消栓肠溶胶囊对缺氧缺糖再灌注损伤PC12细胞的保护作用
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摘要
目的:观察消栓肠溶胶囊对缺氧缺糖再灌注损伤肾上腺嗜铬细胞瘤细胞(PC12细胞)的作用。方法:以PC12细胞缺氧缺糖再灌注损伤模拟脑缺血再灌注损伤,研究消栓肠溶胶囊在0. 01～10. 00 mg/L浓度范围内对其的作用,采用噻唑蓝法(MTT法)检测PC12细胞的存活率;乳酸脱氢酶(LDH)法检测PC12细胞损伤程度;酶联免疫(ELISA)法检测基质金属蛋白酶-9(MMP-9)和凋亡相关因子P53、B细胞淋巴瘤/白血病-2(B-cell lymphoma/leukemia-2,BCL-2)、BCL-2关联X蛋白(bcl2-associated X protein,Bax)和半胱氨酸蛋白水解酶(Caspase-3)的水平;逆转录-聚合酶链反应(RT-PCR)法检测p53 mRNA、Bax mRNA和caspase-3 mRNA的表达;分光光度法检测超氧化歧化酶(SOD)和丙二醛(MDA)的水平,以及采用荧光酶标仪检测活性氧(ROS)的表达。结果:与模型组比较,消栓肠溶胶囊各剂量组干预后,缺氧缺糖再灌注诱导的PC12细胞的存活率显著增加(P <0. 05),细胞外LDH比例显著减少(P <0. 05),MMP-9、P53、Bax、Caspase-3、MDA和ROS表达都显著减少(P <0. 05),而BCL-2表达和SOD活性都显著增加(P <0. 05)。结论:消栓肠溶胶囊对缺氧缺糖再灌注诱导的PC12细胞损伤具有明显保护作用,能减少细胞凋亡相关因子表达,其机制可能与抑制MMP-9表达和减轻氧化应激损伤有关。
Objective: To observe the effects of Xiaoshuan Entric Capsule on adrenal pheochromocytoma cells( PC12 cells) injury induced by hypoxia and glucose deprivation. Methods: The effects of Xiaoshuan Entric Capsule in the concentration range of 0. 01 ～ 10. 00 mg/L on cerebral ischemia-reperfusion injury induced by hypoxia/hypoglycemia injury in PC12 cells,the survival rate of PC12 cells was detected by MTT assay,the injury degree of PC12 cells was detected by lactate dehydrogenase( LDH),the expressions of matrix metalloproteinase-9( MMP-9) and apoptosis factors( P53,BCL-2,Bax and Caspase-3) were detected by ELISA,the expressions of p53,Bax,caspase-3 mRNA were detected by RT-PCR,the superoxide dismutase( SOD) and malondialdehyde( MDA),and the expression of reactive oxygen species( ROS) were detected by microplate reader. Results: Compared with the model group,in the different doses groups of Xiaoshuan Enteric Capsule,the survival rate of PC12 cells induced by hypoxia/glucose deprivation was significantly increased( P < 0. 05),the ratio of extracellular LDH was significantly decreased( P < 0. 05),while the expressions of MMP-9,P53,Bax,Caspase-3, MDA and ROS were significantly decreased( P < 0. 05),while the expressions of BCL-2:and SOD were significantly increased( P < 0. 05). Conclusion: Xiaoshuan Enteric Capsule has obvious protective effect on PC12 cell injury induced by hypoxia and glucose deprivation,which can reduce the expression of apoptosis-related factors,which may be related to the inhibition of MMP-9 expression and the reduction of oxidative stress.
Objective: To observe the effects of Xiaoshuan Entric Capsule on adrenal pheochromocytoma cells( PC12 cells) injury induced by hypoxia and glucose deprivation. Methods: The effects of Xiaoshuan Entric Capsule in the concentration range of 0. 01 ～ 10. 00 mg/L on cerebral ischemia-reperfusion injury induced by hypoxia/hypoglycemia injury in PC12 cells,the survival rate of PC12 cells was detected by MTT assay,the injury degree of PC12 cells was detected by lactate dehydrogenase( LDH),the expressions of matrix metalloproteinase-9( MMP-9) and apoptosis factors( P53,BCL-2,Bax and Caspase-3) were detected by ELISA,the expressions of p53,Bax,caspase-3 mRNA were detected by RT-PCR,the superoxide dismutase( SOD) and malondialdehyde( MDA),and the expression of reactive oxygen species( ROS) were detected by microplate reader. Results: Compared with the model group,in the different doses groups of Xiaoshuan Enteric Capsule,the survival rate of PC12 cells induced by hypoxia/glucose deprivation was significantly increased( P < 0. 05),the ratio of extracellular LDH was significantly decreased( P < 0. 05),while the expressions of MMP-9,P53,Bax,Caspase-3, MDA and ROS were significantly decreased( P < 0. 05),while the expressions of BCL-2:and SOD were significantly increased( P < 0. 05). Conclusion: Xiaoshuan Enteric Capsule has obvious protective effect on PC12 cell injury induced by hypoxia and glucose deprivation,which can reduce the expression of apoptosis-related factors,which may be related to the inhibition of MMP-9 expression and the reduction of oxidative stress.
引文
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[17] Zhao H,Yenari MA,Cheng D,et al. Bc12 overexpression protects against neuron loss within the ischemic margin following experimental stroke and inhibits cytochrome translocation and caspase 3 activity[J]. J Neurochem,2003,85(4):1026-1036.
[18] Guo J,Zhang K,Ji Y,et al. Effects of ethyl pyruvate on myocardial apoptosis and expression of Bc; 12 and Bax proteins after isc; hemia-reperfusion in rats[J]. J Huazhong Univ Sci Technolog Med Sci,2008,28(3):281-283.
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[2] LI Z,ZHANG HJ,LIU HP,et al. Clinical significance of ser-um MMP-9 and NSE in patients with acute cerebral infarction[J]. J Daliarr Med Uriv,2008,30(1):69-72.
[3] Yamaguchi M,Jadhav V,Obenaus A,et al. Matrix metalloproteinase inhibition attenuates brain edema in an in vivo model of surgically-induced brain injury[J]. Neurosurgery,2007,61(5):1067-1076.
[4] Vila-Petroff M,Salas MA,Said M,et al. Ca MK B inhibition protects against necrosis and apoptosis in irreversihle ischemia-reper-fusion injury[J]. Cardiauasc Res,2007,73(4):689-698.
[5]王清任.医林改错[M].北京:人民卫生出版社,2005:36.
[6]陈冬,杨洁红.补阳还五汤抗脑缺血作用的研究进展[J].中华中医药学刊,2010,28(1):72.
[7] ZHANG YB,DONG WW. Development of neuronal ischemic oxygen and glucose deprivation in vitro[J]. J Chorrgqirrg Med Uriv,2001,26(2):116-118.
[8] Huang SL,He XJ,Li ZF,et al. Neuroprotective effects of ginsenoside Rg1 on oxygen-glucose deprivation reperfusion in PC12cells[J]. Pharmazie,2014,69(3):208-211.
[9]袁清洁,王嘉麟,贺立娟,等.补阳还五汤治疗缺血性中风疗效机制研究进展[J].环球中医药,2017,10(12):1537-1542.
[10] Luo SH,Xiao W,Wei XJ,et al. In vitro evaluation of cytotoxicity of silver-containing borate bioaclive glass[J]. J Biomed Mater Res B Appl Biomater,2010,95(2):441-448.
[11] Romanic AM,White RF,Arleth AJ,et al. Matrix metalloproteinase expression increases after cerebral focal ischemia in rats,inhibition of Matrix metalloproteinase-9 reduces infarct size[J]. Stroke,1998:29(5):1020-1030.
[12]张彤,郑晓霞,廖晓凌,等.急性缺血性脑卒中患者超早期基质金属蛋白酶表达与临床预后的关系[J].中华老年心脑血管病杂志,2008,10(2):109-111.
[13]匡宏宇,段鹏,马丽丽,等.高糖及不同幅度高糖波动下牛视网膜毛细血管周细胞增生与凋亡的研究[J].眼科新进展,2007,27(2):87-90.
[14] Vander Heiden MG,Chandel NS,Williamson EK,et al.Bcl-Xl regulates the membrane potential and volume homeostasis of mitochondria[J]. Cell,1997:627-637.
[15] Chang HY,Yang X. Proteases for cell suicide:functions and regulation of caspases[J]. Microbiol Mol Biol Rev,2000,64(4):821-846
[16] Shi Y. Mechanisms of caspase activation and inhibition during apoptosis[J]. Mol Cell,2002,9(3):459-470.
[17] Zhao H,Yenari MA,Cheng D,et al. Bc12 overexpression protects against neuron loss within the ischemic margin following experimental stroke and inhibits cytochrome translocation and caspase 3 activity[J]. J Neurochem,2003,85(4):1026-1036.
[18] Guo J,Zhang K,Ji Y,et al. Effects of ethyl pyruvate on myocardial apoptosis and expression of Bc; 12 and Bax proteins after isc; hemia-reperfusion in rats[J]. J Huazhong Univ Sci Technolog Med Sci,2008,28(3):281-283.
[19] Siesjo BK. Pathophysiology and treatment of focal czrebral ischemia Part 1:Pathothysiology[J]. J Neurosurg,1992,77:169-184.
[20] Anggard L. Nitric oxide:mediator,murderer and medicine[J]. Lancet,1994,343:1199.