适宜浓度短链脂肪酸混合物对小胶质细胞炎症抑制及机制研究
|
摘要
本研究的主要目的是探讨适宜浓度短链脂肪酸(short chain fatty acids,SCFAs)混合物对炎症环境下小胶质细胞的抑炎作用及其机制.采用脂多糖(LPS)刺激小鼠小胶质细胞系BV-2细胞建立神经炎症模型,并利用CCK8试剂盒检测不同浓度单一的乙酸钠、丙酸钠、丁酸钠处理后的细胞活力.设计选取这三种SCFAs对细胞活力无影响、且有抑炎效果的特定浓度进行组合(SCFAs mix),进一步检测SCFAs mix对LPS刺激下BV-2细胞炎症反应的影响及机制,包括:a.用一氧化氮(NO)试剂盒检测NO的释放;b.用ELISA检测炎症因子TNF-α、IL-6的释放;c.用qRT-PCR和Western blot检测炎症因子TNF-α、IL-6、炎症小体NLRP3、炎症通路相关蛋白TLR4、NF-κB等的表达变化.结果表明LPS刺激BV-2细胞4 h后,在体系中添加特定浓度的单一SCFA处理12 h后,不能缓解BV-2细胞的炎症反应,而将上述SCFAs配制成同等终浓度的SCFAs mix处理12 h却能显著降低细胞培养上清液中NO、TNF-α和IL-6 (均P<0.001)的量,还能抑制BV-2细胞内iNOS、TNF-α、IL-6和NLRP3 mRNA的升高(均P<0.001);通过对炎症信号通路关键分子的检测发现,SCFAs mix可以抑制LPS诱导的BV-2细胞内TLR4、MyD88、TRAF6和NF-κB蛋白的表达升高.综上可见:适宜浓度的混合SCFAs可通过调控TLR4/MyD88/TRAF6/NF-κB炎症通路抑制LPS诱导的小胶质细胞的炎症反应,而起到抗炎的保护作用.
To investigate the anti-inflammatory effect and mechanism of proper concentration of short-chain fatty acids(SCFAs) mixture(SCFAs mix) on microglia under inflammatory environment. In this study, the murine microglial cell line BV-2 treated with lipopolysaccharide(LPS) was used as an inflammatory cell model.BV-2 cells were treated with different concentrations of sodium acetate, sodium propionate and sodium butyrate,and SCFAs mix, the cell viability were detected by CCK8 kit. The concentrations of each SCFA and SCFAs mixture(SCFAs mix) will be selected by double standard: having no effect on cell viability and having the antiinflammation effect. The examination of anti-inflammation effect and its mechanism of SCFAs mix in proper concentration on LPS-stimulated BV-2 cells include:(a) the production of NO of BV-2 cells was detected by NO kit,(b) the inflammatory factors such as TNF-α and IL-6 released into the culture supernatant by BV-2 cells were detected by ELISA,(c) the expression of inflammatory factors such as TNF-α and IL-6, inflammasome NLRP3 and some key factors within inflammatory pathways such as TLR4 and NF-κB, were detected by qRT-PCR and Western-blot. We found that after 4 hours of LPS stimulation on BV-2 cells, the addition of a certain concentration of single SCFA to the system for 12 h could not alleviate the inflammatory response of BV-2 cells, but SCFAs mix with the same terminal concentration of each SCFAs could significantly reduce the levels of NO(P<0.001),TNF-α(P<0.001) and IL-6(P<0.001) in cell culture supernatants. Simultaneously, SCFAs mix inhibited the increase of iNOS, TNF-α, IL-6(P<0.001) and inflammasome NLRP3(P<0.001) mRNA in BV-2 cells induced by LPS. Further study showed that SCFAs mix could inhibit LPS-induced high expression of TLR4, MyD88, TRAF6 and NF-κB proteins, which were the key molecules of the inflammatory signaling pathway in BV-2 cells. In conclusion, proper concentration of SCFAs mix could function as a protective role to inhibit LPS-induced microglial inflammatory responses by regulation of the TLR4/MyD88/TRAF6/NF-κB signaling pathway.
To investigate the anti-inflammatory effect and mechanism of proper concentration of short-chain fatty acids(SCFAs) mixture(SCFAs mix) on microglia under inflammatory environment. In this study, the murine microglial cell line BV-2 treated with lipopolysaccharide(LPS) was used as an inflammatory cell model.BV-2 cells were treated with different concentrations of sodium acetate, sodium propionate and sodium butyrate,and SCFAs mix, the cell viability were detected by CCK8 kit. The concentrations of each SCFA and SCFAs mixture(SCFAs mix) will be selected by double standard: having no effect on cell viability and having the antiinflammation effect. The examination of anti-inflammation effect and its mechanism of SCFAs mix in proper concentration on LPS-stimulated BV-2 cells include:(a) the production of NO of BV-2 cells was detected by NO kit,(b) the inflammatory factors such as TNF-α and IL-6 released into the culture supernatant by BV-2 cells were detected by ELISA,(c) the expression of inflammatory factors such as TNF-α and IL-6, inflammasome NLRP3 and some key factors within inflammatory pathways such as TLR4 and NF-κB, were detected by qRT-PCR and Western-blot. We found that after 4 hours of LPS stimulation on BV-2 cells, the addition of a certain concentration of single SCFA to the system for 12 h could not alleviate the inflammatory response of BV-2 cells, but SCFAs mix with the same terminal concentration of each SCFAs could significantly reduce the levels of NO(P<0.001),TNF-α(P<0.001) and IL-6(P<0.001) in cell culture supernatants. Simultaneously, SCFAs mix inhibited the increase of iNOS, TNF-α, IL-6(P<0.001) and inflammasome NLRP3(P<0.001) mRNA in BV-2 cells induced by LPS. Further study showed that SCFAs mix could inhibit LPS-induced high expression of TLR4, MyD88, TRAF6 and NF-κB proteins, which were the key molecules of the inflammatory signaling pathway in BV-2 cells. In conclusion, proper concentration of SCFAs mix could function as a protective role to inhibit LPS-induced microglial inflammatory responses by regulation of the TLR4/MyD88/TRAF6/NF-κB signaling pathway.
引文
[1]Gill S R,Pop M,Deboy R T,et al.Metagenomic analysis of the human distal gut microbiome.Science,2006,312(5778):1355-1359
[2]Qin J,Li R,Raes J,et al.A human gut microbial gene catalogue established by metagenomic sequencing.Nature,2010,464(7285):59-65
[3]De J R D-P V,Forlenza A S,Forlenza O V.Relevance of gutmicrobiota in cognition,behaviour and Alzheimer's disease.Pharmacol Res,2018,136:29-34
[4]Sharon G,Sampson T R,Geschwind D H,et al.The central nervous system and the gut microbiome.Cell,2016,167(4):915-932
[5]Sun M F,Shen Y Q.Dysbiosis of gut microbiota and microbial metabolites in Parkinson's disease.Ageing Res Rev,2018,45:53-61
[6]Petschow B,Dore J,Hibberd P,et al.Probiotics,prebiotics,and the host microbiome:the science of translation.Ann N Y Acad Sci,2013,1306(1):1-17
[7]Hamer H M,Jonkers D,Venema K,et al.Review article:the role of butyrate on colonic function.Aliment Pharmacol Ther,2008,27(2):104-119
[8]Haghikia A,Jorg S,Duscha A,et al.Dietary fatty acids directly impact central nervous system autoimmunity via the small intestine.Immunity,2016,44(4):951-953
[9]Wang L,Christophersen C T,Sorich M J,et al.Elevated fecal short chain fatty acid and ammonia concentrations in children with autism spectrum disorder.Dig Dis Sci,2012,57(8):2096-2102
[10]Unger M M,Spiegel J,Dillmann K U,et al.Short chain fatty acids and gut microbiota differ between patients with Parkinson's disease and age-matched controls.Parkinsonism Relat Disord,2016,32:66-72
[11]Sun M F,Zhu Y L,Zhou Z L,et al.Neuroprotective effects of fecal microbiota transplantation on MPTP-induced Parkinson's disease mice:Gut microbiota,glial reaction and TLR4/TNF-alpha signaling pathway.Brain Behav Immun,2018,70:48-60
[12]Colonna M,Butovsky O.Microglia function in the central nervous system during health and neurodegeneration.Annu Rev Immunol,2017,35:441-468
[13]Dai J N,Zong Y,Zhong L M,et al.Gastrodin inhibits expression of inducible NO synthase,cyclooxygenase-2 and proinflammatory cytokines in cultured LPS-stimulated microglia via MAPKpathways.Plos One,2011,6(7):e21891
[14]Martindale J L,Holbrook N J.Cellular response to oxidative stress:signaling for suicide and survival.J Cell Physiol,2002,192(1):1-15
[15]Salter M W,Stevens B.Microglia emerge as central players in brain disease.Nat Med,2017,23(9):1018-1027
[16]Lu Y,Fan C,Li P,et al.Short chain fatty acids prevent high-fatdiet-induced obesity in mice by regulating G protein-coupled receptors and gut microbiota.Sci Rep,2016,6:37589
[17]Block M L,Zecca L,Hong J S.Microglia-mediated neurotoxicity:uncovering the molecular mechanisms.Nat Rev Neurosci,2007,8(1):57-69
[18]Frank-Cannon T C,Alto L T,Mcalpine F E,et al.Does neuroinflammation fan the flame in neurodegenerative diseases?Mol Neurodegener,2009,4:47
[19]Erny D,Hrabe De Angelis A L,Jaitin D,et al.Host microbiota constantly control maturation and function of microglia in the CNS.Nat Neurosci,2015,18(7):965-977
[20]Miyake S,Kim S,Suda W,et al.Dysbiosis in the gut microbiota of patients with multiple sclerosis,with a striking depletion of species belonging to clostridia XIVa and IV clusters.Plos One,2015,10(9):e0137429
[21]Tian Y,Xu Q,Sun L,et al.Short-chain fatty acids administration is protective in colitis-associated colorectal cancer development.JNutr Biochem,2018,57:103-109
[22]Kasubuchi M,Hasegawa S,Hiramatsu T,et al.Dietary gut microbial metabolites,short-chain fatty acids,and host metabolic regulation.Nutrients,2015,7(4):2839-2849
[23]Fu S P,Li S N,Wang J F,et al.BHBA suppresses LPS-induced inflammation in BV-2 cells by inhibiting NF-kappaB activation.Mediators Inflamm,2014,2014:983401
[24]Molteni M,Gemma S,Rossetti C.The role of Toll-like receptor 4in infectious and noninfectious inflammation.Mediators Inflamm,2016,2016:6978936
[25]Couturier J,Stancu I C,Schakman O,et al.Activation of phagocytic activity in astrocytes by reduced expression of the inflammasome component ASC and its implication in a mouse model ofAlzheimer disease.J Neuroinflammation,2016,13:20
[26]Baldwin A S,Jr.The NF-kappa B and I kappa B proteins:new discoveries and insights.Annu Rev Immunol,1996,14:649-683
[27]Moon D O,Park S Y,Lee K J,et al.Bee venom and melittin reduce proinflammatory mediators in lipopolysaccharide-stimulated BV2 microglia.Int Immunopharmacol,2007,7(8):1092-1101
[28]Gustin A,Kirchmeyer M,Koncina E,et al.NLRP3 inflammasome is expressed and functional in mouse brain microglia but not in astrocytes.Plos One,2015,10(6):e0130624
[2]Qin J,Li R,Raes J,et al.A human gut microbial gene catalogue established by metagenomic sequencing.Nature,2010,464(7285):59-65
[3]De J R D-P V,Forlenza A S,Forlenza O V.Relevance of gutmicrobiota in cognition,behaviour and Alzheimer's disease.Pharmacol Res,2018,136:29-34
[4]Sharon G,Sampson T R,Geschwind D H,et al.The central nervous system and the gut microbiome.Cell,2016,167(4):915-932
[5]Sun M F,Shen Y Q.Dysbiosis of gut microbiota and microbial metabolites in Parkinson's disease.Ageing Res Rev,2018,45:53-61
[6]Petschow B,Dore J,Hibberd P,et al.Probiotics,prebiotics,and the host microbiome:the science of translation.Ann N Y Acad Sci,2013,1306(1):1-17
[7]Hamer H M,Jonkers D,Venema K,et al.Review article:the role of butyrate on colonic function.Aliment Pharmacol Ther,2008,27(2):104-119
[8]Haghikia A,Jorg S,Duscha A,et al.Dietary fatty acids directly impact central nervous system autoimmunity via the small intestine.Immunity,2016,44(4):951-953
[9]Wang L,Christophersen C T,Sorich M J,et al.Elevated fecal short chain fatty acid and ammonia concentrations in children with autism spectrum disorder.Dig Dis Sci,2012,57(8):2096-2102
[10]Unger M M,Spiegel J,Dillmann K U,et al.Short chain fatty acids and gut microbiota differ between patients with Parkinson's disease and age-matched controls.Parkinsonism Relat Disord,2016,32:66-72
[11]Sun M F,Zhu Y L,Zhou Z L,et al.Neuroprotective effects of fecal microbiota transplantation on MPTP-induced Parkinson's disease mice:Gut microbiota,glial reaction and TLR4/TNF-alpha signaling pathway.Brain Behav Immun,2018,70:48-60
[12]Colonna M,Butovsky O.Microglia function in the central nervous system during health and neurodegeneration.Annu Rev Immunol,2017,35:441-468
[13]Dai J N,Zong Y,Zhong L M,et al.Gastrodin inhibits expression of inducible NO synthase,cyclooxygenase-2 and proinflammatory cytokines in cultured LPS-stimulated microglia via MAPKpathways.Plos One,2011,6(7):e21891
[14]Martindale J L,Holbrook N J.Cellular response to oxidative stress:signaling for suicide and survival.J Cell Physiol,2002,192(1):1-15
[15]Salter M W,Stevens B.Microglia emerge as central players in brain disease.Nat Med,2017,23(9):1018-1027
[16]Lu Y,Fan C,Li P,et al.Short chain fatty acids prevent high-fatdiet-induced obesity in mice by regulating G protein-coupled receptors and gut microbiota.Sci Rep,2016,6:37589
[17]Block M L,Zecca L,Hong J S.Microglia-mediated neurotoxicity:uncovering the molecular mechanisms.Nat Rev Neurosci,2007,8(1):57-69
[18]Frank-Cannon T C,Alto L T,Mcalpine F E,et al.Does neuroinflammation fan the flame in neurodegenerative diseases?Mol Neurodegener,2009,4:47
[19]Erny D,Hrabe De Angelis A L,Jaitin D,et al.Host microbiota constantly control maturation and function of microglia in the CNS.Nat Neurosci,2015,18(7):965-977
[20]Miyake S,Kim S,Suda W,et al.Dysbiosis in the gut microbiota of patients with multiple sclerosis,with a striking depletion of species belonging to clostridia XIVa and IV clusters.Plos One,2015,10(9):e0137429
[21]Tian Y,Xu Q,Sun L,et al.Short-chain fatty acids administration is protective in colitis-associated colorectal cancer development.JNutr Biochem,2018,57:103-109
[22]Kasubuchi M,Hasegawa S,Hiramatsu T,et al.Dietary gut microbial metabolites,short-chain fatty acids,and host metabolic regulation.Nutrients,2015,7(4):2839-2849
[23]Fu S P,Li S N,Wang J F,et al.BHBA suppresses LPS-induced inflammation in BV-2 cells by inhibiting NF-kappaB activation.Mediators Inflamm,2014,2014:983401
[24]Molteni M,Gemma S,Rossetti C.The role of Toll-like receptor 4in infectious and noninfectious inflammation.Mediators Inflamm,2016,2016:6978936
[25]Couturier J,Stancu I C,Schakman O,et al.Activation of phagocytic activity in astrocytes by reduced expression of the inflammasome component ASC and its implication in a mouse model ofAlzheimer disease.J Neuroinflammation,2016,13:20
[26]Baldwin A S,Jr.The NF-kappa B and I kappa B proteins:new discoveries and insights.Annu Rev Immunol,1996,14:649-683
[27]Moon D O,Park S Y,Lee K J,et al.Bee venom and melittin reduce proinflammatory mediators in lipopolysaccharide-stimulated BV2 microglia.Int Immunopharmacol,2007,7(8):1092-1101
[28]Gustin A,Kirchmeyer M,Koncina E,et al.NLRP3 inflammasome is expressed and functional in mouse brain microglia but not in astrocytes.Plos One,2015,10(6):e0130624