柚皮苷预处理通过抑制内质网应激凋亡途径减轻心肌细胞缺氧/复氧损伤
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摘要
目的研究柚皮苷(naringin,Nar)对H9c2心肌细胞缺氧/复氧(hypoxia/reoxygenation,H/R)损伤中内质网应激凋亡途径的影响及其分子作用机制。方法体外培养H9c2心肌细胞,并随机分为5组:正常对照组(C)、缺氧/复氧组(H/R)、Nar低剂量组(L)、中剂量组(M)和高剂量组(H)。C组心肌细胞正常培养至实验结束,H/R组行缺氧4 h后复氧24 h,H/R+Nar低、中、高剂量组分别于缺氧前6 h给予10、20、40 mg·L~(-1)的Nar培养,再行缺氧4 h后复氧24 h。实验后,MTT法测定细胞存活率;TUNEL染色检测心肌细胞凋亡;Western blot检测CCAAT/增强子结合蛋白同源蛋白(CHOP)、活化转录因子4(ATF4)、真核翻译起始因子2α(eIF2α)及其磷酸化水平(p-eIF2α)、双链RNA样内质网激酶(PERK)及其磷酸化水平(p-PERK)。结果与C组相比,H/R组明显降低H9c2心肌细胞存活率,诱导细胞凋亡,增加CHOP、ATF4、p-eIF2α/eIF2α和p-PERK/PERK表达(P<0.05);与H/R组比较,Nar 3个剂量组均能提高H9c2心肌细胞存活率,降低细胞凋亡,CHOP、ATF4、p-eIF2α/eIF2α和p-PERK/PERK表达下调,以Nar中、高剂量组效果最明显(P<0.05)。结论 Nar预处理可以减轻心肌细胞H/R损伤所致的心肌细胞凋亡,提示PERK-eIF2α-ATF4-CHOP途径参与Nar减轻内质网应激相关凋亡的作用。
Aim To study the effect of naringin(Nar) on endoplasmic reticulum stress(ERS)-induced apoptosis in H9c2 myocardial cell hypoxia/reoxygenation (H/R) injury and its molecular mechanism.Methods H9c2 cells were cultured in vitro and randomly clas-sified into five groups: normal control(group C), H/R( H/R) injury and its molecular mechanism. Methods H9c2 cells were cultured in vitro and randomly classified into five groups : normal control( group C),H/R group,H/R with Nar 10 mg·L~(-1)( group L),H/R with Nar 20 mg·L~(-1)( group M) and H/R with Nar 40 mg· L~(-1)( group H). Myocardial cells were normally cultured until the end of the experiment in group C.The myocardial cells were treated by hypoxia for 4 h before reoxygenation for 24 h in group H/R. The myocardial cells were cultured with Nar( 10,20,40 mg·L~(-1)) respectively 6 h before hypoxia,and after 6 h they were treated by hypoxia for 4 h before reoxygenation for 24 h in group L,group M and group H. The survival rate of cells was determined by MTT method after experiment. The apoptosis of cardiomyocytes was detected by TUNEL. CCAAT/enhancer-binding protein-homologous protein( CHOP),activating transcription factor-4( ATF4),eukaryotic initiation factor 2α( eIF2α),p-eIF2α,double-stranded RNA like endoplasmic reticulum kinase( PERK) and p-PERK were all assessed by Western blot. Results Compared withgroup C,H9c2 cell viability decreased,induced apoptosis and the protein expression of CHOP,ATF4,peIF2α/eIF2α and p-PERK/PERK in group H/R were significantly increased( P<0.05). Compared with group H/R,H9c2 cell viability increased,the apoptosis and the protein expression of CHOP,ATF4,peIF2α/eIF2α and p-PERK/PERK were reduced in group L,group M and group H. Of the three groups,group M and group H showed the most significant effect( P<0.05). Conclusions The Nar pretreatment can reduce myocardial cell apoptosis caused by H/R injury,suggesting that Nar can help to relieve the ERS-associated apoptosis through the PERK-eIF2α-ATF4-CHOP pathway.
Aim To study the effect of naringin(Nar) on endoplasmic reticulum stress(ERS)-induced apoptosis in H9c2 myocardial cell hypoxia/reoxygenation (H/R) injury and its molecular mechanism.Methods H9c2 cells were cultured in vitro and randomly clas-sified into five groups: normal control(group C), H/R( H/R) injury and its molecular mechanism. Methods H9c2 cells were cultured in vitro and randomly classified into five groups : normal control( group C),H/R group,H/R with Nar 10 mg·L~(-1)( group L),H/R with Nar 20 mg·L~(-1)( group M) and H/R with Nar 40 mg· L~(-1)( group H). Myocardial cells were normally cultured until the end of the experiment in group C.The myocardial cells were treated by hypoxia for 4 h before reoxygenation for 24 h in group H/R. The myocardial cells were cultured with Nar( 10,20,40 mg·L~(-1)) respectively 6 h before hypoxia,and after 6 h they were treated by hypoxia for 4 h before reoxygenation for 24 h in group L,group M and group H. The survival rate of cells was determined by MTT method after experiment. The apoptosis of cardiomyocytes was detected by TUNEL. CCAAT/enhancer-binding protein-homologous protein( CHOP),activating transcription factor-4( ATF4),eukaryotic initiation factor 2α( eIF2α),p-eIF2α,double-stranded RNA like endoplasmic reticulum kinase( PERK) and p-PERK were all assessed by Western blot. Results Compared withgroup C,H9c2 cell viability decreased,induced apoptosis and the protein expression of CHOP,ATF4,peIF2α/eIF2α and p-PERK/PERK in group H/R were significantly increased( P<0.05). Compared with group H/R,H9c2 cell viability increased,the apoptosis and the protein expression of CHOP,ATF4,peIF2α/eIF2α and p-PERK/PERK were reduced in group L,group M and group H. Of the three groups,group M and group H showed the most significant effect( P<0.05). Conclusions The Nar pretreatment can reduce myocardial cell apoptosis caused by H/R injury,suggesting that Nar can help to relieve the ERS-associated apoptosis through the PERK-eIF2α-ATF4-CHOP pathway.
引文
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[12] 李璐,蔡志亮,贺翼飞,等.内质网应激在黄芪甲苷诱导的心肌线粒体保护中的作用[J].中国药理学通报,2017,33(6):854-8. Li L,Cai Z L,He Y F,et al.Role of endoplasmic reticulum stress in astragaloside Ⅳ-induced cardioprotection in H9c2 cardiac cells[J]. Chin Pharmacol Bull,2017,33(6):854-8.
[13] Du Y, Wang M, Liu X, et al. Araloside C prevents hypoxia/reoxygenation-induced endoplasmic reticulum stress via increasing heat shock protein 90 in H9c2 cardiomyocytes[J].Front Pharmacol,2018,9:180.
[14] Wang X,Jia D,Zhang J,et al.Grape seed proanthocyanidins protect cardiomyocytes against hypoxia/reoxygenation injury by attenuating endoplasmic reticulum stress through PERK/eIF2α pathway[J]. Mol Med Rep,2017,16(6):9189-96.
[2] Cerkezkayabekir A,Sanal F,Bakar E,et al.Naringin protects viscera from ischemia/reperfusion injury by regulating the nitric oxide level in a rat model[J].Biotech Histochem,2017,92(4):252-63.
[3] Okuyama S,Yamamoto K,Mori H,et al. Neuroprotective effect of Citrus kawachiensis(Kawachi Bankan) peels, a rich source of naringin, against global cerebral ischemia/reperfusion injury in mice[J]. Biosci Biotechnol Biochem,2018,82(7):1216-24.
[4] 张羽飞,孟娜娜,李厚忠,等.柚皮苷对糖尿病大鼠心肌病氧化应激及内质网应激的影响[J].中国中药杂志,2018,43(3):596-602. Zhang Y F,Meng N N,Li H Z,et al.Effect of naringin on oxidative stress and endoplasmic reticulum stress in diabetic cardiomyopathy[J]. China J Chin Mater Med,2018,43(3):596-602.
[5] 郑晓佳,余婷,张华, 等.表没食子儿茶素没食子酸酯对心肌细胞缺氧/复氧损伤的抑制作用及其机制[J].中国药理学通报,2017,33(11):1584-8. Zheng X J,Yu T,Zhang H,et al.Protective effect of epigallocatechin-3-gallate on hypoxia /reoxygenation injury in cardiac myocytes and its mechanism[J].Chin Pharmacol Bull,2017,33(11):1584-8.
[6] Jian C Y,Ouyang H B,Xiang X H, et al.Naringin protects myocardial cells from doxorubicin-induced apoptosis partially by inhibiting the p38MAPK pathway[J].Mol Med Rep,2017,16(6):9457-63.
[7] Cai L,Wu H,Tu C,et al.Naringin inhibits ovarian tumor growth by promoting apoptosis:an in vivo study[J].Oncol Lett,2018,16(1):59-64.
[8] 简春燕,郭润民,刘丹铭, 等.柚皮苷保护H9c2心肌细胞对抗阿霉素诱导的心肌毒性[J].中国药理学通报,2014,30(2):238-43. Jian C Y,Guo R M,Liu D M,et al.Naringin protects H9c2 cells against doxorubicin-induced cardiotoxicity[J]. Chin Pharmacol Bull,2014,30(2):238-43.
[9] Chen R C,Sun G B,Wang J,et al.Naringin protects against anoxia/reoxygenation-induced apoptosis in H9c2 cells via the Nrf2 signaling pathway[J].Food Funct,2015,6(4):1331-44.
[10] 林溢煌,方莲花,杜冠华.心肌缺血/再灌注中RhoA 的调控作用研究进展[J].中国药理学通报,2015,31(10): 1336-9. Lin Y H,Fang L H,Du G H,et al. Research advances on regulatory effect of RhoA on myocardial ischemia/reperfusion injury[J].Chin Pharmacol Bull,2015,31(10): 1336-9.
[11] Ma L L,Ma X,Kong F J, et al. Mammalian target of rapamycin inhibition attenuates myocardial ischaemia-reperfusion injury in hypertrophic heart[J].J Cell Mol Med,2018,22(3):1708-19.
[12] 李璐,蔡志亮,贺翼飞,等.内质网应激在黄芪甲苷诱导的心肌线粒体保护中的作用[J].中国药理学通报,2017,33(6):854-8. Li L,Cai Z L,He Y F,et al.Role of endoplasmic reticulum stress in astragaloside Ⅳ-induced cardioprotection in H9c2 cardiac cells[J]. Chin Pharmacol Bull,2017,33(6):854-8.
[13] Du Y, Wang M, Liu X, et al. Araloside C prevents hypoxia/reoxygenation-induced endoplasmic reticulum stress via increasing heat shock protein 90 in H9c2 cardiomyocytes[J].Front Pharmacol,2018,9:180.
[14] Wang X,Jia D,Zhang J,et al.Grape seed proanthocyanidins protect cardiomyocytes against hypoxia/reoxygenation injury by attenuating endoplasmic reticulum stress through PERK/eIF2α pathway[J]. Mol Med Rep,2017,16(6):9189-96.