阿司匹林对大鼠脑缺血再灌注后线粒体UCP4和ATPase6表达的影响
摘要
背景目的:脑缺血再灌注损伤(CI/RP)后,神经元细胞的损伤包括坏死和凋亡两种形式,线粒体作为细胞的“能源中心”,在这两种过程中都起着重要的作用。解偶联蛋白4(UCP4)和ATPase6都是存在于线粒体内膜上的离子转运蛋白。UCP4激活后可以使氧化磷酸化与呼吸链解偶联,通过维持线粒体膜电位、调节细胞内Ca2+稳态、减少氧自由基(ROS)生成、影响ATP含量、调节神经元能量等机制起到神经保护作用。ATPase6属于ATP合成酶F0复合体,是ATP生成的必要环节,其变化直接影响ATP的合成。阿司匹林(ASA)是经典的抗甾体药物,近年来发现,ASA具有神经保护作用:包括增加ATP产量,抑制谷氨酸释放,改善脑组织的能量代谢,抑制iNOS表达和促进nNOS表达,抑制NF-κB的激活,清除自由基,增强cdk5/P53活性,抑制离子通道,阻止Zn2+内流等。我们以前的研究证明,ASA抑制凋亡途径中的Fasl和caspase-3,上调Bcl-2/bax,抑制炎症反应以及增加脑组织内源性神经营养因子如BDNF、NGF、bFGF、neurgulin等的表达。本实验目的是,观察大鼠脑缺血/再灌注(CI/RP)损伤后线粒体UCP4和ATPase6的表达规律及其ASA对其表达的影响,探讨ASA神经保护作用的线粒体机制。
方法:采用改良Zea Longa线栓法建立大鼠大脑中动脉(MCA)的CI/RP动物模型。CI/RP后6h,Bederson神经功能缺损评分在1～3分的大鼠为阳性入选标准,其他大鼠排除在外。部分大鼠脑切片行TTC染色,证实模型成功。依照再灌注时间分为A(0h)、B(6h)、C(24h)、D(48h)、E(72h)5个ASA组及相应的对照组(A'～B')。ASA组于CI/RP术后立即腹腔注射ASA 80mg/kg.d作为干预因素,直至预定的处死时间;对照组腹腔注射等量的赖氨酸溶液。各时间点对实验动物进行神经功能缺损评分,HE染色后光镜下观察缺血后不同时间点梗死侧病理改变,利用免疫组化及免疫荧光法观察UCP4和ATPase6的表达情况。
结果:CI/RP后ASA组及对照组术后6h开始,大鼠肢体功能均有不同程度的恢复,ASA组肢体功能恢复情况优于对照组,但是只在72h点有显著差异(P<0.05)。UCP4和ATPase6在大脑皮质及海马神经元内广泛表达,定位到线粒体,主要位于神经元胞浆内,在梗死灶周边区可见强阳性细胞及大量阳性细胞聚集。对照组,UCP4于CI/RP后6h在梗死中心区表达最强(P<0.05),阳性(+～++)细胞数或强阳性(+++～++++)细胞数最多,随后呈下降趋势,24h后表达开始低于正常侧;ASA组,UCP4于CI/RP后48h还可见到第二个表达高峰。对照组,ATPase6自缺血2h后就低于正常侧,在各时间点呈明显的下降趋势,CI/RP后24h下降最显著;ASA组,ATPase6的变化趋势与对照组相似,但是,各时间点的阳性细胞数均高于相应对照组,24h时最明显,强阳性细胞数最多(P<0.01)。ASA对非缺血侧脑组织中的UCP4及ATPase6的含量未见明显影响。
结论:1. ASA能够明显改善脑缺血后大鼠的神经功能缺损。2.脑缺血后,线粒体UCP4的表达增加,ATPase6的表达呈下降趋势。3. ASA可以增加缺血侧脑组织线粒体内UCP4和ATPase6的表达,对非缺血侧脑组织线粒体内UCP4和ATPase6表达未见明显影响。4.增加缺血脑组织线粒体UCP4和ATPase6的表达可能是ASA神经保护机制之一。
Background and purpose: After Ischemia/Reperfusion, damnification of the neuronal cell has two forms necrosis and apoptotic. As the“energy center”of the cell, the mitochondrial play the important role in the two forms. Recently, we distinguish that UCP4 is a kinds of transporters located in the inner membrane of mitochondrion. Actived UCP4 can dissociate oxidative phosphorylation from respiration. UCP4 can protect the neuronal cell by regulating the mitochondrion membrane potential, maintaining the Ca2+ homeostasis, reducing oxidative stress,influencing the ATP production, regulating the energy of cell. ATPase6 is also a transporters presented in the inner membrane of mitochondrion. It is a important part of F0-F1-ATP synthase and the progress of ATP production. In addition, as a hydronium pump, ATPase6 can keep the hydronium balance of the two sides of memdrane, maintain the memdrane′s function, benefit the energy metabolize. The change of ATPase6 must influence the produce, and than relate the survival or death of the cell. So the expression of the ATPase6 can reflect the ATP level. Aspirin (ASA) is a classical non-steroidal anti- inflammatory drug. Recently researches discovered that ASA also had the direct neuroprotective effects, such as increase of the ATP output; suppression of the glutamic acid release, reform of the energy metabolize of cerebra; decrease of Inos expression, and increase of the nNOS expression; inhibition of NF-κB activation; elimination of the ROS; activation of cdk5/P53; inhibition hydronium pore, prevention of Zn2+ inflow; inhibition of Fasl and caspase-3 in apoptotic, enhancement the expression of Bcl-2/bax; increase the BDNF, NGF, bFGF; decrease inflammation. So the main intention of this study is to clearing the expression rule of UCP4 and ATPase6 after CI/RP in rats, and the neuroprotective mechanism of ASA, how ASA effect the expression of UCP4 and ATPase6.
Methods: The rat model of MCA-CI/RP were established with suture occlusion technique according to modified Zea Longa method and the rats of 1-3 score(Bederson measuring scale) was selected into the 10 groups at 6 hours after CI/RP. Some cerebral slices were taken for TTC staining. The rats were divided into five groups according to the time of reperfusion in control groups: A(0h), B(6h), C(24h), D(48h) and E(72h), so did the experimental groups (A'～E'). ASA were given to the experimental groups with the dose of 80mg/kg via an i.p. route, while the same dose of diaminocaproic acid were given to the control groups after the CI/RP and the next three days till killed at assigned time. The neurologic detect scale were used by Bederson scores. The UCP4 and ATPase6 expression were detected by immunohistochemistry and immunofluorescence technique.
Results: Comparing with the control groups, the Bederson measuring scale of experiment groups were conspicuously reduced, but only at 72 hours after CI/RP had statistical significance(P<0.05). The positive UCP4 and ATPase6 could be seen in the cytoplasm in cortex broadly. In the control groups, the expression of UCP4 reached to peak at 6 hours after CI/RP, and descended gradually later; but had two peaks at 6 hours and 48 hours in experiment groups. ATPase6 declined evidently in both control and experiment groups and significance at 24 hours (P<0.01). It is no evidence that ASA can effect on UCP4 and ATPase6 in no-ischemia cerebra.
Conclusion: 1. Ischemia can induce UCP4 expression in neurons overlying in the infarct region after CI/RP and the expression has a peak at 6 hours. 2. ATPase6 expresses descendant and significance at 24 hours after CI/RP. 3. ASA can enhance the expression of UCP4 and ATPase6 in ischemia cerebra and have no influence in no- ischemia cerebra. The expression of UCP4 has 6 hours and 48 hours two peaks. 4. ASA may attenuate the neurologic impairment after CI/RP. The mechanism of neuroprotective effect of ASA maybe due to enhance the expression of UCP4 and ATPase6, decrease the produce of ROS, maintaining the Ca2+ homeostasis and advance the ATP level relatively.
方法:采用改良Zea Longa线栓法建立大鼠大脑中动脉(MCA)的CI/RP动物模型。CI/RP后6h,Bederson神经功能缺损评分在1～3分的大鼠为阳性入选标准,其他大鼠排除在外。部分大鼠脑切片行TTC染色,证实模型成功。依照再灌注时间分为A(0h)、B(6h)、C(24h)、D(48h)、E(72h)5个ASA组及相应的对照组(A'～B')。ASA组于CI/RP术后立即腹腔注射ASA 80mg/kg.d作为干预因素,直至预定的处死时间;对照组腹腔注射等量的赖氨酸溶液。各时间点对实验动物进行神经功能缺损评分,HE染色后光镜下观察缺血后不同时间点梗死侧病理改变,利用免疫组化及免疫荧光法观察UCP4和ATPase6的表达情况。
结果:CI/RP后ASA组及对照组术后6h开始,大鼠肢体功能均有不同程度的恢复,ASA组肢体功能恢复情况优于对照组,但是只在72h点有显著差异(P<0.05)。UCP4和ATPase6在大脑皮质及海马神经元内广泛表达,定位到线粒体,主要位于神经元胞浆内,在梗死灶周边区可见强阳性细胞及大量阳性细胞聚集。对照组,UCP4于CI/RP后6h在梗死中心区表达最强(P<0.05),阳性(+～++)细胞数或强阳性(+++～++++)细胞数最多,随后呈下降趋势,24h后表达开始低于正常侧;ASA组,UCP4于CI/RP后48h还可见到第二个表达高峰。对照组,ATPase6自缺血2h后就低于正常侧,在各时间点呈明显的下降趋势,CI/RP后24h下降最显著;ASA组,ATPase6的变化趋势与对照组相似,但是,各时间点的阳性细胞数均高于相应对照组,24h时最明显,强阳性细胞数最多(P<0.01)。ASA对非缺血侧脑组织中的UCP4及ATPase6的含量未见明显影响。
结论:1. ASA能够明显改善脑缺血后大鼠的神经功能缺损。2.脑缺血后,线粒体UCP4的表达增加,ATPase6的表达呈下降趋势。3. ASA可以增加缺血侧脑组织线粒体内UCP4和ATPase6的表达,对非缺血侧脑组织线粒体内UCP4和ATPase6表达未见明显影响。4.增加缺血脑组织线粒体UCP4和ATPase6的表达可能是ASA神经保护机制之一。
Background and purpose: After Ischemia/Reperfusion, damnification of the neuronal cell has two forms necrosis and apoptotic. As the“energy center”of the cell, the mitochondrial play the important role in the two forms. Recently, we distinguish that UCP4 is a kinds of transporters located in the inner membrane of mitochondrion. Actived UCP4 can dissociate oxidative phosphorylation from respiration. UCP4 can protect the neuronal cell by regulating the mitochondrion membrane potential, maintaining the Ca2+ homeostasis, reducing oxidative stress,influencing the ATP production, regulating the energy of cell. ATPase6 is also a transporters presented in the inner membrane of mitochondrion. It is a important part of F0-F1-ATP synthase and the progress of ATP production. In addition, as a hydronium pump, ATPase6 can keep the hydronium balance of the two sides of memdrane, maintain the memdrane′s function, benefit the energy metabolize. The change of ATPase6 must influence the produce, and than relate the survival or death of the cell. So the expression of the ATPase6 can reflect the ATP level. Aspirin (ASA) is a classical non-steroidal anti- inflammatory drug. Recently researches discovered that ASA also had the direct neuroprotective effects, such as increase of the ATP output; suppression of the glutamic acid release, reform of the energy metabolize of cerebra; decrease of Inos expression, and increase of the nNOS expression; inhibition of NF-κB activation; elimination of the ROS; activation of cdk5/P53; inhibition hydronium pore, prevention of Zn2+ inflow; inhibition of Fasl and caspase-3 in apoptotic, enhancement the expression of Bcl-2/bax; increase the BDNF, NGF, bFGF; decrease inflammation. So the main intention of this study is to clearing the expression rule of UCP4 and ATPase6 after CI/RP in rats, and the neuroprotective mechanism of ASA, how ASA effect the expression of UCP4 and ATPase6.
Methods: The rat model of MCA-CI/RP were established with suture occlusion technique according to modified Zea Longa method and the rats of 1-3 score(Bederson measuring scale) was selected into the 10 groups at 6 hours after CI/RP. Some cerebral slices were taken for TTC staining. The rats were divided into five groups according to the time of reperfusion in control groups: A(0h), B(6h), C(24h), D(48h) and E(72h), so did the experimental groups (A'～E'). ASA were given to the experimental groups with the dose of 80mg/kg via an i.p. route, while the same dose of diaminocaproic acid were given to the control groups after the CI/RP and the next three days till killed at assigned time. The neurologic detect scale were used by Bederson scores. The UCP4 and ATPase6 expression were detected by immunohistochemistry and immunofluorescence technique.
Results: Comparing with the control groups, the Bederson measuring scale of experiment groups were conspicuously reduced, but only at 72 hours after CI/RP had statistical significance(P<0.05). The positive UCP4 and ATPase6 could be seen in the cytoplasm in cortex broadly. In the control groups, the expression of UCP4 reached to peak at 6 hours after CI/RP, and descended gradually later; but had two peaks at 6 hours and 48 hours in experiment groups. ATPase6 declined evidently in both control and experiment groups and significance at 24 hours (P<0.01). It is no evidence that ASA can effect on UCP4 and ATPase6 in no-ischemia cerebra.
Conclusion: 1. Ischemia can induce UCP4 expression in neurons overlying in the infarct region after CI/RP and the expression has a peak at 6 hours. 2. ATPase6 expresses descendant and significance at 24 hours after CI/RP. 3. ASA can enhance the expression of UCP4 and ATPase6 in ischemia cerebra and have no influence in no- ischemia cerebra. The expression of UCP4 has 6 hours and 48 hours two peaks. 4. ASA may attenuate the neurologic impairment after CI/RP. The mechanism of neuroprotective effect of ASA maybe due to enhance the expression of UCP4 and ATPase6, decrease the produce of ROS, maintaining the Ca2+ homeostasis and advance the ATP level relatively.
引文
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1. Pawade T, Ho PW, Kwok kh, et al. Uncoupling proteins: Targets of endocrine disruptors? Mol Cell Endocrinol, 2005, 244(1-2):79-86
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9. Chan SL, Liu D, Kyriazis GA, et al. Mitochondrial uncoupling protein-4 regulates calcium homeostasis and sensitivity to store depletion-induced apoptosis in neural cells. J Biol Chem, 2006, 281(49):37391-37403
10. Diano S, Matthew RT, Patrylo P, et al. Uncoupling protein 2 prevents neuronal death including that occurring during seizures: a mechanism for preconditioning. Endocrinology. Endocrinology, 2003, 144(11):5014-5021
11. Kagawa Y, Cha SH, Hasegawa K, et al. Regulation of energy metabolism in human cells in aging and diabetes: FoF1, mtDNA, UCP, and ROS. Biochem Biophys Res Commun, 1999, 266(3): 662-676
12. Ho PW, Chan DY, Kwok KH, et al. Methyl-4-phenylpyridinium ion modulates expression of mitochondrial uncoupling proteins 2, 4, and 5 in catecholaminergic (SK-N-SH) cells. J Neurosci Res, 2005, 81(2):261-268
13. Gasiora M, Rogawskia MA, Hartmana AL. Neuroprotective and disease- modifying effects of the ketogenic diet. Behav Pharmacol, 2006, 17(5-6): 431-439
14. Liu D, Chan SL, de Souza-Pinto NC, et al. Mitochondrial UCP4 mediates an adaptive shift in energy metabolism and increases the resistance of neurons to metabolic and oxidative stress. Neuromolecular Med,2006,8(3): 389-414
15. Andrews ZB, Diano S, Horvath TL. Mitochondrial uncoupling proteins in the CNS: in support of function and survival. Nat Rev Neurosci, 2005, 6(11): 829-840
16. NaudíA, Caro P, JovéM,et al. Methionine Restriction Decreases Endogenous Oxidative Molecular Damage and Increases Mitochondrial Biogenesis and Uncoupling Protein 4 in Rat Brain. Rejuvenation Res, 2007, [Epub ahead of print]
17. Echtay KS. Mitochondrial uncoupling proteins–what is their physiological role? Free Radic Biol Med, 2007, 43(10):1351-1371
18. Vincent AM, Olzmann JA, Brownlee M, et al. Uncoupling proteins prevent glucose-induced neuronal oxidative stress and programmed cell death. Diabetes, 2004, 53(3):726–734
19. Zhang M, Wang B, Ni YH, et al. Overexpression of uncoupling protein 4 promotes proliferation and inhibits apoptosis and differentiation of preadipocytes. Life Sci, 2006, 79(15):1428-1435
20. Yasuno K, Ando S, Misumi S, et al. Synergistic association of mitochondrial uncoupling protein (UCP) genes with schizophrenia. Am J Med Genet B Neuropsychiatr Genet, 2007, 144(2):250-253