匹诺塞林对缺糖缺氧/复糖复氧诱导的SH-SY5Y细胞损伤的保护作用
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摘要
目的:研究匹诺塞林对缺糖缺氧/复糖复氧(OGD/R)诱导SH-SY5Y细胞损伤的保护作用。方法:SH-SY5Y细胞系制备OGD/R损伤模型,缺糖缺氧2 h,复糖复氧24 h。MTT法检测细胞存活率,通过LDH释放率考察细胞活力,JC-1荧光探针检测线粒体膜电位,应用DCFH-DA和MitoSOX分别检测细胞ROS及线粒体超氧化物水平,Western Blot法测定cytochrome c,Bcl-2,Bax,cleaved caspase 3蛋白表达水平,免疫荧光法测定HO-1和Nrf2的表达。结果:OGD/R导致SH-SY5Y细胞存活率显著降低。匹诺塞林(1和10μmol·L-1)能显著增加SH-SY5Y细胞存活率,降低细胞凋亡率。同时,匹诺塞林能够减少OGD/R诱导ROS的释放,减缓线粒体膜电位去极化,促进HO-1蛋白表达及Nrf2核转位,降低OGD/R损伤所致cytochrome c,Bax,cleaved caspase 3的升高,抑制Bcl-2的降低。结论:匹诺塞林能够抑制OGD/R损伤引起的SH-SY5Y细胞氧化应激与线粒体凋亡。
Objective: To study the protective effect of pinocembrin on SH-SY5 Y cells subjected to oxygen glucose deprivation/reperfusion(OGD/R) injury. Methods: The OGD/R model was established via 2 h of oxygen/glucose deprivation followed by 24 h of reperfusion in SH-SY5 Y cells. The cell viability was measured by MTT assay,and investigated through the release rate of LDH. Mitochondrial membrane potential was detected by JC-1 fluorescence probe. Cellular ROS levels and mitochondria superoxide levels were detected by the DCFH-DA and Mito SOX probes,respectively. Protein expression levels of cytochrome c,Bcl-2,Bax,and cleaved caspase 3 were detected by Western blot method. And the expression of HO-1 and the nuclear translocation of Nrf2 were determined by immunofluorescence assay. Results: OGD/R resulted in significant decrease of cell viability,and pinocembrin could significantly increase the viability of SH-SY5 Y cells. Meanwhile,pinocembrin could reduce the release of ROS induced by OGD/R,and slow down the mitochondrial membrane potential depolarization. Further study showed that it promoted HO-1 expression and Nrf2 nuclear translocation,reduced the increase of cytochrome c,Bax,cleaved caspase 3,and inhibited the decrease of Bcl-2. Conclusion: Pinocembrin can inhibit oxidative stress and cell apoptosis via ROS-mitochondria pathway in SH-SY5 Y cells damaged by OGD/R.
Objective: To study the protective effect of pinocembrin on SH-SY5 Y cells subjected to oxygen glucose deprivation/reperfusion(OGD/R) injury. Methods: The OGD/R model was established via 2 h of oxygen/glucose deprivation followed by 24 h of reperfusion in SH-SY5 Y cells. The cell viability was measured by MTT assay,and investigated through the release rate of LDH. Mitochondrial membrane potential was detected by JC-1 fluorescence probe. Cellular ROS levels and mitochondria superoxide levels were detected by the DCFH-DA and Mito SOX probes,respectively. Protein expression levels of cytochrome c,Bcl-2,Bax,and cleaved caspase 3 were detected by Western blot method. And the expression of HO-1 and the nuclear translocation of Nrf2 were determined by immunofluorescence assay. Results: OGD/R resulted in significant decrease of cell viability,and pinocembrin could significantly increase the viability of SH-SY5 Y cells. Meanwhile,pinocembrin could reduce the release of ROS induced by OGD/R,and slow down the mitochondrial membrane potential depolarization. Further study showed that it promoted HO-1 expression and Nrf2 nuclear translocation,reduced the increase of cytochrome c,Bax,cleaved caspase 3,and inhibited the decrease of Bcl-2. Conclusion: Pinocembrin can inhibit oxidative stress and cell apoptosis via ROS-mitochondria pathway in SH-SY5 Y cells damaged by OGD/R.
引文
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[3] JIANG YF,LIU ZQ,CUI W,et al. Antioxidant effect of salvianolic acid B on hippocampal CA1 neurons in mice with cerebral ischemia and reperfusion injury[J]. Chin J Integr Med,2015,21(7):516-522.
[4]陈红,吴俊杰,薛强,等.以线粒体为靶点抗缺血性脑卒中中药成分研究进展[J].中国新药杂志,2016,25(11):1236-1240.
[5] GAO M,ZHU SY,TAN CB,et al. Pinocembrin protects the neurovascular unit by reducing inflammation and extracellular proteolysis in MCAO rats[J]. J Asian Nat Prod Res,2010,12(5):407-418.
[6] SHI LL,CHEN BN,GAO M,et al. The characteristics of therapeutic effect of pinocembrin in transient global brain ischemia/reperfusion rats[J]. Life Sci,2011,88(11-12):521-528.
[7] WANG SB,PANG XB,GAO M,et al. Pinocembrin protects rats against cerebral ischemic damage through soluble epoxide hydrolase and epoxyeicosatrienoic acids[J]. Chin J Nat Med,2013,11(3):207-213.
[8] MENG F,WANG Y,LIU R,et al. Pinocembrin alleviates memory impairment in transient global cerebral ischemic rats[J]. Exp Ther Med,2014,8(4):1285-1290.
[9] WU CX,LIU R,GAO M,et al. Pinocembrin protects brain against ischemia/reperfusion injury by attenuating endoplasmic reticulum stress induced apoptosis[J]. Neurosci Lett,2013,546(26):57-62.
[10] ZHAO G,ZHANG W,LI L,et al. Pinocembrin protects the brain against ischemia-reperfusion injury and reverses the autophagy dysfunction in the penumbra area[J]. Molecules,2014,19(10):15786-15798.
[11] GAO M,LIU R,ZHU SY,et al. Acute neurovascular unit protective action of pinocembrin against permanent cerebral ischemia in rats[J]. J Asian Na Prod Res,2008,10(5-6):551-558.
[12] MENG F,LIU R,GAO M,et al. Pinocembrin attenuates bloodbrain barrier injury induced by global cerebral ischemia-reperfusion in rats[J]. Brain Res,2011,1391(3):93-101.
[13] WANG Y,ZHANG J,HAN M,et al. SMND-309 promotes neuron survival through the activation of the PI3K/Akt/CREB-signalling pathway[J]. Pharm Biol,2016,54(10):1982-1990.
[14]张雯,宋俊科,杜冠华.缺血再灌注损伤与细胞凋亡信号转导通路[J].中国药学杂志,2015,50(7):565-569.
[15]时丽丽,强桂芬,高梅,等.匹诺塞林对脑线粒体呼吸功能的促进作用[J].药学学报,2011,46(6):642-649.
[16]光红梅,高梅,朱深银,等.匹诺塞林对急性局灶性脑缺血大鼠脑线粒体功能的影响[J].中国药理学通报,2012,28(1):24-29.
[17]张雯,宋俊科,闫蓉,等.丹酚酸A通过Nrf2/HO-1途径减轻大鼠脑缺血再灌注损伤[J].药学学报,2016,51(11):1717-1723.
[18] ZHANG X,DU L,ZHANG W,et al. Therapeutic effects of baicalein on rotenone-induced Parkinson's disease through protecting mitochondrial function and biogenesis[J]. Sci Rep,2017,7(1):9968.