欧前胡素对Aβ所致AD大鼠模型的神经保护作用
|
摘要
目的探讨欧前胡素对Aβ_(1-40)所致阿尔茨海默病(AD)大鼠模型的神经保护作用及其可能机制。方法将50只SD大鼠随机分为5组(假手术组、模型组、欧前胡素低剂量组、欧前胡素中剂量组、欧前胡素高剂量组),每组各10只。模型组、欧前胡素低剂量组、欧前胡素中剂量组、欧前胡素高剂量组大鼠开颅后,向双侧海马组织海马角1(CA1)区注射Aβ_(1-40)制备AD大鼠模型,假手术组大鼠注射等体积生理盐水。随后,欧前胡素低剂量组、中剂量组和高剂量组大鼠灌胃不同剂量的欧前胡素(25mg·kg~(-1)、50mg·kg~(-1)、100mg·kg~(-1)),模型组和假手术组每日给予等体积生理盐水。干预第17天开始进行Morris水迷宫实验检测大鼠空间学习和记忆能力。干预第21天后,处死大鼠,取双侧尾状核组织,利用免疫组织化学染色和Highman刚果红染色检测大鼠大脑海马神经元组织学变化,流式细胞术检测大鼠海马组织细胞内活性氧(ROS)含量,氧化酶法检测超氧化物歧化酶(SOD)活性,硫代巴比妥酸法检测丙二醛(MDA)含量,逆转录—聚合酶链反应(RT-PCR)和蛋白印迹法(Western blotting)检测大鼠海马组织还原型烟酰胺腺嘌呤二核苷酸(NADPH)氧化酶家族成员亚基(gp91~(phox)、p22~(phox)、p47~(phox)、p67~(phox))表达,Western blotting检测p53、Bcl-2、Bax蛋白表达。结果模型组大鼠寻台时间多于其他各组大鼠,平台滞留时间、穿越平台次数少于其他各组大鼠,欧前胡素干预的大鼠呈剂量依赖性缩短寻台时间,增加平台滞留时间和穿越平台次数,差异分别具有统计学意义(P<0.05)。模型组大鼠海马组织神经元排列混乱,细胞数量少于其他各组大鼠,细胞结构模糊,橘红色淀粉样物质沉积较多,欧前胡素干预的大鼠呈剂量依赖性改善海马组织神经元结构和数量,减少淀粉样物质沉积。模型组大鼠ROS、MDA含量均高于其他各组大鼠,SOD活性低于其他各组大鼠,欧前胡素干预的大鼠呈剂量依赖性降低ROS、MDA含量,升高SOD活性,差异均有统计学意义(P<0.05)。模型组大鼠gp91~(phox)、p22~(phox)、p47~(phox)、p67~(phox)表达均高于其他各组大鼠,欧前胡素干预的大鼠呈剂量依赖性降低gp9l~(phox)、p22~(phox)、p47~(phox)、p67~(phox)表达,差异分别具有统计学意义(P<0.05)。模型组大鼠p53、Bax表达高于其他各组大鼠,Bcl-2表达低于其他各组大鼠,欧前胡素干预的大鼠呈剂量依赖性降低p53、Bax表达,增加Bcl-2表达,差异分别有统计学意义(P<0.05)。结论欧前胡素对Aβ所致AD大鼠模型认知障碍具有明显的改善作用,同时具有一定的神经保护作用,其机制可能是通过抑制NADPH氧化酶-ROS-p53线粒体凋亡通路及其下游p53、Bcl-2、Bax蛋白表达,进而减少海马组织神经元细胞氧化应激损伤,起到改善认知障碍和保护神经的作用。
Objective To investigate the neuroprotective effect of imperatorin on A β_(1-40)-induced Alzheimer's(AD)rat model and its possible mechanism.Method Fifty SD rats were randomly divided into 5 groups(sham group,model group,low dose group of imperatorin,middle dose group of imperatorin,and high dose group of imperatorin),with 10 rats in each group.After the rats in the model group,low dose group,middle dose group and high dose group were cranialized,A β_(1-40) was injected into Cornu Ammonis 1(CA1)area of bilateral hippocampus to prepare AD rat model.The sham group were injected with an equal volume of saline.Subsequently,low dose group,middle dose group and high dose group rats were given different doses of imperatorin(25 mg · kg~(-1),50 mg· kg~(-1),100 mg·kg~(-1)),and the model group and sham operation group were given an equal volume of normal saline.On the 17 th day of intervention,the Morris water maze test was conducted to detect spatial learning and memory in rats.After the 21 st day of intervention,rats were sacrificed and bilateral caudate nucleus tissues were obtained.Flow cytometry was used to detect the content of reactive oxygen species(ROS)in the hippocampus of rats.The activity of superoxide dismutase(SOD)was detected by oxidase method.Malondialdehyde(MDA)content was detected by thiobarbituric acid method.Nicotinamide adenine dinucleotide(NADPH)oxidase family member(gp91~(phox),p22~(phox),p47~(phox),p67~(phox))subunits in rat hippocampus was detected by polymerase chain reaction(RT-PCR)and Western blotting.The p53,Bcl-2,Bax protein expressionwere detected by Western blotting.Results Thelatencytime of the modelgroupwassignificantlylongerthanthatofthe other groups,while the time spent in the quadrant,times of platform crossing were significantly shorter thanthatofthe other groups(P<0.05).Rats treated with imperatorin reduced dosedependent time,increased platform retention time,and number of platform crossings.The neurons in the hippocampus of rats in the model group were disordered.The number of cells was less than that of other groups.The structure of the cells was fuzzy,and the amyloid deposition was more.The rats treated with imperatorin in a dose-dependent manner improved hippocampal tissue nerves.The structure and number of elements reduce amyloid deposition.The ROS and MDA contents in the model group were higher than those in the other groups,and the SOD activity was lower than that in other groups.The rats treated with imperatorin reduced the levels of ROS and MDA in dose-dependent manner,and increased SOD activity.The differences were statistically significant(P<0.05).The expression of gp91~(phox),p22~(phox),p47~(phox),and p67~(phox) in the model group was higher than that in other groups.The rats treated with imperatorin decreased the expressions of those in a dose-dependent manner,and the differences were statistically significant(P<0.05).The expression of p53 and Bax in the model group was higher than that in other groups,and the expression of Bcl-2 was lower than that in other groups.The rats treated with imperatorin decreased the expression of p53 and Bax in a dose-dependent manner and increased Bcl-2.Expression,difference were statistically significant(P<0.05).Conclusion Imperatorin can significantly improve cognitive impairment in AD rat model induced by A β,and it also has neuroprotective effect.The mechanism may be through inhibition of NADPH oxidase-ROS-p53 mitochondrial apoptosis pathway and its downstream effect.The expression of p53,Bcl-2,and Bax proteins can reduce the oxidative stress damage of hippocampal neuronal cells and play a role in improving cognitive impairment and neuroprotection.
Objective To investigate the neuroprotective effect of imperatorin on A β_(1-40)-induced Alzheimer's(AD)rat model and its possible mechanism.Method Fifty SD rats were randomly divided into 5 groups(sham group,model group,low dose group of imperatorin,middle dose group of imperatorin,and high dose group of imperatorin),with 10 rats in each group.After the rats in the model group,low dose group,middle dose group and high dose group were cranialized,A β_(1-40) was injected into Cornu Ammonis 1(CA1)area of bilateral hippocampus to prepare AD rat model.The sham group were injected with an equal volume of saline.Subsequently,low dose group,middle dose group and high dose group rats were given different doses of imperatorin(25 mg · kg~(-1),50 mg· kg~(-1),100 mg·kg~(-1)),and the model group and sham operation group were given an equal volume of normal saline.On the 17 th day of intervention,the Morris water maze test was conducted to detect spatial learning and memory in rats.After the 21 st day of intervention,rats were sacrificed and bilateral caudate nucleus tissues were obtained.Flow cytometry was used to detect the content of reactive oxygen species(ROS)in the hippocampus of rats.The activity of superoxide dismutase(SOD)was detected by oxidase method.Malondialdehyde(MDA)content was detected by thiobarbituric acid method.Nicotinamide adenine dinucleotide(NADPH)oxidase family member(gp91~(phox),p22~(phox),p47~(phox),p67~(phox))subunits in rat hippocampus was detected by polymerase chain reaction(RT-PCR)and Western blotting.The p53,Bcl-2,Bax protein expressionwere detected by Western blotting.Results Thelatencytime of the modelgroupwassignificantlylongerthanthatofthe other groups,while the time spent in the quadrant,times of platform crossing were significantly shorter thanthatofthe other groups(P<0.05).Rats treated with imperatorin reduced dosedependent time,increased platform retention time,and number of platform crossings.The neurons in the hippocampus of rats in the model group were disordered.The number of cells was less than that of other groups.The structure of the cells was fuzzy,and the amyloid deposition was more.The rats treated with imperatorin in a dose-dependent manner improved hippocampal tissue nerves.The structure and number of elements reduce amyloid deposition.The ROS and MDA contents in the model group were higher than those in the other groups,and the SOD activity was lower than that in other groups.The rats treated with imperatorin reduced the levels of ROS and MDA in dose-dependent manner,and increased SOD activity.The differences were statistically significant(P<0.05).The expression of gp91~(phox),p22~(phox),p47~(phox),and p67~(phox) in the model group was higher than that in other groups.The rats treated with imperatorin decreased the expressions of those in a dose-dependent manner,and the differences were statistically significant(P<0.05).The expression of p53 and Bax in the model group was higher than that in other groups,and the expression of Bcl-2 was lower than that in other groups.The rats treated with imperatorin decreased the expression of p53 and Bax in a dose-dependent manner and increased Bcl-2.Expression,difference were statistically significant(P<0.05).Conclusion Imperatorin can significantly improve cognitive impairment in AD rat model induced by A β,and it also has neuroprotective effect.The mechanism may be through inhibition of NADPH oxidase-ROS-p53 mitochondrial apoptosis pathway and its downstream effect.The expression of p53,Bcl-2,and Bax proteins can reduce the oxidative stress damage of hippocampal neuronal cells and play a role in improving cognitive impairment and neuroprotection.
引文
[1]杨小花,胡晓.欧前胡素与异欧前胡素的药理学研究进展[J].南昌大学学报:医学版,2012,52(3):95-97.
[2]Zhang J,Zhang M,Fu S,et al.Simultaneous determination of imperatorin and its metabolite xanthotoxol in rat plasma by using HPLC-ESI-MS coupled with hollow fiber liquid phase microextraction[J].J Chromatogr B Analyt Technol Biomed Life Sci,2014,945-946:185-192.
[3]Zhou N,Zhang Y,Wang T,et al.The imperatorin derivative OW1,a new vasoactive compound,inhibits VSMC proliferation and extracellular matrix hyperplasia[J].Toxicol Appl Pharmacol,2015,284(2):125-133.
[4]Budzynska B,Boguszewska-Czubara A,Kruk-Slomka M,et al.Effects of imperatorin on nicotine-induced anxiety-and memoryrelated responses and oxidative stress in mice[J].Physiol Behav,2013,122:46-55.
[5]Raja SB,Murali MR,Roopa K,et al.Imperatorin a furocoumarin inhibits periplasmic Cu-Zn SOD of Shigella dysenteriae their by modulates its resistance towards phagocytosis during host pathogen interaction[J].Biomed Pharmacother,2011,65(8):560-568.
[6]Wang L,Benzinger T L,Su Y,et al.Evaluation of tau imaging in staging Alzheimer disease and revealing interactions betweenβ-amyloid and tauopathy[J].JAMA Neurology,2016,73(9):1070-1077.
[7]Cervellati C, Romani A, Seripa D, et al.Oxidative balance,homocysteine,and uric acid levels in older patients with late onset Alzheimer's disease or vascular dementia[J].J Neurol Sci,2014,337(1-2):156-161.
[8]Farr SA,Ripley JL,Sultana R,et al.Antisense oligonucleotide against GSK-3βin brain of SAMP8 mice improves learning and memory and decreases oxidative stress:Involvement of transcription factor Nrf2 and implications for Alzheimer disease[J].Free Radic Biol Med,2014,67:387-395.
[9]Liu Y,Hettinger CL,Zhang D,et al.The proteasome function reporter GFPu accumulates in young brains of the APPswe/PS1dE9Alzheimer's disease mouse model[J].Cell Mol Neurobiol,2014,34(3):315-322.
[10]Safdar A,Annis S,Kraytsberg Y,et al.Amelioration of premature aging in mtDNA mutator mouse by exercise:the interplay of oxidative stress,PGC-1α, p53,and DNA damage.A hypothesis[J].Curr Opin Genet Dev,2016,38:127-132.
[11]Nadesalingam A,Chen J H K,Farahvash A,et al.hypertonic saline suppresses NADPH oxidase-dependent neutrophil extracellular trap formation and promotes apoptosis[J].Front Immunol,2018,9:359.
[12]Altenh?fer S,Radermacher K A,Kleikers P W,et al.Evolution of NADPH oxidase inhibitors:selectivity and mechanisms for target engagement[J].Antioxid Redox Sign,2015,23(5):406-427.
[13]Karabay A Z,Aktan F, Sunguroglu A,et al.Methylsulfonylmethane modulates apoptosis of LPS/IFN-γ-activated RAW 264.7macrophage-like cells by targeting p53,Bax,Bcl-2,cytochrome c and PARP proteins[J].Immunopharmacol Immunotoxicol,2014,36(6):379-389.
[2]Zhang J,Zhang M,Fu S,et al.Simultaneous determination of imperatorin and its metabolite xanthotoxol in rat plasma by using HPLC-ESI-MS coupled with hollow fiber liquid phase microextraction[J].J Chromatogr B Analyt Technol Biomed Life Sci,2014,945-946:185-192.
[3]Zhou N,Zhang Y,Wang T,et al.The imperatorin derivative OW1,a new vasoactive compound,inhibits VSMC proliferation and extracellular matrix hyperplasia[J].Toxicol Appl Pharmacol,2015,284(2):125-133.
[4]Budzynska B,Boguszewska-Czubara A,Kruk-Slomka M,et al.Effects of imperatorin on nicotine-induced anxiety-and memoryrelated responses and oxidative stress in mice[J].Physiol Behav,2013,122:46-55.
[5]Raja SB,Murali MR,Roopa K,et al.Imperatorin a furocoumarin inhibits periplasmic Cu-Zn SOD of Shigella dysenteriae their by modulates its resistance towards phagocytosis during host pathogen interaction[J].Biomed Pharmacother,2011,65(8):560-568.
[6]Wang L,Benzinger T L,Su Y,et al.Evaluation of tau imaging in staging Alzheimer disease and revealing interactions betweenβ-amyloid and tauopathy[J].JAMA Neurology,2016,73(9):1070-1077.
[7]Cervellati C, Romani A, Seripa D, et al.Oxidative balance,homocysteine,and uric acid levels in older patients with late onset Alzheimer's disease or vascular dementia[J].J Neurol Sci,2014,337(1-2):156-161.
[8]Farr SA,Ripley JL,Sultana R,et al.Antisense oligonucleotide against GSK-3βin brain of SAMP8 mice improves learning and memory and decreases oxidative stress:Involvement of transcription factor Nrf2 and implications for Alzheimer disease[J].Free Radic Biol Med,2014,67:387-395.
[9]Liu Y,Hettinger CL,Zhang D,et al.The proteasome function reporter GFPu accumulates in young brains of the APPswe/PS1dE9Alzheimer's disease mouse model[J].Cell Mol Neurobiol,2014,34(3):315-322.
[10]Safdar A,Annis S,Kraytsberg Y,et al.Amelioration of premature aging in mtDNA mutator mouse by exercise:the interplay of oxidative stress,PGC-1α, p53,and DNA damage.A hypothesis[J].Curr Opin Genet Dev,2016,38:127-132.
[11]Nadesalingam A,Chen J H K,Farahvash A,et al.hypertonic saline suppresses NADPH oxidase-dependent neutrophil extracellular trap formation and promotes apoptosis[J].Front Immunol,2018,9:359.
[12]Altenh?fer S,Radermacher K A,Kleikers P W,et al.Evolution of NADPH oxidase inhibitors:selectivity and mechanisms for target engagement[J].Antioxid Redox Sign,2015,23(5):406-427.
[13]Karabay A Z,Aktan F, Sunguroglu A,et al.Methylsulfonylmethane modulates apoptosis of LPS/IFN-γ-activated RAW 264.7macrophage-like cells by targeting p53,Bax,Bcl-2,cytochrome c and PARP proteins[J].Immunopharmacol Immunotoxicol,2014,36(6):379-389.