Astragaloside Ⅳ pretreatment of umbilical cord mesenchymal stem cell-derived exosomes intervenes in Nrf2/GPX4 pathway regulation of chondrocytes research on the mechanism of iron death

Abstract:Objective To investigate the impact of Astragaloside Ⅳ(AS-Ⅳ) pretreatment of human umbilical cord mesenchymal stem cell-derived exosomes(hUCMSCs-Exos) on ferroptosis in chondrocytes.Methods Experiments were conducted at the Animal Experimental center of Xinjiang Medical University from January 2024 to December 2024, hUCMSCs were pretreated with AS-Ⅳ, followed by the extraction of hUCMSCs-Exos, which were characterized for morphology and size distribution using transmission electron microscopy(TEM) and nanoparticle tracking analysis(NTA). Rat chondrocytes were divided into four groups based on intervention methods: control group(normal chondrocyte culture), model group(IL-1β intervention), exosome group(IL-1β + hUCMSCs-Exos intervention), and AS-Ⅳ combined exosome group(IL-1β + AS-Ⅳ pretreated hUCMSCs-Exos). Mitochondrial morphology in each group was observed using electron microscopy. Cells were collected by centrifugation, and glutathione(GSH), malondialdehyde(MDA), and reactive oxygen species(ROS) were extracted and quantitatively analyzed. mRNA and protein expression levels of Nrf2 and GPX4 in chondrocytes were detected using RT-qPCR and Western blotting, respectively. Results Exosomes pretreated with AS-Ⅳ exhibited similar vesicular structures and diameters compared to untreated exosomes. Compared to the control group, the model group showed significant mitochondrial atrophy and membrane rupture. Both the exosome group and the AS-Ⅳ combined exosome group demonstrated improved mitochondrial morphology compared to the model group, with the AS-Ⅳ combined exosome group showing more pronounced improvements. The model group exhibited decreased GSH content and increased MDA and ROS levels compared to the control group(t/P=34.851/<0.001). The exosome group and AS-Ⅳ combined exosome group showed sequentially decreased GSH content and increased MDA and ROS levels compared to the model group(F/P=6.977/<0.001; 17.249/<0.001), with the AS-Ⅳ combined exosome group having higher GSH content and lower MDA and ROS levels than the exosome group(t/P=7.744/<0.001). The model group showed reduced mRNA and protein expression levels of Nrf2 and GPX4 in chondrocytes compared to the control group(t/P=18.871/<0.001). The exosome group and AS-Ⅳ combined exosome group exhibited sequentially increased mRNA and protein expression levels of Nrf2 and GPX4 compared to the model group(F/P=8.390/<0.001; 4.143/<0.05), with the AS-Ⅳ combined exosome group showing higher expression levels than the exosome group(t/P=6.912/<0.05).Conclusion The regulatory effects of AS-Ⅳ pretreated hUCMSCs-Exos on ferroptosis in chondrocytes may be associated with the upregulation of the Nrf2/GPX4 signaling pathway, thereby inhibiting ferroptotic responses and reducing chondrocyte damage.