异丙酚通过TRPV1离子通道抑制LPS诱导的小胶质细胞炎症反应
|
摘要
目的:探讨异丙酚(propofol,P)对脂多糖(lipopolysaccharide,LPS)诱导的小胶质细胞炎症反应的影响及其机制。方法:LPS(100μg/L)处理建立小鼠小胶质细胞BV2神经炎症损伤模型,将细胞分为对照(C)组、模型(L)组、L+P组和LPS+AMG517(L+A)组。ELISA法检测各组细胞培养液中肿瘤坏死因子α(TNF-α)的含量;real-timePCR法检测各组细胞瞬时受体电位阳离子通道V亚族成员1(TRPV1) mRNA的表达;Western blot法检测各组细胞TRPV1、TNF-α、白细胞介素1β(IL-1β)、IL-6和磷酸化钙/钙调蛋白依赖性蛋白激酶Ⅱ(p-CaMKⅡ)的蛋白表达;荧光探针Fluo-3 AM标记法检测细胞内游离的Ca~(2+)含量。结果:与C组相比,L组细胞培养液中TNF-α水平显著升高(P<0.01);单独propofol处理TNF-α水平无显著差异;与L组相比,L+P组细胞培养液中在4 h内TNF-α水平显著降低(P<0.01)。与C组相比,L组TRPV1 mRNA表达显著上调(P<0.01);与L组相比,L+P组TRPV1 mRNA表达显著下调(P<0.01)。相比L组,L+P和L+A组TNF-α、IL-1β、IL-6和p-CaMKⅡ蛋白表达显著下调(P<0.01)。相比L组,L+P和L+A组细胞内Ca~(2+)浓度显著降低(P<0.01)。结论:Propofol通过下调TRPV1的表达,下调p-CaMKⅡ的表达,降低细胞内Ca~(2+)浓度,从而下调TNF-α、IL-1β和IL-6的表达,抑制小胶质细胞的炎症反应。
AIM: To investigate the effects of propofol(P) on the inflammatory response of microglia induced by lipopolysaccharide(LPS) and the mechanisms. METHODS: Mouse microglia BV2 cells were treated with LPS at 100 μg/L to establish a neuroinflammatory injury model. The BV2 cells were divided into 4 groups: control group(C group), model group(L group), L+P group and LPS+AMG517 group(L+A group). The level of tumor necrosis factor-α(TNF-α) in the cell culture supernatant was measured by ELISA. The mRNA expression of transient receptor potential cation channel subfamily V member 1(TRPV1) was detected by real-time PCR. The protein levels of TRPV1, TNF-α, interleukin-1β(IL-1β), interleukin-6(IL-6) and phosphorylated calcium/calmodulin-dependent protein kinase Ⅱ(p-CaMKⅡ) were determined by Western blot. The content of free Ca~(2+) in the microglia BV2 cells was detected by Fluo-3 AM assay. RESULTS: Compared with C group, the level of TNF-α was significantly increased in L group(P<0.01), but that in P group was not changed. Compared with L group, the level of TNF-α was significantly lower than that in L+P group within 4 h(P<0.01). Compared with C group, the mRNA expression of TRPV1 was significantly increased in L group(P<0.01). Compared with L group, the mRNA expression of TRPV1 was significantly down-regulated in L+P group(P<0.01).Compared with L group, the protein levels of TNF-α, IL-1β, IL-6 and p-CaMKⅡ and intracellular Ca~(2+) concentration were significantly lower than those in L+P group and L+A group(P<0.01). CONCLUSION: Propofol inhibits the inflammatory response of microglia by reducing the expression of TNF-α, IL-1 and IL-6, which may be related to the down-regulation of TRPV1 and p-CaMKⅡ and the reduction of intracellular Ca~(2+) concentration.
AIM: To investigate the effects of propofol(P) on the inflammatory response of microglia induced by lipopolysaccharide(LPS) and the mechanisms. METHODS: Mouse microglia BV2 cells were treated with LPS at 100 μg/L to establish a neuroinflammatory injury model. The BV2 cells were divided into 4 groups: control group(C group), model group(L group), L+P group and LPS+AMG517 group(L+A group). The level of tumor necrosis factor-α(TNF-α) in the cell culture supernatant was measured by ELISA. The mRNA expression of transient receptor potential cation channel subfamily V member 1(TRPV1) was detected by real-time PCR. The protein levels of TRPV1, TNF-α, interleukin-1β(IL-1β), interleukin-6(IL-6) and phosphorylated calcium/calmodulin-dependent protein kinase Ⅱ(p-CaMKⅡ) were determined by Western blot. The content of free Ca~(2+) in the microglia BV2 cells was detected by Fluo-3 AM assay. RESULTS: Compared with C group, the level of TNF-α was significantly increased in L group(P<0.01), but that in P group was not changed. Compared with L group, the level of TNF-α was significantly lower than that in L+P group within 4 h(P<0.01). Compared with C group, the mRNA expression of TRPV1 was significantly increased in L group(P<0.01). Compared with L group, the mRNA expression of TRPV1 was significantly down-regulated in L+P group(P<0.01).Compared with L group, the protein levels of TNF-α, IL-1β, IL-6 and p-CaMKⅡ and intracellular Ca~(2+) concentration were significantly lower than those in L+P group and L+A group(P<0.01). CONCLUSION: Propofol inhibits the inflammatory response of microglia by reducing the expression of TNF-α, IL-1 and IL-6, which may be related to the down-regulation of TRPV1 and p-CaMKⅡ and the reduction of intracellular Ca~(2+) concentration.
引文
[1] McDonough A,Weinstein JR.Neuroimmune response in ischemic preconditioning[J].Neurotherapeutics,2016,13(4):748-761.
[2] Hanisch UK,Kettenmann H.Microglia:active sensor and versatile effector cells in the normal and pathologic brain[J].Nat Neurosci,2007,10(11):1387-1394.
[3] Anttila JE,Whitaker KW,Wires ES,et al.Role of microglia in ischemic focal stroke and recovery:focus on toll-like receptors[J].Prog Neuropsychopharmacol Biol Psychiatry,2017,79(Pt A):3-14.
[4] Xiong XY,Liu L,Yang QW.Functions and mechanisms of microglia/macrophages in neuroinflammation and neurogenesis after stroke[J].Prog Neurobiol,2016,142:23-44.
[5] Zhou JM,Gu SS,Mei WH,et al.Ginkgolides and bilo-balide protect BV2 microglia cells against OGD/reoxyge-nation injury by inhibiting TLR2/4 signaling pathways[J].Cell Stress Chaperones,2016,21(6):1037-1053.
[6] Chidambaran V,Costandi A,D’Mello A.Propofol:a review of its role in pediatric anesthesia and sedation[J].CNS Drugs,2015,29(7):543-563.
[7] Ozkan F,Senayli Y,Ozyurt H,et al.Antioxidant effects of propofol on tourniquet-induced ischemia-reperfusion injury:an experimental study[J].J Surg Res,2012,176(2):601-607.
[8] Demir IE,Schafer KH,Tieftrunk E,et al.Neural plasti-city in the gastrointestinal tract:chronic inflammation,neurotrophic signals,and hypersensitivity[J].Acta Neuropathol,2013,125(4):491-509.
[9] 施孟如,南超,徐丽艳,等.TRPV1激活对内毒素血症小鼠肺组织炎症损伤的影响及机制[J].中国病理生理杂志,2013,29(12):2223-2228.
[10] Zheng X,Huang H,Liu J,et al.Propofol attenuates inflammatory response in LPS-activated microglia by regulating the miR-155/SOCS1 pathway[J].Inflammation,2018,41(1):11-19.
[11] Huang WX,Yu F,Sanchez RM,et al.TRPV1 promotes repetitive febrile seizures by pro-inflammatory cytokines in immature brain[J].Brain Behav Immun,2015,48:68-77.
[12] 张彬,肖献忠,张文辉,等.核仁素对LPS诱导的白细胞介素1β释放的影响[J].中国病理生理杂志,2010,26(9):1796-1800.
[13] Yan YQ,Jiang W,Liu L,et al.Dopamine controls systemic inflammation through inhibition of NLRP3 inflammasome[J].Cell,2015,160(1-2):62-73.
[14] Cheng L,Chen Z,Wang L,et al.Propofol partially atte-nuates complete Freund’s adjuvant-induced neuroinflammation through inhibition of the ERK1/2/NF-κB pathway[J].J Cell Biochem,2019,120(6):9400-9408.
[15] Jang S,Kelley KW,Johnson RW.Luteolin reduces IL-6 production in microglia by inhibiting JNK phosphorylation and activation of AP-1[J].Proc Natl Acad Sci U S A,2008,105(21):7534-7539.
[16] Gavva NR,Bannon AW,Hovland DN Jr,et al.Repeated administration of vanilloid receptor TRPV1 antagonists attenuates hyperthermia elicited by TRPV1 blockade[J].J Pharmacol Exp Ther,2007,323(1):128-137.
[17] Marrs WR,Blankman JL,Horne EA,et al.The serine hydrolase ABHD6 controls the accumulation and efficacy of 2-AG at cannabinoid receptors[J].Nat Neurosci,2010,13(8):951-957.
[18] Sfera A,Gradini R,Cummings M,et al.Rusty microglia:trainers of innate immunity in Alzheimer’s disease[J].Front Neurol,2018,9:1062.
[19] Jain N,Vogel V.Spatial confinement downsizes the inflammatory response of macrophages[J].Nat Mater,2018,17(12):1134-1144.
[20] Pflieger FJ,Hernandez J,Schweighofer H,et al.The role of neutrophil granulocytes in immune-to-brain communication[J].Temperature (Austin),2018,5(4):296-307.
[21] Geiseler SJ,Larson J,Folkow LP.Synaptic transmission despite severe hypoxia in hippocampal slices of the deep-diving hooded seal[J].Neuroscience,2016,334:39-46.
[22] Korvers L,de Andrade Costa A,Mersch M,et al.Spontaneous Ca2+ transients in mouse microglia[J].Cell Calcium,2016,60(6):396-406.
[23] Lam SY,Liu Y,Ng KM,et al.Chronic intermittent hypoxia induces local inflammation of the rat carotid body via functional upregulation of proinflammatory cytokine pathways[J].Histochem Cell Biol,2012,137(3):303-317.
[2] Hanisch UK,Kettenmann H.Microglia:active sensor and versatile effector cells in the normal and pathologic brain[J].Nat Neurosci,2007,10(11):1387-1394.
[3] Anttila JE,Whitaker KW,Wires ES,et al.Role of microglia in ischemic focal stroke and recovery:focus on toll-like receptors[J].Prog Neuropsychopharmacol Biol Psychiatry,2017,79(Pt A):3-14.
[4] Xiong XY,Liu L,Yang QW.Functions and mechanisms of microglia/macrophages in neuroinflammation and neurogenesis after stroke[J].Prog Neurobiol,2016,142:23-44.
[5] Zhou JM,Gu SS,Mei WH,et al.Ginkgolides and bilo-balide protect BV2 microglia cells against OGD/reoxyge-nation injury by inhibiting TLR2/4 signaling pathways[J].Cell Stress Chaperones,2016,21(6):1037-1053.
[6] Chidambaran V,Costandi A,D’Mello A.Propofol:a review of its role in pediatric anesthesia and sedation[J].CNS Drugs,2015,29(7):543-563.
[7] Ozkan F,Senayli Y,Ozyurt H,et al.Antioxidant effects of propofol on tourniquet-induced ischemia-reperfusion injury:an experimental study[J].J Surg Res,2012,176(2):601-607.
[8] Demir IE,Schafer KH,Tieftrunk E,et al.Neural plasti-city in the gastrointestinal tract:chronic inflammation,neurotrophic signals,and hypersensitivity[J].Acta Neuropathol,2013,125(4):491-509.
[9] 施孟如,南超,徐丽艳,等.TRPV1激活对内毒素血症小鼠肺组织炎症损伤的影响及机制[J].中国病理生理杂志,2013,29(12):2223-2228.
[10] Zheng X,Huang H,Liu J,et al.Propofol attenuates inflammatory response in LPS-activated microglia by regulating the miR-155/SOCS1 pathway[J].Inflammation,2018,41(1):11-19.
[11] Huang WX,Yu F,Sanchez RM,et al.TRPV1 promotes repetitive febrile seizures by pro-inflammatory cytokines in immature brain[J].Brain Behav Immun,2015,48:68-77.
[12] 张彬,肖献忠,张文辉,等.核仁素对LPS诱导的白细胞介素1β释放的影响[J].中国病理生理杂志,2010,26(9):1796-1800.
[13] Yan YQ,Jiang W,Liu L,et al.Dopamine controls systemic inflammation through inhibition of NLRP3 inflammasome[J].Cell,2015,160(1-2):62-73.
[14] Cheng L,Chen Z,Wang L,et al.Propofol partially atte-nuates complete Freund’s adjuvant-induced neuroinflammation through inhibition of the ERK1/2/NF-κB pathway[J].J Cell Biochem,2019,120(6):9400-9408.
[15] Jang S,Kelley KW,Johnson RW.Luteolin reduces IL-6 production in microglia by inhibiting JNK phosphorylation and activation of AP-1[J].Proc Natl Acad Sci U S A,2008,105(21):7534-7539.
[16] Gavva NR,Bannon AW,Hovland DN Jr,et al.Repeated administration of vanilloid receptor TRPV1 antagonists attenuates hyperthermia elicited by TRPV1 blockade[J].J Pharmacol Exp Ther,2007,323(1):128-137.
[17] Marrs WR,Blankman JL,Horne EA,et al.The serine hydrolase ABHD6 controls the accumulation and efficacy of 2-AG at cannabinoid receptors[J].Nat Neurosci,2010,13(8):951-957.
[18] Sfera A,Gradini R,Cummings M,et al.Rusty microglia:trainers of innate immunity in Alzheimer’s disease[J].Front Neurol,2018,9:1062.
[19] Jain N,Vogel V.Spatial confinement downsizes the inflammatory response of macrophages[J].Nat Mater,2018,17(12):1134-1144.
[20] Pflieger FJ,Hernandez J,Schweighofer H,et al.The role of neutrophil granulocytes in immune-to-brain communication[J].Temperature (Austin),2018,5(4):296-307.
[21] Geiseler SJ,Larson J,Folkow LP.Synaptic transmission despite severe hypoxia in hippocampal slices of the deep-diving hooded seal[J].Neuroscience,2016,334:39-46.
[22] Korvers L,de Andrade Costa A,Mersch M,et al.Spontaneous Ca2+ transients in mouse microglia[J].Cell Calcium,2016,60(6):396-406.
[23] Lam SY,Liu Y,Ng KM,et al.Chronic intermittent hypoxia induces local inflammation of the rat carotid body via functional upregulation of proinflammatory cytokine pathways[J].Histochem Cell Biol,2012,137(3):303-317.