【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程中將質(zhì)子從內(nèi)膜基質(zhì)轉(zhuǎn)到內(nèi)膜外,形成跨膜質(zhì)子電動勢(△P),驅(qū)動ATP合成酶催化ADP和無機磷合成ATP。質(zhì)子還可通過內(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)中,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“無效氧耗”。高原缺氧時線粒體脫偶聯(lián)增強,膜電位降低,能量生成減少,導致機體功能和代謝障礙。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物腦組織中的UCPs家族成員,占腦中UCPs的84%以上。游離脂肪酸通過與UCPs分子構(gòu)象中某些位點結(jié)合而促進棕色脂肪內(nèi)UCP1,骨骼肌中UCP2和UCP3的活性,是潛在的UCP4、UCP5的激動劑。本實驗通過棕櫚酸腦組織塊體外干預模型和棕櫚酸游離腦線粒體干預模型,觀察外源性游離脂肪酸對UCPs的活性和“含量”的影響及其在高原缺氧大鼠腦線粒體能量合成改變的作用,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及調(diào)控機制。 方法 分別建立棕櫚酸腦組織塊體外干預模型和棕櫚酸腦游離線粒體干預模型,在觀察了棕櫚酸對大鼠腦組織UCP4和UCP5 mRNA和蛋白表達的時效性影響以及對游離腦線粒體氧化磷酸化影響的時效性和量效性基礎(chǔ)上,進一步探討了棕櫚酸對模擬高原缺氧大鼠腦UCPs表達、活性及其與線粒體呼吸氧耗和能量生成的關(guān)系。健康雄性SD大鼠暴露于模擬海拔5000米高原低壓艙內(nèi),23h/d,分別連續(xù)缺氧3天(急性組)和30天(慢性組),同時設立對照組。心臟取血后,分別在平原和模擬高原低壓艙斷頭處死,取大腦半球,分別切成1～2×2～3mm2的組織碎塊和直接分離線粒體,分別以100μmol/L的棕櫚酸進行腦組織塊體外干預和游離腦線粒體體外干預,以Clark氧電極法測定線粒體氧化呼吸活性,TPMP+電極與Clark氧電極結(jié)合測質(zhì)子漏,寡霉素抑制法測定F0F1-ATP酶活性,羅丹明123法測定線粒體膜電位,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)合法測定腦組織UCPs的活性。RT-PCR和Westernblot分別測定腦組織干預后UCP4、UCP5 mRNA和蛋白表達。同時,以銅離子法測定缺氧大鼠血清、腦組織勻漿液和線粒體內(nèi)游離脂肪酸含量。 結(jié)果 1.100μmol/L的棕櫚酸大鼠腦組織塊體外干預時間達30min時,UCP4和UCP5 mRNA表達達其峰值,而蛋白表達亦顯著升高,線粒體氧化磷酸化呼吸效率顯著降低。棕櫚酸大鼠游離腦線粒體體外干預,當濃度在0.1 mmol/L內(nèi),時間在1min內(nèi)時與線粒體氧化呼吸存在量效關(guān)系。 2.缺氧能增強大鼠腦組織UCPs活性,急性缺氧組Kd值降低41.24%,而Bmax值升高1.56倍。棕櫚酸干預能進一步增加各組UCPs的活性,但使急性組增加幅度最低,Kd值僅下降13.96%,而Bmax則僅升高16.01%。 3.缺氧使大鼠血清、腦組織勻漿、線粒體內(nèi)游離脂肪酸含量升高,以急性組升高最明顯,分別達到51.36%, 243.35%和69.49%,慢性組較急性組有所下降,但仍高于對照組。相關(guān)性分析顯示,大鼠血清、腦組織、線粒體游離脂肪酸含量與反映UCPs活性的Kd值呈線性負相關(guān),與反映UCPs“含量”的Bmax呈線性正相關(guān)(腦組織游離脂肪酸含量與UCPs活性的相關(guān)系數(shù)最高)。 4.缺氧組腦線粒體ST3、RCR、OPR、P/O和MMP明顯降低,ST4、質(zhì)子漏則明顯升高。棕櫚酸可進一步增加各組呼吸氧耗和質(zhì)子漏,降低MMP,其中對急性缺氧組的影響最小,ST3和ST4分別升高5.12%和38.69%,RCR、OPR和P/O分別升高24.64%、3.19%和3.96%,MMP僅降低6.91%。 5.缺氧組腦線粒體F0F1-ATP酶活性、ATP含量、ATP/ADP和ATP/總腺苷酸比值顯著降低,以急性組降低最顯著,分別降低43.32%、27.79%、16.39%和28.67%。棕櫚酸可使急性組總腺苷酸池(ATP+ADP+AMP)、(ATP+ADP)池含量分別降低30.90%和49.29%以及各組ATP/ADP均升高,且急性組升高163.04%;同時各組ATP/總腺苷酸池比值均降低,對照組、急性組能荷均分別降低36.91%和13.64%。 6.缺氧組腦UCP4、UCP5mRNA和蛋白表達增高,其中急性組上調(diào)幅度最大,UCP4mRNA和蛋白表達分別為對照組的19.04倍和16.95倍,UCP5 mRNA以及蛋白則分別為對照組的2.17倍和4.71倍。棕櫚酸體外干預進一步增強各組UCP4、UCP5mRNA和蛋白表達,但急性組升高幅度最小。 結(jié)論 1.棕櫚酸可直接影響腦線粒體UCPs活性和UCP4和UCP5 mRNA和蛋白表達,增強質(zhì)子漏和脫偶聯(lián)呼吸,進而影響腦線粒體的氧化磷酸化功能,降低氧化磷酸化效率,且有時間和劑量依賴性。缺氧暴露在一定程度上可削弱棕櫚酸促UCPs活性和含量的效能。 2.缺氧可增加腦線粒體UCPs活性,以及UCP4、UCP5 mRNA和蛋白表達,與缺氧時血及腦內(nèi)游離脂肪酸代謝改變有關(guān)。缺氧時游離脂肪酸升高——UCPs活性增強——解耦聯(lián)——ATP生成下降為缺氧時腦能量合成障礙的中心環(huán)節(jié)。 全文總結(jié) 模擬高原缺氧暴露可使大鼠血清、腦組織及線粒體內(nèi)游離脂肪酸含量升高,腦線粒體UCPs活性和含量增加,腦UCP4、UCP5 mRNA和蛋白表達增強,質(zhì)子漏增強,膜電位降低,從而使“無效氧耗”增加,氧化磷酸化效率降低,線粒體能量合成減少;棕櫚酸則可進一步升高缺氧大鼠腦線粒體UCPs活性與含量,增強腦UCP4、UCP5 mRNA和蛋白表達,從而增加質(zhì)子漏,降低膜電位,增強脫偶聯(lián),具有降低氧化磷酸化效率和能量生成效率的作用。棕櫚酸的這些作用與缺氧暴露有關(guān),缺氧暴露可弱化棕櫚酸的作用。實驗揭示了模擬高原缺氧時游離脂肪酸——UCPs表達、含量及活性——線粒體呼吸氧耗——ATP生成之間的相互關(guān)系,提示游離脂肪酸——UCPs相互作用是缺氧時組織能量代謝障礙的重要環(huán)節(jié)之一。
[Abstract]:Mitochondrial oxidative phosphorylation is the transfer of protons from the endometrial matrix to the outer membrane by the respiratory chain during electron transfer to oxygen, forming a transmembrane proton electromotive force (delta P), which drives ATP synthase to catalyze the synthesis of ATP from ADP and inorganic phosphorus. Back in the matrix, proton leaks are formed, Delta P is reduced, oxidative phosphorylation is decoupled, ATP is reduced, and oxygen use efficiency is reduced. This part of oxygen consumption is "ineffective oxygen consumption". UCP4 and UCP5 are members of the UCPs family specifically present in mammalian brain tissues, accounting for more than 84% of the UCPs in the brain. Free fatty acids promote the activity of UCP1 in brown fat, UCP2 and UCP3 in skeletal muscle by binding to certain sites in the conformation of UCPs. They are potential agonists of UCP4 and UCP5. In order to investigate the effect of exogenous free fatty acids on the activity and content of UCPs and the change of mitochondrial energy synthesis in the brain of hypoxic rats at high altitude, the in vitro intervention model of palmitic acid brain block and the model of palmitic acid free brain mitochondrial intervention were used to observe the effect of exogenous free fatty acids on the activity and content of UCPs. The role and regulation mechanism of efficiency.
Method
The in vitro intervention model of palmitic acid brain tissue block and the model of palmitic acid brain free mitochondria intervention were established. The effect of palmitic acid on the expression of UCP4 and UCP5 mRNA and protein in rat brain tissue and the effect of palmitic acid on the oxidative phosphorylation of free brain mitochondria were observed. Healthy male SD rats were exposed to simulated altitude hypobaric chamber at 5000 m for 23 h/d for 3 days (acute group) and 30 days (chronic group) respectively, and control group was set up. After blood collection, the rats were exposed to simulated altitude hypobaric chamber for 3 days (acute group) and 30 days (chronic group). Cerebral hemisphere was cut into 1-2 *2-3 mm2 tissue fragments and mitochondria were separated directly. Brain tissue fragments and free brain mitochondria were interfered with palmitic acid at 100 micromol/L in vitro. Mitochondrial oxidative respiratory activity was measured by Clark oxygen electrode, proton leakage was detected by TPMP + electrode and Clark oxygen electrode. The activity of F0F1-ATPase was measured by inhibiting factor assay, mitochondrial membrane potential by rhodamine 123, adenylate content in brain mitochondria by high performance liquid chromatography, and UCPs activity by combining [3H]-GTP. The expression of UCP4 and UCP5 mRNA and protein were measured by RT-PCR and Western blot respectively. The contents of free fatty acids in serum, brain homogenate and mitochondria were measured in rats.
Result
The expression of UCP4 and UCP5 mRNA reached its peak value, and the expression of UCP4 and UCP5 protein increased significantly, and the respiratory efficiency of mitochondria oxidative phosphorylation decreased significantly. The free cerebral mitochondria of palmitic acid Rats Intervened in vitro when the concentration of palmitic acid was within 0.1 mmol/L and the time was within 1 minute. There is a dose effect relationship.
2. Hypoxia could enhance the activity of UCPs in rat brain tissue. The Kd value decreased by 41.24% in acute hypoxia group, while the Bmax value increased by 1.56 times. Palmitic acid intervention could further increase the activity of UCPs in each group, but the increase was the lowest in acute group. The Kd value decreased only by 13.96%, while the Bmax value increased only by 16.01%.
3. Hypoxia increased the content of free fatty acids in serum, brain homogenate and mitochondria of rats, the highest in acute group was 51.36%, 243.35% and 69.49% respectively. The content of free fatty acids in serum, brain tissue and mitochondria of chronic group was lower than that of acute group, but still higher than that of control group. There was a linear negative correlation between D value and Bmax reflecting UCPs content (the highest correlation coefficient between free fatty acid content and UCPs activity in brain tissue).
4. Mitochondrial ST3, RCR, OPR, P/O and MMP decreased significantly in hypoxic group, while ST4 and proton leakage increased significantly. Palmitic acid could further increase respiratory oxygen consumption and proton leakage, and reduce MMP. Among them, ST3 and ST4 increased by 5.12% and 38.69%, RCR, OPR and P/O increased by 24.64%, 3.19% and 3.96% respectively, while MMP decreased by only 6.91%.
5. The activity of F0F1-ATPase, ATP content, ATP/ADP and ATP/total adenylate ratio in the hypoxic group were significantly decreased, with the most significant decrease in the acute group, 43.32%, 27.79%, 16.39% and 28.67% respectively. Palmitic acid could decrease the total adenylate pool (ATP+ADP+AMP), ATP+ADP pool (ATP+ADP) content by 30.90% and ATP/ADP ratio by 49.29% in the acute group. At the same time, ATP / total adenylate pool ratio of each group decreased, while energy charge of control group and acute group decreased by 36.91% and 13.64% respectively.
6. The expression of UCP4, UCP5 mRNA and protein in the brain of hypoxic group increased significantly. The expression of UCP4 mRNA and protein was 19.04 and 16.95 times higher in the acute group than in the control group. The expression of UCP5 mRNA and protein was 2.17 and 4.71 times higher in the hypoxic group than that in the control group. The minimum amplitude.
conclusion
1. Palmitic acid can directly affect the activity of UCPs and the expression of UCP4 and UCP5 mRNA and protein in brain mitochondria, enhance proton leakage and decoupling respiration, then affect the oxidative phosphorylation function of brain mitochondria, reduce the efficiency of oxidative phosphorylation, and it is time and dose dependent. Hypoxia exposure can weaken the activity and content of palmitic acid-induced UCPs to a certain extent. Efficiency.
2. Hypoxia can increase the activity of UCPs in brain mitochondria, and the expression of UCP4, UCP5 mRNA and protein, which is related to the changes of free fatty acid metabolism in blood and brain during hypoxia.
A summary of the full text
Simulated high altitude hypoxia exposure can increase the free fatty acid content in serum, brain tissue and mitochondria, increase the activity and content of UCPs in brain mitochondria, increase the expression of UCP4 and UCP5 mRNA and protein, increase the proton leakage and decrease the membrane potential, so that the "ineffective oxygen consumption" increases, the oxidative phosphorylation efficiency decreases, and mitochondrial energy synthesis decreases. Palmitic acid can further increase the activity and content of UCPs in the brain mitochondria of hypoxic rats, enhance the expression of UCP4, UCP5 mRNA and protein, thereby increasing proton leakage, reducing membrane potential, enhancing decoupling, and reducing the efficiency of oxidative phosphorylation and energy production. The experiment reveals the relationship between the expression, content and activity of free fatty acid-UCPs, oxygen consumption of mitochondria and ATP production during simulated altitude hypoxia, suggesting that the interaction of free fatty acid-UCPs is one of the important links of energy metabolism disorder during hypoxia.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R363

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