Effect of Acupuncture on the p38 Signaling Pathway in Several Nervous System Diseases: A Systematic Review

Acupuncture is clinically used to treat various diseases and exerts positive local and systemic effects in several nervous system diseases. Advanced molecular and clinical studies have continually attempted to decipher the mechanisms underlying these effects of acupuncture. While a growing understanding of the pathophysiology underlying several nervous system diseases shows it to be related to inflammation and impair cell regeneration after ischemic events, the relationship between the therapeutic mechanism of acupuncture and the p38 MAPK signal pathway has yet to be elucidated. This review discusses the latest advancements in the identification of the effect of acupuncture on the p38 signaling pathway in several nervous system diseases. We electronically searched databases including PubMed, Embase, and the Cochrane Library from their inception to April 2020, using the following keywords alone or in various combinations: “acupuncture”, “p38 MAPK pathway”, “signaling”, “stress response”, “inflammation”, “immune”, “pain”, “analgesic”, “cerebral ischemic injury”, “epilepsy”, “Alzheimer’s disease”, “Parkinson’s disease”, “dementia”, “degenerative”, and “homeostasis”. Manual acupuncture and electroacupuncture confer positive therapeutic effects by regulating proinflammatory cytokines, ion channels, scaffold proteins, and transcription factors including TRPV1/4, Nav, BDNF, and NADMR1; consequently, p38 regulates various phenomena including cell communication, remodeling, regeneration, and gene expression. In this review article, we found the most common acupoints for the relief of nervous system disorders including GV20, GV14, ST36, ST37, and LI4. Acupuncture exhibits dual regulatory functions of activating or inhibiting different p38 MAPK pathways, contributing to an overall improvement of clinical symptoms and function in several nervous system diseases.


Introduction
Acupuncture is a form of therapy that has been practiced for more than 3000 years in Asia [1,2]. Medical doctors practice acupuncture under the guidance of meridian theory, which was first recorded in detail in The Yellow Emperor's Classic of Internal Medicine [2]. To perform acupuncture, doctors use thin and sterile metal needles to penetrate specific stimulation points termed acupoints, and they manipulate the needle to achieve "de qi" status [2]. Both manual and electroacupuncture (EA) are used in medical practice. Acupuncture is generally a safe, easy to perform, [3][4][5] and economical procedure that provides another choice for those who are concerned about the adverse effects of routine managements such as drug prescription [6,7]. Occasional minor problems, such as needles left in patients by mistake, headaches, and drowsiness have been reported after acupuncture treatment; usually, these symptoms are self-resolved after a short rest [1, 3,6,7]. Due to the limited number of controlled clinical trials that have been published, the efficacy of acupuncture as treatment for has been questioned [8].
The p38 MAPK family comprises p38α, p38β, p38γ, and p38δ (summarized in Table 1). Considering its central role in developmental programming, cellular adaptation to environmental stress, immune responses, inflammation, tissue regeneration, and tumorigenesis, this protein subfamily has gained increasing attention since its initial discovery. Recent studies using new genetic and pharmacological tools have provided essential information regarding the functions of p38 MAPKs in the pathogenesis of prevalent conditions associated with inflammation, diabetes, neurodegeneration, and cancer .
SAPK: stress-activated protein kinase; CNS: central nervous system; mTORC1: mammalian target of rapamycin complex 1 or mechanistic target of rapamycin complex 1; NAFLD: nonalcoholic fatty liver disease. X-ray crystallographic studies have yielded structural insights into the mechanisms underlying the interaction between an MAPKK and an MAPK [103,107]. Both p38α and p38β are well-conserved at both the gene and protein levels and are important in eliciting innate immunity [77,78,84,[101][102][103][104][105]. Some p38α and p38β physiological substrates are summarized in Figure 3; these include transcription factors, transcription factor kinases, cytoskeletal proteins, translational machinery components, and other proteins including metabolic enzymes, glycogen synthase, or cytosolic phospholipase A2 (cPLA2). The p38γ and p38δ MAPK isoforms can phosphorylate typical p38 MAPK substrates including transcription factors activating transcription factor 2 (ATF2), erythroblast transformation specific (ETS) like-1 protein (Elk-1), or stress-activated protein kinase (SAP1). However, they cannot phosphorylate MK2 or MK3, which are suitable substrates for the other two p38 MAPK isoforms [109][110][111]. These four p38 MAPKs are encoded by different genes and display different histotypic expression patterns, with p38α being ubiquitously expressed at significant levels in most cell types, whereas the others potentially display more histotypic expression patterns. For example, p38β is expressed in the brain, p38γ is expressed in skeletal muscle (neuromuscular junction, gap junction), and p38δ is expressed in endocrine glands [108]. The genetic ablation of specific p38 MAPK family members has revealed functional redundancy in this protein subfamily. For example, the osmotic shock-induced phosphorylation of the stress-activated protein 97 (alternatively termed synapseassociated protein 97, SAP97; also known as discs large homolog 1 scaffold protein, hDLG1) is usually mediated by p38γ; however, in the absence of this kinase, other p38 MAPKs can perform this function.
Of the MAPKs, p38γ has a unique short C-terminal sequence, KETXL, which is ideal for binding PDZ domains in proteins, thus accounting for the regulatory role of p38γ in the localization of cellular elements and interactions with cytoskeletal components. Under stress conditions, p38γ interacts with and activates various scaffold proteins including α1-syntrophin, SAP90/PSD95, and SAP97, which are generally targeted to the plasma membrane cytoskeleton at specialized sites, including the neuromuscular junction and gap junctions through protein-protein interactions [111].
With respect to p38δ, it potentially contributes to cytoskeletal regulation because it phosphorylates the cytoplasmic protein stathmin [110,111], which is a crucial regulator of microtubule dynamics and the cell cycle, by promoting the depolymerization of microtubules and/or preventing the polymerization of tubulin heterodimers [109,111]. Furthermore, eukaryotic elongation factor 2 (eEF2) kinase and microtubule-associated protein tau are substrates of p38δ [109,111]. The four p38 isoforms and the substrates are shown in Figure 3.
These four p38 MAPKs are encoded by different genes and display different histotypic expression patterns, with p38α being ubiquitously expressed at significant levels in most cell types, whereas the others potentially display more histotypic expression patterns. For example, p38β is expressed in the brain, p38γ is expressed in skeletal muscle (neuromuscular junction, gap junction), and p38δ is expressed in endocrine glands [108][109][110][111][112][113][114][115]. The genetic ablation of specific p38 MAPK family members has revealed functional redundancy in this protein subfamily. For example, the osmotic shock-induced phosphorylation of the stress-activated protein 97 (alternatively termed synapse-associated protein 97, SAP97; also known as discs large homolog 1 scaffold protein, hDLG1) is usually mediated by p38γ; however, in the absence of this kinase, other p38 MAPKs can perform this function.

MAPK Substrates, Signaling Pathways, and Functions
Nine members of the dual-specificity phosphatases specific for MAPKs, termed MKPs, have been reported. Each member has specific substrates, tissue distribution, and subcellular localization. For example, MAPK phosphatase 7 (MKP-7) binds to and inactivates p38α and p38β through dephosphorylation; however, it does not interact with p38γ and p38δ; MKP-5 and CL100/MKP-1 also bind to p38α and p38β, but not to p38γ or p38δ [115] (Figures 2 and 3).
Several studies have revealed a direct interaction of the N-terminal region of the MAPKK with a docking groove present on the surface of the MAPK distant from the catalytic active site. The second determinant of MAPKK specificity is the structure of the MAPK activation loop harboring the Thr-Xxx-Tyr dual-phosphorylation motif. The specificity of these interactions partly mediates the potential of an individual MAPKK to selectively activate a particular MAPK.
A recent study emphasized that dynamic changes are necessary for enzyme activity [116]. For instance, two MAPKs, ERK2 and p38, are differentially activated owing to differences in dynamism. A comparison of the dynamics of PKA and p38 revealed similarities in their dynamic properties.

Dual Phosphorylation by MKKs
In yeast, only a single MAPKK appears to activate each MAPK, whereas mammalian MAPK signaling modules include more than one MAPKK. The MAPKKs responsible for activating the p38 MAPK pathways appear to be specific to cell type and stimulus [105,107,114,116]. Brancho and Tanaka et al. investigated the mechanism underlying p38 activation in vivo by examining the effect of disruption of the murine Mkk3, Mkk4, and Mkk6 genes on the p38 MAPK signaling pathway [100]; they found that Mkk3 and Mkk6 are essential for tumor necrosis factor (TNF)-mediated p38 activation. By contrast, ultraviolet (UV) radiation-induced p38 activation was mediated by Mkk3, Mkk4, and Mkk6. Furthermore, they reported that the role of Mkk4 in p38 MAPK activation in fibroblasts was largely redundant with those of Mkk3 and Mkk6. Mkk4 is a potentially important p38 MAPK activator in cells with low levels of Mkk3 and Mkk6. These data indicated that p38 MAPK was regulated by the coordinated and selective action of the three different protein kinases MKK3, MKK4, and MKK6 in response to cytokines and exposure to environmental stress. The inactivation of p38 MAPK was reportedly associated with defects in cell cycle arrest and increased tumorigenesis [117][118][119][120][121][122][123].

Autophosphorylation
MKK-independent activation is achieved through autophosphorylation and activation of p38α after interaction with TGFβ-activated kinase 1 (TAB1), which appears to activate p38α via the 5' AMP-activated protein kinase (AMPK) in ischemic heart tissue ( Figure 5). TAB1 sequesters p38α in the cytosol, thus potentially preventing some MKK-activated p38α functions. However, this mechanism does not contribute to p38 MAPK activation in fibroblasts or epithelial cells under the same conditions [134].
activating transcription factor; MSK: mitogen-and stress-activated protein kinase; CREB: cAMP response element-binding protein; AP-1: activator protein 1; MEF: myocyte enhancing factor; CHOP: C/EBP homologous protein, a member of the CCAAT/enhancer-binding proteins; Elk-1: erythroblast transformation specific (ETS) like-1 protein; Stat1: signal transducer and activator of transcription 1; Pax6: a paired-box protein encoded by the master gene Pax-6; Myc: a group of transcription factors coded by a regulator genes and proto-oncogene called Myc; Max: a transcription factor coded by the myc-associated factor X.
Another MKK-independent mechanism underlying p38α activation has been observed in T-cell stimulation, wherein p38α is activated by T-cell antigen receptor (TCR)-mediated stimulation through p38α phosphorylation on a noncanonical activating residue, Tyr323. This activated p38α alters its structural conformation, phosphorylating third-party substrates and its TGY motif [115].

TRPV1 and the p38 Pathway
Transient receptor potential vanilloid receptors (TRPVs) are mechanosensitive channels highly associated with nervous system functions including pain, memory, and mechanical sensations. Furthermore, capsaicin receptor TRPV1 is a key regulator of pain and inflammation and is upregulated in microglia in the brain, especially in the anterior cingulate cortex [138][139][140]. The stimulation of microglial TRPV1 induces cortical microglial activation and indirectly enhances glutamatergic neuronal transmission by promoting the shedding of extracellular microglial microvesicles (release of vesicles in the extracellular space [141]). Moreover, in the cortex of mice with neuropathic pain, TRPV1 affects the intrinsic electrical properties of neurons and synaptic strength [142,143]. This signal transduction can be inhibited by the p38 MAPK inhibitor SB203580. Thus, p38 MAPK is a downstream TRPV1 activator whose phosphorylation plays an essential role in microglial microvesicle shedding by activating P2X purinoceptor 7 (P2X7) ATP receptor [138][139][140][141][142][143]. Whereas TRPV1 mediates communication between microglia and neurons, inhibition of the phosphorylation of its downstream p38 MAPK inhibits sphingosine metabolism [140][141][142][143].
Furthermore, several studies have examined biomarkers of nerve damage, including astrocytic marker glial fibrillary acidic protein (GFAP), microglial marker ionized calcium-binding adapter molecule 1 (Iba-1), S100 calcium-binding protein B (S100B), and the receptor for advanced glycation end-products (RAGE), revealing marked upregulation of these molecules in the dorsal root ganglion (DRG) and spinal cord dorsal horn (SCDH) of Complete Freund's adjuvant (CFA)-treated mice. This inflammatory effect was reversed through electroacupuncture, which achieved an equivalent result to that of TRV1 gene deletion [143].

Brain-Derived Neurotrophic Factor and p38 Pathways
Microglia cells are resident macrophages in the central nervous system (CNS) with a small soma with thin and branched processes. In the case of neural injury, their processes rapidly migrate toward the site of injury. In peripheral nerve injury, spinal cord microglia are activated. Spinal P2X4 receptors (P2X4Rs), phosphorylated p38 MAPK (p-p38-MAPK), and brain-derived neurotrophic factor (BDNF) are upregulated in spared nerve injury (SNI) rats [144]. BDNF signals to neurons in spinal lamina I and increases intracellular chloride concentration, thereby counteracting gamma-aminobutyric acid (GABA)-and glycine-mediated inhibition in these cells. The disinhibition unmasks innocuous inputs to lamina I neurons and facilitates their responses to noxious inputs [145]. In other studies, BDNF/TrkB was reported to promote inflammation in spinal cord injury through the p38 signaling pathway in the murine model [40,146]. Moreover, an upregulation of BDNF expression was observed in rats when applying electroacupuncture (EA) to acupoints GV20 and GV14, which later revealed that EA counteract caspase-3-dependent neuronal apoptosis by activating the Raf-1/MEK1/2/ERK1/2/p90RSK/Bad signaling pathway [147].
These results suggest that increasing the expression of BDNF synthesis, the downregulation of P2X4Rs, and the inhibition of p38 phosphorylation might account for the therapeutic effects induced by acupuncture in nerves injury.

Acupuncture and the Effects of Electric Fields on Nerve Regeneration
Previous in vitro and a few in vivo studies have reported positive nerve growth-promoting effects of electric fields where the cathode was placed toward the distal end of the injured nerve stumps [148][149][150][151][152][153][154]. Chen et al. reported that regenerated nerves in the electrical stimulation group may have a more mature ultrastructure compared with those in the control group [155]. Furthermore, they reported low regeneration success in patients receiving electrical stimulation relative to controls [155].
Together with several previous clinical studies [155][156][157][158], these studies show that patients with nerve anastomosis should not receive any electrical stimulation for rehabilitation until their reconnected nerve stumps have grown into a more mature stage of regeneration. One of the mechanisms underlying electroacupuncture (EA) is the relief of acute pain through the release of opiates to activate µ-, δ-, and κ-opioid receptors; furthermore, it can regulate persistent pain by activating µand δ-opioid receptors [25]. These findings explain the application of EA in treating neuropathic pain when pharmacotherapeutic approaches are ineffective.
Furthermore, the analgesic effect of low-frequency (2 Hz) EA is exerted on the noradrenergic descending pathway, involving the modulation of spinal GABAergic nerves (GABA A ). Administering EA at 2 Hz at acupoint ST36-37 reportedly relieved neuropathic pain through long-term depression (LTD) of the C-fiber [159]. This phenomenon could be blocked by N-methyl-D-aspartate (NMDA) and an opioid receptor antagonist. By contrast, high-frequency (100 Hz) EA induces long-term potentiation (LTP) of endogenous GABAergic (GABA B ) and the serotonergic inhibitory systems through modulation of µ-opioid and 5-hydroxytryptamine 1 (5-HT1) receptors [25,160]. Other studies have reported that delivering EA at acupoints GV14 and GV20 exerts neuroprotective effects by activating CREB, BDNF [161], and α7 nicotinic acetylcholine receptor (α7 nAChR) [162] while simultaneously reducing S100B-mediated neurotoxicity [163]. Moreover, EA at acupoint ST36 can evoke excitatory signals in either the peripheral nervous system or CNS in vivo [163].

Neuropathic Pain
In contrast to physiological pain, pathological pain does not depend on the presence of tissuedamaging stimuli. Neuropathic pain can be agonizing, potentially persisting over long periods, and it is often resistant to known painkillers. Increasing evidence indicates that spinal microglia react and undergo a series of changes that directly influence the establishment of painful peripheral neuropathy [168][169][170]. After nerve damage, purinergic P2X4 receptors (nonselective cation channels activated by extracellular ATP) are upregulated in spinal microglia in a manner dependent on transcription factors interferon regulatory factor 8 and 5 (IRF8 and IRF5), both of which are expressed in microglia after peripheral nerve injury [171,172]. Furthermore, in spinal microglia, the response to extracellular stimuli results in signal transduction through intracellular signaling cascades involving p38 and ERK. Inhibition of the function or expression of these microglial molecules suppresses the aberrant excitability of dorsal horn neurons and neuropathic pain [172].
Since MAPKs significantly contribute to the development of hyperalgesia, the inhibition of any of the three pathways, namely the ERK, p38, and JNK pathways, can rescue inflammatory or neuropathic pain. Several studies have reported that 2-and 15-Hz EA can downregulate cerebral TRPV4 expression and attenuate chronic constriction injury (CCI)-induced neuropathic pain in an animal model [173][174][175][176][177][178][179]. Furthermore, Huang et al. reported that EA modulates both excitatory and inhibitory neurotransmitters to relieve neuropathic pain in the higher brain regions [175]. The hippocampus plays an integral role in the transition from acute to chronic pain in the limbic system by activating NMDA receptors and subsequently prolonging acute nociceptive stimuli that continue activating the descending pathways of pain ( Figure 6). surgery [176,177]. Numerous prospective randomized controlled trials on carpal tunnel syndrome (CTS) have reported that acupuncture at acupoints PC7 and PC6 is as effective and safe as steroid treatment of mild-to-moderate CTS, both in the acute phase and throughout the 1-year follow-up period [187,188]. Based on the analgesic mechanisms of acupuncture and EA, which attenuate the p-p38 signaling pathway, the long-term effect of acupuncture or EA may result from neural modulation through posttranscriptional regulation. The potential mechanism underlying EA-mediated analgesic effects is depicted in Figure 6. The underlying mechanisms and the primary results of these studies are summarized in Table 2. Figure 6. Schematic of the potential mechanism underlying the electroacupuncture (EA)-mediated analgesic effect. Noxious stimuli including heat or proinflammatory cytokines are transduced by the Figure 6. Schematic of the potential mechanism underlying the electroacupuncture (EA)-mediated analgesic effect. Noxious stimuli including heat or proinflammatory cytokines are transduced by the transient receptor potential vanilloid 1 (TRPV1) into the cells, thus phosphorylating and activating p38, which translocates from the cytosol to the nucleus and promotes transcription by affecting cAMP response element-binding protein (CREB), thus upregulating specific proteins including inflammatory cytokines or inducing apoptosis. These noxious stimuli can also directly activate protein kinase C (PKC). Nociception regulation by TRPV1 and Na v 1.7/1.8 receptor stimulation in the peripheral nerves propagates the signal through the ascending pathway and upregulates proinflammatory cytokines including cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNFα) in the central nervous system, and the signal is then perceived as "pain". Chronic pain re-emerges when nociceptive signals cause prolonged stimulation through hippocampal N-methyl-D-aspartate receptor (NMDA) receptors and some other areas of the limbic system, and the signals are transmitted through the descending pathway, stimulating TRPV4 and triggering pain. Endogenic analgesic mechanisms involve the release of opioidergic substances that bind to γ-aminobutyric acidergic (GABAergic) receptors and µor δ-opioid receptors that act locally in the central nervous system and inhibit the descending pain pathway. EA exerts therapeutic effects by inhibiting the ascending pain pathway and intracellular p38-mediated inflammatory pathway by stimulating peripheral opioid receptors. Furthermore, EA promotes endogenic analgesic mechanisms, thus exerting immediate local analgesic effects and rescuing CNS-induced chronic pain.

Post-Operation Pain
Presurgical sham-, low-, and high-frequency EA treatments were reported to significantly reduce the postoperative patient-controlled analgesia morphine requirement; moreover, both low-and high-frequency EA decreased opioid-related side effects including nausea and dizziness throughout the first postoperative day [176,177]. Among these studies, ST36 and LI4 were the most frequent acupoints manipulated to achieve analgesic effects [summarized in Table 2].    Furthermore, overexpression of the voltage-gated sodium channel (Na v ), TRPV1, and acid-sensing ion channel 3 (ASIC3) in DRG neurons in response to proinflammatory cytokines is a key component of inflammatory pain [179][180][181][182][183][184][185][186][187][188][189][190][191][192]. Low-frequency (2-Hz) EA at acupoint ST36 can reduce inflammatory pain by attenuating ASIC3 overexpression in peripheral DRG neurons. ASIC3 downregulation potentially prolongs the clinical benefits of EA. Goldman et al. reported that acupuncture stimulates adenosine A1 receptor and induces adenosine release, thus relieving inflammation and neuropathic pain, which was not observed in mice lacking adenosine A1 receptors [193]. These results suggest that the opioid and adenosine pathways potentially contribute to analgesia in both manual acupuncture and EA.

Migraine
Migraine is a complex disorder; each episode begins with prodromes and aura (transient focal neurological symptoms). The origin of recurrent headache accompanied by visual or sensory symptoms is speculated to involve the hypothalamus, brain stem, and cortex. Current theories suggest that migraine is a neurovascular disorder involving cortical spreading depression, neurogenic inflammation, and vasodilation [194,195]. Owing to the disease itself or its genetic underpinnings, the brains of individuals who experience migraine are altered structurally and functionally; these molecular, anatomical, and functional abnormalities provide a neuronal substrate for extreme sensitivity to fluctuations in homeostasis, decreased adaptability, and recurrent headaches [194].
A randomized controlled trial investigated the efficacy and tolerability of acupuncture in comparison with topiramate treatment in chronic migraine prophylaxis. The effectiveness of acupuncture was similar to or greater than that of prophylactic pharmacotherapy, with fewer side effects in migraine [194]. The selected acupoints were bilateral BL2, GB20, EX-HN5, and EX-HN3, which are all associated with the trigeminal and cervical dermatomes. Although different combinations of peripheral effects, spinal/supraspinal mechanisms, and cortical and psychological mechanisms potentially contribute to the clinical effects of acupuncture, several other studies have reported that acupuncture potentially exerts curative effects not only through local analgesia but also through anti-inflammation, neuropeptide regulation, cytoskeleton remodeling, cell repair, and improvement in overall homeostasis.

Transcutaneous Electrical Nerve Stimulation Versus EA
The difference between transcutaneous electrical nerve stimulation (TENS) and EA is that TENS involves the application of the cathode at the affected area rather than at acupoints. Furthermore, TENS is used to reduce mechanical hyperalgesia in knee joint inflammatory pain [196,197], although its effectiveness is only sustained for a short time (several days) [198,199]. The analgesic effect of low-frequency TENS and EA can be attenuated using µ-opioid receptor blockers, thus indicating the involvement of peripheral opiate release in the underlying mechanism [196]; furthermore, the blockade of β-endorphin and corticotropin-releasing factor also reduces EA-induced analgesia.

Hypothesis Regarding the Long-Term Effect of Acupuncture
Although proinflammatory cytokines are regulated at different levels, the role of the p38α MAPK pathway in posttranscriptional regulation has received increasing attention. Numerous p38 MAPK-regulated mRNAs often contain an adenylate-uridylate-rich element (termed AU-rich element or ARE) in the 3 untranslated region (3 UTR) [200,201]. The regulation of mRNA stability is particularly important for the expression of proteins involved in inflammatory response. These elements target mRNAs for rapid decay. The decay of ARE-containing reporter mRNAs is blocked upon p38 MAPK activation. Numerous ARE-containing endogenous mRNAs of inflammatory proteins are destabilized upon p38 inhibition. Some studies have reported a common mechanism underlying gene regulation via the p38α MAPK pathway for posttranscriptional regulation by the ARE.
These results are consistent with those of several previous animal and clinical studies reporting that EA improves analgesia during the acute phase posttraumatic healing and pain after major surgery [176,177]. Numerous prospective randomized controlled trials on carpal tunnel syndrome (CTS) have reported that acupuncture at acupoints PC7 and PC6 is as effective and safe as steroid treatment of mild-to-moderate CTS, both in the acute phase and throughout the 1-year follow-up period [187,188]. Based on the analgesic mechanisms of acupuncture and EA, which attenuate the p-p38 signaling pathway, the long-term effect of acupuncture or EA may result from neural modulation through posttranscriptional regulation. The potential mechanism underlying EA-mediated analgesic effects is depicted in Figure 6. The underlying mechanisms and the primary results of these studies are summarized in Table 2.

Cerebral Ischemia
During a cerebral ischemia (CI) event, hypoxia is induced in the brain, followed by the upregulation of various cytokines and potentially neurotoxic molecules. Nitric oxide reportedly influences cerebral oxygen vasoreactivity during severe hypoxia [202]. Both manual acupuncture and EA can increase cerebral blood flow [13,14]. Furthermore, 2-and 15-Hz EA at acupoint ST36 increased cerebral blood fluid (CBF) in rats with and without CI [14]. Per the traditional Chinese medicine theory, the Governor Vessel (or the Governor Meridian) directly communicates with the brain. In a previous study, EA at acupoints GV20 and GV14 before artificial mild cerebral ischemia-reperfusion (I/R) injury in an animal model exerted a neuroprotective effect against reperfusion injury [165]. Another neuroprotective mechanism of EA involves the downregulation of astrocytic S100B, which in turn deactivates p38 MAPK and its downstream substrate NF-κB. These effects subsequently reduce oxidative/nitrative stress and inhibit the TNF-α/tumor necrosis factor receptor type 1-associated DEATH domain protein (TRADD)/ Fas-associated protein with death domain (FADD)/cleaved caspase-8/cleaved caspase-3 apoptotic pathway in the ischemic cortical penumbra after reperfusion [89,[201][202][203][204][205][206][207]. Both 5-and 25-Hz EA at acupoints GV20 and GV16 effectively downregulated reactive astrocytosis to exert neuroprotective effects against cerebral infarction-induced neuronal apoptosis, probably by activating the p38 MAPK/CREB signaling pathway [203].
Heat shock proteins (Hsp) significantly contribute to cellular regenerative processes in injured tissues because Hsp is upregulated during stem cell differentiation; however, the depletion or inhibition of Hsp70/Hsc70 impairs this process [206]. Hsp27 and Hsp70 reportedly contribute to intracellular protein repair after acute CI [206][207][208][209][210][211]. The dual phosphorylation of p38 MAPK was increased through early ischemia in an in vitro study wherein Hsp27 served as a terminal substrate of the p38 MAPK cascade. A later study indicated that among the p38 MAPKs, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at Ser326 (S326) in cells (phosphorylation at S326 is a hallmark for HSF1 activation) [212], thus contributing to mitotic progression. A protease-mass spectrometry approach unexpectedly revealed that p38 MAPK also catalyzes the phosphorylation of HSF1 at S303/307, which is a repressive posttranslational modification [212,213]. Hsp70 bound MK2 to regulate MK2-p38MAPK interaction in the stem cells, and the essential regions required for Hsp70-MK2 interaction have been identified [206,[209][210][211][212][213]. Taken together, Hsp can regulate cell differentiation by interacting with MK2 to stabilize p38 MAPK.
Another study administered 2 Hz EA the GV20 and ST36 acupoints in rat models of cerebral I/R injury and observed lowered peak levels of adrenocorticotrophic hormone and HSP70, suggesting that EA may inhibit excessive stress, reduce inflammation, and promote neural repair, thus facilitating healing in ischemic stroke [211]. The mechanisms and main results of the identified articles are summarized in Table 3.
EA significantly reduced the cerebral infarct area and downregulated astrocytic S100B expression and decreased p-p38 NF-kB.

Epilepsy and Seizure
Epilepsy is characterized by an abnormal electric discharge of the brain, especially in the hippocampus. Epilepsy is usually associated with neuronal loss, astrocyte proliferation, mossy fiber sprouting, and synaptic reorganization in the hippocampus [40,[214][215][216][217][218][219]. Pharmacological treatment approaches, including those employing Western medicine and Chinese herbal medicine, have been attempted to treat or prevent the harmful effect of seizures. For example, Uncaria rhynchophylla is a herb used in traditional Chinese medicine for epilepsy management; its therapeutic effect was reportedly associated with attenuated mossy fiber sprouting, astrocyte proliferation, S100B protein overexpression, and increased hippocampal neuronal survival [219].
Acupuncture has been applied for clinically managing epilepsy. Previous studies have reported that auricular acupuncture positively influences drug-resistant epilepsy patients, potentially through vagus nerve stimulation, attenuating the hyperactive hippocampus, regulating inflammatory cytokine pathways, protecting the hippocampus from apoptosis, and ameliorating the sprouting of mossy fibers in the hippocampus [40,218,220,221]. Several antiepileptic mechanisms have been proposed, including the downregulation of JNK or ERK1/2 and pro-inflammatory factors (IL-1β, IL-6, TNF-α) in the cerebral cortex and hippocampus [216]. Another study suggested that auricular EA and EA at ST36-ST37 achieved therapeutic effects by reducing hippocampal hyperactivity and the transient receptor potential cation channel subfamily A member 1 (TRPA1), PKCε, PKCα, and pERK1/2 signaling pathways [40]. Moreover, auricular EA with EA at ST36-ST37 exerted long-term (6-week observation in the study) beneficial effects by reducing the anti-inflammatory response in numerous cyclooxygenase-2 (COX-2) immunoreactive cells and hippocampal COX-2 levels [218].
Acupuncture exerts antiepileptic effects by inducing anatomical changes and changes in neurotransmitter, inflammatory cytokine, and transcription factor levels. Regarding studies to date, the related mechanisms primarily involve the suppression of TRPA1/pERK and TLR4/ERK pathways and activation of the PI3K/Akt pathway. Although some studies have reported the inactivation of the TLR4 pathway, accompanied by a reduction in pCaMKIIα, pERK, pp38, pJNK, and pNFκB levels [40], the role of p38 and its association with the aforementioned signaling pathways remain unclear. These mechanisms and the primary results of the aforementioned studies are summarized in Table 4.

Motion Sickness
External noxious stimulation alters intracellular signal transduction pathways in primary afferents, potentially contributing to pain hypersensitivity. This pathway proceeds via TRPVs, especially TRPV1 and TRPV4, leading to the rapid phosphorylation of p38 MAPK in the afferent neurons and the induction of hyperalgesia. Recent studies have revealed that thermal stimulation may not be the only trigger for TRPV activation [222,223]. Kaolin consumption was quantified as a behavioral response of an emetic reflex (pica behavior) in a murine model. A previous study reported that TRPV1 levels were significantly increased upon stimulation of motion sickness, and EA at PC6 acupoints attenuated hypothalamic TRPV1 levels and exerted an antiemetic effect [222]. The acupoint PC6 is well-known for its therapeutic effect, especially in relieving nausea and vomiting. This study revealed that the mechanism underlying the reduction of motion sickness potentially involves the TRPV1 pathway [222].

Degenerative Nerve Diseases
Microglia, the endogenous macrophages of the CNS, monitor their territory through a constant movement of their elaborated thin processes and responses to local stressors and immune disruptions. Clinical studies and preclinical animal models have implicated the dysregulation and overproduction of proinflammatory cytokines from activated microglia in the CNS as contributors to the pathophysiology of chronic neurodegenerative disorders including Alzheimer's disease [16], Parkinson's disease, and multiple sclerosis and acute neurodegenerative conditions including traumatic brain injury and stroke. The p38α MAPK pathway helps increase the microglial production of proinflammatory cytokines induced by diverse stressors [16,17,223]. These results indicate the feasibility of targeting p38α MAPK to modulate the production of pro-inflammatory cytokines by the CNS.
Few studies have focused on the effects of androgens on neuroinflammation. Yang et al. investigated the neuroprotective role of androgens (including testosterone and its metabolite dihydrotestosterone, DHT) in lipopolysaccharide (LPS)-induced neuroinflammation, neuronal damage, and behavioral dysfunction [18]. DHT potentially inhibits the LPS-induced release of proinflammatory factors in primary microglia by suppressing the TLR4-mediated NF-κB and p38 signaling pathways, thus protecting neurons from inflammatory damage induced by activated microglia.
Sodium ferulate is used in traditional Chinese medicine (such as the root of Ligusticum chuanxiong and Angelica sinensis) and is speculated to help treat cardiovascular and cerebrovascular diseases by preventing thrombosis. Furthermore, sodium ferulate prevents Aβ-induced MKK3/MKK6-p38-Hsp27 signaling and reduces apoptosis in the rat hippocampus [17].

Alzheimer's Disease
The pathological hallmarks of Alzheimer's disease (AD) are the accumulation of extracellular plaques and intracellular neurofibrillary tangles that are composed of filaments of β-amyloid polymers and the neuronal microtubule-associated protein Tau, respectively. Elevated β-amyloid levels in an AD brain are speculated to induce microglial activation and the consequent release of proinflammatory cytokines induced by the p38 MAPK pathway, potentially contributing to AD pathogenesis together with other disorders including neuronal injury, trauma, ischemia, and the accumulation of oxidants associated with brain aging [16,18,224].
Since more than half of the phosphorylation sites in paired helical filament-Tau are serine and threonine residues followed by proline, members of the MAPK family, especially p38α, potentially play an important role in phosphorylating Tau [16]. Considering that aberrantly activated JNK and p38 MAPKs are reportedly associated with cells containing filamentous Tau in some neurodegenerative diseases, these kinases may contribute to Tau hyperphosphorylation. Moreover, the p38 MAPK activator, MKK6, is active in neurodegenerative diseases, indicating a potential contribution to Tau hyperphosphorylation in these diseases. Recent studies have reported that Tau is a suitable in vitro substrate for p38 isoforms p38δ and p38γ, and its phosphorylation by these two enzymes reduces its ability to promote microtubule assembly [18,[225][226][227][228]. Moreover, p38γ overexpression in neuroblastoma induces Tau phosphorylation, which is associated with a reduction in Tau that is associated with the cytoskeleton and an increase in soluble Tau. Furthermore, p38δ is the major Tau kinase in neuroblastoma in response to osmotic shock [227]. This evidence indicates that p38 MAPKs potentially regulate Tau hyperphosphorylation in neurodegenerative diseases and are potentially suitable therapeutic targets for those diseases.
Other studies have focused on the receptor for advanced glycation end products (RAGE), a multiligand member of the immunoglobulin superfamily of cell surface molecules, which serves as a receptor for Aβ on neurons, microglia, astrocytes, and endothelial cells of blood vessels. RAGE is upregulated in brain regions affected by AD. A murine transgenic model revealed that RAGE is a potential therapeutic target for AD [229].
Several studies have reported that EA and manual acupuncture have a therapeutic effect on AD. For example, EA at acupoint GV20 suppresses Aβ generation [228]. Stimulation at acupoints GV14 and BL23 downregulates beta-secretase 1, which is an enzyme that is responsible for Aβ generation in AD and increases hippocampal ATP levels in AD mice [230]. Low-frequency (2-Hz) EA at acupoint GV20 and bilateral acupoints KI3 and ST36 for 15 min once daily for 12 sessions was reported to significantly downregulate p-p38 MAPK and IL-1β mRNA expression in the hippocampus and the frontal lobe [65]. Other studies have reported that EA at GV20 reversed behavioral deficit and LTP impairment, possibly through the N-methyl-D-aspartate receptor 1 (NMDA-R1) and TRPV1 pathway, thus reversing NR1-(one of the NMDA receptor subunits) and TRPV1-mediated neurotoxicity in vascular dementia [208,209]. This implies that 2-Hz EA can rescue p38 MAPK signal transduction, resulting in anti-apoptotic and anti-inflammatory effects that reduce Aβ deposits in the brain and improve learning and memory in AD patients [20].

Parkinson's Disease
Parkinson's disease is a neurodegenerative disorder that causes severe motor impairment owing to loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Several studies have focused on PD pathophysiology and reported potential therapeutic targets for PD.
One of the direct neuroprotective effects is exerted by the protein deglycase DJ-1, which inhibits ASK1 through the nuclear sequestration of death domain-associated protein (Daxx) [230][231][232]. DJ-1 is speculated to scavenge reactive oxidative species (ROS) and subsequently attenuate oxidative stress, thereby reducing cellular ROS burden [233,234]. Furthermore, DJ-1 interacts with p38-regulated/activated kinase (PRAK/MK5) under cellular oxidative stress to facilitate nuclear localization of DJ-1 [235]. Thus, DJ-1 co-localizes with PRAK in the nuclei of NIH3T3 cells. Since DJ-1 lacks both nuclear localization and nuclear export signals, PRAK may be a crucial factor assisting DJ-1 in regulating the cellular localization of Daxx and in ASK1 signaling and concomitant cell death [236].
A recent study reported that EA at acupoint KI3 reduced the excitotoxicity of dopaminergic neurons by regulating the NMDA receptor function and thus may potentially serve as a novel therapeutic approach for PD [237]. The levels of pNR1 and pNR2B, phosphorylated PKA, PKC, α-Ca2+/CaMKII, pERK, and CREB were also reduced following EA [237].

Fibromyalgia
Fibromyalgia is a common chronic pain syndrome characterized by chronic widespread mechanical pain. TRPV1 and TRPV4 are believed to play a crucial role in the pathophysiology of fibromyalgia. EA at 2 Hz at bilateral ST36 acupoints was reported to reduce long-lasting mechanical hyperalgesia by downregulating TRPV4, p-p38, p-JNK, and pCREB in the peripheral nervous system and CNS [238,239].
Taken together, acupuncture exerts anti-inflammation, anti-apoptosis, and neuroprotection effects via modulating proinflammatory cytokines, increasing the levels of neurotrophic factors and modulating signaling pathways, such as p38 MAPKs, Raf/MAPK/ERK1/2, TLR4/ERK, PI3K/AKT, AC/cAMP/PKA, ASK1-JNK/p38, and downstream CREB, JNK, m-TOR, NF-κB, and Bcl-2/Bax balance. EA relieves acute pain through inhibiting the TRPV1 signaling and PI3K/PKC/CaMKII pathway. EA also promote the release of opiates to activate µ-, δ-, and κ-opioid receptors, therefore producing immediate and persistent analgesic effects. Furthermore, EA can downregulate TRPV1-mediated inflammation and the release of proinflammatory cytokines, GFAP, Iba-1, and S100B by inhibiting the phosphorylation of p38 MAPK, therefore inhibiting microglial activation that triggers nerve damage and pain. Combining acupuncture to the treatment of a cerebral ischemic/hemorrhagic event can have a positive effect; an upregulation of BDNF synthesis, downregulation of P2X4Rs, and inhibition of p38 phosphorylation leads to the activation of the Raf-1/MEK1/2/ERK1/2/p90RSK/Bad signaling pathway, which contributes to the downregulation of caspase-3-dependent neuronal apoptosis. Acupuncture also inhibits the TNF-α/TRADD/FADD/cleaved caspase-8/cleaved caspase-3 apoptotic pathway in the ischemic cortical penumbra after reperfusion. In the case of treating epilepsy and seizure, several antiepileptic mechanisms have been proposed, including the downregulation of JNK or ERK1/2 and proinflammatory factors (IL-1β, IL-6, TNF-α) in the cerebral cortex and hippocampus. Auricular EA can reduce hippocampal hyperactivity through TRPA1 and PKCε/PKCα/pERK1/2 signaling pathways. Furthermore, acupuncture can regulate p38γ and p38δ and reduce Tau hyperphosphorylation in neurodegenerative diseases. Acupuncture also exhibits a therapeutic effect that was reported to involve the regulation of DJ-1, ASK1, Daxx, phosphorylated PKA, PKC, α-Ca2+/CaMKII, pERK, and CREB. Acupuncture exerts curative effects in migraine through local analgesia, anti-inflammation, neuropeptide regulation, cytoskeleton remodeling, cell repair, and improvement in overall homeostasis. Furthermore, the downregulation of TRPV4, p-p38, p-JNK, and pCREB in the peripheral nervous system and CNS may account for the therapeutic effect of acupuncture in fibromyalgia.

Conclusions
Among the aforementioned cell pathways, the p38 MAPK signaling pathway plays a role in the therapeutic effect of acupuncture in several nervous system diseases. EA inhibits the ascending pain pathway and intracellular p38-mediated inflammatory pathway by stimulating peripheral opioid receptors. Furthermore, EA promotes endogenic analgesic mechanisms, thus exerting immediate local analgesic effects and rescuing CNS-induced chronic pain. Acupuncture also counteracts p38 MAPK signal transduction, reduces Aβ deposits in the brain, and improves learning and memory in AD patients.
Acupuncture exhibits a dual regulatory function through the activation or inhibition of different p38 MAPK pathways and contributes to an overall improvement of clinical symptoms and physiological functions in several nervous system diseases. The p38 MAPK pathway potentially induces both protective and inhibitory effects in highly similar systems. Further studies are required to identify and characterize the various substrates of these kinases involved in cell differentiation/cell repair, neurotoxicity, and neurodegeneration, thus potentially furthering the current understanding of the mechanism underlying acupuncture in treating these diseases.

Conflicts of Interest:
The authors declare no conflicts of interest.

A1R
adenosine A1 receptor α7 nAChR α7 nicotinic acetylcholine receptor AC adenyl cyclase ACC1 acetyl-CoA carboxylase (ACC) 1 is a biotin-dependent enzyme that catalyzes the irreversible carboxylation of acetyl-CoA to produce malonyl-CoA AD Alzheimer's disease Akt (PKB) protein kinase B (PKB) is also known as Akt Alk5 TGFβ type I receptor kinase AMP adenosine mono-phosphate AMPK 5' AMP-activated protein kinase AP-1 activator protein 1 ARE AU-rich elements ASIC3 acid-sensing ion channel 3 ASK-1 apoptosis signal-regulating kinase 1 ATF activating transcription factor ATP adenosine tri-phosphate BACE1 beta-secretase 1, an enzyme responsible for Aβ generation in Alzheimer's disease Bad BCL2 associated agonist of cell death; a protein involved in initiating apoptosis. Bax Bcl-2 associated X Bac-2 B-cell lymphoma 2 BDNF brain-derived neurotrophic factor CaMKII calmodulin-dependent protein kinase II CCI chronic constriction injury Cd242 The 40-to 42-kiloDalton red cell membrane glycoprotein bearing the ICAM-4 antigen named by the LW blood system. CFA-treated Complete Freund's adjuvant injections produced significant mechanical and thermal hyperalgesia in mice. c-Fos A proto-oncogene that is expressed within some neurons following depolarization. CHOP C/EBP homologous protein; belongs to the family of CCAAT/enhancer-binding proteins (C/EBPs) and is involved in the regulation of genes that encode proteins involved in proliferation c-Jun A proto-oncogene that with c-Fos forms the AP-1 early response transcription factor that regulates gene expression in response to extracellular stimuli.

CL100
The human CL100 gene is induced in skin fibroblasts in response to oxidative/heat stress and growth factors. Interleukin 1 beta is a member of the interleukin 1 family of cytokines produced by activated macrophages. IRF interferon regulatory factor JNK c-Jun amino-terminal kinases LTD long-term depression; a term in neurophysiology describing an activity-dependent reduction in the efficacy of neuronal synapses lasting hours or longer following a long patterned stimulus. LTP long-term potentiation; a persistent strengthening of synapses based on recent patterns of activity. These are patterns of synaptic activity that produce a long-lasting increase in signal transmission between two neurons. Paired-box protein Pax-6, also known as aniridia type II protein (AN2) or oculorhombin; a "master control" gene for the development of eyes and other sensory organs P2RX7 P2X purinoceptor 7 is a protein belonging to the family of purinoceptors for ATP. P2X4Rs P2X4 receptors are a subtype of ionotropic ATP receptors P2Y1, -2 human purinergic G protein-coupled receptors PD Parkinson's disease Pellino A group of proteins extremely well conserved during evolution; Pellinos interact with key mediators in TLR/IL-1R-induced signaling pathways. PHF-tau paired helical filament-tau protein PI3K phosphoinositide 3-kinases; also called phosphatidylinositol 3-kinases PKC protein kinase C PSD postsynaptic density protein PRAK p38-regulated and -activated kinase Rac2 Ras-related C3 botulinum toxin substrate 2 RAF rapidly accelerated fibrosarcoma-related oncogene Raf1 v-raf-1 murine leukemia viral oncogene homolog 1 RAGE The receptor for advanced glycation end products is a member of the immunoglobulin superfamily of cell surface molecules. RCT study randomized controlled trial study Rel A proto-oncogene protein encoded by the REL gene. It is a member of the NF-κB family of transcription factors and contains a Rel homology domain (RHD) at its N-terminus and two C-terminal transactivation domains. Rho Rho (ρ) factor is a protein that acts in bacterial cells to mediate termination of transcription at distinct sites, which mediates the dissociation of the RNA from the very stable ternary transcription complex. Rit Ras-like protein in tissues RIP receptor-interacting protein kinases; a class of serine/threonine protein kinases ROS reactive oxidative species S100B S100 calcium-binding protein B SAP stress-activated protein or synapse-associated protein; SAP90 [(synapse-associated protein 90 is also known as PSD-95 (postsynaptic density-95)] SAPK stress-activated protein kinase Schematic of the literature review search process Figure A1. Schematic of the literature review search process.