δ-联蛋白通过AKT信号通路调控OGD/R后的炎症反应
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摘要
联蛋白(delta-catenin)作为高表达于神经系统的黏附蛋白质,在神经系统的功能发挥中有着至关重要的作用,但其在缺血缺氧性脑病中的研究尚未见报道。本文通过体外培养原代皮层神经元,构建氧糖剥夺/再灌注(oxygen-glucose deprivation/reperfusion,OGD/R)模型。用Western印迹、LDH等方法显示,与对照组相比,δ-联蛋白在OGD/R模型后的不同时间点(0、4、12、24和48 h)表达呈先降低后升高的趋势。在12 h表达量最低(0. 48±0. 08 vs 1. 53±0. 18,P<0. 05),在48 h表达升高到对照组水平(1. 35±0. 15 vs 1. 53±0. 18,P>0. 05)。用siRNA慢病毒干扰δ-联蛋白表达后,ELISA结果显示,和对照组相比,OGD/R后,IL-1β和IL-18升高(24. 80±1. 64 vs 12. 75±0. 87,28. 12±2. 69 vs 12. 99±1. 24,P<0. 05),但在干扰δ-联蛋白表达后,和OGD组相比,IL-1β和IL-18释放降低(12. 81±0. 78 vs 24. 80±1. 64,14. 27±1. 37 vs 28. 12±2. 69,P<0. 05)。Western印迹结果显示,AKT信号通路磷酸化位点Ser 473活化增高(1. 08±0. 04 vs 0. 85±0. 06,P<0. 05),但Thr 308位点活化无改变(1. 17±0. 06 vs 1. 11±0. 08,P>0. 05)。在siRNA慢病毒干扰并且联合使用AKT信号通路抑制剂GSK 690693后,和OGD+siRNA组相比,IL-1β和IL-18释放增高(24. 58±0. 99 vs 12. 81±0. 78,31. 62±2. 23 vs 14. 27±1. 37,P <0. 05)。上述结果显示,δ-联蛋白通过AKT信号通路调控OGD/R后的炎症反应,这可作为δ-联蛋白功能研究的新的实验依据。
As a highly expressed adhesive protein in the nervous system,delta-catenin plays a crucial role in the function of the nervous system,but its role in ischemic and hypoxic encephalopathy has not been reported. In this paper, an oxygen-glucose deprivation/reperfusion(OGD/R) model was established by culturing primary cortical neurons in vitro. Western blotting and LDH showed that the expression of delta-catenin at different times of the OGD/R model(0 h,4 h,12 h,24 h,48 h),decreased first and then increased compared with the control group. The expression level decreased to the lowest level at 12 h(0. 48± 0. 08 vs 1. 53 ± 0. 18,P < 0. 05) and increased to the control level at 48 h(1. 35±0. 15 vs 1. 53±0. 18,P>0. 05). Using siRNA lentivirus interference,ELISA tests showed that compared with the control group,inflammatory factors IL-1β and IL-18 in the OGD/R model(24. 80 ±1. 64 vs 12. 75±0. 87,28. 12±2. 69 vs 12. 99±1. 24,P<0. 05) decreased after inhibition of delta-catenin(12. 81±0. 78 vs 24. 80±1. 64,14. 27±1. 37 vs 28. 12±2. 69,P<0. 05),compared with the OGD group.Moreover,compared with the OGD group,Western blotting showed that activation of phosphorylation Ser473 increased(1. 08±0. 04 vs 0. 85±0. 06,P<0. 05) while activation of phosphorylation Thr308 did not change(1. 17±0. 06 vs 1. 11±0. 08,P>0. 05). ELISA results showed that after inhibition of deltacatenin combined with the AKT pathway inhibitor GSK 690693,release of IL-1β and IL-18 increased compared with the OGD+siRNA group(24. 58±0. 99 vs 12. 81±0. 78,31. 62±2. 23 vs 14. 27±1. 37,P<0. 05). All of the data suggest that delta-catenin regulates inflammation in OGD/R through the AKT pathway,which could be a new basis for the study on the function of delta-catenin.
As a highly expressed adhesive protein in the nervous system,delta-catenin plays a crucial role in the function of the nervous system,but its role in ischemic and hypoxic encephalopathy has not been reported. In this paper, an oxygen-glucose deprivation/reperfusion(OGD/R) model was established by culturing primary cortical neurons in vitro. Western blotting and LDH showed that the expression of delta-catenin at different times of the OGD/R model(0 h,4 h,12 h,24 h,48 h),decreased first and then increased compared with the control group. The expression level decreased to the lowest level at 12 h(0. 48± 0. 08 vs 1. 53 ± 0. 18,P < 0. 05) and increased to the control level at 48 h(1. 35±0. 15 vs 1. 53±0. 18,P>0. 05). Using siRNA lentivirus interference,ELISA tests showed that compared with the control group,inflammatory factors IL-1β and IL-18 in the OGD/R model(24. 80 ±1. 64 vs 12. 75±0. 87,28. 12±2. 69 vs 12. 99±1. 24,P<0. 05) decreased after inhibition of delta-catenin(12. 81±0. 78 vs 24. 80±1. 64,14. 27±1. 37 vs 28. 12±2. 69,P<0. 05),compared with the OGD group.Moreover,compared with the OGD group,Western blotting showed that activation of phosphorylation Ser473 increased(1. 08±0. 04 vs 0. 85±0. 06,P<0. 05) while activation of phosphorylation Thr308 did not change(1. 17±0. 06 vs 1. 11±0. 08,P>0. 05). ELISA results showed that after inhibition of deltacatenin combined with the AKT pathway inhibitor GSK 690693,release of IL-1β and IL-18 increased compared with the OGD+siRNA group(24. 58±0. 99 vs 12. 81±0. 78,31. 62±2. 23 vs 14. 27±1. 37,P<0. 05). All of the data suggest that delta-catenin regulates inflammation in OGD/R through the AKT pathway,which could be a new basis for the study on the function of delta-catenin.
引文
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[16]Ding C,Zhang J,Li B,et al.Cornin protects SH-SY5Y cells against oxygen and glucose deprivation-induced autophagy through the PI3K/Akt/m TOR pathway[J].Mol Med Rep,2018,17(1):87-92
[17]Lee GA,Lai YG,Chen RJ,et al.Interleukin 15 activates Akt to protect astrocytes from oxygen glucose deprivation-induced cell death[J].Cytokine,2017,92:68-74
[18]侯倩伶,王岩,李英博,等.三七皂苷R通过雌激素受体调节ATF6/Akt信号通路减轻OGD/R所导致的神经元损伤[J].中国中药杂志(Hou QL,Wang Y,Li YB,et al.Protective effect of notoginsenoside R1 on neuron injury induced by OGD/R through ATF6/Akt signaling pathway[J].China JChin Mater Med),2017,42(6):1167-1174
[19]Yin W,Signore AP,Iwai M,et al.Preconditioning suppresses inflammation in neonatal hypoxic ischemia via Akt activation[J].Stroke,2007,38(3):1017-1024
[20]Benjelloun N,Renolleau S,Represa A,et al.Inflammatory responses in the cerebral cortex after ischemia in the P7 neonatal Rat[J].Stroke,1999,30(9):1916-1923
[21]Chang Y,Kong R.Ganoderic acid A alleviates hypoxia-induced apoptosis,autophagy,and inflammation in rat neural stem cells through the PI3K/AKT/m TOR pathways[J].Phytother Res,2019,doi:10.1002/ptr.6336.[Epub ahead of print]
[22]Yan J,Li J,Zhang L,et al.Nrf2 protects against acute lung injury and inflammation by modulating TLR4 and Akt signaling[J].Free Radic Biol Med,2018,121:78-85
[23]Chen Z,Wang J,Yang W,et al.FAM3A mediates PPARgamma’s protection in liver ischemia-reperfusion injury by activating Akt survival pathway and repressing inflammation and oxidative stress[J].Oncotarget,2017,8(30):49882-49896
[2]Israely I,Costa RM,Xie CW,et al.Deletion of the neuronspecific protein delta-catenin leads to severe cognitive and synaptic dysfunction[J].Curr Biol,2004,14(18):1657-1663
[3]Qin S,Qin L,Zhang C,et al.p120-Catenin modulating nuclear factor-kappaB activation is partially RhoA/ROCKdependent in scratch injury[J].Wound Repair Regen,2015,23(2):231-240
[4]Kim H,Han JR,Park J,et al.δ-Catenin induced dendritic morphogenesis:An essential role of p190RhoGEF interaction through Akt1-mediated phosphorylation[J].J Biol Chem,2008,283(2):977-987
[5]Ide N,Hata Y,Deguchi M,et al.Interaction of S-SCAM with neural plakophilin-related Armadillo-repeat protein/delta-catenin[J].Biochem Biophys Res Commun,1999,256(3):456-461
[6]Lu Q,Mukhopadhyay NK,Griffin JD,et al.Brain armadillo protein delta-catenin interacts with Abl tyrosine kinase and modulates cellular morphogenesis in response to growth factors[J].J Neurosci Res,2002,67(5):618-624
[7]Jones SB,Lanford GW,Chen YH,et al.Glutamate-induced delta-catenin redistribution and dissociation from postsynaptic receptor complexes[J].Neuroscience,2002,115(4):1009-1021
[8]Bareiss S,Kim K,Lu Q.Delta-catenin/NPRAP:A new member of the glycogen synthase kinase-3beta signaling complex that promotes beta-catenin turnover in neurons[J].J Neurosci Res,2010,88(11):2350-2363
[9]Wang Y,Tu L,Li Y,et al.Notoginsenoside R1 protects against neonatal cerebral hypoxic-ischemic injury through estrogen receptor-dependent activation of endoplasmic reticulum stress pathways[J].J Pharmacol Exp Ther,2016,357(3):591-605
[10]Hagberg H,Gilland E,Bona E,et al.Enhanced expression of interleukin(IL)-1 and IL-6 messenger RNA and bioactive protein after hypoxia-ischemia in neonatal rats[J].Pediatr Res,1996,40(4):603-609
[11]Hedtjarn M,Leverin AL,Eriksson K,et al.Interleukin-18involvement in hypoxic-ischemic brain injury[J].J Neurosci,2002,22(14):5910-5919
[12]Rang Z,Wang ZY,Pang QY,et al.MiR-181a Targets PHLPP2to Augment AKT Signaling and Regulate Proliferation and Apoptosis in Human Keloid Fibroblasts[J].Cell Physiol Biochem,2016,40(3-4):796-806
[13]Andjelkovic M,Alessi DR,Meier R,et al.Role of translocation in the activation and function of protein kinase B[J].J Biol Chem,1997,272(50):31515-31524
[14]Yao R,Cooper GM.Requirement for phosphatidylinositol-3kinase in the prevention of apoptosis by nerve growth factor[J].Science,1995,267(5206):2003-2006
[15]Endo H,Nito C,Kamada H,et al.Activation of the Akt/GSK3beta signaling pathway mediates survival of vulnerable hippocampal neurons after transient global cerebral ischemia in rats[J].J Cereb Blood Flow Metab,2006,26(12):1479-1489
[16]Ding C,Zhang J,Li B,et al.Cornin protects SH-SY5Y cells against oxygen and glucose deprivation-induced autophagy through the PI3K/Akt/m TOR pathway[J].Mol Med Rep,2018,17(1):87-92
[17]Lee GA,Lai YG,Chen RJ,et al.Interleukin 15 activates Akt to protect astrocytes from oxygen glucose deprivation-induced cell death[J].Cytokine,2017,92:68-74
[18]侯倩伶,王岩,李英博,等.三七皂苷R通过雌激素受体调节ATF6/Akt信号通路减轻OGD/R所导致的神经元损伤[J].中国中药杂志(Hou QL,Wang Y,Li YB,et al.Protective effect of notoginsenoside R1 on neuron injury induced by OGD/R through ATF6/Akt signaling pathway[J].China JChin Mater Med),2017,42(6):1167-1174
[19]Yin W,Signore AP,Iwai M,et al.Preconditioning suppresses inflammation in neonatal hypoxic ischemia via Akt activation[J].Stroke,2007,38(3):1017-1024
[20]Benjelloun N,Renolleau S,Represa A,et al.Inflammatory responses in the cerebral cortex after ischemia in the P7 neonatal Rat[J].Stroke,1999,30(9):1916-1923
[21]Chang Y,Kong R.Ganoderic acid A alleviates hypoxia-induced apoptosis,autophagy,and inflammation in rat neural stem cells through the PI3K/AKT/m TOR pathways[J].Phytother Res,2019,doi:10.1002/ptr.6336.[Epub ahead of print]
[22]Yan J,Li J,Zhang L,et al.Nrf2 protects against acute lung injury and inflammation by modulating TLR4 and Akt signaling[J].Free Radic Biol Med,2018,121:78-85
[23]Chen Z,Wang J,Yang W,et al.FAM3A mediates PPARgamma’s protection in liver ischemia-reperfusion injury by activating Akt survival pathway and repressing inflammation and oxidative stress[J].Oncotarget,2017,8(30):49882-49896