Animals

Mice of the C57BL/6 and Balb/c strains were purchased from Charles River (Beijing, China) and were bred in-house at the experimental animal facility of the Affiliated Hospital of Guizhou Medical University. Male mice which were 8–10 weeks old were used at the start of all experiments. All the mice were housed together under specific pathogen-free conditions at the experimental animal facility for at least 2 weeks before the experiments. The animal protocols were approved by the Ethics Committee of the Guizhou Medical University.

Human placental extract

The human placental extract (HPE) used in this study was a commercial medicine (H20046260, Taibang Health, Guizhou, China) extracted from post-partum/post-natal placental tissues, and it has been routinely applied as supplemental therapy on demand in the clinic. The State Food and Drug Administration (SFDA) of China approval number of HPE is H20046260. The lyophilized powder of HPE was dissolved in sterile phosphate buffer saline (PBS) in the experiments.

Cell line and culture

The mouse-derived mast cell line (C57) was obtained from Dr. Gunnar Nilsson, Karolinska Institute, Sweden). The human-derived mast cell line (HMC-1) was provided by Stem Cell Bank, Chinese Academy of Sciences. C57 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, USA), 4 mmol/L L-glutamine (Gibco), 50 μmol/L 2-mercaptoethanol (Sigma-Aldrich, USA) and 100 μg/mL penicillin/streptomycin (Gibco). HMC-1 cells were cultured in Iscove’s Modified Dulbecco’s Medium supplemented with 10% heat-inactivated fetal bovine serum and 2 mmol/L L-glutamine.

Cell proliferation and viability assay

The effect of HPE on cell viability was assessed using a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Osaka, Japan) assay. Briefly, C57 or HMC-1 cells (1 × 104 cells/well) were seeded in a 96-well plate and treated with various concentrations of HPE or PBS and were incubated at 37 °C for 4, 8 or 16 h. The CCK-8 reagent was added for the last 4 h for assessing the value of half maximal inhibitory concentration (IC50) or the proliferative capacity of cells. Or alternatively cells were fixed and permeabilized (BD Cytofix/Cytoperm, BD Biosciences) followed by stained with FITC-conjugated Ki-67 (Clone; 11F6, Biolegend, San Diego, California, USA) and analyzed with a flow cytometer (Navios, Beckman coulter, Brea, California, USA).

Apoptosis assay

Cell apoptosis was measured by flow cytometry to determine the profile of annexin V (BD Biosciences, Franklin Lake, New Jersey, USA) and propidium iodide (PI, BD Biosciences). In brief, 2.5 × 105 C57 or HMC-1 cells were incubated with various concentrations of HPE for 4, 8 or 16 h. Next, cells were stained with FITC-annexin V/PI for 20 min followed by analysis by a flow cytometer (Navios).

For measurement of mast cell apoptosis in vivo, C57BL/6 mice were challenged i.p with 120 ng HPE or an identical volume of PBS (5 mice per group) i.p. for 4, 8 or 16 h. Next, 5 mL PBS was injected into the euthanized mouse peritoneal cavity and peritoneal lavage fluid (PLF) was harvested after 5 min. The PLF cells were stained with BV421-conjugated rat anti-mouse CD117 (cKit) (Clone; 2B8, Biolegend, San Diego, CA), APC- conjugated rat anti-mouse FcεRIα (Clone; MAR-1, Biolegend) and FITC-conjugated annexin V/PI (BD Biosciences) and examined by a flow cytometer (Navios).

Degranulation assay

C57 or HMC-1 cells (2.5 × 105) were pre-treated with 30 ng/mL of HPE for 4, 8 h, and HPE was next removed by extensive washing. Cells were activated with 1.5 μg/mL compound 48/80 (C48/80, Sigma-Aldrich, St. Louis, MO) for 30 min in serum-free medium. Alternatively, cells were treated with 1 μg/mL anti-DNP IgE (clone SPE-7; Sigma-Aldrich) for 4, 8 h and were subsequently washed and challenged with 100 μg/mL DNP-30–40-HSA (Sigma-Aldrich) for 30 min at 37 °C in serum-free medium. The supernatant was collected for β-hexosaminidase (β-hex) measurement as a readout for mast cell degranulation. Briefly, the supernatant was mixed with an identical volume of 4-nitrophenyl N-acetyl-b-D-glucosaminide (Sigma Aldrich), the substrate of β-hex, and incubated for 1 h at 37 °C. The reaction was stopped by the addition of an equal volume of 0.2 M glycine (Sigma-Aldrich) (pH10). The absorbance at 405 nm was measured using a microplate reader [21].

Cytokine measurement

C57 or HMC-1 cells were pre-treated with 30 ng/mL HPE or PBS for 4, 8 or 16 h and were subsequently washed. The cells were then treated with 1.5 μg/mL C48/80 for 3 h and the supernatant was collected. The concentrations of IL-4, IL-6, IL-13 and TNF-α in the supernatant were determined using a cytometric bead array assay (LEGENDplex™ Mouse Inflammation Panel, Biolegend) according to the manufacturer’s instruction. The fluorescence intensity was assessed on a Navios flow cytometer (Beckman, USA), followed by data analysis using LEGENDplex v8.0 software (Biolegend).

Antigen phagocytosis

C57 or HMC-1 cells (2.5 × 105 cells/well) were seeded in a 96-well plate and pre-treated with various concentrations of HPE for 0, 4, 8 or 16 h. After removal of HPE, cells were incubated with Alexa Fluor 488-conjugated OVA (Sigma-Aldrich) for 2 h followed by staining with APC-conjugated anti-mouse FcεRIα (Clone; MAR-1, Biolegend) and then analyzed by flow cytometry or a confocal laser scanning microscope (Zeiss, Jena, Germany), and the fluorescence intensity of engulfed OVA was analyzed by Image J software (National Institute of Mental Health, Bethesda, Maryland, USA).

Chemotaxis assay

C57 or HMC-1 cells were pre-treated with 30 ng/mL HPE or PBS for 4, 8 or 16 h and were subsequently washed, and then were added to 8 μm fibronectin-coated transwells (VWR International, Radnor, PA) at 2 × 106 cells per mL in 200 μL 10% medium with serum. Medium (600 μL) was added to the lower chambers with 25 ng/mL stem cell factor (SCF) [8]. Migration was carried out for 16 h at 37 °C. Plates were then shaken for 10 min to remove adherent cells from the bottom of the transwells [22]. Transwells were removed and DAPI (Solarbio, Beijing, China) was added to each well and cells were incubated at room temperature for 10 min. Cells were observed by a fluorescence microscope (Zeiss) and calculated using Image J software (National Institute of Mental Health).

Passive cutaneous anaphylaxis assay

Anesthetized BALB/c mice were injected i.d. with 100 ng HPE in the right ear and with an identical volume of PBS in the left ear as a control for the traumatic response representing the result of skin injury. After 4, 8 or 16 h, 0.4 μg IgE directed against DNP in 10 μL PBS was i.d injected into both ears of a lightly anesthetized mouse. The PCA reaction was induced 24 h later by an intravenous injection of 10 μg DNP-30–40-HSA and 2% Evans blue in 200 μL of PBS. The euthanized mouse ears were removed 30 min later and the dye extravasation was quantified as previously described [21] with slight modification. In brief, ears were ground by a tissue homogenizer with 1 mL PBS. The exudate was collected and mixed with acetone (3:7, v/v), and incubated at room temperature overnight. After vigorous vortexing, the mixture was centrifuged at 3000 rpm for 15 min. The supernatant was collected for the measurement of extravasated Evans blue with an ELISA reader at 620 nm.

For some of the PCA assays, Evans blue was obviated and ear tissues were sectioned and analyzed with toluidine blue staining for revealing mast cell morphology as previously described [23]. Or alternatively, the ear sections were processed for histamine analysis as previously described [24]. Briefly, the sections were incubated with the rabbit anti-histamine antibody (Fine Test, Wuhan, China), and then with horseradish peroxidase-linked anti-rabbit and anti-mouse universal secondary antibodies (Solarbio, Beijing, China). The sections were counterstained with hematoxylin staining solution (Solarbio, Beijing, China) and photographed under a positive position microscope (Leica, Weztlar, Germany). The relative histamine expression was analysis with Image J software (National Institute of Mental Health).

Statistical analyses

Statistical analyses were performed using GraphPad Prism software version 9.5.0 (GraphPad, San Diego, California, USA). Statistical analysis was performed using unpaired Student t-test, one-way ANOVA with Tukey’s multiple comparison test or two-way ANOVA with Tukey’s multiple comparison test. A P value < 0.05 is considered statistically significant.