Exploring the Mechanisms of Acupuncture-Induced Lowering of Blood Pressure Through Positron Emission Tomography and RNA Sequencing of the PVH


 Manual acupuncture (MA) can be used to manage high blood pressure, however, the underlying molecular mechanism remains unknown. To explore the mechanism of acupuncture in the treatment of hypertension, we used Wistar Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) that were subject to either MA stimulation or the corresponding sham procedure as a negative control (sham-MA) for 1 week. Blood pressure was recorded regularly. After 7 days of treatment, PET-CT scans were used to detect brain areas where glucose metabolism was significantly regulated. Additionally, the differentially expressed genes(DEGs) of a specific brain region—the paraventricular hypothalamic nucleus (PVH)—were evaluated by transcriptomics, and verified with quantitative PCR (qPCR). Eight overlapping DEGs were found between the WKY, SHR, and MA groups. The DEGs were then annotated with the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. qPCR was used to verify the DEGs. These genes may lower blood pressure by regulating angiotensin, endothelial function and inflammation, ect. This study reveals MA regulates multiple biological processes and genes of the PVH, and provides a solid theoretical basis for exploring the mechanisms by which MA manages hypertension.


Introduction
In most international guidelines, the measurement for diagnosing hypertension is that systolic blood pressure is no less than 140 mm Hg and/or diastolic blood pressure is no less than 90 mm Hg 1,2 . So far, the reasons for the development of hypertension have mainly been related to increased exposure to environmental factors that cause high blood pressure, including excessive consumption of salt, alcohol, and calories. Elevated blood pressure remains the largest single factor in the global burden of disease and mortality, killing 94 million people each year, according to the latest data 3 . The global prevalence of high blood pressure is expected to increase by about 10% in the next two decades, corresponding to an estimate of 560 million people that will suffer from hypertension 4 . If hypertension is not diagnosed and managed in time, it can lead to myocardial infarction, stroke, kidney failure, and even death [5][6][7] .
Acupuncture is an effective alternative therapy for the treatment of hypertension [8][9][10] . Our previous animal trials showed that manual acupuncture (MA) at the KI3 position can effectively reduce systolic blood pressure (SBP) and diastolic blood pressure (DBP) in spontaneously hypertensive rats (SHRs) 11 . Moreover, our previous PET-CT study found that acupuncture may regulate blood pressure by changing the brain glucose metabolism of SHRs 12 . However, the underlying molecular mechanisms of acupuncture treatment for hypertension have not been widely described.
RNA sequencing is a new technical method that locates and quanti es the transcriptome through microarrays, which can digitally measure the existence and prevalence of transcripts 13,14 . Recent transcriptomic studies have found that acupuncture affects the mRNA levels of multiple differentially expressed genes (DEG) in the brain of the rat and regulates multiple biological processes, including in ammation, oxidative stress, and vascular endothelial function 15,16 . However, it has not been reported which signaling pathways and genes in speci c brain regions are affected by MA at KI3.
Here, we studied the changes in cerebral glucose metabolism of 15-week-old SHRs after MA at KI3, by isolating the PVH, which is the target brain region of acupuncture, and performing transcriptomic sequencing. First, we determined the DEG. Then, we annotated the identi ed genes according to their Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway classi cation, followed by verifying the expression levels of these DEGs by qPCR. This is a promising transcriptome study of the PVH after acupuncture at KI3, and may help us understand the role of acupuncture in the treatment of hypertension.

Results
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 rst treatment. As shown in Figure1 a and b, the SBP and DBP of the MA group were signi cantly 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 signi cantly reduced the SBP and DBP of rats from the rst day to the seventh day, as shown in Figure1 c and d.

Changes to Glucose Metabolism in the Brain of SHRs
Glucose metabolism was signi cantly 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 signi cantly 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 signi cantly 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 pro le 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 gure 1). After ltering 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) classi cation and functional enrichment were performed. GO covers three domains: biological process, cellular component, and molecular function. Functional enrichment was performed and the GO classi cation results between the WKY, SHR, and MA groups are shown in gure 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 classi cation. 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 classi ed by performing KEGG pathway classi cation and functional enrichment. The terms with false discovery rates (FDRs) no larger than 0.01 are de ned as being signi cantly enriched. As shown in gure 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 gure 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, ve genes, Ednra, Ccr5, Gnb3, Gpr81 and Cyp1b1 were upregulated in the SHR group but downregulated after MA treatment.

Discussion
Essential hypertension is a form of hypertension that accounts for the highest proportion of all hypertension cases 17 . Our results have shown that SBP and DBP were effectively reduced at 30-60 minutes after MA treatment, and a stable blood pressure reduction effect was obtained over continuous treatment for 7 days. This research shows that acupuncture is effective and stable at lowering blood pressure, and this therapeutic effect is unique to the speci c acupoint.
To reveal the central mechanism related to the management of blood pressure by acupuncture, PET-CT was used to explore the speci c regulation of MA on the brain glucose metabolism of SHRs. The glucose metabolism of the PVH increased in the SHR group compared with the WKY group. After MA at KI3, the increased glucose metabolism of the PVH was signi cantly reversed, while the Sham-MA group showed no speci c regulation of the metabolism of this brain area. Many human diseases, such as hypertension, obesity, and diabetes, are regulated by the PVH [18][19][20] . Additionally, the increased sympathetic out ow of the PVH and excessive activation of sympathetic synaptic N-methyl-D-aspartate receptors are essential for hypertension 21 .
According to our PET-CT results, the PVH could be the target brain region for acupuncture at KI3 to regulate blood pressure. The pathological process associated with hypertension in the PVH is susceptible to acupuncture intervention. To further explain the effect of MA intervention on molecular modi cation in the PVH, a PVH transcriptome analysis was performed.
Our results showed that, according to the GO functional classi cation, the DEGs were enriched in the categories of "catalytic activity", "transporter activity", "molecular function regular", and "signal transducer activity". KEGG analysis revealed that the DEGs were involved in several pathways, including the "endocrine system", "cardiovascular diseases", and "neurodegenerative diseases". Hypertension is associated with a reduction in key enzymes that maintain cardiac homeostasis, such as ALDH2 [22][23][24] . However, the ability of these enzymes could be increased by enhanced catalytic activity to counteract the cytotoxic aldehydes produced by lipid peroxidation during hypertension 25 and improved ventricular dysfunction caused by hypertension 26 . One study showed that hypertension induced by angiotensin II increased the activation and abundance of transporters. The targeted therapy of transporters is important for the management of hypertension 27 . Therefore, improvements in catalytic activity and transporter activity may play an important role in the mechanism of MA intervention in hypertension.
The renin-angiotensin-aldosterone system (RAAS) is a complex endocrine system that regulates biological functions such as vasodilation and the contraction of blood vessels; it also tightly binds to the vasopressin system, endothelin, and the sympathetic nervous system. The activation of RASS is the basis upon which essential hypertension develops 28 . An increased level of angiotensin II can cause endothelial dysfunction and vascular remodeling during hypertension 29 . Therefore, we speculated that the effects of MA on the endocrine system, such as RAAS, may help to control high blood pressure in SHRs.

Upregulated DEGs in SHRs with MA
This study validated several DEGs that are upregulated after MA, including angiopoietin-like 2 (Angptl2), which encodes a secreted pro-in ammatory glycoprotein 30 that maintains tissue homeostasis by regulating blood pressure angiogenesis and inducing in ammation 31,32 . A decrease in circulating ANGPTL2 levels leads to an increase in systolic blood pressure in humans and mice. A decrease in blood pressure caused by the overexpression of Angptl2 in mice indicated cardiac dysfunction accompanied by a decrease in Ca 2+ -ATPase (SERCA) 2a signaling and a decrease in cardiac energy metabolism. In contrast, Angptl2 knockdown increased left ventricular contractility and blood pressure by AKT-SERCA2a signaling 33 . Therefore, these results indicate that increased levels of ANGPTL2 improve blood pressure.
Another upregulated DEG, Klotho-a newly discovered antiaging gene-encodes a transmembrane protein with an extracellular domain 34 . Klotho is mainly expressed in the brain and kidney 35,36 . Genetic Klotho defects lead to a premature aging phenotype, including a signi cantly shortened lifespan 37 . The circulating klotho level was found to be negatively correlated with age, and the prevalence of hypertension positively correlated with age 38 .
The incidence of peroxide production and vascular disease in the kidneys increases in SHRs. The inhibition of NOX2 via the Klotho gene reduces the production of peroxides in the blood vessels and kidneys of SHRs and is the mechanism by which Klotho lowers blood pressure 39 . Additionally, another study showed that klotho protein complementation is involved in the inhibition of ANG signaling by binding to the ANG II type 1 receptor, ultimately inactivating the RAAS system 40 . Therefore, MA may exert its antihypertensive effect by increasing Klotho gene expression to inhibit oxidative stress and inactivate RAAS.
Together with neuregulin-1 (NRG-1), ERBB2-a subtype of the ERBB family-regulates many aspects of cardiovascular function, such as blood pressure and angiogenesis 41 . Inhibition of ERBB2 reduces the expression of NOS, thereby increasing blood pressure and heart rate by increasing sympathetic activity. NRG-1 lowers blood pressure by increasing NOS-mediated GABA 42,43 . Therefore, MA may induce NOS expression by increasing the levels of ERBB2, thereby inhibiting the sympathetic impulse to lower blood pressure in SHRs.

Downregulated DEGs in SHRs with MA
Endothelin receptor A (Ednra) encodes a G protein-coupled receptor that is an endogenous factor of vasoconstriction and is linked to endothelial dysfunction 44 . EDNRA binds to vasoconstrictors released by endothelial cells, and endothelin-1 (ET-1) increases blood vessel contraction and sodium retention, resulting in elevated blood pressure [45][46][47] . Increased EDNRA activity and elevated ET-1 levels have been found in the vascular system of hypertensive patients. The incidence of hypertension has been found to be signi cantly correlated with the expression of EDNRA (p = 2.39 × 10 −4 ) 48 . This study found that the Gpr81 gene played a role in cardiovascular control. The ET system is the primary mechanism by which GPR81 agonists induce vasoconstriction. Intense GPR81 agonism transmits stress signals in the tissues of the body, inducing the release of ET-1 that causes vasoconstriction 49 . Correspondingly, the expression level of EDNRA in SHRs was found to be downregulated after MA intervention, and the inactivation of GPR81 resulted in a decrease in ET-1 release, followed by a decrease in the binding of EDNRA to ET-1, which demonstrated that MA may play a role in relieving vasoconstriction and sodium retention through this pathway.
CCR5-a chemokine receptor-is expressed primarily on immune cells, such as T cells, natural killer cells, and monocytes. The recruitment of CCR5 + immune cells has proven to be a prominent feature of many autoimmune diseases [50][51][52] . Hypertension is related to activation of the immune system and the development of an in ammatory response 53 . CCR5 expression was detected in the T cells of mice with hypertension, and high blood pressure was reduced by CCR5 antagonists. Additionally, no difference in systolic blood pressure was found between CCR5 −/− and WT mice, suggesting that CCR5 may be a potential target for hypertension management 54 . Reactive oxygen species (ROS) production induced by CYP1B1 caused in ammation, cardiovascular hypertrophy, and endothelial dysfunction related to hypertension, mediated by the activation of some signaling pathways, including ERK1/2 and c-SRC 55 . The increased activity of NADPH oxidase, p3MAPK and ROS production associated with ANG II-induced hypertension was alleviated in Cyp1b1 knockout mice 56 . Therefore, a signi cant reduction in the expression levels of CCR5 and CYP1B1 after MA intervention in SHRs con rmed that MA has the effect of reducing in ammation in hypertension.
The G protein β3 (GNB3) subunit is involved in G protein-coupled receptor signal transduction. Polymorphisms of GNB3 are related to some diseases, such as hypertension, obesity, and diabetes. However, the pathogenesis of GNB3 in these diseases remains uncharacterized 57 . The Gnb3 C825T polymorphism is a marker for the treatment of hypertension, with a phenotype featuring enhanced sodium-proton reverse motility activity.
Increased essential hypertension is related to the T allele of the Gnb3 C825T polymorphism 58 . Therefore, MA intervention may regulate hypertension by downregulating GNB3 and inactivating the T allele of the Gnb3 C825T polymorphism.

Conclusion
Our data suggest that repeated MA treatment of acupoint KI3 reduces the elevated blood pressure of SHRs. PET-CT scanning results further indicated that PVH could be the target brain area for MA at KI3 to manage blood pressure. Furthermore, whole transcriptome sequencing of PVH was used to explore how DEGs regulated by acupuncture in SHRs are related to neurodegenerative diseases, cardiovascular diseases, and immune system signaling pathways, thereby providing a theoretical basis for the further study of acupuncture treatment in hypertension. Groups and Acupuncture Treatment WKY rats were used as the controls (WKY, n = 10). SHRs were randomly assigned into three groups: SHRs (SHR, n = 10), SHRs with manual acupuncture (MA) treatment at acupoint KI3 (MA, n = 10), and SHRs receiving sham-MA treatment (sham-MA, n = 10). The position of the sham acupoint was selected as the space between the 3rd and 4th toes on the back of the foot. The MA treatment and sham-MA treatment was performed according to a previous study 12 . MA and sham-MA groups received acupuncture for 7 consecutive days for 10 minutes per day.

Blood Pressure Measurement
Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured with a blood pressure monitor (CODA7m, Kent Scienti c Corporation, Torrington, Connecticut, USA), according to the method described previously 12 , at 30, 60, and 90 min after the rst day of therapy, or 30 min after each day's acupuncture treatment in the 7 day therapy.

PET-CT Scanning
All PET-CT scans were performed on the animal molecular imaging research platform of Sun Yat-sen Medical College. After receiving acupuncture therapy, all rats were injected intravenously with 1.5 mci/kg 18 F-FDG, followed by PET-CT scanning. After the FDG-PET image was acquired, it was reconstructed through a 128 × 128 × 159 matrix and ltered projection.
After the PET-CT scan, all rats were anesthetized and sacri ced by intraperitoneal injection of 30 mg/kg sodium pentobarbital.

Tissue Processing
The PVHs that were used for RNA-Seq analysis were the WKY, SHR, and MA groups, because no signi cant difference was found in reducing BP between the SHR and sham-MA groups. At 24 hours after the 7 day treatment, the rats were anesthetized and killed, and the PVH was rapidly removed. The total RNA was extracted from the PVH by using the RNeasy Mini Kit (74104, Qiagen, Beijing, China), according to the manufacturer's protocol, and an Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit) was used to perform quality control for the total RNA.

RNA-Seq Analysis
Transcriptome sequencing was performed with BGI Co., Ltd., Shenzhen, China (http://www.genomics.cn/). Brie y, DNA libraries were constructed using the TruSeq stranded mRNA library preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Paired-end reads of 100 bp were read, and the DNA libraries were sequenced on the BGISEQ-500 platform for sequence data analysis.

Quality Control for Raw Data
The raw data for sequencing included low-quality, adaptor-polluted material, with a high content of unknown base (N) reads. These reads needed to be removed before data analysis to ensure the reliability of the results.
The Q20, Q30, and clean read ratios were calculated, and subsequent analyses were based on these clean reads.

Differential Expression Analysis
Clean reads were mapped to the reference using Bowtie2 59 , and the gene expression level was calculated with RSEM 60 . DEGs with DEseq2 were detected as requested 61 . DEGs were chosen according to the parameters with a fold change ≥ 2 and an adjusted p-value (p ≤ 0.05). Then, the Pearson correlation between all samples was calculated using cor, and hierarchical clustering between all samples was performed using hclust. Gene expression was compared between SHRs and WKYs, and between MA and SHR groups. The overlap in upregulated and downregulated gene expression between the different groups was analyzed using BioVenn 62 .
GO and KEGG classi cation and functional enrichment were performed for all identi ed DEGs.

Real-Time PCR
Eight DEGs were selected for validation using qRT-PCR. cDNA was produced from hypothalamus mRNA (2 μg) using an Invitrogen™ SuperScript™ II Reverse Transcriptase reagent kit (Takara, Shanghai, China). OneStep RT-PCR Enzyme mix (Qiagen, Beijing, China) was used to for qRT-PCR on an ABI ViiA 7 PCR System (Thermo Fisher Scienti c, USA). The Ct value in the reaction was collected using a corrected threshold setting. β-αctin was used as the internal reference gene to con rm gene expression levels, and the relative quanti cation was determined using the 2 −ΔΔCt method. The primers used for qRT-PCR validation are shown in supplementary table 1.

Statistical Analysis
All data were analyzed using a one-way analysis of variance test via SPSS 17.0 (SPSS Inc., Chicago, USA). An LSD post hoc test was used to determine the group differences. Data were expressed as the mean ± SD, and p < 0.05 was considered statistically signi cant.      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 signi cant voxel.

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 signi cant voxel. increased in the SHR group but decreased in the MA group. Three genes decreased in the SHR group but increased in the MA group. increased in the SHR group but decreased in the MA group. Three genes decreased in the SHR group but increased in the MA group.   Validation of the up/downregulated genes using qRT-PCR. The loading control gene β-αctin was used for normalization. Data are expressed as the mean ± SD. * p < 0.05 versus the SHR group. Figure 5