小鼠缺血性脑卒中后磷脂酶D1在自噬和神经损伤中的作用
|
摘要
目的探讨在小鼠缺血性脑卒中模型中,磷脂酶D1(PLD1)在自噬和神经损伤中的作用。方法将6只6~8周龄成年雄性C57鼠采用随机数字表法分为两组,sham组和stroke组,每组各3只。sham组小鼠给予缺血性脑卒中处理但不进行大脑中动脉结扎,stroke组小鼠给予缺血性脑卒中处理。观察两组小鼠脑神经元内PLD1的变化。然后构建条件性基因敲除小鼠,将小鼠采用随机数字表法分为3组:sham PLD1~(fl/fl)组12只,缺血性脑卒中后PLD1~(fl/fl)组(stroke PLD1~(fl/fl)组) 12只,缺血性脑卒中后PLD1-KO组(stroke PLD1-KO组) 12只,然后利用免疫荧光染色、Western blotting、TTC染色法观察sham PLD1~(fl/fl)组小鼠,缺血性脑卒中后PLD1~(fl/fl)小鼠和缺血性脑卒中后PLD1-KO小鼠神经元自噬(LC3-Ⅱ)的情况及自噬对梗死面积的影响,明确PLD1的作用机制。结果在缺血性脑卒中后24 h,缺血半暗带的神经元中,stroke组较sham组PLD1表达明显增多。在条件性敲除小鼠中,stroke PLD1~(fl/fl)组较sham PLD1~(fl/fl)组自噬重要标志物LC3-Ⅱ明显升高(P <0. 05),而条件性敲除神经元内PLD1后,stroke PLD1-KO组较stroke PLD1~(fl/fl)组LC3-Ⅱ明显降低(P <0. 05),同时,梗死面积也明显缩小(P <0. 000 1)。结论缺血性脑卒中后神经元内PLD1表达升高,LC3-Ⅱ增多,抑制PLD1引起的过度自噬可以有效改善缺血性脑卒中后的神经损伤。
Objective To investigate the role of phospholipase D1( PLD1) in autophagy and neuronal damage after ischemic stroke in mice. Methods 6 adult male C57 BL/6( 8 ~ 10 weeks) were randomized into 2 groups: sham group and stroke group( n = 3 in each group).Stroke group was given focal ischemic stroke surgery while sham group was given same surgeries but without ligating MCA branches. Then explore the change of PLD1 after focal ischemic stroke in two groups. PLD1 neuronal conditional knockout mice was engineered using homologous recombination to abate the neuronal PLD1. Then the mice were randomized into three groups: sham PLD1~(fl/fl) group,stroke PLD1~(fl/fl) group and stroke PLD1-KO group( n = 12 in each group). Then immunohistochemistry,western blot and TTC staining were used to verify the change of LC3-Ⅱin neuron and to measure the infarction volume. Results Firstly,compared with sham group,stroke group showed higher expression of PLD1 24 h post-stroke in neuron; Secondly,compared with stroke PLD1~(fl/fl) group,stroke PLD1-KO show that inhibiting of PLD1 could decrease the formation LC3( P < 0. 05) and decrease the infarction volume( P < 0. 0001). Conclusion After focal ischemic stroke,the expression of PLD1 and LC3-Ⅱtend to increase in neuron. The inhibition of PLD1-induced autophagy potentiates neurorehabilitation and improve the neurological function.
Objective To investigate the role of phospholipase D1( PLD1) in autophagy and neuronal damage after ischemic stroke in mice. Methods 6 adult male C57 BL/6( 8 ~ 10 weeks) were randomized into 2 groups: sham group and stroke group( n = 3 in each group).Stroke group was given focal ischemic stroke surgery while sham group was given same surgeries but without ligating MCA branches. Then explore the change of PLD1 after focal ischemic stroke in two groups. PLD1 neuronal conditional knockout mice was engineered using homologous recombination to abate the neuronal PLD1. Then the mice were randomized into three groups: sham PLD1~(fl/fl) group,stroke PLD1~(fl/fl) group and stroke PLD1-KO group( n = 12 in each group). Then immunohistochemistry,western blot and TTC staining were used to verify the change of LC3-Ⅱin neuron and to measure the infarction volume. Results Firstly,compared with sham group,stroke group showed higher expression of PLD1 24 h post-stroke in neuron; Secondly,compared with stroke PLD1~(fl/fl) group,stroke PLD1-KO show that inhibiting of PLD1 could decrease the formation LC3( P < 0. 05) and decrease the infarction volume( P < 0. 0001). Conclusion After focal ischemic stroke,the expression of PLD1 and LC3-Ⅱtend to increase in neuron. The inhibition of PLD1-induced autophagy potentiates neurorehabilitation and improve the neurological function.
引文
[1]Hankey GJ.Stroke[J].Lancet(London,England),2017,389(10069):641-54.
[2]Lie ME,Gowing EK,Johansen NB,et al.GAT3 selective substrate lisoserine upregulates GAT3 expression and increases functional recovery after a focal ischemic stroke in mice[J].J Cereb Bood Flow Metab,2017,271678x17744123.
[3]Soreng K,Knaevelsrud H,Holland P,et al.HS1BP3 inhibits autophagy by regulation of PLD1[J].Autophagy,2017,13(5):985-986.
[4]Dall'armi C,Hurtado-Lorenzo A,Tian H,et al.The phospholipase D1pathway modulates macroautophagy[J].Nat Commun,2010,1:142.
[5]Krishnan B,Kayed R,Taglialatela G.Elevated phospholipase D isoform1 in Alzheimer's disease patients'hippocampus:Relevance to synaptic dysfunction and memory deficits[J].Alzheimers Dement(NY),2018,4:89-102.
[6]Wei L,Cui L,Snider BJ,et al.Transplantation of embryonic stem cells overexpressing Bcl-2 promotes functional recovery after transient cerebral ischemia[J].Neurobiol Dis,2005,19(1-2):183-93.
[7]Li Y,Lu Z,Keogh CL,et al.Erythropoietin-induced neurovascular protection,angiogenesis,and cerebral blood flow restoration after focal ischemia in mice[J].J Cerebral Blood Flow Metab,2007,27(5):1043-54.
[8]Xiong B,Li A,Lou Y,et al.Precise Cerebral Vascular Atlas in Stereotaxic Coordinates of Whole Mouse Brain[J].Front Neuroanat,2017,11:128.
[9]Mizushima N,Levine B,Cuervo AM,et al.Autophagy fights disease through cellular self-digestion[J].Nature,2008,451(7182):1069-1075.
[10]Menzies FM,Fleming A,Caricasole A,et al.Autophagy and Neurodegeneration:Pathogenic Mechanisms and Therapeutic Opportunities[J].Neuron,2017,93(5):1015-1034.
[11]Wang P,Xu TY,Wei K,et al.ARRB1/beta-arrestin-1 mediates neuroprotection through coordination of BECN1-dependent autophagy in cerebral ischemia[J].Autophagy,2014,10(9):1535-1548.
[12]Moskowitz MA.Brain protection:maybe yes,maybe no[J].Stroke,2010,41(10 Suppl):S85-86.
[13]Xin H,Katakowski M,Wang F,et al.MicroRNA cluster miR-17-92Cluster in Exosomes Enhance Neuroplasticity and Functional Recovery After Stroke in Rats[J].Stroke,2017,48(3):747-753.
[14]Holland P,Knaevelsrud H,Soreng K,et al.HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels[J].Nat Commun,2016,7:13889.
[15]Wang P,Guan YF,Du H,et al.Induction of autophagy contributes to the neuroprotection of nicotinamide phosphoribosyltransferase in cerebral ischemia[J].Autophagy,2012,8(1):77-87.
[16]Li H,Wu J,Shen H,et al.Autophagy in hemorrhagic stroke:Mechanisms and clinical implications[J].Prog Neurobiol,2018,163-164:79-97.
[17]Bae EJ,Lee HJ,Jang YH,et al.Phospholipase D1 regulates autophagic flux and clearance of alpha-synuclein aggregates[J].Cell Death Differ,2014,21(7):1132-1141.
[18]Zhu YB,Kang K,Zhang Y,et al.PLD1 Negatively Regulates Dendritic Branching[J].J Neurosci,2012,32(23):7960-7969.
[19]Zhu YB,Gao W,Zhang Y,et al.Astrocyte-derived phosphatidic acid promotes dendritic branching[J].Sci Rep,2016,6:21096.
[20]Thielmann I,Stegner D,Kraft P,et al.Redundant functions of phospholipases D1 and D2 in platelet alpha-granule release[J].JThromb Haemost,2012,10(11):2361-2372.
[21]Stegner D,Thielmann I,Kraft P,et al.Pharmacological inhibition of phospholipase D protects mice from occlusive thrombus formation and ischemic stroke--brief report[J].Arterioscler Thromb Vasc Biol,2013,33(9):2212-2217.
[22]Elvers M,Stegner D,Hagedorn I,et al.Impaired alpha(IIb)beta(3)integrin activation and shear-dependent thrombus formation in mice lacking phospholipase D1[J].Sci Signal,2010,3(103):ra1.
[2]Lie ME,Gowing EK,Johansen NB,et al.GAT3 selective substrate lisoserine upregulates GAT3 expression and increases functional recovery after a focal ischemic stroke in mice[J].J Cereb Bood Flow Metab,2017,271678x17744123.
[3]Soreng K,Knaevelsrud H,Holland P,et al.HS1BP3 inhibits autophagy by regulation of PLD1[J].Autophagy,2017,13(5):985-986.
[4]Dall'armi C,Hurtado-Lorenzo A,Tian H,et al.The phospholipase D1pathway modulates macroautophagy[J].Nat Commun,2010,1:142.
[5]Krishnan B,Kayed R,Taglialatela G.Elevated phospholipase D isoform1 in Alzheimer's disease patients'hippocampus:Relevance to synaptic dysfunction and memory deficits[J].Alzheimers Dement(NY),2018,4:89-102.
[6]Wei L,Cui L,Snider BJ,et al.Transplantation of embryonic stem cells overexpressing Bcl-2 promotes functional recovery after transient cerebral ischemia[J].Neurobiol Dis,2005,19(1-2):183-93.
[7]Li Y,Lu Z,Keogh CL,et al.Erythropoietin-induced neurovascular protection,angiogenesis,and cerebral blood flow restoration after focal ischemia in mice[J].J Cerebral Blood Flow Metab,2007,27(5):1043-54.
[8]Xiong B,Li A,Lou Y,et al.Precise Cerebral Vascular Atlas in Stereotaxic Coordinates of Whole Mouse Brain[J].Front Neuroanat,2017,11:128.
[9]Mizushima N,Levine B,Cuervo AM,et al.Autophagy fights disease through cellular self-digestion[J].Nature,2008,451(7182):1069-1075.
[10]Menzies FM,Fleming A,Caricasole A,et al.Autophagy and Neurodegeneration:Pathogenic Mechanisms and Therapeutic Opportunities[J].Neuron,2017,93(5):1015-1034.
[11]Wang P,Xu TY,Wei K,et al.ARRB1/beta-arrestin-1 mediates neuroprotection through coordination of BECN1-dependent autophagy in cerebral ischemia[J].Autophagy,2014,10(9):1535-1548.
[12]Moskowitz MA.Brain protection:maybe yes,maybe no[J].Stroke,2010,41(10 Suppl):S85-86.
[13]Xin H,Katakowski M,Wang F,et al.MicroRNA cluster miR-17-92Cluster in Exosomes Enhance Neuroplasticity and Functional Recovery After Stroke in Rats[J].Stroke,2017,48(3):747-753.
[14]Holland P,Knaevelsrud H,Soreng K,et al.HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels[J].Nat Commun,2016,7:13889.
[15]Wang P,Guan YF,Du H,et al.Induction of autophagy contributes to the neuroprotection of nicotinamide phosphoribosyltransferase in cerebral ischemia[J].Autophagy,2012,8(1):77-87.
[16]Li H,Wu J,Shen H,et al.Autophagy in hemorrhagic stroke:Mechanisms and clinical implications[J].Prog Neurobiol,2018,163-164:79-97.
[17]Bae EJ,Lee HJ,Jang YH,et al.Phospholipase D1 regulates autophagic flux and clearance of alpha-synuclein aggregates[J].Cell Death Differ,2014,21(7):1132-1141.
[18]Zhu YB,Kang K,Zhang Y,et al.PLD1 Negatively Regulates Dendritic Branching[J].J Neurosci,2012,32(23):7960-7969.
[19]Zhu YB,Gao W,Zhang Y,et al.Astrocyte-derived phosphatidic acid promotes dendritic branching[J].Sci Rep,2016,6:21096.
[20]Thielmann I,Stegner D,Kraft P,et al.Redundant functions of phospholipases D1 and D2 in platelet alpha-granule release[J].JThromb Haemost,2012,10(11):2361-2372.
[21]Stegner D,Thielmann I,Kraft P,et al.Pharmacological inhibition of phospholipase D protects mice from occlusive thrombus formation and ischemic stroke--brief report[J].Arterioscler Thromb Vasc Biol,2013,33(9):2212-2217.
[22]Elvers M,Stegner D,Hagedorn I,et al.Impaired alpha(IIb)beta(3)integrin activation and shear-dependent thrombus formation in mice lacking phospholipase D1[J].Sci Signal,2010,3(103):ra1.