Electroacupuncture intervention relieves pain by stimulating the STING/IFN-I pathway in rat models of cancer-induced bone pain

Abstract

This study aimed to evaluate the effects of electroacupuncture (EA) on cancer-induced bone pain (CIBP) and investigate its interaction with the STING/IFN-I pathway. A CIBP model was established in female rats. EA was administered for six consecutive days at bilateral L3–L5 Jia Ji points (EX-B2). EA-induced antinociception was evaluated through mechanical, thermal, and cold sensitivity assessments. EA significantly increased the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) in rats with CIBP (p < 0.01). In the spinal cord of CIBP model rats, western blot analysis demonstrated that the application of EA upregulated the expression of STING, IRF3, and IFNAR (p < 0.05). The ELISA results indicated that EA significantly increased the expression of IFN-α (p < 0.005) and IFN-β (p < 0.01) and reduced the expression of TNF-α and IL-1β (p < 0.05). Immunofluorescence analysis revealed that STING was predominantly localized in microglia, with a minimal presence in neuronal cells. Furthermore, intrathecal administration of the STING antagonist C-176 attenuated the analgesic effects of EA in CIBP (p < 0.05). Both EA and STING agonist were effective in alleviating pain in rats with CIBP, possibly through the activation of the STING/IFN-I pathway. Notably, EA treatment reduced pro-inflammatory cytokines and increased anti-inflammatory cytokines. In contrast, while the STING agonist exhibited analgesic effects, it was associated with elevated levels of pro-inflammatory cytokines. These finding underscore the therapeutic potential of EA in the management of CIBP.

Introduction

Pain is an aversive sensory experience initiated by nociceptors, which transmit signals to the central nervous system, resulting in discomfort. ¹ A significant proportion of patients with advanced cancer experience pain, ² with approximately 80% of this pain being attributed to cancer-induced bone pain (CIBP).^3,4 Unfortunately, more than 50% of patients with advanced or metastatic cancer receive inadequate pain management due to the limited efficacy and adverse effects associated with current therapeutic options. ³ Available analgesic treatments for CIBP include opioids, bisphosphonates, radiotherapy, denosumab, nerve blocks, and non-steroidal anti-inflammatory drugs (NSAIDs), each with certain limitations in terms of analgesic efficacy.^5,6

Acupuncture therapy, a fundamental aspect of Traditional Chinese Medicine (TCM), has been recognized for its safety and efficacy as an alternative approach to pain management. ⁷ It is endorsed by the European Oncology Nursing Society (EONS) and the American Society of Clinical Oncology in their guidelines as an effective complementary therapy for the treatment of chronic cancer pain in survivors.^7–9 A substantial body of clinical and preclinical research has supported the analgesic effects of electroacupuncture (EA) in various pain disorders.^10–14 Furthermore, a systematic review and meta-analysis provided moderate-level evidence indicating that acupuncture or acupressure significantly alleviates cancer-related pain and reduces the need for analgesics. ¹⁵ In CIBP, foundational studies have shown that EA mitigates mechanical and thermal nociceptive hypersensitivity in CIBP rat models.^16,17 However, the underlying mechanisms by which EA alleviates CIBP remain poorly understood.

The stimulator of interferon genes (STING) pathway, initially identified in 2008, is integral to immune signaling pathways. ¹⁸ Upon activation, STING recruits TANK-binding kinase 1 (TBK1), which subsequently phosphorylates interferon regulatory factor 3 (IRF3) and inhibitory proteins of nuclear factor-κB (NF-κB). The phosphorylated IRF3 and NF-κB then translocate to the nucleus, facilitating the expression of interferon-1 (IFN-I) and cytokines and thus initiating an antigen-specific adaptive immune response. ¹⁹ An increasing body of evidence substantiates the pivotal role of the STING/IFN-I pathway in various physiological and pathological processes,^20–22 including cancer,^23,24 and cancer-induced bone pain. ²⁵ Donnelly et al. ²⁶ elucidated that STING modulates pain perception via the IFN-I signaling pathway in sensory neurons, thereby identifying the STING/IFN-I pathway as a novel target for chronic pain management. Previous research has also indicated that to alleviate postoperative pain (APP) in rats, EA may exert analgesic effects by activating the STING/IFN-I signaling pathway. ²⁷

This study aimed to observe and evaluate the effects of EA on pain and explore potential mechanisms involved. This study aimed to observe and evaluate the effects of EA on pain, with a focus on exploring the underlying mechanisms.

Materials and methods

Experimental animal model

A total of 126 healthy SPF-grade female SD rats, among which 6 rats with a mass 40–60 g were used for ascites culture and 120 rats with a mass 160–180 g were used for experiments, were purchased from Shanghai Bikaikewing Bio-technology Co. Ltd. (License No. SCXK (Shanghai) 2018-0006) and were housed in the SPF-grade rearing room of the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine. The rats were allowed ad libitum access to water and diet, and subjected to alternating light/dark cycles in the morning and evening.

Experimental design

The experiment consisted of three stages. In stage 1, rats were randomly divided into three groups: sham operation (Sham), CIBP model (CIBP), and CIBP model plus electroacupuncture (CIBP+EA). In stage 2, rats were randomly divided into five groups: sham operation (Sham), CIBP model (CIBP), CIBP model plus electroacupuncture (CIBP+EA) group, CIBP model plus STING antagonist (CIBP+C-176), and CIBP model plus electroacupuncture plus STING antagonist (CIBP+EA+C-176). In stage 3, rats were randomly divided into four groups: sham operation (Sham), CIBP model (CIBP), CIBP model plus electroacupuncture (CIBP+EA), and CIBP model plus STING agonist (CIBP+2′3-cGAMP).

CIBP rat models

Using the modeling method described by Shenoy et al., ²⁸ rats were anesthetized with 1% sodium pentobarbital (40 mg/kg body weight, intraperitoneally). The right knee joint was shaved, disinfected, and surgically exposed. A hole was drilled along the longitudinal axis of the tibia to a depth of approximately 1 cm distally. Subsequently, 10 μL of Walker 256 cells (1 × 10⁸ cells/mL) were injected into the bone marrow cavity of the right tibia. The entry site was sealed with bone wax, disinfected, and the incision was sutured. In the sham operation group, 10 μL of phosphate buffered saline (PBS) was injected instead of tumor cells. Successful modeling was confirmed by the development of significant pain hypersensitivity in rats. ²⁹

Electroacupuncture

The rats in the EA group began receiving EA treatment on day 15 after modeling, once daily for a total of 6 days, and the acupuncture points were selected as bilateral L3–L5 Jiaji points (EX-B2). The rats were captured and fixed in black headgear and the backs of the rats were disinfected. Needles were inserted directly from the lower part of the spinous process of L3 and 3 mm from the posterior midline (milli precision needles were 0.25 mm × 25 mm), and the tip of the needles was turned to the caudal side when it touched the vertebral plate, and then penetrated into the spinous process along the side of the spinous process to L5, with a depth of injection of about 15–25 mm. after injection, the needle handle was connected to the Han’s Acupuncture Point Neurostimulation Instrument (HANS-200E, Nanjing Jisheng Medical Technology Co. Medical Technology Co. Ltd), which was used for treatment, with a stimulation frequency of 2/100 Hz, a current intensity of 2 mA, and a treatment time of 30 min. ³⁰ The rats in the remaining groups were similarly captured and immobilized in the headgear starting on day 15 after modeling, but without any intervention, once daily for a total of 6 times. The rats were acclimatized by gently pressing and fixing them in the headgear daily for 3 days prior to treatment.

Administration of drugs

C-176 (MedChemExpress, Cat# HY-112906) a STING antagonist, was dissolved in DMSO and diluted to a concentration of 2 μg/μL in corn oil which was injected intrathecally (i.t.) as in previous studies,^26,31 based on the dose conversion formula between mice and rats based on body surface area. 2′3′-cGAMP (MedChemExpress, Cat# HY-100564), a STING agonist, was dissolved in saline solution and was diluted to a concentration of 1 ug/uL. ³² Both drugs were administered to rats intravenously, using DMSO/saline alone as the vehicle control. Rats were lightly anesthetized with 3% isoflurane and a microliter syringe with a 30-gauge needle was inserted into the intervertebral space between the L5 and L6 vertebrae, and after shaving the lumbar region and disinfecting with iodine povidone, a total volume of 10 μL of the drug was delivered into the cerebrospinal fluid, with rapid movement of the tail indicating that the microsyringe had penetrated the dura mater. The injection rate was approximately 0.5 s/μL, and the microsyringe was held motionless for 1 min after injection.

Behavioral tests

Paw withdrawal threshold

Mechanical reflex thresholds for paw withdrawal (PWT) were assessed in three groups of rats at baseline (day 0) and after surgery on days 3, 7, 10, 14, 16, 18, and 20. Rats were placed in a transparent Plexiglas chamber on a metal mesh pain measurement platform. After 30 min of static acclimatization, an electronic von Frey force transducer with a polypropylene tip (IITC Life Sciences Ltd., USA) was applied perpendicularly to the center of the right hind paw. The pressure was gradually increased and the force intensity (g) at which the paw withdrawal occurred was recorded. Measurements were taken three times per rat at 5-min intervals and the results were averaged across the three trials. PWT was recorded 1 h after EA treatment.

Thermal withdrawal latency of the paw

To assess the withdrawal latency of the paw (PWL), the Hot/Cold Plate Analgesia Meter (Jinan Yiyan Science & Technology Development Co., Ltd., China) was used, the surface was maintained at 53°C. A timer was activated when the rats were placed on the plate and stopped when the animals licked their paws or jumped. A 30-s cut-off time was enforced to prevent tissue damage. The heat test was repeated three times at 10-min intervals and the mean PWL was calculated.

Western blotting assay

The spinal column was dissected bilaterally from the highest point of the iliac crests, and the spinal cord was removed using pre-cooled PBS. Lumbar segments L4–L6 were isolated and the dorsal horn of the spinal cord was processed under a microscope. The dorsal horn tissue was lysed with a protein lysate and the protein concentration was determined using the BCA method (Servicebio, G2026). Equal amounts of protein were separated by SDS-PAGE (Servicebio), transferred to a PVDF membrane (Servicebio), and blocked with 5% skim milk for 2 h. The membrane was then incubated with primary antibody STING (Servicebio, GB111415, diluted 1:1000), IRF3 (Servicebio, GB11368, diluted, 1:1000), IFNAR (ABCLONAL, A18594, diluted, 1:1000), ACTIN (Servicebio, GB11001, diluted, 1:1000), GAPDH (Servicebio, GB15004, diluted, 1:1000) at 4°C for 2 h, rinsed in PBST (Servicebio), and further incubated with HRP labeled secondary antibody (Servicebio, GB23303, diluted, 1:5000) at room temperature for 2 h. Bands were visualized with a chemiluminescent reagent and imaged (Servicebio). The gray values of each band were analyzed using Image J software, with the relative expression of the target protein calculated as the ratio of the target to the GAPDH or β-actin as the internal control.

ELISA

The levels of IFN-α, IFN-β, TNF-α, IL-1β, and IL-10 content in the spinal cord were measured using the respective ELISA kits (ml003203-2, ml102842-2, ml002859-2, ml003057-2, ml102828-2, ml002813-2; Shanghai Enzyme-linked Biotechnology Co, Shanghai, China), following the manufacturer’s instructions.

Immunofluorescence

At 5 h postoperatively, rats deeply anesthetized with sodium pentobarbital were transcranially perfused with 0.9% NaCl, followed by 4% paraformaldehyde. After perfusion, the L1–L2 spinal cord segments were removed and fixed in 4% paraformaldehyde for 72 h at 4°C. The spinal cord tissues were then transferred to a 30% sucrose solution for at least 2 days until completely precipitating to the bottom and cut to a thickness of 30 μm. All sections were washed and incubated overnight at 4°C with the following primary antibodies: STING (1:200, 19851-1-AP+g, Sanying, China), NeuN (1:500, 38-1, Servicebio, China), and Iba-1 (1:300, b15105, Servicebio, China). The sections were then incubated with the corresponding secondary antibodies conjugated with Alexa Fluor 488 (1:300, GB25303, Servicebio, China) for 1 h at 37°C. Finally, DAPI (1:100, G1012, Servicebio, China) was added to stain the cell nuclei. Stained sections were scanned with an Olympus fluorescence microscope system (Nikon Eclipse C1, Nikon, Japan).

Statistical analysis

Data were analyzed using SPSS v.26.0 software. Normally distributed measurement data were expressed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare intergroup differences, with the LSD method applied for homogeneity of variance and the Games-Howell method for heterogeneity. Repeated measures ANOVA was used to assess differences at multiple time points within each group. Statistical significance was set at p < 0.05.

Results

EA alleviated mechanical allodynia in CIBP mode rats

Behavioral assessments were conducted 1 day before surgery and subsequently 3, 7, 10, and 14 days post-operatively, as well as 2, 4, and 6 days after treatment, which corresponded to 16, 18, and 20 days post-operatively (Figure 1(a)). The therapeutic effects of EA on mechanical hyperalgesia in rats with CIBP were investigated. Baseline measurements of PWT in the right hindlimb did not show significant differences between the groups (p > 0.05). CIBP rats exhibited mechanical allodynia, which was alleviated by EA (Figure 1(b)), from day 7 after surgery, PWT was significantly reduced in the CIBP and CIBP+EA groups compared with the sham group (p < 0.001); Compared with the CIBP group, EA treatment significantly increased PWT on day 16 (p < 0.05), day 18 (p < 0.001), and day 20 (p < 0.01).

Figure 1.

EA Alleviated mechanical allodynia and thermal hyperalgesia in CIBP model rats. (a) Experimental design. (b, c) Changes in PWT(g) and PWL(s) values of CIBP models rats with and without EA treatment (n = 10).

EA alleviated thermal hyperalgesia in CIBP model rats

The baseline PWL measurements did not show significant differences between the groups (p > 0.05). CIBP model rats exhibited thermal hyperalgesia (Figure 1(c)), compared with the Sham group, PWL values were significantly reduced in the CIBP and CIBP+EA groups starting from day 7 after surgery (p < 0.05) and persisted through day 20 (p < 0.001). To evaluate the efficacy of EA in alleviating thermal hyperalgesia of CIBP rats, the PWL values were compared between the CIBP and CIBP+EA groups (Figure 1(c)). Compared with the CIBP group, EA treatment significantly increased PWL on days 16 (p < 0.01), 18 (p < 0.01), and 20 (p < 0.01).

EA activated the STING/IFN-I pathway in the spinal cord of CIBP rats

To investigate the underlying mechanisms involved in EA-induced thermal hyperalgesia, we evaluated the expression of STING/IFN-I pathway proteins within the spinal cord. Western blotting analysis demonstrated significantly reduced levels of IRF3 and IFNAR proteins in the CIBP group compared with the Sham group (p < 0.05, p < 0.01). Treatment with EA significantly improved the expression of the STING, IRF3, and IFNAR proteins in the spinal cord of CIBP rats (Figure 2(a)–(c)). Compared with the CIBP group, the expression of spinal STING, IRF3, and IFNAR in CIBP+EA rats increased significantly (p < 0.05). Double label immunostaining was also performed to evaluate the distribution of STING expression in the superficial dorsal horn (SDH). As shown in Figure 2(d) and (e), STING was predominantly localized in Iba1+ microglia and partially in NeuN neuronal cells.

Figure 2.

EA activated the STING/IFN-I pathway in the spinal cord of CIBP model rats (a, b, c) Western blotting images (upper) and quantification of STING (a), IRF3 (b), and IFNAR (c) protein expression (bottom) in SDH of rat models. (d, e) Representative images of double immunostaining of STING with NeuN (d), Iba1 (e) in rats. Arrows indicate typical double-labeled neurons. (f, g) ELISA results of IFN-α and IFN-β levels (pg/mL) in SDH of rats. (h) Results of the ELISA evaluating inflammatory factor levels (TNF-α, IL-1β, IL-10) (pg/mL) in SDH of rats.

The results of the ELISA indicated that EA increased the expression of IFN-α and IFN-β (Figure 2(f) and (g)). Compared with the Sham group, the expression of IFN-β decreased significantly in the CIBP group (p < 0.05); compared with the CIBP group, whereas the expression of IFN-α and IFN-β increased in CIBP+EA group (p < 0.001, p < 0.01).

Effects of EA on the expression of inflammatory cytokines in the lumbar spinal cord

The ELISA showed that CIBP increased pro-inflammatory cytokine levels, whereas EA treatment reduced pro-inflammatory cytokine levels, and increased anti-inflammatory cytokine levels in CIBP model rats (Figure 2(h)). Compared with the Sham group, the levels of TNF-α and IL-1β in the CIBP group were significantly increased (p < 0.001 and p < 0.05, respectively); compared with the CIBP group, the level of TNF-α and IL-1β in the CIBP group were significantly decreased (p < 0.05), and the level of IL-10 in the CIBP group was significantly increased (p < 0.05).

Intrathecal injection of the STING antagonist weaken the analgesic effects of EA

To further validate the role of the STING/IFN-I pathway, we administered C-176, a STING antagonist, intrathecally at a dose of 20 μg, 10 min before EA on day 19, which corresponded to the fifth day of EA treatment (Figure 3(a)). As detailed in Figure 3(b), the mechanical allodynia of CIBP rats, which were elevated after EA treatment, returned to the low thresholds after the intrathecal injection of C-176. Compared with the CIBP+EA group, PWT were significantly reduced in the CIBP+EA+C-176 group on day 20 (p < 0.05); notably, compared to the CIBP group, PWT were significantly increased in the CIBP+C-176 group on day 20 (p < 0.01). As detailed in Figure 3(c), the thermal hyperalgesia of CIBP rats, which were elevated after EA treatment, returned to the low thresholds after the intrathecal injection of C-176. Compared with the CIBP+EA group, PWL were significantly reduced in the CIBP+EA+C-176 group on day 20 (p < 0.05). In particular, compared with the CIBP group, PWL increased significantly in the CIBP+C-176 group on day 20 (p < 0.05).

Figure 3.

Intrathecal injection of the STING antagonist weakened the analgesic effects of experimental EA (a). (b, c) Changes in PWT (g) and PWL (s) of CIBP model rats with and without EA treatment (n = 10). *p < 0.05, **p < 0.01, ***p < 0.001, versus the Sham group; ^#p < 0.05; ^##p < 0.01; ^###p < 0.001, versus the CIBP group; ^&p < 0.05 versus the EA group. (d, e) ELISA results of IFN-α and IFN-β levels (pg/mL) in SDH of rats. (f) ELISA measure of the levels of inflammatory factors (TNF-α, IL-1β, IL-10) (pg/mL) in SDH of rats.

Antinociceptive effects of the EA elicited the STING/IFN-I pathway in the spinal Cord

As illustrated in Figure 3(d) and (e), C-176 partially attenuated the effects of EA and weakened the upregulation of IFN-α and IFN-β expression observed in rats treated with EA. Compared with the CIBP+EA group, the expression of IFN-α and IFN-β were significantly reduced in the CIBP+EA+C-176 group (p < 0.01 and p < 0.05, respectively). Furthermore, compared with the CIBP+EA group, the expression of TNF-α was significantly reduced (p < 0.01) and that of IL-10 was significantly increased (p < 0.01) in the CIBP+EA+C-176 group. In particular, compared with the CIBP group, the expression of TNF-α and IL-1β were also significantly reduced (p < 0.01).

Intrathecal injection of STING agonist-induced analgesic effects similar to those of EA

We conducted a comparative analysis of the effects of a STING agonist and EA treatment in rats with CIBP (Figure 4(a)). Both EA treatment and 2′3-cGAMP exposure increased the mechanical and thermal pain thresholds of CIBP rats (Figure 4(b) and (c)). Compared with the CIBP group, PWT and PWL in the CIBP+EA group increased significantly on days 16 (p < 0.01), 18 (p < 0.01), and 20 (p < 0.01), PWT and PWL in the CIBP+2′3-cGAMP group increased significantly on day 20. No significant differences in PWT and PWL were observed between the CIBP+EA group and the CIBP+2′3-cGAMP group.

Figure 4.

Intrathecal injection of the STING agonist induced analgesic effects similar to EA. (a) Experimental design. (b, c) Changes in PWT (g) and PWL (s) values of CIBP rats with and without EA treatment (n = 10). *p < 0.05, **p < 0.01, ***p < 0.001, versus Sham group; ^#p < 0.05; ^##p < 0.01; ^###p < 0.001, versus CIBP group; ^&p < 0.05 versus EA group. (d, e) ELISA results of IFN-α and IFN-β levels (pg/mL) in SDH of rats. (f) ELISA results of inflammatory factor (TNF-α, IL-1β, IL-10) (pg/mL) in SDH of rats.

Effects of 2′3-cGAMP on the expression of inflammatory cytokines in the lumbar spinal cord

As detailed in Figure 4(d) and (e), compared with the CIBP group, 2′3-cGAMP significantly increased the expression of IFN-β. Furthermore, 2′3-cGAMP increased the expression of pro-inflammatory cytokines (TNF-α and IL-1β), which were decreased by EA treatment and increased the expression of anti-inflammatory cytokines (IL-10). Compared with the CIBP group, the expression of TNF-α, IL-1β, and IL-10 increased significantly in the CIBP+2′3-cGAMP group (p < 0.05, p < 0.05, and p < 0.01, respectively); compared with the CIBP+EA group, the expression of TNF-α and IL-1β were significantly increased in the CIBP+2′3-cGAMP group (p < 0.001).

Discussion

This study demonstrates that CIBP leads to pronounced pain hypersensitivity, as evidenced by reduced PWT and PWL. Similarly to the effect of the STING agonist, EA markedly mitigated pain hypersensitivity in CIBP rats, enhancing pain thresholds through a mechanism likely mediated by activation of the STING/IFN-I pathway. This conclusion is supported by the finding that the analgesic effects of EA were partially weakened by the STING antagonist C-176. Furthermore, although both the EA and STING agonist exhibited analgesic properties, EA also demonstrated additional effects to reduce the inflammatory response in rats with CIBP, highlighting its wider therapeutic potential in the treatment of CIBP.

Numerous experimental investigations have substantiated the effectiveness of EA in managing CIBP.^33–36 Our prior research has indicated that administering 2/100 Hz EA to the Jiaji points produces a significant analgesic effect on CIBP. ³⁷ This effect is potentially attributable to the local distribution of spinal nerve branches associated with subvertebral bones, along with their related actions and venous plexuses in the vicinity of the Jiaji point. ³⁸ Stimulation of the Jiaji point may facilitate signal transmission through peripheral nerve roots to the central nervous system through spinal conduction pathways, thus exerting a regulatory function.^39,40 Consistent with previous reports,^33–37 our behavioral results demonstrated that EA treatment attenuated mechanical anomalous pain and thermal nociceptive hypersensitivity in CIBP rats.

The role of STING in pain modulation has gained increasing recognition. Donnelly et al. ²⁶ demonstrated that intrathecal administration of an STING agonist in normal mice activated STING signaling, leading to analgesia and elevated mechanical paw withdrawal thresholds. Furthermore, the administration of STING antagonists in normal mice resulted in a reduction in pain thresholds and an increase in pain sensitivity, while STING agonists did not induce analgesic effects in STING total knockout mice. ²⁶ Wang et al. ²⁵ extended these findings by investigating a bone cancer pain (BCP) model. Their study demonstrated that intraperitoneal injection of a STING agonists during the early stages of bone cancer (3 and 7 days after cancer cell inoculation) significantly attenuated pain hypersensitivity. The analgesic effect was observed in murine BCP models induced by Lewis lung carcinoma and E0771 breast cancer cells. Furthermore, STING agonists effectively suppressed mechanical and cold pain in a fracture-induced pain model in non-tumor-bearing mice. ²⁵ Notably, although previous studies have indicated that IFN-β, a downstream product of STING, may induce pain^41,42 and that STING antagonists can alleviate pain,^31,43 our findings revealed that activating the STING/IFN-I pathway weakened PWT and PWL in CIBP rats. This discrepancy may be attributed to the differential roles of interferons in the peripheral and central nervous systems. ⁴⁴ Consistent with our findings, several studies have shown that spinal administration of IFN-α/β is generally analgesic in models of inflammatory and neuropathic pain.^26,45,46

EA has been extensively acknowledged for its ability to modulate the nervous system at various levels, including the peripheral, spinal, and supraspinal domains.^47,48 In this study, we found that EA activated the STING/IFN-I pathway, leading to pain alleviation in BCP rats. Immunofluorescence analysis revealed that STING was predominantly localized in Iba1+ microglia and, to a lesser extent, in NeuN neuronal cells. Our findings are consistent with recent research on pain, which has also reported an upregulation of STING expression in microglia following peripheral nerve injury.^26,41,42

To further substantiate the involvement of the STING/IFN-I pathway in EA for CIBP, we incorporated both STING agonist and antagonist into our experimental framework. The analgesic effect of EA was weakened by the STING antagonist C-176, corroborating the findings of Ding et al., ²⁷ who illustrated that EA intervention mitigated pain in rats models with amyloid precursor protein (APP) deposits by activating the STING pathway. In particular, our findings revealed that intrathecal injections of C-176 significantly, although transiently, weakened PWT and PWL in CIBP rats, which can be attributed to the anti-inflammatory properties of C-176.^31,43

Expanding on this premise, we conducted a comparative analysis of the effects of EA and a STING agonist in rats with CIBP-afflicted rats. Both EA and the STING agonist significantly reduced pain; however, no notable differences were observed in their analgesic efficacy. However, EA demonstrated a unique capacity to attenuate the inflammatory response in BCP rats. This differentiation may arise from the observation that STING agonists, such as ADU-S100 and DMXAA, not only activate the STING pathway but also enhance the expression of IFN-I and significantly elevate pro-inflammatory cytokines, including IL-1β, TNFα, and CCL2, particularly in female mice. ⁴⁹ Conversely, EA, as a multimodal therapeutic strategy, exhibits both anti-inflammatory and analgesic properties.^16,50–52

Due to the current scope and experimental limitations, we conducted our study with one STING antagonist and one agonist. The use of a single agonist or antagonist may not fully capture the complex regulatory mechanisms of the pathway under different physiological or pathological conditions. Thus, future studies should consider the incorporation of multiple pharmacological agents to further validate the robustness and generalizability of the findings and integrate genetic knockout or gene intervention approaches to more accurately elucidate the role of this pathway in specific physiological and pathological processes.

Conclusions

Both EA and STING agonist can alleviate pain in CIBP rats, and the mechanism may be related to activation of the STING/IFN-I pathway. Furthermore, EA attenuated the inflammatory response in CIBP rats.