模拟高原低氧环境下运动疲劳大鼠脑皮层单羧酸转运蛋白的变化及其意义

doi:10.12300/j.issn.1674-5817.2022.050

摘要/Abstract

摘要：

目的探讨高原低氧环境下运动疲劳适应性的产生与大脑皮层乳酸转运及代谢状态的关联性。方法 63只清洁级SD大鼠随机分为对照组、常规运动组、急进高原组、高原习服3 d组、高原习服1周组、高原习服2周组及给予乳酸转运体抑制剂组（简称高原抑制剂组）。除对照组外，各组大鼠分别在常压常氧或模拟高原低压低氧条件下建立跑台力竭运动疲劳模型。以平均力竭时间确定大鼠运动疲劳的变化趋势。采用免疫印迹和免疫组织化学法检测运动区皮层单羧酸转运蛋白2（monocarboxylate transporters 2，MCT2）和MCT4的表达。采用尼氏染色进行大脑皮层迟发性神经元病理改变评价，并测定鼠脑乳酸含量。结果急进高原组和高原抑制剂组大鼠平均力竭时间分别为（61.00±6.55）min和（71.25±9.59）min，显著低于常规运动组［（124.75±9.36）min］和高原习服2周组［（100.25±9.74）min，P＜0.05）］。免疫印迹检测提示，高原习服2周组及高原抑制剂组鼠脑运动区皮层MCT2表达水平较对照组明显升高，分别为120.6%和164.4%（P＜0.05）；MCT4的表达分别为174.6%和168.8%（P＜0.05）。免疫组织化学检测印证了免疫印迹结果。模拟高原环境下，各组鼠脑乳酸平均含量较常规运动组［（0.175±0.021）mmoL/g］均明显升高（P＜0.05）。病理学评价提示，急进高原组和高原抑制剂组鼠脑皮层平均神经元密度值（每个高倍视野的神经元数量）分别为46.75±8.65和63.50±7.65，较对照组（135.88±8.59）明显降低（P＜0.05）；高原习服2周组（121.75±16.00）与对照组差异无统计学意义（P >0.05）。结论高原习服后机体对运动疲劳的适应性与脑内MCT2和MCT4表达变化相关联，可作为运动疲劳医学干预的靶点。

关键词: 高海拔, 低氧, 运动疲劳, 单羧酸转运蛋白, 乳酸, 大脑皮层, 大鼠

Abstract:

Objective To explore the correlation between adaptation to exercise-induced fatigue and lactate transport and metabolism in the cerebral cortex under high-altitude hypoxia. Methods A total of 63 SD rats were randomly divided into control, normal exercise, rush-entry-into-altitude, 3-day-altitude acclimatization, 1-week-altitude acclimatization, 2-week-altitude acclimatization, and monocarboxylate-transporter-inhibitor (altitude inhibitor injection) groups. Except for the control group, rats were subjected to exhaustive-load treadmill exercises under either normal pressure and normoxic conditions or low pressure and hypoxic conditions. The average time-to-exhaustion was used to determine variation trends in exercise-induced fatigue. The expression of monocarboxylate transporters (MCT)2 and MCT4 in the cerebral motor cortex was detected using western blotting and immunohistochemistry. The pathological evaluation of neuronal death in the cortex was carried out using Nissl staining, and the lactate content was also determined in rat brains. Results The average time-to-exhaustion in the rush-entry-into-altitude and the altitude inhibitor injection groups were (61.00±6.55) min and (71.25±9.59) min, respectively, which was significantly lower than that in the normal exercise (124.75±9.36) min and the 2-week-altitude acclimatization groups (100.25±9.74) min (P<0.05). Western blotting showed that, compared with the control group, the expression of MCT2 in the motor cortex in the 2-week-altitude acclimatization and the altitude inhibitor injection groups increased significantly by 120.6% and 164.4% (P<0.05), respectively; the expression of MCT4 in the two groups increased significantly by 174.6% and 168.8% (P<0.05). The results of western blotting were confirmed by the results of immunohistochemistry. In the high-altitude environment, the average lactate content in rat brains was significantly higher than that in the normal exercise group (0.175±0.021) mmol/g, P<0.05]. Neuropathological evaluation showed that the average neuronal density [neurons per high power field (HPF)] in the motor cortex in the rush-entry-into-altitude and the altitude inhibitor injection groups were (46.75±8.65) cells/HPF and (63.50±7.65) cells/HPF, respectively, which were significantly lower than that in the control group [(135.88±8.59) cells/HPF](P<0.05). Differences between the 2-week-altitude acclimatization [(121.75±16.00) cells/HPF] and the control groups were not statistically significant (P > 0.05). Conclusion The adaptability of the body to exercise fatigue after altitude acclimatization is correlated with changes in the expression of MCT2 and MCT4 in the brain, which can be used as targets for medical intervention for exercise-induced fatigue.

Key words: High altitude, Hypoxia, Exercise-induced fatigue, Monocarboxylate transporters, Lactate, Cerebral cortex, Rats

中图分类号:

G804.7

高晨,凡春玲,李玉荣,等. 模拟高原低氧环境下运动疲劳大鼠脑皮层单羧酸转运蛋白的变化及其意义[J]. 实验动物与比较医学, 2022, 42(5): 384-392. DOI: 10.12300/j.issn.1674-5817.2022.050.

Chen GAO,Chunling FAN,Yurong LI,et al. Changes in Expression of Monocarboxylate Transporters in the Rat Cerebral Cortex after Exercise-induced Fatigue Under Simulated High-altitude Hypoxia and its Significance[J]. Laboratory Animal and Comparative Medicine, 2022, 42(5): 384-392. DOI: 10.12300/j.issn.1674-5817.2022.050.

图/表 6

表1 各组运动疲劳模型大鼠平均力竭时间 (xˉ±s, n=9)

Table 1 Average time–to–exhaustion in rats under exercise-induced fatigue conditions

分组 Group	平均运动力竭时间 Average exhaustion time /min
常规运动组 NE group	124.75±9.36^#△
急进高原组 REIA group	61.00±6.55^*
高原习服3 d组 3 d AA group	66.38±4.72^*
高原习服1周组 1 week AA group	73.13±7.02^*
高原习服2周组 2 weeks AA group	100.25±9.74^#△
高原抑制剂组 MTI group	71.25±9.59^*

图1 免疫印迹检测各组运动疲劳模型大鼠脑运动皮层中MCT2和MCT4的表达注：MCT2与对照组比较，*P＜0.05；MCT4与对照组比较，#P＜0.05。单因素方差分析采信Bonferroni法结果，n=3。

Figure 1 Western blotting analysis of MCT2 and MCT4 expressions in the cerebral motor cortex of rats in each groupNote：CG: Control group； NE group: Normal exercise group； REIA group: Rush-entry-into-altitude group； 3 d AA group: 3-day-altitude acclimatization group； 1 week AA group: 1-week-altitude acclimatization group； 2 weeks AA group: 2-week-altitude acclimatization group； MTI group: Monocarboxylate transporter inhibitor group. *P＜0.05， versus CG group for MCT2 quantitative analysis; #P<0.05， versus CG group for MCT4 quantitative analysis, ANOVA with post hoc Bonferroni correction. n=3 for each group.

图2 免疫组织化学检测各组运动疲劳模型鼠脑运动皮层中MCT2（A）和MCT4（B）表达（DAB染色法，×400）注：A为大鼠脑运动皮层神经元MCT2表达；B为大鼠脑运动皮层星形胶质细胞MCT4表达。a和a’为对照组；b和b’为常规运动组；c和c’为急进高原组；d和d’为高原习服3 d组；e和e’为高原习服1周组；f和f’为高原习服2周组；g和g’为高原抑制剂组。比例尺大小为25 μm。

Figure 2 Immunohistochemical detection of MCT2 (A) and MCT4 (B) in the cerebral motor cortex of rats in each group (DAB staining, ×400)Note： A, MCT2 expression of neurons in the cerebral motor cortex of rats; B, MCT4 expression in astrocytes in the cerebral motor cortex of rats. a and a’, control group; b and b’, normal exercise group; c and c’, rush-entry-into-altitude group; d and d’, 3-day-altitude acclimatization group; e and e’, 1-week-altitude acclimatization group; f and f’, 2-week-altitude acclimatization group; g and g’, mono-carboxylate transporter inhibitor group. Scale bar = 25 μm.

表2 各组运动疲劳模型鼠脑运动皮层神经元组织学分级数量和平均神经元密度值 (xˉ±s, n=3)

Table 2 Histological grade and average neuronal density in the cerebral motor cortex of rats under exercise-induced fatigue conditions

分组 Group	神经元组织学分级 Neuronal histological grade (count)				平均神经元密度值 Average neuronal density/（cells·HPF^-1）
分组 Group	0级 0 grade	Ⅰ级 Ⅰ grade	Ⅱ级 Ⅱ grade	Ⅲ级 Ⅲ grade	平均神经元密度值 Average neuronal density/（cells·HPF^-1）
对照组 CG	3	0	0	0	135.88±8.59
常规运动组 NE group	2	1	0	0	123.88±6.71
急进高原组 REIA group	0	0	2	1	46.75±8.65^*
高原习服3 d组 3 d AA group	0	0	3	0	54.13±11.33^*
高原习服1周组 1 week AA group	0	2	1	0	119.50±6.99^*
高原习服2周组 2 weeks AA group	0	3	0	0	121.75±16.00
高原抑制剂组 MTI group	0	1	2	0	63.50±7.65^*

图3 各组运动疲劳模型鼠脑运动皮层神经元尼氏染色（×200，局部放大×400）注：a为对照组；b为常规运动组；c为急进高原组；d为高原习服3 d组；e为高原习服1周组；f为高原习服2周组；g为高原抑制剂组。比例尺大小为50 μm。

Figure 3 Nissl staining in the cerebral motor cortex of rats under exercise-induced fatigue conditions (×200，local enlarged image×400 )Note：a, control group; b, normal exercise group; c, rush-entry-into-altitude group; d, 3-day-altitude acclimatization group; e, 1-week-altitude acclimatization group; f, 2-week-altitude acclimatization group; g, monocarboxylate transporter inhibitor group. Scale bar = 50 μm.

表3 各组运动疲劳模型大鼠平均脑组织乳酸含量 (xˉ±s, n=3)

Table 3 Average brain lactate content in rats under exercise-induced fatigue conditions

分组 Group	脑组织乳酸含量 Average brain lactate content/(mmol·g^-1)
对照组 CG	0.163±0.011^#△
常规运动组 NE group	0.175±0.021^#△
急进高原组 REIA group	0.239±0.017^*△
高原习服3 d组 3 d AA group	0.247±0.012^*△
高原习服1周组 1 week AA group	0.397±0.017^*#△
高原习服2周组 2 weeks AA group	0.412±0.024^*#△
高原抑制剂组 MTI group	0.537±0.011^*#

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