The neuro-immune microenvironment of acupoints—initiation of acupuncture effectiveness

Abstract

Acupuncture is a centuried and unfading treatment of traditional Chinese medicine, which has been proved to exert curative effects on various disorders. Numerous works have been put in to uncover the effective mechanisms of acupuncture. And the interdependent interaction between acupuncture and acupoint microenvironment is a crucial topic. As a benign minimally invasive stimulation, the insertion and manipulation of needle at acupoint could cause deformation of local connective tissue and secretion of various molecules, such as high mobility group box 1 and ATP. The molecules are secreted into extracellular space and bind to the corresponding receptors thus active NF-κB, MAPK, ERK pathways on mast cells, fibroblasts, keratinocytes, and monocytes/macrophages, among others. This is supposed to trigger following transcription and translation of immune factors and neural active substance, as well as promote the free ion movement (such as Ca²⁺ influx) and the expansion of blood vessels to recruit more immune cells to acupoint. Finally, acupuncture could enhance network connectivity of local microenvironment at acupoints. The earlier mentioned substances further act on a variety of receptors in local nerve endings, transmitting electrical and biochemical signals to the CNS, and giving full play to the acupuncture action. In conclusion, we portrayed a neuro-immune microenvironment network of acupoints that medicates the acupuncture action, and would lay a foundation for the systematic study of the complex network relationship of acupoints in the future.

Introduction

As one of the traditional and alternative treatments, acupuncture has been approved to be used in 103 member states, and the World Health Organization has recommended 4 categories and 107 indications for acupuncture, including various disorders of nervous system, musculoskeletal system, respiratory system, digestive system, and ophthalmic system.¹ Since modern times, acupuncture treatment has become a comprehensive treatment system represented by manual acupuncture, electroacupuncture, transdermal electrical stimulation, as well as heat acupuncture, needle knife, fire acupuncture, acupoint catgut embedding, among others. Although the mechanism of acupuncture treatment varies in different diseases,² they all share some common characteristics: endogenous regulation and initiation of acupoints microenvironment stimulated by acupuncture. On the one hand, different from exogenous drugs supplementing the shortage or removing the excess of the body, acupuncture taking effects through modulating the intrinsic homeostasis³ thus producing corresponding endogenous substances, in which way could avoid the side effects of exogenous drugs, or even drug resistance. On the other hand, acupuncture takes effect through the stimulation of acupoints’ microenvironment,⁴ a process that includes the changes in anatomic structures, modulation of cell function, and release of various bioactive substances. It was demonstrated that mast cell degranulation in acupoints could be promoted by acupuncture and accompanied with electrical signal transportation and biochemicals’ secretion, such as tryptase, 5-hydroxytryptamine (5-HT) and histamine (HA), substances that have been proven to mediate acupuncture effectiveness.^5,6 After the accumulation of active changes in acupoints, acupuncture signals are transmitted to CNS leading to modulation of organic functions. Taking pain, the most common indication of acupuncture as an example, during acupuncture analgesia, signals are transmitted from the acupoints to the CNS. And then the secretion of neurotransmitters and neuromodulin such as opioid peptide, serotonin, norepinephrine, and dopamine are released from spinal cord and brain, performing a combined action in suppressing the pain.⁷ In this study, we systematically reviewed previous data to draw a systematic map of various components and their crosstalk within acupoints. We portrayed a network of acupoint microenvironment to provide a basis for further systematic study of acupoint microenvironment as well as its network mechanism.

Anatomical Structure of Acupoints

There are 409 acupoints recorded in the classical literature of traditional Chinese medicine (TCM), including 361 acupoints on 14 meridians and collaterals, and 48 extra acupoints outside the meridians.⁸ And there are numerous ashi points whose location is determined by patients’ tenderness points on body surface. Additionally, the majority of acupoints are in interspace between muscles and bones. Most acupoints have similar, 3-D construction that is composed of epidermis, dermis, subcutaneous tissue, muscles, and related structures such as nerve, blood vessels, lymph, tendons, among others. Compared with nonacupoint area, nerves are more densely distributed in acupoints region, and the type, quantity, and combination of nerves varies in different acupoints. Some acupoints are located around nerve endings’ densely distributed region, some near the convergence point of superficial nerve, and some near the nerve plexus.⁹ In addition, it has been demonstrated that abundant neuroreceptors in nerve terminals of acupoint are supposed to be the basis of the needling sensation of patients, such as free nerve terminal muscle spindle, annular corpuscles, Kirschner’s terminal ball, among others. According to different dominant neural structures under the skin, acupoints are classified into five categories: skin points (free nerve endings), muscle points (muscular nerve and free nerve endings), periosteum points (pain-sensing nerves), vessel points (sympathetic nerve), and nerve trunk points (nerve trunk), among others.¹⁰ Furthermore, most acupoints are constituted by thinner skin, higher density and more regular arrangement of blood vessels,¹¹ densely distributed inherent connective tissue (including elastic fibers and collagen fibers), as well as muscles and fascia.¹²

Amid these structures, there are many kinds of cells, such as keratinocytes, mast cells, fibroblasts, macrophages, as well as different molecules, such as ATP, adenosine, HA, free ions, and the like. The earlier mentioned components and their multidirectional interactions constitute the microenvironment of acupoints. Being the targeting sites of acupuncture stimulation, the functional cellular and biochemical response is the initiative mechanism of acupuncture actions.

Acupoints—the initiation link of acupuncture actions

Acupoints play two main roles treating disease: reflecting disorders and receiving acupuncture stimulation to treat diseases. Relative pathologic reactions on acupoints, such as paresthesia, paramorphia, and abnormal biophysical properties (e.g., unusual temperature, electrical, and light characteristics)^13,14 are thought to reflect disorders of relative organs.¹⁵ For instance, it has been reported that increased microcirculatory perfusion was observed in Feishu (BL13, a pair of acupoints considered to reflect lung function and pulmonary diseases) in chronic obstructive pulmonary disease rats.¹⁶ Besides, the electrical characteristics (resistance, voltage, and electric current) would change accordingly with viscera disorders.¹⁷ The internal mechanism mainly includes two theories. One is the spinal segmental sensitization theory. When the pathologic signals of viscera are transmitted into the spinal dorsal horn and synaptically connect with the neurons in the corresponding and adjacent segments, the process would excite dorsal root of the somatosensory nerve and result in pathologic reaction of certain acupoints.¹⁸ The other viewpoint is axonal sensitization. There are some sensory neurons in the spinal ganglion, and their peripheral fibers not only dominate the body, but also emit long lateral branches to a certain viscera.¹⁹ It has been found that abnormal stimulation to the viscera can lead to the release of active substances from the nerve endings on body surface, triggering a neurogenic inflammatory response without going through the CNS. However, this kind of long axon branches is relatively rare, so axonal sensitization could not be the main theory. Thus, acupuncturists tend to use certain acupoints to diagnose, treat, and prognosticate diseases with the earlier mentioned signs of acupoints.

Beyond that, acupoints could sense the outside mechanical stimuli such as acupuncture to treat diseases. Acupuncture is originally defined as solid metal needles pricking into skin at acupoints.²⁰ It is considered as a micro-traumatic mechanical stimulus²¹ using relatively small diameter (ranging from 0.20 to 0.45 mm) needles and the insertion would cause fracture and even necrosis of muscle fibers, as well as the accumulation of red blood cells, immune cells, and cell fragments in the muscle gap.²² One Japanese research group has analyzed the tissues attached to acupuncture needles after withdrawing and found collagen fibers, cell debris of fibroblasts, and adipocyte attached to the needle, which has further confirmed acupuncture as a traumatic stimulus.²³ The local tissue injury would cause the subsequent onset of acute immune response,²⁴ including infiltration of leukocytes, mast cells, and release vasoactive substances such as HA, substance P (SP), and adenosine. While exciting the nerves, these substances would also dilate local blood vessels and enhance blood and lymph circulation so as to recruit more immune factors. However, the inflammation around acupoints is self-controlled because of the activation of cutaneous hypothalamic-pituitary-adrenal (HPA) axis (constitutes of fibroblasts, melanocytes, and keratinocytes), which would release anti-inflammatory glucocorticoid, thus limiting inflammation through autocrine and paracrine effect. More importantly, by the circulation of various elements through liquid transportation, the local inflammatory reactions and upward conduction of nerve signals in acupoints would trigger greater and far-reaching impact on somatic function from the minor and partial forces (acupuncture) applied upon.

Neural activation in acupoints microenvironment is a necessary pathway for acupuncture actions

Sensory nerves are the “scouts” of nervous system in the recognition of acupuncture signal and the absence of local nerve fibers at acupoints would invalidate acupuncture effect.^25,26 For example, injecting 2% lidocaine into acupoint Zusanli (ST36) could inhibit the analgesic effect of acupuncture by blocking the signal transduction from the peroneal nerve to the CNS.²⁷ Torres-Rosas et al. found the expression of transient receptor potential vanilloid 1 (TRPV1) in nerve fibers of acupoint significantly increased after electroacupuncture stimulus,²⁸ and subcutaneous injection of TRPV1 antagonist in Zusanli (ST36) could successfully weaken the anti-inflammatory effect of electroacupuncture in sepsis mice.²⁹ During manual acupuncture stimulation, acupuncturists are intended to rotate the needle in one or both directions at varying speeds, as well as partially extract the needle before pushing it back in repeatedly to elicit the needling sensation of De Qi. De Qi is well recognized as the sign of effectiveness during acupuncture and is characterized by patients’ local or conductive sensations integrating soreness, numbness, heaviness, swelling, and tenderness. It is worth noting that the activation of the nervous system is strongly linked with De Qi. The neural structure types and densities within acupoints, acupuncture stimulation pattern (e.g., electroacupuncture frequency and duration), and puncturing depth are the main influencing factors, but they all come down to activation of various neurosensory apparatus. For instance, numbness is related to the conduction of Aβ/γ fibers, whereas swelling sensation is related to Aδ fibers, and soreness is related to C fibers.³⁰ Nociceptive sensory nerve endings express different receptors, for instance ion-channel receptors such as TRPV1 receptors, excitatory amino acids, TLR, as well as TNFs, IL-6, and prostaglandin receptors, among others. Various receptors would sense acupuncture stimulus and be activated to a certain or lower excitation threshold.³¹ After the excitation of peripheral cutaneous and/or muscular afferent nerve fibers, according receptors are aroused so as to promote the release of local neurotransmitters and neuropeptides (such as HA and SP), and the earlier mentioned substances would work as vasodilation and chemotaxis to recruit immune cells to acupoints.³² As a physical stimulation, acupuncture action varies according to its stimulus parameter (such as frequency, intensity, and time of duration) and eventually affects different afferent nerve fibers. Solid evidence has manifested that both electroacupuncture and manual acupuncture could excite A, B, and C-type afferents and elicit analgesia.^33,34 Acupuncture at Zusanli (ST36) could recover the gastric motility of rats with atropine-induced gastric inhibition and the effects varies with manipulation frequency—the motility of 1–3 Hz stimulation were superior than that of 4 Hz.³⁵ The earlier mentioned finding has confirmed different neural excitation affect target organ function in distinguished manner. Finally, the impulses from exogenous stimulus are integrated with signals of illness at different levels of the CNS³³ and affect target areas via neural pathways as well as endocrine system.³⁶

Components other than nervous tissue induced by acupuncture in acupoints microenvironment

Connective tissue deformation in acupoints after acupuncture

Anatomic researches showed that 80% of acupoints are distributed in the connective tissue¹² of subcutaneous layer, muscles, and regions between muscles and bones, with fascia, periosteum, and joint capsule nearby. Besides, the shape changes of collagen fibers and elastic fibers in the inherent connective tissue are responsible for the pull feeling of needle manipulation³⁷ and the full play of acupuncture actions. Without acupuncture stimulus, fibers in acupoints are curled up, and arrange in bundles with regular direction. During acupuncture manipulation, relative movements between fibers and acupuncture needle are reported to roughen collagen fibers surface,³⁸ distance fibers away³⁹ from the insertion site, and cause the winding, tightening,⁴⁰ deformation, and fracture of fibers, thus forming a spiral pattern with the needle in the center.³⁷ The earlier mentioned morphologic changes would initiate the peripheral cellular and molecular signal transduction, including mast cell degranulation, fibroblasts cytoskeletal remodeling,⁴¹ and the following changes in synthesis and release of various cytokines (including basic fibroblast growth factor [b-FGF], SP, IL, adenosine, etc.). And the breakdown (using collagenases) of normal structure of collagen fibers would inhibit the degranulation of mast cells and other cell reactions in acupoint area, and finally weaken acupuncture effect.⁴² It was speculated that the composition change of matrix as well as cell reactions that take place in connective tissue might mediate remote effects of acupuncture away from the penetration site⁴⁰ even to the CNS.⁴¹

Cellular responses

Plenty studies have revealed that mechanical stimuli would deform microstructure and activate various load-sensitive cells, including smooth muscle cells, fibroblasts, and endothelial cells, among others, to adjust cellular functions and extracellular matrix constituents.⁴³

Immune cells

Acupuncture is supposed to activate the local innate immune response in acupoint area in early phase, concerning activation of mast cells, Langerhans cells, resident macrophages, and outward migration of blood-derived immune cells (neutrophils, monocytes, etc.).⁴ Mast cells are widely distributed in subcutaneous tissue or skin, as well as near blood vessels, nerves, smooth muscle, mucus secreting glands, and hair follicles. They are 55% more densely distributed in intra-epidermis and dermis around the acupoint area than that in the nonacupoint region.⁴³ Besides, mast cells are found to aggregate in close proximity to small blood vessels, small nerve bundles, and nerve endings in the direction of meridians.⁴⁵ As mentioned earlier , the insertion and twirling of acupuncture needle could generate shear force to activate the physical stimulus sensitivity channel TRPV2 as well as stretch-activated chloride channels⁴⁶ on mast cells and simultaneously induce the intracellular influx of Ca²⁺ as well as the activation of adenosine A1 and HA H1 receptors.⁴⁷ This internal flow of Ca²⁺ is supposed to promote the degranulation from mast cells,⁴⁸ a process that contains a variety of neurotransmitters, hormones, and cytokines, such as ATP,⁴⁹ SP, tryptase, HA, IL, and 5-HT granules release in acupoints,⁵⁰ and affect the therapeutic effect of acupuncture.⁵¹ These neuroactive and immunoreactive substances diffuse in extracellular space may in return promote mast cells degranulation, cause autogenous inflammation, as well as mediate acupuncture effects. Therefore, it is believed that mast cells are major cellular participants in triggering neuro-immune response and initiating acupuncture signals.^52,53 In addition, our recent unpublished data has confirmed that acupuncture analgesia in complete Freund’s adjuvant (CFA) rats and the infiltration of CD68⁺ macrophages were found to increase in subcutaneous tissue of acupoints after acupuncture. The directed weighted cell communication network (which we used to calculate the intercellular intensity of secretion/action function of immune cells) manifested that macrophages were the key post-acupuncture cells, which means they interact most with other cells in the local area of acupoints. Macrophages would be differentiated from monocytes in blood that migrate to inflammatory region by the development of inflammation.⁵⁴

Fibroblasts

Fibroblasts are the most common cells in connective tissue, and their main functions include synthesis and reconstruction of extracellular matrix, regulation of inflammation, and repair of tissue injury. Activated fibroblasts could produce a large number of proinflammatory factors (such as IL-6 and IL-8) and chemokines (such as CXCL12), which could attract immune cells migrate to the inflammatory regions.⁵⁵ Fibroblasts are considered to react to a wide range of mechanical stimuli such as acupuncture rotation. The tissue-stretch stimulation of acupuncture rotation could not only double their cross-sectional area and deform them into flake-like bodies with shorter processes in a time-dependent manner,^41,56,57 but also the rotation manipulation could relocate fibroblasts so that they are collinear with collagen fibers.⁴⁰ Besides, acupuncture could accelerate the proliferation of fibroblasts in acupoints possibly through the combination of multiple molecules (e.g., adenosine, ATP, b-FGF, TGF-β1), PGE2, and IL-6 and their receptors on fibroblasts via MAPK and ERK pathway.^58,59

Keratinocytes

It has been demonstrated that many physical and chemical sensitive receptors (such as TRPV1, TRPV3, and TRPV4) are identified in epidermal keratinocytes acting as mechanical stimuli (including acupuncture) sensors. Besides, other neurotransmitters such as glutamate and dopamine,^60,61 as well as their receptors, are also expressed in keratinocytes, which can be activated by acupuncture. Further, the post-acupuncture expression of cannabinoid 2 receptors on the surface of keratinocytes is increased and more endogenous opioid peptides are produced to stimulate local primary neurons in acupoints.⁶² What is more, keratinocytes are also responsible for the endocrine environment alteration in acupoints for releasing HPA axis hormones (such as corticotrophin-releasing hormone [CRH], ACTH, and cortisol) that own anti-inflammatory capacity.^3,63 The hormones would mediate the endocrine-immune regulation of acupuncture on the proinflammatory and anti-inflammatory balance of acupoint microenvironment together with the proinflammatory factors.

Vascular and blood flow reaction in acupoints after acupuncture

The blood vessels in acupoint areas were densely distributed in epidermis, sebaceous gland, hair follicles, and muscle. There are many kinds of vascular structures in the skin layer, among which there is a rich capillary network around sebaceous gland and near hair follicles. The blood vessels at both ends of the capillary bulb are thicker and send out small branches converging to the center of the ball and anastomosing with each other, and there are blood vessels passing through others in parallel to form communicating branches. The superficial dermis is dominated by small capillaries and capillary spheres with a diameter of about 5–10 μm. And the appearance of the blood tube in the middle layer of the dermis is complex. There are not only longitudinal blood vessels and their small transverse branches, but also convoluted capillary spheres, with a diameter of about 300–400 μm. In the deep dermis and subcutaneous tissue, thicker vessels with a diameter of about 80–100 μ m are distributed longitudinally in the direction of meridians and collaterals.

It is recognized that acupuncture could cause vasodilatation and increase capillary permeability so as to up-regulate blood circulation in acupoint region, which would induce the exudation of inflammatory medium. By using laser speckle instrument to detect blood flow, Ding et al. found that acupuncture stimulation could increase the blood flow volume at Yanglingquan (GB34), Zusanli (ST36), and Heding (EX-LE2) specifically, with no change in nonacupoints.⁶⁴ Furthermore, the flare reaction⁶⁵ (reddening, sweating, red line, rash, subcutaneous hemorrhage) as well as local temperature rise are the correspondent manifestation of vessel dilation.⁶⁶ The inner cause of vasodilatation and increased capillary permeability might be up-regulated content of NO and calcitonin gene-related peptide (CGRP) after needle insertion.⁶⁷ More importantly, the dilation of vessels and increased blood flow would bring more immune cells (such as mast cells, monocytes/macrophages, and neutrophil^5,68,69 to acupoints to then participate in neuro-immune network. Meanwhile, it was suggested that needle tip touching the vessels might account for the feeling of pricking in acupuncture because of abundant free nerve endings on the vascular wall.⁷⁰

Signal molecules released from cells to the intercellular matrix in local acupoints after acupuncture

The studies mentioned earlier have revealed that acupuncture could recruit a variety of cells, including immune cells, endothelial cells, fibroblasts, and keratinocytes, among others. Further studies found that through intracellular signal transduction, many soluble factors are transcribed, translated, and released into extracellular matrix, promoting the signal transmission within acupoint. It has been found after acupuncture, several signal pathways are activated, which mediate acupuncture effect near acupoints. Ji-Yeun Park et al. performed a cDNA microarray testing acupoint tissue after needle insertion compared with no acupuncture, and 236 genes were altered and 7 corresponding pathways (such as MAPK, P53, BCR, TCR, TLR receptor signaling pathways, and the circadian rhythm and C21-steroid hormone metabolism) changed significantly. Among them, the change of ERK pathway was the most apparent.⁷¹ Our recent studies demonstrated that the key feature of acupoint microenvironment is proinflammatory. For our gene chip detection showed that the gene expression of proinflammatory substances such as Myd88, Nfkbia, Il1b, Il6, Cxcl1, and Ccl2 of normal rats in local acupoints were dynamically increased by acupuncture stimulus.⁷² Jiang et al. performed electroacupuncture near inguinal region in normal rats and collected corresponding superficial anadesma in acupoint; the results showed that the post-acupuncture expression levels of ERK1/2 and p-ERK1/2 increased at 0, 1, 12, and 36 h after needle insertion.⁷³ It has been previously suggested that acupuncture manipulation on Yanglingquan (GB34) acupoint of mice pain models could promote the phosphorylation of ERK pathway, taking place both on keratinocytes in epidermis and fibroblasts in the dermis at acupoints, and acupuncture analgesia was erased by MEK/MAPK inhibitor U0126. Besides, referring to the results of genome-wide microarray on Yanglingquan (GB34) in skin layer, the gene expression of NF-κB was also up-regulated⁷¹ and because the change of NF-κB and Iκ-Bα was reversed by MEK/MAPK pathway inhibitor U0126, they might have a close relationship with ERK pathway activation. Gao et al. tested the expression of high mobility group box 1 (HMGB1) in Zusanli (ST36) and Yanglingquan (GB34) in chronic constriction injury rats after electroacupuncture and the expression of acetylated-HMBG1, TLR4, and receptor CD24 were up-regulated compared with model group. The application of CD4 neutralizer was observed to prominently reduce HMGB1 and thus weaken analgesia of electroacupuncture.⁷⁴ Therefore, the expression and excitation of the earlier mentioned pathways play a vital role in acupuncture effectiveness.

Neural factors

SP and CGRP are nociceptive neuropeptides that could be promoted by acupuncture expressing in nerve fibers. SP is known as a neuropeptide participating in transmission of algetic signals from the peripheral receptors to the CNS,⁷⁵ and in the meantime dilating skin capillaries⁷⁶ and promoting mast cells activation and degranulation to release bioactive substances. CGRP is considered to act both as the main vasodilator in local acupoints area⁷⁷ to increase muscle blood flow and promoter of collagen proliferation, and the latter might contribute to the healing-promotion effect of acupuncture.⁷⁸ Besides being secreted from local cells, SP and CGRP could also be released from local activated afferents fibers of acupoint, and that is responsible for the high conductance in acupoints.⁷⁹ Moreover, SP and CGRP also show strong chemotaxis characteristic by recruiting congenital and adaptive immune cells.⁸⁰ And by activating endothelial cells and smooth muscle cells, they intend to increase the exudation of inflammatory substances. Thereby neural factors could activate the nociceptive nerve and local nerve endings, resulting in local immune changes, and the network reaction could further expand the nerve input of acupuncture signals.⁸¹ Other bioactive substances such as HA and serotonin, among others, released from mast cells degranulation, are also acupuncture analgesic mediators in acupoints.

Immune factors

It has been verified that acupuncture could significantly up-regulate the content of HMGB1 and TLR4 in acupoints at different time points. HMGB1, as a widely known inflammatory factor as well as damage associated molecular, could be released by a wide range of immune cells to extracellular matrix. And besides participating in systemic or local inflammatory response, it could also stimulate the proliferation and maturation of dendritic cells via combining with its receptors (e.g., TLR2 and TLR4), thus inducing immune response. And as a transmembrane signal transduction receptor, TLR4 could recognize the binding of damage associated molecular and activate cellular NF-κB signaling pathway, which is one of the important inflammatory pathways.⁸² The activation of NF-κB pathway could promote the transcription and translation of several proinflammatory factors (such as TNF- α and IL-6). And the activation would amplify the local inflammatory response so as to transduce acupuncture information in acupoints and perform further chemotaxis on infiltration and differentiation of more immune cells. Several researches also showed that CXCL1, stem cell factor (SCF), MCP-1, and ICAM-1 increased significantly in local acupoints after acupuncture, which could promote the intercellular information transmission to enhance the recognition, activation, proliferation, and migration of cells. For instance,⁸³ it would attract monocytes to inflammatory acupoints, especially near small vessels. Under the action of acupuncture pressure, the deformation of fibroblasts membrane might initiate the synthesis and secretion of PGE2 from infiltrating neutrophils, which might inhibit platelet aggregation, dilate local small blood vessels, and promote local blood circulation. It has been reported that acupuncture could increase NO synthase activity in meridians and acupoints⁸⁴ as well as the concentrations of nitrate and nitrite, coupled with increased blood flow. NO is considered to derive from binding among adenosine, ATP and CGRP, and their receptors on vascular endothelial cells,⁸⁵ as well as the activation of macrophages.⁸⁶ In turn, it could suppress hyperalgesia and allodynia via inhibition of SP release from spinal cord to the painful regions.^87,88

Free ions

Accumulating evidences have verified that acupuncture could increase both cellular and extracellular (along medians and in acupoints) concentration of free ions, including Ca²⁺, K⁺, Na⁺, and Cl⁻, and the increase is related to therapeutic effect on visceral regulation.^89,90 Among these free ions, Ca²⁺ is supposed to matter the most as a second messenger that takes part in different cellular physiologic and biochemical process, including the elevation of K⁺ and Na⁺.⁹¹ Acupuncture could up-regulate the concentration of Ca²⁺ in meridian lines, especially around acupoints.⁹² Subcutaneous infusion of Ca²⁺ could simulate therapeutic effect of acupuncture, and blocking Ca²⁺ channels or antagonizing Ca²⁺-binding protein were proved to weaken acupuncture effect to some degree. Meanwhile, the local Ca²⁺ in acupoints after acupuncture mediates the degranulation of mast cells.⁹³ Therefore, we presume that Ca²⁺ is one of the key factors for acupuncture actions. It was also found out that among the accumulated metal elements (Ca, Fe, Zn, etc.) at acupoints, Fe is the most abundant,⁹⁴ and a strengthened electron exchange between the Fe ion and N(O) atoms took place nearby. Furthermore, Fe in heme showed stronger oxygen-transporting activity along the meridian in acupoints, which might contribute to local energy and substance transportation.⁹⁵

Other factors

Several investigations have showed that extracellular concentration of purines (including ATP, ADP, AMP, and transmitter adenosine) increase in the vicinity of acupoints, possibly released from mast cells, keratinocytes, and fibroblasts. ADP, AMP, and adenosine maintained their growth in extracellular matrix for 1 h after acupuncture stimulation both in mice and human volunteers,⁹⁶ whereas ATP dropped to basal level quickly due to its rapid hydrolysis.⁹⁷ Adenosine concentration was detected to increase rapidly 0.4–0.6 mm away from Zusanli (ST36), up to 24-fold during the half-hour acupuncture manipulation. Besides, the increase of adenosine could only be detected at acupoints after acupuncture manipulation such as rotation.⁹⁶ And exogenous injection of adenosine at Zusanli (ST36) promotes cell proliferation of fibroblasts in local anadesma by activating MAPK pathway.⁹⁸ Besides, ATP would stimulate local sensory nerve endings by combing with receptors P2X2/3, thus transducing the electrical signals.⁹⁹ What Nanna Goldman has verified was that adenosine A1 receptor agonist could perform a local antalgic effect, possibly through unmyelinated C and A∂ fibers near Zusanli (ST36) in the superficial peroneal nerve.^96,97

Discussion and Conclusion

Skin is considered to pose intricate endocrine and immune response to outside stimuli via appropriate production of biologically active compounds such as neurotransmitters, hormones, and immune factors that perform local and systematic action.⁶³ Different sensory receptors are distributed in skin, including Meissner corpuscle, free nerve endings, and Merkel disks, among others. And the receptors would receive information of pressure, pain. and thermal stimulus.^100,101 Followed by nerve excitation, domestic cells in skin layers (for instance, keratinocyte, mast cells, Langerhans, lymphocyte, fibroblasts, macrophages, and endothelial cells, etc.) that would be activated to generate multitudinous molecules into the extracellular matrix. In addition to modulating local microenvironment such as the remodeling cell structure, promoting exocytosis, or firing cutaneous nerve system, the earlier mentioned process also has close connection with the function of the CNS as well as with the internal organs based on theories that include, but are not limited to, brain-skin communication¹⁰² and organ-skin neural connection. Both skin and the CNS originate from neuroectoderm and they share the same characteristics of neural excitation and neuron-endocrine-immune factors secretion when facing outside environmental changes. Skin employs various neuroendocrine structures that are equivalent to the CNS such as HPA axis, hypothalamic-pituitary-thyroid axis, and melatoninergic system, among others. The structures are supposed to act under mental stimulation and have close relationship with several dermatologic diseases.¹⁰³ Therefore, skin and the CNS impose on each other through systematic regulation. Since the 19th century, scientists have noticed changes in blood pressure corresponding to stimulation of the nerves of the limbs; thus they gradually established somatic, visceral, and sympathetic reflexes theories. For instance, the gate control system was proposed in 1965 that explains spinal analgesia mechanism of body surface stimulation therapy. When C fibers from diseased organs conduct nociceptive information to the advanced parts of nervous system to evoke pain, these noxious inputs transmission are dampened by Aβ fibers, which conduct tactile stimulus from same spinal segments. Skin stimulation such as acupuncture, massage, and cupping could activate local type Aβ fibers; thus the transmission of pain information to the brain is blocked to inhibit pain.⁷

Currently, there are a few definitions of acupoints, for instance Kim et al. proposed that acupoints are cutaneous neurogenic inflammation points, which are generated by somatic afferents excitation in diseased visceral organs. Acupoints are located in the overlapping spots that reflect signs of illness of relevant organs. Studies were conducted to verify that cutaneous neurogenic inflammation points share the same electric characteristics such as high electro-conductibility.¹⁰⁴ Zhang et al. defined acupoints as a neural acupuncture unit (NAU) composed of abundant free nerve terminals, sensory receptors, and affiliated afferent fibers in active state.¹⁰⁵ The function of NAU is supposed to be modulated by various acupuncture manipulation patterns, including the direction and depth of needle insertion. The penetration of acupuncture needled would cause a serial of tissue injury and release of inflammatory medium, posing a positive influence on acupuncture effectiveness.⁹⁸ Zhu et al. considered that acupoints are the “sensitization” sites induced by neurogenic response which reflect visceral functional state in the pathologic process of the body. The “sensitization” concludes dynamic changes of acupoint composition, structure, and physicochemical environment.¹⁰⁶ However, according to this review, researches on local acupoint area are relatively scattered and fragmented. Most of them focus only on changes in a certain aspect, such as the changes and functions of neural factors, of certain cells and cytokines before and after acupuncture, instead of the reticular network relationship and interconnection among complex substances. At present, several researchers have put forward the concept of microenvironment for many organs and tissues functioning, such as hematopoietic microenvironment of bone marrow, and tumor microenvironment, among others. Any function is the result of the integrated action of local microenvironment and is affected by the overall environment of the body. Thus, we proposed the concept of post-acupuncture acupoint microenvironment with dynamic changes in physiologic and pathologic conditions. Taking the known local structure of acupoints as a starting point is an effective way to clarify the microenvironment of multiple acupoints by systematically combining the related tissue, cellular, and chemical components, as well as the network interaction among these components.

Acupuncture could treat many diseases, and the traumatic stimulation acting on local microenvironment is the universal initiative mechanism of acupuncture actions. However, referring to different diseases, the acupoints condition is different, and the selection of acupoints and manipulation are diverse; thus the mechanism of signal transduction and integration between acupoint microenvironment and target organs is different. That would lead to different therapeutic effects of acupuncture, for instance, analgesia, anti-inflammation, improving gastrointestinal function, regulating mood changes, and promoting nerve repair, among others. To explore the role of key substances in acupuncture effect, researchers should pay attention to the inner link of acupoint microenvironment alternation and acupuncture efficacy, that is, how the change of a certain substance in the microenvironment causes the following change of other substances, and how they affect acupuncture effect synthetically.

In conclusion, acupuncture causes deformation and winding of local connective tissue, and induces the proliferation of fibroblasts, which up-regulates the release of TGF-β, PGE-2, and other substances. And at the same time, acupuncture could induce the local production of opioid peptides, ACTH, CRH, and glucocorticoid from keratinocyte. In addition, acupuncture would promote calcium influx to induce the degranulation of mast cells, which would secrete ATP, SP, tryptase, HA, IL, 5-HT, and other substances into the extracellular matrix. A wide range of immune cells such as macrophages could produce inflammatory substances such as HMGB1, TLR4, TNF- α, IL-6, IL-1 β, and chemokine such as CXCL1, SCF, MCP-1, and ICAM-1. These active factors would promote the transmission of information among cells thus mediate acupuncture actions. And under the stimulation of acupuncture in acupoints, vascular endothelial cells could produce ATP, adenosine, and other substances, and then produce NO to stimulate local nerves and lead to the production of neural active components such as CGRP. Afterward, various substances mentioned earlier, such as SP, CGRP, serotonin, and HA, are supposed to promote the expansion and permeability of local blood vessels, thus increasing local blood supply, which would consequently recruit immune cells to the local area, and enhance the exudation of inflammatory substances. Finally, the network of acupoint environment induced by acupuncture activates the receptors located on neural terminals and the acupuncture signal is transmitted to the CNS to play therapeutic effect, which would be the universal initiative mechanism of acupuncture actions (graphical abstract and Fig. 1).

Abbreviations

 
• b-FGF

  basic fibroblast growth factor

 
• 5-HT

  5-hydroxytryptamine

 
• CFA

  Complete Freund ’s adjuvant

 
• CGRP

  calcitonin gene related peptide

 
• CRH

  Corticotrophin-releasing hormone

 
• HA

  histamine

 
• HMGB1

  High mobility group box 1

 
• HPA

  hypothalamic-pituitary-adrenal

 
• NAU

  neural acupuncture unit

 
• SCF

  stem cell factor

 
• SP

  substance P

 
• TRPV1

  transient receptor potential vanilloid 1

Acknowledgments

This work was supported by the National Natural Science Foundation of China (NSFC) nos. 81873369 and 81330088 and the Tianjin Natural Science Foundation no. 18JCQNJC82400.

Authorship

The authors contributed in the following manner: Y. Gong: concept design, data collection, and paper writing; N.L.: figures and graphic abstract edit; L.Z., K.Z., Y.Z., T.Y., H.W., and X.Z.: data collection and analysis; Z.C., B.D., B.C., and Yongming Guo: language modification and text check; and Yi Guo and Z.X.: concept design and paper review.

Disclosures

The authors declare no conflicts of interest.

References