10 patients from the Department of Respiratory and Critical Care Medicine of the First Affiliated Hospital of Xi’an Medical University between January 2017 and March 2018 who were diagnosed with steroid-resistant asthma were included in this investigation [8]. All procedures were performed in accordance with the Declaration of Helsinki for medical research involving human subjects, and written consent from the subjects was obtained. The ethics committee of the First Affiliated Hospital of Xi’an Medical University also approved the study procedures.

Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using Ficoll-Paque, as described previously [9]. Furthermore, the PBMCs were isolated from 5 mL of venous blood using Ficoll-Paque PLUS centrifuge, as described previously [10]. After centrifugation, the PBMCs were collected from the interphase layer and washed four times with RPMI-1640 Medium. Then, the PBMCs were gently re-suspended in 10 mL of RMPI-1640 Medium supplemented with 10% FCS and 10-mM EDTA. The PBMCs (1 × 10⁷ cells/mL) suspended in RPMI-1640 Medium supplemented with 10% (v/v) FBS were incubated at 37˚C at 5% CO₂ in air with high humidity.

The Th17 cells were induced by PBMC. Briefly, the PBMCs were cultured for 6 days with anti-CD3 (BioLegend Cat# 300314) plus anti-CD28 mAbs (BD, Cat#555729, at 5 µg/mL and 2 µg/mL, respectively) in the presence of the combination of cytokines and blocking antibodies appropriate for polarization: rhIL-6 (10 ng/mL), rhIL-23 (20 ng/mL), rhTGF-β1 (2 ng/mL), anti-IFN-γ (10 µg/mL), and anti-IL-4 (10 µg/mL).

On day 7, the TH17 cells were harvested via flow cytometry (BD, Cat#560436) and suspended in 200-µL serum-free RPMI-1640 Medium and aliquoted into a 24-well plate (Invitrogen Life Technologies). A total of 1 µg of each ERK2 small interfering RNA (Qiagen, Hilden, Germany; sense5'-CAGUAGGCUGUAUUCUGGCAGAGAU-3' and antisense 5'-GUTTACGUGACACGUUCGGAGAATT-3') and negative control (scrambled) siRNA (sense5'-UAGGCGCAGCUCCGGAUCGDTT-3' and antisense 5'-CGAUCCGGAGCUGCGCCUADTT-3') were incubated separately, with 5-µL Lipofectamine (Genlantis, Inc., San Diego, CA, USA) in 100-µL serum-free RPMI-1640 Medium at room temperature for 5 min. The ERK2-specific and control siRNA mixtures were then added to the respective 200-µL DC cell cultures. After a 4h incubation at 37˚C, an equal volume of 200-µL RPMI-1640 Medium supplemented with 20% FCS was added to the cells. siRNA transfection and silencing of the expression of ERK2 in the TH17 cells were performed according to the methods described in a previous study [11].

Total RNA was isolated from the TH17 cells, which were collected 72 h after the siRNA-ERK2 transfection, using RNeasy Mini Kits (Qiagen, Valencia, CA, USA) containing TRIzol reagent for extraction (Gibco Life Technologies, Carlsbad, CA, USA). The total RNA was then reverse-transcribed into cDNA using a reverse transcription kit at 50˚C for 30 min. The cDNA was amplified using Promega PCR Single-Step Kit (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocols. The following primers (Invitrogen Life Technologies) were used: ERK2, sense 5'-CTAACGTTCTGCACCG TGAC-3' and antisense 5'-GATCACAAGTGGTGTTCAGCAG-3'; β-actin, sense 5'-CCTCATGAAGATCCTGACCG-3', and antisense 5'-ACCGCTCATTGCCGATAGTG-3'. β-actin served as the internal control. Quantification of the PCR products was performed using FTC-2000 (Shanghai Funglyn Biotech Co., Ltd., Shanghai, China). The threshold cycle (Ct) of each sample was recorded as a quantitative measure of the amount of PCR product in the sample. Relative changes in the expression levels were calculated using the 2-ΔΔCT method [12].

The TH17 cells were collected 72 h after the siRNA-ERK2 transfection. Total protein was extracted using ice-cold radioimmunoprecipitation lysis buffer (Santa Cruz, Biotechnology, Inc., Dallas, TX, USA). The protein concentrations were quantified using the Bradford method (Quick Start™; Bio-Rad, Hercules, CA, USA). Cytoplasmic protein samples (20 µg) were separated via 15% SDS-PAGE and semi-dry-transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% bovine serum albumin (Santa Cruz, Biotechnology, Inc.) for 1 h at room temperature. The blots were then incubated at 4˚C overnight with the following primary antibodies: anti-ERK2 (ab231085) and anti-GAPDH (ab8245 Abcam Biotechnology, Inc.) at a 1:1000 dilution in Tris-buffered saline with Tween-20 (TBST). GAPDH was used as a loading control. The blots were washed with TBST and then incubated with an anti-rabbit immunoglobulin G-horseradish peroxidase-conjugated secondary antibody (1:20,000 dilution in TBST; Pierce Biotechnology, Inc., Rockford, IL, USA) for 1 h at room temperature. After further washing with TBST, the blots were visualized using enhanced chemiluminescence (GE Healthcare Life Sciences, Little Chalfont, UK) and densitometrical quantified using a western blotting detection system (GE Healthcare Life Sciences). All results were normalized to GAPDH. Heat-induced epitope retrieval was conducted.

The levels of inflammatory cytokines in the Th17 cell culture supernatants were analyzed via ELISA using ELISA kits for IL-17a and CXCL-1 (R&D Systems, Inc.) according to the manufacturer’s protocols. A total of five mice were used for each group per experiment.

Statistical analysis was conducted using the SPSS software version 20.0 (SPSS Inc., Chicago, IL, USA). Data were analyzed using T-Test, followed by LSD significant difference test. A value of P < 0.05 (two-tailed test) was considered statistically significant. The results are expressed as mean ± standard deviation unless otherwise stated.

To investigate whether ERK2-specific siRNA silenced the expression of ERK2 in the present study, the mRNA expression levels of ERK2 were detected via RT-qPCR. The mRNA expression levels of ERK2 were downregulated in the ERK2-specific siRNA group 72 h after transfection compared with the control group (Figure 1(a) and Figure 1(b)). This indicated that ERK2-specific siRNA effectively inhibited the gene expression of ERK2.

Western blot analysis revealed that the protein expression of ERK2 in the ERK2-siRNA group downregulated compared with the control and scrambled-siRNA group (Figure 2(a) and Figure 2(b)). These results indicated a significant reduction in the expression of ERK2 in the ERK2-siRNA group.

To investigate whether ERK2-specific siRNA affected the gene expression levels of CXCL1, the mRNA levels of CXCL1 were detected via RT-qPCR. The mRNA expression levels of CXCL1 were downregulated in the ERK2-siRNA group 72 h after transfection compared with the control group (Figure 3).

The protein levels of CXCL1 were detected via western blot analysis and were downregulated in the ERK2-siRNA group compared with the control group. A significant difference was observed in the expression levels of CXCL1 between the ERK2-siRNA group and the control group (Figure 4).

The concentration of IL-17A and CXCL1 in the culture supernatants of the TH17 cells significantly decreased in the ERK2–siRNA group compared with the control and scrambled-siRNA group (Figure 5).