Comprehensive evaluation of gene expression signatures in response to electroacupuncture stimulation at Zusanli (ST36) acupoint by transcriptomic analysis

Acupuncture, a traditional therapy in ancient China over thousands of years, has been widely accepted and used in Western society [1, 2]. Acupuncture is believed to balance Yin-Yang, stimulate the circulation of vital energy (qi) and blood, maintain the body health, and prevent the incidence of illness [3]. Electroacupuncture (EA) is a modification of acupuncture that stimulates acupoints with electrical current and displays reproducible in both research and clinical application. Moreover, EA therapy has been used for postoperative analgesia and anesthesia, for the treatment of diverse disorders of internal organs, and for the release of pain [4, 5].

In traditional Chinese medicine, ST36 (Zusanli) is a commonly acupoint that modulates the biological activities of gastrointestinal system, immune system, cardiovascular system, and muscular system. Transcutaneous EA at ST36 reduces gastric accommodation and improves impaired gastric motility in patients with functional dyspepsia [6]. Transcutaneous neuromodulation at ST36 also improves the frequency of spontaneous defecation and increases the bowel movements in patients with chronic constipation [7]. Chronic EA at ST36 improves baroreflex function and hemodynamic parameters in rats with heart failure [8]. Long-term EA stimulation at ST36 and DU20 (BaiHui) also relieves the increased mean arterial pressure and cardiovascular abnormality in both structure and function in spontaneously hypertensive rats [9]. ST36 displays the anti-nociceptive and anti-hyperalgesic effect. EA at ST36 reduces postoperative analgesic requirements and associated side effects in patients undergoing lower abdominal surgery [10]. Treatment of EA at ST36 and GN34 also ameliorates L5 spinal nerve ligation-induced neuropathic pain in rats [11]. Moreover, EA at ST36 and CV4 (Guanyuan) improves clinical curative effects in patients with sepsis in a prospective randomized controlled trial via the regulation of immune system [12]. EA at ST36 promotes myofiber regeneration and restoration of neuromuscular junctions in a rabbit gastrocnemius contusion model [13]. Furthermore, EA at ST36 also improves intestinal mucosal immune barrier in sepsis by increasing the concentration of secretory IgA, the percentage of CD3+, γ/δ, and CD4+ T cells, and the ratio of CD4+/CD8+ T cells [14].

ST36 is an acupoint of Foot’s Yang Supreme Stomach Meridian that targets at gastrointestinal tract [15]. We wondered whether EA stimulation at ST36 altered molecular events in other organs. The genome-wide analysis of ST36-stimulated region (skin) and distant visceral organs or tissues, including cerebral medulla, cerebral cortex, hippocampus, lung, spleen, kidney, uterus and thigh muscle, was therefore performed. Mice were stimulated by EA at ST36, and gene expression signatures of nine organs or tissues were explored by microarray analysis. The process network and disease connection of gene expression profiles were further analyzed to elucidate the molecular events and effects of organs after ST36 stimulation.

In this study, we applied transcriptomic analysis to analyze the gene expression signatures in nine organs or tissues responsive to ST36 stimulation. Microarray analysis has been applied to elucidate the effects of various acupoints in specific organs or tissues. For example, acupuncture at GB34 and LR3 acupoints attenuates the decrease of tyrosine hydroxylase and exhibits the protective effects via affecting the expression of degeneration-related genes in the substantia nigra region in Parkinsonism mouse model [19]. Acupuncture at PC6 acupoint up-regulates the expression of Tph1 gene and down-regulates the expression of Olr883 genes in rat brains, suggesting that the therapeutic effect of acupuncture for ischemic stroke may be closely related to the suppression of post-stroke depression and the regulation of olfactory transduction in middle cerebral artery occlusion rat model [20]. Moreover, EA at PC3 and PC6 acupoints significantly ameliorates the colonic lesions, and affects both the inflammatory pathways in colons and the immunity-associated pathway in spleens in mice with trinitrobenzene sulfuric acid- induced colitis [21]. Gene expression profiles of specific organs or tissues after EA stimulation at ST36 have also been analyzed. For example, gene expression profiles in periaqueductal gray-spinal dorsal horn region of rats after EA stimulation at ST36 and SP6 show that the modulation of neural-immune interaction in the central nervous system plays an important role during EA analgesia [22]. Gene expression profiling of rat arcuate nucleus region responsive to EA at ST36 and SP6 shows that the expression levels of genes are effectively regulated by low-frequency EA, compared with high-frequency EA. It might explain the mechanisms of therapeutic effects of the low-frequency EA [23]. In addition to brain tissues, EA at ST36 affects the expression of cell adhesion molecules in muscle, which might be related to the glucose-lowering effect of ST36 in rats with type 1 diabetes [24]. Acupuncture at ST36, CV12 (Zhongwan), and BL20 (Pishu) acupoints down-regulates nuclear factor-κB p65, miRNA-155, and miRNA-21 and up-regulates miRNA-146a expression in chronic atrophic gastritis rats, suggesting that these genes may play important roles in therapeutic effect of acupuncture in treating chronic atrophic gastritis [25]. Moreover, moxibustion at ST36 affects the biological processes involved in immunity and metabolism in moxibustioned skin under pathological and physiological conditions, respectively [26]. Since ST36 displays various benefit or therapeutic effects in whole bodies, we performed a global and comprehensive study on the gene expression signatures of nine different organs or tissues after ST36 stimulation. Our data showed that EA at ST36 affected the gene expression of different organs or tissues in various degrees. Moreover, EA at ST36 has a more impact on the regulation of gene expression in uterus and has a lesser impact in hippocampus.

By process network and disease connection analysis, we found that EA at ST36 affected the process networks involved in inflammation and immune responses in lung and affected the expression of genes involved in respiratory diseases, such as obstructive pulmonary diseases and microbial infection. Interestingly, a prospective single-blind randomized placebo-controlled study shows that transcutaneous electrical nerve stimulation at ST36, EX-B-1(Dingchuan), BL13 (Feishu), and BL23 (Shenshu) improves lung function on patients with stable chronic obstructive pulmonary disease [27]. Another study shows that EA at ST36 and BL13 improves lung function of rats with chronic obstructive pulmonary disease and displays an anti-inflammatory effect via downregulation of orexin and its receptor [28]. In addition, EA at ST36 displays a potential protective effect on severe thermal injury-induced remote acute lung injury via the limitation of inflammatory responses in rats [29]. Moreover, EA treatment at ST36 and BL13 attenuates lung injury in rats with endotoxic shock-induced acute lung injury through the activation of NF-E2-related factor pathway and the up-regulation of heme oxygenase-1 expression [30]. Acupuncture at ST36 also regulates the disorders of Fas and Bcl-2 mRNA expression, promotes the apoptosis of eosinophils, and consequently inhibits the development of inflammatory reaction of asthma in rats [31].

Acupuncture has shown some benefit effects on Alzheimer’s disease and Parkinson’s disease. Lu et al. [32] showed that acupuncture at ST36 increases blood perfusion and glycol metabolism in certain brain areas in Alzheimer’s disease rat model by Positron Emission Tomography scanning. ST36 stimulation also induces neurogenesis in adult brains via the up-regulations of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, basic fibroblast growth factor and neuropeptide Y, and the activation of the function of primo vascular system [33]. By database searching and screening for articles on clinical trials, Feng et al. [34] found that ST36 combined with GV20 (Baihui) or GV24 (Shenting) is the most frequent and represent potential combination for vascular dementia treatment. In addition, acupuncture at ST36 improves cognitive deficits and increases pyramidal neuron number of hippocampal CA1 area in vascular dementia rats [35]. Moreover, EA at ST36 alleviates dementia via the modulation of interneuron function and the increases of long-term potentiation of hippocampus in rats [36]. By analyzing the gene expression profiling of cerebral cortex, cerebral medulla, and hippocampus after ST36 stimulation, we found that stimulation at ST36 affected the expression of genes involved in neurodegenerative diseases, such as Alzheimer’s disease and Parkinsonian disorder, and mental disorders, such as dementia. In addition, neurophysiological processes, such as GABAergic neurotransmission and long-term potentiation, were also regulated by ST36 stimulation in brains. The connection between gene expression signatures in brain and neurological diseases might provide an explanation on the therapeutic effects of acupuncture for neurological diseases.

In this study, we found that, in addition to brain and lung, EA stimulation at ST36 affected the expression of genes in the local region, such as acupunctured skin, and in the distant regions, like muscle, uterus, kidney, and spleen. How can the stimulation at body surface affect the gene expression in the internal region far from the acupunctured site? Autonomic nervous system is frequently considered to be a mediator of acupuncture. Vagus nerve is a primary target for exploring the possible effect of acupuncture on internal organs because vagus nerve broadly regulates the functions of internal organs. Acupuncture stimulation raises the vagal tone and consequently affects the heart rate and the arterial pressure of cardiovascular system, and the intestinal motility of gastrointestinal system [37]. Acupuncture also exhibits anti-inflammatory effects via vagal modulation of inflammatory responses in internal organs. For example, acupuncture at ST36 activates the splenic nerve via vagus nerve activity to induce anti-inflammatory responses in macrophages of spleens in a lipopolysaccharide-induced inflammation rat model [3]. EA also controls systemic inflammation by inducing vagal activation of aromatic L-amino acid decarboxylase, leading to the production of dopamine in the adrenal medulla and the inhibition of cytokine production [38]. Some neurotransmitters are involved in the transmission of acupuncture stimulation to nerves. Tjen-A-Looi et al. [39] showed that EA at P5 and P6 acupoints restores the blood pressure in phenylbiguanide-induced hypotension and bradycardia cat models through both opioid and GABAergic processing mechanisms. They also showed that EA at P5 and P6 modulates the cardiovascular depressor responses during gastric distention in rats via GABAergic mechanisms [40]. Our data also showed that gene expression signatures responsive to ST36 stimulation connected to the GABAergic neurotransmission network in brain.

In conclusion, we performed a global comprehensive study on the gene expression signatures of nine different organs or tissues after ST36 stimulation. EA at ST36 affected the expression of genes not only in acupunctured site but also in internal organs. Gene expression signatures showed that stimulation at ST36 acupoint commonly affected process networks involved in cytoskeleton and cell adhesion in these organs. However, EA at ST36 also regulated unique process networks in specific organs or tissues. In addition, ST36 stimulation affected the expression of genes related to various diseases. The connection between gene expression signatures and diseases might provide a basis for the prediction and the explanation on the therapeutic potentials of acupuncture in various organs.