基于实时阻抗细胞分析技术评价H9c2心肌细胞缺氧/复氧损伤模型及三七皂苷R_1的干预作用
|
摘要
目的基于实时阻抗细胞分析技术,建立规范标准的H9c2心肌细胞缺氧/复氧损伤模型,并用三七皂苷R_1进行评价。方法将H9c2心肌细胞接种到E-Plate板中,设3个复孔,分别测定生长曲线、不同缺氧时间、不同复氧时间及三七皂苷R_1的干预下细胞指数值,以细胞指数值反映H9c2心肌细胞的生长状态。结果本研究发现,缺氧前更换无血清、无糖DMEM培养基,缺氧4 h,复氧16 h,细胞存活率为(48.82±5.32)%,与MTT法测定结果差异无显著性,且三七皂苷R_1能够保护缺氧/复氧处理H9c2心肌细胞损伤,不存在时间依赖性。结论实时阻抗细胞分析技术能够建立标准规范的缺氧/复氧模型方法,对发现新药物靶点及作用机制也有一定的指导意义。
Aim To establish the standard hypoxia/reoxygenation(H/R) injury model of H9 c2 myocardial cells and evaluate the intervention effect of notoginsenoside R_1 based on RTCA technology. Methods H9 c2 myocardial cells were inoculated into the E-Plate board, 3 wells by a group, then the growth curve and cell index of different hypoxia time, different reoxygenation time and notoginsenoside R_1 intervention were determined respectively, with the growth status of H9 c2 myocardial cells reflected by cell index. Results Cell survival rate was(48.82±5.32)% after hypoxia treatment for 4 h and reoxygenation treatment for 16 h with replacement of serum-free,glucose-free DMEM medium before hypoxia,and no significant difference was found between the results measured with MTT method. Notoginsenoside R1 could protect the hypoxia/reoxygenation injury of H9 c2 myocardial cells without time dependence. Conclusions RTCA technology is an effective means to establish the standard H/R injury model of H9 c2 myocardial cells,which also has some guiding significance in discovering new drug targets and mechanisms.
Aim To establish the standard hypoxia/reoxygenation(H/R) injury model of H9 c2 myocardial cells and evaluate the intervention effect of notoginsenoside R_1 based on RTCA technology. Methods H9 c2 myocardial cells were inoculated into the E-Plate board, 3 wells by a group, then the growth curve and cell index of different hypoxia time, different reoxygenation time and notoginsenoside R_1 intervention were determined respectively, with the growth status of H9 c2 myocardial cells reflected by cell index. Results Cell survival rate was(48.82±5.32)% after hypoxia treatment for 4 h and reoxygenation treatment for 16 h with replacement of serum-free,glucose-free DMEM medium before hypoxia,and no significant difference was found between the results measured with MTT method. Notoginsenoside R1 could protect the hypoxia/reoxygenation injury of H9 c2 myocardial cells without time dependence. Conclusions RTCA technology is an effective means to establish the standard H/R injury model of H9 c2 myocardial cells,which also has some guiding significance in discovering new drug targets and mechanisms.
引文
[1] Kimes B W, Brandt B L. Properties of a clonal muscle cell line from rat heart[J]. Exp Cell Res, 1976,98(2):367-81.
[2] 李光, 邢小燕, 张美双, 等. 基于表观遗传学调控的中医药防治心肌缺血/再灌注损伤的研究进展[J]. 药学学报, 2016,51(7):1047-53.[2] Li G, Xing X Y, Zhang M S, et al. Research progress in epigenetic regulation of myocardial ischemia/reperfusion injury by traditional Chinese medicine[J]. Acta Pham Sin, 2016,51(7):1047-53.
[3] Soumya R S, Prathapan A, Raj P S, et al. Selenium incorporated guar gum nanoparticles safeguard mitochondrial bioenergetics during ischemia reperfusion injury in H9c2 cardiac cells[J]. Int J Biol Macromol, 2017, 107: 254-60.
[4] Sheng M, Huang Z, Pan L, et al.SOCS2 exacerbates myocardial injury induced by ischemia/reperfusion in diabetic mice and H9c2 cells through inhibiting the JAK-STAT-IGF-1 pathway[J]. Life Sci, 2017,188(Supplement C):101-9.
[5] Brand M D, Goncalves R L S, Orr A L, et al. Suppressors of superoxide-H2O2 production at site IQ of mitochondrial complex I protect against stem cell hyperplasia and ischemia-reperfusion injury[J]. Cell Metab, 2016,24(4):582-92.
[6] Ai Q D, Sun G B, Luo Y, et al.Ginsenoside Rb1 prevents hypoxia-reoxygenation-induced apoptosis in H9c2 cardiomyocytes via an estrogen receptor-dependent crosstalk among the Akt, JNK, and ERK 1/2 pathways using a label-free quantitative proteomics analysis[J]. Rsc Adv, 2015,5(33):26346-63.
[7] Li X L, Yang R, Yang X H, et al.Original article paeonol protects H9c2 cardiomyocytes from ischemia/reperfusion injury by activating Notch1 signaling pathway in vitro[J]. Int J Clin Exp Med, 2017,10(2):2866-73.
[8] Yu Y L, Sun G B, Luo Y, et al.Cardioprotective effects of notoginsenoside R1 against ischemia/reperfusion injuries by regulating oxidative stress- and endoplasmic reticulum stress- related signaling pathways[J]. Sci Eep-UK, 2016,6(5):21730.
[9] Chen H B, Zheng H T.MicroRNA-200c represses migration and invasion of gastric cancer SGC-7901 cells by inhibiting expression of fibronectin 1[J]. Eur Rev Med Pharma Co, 2017,21(8):1753-8.
[10] Liu N C, Hsieh P F, Hsieh M K, et al.Capsaicin-mediated tNOX (ENOX2) up-regulation enhances cell proliferation and migration in vitro and in vivo[J]. J Agr Food Chem, 2012,60(10):2758-65.
[11] 俞辰斌, 赵国龙, 于立明, 等.JAK2/STAT3信号通路介导原花青素抗H9c2细胞缺氧/复氧损伤[J]. 生理学报, 2016, 68(5):568-74.[11] Yu C B, Zhao G L, Yu L M, et al. Proanthocyanidin protects H9c2 cells against hypoxia/reoxygenation injury via JAK2/STAT3 signaling pathway[J]. Acta Physiol Sin, 2016, 68(5):568-74.
[12] 刘善新. 羟基红花黄素A对缺氧/复氧诱导的H9c2心肌细胞凋亡的影响及机制研究[D]. 杭州:浙江大学, 2011.[12] Liu S X. The study of effect and mechanisms of hydroxysafflor yellow A on anoxia/reoxygenation-induced apoptosis of H9c2 cardiomyocytes[D]. Hangzhou: Zhejiang University, 2011.
[13] 董曦, 孙桂波, 罗云, 等.异鼠李素对H2O2引起的H9c2细胞氧化应激损伤的保护作用研究[J]. 中国药理学通报, 2015, 31(6):853-60.[13] Dong X, Sun G B, Luo Y, et al. Protective effect of isorhamnetin on H9c2 cell line against oxidative stress[J]. Chin Pharmacol Bull, 2015, 31(6):853-60.
[14] 谢飞, 魏珂, 闵苏, 等.神经生长因子对H9c2心肌细胞缺氧/复氧后凋亡的保护作用及其机制[J]. 中国药理学通报, 2014, 30(4):506-10.[14] Xie F, Wei K, Min S, et al. Anti-apoptotic effect of NGF on H9c2 cardiac myocytes in a hypoxia/reoxygenation injury model [J]. Chin Pharmacol Bull, 2014, 30(4):506-10.
[15] Meng X, Wang M, Wang X, et al. Suppression of NADPH oxidase- and mitochondrion-derived superoxide by notoginsenoside R1 protects against cerebral ischemia-reperfusion injury through estrogen receptor-dependent activation of Akt/Nrf2 pathways[J]. Free Radical Res, 2014,48(7):823-38.
[2] 李光, 邢小燕, 张美双, 等. 基于表观遗传学调控的中医药防治心肌缺血/再灌注损伤的研究进展[J]. 药学学报, 2016,51(7):1047-53.[2] Li G, Xing X Y, Zhang M S, et al. Research progress in epigenetic regulation of myocardial ischemia/reperfusion injury by traditional Chinese medicine[J]. Acta Pham Sin, 2016,51(7):1047-53.
[3] Soumya R S, Prathapan A, Raj P S, et al. Selenium incorporated guar gum nanoparticles safeguard mitochondrial bioenergetics during ischemia reperfusion injury in H9c2 cardiac cells[J]. Int J Biol Macromol, 2017, 107: 254-60.
[4] Sheng M, Huang Z, Pan L, et al.SOCS2 exacerbates myocardial injury induced by ischemia/reperfusion in diabetic mice and H9c2 cells through inhibiting the JAK-STAT-IGF-1 pathway[J]. Life Sci, 2017,188(Supplement C):101-9.
[5] Brand M D, Goncalves R L S, Orr A L, et al. Suppressors of superoxide-H2O2 production at site IQ of mitochondrial complex I protect against stem cell hyperplasia and ischemia-reperfusion injury[J]. Cell Metab, 2016,24(4):582-92.
[6] Ai Q D, Sun G B, Luo Y, et al.Ginsenoside Rb1 prevents hypoxia-reoxygenation-induced apoptosis in H9c2 cardiomyocytes via an estrogen receptor-dependent crosstalk among the Akt, JNK, and ERK 1/2 pathways using a label-free quantitative proteomics analysis[J]. Rsc Adv, 2015,5(33):26346-63.
[7] Li X L, Yang R, Yang X H, et al.Original article paeonol protects H9c2 cardiomyocytes from ischemia/reperfusion injury by activating Notch1 signaling pathway in vitro[J]. Int J Clin Exp Med, 2017,10(2):2866-73.
[8] Yu Y L, Sun G B, Luo Y, et al.Cardioprotective effects of notoginsenoside R1 against ischemia/reperfusion injuries by regulating oxidative stress- and endoplasmic reticulum stress- related signaling pathways[J]. Sci Eep-UK, 2016,6(5):21730.
[9] Chen H B, Zheng H T.MicroRNA-200c represses migration and invasion of gastric cancer SGC-7901 cells by inhibiting expression of fibronectin 1[J]. Eur Rev Med Pharma Co, 2017,21(8):1753-8.
[10] Liu N C, Hsieh P F, Hsieh M K, et al.Capsaicin-mediated tNOX (ENOX2) up-regulation enhances cell proliferation and migration in vitro and in vivo[J]. J Agr Food Chem, 2012,60(10):2758-65.
[11] 俞辰斌, 赵国龙, 于立明, 等.JAK2/STAT3信号通路介导原花青素抗H9c2细胞缺氧/复氧损伤[J]. 生理学报, 2016, 68(5):568-74.[11] Yu C B, Zhao G L, Yu L M, et al. Proanthocyanidin protects H9c2 cells against hypoxia/reoxygenation injury via JAK2/STAT3 signaling pathway[J]. Acta Physiol Sin, 2016, 68(5):568-74.
[12] 刘善新. 羟基红花黄素A对缺氧/复氧诱导的H9c2心肌细胞凋亡的影响及机制研究[D]. 杭州:浙江大学, 2011.[12] Liu S X. The study of effect and mechanisms of hydroxysafflor yellow A on anoxia/reoxygenation-induced apoptosis of H9c2 cardiomyocytes[D]. Hangzhou: Zhejiang University, 2011.
[13] 董曦, 孙桂波, 罗云, 等.异鼠李素对H2O2引起的H9c2细胞氧化应激损伤的保护作用研究[J]. 中国药理学通报, 2015, 31(6):853-60.[13] Dong X, Sun G B, Luo Y, et al. Protective effect of isorhamnetin on H9c2 cell line against oxidative stress[J]. Chin Pharmacol Bull, 2015, 31(6):853-60.
[14] 谢飞, 魏珂, 闵苏, 等.神经生长因子对H9c2心肌细胞缺氧/复氧后凋亡的保护作用及其机制[J]. 中国药理学通报, 2014, 30(4):506-10.[14] Xie F, Wei K, Min S, et al. Anti-apoptotic effect of NGF on H9c2 cardiac myocytes in a hypoxia/reoxygenation injury model [J]. Chin Pharmacol Bull, 2014, 30(4):506-10.
[15] Meng X, Wang M, Wang X, et al. Suppression of NADPH oxidase- and mitochondrion-derived superoxide by notoginsenoside R1 protects against cerebral ischemia-reperfusion injury through estrogen receptor-dependent activation of Akt/Nrf2 pathways[J]. Free Radical Res, 2014,48(7):823-38.