Sage Journals: Discover world-class research

Abstract

Pain, particularly chronic pain, is a major reason patients seek physical therapy. Inflammation plays a crucial role in both the development and persistence of chronic pain. Neuronal PAS domain protein 2 (NPAS2), a core circadian transcriptional regulator, has been implicated in modulating pain-related stress responses. In this study, we first examined NPAS2 expression in nociceptive-sensitized mice following complete Freund’s adjuvant (CFA) administration. We then systematically investigated the effects of CFA on astrocyte activation and inflammatory factor release in NPAS2 knockout (KO) mice. Our results demonstrated that NPAS2 deletion did not alter baseline pain thresholds under normal physiological conditions. However, in CFA-injected mice, NPAS2 KO significantly lowered mechanical and thermal pain thresholds in 50% of subjects, leading to enhanced nociceptive sensitization. This effect may be attributed to the promotion of astrocyte activation and the upregulation of pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α, and NF-κB. These findings highlight NPAS2 as a potential prognostic biomarker for pain chronification and a promising therapeutic target for biologically tailored pain interventions.

Keywords

NPAS2 CFA inflammatory pain cytokine

Introduction

Pain is a common comorbidity of various clinical conditions and has been recognized as the fifth vital sign in recent years.¹ Its occurrence is influenced by sensory, emotional, cognitive, and social factors. The transmission of nociceptive signals is regulated by multiple mechanisms, including immune responses and glial cell activation, both of which contribute to pain sensitization.² Upon exposure to painful stimuli, peripheral neurons transmit nociceptive signals to the dorsal horn of the spinal cord, where neurotransmitters such as calcitonin gene-related peptide (CGRP), substance P, glutamate, γ-aminobutyric acid (GABA), serotonin, and adenosine triphosphate (ATP) are released. These neurotransmitters, in turn, activate glial cells in the synaptic region, further sensitizing postsynaptic neurons and enhancing pain perception.^3,4

Glial cell proliferation is a hallmark of inflammatory responses in the central nervous system (CNS), where inflammatory mediators activate astrocytes, microglia, macrophages, and leukocytes, all of which contribute to nociceptive sensitization.⁵ And nowadays, microglia and astrocytes within the central nervous system have been shown to play a key role in the development and maintenance of neuropathic pain.^6,7 Pain and inflammation are closely linked, previous studies have demonstrated that inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) mediate pain transmission by directly sensitizing nociceptive receptors or by inducing the production of additional pain-sensitizing mediators.^8,9 Furthermore, microglia and astrocytes serve as key sources of neuroinflammatory mediators.¹⁰ The release of inflammatory factors can trigger a cascade of signal transduction pathways that activate or sensitize ion channels and nociceptive receptors on peripheral nerve terminals, leading to neuronal excitation, action potential generation, and ultimately pain perception.^11,12

Neuronal PAS domain protein 2 (NPAS2) is a core circadian gene with known involvement in stress regulation. Studies on anxiety have reported increased NPAS2 expression in response to both acute and chronic stressors, suggesting its role in the stress response.¹³ Moreover, NPAS2 has been identified as one of the most dysregulated circadian genes in immune-inflammatory conditions. Its expression is not only altered in inflammatory states but also directly induced by TNF-α stimulation.¹³ In pain research, NPAS2 knockout (NPAS2^−/−) mice have been shown to exhibit no significant differences in baseline thermal pain thresholds or acute analgesia. However, NPAS2^−/− mice demonstrated altered fentanyl tolerance, indicating a potential role for NPAS2 in pain modulation.¹⁴ This raises important questions: Does pain, as a form of physiological stress, influence NPAS2 expression? Does NPAS2 knockdown impact pain perception? Given the established role of inflammation in pain sensitization, it is critical to determine whether NPAS2 plays a regulatory role in inflammatory pain. This study aims to provide novel insights into the complex interplay among circadian rhythm regulation, inflammation, and pain perception.

Materials and methods

Animals

All animal experiments were conducted in accordance with the regulations of the Animal Experimentation Management Committee of the Second Affiliated Hospital of Air Force Medical University, Tangdu Hospital, and were approved by the Ethics Committee.

NPAS2 knockout (NPAS2^−/−) mice (C57BL/6N, whole-body knockout) were purchased from Cyagen Biosciences and crossed with wild-type C57BL/6J mice (obtained from the Basic Medical Animal Experiment Center, Air Force Medical University, China) to generate heterozygous offspring. Heterozygous males and females were bred to obtain homozygous NPAS2^−/− mice and wild-type littermates. Genotyping was performed via tail biopsy at 3 weeks of age, followed by DNA extraction and polymerase chain reaction (PCR) analysis using specific primers. Eight-week-old male wild-type and NPAS2^−/− mice were randomly assigned to experimental groups.

Mice were housed in individually ventilated cages with standard bedding, under a 12-h light/dark cycle (08:15–20:15 for light, 20:15–08:15 for dark), with controlled temperature (22°C ± 3°C) and humidity (45%–56%). Food and water were available ad libitum, ensuring normal sleep-wake cycles.

Hind paw thickness assessment

Hind paw edema was quantified by measuring the maximum dorsal-ventral thickness using calipers.

Induction of inflammatory pain

Thirty-two C57BL/6J mice were randomly assigned to four experimental groups: Wild-type (WT) saline group (WT-Saline, n = 8), wild-type CFA group (WT-CFA, n = 8), NPAS2 knockout (KO) saline group (KO-Saline, n = 8), and NPAS2 knockout CFA group (KO-CFA, n = 8).

To induce inflammatory pain, Complete Freund’s Adjuvant (CFA) stock solution (F5881; Millipore Sigma, Burlington, MA, USA) was diluted to 50% in 0.9% saline to prevent excessive inflammation and spontaneous pain behaviors. Mice in the CFA groups received a subcutaneous injection of 10 μL of 50% CFA into the right hind paw using a microsyringe, while control mice received an equivalent volume of saline. CFA injection led to localized erythema, edema, and hypersensitivity, but mice continued to exhibit normal grooming behavior and weight gain.

Assessment of hypersensitivity

Thermal hyperalgesia was assessed using the hot plate test, following the protocol of Dirig et al.¹⁵ Mice were acclimated for 30 min prior to testing. The hot plate was set at 52.0°C ± 0.3°C, and each mouse was placed on the heated surface. The Thermal Withdrawal Latency (TWL) in response to radiant heat was recorded over five trials at 5-min intervals, with a 30-s cutoff to prevent tissue damage.

Mechanical pain was examined by the vonFrey hair test, as described by Chaplan et al.¹⁶ The mechanical foot reduction threshold was detected with vonFrey filament 1 day before CFA injection, 0 days after CFA injection, 1, 3, 7, and 14 days after CFA injection. Place the mice in a transparent glass dish on a raised mesh to acclimate for 30 min. Wait until the mouse stops exploring the environment and stands quietly on the raised mesh. Use a Von Frey fiber to stimulate the center of the plantar surface of the right hind paw. Bend the fiber into a C shape as the standard for full stress. Each stimulation lasts for 2 s, with a 60-s interval before the next measurement. Positive manifestations include foot contraction or foot lifting in mice. Each mouse is tested five times. If the result shows three negative responses, a larger fiber is replaced and retested. If the result shows three positive responses, a smaller fiber is retested. The test starts with 1 g. Record the minimum number of fiber grams that can elicit 3 out of 5 positive reactions as the experimental result.

Western blot analysis

After behavioral testing, mice were anesthetized and sacrificed via cervical dislocation. The L4–L6 spinal cord segments were rapidly dissected and homogenized in cold RIPA buffer supplemented with protease and phosphatase inhibitors. The homogenates were centrifuged at 12,000×g for 5 min at 4°C, and the supernatants were collected for protein quantification using the BCA method.

Each sample was run on SDS-PAGE on 10% flow gel and then transferred to Polyvinylidene fluoride (PVDF) membrane. Blocked with 5% buttermilk at room temperature for 1 h, then incubated with the primary antibody at 4°C overnight. Then wash in Tris-buffered saline-Tween solution three times. Anti-rabbits and Anti-mouse antibodies were incubated with horseradish (ab205718; ab205719; Abcam) at room temperature for 1 h. Finally, the Western Blotting Luminol Reagent (sc-2048; Santa Cruz Biotech) for luminescence, adjusted the focus and exposure time of the chemiluminescence instrument, collected the strip image and saved it. The details of the primary antibodies used for Western blot analysis are provided in Supplemental Table 1.

Real-time PCR

After behavioral testing, the mice were anesthetized and were killed by cervical dislocation. The L4-L6 segments of mouse spinal cord were extracted immediately and RNA was extracted by Trizol method. The reaction solution was prepared according to the instructions of the reverse transcription kit and placed in a PCR instrument. The reverse transcription reaction system was set up at 50°C for 15 min and 85°C for 5 min for inactivation of reverse transcriptase. PCR reactions were performed using TB Green^® Premix Ex Taq™ II (RR820A; Takara bio). The results were calculated with the 2^−∆∆Ct method and normalized to β-actin levels. The primer sequences used for quantitative real-time PCR are provided in Supplemental Table 2.

Treatment with BAY-117082

Following the completion of the behavioral assays, mice were administered an intraperitoneal injection of the inhibitor BAY-117082 (5 mg/kg). Ninety minutes after injection, the animals were anesthetized and euthanized by cervical dislocation.¹⁷

Statistical analysis isolation and culture of mouse cortical astrocytes

Data are expressed as the mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 8. Group differences were analyzed using Student’s t-test (for two-group comparisons) or two-way ANOVA, followed by Bonferroni’s post hoc test. Statistical significance was set at p < 0.05, with p > 0.05 considered non-significant (ns).

Results

CFA-evoked inflammation of the hind paw initiates an overproduction of NAPS2 in the spinal dorsal horn of mice

To determine whether pain influences NPAS2 expression, we performed qRT-PCR analysis on WT-CFA group mice following intraplantar CFA injection. Our results revealed a significant increase in NPAS2 mRNA expression compared to the physiological saline control group (Figure 1(a)). Western blot analysis further confirmed this upregulation at the protein level, demonstrating a comparable elevation of NPAS2 in the CFA group (Figure 1(b)). Given the rhythmic nature of NPAS2 expression, we further analyzed its mRNA levels in both wild-type (WT) and NPAS2-knockout (KO) mice over a 48-h period. As expected, NPAS2 in WT mice exhibited pronounced circadian oscillations, whereas rhythmicity was completely abolished in KO mice (Figure 1(c)).

Figure 1.

CFA-evoked inflammation of the hind paw initiates an overproduction of NAPS2 in the spinal dorsal horn of mice: (a) qRT-PCR was employed to quantify NPAS2 mRNA expression in the spinal cord of mice, (b) western blotting was performed to detect the expression of NPAS2 protein in the spinal cord of mice, and (c) temporal expression of NPAS2 mRNA over 48 h in mice of two genotypes (n = 3).

NPAS2 deficiency lowers mechanical and thermal pain thresholds following inflammation

In the 14-day experimental period, no significant differences were observed in hind paw thickness, mechanical withdrawal threshold (MWT), or thermal withdrawal latency (TWL) between WT-Saline and KO-Saline groups, both of which received normal saline injections (Figure 2(b)–(d)). However, in mice injected with complete Freund’s adjuvant (CFA) (WT-CFA and KO-CFA groups), hind paw thickness progressively increased, distinguishing them from the WT-Saline group (Figure 2(a) and (b)).

Figure 2.

The deficiency of NPAS2 reduces the mechanical and thermal pain thresholds of the mice paws after inflammation: (a) images of the hind paw in the KO-CFA group of mice (n = 8), (b) CFA-induced paw edema in wild-type and NPAS2 knockout mice (n = 8), (c) the mechanical withdrawal threshold (MWT) of wild type (WT) and NPAS2 knockout mice (KO) mice receiving different treatments physiological saline injection and Complete Freund’s adjuvant (CFA)-induced (n = 8), and (d) the thermal withdrawal latency (TWL) of wild type (WT) and NPAS2 knockout mice (KO) mice receiving different treatments physiological saline injection and Complete Freund’s adjuvant (CFA)-induced (n = 8).

Following CFA injection, both MWT and TWL declined sharply within the first 3 days, with the most pronounced reduction in MWT occurring on day 3 (Figure 2(c) and (d)). Thereafter, MWT gradually recovered, aligning with previous findings. Notably, at both day 7 and day 14, CFA-treated NPAS2 knockout mice exhibited significantly greater hind paw thickness than their wild-type counterparts. Concurrently, MWT and TWL remained lower in NPAS2 knockout mice compared to wild-type mice (Figure 2(b)–(d)).

These findings indicate that NPAS2 deficiency exacerbates CFA-induced inflammatory pain by further reducing mechanical pain thresholds and thermal pain thresholds in mice.

The absence of NPAS2 exacerbates the release of pro-inflammatory cytokines in the spinal cord of CFA-induced mice

To determine the effect of NPAS2 deficiency on pro-inflammatory cytokine expression in the spinal cord following CFA-induced inflammation, we analyzed the mRNA and protein levels of IL-1β, IL-6, and TNF-α over a 14-day period. qRT-PCR analysis revealed that CFA injection significantly upregulated IL-1β, IL-6, and TNF-α mRNA levels in the spinal cord, peaking on day 3 (Figure 3(a)). Notably, in KO mice subjected to CFA-induced inflammation, the expression levels of these cytokines were significantly higher compared to their WT counterparts. Western blot analysis further confirmed that CFA treatment markedly elevated IL-1β, IL-6, and TNF-α protein levels in the spinal cord of KO mice compared to WT mice (Figure 3(b)). These findings indicate that NPAS2 deficiency amplifies the inflammatory response in the spinal cord by promoting the overproduction of pro-inflammatory cytokines in CFA-induced mice. Given the heightened inflammatory response in NPAS2-deficient mice, we next investigated whether NPAS2 deficiency affects neuroglial activation in the spinal dorsal horn.

Figure 3.

The absence of NPAS2 promotes the release of pro-inflammatory cytokines in the spinal cord of CFA-induced mice: (a) IL-1β, IL-6, and TNF-α mRNA levels were measured in the samples from the wild-type and NPAS2-knockout mice treated with saline and CFA (n = 3) and (b) protein levels of IL-1β, IL-6, and TNF-α were quantified in samples from wild-type (WT) and NPAS2-knockout (KO) mice treated with either saline or CFA (n = 3).

NPAS2 deficiency enhances astrocyte activation in the spinal dorsal horn of CFA-induced mice

To further explore the role of NPAS2 in neuroinflammation, we examined the activation of spinal astrocytes following CFA injection in KO mice. qRT-PCR analysis revealed that, compared to WT-Saline controls, CFA stimulation significantly increased the mRNA expression of GFAP and Iba1 in the spinal cords of WT-CFA mice, indicating the activation of both astrocytes and microglia in response to inflammation. Notably, compared to the WT-CFA group, KO-CFA mice exhibited a significant upregulation of GFAP mRNA, whereas Iba1 mRNA levels remained unchanged (Figure 4(a)). This suggests that NPAS2 deficiency selectively enhances astrocyte activation following CFA-induced inflammation, without significantly affecting microglial activation.

Figure 4.

The deficiency of the NPAS2 gene activates astrocytes in the spinal dorsal horn of CFA-induced mice: (a) GFAP and IBA1 mRNA levels were measured in the samples from the wild-type and NPAS2-knockout mice treated with saline and CFA (n = 3), (b) GFAP and IBA1 protein levels were measured in the samples from the wild-type and NPAS2-knockout mice treated with saline and CFA (n = 3), and (c) GFAP immunohistochemistry in the spinal dorsal horn from the wild-type and NPAS2-knockout mice treated with saline and CFA, magnification ×100, Scale bar, 50 μm (n = 3).

To validate these findings, we performed Western blot and immunohistochemical analyses, both of which confirmed exacerbated astrocyte activation in the spinal dorsal horn of NPAS2-knockout mice following CFA injection (Figure 4(b) and (c)). These results demonstrate that NPAS2 plays a crucial role in regulating astrocyte reactivity in CFA-induced inflammatory conditions, with its deficiency leading to heightened astrocyte activation in the spinal dorsal horn.

NPAS2 deficiency upregulates NF-κB expression in the spinal cord of CFA-induced mice

NF-κB, JNK, ERK, and AKT signaling pathways are all implicated in inflammatory responses. To determine whether NPAS2 deficiency modulates inflammatory pain via these pathways, we performed qRT-PCR and Western blot analyses to examine their expression in the spinal cord. Our results demonstrated that both the mRNA and protein levels of NF-κB were significantly elevated in the spinal cords of WT-CFA mice compared to WT-Saline controls. Furthermore, KO-CFA mice exhibited even higher NF-κB expression than WT-CFA mice, suggesting that NPAS2 deficiency exacerbates NF-κB activation in response to CFA-induced inflammation (Figure 5(a) and (b)).

Figure 5.

The deficiency of NPAS2 increases NF-κB expression in the spinal cord of CFA-induced mice: (a) the mRNA expression levels of NF-κB, JNK, ERK, and AKT were quantified in samples obtained from wild-type and NPAS2-knockout mice following treatment with either saline or CFA (n = 3), (b) the protein expression levels of NF-κB, JNK, ERK, and AKT were determined in samples collected from wild-type and NPAS2-knockout mice following treatment with either saline or CFA (n = 3), (c) mice from the WT and KO groups, pre-treated with CFA, were administered an intraperitoneal injection of BAY-117082 (C + N). Corresponding WT and KO groups, treated with saline, were given an intraperitoneal injection of saline as a control. The L4-L6 spinal cord segments were subsequently dissected, and GFAP mRNA expression levels were evaluated by quantitative PCR (n = 3), and (d) mice from the CWT and KO groups, pre-treated with CFA, were administered an intraperitoneal injection of BAY-117082 (C + N). Mice from the corresponding WT and KO groups, treated with saline, were given an intraperitoneal injection of saline as a control. The L4-L6 spinal cord segments were then dissected, and GFAP protein expression levels were assessed using Western blot analysis (n = 3).

In contrast, the expression levels of JNK, ERK, and AKT remained unchanged across all groups, indicating that NPAS2 knockout selectively influences NF-κB signaling rather than broadly affecting multiple inflammatory pathways (Figure 5(a) and (b)). These findings suggest that NPAS2 may regulate pain-related physiological responses by modulating NF-κB activation in inflammatory conditions, highlighting NF-κB as a potential downstream effector of NPAS2 in CFA-induced inflammatory pain.

Mice from the WT and KO groups, which had been pre-treated with CFA, received an intraperitoneal injection of BAY-117082. Mice from the corresponding WT and KO groups, treated with saline, were administered an intraperitoneal injection of saline as a control. GFAP expression levels were quantified by PCR and Western blot analyses (Figure 5(c) and (d)). BAY-11-7082 treatment resulted in no significant change in GFAP expression, indicating effective suppression of astrocyte activation. These findings suggest that NPAS2 deletion selectively disrupts NF-κB-mediated inflammatory signaling and that NPAS2 modulates pain-related physiological responses by regulating NF-κB activation under inflammatory conditions.

CLOCK is insufficient to fully compensate for the functional loss of NPAS2 under pathological conditions

CLOCK and NPAS2 exhibit similar and overlapping functions in the regulation of circadian rhythms. In our study, mechanical and thermal pain thresholds did not differ significantly between NPAS2 knockout (KO) and wild-type (WT) mice, likely due to compensatory effects of CLOCK under normal physiological conditions. To explore this compensatory mechanism, we assessed CLOCK mRNA and protein expression levels across groups. Under saline treatment, CLOCK expression was significantly upregulated in NPAS2-deficient mice compared to WT controls. However, following CFA administration, CLOCK expression was markedly downregulated in NPAS2-deficient mice (Figure 6(a) and (b)). These results indicate that under inflammatory conditions, NPAS2 deficiency exacerbates pain sensitivity, and CLOCK is insufficient to fully compensate for NPAS2 loss, underscoring the essential role of NPAS2 in pathological states.

Figure 6.

CLOCK is insufficient to fully compensate for NPAS2 deficiency in CFA-treated mice: (a) the mRNA expression levels of CLOCK were quantified in samples obtained from wild-type and NPAS2-knockout mice following treatment with either saline or CFA (n = 3) and (b) the protein expression levels of CLOCK were determined in samples collected from wild-type and NPAS2-knockout mice following treatment with either saline or CFA (n = 3).

Discussion

Previous studies have demonstrated that circadian clock genes play a crucial role in immune-inflammatory responses. Immunohistochemical and molecular analyses in rheumatoid arthritis patients have identified ARNTL2 and NPAS2 as the most significantly altered clock genes under inflammatory conditions.¹⁸ In line with these findings, our study revealed that NPAS2 expression in the spinal cord undergoes substantial changes during CFA-induced inflammatory pain sensitization, supporting its involvement in pain-related circadian regulation. Notably, NPAS2 expression was upregulated in the spinal cord following CFA-induced inflammation, further corroborating its role in pain modulation.

NPAS2 forms an NPAS2/BMAL1 heterodimer that binds to the E-box of target gene promoters, regulating the expression of two other clock genes, PER and CRY, and playing a role in circadian rhythm regulation.¹⁹ The homology between CLOCK and NPAS2 leads to functional similarities and overlaps, as demonstrated by Crumbley et al.,²⁰ who found that mice lacking BMAL1 completely lose their circadian rhythm, while mice lacking CLOCK do not show severe symptoms, likely due to the compensation by its homolog NPAS2. When comparing mice with NPAS2 gene knockout and mice with simultaneous knockout of CLOCK and NPAS2, the latter showed a more obvious loss of circadian rhythm, suggesting overlapping roles in biological function. In line with this, our study showed no significant differences in mechanical and thermal pain latency between NPAS2 KO mice and WT mice, possibly due to compensation by certain substances or mechanisms in normal physiological conditions. However, under inflammatory conditions, NPAS2 deficiency exacerbated pain sensitization, underscoring its functional importance in pathological states.

CFA injection induces robust peripheral and central inflammatory responses, leading to pain sensitization and neuroinflammation. Consistent with previous studies, our results showed that pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were significantly upregulated in the spinal cord following CFA administration. Moreover, in NPAS2 KO mice, cytokine levels were further elevated compared to WT-CFA mice, suggesting that NPAS2 deficiency exacerbates neuroinflammatory responses.

Emerging evidence highlights a bidirectional interaction between the immune and nervous systems.²¹ Activated glial cells, particularly astrocytes and microglia, play a key role in pain modulation. Iba1 and GFAP are well-established markers of microglial and astrocytic activation, respectively, and their expression correlates with pain-related behaviors and neuroinflammation.²² Microglia and astrocytes are also responsible for the production of neuroinflammatory mediators, including TNF-α, IL-1β, etc.¹⁰ Peripheral inflammation and nerve injury lead to spinal astrocyte and microglia activation, which in turn enhances pain perception through pro-inflammatory mediator release.^23–26 Additionally, it is well known that the over activity of Glu is one of the typical pathological changes in pain regulation.²⁷ Astrocytes take up Glu through glutamate transporters (mainly GLT-1) to help maintain the appropriate level of extracellular Glu concentration.²⁸ Therefore, the study of astrocytes is indispensable for understanding the mechanisms of pain. Our study found that both microglia and astrocytes in the spinal cord were affected after CFA stimulation in mice, as evidenced by the significant increase in the expression of GFAP and Iba1 in the spinal cord. Furthermore, when comparing the KO-CFA group and the WT-CFA group, we found a significant increase in GFAP mRNA expression but no difference in Iba1 mRNA expression. Consistent results were obtained through Western blotting and immunohistochemistry, indicating an increase in GFAP expression. This is consistent with the role of astrocytes in early-stage inflammation and their important role in maintaining chronic inflammatory reactions. It suggests that downregulation of NPAS2 promotes the activation of astrocytes in the mouse spinal cord, but has no significant effect on microglia.

NF-κB is a key regulator of inflammatory responses, activated by stress, free radicals, ultraviolet radiation, and antigens. It modulates the expression of pro-inflammatory cytokines and pain-related genes, making it a central player in pain sensitization and neuroinflammation. In our study, NF-κB expression was significantly upregulated in the spinal cord following CFA administration, and its levels were further elevated in NPAS2 KO-CFA mice compared to WT-CFA mice. These findings suggest that NPAS2 deficiency exacerbates NF-κB activation, which may contribute to increased inflammatory pain and enhanced pain sensitization. Interestingly, no significant differences were observed in the expression of other signaling molecules, such as JNK, ERK, and AKT, indicating that NPAS2 primarily modulates inflammatory pain through NF-κB signaling rather than broader MAPK or PI3K/AKT pathways. These results align with behavioral findings, wherein NPAS2 KO mice exhibited increased pain sensitivity.

In summary, our research results indicate that the absence of NPAS2 exacerbates the activation of astrocytes in the dorsal spinal cord after CFA injection in the hind paw, increases the expression of pro-inflammatory factors and NF-κB, and reduces the mechanical and thermal pain thresholds in mice under the CFA model, suggesting the involvement of NPAS2 in the regulation of pain sensitization. In our experiments, we studied NPAS2 knockout and obtained the conclusion that NPAS2 knockout reduces the mechanical and thermal pain thresholds in mice with 50% CFA injection in the hind paw, resulting in pain sensitization. Therefore, we suggest that NPAS2 could represent a promising therapeutic target for the treatment of chronic pain in humans. Studies have demonstrated that the deletion of core clock proteins BMAL1 or CLOCK/NPAS2 significantly suppresses the basal expression of Chi3l1. Additionally, in vitro knockdown of Chi3l1 enhances the phagocytic activity of astrocytes and microglia towards zymosan particles and β-amyloid peptides.²⁹ The potential dependence of astrocyte activation on Chi3l1 following NPAS2 knockdown remains to be investigated and may be explored in future studies. In addition, NF-κB not only participates in immune regulation as a cytokine but also plays a significant role in cell inflammation through its signaling pathway, which has been found to trigger a unique neuroinflammatory response in astrocytes. Can our experimental results establish a connection through this pathway? This will also be a topic for further research in the next steps.

Supplemental Material

sj-docx-1-mpx-10.1177_17448069251351045 – Supplemental material for Circadian gene NPAS2 modulates pain sensitization in CFA-induced inflammatory pain model

Supplemental material, sj-docx-1-mpx-10.1177_17448069251351045 for Circadian gene NPAS2 modulates pain sensitization in CFA-induced inflammatory pain model by Jiaqi Dong, Jingyi Wei, Hongwei Tong, Xiaohua Shi, Menghui Yuan, Yiwei Cao, Mohammed A El-Magd, Qiang Chen, Hongxin Zhang, Peng Yuan and Jiao Mu in Molecular Pain

Footnotes

Abbreviations

NPAS2: neuronal PAS domain protein 2

CFA: complete Freund’s adjuvant

MWT: mechanical withdrawal threshold

TWL: thermal withdrawal latency

GFAP: glial fibrillary acidic protein

Iba1: ionized calcium binding adapter molecule 1

IL-1β: Interleukin-1β

IL-6: Interleukin-6

TNF-α: tumor necrosis factor-α

Authors’ contributions

JD, HT and JW performed most of the experiments and analyzed data; JD, MY and YC participated in the in vitro study. JD, Mohammed A El-Magd, PY and JM designed the overall study, supervised the experiments. JD, XS and QC wrote the paper. PY, HZ and JM revised the paper. PY and JM acquired the funding. All authors read and approved the final manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the ‘The Natural Science Foundation’-Boosting Project Plan of the Second Affiliated Hospital of Air Force Medical University (grants 2021ZTXM-002), and National Science Basic Research Plan in Shaanxi Province of China (grants 2023-JC-YB-671, 2024JC-YBMS-731, and 2024JC-YBQN-0243).

Ethical considerations

This study was approved by the institutional ethics committee of the Air Force Military Medical University.

Consent for publication

All authors have given their consent for the publication of this manuscript.

ORCID iD

Peng Yuan

Supplemental Material

Supplemental material for this article is available online.

References

Woolf

. What is this thing called pain? J Clin Invest 2010; 120: 3742–3744.

Alexander

Jeong

Szabo-Pardi

Burton

. Sex-specific differences in alcohol-induced pain sensitization. Neuropharmacology 2023; 225: 109354.

Luvisetto

. Botulinum neurotoxins beyond neurons: interplay with glial cells. Toxins 2022; 14: 14100704.

Rogoz

Andersen

Kullander

Lagerström

. Glutamate, substance P, and calcitonin gene-related peptide cooperate in inflammation-induced heat hyperalgesia. Mol Pharmacol 2014; 85: 322–334.

Willis

Brooks

Davis

. Chronic inflammatory pain and the neurovascular unit: a central role for glia in maintaining BBB integrity? Curr Pharm Des 2008; 14: 1625–1643.

Wei

Zhou

Zou

Liu

Xiao

Liu

Tan

Cao

. Activation of locus coeruleus-spinal cord noradrenergic neurons alleviates neuropathic pain in mice via reducing neuroinflammation from astrocytes and microglia in spinal dorsal horn. J Neuroinflam 2022; 19: 123.

Miranpuri

Bali

Nguyen

Kim

Modgil

Mehra

Buttar

Brown

Yutuc

Singh

Wood

Singh

Anand

. Role of microglia and astrocytes in spinal cord injury induced neuropathic pain. Ann Neurosci 2021; 28: 219–228.

Omura

Lüdtke

Horewicz

Fernandes

Galassi

Salgado

ASI

Palandi

Baldança

HDS

Bittencourt

Mack

Seim

Martins

Bobinski

. Decrease of IL-1β and TNF in the spinal cord mediates analgesia produced by ankle joint mobilization in complete freund adjuvant-induced inflammation mice model. Front Physiol 2021; 12: 816624.

Akhter

Irfan

Alamgeer

Jahan S

Shahzad

Latif

. Nerolidol: a potential approach in rheumatoid arthritis through reduction of TNF-α, IL-1β, IL-6, NF-kB, COX-2 and antioxidant effect in CFA-induced arthritic model. Inflammopharmacology 2022; 30: 537–548.

10.

Donnelly

Andriessen

Chen

Wang

Jiang

Maixner

. Central nervous system targets: glial cell mechanisms in chronic pain. Neurotherapeutics 2020; 17: 846–860.

11.

Uniyal

Akhilesh, Singh Rathore

Kumari Keshri

Pratap Singh

Singh

Tiwari

. Inhibition of pan-Aurora kinase attenuates evoked and ongoing pain in nerve injured rats via regulating KIF17-NR2B mediated signaling. Int Immunopharmacol 2022; 106: 108622.

12.

Naik

Uniyal

Chouhan

Tiwari

Sahu

. Natural products and some semi-synthetic analogues as potential TRPV1 ligands for attenuating neuropathic pain. Curr Pharm Biotechnol 2022; 23: 766–786.

13.

Ozburn

Kern

Parekh

Logan

Liu

Falcon

Becker-Krail

Purohit

Edgar

Huang

McClung

. NPAS2 Regulation of anxiety-like behavior and GABAA receptors. Front Mol Neurosci 2017; 10: 360.

14.

Puig

Shelton

Barko

Seney

Logan

. Sex-specific role of the circadian transcription factor NPAS2 in opioid tolerance, withdrawal and analgesia. Genes Brain Behav 2022; 21: e12829.

15.

Dirig

Salami

Rathbun

Ozaki

Yaksh

. Characterization of variables defining hindpaw withdrawal latency evoked by radiant thermal stimuli. J Neurosci Methods 1997; 76: 183–191.

16.

Chaplan

Bach

Pogrel

Chung

Yaksh

. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994; 53: 55–63.

17.

Filippone

Scuderi

Basilotta

Lanza

Casili

Bova

Paterniti

Esposito

. BAY-117082-driven NLRP3 inflammasome inhibition resolves nitro-glycerine (NTG) neuronal damage in in vivo model of migraine. Biomed Pharmacother 2022; 156: 113851.

18.

Olkkonen

Kouri

Kuusela

Ainola

Nordström

Eklund

Mandelin

. DEC2 Blocks the effect of the ARNTL2/NPAS2 dimer on the expression of PER3 and DBP. J Circad Rhythms 2017; 15: 6.

19.

McNamara

Seo

Rudic

Sehgal

Chakravarti

FitzGerald

. Regulation of CLOCK and MOP4 by nuclear hormone receptors in the vasculature: a humoral mechanism to reset a peripheral clock. Cell 2001; 105: 877–889.

20.

Crumbley

Wang

Kojetin

Burris

. Characterization of the core mammalian clock component, NPAS2, as a REV-ERBalpha/RORalpha target gene. J Biol Chem 2010; 285: 35386–35392.

21.

Safieh-Garabedian

Poole

Haddad

Massaad

Jabbur

Saadé

. The role of the sympathetic efferents in endotoxin-induced localized inflammatory hyperalgesia and cytokine upregulation. Neuropharmacology 2002; 42: 864–872.

22.

Lai

Iliff

Zaheer

Wang

Gansau

Laudier

Zachariou

Iatridis

. Spinal cord sensitization and spinal inflammation from an in vivo rat endplate injury associated with painful intervertebral disc degeneration. Int J Mol Sci 2023; 24: 24043425.

23.

Colburn

DeLeo

Rickman

Yeager

Kwon

Hickey

. Dissociation of microglial activation and neuropathic pain behaviors following peripheral nerve injury in the rat. J Neuroimmunol 1997; 79: 163–175.

24.

Zhang

De Koninck

. Spatial and temporal relationship between monocyte chemoattractant protein-1 expression and spinal glial activation following peripheral nerve injury. J Neurochem 2006; 97: 772–783.

25.

Baniasadi

Manaheji

Maghsoudi

Danyali

Zakeri

Maghsoudi

Zaringhalam

. Microglial-induced apoptosis is potentially responsible for hyperalgesia variations during CFA-induced inflammation. Inflammopharmacology 2020; 28: 475–485.

26.

Kartha

Weisshaar

Philips

Winkelstein

. Pre-treatment with meloxicam prevents the spinal inflammation and oxidative stress in DRG neurons that accompany painful cervical radiculopathy. Neuroscience 2018; 388: 393–404.

27.

Osikowicz

Mika

Przewlocka

. The glutamatergic system as a target for neuropathic pain relief. Exper Physiol 2013; 98: 372–384.

28.

Pajarillo

Rizor

Lee

Aschner

Lee

. The role of astrocytic glutamate transporters GLT-1 and GLAST in neurological disorders: potential targets for neurotherapeutics. Neuropharmacology 2019; 161: 107559.

29.

Lananna

McKee

King

Del-Aguila

Dimitry

Farias

FHG

Nadarajah

Xiong

Guo

Cammack

Elias

Zhang

Cruchaga

Musiek

. Chi3l1/YKL-40 is controlled by the astrocyte circadian clock and regulates neuroinflammation and Alzheimer’s disease pathogenesis. Sci Transl Med 2020; 12(574): aax3519.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.02 MB