Yang Sijia, Chen Mengyuan, Yang Di, Deng Furong, Guo Xinbiao
Department of Occupational and Environmental Health Sciences, School of Public Health, Peking University, No. 38 Xueyuan Road, Beijing, China.
Arch Toxicol. 2025 Mar 18. doi: 10.1007/s00204-025-04019-x.
In the current study, we investigated the effects of two legacy per- and polyfluoroalkyl substances (PFASs) namely perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) on osteogenesis. The alterations of connexin 43 (Cx43)-mediated gap junctions (GJs) were further explored as a potential mechanism. The two cell models (C3H10T1/2 and MC3T3-E1 cells) differentiated into osteoblasts (OBs) were utilized, and treated with PFOA and PFOS at the doses of 0.25, 2.5, 25, and 75 μM. Real-time PCR and Western blot were applied to assess the mRNA and protein expression of osteogenic-specific markers and Cx43. ALP staining and ARS staining were used to evaluate the osteogenesis process. The scrape-loading dye transfer assay was performed to assess the GJ-mediated intercellular coupling. To investigate the role of gap-junctional intercellular communication (GJIC) in the PFAS-induced osteogenic inhibition, the Cx43-specific GJIC enhancer, rotigaptide (ZP123), was added into the differentiation medium of C3H10T1/2 cells. After the exposure of PFOA and PFOS, the osteogenic molecules were down-regulated and the calcium deposition was reduced in the two cell models, indicating the inhibitory effects of the legacy PFASs. The Cx43 expression and GJIC activity were significantly suppressed, and the usage of ZP123 rescued the adverse impact on osteogenesis, suggesting the remarkable role of GJIC herein.
在本研究中，我们调查了两种传统的全氟和多氟烷基物质（PFASs），即全氟辛酸（PFOA）和全氟辛烷磺酸（PFOS）对成骨作用的影响。作为一种潜在机制，我们进一步探讨了连接蛋白43（Cx43）介导的缝隙连接（GJs）的改变。我们利用两种分化为成骨细胞（OBs）的细胞模型（C3H10T1/2和MC3T3-E1细胞），并用0.25、2.5、25和75μM剂量的PFOA和PFOS进行处理。应用实时PCR和蛋白质印迹法评估成骨特异性标志物和Cx43的mRNA和蛋白质表达。采用碱性磷酸酶（ALP）染色和茜素红染色（ARS染色）评估成骨过程。进行刮擦加载染料转移试验以评估GJ介导的细胞间偶联。为了研究缝隙连接细胞间通讯（GJIC）在PFAS诱导的成骨抑制中的作用，将Cx43特异性GJIC增强剂rotigaptide（ZP123）添加到C3H10T1/2细胞的分化培养基中。暴露于PFOA和PFOS后，两种细胞模型中的成骨分子下调，钙沉积减少，表明传统PFASs具有抑制作用。Cx43表达和GJIC活性显著受到抑制，使用ZP123挽救了对成骨的不利影响，表明GJIC在其中发挥了显著作用。
