All experiments were approved by the Ethics Committee of Rizhao People's Hospital. All subjects signed informed consent forms.
Separation and culture of hDPCs
Healthy permanent premolars for orthodontic or impacted third molars were collected from subjects aged 18–26 years. As mentioned above, hDPCs were separated and cultured using a previously described enzymatic method 52. The dental pulp tissue was separated, cut into small pieces, and digested at 37°C for 20 min with 3 mg/mL of type I collagenase (Gibco, USA). Next, the chopped pulp tissue was cultured at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) containing 20% fetal bovine serum, 100 U/mL of penicillin (Gibco, USA), and 100 mg/mL of streptomycin with 5% CO2. The medium was replaced every 3 days. After the cells achieved 80% confluence, they were separated by trypsin/ethylenediaminetetraacetic acid (Gibco, USA) and sub-cultured at a ratio of 1:2.
Dex (1179333, Sigma, 350 μM) was frozen at −20°C and diluted with DMEM/F-12 to a specified concentration if necessary. Before Dex treatment, cells were allowed to reach approximately 70%–80% confluence. The cells were exposed to Dex at various concentrations (0, 0.1, 1, 5, or 10 μM) for 2 h to determine the optimal concentration. Cells were then divided into the following groups: control group, in which the cells were incubated without Dex treatment in a humidified environment at 37°C with 5% CO2; Car group, in which the cells were incubated with 100 μM of Car for 2 h; and Dex/Car group, in which the cells were pretreated with 5 μM of Dex and 100 μM of Car for 2 h.
To induce an inﬂammatory response, cells were incubated with Car (C1013, Sigma, 100 µM) for 2 h. To promote TRPV1 activity, cells were incubated with capsaicin (Cap, 21750, Sigma, 5 µM) for 2 h. To inhibit PKA activity, cells were treated with H-89 (B1427, Sigma, 10 µM) for 2 h. To inhibitor PKC activity, cells were incubated with Go6983 (ab144414, Abcam, 10 µM) for 2 h.
Immunoﬂuorescence assay (IFA)
hDPCs were inoculated in a 24-well plate and fixed with 4% polyformaldehyde (28906, Thermofisher) for 15 min. hDPCs were permeated for 30 min with 0.1% Triton X-100, cultured at ambient temperature for 15 min with 10% goat serum, and treated at 4°C with the primary antibody overnight. hDPCs were washed with PBS three times and incubated at ambient temperature for 1 h with the secondary Cy3-labeled antibody in dark conditions. Then, the cells were stained with 4’,6-diamidino-2-phenylindole (D1306, Thermofisher) for 15 min. Images were obtained at 400× magnification using a ﬂuorescent microscope.
Cells (1 × 106) were plated on 12-cm dishes and grown for 36 h. Then, the cells were harvested via scraping into 500 μL of cell lysis buffer containing 10 mM HEPES (pH 7.4), 10 mM NaCl, 1 mM KH2PO4, 5 mM NaHCO3, 1 mM CaCl2, 0.5 mM MgCl2, and 5 mM EDTA with complete protease inhibitor cocktail. Cells were allowed to swell for 5 min, followed by Dounce homogenization for 50-time strokes. The cells were then centrifuged at 5,000 rpm for 5 min, generating a pellet containing nuclei and debris and a supernatant of cytosol and plasma. Pellets were resuspended in 1 mL of buffer containing 10 mM Tris (pH 7.5), 300 mM sucrose, 1 mM EDTA, and 0.1% NP40 with complete protease inhibitor cocktail and then pelleted, resuspended, and washed twice. The final pellets obtained were pure nuclei.
Western blotting (WB)
The cell lysis buffer was used for the lysis of hDPCs. Protein was determined using a bicinchoninic analysis kit, separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene fluoride or polyvinylidene difluoride membrane. Tween 20 was added to bovine serum albumin (BSA; 5%) phosphate buffer to block nonbinding sites on the membrane for 1 h. Protein was cultured at 4°C overnight with the primary antibody (p65, ab16502, Abcam, 1:1000; STAT3, ab5073, Abcam, 1:1000; PKC, ab19031, Abcam, 1:2500; PKA, ab187515, Abcam, 1:5000; TRPV1, PA1-748, Thermofisher, 1:1000; Phospho NF-kB p65 (S536), ab86299, Abcam, 1:500; Phospho STAT3 (S727), ab30647, Abcam, 1:500; Phospho PKC (T497), ab59411, Abcam, 1:1000; Phospho PKA alpha (Ser338), PA5-64489, Thermofisher, 1:500; Phospho TRPV1 (Ser503), PA5-64860, Thermofisher, 1:200; Actin, ab8227, Abcam, 1:5000; HSP70, ab2787, Abcam, 1:1000; Lamin B1, ab65986, Abcam, 1:1000), and the secondary antibody was bound to HRP (ab205718, ab205719, Abcam). The protein bands were stained, and the gray values were measured on a C-DiGit Blot Scanner.
RNA extraction and quantitative real-time polymerase chain reaction (qPCR)
Total RNA was extracted with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as an internal standard. Next, under the following conditions, RNA was detected using qPCR (using an SYBR-Green Kit) in a 20-µL system: predenaturation (95°C, 10 min), denaturation (95°C, 15 s, 40 cycles), annealing (60°C, 30 s), and extension (72°C, 30 s). Quantitative analysis was based on the 2−ΔΔCT method and normalized according to GAPDH. The sequences of primers used in this study was displayed as follows: IL-1β F: 5′-CCA CAG ACC TTC CAG GAG AAT G-3′, IL-1β R: 5′-GTG CAG TTC AGT GAT CGT ACA GG-3′; IL-6 F: 5′-AGA CAG CCA CTC ACC TCT TCA G-3′, IL-6 R: 5′-TTC TGC CAG TGC CTC TTT GCT G-3′; TNF-α F: 5′-CTC TTC TGC CTG CTG CAC TTT G-3′; TNF-α R: 5′-ATG GGC TAC AGG CTT GTC ACT C-3′; PKA F: 5′-CAT ATT GCC GAA CAG ATT GG-3′, PKA R: 5′-GCT GGA CTT CAT TGG CTG TA-3′; PKC F: 5′-CGA CTG TCT GTA GAA ATC TGG-3′, PKC R: 5′-CAC CAT GGT GCA CTC CAC GTC-3′; STAT3 F: 5′-CTT TGA GAC CGA GGT GTA TCA CC-3′, STAT3 R: 5′-GGT CAG CAT GTT GTA CCA CAG G-3′; NF-κB F: 5′-GCA GCA CTA CTT CTT GA-3′, NF-κB R: 5′-TCT GCT CCT GAG CAT TG-3′; TRPV1 F: 5′-CCA CAG CGG TGG TGA CGC-3′, TRPV1 R: 5′-GGA GCT GTC AGG TGG CCG-3′; GAPDH F: 5′-GCA CCG TCA AGG CTG AGA A-3′, GAPDH R: 5′-TGG TGA AGA CGC CAG TGG A-3′.
Enzyme-linked immunosorbent assay (ELISA)
According to the manufacturer’s instructions, the concentrations of interleukin (IL)-1β (BMS224-2, Thermofisher), IL-6 (EH2IL6, Thermofisher), TNF-α (KHC3011, Thermofisher), and CGRP (ABIN1095216, Antibodies-online) in the cell culture supernatants were analyzed using an ELISA kit. An automated microplate reader (SpectraMax® M5) was used for the measurement of the optical density (OD) at 450 nm. The concentrations of each sample were detected based on optical density and the concentration of the standard.
The results of our study are presented as means ± standard deviations. Comparisons between two groups or multiple groups were analyzed using one-way ANOVA or a two-tailed Student’s t-test, respectively. A P-value of <0.05 was considered to indicate a statistically significant difference.