PET-CT and RNA sequencing reveal novel targets for acupuncture-induced lowering of blood pressure in spontaneously hypertensive rats

Figures

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  Figure 1. Reducing blood pressure (BP) effects induced by MA in SHRs. Differences in systolic blood pressure (SBP) (A) (H(Pre-acupuncture) = 17.48, H(30 min) = 25.68, H(60 min) = 24.15, H(90 min) = 17.48, Kruskal–Wallis Test) and (C) (F(Day1) = 102.21, F(Day2) = 101.11, F(Day3) = 100.12, F(Day4) = 103.36, F(Day5) = 102.56, F(Day6) = 108.47, F(Day7) = 100.04, repeated measurement ANOVA) and diastolic blood pressure (DBP) (B) (H(Pre-acupuncture) = 17.79, H(30 min) = 25.32, H(60 min) = 21.78, H(90 min) = 18.12, Kruskal–Wallis Test) and (D) (F(Day1) = 96.62, F(Day2) = 97.40, F(Day3) = 94.67, F(Day4) = 95.72, F(Day5) = 94.99, F(Day6) = 98.54, F(Day7) = 93.61, repeated measurement ANOVA) among WKY, SHR, MA and Sham-MA groups were detected at 30, 60 and 90 min after the first day of treatment, or every day of the 7-day treatment. *p < 0.05 versus the SHR group, #p < 0.05 versus the Sham-MA group.

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  Figure 2. Changes of glucose metabolism in the rat brain. Regional glucose metabolism was scanned after the 7-day treatment. Results are overlaid on an axial view of the rat brain and mapped to the Paxinos and Watson rat brain atlas. (A) SHR group versus WKY group, (B) MA group versus SHR group, (C) Sham-MA group versus SHR group. Color bars represent the t-value of each significant voxel.

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  Figure 3. A summary of the original RNA-Seq data. The original data performance of the WKY (A), SHR (B), MA (C) and Sham-MA (D) group is shown in the pie chart (n = 3). N: The total amount of reads that contain more than 5% unknown N base (the N reads ratio); adaptor: The total amount of reads that contain adaptors (the adaptor ratio); low quality: More than 20% of the bases in the total read have a quality score lower than 15 (a low quality read ratio); clean reads: Reads filtered with N reads; reads have adaptors and low quality reads (a clean read ratio).

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  Figure 4. Significantly different mRNA expression in the PVH of WKY, SHR, MA and Sham-MA. The hierarchical clustering (A,D,G), scatter plot (B,E,H), and volcano plot (C,F,I) show differentially expressed genes (DEGs) between the WKY, SHR, MA and Sham-MA groups with the red and blue colors, respectively suggesting upregulated or downregulated expression; (J–M) Venn diagrams of the overlapping DEGs between groups are shown. Five genes increased in the SHR group but decreased in the MA group. Three genes decreased in the SHR group but increased in the MA group. The analysis of Volcano-plots, Scatter-plots and heatmaps were performed using R version 3.6.0 (https://cran.r-project.org/bin/windows/base/old/3.6.0/). The overlap in upregulated and downregulated gene expression between the different groups was analysed using Venn online software VENNY version 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/index.html).

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  Figure 5. The DEGs number of the most enriched Gene Ontology (GO) terms between WKY, SHR, MA and Sham-MA. (A) SHR versus WKY; (B) MA versus SHR; (C) Sham-MA versus SHR. GO analyses were performed using enrichment analysis with the phyper function in R version 3.6.0 (https://cran.r-project.org/bin/windows/base/old/3.6.0/).

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  Figure 6. The KEGG Pathway functional enrichment results for the DEGs between WKY, SHR, MA and Sham-MA. With DEGs, KEGG pathway classification and functional enrichment were performed. (A) SHR versus WKY; (B) MA versus SHR; (C) Sham-MA versus SHR. KEGG analyses were performed using enrichment analysis with the phyper function in R version 3.6.0 (https://cran.r-project.org/bin/windows/base/old/3.6.0/).

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  Figure 7. Validation of the up/downregulated genes using qRT-PCR. The loading control gene β-actin was used for normalization. Data are expressed as the mean ± SD. *p < 0.05 versus the SHR group (F(Ednra) = 30.94, F(Ccr5) = 119.78, F(Angptl2) = 74.93, F(Gnb3) = 336.43, F(Erbb2) = 53.47, F(Klotho) = 215.43, F(Gpr81) = 35.09, F(Cyp1b1) = 54.32, one way-ANOVA).

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  Figure 8. Validation of the up/downregulated genes using western blot. The loading control protein β-actin was used for normalization. Data are expressed as the mean ± SD. *p < 0.05 versus the SHR group (F(Ednra) = 483.27, F(Ccr5) = 82.53, F(Angptl2) = 254.11, F(Gnb3) = 236.63, F(Erbb2) = 507.16, F(Klotho) = 655.65, F(Gpr81) = 60.18, F(Cyp1b1) = 43.76, one way-ANOVA).