钩吻素子对H_2O_2诱导的IPEC-J2细胞氧化应激和凋亡的影响
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摘要
目的:探讨钩吻素子(koumine)对H_2O_2诱导的猪肠道上皮细胞(IPEC-J2)损伤的保护作用及其机制。方法:通过MTT法检测koumine对IPEC-J2细胞增殖的影响,并用H_2O_2诱导IPEC-J2细胞建立细胞损伤模型,通过MTT和LDH比色法检测分析koumine保护IPEC-J2细胞免受H_2O_2损伤的效果;生化方法检测丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性;蛋白免疫印迹(western blot)技术检测凋亡相关蛋白bax和bcl-2的表达情况;用流式细胞仪检测氧自由基(ROS)释放量和细胞凋亡率。结果:检测浓度范围内koumine对IPEC-J2细胞没有毒性;koumine可以通过调控IPEC-J2细胞中SOD活性、LDH漏出率、MDA水平和ROS的产生,保护H_2O_2对IPEC-J2细胞的氧化损伤,抑制H_2O_2引起的IPEC-J2细胞凋亡;koumine在抑制H_2O_2引起的细胞凋亡过程中,减少了促凋亡蛋白Bax表达,增加了抑凋亡蛋白Bcl-2表达,使得Bax/Bcl-2的比值在药物作用时随剂量的增加逐渐降低。结论:koumine能够保护IPEC-J2细胞对抗H_2O_2诱导的氧化损伤,具有一定的时间和剂量依赖关系,其作用机制与抑制细胞凋亡有关。
Objective:The present study was aimed at the anti-oxidative properties of koumine in H2 O2-mediated IPEC-J2 cells injury. Methods:The effect was evaluated by measuring and analyzing the changes on antioxidant capacity(SOD,LDH and MDA). The expression of Bax and Bcl-2 was measured by western blot assay,flow cytometry was used to detect ROS and apoptosis. Results:Treatment with H2 O2 could induce ROS,LDH and MDA accumulation in IPEC-J2 cells,accompanying with losses of activities of SOD. Pretreatment of koumine decreased H2 O2-induced cell apoptosis, and efficiently suppressed the intracellular ROS production LDH and MDA generation induced by H2 O2 treatment. Moreover,a loss of SOD activities were restored to normal level in H2 O2-induced IPEC-J2 cells upon koumine exposure. Also,the tendency of increased protein expression of Bax,as well as decrease of Bcl-2 protein in IPEC-J2 cells challenged with H2 O2 was changed to individual opposite way,thus inhibiting the apoptotic cell. Conclusion:The results demonstrated that koumine protected IPEC-J2 cells against oxidative injury through activation of anti-apoptotic and endogenous antioxidant enzymes signaling pathway.
Objective:The present study was aimed at the anti-oxidative properties of koumine in H2 O2-mediated IPEC-J2 cells injury. Methods:The effect was evaluated by measuring and analyzing the changes on antioxidant capacity(SOD,LDH and MDA). The expression of Bax and Bcl-2 was measured by western blot assay,flow cytometry was used to detect ROS and apoptosis. Results:Treatment with H2 O2 could induce ROS,LDH and MDA accumulation in IPEC-J2 cells,accompanying with losses of activities of SOD. Pretreatment of koumine decreased H2 O2-induced cell apoptosis, and efficiently suppressed the intracellular ROS production LDH and MDA generation induced by H2 O2 treatment. Moreover,a loss of SOD activities were restored to normal level in H2 O2-induced IPEC-J2 cells upon koumine exposure. Also,the tendency of increased protein expression of Bax,as well as decrease of Bcl-2 protein in IPEC-J2 cells challenged with H2 O2 was changed to individual opposite way,thus inhibiting the apoptotic cell. Conclusion:The results demonstrated that koumine protected IPEC-J2 cells against oxidative injury through activation of anti-apoptotic and endogenous antioxidant enzymes signaling pathway.
引文
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[2]王志睿,张忠义,黄昌全,等.钩吻素子对小鼠CD4+T淋巴细胞Th1类及Th2类细胞因子分泌的影响[J].中药材,2005,28(8):693-695.
[3]张兰兰,王志睿,黄昌全,等.钩吻总生物碱中钩吻素子的提取与分离[J].第一军医大学学报,2004,24(9):106-1008.
[4] Yuan Z,Matias F B,Wu J,et al.Koumine attenuates lipopolysaccaride-stimulated inflammation in RAW264.7macrophages,coincidentally associated with inhibition of NF-κB,ERK and p38 pathways[J].Int J Mol Sci,2016,17:430.
[5]许盈,邱宏强,沈洁,等.钩吻素子无吗啡样药物依赖性[J].福建医科大学学报,2013,47(4):210-213.
[6] Matsui Y,Takagi H,Qu X,et al.Distinct roles of autophagy in the heart during ischemia and reperfusion:roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy[J]. Circulation Research,2007,100(6):914-922.
[7] Pelicano H,Carney D,Huang P.ROS stress in cancer cells and therapeutic implications[J].Drug Resist Updates,2004,7(2):97-110.
[8] Scherz-Shouval R,Elazar Z.ROS,mitochondria and the regulation of autophagy[J].Trends in Cell Biology,2007,17(9):422-427.
[9] He H,Xu J,Xu Y,et al.Cardioprotective effects of saponins from Panax japonicus on acute myocardial ischemia against oxidative stress-triggered damage and cardiac cell death in rats[J].J Ethnopharmacol,2012,140(1):73-82.
[10] Torrealba N,Aranguiz P,Alonso C,et al. Mitochondrial oxidative stress and dysfunction in myocardial remodeling[J].Cardiovasc Research,2009,81(3):449-456.
[11] Kawai Y,Taniuchi S,Okahara S,et al.Relationship between cisplatin or nedaplatin-induced nephrotoxicity and renal accumulation[J].Biological and Pharmaceutical Bulletin,2005,28(8):1385-1388.