摘要
化疗药物诱导的心脏毒性近年来广受关注,但有关肺损伤状态下化疗对心脏代谢的影响尚未见报道。本研究采用博莱霉素(BLM)和阿霉素(DOX)构建肺损伤叠加心肌损伤小鼠模型:C57BL/6J小鼠随机分为4组,分别为对照组(CON)、BLM组(单次气管滴注5.0 mg/kg BLM)、DOX组(腹腔注射7.5 mg/kg DOX,每周1次,连续两周)和DOX+BLM组,以血清标志物和组织病理学检查评价心脏损伤程度。采用气质联用(GC-MS)和液质联用(LC-MS)技术对心脏样本进行非靶向代谢组学分析。结果表明,与CON组相比,单独给予BLM可导致小鼠肺损伤,但对心脏代谢轮廓无显著影响;单独给予DOX心脏代谢轮廓发生显著变化,主要差异代谢物为氨基酸、脂肪酸、磷脂等;联合给予BLM和DOX后心脏代谢稳态被严重扰乱,尤其是支链氨基酸蓄积更加严重。研究证实,在肺损伤状态下DOX可导致心脏代谢轮廓发生更显著的变化,并初步聚焦支链氨基酸代谢通路。研究结果为进一步深入探讨化疗药物心脏毒性机制提供了参考。
肿瘤严重危害人类生命健康。过去30年中,肿瘤治疗方法取得长足进展,患者生存时间显著延长、病死率持续下
目前,针对抗肿瘤药心脏毒性的研究大多聚焦于心脏或心肌细胞,而忽视了其他脏器的作用。肺功能异常与心脏疾病的发生紧密关联。如COVID-19患者通常表现为呼吸道感染症状和体征,但包括心肌损伤征象在内的心脏临床表现也十分常
为此,本研究采用经典的、具有肺毒性的化疗药物博莱霉素(bleomycin,BLM)构建小鼠肺损伤模型,继而给予DOX诱导心脏损伤,采用血清标志物和组织病理学检查评价心脏损伤程度;整合运用气相色谱-质谱(gas chromatography-mass spectrometry,GC-MS)和液相色谱-质谱(liquid chromatography-mass spectrometry,LC-MS)联用技术对心脏进行代谢组学分析,通过多元统计分析筛选差异代谢物并聚焦关键代谢通路。研究结果将为深入阐明蒽环类药物对肺损伤状态下的心脏毒性及作用机制提供科学参考。
N-甲基-N-(三甲基硅烷基)三氟乙酰胺(MSTFA,含量大于99.9%)、盐酸甲氧胺(MOX,含量大于99.9%)、吡啶、内标十七酸(美国Sigma-Aldrich公司);甲醇、乙腈及乙酸乙酯(色谱纯,德国Merk公司);内标格列苯脲(中国食品药品检定研究院,批号:121633-201017);注射用盐酸多柔比星(阿霉素,深圳万乐药业有限公司,批号:H44024359);博莱霉素(日本化药株式会社,批号:410730);注射用生理盐水(安徽双鹤药业有限责任公司);水合氯醛(国药集团化学试剂有限公司);多聚甲醛固定液(武汉塞维尔生物科技有限公司);脑利钠肽(brain natriuretic peptide,BNP)和心肌肌钙蛋白(cardiac troponin I,cTn-I)试剂盒(南京森贝伽生物科技有限公司)。
UFLC-IT-TOF/MS液质联用仪、GC/MS-QP2010Ultra气质联用仪(日本岛津公司);CentriVa
小鼠适应性饲养1周后,根据体重随机分为以下4组:对照组(CON,6只)、博莱霉素组(BLM,8只)、阿霉素组(DOX,10只),阿霉素+博莱霉素组(DOX + BLM,12只)。实验方案如

Figure 1 Doxorubicin (DOX) and bleomycin (BLM)-induced toxicity on mouse hearts and lungs
A: Schematic of the animal model; B: Body weight changes of mice during the experiment; C, D: Lung indicators of mice among four groups; E: H&E staining of lung tissues; F: Heart indicators; G, H: Cardiac markers of mice; I: H&E staining of heart tissues , CON, n = 6; BLM, n = 8; DOX, n = 8; DOX+BLM, n = 8). One-way ANOVA
取小鼠心脏底部约1/3部分以及肺右前叶,多聚甲醛中固定,石蜡包埋并切片(5 μm),切片脱蜡水化后经苏木精-伊红(H&E)染色,于倒置显微镜下观察损伤程度。血清脑利钠肽(BNP)和心肌肌钙蛋白(cTn-I)采用酶联免疫分析(ELISA)试剂盒(南京森贝伽生物科技有限公司)测定,实验步骤及计算均按试剂盒说明书操作。取小鼠肺右下叶称重,并在60 ℃条件下干燥72 h后再次称重,以两次重量计算肺湿/干重比。
心脏匀浆液制备:准确称重心脏置于匀浆管中,按1∶10比例加预冷的80%甲醇,利用匀质仪(振动速度6.5 m/s,10 s循环3次,间隔30 min)使心脏样本匀质化,高速离心(14 000 r/min,4 ℃,10 min)两次,取上清液供后续分析。
LC-MS样品前处理:取心脏匀浆液40 µL,加甲醇(含内标格列苯脲10 µg/mL)40 µL,涡旋混匀后,两次高速离心(14 000 r/min,4 ℃,10 min),将上清液转移至进样小瓶,待LC-MS分析。
GC-MS样品前处理:精密量取心脏匀浆液10 µL,加甲醇(含内标十七酸5 µg/mL)100 µL,涡旋混匀后,两次高速离心(14 000 r/min,4 ℃,10 min)。取上清液80 µL至1.5 mL棕色反应管中,加含MOX的吡啶溶液(10 mg/mL)25 µL进行肟化反应,振荡反应1.5 h(37 ℃,1 200 r/min);于50 ℃真空干燥2 h;加MSTFA试剂120 µL(MSTFA-乙酸乙酯,1∶1)进行硅烷化反应,振荡反应2 h(37 ℃,1 200 r/min)。反应完成后,高速离心(14 000 r/min,4 ℃,10 min),将上清液转移至进样小瓶,待GC-MS分析。
质控样本的制备:从每只动物的组织匀浆液中取等体积样本混匀后制得QC样本。分析过程中,每间隔10个真实样本插入1个QC样本。
色谱柱为XSelec
采用Profiling Solution软件(日本岛津公司)对GC-MS和LC-MS采集的原始谱图进行峰提取和峰对齐。将所得数据矩阵导入R Studio(ver.4.2.0)中,使用muma包(ver.1.4)进行单变量统计分析,根据每个变量是否成正态分布(Shapiro-Wilk检验)分别采用t-test或Mann-Whitney U Test检验其组间差异的显著性,并计算组间的倍数变化(fold change,FC);分别利用factoextra包(ver.1.0.7)和ropls包(ver.1.24.0)进行主成分分析(principal component analysis,PCA)和正交偏最小二乘判别分析(orthogonal projection to latent structures-discriminant analysis,OPLS-DA),以变量投影重要性(variable importance in projection,VIP)反映各变量对分组的贡献大小。最终以P < 0.05、VIP > 1以及FC > 1.2或< 1/1.2为阈值,筛选差异变量。通过与HMDB数据库(http://www.hmdb.ca)、NIST11谱库以及实验室自建数据库比对,初步鉴定可能的代谢物结构;利用标准品对部分代谢物进行进一步确证。
脏器系数、生化指标等数据导入Prism 8.0软件(GraphPad,美国)进行统计分析和绘图。采用单因素方差分析(One-way ANOVA)进行显著性差异检验,以P < 0.05认为具有统计学意义。
在临床上,DOX与BLM联用是治疗霍奇金淋巴瘤的一线方案,BLM可损伤肺部造成肺纤维化或严重的间质性肺病,即肺毒性。动物实验过程中,CON组与BLM组未出现动物死亡,DOX组死亡2只,DOX+BLM组死亡4只;因此,3个给药组最终均有8只小鼠存活。动物实验结果发现,BLM对小鼠体重几乎无影响,DOX可导致小鼠体重显著降低(
经“80%规则”筛选、缺失值填补等前处理后,分别从GC-MS和LC-MS的原始图谱(

Figure 2 LC-MS and GC-MS data of untargeted metabolomics analysis
A: Typical total ion chromatograms (TICs) of mouse heart samples; B: PCA score plots from LC-MS and GC-MS data; C, D: Peak area of internal standard in all samples from LC-MS and GC-MS
为寻找DOX诱导肺损伤小鼠心脏毒性相关的代谢特征,首先对CON组和BLM组的样本进行有监督的OPLS-DA和单变量统计分析。结果显示,散点图上这两组样本重叠度很高;在GC-MS数据中未筛选出同时满足P < 0.05、VIP > 1且FC > 1.2或< 1/1.2的变量,在LC-MS数据中仅鉴定出4个差异代谢物(
No | Name | HMDB ID | Category | BLM vs CON | DOX vs CON | DOX+BLM vs DOX |
---|---|---|---|---|---|---|
1 |
Leucin | HMDB0000687 | Amino acids | ↑ | ||
2 |
Isoleucin | HMDB0000172 | Amino acids | ↑ | ↑ | |
3 |
Valin | HMDB0000883 | Amino acids | ↑ | ||
4 |
Tyrosin | HMDB0000158 | Amino acids | ↓ | ||
5 |
Phenylalanin | HMDB0000159 | Amino acids | ↑ | ||
6 |
Aspartic aci | HMDB0000191 | Amino acids | ↑ | ↑ | |
7 |
Threonin | HMDB0000167 | Amino acids | ↑ | ↑ | |
8 |
Lysin | HMDB0000182 | Amino acids | ↑ | ↑ | |
9 |
Methionin | HMDB0000696 | Amino acids | ↑ | ||
10 |
Valin | HMDB0000883 | Amino acids | ↑ | ||
11 |
Serin | HMDB0000187 | Amino acids | ↑ | ||
12 |
Glutamin | HMDB0000641 | Amino acids | ↑ | ||
13 |
Glutamic aci | HMDB0000148 | Amino acids | ↓ | ↓ | |
14 |
Glucos | HMDB0000122 | Carbohydrates | ↓ | ↑ | |
15 | Ribose | HMDB0000283 | Carbohydrates | ↑ | ||
16 | Ribose-5-phosphate | HMDB0001548 | Carbohydrates | ↑ | ||
17 | Glucose 6-phosphate | HMDB0001401 | Carbohydrates | ↓ | ||
18 | Fructose 1,6-bisphosphate | HMDB0001058 | Carbohydrates | ↓ | ||
19 | Gluconic acid | HMDB0000625 | Carbohydrates | ↓ | ||
20 | Threonic acid | HMDB0000943 | Carbohydrates | ↑ | ||
21 |
Succinic aci | HMDB0000254 | Organic acid | ↓ | ||
22 | Pantothenic acid | HMDB0000210 | Vitamins | ↑ | ||
23 |
Inosin | HMDB0000195 | Nucleosides | ↑ | ||
24 | Uridine | HMDB0000296 | Nucleosides | ↓ | ||
25 | Guanosine | HMDB0000133 | Nucleosides | ↑ | ||
26 | Inosinic acid | HMDB0000175 | Nucleotides | ↓ | ||
27 | Adenosine monophosphate | HMDB0000045 | Nucleotides | ↑ | ||
28 | C22:5 | HMDB0001976 | Fatty acids | ↑ | ||
29 | C24:6 | HMDB0013025 | Fatty acids | ↑ | ||
30 |
Acetylcarnitin | HMDB0000201 | Fatty acid esters | ↓ | ||
31 | LysoPE(16:0) | HMDB0011503 | Lysophospholipids | ↑ | ↓ | |
32 | LysoPE(18:0) | HMDB0011130 | Lysophospholipids | ↑ | ↓ | |
33 | LysoPE(18:1) | HMDB0011505 | Lysophospholipids | ↑ | ||
34 | LysoPE(18:2) | HMDB0011507 | Lysophospholipids | ↓ | ||
35 | LysoPE(20:4) | HMDB0011487 | Lysophospholipids | ↑ | ||
36 | LysoPE(22:6) | HMDB0011526 | Lysophospholipids | ↑ |
*The structure of metabolites is confirmed using commercial standards. ↑ indicates an increase of metabolite level in model groups (BLM, DOX, or DOX+BLM) compared to corresponding groups (CON or DOX) while ↓ indicates a decrease trend of metabolite level
OPLS-DA散点图上(

Figure 3 DOX-induces metabolic alterations in mouse heart
A: OPLS-DA score plots from LC-MS and GC-MS data (LC-MS:
进一步比较了DOX组中有肺损伤与无肺损伤小鼠的心脏代谢差异。GC-MS和LC-MS数据的OPLS-DA散点图(

Figure 4 DOX-induced metabolic alterations in the hearts of mice with lung injury
A: OPLS-DA score plots from LC-MS and GC-MS data (LC-MS:

Figure 5 Relative cardiac levels of branched-chain amino acids in the mouse hearts among four groups , CON, n = 6; BLM, n = 8; DOX, n = 8; DOX+BLM, n = 8)
Mann-Whitney U Test,
研究肺损伤状态下的抗肿瘤药心脏毒性,构建合适的动物模型至关重要。作者在前期研究基础
非靶向代谢组学分析发现,在仅发生心肌损伤的小鼠心脏中,DOX毒性相关物质主要集中于氨基酸、脂肪酸、糖类等。长链脂肪酸是健康心脏中主要的能量代谢底物,它们通过线粒体β-氧化磷酸化为心脏提供约40%的三磷酸腺苷(ATP
本研究建立了肺损伤叠加心肌损伤的小鼠模型,对DOX和BLM给药后小鼠心脏样本进行了非靶向代谢组学分析。结果发现在肺损伤状态下,DOX可导致心脏代谢轮廓发生更显著的变化,并初步聚焦于支链氨基酸代谢通路。研究结果为进一步探讨蒽环类抗肿瘤药物心脏毒性机制奠定了基础,也为DOX和BLM的临床合理应用提供参考。
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