Sprague–Dawley adult rats (male, weighing 250–300 g) were obtained from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). Table 1 shows the group. Rats were housed at 23 ± 1°C under a 12 h light/dark cycle. Let the rats adapt to the environment for at least 7 days. The Ethics Committee of the Stomatology Hospital affiliated Chongqing Medical University approved the study (registration no. CQHS-NT022-2022).

All rats were implanted with catheters before receiving drug injections into the temporomandibular joint (TMJ) cavity to observe the effect of pharmacological inhibitors or agonists of GPER1, PLC, and PKC on nociceptive hypersensitivity due to CFA-induced temporomandibular osteoarthritis. All procedures were performed in a large pool. The operation has been described previously (Gao et al., 2017). Before continuing the experiments, the rats were allowed to recover for 1 week.

One week later, the rats were anesthetized and administered separate intracerebral drugs via injection. The GPER1 inhibitor (G15, 10 μM, 5 μL, n = 6, MCE, Monmouth Junction, NJ, USA) or 0.1% dimethyl sulfoxide (DMSO; CFA + Vehicle group; 5 μL, n = 6, Mengbio, Chongqing, China) was injected through the catheter 0.5 h before behavioral testing. The PKC agonist (PMA, 65 μM, 5 μL, MCE, Monmouth Junction, NJ, USA), PKC inhibitor (GF109203x, 4 mM, 5 μL, n = 6, MCE, Monmouth Junction, NJ, USA), PLC agonist (m-3M3FBS, 60 μM, 5 μL, MCE, Monmouth Junction, NJ, USA), and PLC inhibitor (U73122, 5 mM, 5 μL, n = 6, MCE, Monmouth Junction, NJ, USA) were injected through the catheter 24 h before behavioral testing.

One week after intra-articular drug injection, the rats were tested with the von Frey hair test (User Manual, Dan Mic Global, LLC. Camden Avenue, San Jose, CA, USA) to detect the head withdrawal threshold (Fruhstorfer et al., 2001). The rats were placed in acrylic cages and adapted to the environment for approximately 30 min. Von Frey hair was applied on the skin above the TMJ cavities of the rats, and the force applied toward the area increased gradually. Positive responses included (1) stimulated behavior, characterized by the exhibition of rapid backward movement, curling up against the cage wall to avoid further contact with the face, or hiding the head and face from the hairs to avoid the stimulus; (2) aggressive behavior, characterized by the exhibition of rapid grasping and biting of the stimulus, resulting in aggressive movement; and (3) stroking the face with the ipsilateral front paw or making a sound. The presence of one or more of the above responses was considered positive in the stimulation test. The interval for each test was greater than 1 min. The head withdrawal threshold of the rats was measured using von Frey hair 1 h before and after the first CFA injection daily. The test was performed after each catheter infusion (24 h after GF109203x and U73122 injection and 0.5 h after G15 injection) and before injection. The details of test procedures and experimental data processing have been described (Chaplan et al., 1994; Ren, 1999; Cao et al., 2009).

Rats were randomly divided into control (CON) and CFA groups. Rats in the CFA group were anesthetized by isoflurane (same dose as used before), and 50 μL of CFA (Sigma-Aldrich, 3,050 Spruce Street, Saint Louis, USA) was injected bilaterally into the joint cavity using an insulin syringe to cause TMJ inflammation (Wu et al., 2010). One week after CFA injection, the TMJ areas showed severe swelling (Li et al., 2016). Rats in the CON group were injected bilaterally with 50 μL 0.9% saline under the same anesthesia mode. Inflammation was assessed using head withdrawal threshold and joint histopathological examinations.

The TMJ cartilage was collected following sacrifice of the rats. The specimens were fixed in 4% paraformaldehyde (Monbiot, Chongqing, China), decalcified in 10% EDTA decalcifying solution about 1.5 months, dehydrated under the ethanol concentration gradient, and the cartilage was embedded in paraffin. The specimen was then cut into 5 μm sections on a paraffin sectioning machine (Leica RM2245, Wetzlar, Germany). Paraffin sections were dewaxed and joint staining was performed using a modified Safranine O-Fast Green FCF Cartilage Stain Kit (Solarbio, Beijing, China). After staining, the specimen was sealed with resin (Solarbio, Beijing, China). Images were obtained using a microscope (Slideview VS200, Olympus Corporation, Tokyo, Japan).

The expression levels of the total GluA1, GPER1, PKC, PLC, PSD-95, syb-2 and c-Fos were detected by western blot. After administration of G15 for 0.5 h, the head withdrawal threshold was determined. Sacrificed the rats, and the RVM tissue was isolated and homogenized. Measuring the protein concentrations. The proteins were added to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis for 1.5 h (Beyotime, Shanghai, China), and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were sealed with 5% skim milk for 1 h at 37°C and incubated with primary antibodies, including anti-GluA1 (1:1,000, Cell Signaling Technology, Danvers, Massachusetts, USA), anti-GPER1 (1:1,000, Thermo Fisher Scientific, 5,823 Newton Drive, Carlsbad, USA), anti-PKC (1:1,000, Abcam, UK), anti-PLC (1:1,000, Abcam, Cambridge, UK), anti-c-Fos (1:1,000, Affinity, Jiangsu, China), anti-PSD-95 (1:1,000, Cell Signaling Technology, Danvers, Massachusetts, USA), anti-syb-2 (1:1,000, Abcam, Cambridge, UK) and anti-GAPDH (1:10,000, ZEN-Bioscience, Chengdu, China), overnight at 4°C. Membranes were washed with Tris-buffered saline containing Tween 20 (TBST) and incubated with the corresponding secondary antibodies (1,10,000, Mengbio, Chongqing, China) for 2 h at 37°C. The blots were detected using a detection kit (Beyotime, Shanghai, China) and visualized using an imaging system (Bio-Rad, Hercules, California, USA). GAPDH was used as a loading control to normalize protein levels. We used stripping buffer (Solarbio, Beijing, China) to remove the membrane-bound antibody. The next round of western blot experiments was then performed using a different antibody, and using this strategy enabled us to detect proteins on the same membrane multiple times.

Rats were perfused with 0.9% saline and 4% paraformaldehyde via the heart after injecting inhibitor or activator. The brain stem area was immediately separated, fixed in 4% paraformaldehyde for 24 h, and then soaked in sucrose solution with increased concentration (20–30%) in turn until the tissue sank to the bottom. RVM sections were cut into 10 μm-thick sections using a frozen sectioning machine (Leica CM1950, Wetzlar, Germany) and stored at −80°C for subsequent use. Sections were removed from the −80°C refrigerator, brought to room temperature, and then repaired with the EDTA antigen retrieval solution (PH 8.0, Solarbio, Beijing, China) in a microwave oven at a low temperature (approximately 40°C) for 10 min. Next, the slices were cooled to room temperature and washed thrice with phosphate-buffered saline (PBS; Biosharp, Anhui, China), and 0.3% TritonX-100 (Mengbio, Chongqing, China) was added. After incubation at 37°C for 10 min, the slices were washed again with PBS thrice; then, 5% goat serum (Solarbio, Beijing, China) was added. The slices were then incubated at 37°C for a further 30 min. The slices were then incubated at 4°C overnight using anti-neuronal nuclei (NeuN; 1:200, host mouse, Cell Signaling Technology), anti-GFAP (1:200, host mouse, Cell Signaling Technology), anti-Iba1 (1:200, host mouse, Abacam), anti-GPER1 (1:200, host rabbit, Thermo Fisher) and anti-c-Fos (1:200, host mouse, Abcam) antibodies. After washing thrice with PBS, the sections were incubated with fluorescently coupled secondary antibodies (Alexa Fluor 488-labeled goat anti-rabbit IgG and Alexa Fluor 596-labeled goat anti-mouse IgG, ZSGB-Bio, Beijing, China) at 37°C for 90 min. Micrographs were analyzed using a fluorescence confocal microscope (EU 5888; Leica, Wetzlar, Germany). Three sections were randomly selected per rat. Images of cells were captured using a 20× objective. The immunoreactivity of c-Fos and GPER1 in the RVM was captured using a 10× objective. The number of positive cells was calculated as the average number of positive cells obtained from three images.

Following anesthesia, the rats were perfused with 2.5% glutaraldehyde and 2% paraformaldehyde mixed liquid, and their brains were taken out. The RVM was separated and fixed in 4% glutaraldehyde at 4°C for 24 h. The RVM was cut into 1 × 1 × 1 mm tissue blocks, fixed, embedded, sectioned, and stained. Refer to this document for specific methods (Wang et al., 2021). Images were captured using a JEM-1400Plus transmission electron microscope (TEM; JEOL Ltd., Tokyo, Japan) and analyzed using Image Pro Plus 6.2. Synaptic morphological parameters were measured at 50,000× magnification. The width of the synaptic space and the thickness of the postsynaptic density (PSD) were measured using the multipoint average method and Güldner and Ingham’s method (Güldner and Ingham,1980).

The data are presented as the mean ± standard deviation (SD) and were statistically analyzed using GraphPad Prism 8.2.1 (San Diego, CA, USA). The Photoshop software (Adobe Photoshop 2021) was used to generate the illustrations. The results of behavioral tests were analyzed with two-way repeated measure analysis of variance (ANOVA) followed by the Bonferroni post hoc test. One-way ANOVA analysis was followed by the Tukey post hoc test for multiple group comparisons. Differences between two groups were examined using independent-sample t-tests. p-value <0.05 are considered significant differences.

Intense erythema and damage to the articular cartilage in the TMJ region were observed in the CFA-injected rats but not in the CON rats (Figures 1A,B). We used the von Frey hair to determine the head withdrawal threshold. The rats injected with 0.9% normal saline served as the CON group. Before injection, no difference was observed in the head withdrawal threshold between the two groups. The head withdrawal thresholds in the CON group did not change significantly with time, while the thresholds in the orofacial region showed a significant decrease in the CFA group from day 3 onwards. Additionally, the head withdrawal threshold of rats in the CFA group decreased significantly compared with those of rats in the CON group on day 7 (Figure 1C). The expression of c-Fos and GPER1 in the RVM increased significantly after CFA injection (Figures 1D,E). These results showed that GPER1 may be involved in the pathophysiological mechanism of chronic orofacial pain, while the upregulation of c-Fos suggests the activation of neurons in RVM region.

The head withdrawal threshold of rats in the CFA group was reduced 1 week after the bilateral TMJ cavity injection of CFA. To investigate the role of GPER1 in the chronic pain of the orofacial region, we injected G15 intracerebrally in CFA rats. In accordance with previous studies, we found that G15 produced a significant inhibitory effect 30 min after injection (An et al., 2014). Therefore, the RVM of the rats was taken immediately after measuring the orofacial pain threshold 30 min after G15 injection, and the expression of GPER1 and c-Fos was analyzed by protein blotting. The results show that, compared with CFA rats, rats which underwent G15 injection had significantly increased pain thresholds (Figure 2A). Moreover, the protein blotting results suggest that the expression levels of GPER1 and c-Fos in the body were significantly higher in the CFA group than in the CON and CFA + G15 groups (Figures 2B,C). We did not observe significant difference between the CFA and CFA + Vehicle groups, which suggests that the 0.1% DMSO did not change the levels of GPER1 and c-Fos.

c-Fos, a marker of neuronal activation, has been recognized as a reliable marker for central sensitization. The gene c-Fos and its protein product were reported by Hunt et al., and used to study nociception (pain; Harris, 1998; Marvaldi et al., 2020). c-Fos can be used as a cross synaptic marker of neuronal activity after harmful stimulation (Bullitt, 1990). Immunofluorescence results showed that the number of c-Fos-positive (c-Fos⁺) neurons in the RVM of rats in the CFA group was significantly higher than that of rats in the CON and CFA + G15 groups (Figure 3B). The number of GPER1-positive cells was significantly higher in the CFA group than in the CON and CFA + G15 groups (Figure 3C). These results indicate that G15 alleviated the central sensitization of CFA rats. We did not observe significant difference between the CFA and CFA + Vehicle groups.

Immunofluorescence results show that the number of Iba1-positive cells in the RVM of rats in the CFA group was significantly higher than that of rats in the CON and CFA + G15 groups (Figures 4A,C). We did not observe significant difference between the CFA and CFA + Vehicle groups. Additionally, it was observed that GPER1 is expressed in Iba1 labeled microglia and Neu labeled neurons, but not in glial fibrillary acidic protein (GFAP) labeled astrocytes (Figure 4B).

To determine whether G15 influences α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor expression (Figure 5A), we measured the protein expression of total GluA1 by western blotting (Figure 5C). Our results show that the expression of GluA1 in the CFA group was significantly increased compared to the CON group. G15 administration significantly downregulated total GluA1 levels and the phosphorylation of GluA1. We did not observe significant difference between the CFA and CFA + Vehicle groups (Figure 5B).

Since GluA1 regulates synaptic plasticity, which is crucial in the development of chronic orofacial pain, we explored whether G15 regulates the expression of two synaptic-associated proteins; PSD-95 and syb-2 (Figures 6A,B). Western blotting showed that the expressions of PSD-95 and syb-2 in the RVM region of CFA group rats were significantly increased. Injection of G15 reduced the expression of these proteins. We did not observe significant difference between the CFA and CFA + Vehicle groups.

Changes in synaptic structure and function are closely related to synaptic plasticity (10.6084/m9.figshare.20445930). Representative images of synaptic structures in the RVM were observed using TEM (Figure 7). Synaptic spaces were visible in the CON and CFA + G15 groups, and the PSD was appropriate (Figures 7A,D). Conversely, the synaptic space and PSD of the CFA + Vehicle and CFA groups were contracted and blurred (Figures 7B,C). The results displayed that, compared with that in the CON group, the width of the synaptic clefts in the CFA and CFA + Vehicle groups was decreased (Figure 7E). The PSD thickness of the CFA group was significantly greater than that of the CON group. After G15 treatment, these abnormal changes were alleviated (Figure 7F). These results also provide intriguing, albeit indirect, evidence for the mechanism of GPER1 involvement in chronic orofacial pain, which may be related to local sensitization in the RVM region.

In this study, we found that PLC and PKC protein expression was significantly higher in the RVM of rats in the CFA group compared with that of rats in the CON group, while G15 injection significantly downregulated their expression in the RVM of CFA rats (Figures 8A,B). There was no significant difference between the CFA and CFA + Vehicle groups. Rats in the CFA group were randomly selected and divided into a CFA + PLC inhibitor (U73122), CFA + PKC inhibitor (GF109203x), and a CFA + vehicle group, and the pain thresholds of the rats were measured 1 day after drug injection. Compared with the CFA group rats, the CFA + PLC and CFA + PKC inhibitor group rats showed a significant increase in pain thresholds (Figure 8C). Similarly, rats in the G15 group were randomly selected and divided into a G15 + PLC agonist group (m-3M3FBS) and a G15 + PKC agonist group (PMA), and the withdrawal threshold level of the rat heads was measured 1 day after drug injection. Compared with the G15 group rats, G15 + PLC and G15 + PKC agonist group rats showed significantly increased pain thresholds as well (Figure 8D). Therefore, we speculate that GPER1 may cause a decrease in the orofacial mechanical pain threshold in CFA rats through the PLC-PKC signaling pathway.