药学学报  2014, Vol. 49 Issue (8): 1117-1123   PDF    
麦冬皂苷D通过减轻内质网应激对阿霉素所致心肌损伤产生保护作用
孟晨1, 袁彩华1, 张晨晨1, 温明达1, 高艳红1,2, 丁小余1, 张赢予3,4, 张朝1,2     
1. 南京师范大学生命科学学院, 江苏省分子医学生物技术重点实验室、江苏省超分子医用材料及应用重点实验室, 江苏 南京 210023;
2. 南京医科大学, 江苏省心血管病转化医学协同创新中心, 江苏 南京 210029;
3. 江苏大学附属人民医院, 分子生物学和转化医学研究所, 江苏 镇江 212002;
4. 长春医学高等专科学校临床医学系, 吉林 长春 130013
摘要:研究麦冬皂苷D(ophiopogonin D,OP-D) 对阿霉素(doxorubicin,DOX) 所致心肌损伤的保护作用。体外培养H9c2细胞,采用MTT法检测细胞毒性,MitoTracker探针法测定细胞内线粒体中活性氧(reactive oxygen species,ROS) 含量,实时定量PCR和Western blotting分别检测ATF6α,GRP78和CHOP的mRNA及其蛋白表达。结果表明,DOX可诱导H9c2细胞内质网应激相关蛋白的表达量显著上升,并导致细胞活性氧ROS含量增加,细胞活力下降。而OP-D预处理可部分逆转DOX引起的上述变化,siRNA干扰促凋亡转录因子CHOP或抗氧化剂NAC预处理也有类似效应。此外,OP-D可明显减轻DOX所致小鼠心脏超微结构异常。这些结果说明,OP-D通过降低DOX诱导的ROS累积,进而缓解内质网应激而对心肌产生保护作用。
关键词阿霉素     麦冬皂苷D     心肌细胞     内质网应激    
Ophiopogonin D protects cardiomyocytes against doxorubicin-induced injury through suppressing endoplasmic reticulum stress
MENG Chen1, YUAN Cai-hua1, ZHANG Chen-chen1, WEN Ming-da1, GAO Yan-hong1,2, DING Xiao-yu1, ZHANG Ying-yu3,4, ZHANG Zhao1,2     
1. Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Jiangsu Key Laboratory for Supermolecular Medicinal Materials and Applications, College of Life Science, Nanjing Normal University, Nanjing 210023, China;
2. Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing 210029, China;
3. Institute of Molecular Biology and Translational Medicine, the Affiliated People's Hospital, Jiangsu University, Zhenjiang 212002, China;
4. Department of Clinical Medicine, Changchun Medical College, Changchun 130013, China
Abstract: This study aimed to examine whether ophiopogonin D (OP-D) is capable of protecting cardiomyocytes against DOX-induced injury and the mechanisms involved. H9c2 cells were cultured. MTT assay was used to evaluate cell viability and toxicity. Mito-tracker as fluorescence probe was used to measure ROS content raised from mitochondria. The mRNA and protein expression of ATF6α, GRP78 and CHOP were analyzed using real-time PCR and Western blotting, respectively. The results showed that a significant endoplasmic reticulum stress (ERS) was induced upon exposure of H9c2 cells to DOX as indicated by the increase in the expression of ERS related proteins, which was paralleled with the accumulation of reactive oxygen species (ROS) and decrease in the viability of H9c2 cells. Whereas, DOX-induced ROS accumulation and up-regulation of ERS related proteins were partially abolished by pretreatment with OP-D. Consequently, a DOX-induced ERS was mitigated by application of OP-D. Similarly, DOX-induced decrease in cell viability was partially attenuated by either inhibiting CHOP or pretreatment with N-acetylcysteine (NAC), an antioxi-dant. Moreover, cardiac ultrastructural abnormalities seen in mouse receiving DOX injections were obviously ameliorated by pretreatment of OP-D. Taken together, the present study proved that OP-D protects cardiomyocytes against DOX-induced injury, at least in part, through reducing ROS accumulation and alleviating ERS.
Key words: doxorubicin     ophiopogonin D     cardiomyocyte     endoplasmic reticulum stress    

阿霉素 (doxorubicin,DOX) 是一种蒽环类抗肿瘤药物, 广泛用于治疗血液系统恶性肿瘤和实体肿瘤, 具有十分显著的效果。基于其广谱高效性, 以蒽环类药物为基础的化疗方案迄今仍是抗肿瘤治疗的经典一线方案[1, 2]。然而, 因长期使用DOX而致剂量依赖的致命性心肌损伤, 极大程度地限制了其临床应用[3]。因此, 寻找抗DOX诱导心肌损伤的有效药物, 对患者接受有效抗肿瘤治疗的同时降低DOX诱发心脏毒性的风险有十分重要的意义。

DOX导致心肌损伤的原因很复杂, 涉及ROS累积以及由此联系的多重细胞分子机制。新近研究表明,ROS介导的胱硫醚-γ-裂解酶活性抑制, 并由此诱发的内质网应激 (endoplasmic reticulum stress,ERS) 也参与了DOX诱导的心肌细胞损伤[4]。现代中药研究发现, 传统中草药麦冬对心血管系统具有明显的保护作用, 作为生脉饮的重要成分之一, 在各种心血管疾病尤其是心脏疾病的治疗中有长期的历史[5]。麦冬皂苷D (ophiopogonin D,OP-D) 是从麦冬中提取的甾体皂苷类化合物, 具有抗氧化、抗凋亡、抗炎症等生物活性[6]。已有文献报道,OP-D通过抑制ERK1/2的激活、减少ROS累积、抗炎症反应等, 对H2O2诱导的脐静脉内皮细胞损伤有保护作用[7]。但 是,OP-D对DOX所致的心肌细胞损伤是否具有保护作用, 并未见文献报道。本研究以培养的H9c2细胞为模型, 观察OP-D对DOX诱导心肌损伤的影响, 并探讨其是否通过减轻ERS来缓解心肌细胞损伤。

材料与方法 实验材料

OP-D购自上海源叶生物科技有限 公司,DOX和N-乙酰-半胱氨酸 (N-acetylcysteine,NAC) 购自Sigma公司。分别用DMSO助溶OP-D和NAC, 制备成浓度分别为100 μmol·L-1和200 mmol·L-1的储存液。DOX则用DMEM制备成浓度 为100 μmol·L-1的储存液。DMEM培养基和胎牛血清购自Gibco公司; 兔抗ATF6α、GRP78、CHOP抗体购自Cell Signaling Technology公司。线粒体活性氧检测试剂盒购自Invitrogen公司。

细胞培养

H9c2细胞为来源于胚胎期BD1X大鼠心脏组织的亚克隆细胞系, 购自ATCC, 于DMEM培养基及5% CO2、37 ℃恒温箱中培养细胞, 培养基中含有10 μg·mL-1链霉素,10 u·mL-1青霉素和10% 胎牛血清。

MTT法检测细胞活性

将H9c2细胞接种于 96孔板中, 待其贴壁后随机分为对照 (CTL)、DOX、OP-D、OP-D+DOX、NAC和NAC+DOX, 总计6组。每组中加入无双抗无血清培养基, 总体积控制在 100 μL。第1组为空白对照; 第2组加入终浓度为 1 μmol·L-1的DOX, 作为损伤模型; 第3和第5组分别加入1 μmol·L-1 OP-D和1 mmol·L-1 NAC, 作为待测和阳性药物对照; 第4和第6组分别用1 μmol·L-1 OP-D和1 mmol·L-1 NAC预处理12 h, 再加入1 μmol·L-1 DOX持续作用48 h, 之后按照MTT法检测试剂盒说明书操作。

免疫印迹法(Western blotting,WB)

检测蛋白的相对表达量 将H9c2细胞接种于6孔板中, 待细胞充分贴壁之后, 分为6组 (同“MTT法检测细胞活性”), 但各组加入DOX后均作用12 h。12 h后提取细胞总蛋白, 参照文献[8]方法检测各种目标蛋白的表达量。在DOX时间梯度实验中, 也分为6组,1 μmol·L-1 DOX分别处理H9c2细胞0、3、6、12、24及48 h。置于普通显微镜下观察细胞状态, 之后提取细胞总蛋白, 检测目标蛋白的表达量。

线粒体ROS的检测

采用线粒体ROS检测试剂盒, 按照说明书检测线粒体中ROS的产生量。吸弃培养皿中培养基后, 加入用无血清无双抗DMEM培养基配制的500 nmol·L-1 MitoTracker Red CM-H2X Ros 500 μL,37 ℃孵育30 min, 吸弃培养基,PBS漂 洗, 再加入DMEM培养基, 置于荧光显微镜下观察检测。

荧光实时定量PCR的检测

引物设计见表 1。使用SYBR®Premix Ex Taq™ (Tli RNaseH Plus) (Takara DR420A) 检测目的基因的mRNA表达水平。RNA干扰siRNA序列为Negative control: 5'-siRNA UUCUCC GAACGUGUCACGUTT-3'; CHOP siRNA: 5'-CGAAGAGGAAGAAUCAAA-3'。用Lipofectamine 2000TM脂质体转染试剂将上述两种siRNA导入到H9c2细胞中。参照供应商提供的说明书进行转染, 待转染成功, 弃去原培养基, 更换含血清和双抗的完全培养基继续培养48 h。之后提取细胞总蛋白,Western blotting检测靶蛋白的表达情况。

Table 1 Primer design of real-time PCR

透射电镜检测 取健康雄性C57BL/6小鼠 (购自扬州大学比较医学中心, 合格证号: SOXR (苏) 2012-0004) 20只, 随机分为CTL、DOX、OP-D、OP-D+DOX等4组。第1组和第2组隔天分别腹腔注射 (ip) 生理盐水和DOX (2 mg·kg-1); 第3组隔天灌胃给予OP-D (10 mg·kg-1); 第4组在每次腹腔注射DOX之前灌胃给予OP-D。实验持续2周后, 取心脏制备超薄切片并用透射电镜观察其超微结构变化。

统计学分析 细胞活力检测时每组设6个复孔, 用于分子生物学分析的样本均来自3次独立实验。数据采用± s表示, 多组数据间差异的比较用单因素方差分析 (ANOVA) 和q检验, 在Original 8.0分析软件中进行统计学处理并作图。

结果 1 DOX诱导H9c2心肌细胞死亡

在1.0 μmol·L-1 DOX分别作用0、3、6、12、24及48 h后, 将H9c2细胞置于光学相差显微镜下观 察其形态的变化。图 1A显示了各组随机选取视野下的细胞形态, 可以发现随着DOX作用时间的延长, 细胞形态逐渐皱缩变圆,12 h后有一部分细胞丧失贴壁能力而死亡。同时,MTT结果表明, 随DOX作用浓度的增加, 细胞活力显著降低 (图 1B)。这些结果说明DOX对H9c2细胞有浓度和时间依赖的毒性 作用。

Figure 1 Doxorubicin (DOX) induced morphological changes of H9c2 cells and caused the decline of cell viability. A: H9c2 cells were treated with 1.0 μmol·L-1 DOX for indicated time and then photographed under phase-contrast microscope (scale bar, 200 μm); B: H9c2 cells were treated with indicated doses of DOX for 48 h and then cell viability was determined by MTT assay. n = 6, ± s. P < 0.05,**P < 0.01 vs control (CTL) group
2 DOX诱导H9c2心肌细胞产生ERS

Wang等[4]报道在5 μmol·L-1 DOX作用下, 可诱发ERS信号通路的激活, 进而导致心肌细胞损伤。而作者观察到1 μmol·L-1 DOX即可产生这种作用, 不仅表现在随DOX处理H9c2细胞时间的延长, 与内质网应激相关的ATF6α、GRP78和CHOP mRNA水平显著上升, 而且其蛋白表达量也显著增加 (图 2)。提示DOX导致心肌细胞损伤与其诱导ERS相关。

Figure 2 Activation of endoplasmic reticulum stress (ERS) in H9c2 cells in response to DOX application. A: Relative mRNA expression of ERS markers (ATF6α,GRP78,CHOP) were assayed by quantitative real-time PCR after incubating with 1.0 μmol·L-1 DOX for indicated time; B: H9c2 cells were incubated with 1.0 μmol·L-1 DOX for indicated time. The protein expression of ATF6α,GRP78 and CHOP was analyzed by Western blotting; C,D,E: Results of densitometric analysis of B from three independent experiments. n = 3, ± s. P < 0.05,**P < 0.01 vs CTL group
3 干扰CHOP可部分逆转DOX所致心肌细胞损伤

CHOP在ERS介导的细胞死亡中发挥着重要作用。为了证明ERS在DOX诱导心脏毒性中的作用, 利用靶向于CHOP的siRNA, 特异性地干扰CHOP在H9c2细胞中的表达。结果表明, 向H9c2细胞中 转染40 nmol·L-1 CHOP siRNA能显著降低DOX诱 导的CHOP蛋白表达 (图 3A)。相应地,MTT检测发现, 在CHOP表达下调的同时,DOX所致心肌细胞活力下降明显得到改善 (图 3B)。结果说明,ERS的确参与了DOX诱导心脏毒性的过程。

Figure 3 Suppressing CHOP expression partially attenuated DOX-induced cytotoxicity. A: The suppression effect of siRNA on CHOP protein expression was detected by Western blotting; B: H9c2 cells were transfected with negative control or CHOP siRNA (40 nmol·L-1) for 24 h and then cells were treated with 1.0 μmol·L-1 DOX for 48 h,and cell viability was detected by MTT assay. n = 6, ± s. **P < 0.01 vs siCHOP group
4 OP-D缓解DOX诱导的ERS

为了观察OP-D对DOX诱导心肌损伤的保护 作用, 先用OP-D预处理H9c2细胞12 h后, 再加入 1 μmol·L-1 DOX处理12 h, 结果见图 4。OP-D可以显著逆转DOX的诱导作用, 使ERS相关标志的mRNA和蛋白表达水平下调, 这一效应与抗氧化剂NAC预处理类似。提示OP-D通过抗氧化作用抑制DOX诱导的ERS。

Figure 4 Ophiopogonin D (OP-D) relieves DOX-induced ERS in H9c2 cells. A: Effects of OP-D on DOX-induced up-regulation of ATF6α,GRP78 and CHOP in mRNA level. H9c2 cells were treated with 1 μmol·L-1 DOX for 12 h in the absence or presence of preconditioning with 1 μmol·L-1 OP-D or 1 mmol·L-1 NAC for 12 h; B: Western blotting analysis was applied to detect the changes in expressions of ATF6α,GRP78 and CHOP; C,D and E were the results of densitometric analysis of B. n = 3, ± s. P < 0.05 vs DOX group
5 OP-D通过清除DOX诱导的ROS提高心肌细胞的活力

大量研究表明, 线粒体过量产生ROS在DOX致心肌细胞损伤中发挥着核心作用。为确认OP-D通过抗氧化而实现其对心肌细胞的保护作用, 分别检测了不同处理情况下的细胞活力和ROS含量, 其结果见图 5。在OP-D和抗氧化剂NAC预处理显著提高心肌细胞活力的同时 (图 5A), 其ROS含量也显著下降 (图 5B)。这进一步说明,OP-D通过清除ROS而实现对DOX所致心肌损伤的保护。

Figure 5 OP-D attenuates DOX-induced cytotoxicity and ROS accumulation in H9c2 cells. A: H9c2 cells were treated with 1 μmol·L-1 DOX for 12 h in the absence or presence of preconditioning with 1 μmol·L-1 OP-D or 1 mmol·L-1 NAC for 12 h,then cell viability was detected by MTT assay; B: ROS were measured using MitoTracker staining followed by fluorescence microscope for different treatments with A (scale bar: 200 μm). n = 6, ± s. P < 0.05 vs DOX group
6 OP-D减轻DOX引起的小鼠心脏超微结构异常

为了进一步验证OP-D对心肌的保护作用, 对OP-D + DOX组的小鼠心脏进行TEM超微结构观察。结果发现, 与DOX单独处理相比,OP-D预处理可显著减轻DOX引起的心肌超微结构异常 (图 6), 这提供了OP-D对DOX诱导心肌细胞损伤保护作用的体内实验证据。

Figure 6 OP-D reduces the cardiac ultrastructural abnormalities observed in the mice receiving DOX injections. Representative images obtained by the transmission electron microscopy examination for the mouse hearts subjected to the indicated treatments. As compare to CTL,DOX-induced ultrastructural abnormalities were illustrated,including mitochondrial edema,sarcomere disorder and so on. These abnormalities were partially improved by pretreatment with OP-D. Scale bar,500 nm
讨论

业已明确,ERS是真核细胞的一种自我保护机制。在各种刺激条件下, 细胞通过一系列信号转导途径, 对其进行应答, 包括增强蛋白质折叠能力、停滞 大多数蛋白质的翻译、加速蛋白质的降解等, 以减轻或中止ERS反应。但是过强或持续的ERS则造成细胞损伤[9]

近年来,ERS在DOX诱导心脏毒性中的作用备受关注。来自细胞和动物实验的数据表明,DOX可诱导心肌细胞产生过度ERS, 进而通过激活细胞自噬、凋亡而引起心肌细胞的死亡[4, 10]。类似地, 作者也观察到DOX在诱导ERS的同时伴有H9c2细胞的损 伤, 而siRNA干扰CHOP,ERS的重要标志蛋白之 一, 可部分逆转由DOX诱导的H9c2细胞死亡, 这进一步证明DOX诱导过度ERS是其心脏毒性的重要机制之一。需要指出的是, 本研究中所用DOX浓度是1 μmol·L-1, 仅为Wang等[4]报道的1/5, 提示DOX在低浓度范围内即可诱导ERS。

越来越多的实验结果提示,ROS与细胞事件, 诸如蛋白质氧化和折叠之间存在着密切联系[11]。任何原因引起细胞内产生过量ROS或氧化还原反应状态改变, 将直接或间接影响内质网稳态和其中蛋白质折叠, 进而诱发内质网应激[12]。迄今,DOX导致心肌损伤的机制并未完全阐明。广为接受的观点是,DOX通过自身代谢和 (或) 作用于线粒体导致的ROS累积是其致心肌损伤的核心元凶[4, 10, 13]。据此, 本研究中观察到DOX诱导的心肌ERS, 不外乎与细胞内ROS过量累积有关。

鉴于ROS引起的氧化应激在DOX所致心肌损 伤中的重要作用, 针对增强心肌细胞本身的抗氧化能力、帮助心肌清除过量ROS的探索研究, 代表了抗DOX心肌损伤策略发展的主要趋势[14, 15, 16]。传统中草药活性化合物OP-D因其抗炎和抗氧化活性对心血管具有显著保护作用, 如抗缺血、抗心律不齐、抑制血小板聚集、抗氧化应激所致的脐静脉内皮细胞凋亡等[17, 18]。本研究首次发现,OP-D预处理不仅显著缓解DOX诱导的过度ERS, 而且明显降低线粒体ROS产生、提高细胞活力, 与已知的抗氧化剂NAC预处理有类似效果。值得注意的是OP-D可显著改善DOX注射所致的小鼠心肌超微结构异常。综上所述, 本研究用体内外实验证明,OP-D可通过清除细胞内过量ROS, 缓解DOX诱导的ERS及其伴随的心肌损伤。本研究发现内质网可能是OP-D的作用靶点, 这为探索治疗DOX所致心肌损伤的策略提供了新视点。

不可否认, 在本研究中并未观察OP-D完全逆转DOX所致的心肌活力降低和ERS, 增加其用量也如此 (数据未显示)。这从另一角度说明,DOX诱导的心肌损伤是一个多信号通路参与的复杂过程[2]

参考文献
[1] Carvalho C, Santos RX, Cardoso S, et al. Doxorubicin: the good, the bad and the ugly effect [J]. Curr Med Chem, 2009, 16: 3267-3285.
[2] Sterba M, Popelova O, Vavrova A, et al. Oxidative stress, redox signaling, and metal chelation in anthracycline cardiotoxicity and pharmacological cardioprotection [J]. Antioxid Redox Signal, 2013, 18: 899-929.
[3] Scott JM, Khakoo A, Mackey JR, et al. Modulation of anthracycline-induced cardiotoxicity by aerobic exercise in breast cancer: current evidence and underlying mechanisms [J]. Circulation, 2011, 124: 642-650.
[4] Wang XY, Yang CT, Zheng DD, et al. Hydrogen sulfide protects H9c2 cells against doxorubicin-induced cardiotoxicity through inhibition of endoplasmic reticulum stress [J]. Mol Cell Biochem, 2012, 363: 419-426.
[5] Huang KC. The Pharmacology of Chinese Herbs: Vol. 874. [M]. Boca Raton: CRC Press, 2010.
[6] Chang JM, Shen CC, Huang YL, et al. Five new homoisoflavonoids from the tuber of Ophiopogon japonicus [J]. J Nat Prod, 2002, 65: 1731-1733.
[7] Kou J, Tian Y, Tang Y, et al. Antithrombotic activities of aqueous extract from Radix Ophiopogon japonicus and its two constituents [J]. Biol Pharm Bull, 2006, 29: 1267-1270.
[8] Li M, Jin J, Li J, et al. Schisandrin B protects against nephrotoxicity induced by cisplatin in HK-2 cells via Nrf2-ARE activation [J]. Acta Pharm Sin (药学学报), 2012, 47: 1434-1439.
[9] Arensdorf AM, Diedrichs D, Rutkowski DT. Regulation of the transcriptome by ER stress: non-canonical mechanisms and physiological consequences [J]. Front Genet, 2013, 4: 256.
[10] Lai HC, Yeh YC, Ting CT, et al. Doxycycline suppresses doxorubicin-induced oxidative stress and cellular apoptosis in mouse hearts [J]. Eur J Pharmacol, 2010, 644: 176-187.
[11] Higa A, Chevet E. Redox signaling loops in the unfolded protein response [J]. Cell Signal, 2012, 24: 1548-1555.
[12] Malhotra JD, Kaufman RJ. Endoplasmic reticulum stress and oxidative stress: a vicious cycle or a double-edged sword? [J]. Antioxid Redox Signal, 2007, 9: 2277-2293.
[13] Zhang YW, Shi J, Li YJ, et al. Cardiomyocyte death in doxorubicin-induced cardiotoxicity [J]. Arch Immunol Ther Exp (Warsz), 2009, 57: 435-445.
[14] Guo R, Lin J, Xu W, et al. Hydrogen sulfide attenuates doxorubicin-induced cardiotoxicity by inhibition of the p38 MAPK pathway in H9c2 cells [J]. Int J Mol Med, 2013, 31: 644-650.
[15] Yu X, Cui L, Zhang Z, et al. alpha-Linolenic acid attenu-ates doxorubicin-induced cardiotoxicity in rats through suppression of oxidative stress and apoptosis [J]. Acta Biochim Biophys Sin (生物化学与生物物理学报), 2013, 45: 817-826.
[16] Li J, Liu H, Ramachandran S, et al. Grape seed proanthocyanidins ameliorate doxorubicin-induced cardiotoxicity [J]. Am J Chin Med, 2010, 38: 569-584.
[17] Kou J, Sun Y, Lin Y, et al. Anti-inflammatory activities of aqueous extract from Radix Ophiopogon japonicus and its two constituents [J]. Biol Pharm Bull, 2005, 28: 1234-1238.
[18] Qian J, Jiang F, Wang B, et al. Ophiopogonin D prevents H2O2-induced injury in primary human umbilical vein endothelial cells [J]. J Ethnopharmacol, 2010, 128: 438-445.