过劳诱导小鼠血管内皮屏障功能障碍

doi:10.12122/j.issn.1673-4254.2024.09.22

摘要/Abstract

摘要：

目的研究过劳对小鼠血管内皮屏障功能的影响。方法将30只KM小鼠随机分为3组（n=10）：对照（CON）组、过劳2周（W2）组、过劳4周（W4）组。W2组和W4组采用水中站立+束缚实验分别连续造模2周和4周，观察小鼠一般情况。造模结束后，每组选取4只小鼠腹腔注射Evans蓝染料，检测血管通透性。另6只小鼠采取ELISA法检测血清IL-1β含量，获取动脉组织行切片观察，RT-qPCR法检测内皮细胞间紧密连接中occludin、claudin-5、ZO-1、JAM-A和黏附连接中VE-cadherin的mRNA表达水平；免疫荧光法检测内皮细胞间连接复合物中claudin-5、ZO-1、VE-cadherin和血管内皮糖萼中Syndecan-1的蛋白表达情况。结果与CON组相比，W2和W4组小鼠体质量增长缓慢，毛发脱落，活动减少。与CON组相比，W4组血管通透性明显增加。与CON组相比，W2和W4组血清IL-1β含量增加（P<0.05）。与CON组相比，W2组内皮细胞肿胀、脱落，内膜粗糙不规则，W4组出现内膜断裂现象。与CON组相比，W2组动脉组织中occludin、claudin-5、ZO-1、JAM-A的mRNA表达量增加，W4表达均降低（P<0.05）；W2和W4组VE-cadherin的mRNA表达量下降（P<0.05）。与CON组相比，W4组claudin-5、ZO-1、VE-cadherin和Syndecan-1蛋白表达减少（P<0.05）。结论过劳随时间的延长可对动脉内皮细胞间连接复合物以及内皮糖萼造成损伤，破坏其屏障功能，导致血管通透性增加。

关键词: 过劳, 内皮屏障功能, 内皮细胞间连接复合物, 血管内皮糖萼

Abstract:

Objective To investigate the impact of overwork on vascular endothelial barrier function in mice. Methods Thirty KM mice were randomized equally into control, overwork for 2 weeks (W2) group and 4 weeks (W4) group. In the latter two groups, the mice were subjected to continuous standing in water for 8 h followed by restraint for 3 h to simulate overwork on a daily basis for 2 and 4 weeks. After modeling, 4 mice from each group were intraperitoneally injected with Evans blue dye to assess vascular permeability. In the other 6 mice, serum IL-1β levels were measured using ELISA, and arterial tissues were collected for histological examination and detection of mRNA expressions of occludin, claudin-5, ZO-1, JAM-A and VE-cadherin; immunofluorescence assay was used to detect the protein expressions of claudin-5, ZO-1, VE-cadherin, and Syndecan-1. Results The mice in W2 and W4 groups exhibited slower weight gain, hair loss, reduced activity, and significantly increased serum IL-1β levels. Vascular permeability was significantly increased in W4 group. In W2 group, the endothelial cells were swollen and dissociated, and the intima was rough and irregular; arterial intimal rupture was observed in W4 group. The mRNA expressions of occludin, claudin-5, ZO-1 and JAM-A in the arterial tissues were significantly increased in W2 group but decreased in W4 group, while VE-cadherin mRNA expression were reduced in both groups (P<0.05). The protein expressions of claudin-5, ZO-1, VE-cadherin, and Syndecan-1 were all significantly reduced in W4 group. Conclusion Prolonged overwork can cause damage of the intercellular junction complexes in arterial endothelial cells and the endothelial glycocalyx to result in impaired barrier function and increased vascular permeability in mice.

Key words: overwork, endothelial barrier function, endothelial intercellular junction complexes, vascular endothelial glycocalyx

廖阅, 马雪, 邓三春, 陈苏衡, 李玉兰. 过劳诱导小鼠血管内皮屏障功能障碍[J]. 南方医科大学学报, 2024, 44(9): 1814-1820.

Yue LIAO, Xue MA, Sanchun DENG, Suheng CHEN, Yulan LI. Overwork induces vascular endothelial barrier dysfunction in mice[J]. Journal of Southern Medical University, 2024, 44(9): 1814-1820.

图/表 7

表1 PCR引物序列及产物大小

Tab.1 Primer sequence for RT-qPCR and the product size

Gene	Sequence of primers	Length (bp)
occludin	F: CCTCCACCCCCATCTGACTA	79
occludin	R: GCTTGCCATTCACTTTGCCA	79
claudin-5	F: CTTTGTTACCTTGACCGGCG	174
claudin-5	R: GGCACCGTCGGATCATAGAA	174
ZO-1	F: TCAAGTTCCTGAAGCCCGTG	117
ZO-1	R: GGACAGAAACACAGTTGGCT	117
JAM-A	F: CAAGGCAAGGGTTCGGTGTA	117
JAM-A	R: GAACTTCCACTCCACTCGGG	117
VE-cadherin	F: CTTAATTCTGTGCGCGGTGG	240
VE-cadherin	R: GGACTCTGCGATGGACTCTG	240
GAPDH	F: TGTTTCCTCGTCCCGTAG	108
GAPDH	R: CAATCTCCACTTTGCCACT	108

表2 3组小鼠体质量情况

Tab.2 Body weight changes of the mice in the 3 groups (g, n=10, Mean±SD)

Group	Molding time (week)
Group	0	1	2	3	4
CON	31.42±1.27	35.72±1.14^b	37.92±0.86^b	40.47±1.35^b	43.26±1.04^b
W2	31.57±0.99	31.70±1.32^a	33.16±0.85^ab	-	-
W4	30.26±0.70	31.18±1.11^a	34.69±0.65^ab	36.70±1.08^ab	37.15±1.15^a

图1 过劳对小鼠血管通透性的影响

Fig.1 Effect of overwork on vascular permeability in mice. A: Evans blue staining of arteries of the mice in the 3 groups. B: Evans blue staining of the cross sections of the arteries. C: Evans blue fluorescence intensity in the arterial cross-sections in the 3 groups (*P<0.05 vs control group).

图2 3组小鼠血清IL-1β含量

Fig.2 Serum IL-1β levels in 3 groups of mice. *P<0.05 vs control group, #P<0.05 vs W2 group.

图3 3组小鼠动脉内膜病理学变化

Fig.3 Pathological changes in the arterial intima of the 3groups of mice (The arrow indicates rupture of the arterial intima).

表3 内皮细胞间连接复合物相关基因表达水平

Tab.3 Expression levels of endothelial intercellular junction complexes at the mRNA level (n=6, Mean±SD)

Group	occludin	claudin-5	ZO-1	JAM-A	VE-cadherin
CON	1.00±0.03	1.00±0.05	1.00±0.04	1.00±0.06	1.00±0.07
W2	1.38±0.11^a	1.05±0.05^a	1.31±0.07^a	1.41±0.09^a	0.79±0.05^a
W4	0.73±0.06^ab	0.78±0.04^ab	0.87±0.05^ab	0.79±0.05^ab	0.68±0.09^ab

图4 过劳对3组小鼠内皮细胞间连接复合物和血管内皮糖萼相关蛋白的影响

Fig.4 Impact of overwork on the proteins in the intercellular junction complexes and vascular endothelial glycocalyx in mice. A: Fluorescence of arterial claudin-5 protein in the 3 groups ("in" denotes the luman). B: Fluorescence of arterial ZO-1 protein in the 3 groups. C: Fluorescence of arterial VE-cadherin protein in the 3 groups. D: Fluorescence of arterial Syndecan-1 protein in the 3 groups. E: Analysis of the average fluorescence intensity of claudin-5, ZO-1, VE-cadherin, and Syndecan-1 proteins in the arteries in the 3 groups. *P<0.05 vs control group.

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