玄参环烯醚萜苷对氧糖剥夺再灌注诱导的原代皮层神经元细胞内质网应激的作用研究
|
摘要
目的探讨玄参环烯醚萜苷通过内质网应激介导的细胞凋亡通路对氧糖剥夺再灌注(OGD/R)诱导的原代皮层神经元细胞的作用及机制。方法分离SD大鼠原代皮层神经元,用玄参环烯醚萜苷(50、100、200μg/mL)对原代皮层神经元进行预处理24 h,采用OGD/R诱导制备脑缺血再灌注细胞模型,倒置显微镜下鉴定细胞纯度,观察细胞形态变化;MTT法检测细胞存活率;试剂盒检测细胞内乳酸脱氢酶(LDH)水平和超氧化物歧化酶(SOD)活性;流式细胞术检测细胞凋亡率;Western blotting法检测C/EBP同源蛋白(CHOP)、葡萄糖调节蛋白-78(GRP78)和Caspase-12蛋白表达。结果培养的原代皮层神经元胞体饱满,状态良好,纯度较高。与对照组比较,经OGD/R处理后原代皮层神经元整体回缩变圆,胞体表面变粗糙;细胞存活率、SOD活性显著降低;LDH水平、细胞凋亡率显著升高;CHOP、Caspase-12和GRP78蛋白表达水平显著升高。与模型组比较,玄参环烯醚萜苷预处理能改善细胞损伤情况;提高细胞存活率、SOD活性;降低LDH水平和细胞凋亡率;降低CHOP、Caspase-12和GRP78蛋白表达水平。结论玄参环烯醚萜苷能拮抗OGD/R诱导的原代皮层神经元神经损伤,其作用机制与抑制内质网应激介导的细胞凋亡有关。
Objective To investigate the effects and mechanisms of iridoid glycosides of Scrophulariae Radix(IGRS) via endoplasmic reticulum stress-mediated apoptosis pathway on the primary cortical neurons induced by oxygen glucose deprivation/reperfusion(OGD/R). Methods Newborn SD rats were performed primary cortical neurons culture. And the primary cortical neurons were pretreated with IGRS(50, 100, and 200 μg/m L) for 24 h, and the in vitro model of oxygen-glucose deprivation/reoxygenation(OGD/R)was applied. The neurons purity and morphology were observed under inverted microscope, the cell viability was detected by MTT assay; the intracellular lactate dehydrogenase(LDH) level and superoxide dismutase(SOD) activity were detected by commercial kit.The apoptotic rate was detected by flow cytometry. The expression of C/EBP homologous protein(CHOP), glucose-regulated protein-78(GRP78) and Caspase-12 protein were detected by western blotting. Results The cultured primary cortical neurons were plump with high purity in good condition. Compared with the control group, the primary cortical neurons were retracted and rounded after OGD/R treatment, and the surface of the neurons became rough; The cell viability and SOD activity were significantly decreased;The LDH level and apoptotic rate were evidently increased; The expression of CHOP, Caspase-12, and GRP78 were significantly increased. Compared with the model group, IGRS could relieve neurons damage, increase cell viability and SOD activity, decrease LDH level and apoptotic rate, and down-regulate the expression of CHOP, Caspase-12, and GRP78. Conclusion IGRS can antagonize the neuronal damage induced by OGD/R in primary cortical neurons, and its mechanism is related to the inhibition of endoplasmic reticulum stress-mediated apoptosis.
Objective To investigate the effects and mechanisms of iridoid glycosides of Scrophulariae Radix(IGRS) via endoplasmic reticulum stress-mediated apoptosis pathway on the primary cortical neurons induced by oxygen glucose deprivation/reperfusion(OGD/R). Methods Newborn SD rats were performed primary cortical neurons culture. And the primary cortical neurons were pretreated with IGRS(50, 100, and 200 μg/m L) for 24 h, and the in vitro model of oxygen-glucose deprivation/reoxygenation(OGD/R)was applied. The neurons purity and morphology were observed under inverted microscope, the cell viability was detected by MTT assay; the intracellular lactate dehydrogenase(LDH) level and superoxide dismutase(SOD) activity were detected by commercial kit.The apoptotic rate was detected by flow cytometry. The expression of C/EBP homologous protein(CHOP), glucose-regulated protein-78(GRP78) and Caspase-12 protein were detected by western blotting. Results The cultured primary cortical neurons were plump with high purity in good condition. Compared with the control group, the primary cortical neurons were retracted and rounded after OGD/R treatment, and the surface of the neurons became rough; The cell viability and SOD activity were significantly decreased;The LDH level and apoptotic rate were evidently increased; The expression of CHOP, Caspase-12, and GRP78 were significantly increased. Compared with the model group, IGRS could relieve neurons damage, increase cell viability and SOD activity, decrease LDH level and apoptotic rate, and down-regulate the expression of CHOP, Caspase-12, and GRP78. Conclusion IGRS can antagonize the neuronal damage induced by OGD/R in primary cortical neurons, and its mechanism is related to the inhibition of endoplasmic reticulum stress-mediated apoptosis.
引文
[1]Donnan G,Fisher M,Macleod M,et al.Stroke[J].Lancet,2008,371(9624):1612-1623.
[2]Ozaffarian D,Benjamin E J,Go A S,et al.Executive summary:Heart disease and stroke statistics-2016update:A report from the American heart association[J].Circulation,2016,133(4):447-454.
[3]王陇德,刘建民,杨弋,等.我国脑卒中防治仍面临巨大挑战--《中国脑卒中防治报告2018》概要[J].中国循环杂志,2019,34(2):105-119.
[4]Molina C A.Reperfusion therapies for acute ischemic stroke:Current pharmacological and mechanical approaches[J].Stroke,2011,42(Suppl 1):S16.
[5]武彩霞,刘睿,杜冠华.内质网应激与脑缺血/再灌注损伤[J].中国药理学通报,2013,29(5):601-605.
[6]Zhang H Y,Wang Z G,Lu X H,et al.Endoplasmic reticulum stress:Relevance and therapeutics in central nervous system diseases[J].Mol Neurobiol,2015,51(3):1343-1352.
[7]Roussel B D,Kruppa A J,Miranda E,et al.Endoplasmic reticulum dysfunction in neurological disease[J].Lancet Neurol,2013,12(1):105-118.
[8]中国药典[S].一部.2015.
[9]李静,陈长勋,高阳,等玄参提取物抗炎与抗动脉硬化作用的探索[J].时珍国医国药,2010,21(3):532-534.
[10]倪正,蔡雪珠,黄一平,等.玄参提取物对大鼠血液流变性、凝固性和纤溶活性的影响[J].中国微循环,2004,8(3):152-153.
[11]李娟,萧伟,刘涛,等.玄参脑保护作用提取物的工艺优化[J].中成药,2011,33(6):1057-1059.
[12]Huang Q,Gong Q,Yao M,et al.Protective effect of Scrophularia ningpoensis extracts on cerebral ischemia injury in rats[J].Chin J New Drugs Clin Remed,2004,23(6):323-327.
[13]李媛,宋宝安,杨松,等.中草药玄参化学成分的研究[J].天然产物研究与开发,2012,24(1):47-51.
[14]李祚丹,季金苟,楚莎莎,等.玄参中环烯醚萜类物质的分离纯化工艺[J].中成药,2015,37(6):1367-1369.
[15]陈梦静,龚雪媛,龚恒佩,等.芍药苷对皮质酮致原代皮层细胞神经损伤的保护作用及机制研究[J].中华中医药杂志,2018,33(4):1507-1511.
[16]周晓楠,宋雪兰,李艳,等.对羟基苯甲醛对脑缺血/再灌注损伤的保护作用研究[J].中国药理学通报,2017,33(12):1729-1735.
[17]Wang K,Chen M,Gong H,et al.Calcium homeostasis disruption and endoplasmic reticulum stress mediats ischemia/reperfusion-induced PC12 cells apoptosis[J].Int J Clin Exp Med,2017,10(9):14121-14129.
[18]张蔺珊,李娟娟,吴春云.脑缺血的损伤机制及相关信号通路的研究进展[J].神经解剖学杂志,2014,30(6):729-732.
[19]张忠胜.大鼠皮层神经元内质网应激相关凋亡的实验研究[D].武汉:华中科技大学,2013.
[20]姜茜,姜玉武,王静敏,等.一种改进的大鼠皮层神经元原代培养方法及其性质鉴定[J].北京大学学报:医学版,2009,41(2):212-216.
[21]Blankenberg F.In vivo detection of apoptosis[J].J Nucl Med,2008,doi:10.1002/ejoc.200500912.
[22]Sun M S,Jin H,Sun X,et al.Free radical damage in ischemia-reperfusion injury:An obstacle in acute ischemic stroke after revascularization therapy[J].Oxid Med Cell Longev,2018,doi:10.1155/2018/3804979.
[23]俞静静,陈素红,吕圭源.玄参“凉血滋阴”药效相关研究概况[J].中国实验方剂学杂志,2007,13(9):63-66.
[24]Chen B,Liu Y,Liu H W,et al.Iridoid and aromatic glycosides from Scrophularia ningpoensis Hemsl.and their inhibition of[Ca2+]i increase induced by KCl[J].Chem Biodivers,2008,5(9):1723-1735.
[25]Meng X,Xie W,Xu Q,et al.Neuroprotective Effects of Radix Scrophulariae on cerebral ischemia and reperfusion injury via MAPK pathways[J].Molecules,2018,doi:10.3390/molecules23092401.
[2]Ozaffarian D,Benjamin E J,Go A S,et al.Executive summary:Heart disease and stroke statistics-2016update:A report from the American heart association[J].Circulation,2016,133(4):447-454.
[3]王陇德,刘建民,杨弋,等.我国脑卒中防治仍面临巨大挑战--《中国脑卒中防治报告2018》概要[J].中国循环杂志,2019,34(2):105-119.
[4]Molina C A.Reperfusion therapies for acute ischemic stroke:Current pharmacological and mechanical approaches[J].Stroke,2011,42(Suppl 1):S16.
[5]武彩霞,刘睿,杜冠华.内质网应激与脑缺血/再灌注损伤[J].中国药理学通报,2013,29(5):601-605.
[6]Zhang H Y,Wang Z G,Lu X H,et al.Endoplasmic reticulum stress:Relevance and therapeutics in central nervous system diseases[J].Mol Neurobiol,2015,51(3):1343-1352.
[7]Roussel B D,Kruppa A J,Miranda E,et al.Endoplasmic reticulum dysfunction in neurological disease[J].Lancet Neurol,2013,12(1):105-118.
[8]中国药典[S].一部.2015.
[9]李静,陈长勋,高阳,等玄参提取物抗炎与抗动脉硬化作用的探索[J].时珍国医国药,2010,21(3):532-534.
[10]倪正,蔡雪珠,黄一平,等.玄参提取物对大鼠血液流变性、凝固性和纤溶活性的影响[J].中国微循环,2004,8(3):152-153.
[11]李娟,萧伟,刘涛,等.玄参脑保护作用提取物的工艺优化[J].中成药,2011,33(6):1057-1059.
[12]Huang Q,Gong Q,Yao M,et al.Protective effect of Scrophularia ningpoensis extracts on cerebral ischemia injury in rats[J].Chin J New Drugs Clin Remed,2004,23(6):323-327.
[13]李媛,宋宝安,杨松,等.中草药玄参化学成分的研究[J].天然产物研究与开发,2012,24(1):47-51.
[14]李祚丹,季金苟,楚莎莎,等.玄参中环烯醚萜类物质的分离纯化工艺[J].中成药,2015,37(6):1367-1369.
[15]陈梦静,龚雪媛,龚恒佩,等.芍药苷对皮质酮致原代皮层细胞神经损伤的保护作用及机制研究[J].中华中医药杂志,2018,33(4):1507-1511.
[16]周晓楠,宋雪兰,李艳,等.对羟基苯甲醛对脑缺血/再灌注损伤的保护作用研究[J].中国药理学通报,2017,33(12):1729-1735.
[17]Wang K,Chen M,Gong H,et al.Calcium homeostasis disruption and endoplasmic reticulum stress mediats ischemia/reperfusion-induced PC12 cells apoptosis[J].Int J Clin Exp Med,2017,10(9):14121-14129.
[18]张蔺珊,李娟娟,吴春云.脑缺血的损伤机制及相关信号通路的研究进展[J].神经解剖学杂志,2014,30(6):729-732.
[19]张忠胜.大鼠皮层神经元内质网应激相关凋亡的实验研究[D].武汉:华中科技大学,2013.
[20]姜茜,姜玉武,王静敏,等.一种改进的大鼠皮层神经元原代培养方法及其性质鉴定[J].北京大学学报:医学版,2009,41(2):212-216.
[21]Blankenberg F.In vivo detection of apoptosis[J].J Nucl Med,2008,doi:10.1002/ejoc.200500912.
[22]Sun M S,Jin H,Sun X,et al.Free radical damage in ischemia-reperfusion injury:An obstacle in acute ischemic stroke after revascularization therapy[J].Oxid Med Cell Longev,2018,doi:10.1155/2018/3804979.
[23]俞静静,陈素红,吕圭源.玄参“凉血滋阴”药效相关研究概况[J].中国实验方剂学杂志,2007,13(9):63-66.
[24]Chen B,Liu Y,Liu H W,et al.Iridoid and aromatic glycosides from Scrophularia ningpoensis Hemsl.and their inhibition of[Ca2+]i increase induced by KCl[J].Chem Biodivers,2008,5(9):1723-1735.
[25]Meng X,Xie W,Xu Q,et al.Neuroprotective Effects of Radix Scrophulariae on cerebral ischemia and reperfusion injury via MAPK pathways[J].Molecules,2018,doi:10.3390/molecules23092401.