全氟辛酸暴露小鼠胚胎成骨细胞前体细胞作用及其分子机制研究

薛黎明; 徐佳乐; 林元杰; 金玉娥; 卢大胜; 汪国权

doi:10.19428/j.cnki.sjpm.2025.24804

全氟辛酸暴露小鼠胚胎成骨细胞前体细胞作用及其分子机制研究

Effectes of perfluorooctanoic acid exposure on mouse embryonic osteoblast precursor cells and its molecular mechanisms

摘要

摘要:
目的通过转录组学分析探索全氟辛酸（PFOA）引起骨丢失的生物学机制，为全氟化合物应用和骨健康危害预防提供思路。
方法采用0.1、1、10和100 μmol‧L^-1的PFOA染毒小鼠胚胎成骨细胞前体细胞（MC3T3⁃E1）24 h后，观察对细胞活力和碱性磷酸酶（ALP）活性的影响，筛出PFOA毒性临界浓度。采用转录组测序（RNA⁃seq）找出PFOA引起的差异表达基因（DEG），采用基因本体论（GO）和基因集富集分析（GSEA）对影响的基因通路进行富集分析。采用Seahorse XF代谢表型和逆转录聚合酶联反应（RT⁃PCR）方法对关键通路进行验证。
结果 10和100 μmol‧L^-1PFOA染毒后可降低MC3T3⁃E1细胞活力和ALP活性。选定10 μmol‧L^-1 PFOA暴露进行转录组学分析，结果共发现DEG有80个，其中上调基因32个，下调基因48个。GO分析结果显示，主要影响线粒体和细胞核体等细胞组分，GTP酶活性、鸟苷三磷酸（GTP）结合等分子功能和mRNA加工生物过程。GSEA分析发现，PFOA下调线粒体内脂肪酸β氧化通路。代谢表型研究发现，PFOA降低了线粒体有氧呼吸能力和腺苷三磷酸（ATP）产生量，且细胞有氧呼吸与糖酵解产生ATP的比值下降。糖代谢相关基因GK、G6PD和CS与脂肪酸代谢相关基因CPT1A和CPT2的mRNA表达下调。
结论 PFOA通过下调成骨细胞能量代谢和脂肪酸β氧化通路抑制骨形成作用，为揭示PFOA引起骨丢失作用机制奠定基础。

Abstract:
Objective To explore the biological mechanism of bone loss caused by perfluorooctanoic acid (PFOA) through transcriptomic analysis, and to provide new insights into regulating perfluoroalkyl substances (PFAS) applications and the prevention of hazards affecting bone health.
Methods Mouse embryonic osteoblast precursor cells (MC3T3-E1) were exposed to 0.1, 1, 10, and 100 μmol·L^-¹ PFOA for 24 hours to assess the effects on cell viability and alkaline phosphatase (ALP) activity, and to determine the critical concentration of PFOA toxicity. The transcriptome sequencing (RNA-seq) was performed to identify differentially expressed genes (DEGs) induced by PFOA. Gene ontology (GO) analysis and gene set enrichment analysis (GSEA) were conducted to identify significantly affected gene pathways. Additionally, Seahorse XF metabolic phenotyping and reverse transcription polymerase chain reaction (RT-PCR) were used to validate the key pathways.
Results Exposure to 10 and 100 μmol·L^-¹ PFOA significantly reduced the cell viability and ALP activity of MC3T3-E1 cells. Therefore, the results of transcriptomic analysis for 10 μmol‧L^-1 PFOA exposure found that a total of 80 DEGs were identified, including 32 upregulated genes and 48 downregulated genes. According to GO analysis, PFOA mainly affected cellular components such as mitochondrion and nucleus, molecular functions involving GTPase activity and GTP binding, as well as biological process related to mRNA processing. GSEA identified the downregulation of the β-oxidation of fatty acid pathway in mitochondria. Metabolic phenotyping reserches showed that PFOA indeed reduced mitochondrial aerobic respiration capacity and adenosine triphosphate (ATP) production, and the ratio of ATP production from cellular aerobic respiration to glycolysis was significantly decreased as well. The mRNA expression of glucose metabolism-related genes (GK, G6PD, and CS), as well as fatty acid metabolism-related genes (CPT1A and CPT2), were significantly downregulated.
Conclusion PFOA reduces bone formation by inhibiting energy metabolism and β-oxidation of fatty acid pathways in osteoblasts, whihc lays the foundation for revealing the mechanism of PFOA exposure induced bone loss.

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