紫草素通过PI3K/Akt通路抑制氧糖剥夺诱导的大鼠原代皮层神经元凋亡
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摘要
目的:探讨紫草素对氧糖剥夺(OGD)损伤模型中大鼠原代皮层神经元的作用及机制。方法:用不同浓度(0. 02、0. 2、2和20μmol/L)紫草素对大鼠原代皮层神经元经进行预处理,再经OGD损伤处理,用乳酸脱氢酶(LDH)释放法和荧光素二乙酸酯/碘化丙啶(FDA/PI)双染法分别检测神经元活性和凋亡情况,选择最适紫草素浓度。然后,在加入紫草素之前提前加入LY294002(PI3K/Akt信号通路抑制剂,1μmol/L),用Wesern blot法检测神经元p-Akt(Ser473)水平变化,用LDH法和FDA/PI双染法检测神经元活性和凋亡率变化。结果:0. 2、2及20μmol/L的紫草素可显著提高神经元存活率(P <0. 05),同时还可使神经元内p-Akt(Ser473)水平显著升高(P <0. 05); LY294002可显著阻断紫草素对神经元p-Akt(Ser473)水平和凋亡率的影响(P <0. 05)。结论:紫草素可通过激活PI3K/Akt通路来减少OGD诱导的大鼠原代皮层神经元凋亡。
AIM: To explore the effect of shikonin on rat primary cortical neurons in oxygen-glucose deprivation( OGD)-induced injury model. METHODS: The neurons were pretreated with shikonin at different concentrations( 0. 02,0. 2,2 and 20 μmol/L) followed by treatment with OGD. Lactate dehydrogenase( LDH) release assay and fluorescein diacetate/propidium iodide( FDA/PI) double staining were used to detect neuronal viability and apoptosis,and then the optimal concentration of shikonin was determined. LY294002( PI3K/Akt signaling pathway inhibitor,1 μmol/L)was added before the addition of shikonin,and the protein level of p-Akt( Ser473) in the neurons was determined by Western blot. LDH release assay and FDA/PI double staining were also used to detect neuronal viability and apoptosis. RESULTS: A certain concentration( 0. 2 ～ 20 μmol/L) of shikonin increased the viability of impaired neurons( P < 0. 05)and the protein level of p-Akt( Ser473) in the neurons( P < 0. 05). The effect of shikonin on neuronal p-Akt( Ser473)levels and the cell death were blocked by LY294002( P < 0. 05). CONCLUSION: A certain concentration of shikonin reduces OGD-induced apoptosis of rat primary cortical neurons by activating PI3K/Akt signaling pathway.
AIM: To explore the effect of shikonin on rat primary cortical neurons in oxygen-glucose deprivation( OGD)-induced injury model. METHODS: The neurons were pretreated with shikonin at different concentrations( 0. 02,0. 2,2 and 20 μmol/L) followed by treatment with OGD. Lactate dehydrogenase( LDH) release assay and fluorescein diacetate/propidium iodide( FDA/PI) double staining were used to detect neuronal viability and apoptosis,and then the optimal concentration of shikonin was determined. LY294002( PI3K/Akt signaling pathway inhibitor,1 μmol/L)was added before the addition of shikonin,and the protein level of p-Akt( Ser473) in the neurons was determined by Western blot. LDH release assay and FDA/PI double staining were also used to detect neuronal viability and apoptosis. RESULTS: A certain concentration( 0. 2 ～ 20 μmol/L) of shikonin increased the viability of impaired neurons( P < 0. 05)and the protein level of p-Akt( Ser473) in the neurons( P < 0. 05). The effect of shikonin on neuronal p-Akt( Ser473)levels and the cell death were blocked by LY294002( P < 0. 05). CONCLUSION: A certain concentration of shikonin reduces OGD-induced apoptosis of rat primary cortical neurons by activating PI3K/Akt signaling pathway.
引文
[1]Yang JL,Mukda S,Chen SD.Diverse roles of mitochondria in ischemic stroke[J].Redox Biol,2018,16:263-275.
[2]Costantino I,Josef A.The immunology of stroke:from mechanisms to translation[J].Nat Med,2011,17(7):796-808.
[3]Kourounakis AP,Assimopoulou AN,Papageorgiou VP,et al.Alkannin and shikonin:effect on free radical processes and on inflammation-a preliminary pharmacochemical investigation[J].Arch Pharm,2002,335(6):262-266.
[4]Papageorgiou VP,Assimopoulou AN,Ballis AC.Alkannins and shikonins:a new class of wound healing agents[J].Curr Med Chem,2008,15(30):3248-3267.
[5]Esmaeilzadeh E,Gardaneh M,Gharib E,et al.Shikonin protects dopaminergic cell line PC12 against 6-hydroxydopamine-mediated neurotoxicity via both glutathione-dependent and independent pathways and by inhibiting apoptosis[J].Neurochem Res,2013,38(8):1590-1604.
[6]Yu JS,Cui W.Proliferation,survival and metabolism:the role of PI3K/Akt/m TOR signalling in pluripotency and cell fate determination[J].Development,2016,143(17):3050-3060.
[7]Kitagishi Y,Matsuda S.Redox regulation of tumor suppressor PTEN in cancer and aging(review)[J].Int J Mol Med,2013,31(3):511-515.
[8]Martelli AM,Faenza I,Billi AM,et al.Intranuclear 3’-phosphoinositide metabolism and Akt signaling:new mechanisms for tumorigenesis and protection against apoptosis?[J].Cell Signal,2006,18(8):1101-1107.
[9]梁俊清,徐海波,陈小娟,等.通心络通过PI3K/Akt/HIF信号通路改善血管内皮细胞缺氧损伤[J].中国病理生理杂志,2012,28(5):846-851.
[10]Chang N,El-Hayek YH,Gomez E,et al.Phosphatase PTEN in neuronal injury and brain disorders[J].Trends Neurosci,2007,30(11):581-586.
[11]Rommel C,Camps M,Ji H.PI3Kδand PI3Kγ:partners in crimein inflammation in rheumatoid arthritis and beyond?[J].Nat Rev Immunol,2007,7(3):191-201.
[12]丁煌,李静娴,杨筱倩,等.黄芪甲苷与人参皂苷Rg1配伍对PC12细胞氧糖剥夺复糖复氧后细胞自噬和PI3K信号通路的影响[J].中国病理生理杂志,2016,32(11):2003-2009.
[13]Horwood JM,Dufour F,Laroche S,et al.Signalling mechanisms mediated by the phosphoinositide 3-kinase/Akt cascade in synaptic plasticity and memory in the rat[J].Eur J Neurosci,2006,23(12):3375-3384.
[14]Noshita N,Lewén A,Sugawara T,et al.Evidence of phosphorylation of Akt and neuronal survival after transient focal cerebral ischemia in mice[J].J Cereb Blood Flow Metab,2001,21(12):1442-1450.
[15]Liu R,Tang JC,Pan MX,et al.ERK 1/2 activation mediates the neuroprotective effect of bp V(pic)in focal cerebral ischemia-reperfusion injury[J].Neurochem Res,2018,43(7):1424-1438.
[16]Zhang ZF,Chen J,Han X,et al.Bisperoxovandium(pyridin-2-squaramide)targets both PTEN and ERK1/2to confer neuroprotection[J].Br J Pharmacol,2017,174(8):641-656.
[17]Wang S,Zhu Y,Qiu R.Shikonin protects H9c2 cardiomyocytes against hypoxia/reoxygenation injury through activation of PI3K/Akt signaling pathway[J].Biomed Pharmacother,2018,104:712-717.
[18]Liu T,Zhang Q,Mo W,et al.The protective effects of shikonin on hepatic ischemia/reperfusion injury are mediated by the activation of the PI3K/Akt pathway[J].Sci Rep,2017,7:44785.
[19]Zhong C,Chen Y,Tao B,et al.Lim and SH3 protein 1regulates cell growth and chemosensitivity of human glioblastoma via the PI3K/AKT pathway[J].BMC Cancer,2018,18:722.
[2]Costantino I,Josef A.The immunology of stroke:from mechanisms to translation[J].Nat Med,2011,17(7):796-808.
[3]Kourounakis AP,Assimopoulou AN,Papageorgiou VP,et al.Alkannin and shikonin:effect on free radical processes and on inflammation-a preliminary pharmacochemical investigation[J].Arch Pharm,2002,335(6):262-266.
[4]Papageorgiou VP,Assimopoulou AN,Ballis AC.Alkannins and shikonins:a new class of wound healing agents[J].Curr Med Chem,2008,15(30):3248-3267.
[5]Esmaeilzadeh E,Gardaneh M,Gharib E,et al.Shikonin protects dopaminergic cell line PC12 against 6-hydroxydopamine-mediated neurotoxicity via both glutathione-dependent and independent pathways and by inhibiting apoptosis[J].Neurochem Res,2013,38(8):1590-1604.
[6]Yu JS,Cui W.Proliferation,survival and metabolism:the role of PI3K/Akt/m TOR signalling in pluripotency and cell fate determination[J].Development,2016,143(17):3050-3060.
[7]Kitagishi Y,Matsuda S.Redox regulation of tumor suppressor PTEN in cancer and aging(review)[J].Int J Mol Med,2013,31(3):511-515.
[8]Martelli AM,Faenza I,Billi AM,et al.Intranuclear 3’-phosphoinositide metabolism and Akt signaling:new mechanisms for tumorigenesis and protection against apoptosis?[J].Cell Signal,2006,18(8):1101-1107.
[9]梁俊清,徐海波,陈小娟,等.通心络通过PI3K/Akt/HIF信号通路改善血管内皮细胞缺氧损伤[J].中国病理生理杂志,2012,28(5):846-851.
[10]Chang N,El-Hayek YH,Gomez E,et al.Phosphatase PTEN in neuronal injury and brain disorders[J].Trends Neurosci,2007,30(11):581-586.
[11]Rommel C,Camps M,Ji H.PI3Kδand PI3Kγ:partners in crimein inflammation in rheumatoid arthritis and beyond?[J].Nat Rev Immunol,2007,7(3):191-201.
[12]丁煌,李静娴,杨筱倩,等.黄芪甲苷与人参皂苷Rg1配伍对PC12细胞氧糖剥夺复糖复氧后细胞自噬和PI3K信号通路的影响[J].中国病理生理杂志,2016,32(11):2003-2009.
[13]Horwood JM,Dufour F,Laroche S,et al.Signalling mechanisms mediated by the phosphoinositide 3-kinase/Akt cascade in synaptic plasticity and memory in the rat[J].Eur J Neurosci,2006,23(12):3375-3384.
[14]Noshita N,Lewén A,Sugawara T,et al.Evidence of phosphorylation of Akt and neuronal survival after transient focal cerebral ischemia in mice[J].J Cereb Blood Flow Metab,2001,21(12):1442-1450.
[15]Liu R,Tang JC,Pan MX,et al.ERK 1/2 activation mediates the neuroprotective effect of bp V(pic)in focal cerebral ischemia-reperfusion injury[J].Neurochem Res,2018,43(7):1424-1438.
[16]Zhang ZF,Chen J,Han X,et al.Bisperoxovandium(pyridin-2-squaramide)targets both PTEN and ERK1/2to confer neuroprotection[J].Br J Pharmacol,2017,174(8):641-656.
[17]Wang S,Zhu Y,Qiu R.Shikonin protects H9c2 cardiomyocytes against hypoxia/reoxygenation injury through activation of PI3K/Akt signaling pathway[J].Biomed Pharmacother,2018,104:712-717.
[18]Liu T,Zhang Q,Mo W,et al.The protective effects of shikonin on hepatic ischemia/reperfusion injury are mediated by the activation of the PI3K/Akt pathway[J].Sci Rep,2017,7:44785.
[19]Zhong C,Chen Y,Tao B,et al.Lim and SH3 protein 1regulates cell growth and chemosensitivity of human glioblastoma via the PI3K/AKT pathway[J].BMC Cancer,2018,18:722.