4-苯基丁酸钠通过抑制内质网应激减轻皮层神经元氧糖剥夺/再灌注损伤
|
摘要
4-苯基丁酸钠(4-phenylbutyric acid,4-PBA)是协助内质网中蛋白质转录后修饰和折叠的分子伴侣,故可减轻非折叠蛋白反应(unfolded protein response,UPR)及其介导的细胞凋亡。既往研究表明,4-PBA可以减轻脑组织的缺血性损伤,但采用原代皮层神经元构建氧糖剥夺/再灌注(oxygen-glucose deprivation/reoxygenation,OGD/R)损伤模型,来研究4-PBA对神经元损伤的保护作用及其机制尚未见报道。本文采用原代培养的皮层神经元OGD/R损伤模型,同时给予4-PBA处理,探讨4-PBA对OGD/R诱导的神经元内质网应激(endoplasmic reticulum stress,ERS)的作用及其机制。分别采用MTT、LDH和Hoechst 33342染色法检测神经元存活率、细胞膜完整性和细胞凋亡情况。Western印迹检测ERS标志物葡萄糖调节蛋白78(glucose regulated protein 78,GRP78),以及肌醇必需酶1(inositol-requiring enzyme 1,IRE1)通路相关蛋白质的表达。Western印迹结果显示,在OGD/R后0~48 h,GRP78的表达较对照组明显升高。MTT、LDH漏出率和Hoechst 33342染色法检测显示,4-PBA显著改善OGD/R所导致的神经元存活率下降、LDH漏出率升高和细胞凋亡增加,且具有明显的剂量依赖性。通过Western印迹检测发现,4-PBA显著逆转OGD/R所致GRP78蛋白表达水平的上调。此外,对肌醇必需酶1通路相关蛋白质的检测显示,4-PBA下调氧糖剥夺/再灌注组神经元p-IRE1和p-JNK的表达,增加抗凋亡蛋白Bcl-2表达。上述研究结果表明,4-PBA在氧糖剥夺/再灌注情况下对神经元具有保护作用,该保护作用可能是通过抑制肌醇必需酶1信号通路介导的非折叠蛋白反应和内质网应激实现的。
4-Phenylbutyric acid(4-PBA),a chemical chaperone that contributes to post-transcriptional modification and folding of proteins in endoplasmic reticulum(ER),could inhibit unfolded protein response(UPR) and UPR-induced apoptosis. Although previous studies show that 4-PBA could alleviate the ischemic brain damage,the effect and mechanism of 4-PBA on the injury of primary cortical neurons exposed to oxygen-glucose deprivation/reoxygenation(OGD/R) has not been reported. To investigate theeffect of 4-PBA on OGD/R-induced endoplasmic reticulum stress(ERS) and its mechanisms,primary cortical neurons were subjected to OGD/R and treated simultaneously with 4-PBA in the present study.The cell viability,cell membrane integrity,and apoptosis were detected by MTT assay,LDH test,and Hoechst 33342 staining respectively. Moreover,the expression levels of glucose regulated protein 78(GRP78),an ERS marker protein,and the inositol essential enzyme 1(IRE1) pathway-associated proteins were detected by Western blotting. Our results revealed that the expression of GRP78 was significantly increased after OGD/R(0 ~ 48 h). 4-PBA could remarkably ameliorate the decrease of neurons viability and the increase of LDH leakage and neuronal apoptosis. In addition,Western blotting analysis showed that 4-PBA down-regulated the elevated expression of GRP78,p-IRE1 and p-JNK,and up-regulated the suppressed anti-apoptotic protein Bcl-2 expression in OGD/R neurons. The results confirm that 4-PBA has a protective effect on neuron against OGD/R induced injury,by which the effect may be achieved by inhibiting IRE1 signaling-mediated UPR and ERS.
4-Phenylbutyric acid(4-PBA),a chemical chaperone that contributes to post-transcriptional modification and folding of proteins in endoplasmic reticulum(ER),could inhibit unfolded protein response(UPR) and UPR-induced apoptosis. Although previous studies show that 4-PBA could alleviate the ischemic brain damage,the effect and mechanism of 4-PBA on the injury of primary cortical neurons exposed to oxygen-glucose deprivation/reoxygenation(OGD/R) has not been reported. To investigate theeffect of 4-PBA on OGD/R-induced endoplasmic reticulum stress(ERS) and its mechanisms,primary cortical neurons were subjected to OGD/R and treated simultaneously with 4-PBA in the present study.The cell viability,cell membrane integrity,and apoptosis were detected by MTT assay,LDH test,and Hoechst 33342 staining respectively. Moreover,the expression levels of glucose regulated protein 78(GRP78),an ERS marker protein,and the inositol essential enzyme 1(IRE1) pathway-associated proteins were detected by Western blotting. Our results revealed that the expression of GRP78 was significantly increased after OGD/R(0 ~ 48 h). 4-PBA could remarkably ameliorate the decrease of neurons viability and the increase of LDH leakage and neuronal apoptosis. In addition,Western blotting analysis showed that 4-PBA down-regulated the elevated expression of GRP78,p-IRE1 and p-JNK,and up-regulated the suppressed anti-apoptotic protein Bcl-2 expression in OGD/R neurons. The results confirm that 4-PBA has a protective effect on neuron against OGD/R induced injury,by which the effect may be achieved by inhibiting IRE1 signaling-mediated UPR and ERS.
引文
[1]Gerschenfeld G,Muresan IP,Blanc R,et al.Two paradigms for endovascular thrombectomy after intravenous thrombolysis for fcute ischemic stroke[J].JAMA Neurol,2017,74(5):549-556
[2]Reis C,Akyol O,Ho WM,et al.Phase I and phase II therapies for acute ischemic stroke:an update on currently studied drugs in clinical research[J].Biomed Res Int,2017,2017:4863079
[3]Sveinsson OA,Kjartansson O,Valdimarsson EM.Cerebral ischemiainfarction-epidemiology,causes and symptoms[J].Laeknabladid,2014,100(5):271-279
[4]Yang S,Ning F,Li J,et al.Therapeutic effect analysis of sinomenine on rat cerebral ischemia-reperfusion injury[J].J Stroke Cerebrovasc Dis,2016,25(5):1263-1269
[5]Yang Z,Weian C,Susu H,et al.Protective effects of mangiferin on cerebral ischemia-reperfusion injury and its mechanisms[J].Eur J Pharmacol,2016,771:145-151
[6]Jo F,Jo H,Hilzendeger AM,et al.Brain endoplasmic reticulum stress mechanistically distinguishes the saline-intake and hypertensive response to deoxycorticosterone acetate-salt[J].Hypertension,2015,65(6):1341-1348
[7]Zhang XY,Yang SM,Zhang HP,et al.Endoplasmic reticulum stress mediates the arsenic trioxide-induced apoptosis in human hepatocellular carcinoma cells[J].Int J Biochem Cell Biol,2015,68:158-165
[8]Smith HL,Mallucci GR.The unfolded protein response:mechanisms and therapy of neurodegeneration[J].Brain,2016,139(Pt 8):2113-2121
[9]Xin Q,Ji B,Cheng B,et al.Endoplasmic reticulum stress in cerebral ischemia[J].Neurochem Int,2014,68:18-27
[10]Cao G,Zhou H,Jiang N,et al.Yi Qi Fu Mai powder injection ameliorates cerebral ischemia byinhibitingendoplasmic reticulum stress-mediated neuronal apoptosis[J].Oxid Med Cell Longev,2016,2016:5493279
[11]Yang RX,Lei J,Wang BD,et al.Pretreatment with sodium phenylbutyrate alleviates cerebral ischemia/reperfusion injury by upregulating DJ-1 protein[J].Front Neurol,2017,8:256
[12]Srinivasan K,Sharma SS.Sodium phenylbutyrate ameliorates focal cerebral ischemic/reperfusion injury associated with comorbid type 2 diabetes by reducing endoplasmic reticulum stress and DNA fragmentation[J].Behav Brain Res,2011,225(1):110-116
[13]Tung WF,Chen WJ,Hung HC,et al.4-Phenylbutyric acid(4-PBA)and lithium cooperatively attenuate cell death during oxygen-glucose deprivation(OGD)and reoxygenation[J].Cell Mol Neurobiol,2015,35(6):849-859
[14]Qi X,Hosoi T,Okuma Y,et al.Sodium 4-phenylbutyrate protects against cerebral ischemic injury[J].Mol Pharmacol,2004,66(4):899-908
[15]Wang HF,Wang ZQ,Ding Y,et al.Endoplasmic reticulum stress regulates oxygen-glucose deprivation-induced parthanatos in human SH-SY5Y cells via improvement of intracellular ROS[J].CNS Neurosci Ther,2017 Oct 16.doi:10.1111/cns.12771.[Epub ahead of print]
[16]Cameron NE.Role of endoplasmic reticulum stress in diabetic neuropathy[J].Diabetes,2013,62(3):696-697
[17]Song S,Tan J,Miao Y,et al.Crosstalk of ER stress-mediated autophagy and ER-phagy:involvement of UPR and the core autophagy machinery[J].J Cell Physiol,2017 Aug 4.doi:10.1002/jcp.26137.[Epub ahead of print]
[18]Liu C,Huang Y,Zhang Y,et al.Intracellular methylglyoxal induces oxidative damage to pancreatic beta cell line INS-1 cell through Ire1alpha-JNK and mitochondrial apoptotic pathway[J].Free Radic Res,2017,51(4):337-350
[19]Stengel S,Messner B,Falk-Paulsen M,et al.Regulated proteolysis as an element of ER stress and autophagy:implications for intestinal inflammation[J].Biochim Biophys Acta,2017,1864(11 Pt B):2183-2190
[20]Xu Z,Bu Y,Chitnis N,et al.miR-216b regulation of c-Jun mediates GADD153/CHOP-dependent apoptosis[J].Nat Commun,2016,7:11422
[21]Wang F,Weng H,Quon MJ,et al.Dominant negative FADD dissipates the proapoptotic signalosome of the unfolded protein response in diabetic embryopathy[J].Am J Physiol Endocrinol Metab,2015,309(10):E861-873
[22]Sukumaran P,Schaar A,Sun Y,et al.Functional role of TRP channels in modulating ER stress and Autophagy[J].Cell Calcium,2016,60(2):123-132
[23]Mozzini C,Cominacini L,Garbin U,et al.Endoplasmic reticulum stress,NRF2 signalling and cardiovascular diseases in a nutshell[J].Curr Atheroscler Rep,2017,19(8):33
[24]Yu H,Zhen J,Yang Y,et al.Ginsenoside Rg1 ameliorates diabetic cardiomyopathy by inhibiting endoplasmic reticulum stress-induced apoptosis in a streptozotocin-induced diabetes rat model[J].J Cell Mol Med,2016,20(4):623-631
[25]Pavan Grandhi TS,Potta T,Nitiyanandan R,et al.Chemomechanically engineered 3D organotypic platforms of bladder cancer dormancy and reactivation[J].Biomaterials,2017,142:171-185
[26]Nakano M,Imamura H,Sasaoka N,et al.ATP maintenance via two types of ATP regulators mitigates pathological phenotypes in mouse models of Parkinson’s disease[J].EBio Medicine,2017,22:225-241
[27]Mota SI,Costa RO,Ferreira IL,et al.Oxidative stress involving changes in Nrf2 and ER stress in early stages of Alzheimer’s disease[J].Biochim Biophys Acta,2015,1852(7):1428-1441
[28]Hetz C,Saxena S.ER stress and the unfolded protein response in neurodegeneration[J].Nat Rev Neurol,2017,13(8):477-491
[29]Mohammed-Ali Z,Lu C,Marway MK,et al.Endoplasmic reticulum stress inhibition attenuates hypertensive chronic kidney disease through reduction in proteinuria[J].Sci Rep,2017,7:41572
[30]Mizukami T,Orihashi K,Herlambang B,et al.Sodium 4-phenylbutyrate protects against spinal cord ischemia by inhibition of endoplasmic reticulum stress[J].J Vasc Surg,2010,52(6):1580-1586
[31]Chen M,Liu Q,Chen L,et al.Remifentanil postconditioning ameliorates histone H3 acetylation modification in H9c2cardiomyoblasts after hypoxia/reoxygenation via attenuating endoplasmic reticulum stress[J].Apoptosis,2017,22(5):662-671
[32]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
[2]Reis C,Akyol O,Ho WM,et al.Phase I and phase II therapies for acute ischemic stroke:an update on currently studied drugs in clinical research[J].Biomed Res Int,2017,2017:4863079
[3]Sveinsson OA,Kjartansson O,Valdimarsson EM.Cerebral ischemiainfarction-epidemiology,causes and symptoms[J].Laeknabladid,2014,100(5):271-279
[4]Yang S,Ning F,Li J,et al.Therapeutic effect analysis of sinomenine on rat cerebral ischemia-reperfusion injury[J].J Stroke Cerebrovasc Dis,2016,25(5):1263-1269
[5]Yang Z,Weian C,Susu H,et al.Protective effects of mangiferin on cerebral ischemia-reperfusion injury and its mechanisms[J].Eur J Pharmacol,2016,771:145-151
[6]Jo F,Jo H,Hilzendeger AM,et al.Brain endoplasmic reticulum stress mechanistically distinguishes the saline-intake and hypertensive response to deoxycorticosterone acetate-salt[J].Hypertension,2015,65(6):1341-1348
[7]Zhang XY,Yang SM,Zhang HP,et al.Endoplasmic reticulum stress mediates the arsenic trioxide-induced apoptosis in human hepatocellular carcinoma cells[J].Int J Biochem Cell Biol,2015,68:158-165
[8]Smith HL,Mallucci GR.The unfolded protein response:mechanisms and therapy of neurodegeneration[J].Brain,2016,139(Pt 8):2113-2121
[9]Xin Q,Ji B,Cheng B,et al.Endoplasmic reticulum stress in cerebral ischemia[J].Neurochem Int,2014,68:18-27
[10]Cao G,Zhou H,Jiang N,et al.Yi Qi Fu Mai powder injection ameliorates cerebral ischemia byinhibitingendoplasmic reticulum stress-mediated neuronal apoptosis[J].Oxid Med Cell Longev,2016,2016:5493279
[11]Yang RX,Lei J,Wang BD,et al.Pretreatment with sodium phenylbutyrate alleviates cerebral ischemia/reperfusion injury by upregulating DJ-1 protein[J].Front Neurol,2017,8:256
[12]Srinivasan K,Sharma SS.Sodium phenylbutyrate ameliorates focal cerebral ischemic/reperfusion injury associated with comorbid type 2 diabetes by reducing endoplasmic reticulum stress and DNA fragmentation[J].Behav Brain Res,2011,225(1):110-116
[13]Tung WF,Chen WJ,Hung HC,et al.4-Phenylbutyric acid(4-PBA)and lithium cooperatively attenuate cell death during oxygen-glucose deprivation(OGD)and reoxygenation[J].Cell Mol Neurobiol,2015,35(6):849-859
[14]Qi X,Hosoi T,Okuma Y,et al.Sodium 4-phenylbutyrate protects against cerebral ischemic injury[J].Mol Pharmacol,2004,66(4):899-908
[15]Wang HF,Wang ZQ,Ding Y,et al.Endoplasmic reticulum stress regulates oxygen-glucose deprivation-induced parthanatos in human SH-SY5Y cells via improvement of intracellular ROS[J].CNS Neurosci Ther,2017 Oct 16.doi:10.1111/cns.12771.[Epub ahead of print]
[16]Cameron NE.Role of endoplasmic reticulum stress in diabetic neuropathy[J].Diabetes,2013,62(3):696-697
[17]Song S,Tan J,Miao Y,et al.Crosstalk of ER stress-mediated autophagy and ER-phagy:involvement of UPR and the core autophagy machinery[J].J Cell Physiol,2017 Aug 4.doi:10.1002/jcp.26137.[Epub ahead of print]
[18]Liu C,Huang Y,Zhang Y,et al.Intracellular methylglyoxal induces oxidative damage to pancreatic beta cell line INS-1 cell through Ire1alpha-JNK and mitochondrial apoptotic pathway[J].Free Radic Res,2017,51(4):337-350
[19]Stengel S,Messner B,Falk-Paulsen M,et al.Regulated proteolysis as an element of ER stress and autophagy:implications for intestinal inflammation[J].Biochim Biophys Acta,2017,1864(11 Pt B):2183-2190
[20]Xu Z,Bu Y,Chitnis N,et al.miR-216b regulation of c-Jun mediates GADD153/CHOP-dependent apoptosis[J].Nat Commun,2016,7:11422
[21]Wang F,Weng H,Quon MJ,et al.Dominant negative FADD dissipates the proapoptotic signalosome of the unfolded protein response in diabetic embryopathy[J].Am J Physiol Endocrinol Metab,2015,309(10):E861-873
[22]Sukumaran P,Schaar A,Sun Y,et al.Functional role of TRP channels in modulating ER stress and Autophagy[J].Cell Calcium,2016,60(2):123-132
[23]Mozzini C,Cominacini L,Garbin U,et al.Endoplasmic reticulum stress,NRF2 signalling and cardiovascular diseases in a nutshell[J].Curr Atheroscler Rep,2017,19(8):33
[24]Yu H,Zhen J,Yang Y,et al.Ginsenoside Rg1 ameliorates diabetic cardiomyopathy by inhibiting endoplasmic reticulum stress-induced apoptosis in a streptozotocin-induced diabetes rat model[J].J Cell Mol Med,2016,20(4):623-631
[25]Pavan Grandhi TS,Potta T,Nitiyanandan R,et al.Chemomechanically engineered 3D organotypic platforms of bladder cancer dormancy and reactivation[J].Biomaterials,2017,142:171-185
[26]Nakano M,Imamura H,Sasaoka N,et al.ATP maintenance via two types of ATP regulators mitigates pathological phenotypes in mouse models of Parkinson’s disease[J].EBio Medicine,2017,22:225-241
[27]Mota SI,Costa RO,Ferreira IL,et al.Oxidative stress involving changes in Nrf2 and ER stress in early stages of Alzheimer’s disease[J].Biochim Biophys Acta,2015,1852(7):1428-1441
[28]Hetz C,Saxena S.ER stress and the unfolded protein response in neurodegeneration[J].Nat Rev Neurol,2017,13(8):477-491
[29]Mohammed-Ali Z,Lu C,Marway MK,et al.Endoplasmic reticulum stress inhibition attenuates hypertensive chronic kidney disease through reduction in proteinuria[J].Sci Rep,2017,7:41572
[30]Mizukami T,Orihashi K,Herlambang B,et al.Sodium 4-phenylbutyrate protects against spinal cord ischemia by inhibition of endoplasmic reticulum stress[J].J Vasc Surg,2010,52(6):1580-1586
[31]Chen M,Liu Q,Chen L,et al.Remifentanil postconditioning ameliorates histone H3 acetylation modification in H9c2cardiomyoblasts after hypoxia/reoxygenation via attenuating endoplasmic reticulum stress[J].Apoptosis,2017,22(5):662-671
[32]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