卵泡抑素样蛋白1对阿霉素所致小鼠急性心肌损伤的改善作用及其机制

doi:10.13481/j.1671-587X.20230303

摘要/Abstract

摘要：

目的探讨卵泡抑素样蛋白1（FSTL1）对阿霉素（DOX）诱导的小鼠急性心肌损伤的保护作用，并阐明其相关机制。方法 80只C57BL/6J小鼠采用单次腹腔内注射DOX建立小鼠急性心肌损伤模型，小鼠按DOX不同剂量（0、5、10、15和20 mg·kg^－1）分为5组（n=8）；按不同干预时间（0、0.5、1.0、2.0和3.0 d）分为5组（n=8）。另取32只小鼠随机分为生理盐水组、FSTL1组、DOX组和DOX+FSTL1组。测定各组小鼠心脏超声心动图和血流动力学指标，酶联免疫吸附测定（ELISA）法检测各组小鼠血清中肿瘤坏死因子α（TNF-α）、N端脑钠肽体（NT-proBNP）和肌钙蛋白T（cTn-T）水平，氧化应激试剂盒检测各组小鼠心肌组织中超氧化物歧化酶（SOD）活性和丙二醛（MDA）、4-羟基壬烯醛（4-HNE）及15-F_2t-isoprostane水平，实时荧光定量PCR（RT-qPCR）法检测各组小鼠心肌组织中FSTL1 mRNA表达水平，Western blotting法检测各组小鼠心肌组织中核因子E2相关因子2（Nrf2）和FSTL1蛋白表达水平。结果与生理盐水组比较，DOX组和DOX+FSTL1组小鼠左心室射血分数（LVEF）、左心室短轴缩短率（LVFS）、左心室收缩压（LVSP）、左心室内压最大上升速率（+dP/dt_max）和左心室内压最大下降速率（－dP/dt_max）降低（P<0.05），心率（HR）、左心室舒张末期容积（LVEDV）和左心室舒张压（LVDP）升高（P<0.05）；与DOX组比较，DOX+FSTL1组小鼠LVEF、+dP/dt_max和－dP/dt_max升高（P<0.05），LVEDV降低（P<0.05）。与生理盐水组比较，DOX组和DOX+FSTL1组小鼠血清中TNF-α、NT-proBNP和cTn-T水平升高（P<0.05）；与DOX组比较，DOX+FSTL1组小鼠血清中NT-proBNP和cTn-T水平降低（P<0.05）。与生理盐水组比较，DOX组小鼠心肌组织中SOD活性降低（P<0.05），MDA、4-HNE和15-F_2t-isoprostane水平升高（P<0.05），DOX+FSTL1组小鼠心肌组织中15-F_2t-isoprostane水平升高（P<0.05）；与DOX组比较，DOX+FSTL1组小鼠心肌组织中SOD活性升高（P<0.05），MDA、4-HNE和15-F_2t-isoprostane水平降低（P<0.05）。与0 mg·kg^－1DOX组比较，随着DOX剂量增加，其他各组小鼠心肌组织中FSTL1 mRNA表达水平降低（P<0.05）；与DOX 0 d组比较，随着DOX应用时间延长，其他各组小鼠心肌组织中FSTL1 mRNA表达水平降低（P<0.05）。与生理盐水组比较，DOX组和DOX+FSTL1组小鼠心肌组织中FSTL1及Nrf2蛋白表达水平降低（P<0.05）；与DOX组比较，DOX+FSTL1组小鼠心肌组织中FSTL1和Nrf2蛋白表达水平升高（P<0.05）。结论 FSTL1通过调节机体氧化应激反应减轻DOX诱导的小鼠急性心肌损伤和改善心脏功能，其机制与上调Nrf2表达有关。

关键词: 卵泡抑素样蛋白1, 阿霉素, 心肌损伤, 心脏毒性, 氧化应激

Abstract:

Objective To discuss the protective effectof follistatin-like 1 （FSTL1） on doxorubicin （DOX）-induced acute myocardial injury of the mice，and to clarify its related mechanism. Methods A total of 80 C57BL/6J mice were used to establish the acute myocardial injury models by intraperitoneal injection of DOX for one time.The mice were divided into 5 groups （n=8） according to the different doses （0，5，10，15， and 20 mg·kg^－1） of DOX，and didvided into 5 groups（n=8） according to the different intervention time（0，0.5，1.0，2.0，and 3.0 d）.The other 32 mice were randonly divided into normal saline group，FSTL1 group，DOX group， and DOX-FSTL1 group.The echocardiographic and hemodynamic indexes of the mice in various groups were detected；enzyme-linked immunosorbent assay（ELISA） method was used to detect the levels of tumor necrosis factor-α（TNF-α），N-terminal pro-brain natriuretic peptide（NT-proBNP） and cardiae troponin-T（cTn-T） in serum of the mice in various groups；the activities of superoxide dismutase （SOD）， the levels of malondialdehyde （MDA）， 4-hydroxynonenal （4-HNE） and 15-F_2t isoprostane in myocardium tissue of the mice in various groups were detected by oxidative stress kit；the expression levels of FSTL1 mRNA in myocardium tissue of the mice in various groups were detected by real-time fluorescence quantitative PCR （RT-qPCR） method； the expression levels of nuclear factor E2 related factor 2 （Nrf2） and FSTL1 proteins in myocardium tissue of the mice in various groups were detected by Western blotting method. Results Compared with normal saline group， the left ventricular ejection fraction（LVEF），left ventricular fraction shortening（ LVFS）， left ventricular systolic pressure（LVSP）， maximum rise rate of left ventricular pressure（+dP/dt_max），and maximum drop rate of left ventricular pressure（－dp /dt_max） of the mice in DOX group and DOX+FSTL1 group were significantly decreased（P<0.05）， and the heart rate（HR），left ventricular end-diastolic volume（LVEDV），and left ventricular diastolic pressure were increased （P<0.05）； compared with DOX group， the LVEF，+dP/dt_max， and －dp /dt_max of the mice in DOX+FSTL1 group were significantly increased（P<0.05），and the LVEDV was significantly decreased （P<0.05）. Compared with normal saline group， the serum levels of TNF-α， NT-proBNP and cTn-T in serum of the mice in DOX group and DOX+FSTL1 group were significantly increased （P<0.05）； compared with DOX group， the serum levels of NT-proBNP and cTn-T in serum of the mice in DOX+FSTL1 group were significantly decreased （P<0.05）. Compared with normal saline group， the activity of SOD in myocardium tissue of the mice in DOX group was significantly decreased （P<0.05）， and the levels of MDA， 4-HNE， and 15-F_2t-isoprostane were significantly increased （P<0.05），the level of 15-F_2t-isoprostane in serum of the mice in DOX+FSTL1 group was increased （P<0.05）； compared with DOX group， the SOD activity in myocardium tissue of the mice in DOX+FSTL1 group was increased （P<0.05）， and the levels of MDA， 4-HNE， and 15-F_2t-isoprostane were decreased （P<0.05）.Compared with 0 mg·kg^－1 DOX group， the levels of FSTL1 mRNA in myocardium tissue of the mice in the other groups were significantly decreased with the increasing of the doxorubicin dose （P<0.05）. Compared with DOX 0 d group， the levels of FSTL1 mRNA in myocardium tissue of the mice in the other groups were significantly decreased with the prolongation of the doxorubicin intervention time （P<0.05）. Compared with normal saline group，the expresison levels of FSTL1 and Nrf2 proteins in myocardium tissue of the mice in DOX group was decreased（P<0.05）；compared with DOX group， the expression levels of FSTL1 and Nrf2 proteins in myocardium tissue of the mice in DOX+FSTL1 group were increased （P<0.05）. Conclusion FSTL1 can alleviate the DOX-induced acute myocardial injury and improve the cardiac function by regulating the oxidative stress， and its mechanism may be reated to up-regulating the Nrf2 expression.

Key words: Follistatin-like 1, Doxorubicin, Myocardial injury, Cardiotoxicity, Oxidative stress

中图分类号:

R542.2

赵荫涛, 杨莹莹, 张相钦, 郑璐, 徐亚威, 杨海波, 刘源. 卵泡抑素样蛋白1对阿霉素所致小鼠急性心肌损伤的改善作用及其机制[J]. 吉林大学学报(医学版), 2023, 49(3): 565-572.

Yintao ZHAO, Yingying YANG, Xiangqin ZHANG, Lu ZHENG, Yawei XU, Haibo YANG, Yuan LIU. Improvement effect of follistatin-like 1 on doxorubicin-induced acute myocardial injury in mice and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2023, 49(3): 565-572.

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Group	HR (beat·min^－1)	LVEDV (V/μL)	LVEF (η/%)	LVFS (η/%)
Normal saline	424±27	52.78±5.12	95.73±10.10	74.12±8.02
FSTL1	438±31	53.29±4.84	94.36±9.84	75.95±8.46
DOX	491±45 ^*	68.34±5.05 ^*	75.45±9.69 ^*	49.43±5.13 ^*
DOX+FSTL1	476±40 ^*	62.44±4.83 ^*^△	84.37±8.67 ^*^△	47.11±4.54 ^*
F	52.32	102.44	32.85	89.63
P	0.021	0.004	0.040	0.012

Group	LVSP (P/mmHg)	LVDP (P/mmHg)	+dP/dt_max (mmHg·s^－1)	－dP/dt_max (mmHg·s^－1)
Normal saline	122.31±12.85	6.07±1.81	6 400.07±366.45	5 217.45±295.19
FSTL1	132.47±12.56	5.97±1.50	6 548.54±356.22	5 102.75±295.04
DOX	90.89±13.93 ^*	11.55±2.66 ^*	4 000.35±292.07 ^*	2 982.70±228.14 ^*
DOX+FSTL1	94.76±14.36 ^*	9.79±2.03 ^*	5 178.07±358.12 ^*^△	4 193.57±188.41 ^*^△
F	74.52	65.12	118.17	98.74
P	0.017	0.029	<0.01	0.009

Group	TNF-α	NT-proBNP	cTn-T
Normal saline	13.65±3.04	83.65±11.67	88.82±9.34
FSTL1	14.05±2.93	90.50±12.08	82.29±10.16
DOX	64.67±16.92 ^*	256.48±26.76 ^*	301.14±27.64 ^*
DOX+FSTL1	50.77±13.14 ^*	139.19±20.50 ^*^△	143.89±18.56 ^*^△
F	55.22	139.06	148.43
P	0.027	<0.01	<0.01

Group	SOD activity	MDA level	4-HNE level	15-F_2t-isoprostane level
Normal saline	1.00±0.03	1.00±0.14	1.00±0.03	1.00±0.12
FSTL1	1.23±0.06	0.98±0.15	1.07±0.03	1.12±0.14
DOX	0.71±0.02^*	3.55±0.53^*	2.86±0.04^*	2.19±0.16 ^*
DOX+FSTL1	0.86±0.03^△	1.74±0.03^△	1.28±0.06^△	1.88±0.11 ^*^△
F	37.12	60.22	75.18	34.97
P	0.031	0.028	0.016	0.034

Group	FSTL1 mRNA
DOX( mg·kg^－1)
0	1.00±0.17
5	0.78±0.05^*
10	0.57±0.11^*
15	0.49±0.13^*
20	0.38±0.10^*
F	64.12
P	0.028
^*P<0.05 compared with 0 mg·kg^－1 DOX group.