玛咖水提物在PC12细胞对抗化学性缺氧损伤中作用
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摘要
目的探讨玛咖水提物在PC12细胞对抗二氯化钴(CoCl_2)诱导化学性缺氧损伤中的作用及机制。方法应用不同浓度的CoCl_2处理PC12细胞不同时间,建立化学性缺氧损伤模型。应用细胞增殖检验试剂盒(CCK-8)检测细胞存活率;试剂盒法测定细胞培养液中超氧化物歧化酶(SOD)和一氧化氮合酶(NOS)的活性,检测丙二醛、一氧化氮(NO)及乳酸脱氢酶(LDH)的含量;NO荧光探针(DAF-FM DA)染色流式细胞术检测细胞内NO含量;Annexin V-FITC/PI染色检测细胞凋亡率。结果与对照组比较,CoCl_2组PC12细胞存活率明显受到抑制,且呈现一定的浓度与时间依赖性;与对照组比较,300μmol/L CoCl_2组PC12细胞存活率[(51.55±2.71)%]明显降低,细胞培养液中SOD、NOS活性升高,丙二醛、NO和LDH含量增加;与CoCl_2组比较,60、120μg/mL玛咖组PC12细胞存活率[分别为(63.99±3.24)%、(69.95±2.25)%]明显升高,细胞培养液中NOS活性降低,丙二醛、NO和LDH含量降低。与对照组比较,500μmol/L CoCl_2组PC12细胞内NO含量[(61.5±5.7)%]明显升高,细胞凋亡率[(41.33±3.83)%]明显增加;与500μmol/L CoCl_2组比较,60、120μg/mL玛咖组细胞内NO含量[分别为(36.5±4.7)%、(33.4±3.8)%]明显降低,细胞凋亡率[分别为(34.87±2.50)%、(31.33±3.44)%]明显下降。结论玛咖水提物对CoCl_2诱导的化学性缺氧损伤具有一定拮抗作用,其机制可能与抑制NO的产生、减轻氧化应激反应,抑制细胞凋亡有关。
Objective To explore the effect of maca water extract on cobalt chloride(CoCl_2)-induced hypoxia injury in PC12 cells. Methods PC12 cells were exposed to CoCl_2 at different dose and for different time duration to set up the chemical hypoxia injury model. Cell viability was determined with cell counter kit-8(CCK-8). The activity of superoxide dismutase(SOD) and nitric oxide synthase(NOS), the quantity of malondialdehyde(MDA), nitric oxide(NO), and lactate dehydrogenase(LDH) in culture medium were measured with kit assay. The intracellular NO was detected using a fluorescent probe(2 ′,7 ′-dichlorodihydrofluorescein diacetate, DCFH-DA). The cell apoptotic rate was determined with annexin V-FITC/PI staining. Results Compared with that of the control cells, the viability of PC12 cells exposed to CoCl_2 was inhibited significantly in a dose-and time-dependent manner. Significantly decreased cell viability rate(51.55% ±2.71%) and increased of SOD and NOS and content of MDA, NO, and LDH in culture medium were observed in PC12 cells exposed to 300 μmol/L CoCl_2 for 24 hours in comparison with those of the control cells. For the PC12 cells treated with maca water extraction of 60 and 120 μg/ml, the viability rate increased obviously(63.99% ± 3.24% and 69.95% ± 2.25%) and the NOS activity and content of MDA, NO, and LDH in culture medium decreased compared to the CoCl_2 exposed PC12 cells.Obviously increased intracellular NO content(61.5% ± 5.7%) and cell apoptotic rate(41.33% ± 3.83%) were observed in the PC12 cells treated with 500 μmol/L CoCl_2 for 24 hours compared to the control cells. Obviously decreased intracellular NO content(36.50% ± 4.70% and 33.40% ± 3.80%) and cell apoptotic rate(34.87 ± 2.50% and 31.33% ± 3.44%) were observed in the PC12 cells treated with maca water extraction of 60 and 120 μg/ml compared to those in the cells exposed to 500 μmol/L CoCl_2. Conclusion Maca water extract has protective effect on cobalt chloride-induced hypoxia injury by inhibiting NO generation, oxidative stress and cell apoptosis in PC12 cells.
Objective To explore the effect of maca water extract on cobalt chloride(CoCl_2)-induced hypoxia injury in PC12 cells. Methods PC12 cells were exposed to CoCl_2 at different dose and for different time duration to set up the chemical hypoxia injury model. Cell viability was determined with cell counter kit-8(CCK-8). The activity of superoxide dismutase(SOD) and nitric oxide synthase(NOS), the quantity of malondialdehyde(MDA), nitric oxide(NO), and lactate dehydrogenase(LDH) in culture medium were measured with kit assay. The intracellular NO was detected using a fluorescent probe(2 ′,7 ′-dichlorodihydrofluorescein diacetate, DCFH-DA). The cell apoptotic rate was determined with annexin V-FITC/PI staining. Results Compared with that of the control cells, the viability of PC12 cells exposed to CoCl_2 was inhibited significantly in a dose-and time-dependent manner. Significantly decreased cell viability rate(51.55% ±2.71%) and increased of SOD and NOS and content of MDA, NO, and LDH in culture medium were observed in PC12 cells exposed to 300 μmol/L CoCl_2 for 24 hours in comparison with those of the control cells. For the PC12 cells treated with maca water extraction of 60 and 120 μg/ml, the viability rate increased obviously(63.99% ± 3.24% and 69.95% ± 2.25%) and the NOS activity and content of MDA, NO, and LDH in culture medium decreased compared to the CoCl_2 exposed PC12 cells.Obviously increased intracellular NO content(61.5% ± 5.7%) and cell apoptotic rate(41.33% ± 3.83%) were observed in the PC12 cells treated with 500 μmol/L CoCl_2 for 24 hours compared to the control cells. Obviously decreased intracellular NO content(36.50% ± 4.70% and 33.40% ± 3.80%) and cell apoptotic rate(34.87 ± 2.50% and 31.33% ± 3.44%) were observed in the PC12 cells treated with maca water extraction of 60 and 120 μg/ml compared to those in the cells exposed to 500 μmol/L CoCl_2. Conclusion Maca water extract has protective effect on cobalt chloride-induced hypoxia injury by inhibiting NO generation, oxidative stress and cell apoptosis in PC12 cells.
引文
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[15]廖新学,阮经文,兰爱平,等.p38 MAPK-iNOS-NO通路介导化学性缺氧对PC12细胞的损伤作用[J].中国病理生理杂志,2010,26(12):2410-2414.
[2]Lee MS,Shin BC,Yang EJ,et al.Maca(Lepidium meyenii)for treatment of menopausal symptoms:a systematic review[J].Maturitas,2011,70(3):227-233.
[3]Gonzales GF,Cordova A,Gonzales C,et al.Lepidium meyenii(Maca)improved semen parameters in adult men[J].Asian Journal of Andrology,2001,3(4):301-303.
[4]张静,李慧,周雯,等.玛咖粉对小鼠的抗疲劳作用及其机制研究[J].卫生研究,2013,42(6):1046-1049.
[5]Vecera R,Orolin J,SkottováN,et al.The influence of Maca(Lepidium meyenii)on antioxidant status,lipid and glucose metabolism in rat[J].Plant Foods for Hum Nutr,2007,62(2):59-63.
[6]Liu B,Yang P,Ye Y,et al.Role of ROS in the protective effect of silibinin on sodium nitroprusside-induced apoptosis in rat pheochromocytoma PC12 cells[J].Free Radic Res,2011,45(7):835-847.
[7]曾季平,王立祥,胡晓燕,等.氯化钴诱导PC12细胞凋亡的分子机制[J].毒理学杂志,2005,19(3):193-195.
[8]曾克武,王学美,富宏,等.金丝桃苷对氯化钴所致的PC12细胞损伤模型的保护作用和机制研究[J].中国中药杂志,2011,36(17):2409-2412.
[9]姚娟,马慧萍,杨燕,等.缺氧环境对PC12细胞损伤及HIF-1m RNA表达水平的影响[J].中国药理学通报,2011,27(2):162-166.
[10]Petrik MS,Wilson JM,Grant SC,et al.Magnetic resonance microscopy and immunohistochemistry of the CNS of the mutant SOD murine model of ALS reveals widespread neural deficits[J].Neuromolecular Med,2007,9(3):216-229.
[11]Kowalczuk K,Stryjecka-Zimmer M.The influence of oxidative stress on the level of malondialdehyde(MDA)in different areas of the rabbit brain[J].Annales Universitatis Mariae CurieSklodowska,2002,57(2):160-164.
[12]Ferdinandy P,Danial H,Ambrus I,et al.Peroxynitrite is a major contributor to cytokine-induced myocardial contractile failure[J].Circ Res,2000,87(3):241-247.
[13]Mitra S,Goyal T,Mehta JL.Oxidized LDL,LOX-1 and aterosclerosis[J].Cardiovascular Drugs and Therapy,2011,25(5):419-429.
[14]Gielis JF,Lin JY,Wingler K,et al.Pathogenetic role of eNOS uncoupling in cardiopulmonary disorders[J].Free Radic Biol Med,2011,50(7):765-776.
[15]廖新学,阮经文,兰爱平,等.p38 MAPK-iNOS-NO通路介导化学性缺氧对PC12细胞的损伤作用[J].中国病理生理杂志,2010,26(12):2410-2414.