抑制PI3K/AKt信号通路对帕金森病炎症细胞模型反应的影响
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摘要
目的探讨LY294002(LY)抑制磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(AKt)信号通路后对帕金森病(PD)细胞模型中炎症反应的影响。方法体外培养小鼠小胶质瘤细胞(BV2),分为对照组、脂多糖(LPS)组、LY+LPS组及LY组。LPS刺激BV2细胞来诱导炎症反应,运用Western印迹检测PI3K/AKt信号通路标志物磷酸化(p)-AKt蛋白表达情况;LY抑制PI3K/AKt信号通路后,运用荧光定量PCR (Q-PCR)检测炎症因子白细胞介素(IL)-1β、肿瘤坏死因子(TNF)-α的mRNA的表达情况,Western印迹检测诱导型一氧化氮合酶(iNOS)蛋白表达情况。结果与对照组相比,LPS组p-AKt蛋白表达显著下降(P<0.01);与LPS组相比,LY+LPS组p-AKt蛋白下降更明显(P<0.01);与对照组相比,LPS组IL-1β、TNF-αmRNA及iNOS蛋白表达均显著升高(均P<0.01);与LPS组相比,LY+LPS组中IL-1β和TNF-αmRNA及iNOS蛋白表达均显著下降(均P<0.01)。结论 LY抑制PI3K/AKt信号通路对BV2细胞中的LPS诱导的炎症反应有一定的抑制作用。
Objective To investigate the effects of PI3 K/AKt signaling pathway inhibited by LY294002 on the inflammatory response in the cell model of Parkinson′s disease(PD). Methods The routinely cultured BV2 microglial in vitro were divided into control, lipopolysaccharide(LPS), LY294002 + LPS and the LY294002 groups. LPS was used to stimulate the BV2 cell to induce inflammatory reaction, Western blot was used to detect the expression of p-AKt protein, which was the marker of PI3 K/AKt signal pathway. Inhibition of PI3 K/AKt signaling pathway by LY294002, real time quantitative PCR was used to detect the expressions of inflammatory factor IL-1β, TNF-α mRNA and Western blot was used to detect the expression of iNOS protein. Results Compared with control group, the expression of p-AKt protein was significantly decreased after LPS stimulation(P<0.01), compared with the LPS group, the expression of p-AKt protein was decreased more significantly after treated by LY294002 and LPS(P<0.01). Compared with control group, the expressions of IL-1β, TNF-α mRNA and the expression of p-AKt protein were significantly increased after stimulated by LPS(all P<0.01). Compared with the LPS group, the expressions of IL-1β, TNF-α mRNA and p-AKt protein were decreased after treated by LY294002 and LPS(all P<0.01).Conclusions Inhibition of PI3 K/AKt signaling pathway by LY294002 could attenuate inflammatory response in BV2 induced by LPS.
Objective To investigate the effects of PI3 K/AKt signaling pathway inhibited by LY294002 on the inflammatory response in the cell model of Parkinson′s disease(PD). Methods The routinely cultured BV2 microglial in vitro were divided into control, lipopolysaccharide(LPS), LY294002 + LPS and the LY294002 groups. LPS was used to stimulate the BV2 cell to induce inflammatory reaction, Western blot was used to detect the expression of p-AKt protein, which was the marker of PI3 K/AKt signal pathway. Inhibition of PI3 K/AKt signaling pathway by LY294002, real time quantitative PCR was used to detect the expressions of inflammatory factor IL-1β, TNF-α mRNA and Western blot was used to detect the expression of iNOS protein. Results Compared with control group, the expression of p-AKt protein was significantly decreased after LPS stimulation(P<0.01), compared with the LPS group, the expression of p-AKt protein was decreased more significantly after treated by LY294002 and LPS(P<0.01). Compared with control group, the expressions of IL-1β, TNF-α mRNA and the expression of p-AKt protein were significantly increased after stimulated by LPS(all P<0.01). Compared with the LPS group, the expressions of IL-1β, TNF-α mRNA and p-AKt protein were decreased after treated by LY294002 and LPS(all P<0.01).Conclusions Inhibition of PI3 K/AKt signaling pathway by LY294002 could attenuate inflammatory response in BV2 induced by LPS.
引文
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11 Yang X,Ren H,Wood K,et al.Depletion of microglia augments the dopaminergic neurotoxicity of MPTP[J].FASEB J,2018;32(6):3336-45.
12 Dong L,Li YZ,An HT,et al.The E3 ubiquitin ligase c-cbl inhibits microglia-mediated cns inflammation by regulating PI3K/Akt/NF-κB pathway[J].CNS Neurosci Ther,2016;22(8):661-9.
13 Zhao Y,Zhang Q,Xi J,et al.Neuroprotective effect of fasudil on inflammation through PI3K/Akt and Wnt/β-catenin dependent pathways in a mice model of Parkinson′s disease[J].Int J Clin Exp Pathol,2015;8(3):2354-64.
14 Dilshara MG,Lee KT,Jayasooriya RG,et al.Downregulation of NO and PGE2 in LPS-stimulated BV2 microglial cells by trans-isoferulic acid via suppression of PI3K/Akt-dependent NF-κB and activation of Nrf2-mediated HO-1[J].Int Immunopharmacol,2014;18(1):203-11.
2 Heckman MG,Kasanuki K,Diehl NN,et al.Parkinson′s disease susceptibility variants and severity of Lewy body pathology[J].Parkinsonism Relat Disord,2017;44:79-84.
3 Kurnik M,Thor P.The non-motor complications in Parkinson′s disease-what can we learn from animal models[J].Folia Med Cracov,2015;55(2):69-84.
4 Gustot A,Gallea JI,Sarroukh R,et al.Amyloid fibrils are the molecular trigger of inflammation in Parkinson′s disease[J].Biochem J,2015;471(3):323-33.
5 Saijo K,Winner B,Carson CT,et al.A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death[J].Cell,2009;137(1):47-59.
6 Liu Z,Chen HQ,Huang Y,et al.Transforming growth factor-beta1 acts via TbetaR-I on microglia to protect against MPP(+)-induced dopaminergic neuronal loss[J].Brain Behav Immun,2016;51:131-43.
7 Jia Y,Mo SJ,Feng QQ,et al.EPO-dependent activation of PI3K/Akt/FoxO3a signalling mediates neuroprotection in in vitro and in vivo models of Parkinson′s disease[J].J Mol Neurosci,2014;53(1):117-24.
8 Yang PH,Zhu JX,Huang YD,et al.Human basic fibroblast growth factor inhibits tau phosphorylation via the PI3K/Akt-GSK3β signaling pathway in a 6-hydroxydopamine-induced model of parkinson′s disease[J].Neurodegener Dis,2016;16(5-6):357-69.
9 Han MH,Lee WS,Nagappan A,et al.Flavonoids isolated from flowers of lonicera japonica Thunb.Inhibit inflammatory responses in BV2 microglial cells by suppressing TNF-α and IL-β through PI3K/Akt/NF-kb signaling pathways[J].Phytother Res,2016;30(11):1824-32.
10 洪乐鹏,邵帅,汪光亮.Purmorphamine对BV2细胞中帕金森病相关基因Nurr1表达的影响[J].中国神经免疫学和神经病学杂志,2016;23(3):195-8.
11 Yang X,Ren H,Wood K,et al.Depletion of microglia augments the dopaminergic neurotoxicity of MPTP[J].FASEB J,2018;32(6):3336-45.
12 Dong L,Li YZ,An HT,et al.The E3 ubiquitin ligase c-cbl inhibits microglia-mediated cns inflammation by regulating PI3K/Akt/NF-κB pathway[J].CNS Neurosci Ther,2016;22(8):661-9.
13 Zhao Y,Zhang Q,Xi J,et al.Neuroprotective effect of fasudil on inflammation through PI3K/Akt and Wnt/β-catenin dependent pathways in a mice model of Parkinson′s disease[J].Int J Clin Exp Pathol,2015;8(3):2354-64.
14 Dilshara MG,Lee KT,Jayasooriya RG,et al.Downregulation of NO and PGE2 in LPS-stimulated BV2 microglial cells by trans-isoferulic acid via suppression of PI3K/Akt-dependent NF-κB and activation of Nrf2-mediated HO-1[J].Int Immunopharmacol,2014;18(1):203-11.
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