纤维状α-突触核蛋白聚集体激活NLRP3炎症小体的机制研究

doi:10.19803/j.1672-8629.20220710

中国药物警戒 ›› 2023, Vol. 20 ›› Issue (4): 409-415.
DOI: 10.19803/j.1672-8629.20220710

纤维状α-突触核蛋白聚集体激活NLRP3炎症小体的机制研究

马一丹^1,2, 闫加庆^3Δ, 楚世峰², 张钊², 刘杨波⁴, 陈乃宏^2#, 彭灿^1,5,6,*

¹安徽中医药大学药学院,安徽合肥 230012;
²中国医学科学院北京协和医学院药物研究所,北京 100050;
³国家癌症中心/国家肿瘤临床医学研究中心/中国医学科学院北京协和医学院肿瘤医院药剂科,北京 100021;
⁴湖南中医药大学药学院,湖南长沙 410208;
⁵省部共建安徽道地中药材品质提升协同创新中心,安徽合肥 230012;
⁶药物制剂技术与应用安徽省重点实验室,安徽合肥 230012

收稿日期:2022-12-08 出版日期:2023-04-15 发布日期:2023-04-20
通讯作者: ^*彭灿,男,副教授,药剂学与药物分析。E-mail:pengcanking@hotmail.com;^#为共同通信作者。
作者简介:马一丹,女,硕士,药剂学与神经药理学。^Δ为并列第一作者。
基金资助:
国家自然科学基金资助项目（82003973）

Mechanism of activation of NLRP3 inflammasome by α-synuclein performed fibrils

MA Yidan^1,2, YAN Jiaqing^3Δ, CHU Shifeng², ZHANG Zhao², LIU Yangbo⁴, CHEN Naihong^2#, PENG Can^1,5,6,*

¹School of Pharmacy, Anhui University of Traditional Chinese Medicine, Hefei Anhui 230012, China;
²Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China;
³Department of Pharmacy, National Cancer Center, National Clinical Research Center for Cancer, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China;
⁴School of Pharmacy, Hunan University of Chinese Medicine, Changsha Hunan 410208, China;
⁵MOE-Anhui Joint Collaborative Innovation Center for Quality Improvement of Anhui Genuine Chinese Medicinal Materials, Hefei Anhui 230012, China;
⁶Anhui Province Key Laboratory of Pharmaceutical Technology and Application, Hefei Anhui 230012, China

Received:2022-12-08 Online:2023-04-15 Published:2023-04-20

摘要/Abstract

摘要： 目的探索纤维状α-突触核蛋白（α-synuclein）聚集体激活NLRP3炎症小体诱导神经炎症的机制。方法构建纤维状α-synuclein聚集体,采用纤维状α-synuclein聚集体刺激BV-2小胶质细胞,检测白介素（IL）-1β、IL-18、IL-6和肿瘤坏死因子α（TNF-α）等相关炎症因子和NLRP3、caspase-1、ASC等蛋白的表达和mRNA水平评价NLRP3炎症小体激活;采用乳酸脱氢酶释放（LDH）实验检测细胞焦亡的发生。机制研究部分,检测Toll样受体（TLR）2和TLR4的激活,并分别加入TLR2和TLR4抑制剂C29和TAK-242检测对纤维状α-synuclein聚集体诱导的NLRP3炎症小体激活的影响及核转录因子-κB（NF-κB）的入核情况。结果 Western blot和硫黄素T染色实验结果显示,纤维状α-synuclein聚集体成功制备。纤维状α-synuclein聚集体刺激BV-2小胶质细胞24 h后可激活NLRP3炎症小体,表现为IL-1β释放增加,相关蛋白NLRP3、caspase-1表达升高,NLRP3、ASC和IL-1β的mRNA水平显著增加,且核内NF-κB蛋白表达显著增加。LDH实验显示,纤维状α-synuclein聚集体不引起BV-2小胶质细胞焦亡。机制研究表明,纤维状α-synuclein聚集体可显著激活TLR2,但对TLR4表达无显著影响。TLR2抑制剂C29可显著抑制纤维状α-synuclein聚集体诱导的NLRP3炎症小体激活,但TLR4抑制剂TAK-242对α-synuclein聚集体诱导的NLRP3炎症小体激活无显著影响。结论纤维状α-synuclein聚集体可激活NLRP3炎症小体,其机制可能与激活TLR2有关。

关键词: 纤维状α-突触核蛋白聚集体, NLRP3炎症小体, Toll样受体2, Toll样受体4

Abstract: Objective To investigate the mechanism by which fibrillar α-synuclein aggregates induce neuroinflammation by activating NLRP3 inflammasomes. Methods α-synuclein performed fibrils (PFF) was established and added to BV-2 cells. Levels of such inflammatory factors as IL-1β, IL-18, IL-6 and TNF-α as well as protein and mRNA levels of NLRP3, caspase-1 and ASC were detected. LDH release assay was used to investigate pyroptosis. For mechanism research, activation of TLR2 and TLR4 was measured. C29 and TAK-242, inhibitors for TLR2 and TLR4, were used to detect the influence on NLRP3 inflammasome activation mediated by PFF. Results PFF was established as revealed by Western blot and Thioflavin T (ThT) staining. After PFF treatment, NLRP3 inflammasomes were activated in BV-2 cells as evidenced by the release of IL-1β, increased protein levels of NLRP3 and caspase-1 as well as significantly higher mRNA levels of NLRP3, caspase-1 and ASC in the PFF group compared with the control group. However, pyroptosis was not detected in either the PFF group or in the control group. TLR2, but not TLR4, was activated by PFF treatment. Inhibition of TLR2 rather than TLR4 could block PFF induced activation of NLRP3 inflammasomes. Conclusion Fibrillaraα-synuclein aggregates can activate NLRP3 inflammasomes, and the mechanism may be related to the activation of TLR2.

Key words: α-synuclein performed fibrils, NLRP3 inflammasome, TLR2, TLR4

中图分类号:

马一丹, 闫加庆, 楚世峰, 张钊, 刘杨波, 陈乃宏, 彭灿. 纤维状α-突触核蛋白聚集体激活NLRP3炎症小体的机制研究[J]. 中国药物警戒, 2023, 20(4): 409-415.

MA Yidan, YAN Jiaqing, CHU Shifeng, ZHANG Zhao, LIU Yangbo, CHEN Naihong, PENG Can. Mechanism of activation of NLRP3 inflammasome by α-synuclein performed fibrils[J]. Chinese Journal of Pharmacovigilance, 2023, 20(4): 409-415.

参考文献

[1] WANG SH, WEN L, ZHANG CL, et al.Changes of neurotransmitters in the striatum of MPTP-induced wild-type and A53T transgenic mouse Parkinson's disease models[J]. Chinese Journal of Pharmacovigilance(中国药物警戒), 2021, 18(4): 315-319.
[2] POEWE W, SEPPI K, TANNER CM, et al.Parkinson disease[J]. Nat Rev Dis Primers, 2017, 23(3): 17013.
[3] BARTELS T, DE SCHEPPER S, HONG S.Microglia modulate neurodegeneration in Alzheimer's and Parkinson's diseases[J]. Science, 2020, 370(6512): 66-69.
[4] HENEKA MT, MCMANUS RM, LATZ E.Inflammasome signalling in brain function and neurodegenerative disease[J]. Nat Rev Neurosci, 2018, 19(10): 610-621.
[5] GORDON R, ALBORNOZ EA, CHRISTIE DC, et al.Inflammasome inhibition prevents α-synuclein pathology and dopaminergic neurodegeneration in mice[J]. Sci Transl Med, 2018, 10(465): 4066.
[6] SWANSON KV, DENG M, TING JP.The NLRP3 inflammasome: molecular activation and regulation to therapeutics[J]. Nat Rev Immunol, 2019, 19(8): 477-489.
[7] FAN Z, PAN YT, ZHANG ZY, et al.Systemic activation of NLRP3 inflammasome and plasma α-synuclein levels are correlated with motor severity and progression in Parkinson's disease[J]. J Neuroinflammation, 2020, 17(1): 11.
[8] BROZ P, PELEGRIN P, SHAO F.The gasdermins, a protein family executing cell death and inflammation[J]. Nat Rev Immunol, 2020, 20(3): 143-157.
[9] FITZGERALD KA, KAGAN JC.Toll-like receptors and the control of immunity[J]. Cell, 2020, 180(6): 1044-1066.
[10] VOLPICELLI-DALEY LA, LUK KC, LEE VM.Addition of exogenous α-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous α-synuclein to Lewy body and Lewy neurite-like aggregates[J]. Nat Protoc, 2014, 9(9): 2135-2146.
[11] HENEKA MT, KUMMER MP, STUTZ A, et al.NLRP3 is activated in Alzheimer's disease and contributes to pathology in APP/PS1 mice[J]. Nature, 2013, 493(7434): 674-678.
[12] LEE E, HWANG I, PARK S, et al.MPTP-driven NLRP3 inflammasome activation in microglia plays a central role in dopaminergic neurodegeneration[J]. Cell Death Differ, 2019, 26(2): 213-228.
[13] WONG YC, KRAINC D.α-synuclein toxicity in neurodegeneration: mechanism and therapeutic strategies[J]. Nat Med, 2017, 23(2): 1-13.
[14] HAN QW, WEN L, CHEN NH, et al.Changes of α-Synuclein in A53T transgenic mice[J]. Chinese Journal of Pharmacovigilance(中国药物警戒), 2021, 18(4): 312-314.
[15] PRINZ M, JUNG S, PRILLER J.Microglia biology: one century of evolving concepts[J]. Cell, 2019, 179(2): 292-311.
[16] WANG W, NGUYEN LT, BURLAK C, et al.Caspase-1 causes truncation and aggregation of the Parkinson's disease-associated protein α-synuclein[J]. Proc Natl Acad Sci U S A, 2016, 113(34): 9587-9592.
[17] BORGHI A, VERSTREPEN L, BEYAERT R.TRAF2 multitasking in TNF receptor-induced signaling to NF-κB, MAP kinases and cell death[J]. Biochem Pharmacol, 2016, 116(15): 1-10.
[18] QIN ZH, TAO LY, CHEN X.Dual roles of NF-kappa B in cell survival and implications of NF-kappaB inhibitors in neuroprotec-tive therapy[J]. Acta Pharmacol. Sin, 2007, 28(12): 1859-1872.
[19] TAK PP, FIRESTEIN GS.NF-kappa B: A key role in inflammatory diseases[J]. The Journal of Clinical Investigation, 2001, 107(1): 7-11.
[20] KESAVARDHANA S, MALIREDDI RKS, KANNEGANTI TD.Caspases in cell death, inflammation, and pyroptosis[J]. Annu Rev Immunol, 2020, 38: 567-595.
[21] KIM C, HO DH, SUK JE, et al.Neuron-released oligomeric α-synuclein is an endogenous agonist of TLR2 for paracrine activation of microglia[J]. Nat Commun, 2013, 4: 1562.
[22] KIM C, ROCKENSTEIN E, SPENCER B, et al.Antagonizing neuronal toll-like receptor 2 prevents synucleinopathy by activating autophagy[J]. Cell Rep, 2015, 13(4): 771-782.

纤维状α-突触核蛋白聚集体激活NLRP3炎症小体的机制研究

Mechanism of activation of NLRP3 inflammasome by α-synuclein performed fibrils

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 1

编辑推荐

Metrics