沉默P2X4R基因对P2X4/NLRP3炎性小体通路介导的BV2小胶质细胞中肿瘤坏死因子α表达的影响
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摘要
目的探讨使用慢病毒(LV-P2rx4)沉默P2X4嘌呤受体(P2X4R)对活化状态小胶质细胞释放肿瘤坏死因子α(TNF-α)的影响。方法常规建立脂多糖(LPS)和三磷酸腺苷(ATP)双信号诱导的小胶质细胞活化模型,随机分为(1)空白对照组(常规DMEM培养液培养);(2)活化模型组(细胞种板后待密度接近80%后先给予LPS 1μg·m L-1处理6 h后弃原培养液,再加ATP 2 mmol·L-1处理3 h);(3)目的基因阴性对照组(种板1 d,先加入病毒处理12 h后弃培养液,再加入常规培养液,间隔12 h更换培养液);(4)目的基因阴性对照+活化模型组(种板1 d后,先加入病毒处理12 h后弃培养液,再加常规培养液,期间每隔12 h更换培养液,待细胞密度达80%后,予LPS 1μg·m L-1处理6 h后弃原培养液,再加ATP 2 mmol·L-1处理3 h);(5)空载病毒阴性对照组(处理方法同目的基因阴性对照组);(6)空载病毒阴性对照+活化模型组(处理方法同目的基因阴性对照+活化模型组)。各组均n=6。细胞处理后每组取3孔分别提取核糖核酸(RNA)和蛋白,应用Real time PCR和Western blot法分别检测NLRP3、TNF-α的mRNA及蛋白的相对表达。结果与活化模型组和空载病毒阴性对照+活化模型组比较,目的基因阴性对照+活化模型组TNF-αmRNA相对表达水平明显减少(P <0. 001,F=195. 4),TNF-α蛋白相对表达水平显著降低(P <0. 001,F=60. 10)。结论沉默小胶质细胞中的P2X4R基因,可下调细胞中TNF-α表达,继而可能削弱小胶质细胞的炎症反应。
Aim To investigate the effect of lentivirus( LV-P2rx4) silencing P2X4 receptor( P2X4R) on the release of tumor necrosis factor alpha( TNF-α) in activated microglia. Methods The models of microglia activation induced by dual-signaling of lipopolysaccharide( LPS) and adenosine triphosphate( ATP) were routinely established,and randomly divided into the following groups:(1)Blank control group( n = 6,conventional DMEM culture medium);(2)Activation model group( n = 6,when cell seed plate was close to 80%,first treated with 1 μg·m L~(-1) LPS for 6 h,discarded the original culture solution,and then ATP 2 mmol·L~(-1) was added for 3 h);(3)Target gene negative control group( n = 6;seed plate for 1 day,first treated with virus for 12 h,then discarded the culture solution,and then added the conventional culture solution,changed the culture solution at intervals of 12 h;(4)Target gene negative control plus activation model group( n = 6,after seeding for 1 day,first treated with virus for 12 h,then discarded the culture solution,and then added the normal culture solution,and changed the culture solution every 12 h,until the cell density reached 80%,treated with 1 μg·m L~(-1) LPS for 6 h,then discarded the original culture solution,and treated with 2 mmol·L~(-1) ATP for 3 h;(5)Empty virus negative control group( n = 6,the treatment method is the same as the target gene negative control group);(6)Empty virus negative control plus activation model group( n = 6,the treatment method is the same as the target gene negative control plus activation model group). After the treatment,RNA and proteins were extracted from each well by 3 wells. The relative expression of NLRP3 and TNF-α mRNA and protein were detected by real time PCR and Western blot. Results Compared with the activation model group and the empty virus negative control plus activation model group,the relative expression level of TNF-α mRNA in the target gene negative control plus activation model group was significantly decreased( P < 0. 001,F =195. 4),and the relative expression level of TNF-α protein significantly decreased( P < 0. 001,F =60. 10). Conclusion Silencing the P2X4R gene in microglia can down-regulate TNF-α expression in cells,which in turn may impair the inflammatory response of microglia.
Aim To investigate the effect of lentivirus( LV-P2rx4) silencing P2X4 receptor( P2X4R) on the release of tumor necrosis factor alpha( TNF-α) in activated microglia. Methods The models of microglia activation induced by dual-signaling of lipopolysaccharide( LPS) and adenosine triphosphate( ATP) were routinely established,and randomly divided into the following groups:(1)Blank control group( n = 6,conventional DMEM culture medium);(2)Activation model group( n = 6,when cell seed plate was close to 80%,first treated with 1 μg·m L~(-1) LPS for 6 h,discarded the original culture solution,and then ATP 2 mmol·L~(-1) was added for 3 h);(3)Target gene negative control group( n = 6;seed plate for 1 day,first treated with virus for 12 h,then discarded the culture solution,and then added the conventional culture solution,changed the culture solution at intervals of 12 h;(4)Target gene negative control plus activation model group( n = 6,after seeding for 1 day,first treated with virus for 12 h,then discarded the culture solution,and then added the normal culture solution,and changed the culture solution every 12 h,until the cell density reached 80%,treated with 1 μg·m L~(-1) LPS for 6 h,then discarded the original culture solution,and treated with 2 mmol·L~(-1) ATP for 3 h;(5)Empty virus negative control group( n = 6,the treatment method is the same as the target gene negative control group);(6)Empty virus negative control plus activation model group( n = 6,the treatment method is the same as the target gene negative control plus activation model group). After the treatment,RNA and proteins were extracted from each well by 3 wells. The relative expression of NLRP3 and TNF-α mRNA and protein were detected by real time PCR and Western blot. Results Compared with the activation model group and the empty virus negative control plus activation model group,the relative expression level of TNF-α mRNA in the target gene negative control plus activation model group was significantly decreased( P < 0. 001,F =195. 4),and the relative expression level of TNF-α protein significantly decreased( P < 0. 001,F =60. 10). Conclusion Silencing the P2X4R gene in microglia can down-regulate TNF-α expression in cells,which in turn may impair the inflammatory response of microglia.
引文
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[23]Polazzi E,Contestabile A.Reciprocal interactions between microglia and neurons:from survival to neuropathology[J].Rev Neurosci,2002,13:221-242
[24]Lee EJ,Han JE,Woo MS,et al.Matrix metalloproteinase-8plays a pivotal role in neuroinflammation by modulating TNF-αactivation[J].J Immunol,2014,193:2384-2393
[25]Welser-Alves JV,Milner R.Microglia are the major source of TNF-αand TGF-β1 in postnatal glial cultures;regulation by cytokines,lipopolysaccharide,and vitronectin[J].Neurochem Int,2013,63:47-53
[26]Francesca RR,Alessandra M,Francesca DV,et al.Tumor Necrosis Factor and Interleukin-1 Modulate Synaptic Plasticity during Neuroinflammation[J].Neural Plast,2018,2018:8430123
[27]邓小园,陈博,刘红亮,等.TNF-α在丙泊酚诱发的神经元凋亡及认知功能障碍中的作用[J].中国药理学通报,2016,32:945-949
[28]Frankola KA,Greig NH,Luo W,et al.Targeting TNF-αto elucidate and ameliorate neuroinflammation in neurodegenerative diseases[J].CNS Neurol Disord Drug Targets,2011,10:391-403
[2]陈汉泽,田力,薛维爽,等.阿尔茨海默病中炎症反应的研究进展[J].中国临床神经科学,2017,25:342-347
[3]Babelova A,Moreth K,Tsalastra-Greul W,et al.Biglycan,a danger signal that activates the NLRP3 inflammasome via toll-like and P2X receptors[J].J Biol Chem,2009,284:24035-24048
[4]Beamer E,G9l9ncsér F,Horváth G,et al.Purinergic mechanisms in neuroinflammation:An update from molecules to behavior[J].Neuropharmacology,2016,104:94-104
[5]Fiebich BL,Akter S,Akundi RS.The two-hit hypothesis for neuroinflammation:role of exogenous ATP in modulating inflammation in the brain[J].Front Cell Neurosci,2014,8:260
[6]Holmin S,S9derlund J,Biberfeld P,et al.Intracerebral Inflammation after human brain contusion[J].Neurosurgery,1998,43:291-298
[7]Zhang Y,Chi D,Overexpression of SIRT2 Alleviates Neuropathic Pain and Neuroinflammation Through Deacetylation of Transcription Factor Nuclear Factor-Kappa B[J].Inflammation,2018,41:569-578
[8]Chen K,Zhang J,Zhang W,et al.ATP-P2X4 signaling mediates NLRP3 inflammasome activation:a novel pathway of diabetic nephropathy[J].Int J Biochem Cell Biol,2013,45:932-943
[9]Dunton CL,Purves JT,Hughes FM Jr,et al.Elevated hydrostatic pressure stimulates ATP release which mediates activation of the NLRP3 inflammasome via P2X4 in rat urothelial cells[J].Int Urol Nephrol,2018,50:1607-1617
[10]Hasan D,Satalin J,van der Zee P,et al.Excessive Extracellular ATP Desensitizes P2Y2 and P2X4 ATP Receptors Provoking Surfactant Impairment Ending in Ventilation-Induced Lung Injury[J].Int J Mol Sci,2018,19:E1185
[11]蔡敏,薛莉,王静,等.P2X4/NLRP3炎性小体通路介导小胶质细胞白细胞介素-1β的释放[J].中国临床神经科学,2018,26:126-132
[12]宫黎明,周满红,廖富团,等.HLA-G5慢病毒过表达载体构建和鉴定及对人羊膜间充质干细胞的感染效率分析[J].遵义医学院学报,2018,41:165-169,175
[13]何承文,何玉龙,吴月红,等.不同转染试剂对慢病毒包装及包装后病毒感染豚鼠成纤维细胞效率的影响[J].黑龙江畜牧兽医,2015,484:34-38
[14]Liang Y,Jing X,Zeng Z,et al.Rifampicin attenuates rotenone-induced inflammation via suppressing NLRP3inflammasome activation in microglia[J].Brain Res,2015,1622:43-50
[15]Gicquel T,Victoni T,Fautrel A,et al.Involvement of purinergic receptors and NOD-like receptor-family protein 3-inflammasome pathway in the adenosine triphosphate-induced cytokine release from macrophages[J].Clin Exp Pharmacol Physiol,2014,41:279-286
[16]Gicquel T,Robert S,Loyer P,et al.IL-1beta production is dependent on the activation of purinergic receptors and NLRP3pathway in human macrophages[J].FASEB J,2015,29:4162-4173
[17]徐瑞,刘贤,牛梦月,等.MiR-133b对MPP+诱导的帕金森病多巴胺能神经元模型中酪氨酸羟化酶表达的影响[J].中国临床神经科学,2016,24:133-138
[18]Soto F,Garcia-Guzman M,Gomez-Hernandez JM,et al.P2X4:an ATP-activated ionotropic receptor cloned from rat brain[J].Proc Natl Acad Sci U S A,1996,93:3684-3688
[19]Zabala A,Vazquez-Villoldo N,Rissiek B,et al.P2X4 receptor controls microglia activation and favors remyelination in autoimmune encephalitis[J].EMBO Mol Med,2018,10:e8743
[20]Sa6d-Sadier N,Ojcius DM.Alarmins,inflammasomes and immunity[J].Biomed J,2012,35:437-449
[21]Burnstock G.P2X ion channel receptors and inflammation[J].Purinergic Signal,2016,12:59-67
[22]Veit Hornung,Franz Bauernfeind,Annett Halle,et al.Silica crystals and aluminum salts mediate NALP-3 inflammasome activation via phagosomal destabilization[J].Nat Immunol,2008,9:847-856
[23]Polazzi E,Contestabile A.Reciprocal interactions between microglia and neurons:from survival to neuropathology[J].Rev Neurosci,2002,13:221-242
[24]Lee EJ,Han JE,Woo MS,et al.Matrix metalloproteinase-8plays a pivotal role in neuroinflammation by modulating TNF-αactivation[J].J Immunol,2014,193:2384-2393
[25]Welser-Alves JV,Milner R.Microglia are the major source of TNF-αand TGF-β1 in postnatal glial cultures;regulation by cytokines,lipopolysaccharide,and vitronectin[J].Neurochem Int,2013,63:47-53
[26]Francesca RR,Alessandra M,Francesca DV,et al.Tumor Necrosis Factor and Interleukin-1 Modulate Synaptic Plasticity during Neuroinflammation[J].Neural Plast,2018,2018:8430123
[27]邓小园,陈博,刘红亮,等.TNF-α在丙泊酚诱发的神经元凋亡及认知功能障碍中的作用[J].中国药理学通报,2016,32:945-949
[28]Frankola KA,Greig NH,Luo W,et al.Targeting TNF-αto elucidate and ameliorate neuroinflammation in neurodegenerative diseases[J].CNS Neurol Disord Drug Targets,2011,10:391-403