Systematic acupuncture explains acupuncture at Baihui (GV20) and Fengchi (GB20) targeting the inflammatory response to regulate migraine

Abstract

OBJECTIVE:

To take Baihui (GV20) and Fengchi (GB20) targeting inflammatory response to regulate migraine as an example to describe a new method for studying the mechanism of stimulating acupoints.

METHODS:

The target information of Baihui (GV20) and Fengchi (GB20) was retrieved, and after intersection with migraine, Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, and UniProt Keywords were used for functional enrichment. After selecting the main pathway, rats were selected and nitroglycerin was used for modeling, and the behavioral scores, inflammatory factors, heme oxygenase 1 (HMOX1), protein kinase B (AKT1), signal transducer and activator of transcription 3 (STAT3), phosphorylated extracellular signal-regulated kinase 1/2 (P-ERK1/ERK2) and other states of the rats in the acupuncture, twisting, and electroacupuncture groups were compared.

RESULTS:

A total of 135 Baihui (GV20) targets and 27 Fengchi (GB20) targets were collected. A total of 73 target information were obtained after the intersection of these targets in migraine. These 73 targets have three main pathways: hypoxia-inducible factor 1 (HIF-1) signaling pathway, signaling by interleukins and inflammatory response. The main targets in the pathway were verified and found that interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and HMOX1, AKT1, STAT3, P-ERK1/ERK2 can be regulated by Baihui (GV20) and Fengchi (GB20).

CONCLUSION:

Baihui (GV20) and Fengchi (GB20) can regulate migraine by regulating inflammatory factors and HMOX1, AKT1, STAT3, P-ERK1/ERK2 and other changes in HIF-1 signaling pathway, Signaling by Interleukins and Inflammatory response pathways. Based on systems biology and network pharmacology, and with the model of "acupoint-target-disease", explore the research methods of systematic acupuncture and moxibustion. We believe this is a usable research direction for exploring the mechanism of acupuncture stimulation.

1. INTRODUCTION

Migraine is a neurovascular headache associated with periodic episodic attacks that often presents as repeated severe pain on the side of the head accompanied by nausea, vomiting, photophobia and other symptoms. In addition to the damage caused by the disease itself, migraine can also increase the incidence of anxiety, depression and other diseases and bring many inconveniences to the patient's work and life. The incidence of migraine is 9.3% in China and approximately 15% in the United States,^1,2 and the incidence is greater in women than in men.³ In recent years, drugs targeting calcitonin gene-related peptide (CGRP) and 5-hydroxytryptamine (5-HT) 1F receptors have become new methods for the treatment of acute migraine. Human-derived anti-CGRP monoclonal antibodies (mAbs) and anti-CGRP receptor mAbs have also been approved for the prophylactic treatment of migraine. However, the above drugs all have certain downsides in terms of compliance, cardiovascular system safety and economic burden, and CGRP receptor antagonists and 5-HT1F receptor agonists, as new types of migraine drugs, still need time to be tested.^4-7 Traditional Chinese Medicine (TCM), which has been used for thousands of years, is a unique treatment method that has been tested over time. Acupuncture is an advantageous branch of TCM that generally has good efficacy in treating migraine. Unfortunately, there is still little evidence of acupuncture treatment for migraine, and the relevant research models are relatively traditional. However, wisdom and courage are still needed to propose innovative research methods. Based on this, we referred to network pharmacology, systematic pharmacology, integrated pharmacology and other methods and used "acupoint-target-disease" as the model to explore the research methods of systematic acupuncture and explored the inheritance,⁸ addition and emergence of the overall function after acupuncture stimulation. The selection of acupuncture points was based on the "head is the meeting of all Yang" theory in Traditional Chinese Medicine, and according to the circulation parts of the Du meridian and the foot Shaoyang meridian, we selected Baihui (GV20) of the Du meridian and Fengchi (GB20) of the gallbladder meridian of the foot Shaoyang to treat migraine. This treatment has also been written into the Chinese Traditional Chinese Medicine textbook "Acupuncture Therapy".⁹

In conclusion, this study used a systems biology approach to construct a Baihui (GV20) and Fengchi (GB20) target database. We subsequently searched for common targets of acupoints and migraine and verified them via animal experiments. Our ultimate goal was to investigate the effects of acupuncture at Baihui (GV20) and Fengchi (GB20), which target the inflammatory response to regulate migraine, as an example and to identify a new method for studying the mechanism of stimulating acupoints.

2. MATERIALS AND METHODS

2.1. Acquisition of acupoints and disease targets

Through a massive literature search of the China National Knowledge Infrastructure (CNKI), Wanfang, China Science and Technology Journal Database (VIP), PubMed, and Web of Science databases, the Baihui (GV20) and Fengchi (GB20)-target databases were established and uploaded to https://www.tcmmodel.com/. The acupoint targets were searched for by subject headings and free words. Using the Fengchi (GB20) as an example, when searching PubMed, we searched for Acupuncture Points, Electroacupuncture, Moxibustion and Acupuncture Therapy as the MeSH keywords; Acupuncture Point, Acupuncture, Points, Acupuncture, Acupoints, Acupoints, Acupuncture Treatment, Acupuncture Treatments, Treatment Acupuncture, Therapy, Acupuncture, Pharmacoacupuncture Treatment, Pharmacoacupuncture, Pharmacoacupuncture Therapy, Pharmacoacupuncture Therapy, Pharmacoacupuncture, Acupotomy, and Acupotomies as subsubject words. The above keywords included Fengchi (GB20) and Baihui (GV20). Special personnel reviewed, debugged, and checked duplicates and filled omissions in the results. All the targets were entered into the UniProt database one by one for correction and were subsequently converted into gene names. Migraine gene sequences were obtained from the GeneCards database and cross-linked with the above acupoint targets to obtain common targets.

2.2. Protein interaction network

The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database is a database for identifying protein-protein interactions (for which gene names can be entered). Both direct physical interactions between proteins and indirect functional correlations between proteins are included. In this study, the protein interaction network in STRING was used to evaluate the element relationships, the confidence (interaction score) was set as the mediation value of 0.4, and the STRING database was directly used to construct graphs. The function of STRING was used to download Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, and UniProt keyword data, and the Weishengxin website was used to construct graphs. KEGG, Reactome, and UniProt Keywords are three sources of target analysis data, and their specific information is listed in the Help Center of the String database.

2.3. Network diagram drawing

Institute for Systems Biology [Cytoscape (version3.7.2), Seattle, WA, USA] was used to construct the interaction network between acupoint targets, and a Venn diagram was generated to illustrate the intersection of acupoints and diseases.

2.4. Experimental animals and models

Fifty healthy specific pathogen free (SPF) male Wistar rats aged 6-8 weeks and mass (200 ± 20) g were purchased from Liaoning Changsheng Biotechnology Co., Ltd., Benxi, China. The animals were reared in a standard laboratory animal room with an ambient humidity of 50%-60%, a temperature of 20-25 ℃, and alternating light and dark conditions for 12 h. After 1 week of adaptive feeding, the rats were randomly divided into 10 rats in the control group, 10 rats in the model group, 10 rats in the acupuncture group, 10 rats in the twist group, and 10 rats in the electroacupuncture group. All the rats infection assays were approved by the ethics committee of Guangzhou Miles Biosciences Application for Laboratory Animal Welfare and Ethical.

2.5. Modeling and intervention

Before modeling, all rats were adaptively immobilized for 0.5 h per day for 8 consecutive days, and the model was established on the 8th day. The rats in the control group were subcutaneously injected with 0.9% sodium chloride solution (5 mg/kg), and the rats in the other groups were subcutaneously injected with nitroglycerin (5 mg/kg) in the flat area of the back of the neck.¹⁰ Modeling success criteria included the following: 20 to 30 min after injection, the rats exhibited red ears, agitation, repeated scratching of their forelimbs, crawling in their cage and tail biting.¹¹ After 30 min of successful modeling, the rats were immobilized. In the acupuncture group, "Baihui (GV20)" and "Fengchi (GB20)" were selected for acupuncture. As shown in supplementary Figures 1 and 2, acupoint selection refers to the names and locations of commonly used acupoints in experimental animals¹² and Experimental Acupuncture Science.¹³ The depth of needle insertion was approximately 2 mm, and the needle was retained for 30 min. The needles were manually twisted for 1 min with flat tonic and flat diarrhea, twisting once every 5 min for 1 min each time for 30 min. The electroacupuncture group was treated with a parse and dense waveform, frequency 2/15 Hz, and electroacupuncture intensity 1 mA, which lasted for 30 min. The model group and the control group only received routine immobilization for 0.5 h without any acupuncture intervention.

Figure 1. Baihui (GV20) and Fengchi (GB20) target information and intersection of acupoints with migraine targets.

A: the Venn diagram of Fengchi (GB20) and Baihui (GV20); B: the intersection of acupoints and migraine targets, in which red is the acupoint, gray and light green are the targets, and dark green are the intersecting targets; C: the target information of Fengchi (GB20) and Baihui (GV20).

Figure 2. Behavioral scores and changes in inflammatory factor levels in rats.

A: behavioral score; B: levels of IL-1β; C: levels of IL-6; D: levels of TNF-α, respectively, as measured via ELISA. After 30 min of successful modeling, the rats were immobilized. In the acupuncture group, "Baihui (GV20)" and "Fengchi (GB20)" were selected for acupuncture. The depth of needle insertion was approximately 2 mm, and the needle was retained for 30 min. The needles were manually twisted for 1 min with flat tonic and flat diarrhea, twisting once every 5 min for 1 min each time for 30 min. The electroacupuncture group was treated with a parse and dense waveform, frequency 2/15 Hz, and electroacupuncture intensity 1 mA, which lasted for 30 min. The model group and the control group only received routine immobilization for 0.5 h without any acupuncture intervention. IL-1β: interleukin-1 beta; IL-6: interleukin-6; TNF-α: tumor necrosis factor-alpha; ELISA: enzyme linked immunosorbent assay. Comparisons between groups were analyzed by Student's t tests, and the values are shown as the mean ± standard deviation (n = 10). Compared with the control group, ^aP < 0.05; compared with the Model group, ^bP < 0.05.

2.6. Observation indicators and detection methods

Behavioral testing and evaluation of model preparation: two parts of the experiment were performed before and after modeling and 30 min after intervention to observe the total amount of scratching, tail biting, cage climbing and back-and-forth movements of the rats in each group, with 1 point for each occurrence.¹⁴ All rats were anesthetized with 3% sodium pentobarbital (0.1 mL/100 g) by intraperitoneal injection after completing the behavioral test. Five milliliters of blood was collected from the abdominal aorta, left at room temperature for 30 min, centrifuged for 5 min to obtain the supernatant, and stored at －80 ℃. The instructions of the kit were strictly followed, the absorbance was read at 450 nm with a microplate reader, and the levels of interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in the measured serum were calculated. After the blood was collected from all the rats, the whole brain was completely removed and placed on ice. The range of sampling was determined according to the stereotaxic atlas of the rat brain: within the cerebral cortex, with bregma as the marker point, 2.2-1.42 mm forward, 1 mm laterally and 1-2 mm deep. The left and right sides of the frontal cortex were separated from the middle. The anterior cingulate cortex was washed 3 times with precooled phosphate buffered saline and thoroughly ground. A whole protein extraction kit was used to extract tissue protein, and the protein concentration was measured by the bicinchoninic acid (BCA) method. After loading buffer was added, the proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a 0.45 μm polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk, and primary antibody (1:1000) was added and incubated at 4 ℃ overnight. After the membrane was washed 3 times with Tris buffered saline with Tween, a horseradish peroxidase-labeled secondary antibody (1:1000) was added, and the mixture was incubated at room temperature for 1 h, followed by exposure to enhanced chemiluminescence for color development. Radio immunoprecipitation assay lysis buffer (Wuhan Boster Biological Company, Wuhan, China, batch No. AR0102) is a traditional rapid lysis solution for cell tissue. PVDF membranes (Millipore Company, headquartered in Burlington, MA, USA; batch number: IPVH00010), SDS-PAGE gels (Xi'an Jingcai Biological Company, Xi'an, China; batch No. JCPE022), GAPDH antibodies (Cat. No. ab8245), toll-like receptor 4 (TLR4) antibodies (Cat. No. ab218987), protein kinase B (AKT1) antibodies (Cat. No. ab179463), heme oxygenase-1 (HMOX1) antibodies (Cat. No. ab137749), and signal transducer and activator of transcription 3 (STAT3) antibodies (Cat. No. ab119352) were purchased from ABCAM, Cambridge, UK. Rat IL-1β, IL-6, and TNF-α kits were purchased from Beijing Solarbio Technology Co., Ltd., Beijing, China. Huatuo brand acupuncture needles (0.30 mm × 15 mm) were purchased from Suzhou Medical Products Factory Co., Ltd., Suzhou, China. The main equipment used was an SDS-PAGE system (Bio-Rad, Hercules, CA, USA; Model Mini-PROTEAN), a protein wet transfer instrument (Bio-Rad, Hercules, CA, USA; Model Mini Trans-Blot), a Western blot imaging system (Bio-Rad, Hercules, CA, USA; Model ChemiDoc XRS+), an electronic needle instrument (SDZ-Il, Suzhou Medical Products Factory Co., Ltd. Suzhou, China), an electronic balance (FA2004N, Shanghai Jinghai Instrument Co., Ltd., Shanghai, China), a microplate reader (RT6000, Shenzhen Leidu Life Science Co., Ltd., Shenzhen, China), and a centrifuge (LABOFUGE400R, Thermo, Waltham, MA, USA) and a microscope (BX 51, Olympus, Tokyo, Japan).

2.7. Statistical analyses

The data were counted and plotted using R 4.2.1 and GraphPad Prism 8.0 (San Diego, CA, USA). KEGG, Reactome and UniProt Keyword pathway analyses were performed using the STRING database, and secondary mapping was performed using R. KEGG is a database for systematic analysis of gene function and genomic information that integrates genomic, biochemical and functional omics information and helps researchers study gene and expression information as a whole. The Reactome is a free, open-source, data-edited and peer-reviewed knowledge database of biomolecular pathways. The goal is to provide intuitive bioinformatic tools for the visualization, interpretation, and analysis of biomolecular pathway information. UniProtKB is a controlled vocabulary of keywords that can be used to retrieve subsets of protein entries or generate entry indices based on function, structure, or other categories. The above three enrichment methods have been widely used for bioinformatics analysis. Comparisons between groups were analyzed by Student's t tests, and the values are shown as the mean ± standard deviation. Differences were considered statistically significant at P < 0.05.

3. RESULTS

3.1. Baihui (GV20) and Fengchi (GB20) targets

A total of 162 Baihui (GV20) and Fengchi (GB20) targets were identified, including 135 targets for Baihui (GV20) and 27 targets for Fengchi (GB20). Interestingly, these two acupoints have 12 common targets, which seems to imply that Baihui (GV20) and Fengchi (GB20) may have a common mechanism of action (Figures 1A, 1B).

We continued to use GeneCards to obtain genetic information on migraine and observed the intersection with the acupoint targets (Figure 1C). A total of 73 intersecting targets were obtained.

3.2. Functional enrichment of intersecting targets

To comprehensively describe the functions of the intersecting genes, we used three enrichment methods, namely, KEGG, Reactome and UniProt, and plotted the top 15 results of each method as the main enrichment pathway (supplementary Figures 2A-2C). The hypoxia-inducible factor-1 (HIF-1) signaling pathway, signaling by interleukins, and the inflammatory response were the primary pathways in the three enrichment pathways. To more intuitively discover the main targets of the three primary pathways, we constructed a PPI network diagram There are 12 targets in two colours in the figure, namely, interleukin-1 alpha (IL1A), chemokine (C-C motif) ligand 4 (CCL4), myeloid differentiation factor 88 (MYD88), IL18, IL1B, TLR4, mitogen-activated protein kinase 3 (MAPK3), AKT1, STAT3, HMOX1, IL6, and inducible nitric oxide synthase (NOS2). These targets can be considered the main targets of the HIF-1 signaling pathway, signaling by interleukins, and the inflammatory response, which also indicates that this is the main mechanism of action of Baihui (GV20) and Fengchi (GB20).

3.3. Changes in behavioral scores and inflammatory factor levels in rats after modeling

To determine the accuracy of the enrichment results obtained above, we continued to use experimental validation. Figure 3A means that the migraine model has been successfully established. These behavioral changes were significantly reduced after Baihui (GV20) and Fengchi (GB20) stimulation. In addition, IL-1β (Figures 2B), IL-6 (Figures 2C), and TNF-α (Figures 2D) can also be regulated by Baihui (GV20) and Fengchi (GB20).

Figure 3. Effect of acupuncture on target proteins HMOX1, AKT1, STAT3, P-ERK1/ERK2 and TLR4.

A: effects of acupuncture on target proteins HMOX1, AKT1 and STAT3; B: effects of acupuncture on target proteins P-ERK1/ERK2 and TLR4. After 30 min of successful modeling, the rats were immobilized. In the acupuncture group, "Baihui (GV20)" and "Fengchi (GB20)" were selected for acupuncture. The depth of needle insertion was approximately 2 mm, and the needle was retained for 30 min. The needles were manually twisted for 1 min with flat tonic and flat diarrhea, twisting once every 5 min for 1 min each time for 30 min. The electroacupuncture group was treated with a parse and dense waveform, frequency 2/15 Hz, and electroacupuncture intensity 1 mA, which lasted for 30 min. The model group and the control group only received routine immobilization for 0.5 h without any acupuncture intervention. All increased after modeling after acupuncture, twist or electroacupuncture intervention, the expression of the above proteins was reduced. HMOX1: heme oxygenase 1; AKT1: protein kinase B; STAT3: signal transducer and activator of transcription 3; P-ERK1/ERK2: extracellular signal-regulated kinase 1/extracellular signal-regulated kinase 2; TLR4: toll-like receptor 4; GAPDH: glyceraldehyde-3-phosphate dehydrogenase. Comparisons between groups were analyzed by Student's t tests, and the values are shown as the mean ± standard deviation (n = 10). Differences were considered statistically significant at P < 0.05.

3.4. Effect of acupuncture on target proteins

We continued to perform Western blot analysis of the main targets, HMOX1, AKT1, STAT3, P-ERK1/ERK2 and TLR4, in brain tissue. Like in a previous study, we found that the levels of HMOX1, AKT1, STAT3, P-ERK1/ERK2 and TLR4 changed to a certain extent after acupoint stimulation (Figure 3).

4. DISCUSSION

4.1. Baihui (GV20) and Fengchi (GB20) regulate migraine by regulating the inflammatory response

In this study, we found that Baihui (GV20) and Fengchi (GB20) can regulate three main pathways, the HIF-1 signaling pathway, signaling by interleukins, and the inflammatory response. HIF is a major regulator of intracellular oxygen homeostasis¹⁵ and plays a central role in physiological development and disease pathogenesis. Among the HIF subtypes, HIF-1α is the most studied.¹⁶ In certain cell types, HIF-1α is involved in the angiotensin II-induced inflammatory response, endothelial cell dysfunction, proliferation, angiogenesis, and inflammation, and activation of the NF-κB pathway in endothelial cells is an important contributor to inflammation. HIF-1α has a positive feedback effect on HIF-1, and the downregulation of HIF-1α may partially protect or reverse cellular inflammatory damage.¹⁷ Our study revealed that Baihui (GV20) and Fengchi (GB20) regulate the HIF-1 signaling pathway. Moreover, we also found that signaling by interleukins and the inflammatory response are closely related to the inflammatory response. Interleukins (ILs) are cytokines involved in systemic inflammation and regulation of the immune system. They are produced by a variety of cells and can act on a variety of cells. Thirty-eight species were found, and they were named IL-1 to IL-38 according to the sequence number of their discovery. ILs are generally divided into the following categories: the IL-1 family, the IL-2 family, the chemokine family, the IL-6 family, the IL-10 family, the IL-12 family, the IL-17 family and others. IL is the most important inflammatory response mediator in cells. Our study also revealed that the IL plays an important role in migraine by transmitting information and regulating immunity and other functions. There is evidence that an inflammatory pain system may increase the sensitivity of migraine, and because of this sensitivity, pain may persist even in the absence of any migraine attack triggers.¹⁸ In contrast, the frequency of migraine in chronic migraine patients with neurogenic neuroinflammation may increase cytokine expression by activating protein kinases in neurons and glial cells of the trigeminal vasculature.¹⁹ In conclusion, the inflammatory response is currently believed to play a role in migraine, and our study illustrates that Baihui (GV20) and Fengchi (GB20) can reduce migraine by regulating the inflammatory response.

4.2. Baihui (GV20) and Fengchi (GB20) can directly regulate migraine through HMOX1, protein kinase B1 (Akt1), STAT3, phosphorylated extracellular signal-regulated kinase 1/2 (P-ERK1/2), and TLR4

HO-1, encoded by the HMOX1 gene on chromosome 22q12.3, is widely distributed in various mammalian tissue cells. It is only expressed in a small amount under physiological conditions. Factors such as ischemia, hypoxia, heme, and stress can induce significant expression. HO-1 has binding sites for HIF-1, nuclear factor kappa-light-chain-enhancer of activated B cells, and activator protein-1 and participates in the regulation of cellular redox status.²⁰ Akt1 is a serine/threonine-specific protein kinase that is a member of the protein kinase B family and a key protein in the phosphoinositide 3-kinase/Akt signaling pathway. Akt1 is a key mediator of angiogenesis and metabolism and is widely present in vascular endothelium and smooth muscle cells. The release of vasoactive substances causes Akt1-dependent phosphorylation of endothelial nitric oxide synthase, promoting NO release and leading to migraine.²¹ Several scholars have conducted in-depth research on the STAT3 pathway, the most important pathway in the STAT family.²² It was found to be effective at reducing the increase in the number of inflammatory cells, such as neutrophils, in white blood cells.²³ ERK1/2 is an important intracellular signaling molecule that is widely expressed in central nervous system cells. Its phosphorylated form can not only act on substrates in the cytoplasm but also translocate into the nucleus, where it can act on transcription factors and regulate gene expression. In recent years, studies have shown that PERK1/2 can reflect rapid changes in neurons in the brain induced by various stimuli and have been used as a new functional morphological indicator of neuronal activity. Many studies have shown that ERK regulates important functions, such as the death, differentiation and synaptic plasticity of neural tissue cells. Learning and memory also require the activation and participation of ERK.²⁴ The TLR4/MyD88/NF-κB signaling pathway is considered an important pathway in regulating disease development. Among them, TLR4 was the first member of the Toll family discovered in humans. Many studies have shown that overexpression of TLR4 can cause the development of inflammation in the body and aggravate this disease. In the central nervous system (CNS), TLR4 is most abundantly expressed on microglia.²⁵ TLR4 is also expressed on other myeloid cells, astrocytes, neurons and epithelial cells, and a recent intraventricular hemorrhage rat model showed that blood in the ventricles triggers the choroid plexus epithelium (CPe) to trigger TLR4-mediated cerebrospinal fluid hypersecretion, resulting in posthemorrhagic hydrocephalus of prematurity.²⁶ Recent studies have shown that in addition to microglia, MyD88-dependent TLR4 signaling may also play a key role as an inflammatory mediator in other CNS cell types. The above studies demonstrated the relationships between inflammation and migraine and the expression of HMOX1, Akt1, STAT3, P-ERK1/2, and TLR4. The mechanism of action of Baihui (GV20) and Fengchi (GB20) on the above targets directly proves the multitarget and complex role of acupuncture in regulating migraine.

4.3. Systematic acupuncture and moxibustion combined with systems biology may provide directions for research on acupuncture and moxibustion

The holistic concept is the main way for Chinese medicine to understand the world. Humans are a whole, and humans and nature are also a whole. Stimulating one part of the human body or using a certain decoction will lead to changes in the whole body, either good or bad. In view of this, based on the development of systems biology, network pharmacology and bioinformatics, the field of Chinese medicine has been developed systematically. An increasing number of scholars have obtained as much information as possible before conducting research and are striving to observe changes in the whole body from the field of collation. Databases such as the TCMSP and TCMID established for traditional Chinese medicines, prescriptions, and proprietary Chinese medicines have also gradually been widely used. The development of network pharmacology, systems pharmacology, integrative pharmacology and other fields has promoted the development of molecular biology, pharmacology and other related disciplines combined with systems science theory. These methods and technologies focus on studying the interaction between the body and drugs from a holistic level, which has led to the initial establishment of systematic Chinese pharmacy theory. Systems biology methods seem to have become important tools for studying the underlying mechanisms of TCM, but unfortunately, as an important part of TCM, acupuncture is not included in this list. Based on the concept of being an important part of the whole body, stimulating acupuncture points will inevitably lead to further changes in the biological functions of the whole body. Therefore, this study compared systems biology theory to determine the mechanism of action of acupoints and meridians. The concept of systemic acupuncture is used. This method involves cross-integration of acupuncture and systemic science, explains the efficacy of acupuncture from a holistic perspective, and explains the changes in overall functions and physiological and pathological states caused by stimulating the human body.⁸ The "acupoint-target-disease" model that intersects with diseases can reveal biological changes in the whole body more comprehensively and systematically than can the traditional single mechanism and single target model. We think this may constitute a new research method to elucidate the black box of the mechanism of acupuncture.

In conclusion, this study focused on exploring the mechanism by which Baihui (GV20) and Fengchi (GB20) regulate migraine through three pathways: the HIF-1 signaling pathway, signaling by ILs, and the inflammatory response. We also explored a new approach to studying acupuncture targets, systemic acupuncture. We believe that this study may provide new ideas for exploring the mechanism of action of acupuncture.

5. SUPPORTING INFORMATION

Supporting data to this article can be found online at http://journaltcm.com.

Funding Statement

Supported by the Study on the Mechanism of Action of Dutong and Tiaoshen Acupuncture in Improving Upper Limb Motor Function After Ischemic Stroke based on Functional Near-infrared Spectroscopy Technology (Science and Technology Program of Panyu, 2022-Z04-107)