Effects of Manual Acupuncture (MA) on Blood Pressure (BP)
To evaluate the antihypertensive effect of MA, the BP of the rats was measured at 30, 60, and 90 minutes after the first treatment. As shown in Figure1 a and b, the SBP and DBP of the MA group were significantly reduced at 30 and 60 minutes after acupuncture compared with the SHR group. Therefore, for the following 6 days, the BP of the rats was repeatedly measured 30 minutes after daily acupuncture. Compared with the SHR group, acupuncture at KI3 significantly reduced the SBP and DBP of rats from the first day to the seventh day, as shown in Figure1 c and d.
Changes to Glucose Metabolism in the Brain of SHRs
Glucose metabolism was significantly increased in the hypothalamus, thalamus, dorsal thalamus and olfactory bulb of the SHR group compared with the WKY group, but decreased in other regions including the cingulate cortex, cingulate gyrus, and motor cortex(Table 1 and Figure2 a). However, compared with the SHR group, the MA group showed significantly reduced glucose metabolism in the accumbens nucleus, hypothalamus, olfactory bulb, thalamus, anterior commissure, dorsal thalamus, and hypothalamus tuberal regions, and increased glucose metabolism in the cerebellum posterior lobe and visual cortex(Table 2 and Figure2 b). Furthermore, compared with the SHR group, the Sham-MA group showed significantly reduced glucose metabolism in the basal ganglia, caudate putamen, olfactory bulb, prefrontal cortex and orbital cortex, while this increased in the medulla oblongata(Table 3 and Figure2 c).
Filter of sequencing raw reads
Transcriptomic sequencing was conducted to analyze the expression profile of the hypothalamus in the WKY, SHR group, and MA groups. A total of 955.27 Mbp of raw reads were obtained. The percentage of clean reads Q20 and Q30 (the ratio of the quality values of the reads that were respectively larger than 20 and 30 compared to the total reads) indicated that the sequence was of high quality and could be used for subsequent analyses (supplementary table 2 and supplementary figure 1). After filtering the invalid readings, 267.75, 267.54, and 266.12 Mbp of clean readings were respectively obtained from the WKY, SHR group, and MA group. The proportion of clean reads were 82.9%–84.15% in the WKY group, 84.15%–86.29% in the SHR group, and 81.2%–84.69% in the MA group. The clean read ratio of the rat genome suggested that the sequencing depth was satisfactory for the analysis of the differentially expressed genes between the groups of rats.
Differentially expressed genes in the hypothalamus of WKY, SHR, and MA
In order to study the regulation of DEGs between the WKY, SHR, and MA groups, DEGs in the hypothalamus were analyzed. DEseq2 algorithms were based on negative binomial distribution to detect the DEGs by applying a fold change ≥2.00 and an adjusted p-value (p ≤ 0.05). The statistics of the number of DEGs are shown by heatmap (Figure 3 a, d), scatter plot (Figure 3 b, e), and volcano plot (Figure 3 c, f). There were 695 DEGs in the SHRs relative to the WKY rats, with 375 upregulated genes and 320 downregulated genes. A total of 120 DEGs were found in the MA rats relative to the SHRs, with 72 upregulated genes and 48 downregulated genes. A Venn analysis was used to study the possible genes involved in reducing blood pressure as a result of acupuncture. We found that when applying MA treatment, we could abolish the upregulation of 5 of the 375 genes upregulated in SHRs compared to wild type rats. Correspondingly, MA treatment was able to counteract the downregulation of 3 of the 320 downregulated genes in SHRs (Figure 3 g, h).
Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis
To investigate the function of the DEGs, Gene Ontology (GO) classification and functional enrichment were performed. GO covers three domains: biological process, cellular component, and molecular function. Functional enrichment was performed and the GO classification results between the WKY, SHR, and MA groups are shown in figure 4 a and b. Regarding biological processes, the categories “cellular process”,
“single-organism process”, “metabolic process”, and “biological regulation” showed large enrichment. The DEGs were involved in the “cell”, “cell part”, “organelle”, and “membrane”, according to their cellular component classification. In terms of the molecular functions, the “molecular transducer activity”, “binding”, “catalytic activity”, and “nucleic acid-binding transcription factor activity” showed large enrichment.
To further investigate the possible pathways affected directly by MA treatment in SHRs, DEGs were classified by performing KEGG pathway classification and functional enrichment. The terms with false discovery rates (FDRs) no larger than
0.01 are defined as being significantly enriched. As shown in figure 4 c and d, DEGs were found to be enriched in several signaling pathways, including “endocrine and metabolic diseases”, “neurodegenerative diseases”, “cardiovascular diseases”, “energy metabolism”, and “signaling molecules and interaction”.
Validation of the differentially expressed genes using real-time PCR
To further verify the reliability of the RNA-Seq data, real-time PCR was used to examine eight DEGs: Angptl2, Erbb2, Klotho, Ednra, Ccr5, Gnb3, Gpr81, and Cyp1b1. As shown in figure 5, trends in the expression data of the examined DEGs were the same as observed for RNA-Seq. Three genes, Angptl2, Erbb2, and Klotho, were downregulated in the SHR group but upregulated after the MA treatment. On the other hand, five genes, Ednra, Ccr5, Gnb3, Gpr81 and Cyp1b1 were upregulated in the SHR group but downregulated after MA treatment.