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. 2025 Jul 8;20:622. doi: 10.1186/s13018-025-05986-8

Acupressure attenuates fibromyalgia central sensitization via MAPK phosphorylation-mediated neuroinflammation regulation

Kezhi Liu 1,#, Yuye Lin 1,#, Tianyu Rao 1, Yuting Huang 1,✉, Yu Kan 1,✉, Jun Liao 1,✉
PMCID: PMC12235862  PMID: 40629417

Abstract

Background

Fibromyalgia syndrome (FMS) is a chronic musculoskeletal disorder with unclear pathogenesis and a lack of targeted therapies. Central sensitization has emerged as a key mechanism that drives its pathophysiology. Although clinical studies support the efficacy of acupressure (ACP) in alleviating FMS symptoms, its underlying mechanisms remain poorly understood. This study investigates how ACP modulates central sensitization and inflammatory pathways in a rat model of FM.

Methods

An FM rat model was developed to evaluate the efficacy of ACP therapy. Pain sensitivity and emotional distress were assessed using behavioral tests. Molecular analyses, including Western blot and immunofluorescence, were used to measure MAPK pathway activity (phosphorylation of p38 MAPK and JNK MAPK), glial cell activation (microglia and astrocytes), and inflammatory cytokine levels (TNF-ɑ, IL-6). Post-translational modifications were analyzed to explore anti-inflammatory mechanisms.

Results

ACP therapy significantly reduced pain sensitivity in FM rats. Mechanistically, ACP inhibited phosphorylation of p38 MAPK and JNK MAPK, suppressed glial cell activation, reduced pro-inflammatory cytokine release, and eventually attenuated central sensitization. The anti-inflammatory effects were mediated primarily by regulating post-translational modifications rather than altering protein synthesis or degradation. Additionally, ACP restored muscle function and demonstrated therapeutic effects on somatic manifestations of FMS.

Conclusion

ACP exerts multimodal therapeutic effects on FMS through dual modulation of MAPK signaling, which targets central sensitization and inflammation through precision-driven post-translational regulation. Its ability to alleviate physical symptoms highlights its potential as a targeted intervention for FMS. These findings provide mechanistic insights into ACP’s clinical efficacy and support its integration into FMS management strategies.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13018-025-05986-8.

Keywords: Fibromyalgia syndrome, Acupressure, MAPK, Phosphorylated

Introduction

Fibromyalgia syndrome (FMS) is a chronic musculoskeletal disorder characterized by persistent pain and complex clinical manifestations that severely impair patients’ quality of life [1]. Although the pathogenesis of FMS remains elusive, central sensitization is recognized as the primary pathological mechanism [2]. During this process, Substance P (SP) serves as a key mediator, particularly in spinal glial cell activation and pain signal transmission [3]. Its pathological role is further supported by elevated SP levels in the cerebrospinal fluid of FMS patients [4]. Nociceptive nerve fibers release SP and activate the Neurokinin 1 (NK-1) receptor to facilitate the propagation of pain signals [5]. Binding to the NK-1 receptor, SP activates astrocytes, downregulates the expression of anti-inflammatory IL-10, and ultimately promotes spinal central sensitization [6]. Concurrently, SP stimulates microglial cells to produce pro-inflammatory cytokines, such as TNF-α and IL-1β [7]. Microglial-derived IL-10 binds to astrocytic IL-10 receptors and then triggers the release of TGF-β. This feedback loop suppresses the production of microglial IL-1β, which highlights the dynamic interplay between glial cells in the progression of FMS [8].

The MAPK signaling pathway is a key mediator of chronic pain and central sensitization in FMS, with its activation closely linked to inflammatory cascades [9]. Notably, the P38 and JNK subfamilies of MAPK regulate the progression of neuropathic pain by modulating pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6. Clinical evidence further confirms that P38 MAPK is a potential biomarker and therapeutic target in FMS, which reflects its dual role in nociception and inflammation [10–12]. Mechanistically, SP binds to the NK-1 receptor on macrophages, induces P38 phosphorylation, triggers the release of cytokines and amplifies inflammatory pain signaling [13, 14]. This cytokine-driven MAPK activation forms a self-sustaining loop that perpetuates chronic pain [15]. The hierarchical regulation of the MAPK pathway—via the upstream kinases MKK3/6 (P38-specific) and MKK7 (JNK-specific)—provides a framework for understanding the pathophysiology of FMS and developing therapeutic strategies [16–19].

FMS remains a clinical challenge due to its unclear pathogenesis and the absence of universally effective therapies [20]. Current pharmacological interventions, including tricyclic antidepressants and anticonvulsants, are limited by suboptimal efficacy and adverse effects, which restrict their clinical utility. In contrast, the European League Against Rheumatism (EULAR) prioritizes non-pharmacological approaches as first-line management [21]. Acupressure (ACP) therapy has emerged as a complementary intervention with clinical benefits in pain relief, anxiety reduction, and quality of life improvement [22–24]. However, the therapeutic mechanisms of ACP and the fundamental pathophysiology of FMS require deeper exploration. Therefore, this study investigates the anti-inflammatory and analgesic mechanisms of ACP in a rat model of FMS by analyzing spinal glial cell activation, inflammatory cytokine profiles, and central sensitization status. The expression patterns of MAPK signaling components and their upstream kinases are further evaluated to elucidate how ACP attenuates central sensitization in FMS.

Methods

Animal

Male Sprague Dawley (SD) rats, weighing (150 ± 20) g and aged 5–6 weeks, were provided and housed by the Animal Experiment Center of Fujian University of Traditional Chinese Medicine (Qishan Campus). The license number of experimental animals was SYXK (Min) 2019-0007. The rats were housed in an SPF-grade animal laboratory under controlled conditions at a temperature of 22℃-26℃, a humidity level of 40-70%, and a 12-hour light/12-hour dark cycle. In this study, all procedures strictly adhered to the “Guidelines on the Humane Treatment of Laboratory Animals” and the relevant regulations of the Animal Ethics Committee of Fujian University of Traditional Chinese Medicine.

After one week of the acclimatization period, 45 SD rats were stratified by body weight and randomized into three groups (n = 15 per group) through the Proc Plan randomization procedure in SPSS 22.0: (i)Control group: No FM model induction or ACP intervention; rats were subjected to restraint only; (ii)Model group: FM model induction without ACP intervention; rats were subjected to restraint only; (iii)ACP group: FM model induction followed by daily ACP intervention for 7 consecutive days.

FM model

An animal model was established to induce pain hypersensitivity in FM rats using the method of Rasha B. Abd-ellatief et al. [25]. On day 0, the left hindlimb of the rats was shaved and prepared using a small animal shaver under isoflurane anesthesia (flow rate: 0.8 L/min, concentration: 1%). A 0.45 × 16 mm needle was inserted into the belly of the left tibialis anterior muscle at a depth of approximately 2–4 mm. Then, 0.1 ml of 2-morpholinoethanesulfonic acid (10 mmol/L, pH = 4.0) was injected into different directions within the muscle belly. On day 5, the second intramuscular injection of 2-morpholinoethanesulfonic acid was given using the same method. Subsequently, the rats were kept under normal conditions until day 14 to complete the model replication(Fig. 1.A).

Fig. 1.

Fig. 1

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Establishment of FM rats. (A) Experimental procedures in the rat model of FM. (a) Injection. (b) Intervention. (B) Design and real-life images of the ACP strength tester used in animal experiments. (C) Schematic diagram of the actual operation. (D-E) After modeling, three rats were randomly selected from each group to detect the transverse section (TS) and longitudinal section (LS) of tibialis anterior muscle

ACP intervention

ACP intervention was administered at the Zusanli acupoint (ST36). To minimize experimental variability, a customized force-feedback device was employed throughout the animal experiments. The force curve data generated by an integrated computer visualization module were used to dynamically adjust the force (4 ± 0.1 N) and compression frequency of the acupressure in real time. This system ensured standardized intervention parameters across all sessions, which effectively mitigated operator-dependent variability in manual techniques(Fig. 1.B-C).

Behavioral test

The Von Frey test was employed to measure the mechanical paw withdrawal threshold of rats before modeling, after modeling, and after intervention. The test was conducted using Von Frey filaments at the bending forces of 0.6, 1, 1.4, 2, 4, 6, 8, 10, and 15 g. These filaments were applied perpendicularly to the plantar surface of the rat’s hind paw to observe its response to the mechanical stimulus. A positive response characterized by paw withdrawal was recorded. The up-and-down method was used to determine the 50% mechanical paw withdrawal threshold. The filament size was gradually adjusted according to the response of the rats. The threshold value was calculated through an online tool [26].

The Open Field test involved placing rats in an open, enclosed arena (100 cm × 100 cm × 50 cm) with a black floor, and recording their behavior in a video tracking system. Parameters such as total distance traveled, head movement latency, and time spent in a stationary position were assessed. During the trial, the arena was thoroughly cleaned with 75% ethanol to eliminate olfactory cues. This test was performed before modeling, after modeling, and after intervention to evaluate the effects of the intervention on anxiety and locomotor activity.

The Gait analysis was conducted using the Runway Scan™ 3.0 platform gait analysis system, and rats were trained before testing. During the test, rats were allowed to freely traverse a pre-set length of the testing corridor. Their paw prints were captured using a light source and analyzed via video to assess movement patterns. Key gait parameters, such as stride length, swing time, stance time, stride time, brake time and propulsion time, were evaluated under natural walking conditions. All tests were conducted in a darkroom environment, and each rat underwent at least three trials for measurement.

HE staining

The left tibialis anterior muscle tissue of the rats was fixed in 4% paraformaldehyde solution. After dehydration, the tissue was embedded in paraffin with xylene and hydration in various graded ethanol solutions. Paraffin Sect. (5 μm thick) of the tibialis anterior muscle were prepared. To examine the morphology of the tissue, the sections were stained with hematoxylin and eosin, mounted with neutral gum, and observed under a microscope.

Enzyme linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) kits were used to detect the expression of inflammatory factors, including TNF-α, IL-1β, PGE2, IL-10, and TGF-β, in the lumbar spinal cord of the rats. The CurveExpert 2.20 software was used for standard curve fitting. The concentrations of sample indicators were calculated for statistical analysis.

Immunohistochemistry

Immunofluorescence staining was performed to detect the expression of SP, OX-42 (microglia marker), and GFAP (astrocyte marker) in the rat lumbar spinal cord. Co-localization analysis was further conducted between OX-42 and p-P38, as well as GFAP and p-JNK. After embedding the lumbar spinal cord tissue of the rats, 20 μm thick spinal cord sections were prepared with a cryostat, and stored at 4℃. After warming to room temperature, the blocking solution was applied for 30 min. Then, the blocking solution was discarded, and primary antibodies (SP 1:500, GFAP 1:500, OX-42 1:500) were added and incubated at 4℃ overnight in the dark. For GFAP/p-JNK co-localization, sections were incubated with a mixture of mouse anti-GFAP (1:500) and rabbit anti-phospho-JNK (1:1000); for OX-42/p-P38 co-localization, sections were treated with mouse anti-OX-42 (1:500) and rabbit anti-phospho-P38 (1:1000). After discarding the primary antibodies and washing, secondary antibodies were added, and sections were incubated for 60 min at 37 °C in the dark. After discarding the secondary antibodies, GFAP and OX-42 sections were mounted with the anti-fade mounting medium containing DAPI. The fluorescence intensity of SP, OX-42, and GFAP in the lumbar spinal cord of the rats was assessed.

Western blot

The expression of MKK3/6, P38 MAPK, p-P38 MAPK, MKK4/7, JNK MAPK, and p-JNK MAPK proteins in the lumbar spinal cord of the rats was detected using Western blot. The total cell protein was extracted by lysing the tissue on ice for 30 min, followed by centrifugation for 10 min at 12,000 r/min. The supernatant was collected as the protein sample. The samples were heated in a boiling water bath for 10 min, followed by cooling. The protein samples were loaded, subjected to electrophoresis, transferred onto membranes, and blocked. The gel was washed with transfer buffer, while filter paper and PVDF membranes of a similar size to the gel were cut and immersed in transfer buffer for 5–10 min. The diluted primary antibodies were added to the PVDF membranes: MKK3 (1:50,000), MKK6 (1:2,000), P38 MAPK (1:50,000), p-P38 MAPK (1:5,000), MKK4 (1:6,000), MKK7 (1:6,000), JNK MAPK (1:50,000), p-JNK MAPK (1:20,000), followed by incubation at 4 °C overnight on a shaker. The next day, the primary antibodies were washed off, followed by adding the secondary antibodies (goat anti-rabbit IgG 1:5,000, goat anti-mouse IgG 1:5,000). The mixture was then incubated at room temperature for 1 h. After adding a rapid chemiluminescent substrate for color development and exposure, the bands were analyzed and quantified using the ImageJ software. The ratio of the grayscale values of the target protein to the internal reference protein was calculated to determine the relative expression level of the target protein.

Statistical analysis

The SPSS 22.0 statistical software was used to analyze data. The results were expressed as mean ± standard deviation (Mean ± SD). Intergroup differences were analyzed using ANOVA. Comparisons before and after treatment were performed using the t-test. Differences between groups were compared using a one-way analysis of variance (ANOVA). For longitudinal analysis of two factors, two-way repeated-measures ANOVA (RM-ANOVA) was applied. P < 0.05 was set as the significance threshold. Pairwise comparisons between multiple groups were also conducted.

Results

ACP effectively alleviated hyperalgesia in the FM model rats

Before the intervention, three rats from the control group, the model group, and the ACP group were selected, and HE staining confirmed successful modeling (Fig. 1.D-E). To assess changes in the pain threshold in the FM model rats, von Frey mechanical threshold testing was performed before modeling, after modeling, and after ACP intervention. The 50% paw withdrawal mechanical threshold (PWMT) was observed. The results showed that the FM rats exhibited a reduced pain threshold after modeling. However, after ACP intervention, hyperalgesia was improved, as shown in Fig. 2.A.

Fig. 2.

Fig. 2

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ACP promotes functional recovery in FM rats. Data are presented as mean ± SD (n = 6). (A) Changes in 50% PWMT of FM rats. Data are presented as mean ± SD (n = 6). Day 14: ***p < 0.001 vs. Control group; ###p < 0.001 vs. Control group. Day 21: ***p < 0.001 vs. Control group; ###p < 0.001 vs. Model group. (B) Standing time: Day 14: ***p < 0.001 vs. Control group; ###p < 0.001 vs. Control group. Day 21: ***p < 0.001 vs. Control group; ###p < 0.001 vs. Model group. (C) Braking time: Day 14: **p < 0.01 vs. Control group; ##p < 0.01 vs. Control group. Day 21: ***p < 0.001 vs. Control group; ###p < 0.001 vs. Model group. (D) Propulsion time: Day 14: ***p < 0.001 vs. Control group; ###p < 0.001 vs. Control group. Day 21: ***p < 0.001 vs. Control group; ###p < 0.001 vs. Model group. (E) Swing time: No significant differences on day 14 and day 21. (F) Stride time: Day 14: ***p < 0.001 vs. Control group; ###p < 0.001 vs. Control group. Day 21: **p < 0.01 vs. Control group; ##p < 0.01 vs. Model group. (G) Total distance: Day 14: **p < 0.01 vs. Control group; ##p < 0.01 vs. Control group. Day 21: ***p < 0.001 vs. Control group; ###p < 0.001 vs. Model group. (H) Resting time: No significant differences on day 14 and day 21. (I) Head exploration latency: No significant differences on day 14 and day 21. (J) Track map of rat open field experiment. (K) TS of tibialis anterior muscle HE staining. Red arrows indicate positive reaction sites with disrupted muscle fibers. (L) LS of tibialis anterior muscle HE staining. White arrows indicate positive reaction sites with muscle fiber atrophy and nuclear translocation

ACP effectively promoted the morphological and functional repair of the tibialis anterior muscle tissue in the FM model rats

HE staining demonstrated that the tibialis anterior muscle fibers in the control group were tightly arranged in the transverse sections, with uniform nucleus size and staining, which indicated normal muscle structure and function. In contrast, the muscle fibers in the model group were loosely arranged, with an irregular widening of the intermuscular space, inconsistent nuclear morphology, and some ruptured nuclei with uneven staining. These changes may be associated with the muscle damage and inflammation caused by FMS. Additionally, perimysium and endomysium were significantly thickened, which suggested the occurrence of inflammation or fibrosis. The structure of the ACP group was clearer than that of the model group, with slightly widened intermuscular spaces and a few displaced nuclei. However, the nucleus size and staining remained uniform, which indicated that the ACP intervention helped to restore normal muscle morphology and reduced pathology (Fig. 2.K). These changes were further confirmed by longitudinal section analysis. Unlike the control group, which showed neatly and continuously arranged muscle fibers, the model group exhibited disorganized fiber alignment and significant inflammatory infiltration, which indicated structural damage to the muscle tissues caused by FMS. After intervention, the ACP group displayed relatively orderly muscle fiber alignment, with uniform fiber thickness and a small amount of inflammatory infiltration. However, the overall structure remained intact, which suggested that ACP alleviated inflammation and promoted tissue repair to some extent (Fig. 2.L).

Gait analysis revealed the negative impact of FMS on motor functions in the model rats(Fig. 2.B-F). After modeling, the resting time, propulsion time, and braking time in all groups were significantly increased, while the stride time was decreased. After intervention, the resting time, propulsion time, and braking time were significantly reduced in the ACP group. This alleviated the motor dysfunction induced by FMS and highlighted the effectiveness of ACP in improving motor ability. There were no significant differences in the swing time between the groups.

The open field test (Fig. 2.G-J) showed that the total distance traveled by FMS model rats was significantly reduced compared to the control group, which supported that FMS induces movement restriction. After the ACP intervention, the total distance traveled by rats from the ACP group was significantly increased, which indicated that ACP could promote exploratory behavior and improve activity levels. No significant differences in head movement latency and stationary time were observed before and after intervention.

ACP-Mediated MAPK phosphorylation signaling in astrocytes

SP is a neuropeptide that acts as a key activator of both astrocytes and microglia, which was prominently expressed in the spinal dorsal horn layer II of FM model rats. ACP intervention significantly suppressed overexpression of SP (Fig. 3.A) and interrupted its dual glial-activating effects. GFAP and OX-42 serve as markers of astrocyte and microglial cell activation, respectively, which reflect changes in cell proliferation and activation [27, 28]. Astrocyte activation is characterized by elevated GFAP expression and hypertrophic morphological changes such as enlarged cell bodies and increased protrusions, which was observed in the spinal dorsal horn of the model group. ACP treatment significantly reduced GFAP expression and restored astrocytic morphology to a quiescent state. Co-localization analysis revealed that phosphorylated JNK, a critical mediator of the MAPK pathway, was predominantly localized to GFAP-positive astrocytes, which indicated JNK-driven neuroinflammatory signaling during fibromyalgia progression. ACP significantly attenuated p-JNK/GFAP co-localization, which demonstrated selective inhibition of JNK-mediated astrocytic activation (Fig. 4.A-B). Notably, the pro-inflammatory cytokine IL-1β exhibited an expression pattern similar to anti-inflammatory factors TGF-β and IL-10 in the ACP group (Fig. 3B-D). However, this coordinated modulation involved MKK7 upregulation—a kinase against the canonical MAPK cascade—rather than MKK4, which showed no significant changes across groups. These findings suggest that ACP alleviates pain by regulating the astrocyte-specific MAPK pathway (Fig. 4C-D).

Fig. 3.

Fig. 3

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ACP modulates inflammatory factor expression via glial cell regulation. (A) Immunofluorescence analysis of SP expression in the lumbar spinal cord after ACP intervention. SP (green), DAPI (blue) (scale bar: 100 μm). Data are presented as mean ± SD (n = 6). **p < 0.01 vs. Control group; #p < 0.05 vs. Model group. (B-F) Changes in inflammatory cytokine expression in the lumbar spinal cord after ACP intervention. (B) TGF-β expression. ****p < 0.0001 vs. Control group; ##p < 0.01 vs. Model group; (C) IL-1β expression. **p < 0.01 vs. Control group; ##p < 0.01 vs. Model group; (D) IL-10 expression. **p < 0.01 vs. Control group; #p < 0.05 vs. Model group; (E) TNF-α expression. **p < 0.01 vs. Control group; ##p < 0.01 vs. Model group; (F) PGE2. Data are presented as mean ± SD (n = 6)

Fig. 4.

Fig. 4

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ACP modulates astrocytes via p-JNK regulation. (A) Co-localization of GFAP and p-JNK in the lumbar spinal cord after ACP intervention. Immunofluorescence staining shows GFAP (green), p-JNK (red), and DAPI (blue). Merged images indicate co-localization of GFAP and p-JNK. Scale bar: 100 μm. Data are presented as mean ± SD (n = 6). ****p < 0.0001 vs. Control group; ####p < 0.0001 vs. Model group. (B) Expression of JNK and p-JNK proteins in the lumbar spinal cord. ***p < 0.001 vs. Control group; ##p < 0.01 vs. Model group. (C) Expression of MKK7 protein in the lumbar spinal cord. ***p < 0.001 vs. Control group; ###p < 0.001 vs. Model group. (D) Expression of MKK4 protein in the lumbar spinal cord. Data are presented as mean ± SD (n = 3)

ACP-Mediated MAPK phosphorylation signaling in microglia

Concurrent with astrocytic changes, microglial activation in FM rats was evidenced by the upregulation of OX-42 expression along with morphological hypertrophy and process ramification. ACP intervention effectively normalized microglial morphology and reduced OX-42 levels, which further corroborated the role of SP as a dual glial activator. Mechanistically, phosphorylated P38 showed strong co-localization with OX-42-positive microglia, which implicated that P38 MAPK is a central driver of microglial activation (Fig. 5A). ACP treatment significantly suppressed p-P38/OX-42 co-localization, which indicated targeted blockade of P38 signaling. This inhibition aligned with a distinct cytokine shift: reduced TNF-α and elevated IL-10 levels (Fig. 3D-E), which highlighted ACP’s dual control over microglia-dependent pro- and anti-inflammatory networks. Critically, unaltered PGE2 levels across groups excluded the involvement of the prostaglandin pathway (Fig. 3F), which emphasized the specificity of SP-MAPK crosstalk. Downstream analysis revealed that ACP reduced phosphorylation of MKK6/7, upstream activators of P38 and JNK, without altering total MAPK protein levels, which was consistent with post-translational regulation (Fig. 5B-D). The attenuated glial-MAPK interaction, encompassing both astrocytes and microglia, correlated with improved mechanical hypersensitivity and motor function recovery. This underscored the necessity of coordinated glial deactivation in reversing central sensitization and pain pathway dysregulation.

Fig. 5.

Fig. 5

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ACP modulates microglia via p-P38 regulation. (A) Co-localization of OX-42 and p-P38 in the lumbar spinal cord after ACP intervention. Immunofluorescence staining shows OX-42 (green), p-P38 (red), and DAPI (blue). Merged images indicate co-localization of OX-42 and p-P38. Scale bar: 100 μm. Data are presented as mean ± SD (n = 6). ****p < 0.0001 vs. Control group; ###p < 0.001 vs. Model group; **p < 0.01 vs. Control group; ##p < 0.01 vs. Model group. (B) Expression of P38 and p-P38 proteins in the lumbar spinal cord. ***p < 0.001 vs. Control group; ##p < 0.01 vs. Model group. (C) Expression of MKK6 protein in the lumbar spinal cord. ***p < 0.001 vs. Control group; ##p < 0.01 vs. Model group. (D) Expression of MKK3 protein in the lumbar spinal cord. **p < 0.01 vs. Control group; ##p < 0.01 vs. Model group. Data are presented as mean ± SD (n = 3)

Discussion

This study demonstrates that ACP can effectively ameliorate adverse effects in FM model rats. The histopathological evaluation of tibialis anterior muscle sections via HE staining visually revealed the robust potential of ACP in repairing FMS-damaged muscle groups. Gait analysis further indicated that FMS impaired motor performance, including locomotion capacity, balance ability, and overall motor function, while these deficits were significantly alleviated by ACP intervention. These findings suggest that the tissue-repairing capacity of ACP may be mediated through direct actions on muscle tissues to restore structural and functional integrity.

In animal models, antinociceptive effects can be observed in substances that inhibit the SP signaling pathway [29]. In this study, ACP demonstrated superior analgesic efficacy, which significantly reduced the expression levels of SP. Activation of the SP-NK1 receptor axis drives P38 MAPK and JNK MAPK phosphorylation cascades, which exacerbates central sensitization. Western blot results revealed that ACP suppressed the expression of p-P38 MAPK by downregulating the upstream kinases MKK3 and MKK6, which attenuated central sensitization. At the same time, it inhibited the p-JNK MAPK pathway via MKK7 reduction. Notably, no intergroup differences were observed in MKK4 expression. Previous studies have indicated that MKK4 expression in the central nervous system peaks at postnatal day 18 [30]. However, the tissue sampling occurred well beyond this developmental window, which suggested that MKK4 may not be involved in the pathogenesis of FMS. Although it has been reported that MKK4 activates P38 MAPK and its deficiency reduces JNK and P38 MAPK activation [15, 31], this trend was not reflected in this study. Intriguingly, prior research has proposed an alternative MKK4 activation mechanism linked to TNF-α secretion [15]. This hypothesis is consistent with the findings, as the expression trend of p-P38 is correlated closely with TNF-α levels, which offers novel insights into MAPK signaling dynamics in FMS.

Glial cell activation plays an important role in central sensitization, as evidenced by their aberrant expression in FM model rats. ACP intervention normalized the expression of OX-42 and GFAP, accompanied by a significant reduction in glial process density. This is consistent with previous studies showing that P38/JNK kinase inhibitors suppress glial activation and neuroinflammation [10, 12]. These results indicate that ACP alleviates hyperalgesia and inflammation by inhibiting the MAPK signaling pathway (P38 and JNK).

Pro-inflammatory cytokines sensitize and activate nociceptors to induce pain in humans and hyperalgesia in animals [32]. Notably, microglia-derived IL-1β [7] exhibited a paradoxical trend and expression levels mirrored anti-inflammatory cytokines. This aligns with the reports of similar IL-1β dynamics in cerebrospinal fluid but contrasts patterns in serum [21, 33, 34]. Intriguingly, this phenomenon may be explained by a non-canonical IL-1β secretion pathway—unaffected by brefeldin A and monensin (which suppress TNF-α release) [35]. It can be hypothesized that microglial polarization drives these observations. Activated microglia can be polarized into M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes: M1 releases TNF-α, IL-6, and IL-1β, while M2 secretes IL-4 and IL-10, counteracting M1 activation [36, 37]. In FMS model rats, the central nervous system may exhibit a predominant M2-polarized microglial state, which leads to reduced IL-1β release compared to the control group. Prostaglandin E2 (PGE2) is closely associated with inflammatory pain and can induce hyperalgesia, which is primarily related to peripheral sensitization [38, 39]. The results show no significant difference in PGE2 expression between the lumbar spinal cord groups. This suggests that PGE2 may not be involved in the central sensitization mechanism of the FM model, particularly at the spinal cord level.

Based on the above results, these findings suggest that pain hypersensitivity in the FM model is jointly modulated by glial cells and inflammatory mediators through crosstalk mechanisms, both of which are controlled by the MAPK signaling pathway (Fig. 6). The release of IL-1β plays a specific role and cannot be simply considered as a pro-inflammatory factor released by glial cells. However, its complex mechanism remains unexplored. ACP can effectively alleviate pain and central sensitization by inhibiting the expression of P38 MAPK and JNK MAPK signaling pathways, which significantly reduces the expression of inflammatory factors.

Fig. 6.

Fig. 6

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Schematic illustration of ACP-mediated MAPK signaling in astrocytes and microglia

Conclusion

This study demonstrates that ACP effectively alleviates pain hypersensitivity and central sensitization in FM model rats by modulating inflammatory pathways, including MAPK signaling cascades. These findings demonstrate that the synergistic interaction between glial cells and inflammatory mediators plays an important role in pain hypersensitivity. Compared to normal physiological conditions, the release of IL-1β exhibits distinctive features in this process. Furthermore, ACP regulates glial cell activation and attenuates inflammatory cytokine expression, predominantly by targeting post-translational modifications rather than altering protein synthesis or degradation. This precision in disrupting activated signaling hubs while maintaining basal cellular homeostasis provides strong evidence for the potential of ACP as an effective therapeutic intervention for FMS.These results support the hypothesis that ACP may provide a novel approach for FMS-related pain treatment.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (4.6MB, docx)

Acknowledgements

This work was supported by the National Natural Science Foundation of China (NO. 82074181, NO.82204908, NO.82374202).

Author contributions

K.L. and Y.L. wrote the main manuscript text. K.L., Y.L., T.R., J.L., Y.K., Y.H. participated in experimental procedures. J.L., Y.K., and Y.H. provided guidance on experimental design and implementation and secured funding. All authors reviewed and approved the final manuscript.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Clinical trial number

Not applicable.

Ethics approval

All animal experiments in this study strictly adhered to the “Guidelines on the Humane Treatment of Laboratory Animals” and the relevant regulations of the Animal Ethics Committee of Fujian University of Traditional Chinese Medicine.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Kezhi Liu and Yuye Lin have contributed equally to the work.

Contributor Information

Yuting Huang, Email: 836529518@qq.com.

Yu Kan, Email: 2019035@fjtcm.edu.cn.

Jun Liao, Email: 2007065@fjtcm.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (4.6MB, docx)

Data Availability Statement

No datasets were generated or analysed during the current study.

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