川芎嗪对t-BHP致PC12细胞损伤的保护及机制
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摘要
目的:研究川芎嗪(TMP)对叔丁基过氧化氢(t-BHP)诱导的PC12细胞损伤的保护作用及对磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/雷帕霉素靶蛋白(m TOR)信号通路的影响。方法:培养PC12细胞,孵育不同浓度的川芎嗪(10,25,50,100,200μmol·L~(-1)),12 h后采用细胞增殖毒性检测试剂盒(CCK-8)确定川芎嗪的保护浓度。随后将PC12细胞分为空白组,t-BHP组,TMP组。空白组加入培养基,t-BHP组加入t-BHP(200μmol·L~(-1)),TMP组加入TMP(25,50,100μmol·L~(-1))预处理12 h后加入t-BHP(200μmol·L~(-1))。共作用6 h后采用酶联免疫吸附测定(ELISA)检测乳酸脱氢酶(LDH)漏出量、丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、活性氧(ROS)含量和谷胱甘肽过氧化物酶(GSH-Px)活性。采用蛋白免疫印迹法(Western blot)检测B淋巴细胞瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax),Akt,磷酸化Akt(p-Akt)及m TOR,p-mTOR的表达情况。结果:与空白组比较,t-BHP组细胞生存率降低(P<0.01),与t-BHP组比较,25,50,100μmol·L~(-1)的川芎嗪促进细胞增殖(P<0.05,P<0.01),其中50μmol·L~(-1)组的细胞增殖率最高(P<0.01);与空白组比较,t-BHP组LDH的释放量,ROS和MDA含量显著增加(P<0.01),SOD和GSH-Px的活性显著降低(P<0.01),与t-BHP组比较,TMP组LDH的释放量,ROS和MDA含量显著降低(P<0.05,P<0.01),SOD和GSH-Px的活性显著升高(P<0.05,P<0.01);Western blot表明与空白组比较,t-BHP组中p-Akt,p-mTOR蛋白表达水平明显下降(P<0.01),Bcl-2/Bax值明显降低(P<0.01),与t-BHP组比较,TMP(50μmol·L~(-1))组中p-Akt,p-mTOR蛋白表达水平明显上升(P<0.05),Bcl-2/Bax值明显上升(P<0.05)。另外LY294002(PI3K蛋白抑制剂)处理减弱了这些变化。结论:川芎嗪通过激活PI3K/Akt/m TOR信号通路保护t-BHP诱导的PC12细胞损伤。
Objective:To study the protective effect of tetramethylpyrazine(TMP)on PC12 cells induced by tert-butyl hydroperoxide(t-BHP)and the regulatory mechanism on signaling pathway of phosphatidylinositol-3-kinases(PI3K)/kinase B(Akt)/mammalian target of rapamycin(m TOR).Method:PC12 cells cultured in vitro were treated with t-BHP(200μmol·L~(-1))for 6 h to establish a model of oxidative damage in PC12 cells.The experiment was divided into blank group,model group(200μmol·L~(-1)t-BHP),TMP group.PC12 cells were pretreated with TMP(25,50,100μmol·L~(-1))for 12 h,and then treated with t-BHP for 6 h.The cell viability was detected by cell counting kit-8(CCK-8)method,and lactate dehydrogenase(LDH)leakage,malondialdehyde(MDA)content,superoxide dismutase(SOD)activity,reactive oxygen species(ROS)and glutathione peroxidase(GSH-Px)activity were detected by enzyme-linked immunosorbent assay(ELISA).Apoptosis was observed by annexin V-FITC/PI double staining.B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),total protein kinase B(Akt),and phosphorylated protein kinase B(p-Akt),m TOR and p-mTOR expressions were detected by Western blot.Result:The cell viability of PC12 cells treated with 200μmol·L~(-1)tBHP decreased to about 50%after 6 h.This condition was suitable for the establishment of oxidative damage model.Compared with the model group,TMP(25,50,100μmol·L~(-1))pretreatment for 12 h significantly increased the survival rate of PC12 cells(P<0.05,P<0.01),decreased the release of LDH and MDA(P<0.05,P<0.01),significantly reduced the ROS content in PC12 cells(P<0.01),significantly increased the activity of SOD,GSH-Px(P<0.05,P<0.01),and inhibited the cell apoptosis.Western blot showed that compared with the model group,the protein expressions of p-Akt and p-mTOR in TMP(50μmol·L~(-1))pretreatment group increased significantly(P<0.01),and the Bcl-2/Bax ratio increased significantly.Treatment with LY294002(PI3K protein inhibitor)also attenuated these changes.Conclusion:Ligustrazine protects PC12cell injury induced by t-BHP by activating PI3K/Akt/m TOR signaling pathway.
Objective:To study the protective effect of tetramethylpyrazine(TMP)on PC12 cells induced by tert-butyl hydroperoxide(t-BHP)and the regulatory mechanism on signaling pathway of phosphatidylinositol-3-kinases(PI3K)/kinase B(Akt)/mammalian target of rapamycin(m TOR).Method:PC12 cells cultured in vitro were treated with t-BHP(200μmol·L~(-1))for 6 h to establish a model of oxidative damage in PC12 cells.The experiment was divided into blank group,model group(200μmol·L~(-1)t-BHP),TMP group.PC12 cells were pretreated with TMP(25,50,100μmol·L~(-1))for 12 h,and then treated with t-BHP for 6 h.The cell viability was detected by cell counting kit-8(CCK-8)method,and lactate dehydrogenase(LDH)leakage,malondialdehyde(MDA)content,superoxide dismutase(SOD)activity,reactive oxygen species(ROS)and glutathione peroxidase(GSH-Px)activity were detected by enzyme-linked immunosorbent assay(ELISA).Apoptosis was observed by annexin V-FITC/PI double staining.B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),total protein kinase B(Akt),and phosphorylated protein kinase B(p-Akt),m TOR and p-mTOR expressions were detected by Western blot.Result:The cell viability of PC12 cells treated with 200μmol·L~(-1)tBHP decreased to about 50%after 6 h.This condition was suitable for the establishment of oxidative damage model.Compared with the model group,TMP(25,50,100μmol·L~(-1))pretreatment for 12 h significantly increased the survival rate of PC12 cells(P<0.05,P<0.01),decreased the release of LDH and MDA(P<0.05,P<0.01),significantly reduced the ROS content in PC12 cells(P<0.01),significantly increased the activity of SOD,GSH-Px(P<0.05,P<0.01),and inhibited the cell apoptosis.Western blot showed that compared with the model group,the protein expressions of p-Akt and p-mTOR in TMP(50μmol·L~(-1))pretreatment group increased significantly(P<0.01),and the Bcl-2/Bax ratio increased significantly.Treatment with LY294002(PI3K protein inhibitor)also attenuated these changes.Conclusion:Ligustrazine protects PC12cell injury induced by t-BHP by activating PI3K/Akt/m TOR signaling pathway.
引文
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[12]Bajpai V K,Alam M B,Ju M K,et al. Antioxidant mechanism of polyphenol-rich Nymphaea nouchali leaf extract protecting DNA damage and attenuating oxidative stress-induced cell death via Nrf2-mediated hemeoxygenase-1 induction coupled with ERK/p38 signaling pathway[J]. Biomed Pharmacother,2018,103:1397-1407.
[13]陈剑平,李中桂,张尚斌,等.大枣神经保护作用的活性组分筛选及其作用机制研究[J].中国药房,2016. 27(25):3495-3498.
[14]高婷,王子旭,陈祝茗,等. ROS介导的氧化应激与自噬[J].中国畜牧兽医,2018,45(3):656-662.
[15]ZHOU J,MA X,CUI Y,et al. Methyleugenol protects against t-BHP-triggered oxidative injury by induction of Nrf2 dependent on AMPK/GSK3beta and ERK activation[J]. J Pharmacol Sci,2017,135(2):55-63.
[16]SHEN C,MA W,ZHENG W,et al. The antioxidant effects of riluzole on the APRE-19 celll model injuryinduced by t-BHP[J]. BMC Ophthalmol,2017,17(1):210.
[17]高健美,李德芬,牛爽,等.金丝桃苷对过氧化氢诱导的A549细胞氧化损伤的保护作用[J].中国实验方剂学杂志,2017,23(11):128-133.
[18]刘亭,宋菲,杨健,等.基于HSF1高表达H9c2细胞的参芎葡萄糖注射液抗氧化损伤作用探讨[J].中国实验方剂学杂志,2018,24(8):85-90.
[19]张建武,闵冬雨,周云,等.番茄红素对H2O2致乳鼠心肌细胞氧化应激损伤的保护作用[J].中国实验方剂学杂志,2014,20(12):160-164.
[20]林文新,马阮昕,冯真英.川芎嗪对过氧化氢诱导PC12细胞氧化应激的作用[J].江西中医药,2018,49(2):29-31.
[21]Aydemir T,Oztürk R,Bozkaya L A,et al. Effects of antioxidant vitamins A,C,E and trace elements Cu,Se on Cu Zn SOD,GSH-Px,CAT and LPO levels in chicken erythrocytes[J]. Cell Biochem Funct,2000,18(2):109-115.
[22]陈丹丹,彭成,李梦婷,等.自噬参与氢溴酸樟柳碱对缺氧状态下PC12细胞损伤的保护作用[J].中国实验方剂学杂志,2017,23(20):144-149.
[23]卢春凤,杨玉,商宇,等. ROS介导的氧化应激在INH诱导的细胞毒性中的作用及槲皮素的干预[J].生态毒理学报,2015,10(3):209-215.
[24]严钢莉,李朝武,聂海岭,等.丹参川芎嗪注射液对Aβ损伤的PC12细胞保护作用及机制研究[J].卒中与神经疾病,2016,23(1):15-20.
[25]TANG F,ZHOU X,WANG L,et al. A novel compound DT-010 protects against doxorubicin-induced cardiotoxicity in zebrafish and H9c2 cells by inhibiting reactive oxygen species-mediated apoptotic and autophagic pathways[J]. Eur J Pharmacol,2018,820:86-96.
[26]WANG L, ZHANG H Y, GAO B, et al.Tetramethylpyrazine protects against glucocorticoidinduced apoptosis by promoting autophagy in mesenchymal stem cells and improves bone mass in glucocorticoid-induced osteoporosis rats[J]. Stem Cells Dev,2017,26(6):419-430.
[2]Sandra F I,Miquel V,Celine P. Mitochondrial quality control in neurodegenerative diseases:focus on parkinson's disease and huntington's disease[J]. Front Neurosci,2018,12:1-25.
[3]CHEN L, LIU B. Relationships between stress granules, oxidative stress, and neurodegenerative diseases[J]. Oxid Med Cell Longev,2017,doi:10.1155/2017/1809592.
[4]傅瑜,廖琴,樊东升.神经保护剂治疗缺血性脑卒中的研究进展[J].中国新药杂志,2011,20(11):973-977.
[5]周惠芬,何昱,张宇燕,等.川芎和黄芪有效部位组合给药后川芎嗪在脑缺血再灌注大鼠体内的PK-PD结合研究[J].中草药,2016,47(19):3463-3468.
[6]吴平勇,曾宪涛,王学军,等.川芎嗪对心肌缺血再灌注损伤防治作用的系统评价与Meta分析[J].中国循证心血管医学杂志,2014,6(6):653-657.
[7]李健琳,韩江全,陈玲.川芎嗪和葛根素配伍对脑缺血大鼠神经细胞调亡及Caspase-3影响[J].齐齐哈尔医学院学报,2016,37(4):423-425.
[8]林文新,马阮昕,冯真英.川芎嗪对过氧化氢诱导PC12细胞氧化应激的作用[J].江西中医药,2018,49(2):29-31.
[9]李杰.川芎嗪联合血栓通治疗缺血性脑卒中的疗效分析[J].中国实用神经疾病杂志,2014,17(17):108-109.
[10]刘文铠,肖辉亮,周徐州.丹参川芎嗪注射液辅助辨治缺血性脑卒中的临床研究[J].中国生化药物杂志,2014,34(2):120-121.
[11]Ghosh M,Manna P,Sil P C. Protective role of a coumarin-derived schiff base scaffold against tertiary butyl hydroperoxide(TBHP)-induced oxidative impairment and cell death via MAPKs,NF-kappaB and mitochondria-dependent pathways[J]. Free Radic Res,2011,45(5):620-637.
[12]Bajpai V K,Alam M B,Ju M K,et al. Antioxidant mechanism of polyphenol-rich Nymphaea nouchali leaf extract protecting DNA damage and attenuating oxidative stress-induced cell death via Nrf2-mediated hemeoxygenase-1 induction coupled with ERK/p38 signaling pathway[J]. Biomed Pharmacother,2018,103:1397-1407.
[13]陈剑平,李中桂,张尚斌,等.大枣神经保护作用的活性组分筛选及其作用机制研究[J].中国药房,2016. 27(25):3495-3498.
[14]高婷,王子旭,陈祝茗,等. ROS介导的氧化应激与自噬[J].中国畜牧兽医,2018,45(3):656-662.
[15]ZHOU J,MA X,CUI Y,et al. Methyleugenol protects against t-BHP-triggered oxidative injury by induction of Nrf2 dependent on AMPK/GSK3beta and ERK activation[J]. J Pharmacol Sci,2017,135(2):55-63.
[16]SHEN C,MA W,ZHENG W,et al. The antioxidant effects of riluzole on the APRE-19 celll model injuryinduced by t-BHP[J]. BMC Ophthalmol,2017,17(1):210.
[17]高健美,李德芬,牛爽,等.金丝桃苷对过氧化氢诱导的A549细胞氧化损伤的保护作用[J].中国实验方剂学杂志,2017,23(11):128-133.
[18]刘亭,宋菲,杨健,等.基于HSF1高表达H9c2细胞的参芎葡萄糖注射液抗氧化损伤作用探讨[J].中国实验方剂学杂志,2018,24(8):85-90.
[19]张建武,闵冬雨,周云,等.番茄红素对H2O2致乳鼠心肌细胞氧化应激损伤的保护作用[J].中国实验方剂学杂志,2014,20(12):160-164.
[20]林文新,马阮昕,冯真英.川芎嗪对过氧化氢诱导PC12细胞氧化应激的作用[J].江西中医药,2018,49(2):29-31.
[21]Aydemir T,Oztürk R,Bozkaya L A,et al. Effects of antioxidant vitamins A,C,E and trace elements Cu,Se on Cu Zn SOD,GSH-Px,CAT and LPO levels in chicken erythrocytes[J]. Cell Biochem Funct,2000,18(2):109-115.
[22]陈丹丹,彭成,李梦婷,等.自噬参与氢溴酸樟柳碱对缺氧状态下PC12细胞损伤的保护作用[J].中国实验方剂学杂志,2017,23(20):144-149.
[23]卢春凤,杨玉,商宇,等. ROS介导的氧化应激在INH诱导的细胞毒性中的作用及槲皮素的干预[J].生态毒理学报,2015,10(3):209-215.
[24]严钢莉,李朝武,聂海岭,等.丹参川芎嗪注射液对Aβ损伤的PC12细胞保护作用及机制研究[J].卒中与神经疾病,2016,23(1):15-20.
[25]TANG F,ZHOU X,WANG L,et al. A novel compound DT-010 protects against doxorubicin-induced cardiotoxicity in zebrafish and H9c2 cells by inhibiting reactive oxygen species-mediated apoptotic and autophagic pathways[J]. Eur J Pharmacol,2018,820:86-96.
[26]WANG L, ZHANG H Y, GAO B, et al.Tetramethylpyrazine protects against glucocorticoidinduced apoptosis by promoting autophagy in mesenchymal stem cells and improves bone mass in glucocorticoid-induced osteoporosis rats[J]. Stem Cells Dev,2017,26(6):419-430.
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