低氧暴露下高原鼠兔肺组织间隙连接蛋白40表达分析

doi:10.16829/j.slxb.150686

兽类学报 ›› 2022, Vol. 42 ›› Issue (5): 572-578.DOI: 10.16829/j.slxb.150686

低氧暴露下高原鼠兔肺组织间隙连接蛋白40表达分析

张湑泽¹(), 付林²^,⁵, 邹小艳³, 都玉蓉⁴()

^1.青海民族大学生态环境与资源学院，国家民委青藏高原资源化学与生态环境保护重点实验室，西宁 810007
^2.云南大学生命科学学院，昆明 650091
^3.长沙医学院药学院，长沙 410219
^4.青海师范大学生命科学学院，青海省青藏高原生物多样性形成机制与综合利用重点实验室，西宁 810008
^5.中国科学院西北高原生物研究所，中国科学院高原生物适应与进化重点实验室，西宁 810001

收稿日期:2022-04-23 接受日期:2022-08-19 出版日期:2022-09-30 发布日期:2022-09-21
通讯作者: 张湑泽,都玉蓉
作者简介:付林 (1991- )，男，博士研究生，主要从事为动物功能代谢研究.第一联系人：共同第一作者
基金资助:
中国科学院青海省人民政府三江源国家公园联合研究专项(LHZX-2020-01);青海省自然科学基金(2020-ZJ-965Q);青海民族大学校级规划项目(2021XJGH15)

Expression analysis of gap junction protein 40 in lung of plateau pika exposed to hypoxia

Xuze ZHANG¹(), Lin FU²^,⁵, Xiaoyan ZOU³, Yurong DU⁴()

^1.College of Eco-Environment and Resources, Key Laboratory of Resource Chemistry and Eco-environmental Protection in Tibetan Plateau of State Ethnic Affairs Commissionm, Qinghai Minzu University, Xining 810007, China
^2.College of Life Sciences, Yunnan University, Kunming 650091, China
^3.College of Pharmaceutical, Changsha Medical University, Changsha 410219, China
^4.College of Life Sciences Qinghai Normal University, Key Laboratory of Biodiversity Formation Mechanism and Comprehensive Utilization of Qinghai-Tibetan Plateau in Qinghai Province, Xining 810008, China
^5.Key Laboratory of Adaptation and Evolutionof Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China

Received:2022-04-23 Accepted:2022-08-19 Online:2022-09-30 Published:2022-09-21
Contact: Xuze ZHANG,Yurong DU

摘要/Abstract

摘要：

低氧性肺血管收缩反应钝化是高原鼠兔适应低氧环境的重要策略，但参与该生理代偿反应的功能基因尚不明确。间隙连接蛋白40 (Connexin40, Cx40) 在哺乳动物肺血管内皮表达。本研究对生活在海拔3 200 m的高原鼠兔进行28 d模拟海拔5 000 m低氧处理，以Sprague Dawley (SD) 大鼠为对照，采用免疫组化法分析模拟低氧处理后高原鼠兔和SD大鼠肺组织形态结构，qPCR和蛋白印记法检测Cx40基因和蛋白表达量变化，探究Cx40在高原鼠兔低氧性肺血管收缩反应钝化中的潜在作用。结果显示，低氧处理后，高原鼠兔肺泡呈空泡囊状，Cx40蛋白在支气管和肺血管中均表达，Cx40 基因mRNA水平随着低氧暴露而升高，但其蛋白质水平呈下降趋势，肺支气管Cx40蛋白无明显变化。SD大鼠肺血管和肺支气管表达的Cx40蛋白均无明显变化。暗示生活在高海拔低氧环境中的高原鼠兔，Cx40蛋白下调可抑制血管收缩信号，减弱低氧性肺血管收缩反应，使低氧性肺血管收缩反应钝化，以适应高原缺氧环境。研究结果可为高原土著动物适应高寒缺氧环境提供基础理论数据。

关键词: 青藏高原, 低氧, 低氧性肺血管收缩反应钝化, 高原鼠兔, 间隙连接蛋白40

Abstract:

Blunted hypoxic pulmonary vasoconstriction response is a major adaptation to hypoxia in plateau pika (Ochotona curzoniae). However, the genes involved in this physiological compensation remained unclear. In the present study, plateau pikas and Sprague Dawley (SD) rats were challenged with a hypoxia condition simulating an altitude of 5 000 m for 28 days. Morphological structure analysis by immunohistochemistry was carried out for the pulmonary in both species and the expression levels of Connexin40 (Cx40), a member of the Connexon family expressed in pulmonary vascular endothelia in mammals, were determined by both qPCR and Western blot. The results showed that upon hypoxia challenge the alveoli of plateau pika appeared to be vacuolated sacs. Cx40 expression was detected in both bronchi and pulmonary blood vessels in both species. In plateau pikas, mRNA level of Cx40 increased after hypoxia challenge, while its protein level decreased. In contrast, protein levels of Cx40 had no significant change with prolonged hypoxia exposure in pulmonary blood vessels of SD rats. No significant change in the Cx40 protein in the lung bronchi was observed in both species. Our results indicate that with hypoxia exposure, pulmonary vasoconstriction response in plateau pikas could be suppressed by down-regulation of Cx40, contributing to their adaptation to a hypoxic environment on the plateau. Our results can provide a reference for the expression analysis of gap junction proteins in the lungs of indigenous animals living on the Qinghai-Tibet Plateau.

Key words: Qinghai-Tibet Plateau, Hypoxia, Blunted hypoxia pulmonary vasoconstriction, Plateau pika (Ochotona curzoniae), Connexin40

中图分类号:

张湑泽, 付林, 邹小艳, 都玉蓉. 低氧暴露下高原鼠兔肺组织间隙连接蛋白40表达分析[J]. 兽类学报, 2022, 42(5): 572-578.

Xuze ZHANG, Lin FU, Xiaoyan ZOU, Yurong DU. Expression analysis of gap junction protein 40 in lung of plateau pika exposed to hypoxia[J]. ACTA THERIOLOGICA SINICA, 2022, 42(5): 572-578.

图/表 3

图1 低氧处理前后高原鼠兔和SD大鼠肺组织Cx40蛋白的免疫组化定位. A ~ B：SD大鼠肺组织切片；C ~ D：高原鼠兔肺组织切片；E ~ F：SD大鼠低氧处理后肺组织切片；G ~ H：高原鼠兔低氧处理后肺组织切片. 图中红色箭头指向血管，蓝色箭头指向支气管

Fig. 1 Cx 40 localization in the lung tissues of plateau pika and SD Rat between hypoxia group and control group. A - B: refers lung tissue sections of SD rats; C - D: refers lung tissue sections of plateau pika; E - F: refers lung tissue sections of SD rats treated with hypoxia; G - H: refers lung tissue sections of plateau pika treated with hypoxia. The red arrow point to vascular, the blue arrow points to the bronchus. The red arrow point to vascular, the blue arrow points to the bronchus

图2 高原鼠兔低氧处理前后Cx40基因mRNA表达量 (n = 6). mRNA定量以GAPDH为内参基因，结果以mean ± SE表示，*表示显著差异 (P < 0.05)

Fig. 2 Cx40 mRNA expression level in plateau pikas treated with hypoxia (n = 6). GAPDH was used as the reference gene for mRNA quantification, and the result was expressed by mean ± SE. * represents significant difference (P < 0.05)

图3 高原鼠兔和SD大鼠在低氧处理前后Cx40蛋白水平的变化(n = 3). 蛋白定量以β-actin为内参，结果以mean ± SE表示. **表示极显著差异 (P < 0.01)，ns表示无显著差异

Fig. 3 Changes of Cx40 protein levels in plateau pika and SD rat under hypoxia treatment (n = 3). β-actin was used as internal reference for protein quantification, and the results were expressed as mean ± SE; ** represented extremely significant difference (P < 0.01), while ns represented no significant difference

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低氧暴露下高原鼠兔肺组织间隙连接蛋白40表达分析

Expression analysis of gap junction protein 40 in lung of plateau pika exposed to hypoxia

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