慢性间歇低氧和复氧对大鼠胰岛素抵抗及骨骼肌miR-27a-3p/PPARγ/IRS1/PI3K/AKT表达的影响

doi:10.12122/j.issn.1673-4254.2024.09.13

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (9): 1729-1737.doi: 10.12122/j.issn.1673-4254.2024.09.13

慢性间歇低氧和复氧对大鼠胰岛素抵抗及骨骼肌miR-27a-3p/PPARγ/IRS1/PI3K/AKT表达的影响

周雪利¹(), 李华³, 陈青宇², 靳美娜¹, 李海波¹, 白炜¹, 贾楚璇¹, 魏翠英¹()

^1.内蒙古科技大学包头医学院第一附属医院，老年医学科，内蒙古包头 014010
^2.内蒙古科技大学包头医学院第一附属医院，口腔科，内蒙古包头 014010
^3.内蒙古包头市固阳县人民医院公共卫生科，内蒙古包头 014010

收稿日期:2024-03-05 出版日期:2024-09-20 发布日期:2024-09-30
通讯作者: 魏翠英 E-mail:15540887135@163.com;weicuiying9@163.com
作者简介:周雪利，硕士，E-mail: 15540887135@163.com
基金资助:
国家自然科学基金(81660020);内蒙古自治区科技计划项目(2021GG0219);包头市卫生健康科技计划项目(wsjkkj2022008)

Effects of chronic intermittent hypoxia and reoxygenation on insulin resistance and skeletal muscle miR-27a-3p/PPARγ/IRS1/PI3K/AKT expressions in rats

Xueli ZHOU¹(), Hua LI³, Qingyu CHEN², Meina JIN¹, Haibo LI¹, Wei BAI¹, Chuxuan JIA¹, Cuiying WEI¹()

^1.Department of Geriatrics, Guyang County People's Hospital, Baotou 014010, China
^2.Department of Stomatology, First Affiliated Hospital of Baotou Medical College, Inner Mongolia University of Science and Technology, Baotou 014010, China, Guyang County People's Hospital, Baotou 014010, China
^3.Department of Public Health, Guyang County People's Hospital, Baotou 014010, China

Received:2024-03-05 Online:2024-09-20 Published:2024-09-30
Contact: Cuiying WEI E-mail:15540887135@163.com;weicuiying9@163.com
Supported by:
National Natural Science Foundation of China(81660020)

摘要/Abstract

摘要：

目的探讨慢性间歇低氧（CIH）和复氧对大鼠胰岛素抵抗（IR）及骨骼肌miR-27a-3p/PPARγ/IRS1/PI3K/AKT表达的影响。方法从基因表达数据库（GEO）中下载CIH条件下miRNA数据集，运用DESeq2筛选差异表达最显著的miRNA作为研究对象，使用miRNA walk网站对差异miRNA的靶基因进行基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析，并Cytoscape软件构建miRNA-mRNA-pathway调控网络。将48只雄性SD大鼠随机分为对照（CON）组和CIH组，24只/组，CON组常氧环境饲养12周，CIH组先予CIH环境饲养8周，再恢复常氧饲养4周。两组均在基线、8周、12周时留取血样和骨骼肌组织，检测空腹血糖（FBG）、空腹胰岛素（FINS）水平，HE染色观察骨骼肌病理改变并计算肌纤维横截面积，实时定量聚合酶链反应（RT-qPCR）和Western blotting法检测骨骼肌miR-27a-3p、过氧化物酶体增殖物激活受体γ（PPARγ）、葡萄糖转运蛋白4（GLUT4）、胰岛素受体1（IRS1）、p-IRS1、磷脂酰肌醇3激酶（PI3K）、磷酸激酶B（AKT）、p-AKT表达，比较组间差异。结果生信分析发现，GEO数据库未筛选出CIH状态下肌肉组织相关的miRNA数据集，仅得到肾脏组织差异表达miRNA的GSE202480数据集，从CON组和CIH组样本中筛选出165个差异表达的miRNA，选最显著miR-27a-3p作为研究对象。GO和KEGG分析显示，差异miRNA的靶基因主要参与肌肉调节和胰岛素信号传导。miRNA-mRNA-pathway调控网络显示miR-27a-3p是PPAR信号通路和PI3K/AKT信号通路的关键调控因子。动物实验发现，基线时，两组各项观察指标均无统计学意义（P>0.05）；8周时，与CON组相比， CIH组FBG、FINS、HOMA-IR、PPARγ显著升高（P<0.05或P<0.01），肌纤维排列疏松，肌纤维横截面积、miR-27a-3p、p-IRS1/IRS1、PI3K、p-AKT/AKT显著降低（P<0.05或P<0.01），GLUT4表达呈升高趋势但无统计学意义（P>0.05）；12周时，两组各项观察指标均无统计学意义（P>0.05）。结论 CIH可导致大鼠IR增加和骨骼肌病理改变，而复氧可以逆转；CIH可致骨骼肌miR-27a-3p表达下调，PPARγ表达上调，IRS1/PI3K/AKT胰岛素信号转导受到抑制，而复氧干预可以逆转；miR-27a-3p可能通过调控PPARγ/IRS1/PI3K/AKT信号通路参与了CIH所致的IR。

关键词: 慢性间歇低氧-复氧, 胰岛素抵抗, GEO, miR-27a-3p, IRS1/PI3K/AKT通路

Abstract:

Objective To investigate the effects of chronic intermittent hypoxia (CIH) and reoxygenation on insulin resistance (IR) and expressions of miR-27a-3p/PPARγ/IRS1/PI3K/AKT in rat skeletal muscle. Methods GEO database was used for screening the differentially expressed miRNAs in CIH, and their target genes were subjected to GO and KEGG enrichment analysis followed by construction of the miRNA-mRNA-pathway regulatory network using Cytoscape. In the animal experiment, 48 male SD rats were randomly divided into normoxia group and CIH group (8 weeks of CIH followed by 4 weeks of normoxic recovery). Blood and skeletal muscle samples were collected at baseline, 8 weeks, and 12 weeks to evaluate the changes in fasting blood glucose (FBG) and fasting insulin (FINS) levels and muscular pathology. RT-qPCR and Western blotting were used to detect the changes in the expressions of miR-27a-3p, PPARγ, GLUT4, IRS1, p-IRS1, PI3K, p-AKT and AKT in the muscular tissues. Results No muscular miRNA datasets for CIH were available in GEO database, from which only a kidney-related dataset (GSE202480) was obtained, based on which a total of 165 differentially expressed miRNAs were identified. GO/KEGG analysis suggested that these miRNAs were involved in muscular regulation and insulin signaling. The miRNA-mRNA-pathway network highlighted miR-27a-3p as a crucial regulator in the PPAR and PI3K/AKT pathway. In the animal experiment, the rats subjected to CIH for 8 weeks showed significantly increased FBG, FINS, HOMA-IR, and PPARγ levels, loose muscle fiber arrangement, decreased cross-sectional area of the muscle fibers, and lowered expressions of miR-27a-3p, p-IRS1/IRS1, PI3K, and p-AKT/AKT in the skeletal muscles. Conclusion CIH increases IR, causes skeletal muscle pathology, downregulates miR-27a-3p expression, upregulates PPARγ expression, and inhibits IRS1/PI3K/AKT insulin signaling in the skeletal muscles of rats, and these changes can be reversed by reoxygenation. MiR-27a-3p may participate in CIH-induced IR by modulating the PPAR γ/IRS1/PI3K/AKT signaling pathway.

Key words: chronic intermittent hypoxia-reoxygenation, Insulin resistance, GEO database, miR-27a-3p, IRS1/PI3K/AKT pathway

周雪利, 李华, 陈青宇, 靳美娜, 李海波, 白炜, 贾楚璇, 魏翠英. 慢性间歇低氧和复氧对大鼠胰岛素抵抗及骨骼肌miR-27a-3p/PPARγ/IRS1/PI3K/AKT表达的影响[J]. 南方医科大学学报, 2024, 44(9): 1729-1737.

Xueli ZHOU, Hua LI, Qingyu CHEN, Meina JIN, Haibo LI, Wei BAI, Chuxuan JIA, Cuiying WEI. Effects of chronic intermittent hypoxia and reoxygenation on insulin resistance and skeletal muscle miR-27a-3p/PPARγ/IRS1/PI3K/AKT expressions in rats[J]. Journal of Southern Medical University, 2024, 44(9): 1729-1737.

图/表 7

表1 用于RT-qPCR的引物序列

Tab.1 Primer sequences for RT-qPCR

Gene	Primer sequence 5'-3'
miR-27a-3p-F	ACACTCCAGCTGGGTTCACAGTGGCTAAG
miR-27a-3p-R	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCGGAACT
U6-F	CTCGCTTCGGCAGCACA
U6-R	AACGCTTCACGAATTTGCGT
PPARγ-F	TTTCAAGGGTGCCAGTTTCG
PPARγ-R	GGAGGCCAGCATGGTGTAGAT
GAPDH-F	CTGGAGAAACCTGCCAAGTATG
GAPDH-R	GGTGGAAGAATGGGAGTTGCT

图1 慢性间歇性低氧(CIH)中差异表达mRNA的生物信息学分析

Fig.1 Bioinformatics analysis of differentially expressed mRNAs in chronic intermittent hypoxia (CIH). A: CIH exposure regulates miR-27a-3p. B: Heat map showing significant differences in miRNAs between CIH group and control group. C: GO enrichment analysis of the differentially expressed mRNAs. D: KEGG enrichment analysis of the differentially expressed mRNAs. E: Venn diagram of intersecting mRNAs. F: The miR-27a-3p-mRNA pathway regulatory network.

图2 不同时间点两组大鼠骨骼肌肌纤维HE染色及分析

Fig.2 HE staining of the skeletal muscle and analysis of cross-sectional area of the muscle fibers in the two groups (Original magnification: ×400). A: Control group at baseline. B: CIH group at baseline. C: Control group at 8 weeks. D: CIH group at 8 weeks. E: Control group at 12 weeks. F: CIH group at 12 weeks. G: Comparison of the cross-sectional area of the skeletal muscle fibers. aP<0.05 vs control group.

图3 不同时间点两组大鼠骨骼肌中miR-27a-3p、PPARmRNA水平的比较

Fig.3 Comparison of miR-27a-3p (A) and PPAR mRNA (B) levels in skeletal muscles in the two groups at different time points detected by RT-qPCR. aP< 0.05 vs control group.

图4 TargetScan数据库显示miR-27a-3p靶向PPARG和IRS1的结合位点

Fig.4 TargetScan database showing the binding site of miR-27a-3p for targeting PPARG and IRS1.

图5 Western blotting检测不同时间两组大鼠骨骼肌中PPARγ、GLUT4、p-IRS1/IRS1、PI3K和p-AKT/AKT蛋白表达水平

Fig.5 Western blotting for detecting expression levels of PPARγ, GLUT4, p-IRS1/IRS1, PI3K and p-AKT/AKT proteins in the skeletal muscles in the two groups at different time points. A: Western blots of PPARγ, GLUT4, p-IRS1/IRS1, PI3K and p-AKT/AKT in the skeletal muscles. B-F: Relative protein expression levels of PPARγ, GLUT4, p-IRS1/IRS1, PI3K and p-AKT/AKT, respectively. aP<0.05,bP<0.01 vs control group.

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慢性间歇低氧和复氧对大鼠胰岛素抵抗及骨骼肌miR-27a-3p/PPARγ/IRS1/PI3K/AKT表达的影响

Effects of chronic intermittent hypoxia and reoxygenation on insulin resistance and skeletal muscle miR-27a-3p/PPARγ/IRS1/PI3K/AKT expressions in rats

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