α-突触核蛋白激活NLRP3炎性小体介导神经细胞焦亡的发生
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摘要
目的:研究α-突触核蛋白(α-synuclein)对于神经细胞焦亡发生的影响和机制。方法:以小鼠海马神经元细胞HT22细胞株为对象,体外培养后用α-synuclein干预细胞,以CCK-8法检测细胞活力,确定IC_(50)值。设置对照组(Con组)后,采用TUNEL染色法检测细胞焦亡水平,免疫荧光法观察Gasdermin-D-N(GSDMD-N)的表达,Western blot法检测细胞中NOD样受体热蛋白结构域相关蛋白3(NLRP3)、细胞凋亡相关斑点样蛋白(ASC)、Gasdermin-D(GSDMD)、GSDMD-N、Caspase-1的表达水平,酶联免疫吸附法检测培养基中IL-18、IL-1β的表达水平。采用Caspase-1抑制剂预处理HT22后,同样使用IC_(50)值的α-synuclein干预细胞,同时设置α-synuclein组(单纯α-synuclein干预)和α-synuclein+ML132组,检测细胞焦亡的水平。结果:α-synuclein的IC_(50)值为50 nmol/L。α-synuclein干预后细胞焦亡数目显著增高,TUNEL染色显示阳性细胞数目多于Con组,免疫荧光染色结果显示细胞中GSDMD-N的表达水平上调,细胞中NLRP3、ASC、GSDMD-N、Caspase-1的表达水平显著高于Con组,而GSDMD水平下调,同时细胞培养基中IL-18、IL-1β的水平上调。Caspase-1抑制剂处理后,50 nmol/L的α-synuclein干预后,细胞焦亡水平显著低于α-synuclein组,同时免疫荧光染色结果显示GSDMD-N的水平下调,细胞中NLRP3、ASC、GSDMD-N、Caspase-1的表达水平显著低于α-synuclein组,而GSDMD水平上调,培养基中IL-18和IL-1β的水平下调。结论:α-synuclein可以通过激活NLRP3炎性小体活化继发神经细胞焦亡的发生,在帕金森病的发生发展中具有一定的作用。
AIM: To study the effect and mechanism of α-synuclein on nerve cell pyroptosis. METHODS: HT22 cell was cultured in vitro and interfered by α-synuclein. Cell viability was measured by CCK-8 method and IC_(50) value was determined. After setting up the control group(Con group), TUNEL staining was used to detect the pyroptosis level of cells, immunofluorescence was used to observe the expression of GSDMD-N, Western blot was used to detect the expression levels of NLRP3, ASC, GSDMD, GSDMD-N and Caspase-1 in cells, and enzyme-linked immunosorbent assay was used to detect the expression levels of IL-18 and IL-1β in culture medium. After HT22 was pretreated with Caspase-1 inhibitor, the cells were also treated with α-synuclein of IC_(50) value, and the α-synuclein group(only α-synuclein intervention) and α-synuclein+ML132 group were set up to detect the level of pyroptosis. RESULTS:The IC_(50) value of α-synuclein was 50 nmol/L. After the intervention of α-synuclein, the number of nerve cells increased significantly. TUNEL staining showed that the number of positive cells was more than that of Con group. Immunofluorescence staining showed that the expression level of GSDMD-N in cells was up-regulated. The expression levels of NLRP3, ASC, GSDMD-N and Caspase-1 in cells were significantly higher than those in Con group, while the level of GSDMD was down-regulated in cell culture medium. The levels of IL-18 and IL-1β were up-regulated. After treatment with Caspase-1 inhibitor, the pyroptotic level of 50 nmol/L α-synuclein was significantly lower than that of α-synuclein group. Immunofluorescence staining showed that the level of GSDMD-N was down-regulated. The expression levels of NLRP3, ASC, GSDMD-N and Caspase-1 in cells were significantly lower than those in α-synuclein group, while the level of GSDMD was up-regulated. The levels of IL-18 and IL-1β in culture medium were down regulation. CONCLUSION:α-synuclein may play a role in the development of Parkinson's disease by activating NLRP3 inflammatory bodies.
AIM: To study the effect and mechanism of α-synuclein on nerve cell pyroptosis. METHODS: HT22 cell was cultured in vitro and interfered by α-synuclein. Cell viability was measured by CCK-8 method and IC_(50) value was determined. After setting up the control group(Con group), TUNEL staining was used to detect the pyroptosis level of cells, immunofluorescence was used to observe the expression of GSDMD-N, Western blot was used to detect the expression levels of NLRP3, ASC, GSDMD, GSDMD-N and Caspase-1 in cells, and enzyme-linked immunosorbent assay was used to detect the expression levels of IL-18 and IL-1β in culture medium. After HT22 was pretreated with Caspase-1 inhibitor, the cells were also treated with α-synuclein of IC_(50) value, and the α-synuclein group(only α-synuclein intervention) and α-synuclein+ML132 group were set up to detect the level of pyroptosis. RESULTS:The IC_(50) value of α-synuclein was 50 nmol/L. After the intervention of α-synuclein, the number of nerve cells increased significantly. TUNEL staining showed that the number of positive cells was more than that of Con group. Immunofluorescence staining showed that the expression level of GSDMD-N in cells was up-regulated. The expression levels of NLRP3, ASC, GSDMD-N and Caspase-1 in cells were significantly higher than those in Con group, while the level of GSDMD was down-regulated in cell culture medium. The levels of IL-18 and IL-1β were up-regulated. After treatment with Caspase-1 inhibitor, the pyroptotic level of 50 nmol/L α-synuclein was significantly lower than that of α-synuclein group. Immunofluorescence staining showed that the level of GSDMD-N was down-regulated. The expression levels of NLRP3, ASC, GSDMD-N and Caspase-1 in cells were significantly lower than those in α-synuclein group, while the level of GSDMD was up-regulated. The levels of IL-18 and IL-1β in culture medium were down regulation. CONCLUSION:α-synuclein may play a role in the development of Parkinson's disease by activating NLRP3 inflammatory bodies.
引文
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[13] MartínSánchez F,MartínezGarcía JJ,MuíozGarcía M,et al.Lytic cell death induced by melittin bypasses pyroptosis but induces NLRP3 inflammasome activation and IL-1β release [J].Cell Death Dis,2017,8(8):e2984.
[14] Bai Y,Sun X,Chu Q,et al.Caspase-1 regulates Ang II-induced cardiomyocyte hypertrophy via up-regulation of IL-1β [J].Biosci Rep,2018,38(2):241-250.
[15] Chen X,He WT,Hu L,et al.Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis [J].Cell Res,2016,26(9):1007-1020.
[16] Schroder K,Zhou R,Tschopp J.The NLRP3 inflammasome:A sensor for metabolic danger [J].Science,2010,327(5963):296-300.
[17] 郑远鹏,臧彩霞,王璐,等.脂多糖加重α-synuclein基因突变小鼠多巴胺能神经元损伤机制的研究[J].药学学报,2017,52(5):75-82.
[18] Soldner F,Stelzer Y,Shivalila CS,et al.Parkinson-associated risk variant in distal enhancer of -synuclein modulates target gene expression [J].Nature,2016,533(7601):95-112.
[19] 杨毅,官俏兵,郭丽,等.樟芝多糖通过降低NLRP3-Caspase1炎性小体表达改善帕金森小鼠神经行为学的机制研究[J].中国临床药理学与治疗学,2018,23(4):406-412.
[20] 屈洪党,杨维.炎症机制介导的胡椒碱对帕金森病小鼠模型的神经保护作用[J].中国临床药理学与治疗学,2016,21(6):630-635.
[2] Aglietti RA ,Estevez A ,Gupta A,et al.GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes [J].Proc Natl Acad Sci U S A,2016,113(28):7858-7863.
[3] Liu X,Zhang Z,Ruan J,et al.Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores [J].Nature,2016,535(7610):153-163.
[4] Miao EA,Leaf IA,Treuting PM,et al.Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria [J].Nat Immunol,2010,11(12):1136-1142.
[5] Bergsbaken T,Cookson BT.Macrophage activation redirects yersinia-infected host cell death from apoptosis to caspase-1-dependent pyroptosis [J].Plos Pathogens,2007,3(11):e161.
[6] Liu Z,Gan L,Xu Y,et al.Melatonin alleviates inflammasome-induced pyroptosis through inhibiting NF-κB/GSDMD signal in mice adipose tissue [J].J Pineal Res,2017,63(1):254-259.
[7] Schneider KS,Gron CJ,Dreier RF,et al.The inflammasome drives GSDMD-independent secondary pyroptosis and IL-1 release in the absence of caspase-1 protease activity [J].Cell Rep,2017,21(13):3846-3859.
[8] Dumitriu A,Pacheco CD,Wilk JB,et al.Cyclin-G-associated kinase modifies α-synuclein expression levels and toxicity in Parkinson's disease:results from the GenePD Study[J].Hum Mol Genet,2011,20(8):1478-1487.
[9] Reeve AK,Tae-Kyung P,Evelyn J,et al.Relationship between mitochondria and α-synuclein:a study of single substantia nigra neurons [J].Arch Neurol,2012,69(3):385-392.
[10] Chen KW,Grob CJ,Sotomayor FV,et al.The neutrophil NLRC4 inflammasome selectively promotes IL-1β maturation without pyroptosis during acute Salmonella challenge [J].Cell Rep,2014,8(2):570-582.
[11] Lee S,Choi E,Cha MJ,et al.Looking for pyroptosis-modulating miRNAs as a therapeutic target for improving myocardium survival [J].Mediators Inflamm,2015,2015,254871.
[12] Whitfield NN,Byrne BG,Swanson MS.Mouse macrophages are permissive to motile legionella species that fail to trigger pyroptosis[J].Infect Immun,2010,78(1):423-432.
[13] MartínSánchez F,MartínezGarcía JJ,MuíozGarcía M,et al.Lytic cell death induced by melittin bypasses pyroptosis but induces NLRP3 inflammasome activation and IL-1β release [J].Cell Death Dis,2017,8(8):e2984.
[14] Bai Y,Sun X,Chu Q,et al.Caspase-1 regulates Ang II-induced cardiomyocyte hypertrophy via up-regulation of IL-1β [J].Biosci Rep,2018,38(2):241-250.
[15] Chen X,He WT,Hu L,et al.Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis [J].Cell Res,2016,26(9):1007-1020.
[16] Schroder K,Zhou R,Tschopp J.The NLRP3 inflammasome:A sensor for metabolic danger [J].Science,2010,327(5963):296-300.
[17] 郑远鹏,臧彩霞,王璐,等.脂多糖加重α-synuclein基因突变小鼠多巴胺能神经元损伤机制的研究[J].药学学报,2017,52(5):75-82.
[18] Soldner F,Stelzer Y,Shivalila CS,et al.Parkinson-associated risk variant in distal enhancer of -synuclein modulates target gene expression [J].Nature,2016,533(7601):95-112.
[19] 杨毅,官俏兵,郭丽,等.樟芝多糖通过降低NLRP3-Caspase1炎性小体表达改善帕金森小鼠神经行为学的机制研究[J].中国临床药理学与治疗学,2018,23(4):406-412.
[20] 屈洪党,杨维.炎症机制介导的胡椒碱对帕金森病小鼠模型的神经保护作用[J].中国临床药理学与治疗学,2016,21(6):630-635.
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