Fig. 4

hNSC-Exos rescued mitochondrial dysfunction by promoting mitophagy. A Mito Tracker Red CMXRos, a red fluorescent probe dependent on mitochondrial membrane potential, and Annexin V-FITC, a green fluorescent probe for cell apoptosis, were used to jointly detect mitochondrial membrane potential level (red) and apoptosis (green), scale bar = 20 μm. The ratio of red/green fluorescence was quantified by ImageJ. B Representative fluorescence image and quantification of reactive oxygen species (ROS) (green), scale scale = 50 μm. The mean ROS fluorescence was quantified by ImageJ. C RNA-seq analysis revealed increased lysosomal gene expression in OS-treated neurons, suggesting a role for lysosomes in mitophagy activation ('NC1' refers to the 'Control' group, 'NC2' refers to the 'H₂O₂' group)". D Monodansylcadavrine (MDC) autophagy fluorescent staining (green), scale bar = 20 μm. The percentage of proportion of MDC-positive cells was quantified by ImageJ. E Representative image of HT22 cells transfected with Ad-mCherry-GFP-LC3B adenovirus. Autophagosomes are labeled by red and green fluorescence (yellow dots), whereas autophagic lysosomes are labeled by red fluorescence (red dots). The expression of red and yellow dots was observed in each cell. The bottom left corner panel magnifies the boxed area in the top panel, scale bar = 50 μm. The percentage of autophagosomes & autolysosomes per cell was quantified by ImageJ. F Mitochondrial morphology and the number of autophagosomes were observed and quantified through TEM, red arrow indicates mitochondria, and circle indicates autophagosomes, scale bar = 500 nm. The numbers of autophagosome per filed were quantified by ImageJ. WB analysis showed the expression of mitophagy-related proteins (PINK1, Parkin, Beclin, and LC3B) in vitro (G) and in vivo (H). hNSC-Exos significantly increased mitophagy-related proteins expression compared to H₂O₂-treated cells. Data were normalized to GAPDH protein expression (n = 6 rats/group). Representative images of (I) PINK1, (J) Beclin staining (green) and (K) LC3B staining (red) with DAPI-stained nuclei (blue), scale bar = 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data were presented as mean ± SD from three biological replicates, each performed in triplicate. Experimental groups ('Control,' 'H₂O₂,' and 'hNSC-Exos') were as described in Fig. 2. Experimental groups (‘Sham,’ ‘MCAO,’ and ‘hNSC-Exos’) were as described in Fig. 3