Animals

Specific pathogen-free (SPF) male Sprague Dawley rats, weighing 180–220 g, were purchased from Guangzhou Ruige Biotechnology Co., LTD (license number: SCXK [Guangdong] 2021–0059; experimental unit use license number: SYXK [Guangdong] 2018–0186).

The study was conducted in accordance with the Animals in Research: Reporting In Vivo Experiments guidelines (Kilkenny et al. 2010) and was approved by the Animal Management Committee of the Southern University of Science and Technology (SUSTech-JY202104012).

Chronic inflammatory pain model and CAP administration

After 1 week of adaptive feeding, the rats were randomly divided into four groups: control (five rats), model (seven rats), low-dose (seven rats), and high-dose (seven rats). A total of 21 animals in the model and administration groups were subcutaneously injected with 100 μL of complete Freund’s adjuvant (CFA) to establish a chronic inflammatory pain (CIP) model. Epidural administration was performed on the first day after modeling (day 1 after CFA injection). The low-dose administration group (L-CAP) was injected with 50 μL of normal saline solution containing CAP (40 mg/kg), and the high-dose administration group (H-CAP) was injected with 50 μL of normal saline solution containing CAP (100 mg/kg).

Mechanical withdrawal threshold

A 30 cm-high metal grid (0.8 × 0.8 cm2/ grid) was placed on the table, and the rats were covered with a transparent plex glass box on the metal grid. The rats were allowed to acclimate to the environment for approximately 15 min or until the exploratory behavior terminated. The mechanical paw withdrawal threshold of the left hind paw of the rats was measured, and the interval between each measurement was 30 s; an average of six measurements were taken. The mechanical pain threshold was recorded before and after modeling and after treatment. It was tested once before modeling and once a day after modeling, resulting in a total of eight times.

Cold pressor test

Pain was induced using the acetone spray method, and the degree of pain was scored. A 30-cm high metal grid (0.8 × 0.8 cm2/ grid) was placed on a table, and the rats were covered with a transparent plex glass box over the metal grid. The rats were allowed to acclimate to the environment for approximately 15 min or until the end of exploratory behavior. Three groups were tested each time at an interval of 10 min. Testing was done once before modeling and once a day after modeling, resulting in a total of eight times.

Cell culture

Mice microglia cells (BV2; BNCC337749, BeNa Culture Collection, China) and mice brain neurotumor cells (N2a; BNCC338529, BeNa Culture Collection, China) were used for in vitro experiments. Both cells were cultured in a complete DMEM culture medium (Gibco, USA), which contained 1% penicillin (100 U/mL) with streptomycin (100 μg/mL) and 10% fetal bovine serum (Gibco, USA). The culture flasks were placed in a thermostat incubator (5% CO2, 37℃).

Samples collection

Peripheral blood samples (300 μL) were collected at four time points: on days 1, 2, 4, and 6 after CAP administration (corresponding to days 2, 3, 5, and 7 after CFA injection). The supernatants were collected after centrifugation at 1000 g for each sample. Upon completion of the assessment on day 7, DEG tissues were collected. A portion was embedded in paraffin, another portion was prepared as frozen sections, and the remaining tissues were stored at -80 °C.

Enzyme-linked immunoassay (ELISA)

Levels of 5-hydroxytryptamine (5-HT), substance P (SP), bradykinin (BK), IL-6, IL-1β, C–C motif ligand 2 (CCL2), and TNF-α in the samples were measured using the corresponding kits purchased from CHUNDUBIO in Wuhan, China. Tests were performed according to the kit instructions, with each sample subjected to triplicate measurements.

Immunohistochemical staining

The DRG tissue was dehydrated in gradient alcohol, embedded in paraffin, and cut into 3-µm sections. The sections were deparaffinized, hydrated, repaired antigen, blocked, and incubated with TRPV1 antibodies (66,983, Proteintech, China) at 4℃ overnight and incubated with secondary antibodies at 37℃ for 1 h. The Diaminobenzidine Peroxidase Substrate Kit (KIT-9701, Maixin Biotechnology, China) was used to detect positive signals and then counterstained with hematoxylin (G1040, Servicebio, China). Visualization was performed using a microscope (ML31, Mshot, China).

Immunofluorescence staining

The sections were fixed with 4% paraformaldehyde for 20 min, washed with phosphate-buffered saline (PBS), and subjected to antigen retrieval using citrate buffer. The procedure followed the instructions provided in the kit for the tissue autofluorescence quencher (G1221, Servicebio, China). A total of 100 µL of primary antibodies OX-42 (BS6640, Bioworld, USA) and NF-κB (BS-3332R, Bioss, China) solution was added, followed by overnight incubation at 4 °C. After thorough washing, 100 µL of secondary antibody (GB21403 and GB22403, Servicebio, China) was added, and the sample was incubated at 25℃ in darkness for 60 min. After three washes with PBS, the sections were stained with DAPI solution (G1012, Servicebio, China). Ultimately, images were captured using a fluorescence microscope (MF53, Mshot, China).

Western blot

Protein samples for Western blotting were prepared by fully disrupting the DEG tissue and BV2 cells. Samples were denatured by boiling for 8 min at 100℃ in a loading buffer containing 10% SDS and 100 mM dithiothreitol. The Western blot procedure was performed according to the instructions provided in the PAGE Gel Fast Preparation Kit (PG112, Yamei, China). Samples were immunoblotted with primary antibodies against p38 (8690, CST, USA), p-p38 (4511, CST, USA), NF- κB (YM3111, ImmunoWay, USA), p- NF- κB (YP0191, ImmunoWay, USA), TLR4 (BS-20594R, Bioss, China), AKT (4691S, CST, USA), p-AKT (AF0016, Affbiotech, USA), TRPV1 (66,983, Proteintech, China), and secondary antibodies (BA1055 and BA1051, Boster, USA). Proteins were visualized using Tanon’s fully automated chemiluminescence image analysis system (5200, Tanon, China). Three replicate experiments were conducted for each sample.

Cell counting kit-8

The cell suspension (100 μL/well) was prepared based on the count and inoculum (5000 cells/well) and subsequently seeded into 96-well plates. Control BV2 cells were cultured untreated for 72 h, whereas model cells were treated with 1 μg/mL LPS (S1732, Beyotime, China) for 24 h, followed by 48 h of culture in fresh medium. Drug administration groups were pretreated with LPS for 24 h and then with CAP (10 μM or 25 μM, HY-10448, MCE, USA) for 48 h. The plates were then incubated at 37℃ with 5% CO2 for 6, 24, 48, and 72 h. Thereafter, 10 μL of cell counting kit-8 solution was added per well, and plates were incubated for 2 h before measuring absorbance at 450 nm using a microplate reader.

Transwell migration assays

Control group BV2 cells were cultured untreated for 48 h. Model group cells were treated with 1 μg/mL LPS for 24 h and then cultured for another 24 h in fresh medium. Drug administration groups were pretreated with LPS for 24 h, followed by CAP (10 μM or 25 μM) treatment for 24 h. After seeding 5 × 104 BV2 cells in the upper chamber, plates were incubated (37℃, 5% CO2) for 24 h. Cells were washed with PBS buffer and fixed with paraformaldehyde for 15 min. After washing, the cells were stained with 0.1% crystal violet staining solution (M186781, Guanghua Sci-Tech, China) for 20 min. Membranes of the chamber were cut off, placed on slides, and observed under a microscope. Cell counts were conducted in three random fields at 400 × magnification.

Flow cytometry

The culture conditions for the four groups of BV2 cells were consistent with those described above. Upon completion of treatments, BV2 supernatants from each group were collected and cultured with corresponding N2a cells for 24 h. The cells were harvested to prepare cell suspensions, with each tube containing 1 × 106 cells. Subsequently, the Annexin V-APC and 7-aminoactinomycin D (7AAD) assay kit (AP104-100-AAD, Lianke Biotech, China) were added, gently vortexed for mixing, and incubated at 25℃ in the dark for 15 min. Precooled 1 × Binding Buffer was then added to each tube, resuspended, and mixed. The single-cell suspension was analyzed using flow cytometry, with an excitation peak at 488 nm and an emission peak at 575 nm. Data analysis was performed using NovoExpress.

Calcium ion measurement

The supernatants from the four groups of BV2 cells were collected and co-cultured with N2a cells under conditions consistent with the sample processing method for flow cytometry analysis. The concentration of intracellular calcium ions in N2a cells was measured according to the instructions of the Cell Calcium Assay Kit (GMS 50097.1, Genmed, China).

Statistical analysis

All statistical analyses were performed using SPSS software (version 26.0), and graphs were created using GraphPad Prism 10.1.2 software. Data are presented as mean ± standard deviation. The Shapiro–Wilk normality test was used to assess normality, and Levene’s test was applied to evaluate the homogeneity of variance. For comparisons among multiple groups, a one-way analysis of variance was conducted for data that satisfied the assumptions of normality and homogeneity of variance.