zhuǎn)到內(nèi)膜外,從而在線粒體內(nèi)膜兩側(cè)形成質(zhì)子梯度(△H+)和電位梯度(△Ψ),兩者共同構(gòu)成驅(qū)動(dòng)ATP合成酶的跨膜質(zhì)子電動(dòng)勢(shì)(△P ),催化ADP和無(wú)機(jī)磷合成ATP。質(zhì)子還可通過(guò)內(nèi)膜上的另一質(zhì)子通道——脫偶聯(lián)蛋白(Uncoupling Proteins,UCPS)漏回到基質(zhì),形成質(zhì)子漏,降低△P,使氧化磷酸化脫偶聯(lián),減少ATP的生成,從而降低用氧效率,這部分氧耗為“無(wú)效氧耗”。高原缺氧時(shí)線粒體中脫偶聯(lián)程度增加,線粒體膜電位降低,能量生成減少,導(dǎo)致機(jī)體功能和代謝障礙。脫偶聯(lián)蛋白4(Uncoupling Proteins 4,UCP4)和脫偶聯(lián)蛋白5(Uncoupling Proteins 5,UCP5)是特異存在于哺乳動(dòng)物腦組織中的UCPs家族成員,占腦中UCPs的84%以上。嘌呤核苷酸(GDP)是UCPs的特異抑制劑。本實(shí)驗(yàn)利用體外線粒體呼吸氧耗測(cè)定體系,觀察GDP對(duì)UCPs的活性和“含量”以及對(duì)高原缺氧大鼠腦線粒體呼吸氧耗和能量代謝的影響,以探討UCPs在缺氧大鼠腦線粒體能量生成和氧利用效率中的作用。 方法 健康雄性SD大鼠暴露于模擬海拔5000米高原低壓艙內(nèi),23小時(shí)/天,分別連續(xù)缺氧3天(急性缺氧組)和30天(慢性缺氧組),同時(shí)設(shè)立平原對(duì)照組。分別在平原和模擬高原低壓艙斷頭處死,分離大鼠腦線粒體。Clark氧電極法測(cè)定線粒體氧化呼吸活性,寡霉素抑制法測(cè)定F0F1-ATP酶活性,羅丹明123法測(cè)定線粒體膜電位,高壓液相色譜法分析腦組織線粒體內(nèi)腺苷酸含量,[3H]-GTP結(jié)合法測(cè)定腦組織UCPs的活性,RT-PCR和Western blot分別測(cè)定UCP4、UCP5mRNA和蛋白表達(dá)。 結(jié)果 1.缺氧組UCPs活性明顯升高,其中急性組升高程度最大,Kd值降低43.08%,Bmax升高1.7倍;同時(shí)腦組織中UCP4和UCP5的mRNA和蛋白質(zhì)表達(dá)也明顯升高,其中急性組UCP4和UCP5的mRNA量分別升高1.47倍和3.69倍,蛋白質(zhì)表達(dá)升高 2.44倍和3.62倍;GDP能顯著抑制UCPs活性,對(duì)急性缺氧組抑制率最大,達(dá)83.13%。但對(duì)各組UCP4和UCP5的mRNA和蛋白質(zhì)表達(dá)改變無(wú)統(tǒng)計(jì)學(xué)意義。 2.缺氧組ST3、RCR、OPR、P/O和MMP明顯降低,其中急性缺氧組分別降低16.96%、38.98%、23.55%、8%和18.04%;但ST4則明顯升高36.12%。GDP可使各組呼吸氧耗顯著降低, MMP顯著升高,其中對(duì)急性缺氧組的影響最顯著,ST3和ST4分別降低31.54%和60.91%,RCR、OPR和P/O分別升高75.12%、15.08%和24.24%, MMP提高39.73%。 3.缺氧組F0F1-ATP酶活性、ATP含量、ATP/ADP和ATP/總腺苷酸比值顯著降低,以急性缺氧組降低最顯著,F0F1-ATP酶活性降低39.06%,ATP含量、ATP/ADP以及ATP/總腺苷酸比值分別降低47.67%、51.14%和47.83%。GDP可顯著升高F0F1-ATP酶活性、ATP含量、ATP/ADP和ATP/總腺苷酸比值以及能荷,其中急性組F0F1-ATP酶活性升高22.29%,ATP含量升高70.19%,ATP/ADP和ATP/總腺苷酸比值分別升高49.41%和70.85%,能荷升高30.16%。 結(jié)論 1.模擬高原缺氧暴露可顯著升高大鼠腦線粒體UCPs活性,增強(qiáng)UCP4和UCP5的mRNA和蛋白表達(dá),并且急性缺氧組更顯著;GDP可抑制正常以及缺氧暴露的大鼠腦線粒體UCPs活性,尤以急性缺氧組更明顯,但在體外對(duì)UCP4和UCP5mRNA和蛋白表達(dá)無(wú)影響。 2.模擬高原缺氧暴露可顯著增加大鼠腦線粒體脫偶聯(lián)呼吸氧耗,降低呼吸控制率和氧化磷酸化效率及膜電位;GDP具有減輕缺氧大鼠腦線粒體脫偶聯(lián)效應(yīng),降低“無(wú)效氧耗”的作用,從而提高呼吸控制率和氧化磷酸化效率。 3.模擬高原缺氧暴露可降低大鼠腦線粒體中F0F1-ATP酶活性、ATP含量、ATP/ADP和ATP/總腺苷酸比值; GDP具有升高F0F1-ATP酶活性和ATP含量的作用。 綜上所述 模擬高原缺氧暴露可使大鼠腦線粒體UCPs活性和含量增加,膜電位降低,從而使“無(wú)效氧耗”增加,氧化磷酸化效率降低,線粒體能量合成減少;GDP則具有抑制缺氧大鼠腦線粒體UCPs活性,從而提高膜電位,降低“無(wú)效氧耗”,增加氧化磷酸化效率和能量生成效率的作用,且作用程度與缺氧暴露時(shí)間有關(guān)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363
本文編號(hào)：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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363
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