Female BALB/c mice were purchased from Orient Bio (Seongnam-si, Korea) and maintained in individual cages under specific pathogen-free conditions at 22 ± 1 °C with 12 h of light/dark cycle. Six-week-old mice with an average body weight of 18 g were employed for the study and euthanized by CO2 inhalation after the termination of each experiment. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Konkuk University.
Preparation of PPE
Placenta extracts were isolated from pig placenta, kindly provided by KR Biotech (Seoul, Korea). Pig placenta were obtained immediately after vaginal deliveries, and the umbilical cord and amnion were discarded. Remaining tissue was exhaustively washed with ice-cold phosphate buffered saline (PBS) to remove all traces of blood. Porcine placenta tissues were disrupted using a tissue homogenizer (Tissue Tearor, Biospec Products Inc., Bartlesville, OK, USA) in cold PBS. Tissue homogenates were centrifuged at 6000 g for 15 min, and the supernatants were lyophilized. Sterilization of placenta extracts was performed via γ-irradiation in air using an isotope source of Cobalt-60 at Greenpia Technology Co. (Seoul, Korea). The absorbed γ-irradiation dose was 25 kGy at a rate of 1 kGy/h.
Induction of contact dermatitis with a DNCB sensitizer and topical PPE treatment
One day prior to sensitization, mice dorsal hairs were completely shaved with an electric trimmer and depilatory cream applied (total 30 mice). For sensitization, 0.7 cm2 gauze-attached patches (Tegaderm™, 3M™ Health Care, St. Paul, MN, USA) were treated with 150 μl of 1% DNCB and applied to the back skin of mice for 20 h on days 0, 3, and 6. For elicitation of responses, patches were treated with 100 μl of 0.5% DNCB and applied to back skin of mice for 20 h on days 9, 12, and 15. DNCB (99% purity, Sigma-Aldrich, St. Louis, MO, USA) was resolved in Acetone–olive oil (AOO) (Sigma Aldrich, St. Louis, MO, USA). Mice were randomly divided into 5 groups representing different experimental conditions (n = 6 per group). To examine the potential beneficial effects of PPE on contact dermatitis, back skin of mice were topically treated with PPE or dexamethasone (DEX, a positive control) on a daily basis after removal of DNCB patches. PPE (10 mg) was thoroughly mixed with 1 g of base cream and 3 mg DEX (0.3 g daily) with 1 kg of base cream using a homogenizer. PPE or DEX cream mixtures were applied to back skin of mice with until the end of the experiment (day 17). Mice were treated with cream alone as a negative control group to determine the effect of vehicle itself on inflammatory responses.
Histopathological and immunohistochemical (IHC) analyses
Back skin tissues of mice (1 cm2) were collected, fixed with neutral buffered formalin (NBF) for 24 h, and embedded in paraffin. Paraffin blocks were cut into 3 μM thick sections and stained with hematoxylin and eosin (H&E) using a standard protocol (Abion CRO, Seoul, Korea). Epidermal thickness was measured at the thickest part of five different dorsal epidermis specimens per mouse group, and significant differences between each pair of conditions were analyzed.
Measurement of DNCB-induced serum IgE and IgG
DNCB-induced serum IgE and IgG levels were assessed using an enzyme-linked immunosorbent assay (ELISA) kit. Briefly, blood samples of mice were obtained from facial veins on the last day of the experiment (day 17) (n = 3 per group). Collected samples were incubated at room temperature for 30 min and centrifuged at 3500 rpm for 25 min, and supernatants were stored at − 80 °C until use. The 96-well plates were pre-coated with 0.17 mg/ml DNP-ovalbumin (Santa Cruz, Dallas, TX, USA), and then blocked with 2% BSA for 1 h at 37 °C, followed by washing with PBS-T. Diluted serum samples and standards were added to each well and incubated for 2 h at room temperature. After washing with PBS-T, goat anti-mouse IgG (Santa Cruz, Dallas, TX, USA) and goat anti-mouse IgE (Southern Biotech, Birmingham, AL, USA) in 1% PBS were added at ratios of 1:4000 and 1:2000, respectively. Following several wash steps, TMB Peroxidase EIA Substrate (Bio-Rad, Hercules, CA, USA) was added to each well for colorimetric development, and the reaction was subsequently terminated with 1 N H2SO4. Absorbance was detected at 450 nm using a microplate reader (Epoch Microplate Spectrophotometer, BioTek, Winooski, VT, USA).
The human keratinocyte cell line, HaCaT, was obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were grown in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin.
Cellular ROS detection assay using H2DCFDA
The DNCB-induced total ROS content in HaCaT cells was measured using 2′,7′-dichlorofluorescein diacetate (H2DCFDA), a ROS detection reagent (Thermo Fisher Scientific, Waltham, MA, USA). H2DCFDA, a cell-permeable non-fluorescent probe, is oxidized into a highly fluorescent compound in cells. Intracellular fluorescence intensity is proportional to the amount of ROS generated in cells. Prior to the experiment, HaCaT cells were pre-incubated with serum-free DMEM containing PPE or DEX for 24 h. Incubation medium was collected and stored at − 20 °C until use. For the ROS detection assay, HaCaT cells were plated on 96-well plates (Sigma Aldrich, St. Louis, MO, USA) at a density of 2 × 104 cells per well. The next day, PPE or DEX pre-incubated medium was gently mixed with 40 μM DNCB-treated medium, added to each well, and incubated for different time periods (3, 6, 12, 24, and 48 h). After washing with PBS, cells were treated with 5 μM H2DCFDA for 30 min at 37 °C in the dark and cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) in mounting solution for 30 min at RT. Following several washes, DCF fluorescence and DAPI staining were visualized under a fluorescence microscope (Eclipse Ti, Nikon, Tokyo, Japan), and the representative images were captured using a NIS-Elements image browser.
Ex vivo ROS detection assay using dihydroethidium (DHE)
Ears of mice were topically pre-treated with PPE or DEX mixed with cream for 15 min and subsequently irritated with 5% DNCB to trigger ROS production. After 15 min, mice were euthanized and 0.5 cm2 ear sections were obtained for the ROS detection assay. Ear sections were incubated ex vivo with 5 mM DHE (Sigma Aldrich, St. Louis, MO, USA) in DMSO for 30 min in the dark. DHE is an ethidium-based fluorescent dye that intercalates within DNA of cells upon oxidization, staining the nucleus a bright fluorescent red. After washing with PBS, ear tissues were placed on glass slides and ROS production were monitored by visualization under a fluorescence microscope (Eclipse Ti, Nikon, Tokyo, Japan).
Significant differences between pairs of groups were analyzed by two-tailed Student’s t-tests or one-way ANOVA followed by Tukey’s multiple comparisons tests. P < 0.05 was considered statistically significant.