Acupoint Catgut Embedding Diminishes Fibromyalgia Pain through TRPV1 in the Mouse Brain

Background : Chronic pain refers to pain that persists for over three months. Chronic pain may restrict activities of daily living, including work, learning, social life, and can lead to anxiety, depression


Introduction
Acute pain typically accompanies injury, such as wounds, tissue damage, or fractures. Various pharmacological agents are available to relieve acute pain. In contrast, chronic pain persists long after recovery from the injury or damage. About 1/4 of adults suffer from chronic pain, identified as discomfort sustained for over three months, and accounts for a high proportion of insurance claims [1]. Chronic pain can result from various etiologies including arthritis, cancer pain, low back pain, headaches, muscle pain, and fibromyalgia (FM). FM is presently described as a complex disease characterized by chronic widespread muscle pain, fatigue, insomnia, anxiety, and depression [2]. FM is a growing issue affecting individuals, insurance providers, healthcare systems, economies, and societies. There are no curative therapies for FM and methods to relieve FM pain are lacking due to its unclear fundamental mechanisms. The occurrence of FM is around 2-8% of people, depending on the analytic principles used [2]. New di-agnostic principles for FM to include widespread pain index (WPI) and symptom severity scale (SS). The WPI examines 19 common body pain parts for two weeks. The SS calculates the degree of fatigue, waking, and cognitive symptoms. FM is characterized as WPI ≥7 and SS ≥5 or WPI 3-6 and SS ≥9 in the last three months. Recently, three medications of FM were permitted by the U.S. Food and Drug Administration (FDA), including duloxetine (Cymbalta), milnacipran (Savella), and pregabalin (Lyrica) [3][4][5].
Acupuncture is an ancient Chinese medicine technique that involves implanting a steel needle into an acupoint. Acupuncture is helpful in managing FM due to its ability to reduce pain, anxiety, depression, and insomnia [6,7]. In addition, electroacupuncture (EA) has been described to attenuate numerous kinds of pain [8][9][10][11][12]. Recently, acupoint catgut embedding (ACE) was developed as a novel technique to establish constant acupoint stimulation. ACE treatment has been shown to be more effective and its benefits prolonged than acupuncture or EA in man-aging obesity [13]. ACE has the advantages of long-term effects and low cost, and has been broadly utilized in the management of acute or chronic pain [14,15]. Recent articles showed that ACE activates the immune system because the absorbable chromic catgut sutures are recognized as a non-self protein [16]. ACE can induce long-term immune responses such as macrophage phagocytosis and neutrophil infiltration [17]. ACE can also reliably diminish postoperative pain and decrease analgesic medicine intake [18].
Transient Receptor Potential Vanilloid (TRPV) is an ion channel chiefly located on the neuronal membrane. TRPV can be activated by vanilloid compounds and has six subtypes. TRPV1 was first recognized as a receptor of capsaicin, the major constituent of chili pepper [19]. TRPV1 exists in the dorsal root ganglion, spinal cord, and trigeminal ganglion for pain sensation and transmission. In peripheral regions, TRPV1 greatly exists in A-and C-type sensory neurons to detect inflammation and painful stimuli. TRPV1 was also described to exists in the central nerve system (CNS) for thermoregulation, synaptic transmission, and detection of inflammation [19]. Furthermore, TRPV1 is expressed in non-neuronal astrocytes or glial cells for pain signaling [20]. TRPV1 is expressed in several brain areas including thalamus, somatosensory cortex, prefrontal cortex, hippocampus, amygdala, hypothalamus (Hypo), and cerebellum (CB). TRPV1 activation sequentially activates kinases, such as protein kinase A (PKA), phosphoinositide 3-kinases (PI3K), protein kinase C (PKC), and Ca 2+ /calmodulin-dependent kinase II (CaMKII) [21,22]. Furthermore, these kinases then trigger mitogen-activated protein kinase (MAPK), pAkt, pmTOR, and further increase transcriptional factors, such as cAMP-response element binding protein (CREB) and nuclear factor kappalight-chain-enhancer of activated B cells (NFκB). These kinases also play a role in sensitizing peripheral afferents resulting in mechanical and thermal hyperalgesia [21,22]. A recent article indicated that immune cells release several factors to activate individual receptors on the membrane of the nerve terminals during pain development and treatment [23].
The plan of the current study was to verify our hypotheses that FM is related to augmenting TRPV1 signaling in the mouse brain and that ACE ameliorates both mechanical and thermal nociception through suppression of TRPV1 pathway. To address these questions, we used a mouse FM through intermittent cold stress (ICS). Consistent with our hypothesis, mice subjected to ICS exhibited significant chronic mechanical and thermal pain that could be abridged by ACE or TRPV1 gene deletion. TRPV1 pathway was amplified in the mouse Hypo and CB and can be relieved by ACE and TRPV1 gene loss. These data suggest the therapeutic effect of ACE is connected with TRPV1 in the mouse brain. We propose that the TRPV1 signaling pathway is a potential target for FM treatment.

Experimental Animals
The treatment of mice was permitted by China Medical University Committee (CMUIACUC-2022-424), Taiwan, subsequent the guideline for the practice of experimental animals (National Academy Press). The current work is indicated according to the ARRIVE guidelines. Totally, 40 female mice were used in the current study, containing 30 normal mice (BioLASCO, Co., Ltd, Taipei, Taiwan) and 10 Trpv1 −/− mice. The animals were hosted in the experimental animal center (12/12 h) with all diet and water provided free. The estimated model size of 10 mice in each group was designed for an α value of 0.05 with the influence of 80%. Mice used in the present study and their misery were minimalized. The experimental staffs were blind to group distribution through the examinations. Animals were further subgrouped into four groups: Normal mice (Group 1: Normal); ICS-initiated FM group (Group 2: FM); FM cured with ACE group (Group 3: FM + ACE), and FM in Trpv1 −/− group (Group 4: FM + Trpv1 −/− ).

FM Induction and Multiplex Enzyme-Linked Immunosorbent Assay
All animals were accommodated at 24 ± 1°C before FM induction. In this ICS model, the animals were placed at ± 1°C temperature overnight (starting from 4 PM-10 AM). Animals were next transported to 24 ± 1°C temperature for a 30-minute duration in the next morning. The animals were further relocated back to 4 ± 1°C temperature for another 30 minutes. The challenge of environmental temperatures induced a murine FM model. Normal mice were placed in the same place at room temperature. After all experimental treatment of FM at day 14, the mice plasma was collected by retro-orbital sinus puncture and examined through examined multiplex enzyme-linked immunosorbent assay (ELISA: lot. MA1M191126, Quansys Biosciences, Logan, UT, USA) through Q-view 1.5 (Quansys Biosciences, Logan, UT, USA).

ACE Treatment
ACE mice had received ACE treatment at the bilateral stomach 36 (ST36) acupoint at days 0 and 7. Like human anatomy, the ST36 acupoint is sited longitudinally at 3 mm under the apex of patella bone intersecting horizontally 1 mm lateral to anterior the tibia bone at the central of the tibialis anterior muscle. Sterilized needle 0.6 × 25 mm (160925, Terumo Corporation, Tokyo, Japan), steel needle 0.35 × 40 mm (Suzhou Medical Appliance, Suzhou, China), or catgut suture 0.2 × 4 mm (CP Medical Inc, Norcross, GA, USA) were utilized for ACE. Mice were engaged into a chamber for anesthesia. Both ST36 acupoints were decontaminated with 75% alcohol and the commercial syringe needle was implanted, followed by the catgut inserted at the 5 mm depth.

Pain Behavior Test
Mechanical or thermal nociceptive performances were measured 7 periods from day 0 to 14 after FM induction. We first verified the von Frey filament examination, mechanical pain was tested by calculating the strength of replies to stimuli within 3 performances of von Frey test (IITC Life Science Inc, Los Angeles, CA, USA). Animals were then placed to a steel net (75 × 25 × 45 cm) and then quarantined under plastic cage (10 × 6 × 11 cm). Animals were examined with von Frey at center plantar of hind paw. The values were calculated as gram and were documented when the mice withdraw the paw. Besides, the Hargreaves' examination was achieved to quantity the thermal hyperal-gesia via conniving the latency to thermal stimuli with three tests through Hargreaves' test (IITC Life Sciences, Los Angeles, CA, USA). Animals were then engaged in a flexible cage on the top of IITC analgesiometer. The thermal examiner was placed under the IITC analgesiometer. Animals were placed to the behavior room for more than 30 minutes earlier examinations. The tests were performed when the individuals were quiet rather than grooming or sleeping.

Western Blot Analysis
Animals were sedated under isoflurane and suffered cervical dislocation. Mice Hypo and CB samples were separated to draw out proteins and further kept at -80°C re-

Immunofluorescence
Animals had been anaesthetized with isoflurane and intracardially filled with 0.9% normal saline and then 4% paraformaldehyde. The mice brain was instantly cut apart and next fixed with 4% paraformaldehyde in refrigerator for 2-3 days. The brains were next engaged in 30% sucrose in 4

Statistical Analysis
Statistical differences were accomplished by using the SPSS software (SPSS 21, IBM Corp., Chicago, IL, USA).
All results are offered as the mean with standard error (SEM). Shapiro-Wilk examination was achieved to examination the familiarity of results. Statistical differences in four groups were confirmed via the analysis of variance (ANOVA) test and a post hoc Tukey's test. p < 0.05 were identified as statistical differences.

ICS Induced Mechanical and Thermal Chronic Pain Which Could Be Relieved by ACE or TRPV1 Gene Loss
We induced a mice FM and determined their analgesic outcome of ACE and TRPV1 gene deletion. All mice showed similar mechanical threshold tendencies, which was normally distributed at baseline and was not statistically different among all groups. Cold stress reliably induced mechanical hyperalgesia (Fig. 1A, red circle, D14: 1.96 ± 0.13, * p ˂ 0.05, n = 10) via von Frey filament test. Mechanical hyperalgesia was markedly alleviated by ACE treatment and TRPV1 gene deletion (Fig. 1A, blue and green circles, D14: 4.05 ± 0.14 and 4.26 ± 0.17, # p ˂ 0.05, n = 10, respectively). ICS also induced thermal hyperalge-

Antinociceptive Effect of ACE or TRPV1 Gene Deletion Involved TRPV1 in the Mice Hypothalamus
Hypothalamic dysfunction was reported in FM patients. We performed Western blot to count the protein intensities of molecules in the TRPV1 in the mice hypothalamus. The level of TRPV1 was detected in mice hypothalamus and increased in FM mice ( Fig. 2A, black column, * p ˂ 0.05, n = 6). Further data showed that ACE reliably reversed the overexpression of TRPV1 ( Fig. 2A, black column, # p ˂ 0.05, n = 6). TRPV1 was disappeared in hypothalamus of Trpv1 −/− mice. The level of pPKA, pPI3K, and pPKC were amplified in FM group ( Fig. 2A, * p ˂ 0.05, n = 6) and were reduced by ACE treatment or TRPV1 gene deletion ( Fig. 2A, # p ˂ 0.05, n = 6). The level of pAkt and pmTOR were increased in the hypothalamus ( Fig. 2A, * p ˂ 0.05, n = 6) but decreased in AC-treated and Trpv1 −/− mice ( Fig. 2A, # p ˂ 0.05, n = 6). A similar pattern regarding pERK, pp38, and pJNK was also observed in the FM mice. By contrast, this increase was attenuated in mice treated with ACE or Trpv1 −/− mice (Fig. 2B, # p ˂ 0.05, n = 6). pCREB and pNFκB were both increased in the mouse hypothalamus (Fig. 2B, * p ˂ 0.05, n = 6) and were reduced by ACE or TRPV1 gene loss (Fig. 2B, # p ˂ 0.05, n = 6).
We next used immunofluorescence staining to measure TRPV1 in the hypothalamus. We detected low levels of TRPV1 in normal mice and increased levels in FM mouse hypothalamus (Fig. 2C, green color, n = 4). ACE and TRPV1 loss consistently abolished the augmentation of TRPV1 (Fig. 2C, green color, n = 4). A similar tendency was also observed in pERK appearance (Fig. 2C, red color, n = 4). TRPV1 and pERK colocalization increased in the FM mice but was diminished in ACE and Trpv1 −/− mice (Fig. 2C, yellow color, n = 4).

ACE or Loss of TRPV1 Attenuated FM through TRPV1 in the Mouse CB5
We investigated protein levels in the CB5. TRPV1 protein was significantly augmented after FM induction (Fig. 3A, * p ˂ 0.05, n = 6). In contrast, it was diminished by ACE handling and TRPV1 gene deletion, compared to the FM group. (Fig. 3A, # p ˂ 0.05, n = 6). We observed similar overexpression patterns of pPKA, pPI3K, and pPKC compared to the normal group that was attenuated in the ACE and Trpv1 −/− individuals (Fig. 3A, # p ˂ 0.05, n = 6). pERK, pp38, pJNK, pCREB, and pNFκB levels were amplified after FM induction in the CB5 region (Fig. 3B, * p ˂ 0.05, n = 6). These effects were relieved after ACE treatment or TRPV1 gene deletion (Fig. 3B, # p ˂ 0.05, n = 6). Using immunofluorescence, we showed TRPV1 is expressed in the CB5 of normal mice. TRPV1 was increased after FM induction in the mouse CB5 (Fig. 3C, green color, n = 4). The level was then reduced by ACE and TRPV1 gene deletion groups (Fig. 3C, green, n = 4). A comparable finding was perceived for pERK (Fig. 3C, red color, n = 4). Colocolization of overexpressed TRPV1 or pERK had been detected in the CB5 of FM mice (Fig. 3C, yellow color, n = 4). The amplified pattern was decreased in ACEor TRPV1 groups (Fig. 3C, yellow color, n = 4).
We used immunofluorescence to determine the localization of TRPV1 and pERK in CB6 and CB7 following FM induction. TRPV1 levels were significantly increased in the CB6 and CB7 in FM mice (Figs. 4,5C, green fluorescence, n = 4), while the result was diminished in the ACE and Trpv1 −/− groups (Figs. 4,5C). These expression profiles were also observed for pERK (Figs. 4,5C, red fluorescence, n = 4). A parallel trend was similarly detected for TRPV1 and pERK colocalization (Figs. 4,5C, yellow fluorescence, n = 4).

Discussion
We reveal that the FM model mimics the clinical symptoms of chronic mechanical and thermal nociception in FM mice. We then presented that ACE at ST36 site reliably attenuated either mechanical or thermal pain. Similar decreases in pain-related behaviors were also found in mice lacking TRPV1 receptors. TRPV1 and related kinases were all increased in the mice Hypo and CB, which are brain areas involved in pain modulation. These molecules were sequentially diminished by ACE and TRPV1 gene deletion, signifying that the therapeutic effect of ACE is linked with TRPV1 in the mice Hypo and CB. Our data provide suggestion that TRPV1 signaling pathway molecules could potentially be beneficial targets for FM treatment.
Peripheral nerve stimulation (PNS) is extensively used in several clinical scenarios, especially for pain control. ACE shares similar effects as PNS and transcutaneous electrical nerve stimulation for chronic pain management. PNS is utilized in several chronic pain situations, such as peripheral nerve injury, local pain syndrome, and FM [24,25]. Gilmore et al. [26] reported that chronic neuropathic pain is an underdiagnosed nociceptive condition following elimination. They verified the possibility of inserting fine-wire PNS inserts into the sciatic and femoral nerves for pain control. They suggest that PNS may deliver noteworthy pain assistance and facilitate recovery in chronic neuropathic pain patients. They also showed that 60-day PNS stimulation achieves continuous control of chronic pain [26]. A recent article showed that PNS works through both peripheral and central mechanisms. In its central mechanism, PNS can activate dorsal horn interneurons to block pain signals, attenuating central sensitization and hyperalgesia. In the spinal cord, PNS may increase the release of neurotransmitters, including Gamma-aminobutyric acid (GABA), glycine, serotonin, and cannabinoid pathways  [27]. PNS in peripheral sites can attenuate nociceptive afferent signals via changes in the local microenvironment, decrease in neurotransmitters and inflammatory cytokines, and frequency of neural discharge [28,29].
Cui et al. [30] showed that ACE can alleviate the overexpression of N-methyl-D-aspartate receptor (NMDA), pCaMKII, pERK, and pCREB by CFA-treated rats. The shot of the serotonin 1A receptor agonist mimicked the properties of ACE and was further blocked by the serotonin 1A receptor blocker WAY-100635 [30]. Du et al. [31] reported that ACE has analgesic effects measured as withdrawal thresholds and can decrease paw edema in a mouse inflammatory pain model. ACE markedly ameliorated the increased protein level of the sigma receptor in the mouse lumbar spinal cord. ACE also attenuated the increase in pERK and pp38, and injection of the sigma receptor agonist PRE084 significantly reversed the phenomena [31]. Our recent publications show that EA can relieve mice FM pain through TRPV1 pathway [8,10]. The current study further determined that ACE has an analgesic effect through suppression of TRPV1 activity similar to EA. These results indicate the beneficial effect of ACE for continuous and prolonged treatment for FM.
Recently, we showed that EA can relieve both acute and chronic pain through the TRPV1 pathway. In this study, we provide evidence that cold stress chronic pain may be associated with increased TRPV1 in the mouse brain and that ACE exerts its analgesic effect via suppression of TRPV1 activity in these areas. To address this issue, we induced chronic pain by cold stress and observed painrelated behaviors in mice. Consistent with our hypothesis, cold stress meaningfully convinced mechanical and thermal hyperalgesia, and ACE reliably reversed this nociception. In addition, chronic pain induction increased TRPV1 signaling pathway and correlated kinases in the mouse Hypo and CB. Furthermore, ACE and TRPV1 gene deletion dramatically reduced these molecular changes, suggesting that TRPV1-induced central sensitization can be reversed.

Conclusions
We demonstrate that the antinociceptive effect of ACE is dependent on TRPV1 pathway in the mouse brain and that TRPV1 signaling pathways participated in pain control in the FM model (Fig. 6). We thereby propose that these could be possible novel targets for the management of chronic pain.

Availability of Data and Materials
The data used to support the findings of this study are available from the corresponding author upon request.

Author Contributions
PCL, CMY, and IHH performed the experiments, analyzed and interpreted the data, and wrote the draft manuscript. YHC advice on the Western blot and ELISA experiments. YWL analyzed the data and draw the figures. YHC and YWL designed the research study and revised and submitted the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Ethics Approval and Consent to Participate
The treatment of mice was permitted by China Medical University Committee (CMUIACUC-2022-424), Taiwan, subsequent the guideline for the practice of experimental animals (National Academy Press).

Funding
This research was funded by Tzu Chi University TTCRD112-14, China Medical University CMU111-S-25, 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.