Effects of Exogenous Spermine on Radiation-induced Oxidative Injury of H9c2 Cardiomyocytes

doi:10.12114/j.issn.1007-9572.2021.01.017

Abstract

Abstract: Background　During radiotherapy，a therapy for increasing number of cancer patients，the normal organ may be injured unavoidably due to exposure to radiation. Spermine is a polyamine with high biological activity. Studies have proved that exogenous spermine has a certain ROS-like function. We attempted to explore the effect of exogenous spermine on cardiomyocytes of the heart，an organ often damaged by radiation in radiotherapy. Objective　To explore the effect of exogenous spermine on radiation-induced oxidative injury of H9c2 cardiomyocytes. Methods　This study was carried out from December 2018 to December 2020. H9c2 cardiomyocytes in the logarithmic growth phase were divided into three spermine groups and a blank group： 100 μmol/L，200 μmol/L，400 μmol/L spermine groups，and a blank group（treated with culture medium alone）. CCK8 assay was used to determine the number of viable cells at 12，24，48 h in each group. Then according to previous screening results，the H9c2 cardiomyocytes cultured in vitro were taken and divided into four groups： blank group（DMEM medium），radiation damage group（X-ray irradiation only），low drug concentration combined radiation group（100 μmol/L spermine-pretreatment，followed by X-ray irradiation）and high drug concentration combined radiation group（200 μmol/L spermine-pretreatment，followed by X-ray irradiation）. Cells in each group were irradiated vertically by a medical linear accelerator，then were cultured for 0，12，24 and 48 hours after processing to X-ray irradiation. Apoptosis was detected by flow cytometry. Fluorescence microscope was used to observe the state of cells after X-ray irradiation. Malondialdehyde（MDA） ELISA Kit was used to measure the MDA of cells. Total superoxide dismutase（T-SOD） kits were used to detect the SOD vitality of cells. Results　The survival rate of cells in 100 μmol/L，200 μmol/L or 400 μmol/L group at 24 h was higher than that at 12 h and 48 h（P<0.05）. At 12 hours after treatment ： the survival rate of blank group was higher than that of three spermine groups（100 μmol/L，200 μmol/L and 400 μmol/L）（P<0.05）. The survival rate of 100 μmol/L or 200 μmol/L group was higher than that of 400 μmol/L group（P<0.05）. At 24 hours after treatment： the survival rate of blank group was higher than that of 100 μmol/L group and 400 μmol/L group（P<0.05）. The survival rate of 100 μmol/L group was lower than that of 200 μmol/L group，but higher than that of 400 μmol/L group（P<0.05）. The survival rate of 200 μmol/L group was higher than that of 400 μmol/L group（P<0.05）. At 48 hours after treatment： the survival rate of blank group was higher than that of 100 μmol/L，200 μmol/L and 400 μmol/L groups（P<0.05）. The survival rate of 100 μmol/L group was lower than that of 200 μmol/L group，but higher than that of 400 μmol/L group（P<0.05）. The survival rate of 200 μmol/L group was higher than that of 400 μmol/L group（P<0.05）. Apoptosis at 0 hour after treatment： the blank group had lower apoptosis rate than radiation+100 μmol/L spermine treatment group，and radiation +200 μmol/L spermine treatment group（P<0.05）. The apoptosis rate of radiation group was lower than that of radiation +100 μmol/L spermine group and radiation +200 μmol/L spermine group（P<0.05）. The apoptosis rate of radiation +200 μmol/L spermine group was lower than that of radiation +100 μmol/L spermine group（P<0.05）. Similar inter-group comparative results of apoptosis were also found at 12，24 and 48 hours after treatment. In terms of comparison for single group，as for the blank group，the apoptosis rate at 0 hour after treatment was lower than that at 12 and 48 hours after treatment（P<0.05），the apoptosis rate at 12 hours after treatment was lower than that at 24 hours after treatment（P<0.05），and the apoptosis rate at 24 hours after treatment was lower than that at 48 hours after treatment（P<0.05）. For the radiation +200 μmol/L spermine group，the apoptosis rate at 24 hours after treatment was lower compared with that at 0，12 and 48 hours after treatment（P<0.05）；the apoptosis rate at 12 hours after treatment was lower compared with that at 0，and 48 hours after treatment（P<0.05）；the apoptosis rate at 24 hours after treatment was lower compared with that at 48 hours after treatment（P<0.05）. For radiation +100 μmol/L spermine group，the apoptosis rate at 24 hours after treatment was lower than that at 0 and 48 hours after treatment（P<0.05）；the apoptosis rate at 12 hours after treatment was lower than that at 0 and 48 hours after treatment（P<0.05）. For the radiation group，the apoptosis rate at 0 hour after treatment was lower than that at 12，24 and 48 hours after treatment（P<0.05），the apoptosis rate at 12 hours after treatment was lower than that at 24 and 48 hours after treatment（P<0.05），and the apoptosis rate at 24 hours after treatment was lower than that at 48 hours after treatment（P<0.05）. At 12 hours after treatment，the blank group had significantly different SOD activity and MDA levels compared to radiation +100 μmol/L spermine group，radiation +200 μmol/L spermine group and radiation group（P<0.05）. The radiation group had higher SOD activity level and lower MDA level than radiation +100 μmol/L spermine group（P<0.05）. The radiation group had higher SOD activity level than radiation +200 μmol/L spermine group（P<0.05）. The radiation +100 μmol/L spermine group had lower SOD activity level and higher MDA level than radiation +200 μmol/L spermine group（P<0.05）. At 24 hours after treatment，the blank group showed significantly different SOD activity and MDA levels compared to radiation +100 μmol/L spermine group，radiation +200 μmol/L spermine group and radiation group（P<0.05）. The SOD activity level in the radiation group was lower than that in radiation +100 μmol/L spermine group and radiation +200 μmol/L spermine group（P<0.05）. The radiation group had higher MDA level than radiation +200 μmol/L spermine group（P<0.05）. The radiation +100 μmol/L spermine group had lower SOD activity level and higher MDA level than radiation +200 μmol/L spermine group（P<0.05）. At 48 hours after treatment，the blank group demonstrated significantly different SOD activity and MDA levels compared to radiation +100 μmol/L spermine group，radiation +200 μmol/L spermine group and radiation group（P<0.05）. The radiation group had lower SOD activity level and higher MDA level than radiation +200 μmol/L spermine group（P<0.05）. Compared with radiation +200 μmol/L spermine group，the SOD activity level was lower and MDA level was higher in radiation +100 μmol/ L spermine group，with statistical difference（P<0.05）. After 24-hour X-ray irradiation，H9c2 cardiomyocytes in the blank group were long spindle-shaped，with complete nucleus and uniform staining. In contrast，H9c2 cardiomyocytes in radiation +100 μmol/L spermine group，radiation +200 μmol/L spermine group and radiation group showed different degrees of changes，such as rounded shape with nuclear breakage，uneven staining，and so on，and the cell morphology of the 200 μmol/L spermine group had the slightest changes，which was most similar to that of blank group. Conclusion　Exogenous spermidine could protect H9c2 cardiomyocytes against radiation-induced oxidative injury，and the protective effect may be dependent on concentration and time. Within a certain concentration range，the protective effect of spermine was stronger when it was at a high concentration. And its protective effect was the strongest when the cells were treated at each concentration for 24 hours.

Key words: Radiation Injuries；Spermine；Radiation protection；Cardiomyocytes；Myocytes, cardiac

摘要： 背景　恶性肿瘤的发病率不断上升，放疗所致的正常器官的辐射损伤难以避免，精胺是具有极高生物活性的一种多胺类物质，研究证明外源性精胺具有一定的活性氧清除剂作用，而心脏作为常见的辐射损伤器官，外源性精胺对辐射损伤的心肌细胞的影响研究较少。目的　探讨精胺对辐射所致氧化损伤H9c2心肌细胞的影响。方法　2018年12月至2020年12月，取对数生长期的H9c2心肌细胞加入精胺，分为100 μmol/L组、200 μmol/L组、400 μmol/L组，空白对照组为加等量不含药物的培养液，采用CCK8溶液计算各组细胞培养12、24、48 h的存活率。根据前期筛选结果将体外培养的H9c2心肌细胞分为空白对照组（单纯DMEM培养基）、单纯辐射组（单纯X线照射）、辐射+100 μmol/L精胺组及辐射+200 μmol/L精胺组；各组用医用直线加速器进行照射，X线照射后继续培养0、12、24、48 h，计算细胞凋亡率。使用流式细胞仪检测细胞凋亡情况，荧光显微镜拍摄各组细胞照射后状态，丙二醛（MDA）试剂盒检测各组细胞MDA水平，总超氧化物歧化酶（T-SOD）试剂盒检测各组细胞的SOD活性。结果　100 μmol/L组、200 μmol/L组、400 μmol/L组24 h细胞存活率高于12、48 h（P<0.05）。处理时间12 h：空白对照组细胞存活率高于100 μmol/L组、200 μmol/L组及400 μmol/L组（P<0.05）；100 μmol/L组、200 μmol/L组细胞存活率高于400 μmol/L组（P<0.05）。处理时间24 h：空白对照组细胞存活率高于100 μmol/L组、400 μmol/L组（P<0.05）；100 μmol/L组细胞存活率低于200 μmol/L组，高于400 μmol/L组（P<0.05）；200 μmol/L细胞存活率高于400 μmol/L组（P<0.05）。处理时间48 h：空白对照组细胞存活率高于100 μmol/L组、200 μmol/L组及400 μmol/L组（P<0.05）；100 μmol/L组细胞存活率低于200 μmol/L组，高于400 μmol/L组（P<0.05）；200 μmol/L组细胞存活率高于400 μmol/L组（P<0.05）。处理时间0 h：辐射+200 μmol/L精胺组、辐射+100 μmol/L精胺组细胞凋亡率高于空白对照组（P<0.05）；辐射+200 μmol/L精胺组、辐射+100 μmol/L精胺组细胞凋亡率高于单纯辐射组（P<0.05）；辐射+200 μmol/L精胺组细胞凋亡率低于辐射+100 μmol/L精胺组（P<0.05）。处理时间为12、24、48 h细胞凋亡情况同处理时间为0 h。空白对照组0 h细胞凋亡率低于12、48 h（P<0.05），12 h细胞凋亡率低于24 h（P<0.05），24 h细胞凋亡率低于48 h（P<0.05）。辐射+200 μmol/L精胺组24 h细胞凋亡率低于0 h、12 h和48 h（P<0.05），12 h细胞凋亡率低于0、48 h（P<0.05），24 h细胞凋亡率低于48 h（P<0.05）。辐射+100 μmol/L精胺组24 h细胞凋亡率低于0、48 h（P<0.05），12 h细胞凋亡率低于0、48 h（P<0.05）。单纯辐射组0 h细胞凋亡率低于12、24、48 h（P<0.05）；12 h细胞凋亡率低于24、48 h（P<0.05）；24 h细胞凋亡率比48 h低（P<0.05）。处理时间为12 h时：空白对照组与辐射+200 μmol/L精胺组、辐射+100 μmol/L精胺组、单纯辐射组SOD活性、MDA水平比较，差异有统计学意义（P<0.05）；单纯辐射组比辐射+100 μmol/L精胺组细胞及辐射+200 μmol/L精胺组细胞SOD活性高，比辐射+100 μmol/L精胺组细胞MDA水平低（P<0.05）；辐射+100 μmol/L精胺组比辐射+200 μmol/L精胺组细胞SOD活性低、MDA水平高（P<0.05）。处理时间为24 h时：空白对照组与辐射+200 μmol/L精胺组、辐射+100 μmol/L精胺组、单纯辐射组SOD活性、MDA水平比较，差异有统计学意义（P<0.05）；单纯辐射组比辐射+100 μmol/L精胺组及辐射+200 μmol/L精胺组细胞SOD活性低，比辐射+200 μmol/L精胺组细胞MDA水平高（P<0.05）；辐射+100 μmol/L精胺组比辐射+200 μmol/L精胺组细胞SOD活性低、MDA水平高（P<0.05）。处理时间为48 h时：空白对照组与辐射+200 μmol/L精胺组、辐射+100 μmol/L精胺组、单纯辐射组SOD活性、MDA水平比较，差异有统计学意义（P<0.05）；单纯辐射组比辐射+200 μmol/L精胺组细胞SOD活性低、MDA水平高（P<0.05）；辐射+100 μmol/L精胺组比辐射+200 μmol/L精胺组细胞SOD活性低、MDA水平高（P<0.05）。给予X线照射24 h后，空白对照组H9c2心肌细胞形态呈长梭形，细胞核形态完整，染色均匀；辐射+200 μmol/L精胺组、辐射+100 μmol/L精胺组、单纯辐射组的H9c2心肌细胞出现不同程度的变化，如细胞形态变圆、细胞核碎裂、染色不均匀等，其中200 μmol/L精胺组的细胞形态最接近空白对照组细胞，变化程度较轻。结论　精胺对辐射导致的心肌细胞的氧化损伤具有保护作用，且其保护作用与浓度及时间有关，在一定浓度范围内高浓度精胺的保护能力更强，各浓度精胺处理24 h时的细胞保护能力最强。

关键词: 辐射损伤；精胺；辐射防护；心肌细胞；肌细胞, 心脏

DU Xiao,XU Chao,TU Yu, et al. Effects of Exogenous Spermine on Radiation-induced Oxidative Injury of H9c2 Cardiomyocytes　[J]. Chinese General Practice, 2021, 24(29): 3716-3723. DOI: 10.12114/j.issn.1007-9572.2021.01.017.
杜霄,许超,涂彧等. 精胺对辐射所致氧化损伤H9c2心肌细胞的影响研究[J]. 中国全科医学, 2021, 24(29): 3716-3723. DOI: 10.12114/j.issn.1007-9572.2021.01.017.

References

［1］NOWOTARSKI S L，WOSTER P M，CASERO R A Jr. Polyamines and cancer： implications for chemotherapy and chemoprevention［J］. Expert Rev Mol Med，2013，15： e3. DOI：10.1017/erm.2013.3.
［2］PEGG A E. Mammalian polyamine metabolism and function［J］. IUBMB Life，2009，61（9）： 880-894. DOI：10.1002/iub.230.
［3］HE Y，YANG J，LI H，et al. Exogenous spermine ameliorates high glucose-induced cardiomyocytic apoptosis via decreasing reactive oxygen species accumulation through inhibiting p38/JNK and JAK2 pathways［J］. Int J Clin Exp Pathol，2015，8（12）： 15537-15549.
［4］杨金霞，徐长庆，王秀丽，等.外源性精胺对大鼠离体胸主动脉环的收缩作用［J］.哈尔滨医科大学学报，2007（2）：98-101.
YANG J X，XU C Q，WANG X L，et al. The contracting effects of exogenous spermine on rat's thoracic aortic artery ring［J］.Journal of Harbin Medical University，2007（2）：98-101.
［5］HONG M，HE J，LI D，et al. Runt-related transcription factor 1 promotes apoptosis and inhibits neuroblastoma progression in vitro and in vivo［J］. J Exp Clin Cancer Res，2020，39（1）： 52. DOI：10.1186/s13046-020-01558-2.
［6］KUCHERYAVYKH L Y，BENEDIKT J，CUBANO L A，et al. Polyamines preserve connexin 43-mediated gap junctional communication during intracellular hypercalcemia and acidosis［J］. Neuroreport，2017，28（4）： 208-213. DOI：10.1097/WNR.0000000000000746.
［7］TALMON G，FINK D L，HOROWITZ Y，et al. The prevalence of subclinical myocarditis among young children with acute viral infection［J］. Harefuah，2015，154（10）： 641-645，676，675.
［8］MASSARO C，THOMAS J，PHANSTIEL IV O. Investigation of polyamine metabolism and homeostasis in pancreatic cancers［J］. Med Sci （Basel），2017，5（4）： E32. DOI：10.3390/medsci5040032.
［9］GEVREKCI A ?. The roles of polyamines in microorganisms［J］. World J Microbiol Biotechnol，2017，33（11）： 204. DOI：10.1007/s11274-017-2370-y.
［10］HINDERER C，KATZ N，LOUBOUTIN J P，et al. Abnormal polyamine metabolism is unique to the neuropathic forms of MPS： potential for biomarker development and insight into pathogenesis［J］. Hum Mol Genet，2017，26（19）： 3837-3849. DOI：10.1093/hmg/ddx277.
［11］TANTINI B，FIUMANA E，CETRULLO S，et al. Involvement of polyamines in apoptosis of cardiac myoblasts in a model of simulated ischemia［J］. J Mol Cell Cardiol，2006，40（6）： 775-782. DOI：10.1016/j.yjmcc.2006.03.002.
［12］MADEO F，EISENBERG T，BüTTNER S，et al. Spermidine： a novel autophagy inducer and longevity elixir［J］. Autophagy，2010，6（1）： 160-162. DOI：10.4161/auto.6.1.10600.
［13］WEI C，LI H Z，WANG Y H，et al. Exogenous spermine inhibits hypoxia/ischemia-induced myocardial apoptosis via regulation of mitochondrial permeability transition pore and associated pathways［J］. Exp Biol Med （Maywood），2016，241（14）： 1505-1515. DOI：10.1177/1535370216643417.
［14］PEGG A E. Toxicity of polyamines and their metabolic products［J］. Chem Res Toxicol，2013，26（12）： 1782-1800. DOI：10.1021/tx400316s.
［15］VON DEUTSCH A W，MITCHELL C D，WILLIAMS C E，et al. Polyamines protect against radiation-induced oxidative stress［J］. Gravitational Space Biol Bull，2005，18（2）： 109-110.
［16］AN N，LIU T T，ZHU B L，et al. A bidirectional effect of Rac1 inhibition-Protects radiation-induced intestinal injury while inhibits tumor［J］. Life Sci，2020，240： 117105. DOI：10.1016/j.lfs.2019.117105.
［17］GRANCARA S，OHKUBO S，ARTICO M，et al. Milestones and recent discoveries on cell death mediated by mitochondria and their interactions with biologically active amines［J］. Amino Acids，2016，48（10）： 2313-2326. DOI：10.1007/s00726-016-2323-z.
［18］DOUKI T，BRETONNIERE Y，CADET J. Protection against radiation-induced degradation of DNA bases by polyamines［J］. Radiat Res，2000，153（1）： 29-35. DOI：10.1667/0033-7587（2000）153.
［19］YANG J X，MA Y，MU S，et al. Damaging effects of exogenous spermine on HUVECs and its possible mechanism ［J］. Chin J Pathophysiol，2009，25（10）：1903-1906.
［20］HA K C，CHAE H J，PIAO C S，et al. Dendroaspis natriuretic peptide induces the apoptosis of cardiac muscle cells［J］. Immunopharmacol Immunotoxicol，2005，27（1）： 33-51. DOI：10.1081/iph-51292.
［21］CAO W，WU X J，JIA G，et al. Effects of dietary spermine supplementation on cell cycle，apoptosis，and amino acid transporters of the Thymus and spleen in piglets［J］. Asian-Australas J Anim Sci，2018，31（8）： 1325-1335. DOI：10.5713/ajas.17.0799.
［22］ABU-KHUDIR R，HABIEB M E，MOHAMED M A，et al. Anti-apoptotic role of spermine against lead and/or gamma irradiation-induced hepatotoxicity in male rats［J］. Environ Sci Pollut Res Int，2017，24（31）： 24272-24283. DOI：10.1007/s11356-017-0069-0.
［23］ZHANG P，YANG P，HONG L，et al. Oxidative stress in radiation-induced cardiotoxicity［J］. Oxidative Med Cell Longev，2020，2020： 3579143. DOI：10.1155/2020/3579143.
［24］KADIVAR F，HADDADI G，MOSLEH-SHIRAZI M A，et al. Protection effec of cerium oxide nanoparticles against radiation-induced acute lung injuriesin rats［J］.Rep Pract Oncol Radiother，2020，25（2）：206-211. DOI：10.1016/j.rpor.2019.12.023.
［25］AZZAM E I，JAY-GERIN J P，PAIN D. Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury［J］. Cancer Lett，2012，327（1/2）： 48-60. DOI：10.1016/j.canlet.2011.12.012.
［26］WANG R，XU C Q，ZHAO W M，et al. Calcium and polyamine regulated calcium-sensing receptors in cardiac tissues［J］. Eur J Biochem，2003，270（12）： 2680-2688. DOI：10.1046/j.1432-1033.2003.03645.x.

[1]	ZHANG Yu, WANG Meixia, ZHANG Jing, WANG Fei, LI Zhonglei, ZHANG Yanyun, JIANG Zhenzhen. Evaluation of Cardiac Structure and Function in Patients with Wilson Disease Based on RT-3DE: a Clinical Study [J]. Chinese General Practice, 2023, 26(29): 3689-3697.
[2]	Expert group of expert consensus on the application of ulinastatin in common clinical critical illness. Expert Consensus on the Application of Ulinastatin in Common Clinical Critical Illness [J]. Chinese General Practice, 2023, 26(26): 3207-3219.
[3]	ZHANG Dufei, CHEN Renwei, MO Zelai, YANG Ling, WANG Yazhou, WANG Haifan. Application of Appropriate Technology for Screening, Diagnosis and Evaluation of Congenital Heart Disease in Neonates in Hainan Province [J]. Chinese General Practice, 2023, 26(25): 3170-3177.
[4]	LIU Jingtao, SU He, QIN Xiaojin, LAN Yunxia, ZHANG Jinzhi. Guidelines on Cardiac Rehabilitation in Patients with Coronary Heart Disease: a Systematic Review [J]. Chinese General Practice, 2023, 26(19): 2323-2331.
[5]	LIU Lu, JIANG Yunlan, PENG Hanmei, LU Yutong, LIU Mingting, LIAO Shiqin. Dysphagia Incidence after Cardiac Surgery: a Meta-analysis [J]. Chinese General Practice, 2023, 26(18): 2293-2300.
[6]	CHEN Yilin, LIN Ping, HAN Yongkui, WANG Yini. Research on the Construction of Adherence and Its Influencing Factors of Patients with Percutaneous Coronary Intervention in Phase Ⅰ Cardiac Rehabilitation [J]. Chinese General Practice, 2023, 26(18): 2209-2216.
[7]	YU Xinyan, ZHAO Xudong, ZHAO Xiaoye, LIU Haipeng, JIANG Qingru, ZHANG Haicheng. Diagnostic Accuracies of Three Schemes for Arrhythmia Screening Using a Wearable Single-lead ECG Monitoring System: a Real-world Community-based Study [J]. Chinese General Practice, 2023, 26(02): 192-200.
[8]	WANG Zhe, ZHAO Haibin, WANG Guoliang, MA Xiaojuan, YIN Huijun. Complete Revascularization for Acute Myocardial Infarction and Multivessel Disease: an Overview of Systematic Reviews [J]. Chinese General Practice, 2023, 26(02): 142-153.
[9]	JIA Siqi, WANG Di, LU Yan, JIA Yongping. Effects of High-intensity Interval Training on Cardiac Structure and Function in a Rat Model of Myocardial Infarction [J]. Chinese General Practice, 2022, 25(33): 4159-4167.
[10]	LI Yong, WANG Ying, CHEN Xun, LIN Wei, ZHANG Kai, DING Ning. Relationship between Pulse Oxygen Desaturation Rate and Heart Rate Fluctuation Rate and Arrhythmias in Patients with Obstructive Sleep Apnea Hypopnea Syndrome [J]. Chinese General Practice, 2022, 25(33): 4185-4190,4195.
[11]	Jie LIU, Qiaozhi LIU, Shengjue XIAO, Defeng PAN. SPECT in Assessment of Left Ventricular Diastolic Function in Coronary Heart Disease [J]. Chinese General Practice, 2022, 25(29): 3646-3651,3657.
[12]	Husheng LI, Feng HUANG, Yuting GAO, Yiyan WANG, Jiaqi WANG, Jing WU. Association of Preoperative Glycosylated Hemoglobin with the Prognosis of Adult Patients Undergoing Cardiac Surgery: a Meta-analysis [J]. Chinese General Practice, 2022, 25(17): 2135-2144.
[13]	Lida YIN, Jiasen WANG, Yongchao HE, Aiqun LI. A Meta-analysis of the Effect of Unprofessional Early Defibrillation on the Survival Rate of Patients with Out-of-hospital Cardiac Arrest [J]. Chinese General Practice, 2022, 25(14): 1757-1764.
[14]	GUO Yuyang, ZENG Qingchun. Actively Managing Obesity to Reduce the Risk of Cardiovascular Disease [J]. Chinese General Practice, 2022, 25(06): 643-650.
[15]	HUANG Baotao. Prediction and Management of People at High Risk of Sudden Death after Myocardial Infarction Who are not Covered by Guidelines [J]. Chinese General Practice, 2022, 25(05): 515-521.