Electroacupuncture alters cannabinoid receptor 1 and transient receptor potential V1 expression on individual brain regions in a mouse fibromyalgia model

doi:10.21203/rs.3.rs-4653222/v1

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Electroacupuncture alters cannabinoid receptor 1 and transient receptor potential V1 expression on individual brain regions in a mouse fibromyalgia model

https://doi.org/10.21203/rs.3.rs-4653222/v1

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Fibromyalgia, one of the most challenging pains to treat, lacks impartial considerations for diagnosis and useful assessment. The core symptoms are persistent extensive pain accompanied by fatigue, psychological disorders, sleep disturbance, and obesity. The current study aims to explore the role of cannabinoid receptor 1 (CB1) signaling pathways in a mouse model of fibromyalgia. This model was subjected to intermittent cold stress (ICS) to induce fibromyalgia, as measured by the nociceptive behavior determined by von Frey and Hargraves’ tests. Our results showed lower mechanical threshold (2.32 ± 0.12 g) and thermal latency (4.14 ± 0.26 s) in ICS-induced fibromyalgia mice. The hyperalgesia could be alleviated by electroacupuncture (EA) or by transient receptor potential V1 (TRPV1) knockout. We found differential expression of CB1 and TRPV1 signaling molecules in specific brain regions: upregulated TRPV1 and related kinases in the dorsal root ganglion, spinal cord, hypothalamus and periaqueductal gray region, and decreased CB1 receptor composition. EA reversed these effects associated with fibromyalgia, aligning with observations in Trpv1^−/− mice. Peripheral acupoint or intracerebral ventricle injection of a CB1 agonist or antagonist significantly regulated hyperalgesia through the CB1 signaling pathway. Our discoveries shed light on the involvement of CB1 on the TRPV1 pathway in the effects of EA in fibromyalgia, suggesting its potential as a treatment target.

Electroacupuncture

Fibromyalgia

CB1

TRPV1

DRG

PAG.

Cannabinoid receptors (CB) are 7-transmembrane domain receptors that can be activated at both peripheral or central sites with cannabis plant. CBs are considered G-protein-coupled receptors with two distinguished subtypes: CB1 and CB2 receptors, encoded by CNR1 and CNR2, respectively. Cannabinoids can bind at different receptors such as CB1 in neurons and CB2 in immune cells. CB1 is mostly found in the brain, especially in the hippocampus, cerebellum, hypothalamus (Hypo), amygdala, periaqueductal grey (PAG), and cerebral cortex (1, 2). Endocannabinod ligands such as 2-arachidonoylglycerol (2-AG) or arachidonylethanolamide (AEA) can bind at presynaptic CB1 receptors to inhibit adenylate cyclase, cAMP, A-type K + channels, as well as voltage-gated Ca²⁺ channels to reduce synaptic transmission. CBs have been implicated in physiological and pathophysiological conditions involved in regulation of mood, appetite, pain sensation, and immune response (3, 4). The sequential signaling possessions of CB1 were next modified by Src homology 3-domain growth factor receptor-bound 2-like endophilin interacting protein 1 (SGIP1), which can decrease ERK1/2 signaling. In addition, CB1 selective inhibitors had been used for weight reduction and smoking cessation (5).

On the other hand, transient receptor potential V1 (TRPV1) has been implicated in inflammation, cancer and immunity progress (6). TRPV1 has been used as target of drugs for human pain conditions. However, the identification of new targets for TRPV1 structure, agonists, and mechanisms is urgently required (7, 8). TRPV1 activation cooperate with protein kinase A (PKA), phosphoinositide 3-kinase (PI3K) and PKC in the modulation of pain, indicating its crucial role in central sensitization linked with fibromyalgia pain at both peripheral and central nervous systems (9, 10). The epsilon isoform of protein kinase C (PKCɛ) is used as a transient insult to deliver hyperalgesia in nociceptors to form long-lasting central sensitization changes, a well-known factor involved in fibromyalgia pain. MAPK is considered to be involved in the inflammation and pain signaling pathway including extracellular signal-regulated protein kinase (ERK), p38, and c-Jun N-terminal kinase/stress-activated protein kinase (JNK) (11, 12). These components have been implicated in nociceptive processes associated with painful sensation, neuronal plasticity, central sensitization and certain cognitive function (12, 13). Further, the PI3K-Akt-mTOR signaling pathway is involved in changes in both peripheral and central nociceptive response for central sensitization. Moreover, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) seems to be involved in fibromyalgia pain development, while its suppression appears to have therapeutic benefits for pain (14, 15).

Fibromyalgia is a chronic widespread pain in the body arising from multiple sites. Fibromyalgia pain is frequently sensed in the arms, legs, head, chest, abdomen, and back. Fibromyalgia patients usually describe it as painful, burning, or as soreness. Fibromyalgia often occurs in women with tension headaches, irritable bowel syndrome, anxiety and depression (16). So far, there is no cure for fibromyalgia pain, as current medications can only manage the symptoms. In addition, nutrients, exercise, meditation, sleep therapy, yoga, and relaxation may be beneficial. Fibromyalgia may result from repeated nerve stimulation–central sensitization–or overexpression of neurotransmitters or neuromodulators in the brain (17). These phenomena may result from genetic mutations, infections, or psychological stress. Fibromyalgia affects about 2–8% of the population (16, 18). The American College of Rheumatology defined the diagnostic principles for fibromyalgia, including use of the widespread pain index (WPI) and the symptom severity scale (SS) to evaluate fibromyalgia. WPI comprises 19 common pain points experienced in the previous two weeks. The SS measures the degree of fatigue, waking, and cognitive symptoms. Fibromyalgia pain is considered for WPI ≥ 7 and SS ≥ 5 or WPI 3–6 and SS ≥ 9 in the last three months (19, 20). Duloxetine (Cymbalta), milnacipran (Savella), and pregabalin (Lyrica) are drugs currently used for treating fibromyalgia and approved by the U.S. Food and Drug Administration (FDA). Gabapentinoids, sedatives, selective serotonin reuptake inhibitors, serotonin norepinephrine reuptake inhibitors, and tricyclic compounds have also been applied for fibromyalgia, with several side effects. For all the above, fibromyalgia has a high economic impact (21–23).

Acupuncture, a main component of traditional Chinese medicine, is based on inserting steel needles into specific acupoints on the body and usually reported to relieve pain. In Chinese medicine, doctors consider that acupuncture can control the meridians to control the qi sensation and balance the energy flow within the body. Recently, the development of neuroanatomy demonstrated that acupoints are specific regions which can be used to easily stimulate connective tissue, muscle, and peripheral nerves for medical purposes. A recent study suggests that electroacupuncture (EA), via optogenetic stimulation of nerve terminals over the ST36 acupoint, can activate the vagal-adrenal axis in mice (24). Another study indicated a novel anti-inflammatory effect of EA in a mouse sepsis model (25). We previously demonstrated that EA could trigger the release of adenosine triphosphate, interleukin-1β, interleukin-6, glutamate, substance P, and histamine in local acupoints (26). Further, EA was reported to decrease inflammatory, neuropathic, and fibromyalgia pain in various mouse models (27–33). It appears that EA can decrease fibromyalgia pain by decreasing the levels of inflammatory cytokines such as interleukins, TNF-α and IFN-γ in mouse plasma (11). However, the underlying mechanisms for the effects of EA remain unclear.

In the current research, our hypothesis was that EA could diminish fibromyalgia symptoms via CB1 signaling. In addition, novel and detailed cellular mechanisms behind pain-relieving properties of EA to treat fibromyalgia pain would be obtained. We found that hyperalgesia increased the expression of TRPV1 and related kinases in a mouse model of fibromyalgia pain. Such effects were observed in the mouse dorsal root ganglion (DRG), spinal cord dorsal horn (SCDH), Hypo and periaqueductal gray (PAG). These effects were reversed by EA treatment, even in Trpv1^−/− mice. Further, peripheral acupoint injection of a CB1 agonist or antagonist significantly attenuated hyperalgesia through CB1 signaling. In addition, intracerebral ventricle injection of a CB1 agonist or antagonist significantly attenuated hyperalgesia through central, but not peripheral CB1 signaling. Based on the above, these data provide a novel indication for EA in mice fibromyalgia pain.

Animals

The experimental subjects were 8–12-week-old female C57B/L6 wild type mice (18–20 g) bought from BioLasc Taiwan Ltd (Yilan, Taiwan). After their arrival, mice were placed in a home cage under a 12-h light and dark cycle, light beginning from 6 a.m. to 6 p.m., at 25℃ and 60% humidity. We used statistic process to determine the sample size. Nine mice per group was considered as the minimum number required for a significance alpha level of 0.05 and a power of 80%. Mouse experiments were approved by the Institute of Animal Care and Use Committee of China Medical University (Permit no. CMUIACUC-2023-071), Taiwan, following the Guide for the use of Laboratory Animals (National Academy Press).

Mouse fibromyalgia pain induction

Mice were assigned at random to one of four groups including normal (Normal), ICS-induced fibromyalgia pain (FM), ICS-induced fibromyalgia pain with EA (FM + EA), ICS-induced fibromyalgia pain in Trpv1^−/− mice (FM + Trpv1^−/−). To induce mice fibromyalgia pain, mice were placed in a 4℃ environment, while the normal group was maintained at normal temperature. At 10 a.m. the following day, the fibromyalgia groups were relocated to the normal environment at 25ºC for 30 min, then back to 4℃ for 30 min. This process was carried out until 4 p.m. to a total of 6 h, before they were repositioned again overnight from 4 p.m. during the first 3 days.

Electroacupuncture

After anesthesia with 5% isoflurane for induction, the mice were placed in 1% gas via a head-stuffed tube for inhalation. A pair of 1-inch-long acupuncture needles (32G, Yu Kuang Chem. Ind. Corp., Taiwan) were inserted bilaterally into the mouse ST36 acupoint. The murine ST36 is found 3–4 mm lower the patella, between the fibula and tibia, and on the anterior side of the anterior tibial muscle. In addition, electrical stimulation was delivered through a constant square pulse a depth of 3–4 mm with 1 mA intensity, 2 Hz frequency, and 150 s width for 20 min utilizing an electronic source from a Trio 300 stimulator (Ito, Japan).

Pain behavior test

Mechanical and thermal nociceptive behaviors were evaluated 3 times at day 0, 3, and 4 before and after fibromyalgia pain induction. Mice were enclosed in plexiglas boxes (9.5 × 11 × 6.5 cm³) above a steel mesh in a dark, noiseless, room-temperature room to allow them to adapt to their new surroundings. When mice were not sleeping, scratching, or grooming, the von Frey test was performed three times per section, separated 10 min (IITC Life Science Inc., USA). Then, the Hargreaves’ test was applied to measure mouse thermal responses. Mice were separated in various plexiglas enclosures to restrict interaction. After 30 min of environmental habituation, we began with the examination. The IITC Plantar Analgesia Meter (IITC Life, Sciences, SERIES8, Model 390G) determined the withdrawal delay of the mouse foot from radiant thermal light, placed under the tempered glass and slid across the surface to the exact middle of the plantar’s right hind paw. Each phase was repeated three times per mouse. The device was programmed to stop after 20 s to prevent harming their paws.

Western blot analysis

The DRG, SC, Hypo, and PAG tissues were excised to extract protein. Tissues were placed on ice and stored at − 80℃ until protein extraction. Total proteins were homogenized in a cold radioimmunoprecipitation (RIPA) lysis buffer containing 50 mM Tris-HCl pH 7.4, 250 mM NaCl, 1% NP-40, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 0.02% NaN3, and 1× protease inhibitor cocktail (AMRESCO). The extracted proteins were subjected to 8% SDS-Tris glycine gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk in TBS-T buffer (10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20), and incubated with a primary antibody in TBS-T with 1% bovine serum albumin (BSA) for 1 h at room temperature. Then, a peroxidase-conjugated anti-rabbit antibody, anti-mouse antibody, or anti-goat antibody (1: 5000) was used as the appropriate secondary antibody. The bands were visualized using an enhanced chemiluminescent substrate kit (PIERCE) with LAS-3000 Fujifilm (Fuji Photo Film Co., Ltd.). Where applicable, the image intensity of specific bands was quantified with NIH ImageJ software (Bethesda, MD, USA). β-actin or α-tubulin served as internal control.

Immunofluorescence

Mice were euthanized with 5% isoflurane and intracardially perfused with normal saline followed by 4% paraformaldehyde. The brain was immediately dissected and post fixed with 4% paraformaldehyde at 4℃ for 3 days. Then, the tissues were placed overnight in 30% sucrose for cryoprotection at 4℃. Next, the brain was embedded in an Optimal cutting temperature compound and rapidly frozen using liquid nitrogen before storing the tissues at − 80℃. Frozen segments were cut into 20 mm sections using a cryostat and placed on glass slides. The samples were fixed with 4% paraformaldehyde, and incubated with a blocking solution consisting of 3% BSA, 0.1% Triton X-100, and 0.02% sodium azide for 1 h at room temperature. After blocking, the samples were incubated with the primary antibody (1:200, Alomone), CB1 receptor, and pERK, prepared in 1% BSA overnight. The samples were then incubated with the secondary antibody (1:500), 488-conjugated AffiniPure donkey anti-rabbit IgG (H + L), 594-conjugated AffiniPure donkey anti-goat IgG (H + L) and peroxidase-conjugated AffiniPure donkey anti-mouse IgG (H + L), for 2 h at room temperature before fixation with cover slips for immunofluorescence visualization.

CB1 receptor agonist and antagonist administration

Adult C57BL/6 female mice (n = 6) were used for the CB1 agonist or antagonist test. After mice fibromyalgia induction, the CB1 agonist AEA (Sigma, St. Louis, MO, USA) was administered at the acupoint or i.c.v at 100 µM. Alternatively, the CB1 antagonist AM251 (Sigma, St. Louis, MO, USA; in 10 µL of saline) was immediately administered at the acupoint or i.c.v at 5 µg. Under light isoflurane anesthesia (1%), AEA and AM251 were administered after fibromyalgia induction.

Statistical analysis

Statistical analysis was performed using SPSS 21.0 software. All statistic data are shown as the mean ± standard error (SEM). Statistical significance was verified using ANOVA test, followed by a post hoc Tukey’s test. A P < 0.05 indicated statistical significance.

Intermittent cold stress-induced fibromyalgia pain in mice, reversed by electroacupuncture

EA regulated CB1 signaling of fibromyalgia pain in the mouse peripheral DRG

Next, we used Western blot to clarify how CB1 affect fibromyalgia pain in peripheral DRG areas. We examined CB1 expression in the murine DRG region. After ICS induction, fibromyalgia pain was observed together with decreased CB1 expression in the DRG, suggesting CB1 downregulation (Fig. 2A, *P < 0.05, n = 6). In contrast, 3 secessions of EA decreased fibromyalgia pain by increasing CB1 protein levels in the DRG (Fig. 2A, ^#P < 0.05, n = 6). In the Trpv1^−/− group, CB1 protein levels were almost normal (Fig. 2A, ^#P < 0.05, n = 6). In addition, fibromyalgia pain increased TRPV1 expression, while 2 Hz EA significantly suppressed such overexpression after 3 sessions. Moreover, TRPV1 expression in the DRG was almost undetectable (Fig. 2B, ^#P < 0.05, n = 6). Next, we determined the expression of downstream molecules such as phosphorylated PKA (pPKA), pPI3K, and pPKC, all kinases associated with TRPV1. Increased expression of these kinases was observed in fibromyalgia pain mice than in the normal group; being diminished by 2 Hz EA treatment at ST36 acupoints as well as in Trpv1^−/− mice (Fig. 2C-E, ^#P < 0.05, n = 6). In addition, pAkt and pmTOR levels increased in fibromyalgia mice, as did those of pERK and pNKκB (significantly so). EA and Trpv1 knockout reverted these upregulations (Fig. 2F-I, ^#P < 0.05, n = 6).

EA at the ST36 acupoint decreased cold stress-induced fibromyalgia pain through TRPV1-CB1 in the SCDH

As in the previous subsection, we investigated the effects of EA after fibromyalgia induction in the SCDH region. Interestingly, in this region fibromyalgia pain induction decreased CB1 protein levels (Fig. 3A, ^*P < 0.05, n = 6). This effect was reverted by 2 Hz EA, which not only relieved fibromyalgia but increased CB1 levels in the SCDH. A similar result was obtained in Trpv1^−/− mice (Fig. 3A, ^#P < 0.05, n = 6). The low TRPV1 expression in normal mice significantly increased in the SCDH of fibromyalgia pain mice (Fig. 3B, ^*P < 0.05, n = 6). A phenomenon alleviated by 2 Hz EA treatment and also observed in Trpv1^−/− mice (Fig. 3B, ^#P < 0.05, n = 6). At the protein level, we observed a significantly increased expression of pPKA, pPI3K, pPKC in the SCDH of mice in the fibromyalgia pain group. Those increased levels were attenuated by EA as well as in Trpv1^−/− mice (Fig. 3C-E, ^#P < 0.05, n = 6). Moreover, levels of pAkt and pmTOR, downstream mediators of pPI3K for pain signaling, were increased in the SCDH of fibromyalgia mice. A similar tendency was observed for pERK and pNKκB. These trends were reversed by EA and in Trpv1^−/− mice (Fig. 3F-I, ^#P < 0.05, n = 6).

EA at the ST36 acupoint decreased cold stress-induced fibromyalgia pain and regulated TRPV1-CB1 signaling pathway in the hypothalamus

After the final behavioral tests on day 5, we collected the total Hypo from all mouse groups and measured protein expression via Western blotting. CB1 levels were reduced in mice suffering from fibromyalgia pain (Fig. 4A, ^*P < 0.05, n = 6). This indicated a lower antinociceptive effect, which can be reversed by EA and Trpv1 gene loss (Fig. 4A, ^#P < 0.05, n = 6). In the Hypo of fibromyalgia pain mice, we observed increased TRPV1 levels. An effect attenuated by EA and observed in Trpv1^−/− mice (Fig. 4B, ^#P < 0.05, n = 6). Similar to other tissues, levels of TRPV1-associated kinases such as pPKA, pPI3K, and pPKC all increased after ICS- fibromyalgia induction; effects alleviated by EA and in Trpv1^−/− mice (Fig. 4C-E, ^#P < 0.05, n = 6). Sham EA could not alter the responses. Establishment of the fibromyalgia pain also increased pAkt and pmTOR expression in the Hypo suggesting its crucial role in pain development. EA treatment or Trpv1 gene deletion reduced the overexpression of these molecules. Likewise, ICS increased pERK and pNFκB expression. Furthermore, EA or TRPV1 protein remove reliably diminished the overexpression of these factors (Fig. 4F-I, ^#P < 0.05, n = 6).

TRPV1 and related factors were inhibited in the PAG, an effect reversed by EA and Trpv1 gene deletion

Finally, to determine the influence of ICS-induced fibromyalgia pain and EA on fibromyalgia pain signaling in the descending pathway, we subjected the PAG to protein analysis. We observed a significant decrease in CB1 expression in mice suffering from ICS-initiated fibromyalgia pain (Fig. 5A, ^*P < 0.05, n = 6). An effect reversed by EA or TRPV1 dysfunction (Fig. 5A, ^#P < 0.05, n = 6). In addition, ICS significantly increased TRPV1 expression in the PAG. Such increase was reduced by EA or Trpv1 gene deletion (Fig. 5B, ^#P < 0.05, n = 6). A comparable trend was perceived in pPKA, pPI3K, and pPKC protein content in fibromyalgia mice related to normal mice (Fig. 5C-E, ^#P < 0.05, n = 6). This effect was significantly reversed by EA and Trpv1 gene loss. Similar tendencies were observed for pAkt, pmTOR, pERK, and pNFκB (Fig. 5F-I, ^#P < 0.05, n = 6).

CB1 and pERK expression in peripheral DRG and central PAG regions can be modulated by EA or Trpv1 gene deletion

We next examined if ICS could alter CB1 expression in DRG neurons after fibromyalgia induction. Here, we confirmed that fibromyalgia induction significantly decreased CB1 expression in peripheral DRG neurons through CB1 specific immunohistochemistry staining (Fig. 6, n = 3). Consistent with Western blot findings, animals that received EA or Trpv1 gene deletion showed increased CB1 expression. In contrast, pERK levels increased after ICS, being alleviated in EA or Trpv1 gene deletion groups. Subsequently, we checked the role of CB1 and pERK in modulation of ICS-induced fibromyalgia pain in central PAG areas (Fig. 6, n = 3). Our results revealed that ICS also decreased CB1 expression in the central PAG region. In addition, EA can reverse CBA expression in the PAG regions. A similar tendency was obtained in Trpv1^−/− mice (Fig. 6, n = 3).

EA diminished fibromyalgia directly targets the CB1 receptor in the mice acupoint or brain

We further examined if EA can regulate CB1 receptor expression to relieve mice fibromyalgia pain. We first confirmed that ICS significantly induced mechanical hyperalgesia in mouse fibromyalgia pain (Fig. 7A, day 4: 1.94 ± 0.15 g, n = 6). To test our hypothesis that both peripheral and central CB1 activation could abolish mechanical hyperalgesia, we injected the CB1 agonist anandamide (AEA) into the acupoint and intracerebral ventricle. In von Frey filament tests, our data indicated that either acupoint administration (Fig. 7A, day 4: 3.82 ± 0.18 g, n = 6) or intracerebral ventricle injection (Fig. 7A, day 4: 4.04 ± 0.19 g, n = 6) significantly attenuated mechanical hyperalgesia. We next used AM251, a CB1 receptor antagonist, to check if EA delivers its analgesic effect through CB1. We demonstrated that EA’s antinociceptive effect was abolished during AM251 antagonism (Fig. 7A, day 4, n = 6). Similar trends were also observed in thermal latency (Fig. 7B, n = 6).

After confirming the analgesic effect of EA, we performed Western blotting to further examine changes to CB1 signaling molecules in peripheral DRG regions. Injecting AEA into the acupoint significantly increased CB1 levels in fibromyalgia mouse DRG neurons (Fig. 7C, day 4: 132.70 ± 3.41%, ^*P < 0.05, n = 6). In contrast, injecting AEA in the central cerebral ventricle did not affect CB1 levels in the DRG (Fig. 7C, day 4: 100.50 ± 2.34%, P > 0.05, n = 6). Similar phenomena were obtained when AM251 was injected into the acupoint (Fig. 7C, day 4: 104.22 ± 3.37%, P > 0.05, n = 6). Further, AM251 intracerebral injection did not affect EA’s effects (Fig. 7C, day 4: 139.84 ± 5.85%, ^*P < 0.05, n = 6). In contrast, TRPV1 and pPKA, pPI3K, pPKC levels all decreased after acupoint injection of AEA, but not when AEA was injected in the intracerebral ventricle. This suggest a central role of CB1. Similar results were obtained in EA treated-mice after AM251 acupoint administration. Furthermore, intracerebral injection of AM251 did not alter the effect of EA on TRPV1 and related molecules. The same trends were obtained in TRPV1 downstream molecules including pAkt, pmTOR, pERK, and pCREB (Fig. 7C, day 4, n = 6).

To better explore the direct effect of EA on CB1 at a central level, both AEA and AM251 were injected at acupoint or intracerebral ventricle after ICS fibromyalgia pain induction. We further used Western blotting to investigate the protein alterations of CB1 signaling in central PAG regions. AEA, injected into acupoint or ventricle, significantly increased CB1 levels in fibromyalgia mice PAG neurons (Fig. 7D, day 4, ^*P < 0.05, n = 6). In addition, CB1 levels were not altered in PAG from mice receiving EA and AM251 administration in either the acupoint or ventricle (Fig. 7D, day 4, P > 0.05, n = 6). Conversely, TRPV1 and related molecules such as pPKA, pPI3K, pPKC all decreased after acupoint or intracerebral AEA injection. These factors were not altered in EA treated-mice after AM251 administration. The same trends were obtained for TRPV1 downstream molecules including pAkt, pmTOR, pERK, and pCREB (Fig. 7D, day 4, n = 6).

To our knowledge, this is the first study to show that ICS-induced mice fibromyalgia pain can be improved by 2 Hz EA or Trpv1 gene deletion. CB1 protein levels decreased after fibromyalgia induction. TRPV1 and downstream kinases were increased in the mouse DRG, SCDH, Hypo, and PAG. These trends could be reversed by 2 Hz EA treatment and TRPV1 knock out. The turnover of these protein alterations in signaling protein kinases through the use of EA suggests that these managements can be used as an efficient beneficial intervention.

Cannabidiol is the major nonaddictive component of cannabis, and reported to interact with several neurotransmitters to produce its analgesic effects. De Gregorio et al. reported that acute intravenous cannabidiol injection could reliably decrease the firing rate of serotoninergic (5-HT) neurons in the dorsal raphe nucleus. This decrease could be prevented by administration of 5-HT_1A and a TRPV1 antagonist, suggesting its critical involvement in this phenomenon. In addition, cannabidiol appears to induce analgesia mainly through TRPV1, decreasing anxiety through the 5-HT_1A receptor (34). The high co-localization of CB1 and Kv1.4 in nociceptive DRG neurons indicates an efficient synergistic action between the two. As the Kv1.4 potassium channel appears involved in the control of repetitive discharges at peripheral terminals for pain relief, it maybe serve as a downstream site of CB1 for the control of presynaptic transmitter release at central terminals (35). Moreover, Liu et al. proposed that a cannabinoid receptor agonist can significantly diminish the functional current of acid-sensing ion channels in rat DRG neurons. The same result was obtained when using a CB1 receptor antagonist but not a CB2 receptor, suggesting the crucial role of CB1 in the peripheral DRG (36). Pharmacological description of cannabinoid agonists and antagonists indicated that potent agonists trigger in adenylate cyclase and MAPK mainly via CB1 receptors (37). In the current study, we showed that CB1 expression was reduced in fibromyalgia DRG leading to overexpression of elements in the TRPV1 nociceptive signaling pathway. Conversely, EA or Trpv1 deletion increased CB1 expression and diminished that of TRPV1 and associated molecules.

A recent study reported cannabidiol could alleviate rat spinal cord injury-induced chronic pain, through dose-dependent reductions in tactile and cold hypersensitivity with benign side effects. This antinociceptive effect was inhibited by the CB1 antagonist AM251 suggesting an innovative CB1 mechanisms in the SCI pain state (38). Further, Zhang et al. found that miR-338-5p from bone marrow-derived mesenchymal stromal cells, increased the levels of neurofilament protein-M, growth-associated protein-43, and decreased myelin-associated glycoprotein and glial fibrillary acidic protein, providing neuroprotective effects from spinal cord injury. They next demonstrated that CB1 is the target receptor of miR-338-5p, which can reliably attenuate cell apoptosis and promote neuron survival (39). In addition, Nerandzic suggested that endogenous lipid precursor N-arachidonoylphosphatidylethanolamine (20:4-NAPE) treatment can inhibit excitatory synaptic transmission in both naive and inflammatory rat models. 20:4-NAPE application further activated CB1 having inhibitory effects in naive animals, while TRPV1-mediated mechanisms were involved after inflammation (40). Jiang found that combined-acupoint treatment could deliver stronger analgesic effect than a single one, in addition to resulting in better inhibition of synaptic transmission at the spinal cord level. Naloxone, an opioid receptor antagonist, blocked the analgesic effect of single-acupoint EA (41). Further, coadministration of naloxone and a CB1 antagonist significantly reduced the analgesic effect of combined-acupoint EA. This suggests a beneficial effect of combined EA due to initiating diverse mechanisms from different acupoints (41). We also demonstrated that 2 Hz EA increased CB1 expression, alleviating fibromyalgia pain through downstream TRPV1 and associated molecules at the spinal cord level.

Previous work indicated the relevance of mechanisms of peripheral and central sensitization in migraine, which a higher-level center the Hypo have a vital import in the initiation of migraine. Watanabe et al. determined stress-induced activation of hypothalamic pain responses, suggesting a mechanistic link between stress-induced hypothalamic activation and trigeminal nociceptor effectors in migraine (42). Stress-induced activation of the hypothalamic–pituitary–adrenal (HPA) axis is triggered by the release of corticotropin-releasing hormone reported in the pathogenesis of fibromyalgia. The affective measurement of pain initiated at SC superficial dorsal horn and ascends pain signals in the brain associated with unpleasantness including Hypo (43). Smith et al. determined that patients with pain syndromes often present dysfunction of the HPA axis with increased mast cell infiltration and activation, leading to nociceptive responses. Chronic stress significantly resulted in rodent depression via decreased glutamatergic transmission in the ventral lateral PAG (44). A recent study confirmed the role of vlPAG in analgesia (45), while another reported the association of vlPAG excitatory transmission in a murine model of visceral pain (46). Yin et al. demonstrated that precise activation of the dmPFC/vlPAG neural pathway through optogenetics caused an analgesic effect in a mouse model of chronic pain. Further suppressing the dmPFC/vlPAG neural circuit resulted in significant maintenance of chronic pain (47). Here, we showed decreased TRPV1 signaling and increased CB1 expression in the vlPAG in a fibromyalgia pain mouse model. These phenomena could be reversed by 2 Hz EA and TRPV1 deletion, suggesting the crucial role of TRPV1-CB1 signaling in fibromyalgia pain.

In conclusion, our findings provide precise evidence in necessary possessions of 2 Hz EA in mice fibromyalgia pain, especially via CB1-TRPV1 signaling pathway. We characterized the antinociceptive effect of 2 Hz EA on mouse fibromyalgia pain and its underlying cellular mechanisms. Our data revealed that TRPV1 and related kinases were overexpressed in our mouse model of fibromyalgia pain. In contrast, we observed decreased CB1 expression in the DRG, SCDH, Hypo, and PAG. These effects were abolished by EA treatment and observed in Trpv1^−/− mice. Consequently, our results provide indications for the use of EA in fibromyalgia pain and recommend the clinical use of EA.

Ethics approval and consent to participate

All ethics are approved and consent to participate in this manuscript.

Consent for publication

All authors have read the manuscript and consent for publication.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article.

Conflict of Interest

There are no financial or other relationships that might lead to a conflict of interest for all authors.

Funding

This work was supported by the following grants: NSTC 112-2314-B-039-021, CMUHCH-DRM-113-025, and the "Chinese Medicine Research Center, China Medical University" from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan.

Authors' contributions

HC Lin and HC Hsu: Conceptualization, Methodology, Software, Data curation, Writing - original draft, Visualization, Investigation. HC Hsu and YW Lin: Supervision, Validation, Writing - review & editing

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Electroacupuncture alters cannabinoid receptor 1 and transient receptor potential V1 expression on individual brain regions in a mouse fibromyalgia model

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Abstract

Figures

Introduction

Methods and materials

Animals

Mouse fibromyalgia pain induction

Electroacupuncture

Pain behavior test

Western blot analysis

Immunofluorescence

CB1 receptor agonist and antagonist administration

Statistical analysis

Results

Intermittent cold stress-induced fibromyalgia pain in mice, reversed by electroacupuncture

EA regulated CB1 signaling of fibromyalgia pain in the mouse peripheral DRG

EA at the ST36 acupoint decreased cold stress-induced fibromyalgia pain through TRPV1-CB1 in the SCDH

EA diminished fibromyalgia directly targets the CB1 receptor in the mice acupoint or brain

Discussion

Declarations

References

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