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. Author manuscript; available in PMC: 2024 Mar 11.
Published in final edited form as: Brain Res Bull. 2024 Jan 28;208:110889. doi: 10.1016/j.brainresbull.2024.110889

Valproate attenuates somatic hyperalgesia induced by orofacial inflammation combined with stress through inhibiting spinal IL-6 and STAT1 phosphorylation

Chen-Xi Xu a, Xin-Yi Qiu a, Yi Guo a, Tian-Ming Xu b, Richard J Traub c, Hai-Nan Feng b,*, Dong-Yuan Cao a,c,**
PMCID: PMC10926348  NIHMSID: NIHMS1971975  PMID: 38290590

Abstract

Temporomandibular disorder (TMD) and fibromyalgia syndrome (FMS) may present as comorbid conditions, but treatment options are ineffective. The purpose of this study was to investigate whether valproate (VPA) attenuates somatic hyperalgesia induced by orofacial inflammation combined with stress, which represents a model of pain associated with TMD and FMS comorbidity, and to explore the potential mechanisms. The results showed that VPA inhibited somatic hyperalgesia induced by orofacial inflammation combined with stress, and down-regulated the interleukin-6 (IL-6) expression in the L4-L5 spinal dorsal horn of female rats. The anti-nociceptive effect of VPA was blocked by single or 5 consecutive day intrathecal administration of recombinant rat IL-6. Orofacial inflammation combined with stress up-regulated the ratio of phosphorylated signal transducer and activator of transcription 1 (p-STAT1) to STAT1 (p-STAT1/STAT1) in the spinal cord. VPA did not affect the STAT1 expression, while it down-regulated the ratio of p-STAT1/STAT1. The expression of STAT3 and the ratio of p-STAT3/STAT3 were not affected by orofacial inflammation combined with stress and VPA treatment. Intrathecal administration of exogenous IL-6 up-regulated the ratio of p-STAT1/STAT1. These data indicate that VPA attenuated somatic hyperalgesia induced by orofacial inflammation combined with stress via inhibiting spinal IL-6 in female rats, and the mechanism may involve the alteration of activation status of spinal STAT1. Thus, VPA may be a new candidate analgesic that targets IL-6 and STAT1 for the treatment of pain associated with the comorbidity of TMD and FMS.

Keywords: Valproate, IL-6, STAT1, Stress, Hyperalgesia, Comorbidity

1. Introduction

Chronic primary pain, such as temporomandibular disorder (TMD), fibromyalgia syndrome (FMS) and irritable bowel syndrome, is a massive global health problem (Cohen et al., 2021). TMD is a group of musculoskeletal and neuromuscular conditions affecting the masticatory muscles, the temporomandibular joint and the other associated structures. FMS presents with a series of symptoms including wide-spread musculoskeletal pain. It has been reported that the occurrence and development of TMD and FMS are closely associated with stress (Fischer et al., 2016; Park et al., 2022) and has a high female predominance (Cabo-Meseguer et al., 2017; Ferreira et al., 2016). In addition, clinical observation shows that these painful problems frequently arise together or overlap eventually (Moreno-Fernández et al., 2017). Unfortunately, the mechanisms of TMD and FMS comorbidity are inadequately understood (Nicholas et al., 2019), and there is no effective medicine to cure this comorbidity.

Accumulating evidence shows the critical role of the immune system in pain states. Using an animal model of comorbidity of TMD and FMS through unilateral anterior crossbite, we found that the proinflammatory cytokine interleukin-18 was upregulated in the spinal dorsal horn which contributed to the development of the comorbidity of TMD and FMS (Xiang et al., 2022). In addition, orofacial muscle inflammation combined with stress induced widespread pain hypersensitivity in female rats, establishing another comorbid animal model to investigate the mechanisms of the TMD and FMS (Duan et al., 2021; Li et al., 2020a; Xue et al., 2020). Valproate (VPA) is a traditional antiepileptic drug. VPA has also been reported to exert potent anti-nociceptive properties in many preclinical and clinical studies (Ximenes et al., 2013; Xu et al., 2020; Yancey et al., 2014). In the central nervous system (CNS), VPA has neuroprotective and anti-inflammatory effects in diverse animal models of neuropathological conditions (Chen et al., 2018a, 2018b; Guo et al., 2021; Zhao et al., 2020). These effects are associated with the down-regulation of elevated expression of inflammatory factors including interleukin-6 (IL-6). However, whether VPA attenuates hyperalgesia in the comorbidity of TMD and FMS, and whether IL-6 is involved are unknown.

The signal transducer and activator of transcription (STAT) is identified as a transcription factor, which is vital in mediating cytokine driven signaling. STAT1 and STAT3 are two important members of the STAT protein family. STAT1 and STAT3 signaling can be activated by either distinct or by the same set of cytokines and growth factors including IL-6 (Butturini et al., 2020). It has been reported that the spinal STAT1 and STAT3 signaling cascade is involved in neuropathic pain (Chen et al., 2018a; Dominguez et al., 2008; Guo et al., 2021) and inflammatory pain (Li et al., 2022). Therefore, in the current study, we investigated the potential effect of VPA on somatic hyperalgesia induced by orofacial inflammation combined with stress, which represents a model of TMD and FMS comorbidity (Duan et al., 2021; Li et al., 2020a; Xue et al., 2020), and the effect of VPA on IL-6, STAT1, and STAT3 signaling in the spinal dorsal horn was determined.

2. Materials and methods

2.1. Animals

Female Sprague-Dawley rats (180–240 g) were obtained from Xi’an Jiaotong University Laboratory Animal Center (Xi’an, Shaanxi, China). Given pain syndromes, including TMD and FMS, have a significant female predominance (Cabo-Meseguer et al., 2017; Ferreira et al., 2016), it is reasonable to choose female animals to simulate the diseases in human. All animals were housed in a room (temperature 21–25 °C, and humidity 40–60%), with a 12-h light-dark cycle (lights on at 7 am). Food pellets and water could be accessed ad libitum. Experimental protocols were approved by the Institutional Animal Care and Use Committees of Xi’an Jiaotong University, China (No. 2019–950) and in accordance with the guidelines of the International Association for the Study of Pain. All efforts were made to minimize the numbers and any discomfort of animals used in the study. The behavioral examiners were blinded to the treatment administered and all behavioral experiments were performed at the same time in a quiet room between 9:00 am and 1:00 pm.

2.2. Animal model development

A rat model of orofacial inflammation combined with stress was established as described previously (Duan et al., 2021; Li et al., 2020a; Traub et al., 2014; Xue et al., 2020; Zhao et al., 2018). Briefly, female rats were anesthetized with isoflurane (5% induction reduced to 2–3%, RWD Life Science, Shenzhen, China), then the complete Freund’s adjuvant (CFA, 150 μL, 1:1 in saline, Sigma-Aldrich, St. Louis, MO, United States) was injected bilaterally into the masseter muscles. Saline injection was used as the control for CFA. Forced swim (FS) stress was produced by placing the rat in a cylindrical container (20 cm diameter, 45 cm height) containing 30 cm deep water (24–26 °C) for 10 min on the first day and 20 min on the next 2 days, starting the day following CFA injection as described previously (Duan et al., 2021; Li et al., 2020a; Quintero et al., 2000; Traub et al., 2014; Xue et al., 2020; Zhao et al., 2018). The cylinders were filled with fresh water for each rat. After each swim session, the rats were dried with a towel and placed in a cage partially heated with a fan before being returned to their home cages. Non-FS rats were remained in their cages without any additional treatment.

2.3. Drug administration

VPA (Sigma, St Louis, MO, USA) was dissolved with saline (0.9%), which was administered intraperitoneally in the dose of 300 mg/kg. Recombinant rat IL-6 protein (rrIL-6, Cloud-Clone Corp., Wuhan, China) was dissolved into the dose of 20 ng/10 μL with saline (0.9%), which was administered intrathecally. An equal amount of saline was injected in the control groups.

2.4. Intrathecal injection

Drugs were administered through a lumbar puncture as described previously (Li et al., 2020a; Xue et al., 2020). Briefly, the rats were anesthetized with isoflurane inhalation (5% induction reduced to 2–3%, RWD Life Science, Shenzhen, China). A 25-gauge stainless steel needle attached to a 25 μL Hamilton syringe was inserted into the L4-L5 interspace, and the needle was allowed to penetrate the dura. The presence of a quick flick of the tail indicated the needle was successfully inserted into the intrathecal space, then 10 μL of drug was slowly injected over 1 min.

2.5. Behavioral tests

2.5.1. Mechanical withdrawal threshold

The mechanical sensitivity of the hindpaw was measured with von Frey filaments. The rats were placed in plexiglass test cages equipped with a metal mesh floor and acclimated for 30 min. Once the rats exhibited a calm state, mechanical withdrawal threshold (MWT) was measured. The von Frey filaments (Stoelting Co., Wood Dale, IL, USA) were applied to the middle plantar surface of the left hindpaw and the withdrawal threshold was evaluated by applying force ranging from 0.41 to 26 g (4 – 255 mN). The cutoff force was set to 26 g to avoid the potential injury. Six values were recorded by up-down paradigm (Li et al., 2020a; Xue et al., 2020) with a 5 min interval between tests, and the mechanical pain threshold was calculated using the threshold calculation software (JFlashDixon Calculator, University of Arizona, USA). The baseline data were collected 1 and 2 days prior to the CFA injection and the average of the two tests was used for baseline value.

2.5.2. Thermal withdrawal latency

Thermal hyperalgesia was assessed by measuring the withdrawal latency of the hindpaw from a radiant heat source, as reported previously (Li et al., 2020a; Xue et al., 2020). A rat was placed in a transparent plexiglass chamber and acclimated for 30 min. An infrared beam was applied from beneath the glass floor and focused on the middle plantar surface of right hindpaw, and the paw withdrawal latency was recorded. In all experiments, a fixed infrared stimulus (90 infrared intensity), was chosen. A 20 s cutoff was used to avoid tissue injury. The tests were repeated 3 times, with an interval of 5–8 min to avoid possible sensitization. The average of three tests was used as the thermal withdrawal latency (TWL) on that day. The baseline data were collected 1 and 2 days prior to the CFA injection and the average of the two tests was used for baseline value.

2.6. Euthanasia and tissue harvest

Rats were deeply anesthetized with isoflurane (5%, RWD Life Science, Shenzhen, China) and euthanized by decapitation. The spinal cord was removed by pressure ejection with ice cold saline as described previously (Li et al., 2020a; Xue et al., 2020). Briefly, the lumbar enlargement part of the rat’s spinal cord was taken, and the dorsal half of L4-L5 segments were excised, rapidly frozen on dry ice and stored at −80 °C until use.

2.7. Western blot

The tissue was homogenized in RIPA buffer (1% NP-40, 1% Sodium deoxycholate, 0.1% SDS) and protease inhibitor cocktail (Boster, Wuhan, China). The samples were centrifuged at 10,000 g for 15 min at 4 °C. Protein concentrations in the supernatant were measured by using the bicinchoninic acid method. Protein samples (20 μg) fractionated per lane on 4–12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Boster, AR0138, Wuhan, China) by electrophoresis and transferred onto the polyvinylidene fluoride membranes (Millipore, IPVH00010, Darmstadt, Germany). Membranes were blocked with 5% nonfat milk for 2 h at room temperature, and then were incubated with the primary antibody at 4 °C overnight respectively. The following primary antibodies were used: anti-IL-6 (1:1000, Proteintech, Wuhan, China), anti-p-STAT1 (1:500, ab30645, Abcam, Cambridge, UK), anti-p-STAT3 (1:1000, ab30647, Abcam), and anti-GAPDH (1:5000, Boster, BA1054, Wuhan, China). After washing with TBST (10 mM Tris, 150 mM and 1 mL Tween-20 dissolved in 1 L distilled water with pH = 7.4–7.6) for 3 times, the membranes were incubated with goat anti-rabbit secondary antibody (1:4000, Boster, BA1054) for 2 h at room temperature. The antigen-antibody complexes were visualized by chemiluminescence. The immunoreactive band densities were computed using Image J software (National Institutes of Health, Bethesda, MA, USA). Following the detection of phosphorylated STAT1 (p-STAT1) and p-STAT3, the blots within the same membrane were stripped for 30 min and reprobed with antibodies against STAT1 (1:500, ab31369, Abcam) and STAT3 (1:5000, ab119352, Abcam) served as the internal control for p-STAT1 and p-STAT3 respectively, and then GAPDH for STAT1 and STAT3.

2.8. Experimental design

2.8.1. Experiment 1

To evaluate the effects of VPA on somatic hyperalgesia induced by orofacial inflammation combined with 3 day FS stress, rats were divided into two groups: the CFA+FS+Saline group, and CFA+FS+VPA group. The administration time and dose was based on our and other previous studies (Winkler et al., 2005; Xu et al., 2020). The behavioral tests were performed as shown in Fig. 1A. MWT and TWL were measured before (baseline), the 1st and 3rd day after the last FS and then every 4 days.

Fig. 1.

Fig. 1.

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VPA inhibited somatic hyperalgesia induced by orofacial inflammation combined with FS stress. (A) Experimental design and timeline of the thermal withdrawal latency and mechanical withdrawal threshold. Rats were treated with VPA or saline intraperitoneally for 5 days. The behavioral tests for thermal withdrawal latency and mechanical withdrawal threshold were assessed before (baseline) and after stress. d, day; T, thermal withdrawal latency; M, mechanical withdrawal threshold; CFA, complete Freund’s adjuvant; FS, forced swim; VPA, valproate. (B) Mechanical withdrawal threshold test. (C) Thermal withdrawal latency test. n = 7 per group. **, ***, **** P < 0.01, 0.001, 0.0001 vs baseline, respectively. ##, ###, #### P < 0.01, 0.001, 0.0001 vs the CFA+FS+VPA group at the same time point.

2.8.2. Experiment 2

To evaluate the effects of VPA on the expression of IL-6 in the spinal cord in orofacial inflammation combined with 3 day FS stress rats and naïve rats, rats were divided into four groups: CFA+FS+Saline, CFA+FS+VPA, Saline, VPA. Tissue collection followed the scheme in Fig. 2A and Fig. 2C. Three hours after the last intraperitoneal injection (i.p.) of VPA/saline, all rats were euthanized. The dorsal parts of L4-L5 spinal cord segments were excised for the IL-6 expression using Western blot.

Fig. 2.

Fig. 2.

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IL-6 expression in the L4-L5 segments of spinal dorsal horn following VPA treatment. (A) and (C) represent each experimental design and time of tissue collection. (B) VPA down-regulated IL-6 expression in the L4-L5 segments of spinal dorsal horn induced by orofacial inflammation combined with FS stress. * P < 0.05 vs the CFA+FS+Saline group. (D) VPA did not affect the expression of IL-6 in the spinal dorsal horn of naïve rats. ns, non-significant.

2.8.3. Experiment 3

To further clarify the role of IL-6 in VPA’s analgesic effects, rats were divided into two groups: the CFA+FS+VPA+Saline group, and CFA+FS+VPA+rrIL-6 group. All animals received CFA, FS and VPA treatment, the difference between two groups was intrathecal injection (i.t.) of rrIL-6 or saline after VPA treatment. Injection of rrIL-6 was performed 1 h after last VPA administration for single injection or 5 consecutive days after VPA administration. The dose of rrIL-6 was chosen based on a previous study (Dubový et al., 2019) and our preliminary experiments. The behavioral tests were conducted 30 min before rrIL-6 injection (pre-rrIL-6), hourly after single rrIL-6 injection and daily after 5 consecutive day injection (Fig. 3A and Fig. 3B).

Fig. 3.

Fig. 3.

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Intrathecal injection of rrIL-6 reversed anti-nociceptive effect of VPA on somatic hyperalgesia induced by orofacial inflammation combined with FS stress. (A) Experimental design for single injection of rrIL-6 and behavior tests of the thermal withdrawal latency and mechanical withdrawal threshold. (B) Experimental design for consecutive injection of rrIL-6 and timeline of the thermal withdrawal latency and mechanical withdrawal threshold. Rats were treated with VPA or saline intraperitoneally for 5 days, rrIL-6/saline were administered intrathecally 1 h after last VPA injection for single injection of rrIL-6 or 5 day consecutive injection of rrIL-6 after last FS. The behavioral tests for thermal withdrawal latency and mechanical withdrawal threshold were assessed before and after rrIL-6 injection. d, day; T, thermal withdrawal latency; M, mechanical withdrawal threshold; CFA, complete Freund’s adjuvant; FS, forced swim; VPA, valproate; rrIL-6, recombinant rat IL-6. (C, D) The mechanical withdrawal threshold (C) and thermal withdrawal latency (D) were significantly decreased after single injection of rrIL-6. (E, F) The mechanical withdrawal threshold (E) and thermal withdrawal latency (F) were significantly decreased after consecutive injection of rrIL-6. *, ***, **** P < 0.05, 0.001, 0.0001 vs pre-IL-6, respectively. #, ##, ###, #### P < 0.05, 0.01, 0.001, 0.0001 vs the CFA+FS+VPA+Saline group at the same time point, respectively.

2.8.4. Experiment 4

To further verify the underlying mechanism of VPA in the spinal cord in attenuating somatic hyperalgesia induced by orofacial inflammation combined with stress, rats were divided into four groups: saline (masseter muscle)+non-FS, CFA+FS, CFA+FS+Saline, CFA+FS+VPA. The protocols followed the schemes in Fig. 4A and Fig. 2A, respectively. The dorsal parts of L4-L5 spinal cord segments were harvested for examining the expression of STAT1, p-STAT1, STAT3 and p-STAT3 using Western blot.

Fig. 4.

Fig. 4.

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The expression of STAT1 and STAT3 in the L4-L5 segments of spinal dorsal horn. (A) Experimental design and time of tissue collection. (B) The expression of STAT1 protein significantly decreased in orofacial inflammation combined with stress rats compared to the control group, while the relative expression level of p-STAT1 increased in the model group compared to the control, and the expression level of p-STAT1 (p-STAT1/GAPDH) did not significantly alter. (C) The expression levels of STAT3 and p-STAT3 did not change in orofacial inflammation combined with stress rats compared to the control group. ns, non-significant, * P < 0.05.

2.8.5. Experiment 5

To further verify the relationship between IL-6 and STAT1, rats were divided into two groups: the CFA+FS+VPA+rrIL-6 group, and CFA+FS+VPA+Saline group. All animals received CFA and FS with VPA treatment, the difference between two groups was i.t. rrIL-6 or saline for 5 consecutive days after last FS (Fig. 6A). The administration time and dose was same as used in above-mentioned Experiment 3. Three hours after the last i.t. rrIL-6/saline, all rats were euthanized. The dorsal parts of L4-L5 spinal cord segments were harvested for the expression of STAT1 and p-STAT1 using Western blot.

Fig. 6.

Fig. 6.

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The expression of STAT1 and p-STAT1 in the L4-L5 segments of spinal dorsal horn following rrIL-6 was administered intrathecally in VPA treatment on orofacial inflammation combined with stress rats. (A) Experimental design and time of tissue collection. (B) STAT1 protein expression significantly decreased in the rrIL-6 injection group compared to the vehicle control group, while the relative expression level of p-STAT1 (p-STAT1/STAT1 ratio) increased in the rrIL-6 injection group. The p-STAT1 expression level (p-STAT1/GAPDH) did not significantly change. ns, non-significant, * P < 0.05.

2.9. Statistical analysis

All data are presented as the mean ± SEM. Statistical analysis was performed using GraphPad Prism 6 software (San Diego, CA, USA). Behavioral data were compared using two-way analysis of variance (ANOVA) followed by Sidak post hoc test. Western blot data were compared using unpaired t-test. A value of P < 0.05 was considered statistically significant.

3. Results

3.1. VPA inhibits somatic hyperalgesia induced by orofacial inflammation combined with FS stress

VPA inhibited mechanical hyperalgesia induced by orofacial inflammation combined with FS stress (Two way ANOVA, F4,60 = 3.023, P = 0.0245 for interaction; F4,60 = 4.780, P = 0.0021 for time factor; F1,60 = 22.16, P < 0.0001 for group factor, Fig. 1B). The MWT decreased for 7 days post FS compared to baseline in the vehicle group, indicating that mechanical hyperalgesia is induced by orofacial inflammation combined with FS stress. Preventive i.p. administration of VPA blocked the decrease of MWT (P > 0.05 compared to baseline, n = 7). In addition, the MWT was significantly lower on day 1 and day 3 post FS in the CFA+FS+Saline group compared to the CFA+FS+VPA group. These data suggest that administration of VPA prevents the development of mechanical hyperalgesia induced by orofacial inflammation combined with FS stress.

As shown in Fig. 1C, VPA inhibited thermal hyperalgesia induced by orofacial inflammation combined with FS stress (Two way ANOVA, F4,60 = 6.392, P = 0.0002 for interaction; F4,60 = 2.773, P = 0.0350 for time factor; F1,60 = 35.34, P < 0.0001 for group factor, Fig. 1C). The TWL significantly decreased on day 1, day 3, and day 7 post FS compared to baseline in the vehicle group. Pre-treatment of VPA blocked the decrease of TWL post FS (P > 0.05 compared to baseline, n = 7). The TWL was significantly lower on day 1, day 3, and day 7 post FS in the CFA+FS+Saline group compared to the CFA+FS+VPA group, indicating that administration of VPA prevents the development of thermal hyperalgesia induced by orofacial inflammation combined with FS stress.

3.2. The effects of VPA on the expression of IL-6 in the spinal cord

In the aforementioned experiment, VPA inhibited somatic hyperalgesia induced by orofacial inflammation combined with FS, but the exact mechanism is unclear. Inspired by previous literature (Chen et al., 2018a; Guo et al., 2021), we speculate one possibility is VPA decreases the proinflammatory cytokine IL-6 in the spinal cord. Western blot data showed that IL-6 expression significantly decreased in the CFA+FS+VPA group (n = 5) compared to the vehicle control group (CFA+FS+Saline, n = 6, P = 0.0469, Fig. 2B). The results suggest that the neuroinflammation (manifested as an increase in IL-6 expression) in the spinal cord following orofacial inflammation combined with FS stress is alleviated by i.p. administration of VPA.

As showed in Fig. 2D, no difference was observed between the VPA group (n = 9) and Saline group (n = 7) in the expression of IL-6 in the spinal cord (P > 0.05, Fig. 2D), suggesting that VPA does not affect the expression of IL-6 in naïve rats.

3.3. rrIL-6 reverses anti-nociceptive effect of VPA on somatic hyperalgesia induced by orofacial inflammation combined with FS stress

Single injection of rrIL-6 reversed the anti-nociceptive effect of VPA on mechanical hyperalgesia induced by orofacial inflammation combined with FS stress (Two way ANOVA, F5,60 = 4.433, P = 0.0017 for interaction; F5,60 = 4.087, P = 0.0029 for time factor; F1,60 = 67.09, P < 0.0001 for group factor, Fig. 3C). After single injection of rrIL-6, the MWT was significantly lower at 0.5, 1.5, 2.5 h in the CFA+FS+V-PA+rrIL-6 group compared to the CFA+FS+VPA+Saline group (n = 6 for each group). Single injection of rrIL-6 also reversed the anti-nociceptive effect of VPA on thermal hyperalgesia induced by orofacial inflammation combined with FS stress (Two way ANOVA, F5,60 = 2.820, P = 0.0236 for interaction; F5,60 = 2.215, P = 0.0644 for time factor; F1,60 = 21.51, P < 0.0001 for group factor, Fig. 3D). After single injection of rrIL-6, the TWL was significantly lower at 1–3 h in the CFA+FS+VPA+rrIL-6 group compared to the CFA+FS+VPA+Saline group (n = 6 for each group). I.t. administration of saline did not significantly change the MWT and TWL in the CFA+FS+VPA+Saline group (P > 0.05 compared to pre-rrIL-6). These results indicate that single injection of rrIL-6 reverses the anti-nociceptive effect of VPA on the mechanical and thermal hyperalgesia induced by orofacial inflammation combined with FS stress.

Injections of rrIL-6 for 5 consecutive days also reversed the anti-nociceptive effect of VPA on mechanical hyperalgesia (Two way ANOVA, F10,110 = 2.708, P = 0.0052 for interaction; F10,110 = 2.492, P = 0.0098 for time factor; F1110 = 256.9, P < 0.0001 for group factor, Fig. 3E) and thermal hyperalgesia (Two way ANOVA, F10,110 = 4.367, P < 0.0001 for interaction; F10,110 = 2.982, P = 0.0023 for time factor; F1110 = 101.6, P < 0.0001 for group factor, Fig. 3F) induced by orofacial inflammation combined with FS stress. After 5 consecutive days of rrIL-6 injection, the MWT was significantly lower on day 1 till day 9 post FS in the CFA+FS+VPA+rrIL-6 group compared to the CFA+FS+VPA+Saline group (Fig. 3E), and the TWL was significantly lower on day 1 to day 3 and day 5 post FS in the CFA+FS+VPA+rrIL-6 group compared to the CFA+FS+VPA+Saline group (Fig. 3F). I.t. administration of saline for 5 consecutive days did not significantly change the MWT and TWL in the CFA+FS+VPA+Saline group (P > 0.05 compared to pre-rrIL-6). These results indicate that multiple administration of rrIL-6 also reverses the anti-nociceptive effect of VPA on the mechanical and thermal hyperalgesia induced by orofacial inflammation combined with FS stress and the effects last longer.

3.4. The expression of STAT1 and STAT3 in the spinal cord following orofacial inflammation combined with FS stress and additional VPA treatment

IL-6 drives a signaling cascade involving phosphorylation of STAT1 and STAT3 proteins (Heinrich et al., 2003). To determine whether spinal STAT1 and STAT3 contribute to somatic hyperalgesia induced by orofacial inflammation combined with FS stress, we examined STAT1 and STAT3 and their phosphorylated forms in the L4-L5 spinal dorsal horn by Western blot. Though the expression of p-STAT1 (p-STAT1/GAPDH) was not significantly altered, the expression of STAT1 (STAT1/GAPDH) was significantly decreased following orofacial inflammation combined with stress (CFA+FS group, n = 6) compared to the control (Saline+non-FS group, n = 5) group (P = 0.0402, Fig. 4B), and the relative expression level of p-STAT1 (p-STAT1/STAT1 ratio) increased in the CFA+FS group compared to the control group (P = 0.0395, Fig. 4B). The expression levels of p-STAT3/GAPDH, STAT3/GAPDH and p-STAT3/-STAT3 ratio did not alter in the CFA+FS group compared to the control group (P > 0.05, n = 5 – 6, Fig. 4C). These results suggest that the alteration of activation status of spinal STAT1 may be involved in the somatic hyperalgesia development following orofacial inflammation combined with FS stress.

To determine whether the analgesic effect of VPA was associated with the STAT signaling pathways, the expression levels of STAT1, p-STAT1, STAT3 and p-STAT3 in the L4-L5 spinal dorsal horn were measured. Western blot data showed that there was no difference in the STAT1 expression (STAT1/GAPDH) between the two groups (P > 0.05, Fig. 5A), while p-STAT1/GAPDH and relative expression level of p-STAT1 (p-STAT1/STAT1 ratio) significantly decreased in the CFA+FS+VPA group (n = 6) compared to the control group (CFA+FS+Saline group, n = 6, P = 0.0373 and 0.0147, respectively, Fig. 5A). These results suggest that VPA inhibits the phosphorylation of STAT1 in the L4-L5 spinal dorsal horn. In contrast, the protein expression levels of STAT3 and p-STAT3/STAT3 ratio did not change in response to VPA treatment (P > 0.05, Fig. 5B).

Fig. 5.

Fig. 5.

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Effects of VPA on the STAT protein expression in the L4-L5 spinal dorsal horn. (A) The expression level of STAT1 (STAT1/GAPDH) did not significantly change, while p-STAT1/GAPDH and the relative expression level of p-STAT1 (p-STAT1/STAT1 ratio) significantly decreased in VPA treatment group compared to the vehicle control group. (B) The expression levels of STAT3 and p-STAT3 did not change in VPA treatment on orofacial inflammation combined with stress rats compared to the vehicle control group. ns, non-significant, * P < 0.05.

3.5. IL-6 down-regulates the expression of STAT1 under VPA treatment

To determine whether IL-6 affects spinal STAT1 signaling pathways in VPA inhibiting somatic hyperalgesia induced by orofacial inflammation combined with FS stress, we examined the protein expression of STAT1 and p-STAT1 in the L4-L5 spinal dorsal horn following i.t. administrated of exogenous rrIL-6. Western blot data indicated that the expression of STAT1 significantly decreased in the CFA+FS+VPA+rrIL-6 group (n = 5) compared to the CFA+FS+VPA+Saline group (n = 5, P = 0.0120, Fig. 6B). There was no difference in the p-STAT1 expression (p-STAT1/GAPDH), on its own, between the two groups. However, the relative expression level of p-STAT1 (p-STAT1/STAT1 ratio) significantly increased in the CFA+FS+VPA+rrIL-6 group compared to the vehicle control group (P = 0.0441, Fig. 6B). Taken together, these results suggest that IL-6 reverses the decrease of p-STAT1/STAT1 ratio by VPA in this pain model.

4. Discussion

In the present study, we found that VPA attenuated somatic hyperalgesia induced by orofacial inflammation combined with stress by inhibiting spinal IL-6 in female rats, and the mechanisms may involve the alteration of activation status of spinal STAT1. Here, masseter muscle inflammation modeled pain associated with TMD, and then the rats were given subchronic stress, thus somatic hyperalgesia induced by orofacial inflammation combined with stress was consider a model of the comorbidity of TMD and FMS (Li et al., 2020a). These results suggest that VPA may be a new candidate through targeting IL-6 and STAT1 for the treatment of the comorbidity of TMD and FMS.

In the current study, we used intact rats to simulate the whole female population in the orofacial inflammation combined with stress situation. To a certain extent, this is due to the higher number of female patients with these diseases (Cabo-Meseguer et al., 2017; Ferreira et al., 2016). It is well known that hormonal levels may affect pain sensitivity. Our previous studies have used ovariectomized rats with estradiol supplements to maintain the same stage of rats’ estrous cycle (Duan et al., 2021; Li et al., 2020a; Traub et al., 2014; Xue et al., 2020; Zhao et al., 2018). Based on our preliminary studies, we found that stress might affect the estrous cycle so that very few rats were in proestrus and estrus stages following stress, i.e. most stressed rats were in metestrus or diestrus stages (data not show). Therefore, in the present study, we did not specifically determine the stages of the estrous cycle in rats prior to the behavior tests.

4.1. VPA attenuates somatic hyperalgesia induced by orofacial inflammation combined with stress by down-regulating spinal IL-6 expression

Chronic pain is maintained in part by central sensitization, a phenomenon of synaptic plasticity. Accumulating evidence suggests that central sensitization is also driven by neuroinflammation in the CNS (Ji et al., 2018). IL-6 is a well-known proinflammatory cytokine, and IL-6 has been demonstrated to contribute to central sensitization (Kawasaki et al., 2008; Vazquez et al., 2012). Administration of IL-6 to naïve animals through intrathecal injection (Avona et al., 2021; DeLeo et al., 1996; Li et al., 2022; Vazquez et al., 2012), or to a supraspinal site (Avona et al., 2021; Oka et al., 1995; Yang et al., 2022) induces somatic allodynia or hyperalgesia. Blocking spinal IL-6 signal with neutralizing antibodies relieves somatic hyperalgesia (Dominguez et al., 2008; Kwok et al., 2020; Yang et al., 2022).

IL-6 represents a biomarker of neuroinflammation in the spinal cord and supraspinal sites. During orofacial inflammation induced by CFA injection, IL-6 significantly increased in the brainstem (Scarabelot et al., 2019). In addition, repeated FS stress induced neuroinflammation presented as increasing IL-6 (Lovelock and Deak, 2017) in the brain and increasing IL-1β in the spinal cord (Suarez-Roca et al., 2014). Single-prolonged stress including 2 h restraint stress followed immediately by 20 min FS enhanced the increase of the spinal IL-6 by CFA injection into hindpaw (Qi et al., 2016). These studies suggest that neuroinflammation may be induced in the CNS under peripheral inflammatory pain and stress.

VPA produces analgesic effects in animal and human studies (Ximenes et al., 2013; Xu et al., 2020; Yancey et al., 2014), but the exact mechanism is still unclear. A previous study reported that the anti-nociceptive effect of VPA involved reducing inflammatory factors released in peripheral tissues (Ximenes et al., 2013). In the present study, VPA down-regulated spinal IL-6, and alleviated somatic hyperalgesia induced by orofacial inflammation combined with FS stress. Moreover, i.t. administration of rrIL-6 reversed the anti-nociceptive effects of VPA. These results suggest that VPA alleviates somatic hyperalgesia through down-regulating IL-6 in the spinal cord. These findings are in accordance with previous preclinical studies that the anti-nociceptive and anti-inflammatory properties of VPA rely on the down-regulation of spinal IL-6 expression in neural injury models (Chen et al., 2018a; Guo et al., 2021).

Although peripheral inflammation is important in heightening nociception, electrophysiological studies indicate the contribution of immune mediators in the CNS to nociceptive hypersensitivity by modulating excitatory and inhibitory synaptic transmission, inducing central sensitization and hyperalgesia (Kawasaki et al., 2008). There is evidence showing that IL-6 has a role in pain development by involving nociceptive plasticity and sensitization (Melemedjian et al., 2014). Some clinical studies have realized the important role of IL-6 in chronic primary pain such as FMS (Theoharides et al., 2019) and TMD (Zwiri et al., 2022). Targeting the related processes and molecules, such as IL-6, which is involved in neuroinflammation may lead to better treatments for these chronic pain.

4.2. The roles of STAT1 and STAT3 in VPA’s analgesia

In the rat spinal cord injury model, STAT1 was elevated in the spinal cord, VPA further increased the expression of STAT1 and acetylated STAT1, suggesting that the anti-inflammatory effect of VPA is dependent on STAT1 expression and acetylated STAT1 (Chen et al., 2018a). Similarly, in another neuropathic pain model generated by spinal nerve ligation (SNL), VPA inhibited the neuroinflammatory response and attenuated mechanical allodynia by activating the STAT1 signaling pathway. The expression of p-STAT1 was up-regulated in the SNL group, VPA down-regulated p-STAT1 expression (Guo et al., 2021). However, we did not observe an increase in the expression of STAT1 in the spinal cord following orofacial inflammation combined with FS stress. In contrast, the expression of STAT1 was decreased, while the relative phosphorylation level (p-STAT1/STAT1 ratio) was significantly elevated. After VPA treatment, STAT1 was unaffected, which was inconsistent with previous studies (Chen et al., 2018a; Gao et al., 2020; Guo et al., 2021), while the phosphorylation of STAT1 was significantly decreased in VPA treatment group, which was consistent with the previous SNL model (Guo et al., 2021). The different results between these studies may be due to the differences in the pain models, sex, and detection time.

In the present study, orofacial inflammation combined with stress induced somatic hyperalgesia, accompanied by the elevated activation of spinal STAT1. Somatic hyperalgesia was inhibited by VPA, accompanied by the reduced activation of spinal STAT1. Given that spinal STAT1 activation contributed to bone cancer pain and inhibiting spinal STAT1 activation by fludarabine (the STAT1 antagonist/inhibitor) treatment effectively alleviated somatic hyperalgesia induced by bone cancer (Song et al., 2017), it is possible that VPA attenuates somatic hyperalgesia induced by orofacial inflammation combined with stress through down-regulation of STAT1 phosphorylation.

We noted that the protein expression levels of p-STAT3 and STAT3 did not change after orofacial inflammation combined with stress or VPA treatment. These results differ from previous studies which demonstrate that inhibiting the activation of JAK/STAT3 pathway alleviates neuropathic pain (Dominguez et al., 2010; Dominguez et al., 2008; Ono et al., 2020), and VPA partly inhibits the inflammatory response by down-regulating the JAK2/STAT3 signaling pathway (Guo et al., 2021). Our results indicate that the STAT3 pathway is not involved in the development of hyperalgesia and analgesic mechanism of VPA in this pain model.

4.3. IL-6 down-regulates STAT1 under VPA treatment

STAT proteins play an important role in cytokine signaling. Different STATs become activated by different cytokine receptors. IL-6 most potently activates STAT3, and to a lesser extent activates STAT1 (Heinrich et al., 2003). Increasing evidence indicates that the involvement of an IL-6/JAK/STAT3 signaling pathway in the development of pain, including neuropathic pain and inflammatory pain. Intrathecal administration of IL-6 elevated the level of p-STAT3 in the spinal cord in SNL rats (Wang et al., 2016). Intrathecal injection of anti-IL-6 antibody prevented accumulation of p-STAT3 in microglia in SNL rats (Dominguez et al., 2008). Peripheral inflammation induced by carrageenan or CFA evoked hyperalgesia and STAT3 activation, and anti-IL-6 antibody or anti-IL-6 receptor antibody exerted anti-nociceptive effects and decreased the spinal p-STAT3 (Li et al., 2020b, 2022; Oka et al., 2007). In naïve rats, intrathecal administration of IL-6 rapidly activated microglial JAK/STAT3 (Dominguez et al., 2010). However, our results indicate that STAT3 pathway is not involved in the development of this comorbidity pain model, the difference between these conditions needs further exploration. In the present study, different from these traditional studies, under the condition of VPA treatment, exogenous IL-6 induced STAT1 activation by elevating the STAT1 phosphorylation, which is similar to the trend in model group, suggesting that IL-6 activates the STAT1 signal. The relationship between IL-6 and STAT1 was consistent with the previous study which reported that IL-6 activated STAT1 in normal peripheral blood leukocytes and rheumatoid synovial fluid cells, and the absence of STAT1 activity in the presence of anti-IL-6 antibody in rheumatoid arthritis (Yokota et al., 2001), while other studies suggest that IL-6 activated STAT1 played minimal role in the IL-6 signaling and actions (Heinrich et al., 2003; Sanz et al., 2008).

5. Conclusion

In the present study, we demonstrated that VPA attenuated somatic hyperalgesia induced by orofacial inflammation combined with stress via down-regulating spinal proinflammatory cytokine IL-6 in female rats. The possible mechanisms may involve the alteration of activation status of spinal STAT1. Thus, VPA may be a new candidate for the treatment of the comorbidity of TMD and FMS through targeting IL-6 and STAT1.

Funding

This work was supported by the National Natural Science Foundation of China (81971049, 81671097) to DYC and partially by the National Institutes of Health, USA (R01 DE029074) to RJT.

Footnotes

Author statement

This manuscript has not been published or presented elsewhere in part or in entirety, and is not under consideration by another journal. All authors have approved the manuscript and have agreed to submit it to this esteemed journal. If the manuscript is accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder.

CRediT authorship contribution statement

Xu Chen-Xi: Writing – original draft, Validation, Methodology, Investigation. Qiu Xin-Yi: Writing – original draft, Methodology, Investigation. Guo Yi: Methodology, Investigation. Xu Tian-Ming: Methodology, Investigation. Traub Richard J: Writing – review & editing. Feng Hai-Nan: Writing – review & editing, Supervision, Methodology, Investigation. Cao Dong-Yuan: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Declaration of Competing Interest

All authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability

The datasets generated and analyzed during the present study are available from the corresponding author on reasonable request.

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Associated Data

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

Data Availability Statement

The datasets generated and analyzed during the present study are available from the corresponding author on reasonable request.

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