NECs were purchased from Jennio Biotech Co., Ltd. (Guangzhou, China) and maintained in a humidified chamber with 5% CO2/95% air at 37 ℃. NECs were incubated in BEGM medium (Lonza, Walkersville, Md., USA) contained with 100 U/ml penicillin (Sigma-Aldrich, MO, USA), 100 μg/ml streptomycin (Sigma-Aldrich, MO, USA), and 10% fetal bovine serum (Sigma-Aldrich, MO, USA).
Cell viability measurement
3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method was performed to evaluate the cell viability. Briefly, NECs were seeded into 96-well plates (5 × 103 cells/well). After 24 h of pre-incubation, NECs were cultured with different concentrations of His (0, 0.025, 0.05, and 0.1 µM; Sigma-Aldrich, MO, USA) or AS-IV (0, 20, 40, 60, and 80 µM; > 98% purity; Aladdin, Shanghai, China). After 24 and 48 h of incubation, 20 µL of MTT solution (Sigma-Aldrich, MO, USA) was transferred to each well and cultured at 37 ℃ for another 4 h. Then, the medium was discarded and the generated formazan crystal was dissolved in 200 µL of dimethyl sulfoxide (Sigma-Aldrich, MO, USA). Finally, the absorbance was measured in a microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm.
Treatment of NECs with AS-IV and His stimulation
AS-IV (Figure 1A, purity > 98%) was purchased from Sigma-Aldrich (MO, USA). The NECs were pretreated with AS-IV (0, 20, 40, and 60 µM) for 30 min. Subsequently, NECs were either unstimulated or stimulated with His (0.1 µM) for 24 h in BEGM medium. His and AS-IV were dissolved in 0.1% dimethyl sulfoxide before the experiment. NECs were dissolved in the same amount of 0.1% dimethyl sulfoxide in the control group. After 24 h of incubation, cell pellets were collected for further analysis.
The NECs were seeded in cell plates for 24 h. The NECs were pretreated with AS-IV (60 µM) for 30 min. Subsequently, NECs were either unstimulated or stimulated with His (0.1 µM) for 24 h in BEGM medium. Total RNA was extracted from NECs using Trizol reagent (Invitrogen, CA, USA). The PCR amplification and sequencing were performed on the GPL16791 Illumina HiSeq 2500 (Homo sapiens). Fold change ≥ 2 and p < 0.05 were used as the criteria for the differentially expressed genes (DEGs), and enriched via KEGG analysis.
Measurement of inflammatory cytokines releasing from NECs cells
NECs cell supernatant was collected by centrifugation at 4,000 g for 5 min at 4 ℃ after 24 h of co-treatment for inflammatory cytokines measurement. The levels of IL-6, IL-8, MCP-1, IL-1β, GM-CSF, and eotaxin in cell culture supernatant were detected by ELISA using commercially available ELISA kits (R&D Systems, MN, USA) and conducted according to the manufacturer’s protocols.
Assay of MUC5AC mucin
NECs cell supernatant was obtained by centrifugation at 4,000 g for 5 min at 4 ℃ after 24 h of co-treatment for MUC5AC assay. MUC5AC protein released from cell culture supernatant was detected using Human MUC5AC ELISA kits (NeoMarkers, CA, USA) and performed according to the manufacturer’s protocols.
RNA extraction and real-time quantitative reverse transcription-PCR (qRT-PCR)
Total RNA from NECs was extracted using Trizol reagent (Invitrogen, CA, USA) and purified by the RNeasy kit (Qiagen Inc, CA, USA) according to the manufacturer’s instructions. Then, reverse-transcription of total RNA was performed by SuperScript III Reverse Transcriptase (Invitrogen, CA, USA). Then, the qRT-PCR was implemented on the ABI Prism 7300 Detection System (Applied Biosystems, CA, USA) by the SYBR Green qPCR Super Mix-UDG kit (Invitrogen, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for internal calibration. The comparative 2−ΔΔCt method was used to calculate the relative mRNA expression. Primer sequences were displayed in Table 1.
All results were shown as mean ± SD. Data analysis was carried out using statistical software (GraphPad Software, Inc., La Jolla, CA, USA). The statistical significance of differences among experimental groups was evaluated using one-way ANOVA followed by Dunnett’s post hoc test. A value of P < 0.05 was considered statistical significance.