梓葛冻干粉针对体外氧糖剥夺/再灌注损伤大鼠大脑皮层星形胶质细胞的保护作用

童杰, 曾杰, 孙文威, 刘杨, 魏述永, 徐晓玉

中国药学杂志 ›› 2016, Vol. 51 ›› Issue (13) : 1124-1130.

PDF(3331 KB)
PDF(3331 KB)
中国药学杂志 ›› 2016, Vol. 51 ›› Issue (13) : 1124-1130. DOI: 10.11669/cpj.2016.13.016
论著

梓葛冻干粉针对体外氧糖剥夺/再灌注损伤大鼠大脑皮层星形胶质细胞的保护作用

  • 童杰1, 曾杰1, 孙文威1, 刘杨1, 魏述永2, 徐晓玉1*
作者信息 +

Neuroprotective Effect of Lyophilized Powder of Catalpol and Puerarin on Oxygen-glucose Deprivation/reperfusion Injury in Astrocytes of Rat Cerebral Cortex in Vitro

  • TONG Jie1, ZENG Jie1, SUN Wen-wei1, LIU Yang1, WEI Shu-yong2, XU Xiao-yu1*
Author information +
文章历史 +

摘要

目的 探讨梓葛冻干粉针(C-P)对体外氧糖剥夺/再灌注(OGD/R)损伤大鼠大脑皮层星形胶质细胞的保护作用及可能的机制。方法 原代分离培养大鼠大脑皮层星形胶质细胞,神经胶质纤维酸性蛋白(GFAP)免疫荧光鉴定星形胶质细胞的纯度。实验分正常组、模型组、辅料组、C-P:12.25、24.50、49.00 μg·mL-1组。对星形胶质细胞进行OGD 6h/R 12h损伤并同时给予C-P。噻唑蓝(MTT)法检测细胞存活率、比色法测定乳酸脱氢酶(LDH)漏出率、TUNEL荧光染色法检测细胞凋亡率。利用试剂盒的方法分别检测细胞中超氧化物歧化酶(SOD)的活性,谷胱甘肽(GSH)、活性氧(ROS)和丙二醛(MDA)的含量以及一氧化氮(NO)的释放量,ELISA法测定细胞培养液中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β和前列腺素(PG)E2的含量,Western blot检测细胞内caspase-3,诱导型一氧化氮合酶(iNOS),环氧酶(COX)-2,核因子(NF)-κB p65,p-NF-κB p65,IκB-α,p-IκB-α蛋白表达量。结果 原代培养的星形胶质细胞纯度在97%以上。与正常组相比,模型组细胞存活率显著降低,LDH漏出率及细胞凋亡率明显增高;细胞内SOD活性和GSH含量明显降低,MDA和ROS含量显著升高;细胞培养液中NO、TNF-α、IL-1β和PGE2的释放量也显著增加。而以上相关生化指标均可被不同剂量的C-P所抑制。辅料组与模型组无显著差异。进一步研究发现,C-P能显著抑制OGD/R损伤引起的细胞中caspase-3、iNOS、COX-2蛋白表达量及NF-κB p65和IκB-α的磷酸化水平的增高。结论 C-P对体外OGD/R损伤的星形胶质细胞具有保护作用,其保护机制与其抗炎、抗氧化以及抑制细胞凋亡和NF-κB信号通路的激活有关。

Abstract

OBJECTIVE To investigate the protection of lyophilized powder of catalpol and puerarin (C-P) on oxygen-glucose deprivation/reperfusion(OGD/R)-injured astrocytes and the possible mechanism in vitro.METHODS Primary astrocytes were isolated from the cerebral cortex of neonatal rats. The purity of astrocytes was identified by GFAP immunofluorescence. Astrocytes were divided into 6 groups:normal group, model group, excipients group, and three groups with gradient doses of C-P (12.25, 24.50, 49.00 μg·mL-1). After astrocytes suffered from OGD/R (OGD 6 h/R 12 h) with or without C-P, cell survival, LDH leakage, and apoptosis were analyzed by MTT, colorimetry, and TUNEL respectively. The apoptotic protein, caspase-3, was evaluated by Western blot. Meanwhile, oxidative indexes including SOD, GSH, ROS, MDA, and NO were detected by assay kits. In terms of inflammation, TNF-α, IL-1β, and PGE2 in medium were evaluated via ELISA. iNOS, COX-2, NF-κB p65, p-NF-κB p65, IκB-α, and p-IκB-α were examined by Western blot. RESULTS The purity of primary astrocytes was more than 97%. Compared with normal group, the cell survival rate of model group decreased significantly, while the LDH leakage rate and cell apoptosis rate markedly increased after OGD/R injury in model group. Meanwhile, SOD activity and GSH level in astrocytes markedly decreased. The level of MDA and ROS significantly increased. The content of NO, TNF-α, IL-1β, and PGE2 released in medium also sharply increased. However, those changes of related biochemical indexes above could be reversed by different gradient doses of C-P. There was no significant difference between excipients group and model group. Further study found that C-P could inhibit the protein expression of caspase-3, iNOS, and COX-2, as well as the increase of phosphorylation level of NF-κB p65 and IκB-α significantly. CONCLUSION C-P shows a protective effect on the OGD/R injured astrocytes in vitro, and its protection mechanism involves in anti-inflammation, antioxidant, inhibiting the cell apoptosis and activation of NF-κB signaling pathway.

关键词

梓葛冻干粉针 / 星形胶质细胞 / 氧糖剥夺/再灌注损伤 / 炎症反应 / 氧化应激 / 细胞凋亡

Key words

lyophilized powder of catalpol and puerarin / astrocytes / oxygen-glucose deprivation/reperfusion injury / inflammatory response / oxidative stress / cell apoptosis

引用本文

导出引用
童杰, 曾杰, 孙文威, 刘杨, 魏述永, 徐晓玉. 梓葛冻干粉针对体外氧糖剥夺/再灌注损伤大鼠大脑皮层星形胶质细胞的保护作用[J]. 中国药学杂志, 2016, 51(13): 1124-1130 https://doi.org/10.11669/cpj.2016.13.016
TONG Jie, ZENG Jie, SUN Wen-wei, LIU Yang, WEI Shu-yong, XU Xiao-yu. Neuroprotective Effect of Lyophilized Powder of Catalpol and Puerarin on Oxygen-glucose Deprivation/reperfusion Injury in Astrocytes of Rat Cerebral Cortex in Vitro[J]. Chinese Pharmaceutical Journal, 2016, 51(13): 1124-1130 https://doi.org/10.11669/cpj.2016.13.016
中图分类号: R965   

参考文献

[1] GO A S, DARIUSH M, ROGER V L, et al. Heart disease and stroke statistics--2014 update:a report from the American Heart Association[J]. Circulation, 2014, 129(2):e28-e292.
[2] DOYLE K P, SIMON R P, STENZEL-POORE M P. Mechanisms of ischemic brain damage[J]. Neuropharmacology, 2008, 55(3):310-318.
[3] BARRETO G, WHITE R E, OUYANG Y, et al. Astrocytes:targets for neuroprotection in stroke[J]. Cent Nerv Syst Agents Med Chem, 2011, 11(2):164-173.
[4] ZHANG F. The Experimental Investigation of the ZG Compatibility Effect to Cerebral Edema of Rat[D]. Chongqing:Univ of Southwest, 2011.
[5] LIU J L. Effects of CPFPI on the neural function of cerebral ischemia in mices and neural stem cells proliferation in cerebral ischemia in rats[D]. Chongqing:Univ of Southwest, 2012.
[6] LIU Y, XUE Q, LI X, et al. Amelioration of stroke-induced neurological deficiency by lyophilized powder of catalpol and puerarin[J]. Int J Biol Sci, 2014, 10(4):448-456.
[7] WANG Q. Pharmacokinetic Study of Zige Freeze-dried Powder and Mechanism Study of improving permeability of BBB[D]. Chongqing:University of Southwest, 2012.
[8] SHANG Y H, WANG H J, TIAN J F, et al. Effect of zige lyophilized powder for injection in improving acute cerebral microcirculation disturbance in rats[J]. China J Chin Mater Med(中国中药杂志), 2014, 39(4):733-737.
[9] XUE Q, LIU Y, QI H Y, et al. A novel brain neurovascular unit model with neurons, astrocytes and microvascular endothelial cells of rat [J]. Int J Biol Sci, 2013, 9(2):174-189.
[10] BROUNS R, DEYN P P D. The complexity of neurobiological processes in acute ischemic stroke[J]. Clin Neurol Neurosur, 2009, 111(6):483-495.
[11] ABAS F, ALKAN T, GOREN B, et al. Neuroprotective effects of postconditioning on lipid peroxidation and apoptosis after focal cerebral isnhemia/repedusion injury in rats[J]. Turk Neurosurg, 2010,20(1):1-8.
[12] SOFRONIEW M V, VINTERS H V. Astrocytes:biology and pathology[J]. Acta Neuropathol, 2010, 119(1):7-35.
[13] GOUIX E, BUISSON A, NIEOULLON A, et al. Oxygen glucose deprivation-induced astrocyte dysfunction provokes neuronal death through oxidative stress[J]. Pharmacol Res, 2014, 87(9):8-17.
[14] GU L Z, HUANG B S, SHEN W, et al. Early activation of nSMase2/ceramide pathway in astrocytes is involved in ischemia-associated neuronal damage via inflammation in rat hippocampi. [J]. J Neuroinflamm, 2013, 10(1):1-16.
[15] MOSKOWITZ M A, LO E H, IADECOLA C, et al. The science of stroke:mechanisms in search of treatments[J]. Neuron, 2010, 67(2):181-198.
[16] MUOIO V, PERSSON P B, SENDESKI M M. The neurovascular unit-concept review [J]. Acta Physiol, 2014, 210(4):790-798.
[17] ZHANG C C, MA Q, XUE Q, et al. Protective effect of zige lyophilized powder on hypoxia/reoxygenation injury of human umbilical vein endothelial cells[J]. China J Chin Mater Med(中国中药杂志), 2012,37(7):1997-2001.

基金

国家自然科学基金面上项目(81473549);国家青年科学基金项目(31402237);中国博士后基金资助项目(2014M562272)
PDF(3331 KB)

Accesses

Citation

Detail

段落导航
相关文章

/