Fig. 4. MSC-Exo mediated the effect of MSCs against CCl₄-induced ferroptosis in vitro.

A Relative ROS levels were measured by H₂DCFDA staining in primary hepatocytes treated with control (PBS), dimethyl sulfoxide (DMSO), or 10 mM CCl₄ for 48 h with or without MSCs coculture. B The increased MDA level induced by CCl₄ was downregulated by MSCs coculture and Fer-1 treatment in primary hepatocytes. The increased MDA level induced by erastin was also downregulated by MSCs coculture in primary hepatocytes. C Refrigerated transmission electron microscopy was used to assess exosomes isolated from MSCs. D Exosomes collected from MSC-conditioned medium were labeled with PKH67 and incubated with CCl₄-induced acute injured hepatocytes for 24 h. The cell membrane was stained with Dil. The nuclei were stained with DAPI. PKH67 lipid dye was detected in damaged hepatocytes treated with PKH67-labeled exosomes in the CCl₄ group. E Isolated hepatocytes were incubated with C11-BODIPY^581/791 and then analyzed by using laser confocal focus. Cell membrane was stained with Dil. The nuclei were stained with DAPI. C11-BODIPY^581/591 staining showed a significant increase in lipid peroxidation in hepatocytes following CCl₄ and erastin treatment, while MSC-Exo and Fer-1 downregulated the increased lipid peroxidation induced by CCl₄ and erastin. F The increased MDA level induced by CCl₄ and erastin were downregulated by MSC-Exo and Fer-1 treatments in primary hepatocytes. Significance was calculated by one-way ANOVA with Tukey’s post hoc test. *p < 0.05 or **p < 0.001 indicated a significant difference between groups.