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The Korean Journal of Pain logoLink to The Korean Journal of Pain
. 2026 Oct 1;39(4):521–531. doi: 10.3344/kjp.26061

cGAS–STING signaling in the anterior cingulate cortex modulates electroacupuncture analgesia in a rat neuropathic pain model

Ang Li 1,2,*, Mei Li 2,*, Zhen-Ping Hu 2, Meng-Jing Yang 2, Hui Zhang 2, Xu-Wu Xiang 2, Yong-Xing Yao 2,✉
PMCID: PMC13628125  PMID: 42680681

Abstract

Background

The cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) signaling pathway is associated with different neurological dysfunctions in the nervous system. Although electroacupuncture (EA) has been proven to alleviate neuropathic pain (NP), its underlying mechanisms remain unclear. The role of the cGAS–STING signaling pathway in the anterior cingulate cortex (ACC) during NP and its potential association with EA-mediated analgesic effects remain unknown.

Methods

The authors established an NP model by applying chronic constriction injury (CCI) to the sciatic nerve and evaluated behavioral hyperalgesia using the acetone test score and mechanical threshold. Protein distribution and expression were assessed using immunofluorescence and Western blotting, respectively.

Results

cGAS and STING were predominantly expressed in neurons in the ACC of rats, and the total abundance of cGAS and STING was upregulated on both sides of the ACC following CCI. Intra-ACC administration of either RU.521 or C-176 alleviated the pain hypersensitivity caused by CCI. Moreover, EA reduced pain hypersensitivity and inhibited the CCI-induced upregulation of cGAS and STING in the ACC.

Conclusions

This study demonstrates that the cGAS–STING signaling pathway is involved in the occurrence and development of NP in the ACC following peripheral nerve injury, and that EA may alleviate NP by suppressing this pathway in rats.

Keywords: Electroacupuncture, Gyrus Cinguli, Membrane Proteins, Neuralgia, Nucleotidyltransferases, Peripheral Nerve Injuries, Sciatic Nerve, Signal Transduction

INTRODUCTION

Neuropathic pain (NP), which is characterized by hyperalgesia, allodynia, and spontaneous pain, arises from nerve injuries, metabolic disorders, viral infections, and chemotherapy. It affects approximately 7%–10% of adults and remains a significant societal burden with limited effective therapeutic options [1,2]. The essential pathogenesis of NP includes central and peripheral sensitization, and current treatments are often ineffective because of the incomplete elucidation of the etiology and pathogenesis of NP [3,4]. Located in the frontal lobe of the cerebral hemisphere, the anterior cingulate cortex (ACC) is a vital part of the limbic system, and electrical stimulation of the ACC triggers internal responses such as changes in breathing, gastrointestinal peristalsis, glandular secretion, and mydriasis [5,6]. Moreover, accumulating evidence, including the authors’ previous reports, indicates that the ACC is a key brain region involved in pain processing and pain-related emotional regulation [7–9]. However, the specific mechanisms underlying these ACC functions remain unclear.

The cyclic GMP–AMP synthase (cGAS) detects pathological DNA fragments produced by foreign invading bacteria, viruses, or those generated by the cells themselves. The cGAS undergoes conformational changes and becomes activated after binding to cytoplasmic DNA fragments, subsequently synthesizing cyclic guanosine monophosphate adenosine monophosphate (cGAMP) [10,11]. Under resting conditions, stimulator of interferon genes (STING) is localized in the endoplasmic reticulum. STING mediates downstream signaling activation after binding to cGAMP, promotes autophagosome formation, stimulates the production of interferons and other cytokines, and maintains intracellular homeostasis [12,13]. Previous studies demonstrated that STING regulates physiological pain via interferon-mediated mechanisms in the spinal cord [14]. The cGAS–STING pathway has also been demonstrated by the authors’ previous study to participate in NP as a downstream effector of TAOK2, and microglial STING regulates interleukin (IL)-6 to mediate NP in the spinal dorsal horn [15,16]. Moreover, pharmacological inhibition of the cGAS–STING signaling pathway in the spinal cord attenuates mechanical allodynia induced by nerve injury by suppressing microglial polarization [17]. However, whether cGAS and STING signaling in the ACC contribute to the occurrence and development of NP requires further elucidation. Notably, a recent study has demonstrated that mitochondrial stress in the hippocampus of mice leads to increased cytoplasmic DNA fragments in neurons following peripheral nerve injury [18], providing a theoretical basis for the activation of the cGAS–STING signaling pathway in the ACC after peripheral nerve injury.

The literature has documented that electroacupuncture (EA) alleviates NP through multiple pathways [19,20]. For example, the ACC plays a role in pain perception and pain-related emotional regulation, and EA relieves anxiety-like behaviors caused by inflammatory pain by regulating the excitability of glutamatergic neurons in the ACC [21]. Furthermore, recent studies have demonstrated that EA attenuates hippocampal neuroinflammation by inhibiting the activation of the cGAS–STING signaling pathway [22], suggesting that EA may exert a significant regulatory effect on the cGAS–STING pathway within the central nervous system. However, it remains unknown whether the analgesic effects of EA involve the modulation of the cGAS–STING pathway in the ACC.

MATERIALS AND METHODS

1. Animal and housing

Specific pathogen-free male Sprague–Dawley rats, aged 6–8 weeks and weighing 200 ± 20 g, were purchased from the Laboratory Animal Center of the Zhejiang Academy of Medical Sciences. Animals were kept with 4–5 rats per cage, having unrestricted access to forage and water, with a 12-hour light/dark cycle with 50% ± 5% humidity and temperature of 24°C ± 2°C. All procedures involving animals were performed in compliance with the ARRIVE guidelines for animal research [23]. The experimental protocol was approved by the Ethics Committee of the First Affiliated Hospital of the Zhejiang University School of Medicine (no. 2022-883).

2. Establishment of NP model

As described by Bennett and Xie [24] and in the authors’ previous reports [9], chronic constriction injury (CCI) of the sciatic nerve was used to establish an NP model. Under isoflurane anesthesia, hair in the surgical area of the lateral region of the left hind limb was cleaned. After disinfection with iodophor, an approximately 1 cm longitudinal incision was made, and the biceps femoris was gently dissected by blunt dissection to expose the sciatic nerve. Three strands of 4–0 chromic catgut sutures (Pudong Jinhuan Co. Ltd.) were used to ligate the sciatic nerve at an interval of 1 mm for each ligature. The incision was sutured layer by layer after ligation. The rats in the sham group were exposed in an identical manner to those in the CCI group, but not ligated.

3. Behavioral tests

1) Mechanical threshold

Mechanical threshold (MT) in response to mechanical stimuli was tested using a set of von Frey filaments. Filaments with increasing force (0.4, 1, 2, 4, 6, 8, 10, 15, and 26 g) were applied to the plantar surface of the left paw through a wire mesh floor until a rapid withdrawal or licking response was elicited. The force of the filament at the response threshold was recorded as the MT. The final MT was calculated as the average of three consecutive tests with at least 5-minute intervals.

2) Acetone test score

As described in Yoon et al. [25]’s study, cold hyperalgesia was evaluated using acetone and recorded as the acetone test score (ATS). The same equipment used for MT tests was employed. Briefly, 100 µL of acetone was tenderly sprayed onto the sole of the left paw via a wire mesh bottom, and behavioral reactions were observed for 20 seconds. Responses were recorded using a 4-point scale: 3 points, repetitive withdrawal accompanied by licking; 2 points, prolonged paw withdrawal; 1 point, slight paw withdrawal; and 0 points, no response. The final ATS was calculated as the average of three consecutive tests with at least a 5-minute interval.

4. ACC catheterization and agent administration

A customized catheter was inserted into the bilateral ACC for intra-ACC injection as described in the authors’ previous study [9]. Briefly, the rats were anesthetized using pentobarbital (60 mg/kg, i.p.), stereotactic equipment (RWD Life Science Co., Ltd.) was used to fix the head and two holes (anterior to bregma, 1.6 mm; lateral, 0.5 mm) were drilled in the frontal bone on each side, and the catheter was installed and subsequently fixed onto the skull using polymethyl methacrylate. Seven days after catheterization, rats that did not exhibit abnormal physical behavior were subjected to CCI. The cGAS inhibitor RU.521 (Selleck) and the STING inhibitor C-176 (Selleck) were dissolved in 10% dimethyl sulfoxide (DMSO) at a concentration of 20 µg/µL, based on previous reports and the authors’ pilot experiments [14,26,27]. The rats were allocated to four groups: CCI, CCI + DMSO, CCI + RU.521, and CCI + C-176. On days 7–9 after CCI, 1 µL of DMSO, RU.521, or C-176 was micro-injected into the bilateral ACC with a micro pump according to their group allocation.

5. EA application

EA was performed as described in the authors’ recent study [28]. In the CCI + EA group, EA was performed at the left hind limb Zusanli (ST36; anterior lateral side of the hind limb, 6 mm below the depression on the lateral side of the patella) and Kunlun (BL60; depression between the Achilles tendon and lateral malleolus) acupoints. EA was operated using a Hua Tuo acupuncture device (Suzhou Medical Equipment Co., Ltd.), and the stimulation parameter was set as follows: frequency of 2 Hz, continuous output wave for 30 minutes, current intensity starting from 0.5 mA, increasing by 0.5 mA every 10 minutes. Rats in the CCI + sham EA (SEA) group were subjected to acupuncture needles inserted at the ST36 and BL60 acupoints, but no stimulation was applied. EA was performed once a day for 7 consecutive days, from the 7th to 13th days after CCI.

6. Immunofluorescence assay

Naive rats were deeply anesthetized with pentobarbital. After fixation on a foam board, the thoracic cavity was opened, and the heart was fully exposed. Next, 200 mL of 4°C 1× phosphate-buffered saline was perfused into the ascending aorta, followed by 200 mL of 4°C 4% paraformaldehyde. The ACC was harvested, fixed in 4°C 4% paraformaldehyde for 24 hours, and subsequently cryoprotected with 20% saccharose at 4°C for 2 days. After that, the ACC was cut into 30 μm sections using a cryostat and stored at −20°C. For immunofluorescence, 0.3% phosphate-buffered saline–Triton was used to permeabilize sections, and treated with blocking buffer for 1 hour at 37°C and incubated with the mouse monoclonal anti-neuron-specific nuclear protein (NeuN, 1:1000; Abcam), mouse monoclonal anti-ionized calcium-binding adaptor molecule 1 (Iba1, 1:200; Abcam), mouse monoclonal anti-glial fibrillary acidic protein (GFAP, 1:200; Cell Signaling Technology [CST]), rabbit monoclonal anti-cGAS (1:500; CST), and rabbit monoclonal anti-STING (1:100; CST) antibodies 24 hours at 4°C constant temperature. After washing thrice (10 minutes each time on the horizontal shaker) with 1× phosphate-buffered saline, a fluorescence secondary antibody was used to bind the primary antibody and detect the target protein in a dark room for 1 hour at 37°C. Images were captured using a confocal fluorescence microscope (Olympus). For percentage analysis, images from three randomly selected sections per animal were analyzed and averaged to obtain a mean value. The percentage of NeuN-, Iba1-, or GFAP- positive cells among cGAS- or STING- positive cells was calculated using the ImageJ software.

7. Western blotting

As described in the authors’ previous study [9], rats were anesthetized with pentobarbital and euthanized. The ACC tissue was separated into left (ipsilateral to CCI) and right (contralateral to CCI) regions and stored at −80°C for later protein expression analysis. Tissues were homogenized with RIPA buffer containing phenylmethylsulfonyl fluoride and phosphatase inhibitors as protein samples. Next, the protein samples were separated by sodium dodecyl–polyacrylamide gel electrophoresis under a constant voltage of 100 V and transferred onto 0.45 μm polyvinylidene fluoride membranes under a constant current of 300 mA. Membranes were pre-absorbed with blocking buffer on a horizontal shaker at room temperature for 1 hour and incubated with rabbit monoclonal anti-cGAS (1:1000; CST), rabbit monoclonal anti-STING (1:1000; CST), mouse monoclonal anti-GAPDH (1:1000; Beyotime Biotechnology Co., Ltd.), and mouse monoclonal anti-Tubulin (1:1000; Beyotime Biotechnology Co., Ltd.) antibodies for 24 hours at 4°C. After washing 3 times (10 minutes each time on a horizontal shaker) with Tris-buffered saline Tween, secondary antibodies were used to bind the primary antibody and detect the target protein. Finally, the immunoreactivity of the membranes was examined using a ChemiDoc MP imaging system (Bio-Rad).

8. Statistical analysis

All data are expressed as mean and standard errors of the mean and were analyzed using GraphPad Prism version 10.0. Independent t-tests were used to analyze the behavioral data between the two groups. When behavioral data were tested at different time points, repeated-measures two-way analysis of variance (ANOVA), was used followed by Tukey’s post-hoc test. One-way ANOVA was used to analyze Western blot data, followed by Dunnett’s multiple comparison test. For all tests, a P value < 0.05 was considered statistically significant.

RESULTS

1. Cellular localization of cGAS and STING in the rat ACC

The distributions of cGAS and STING in the ACC were explored using double-staining immunofluorescence. NeuN was used as a neuronal marker, Iba1 was used as a microglial marker, and GFAP was used for astrocytes. The immunofluorescence results revealed that cGAS and STING predominantly colocalized with NeuN, with little colocalization with Iba1 or GFAP (Fig. 1A–C). Quantitative analysis revealed that 94% of cGAS- and 95% of STING-positive cells were NeuN-positive (n = 3; Fig. 1D). In contrast, a small number of Iba1 positive (1% cGAS- and 2% STING-positive) or GFAP-positive (1% cGAS- and 2% STING-positive) cells were observed (n = 3, Fig. 1E, F), suggesting that cGAS and STING are predominantly expressed in neurons in the ACC.

Fig. 1.

Fig. 1

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Cellular co-localization of cGAS and STING in the ACC of rats. (A) The co-localization of cGAS (red) or STING (red) with NeuN (neuronal marker, green). Yellow signals indicate co-localization (arrows). (B) The co-localization of cGAS (red) or STING (red) with Iba1 (microglial marker, green). (C) The co-localization of cGAS (red) or STING (red) with GFAP (astrocyte marker, green). Immunofluorescence staining for (A), (B), and (C). Scale bar = 100 μm. (D–F) Percentage of NeuN-, Iba1-, or GFAP-positive cells among cGAS- or STING-positive cells (results from three animals). Data are presented as mean ± standard error of the mean. cGAS: GMP–AMP synthase, STING: stimulator of interferon genes, GFAP: glial fibrillary acidic protein, ACC: anterior cingulate cortex.

2. CCI caused pain hypersensitivity and overexpression of cGAS and STING in the ACC

To investigate whether CCI leads to changes in the expression of cGAS and STING in the ACC, the authors examined their protein levels by Western blotting after the NP model was established. The behavioral tests revealed that, compared with sham surgery, CCI significantly reduced MT (n = 7, t-test, P < 0.001, Fig. 2A) and significantly increased ATS (n = 7, t-test, P < 0.001, Fig. 2B) on day 7 following CCI, indicating that CCI successfully induced mechanical and cold hyperalgesia. After the behavioral test on the seventh day following CCI, the rats were euthanized, and the ACC was harvested for Western blotting. The results revealed that CCI upregulated the abundance of cGAS (n = 4, ANOVA, P = 0.010 or P = 0.006, Fig. 2C) and STING (n = 4, ANOVA, P < 0.001, Fig. 2D) compared to sham surgery. These findings indicated the activation of the ACC cGAS–STING pathway following CCI.

Fig. 2.

Fig. 2

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The establishment of the NP model and the activated cGAS–STING in the ACC. (A) CCI significantly lowered MT compared to sham surgery (n = 7, t-test, ***P < 0.001, vs. sham). (B) CCI significantly increased ATS compared to sham surgery (n = 7, t-test, ***P < 0.001, vs. sham). (C) Western blotting revealed the upregulated level of cGAS in the ACC in the CCI group compared to that in the sham group (n = 4, ANOVA, *P = 0.010; ##P = 0.006). (D) Western blotting revealed the upregulated level of STING in the ACC in the CCI group compared to that in the sham group (n = 4, ANOVA, ***P < 0.001; ###P < 0.001). Data are presented as mean ± standard error of the mean. CCI: chronic constriction injury, MT: mechanical threshold, ATS: acetone test score, cGAS: GMP–AMP synthase, STING: stimulator of interferon genes, NP: neuropathic pain, ACC: anterior cingulate cortex, ANOVA: analysis of variance.

3. RU.521 and C-176 reversed CCI caused pain hypersensitivity

Next, the authors explored whether the ACC cGAS–STING pathway was involved in the progression of NP by bilaterally injecting RU.521 or C-176 into ACC to inhibit the cGAS–STING pathway from the seventh to the ninth day after CCI (Fig. 3A, B). Behavioral tests revealed that, compared to DMSO, RU.521 and C-176 significantly alleviated the response to mechanical (n = 7, ANOVA, P < 0.001, Fig. 3C) and cold stimuli (n = 7, ANOVA, P < 0.001, Fig. 3D) from day 9 after CCI onward. These results demonstrated that inhibition of the ACC cGAS–STING pathway reversed CCI-induced mechanical and cold hyperalgesia, indicating a crucial role of the ACC cGAS–STING pathway in the progression of NP after CCI.

Fig. 3.

Fig. 3

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Antagonism of cGAS or STING in the ACC reversed the hyperalgesia caused by CCI. (A) Experimental flow chart. (B) Verification of the ACC catheterization with methylene blue. (C) RU.521 and C-176 (Selleck) significantly increased MT as compared with DMSO (n = 7, ANOVA, ***P < 0.001; ###P < 0.001). (D) RU.521 and C-176 significantly decreased ATS compared with DMSO (n = 7, ANOVA, ***P < 0.001; ###P < 0.001). Data are presented as mean ± standard error of the mean. CCI: chronic constriction injury, MT: mechanical threshold, DMSO: dimethyl sulfoxide, ATS: acetone test score, cGAS: GMP–AMP synthase, STING: stimulator of interferon genes, ACC: anterior cingulate cortex, ANOVA: analysis of variance.

4. EA attenuated pain hypersensitivity and suppressed cGAS–STING pathway activation in ACC

To further investigate the analgesic effects and potential mechanisms of EA, continuous EA treatments were administered from the 7th to 13th days following CCI. Behavioral tests were performed before EA administration and once daily during EA treatment. The results revealed that, compared to the CCI + SEA group, the response to mechanical stimuli in the CCI + EA group was significantly alleviated from day 10 after CCI onward, whereas the response to cold stimuli was significantly alleviated from day 11 after CCI onward (n = 7, ANOVA, P < 0.001, Fig. 4A, B), indicating that EA effectively attenuated CCI-induced hyperalgesia. After the behavioral test on the 14th day following CCI, the rats were euthanized, and the ACC was collected for Western blotting. The blot results demonstrated that, compared to SEA, EA significantly downregulated the levels of cGAS and STING (n = 4, ANOVA, P < 0.001, Fig. 4C, D) in the ACC. These results demonstrated that EA alleviated NP by inhibiting the cGAS–STING pathway, indicating that the cGAS–STING pathway in the ACC may be the cortical mechanism of the analgesic effects of EA.

Fig. 4.

Fig. 4

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EA alleviated hyperalgesia and decreased cGAS and STING expression in ACC. (A, B) EA significantly increased the MT and decreased ATS compared to the SEA (n = 7, ANOVA, ***P < 0.001). (C, D) EA significantly decreased the protein level of cGAS and STING compared to SEA (n = 4, ANOVA, ***P < 0.001). Data are presented as mean ± standard error of the mean. CCI: chronic constriction injury, MT: mechanical threshold, ATS: acetone test score, EA: electroacupuncture, SEA: sham EA, cGAS: GMP–AMP synthase, STING: stimulator of interferon genes, ACC: anterior cingulate cortex, ANOVA: analysis of variance.

DISCUSSION

This study demonstrated that cGAS and STING were mainly enriched in ACC neurons, but not in astrocytes or microglia. Consistent with the development of behavioral hyperalgesia, the expression of cGAS and STING increased bilaterally in the ACC after CCI. Microinjection of cGAS or STING inhibitors into the ACC alleviated CCI-induced NP, suggesting that the activated cGAS–STING pathway contributed to the occurrence of NP following peripheral nerve injury. Moreover, EA alleviated CCI-induced hyperpathia and impeded the upregulation of cGAS and STING caused by CCI, indicating that EA may alleviate NP by inhibiting the cGAS–STING pathway in the ACC.

cGAS- and STING- mediated innate immune responses are important for mammalian cells to detect pathological DNA fragments and maintain cellular homeostasis. The cGAS–STING pathway is a key factor in the occurrence and development of several neurological diseases [29], indicating its important role in neurological disorders. It has been revealed that, in rodent models, peripheral nerve injury triggers the release of mitochondrial or nuclear DNA into the cytoplasm of spinal neurons and glial cells, activating the cGAS–STING pathway, promoting pro-inflammatory signaling, and contributing to the development of NP [30]. However, the specific function of STING in NP and its association with cGAS remains controversial [13,14]. Previous studies have demonstrated that microglial STING mediates IL-6 induction and maintains a local inflammatory state, or promotes microglial polarization, enhancing pain signal transmission in the spinal dorsal horn, which leads to an increase in the excitability of spinal neurons and induces NP [14,16]. To date, studies investigating the cGAS–STING pathway in the ACC have been limited. The activation of the cGAS–STING pathway in the central nervous system upregulates inflammatory cytokines, including tumor necrosis factor (TNF), ILs, and interferons, thereby promoting the development of neurodegenerative diseases, such as ataxia telangiectasia and Parkinson’s disease [31,32]. These findings suggest a potential role of the cGAS–STING pathway in mediating neuroinflammation in the central nervous system. However, it remains unclear whether similar mechanisms operate in specific brain regions, such as the ACC. Therefore, it is hypothesized that the activation of the cGAS–STING pathway in the ACC may contribute to the expression of inflammatory factors following peripheral nerve injury.

In the present study, a CCI-induced NP model was used to investigate the cGAS–STING cascade signaling in the ACC. Western blotting revealed that cGAS and STING expression increased bilaterally following peripheral nerve injury, unlike in the spinal cord, in which cGAS and STING were only upregulated on the injured side [15]. This discrepancy may be attributed to several factors. The crossover of the spinothalamic tracts in the spinal cord and the afferent projections from the contralateral pain-transmitting area in the hypothalamus result in a potential cross-afferent in the ACC [33,34]. Moreover, microglia in the ACC release chemokine C-C motif ligand 2 on the side of peripheral nerve injury, which spreads to the opposite side through the corpus callosum and subsequently activates the microglia of the opposite ACC to release IL-1β or TNF-α, thereby activating the neurons [35]. Bilateral function has been verified in clinical studies, demonstrating that the degree of bilateral ACC activation correlates with pain duration in patients with fibromyalgia [36]. However, in terms of pain and emotion, it appears that the function of the left ACC is more correlated with pain, whereas that of the right is more correlated with mood [37], implying a lateral-dominant process of pain and emotion in the ACC.

In the present study, the authors found that local pharmacological inhibition of cGAS or STING alleviated CCI-induced hyperpathia, suggesting that the activation of the cGAS–STING signaling pathway in the ACC contributes to the development and progression of NP. Previous studies have demonstrated that peripheral nerve injury induces the upregulation of TNF-α and IL-6 in the ACC, which activates the NF-κB pathway, leading to the release of cytokines and chemokines, enhancing the excitability of neurons and aggravating synaptic plasticity and pain transmission [38]. Furthermore, IL-6 promotes microglial activation, thereby increasing the sensitivity of neurons to noxious stimuli and enhancing the excitability of neurons and the transmission of pain signals by activating the transient receptor potential vanilloid 1 ion channel or phosphorylating the N-methyl-d-aspartate receptor in the ACC [39,40]. However, the specific molecular or neural circuit mechanisms underlying the cGAS–STING pathway-mediated NP require further investigation.

Accumulating evidence suggests that EA mainly exerts its analgesic effect by regulating noradrenaline, opioids, or acetylcholine in the periventricular gray matter, spinal cord, or hypothalamus [19,41]. Recent studies have demonstrated that EA alleviates NP by inhibiting pain-induced alterations in glucose metabolism in the prefrontal cortex [42]. It has also been reported that, in the ACC, EA may affect inhibitory interneuron populations (such as GABAergic neurons) and downregulate excitatory neurotransmission, thereby reducing pain sensitivity and alleviating pain-associated negative emotions [43,44]. Although the precise mechanisms underlying the EA-induced modulation of the ACC remain unclear, the above evidence supports the role of ACC hyperactivity in contributing pain-related behaviors. In the present study, the authors found that EA not only relieved hyperpathia but also suppressed the overexpression of cGAS and STING induced by CCI, suggesting that inhibiting the ACC cGAS–STING pathway contributes to the cortical mechanism of the analgesic effect of EA. Nevertheless, this study has several limitations. First, the downstream molecular mechanisms of cGAS–STING signaling in the ACC remain unknown. In particular, the proposed involvement of cytokines and related signaling pathways is largely extrapolated from studies conducted in other regions; thus, the present results should be interpreted with caution. Second, this study focused on a neuron-centered mechanism; however, the potential contribution of non-neuronal cells to cGAS–STING signaling in the ACC was not directly examined. Third, although pharmacological inhibitors are effective, they may produce off-target effects and do not fully recapitulate cell-type or circuit-specific modulation of cGAS–STING signaling.

In conclusion, the authors’ findings suggest that the activation of the cGAS–STING pathway in the ACC may contribute to the development and progression of NP following peripheral nerve injury, and that EA may alleviate NP, at least in part, by modulating cGAS–STING signaling in the ACC of rats.

Footnotes

DATA AVAILABILITY

Data files are available from Harvard Dataverse: https://doi.org/10.7910/DVN/8BYU6P. Further inquiries can be directed to the corresponding author.

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

FUNDING

This work was supported by the National Natural Science Foundation of China (82401395), a clinical study fund from the First Affiliated Hospital, Zhejiang University School of Medicine (BL202234) and the Natural Science Foundation of Zhejiang Province, China (LQ23H090013 and LBY22H270007).

AUTHOR CONTRIBUTIONS

Ang Li: Methodology, Formal analysis, Data curation, Manuscript preparation; Mei Li: Methodology, Computation, Formal analysis, Data curation; Zhen-Ping Hu: Critical revision; Meng-Jing Yang: Methodology, Data curation; Hui Zhang: Investigation, Resources; Xu-Wu Xiang: Supervision, Funding acquisition; Yong-Xing Yao: Study conception, Critical review, Supervision, Funding acquisition.

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