7,8-二羟基-4-甲基香豆素对谷氨酸诱导的PC12细胞损伤的神经保护作用
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摘要
目的探讨7,8-二羟基-4-甲基香豆素(Dhmc)对谷氨酸诱导的PC12细胞损伤的神经保护作用。方法采用谷氨酸诱导PC12细胞损伤凋亡模型,给予Dhmc处理后,MTT法检测细胞增殖;化学检测法测定乳酸脱氢酶(LDH)漏出量、细胞上清液中丙二醛(MDA)含量及超氧化物歧化酶(SOD)水平;采用Annexin-V-FITC双染法检测细胞凋亡率;采用荧光法检测活性氧(ROS)的生成;采用流式细胞仪检测线粒体膜电位(MMP);Western blot法检测Bcl-2、Bax、Cyt C、cleaved caspase-3蛋白表达。结果 Dhmc可显著降低由谷氨酸诱导的细胞损伤,提高了细胞存活率,减少LDH漏出量,降低MDA含量,增加SOD活性,抑制ROS的生成,维持线粒体膜电位水平,下调Bcl-2/Bax比率,减少Cyt C的释放,降低Caspase-3活性。结论 Dhmc具有减轻由谷氨酸诱导的PC12细胞损伤的作用,其机制可能与抗氧化应激和减轻线粒体损伤有关。
Objective To determine the neuroprotective effect of 7, 8-dihydroxy-4-methylo-umarin against glutamate induced PC12 cell injury. Methods PC12 cells were treated with glutamate to establish the cell model. After Dhmc treatment, MTT assay was used to analyze the PC12 cell viability. The contents of LDH, MDA and the level of SOD in the cells were determined by kit respectively. Cell apoptosis detection was performed with an Annexin-V-FITC Apoptosis Detection Kit. The production of ROS was determined by fluorescence method. The mitochondrial membrane potential(MMP) was measured by flow cytometer. Western blot was carried out to detect the protein expression. Results Dhmc significantly decreased the contents of LDH and MDA, enhanced the activity of SOD induced by glutamate. In addition, Dhmc treatment decreased the intracellular ROS generation and maintained the level of MMP. Finally, Dhmc treatment increased the ratio of Bcl-2/Bax, reduced the release of Cyt C, decreased the caspase-3 activity. Conclusion Dhmc can significantly ameliorate glutamate induced PC12 cell injury. The mechanism may be partly through anti-oxidative stress and alleviating mitochondrial damage.
Objective To determine the neuroprotective effect of 7, 8-dihydroxy-4-methylo-umarin against glutamate induced PC12 cell injury. Methods PC12 cells were treated with glutamate to establish the cell model. After Dhmc treatment, MTT assay was used to analyze the PC12 cell viability. The contents of LDH, MDA and the level of SOD in the cells were determined by kit respectively. Cell apoptosis detection was performed with an Annexin-V-FITC Apoptosis Detection Kit. The production of ROS was determined by fluorescence method. The mitochondrial membrane potential(MMP) was measured by flow cytometer. Western blot was carried out to detect the protein expression. Results Dhmc significantly decreased the contents of LDH and MDA, enhanced the activity of SOD induced by glutamate. In addition, Dhmc treatment decreased the intracellular ROS generation and maintained the level of MMP. Finally, Dhmc treatment increased the ratio of Bcl-2/Bax, reduced the release of Cyt C, decreased the caspase-3 activity. Conclusion Dhmc can significantly ameliorate glutamate induced PC12 cell injury. The mechanism may be partly through anti-oxidative stress and alleviating mitochondrial damage.
引文
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[2]张雨薇,刘宏,张博,等.益气活血方药对缺血性脑损伤保护作用的实验研究进展[J].中国医学装备,2016,13(1):122-125.
[3]Wang S,Xing Z,Vosler PS,et al.Cellular NAD replenishment confers marked neuroprotection against ischemic cell death:role of enhanced DNA repair[J].Stroke,2008,39(9):2587-2595.
[4]Baines CP.How and when do myocytes die during ischemia and reperfusion:the late phase[J].J Cardiovasc Pharmacol Ther,2011,16(3-4):239-243.
[5]曹丽霞,杨玉梅.缺血性脑损伤神经细胞凋亡及其相关基因的表达[J].包头医学院学报,2016,32(5):170-172
[6]Radak D,Resanovic I,Isenovic ER.Link between oxidative stress and acute brain ischemia[J].Angiology,2014,65(8):667-676.
[7]Hussein BHM.Spectroscopic studies of 7,8-dihydroxy-4-methylcoumarin and its interaction with bovine serum albumin[J].J Lumin,2011,131(5):900-908.
[8]Todorov L,Traykova M,Traykov T.In vitro evaluation of the antioxidant effect of yohimbine[J].Toxicol Lett,2005,158:S182.
[9]田元祥,杨牧祥.血管性痴呆学习记忆障碍小鼠行为学变化及醒脑启智胶囊的影响[J].中国病理生理杂志,2004,20(6):1104-1106.
[10]李峥,高项羽,刘喆,等.丙泊酚通过MAPK/ERK信号通路对谷氨酸诱导的神经PC12细胞损伤的抑制作用[J].中国药房,2016,27(1):61-63.
[11]Jiang G,Wu H,Hu Y,et a1.Gastrodin inhibits glutamate-induced apoptosis of PC 12 cells via inhibition of Ca MKII/ASK-I/p38 MAPK/p53 signaling cascade[J].Cell Mol Neurobiol,2014,34(4):591.
[12]彭晓明,高莉,甘萍,等.类叶升麻苷抗H2O2诱导的PC12细胞氧化损伤的保护作用[J].中药药理与临床,2013,29(3):35-38.
[13]Hyman BT,Yuan J.Apoptotic and non-apoptotic roles of caspases in neuronal physiology and pathophysiology[J].Nat Rev Neurosci,2012,13(6):395-406.