Hypoxia induced neurological dysfunction and nerve injury in mice.

(A) Schematic diagram of chronic hypoxic mouse model construction. Recognition coefficient in Con and H28d mice in (B) Y-maze test (n = 6) and (C) new object recognition test (n = 10). (D) Trajectories, (E) movement index statistics, including movement distance, speed, and time (n = 10), and (F) cognitive indicators, including length of exploration and number of crossings in the central area (n = 10) for Con and H28d mice in open-field tests. (G) Nissl staining showing arrangement of neurons in the hippocampus in Con and H28d mice. (H)The area of the hippocampus was counted in each group (n = 5). (I) FJB staining showing neurodegeneration in the cerebral cortex and hippocampus in Con and H28d mice. (J) Golgi staining showing dendritic spines of neurons in the hippocampus in Con and H28d mice. (K)The number of dendritic spines on a single axon was counted (n = 5). In (E) and (K), data expressed as mean ± SEM (Welch’s t-test); other data expressed as mean ± SEM (unpaired t-test). ns: not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Con: control group; H28d: mice treated with hypoxia for 28 days.

Chronic hypoxia increased hemoglobin transcription in mouse brain.

(A) Transcription profiles of cell subsets in the brain in Con and H28d mice visualized by UMAP. (B) Proportions of different cells in the brain in Con and H28d mice. (C) Histogram showing numbers of differentially expressed genes (DEGs) in different cell types in mouse brain after hypoxia. Up- and down-regulated forms displayed separately. (D) DEGs in different cell types in mouse brain after hypoxia shown in stacked volcano plots. Top 10 DEGs marked based on absolute log2FC value. (E) Circle diagram showing number and intensity of intercellular communications among different cell types in the brain in Con and H28d mice. Red indicates increase and blue indicates decrease. (F) Communications among cells in the H28d and Con groups demonstrated by combination of heat map and histogram. Con: control group; H28d: mice treated with hypoxia for 28 days. AST: Astrocyte; Endo: Endothelial cell; EPC: Ependymocyte; EX: Excitatory neuron; Granule: Granule cell; IN: Inhibitory neuron; Microgila: Microgila; mt-rich: Mitochondrial-rich cell; Nend: Neuronendocrine cell; OLI: Oligodendrocyte; OPC: Oligodendrocyte precursor cell; Purkinje: Purkinje cell; VLMC: Vascular lepotomeningeal cell.

Hypoxia increased release of EVs carrying hemoglobin mRNA in mouse brain.

(A) Schematic diagram of EV extraction and transcriptome sequencing in brain tissue from Con and H28d mice. (B) Typical electron microscope images of brain tissue EVs (scale bar = 0.5 μm). (C) Western blot analysis of EVs markers TSG101, HSP70, and CD9 and EVs negative marker calnexin in EVs isolated from cell lysates and brain tissues. (D) NTA analysis of brain tissue EVs isolated from Con and H28d mice. (E) Differentially expressed genes (DEGs) carried by mouse brain tissue EVs after hypoxia displayed by volcanic maps. Hemoglobin subunits labeled. (F) Histogram showing enriched signaling pathways for top five up- and down-regulated DEGs in EVs-RNA-seq. (G) The proportion of cells releasing EVs in each group. Data expressed as mean ± SEM (two-way ANOVA). *: up-regulated, #: down-regulated. ns: not significant, **P<0.01, # P<0.05, ## P<0.01. (H) Histogram illustrating the frequencies of cell types secreting EVs containing the same DEG mRNAs. The mRNAs co-carried by EVs secreted by seven, eight, and nine cells are displayed. (I) mRNA levels of each hemoglobin subunit in brain tissue EVs from Con and H28d mice (n = 3). Data expressed as mean ± SEM (two-way ANOVA). ***P<0.001, ****P<0.0001. (J) Heat map showing cell sources of EVs carrying hemoglobin subunit mRNAs. (K) GO enrichment analysis of DEGs obtained by combined snRNA-seq and EVs-RNA-seq. Signal pathways of more than three cell types displayed in a bubble map. Con: control group; H28d: mice treated with hypoxia for 28 days. AST: Astrocyte; Endo: Endothelial cell; EPC: Ependymocyte; EX: Excitatory neuron; Granule: Granule cell; IN: Inhibitory neuron; Microgila: Microgila; mt-rich: Mitochondrial-rich cell; Nend: Neuronendocrine cell; OLI: Oligodendrocyte; OPC: Oligodendrocyte precursor cell; Purkinje: Purkinje cell; VLMC: Vascular lepotomeningeal cell.

Hypoxia increased of hemoglobin expression in mouse neurons.

(A) Brains of Con and H28d mice. (B) Expression levels of hemoglobin subunits in snRNA-seq in different cell types in the brain shown by violin maps. (C) Hba-a1 mRNA levels in brain in Con and H28d mice detected by real-time qPCR (n = 5). (D and E) Hba expression levels in the brain in Con and H28d mice detected by western blotting (n = 3). (F and G) Hba expression levels in the brain in Con and H28d mice detected by immunofluorescence (scale bar = 20 μm, n = 6). (H) Expression levels of Hba and Map2 in the brain in Con and H28d mice detected by immunofluorescence (scale bar = 20 μm, n = 5). (I) Fluorescence intensity of Map2 in the brain in Con and H28d mice. (J) Co-localization of Hba and Map2 in the brain in Con and H28d mice. (K to M) Expression levels of mitochondrial complex I marker NDUFB8 and mitochondrial complex IV marker citrate synthase in the brain in Con and H28d mice detected by western blotting (n = 4-5). (N) Activity of mitochondrial complex I in the brain in Con and H28d mice (n = 4-5). In (C), (E) and (G), data expressed as mean ± SEM (Welch’s t-test); other data expressed as mean ± SEM (unpaired t-test). ns: not significant, *P<0.05, **P<0.01, ***P<0.001. Con: control group; H28d: mice treated with hypoxia for 28 days.

Hypoxia increased hemoglobin transcription in endothelial cells and induced release of EVs.

(A) Viability of hcMEC/D3 cells detected by CCK-8 assay in H0h, H4h, H8h, and H12h groups (n = 8) and (B) cytotoxicity in each group detected by lactate dehydrogenase (LDH) assay (n = 6). (C) Viability of SH-SY5Y cells in each group detected by CCK-8 assay (n =8) and (D) cytotoxicity in each group detected by LDH assay (n = 8). (E-F) Mitochondria were labeled in SH-SY5Y groups using MitoTracker staining, and the number of mitochondria in each group was analyzed and counted by flow cytometry (n = 5). (G) Mitochondrial membrane potential in each SH-SY5Y group was detected by JC-1 labeling and analyzed by flow cytometry (n = 5). HBA1 mRNA levels in (H) hcMEC/D3 cells and (I) SH-SY5Y cells detected by real-time qPCR (n = 5 each). (J) NTA analysis of EVs in hcMEC/D3 cells treated with hypoxia for 0 and 8 h. (K) Mechanism diagram. In (B), data expressed as mean ± SEM (Brown-Forsythe ANOVA test); other data as mean ± SEM (one-way ANOVA). ns: not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. H0, 4, 8, 12h: cells treated with hypoxia for 0, 4, 8, 12 hours.

Hypoxia induced neuronal uptake of endothelial cell-derived EVs and expression of hemoglobin.

(A) EVs in medium from H8h hcMEC/D3 cells were labeled with Dil dye and added to SH-SY5Y cells labeled with Calcein AM for 24 h. Uptake of hcMEC/D3 EVs by SH-SY5Y cells was observed by confocal microscopy (scale bar = 5 μm). (B) HBA1 mRNA levels in SH-SY5Y cells detected by real-time qPCR (n = 5). (C and D) Hba1 expression levels in SH-SY5Y cells detected by immunofluorescence (scale bar = 10 μm) (n = 10). (E) hcMEC/D3 conditioned medium treatment diagram. (F) Viability of SH-SY5Y cells (n = 8-10). (G) Numbers of mitochondria in SH-SY5Y cells (n = 5). (H) Mitochondrial membrane potential in SH-SY5Y cells (n = 5). (I) EVs in medium from H8h hcMEC/D3 cells were labeled with Dil and then added to calcein AM-labeled primary neurons for 24 h. Uptake of hcMEC/D3 EVs by primary neurons was observed by confocal microscopy (scale bar = 5 μm). (J) Hba-a1 mRNA levels in primary neurons detected by real-time qPCR (n = 5). (K and L) Hba expression levels in primary neurons detected by immunofluorescence (scale bar = 10 μm) (n = 10). (M) Viability of primary neurons in each group characterized by PI/Hoechst staining and photographed by confocal microscopy (n = 8). (N) Number of mitochondria in primary neurons in each group characterized by MitoTracker staining and photographed by confocal microscopy (n = 9). (O) Mitochondrial membrane potential of primary neurons in each group characterized by JC-1 staining and photographed by confocal microscopy (n = 9). (P) Mechanism diagram. In (L), data expressed as mean ± SEM (Brown-Forsythe ANOVA test); other data as mean ± SEM (one-way ANOVA). ns: not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Exosomal hemoglobin helped neurons to resist hypoxic injury by maintaining neuronal mitochondrial homeostasis.

(A) hcMEC/D3 conditioned medium EV removal pretreatment diagram. (B to D) Viability, mitochondrial number, and mitochondrial membrane potential of SH-SY5Y cells (n = 5-8). (E) HBA RNAi pretreatment diagram of hcMEC/D3 conditioned medium. (F to H) Viability, mitochondrial number, and mitochondrial membrane potential of SH-SY5Y cells (n = 3-8). (I) Mechanism diagram. In (H), data expressed as mean ± SEM (Brown-Forsythe ANOVA test); other data as mean ± SEM (one-way ANOVA). ns: not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

SnRNA-seq analysis identified 13 cell types in the mouse brain.

(A) Transcription profiles in the brain in Con and H28d mice visualized by UMAP. (B) Bubble map showing marker expression of each cell type in mouse brain. (C to L) Histogram showing enriched signaling pathways for top five up- and down-regulated DEGs in 10 kinds of cells in the brain. Con: control group; H28d: mice treated with hypoxia for 28 days. AST: Astrocyte; Endo: Endothelial cell; EPC: Ependymocyte; EX: Excitatory neuron; Granule: Granule cell; IN: Inhibitory neuron; Microgila: Microgila; mt-rich: Mitochondrial-rich cell; Nend: Neuronendocrine cell; OLI: Oligodendrocyte; OPC: Oligodendrocyte precursor cell; Purkinje: Purkinje cell; VLMC: Vascular lepotomeningeal cell.

EVs-RNA-seq analysis showed that mitochondrial function was impaired in the mouse brain after hypoxia.

(A) Levels of gene expression in each sample. (B) The average expression distribution heat map of DEGs top1000 in EVs-RNA-seq. (C to D) Histogram diagram showing signaling pathways enriched in differentially up- and down-regulated genes in EVs. (E to F) Cluster diagram showing signaling pathways enriched in differentially up- and down-regulated genes in EVs. (G to H) Cluster diagram showing protein–protein interaction network of up-regulated DEGs in EVs. (I to J) GSEA analysis of EVs-RNA-seq.

HBA1 siRNA treatment disrupts the transfer of hemoglobin mRNA in EC-conditioned medium.

(A to B) NTA analysis of EVs in hcMEC/D3 cells treated with hypoxia for 0, 4 and 12 h. (C) NTA analysis of EC-derived EVs. (D) Safety detection of endothelial cell conditioned medium. (E) Viability of SH-SY5Y cells (n = 8-10). (F) Numbers of mitochondria in SH-SY5Y cells (n = 5). (G) Mitochondrial membrane potential in SH-SY5Y cells (n = 5-6). (H) HBA1 mRNA levels in hcMEC/D3 cells detected by real-time qPCR. (I and J) hcMEC/D3 cells were labeled with calcein AM and the ratio of calcein AM-positive to mCherry-positive cells carried by the plasmid was observed by confocal microscopy. The HBA1 RNAi plasmid was transfected and screened. (K) HBA1 mRNA levels in hcMEC/D3 cells detected by real-time qPCR. In (K), data expressed as mean ± SEM (Brown-Forsythe ANOVA test); other data as mean ± SEM (one-way ANOVA). ns: not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.