过度劳累损伤小鼠心肌能量代谢稳态

doi:10.12122/j.issn.1673-4254.2025.12.07

摘要/Abstract

摘要：

目的探讨过度劳累对小鼠心肌能量代谢的影响。方法 32只C57BL/6J小鼠被随机分为4组（n=8）：对照组、过劳2周（W2）组、过劳4周（W4）组和过劳6周（W6）组，W2、W4和W6组每天强迫水中站立8 h+束缚3 h，分别连续造模2周、4周和6周，每周记录小鼠体质量，并观察一般情况。造模结束后行超声心动图，并检测血脂和血糖水平，HE染色观察心肌组织病理学变化，ELISA法检测心肌甘油三酯（TG）、三磷酸腺苷（ATP）水平，免疫组化染色检测心肌组织分化簇36（CD36）和葡萄糖易化扩散转运蛋白1（GLUT1）的表达，RT-qPCR和Western blotting分析心肌脂代谢调控因子肉碱棕榈酰转移酶1B（CPT1B）和过氧化物酶体增殖物激活受体α（PPARα），糖酵解酶磷酸果糖激酶（PFKM）和丙酮酸激酶 M2（PKM2）的mRNA表达水平和蛋白相对表达量。结果小鼠随着过劳时间延长逐渐表现出活动减少、毛发脱落、皮毛晦暗以及体质量增长迟缓现象，但心脏指数及心功能无显著改变（P>0.05）；与CON组相比，W2和W4组血糖水平升高（P<0.001），而W6组降低（P<0.001），W4和W6组血清TG水平持续上升（P<0.001），而TC、HDL和LDL水平均降低（P<0.01）；过劳导致心肌细胞肿胀、排列紊乱、疏松和空泡化，与CON组相比，W4和W6组心肌TG水平升高（P<0.001），W6组ATP水平降低（P<0.05）；与CON组相比，W4和W6组CPT1B和PPARαmRNA及蛋白表达持续下调（P<0.05或P<0.01或P<0.001），W4组CD36蛋白反而表达增加（P<0.05）；此外，W2组GLUT1蛋白及PFKM、PKM2mRNA和蛋白表达降低（P<0.05或P<0.01或P<0.001），但W4和W6组较W2组又有所增加（P<0.01）。结论过劳初期小鼠血糖水平升高，并伴有心肌糖酵解受到抑制；持续过劳导致血糖水平下降，血TG水平升高，而TC、HDL和LDL水平降低，同时心肌脂肪酸氧化能力受损、加之糖酵解供能有限，最终引起心肌结构损伤、TG堆积和ATP水平下降。

关键词: 过度劳累, 心肌, 能量代谢, 脂肪酸氧化, 糖酵解

Abstract:

Objective To investigate the effect of overwork on myocardial energy metabolism in mice. Methods Thirty-two C57BL/6J mice were randomized equally into a control group and 3 overwork groups with overwork for 2, 4, and 6 weeks (W2, W4, and W6 groups, respectively). The mice in overwork groups were subjected to daily forced water standing and restraint. The changes in body weight and general condition of the mice were observed weekly. After successful modeling, the mice were examined for changes in echocardiography, blood glucose/lipid profiles, myocardial pathologies, myocardial TG and ATP levels, and expressions in CD36, GLUT1, CPT1B, PPARα, PFKM, and PKM2 using immunohistochemistry, RT-qPCR or Western blotting. Results The mice with prolonged overwork exhibited reduced activity with hair loss, dull fur, and slowed body weight gain without significant changes in cardiac index or function. Blood glucose levels increased significantly in W2 and W4 groups but decreased in W6 group. Serum TG level increased significantly while TC, HDL, and LDL decreased in W4 and W6 groups. HE staining revealed myocardial swelling, disorganization, and vacuolation in the mouse models. Myocardial TG was elevated in W4 and W6 groups and ATP level decreased in W6 group. The mRNA and protein expressions of CPT1B and PPARα were downregulated in W4 and W6 group, and CD36 expression increased significantly in W4 group. GLUT1 and PFKM/PKM2 expressions decreased obviously in W2 group but increased in W4 and W6 group compared with that in W2 group. Conclusion Short-term overwork causes elevation of blood glucose and suppresses glycolysis in mice, while prolonged overwork reduces glucose, increases TG, impairs fatty acid oxidation, and limits glycolytic compensation to eventually result in myocardial damage, lipid accumulation, and ATP deficiency.

Key words: overwork, myocardium, energy metabolism, fatty acid oxidation, glycolysis

崔俊杰, 赖睿茵, 陈苏衡, 瞿珊珊, 廖阅, 马雪, 李玉兰. 过度劳累损伤小鼠心肌能量代谢稳态[J]. 南方医科大学学报, 2025, 45(12): 2598-2606.

Junjie CUI, Ruiyin LAI, Suheng CHEN, Shanshan QU, Yue LIAO, Xue MA, Yulan LI. Overwork damages myocardial energy metabolism homeostasis in mice[J]. Journal of Southern Medical University, 2025, 45(12): 2598-2606.

图/表 9

表1 PCR引物序列

Tab.1 Primer sequence for RT-qPCR

Gene	Sequence of primers
CPT1B	F: GCACACCAGGCAGTAGCTTT
CPT1B	R: CAGGAGTTGATTCCAGACAGGTA
PPARα	F: AGAGCCCCATCTGTCCTCTC
PPARα	R: ACTGGTAGTCTGCAAAACCAAA
PFKM	F: TGTGGTCCGAGTTGGTATCTT
PFKM	R: GCACTTCCAATCACTGTGCC
PKM2	F: GCCGCCTGGACATTGACTC
PKM2	R: CCATGAGAGAAATTCAGCCGAG
ACTB	F: GGCTGTATTCCCCTCCATCG
ACTB	R: CCAGTTGGTAACAATGCCATGT

表2 各组小鼠体质量随造模时间变化情况

Tab.2 Changes of body weight of the mice in each group during modeling (g, Mean±SD, n=8)

Group	Modeling time (weeks)
Group	0	1	2	3	4	5	6
CON	20.45±0.29	21.13±0.23	22.02±0.31^a	22.54±0.47^a	23.18±0.47^a	23.96±0.37^a	24.3±0.38^a
W2	20.00±0.37	19.94±0.50	20.13±0.40^d
W4	20.50±0.19	20.24±0.24	20.41±0.19^d	20.90±0.24^b	21.14±0.30^c
W6	19.89±0.19	20.51±0.20	20.85±0.25^c	20.35±0.48^c	20.80±0.49^d	21.29±0.52^d	21.54±0.45^d

图1 各组心脏质量指数和超声心动图

Fig.1 Heart weight index (A) and echocardiography (B) in each group (mg/g, Mean±SD, n=8). #P>0.05 vs CON group. CON: Control group. W2: overwork for 2 weeks group; W4: overwork for 4 weeks group; W6: overwork for 6 weeks group.

表3 各组心功能变化

Tab.3 Cardiac function changes in each group (Mean±SD, n=3)

Group	LVIDs (mm)	LVIDd (mm)	EF%	FS%	LVPWd (mm)
CON	2.06±0.52	3.03±0.02	65.29±0.32	35.01±0.50	0.84±0.03
W2	2.01±0.33	3.06±0.03	65.76±0.44	35.14±0.79	0.84±0.01
W4	2.10±0.09	3.11±0.06	64.99±0.79	36.26±0.21	0.85±0.05
W6	2.11±0.06	3.18±0.01	64.87±0.55	35.28±0.43	0.85±0.01

图2 各组血糖和血脂变化

Fig.2 Changes of blood glucose and blood lipids in each group (Mean±SD, n=6). A-E: Blood glucose (Glu), triglycerides (TG), cholesterol (TC), high-density lipoprotein cholesterol (HDL), and low-density lipoprotein cholesterol (LDL) levels, respectively. **P<0.01, ***P<0.001 vs CON group; ##P<0.01 vs W2 group; &&&P<0.001 vs W4 group.

图3 各组小鼠心肌组织病理学,TG和ATP浓度变化

Fig.3 Histopathological changes of the myocardial tissues and changes in myocardial TG and ATP levels in each group. A: HE staining (Scale bar=50 μm). B: Myocardial TG level (n=6; ***P<0.001 vs CON group). C: Myocardial ATP level (n=6; *P<0.05 vs CON group).

图4 各组心肌CPT1B、PPARα、PFKM、PKM2 mRNA相对表达量

Fig.4 Relative expression levels of CPT1B (A), PPARα (B), PFKM (C), and PKM2 (D) mRNA in each group (n=3). *P<0.05, **P<0.01, ***P<0.001 vs CON group; #P<0.05, ###P<0.001 vs W2 group.

图5 各组心肌CD36、GLUT1蛋白表达及阳性细胞所占面积

Fig.5 Expression of CD36 and GLUT1 protein and the area of positive cells (%) in the myocardium in each group. A, B: Immunohistochemical staining (Scale bar=100 μm). C: CD36 and GLUT1 protein expression levels. *P<0.05 vs CON group.

图6 各组心肌CPT1B、PPARα、PFKM、PKM2 蛋白相对表达量

Fig.6 Relative protein expressions of CPT1B, PPARα, PFKM and PKM2 in the myocardium in each group. A, B: Protein bands in Western blotting. C, D: CPT1B, PPARα, PFKM, and PKM2 protein expression levels in each group. *P<0.05, **P<0.01, ***P<0.001 vs CON group; ##P<0.01, ###P<0.001 vs W2 group.

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