PPARγ通路激活可增强细胞抗氧化能力和保护长程培养原代神经细胞
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摘要
目的研究PPARγ通路增强对长程培养原代神经细胞凋亡的保护作用。方法以长程培养22 d的原代神经细胞建立自然衰老模型。根据不同干预方法分为衰老对照组(C组)、GW9662干预组(G组)和吡格列酮(Pioglitazone)干预组(P组)。其中GW9662干预组(G组)根据GW9662干预终浓度分为4个亚组:G(0.1):0.1μmol/Lol/L,G(1):1μmol/L,G(5)5μmol/L,G(10):10μmol/L。吡格列酮干预组(P组):根据吡格列酮干预终浓度分为4个亚组:P(0.1):0.1μmol/L,P(1):1μmol/L,P(5)5μmol/L,P(10):10μmol/L。首先应用MTT法观察各组细胞活力,应用倒置显微镜观察各组神经细胞形态,确定下一步实验的G组和P组的最终干预浓度。其次应用免疫荧光双标染色、流式细胞仪观察各组神经细胞计数、凋亡细胞比率的变化,明确PPARγ通路增强对长程培养原代神经细胞凋亡的保护作用。最后应用免疫化学法观察各组细胞抗氧化能力的变化,探讨PPARγ通路增强保护原代神经细胞的机制。结果随着GW9662干预浓度的增加,细胞活力进行性下降,其中G(1)、G(5)和G(10)组细胞活力均较C组显著降低(P<0.05)。G(1)组神经细胞形态尚完整,部分突触交织成网,最终确定后续实验G组的GW9662干预浓度为1μmol/L。加用吡格列酮干预后细胞活力较C组提高,其中P(1)、P(5)和P(10)组细胞活力较C组显著提高,差异具有统计学意义(P<0.05),但P(1)、P(5)、P(10)组3组之间比较无明显差异(P>0.05),最终确定后续实验P组的吡格列酮干预浓度为1μmol/L。G组细胞总数及神经细胞计数均较C组显著下降(P<0.05)。P组细胞总数及神经细胞计数均高于C组(P<0.05)。各组神经细胞比率均维持在80%左右,差异无统计学意义(P>0.05)。G组凋亡细胞比率明显高于C组(P<0.05),而P组凋亡细胞比率显著低于C组(P<0.05)。G组SOD活性及GSH含量较C组均显著降低(P<0.05),而MDA含量显著增加(P<0.05)。P组SOD活性及GSH含量均较C组显著升高(P<0.05),而MDA含量较C组显著减少(P<0.05)。结论 PPARγ通路是长程培养原代神经细胞的保护性通路,该通路的激活可以增强细胞抗氧化能力减少细胞凋亡保护长程培养原代神经细胞,提示针对PPARγ通路进行的药物干预有可能是临床抗神经系统衰老治疗的有效途径之一。
Objective To study the protective effect of enhanced peroxisome proliferator activated receptor γ(PPARγ) pathway against apoptosis of long-term cultured primary nerve cells. Methods A natural aging model was established in primary rat nerve cells by long-term culture for 22 days. The cells were divided into control group, 0.1, 1.0, 5.0, and 10 μmol/L GW9662 intervention groups, and 0.1, 1.0, 5.0, and 10 μmol/L pioglitazone intervention groups. The cell viability was assessed using MTT assay and the cell morphological changes were observed after the treatments to determine the optimal concentrations of GW9662 and pioglitazone. Double immunofluorescence labeling and flow cytometry were used to observe the changes in the number of viable cells and cell apoptosis following the treatments; immunocytochemical staining was used to assess the changes in the anti-oxidation ability of the treated cells. Results The optimal concentrations of GW9662 and pioglitazone determined based on the cell viability and morphological changes were both 1 μmol/L. Compared with the control group,GW9662 treatment significantly lowered while pioglitazone significantly increased the total cell number and nerve cell counts(P<0.05), and nerve cells in the cell cultures maintained a constant ratio at about 80% in all the groups(P>0.05). GW9662 significantly enhanced while pioglitazone significantly lowered the cell apoptosis rates compared with the control group(P<0.05). GW9662 obviously lowered SOD activity and GSH content in G group(P<0.05) and increased MDA content in the cells(P<0.05), and pioglitazone resulted in reverse changes in SOD, GSH and MDA contents in the cells(P<0.05). Conclusion Activation of PPARγ pathway protects long-term cultured primary nerve cells by enhancing cellular anti-oxidant capacity and reducing cell apoptosis, suggesting a potential strategy for anti-aging treatment of the nervous system through intervention of the PPARγ pathway.
Objective To study the protective effect of enhanced peroxisome proliferator activated receptor γ(PPARγ) pathway against apoptosis of long-term cultured primary nerve cells. Methods A natural aging model was established in primary rat nerve cells by long-term culture for 22 days. The cells were divided into control group, 0.1, 1.0, 5.0, and 10 μmol/L GW9662 intervention groups, and 0.1, 1.0, 5.0, and 10 μmol/L pioglitazone intervention groups. The cell viability was assessed using MTT assay and the cell morphological changes were observed after the treatments to determine the optimal concentrations of GW9662 and pioglitazone. Double immunofluorescence labeling and flow cytometry were used to observe the changes in the number of viable cells and cell apoptosis following the treatments; immunocytochemical staining was used to assess the changes in the anti-oxidation ability of the treated cells. Results The optimal concentrations of GW9662 and pioglitazone determined based on the cell viability and morphological changes were both 1 μmol/L. Compared with the control group,GW9662 treatment significantly lowered while pioglitazone significantly increased the total cell number and nerve cell counts(P<0.05), and nerve cells in the cell cultures maintained a constant ratio at about 80% in all the groups(P>0.05). GW9662 significantly enhanced while pioglitazone significantly lowered the cell apoptosis rates compared with the control group(P<0.05). GW9662 obviously lowered SOD activity and GSH content in G group(P<0.05) and increased MDA content in the cells(P<0.05), and pioglitazone resulted in reverse changes in SOD, GSH and MDA contents in the cells(P<0.05). Conclusion Activation of PPARγ pathway protects long-term cultured primary nerve cells by enhancing cellular anti-oxidant capacity and reducing cell apoptosis, suggesting a potential strategy for anti-aging treatment of the nervous system through intervention of the PPARγ pathway.
引文
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[12]Pilipovic K,Zupan Z,Dolenec PA,et al.A single dose of PPARgamma agonist pioglitazone reduces cortical oxidative damage and microglial reaction following lateral fluid percussion brain injury in rats[J].Prog Neuropsychopharmacol Biol Psychiatry,2015,59(2015):8-20.
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[14]Wang HQ,Fan JX,Chen MY,et al.rhEPO enhances cellular antioxidant capacity to protect long-term cultured aging primary nerve cells[J].JMol Neurosci,2017,62(3/4):291-303.
[15]Zeng Y,Xie K,Dong H,et al.Hyperbaric Oxygen preconditioning protects cortical neurons against oxygen-glucose deprivation injury:role of peroxisome proliferator-activated receptor-gamma[J].Brain Res,2012,1452(13):140-50.
[16]Gray E,Ginty M,Kemp K,et al.The PPAR-γagonist pioglitazone protects cortical neurons from inflammatory mediators via improvement in peroxisomal function[J].J Neuroinflammation,2012,9(1):63.
[17]Dong WG,Cheng SW,Huang F,et al.Mitochondrial dysfunction in long-term neuronal cultures mimics changes with aging[J].Med Sci Monit,2011,17(4):BR91-6.
[18]Bertrand SJ,Aksenova MV,Aksenov MY,et al.Endogenous amyloidogenesis in long-term rat hippocampal cell cultures[J].BMC Neurosci,2011,12(1):38.
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[23]章红妍,蒙思颖,林利芳,等.PPARγ在神经系统中能够促进IRS-4基因表达[J].南方医科大学学报,2013,33(10):1463-6.
[24]Huang T,Gao DK,Hei YE,et al.D-allose protects the blood brain barrier through PPAR gamma-mediated anti-inflammatory pathway in the mice model of ischemia reperfusion injury[J].Brain Res,2016,1642(2016):478-86.
[25]Liu J,Wang LN,Jia JP.Peroxisome proliferator-activated receptorgamma agonists for alzheimer's disease and amnestic mild cognitive impairment:a systematic review and meta-analysis[J].DrugsAging,2015,32(1):57-65.
[26]Vallée A,Lecarpentier Y.Alzheimer disease:crosstalk between the canonical wnt/beta-catenin pathway and PPARs alpha and gamma[J].Front Neurosci,2016,10(429):459.
[27]郭鹏,王晓民,王德生.过氧化物酶体增殖物激活受体γ在神经系统退行性疾病中的作用研究进展[J].生理科学进展,2015,27(5):379-82.
[28]Denner LA,Rodriguez-Rivera J,Haidacher SJ,et al.Cognitive enhancement with rosiglitazone links the hippocampal PPARγand ERK MAPK signaling pathways[J].J Neurosci,2012,32(47):1635a-725.
[29]Dineley KT,Jahrling J,Hernandez CA,et al.Ppara recruitment to active ERK during memory consolidation is required for alzheimer's disease-related cognitive enhancement[J].J Neurosci,2014,35(3,S):4054-63.
[30]Nenov MN,Tempia F,Denner L,et al.Impaired firing properties of dentate granule neurons in an Alzheimer's disease animal model are rescued by PPAR gamma agonism[J].J Neurophysiol,2015,113(6):1712-26.
[31]何兰英,张蓓,罗勇.吡格列酮对培养大鼠大脑皮质神经元氧-葡萄糖剥夺/复氧后PPARγ,NF-κB c-Rel和Bcl-xL表达的影响[J].国际脑血管病杂志,2013,21(5):357-62.
[32]李键,冯江,段宇,等.15d-PGJ_2对神经元氧糖剥夺/复氧损伤的影响[J].中风与神经疾病杂志,2016,33(10):873-7.
[33]Lee JE,Park JH,Jang SJ,et al.Rosiglitazone inhibits chlorpyrifosinduced apoptosis via modulation of the oxidative stress and inflammatory response in SH-SY5Y cells[J].Toxicol Appl Pharmacol,2014,278(2):159-71.
[34]Ko J,Park JH,Park YS,et al.PPAR-gamma activation attenuates deltamethrin-induced apoptosis by regulating cytosolic PINK1 and inhibiting mitochondrial dysfunction[J].Toxicol Lett,2016,260(2016):8-17.
[35]Ermini F,Jucker M.Nerve cell loss and neurogenesis in the aging brain[J].Praxis,2000,89(40):1605-8.
[36]Aging AY.Disease and nerve cell death[J].Bull Acad Natl Med,1995,179(6):1193-203.
[37]刘师兵,徐冶,于洋,等.人胎盘组织液对自然衰老大鼠海马区细胞凋亡和氧化应激的影响[J].中国老年学杂志,2016,36(2):282-4.
[38]Wood KA,Youle RJ.The role of free radicals and p53 in neuron apoptosis in vivo[J].J Neurosci,1995,15(8):5851-7.
[39]Wang ZM,Qiu ZW,Gao CX,et al.2,5-hexanedione downregulates nerve growth factor and induces neuron apoptosis in the spinal cord of rats via inhibition of the PI3K/Akt signaling pathway[J].PLoSOne,2017,12(6):e0179388.
[40]Tian K,Wang YX,Li LX,et al.Neuronal death/apoptosis induced by intracellular Zinc deficiency associated with changes in amino-acid neurotransmitters and glutamate receptor subtypes[J].J Inorg Biochem,2018,179(2018):54-9.
[41]Samson FE,Nelson SR.The aging brain,metals and Oxygen free radicals[J].CellMol Biol(Noisy-le-grand),2000,46(4):699-707.
[42]Sims-Robinson C,Hur J,Hayes JM,et al.The role of oxidative stress in nervous system aging[J].PLoS One,2013,8(7):e68011.
[43]Feng X,Yu W,Li X,et al.Apigenin,a modulator of PPAR款,attenuates HFD-induced NAFLD by regulating hepatocyte lipid metabolism and oxidative stress via Nrf2 activation[J].Biochem Pharmacol,2017,136(2017):136-49.
[44]ChiangMC,Nicol CJ,Yc C,et al.Rosiglitazone activation of PPARγ-dependent pathways is neuroprotective in human neural stem cells against amyloid-beta-induced mitochondrial dysfunction and oxidative stress[J].NeurobiolAging,2016,40(2016):181-90.
[45]Zhang ZX,Li YB,Zhao RP.Epigallocatechin gallate attenuatesβ-Amyloid Generation and oxidative stress involvement of PPARγin N2a/APP695 cells[J].Neurochem Res,2017,42(2):468-80.
[2]Harada S,Hiromori Y,Nakamura S,et al.Structural basis for PPARgamma transactivation by endocrine-disrupting organotin compounds[J].Sci Rep,2015,5:doi:10.1038/srep08520.
[3]Plutzky J.The PPAR-RXR transcriptional complex in the vasculature energy in the balance[J].Circ Res,2011,108(8):1002-16.
[4]álvarez-Almazán S,Bello M,Tamaycach F,et al.Study of new interactions of glitazone's stereoisomers and the endogenous ligand15d-PGJ2 on six different PPAR gamma proteins[J].Biochem Pharmacol,2017,142(2017):168-93.
[5]Agarwal S,Yadav A,Chaturvedi RK.Peroxisome proliferatoractivated receptors(PPARs)as therapeutic target in neurodegenerative disorders[J].Biochem Biophys Res Commun,2017,483(4):1166-77.
[6]Pang T,Sun LX,Wang T,et al.Telmisartan protects central neurons against nutrient deprivation-induced apoptosis in vitro through activation of PPARy and the Akt/GSK-beta pathway[J].Acta Pharmacol Sin,2014,35(6):727-37.
[7]Taheri M,Salamian A,Ghaedi K,et al.A ground state of PPARgamma activity and expression is required for appropriate neural differentiation of hESCs[J].Pharmacol Rep,2015,67(6):1103-14.
[8]董燕,丁奇,金英,等.吡格列酮对Aβ_(25-35)引起的皮层神经元损伤保护作用部分机制的研究[J].中国药理学通报,2010,26(12):1652-7.
[9]Landreth G,Jiang Q,Mandrekar S,et al.PPARgamma agonists as therapeutics for the treatment of Alzheimer's disease[J].Neurotherapeutics,2008,5(3):481-9.
[10]Swanson CR,Joers V,Bondarenko VA,et al.The PPAR-gamma agonist pioglitazone modulates inflammation and induces neuroprotection in parkinsonian monkeys[J].J Neuroinflammation,2011,8(1):1-14.
[11]Huot P,Johnston TH,Fox SH,et al.Pioglitazone May impair LDOPAanti-parkinsonian efficacy in the MPTP-lesioned macaque:results of a pilot study[J].Synapse,2015,69(3):99-102.
[12]Pilipovic K,Zupan Z,Dolenec PA,et al.A single dose of PPARgamma agonist pioglitazone reduces cortical oxidative damage and microglial reaction following lateral fluid percussion brain injury in rats[J].Prog Neuropsychopharmacol Biol Psychiatry,2015,59(2015):8-20.
[13]Lesuisse C,Martin LJ.Long-term culture of mouse cortical neurons as a model for neuronal development,aging,and death[J].JNeurobiol,2002,51(1):9-23.
[14]Wang HQ,Fan JX,Chen MY,et al.rhEPO enhances cellular antioxidant capacity to protect long-term cultured aging primary nerve cells[J].JMol Neurosci,2017,62(3/4):291-303.
[15]Zeng Y,Xie K,Dong H,et al.Hyperbaric Oxygen preconditioning protects cortical neurons against oxygen-glucose deprivation injury:role of peroxisome proliferator-activated receptor-gamma[J].Brain Res,2012,1452(13):140-50.
[16]Gray E,Ginty M,Kemp K,et al.The PPAR-γagonist pioglitazone protects cortical neurons from inflammatory mediators via improvement in peroxisomal function[J].J Neuroinflammation,2012,9(1):63.
[17]Dong WG,Cheng SW,Huang F,et al.Mitochondrial dysfunction in long-term neuronal cultures mimics changes with aging[J].Med Sci Monit,2011,17(4):BR91-6.
[18]Bertrand SJ,Aksenova MV,Aksenov MY,et al.Endogenous amyloidogenesis in long-term rat hippocampal cell cultures[J].BMC Neurosci,2011,12(1):38.
[19]Brody H.Organization of the cerebral cortex.III.A study of aging in the human cerebral cortex[J].J Comp Neurol,1955,102(2):511-6.
[20]Roberts DE,Killiany RJ,Rosene DL.Neuron numbers in the hypothalamus of the normal aging rhesus monkey:stability across the adult lifespan and between the sexes[J].J Comp Neurol,2012,520(6):1181-97.
[21]Uylings HB,de Brabander JM.Neuronal changes in normal human aging andAlzheimer's disease[J].Brain Cogn,2002,49(3):268-76.
[22]Berger J,Moller D.The mechanisms of action of PPARs[J].Annu RevMed,2002,53(1):409-35.
[23]章红妍,蒙思颖,林利芳,等.PPARγ在神经系统中能够促进IRS-4基因表达[J].南方医科大学学报,2013,33(10):1463-6.
[24]Huang T,Gao DK,Hei YE,et al.D-allose protects the blood brain barrier through PPAR gamma-mediated anti-inflammatory pathway in the mice model of ischemia reperfusion injury[J].Brain Res,2016,1642(2016):478-86.
[25]Liu J,Wang LN,Jia JP.Peroxisome proliferator-activated receptorgamma agonists for alzheimer's disease and amnestic mild cognitive impairment:a systematic review and meta-analysis[J].DrugsAging,2015,32(1):57-65.
[26]Vallée A,Lecarpentier Y.Alzheimer disease:crosstalk between the canonical wnt/beta-catenin pathway and PPARs alpha and gamma[J].Front Neurosci,2016,10(429):459.
[27]郭鹏,王晓民,王德生.过氧化物酶体增殖物激活受体γ在神经系统退行性疾病中的作用研究进展[J].生理科学进展,2015,27(5):379-82.
[28]Denner LA,Rodriguez-Rivera J,Haidacher SJ,et al.Cognitive enhancement with rosiglitazone links the hippocampal PPARγand ERK MAPK signaling pathways[J].J Neurosci,2012,32(47):1635a-725.
[29]Dineley KT,Jahrling J,Hernandez CA,et al.Ppara recruitment to active ERK during memory consolidation is required for alzheimer's disease-related cognitive enhancement[J].J Neurosci,2014,35(3,S):4054-63.
[30]Nenov MN,Tempia F,Denner L,et al.Impaired firing properties of dentate granule neurons in an Alzheimer's disease animal model are rescued by PPAR gamma agonism[J].J Neurophysiol,2015,113(6):1712-26.
[31]何兰英,张蓓,罗勇.吡格列酮对培养大鼠大脑皮质神经元氧-葡萄糖剥夺/复氧后PPARγ,NF-κB c-Rel和Bcl-xL表达的影响[J].国际脑血管病杂志,2013,21(5):357-62.
[32]李键,冯江,段宇,等.15d-PGJ_2对神经元氧糖剥夺/复氧损伤的影响[J].中风与神经疾病杂志,2016,33(10):873-7.
[33]Lee JE,Park JH,Jang SJ,et al.Rosiglitazone inhibits chlorpyrifosinduced apoptosis via modulation of the oxidative stress and inflammatory response in SH-SY5Y cells[J].Toxicol Appl Pharmacol,2014,278(2):159-71.
[34]Ko J,Park JH,Park YS,et al.PPAR-gamma activation attenuates deltamethrin-induced apoptosis by regulating cytosolic PINK1 and inhibiting mitochondrial dysfunction[J].Toxicol Lett,2016,260(2016):8-17.
[35]Ermini F,Jucker M.Nerve cell loss and neurogenesis in the aging brain[J].Praxis,2000,89(40):1605-8.
[36]Aging AY.Disease and nerve cell death[J].Bull Acad Natl Med,1995,179(6):1193-203.
[37]刘师兵,徐冶,于洋,等.人胎盘组织液对自然衰老大鼠海马区细胞凋亡和氧化应激的影响[J].中国老年学杂志,2016,36(2):282-4.
[38]Wood KA,Youle RJ.The role of free radicals and p53 in neuron apoptosis in vivo[J].J Neurosci,1995,15(8):5851-7.
[39]Wang ZM,Qiu ZW,Gao CX,et al.2,5-hexanedione downregulates nerve growth factor and induces neuron apoptosis in the spinal cord of rats via inhibition of the PI3K/Akt signaling pathway[J].PLoSOne,2017,12(6):e0179388.
[40]Tian K,Wang YX,Li LX,et al.Neuronal death/apoptosis induced by intracellular Zinc deficiency associated with changes in amino-acid neurotransmitters and glutamate receptor subtypes[J].J Inorg Biochem,2018,179(2018):54-9.
[41]Samson FE,Nelson SR.The aging brain,metals and Oxygen free radicals[J].CellMol Biol(Noisy-le-grand),2000,46(4):699-707.
[42]Sims-Robinson C,Hur J,Hayes JM,et al.The role of oxidative stress in nervous system aging[J].PLoS One,2013,8(7):e68011.
[43]Feng X,Yu W,Li X,et al.Apigenin,a modulator of PPAR款,attenuates HFD-induced NAFLD by regulating hepatocyte lipid metabolism and oxidative stress via Nrf2 activation[J].Biochem Pharmacol,2017,136(2017):136-49.
[44]ChiangMC,Nicol CJ,Yc C,et al.Rosiglitazone activation of PPARγ-dependent pathways is neuroprotective in human neural stem cells against amyloid-beta-induced mitochondrial dysfunction and oxidative stress[J].NeurobiolAging,2016,40(2016):181-90.
[45]Zhang ZX,Li YB,Zhao RP.Epigallocatechin gallate attenuatesβ-Amyloid Generation and oxidative stress involvement of PPARγin N2a/APP695 cells[J].Neurochem Res,2017,42(2):468-80.