Ethics
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 an enzymatic method 39. The dental pulp tissue was separated, cut into small pieces, and digested at 37℃ 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 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 incubation
Dex (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 different concentrations (0, 0.1, 1, 5, or 10 μM) for 2 h to determine the most appropriate 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; the Car group, in which the cells were incubated with 100 μM of Car for 2 h; and the Dex/Car group, in which the cells were pretreated with 5 μM of Dex and 100 μM of Car for 2 h.
Drug administration
To induce an inflammatory response, Car (100 µM) was incubated with cells for 2 h. To promote TRPV1 activity, Cap (5 µM) was incubated with cells for 2 h.
Immunofluorescence assay (IFA)
hDPCs were inoculated in a 24-well plate and fixed with 4% polyformaldehyde 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 thrice and incubated at ambient temperature for 1 h with the secondary Cy3-labeled antibody in dark conditions. Then, the cells were dyed with 4’,6-diamidino-2-phenylindole for 15 min. Images were obtained at 400× magnification using a fluorescent microscope.
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, and the secondary antibody was bound to peroxidase (Amersham ECL). 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)
The total RNA was extracted with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as an internal standard. Next, under the following conditions, RNA was detected by 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.
Enzyme-linked immunosorbent assay (ELISA)
According to the manufacturer’s protocol, the concentrations of interleukin (IL)-1β, IL-6, TNF-α, and CGRP 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. Based on the OD value and the standard substance concentration, each sample concentration was detected.
Statistical analysis
The results of our study are presented as means ± standard deviations. Comparisons were analyzed by analysis of variance or a two-tailed Student’s t-test. A P-value of <0.05 was considered to indicate a statistically significant difference.