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${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ 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 $\alpha$ 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${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ group (Group 4: FM + Trpv1${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$).

All animals were accommodated at 24 $\pm$ 1 °C before FM induction. In this ICS model, the animals were placed at $\pm$ 1 °C temperature overnight (starting from 4 PM–10 AM). Animals were next transported to 24 $\pm$ 1 °C temperature for a 30-minute duration in the next morning. The animals were further relocated back to 4 $\pm$ 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 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 $\times$ 25 mm (160925, Terumo Corporation, Tokyo, Japan), steel needle 0.35 $\times$ 40 mm (Suzhou Medical Appliance, Suzhou, China), or catgut suture 0.2 $\times$ 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.

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 $\times$ 25 $\times$ 45 cm) and then quarantined under plastic cage (10 $\times$ 6 $\times$ 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 hyperalgesia 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.

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 refrigerator for protein mining. All extracted samples were standardized with radioimmunoprecipitation (RIPA) with: 1% NP-40, 50 mM Tris-HCl, 0.02% NaN${}_3$, 250 mM NaCl, 1 mM Na${}_3$VO${}_4$, 5 mM EDTA, 50 mM NaF, and 1$\times$ protease blocker set (AMRESCO, Radnor, PA, USA). Total components were undergoing 8% SDS-Tris electrophoresis and conveyed to commercial polyvinylidene difluoride (PVDF). The membrane was rinsed with 3% bovine serum albumin (BSA, Merck, St. Louis, MO, USA) in TBS-T buffer (100 mM NaCl, 10 mM Tris pH 7.5, 0.1% Tween 20), nurtured with a primary antibody in TBS-T for 1 hour against anti-tubulin ($\sim$55 kDa, 1:5000, Merck, St. Louis, MO, USA), TRPV1 ($\sim$95 kDa, 1:1000, Alomone, Jerusalem, Israel), pPKA ($\sim$40 kDa, 1:1000, Alomone, Jerusalem, Israel), pPKC ($\sim$100 kDa, 1:1000, Millipore, St. Louis, MO, USA), pPI3K ($\sim$125 kDa, 1:1000, Millipore, St. Louis, MO, USA), pERK1/2 ($\sim$42–44 kDa, 1:1000, Millipore, St. Louis, MO, USA), pp38 ($\sim$41 kDa, 1:1000, Millipore, St. Louis, MO, USA), pJNK ($\sim$42 kDa, 1:1000, Millipore, St. Louis, MO, USA), pAkt ($\sim$60 kDa, 1:1000, Millipore, St. Louis, MO, USA), pmTOR ($\sim$180 kDa, 1:500, Millipore, St. Louis, MO, USA), pNF$\kappa$B ($\sim$65 kDa, 1:1000, Millipore, St. Louis, MO, USA), and pCREB ($\sim$55 kDa, 1:1000, Millipore, St. Louis, MO, USA) in TBS-T (1% BSA). The PVDF membranes were then edited for incubation with individual antibodies. Peroxidase-conjugated anti-mouse, anti-rabbit, or anti-goat antibodies (1:5000) were utilized as a secondary antibody (Merck, St. Louis, MO, USA). The western blot signals were imagined with improved chemiluminescent (PIERCE) with LAS-3000 Fujifilm (Fuji Photo Film Co., Ltd., Tokyo, Japan). Visible bands concentrations of precise molecular weight were counted with Image J software (NIH, Bethesda, MD, USA). The $\beta$-actin band was used as control.

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 °C refrigerator for 2–3 days. The mice tissue was fixed with optimal cutting temperature compound and quickly stored at –80 °C refrigerator. Mice brains were sliced and then placed on glass slides (20 µm). The tissues were further rinsed with 3% BSA, 0.1% Triton X-100, and 0.02% NaN${}_3$ (1 h). The tissues were then nurtured through the first antibody (1:100, Alomone, Jerusalem, Israel), anti-TRPV1 or anti-phosphorylated endoplasmic reticulum kinase (pERK) primed in 1% BSA in 4 °C refrigerator. Tissue slices were further nurtured with the secondary antibody (1:500), 488-conjugated AffiniPure donkey anti-rabbit IgG (Merck, St. Louis, MO, USA), 594-conjugated AffiniPure donkey anti-goat IgG (Merck, St. Louis, MO, USA) for 2 h at room temperature. The tissues were further examined with a microscope (BX-51, Olympus, Tokyo, Japan) with 20 $\times$ objective. The photos were examined with Image J software (NIH, Bethesda, MD, USA).

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.

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 $\pm$ 0.13, * p $\mathrm{˂}$ 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 $\pm$ 0.14 and 4.26 $\pm$ 0.17, ${}^{\mathrm{\#}}$ p $\mathrm{˂}$ 0.05, n = 10, respectively). ICS also induced thermal hyperalgesia, as observed in the Hargraves’ test (Fig. 1B, red circle, D14: 4.86 $\pm$ 0.2, * p $\mathrm{˂}$ 0.05, n = 10). Furthermore, ACE decreased thermal hyperalgesia in the FM mice (Fig. 1B, blue and green circles, D14: 7.15 $\pm$ 0.38 and 7.96 $\pm$ 0.17, ${}^{\mathrm{\#}}$ p $\mathrm{˂}$ 0.05, n = 10, respectively).

Fig. 1.

Mechanical, thermal pain, and inflammatory cytokines in normal, FM, FM+ACE, and FM+Trpv1${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ groups. (A) Mechanical force obtained via von Frey examinations. (B) Thermal time observed via the Hargreaves’ test. (C) IL-1$\alpha$, IL-1$\beta$, IL-2, IL-5. (D) IL-6, IL-9, IL-10, IL-12(p70). (E) IL-13, IL-17A, TNF-$\alpha$, IFN-$\gamma$. (F) MCP-1, and MIP-1$\alpha$. MIP-1$\beta$, RANTES. *shows statistical differences compared with normal mice. ${}^{\mathrm{\#}}$shows statistical differences compared with FM groups. n = 10 in four groups. FM, fibromyalgia; ACE, acupoint catgut embedding; Trpv1, Transient receptor potential vanilloid 1.

Neuroinflammation was detected in the blood and cerebral spinal fluid in FM patients. Accordingly, we determined the concentration of inflammatory cytokines in mouse plasma through multiplex ELISA technique. We measured interleukin-1 alpha (IL-1$\alpha$), IL-1$\beta$, IL-2, IL-5, IL-6, IL-9, IL-10, IL-12(p70), IL-13, IL-17A, tumor necrosis factor alpha (TNF-$\alpha$), interferon gamma (IFN-$\gamma$), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1$\alpha$), MIP-1$\beta$, and regulated on activation, normal T cell expressed and secreted (RANTES). Inflammatory cytokines were low in normal mice at basal conditions. IL-1$\alpha$, IL-1$\beta$, IL-2, IL-5, IL-6, IL-12, IL-17A, TNF-$\alpha$, IFN-$\gamma$, and MCP-1 were increased at 14 days after FM induction (Fig. 1C–F, red column, * p 0.05, n = 10). ACE meaningfully reduced inflammatory cytokines levels (Fig. 1C–F, blue column, ${}^{\mathrm{\#}}$ p 0.05, n = 10). These cytokines were next reduced in Trpv1${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ group (Fig. 1C–F, ${}^{\mathrm{\#}}$ p 0.05, n = 10, green column).

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 $\mathrm{˂}$ 0.05, n = 6). Further data showed that ACE reliably reversed the overexpression of TRPV1 (Fig. 2A, black column, ${}^{\mathrm{\#}}$ p $\mathrm{˂}$ 0.05, n = 6). TRPV1 was disappeared in hypothalamus of Trpv1${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice. The level of pPKA, pPI3K, and pPKC were amplified in FM group (Fig. 2A, * p $\mathrm{˂}$ 0.05, n = 6) and were reduced by ACE treatment or TRPV1 gene deletion (Fig. 2A, ${}^{\mathrm{\#}}$ p $\mathrm{˂}$ 0.05, n = 6). The level of pAkt and pmTOR were increased in the hypothalamus (Fig. 2A, * p $\mathrm{˂}$ 0.05, n = 6) but decreased in AC-treated and Trpv1${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice (Fig. 2A, ${}^{\mathrm{\#}}$ p $\mathrm{˂}$ 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${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice (Fig. 2B, ${}^{\mathrm{\#}}$ p $\mathrm{˂}$ 0.05, n = 6). pCREB and pNF$\kappa$B were both increased in the mouse hypothalamus (Fig. 2B, * p $\mathrm{˂}$ 0.05, n = 6) and were reduced by ACE or TRPV1 gene loss (Fig. 2B, ${}^{\mathrm{\#}}$ p $\mathrm{˂}$ 0.05, n = 6).

Fig. 2.

TRPV1 and linked kinases in the mice hypothalamus. The western blot signals of Normal, FM, ACE, and Trpv1${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$. (A) TRPV1, pPKA, pPI3K, pPKC, pAkt, and pmTOR. (B) pERK, pp38, pJNK, pCREB, and pNF$\kappa$B. *shows statistical differences compared with normal mice. ${}^{\mathrm{\#}}$shows statistical differences compared with FM groups. n = 10 in four groups. n = 6 in four groups. (C) Immunofluorescence signals of TRPV1, pERK, or colocalized labeling in the mice hypothalamus (green, red or yellow). Scale bar = 100 µm. n = 4 in four groups.

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${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice (Fig. 2C, yellow color, n = 4).

We investigated protein levels in the CB5. TRPV1 protein was significantly augmented after FM induction (Fig. 3A, * p $\mathrm{˂}$ 0.05, n = 6). In contrast, it was diminished by ACE handling and TRPV1 gene deletion, compared to the FM group. (Fig. 3A, ${}^{\mathrm{\#}}$ p $\mathrm{˂}$ 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${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ individuals (Fig. 3A, ${}^{\mathrm{\#}}$ p $\mathrm{˂}$ 0.05, n = 6). pERK, pp38, pJNK, pCREB, and pNF$\kappa$B levels were amplified after FM induction in the CB5 region (Fig. 3B, * p $\mathrm{˂}$ 0.05, n = 6). These effects were relieved after ACE treatment or TRPV1 gene deletion (Fig. 3B, ${}^{\mathrm{\#}}$ p $\mathrm{˂}$ 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).

Fig. 3.

The intensities of TRPV1 and associated factors in CB5. The western blot signals of Normal, FM, FM+ACE, and Trpv1${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ groups. We found intensifications of (A) TRPV1, pPKA, pPI3K, pPKC, pAkt, and pmTOR. (B) pERK, pp38, pJNK, pCREB, and pNF$\kappa$B. *shows statistical differences compared with normal mice. ${}^{\mathrm{\#}}$shows statistical differences compared with FM groups. n = 10 in four groups. n = 6 in all groups. (C) Immunofluorescence labeling of TRPV1, pERK, and colocalized labeling in CB5 (green, red or yellow). Scale bar = 100 µm. n = 4 in four groups.

Our data determined the TRPV1 and correlated kinases in the CB6 and CB7 in mice. TRPV1 proteins were significantly augmented after FM stimulation, compared with normal mice (Figs. 4,5A, black column, * p $\mathrm{˂}$ 0.05, n = 6). TRPV1 proteins were reduced in the ACE group and were not detected in Trpv1${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ mice (Figs. 4,5A, black column, ${}^{\mathrm{\#}}$ p $\mathrm{˂}$ 0.05, n = 6). FM mice showed augmented pPKA, pPI3K, or pPKC protein in the CB6 and CB7 (Figs. 4,5A, * p $\mathrm{˂}$ 0.05, n = 6). These proteins were reduced in the EA and Trpv1${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$groups (Figs. 4,5A, ${}^{\mathrm{\#}}$ p $\mathrm{˂}$ 0.05, n = 6). Similar results were observed for pAkt-pmTOR molecules (Figs. 4,5A, * p $\mathrm{˂}$ 0.05, n = 6). EA and Trpv1${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ also reduced aforementioned molecules (Figs. 4,5A, ${}^{\mathrm{\#}}$ p $\mathrm{˂}$ 0.05, n = 6). Overexpression of pERK, pp38, and pJNK was obtained in FM group (Figs. 4,5B, * p $\mathrm{˂}$ 0.05, n = 6) and was abrogated by EA and TRPV1 loss (Figs. 4,5B, ${}^{\mathrm{\#}}$ p $\mathrm{˂}$ 0.05, n = 6). Our data specified a similar profile for the expression of pCREB and pNF$\kappa$B (Figs. 4,5B, * p $\mathrm{˂}$ 0.05, n = 6).

Fig. 4.

TRPV1 and associated kinases in CB6. The western blot signals of Normal, FM, FM+ACE, and Trpv1${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$. (A) TRPV1, pPKA, pPI3K, pPKC, pAkt, and pmTOR. (B) pERK, pp38, pJNK, pCREB, and pNF$\kappa$B. *shows statistical differences compared with normal mice. ${}^{\mathrm{\#}}$shows statistical differences compared with FM groups. n = 10 in four groups. n = 6 in all groups. (C) Immunofluorescence labeling of TRPV1, pERK, and colocalized labeling in CB6 (green, red or yellow). Scale bar = 100 µm. n = 4 in four groups.

Fig. 5.

TRPV1 and associated kinases in CB7. The western blot signals of Normal, FM, FM+ACE, and Trpv1${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$. (A) TRPV1, pPKA, pPI3K, pPKC, pAkt, and pmTOR. (B) pERK, pp38, pJNK, pCREB, and pNF$\kappa$B. *shows statistical differences compared with normal mice. ${}^{\mathrm{\#}}$shows statistical differences compared with FM groups. n = 10 in four groups. n = 6 in four groups. (C) Immunofluorescence staining of TRPV1, pERK, and double staining in the mice CB7 (green, red or yellow). Scale bar = 100 µm. n = 4 in four groups.

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${}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}}$ 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).