银杏提取物降低丙烯酰胺诱导的小鼠小胶质细胞炎性反应及机制
|
摘要
目的研究低浓度丙烯酰胺(ACR)长期刺激下,银杏提取物(GBE)调控小胶质细胞炎性反应的分子机制。方法小胶质细胞取自新生小鼠大脑,采用(0.01～10)mmol/L ACR刺激48 h,用CCK-8法检测细胞增殖、免疫荧光细胞化学染色法检测裂解型胱天蛋白酶3(c-caspase-3)确定ACR最优浓度为0.1 mmol/L。GBE给药方法为100 mg/L GBE预处理2 h。ACR刺激48 h后,免疫荧光细胞化学染色法检测小胶质细胞离子钙接头蛋白分子1 (IBA-1)、核因子κB (NF-κB)的表达及分布情况, Griess反应检测培养液中一氧化氮(NO)水平, ELISA检测培养液白细胞介素6(IL-6)、 IL-1β、肿瘤坏死因子α(TNF-α)含量;实时定量PCR检测IBA-1和IL-6、 IL-1β、 TNF-α、诱导型一氧化氮合酶(iNOS) mRNA水平,染色质免疫共沉淀法检测p65、核受体相关蛋白1 (Nurr1)、 RE-1沉默转录因子共抑制物(CoREST)等转录因子在炎症细胞因子基因启动子区域NF-κB结合位点的结合程度变化;蛋白免疫共沉淀法检测Nurr1与CoREST蛋白的相互作用。结果低浓度ACR长期刺激将静息态小胶质细胞转变为激活态,通过NF-κB通路激活IL-6、 IL-1β、 TNF-α基因转录表达,促使小胶质细胞释放大量炎症细胞因子。GBE预处理显著改善小胶质细胞的炎症态应激,减少其炎症细胞因子的释放。在炎症细胞因子基因启动子区域的NF-κB结合位点上, GBE能够减少招募NF-κB亚单位p65,并增加招募Nurr1-CoREST复合物以抑制基因的转录表达。结论 GBE通过Nurr1/CoREST反式抑制NF-κB通路,改善小胶质细胞因长期低浓度ACR刺激引发的炎症反应。
Objective To investigate the effect of Gingko biloba extract(GBE) on microglial inflammatory response due to long-term low-dose arylamide(ACR) exposure and its underlying mechanism. Methods Primary microglial cells were extracted from neonatal mouse brain cortex. Using CCK-8 assay to detect cell proliferation and immunofluorescence cytochemistry to detect cleaved-caspase 3, the optimal concentration of ACR(0.1 mmol/L) treatment was determined in order to minimize the apoptotic toxicity of ACR(0.01～10 mmol/L, for 48 hours). GBE(100 mg/L) pre-treatment was given 2 hours before ACR treatment. After 48 hours of ACR treatment, immunocytochemistry was used to detect the expression of ionized calcium-binding adaptor molecule-1(IBA-1) and nuclear factor κB(NF-κB) for observing the morphological alteration of microglia and NF-κB nuclear translocation, respectively. The concentrations of nitric oxide(NO) and three inflammatory cytokines(IL-6, IL-1β, TNF-α) in the culture media were examined with Griess reaction and ELISA kits. Real-time PCR was applied to measure mRNA expression levels of IBA-1 and other inflammatory cytokine genes. Recruitments of p65, Nurr1 and CoREST onto the promoter regions of those inflammatory cytokine genes were examined by chromatin immunoprecipitation. Protein interaction between Nurr1 and CoREST was determined by protein immunoprecipitation. Results Long-term low-dose treatment of ACR transformed resting microglia towards activated morpholgy, activated the expression of inflammatory cytokine genes through NF-κB pathway, and resulted in extracellular release of those cytokines. Pre-treatment of GBE greatly prevented microglial activation and its adverse consequences. GBE decreased the recruitment of p65 on the promoter regions of inflammatory cytokine genes, while increased the recruitment of Nurr1-CoREST complex. Conclusion GBE can alleviate microglial inflammatory response induced by long-term low-dose ACR exposure by transrepression of inflammatory cytokine genes, which involves the expelling of p65 and the recruitment of Nurr1-CoREST complex onto NF-κB binding site in their promoters.
Objective To investigate the effect of Gingko biloba extract(GBE) on microglial inflammatory response due to long-term low-dose arylamide(ACR) exposure and its underlying mechanism. Methods Primary microglial cells were extracted from neonatal mouse brain cortex. Using CCK-8 assay to detect cell proliferation and immunofluorescence cytochemistry to detect cleaved-caspase 3, the optimal concentration of ACR(0.1 mmol/L) treatment was determined in order to minimize the apoptotic toxicity of ACR(0.01～10 mmol/L, for 48 hours). GBE(100 mg/L) pre-treatment was given 2 hours before ACR treatment. After 48 hours of ACR treatment, immunocytochemistry was used to detect the expression of ionized calcium-binding adaptor molecule-1(IBA-1) and nuclear factor κB(NF-κB) for observing the morphological alteration of microglia and NF-κB nuclear translocation, respectively. The concentrations of nitric oxide(NO) and three inflammatory cytokines(IL-6, IL-1β, TNF-α) in the culture media were examined with Griess reaction and ELISA kits. Real-time PCR was applied to measure mRNA expression levels of IBA-1 and other inflammatory cytokine genes. Recruitments of p65, Nurr1 and CoREST onto the promoter regions of those inflammatory cytokine genes were examined by chromatin immunoprecipitation. Protein interaction between Nurr1 and CoREST was determined by protein immunoprecipitation. Results Long-term low-dose treatment of ACR transformed resting microglia towards activated morpholgy, activated the expression of inflammatory cytokine genes through NF-κB pathway, and resulted in extracellular release of those cytokines. Pre-treatment of GBE greatly prevented microglial activation and its adverse consequences. GBE decreased the recruitment of p65 on the promoter regions of inflammatory cytokine genes, while increased the recruitment of Nurr1-CoREST complex. Conclusion GBE can alleviate microglial inflammatory response induced by long-term low-dose ACR exposure by transrepression of inflammatory cytokine genes, which involves the expelling of p65 and the recruitment of Nurr1-CoREST complex onto NF-κB binding site in their promoters.
引文
[1] Salter M W, Stevens B. Microglia emerge as central players in brain disease[J]. Nat Med, 2017, 23(9): 1018-1027.
[2] Nimmerjahn A, Kirchhoff F, Helmchen F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo[J]. Science, 2005, 308(5726): 1314-1318.
[3] Mottram D S, Wedzicha B L, Dodson A T. Acrylamide is formed in the Maillard reaction: Food chemistry[J]. Nature, 2002, 419(6906): 448-449.
[4] Semla M, Goc Z, Martiniaková M, et al. Acrylamide: a common food toxin related to physiological functions and health[J]. Physiol Res, 2017, 66(2): 205-217.
[5] Attoff K, Kertika D, Lundqvist J, et al. Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y[J]. Toxicol In Vitro, 2016, 35: 100-111.
[6] LoPachin R M, Gavin T. Molecular mechanism of acrylamide neurotoxicity: Lessons learned from organic chemistry[J]. Environ Health Perspect, 2012, 120(12): 1650-1657.
[7] Tian S M, Ma Y X, Shi J, et al. Acrylamide neurotoxicity on the cerebrum of weaning rats[J]. Neural Regen Res, 2015, 10(6): 938-943.
[8] Li J, Li D, Yang Y, et al. Acrylamide induces locomotor defects and degeneration of dopamine neurons in Caenorhabditis elegans: Acrylamide induces locomotor defects and dopamine neurodegeneration[J]. J Appl Toxicol, 2016, 36(1): 60-67.
[9] Lee S, Park H R, Lee J Y, et al. Learning, memory deficits, and impaired neuronal maturation attributed to acrylamide[J]. J Toxicol Environ Health A, 2018, 81(9): 254-265.
[10] Chen J H, Tsou T C, Chiu I M, et al. Proliferation inhibition, DNA damage, and cell-cycle arrest of human astrocytoma cells after acrylamide exposure[J]. Chem Res Toxicol, 2010, 23(9): 1449-1458.
[11] Lee J G, Wang Y S, Chou C C. Acrylamide-induced apoptosis in rat primary astrocytes and human astrocytoma cell lines[J]. Toxicol In Vitro, 2014, 28(4): 562-570.
[12] Chen J H, Wu K Y, Chiu I M, et al. Acrylamide-induced astrogliotic and apoptotic responses in human astrocytoma cells[J]. Toxicol In Vitro, 2009, 23(5): 855-861.
[13] Zhao M, Lewis Wang F S, Hu X, et al. Acrylamide-induced neurotoxicity in primary astrocytes and microglia: Roles of the Nrf2-ARE and NF-κB pathways[J]. Food Chem Toxicol, 2017, 106(PtA): 25-35.
[14] Zhao M, Wang F S L, Hu X S, et al. Effect of acrylamide-induced neurotoxicity in a primary astrocytes/microglial co-culture model[J]. Toxicol In Vitro, 2017, 39: 119-125.
[15] Gargouri B, Carstensen J, Bhatia H S, et al. Anti-neuroinflammatory effects of Ginkgo biloba extract EGb761 in LPS-activated primary microglial cells[J]. Phytomedicine, 2018, 44: 45-55.
[16] Liu F Q, Gao Q, Wang D D, et al. Effects of GBE50 on LPS/ATP induced NLRP3 inflammasome activation in primary rat microglia[J]. Zhongguo Zhong Yao Za Zhi, 2018, 43(16): 3346-3352.
[17] Wan F, Zang S, Yu G, et al. Ginkgolide B suppresses methamphetamine-induced microglial activation through TLR4-NF-κB signaling pathway in BV2 cells[J]. Neurochem Res, 2017, 42(10): 2881-2891.
[18] Zhou J M, Gu S S, Mei W H, et al. Ginkgolides and bilobalide protect BV2 microglia cells against OGD/reoxygenation injury by inhibiting TLR2/4 signaling pathways[J]. Cell Stress Chaperones, 2016, 21(6): 1037-1053.
[19] Li Z Y, Chung Y H, Shin E J, et al. YY-1224, a terpene trilactone-strengthened Ginkgo biloba, attenuates neurodegenerative changes induced by β-amyloid (1-42) or double transgenic overexpression of APP and PS1 via inhibition of cyclooxygenase-2[J/OL]. J Neuroinflammation, 2017, 14(1): 94. DOI: 10.1186/s12974-017-0866-x.
[20] Wan W, Zhang C, Danielsen M, et al. EGb761 improves cognitive function and regulates inflammatory responses in the APP/PS1 mouse[J]. Exp Gerontol, 2016, 81: 92-100.
[21] Luo C, Fan L H, Zhang H, et al. Effects of Ginkgo biloba extract on the cognitive function and expression profile of inflammatory factors in a rat model of hemorrhagic stroke[J]. Neuroreport, 2018, 29(15): 1239-1243.
[22]Chen A, Xu Y, Yuan J. Ginkgolide B ameliorates NLRP3 inflammasome activation after hypoxic-ischemic brain injury in the neonatal male rat[J]. Int J Dev Neurosci, 2018, 69: 106-111.
[23] Saijo K, Winner B, Carson C T, et al. A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death[J]. Cell, 2009, 137(1): 47-59.
[24] Saura J, Tusell J M, Serratosa J. High-yield isolation of murine microglia by mild trypsinization[J]. Glia, 2003, 44(3): 183-189.
[25] Li L, Liu Y, Chen H Z, et al. Impeding the interaction between Nur77 and p38 reduces LPS-induced inflammation[J]. Nat Chem Biol, 2015, 11(5): 339-346.
[26] Kim C H, Han B S, Moon J, et al. Nuclear receptor Nurr1 agonists enhance its dual functions and improve behavioral deficits in an animal model of Parkinson’s disease[J]. Proc Natl Acad Sci U S A, 2015, 112(28): 8756-8761.
[27] Spathis A D, Asvos X, Ziavra D, et al. Nurr1: RXRα heterodimer activation as monotherapy for Parkinson’s disease[J]. Proc Natl Acad Sci U S A, 2017, 114(15): 3999-4004.
[28] Loppi S, Kolosowska N, K?rkk?inen O, et al. HX600, a synthetic agonist for RXR-Nurr1 heterodimer complex, prevents ischemia-induced neuronal damage[J]. Brain Behav Immun, 2018, 73: 670-681.
[2] Nimmerjahn A, Kirchhoff F, Helmchen F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo[J]. Science, 2005, 308(5726): 1314-1318.
[3] Mottram D S, Wedzicha B L, Dodson A T. Acrylamide is formed in the Maillard reaction: Food chemistry[J]. Nature, 2002, 419(6906): 448-449.
[4] Semla M, Goc Z, Martiniaková M, et al. Acrylamide: a common food toxin related to physiological functions and health[J]. Physiol Res, 2017, 66(2): 205-217.
[5] Attoff K, Kertika D, Lundqvist J, et al. Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y[J]. Toxicol In Vitro, 2016, 35: 100-111.
[6] LoPachin R M, Gavin T. Molecular mechanism of acrylamide neurotoxicity: Lessons learned from organic chemistry[J]. Environ Health Perspect, 2012, 120(12): 1650-1657.
[7] Tian S M, Ma Y X, Shi J, et al. Acrylamide neurotoxicity on the cerebrum of weaning rats[J]. Neural Regen Res, 2015, 10(6): 938-943.
[8] Li J, Li D, Yang Y, et al. Acrylamide induces locomotor defects and degeneration of dopamine neurons in Caenorhabditis elegans: Acrylamide induces locomotor defects and dopamine neurodegeneration[J]. J Appl Toxicol, 2016, 36(1): 60-67.
[9] Lee S, Park H R, Lee J Y, et al. Learning, memory deficits, and impaired neuronal maturation attributed to acrylamide[J]. J Toxicol Environ Health A, 2018, 81(9): 254-265.
[10] Chen J H, Tsou T C, Chiu I M, et al. Proliferation inhibition, DNA damage, and cell-cycle arrest of human astrocytoma cells after acrylamide exposure[J]. Chem Res Toxicol, 2010, 23(9): 1449-1458.
[11] Lee J G, Wang Y S, Chou C C. Acrylamide-induced apoptosis in rat primary astrocytes and human astrocytoma cell lines[J]. Toxicol In Vitro, 2014, 28(4): 562-570.
[12] Chen J H, Wu K Y, Chiu I M, et al. Acrylamide-induced astrogliotic and apoptotic responses in human astrocytoma cells[J]. Toxicol In Vitro, 2009, 23(5): 855-861.
[13] Zhao M, Lewis Wang F S, Hu X, et al. Acrylamide-induced neurotoxicity in primary astrocytes and microglia: Roles of the Nrf2-ARE and NF-κB pathways[J]. Food Chem Toxicol, 2017, 106(PtA): 25-35.
[14] Zhao M, Wang F S L, Hu X S, et al. Effect of acrylamide-induced neurotoxicity in a primary astrocytes/microglial co-culture model[J]. Toxicol In Vitro, 2017, 39: 119-125.
[15] Gargouri B, Carstensen J, Bhatia H S, et al. Anti-neuroinflammatory effects of Ginkgo biloba extract EGb761 in LPS-activated primary microglial cells[J]. Phytomedicine, 2018, 44: 45-55.
[16] Liu F Q, Gao Q, Wang D D, et al. Effects of GBE50 on LPS/ATP induced NLRP3 inflammasome activation in primary rat microglia[J]. Zhongguo Zhong Yao Za Zhi, 2018, 43(16): 3346-3352.
[17] Wan F, Zang S, Yu G, et al. Ginkgolide B suppresses methamphetamine-induced microglial activation through TLR4-NF-κB signaling pathway in BV2 cells[J]. Neurochem Res, 2017, 42(10): 2881-2891.
[18] Zhou J M, Gu S S, Mei W H, et al. Ginkgolides and bilobalide protect BV2 microglia cells against OGD/reoxygenation injury by inhibiting TLR2/4 signaling pathways[J]. Cell Stress Chaperones, 2016, 21(6): 1037-1053.
[19] Li Z Y, Chung Y H, Shin E J, et al. YY-1224, a terpene trilactone-strengthened Ginkgo biloba, attenuates neurodegenerative changes induced by β-amyloid (1-42) or double transgenic overexpression of APP and PS1 via inhibition of cyclooxygenase-2[J/OL]. J Neuroinflammation, 2017, 14(1): 94. DOI: 10.1186/s12974-017-0866-x.
[20] Wan W, Zhang C, Danielsen M, et al. EGb761 improves cognitive function and regulates inflammatory responses in the APP/PS1 mouse[J]. Exp Gerontol, 2016, 81: 92-100.
[21] Luo C, Fan L H, Zhang H, et al. Effects of Ginkgo biloba extract on the cognitive function and expression profile of inflammatory factors in a rat model of hemorrhagic stroke[J]. Neuroreport, 2018, 29(15): 1239-1243.
[22]Chen A, Xu Y, Yuan J. Ginkgolide B ameliorates NLRP3 inflammasome activation after hypoxic-ischemic brain injury in the neonatal male rat[J]. Int J Dev Neurosci, 2018, 69: 106-111.
[23] Saijo K, Winner B, Carson C T, et al. A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death[J]. Cell, 2009, 137(1): 47-59.
[24] Saura J, Tusell J M, Serratosa J. High-yield isolation of murine microglia by mild trypsinization[J]. Glia, 2003, 44(3): 183-189.
[25] Li L, Liu Y, Chen H Z, et al. Impeding the interaction between Nur77 and p38 reduces LPS-induced inflammation[J]. Nat Chem Biol, 2015, 11(5): 339-346.
[26] Kim C H, Han B S, Moon J, et al. Nuclear receptor Nurr1 agonists enhance its dual functions and improve behavioral deficits in an animal model of Parkinson’s disease[J]. Proc Natl Acad Sci U S A, 2015, 112(28): 8756-8761.
[27] Spathis A D, Asvos X, Ziavra D, et al. Nurr1: RXRα heterodimer activation as monotherapy for Parkinson’s disease[J]. Proc Natl Acad Sci U S A, 2017, 114(15): 3999-4004.
[28] Loppi S, Kolosowska N, K?rkk?inen O, et al. HX600, a synthetic agonist for RXR-Nurr1 heterodimer complex, prevents ischemia-induced neuronal damage[J]. Brain Behav Immun, 2018, 73: 670-681.