人参皂苷Rg1对氧糖剥夺/复氧复糖损伤PC12细胞的保护机制
|
摘要
背景:人参皂苷具有抗肿瘤、抗氧化、抗疲劳、抗衰老、降血脂、增强记忆力、提高免疫力等作用,其中Rb1和Rg1是人参皂苷的最主要的活性成分。但人参皂苷Rg1对氧糖剥夺诱导的PC12细胞凋亡是否也有保护作用,尚不清楚。目的:观察人参皂苷Rg1预处理对于PC12细胞氧糖剥夺/复氧复糖模型中PC12细胞活力、survivin及caspase-3表达和细胞凋亡的影响。方法:采用构建的PC12细胞氧糖剥夺/复氧复糖模型,并使用5,10,20,40μmol/L的人参皂苷Rg1对其进行预处理,以正常细胞做对照。通过MTT法检测细胞活性,通过免疫细胞化学技术检测survivin及caspase-3蛋白,通过TUNEL法检测细胞凋亡。结果与结论:(1)细胞活力检测氧糖剥夺/复氧复糖组细胞活性明显下降(P <0.05),人参皂苷Rg1干预后药物组各亚组细胞活性均较氧糖剥夺/复氧复糖组有所恢复,但仍较正常组为低(P <0.05);(2)人参皂苷Rg1干预使survivin阳性细胞增多,caspase-3阳性细胞和TUNEL阳性细胞计数值减少;(3)神经元凋亡出现与人参皂苷Rg1诱导的survivin成负相关,与caspase-3成正相关。(4)结果说明,人参皂苷Rg1对于PC12细胞氧糖剥夺/复氧复糖处理后的survivin蛋白表达具有促进作用,并通过促进survivin蛋白的表达来对抗caspase-3的表达,从而抑制细胞凋亡,且该作用具有剂量依赖关系。
BACKGROUND: Ginsenoside has anti-tumor, anti-oxidative, anti-fatigue, anti-aging, hypolipidemic, memory enhancement, immunity enhancement effects, and Rb1 and Rg1 are the most important active components of ginsenosides. However, it is unclear whether ginsenoside Rg1 also protects against apoptosis in PC12 cells induced by oxygen-glucose deprivation(OGD). OBJECTIVE: To investigate the effects of pretreatment with ginsenoside Rg1 on PC12 cell viability, survivin and caspase-3 expression and apoptosis in PC12 cells induced by OGD/reperfusion. METHODS: In the OGD/reperfusion model, the PC12 cells were pretreated with 5, 10, 20, 40 μmol/L ginsenoside Rg1. Normal cells were used as controls. Cell viability was determined by MTT assay. Expression of survivin and caspase-3 proteins was measured by immunocytochemistry. Apoptosis in PC12 cells was detected by TUNEL assay. RESULTS AND CONCLUSION:(1) The cell viability was significantly decreased after OGD/reperfusion(P < 0.05). Pretreatment with ginsenoside Rg1 could elevate the cell viability, but it was still lower than the normal level(P < 0.05).(2) Pretreatment with ginsenoside Rg1 increased survivin positive cells, but decreased caspase-3 positive cells and TUNEL positive cells.(3) Neuronal apoptosis was negatively correlated with the ginsenoside Rg1-induced survivin, but positively correlated with the caspase-3. All these findings indicate that ginsenoside Rg1 pretreatment can promote the expression of survivin in PC12 cells after OGD/reperfusion, and moreover, this promotion effect on survivin can further inhibit the expression of caspase-3, thereby suppression apoptosis in PC12 cells, in a dose-dependent manner.
BACKGROUND: Ginsenoside has anti-tumor, anti-oxidative, anti-fatigue, anti-aging, hypolipidemic, memory enhancement, immunity enhancement effects, and Rb1 and Rg1 are the most important active components of ginsenosides. However, it is unclear whether ginsenoside Rg1 also protects against apoptosis in PC12 cells induced by oxygen-glucose deprivation(OGD). OBJECTIVE: To investigate the effects of pretreatment with ginsenoside Rg1 on PC12 cell viability, survivin and caspase-3 expression and apoptosis in PC12 cells induced by OGD/reperfusion. METHODS: In the OGD/reperfusion model, the PC12 cells were pretreated with 5, 10, 20, 40 μmol/L ginsenoside Rg1. Normal cells were used as controls. Cell viability was determined by MTT assay. Expression of survivin and caspase-3 proteins was measured by immunocytochemistry. Apoptosis in PC12 cells was detected by TUNEL assay. RESULTS AND CONCLUSION:(1) The cell viability was significantly decreased after OGD/reperfusion(P < 0.05). Pretreatment with ginsenoside Rg1 could elevate the cell viability, but it was still lower than the normal level(P < 0.05).(2) Pretreatment with ginsenoside Rg1 increased survivin positive cells, but decreased caspase-3 positive cells and TUNEL positive cells.(3) Neuronal apoptosis was negatively correlated with the ginsenoside Rg1-induced survivin, but positively correlated with the caspase-3. All these findings indicate that ginsenoside Rg1 pretreatment can promote the expression of survivin in PC12 cells after OGD/reperfusion, and moreover, this promotion effect on survivin can further inhibit the expression of caspase-3, thereby suppression apoptosis in PC12 cells, in a dose-dependent manner.
引文
[1]Ahmadvand H,Ahmadi SAY,Sayahi A,et al.Role of Apoptosis in CNS Emphasizing Spinal Cord Injuries:A Commentary.2016:30-31.10.18869/acadpub.irjns.1.4.29.
[2]Karimipour M,Shojaei ZS,Mohajer MM,et al.Pre-Treatment with Metformin in Comparison with Post-Treatment Reduces Cerebral Ischemia Reperfusion Induced Injuries in Rats.Bull Emerg Trauma.2018;6(2):115-121.
[3]Sahin MA,Onan B,Guler A,et al.Cilostazol,a type IIIphosphodiesterase inhibitor,reduces ischemia/reperfusioninduced spinal cord injury.Heart Surg Forum.2011;14(3):E171-E177.
[4]Wilcox CS.Effects of tempol and redox-cycling nitroxides in models of oxidative stress.PharmacolTher.2010;126(2):119-145.
[5]Esposito E,Cuzzocrea S.Antiinflammatory activity of melatonin in central nervous system.CurrNeuropharmacol.2010;8(3):228-242.
[6]Chen WF,Sung CS,Jean YH,et al.Suppressive effects of intrathecal granulocyte colony-stimulating factor on excessive release of excitatory amino acids in the spinal cerebrospinal fluid of rats with cord ischemia:role of glutamate transporters.Neuroscience.2010;165(4):1217-1232.
[7]Zhao H,Alam A,San CY,et al.Molecular mechanisms of brain-derived neurotrophic factor in neuro-protection:Recent developments.Brain Res.2017;1665:1-21.
[8]Fadeel B,Orrenius S.Apoptosis:a basic biological phenomenon with wide-ranging implications in human disease.J Intern Med.2005;258(6):479-517.
[9]Yu Q,Zhou Q,Huang H,et al.Protective effect of etomidate on spinal cord ischemia-reperfusion injury induced by aortic occlusion in rabbits.Ann Vasc Surg.2010;24(2):225-232.
[10]Kolesar D,Kolesarova M,Pavel J,et al.Region-specific sensitivity of the spinal cord to ischemia/reperfusion:the dynamic of changes in catalytic NOS activity.J Physiol Sci.2009;59(2):97-103.
[11]Zhang DX,Zhang LM,Zhao XC,et al.Neuroprotective effects of erythropoietin against sevoflurane-induced neuronal apoptosis in primary rat cortical neurons involving the EPOR-Erk1/2-Nrf2/Bach1 signal pathway.Biomed Pharmacother.2017;87:332-341.
[12]Zipfel GJ,Babcock DJ,Lee JM,et al.Neuronal apoptosis after CNS injury:the roles of glutamate and calcium.J Neurotrauma.2000;17(10):857-869.
[13]Yang P,Ling L,Sun W,et al.Ginsenoside Rg1 inhibits apoptosis by increasing autophagy via the AMPK/mTOR signaling in serum deprivation macrophages.ActabiochimicaetbiophysicaSinica.2018;50(2):144-155.
[14]Meiri KF,Saffell JL,Walsh FS,et al.Neurite outgrowth stimulated by neural cell adhesion molecules requires growth-associated protein-43(GAP-43)function and is associated with GAP-43phosphorylation in growth cones.J Neurosci.1998;18(24):10429-10437.
[15]Greene LA,Tischler AS.Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.ProcNatlAcadSci U S A.1976;73(7):2424-2428.
[16]Vaudry D,Stork PJ,Lazarovici P,et al.Signaling pathways for PC12 cell differentiation:making the right connections.Science.2002;296(5573):1648-1649.
[17]Fan CD,Li Y,Fu X T,et al.Reversal of Beta-Amyloid-Induced Neurotoxicity in PC12 Cells by Curcumin,the Important Role of ROS-Mediated Signaling and ERK Pathway.Cell MolNeurobiol.2017;37(2):211-222.
[18]靳晓飞,张颖,周晓红,等.黄芪甲苷对缺氧缺糖/复氧复糖PC12细胞凋亡的影响[J].中国药理学通报,2016,32(10):1411-1415.
[19]赵培,岳少乾,马妍,等.苏合香对脑微血管内皮细胞氧糖剥夺/复氧损伤的影响[J].中国临床药理学杂志,2016,32(9):847-849.
[20]Globus MY,Busto R,Dietrich WD,et al.Effect of ischemia on the in vivo release of striatal dopamine,glutamate,and gamma-aminobutyric acid studied by intracerebralmicrodialysis.JNeurochem.1988;51(5):1455-1464.
[21]Huang SL,He XJ,Lin L,et al.Neuroprotective effect of ginsenoside Rg1 against spinal cord ischemia and reperfusion injury in rats.Neurochemical Journal.2014;8(3):199-204.
[22]Ambrosini G,Adida C,Altieri DC.A novel anti-apoptosis gene,survivin,expressed in cancer and lymphoma.Nat Med.1997;3(8):917-921.
[23]Chantalat L,Skoufias DA,Kleman JP,et al.Crystal structure of human survivin reveals a bow tie-shaped dimer with two unusual alpha-helical extensions.Mol Cell.2000;6(1):183-189.
[24]Sokolowska J,Urbanska K.Survivin expression in correlation with apoptotic activity in canine lymphomas.Pol J Vet Sci.2017;20(2):329-338.
[25]Dizdar L,Junemann LM,Werner TA,et al.Clinicopathological and functional implications of the inhibitor of apoptosis proteins survivin and XIAP in esophageal cancer.OncolLett.2018;15(3):3779-3789.
[26]Caldas H,Jiang Y,Holloway MP,et al.Survivin splice variants regulate the balance between proliferation and cell death.Oncogene.2005;24(12):1994-2007.
[27]Tamm I,Wang Y,Sausville E,et al.IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas(CD95),Bax,caspases,and anticancer drugs.Cancer Res.1998;58(23):5315-5320.
[28]Eckelman BP,Salvesen GS,Scott FL.Human inhibitor of apoptosis proteins:why XIAP is the black sheep of the family.EMBO Rep.2006;7(10):988-994.
[29]Salvesen GS,Duckett CS.IAP proteins:blocking the road to death's door.Nat Rev Mol Cell Biol.2002;3(6):401-410.
[30]Zumbragel FK,Machtens DA,Curth U,et al.Survivin does not influence the anti-apoptotic action of XIAP on caspase-9.BiochemBiophys Res Commun.2017;482(4):530-535.
[31]Altieri DC.Survivin,cancer networks and pathway-directed drug discovery.Nat Rev Cancer.2008;8(1):61-70.
[32]Kleinberg L,Florenes VA,Silins I,et al.Nuclear expression of survivin is associated with improved survival in metastatic ovarian carcinoma.Cancer.2007;109(2):228-238.
[33]陈大艳,马静萍.不同剂量丁苯酞对大鼠急性脑缺血再灌注损伤后缺氧诱导因子-1α、存活素表达及血管再生的影响[J].中华临床医师杂志(电子版)2017,11(6):957-962.
[34]Baturcam E,Snape N,Yeo T H,et al.Human Metapneumovirus Impairs Apoptosis of Nasal Epithelial Cells in Asthma via HSP70.J Innate Immun.2017;9(1):52-64.
[35]Kang SJ,Wang S,Hara H,et al.Dual role of caspase-11 in mediating activation of caspase-1 and caspase-3 under pathological conditions.J Cell Biol.2000;149(3):613-622.
[36]Zhang Q,Liu J,Chen S,et al.Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress.Apoptosis.2016;21(4):432-442.
[37]Sakurai M,Nagata T,Abe K,et al.Survival and death-promoting events after transient spinal cord ischemia in rabbits:induction of Akt and caspase3 in motor neurons.J ThoracCardiovasc Surg.2003;125(2):370-377.
[2]Karimipour M,Shojaei ZS,Mohajer MM,et al.Pre-Treatment with Metformin in Comparison with Post-Treatment Reduces Cerebral Ischemia Reperfusion Induced Injuries in Rats.Bull Emerg Trauma.2018;6(2):115-121.
[3]Sahin MA,Onan B,Guler A,et al.Cilostazol,a type IIIphosphodiesterase inhibitor,reduces ischemia/reperfusioninduced spinal cord injury.Heart Surg Forum.2011;14(3):E171-E177.
[4]Wilcox CS.Effects of tempol and redox-cycling nitroxides in models of oxidative stress.PharmacolTher.2010;126(2):119-145.
[5]Esposito E,Cuzzocrea S.Antiinflammatory activity of melatonin in central nervous system.CurrNeuropharmacol.2010;8(3):228-242.
[6]Chen WF,Sung CS,Jean YH,et al.Suppressive effects of intrathecal granulocyte colony-stimulating factor on excessive release of excitatory amino acids in the spinal cerebrospinal fluid of rats with cord ischemia:role of glutamate transporters.Neuroscience.2010;165(4):1217-1232.
[7]Zhao H,Alam A,San CY,et al.Molecular mechanisms of brain-derived neurotrophic factor in neuro-protection:Recent developments.Brain Res.2017;1665:1-21.
[8]Fadeel B,Orrenius S.Apoptosis:a basic biological phenomenon with wide-ranging implications in human disease.J Intern Med.2005;258(6):479-517.
[9]Yu Q,Zhou Q,Huang H,et al.Protective effect of etomidate on spinal cord ischemia-reperfusion injury induced by aortic occlusion in rabbits.Ann Vasc Surg.2010;24(2):225-232.
[10]Kolesar D,Kolesarova M,Pavel J,et al.Region-specific sensitivity of the spinal cord to ischemia/reperfusion:the dynamic of changes in catalytic NOS activity.J Physiol Sci.2009;59(2):97-103.
[11]Zhang DX,Zhang LM,Zhao XC,et al.Neuroprotective effects of erythropoietin against sevoflurane-induced neuronal apoptosis in primary rat cortical neurons involving the EPOR-Erk1/2-Nrf2/Bach1 signal pathway.Biomed Pharmacother.2017;87:332-341.
[12]Zipfel GJ,Babcock DJ,Lee JM,et al.Neuronal apoptosis after CNS injury:the roles of glutamate and calcium.J Neurotrauma.2000;17(10):857-869.
[13]Yang P,Ling L,Sun W,et al.Ginsenoside Rg1 inhibits apoptosis by increasing autophagy via the AMPK/mTOR signaling in serum deprivation macrophages.ActabiochimicaetbiophysicaSinica.2018;50(2):144-155.
[14]Meiri KF,Saffell JL,Walsh FS,et al.Neurite outgrowth stimulated by neural cell adhesion molecules requires growth-associated protein-43(GAP-43)function and is associated with GAP-43phosphorylation in growth cones.J Neurosci.1998;18(24):10429-10437.
[15]Greene LA,Tischler AS.Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.ProcNatlAcadSci U S A.1976;73(7):2424-2428.
[16]Vaudry D,Stork PJ,Lazarovici P,et al.Signaling pathways for PC12 cell differentiation:making the right connections.Science.2002;296(5573):1648-1649.
[17]Fan CD,Li Y,Fu X T,et al.Reversal of Beta-Amyloid-Induced Neurotoxicity in PC12 Cells by Curcumin,the Important Role of ROS-Mediated Signaling and ERK Pathway.Cell MolNeurobiol.2017;37(2):211-222.
[18]靳晓飞,张颖,周晓红,等.黄芪甲苷对缺氧缺糖/复氧复糖PC12细胞凋亡的影响[J].中国药理学通报,2016,32(10):1411-1415.
[19]赵培,岳少乾,马妍,等.苏合香对脑微血管内皮细胞氧糖剥夺/复氧损伤的影响[J].中国临床药理学杂志,2016,32(9):847-849.
[20]Globus MY,Busto R,Dietrich WD,et al.Effect of ischemia on the in vivo release of striatal dopamine,glutamate,and gamma-aminobutyric acid studied by intracerebralmicrodialysis.JNeurochem.1988;51(5):1455-1464.
[21]Huang SL,He XJ,Lin L,et al.Neuroprotective effect of ginsenoside Rg1 against spinal cord ischemia and reperfusion injury in rats.Neurochemical Journal.2014;8(3):199-204.
[22]Ambrosini G,Adida C,Altieri DC.A novel anti-apoptosis gene,survivin,expressed in cancer and lymphoma.Nat Med.1997;3(8):917-921.
[23]Chantalat L,Skoufias DA,Kleman JP,et al.Crystal structure of human survivin reveals a bow tie-shaped dimer with two unusual alpha-helical extensions.Mol Cell.2000;6(1):183-189.
[24]Sokolowska J,Urbanska K.Survivin expression in correlation with apoptotic activity in canine lymphomas.Pol J Vet Sci.2017;20(2):329-338.
[25]Dizdar L,Junemann LM,Werner TA,et al.Clinicopathological and functional implications of the inhibitor of apoptosis proteins survivin and XIAP in esophageal cancer.OncolLett.2018;15(3):3779-3789.
[26]Caldas H,Jiang Y,Holloway MP,et al.Survivin splice variants regulate the balance between proliferation and cell death.Oncogene.2005;24(12):1994-2007.
[27]Tamm I,Wang Y,Sausville E,et al.IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas(CD95),Bax,caspases,and anticancer drugs.Cancer Res.1998;58(23):5315-5320.
[28]Eckelman BP,Salvesen GS,Scott FL.Human inhibitor of apoptosis proteins:why XIAP is the black sheep of the family.EMBO Rep.2006;7(10):988-994.
[29]Salvesen GS,Duckett CS.IAP proteins:blocking the road to death's door.Nat Rev Mol Cell Biol.2002;3(6):401-410.
[30]Zumbragel FK,Machtens DA,Curth U,et al.Survivin does not influence the anti-apoptotic action of XIAP on caspase-9.BiochemBiophys Res Commun.2017;482(4):530-535.
[31]Altieri DC.Survivin,cancer networks and pathway-directed drug discovery.Nat Rev Cancer.2008;8(1):61-70.
[32]Kleinberg L,Florenes VA,Silins I,et al.Nuclear expression of survivin is associated with improved survival in metastatic ovarian carcinoma.Cancer.2007;109(2):228-238.
[33]陈大艳,马静萍.不同剂量丁苯酞对大鼠急性脑缺血再灌注损伤后缺氧诱导因子-1α、存活素表达及血管再生的影响[J].中华临床医师杂志(电子版)2017,11(6):957-962.
[34]Baturcam E,Snape N,Yeo T H,et al.Human Metapneumovirus Impairs Apoptosis of Nasal Epithelial Cells in Asthma via HSP70.J Innate Immun.2017;9(1):52-64.
[35]Kang SJ,Wang S,Hara H,et al.Dual role of caspase-11 in mediating activation of caspase-1 and caspase-3 under pathological conditions.J Cell Biol.2000;149(3):613-622.
[36]Zhang Q,Liu J,Chen S,et al.Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress.Apoptosis.2016;21(4):432-442.
[37]Sakurai M,Nagata T,Abe K,et al.Survival and death-promoting events after transient spinal cord ischemia in rabbits:induction of Akt and caspase3 in motor neurons.J ThoracCardiovasc Surg.2003;125(2):370-377.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |