Effects and Mechanisms of Wrist-Ankle Acupuncture on Inflammatory Pain in the Rat Gastrocnemius Muscle

Abstract

Objective

To compare the therapeutic effects of wrist-ankle acupuncture stimulation (WAA-AS) and wrist-ankle acupuncture theory-based transcutaneous electrical nerve stimulation (WAA-TENS), and explore the mechanism of wrist-ankle acupuncture (WAA) using transcriptomic analysis.

Methods

Sprague-Dawley rats were randomly divided into control (CON), model (MOD), WAA-TENS and WAA-AS groups. An inflammatory pain model was established by complete Freund’s adjuvant (CFA) injection into the gastrocnemius muscle. Mechanical pain thresholds, inflammatory infiltration and collagen deposition were evaluated. Inflammatory factors were measured by ELISA. Transcriptomic sequencing, PPI network analysis and RT-qPCR were performed.

Results

Compared with the model group, both WAA-TENS and WAA-AS significantly increased pain thresholds: post-intervention pain thresholds increased to ~7 g (WAA-AS) and ~7–8 g (WAA-TENS), a 2.2–3-fold elevation vs the persistently low ~2–3 g in the MOD group (p < 0.01), reduced inflammatory infiltration and collagen deposition, downregulated IL-1β, IL-6 and TNF-α, and upregulated TGF-β. Core circadian rhythm-related genes (Cd74, Cry1, Per2, Nfil3, Nr1d2, Npas2) were screened. RT-qPCR verified that WAA-TENS upregulated Per2 and Nr1d2 and downregulated Nfil3, while WAA-AS decreased Cry1 and Nfil3.

Conclusion

WAA-TENS and WAA-AS exert comparable analgesic and anti-inflammatory effects and inhibit excessive collagen synthesis. WAA-TENS may represent a promising alternative to WAA-AS for acute-phase intervention, based on our preliminary findings. The mechanism may involve regulating circadian rhythm-related genes including Cry1, Per2, Nfil3 and Nr1d2.

Introduction

Musculoskeletal pain is one of the most prevalent clinical complaints, affecting approximately 20% of adults and presenting a substantial public health challenge.^1,2 Inflammatory muscle pain, as a primary subtype, is closely associated with muscle injury and the release of inflammatory mediators.^3,4 Without timely intervention, it can lead to impaired motor function, reduced quality of life, and potentially progress into refractory chronic musculoskeletal disorders such as fibromyalgia, driven by persistent inflammation and ongoing pain.⁵ This progression places a significant economic burden on patients. Current pharmacological approaches, including nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids, can alleviate symptoms. However, long-term use is associated with gastrointestinal risks and the potential for addiction.^6,7 Therefore, developing safe, effective, rapid-acting, and easy-to-administer non-pharmacological treatments has become an urgent clinical priority.

Among numerous non-pharmacological therapies, wrist-ankle acupuncture (WAA) is a superficial needling technique⁸ that demonstrates unique advantages in pain management due to its safety, efficacy, and rapid onset of action. Clinical studies have confirmed that WAA acupuncture stimulation (WAA-AS) can significantly alleviate various acute and chronic pain conditions.⁹ However, as an invasive procedure, WAA-AS carries a risk of infection. Moreover, its therapeutic effect is highly operator-dependent, which hinders its standardized application in clinical practice.

Transcutaneous electrical nerve stimulation (TENS) is a non-invasive therapy that alleviates pain by delivering electrical currents to affected areas through surface electrodes.¹⁰ It is widely used in clinical practice for managing various acute and chronic pain conditions due to its ease of operation.¹¹ In recent years, transcutaneous electroacupuncture stimulation (TEAS) has garnered increasing interest. As a form of TEAS, wrist-ankle acupuncture-based TENS (WAA-TENS) integrates the standardized application of TENS with the analgesic benefits of WAA. Preliminary studies by our team indicate that WAA-TENS effectively relieves various pain conditions, including myofascial pain syndrome,¹² rotator cuff injuries,¹³ and pain during non-sedated colonoscopies,¹⁴ with no significant adverse effects reported. However, the clinical adoption of WAA has been hindered by its unclear mechanism of action. Existing studies have demonstrated that WAA exerts analgesic effects by elevating cerebral serotonin levels and increasing pain thresholds,¹⁵ which may be associated with enhanced plasma β‑endorphin release and suppressed substance P production.¹⁶ Nevertheless, previous research by the team has primarily focused on efficacy evaluation, leaving its deeper underlying mechanism of action unclear. Elucidating the mechanism of WAA could provide new insights for the clinical treatment of inflammatory muscle pain.

Emerging evidence has revealed that symptom severity of multiple inflammatory diseases exhibits diurnal fluctuations. In mouse models of arthritis, altered expression of intra‑articular clock genes exacerbates inflammatory responses.¹⁷ Disruption of core clock components (eg, BMAL1 and REV‑ERB) abolishes circadian regulation of immune‑inflammatory reactions, mechanistically amplifying oxidative stress, inducing immune cell dysfunction, and triggering inflammatory cascades^18–20 These changes result in sustained inflammatory cell activation and prolonged expression of inflammatory mediators, ultimately disrupting immune homeostasis.^21,22 Given the pivotal role of molecular clocks in inflammatory modulation, targeting circadian rhythms is regarded as a promising strategy for immune intervention, yet its precise mechanisms remain incompletely clarified.

To this end, this study established a rat model of inflammatory muscle pain by injecting complete Freund’s adjuvant into the gastrocnemius muscle. We aimed to compare the therapeutic effects and underlying mechanisms of WAA‑TENS with those of WAA‑AS, particularly focusing on circadian rhythm‑related regulatory pathways. This work presents a novel, standardized treatment protocol for inflammatory muscle pain, laying the groundwork for its future clinical application.

Materials and Methods

Reagents and Instruments

Complete freund’s adjuvant (CFA) (Sigma, USA, F5881), trichloroethanol (Aibei, Nanjing, CN, M2820), acupuncture needles (Jiajian Medical, CN), TENS-WAA equipment (Shanghai MicroPort Scientific Co, CN), Von Frey hairs mechanical stimulation needles (Yuyan Instrument, CN), Von Frey test cage (Yuyan Instrument, CN), Rat IL-6 ELISA Kit (Servicebio, CN, GER0001-96T), Rat IL-1β ELISA Kit (Servicebio, CN, GER0002-96T), Rat TNF-α ELISA Kit (Servicebio, CN, GER0004-96T), Rat TGF-β1 ELISA Kit (Servicebio, CN, GER0051-96T), BCA Protein Assay Kit (Servicebio, CN, G2026-1000T), TRIzol^® reagent (China, Servicebio, CN, G3013) SweScript All-in-One SuperMix for qPCR (Servicebio, CN, G3337), Blue SYBR Green qPCR Master Mix (Servicebio, CN, G3326).

Animals

Sixteen healthy male SD rats, aged 8 weeks and specific pathogen-free (SPF) grade, were purchased from Zhejiang VTLH Laboratory Animal Center. All rats were randomly housed in standard polycarbonate ventilated cages at the laboratory animal center of Changhai Hospital, Naval Medical University. The housing environment was maintained with a 12 h light-dark cycle, and all rats were provided with ad libitum access to standard laboratory chow and sterile drinking water throughout the experimental period. All animal housing and experimental procedures were performed under optimal standardized laboratory animal care conditions, and all efforts were made to minimize animal suffering, with pain and distress controlled to the indispensable minimum level in accordance with the principle of 3Rs. All animal experimentation procedures and care have been approved by the Animal Ethics Committee of Changhai Hospital (CHEC (A.E)2025-014). Experiments commenced after a one-week acclimatization period in the SPF environment. Sample size was calculated using a two-sample t-test formula: , With a mean difference of 30%, pooled standard deviation of 18%, α=0.05 (two-tailed), and a target power of 0.80, the required sample size was n=4 per group. Rats were randomly assigned to four groups (n=4) using a random number table: CON, MOD, WAA-AS, and WAA-TENS.

Modeling

The inflammatory muscle pain model was established in the gastrocnemius muscle as previously described by Shi et al.²³ Briefly, after body weight measurement, rats were anesthetized by an intraperitoneal injection of tribromoethanol (1.2 mL/100 g). CFA was then injected bilaterally into the gastrocnemius muscles (200 μL per side) to induce inflammatory pain (Figure 1). The mechanical pain threshold was assessed on the plantar surface of the hind paws on day 3 after modeling. A significant decrease in the pain threshold was considered indicative of successful model establishment.

Figure 1.

Schematic Diagram of Construction and Treatment Procedures for the Gastrocnemius Inflammation Model. Created with BioGDP.com, and permission has been obtained.

Intervention Methods

WAA divides the body into six longitudinal zones, with six zones and acupuncture points on each wrist and ankle. Subcutaneous puncture at different acupuncture points can treat pain in the corresponding tissues and organs. According to the WAA theory, the lower 1st zone primarily treats gastrocnemius muscle pain²⁴ Therefore, both the WAA-AS group and the WAA-TENS group selected bilateral lower 1st zones in rats for intervention. The WAA-AS group administered single-use sterile acupuncture needles directed toward the modeled site. Upon successful insertion, the needle shaft remained visible beneath the skin. Treatment was performed once daily for 20 minutes over three consecutive days.²⁵ The WAA-TENS group applied electrode pads with a diameter of 1 cm to the lower 1 region (ie, 1 cm above the medial malleolus of rats, along the medial edge of the Achilles tendon) with dense‑sparse wave stimulation at parameters of 2 Hz (200 μs), 100 Hz (150 μs), and 1 mA current intensity Each session lasted 20 minutes, administered once daily for three consecutive days,¹⁴ as shown in Figure 1. The CON and MOD groups followed identical protocols to the intervention groups, except for receiving no intervention.

Mechanical Pain Threshold Measurement

Mechanical pain thresholds in rat plantar regions were measured using Von Frey filaments at three time points: pre-modeling, 3 days post-modeling, and 3 hours after the final intervention. Rats were acclimatized in glass cages for 20 minutes before testing. Von Frey filaments with calibrated forces of 0.4g, 0.6g, 1.4g, 2.0g, 4.0g, 6.0 g, 8.0 g, 10.0 g, and 15.0 g. Starting with the 4.0 g filament, the mid-sole of the hind paw was stimulated for 6 seconds, observing and recording paw withdrawal responses. If the rat showed no obvious reaction, it was recorded as (O), and the next highest gauge fiber was used. If the rat rapidly withdrew its hind paw, it was recorded as (X), and the next lower gauge fiber was used. When an OX/XO pattern emerged, four additional measurements were taken to obtain a sequence combining O and X. Import this sequence into the Up-Down Reader analysis software to determine the 50% mechanical withdrawal threshold.

Gastrocnemius Muscle Harvesting

After the final behavioral test, the rats were euthanized in a CO₂ asphyxiation chamber. After dissection, the gastrocnemius muscle was exposed, and a 1×1 cm tissue sample was taken from the middle portion of the muscle belly. The sample was divided into three parts: one was fixed in 4% paraformaldehyde for histology, one was homogenized, the supernatant was collected for ELISA analysis, and the remainder was snap-frozen at −80°C for further analysis.

ELISA

Tissue expression of IL-1β, IL-6, TNF-α, and TGF-β was measured according to the kit instructions. After reaction termination, absorbance was measured using an ELISA reader, and concentrations of each marker were calculated based on the established standard curve.

Histological Analysis

To evaluate inflammatory infiltration and collagen deposition in the gastrocnemius muscle, tissue sections were subjected to hematoxylin and eosin (H&E) and Masson’s trichrome staining. Stained sections were examined under a light microscope, and images from three randomly selected fields per section were captured for quantitative analysis. The extent of inflammatory infiltration and collagen deposition was assessed using image analysis software.

Transcriptomic Sequencing

Total RNA was isolated using TRIzol reagent. Then, RNA quality was determined by the 5300 Bioanalyzer and quantified using the ND-2000. Only high-quality RNA sample (OD260/280=1.8~2.2, OD260/230≥2.0, RQN≥6.5, 28S:18S≥1.0, >1μg) was used to construct sequencing library.

The gastrocnemius RNA-seq transcriptome library was prepared using the Illumina Stranded mRNA Prep and Ligation protocol with 1 μg of total RNA. Shortly, messenger RNA was isolated using the polyA selection method with oligo (dT) beads, and then fragmented with the fragmentation buffer. Secondly, double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit with random hexamer primers. Then the synthesized cDNA was subjected to end-repair, phosphorylation, and adapter addition according to the library construction protocol. Libraries were size-selected for cDNA target fragments of 300 bp on 2% Low Range Ultra Agarose, followed by PCR amplification using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantification by Qubit 4.0, the sequencing library was performed on the NovaSeq X Plus platform (PE150) using the NovaSeq Reagent Kit.

To identify DEGs (differential expression genes) between two different samples, the expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. DEGs with |log2FC|≧1 and FDR< 0.05 (DESeq2) or FDR < 0.001 (DEGseq) were considered to be significantly differentially expressed genes. In addition, functional-enrichment analysis, including GO and KEGG, was performed to identify which DEGs were significantly enriched in GO terms and metabolic pathways at a Bonferroni-corrected P-value < 0.05 compared with the whole-transcriptome background. GO functional enrichment and KEGG pathway analysis were carried out by Goatools and Python scipy software, respectively.

Reverse Transcription-Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR)

Total RNA was extracted from gastrocnemius muscle tissues using TRIzol^® reagent. Three biological replicates and three technical replicates were included for RNA‑sequencing and subsequent RT‑qPCR validation. The RNA concentration and purity were assessed using a spectrophotometer. The RNA was then converted into cDNA utilizing a reverse transcription kit and was analyzed by Universal Blue SYBR Green qPCR Master Mix. The PCR conditions consisted of 30 seconds of pre-denaturation at 95 °C, 15 seconds of denaturation at 95 °C, and annealing at 60 °C for 30 seconds for 40 cycles. The mRNA expression levels were assessed using the comparative 2-ΔΔCT method, with normalization to β-actin as an internal control. The RT-qPCR method was utilized to validate the expression of the target genes. The primer sequences are listed in Table 1.

Table 1.

Primer Sequences

Gene	Primer Sequences (5′-3′)	Length (bp)
Cry1	forward 5′-GTCCGACGACCATGATGAGAA-3′	196
	reverse 5′-GCTTGCGAGCAGGGAGTTT-3′
Per2	forward 5′-GCAGGCAGCAGTGATACAAGTC-3′	130
	reverse 5′-CCTGCAAGACGTACTTAATGAACTG-3′
Nfil3	forward 5′-TCTTTCTCCACCTACTCCCACTC-3′	113
	reverse 5′-CCATCAGAAGACTTGCCCACC-3′
Nr1d2	forward 5′-TCTGTCCGTGGGAATGTCG-3′	116
	reverse 5′-ACTGGGTGCTCATCATGGTCTT-3′
Cd74	forward 5′-CAAACCTGTGAGCCCGATG-3′	204
	reverse 5′-TTAAGGTGCTTCAGATTCTCCGG-3′
Npas2	forward 5′-TCGACAAGGCGTCCATCATT-3′	138
	reverse 5′-GCCTTCGTTGTGCACCTTTT-3′

Statistical Analysis

All data are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism software (version 9.0, San Diego, USA). Differences between groups were compared using one-way analysis of variance (ANOVA), and comparisons between two groups were performed using the independent samples t-test. Image analysis was conducted using ImageJ software. Differences were considered statistically significant at p < 0.05.

Results

WAA-AS and WAA-TENS Significantly Increase Mechanical Pain Threshold in Rat Paws

After model establishment, the mechanical pain thresholds in the MOD, WAA-AS, and WAA-TENS groups were significantly lower than those in the CON group (p < 0.01), with no significant differences among these three groups (p > 0.05; Figure 2a). Following three days of intervention, both the WAA-TENS and WAA-AS groups showed significantly increased pain thresholds compared to the MOD group (p < 0.01; Figure 2b). Intragroup comparison revealed that the mechanical pain thresholds in the WAA-TENS and WAA-AS groups were significantly elevated after intervention compared to pre-intervention levels, whereas the MOD group exhibited a further decrease in pain threshold (Figure 2c).

Figure 2.

Pain threshold in rat paws. WAA-AS and WAA-TENS treatment increased the mechanical pain threshold in rat plantar regions. (a) Comparison of pain thresholds among groups before intervention. (b) Comparison of pain thresholds among groups after intervention. (c) Trend of pain threshold over time in rats across groups. Data are expressed as mean ± SEM. Differences between groups were compared using one-way ANOVA. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

WAA-AS and WAA-TENS Significantly Improve Inflammatory Infiltration and Collagen Deposition in the Gastrocnemius Muscle

H&E staining showed regularly arranged myocytes with intact morphology in the CON group, while the MOD group exhibited disrupted myocyte architecture and extensive inflammatory cell infiltration. Both WAA-AS and WAA-TENS treatments improved myocyte morphology and arrangement, accompanied by significantly reduced inflammatory infiltration (Figure 3a and c; p < 0.05). Masson’s trichrome staining revealed minimal blue collagen deposition and intact muscle structural morphology in the CON group. In contrast, the MOD group displayed severe skeletal muscle damage characterized by disordered myofiber arrangement, interstitial widening, and a marked increase in collagen fiber area, indicating excessive collagen deposition (Figure 3b). Both intervention groups showed significant amelioration of muscle tissue injury and collagen deposition, with well‑preserved myofiber integrity and markedly reduced collagen fiber area (Figure 3d; p < 0.01).

Figure 3.

Pathological sections of the gastrocnemius muscle in rats from each group. (a) H&E staining (20×). (b) Masson’s trichrome staining (20×). (c) Percentage of inflammatory infiltration areas in H&E staining (n = 3). (d) Percentage of collagen fiber deposition areas in Masson’s staining (n = 3). Data are expressed as mean ± SEM. Differences between groups were compared using one-way ANOVA. ***p < 0.001, **p < 0.01, *p < 0.05.

WAA-AS and WAA-TENS Reduce Production of Inflammatory Factors in the Gastrocnemius Muscle

Compared with the MOD group, WAA‑AS significantly reduced IL‑1β levels (p < 0.05) and increased TGF‑β levels (p < 0.001); WAA‑TENS significantly reduced IL‑1β and IL‑6 levels (p < 0.05) and elevated TGF‑β levels (p < 0.05), as shown in Figure 4. Notably, TGF‑β exerts dual regulatory roles of anti‑inflammation and pro‑collagen deposition in a time‑ and cell‑context‑dependent manner. The upregulated TGF‑β observed here mainly functions to suppress early‑stage inflammatory responses in acute muscle injury,^26,27 which may explain the reduced collagen deposition identified by Masson’s trichrome staining despite elevated TGF‑β expression.

Figure 4.

Inflammatory cytokine levels in the gastrocnemius muscle of rats in each group. (a) IL-1β; (b) IL-6; (c) TNF-α; (d) TGF-β (n = 4). Data are presented as mean ± SEM. Differences between groups were compared using one-way ANOVA. *p < 0.05, ns p > 0.05.

GO Functional and KEGG Pathway Enrichment Analysis of Transcriptomes in Inflammatory Pain Rats Following WAA-AS and WAA-TENS Treatment

Transcriptomic sequencing identified 18 upregulated and 45 downregulated genes in WAA-TENS vs MOD groups, and 6 upregulated and 41 downregulated genes in WAA-AS vs MOD groups (Figure 5a). GO enrichment analysis suggested that WAA-TENS modulates immune response, response to stimulus, immunoglobulin complex, and adaptive immunity, while WAA-AS is associated with immune response, response to stimulus, and IgG protein complex (Figure 5b). KEGG pathway analysis revealed that WAA-TENS may exert its effects through circadian rhythm, antigen processing and presentation, and Th1/Th2/Th17 cell differentiation pathways, whereas WAA-AS is linked to circadian rhythm, Toll-like receptor signaling, and osteoclast differentiation pathways (Figure 5c).

Figure 5.

Effects of WAA-AS and WAA-TENS interventions on the gastrocnemius muscle. (a) Differential gene visualization: red indicates significantly upregulated genes, blue indicates significantly downregulated genes. (b) GO enrichment analysis (c) KEGG enrichment analysis.

Core Gene Screening for Inflammatory Pain Treatment

Differentially expressed genes common to both the WAA-AS vs MOD and WAA-TENS vs MOD comparisons were defined as target genes (Figure 6a). Their expression patterns across groups are displayed in Figure 6b. GO enrichment analysis showed that these target genes were significantly associated with MHC class II protein complex, response to stimulus, antigen processing and presentation, immune response, and immune cell differentiation (Figure 6c). KEGG pathway analysis further revealed enrichment in circadian rhythm, antigen processing and presentation, and Th1/Th2/Th17 cell differentiation pathways (Figure 6d). Based on protein-protein interaction (PPI) network node degree analysis, ten core genes were identified: Cry1, Per2, Nfil3, Nr1d2, Npas2, Cd74, RT1-Bb, RT1-Ba, RT1-Db1, and RT1-Da (Figure 6e).

Figure 6.

Analysis of core mechanisms in WAA-AS and WAA-TENS treatment of inflammatory pain in the gastrocnemius muscle. (a) Target gene screening. (b) Expression of target genes in the MOD group, WAA-AS group, and WAA-TENS group. (c) GO enrichment analysis of target gene sets. (d) KEGG enrichment analysis of target genes. (e) Core gene screening, where larger nodes indicate more central genes.

Validation of Core Genes for WAA-AS and WAA-TENS Treatment of Inflammatory Pain in the Gastrocnemius Muscle

Based on sequencing data and literature reports, RT1-Bb, RT1-Ba, RT1-Db1, and RT1-Da were identified as functionally related to Cd74.¹⁸ We therefore selected Cd74, Cry1, Per2, Nfil3, Nr1d2, and Npas2 for RT-qPCR validation. Compared with the MOD group, the WAA-TENS group showed significantly upregulated Per2 and Nr1d2 expression (p < 0.05) and downregulated Nfil3 expression (p < 0.05). The WAA-AS group exhibited significantly decreased expression of Cry1 and Nfil3 (p < 0.05; Figure 7). These expression patterns were consistent with the transcriptomic sequencing results.

Figure 7.

RT-qPCR validation of core genes for treating inflammatory pain in the gastrocnemius muscle. (a) Cry1; (b) Per2; (c) Nfil3; (d) Npas2; (e) Nr1d2; (f) Cd74 (n=3). Data are expressed as mean ± SEM. Differences between groups were compared using one-way ANOVA. **p < 0.01, *p < 0.05, ns p > 0.05.

Discussion

Muscle pain represents a significant global health challenge that markedly compromises patients’ quality of life.^28,29 While nonsteroidal anti-inflammatory drugs and opioids offer effective analgesia, their long-term use is associated with considerable adverse effects.³⁰ Our previous clinical trials have demonstrated that WAA-TENS intervention effectively elevates pain thresholds and reduces pain levels, with its therapeutic effects potentially mediated through the inhibition of pain perception activation in the anterior cingulate cortex and dorsolateral prefrontal cortex.^12–14,31 However, the molecular mechanisms underlying this therapy remain incompletely understood. Therefore, we compared the analgesic efficacy of two non-pharmacological interventions—WAA-AS and WAA-TENS—in an animal model of inflammatory pain. Our results indicate that both interventions significantly elevated mechanical pain thresholds and suppressed local inflammation and collagen deposition in rats with inflammatory pain. Notably, WAA-TENS achieves its effects through non-invasive transcutaneous electrical stimulation that mimics WAA acupuncture, offering a standardized treatment protocol while maintaining therapeutic equivalence to traditional WAA-AS. These findings present a safe, effective, and readily applicable new strategy for the clinical management of inflammatory pain.

Current evidence indicates that the inflammatory response, particularly the infiltration and activation of immune cells, plays a critical role in the initiation and maintenance of pain.³² Tissue injury often triggers pain,³³ during which immune cells migrate to the injury site under the influence of chemokines. IL-1β and IL-6 are core cytokines mediating inflammatory pain, enhancing pain transmission by directly or indirectly sensitizing peripheral nociceptors, thereby lowering the body’s pain threshold.³⁴ TGF-β exerts immunosuppressive effects by inhibiting the production of pro-inflammatory cytokines such as IL-1β and IL-6.^35,36 Notably, TGF‑β exhibits dual time‑ and cell‑context‑dependent regulatory roles: it primarily exerts anti‑inflammatory effects in the early acute phase of muscle injury, whereas its pro‑fibrotic activity predominates in the late chronic remodeling stage,^26,27 which explains the elevated TGF‑β expression accompanied by reduced collagen deposition observed in our 3‑day acute‑phase intervention. Our ELISA results showed that both WAA-AS and WAA-TENS significantly decreased IL-1β and IL-6 levels while increasing TGF-β expression in gastrocnemius muscle tissue. HE-stained sections revealed reduced inflammatory cell infiltration. Based on these findings, we propose that both therapies may alleviate pain by modulating the local inflammatory microenvironment, shifting the balance from a pro-inflammatory to an anti-inflammatory state, thereby fundamentally reducing inflammatory stimulation of nerves in the locally injured area.

Transcriptomic analysis identified the circadian rhythm pathway as a common target enriched with differentially expressed genes following both WAA-AS and WAA-TENS treatments. Growing evidence indicates a close relationship between circadian rhythms and the regulation of pain perception and inflammatory processes.³² Pain and inflammatory responses may disrupt circadian rhythms, while circadian rhythm disruption may also exacerbate pain sensitivity and inflammatory responses.^37–39 Clinical observations have shown that circadian rhythm disturbance lowers pain thresholds⁴⁰ and promotes activation of the inflammatory transcription factor NF-κB, leading to upregulated expression of pro-inflammatory cytokines, including IL-1β and IL-6.^41,42 Herein, we identified correlated changes between the expression of key circadian genes and improvements in analgesia; however, we did not establish direct causal evidence that WAA intervention exerts its analgesic effects strictly through circadian regulation. Further mechanistic validation is needed to elucidate the direct causal relationship between circadian gene regulation and pain relief.

RT-qPCR validation confirmed the key involvement of Cry1, Per2, Nr1d2, and Nfil3—all core components of the circadian regulatory network. Intriguingly, the two interventions exhibited distinct circadian gene‑targeting profiles: WAA‑TENS preferentially upregulated Per2 and Nr1d2 and suppressed Nfil3, whereas WAA‑AS mainly downregulated Cry1 and Nfil3. This divergence may reflect differential neurobiological pathways triggered by transcutaneous electrical stimulation versus invasive acupuncture needling. Electrical stimulation of peripheral nerve afferents in WAA‑TENS may preferentially engage peripheral clock‑regulated mechanosensitive signaling cascades centered on Per2‑Nr1d2, while direct mechanical needling in WAA‑AS primarily modulates Cry1‑dependent inflammatory suppression pathways. Specifically, Cry1 and Per2 regulate inflammatory responses by influencing downstream Nr1d2 and Nfil3, providing a crucial molecular basis for elucidating the circadian fluctuations of inflammatory responses and the mechanisms underlying their dysregulation.

Beyond circadian pathways, our analysis identified antigen processing and presentation as well as Th1/Th2/Th17 cell differentiation as common signaling pathways modulated by both WAA-AS and WAA-TENS interventions. Following muscle injury, damaged cells typically release damage-associated molecular patterns (DAMPs), which activate macrophages and other immune cells. These cells then phagocytose antigens and present them to helper T cells via MHC class II complexes, thereby promoting T cell activation and differentiation.^43–45 Rather than mediating systemic immune regulation, these pathways are primarily localized within the injured gastrocnemius microenvironment, where infiltrating pro‑inflammatory macrophages and resident muscle stem cells serve as key functional executors. Macrophage‑driven antigen presentation and T‑helper cell polarization dominate early inflammatory cascades, while muscle stem cell activation modulates local immune homeostasis to facilitate tissue repair.^46,47 Emerging evidence indicates that circadian rhythm genes play regulatory roles in T helper cell differentiation. Cry1 functions as a key suppressor of proinflammatory factors,⁴⁸ while Nr1d2 inhibits inflammatory responses by suppressing IL-6 production.⁴⁹ Furthermore, Nr1d2 antagonizes Th17 cell differentiation by competing with RORγt for binding to critical genomic regions, consequently reducing the production of IL-17 and other proinflammatory cytokines, including IL-6 and IFN-γ.⁵⁰ Nfil3 plays a vital role in regulating immune cell development and influencing T cell differentiation.^51,52 It can suppress proinflammatory cytokine production by modulating Th17 cell differentiation.^53,54 Collectively, these pathways appear to interact synergistically to inhibit inflammatory cytokine secretion and produce analgesic effects. This preliminary finding sheds light on the scientific basis underlying WAA and its derivative therapies.

Early rehabilitation achieves optimal outcomes by actively regulating the healing process. Compared to the model group’s rest-and-wait approach, histopathological analysis revealed that early acupoint stimulation in the intervention groups promoted tissue repair while suppressing excessive collagen deposition at the lesion site, providing experimental support for the early application of WAA. Mechanistically, existing studies indicate that Per2 activates myoblast proliferation by regulating the cell cycle during myogenesis and influences subsequent myocyte differentiation and fusion, thereby facilitating muscle repair.^50,55 In the present study, WAA-TENS intervention significantly increased Per2 expression. Additionally, the Cry1 gene modulates myogenesis by acting on downstream cell cycle components in myoblasts.⁵⁶ Furthermore, TGF-β serves as a key regulatory factor in tissue repair.⁵⁷ It effectively suppresses inflammatory responses within the injury microenvironment while activating fibroblast function and promoting the directed differentiation of muscle stem cells,^58–60 collectively advancing the repair process. Our results demonstrate that both WAA-AS and WAA-TENS significantly elevate TGF-β levels, thereby enriching the scientific understanding of how early acupoint stimulation in WAA promotes muscle tissue regeneration.

Conclusion

In summary, this study provides novel evidence that wrist-ankle acupuncture (WAA) point stimulation effectively alleviates inflammatory muscle pain in acute inflammatory injury models, reduces inflammatory cell infiltration and cytokine expression at the lesion site, promotes muscle fiber repair, and inhibits excessive collagen deposition. The therapeutic mechanism may involve the modulation of circadian rhythm pathways, while direct causal relationships remain to be verified. Notably, our exploratory findings support WAA‑TENS as a promising standardized non‑pharmacological candidate for acute inflammatory muscle pain intervention; however, several limitations should be acknowledged, including the small sample size, short‑term 3‑day intervention period, and animal‑model‑derived observations that require further clinical validation before widespread clinical promotion. Future work will focus on clinical translational evaluation of WAA‑TENS efficacy in patients with inflammatory muscle pain, as well as mechanistic verification of circadian rhythm pathways using genetic and pharmacological approaches, to further consolidate its clinical application potential.

Funding Statement

This work was supported by the National Health Commission of the People’s Republic of China under Grant SZ2024HL010 and the National Key Research and Development Program of China under Grant 2019YFC1711803.

Ethical Statement

All animal procedures and care were approved by the Animal Ethics Subcommittee of the Medical Ethics Committee at Shanghai Changhai Hospital (CHEC (A.E)2025-014) and performed in accordance with the guidelines for the ethical use of conscious animals in pain studies published by the International Association for the Study of Pain (IASP). The model was established using tribromoethanol anesthesia, and tissue samples were collected after euthanasia using a CO₂ asphyxiation chamber.

Disclosure

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.