高原低氧暴露导致小鼠脾脏组织脂代谢发生紊乱的分子机制

doi:10.12122/j.issn.1673-4254.2024.10.21

摘要/Abstract

摘要：

目的探究高原低氧致使小鼠脾脏组织脂代谢发生紊乱的分子机制。方法本研究以C57BL/6雄性小鼠脾脏组织为对象，随机分为两组，5只/组：平原常氧组（PSC组），饲养于海拔400 m；高原低氧组（HST组），饲养于海拔4200 m，构建30 d的低氧动物模型。通过脂质组学分析脂类代谢产物的变化，采用液相色谱-质谱联用（LC-MS）技术对脾脏组织进行代谢组学分析，筛选差异代谢物，并对其进行KEGG富集注释分析和KEGG通路分析，进一步通过转录组测序筛选出差异基因。最后，运用生物信息学分析将代谢组学和转录组学联合，重点关注了类固醇激素的生物合成、花生四烯酸代谢和嘌呤代谢通路中差异代谢物的上游靶基因。通过RT-qPCR检测11β-羟化类固醇脱氢酶1（HSD11B1）、类固醇5α还原酶1（SRD5A1）、前列腺素-内过氧化物合酶 1（PTGS1）、造血前列腺素D合成酶（HPGDS）、黄嘌呤脱氢酶（XDH）、嘌呤核苷磷酸化酶（PNP）、次黄嘌呤鸟嘌呤-磷酸核糖基转移酶（HPRT）、胞外5′-核苷酸酶（NT5E）的mRNA的表达量，通过Western blotting检测HSD11B1、SRD5A1、XDH、PNP、HPRT的蛋白表达量。结果脂质组学富集到41种差异脂类代谢物，通过代谢组学和转录组学联合分析，发现差异代谢物和差异基因显著富集于类固醇激素的生物合成、花生四烯酸代谢和嘌呤代谢。与平原常氧组相比，高原低氧组的差异代谢物肾上腺甾酮、雄甾酮、前列腺素D2、前列腺素J2、黄嘌呤、黄嘌呤碱、尿酸的表达量显著上调（P<0.05），且代谢物上游的关键基因HSD11B1、SRD5A1、PTGS1、HPGDS、XDH、PNP、HPRT、NT5E的mRNA表达量显著上调或下调（P<0.05），差异蛋白HSD11B1、SRD5A1、XDH、PNP、HPRT的表达量上调或下调（P<0.05）。结论高原低氧通过影响类固醇激素的生物合成、花生四烯酸代谢、嘌呤代谢三条通路来致使小鼠脾脏组织脂代谢发生紊乱。

关键词: 脂代谢, 高原低氧, 代谢组学, 转录组学, 脾脏, 脂质组学

Abstract:

Objective To investigate the molecular mechanism of lipid metabolism disorder in mouse spleen tissues due to high-altitude hypoxia. Methods Ten C57BL/6 male mice were randomly divided into normoxia group (maintained at an altitude of 400 m) and high-altitude hypoxia group (maintained at 4200 m) for 30 days (n=5). Lipidomics and metabolomics analyses of the spleen tissue of the mice were conducted using liquid chromatography-mass spectrometry (LC-MS) to identify the differential metabolites, which were further analyzed by KEGG enrichment and pathway analyses, and the differential genes were screened through transcriptome sequencing. Bioinformatics analysis was conducted to identify the upstream target genes of the differential metabolites in specific metabolic pathways. RT-qPCR and Western blotting were used to detect mRNA expressions of 11β‑hydroxysteroid dehydrogenase 1 (HSD11B1), steroid 5α reductase 1 (SRD5A1), prostaglandin-endoperoxide synthase 1 (PTGS1), hematopoietic prostaglandin D synthetase (HPGDS), xanthine dehydrogenase (XDH), purine nucleoside phosphorylase (PNP), hypoxanthine guanine-phosphoribosyltransferase (HPRT) and extracellular 5'-nucleotidase (NT5E) and protein expressions of HSD11B1, SRD5A1, XDH, PNP and HPRT in the mouse spleens. Results We identified a total of 41 differential lipid metabolites in the mouse spleens, and these metabolites and the differential genes were enriched in steroid hormone biosynthesis, arachidonic acid metabolism, and purine metabolism pathways. Compared to the mice kept in normoxic conditions, the mice exposed to high-altitude hypoxia showed significantly upregulated expressions of adrenosterone, androsterone, prostaglandin D2, prostaglandin J2, xanthine, xanthosine, and uric acid in the spleen with also changes in the expression levels of HSD11B1, SRD5A1, PTGS1, HPGDS, XDH, PNP, HPRT, and NT5E. Conclusion High-altitude hypoxia can result in lipid metabolism disorder in mouse spleen tissue by affecting steroid hormone biosynthesis, arachidonic acid metabolism, and purine metabolism pathways.

Key words: lipid metabolism, high-altitude hypoxia, metabolomics, transcriptomics, spleen, lipidomics

崔成玲, 许玉珍, 唐超群, 蒋佳颖, 胡英, 双杰. 高原低氧暴露导致小鼠脾脏组织脂代谢发生紊乱的分子机制[J]. 南方医科大学学报, 2024, 44(10): 2024-2032.

Chengling CUI, Yuzhen XU, Chaoqun TANG, Jiaying JIANG, Ying HU, Jie SHUANG. Molecular mechanism of high-altitude hypoxia-induced lipid metabolism disorder in mouse spleen tissue[J]. Journal of Southern Medical University, 2024, 44(10): 2024-2032.

图/表 12

表1 RT-qPCR引物信息

Tab.1 RT-qPCR primer sequences

Gene	Primer sequence (5′→3′)	bp
β-Actin	F: CATCCGTAAAGACCTCTATGCCAAC	25
	R: ATGGAGCCACCGATCCACA	19
HSD11B1	F: GCCAGGTCGGAGGAAGGTC	19
	R:GCCAGCAATGTAGTGAGCAGAG	22
SRD5A1	F: TCGTGGAGTGGTGTGGCTTTG	21
	R: TGATGATGCTGCCTCGCTCTG	21
PTGS1	F: TGTTGTTACTATCCGTGCCAGAAC	24
	R: TTCCGAAGCCAGGTCCAGATC	21
HPGDS	F: TGGTGGACACGCTGGATGAC	20
	R: CAGAAGGCGAGGTGCTTGATG	21
XDH	F: TTGCGAAGGATGAGGTTACTTGTG	24
	R: CTGGATTGTGATAATGGCTGGAAGG	25
NT5E	F: GACCAGTACCAGGGCACCATC	21
	R: CAATCAGTCCTTCCACACCGTTATC	25
HPRT	F: AGTCCCAGCGTCGTGATTAGC	21
	R: CGAGCAAGTCTTTCAGTCCTGTC	23
PNP	F: CAAAGGTGACATTCCCAGTGAGAG	24
	R: GTTGAGTCCTCCAGCAGCATTG	22

表2 小鼠脾脏指数检测

Tab.2 Detection of spleen index of the mice in the two groups (Mean±SD)

Group	Body weight (g)	Spleen quality (g)	Spleen index (%)
PSC	22.890±0.265	0.0632±0.002	0.276±0.009
HST	20.280±0.347	0.0491±0.004	0.240±0.023
t	11.97****	6.722***	2.879*

图1 脂质组学数据分析

Fig.1 Analysis of the lipidomics data of the mice. A: PCA analysis. B: Volcano map. C: Type and number of the enriched lipid metabolites. D: Correlation analysis of the differential lipids.

表3 富集到的差异代谢物数目

Tab.3 Number of the enriched differential metabolites

Compared samples

Total metabolite identification results

Total number of metabolites

with significant differences

Significantly

upregulated metabolites

Significantly downregulated metabolites

图2 Z-score 图

Fig.2 Z-score plot.

图3 KEGG通路图

Fig.3 KEGG pathway diagram. A: KEGG annotation analysis plot. B: KEGG enrichment bubble diagram.

图4 LIPID MAPS分类注释

Fig.4 LIPID MAPS classification annotation.

图5 转录组学数据分析

Fig.5 Transcriptomics data analysis. A: Volcano map. B: KEGG pathway diagram.

图6 转录代谢联合KEGG通路图

Fig.6 Transcriptional metabolism combined with KEGG pathway analysis.

图7 脂代谢相关通路中差异代谢物的相对丰度

Fig.7 Relative abundance of the differential metabolites in lipid metabolism-related pathways. *P<0.05, **P<0.01, ***P<0.001.

图8 脂代谢相关通路中关键基因的表达量

Fig.8 Expression of the key genes in lipid metabolism-related pathways. *P<0.05, **P<0.01,***P<0.001.

图9 脂代谢相关通路中基因的蛋白表达量

Fig.9 Protein expression of the genes in lipid metabolism-related pathways in the mouse spleens detected by Western blotting. A: Protein bands. B: Relative protein expression levels. *P<0.05, **P<0.01, ***P<0.001.

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