黄芩茎叶黄酮减轻复合Aβ所致大鼠记忆障碍及抑制神经细胞凋亡的调节机制
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摘要
目的:探讨黄芩茎叶黄酮(SSF)对β-淀粉样蛋白25-35(Aβ25-35)结合三氯化铝(Al Cl3)及重组人类转化生长因子-β1(RHTGF-β1)(复合Aβ)所致大鼠记忆障碍及抑制神经细胞凋亡的调节机制。方法:SD雄性大鼠手术d 1脑室注射RHTGF-β1,手术d 2开始上午脑室注射Aβ25-35,连续注射14 d,下午脑室注射Al Cl3,连续注射5 d,建立记忆障碍模型,术后d 45用Morris水迷宫进行记忆障碍模型筛选,术后d 49模型成功大鼠随机分为模型组和3个剂量SSF药物组。药物组大鼠分别灌胃35,70和140 mg·kg-1SSF,qd,在给药d 31~37进行Morris水迷宫学习、记忆能力测定。Western blot法测定神经细胞线粒体膜B细胞淋巴瘤/白血病-2(Bcl-2)和Bcl-2相关X蛋白(Bax)及胞液中COX4的蛋白表达;RT-PCR法测定神经细胞胞液中CytC和Caspase-9的mRNA的表达。结果:Morris水迷宫模型筛选结果显示,本实验大鼠模型成功率为98.3%。学习记忆能力测试显示,脑室注射复合Aβ明显引起大鼠记忆获得、记忆保持和记忆再现障碍;使神经细胞线粒体膜上Bcl-2蛋白表达水平明显降低、Bax蛋白表达水平及胞液中Cyto C和Caspase-9 mRNA表达水平都明显增加。而灌胃35,70和140 mg·kg-1SSF不同程度地逆转复合Aβ所致大鼠上述学习记忆障碍及线粒体膜Bcl-2和Bax及胞液中Cyto C和Caspase-9表达的异常改变。线粒体膜和胞液中COX4在各组中变化不明显。结论:SSF能够改善复合Aβ所致大鼠记忆障碍,其作用机制可能是通过抑制神经细胞凋亡和调节线粒体凋亡通路中凋亡因子完成的。
Objective: To investigate the effects of flavonoid from Scutellaria stems and leaves( SSF) on memory impairment and regulation mechanism by inhibiting apoptosis in rats induced by amyloid beta protein 25-35( Aβ25- 35) in combination with Al Cl3 and RHTGF-β1( composited Aβ). Methods: The memory impairment model was established by microinjection of RHTGF-β1into cerebral ventricle of male Sprague Dawley( SD) rats on the first day of operation,accompanied by daily microinjection of Aβ25- 35 for 14 d in the morning and Al Cl3 for 5 d in the afternoon,respectively from the second day of the operation. The successful model screening was performed for4 d with Morris water maze since day 45 of operation. On the day 49 of operation,the successful model rats were randomly divided into model group and three SSF-treatment groups. The rats in the drug groups were orally given35,70 and 140 mg·kg- 1SSF daily. The ability of learning and memory was measured with Morris water maze from day 31 to 37 of drug treatment. Levels of protein expression of Bcl-2 and Bax on mitochondrial membrane and COX4 in cytoplasm were detected with western blotting,and mRNA expressions of Cyt-C and Caspase-9 in cytoplasm were assayed by RT-PCR. Results: The rat model screening by Morris water maze showed a successful model rate of 98. 30% and the ability of learning and memory tests showed that the composited Aβ given by intracerebral ventricle injection obviously caused memory acquirement,memory retention and memory re-extract disorders to rats. The protein expression of Bcl-2 on mitochondrial membrane of neuron dramatically decreased and Bax protein expression and mRNA expression of Cyt-C and Caspase-9 in cytochylema were markedly increased.However,the three doses of SSF 35,70 and 140 mg·kg- 1can differently reversed the above abnormal changes induced by composited Aβ in rats. The changes of COX4 in mitochondrial membrane and cytochylema were not obvious. Conclusion: SSF can ameliorate rats' memory impairment induced by composited Aβ,and the action mechanism of SSF is primarily derived from inhibiting apoptosis and regulating the apoptosis factor in the mitochondrial apoptotic pathway.
Objective: To investigate the effects of flavonoid from Scutellaria stems and leaves( SSF) on memory impairment and regulation mechanism by inhibiting apoptosis in rats induced by amyloid beta protein 25-35( Aβ25- 35) in combination with Al Cl3 and RHTGF-β1( composited Aβ). Methods: The memory impairment model was established by microinjection of RHTGF-β1into cerebral ventricle of male Sprague Dawley( SD) rats on the first day of operation,accompanied by daily microinjection of Aβ25- 35 for 14 d in the morning and Al Cl3 for 5 d in the afternoon,respectively from the second day of the operation. The successful model screening was performed for4 d with Morris water maze since day 45 of operation. On the day 49 of operation,the successful model rats were randomly divided into model group and three SSF-treatment groups. The rats in the drug groups were orally given35,70 and 140 mg·kg- 1SSF daily. The ability of learning and memory was measured with Morris water maze from day 31 to 37 of drug treatment. Levels of protein expression of Bcl-2 and Bax on mitochondrial membrane and COX4 in cytoplasm were detected with western blotting,and mRNA expressions of Cyt-C and Caspase-9 in cytoplasm were assayed by RT-PCR. Results: The rat model screening by Morris water maze showed a successful model rate of 98. 30% and the ability of learning and memory tests showed that the composited Aβ given by intracerebral ventricle injection obviously caused memory acquirement,memory retention and memory re-extract disorders to rats. The protein expression of Bcl-2 on mitochondrial membrane of neuron dramatically decreased and Bax protein expression and mRNA expression of Cyt-C and Caspase-9 in cytochylema were markedly increased.However,the three doses of SSF 35,70 and 140 mg·kg- 1can differently reversed the above abnormal changes induced by composited Aβ in rats. The changes of COX4 in mitochondrial membrane and cytochylema were not obvious. Conclusion: SSF can ameliorate rats' memory impairment induced by composited Aβ,and the action mechanism of SSF is primarily derived from inhibiting apoptosis and regulating the apoptosis factor in the mitochondrial apoptotic pathway.
引文
[1]曲艳,刘萍,李晓.阿尔茨海默病生物学标志物研究进展[J].医学与哲学,2013,34(10B):55-57.
[2]蒋术一,毛凤菊,李兴强,等.Aβ的神经毒性及治疗[J].中国当代医药,2011,18(12):21-22.
[3]SADIGH-ETEGHAD S,SABERMAROUF B,MAJDI A,et al.Amyloid-beta:a crucial factor in Alzheimer's disease[J].Med Princ Pract,2015,24(1):1-10.
[4]SHUCHANG H,QIAO N,PIYE N,et al.Protective effects of gastrodia elata on aluminium-chloride-induced learning impairments and alterations of amino acid neurotransmitter release in adult rats[J].Restor Neurol Neurosci,2008,26(6):467-473.
[5]RUI D,YONGJIAN Y.Aluminum chloride induced oxidative damage on cells derived from hippocampus and cortex of ICR mice[J].Brain Res,2010,1324:96-102.
[6]SHI-LEI S,GUANG-YU MA,BACHELOR LH,et al.Effect of naloxone on aluminum-induced learning and memory impairment in rats[J].Neurol India,2005,53(1):79-82.
[7]曾芳,余曙光,唐勇,等.老年性痴呆复合动物模型研究概况[J].中国神经免疫学和神经病学杂志,2007,14(4):197-200.
[8]SMALL DH,GASPERINI R,VINCENT AJ,et al.The role of A beta-induced calcium dysregulation in the pathogenesis of Alzheimer's disease[J].J Alzheimer's Dis,2009,16(2):225-233.
[9]DAJAS F,RIVERA-MEGRET F,BLASINA F,et al.Neuroprotection by flavonoids[J].Braz J Med Biol Res,2003,36(12):1613-1620.
[10]商亚珍,苏丙凡,王永利.黄芩根及其茎叶成分的药理学研究进展[J].承德医学院学报,2005,22(2):154-155.
[11]王玉梅,刘永平,曹凯,等.黄芩茎叶黄酮对大鼠缺血性记忆障碍和神经炎症的作用[J].中国药理学与毒理学杂志,2011,25(2):135-140.
[12]赵泓翔,郭可,崔亚迪,等.复合Aβ25-35引起线粒体膜Bcl-2、Bax、Bcl-x L及Bak的异常及半枝莲黄酮的干预作用[J].中国病理生理杂志,2014,30(12):2262-2266.
[13]SHANG Y,CHENG J,QI J,et al.Scutellaria flavonoid reduced memory dysfunction and neuronal injury caused by permanent global ischemia in rats[J].Pharmacol Biochem Behav,2005,82(1):67-73.
[14]刘海洋,韩金利,孙征,等.Giemsa染色法标记脑组织凋亡细胞的可行性验证[J].解剖学研究,2013,35(2):152-153.
[15]王玲,朱鸣峰.阿尔茨海默病发病机制的研究进展[J].吉林医学,2013,4(3):531-532.
[16]FINDER VH,GLOCKSHUBER R.Amyloid-beta aggregation[J].Neurod Egener Dis,2007,4(1):13-27.
[17]BUCHHAVE P,MINTHON L,ZETTERBERG H,et al.Cerebrospinal fluid levels ofβ-amyloid 1-42,but not of tau,are fully changed already 5 to10 years before the onset of Alzheimer dementia[J].Arch Gen Psychiatry,2012,69(1):98-106.
[18]JIN M,SHEPARDSON N,YANG T,et al.Soluble amyloidβ-protein dimmers isolated from Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration[J].Proc Natl Acad Sci USA,2011,108(14):5819-5824.
[19]张璇,杨叶雅,蔡青青.BACE1-siRNA纳米复合物对阿尔茨海默病模型小鼠学习记忆和相关蛋白表达的影响[J].中国新药杂志,2015,24(22):2012-2017.
[20]GALANTE D,CORSARO A,FLORIO T,et al.Differential toxicity conformation and morphology of typical initial aggregation states of Aβ1-42 and Aβpy3-42 beta-amyloids[J].Int J Biochem Cell Biol,2012,44(11):2085-2093.
[21]AKIYAMA H,HOSOKAWA M,KAMETANI F,et al.Long-term oral intake of aluminium or zinc does not accelerate Alzheimer pathology in AβPP and AβPP/tau transgenic mice[J].Neuropathology,2012,32(4):390-397.
[22]GHRIBI O,HERMAN MM,FORBES MS,et al.GDNF protects against aluminum-induced apoptosis in rabbits by upregulating Bcl-2 and Bcl-xl and inhibiting mitochondrial Bax translocation[J].Neurobiol Dis,2001,8(5):764-773.
[23]KAWAHARA M,KATO M,KURODA Y.Effect s of aluminum on the neurotoxicity of primary cultured neurons and on the aggregation of beta-amyloid protein[J].Brain Res Bull,2001,55(2):211-217.
[24]方芳,晏勇,冯占辉,等.多因素损伤的老年性痴呆动物模型的实验研究[J].重庆医学,2007,36(2):146-151.
[25]SCHINDLER CK,SHINODA S,SIMON RP,et al.Subcellular distribution of Bcl-2 family proteins and 14-3-3 within the hippocampus during seizure-induced neuronal death in the rat[J].Neurosci Lett,2004,356(3):163-166.
[26]SHI Y.Mechanisms of Caspase activation and inhibition during apoptosis[J].Mol Cell,2002,9(3):459-470.
[27]ZHAO H,YENARI MA,CHENG D,et al.Bcl-2 overexpression protects against neuron loss within the ischemic margin following experimental stroke and inhibits cytochrome C translocation and caspase-3 activity[J].J Neurochem,2003,85(9):1026-1036.
[28]史静宇,陈思,孙岩,等.胰岛素对Aβ25-35损伤的神经元的保护作用[J].中国新药杂志,2014,23(10):1192-1196.
[2]蒋术一,毛凤菊,李兴强,等.Aβ的神经毒性及治疗[J].中国当代医药,2011,18(12):21-22.
[3]SADIGH-ETEGHAD S,SABERMAROUF B,MAJDI A,et al.Amyloid-beta:a crucial factor in Alzheimer's disease[J].Med Princ Pract,2015,24(1):1-10.
[4]SHUCHANG H,QIAO N,PIYE N,et al.Protective effects of gastrodia elata on aluminium-chloride-induced learning impairments and alterations of amino acid neurotransmitter release in adult rats[J].Restor Neurol Neurosci,2008,26(6):467-473.
[5]RUI D,YONGJIAN Y.Aluminum chloride induced oxidative damage on cells derived from hippocampus and cortex of ICR mice[J].Brain Res,2010,1324:96-102.
[6]SHI-LEI S,GUANG-YU MA,BACHELOR LH,et al.Effect of naloxone on aluminum-induced learning and memory impairment in rats[J].Neurol India,2005,53(1):79-82.
[7]曾芳,余曙光,唐勇,等.老年性痴呆复合动物模型研究概况[J].中国神经免疫学和神经病学杂志,2007,14(4):197-200.
[8]SMALL DH,GASPERINI R,VINCENT AJ,et al.The role of A beta-induced calcium dysregulation in the pathogenesis of Alzheimer's disease[J].J Alzheimer's Dis,2009,16(2):225-233.
[9]DAJAS F,RIVERA-MEGRET F,BLASINA F,et al.Neuroprotection by flavonoids[J].Braz J Med Biol Res,2003,36(12):1613-1620.
[10]商亚珍,苏丙凡,王永利.黄芩根及其茎叶成分的药理学研究进展[J].承德医学院学报,2005,22(2):154-155.
[11]王玉梅,刘永平,曹凯,等.黄芩茎叶黄酮对大鼠缺血性记忆障碍和神经炎症的作用[J].中国药理学与毒理学杂志,2011,25(2):135-140.
[12]赵泓翔,郭可,崔亚迪,等.复合Aβ25-35引起线粒体膜Bcl-2、Bax、Bcl-x L及Bak的异常及半枝莲黄酮的干预作用[J].中国病理生理杂志,2014,30(12):2262-2266.
[13]SHANG Y,CHENG J,QI J,et al.Scutellaria flavonoid reduced memory dysfunction and neuronal injury caused by permanent global ischemia in rats[J].Pharmacol Biochem Behav,2005,82(1):67-73.
[14]刘海洋,韩金利,孙征,等.Giemsa染色法标记脑组织凋亡细胞的可行性验证[J].解剖学研究,2013,35(2):152-153.
[15]王玲,朱鸣峰.阿尔茨海默病发病机制的研究进展[J].吉林医学,2013,4(3):531-532.
[16]FINDER VH,GLOCKSHUBER R.Amyloid-beta aggregation[J].Neurod Egener Dis,2007,4(1):13-27.
[17]BUCHHAVE P,MINTHON L,ZETTERBERG H,et al.Cerebrospinal fluid levels ofβ-amyloid 1-42,but not of tau,are fully changed already 5 to10 years before the onset of Alzheimer dementia[J].Arch Gen Psychiatry,2012,69(1):98-106.
[18]JIN M,SHEPARDSON N,YANG T,et al.Soluble amyloidβ-protein dimmers isolated from Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration[J].Proc Natl Acad Sci USA,2011,108(14):5819-5824.
[19]张璇,杨叶雅,蔡青青.BACE1-siRNA纳米复合物对阿尔茨海默病模型小鼠学习记忆和相关蛋白表达的影响[J].中国新药杂志,2015,24(22):2012-2017.
[20]GALANTE D,CORSARO A,FLORIO T,et al.Differential toxicity conformation and morphology of typical initial aggregation states of Aβ1-42 and Aβpy3-42 beta-amyloids[J].Int J Biochem Cell Biol,2012,44(11):2085-2093.
[21]AKIYAMA H,HOSOKAWA M,KAMETANI F,et al.Long-term oral intake of aluminium or zinc does not accelerate Alzheimer pathology in AβPP and AβPP/tau transgenic mice[J].Neuropathology,2012,32(4):390-397.
[22]GHRIBI O,HERMAN MM,FORBES MS,et al.GDNF protects against aluminum-induced apoptosis in rabbits by upregulating Bcl-2 and Bcl-xl and inhibiting mitochondrial Bax translocation[J].Neurobiol Dis,2001,8(5):764-773.
[23]KAWAHARA M,KATO M,KURODA Y.Effect s of aluminum on the neurotoxicity of primary cultured neurons and on the aggregation of beta-amyloid protein[J].Brain Res Bull,2001,55(2):211-217.
[24]方芳,晏勇,冯占辉,等.多因素损伤的老年性痴呆动物模型的实验研究[J].重庆医学,2007,36(2):146-151.
[25]SCHINDLER CK,SHINODA S,SIMON RP,et al.Subcellular distribution of Bcl-2 family proteins and 14-3-3 within the hippocampus during seizure-induced neuronal death in the rat[J].Neurosci Lett,2004,356(3):163-166.
[26]SHI Y.Mechanisms of Caspase activation and inhibition during apoptosis[J].Mol Cell,2002,9(3):459-470.
[27]ZHAO H,YENARI MA,CHENG D,et al.Bcl-2 overexpression protects against neuron loss within the ischemic margin following experimental stroke and inhibits cytochrome C translocation and caspase-3 activity[J].J Neurochem,2003,85(9):1026-1036.
[28]史静宇,陈思,孙岩,等.胰岛素对Aβ25-35损伤的神经元的保护作用[J].中国新药杂志,2014,23(10):1192-1196.