Single-cell transcriptome sequencing for opening the blood-brain barrier through specific mode electroacupuncture stimulation
- The Third Affiliated Hospital of Zhejiang Chinese Medical University, China
- The Third Clinical Medical College, Zhejiang Chinese Medical University, China
- Key Laboratory of Acupuncture and Neurology of Zhejiang Province, China
- Department of Rehabilitation, Lishui Central Hospital, China
Figures
Figure 1
Overview of single-cell analysis of the experimental samples.
(a) Schematic overview of the study design. (b) Results of effective cell identification. (c) Percentage of 23 clusters in EA and CON groups. (d) T-Distributed Stochastic Neighbor Embedding (t-SNE) of the transcriptome from cells of the CON and EA groups. CON, control; EA, electroacupuncture.
Figure 2
Results of the cell type identification.
(a) Dot plot heatmap of the marker genes in individual clusters. (b) Cell type identification T-Distributed Stochastic Neighbor Embedding (t-SNE) diagram. (c) The violin plot shows the expression of marker genes of eight cells. CON, control; EA, electroacupuncture.
Figure 3
Differential gene and enrichment analysis results of the endothelial cells (electroacupuncture, EA vs control group, CON).
(a) Differential gene volcano map. (b) Histogram of the KEGG analysis results. (c) Histogram of the GO analysis results. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 4
Gene set enrichment analysis results (electroacupuncture, EA vs control group, CON).
(a-h) Eight pathways of GSEA results, EA vs CON.
Figure 5
Differential gene and enrichment analysis results of astrocytes (electroacupuncture, EA vs control group, CON).
(a) Differential gene volcano map. (b) Histogram of KEGG analysis results. (c) Histogram of GO analysis results. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 6
Differential gene and enrichment analysis results of the microglia (electroacupuncture, EA vs control group, CON).
(a) Differential gene volcano map. (b) Histogram of KEGG analysis results. (c) Histogram of GO analysis results. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 7
Subgroup clustering and functional enrichment of endothelial cells (electroacupuncture, EA vs control group, CON).
(a) Endothelial cell subset analysis in the two groups. (b) The top 10 characteristic expression genes of the four subgroups. (c–f) The GO and KEGG results of the characteristic expression genes of the four subgroups. The histogram of GO results show that the number of enriched genes was ranked in the top 20, and q<0.05. The histogram and bubble diagram of the KEGG results show the top 20 terms of the number of enriched genes and the 20 terms with the smallest p-value, respectively. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 8
Differential gene and enrichment analysis results of EC_Cluster0 and EC_Cluster2.
(a) KEGG enrichment results for down-regulated genes in EC_Cluster0. (b) EC_Cluster0 differential gene volcano map. (c) KEGG results of EC_Cluster0 upregulated genes. (d) GO results of EC_Cluster 0 downregulated genes. (e) GO results of EC_Cluster0 upregulated genes. (f) KEGG results of EC_Cluster2 downregulated genes. (g) EC_Cluster2 differential gene volcano map. (h) KEGG results of EC_Cluster2 upregulated genes. (i) GO results of EC_Cluster2 downregulated genes. (j) GO results of EC_Cluster2 upregulated genes. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 9
Differential gene and enrichment analysis results of EC_Cluster4 and EC_Cluster5.
(a) KEGG results of EC_Cluster4 downregulated genes. (b) EC_Cluster4 differential gene volcano map. (c) KEGG results of EC_Cluster4 upregulated genes. (d) GO results of EC_Cluster4 downregulated genes. (e) GO results of EC_Cluster4 upregulated genes. (f) KEGG results of EC_Cluster5 downregulated genes. (g) EC_Cluster5 differential gene volcano map. (h) KEGG results of EC_Cluster5 upregulated genes. (i) GO results of EC_Cluster5 downregulated genes. (j) GO results of EC_Cluster5 upregulated genes. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 10
Venn diagram showing the differential gene intersection and uniqueness of the four clusters of interest in endothelial cells.
(a) Intersection and uniqueness of upregulated genes among the four clusters of interest in endothelial cells. (b) Intersection and uniqueness of downregulated genes among the four clusters of interest in endothelial cells.
Figure 11
Subgroup clustering and functional enrichment of microglia.
(a, b) Microglia subset analysis in the two groups. (c–h) The top 10 characteristic expression genes of the six clusters of interest.
Figure 12
The GO and KEGG results of the characteristic expression genes of the six subgroups of microglia.
(a–f) represent the enrichment analysis results of MG_Cluster0, MG_Cluster1, MG_Cluster2, MG_Cluster3, MG_Cluster5, and MG_Cluster6, respectively. The histogram of GO results shows that the number of enriched genes was ranked in the top 20, and q<0.05. The histogram and bubble diagram of the KEGG results show the top 20 terms of the number of enriched genes and the 20 terms with the smallest p-value, respectively. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 13
Upset map of crossover and uniqueness of the enrichment function of the characteristic genes of 6 MG_Clusters of interest.
(a) GO results. (b, c) KEGG results with the largest number of genes and the most significant enrichment, respectively. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 14
Functional enrichment analysis of unique upregulated genes in MG_clusters after electroacupuncture.
(a) Identical and different upregulated genes in six subpopulations of microglia. The histograms of different colors represent different clusters and gene numbers. Colored circles represent genes that are only upregulated in the corresponding clusters, and these genes are listed in their respective colors. (b, i) Crossover and separation of upregulated genes GO and KEGG in six clusters of microglia. (c–h) Unique important GO entries and their genes in the six clusters are displayed. (j, k) Unique key KEGG pathways and their genes in the five clusters (MG_Cluster0 has no unique enrichment pathway). GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 15
Functional enrichment analysis of unique downregulated genes in MG_clusters after electroacupuncture (EA).
(a) Crossover and separation of downregulated genes in six clusters of microglia. The histograms with different colors represent different clusters and gene numbers. Colored circles represent genes that are only downregulated in the corresponding clusters, and these genes are listed in their respective colors. (b, i) Same and different GO and KEGG for down-regulated gene enrichment in six clusters of microglia. (c–h) Unique key GO entries and their genes in the six clusters are displayed. (j–l) Unique key KEGG pathways and their genes in the five clusters (MG_Cluster0 has no unique enrichment pathway). GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 16
The interactions between cells, related to the section ‘Coordination of cell communication in the blood-brain barrier (BBB) following EA intervention’.
(a–c, f) Heatmap of the density of ligand-receptor pairs in the EA and CON groups. The redder the color, the closer the relationship between the cell groups; (c) and (b) are the original values; (a) and (f) are the values after taking the log value. (b, e, g, h) Network diagram of the active degree of the ligand-receptor relationship between two cells. (b) and (e) show the overall interaction network of the two groups. (g) and (h) show interactions of individual cells with other cells in both groups. The thicker the connection, the closer the correlation between the cells on the surface. CON, control; EA, electroacupuncture.
Figure 17
Cell communication with endothelial cells, related to the section ‘Coordination of cell communication in the blood-brain barrier (BBB) following electroacupuncture (EA) intervention’.
(a) The top 20 ligand receptor pairs with mean value in the EA and CON groups, respectively. Different colored boxes are used to indicate pairs with differences between the groups. (b–h) Detailed ligand-receptor pairs for communication between other cells to endothelial cells. The depth of the colors in the image represents the strength of the corresponding interaction, and the size of the dots represents the significance of the p-value. The green box represents pairs that are only present in a specific group (EA or CON), the red box represents pairs that are upregulated in the EA group (mean or p-value), and the blue box represents pairs that are downregulated in the EA group. CON, control; EA, electroacupuncture.
Additional files
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Supplementary file 1
Pathway analysis for genes downregulated by SMES only in EC_cluster0.
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp1-v1.docx
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Supplementary file 2
Pathway analysis for genes upregulated by SMES only in EC_cluster0.
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp2-v1.docx
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Supplementary file 3
Pathway analysis for genes upregulated only in EC_cluster2.
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp3-v1.docx
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Supplementary file 4
Pathway analysis for genes downregulated only in EC_cluster2.
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp4-v1.docx
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Supplementary file 5
Pathway analysis for genes downregulated only in EC_cluster4.
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp5-v1.docx
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Supplementary file 6
Pathway analysis for genes upregulated only in EC_cluster4.
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp6-v1.docx
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Supplementary file 7
Pathway analysis for genes downregulated only in EC_cluster5.
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp7-v1.docx
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Supplementary file 8
Pathway analysis for genes upregulated only in EC_cluster5.
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp8-v1.docx
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Supplementary file 9
Gene Ontology (GO) analysis for MG_cluster1 top genes only (S≥2).
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp9-v1.docx
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Supplementary file 10
Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for MG_cluster1 top genes only (counts top 20).
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp10-v1.docx
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Supplementary file 11
Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for MG_cluster1 top genes only (20 smallest p-values).
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp11-v1.docx
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Supplementary file 12
Gene Ontology (GO) analysis for MG_cluster0 top genes only (S≥2).
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp12-v1.docx
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Supplementary file 13
Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for MG_cluster0 top genes only (counts top 20).
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp13-v1.docx
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Supplementary file 14
Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for MG_cluster0 top genes only (20 smallest p-values).
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp14-v1.docx
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Supplementary file 15
Gene Ontology (GO) analysis for MG_cluster2 top genes only (S≥2).
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp15-v1.docx
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Supplementary file 16
Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for MG_cluster2 top genes only (counts top 20).
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp16-v1.docx
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Supplementary file 17
Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for MG_cluster2 top genes only (20 smallest p-values).
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp17-v1.docx
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Supplementary file 18
Gene Ontology (GO) analysis for MG_cluster3 top genes only (S≥2).
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp18-v1.docx
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Supplementary file 19
Gene Ontology (GO) analysis for MG_cluster5 top genes only (S≥2).
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp19-v1.docx
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Supplementary file 20
Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for MG_cluster5 top genes only (counts top 20).
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp20-v1.docx
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Supplementary file 21
Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for MG_cluster5 top genes only (20 smallest p-values).
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp21-v1.docx
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Supplementary file 22
Gene Ontology (GO) analysis for MG_cluster6 top genes only (S≥2).
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp22-v1.docx
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Supplementary file 23
Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for MG_cluster3 top genes only (counts top 20).
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp23-v1.docx
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Supplementary file 24
Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for MG_cluster3 top genes only (20 smallest p-values).
- https://cdn.elifesciences.org/articles/107938/elife-107938-supp24-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/107938/elife-107938-mdarchecklist1-v1.docx
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Single-cell transcriptome sequencing for opening the blood-brain barrier through specific mode electroacupuncture stimulation
eLife 14:RP107938.
https://doi.org/10.7554/eLife.107938.3
