Normal human lung epithelial BEAS-2B cells were provided by BLUEFBIO (BFN608009328, China) and cultured in DMEM high glucose medium with 10% FBS and 1% double antibiotics. A COVID-19-type proinflammatory cell model was constructed [13, 17, 18], and the cells were grouped into the control group (normal cultured cells + solvent control for 12 and 24 h), RBD-500 group (BEAS-2B cells treated with 500 ng/mL recombinant SARS-CoV-2 spike RBD protein with His tag (RBD) for 12 and 24 h), and RBD-1000 group (BEAS-2B cells treated with 1000 ng/mL RBD for 12 and 24 h). To study the effects of GH/estrogen/androgen on COVID-19-type proinflammatory responses in BEAS-2B cells, the cells were further divided into the control group (normal cultured cells + solvent control treatment for 24 h), RBD group (cells treated with 1000 ng/mL RBD for 24 h), RBD + GH group (cells treated with 1000 ng/mL RBD and 200 ng/mL GH simultaneously for 24 h), RBD + E2 group (cells treated with 1000 ng/mL RBD and 1 nM 17β-estradiol (E2) simultaneously 24 h [6]), and RBD + Tes group (cells treated with 1000 ng/mL RBD and 10 nM Tes simultaneously treated for 24 h [6]).

Different groups of BEAS-2B cells were digested, counted and seeded in a 96-well plate at a density of 1 × 104 cells/well, with 100 µL per well. The cell count assay was performed in replicates. Each group had 3 replicate wells. After culturing adherent cells, 10 µL/well of CCK-8 (#NU679, DOJINDO, Japan) was added, and CCK-8 solution was prepared with complete medium. After incubation at 37 °C and 5% CO2, the absorbance value at 450 nm was analyzed on a Bio-Tek microplate reader.

Total RNA was extracted by TRIzol (#15596026, Thermo, USA) and reverse transcribed into cDNA with a cDNA reverse transcription kit (#CW2569, Beijing ComWin Biotech, China). Subsequently, real-time PCR was performed on a fluorescence quantitative PCR instrument (QuantStudio1, Thermo, USA) using Ultra SYBR Mixture (#CW2601, Beijing ComWin Biotech, China). The reaction system was 30 µL, including 2 µL reverse transcription product cDNA, 1 µL Primer R (10 µM), 1 µL Primer F (10 µM), 11 µL ddH2O, and 15 µL 2× SYBR Green PCR Master Mix. The reaction conditions were as follows: predenaturation at 95°C for 10 min, and 40 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 30 s. The experiment was repeated three times. Using ACTB as the internal reference gene, the gene levels were calculated by the 2−ΔΔCt method, where △△Ct=△Ct experimental group-△Ct control group, and △Ct = Ct (target gene)-Ct (internal reference). The primer sequences were as follows: ACE2-F: 5’-GGACACTGAGCTCGCTTCTG-3’, ACE2-R: 5’-CCTTTGAACTTGGGTTGGGC-3’; AGTR1-F: 5’-CGGGGCGCGGGTTTG-3’, AGTR1-R: 5’-TACACCTGGTGCCGACTTTC-3’; TMPRSS2-F: 5’-ATACAAGCTGGGGTTCTGGC-3’, TMPRSS2-R: 5’-TAGCCGTCTGCCCTCATTTG-3’; ISG15-F: 5’-GGTGGACAAATGCGACGAAC-3’, ISG15-R: 5’-TCGAAGGTCAGCCAGAACAG-3’; MAPK14-F: 5’-ACAGGATGCCAAGCCATGAG-3’, MAPK14-R: 5’-GATCGGCCACTGGTTCATCA-3’; RELA -F: 5’-CGCCTGTCCTTTCTCATCCCAT-3’, RELA-R: 5’-CCTCTTTCTGCACCTTGTCACAC-3’; ACTB-F: 5’-ACCCTGAAGTACCCCATCGAG-3’, ACTB-R: 5’-AGCACAGCCTGGATAGCAAC-3’.

Total protein was extracted from 5.3 × 106 BEAS-2B cells using a RIPA kit (P0013B, Beyotime, China). After the protein concentration was determined, proteins were separated by 10% SDS‒PAGE electrophoresis. Proteins were transferred to PVDF membranes by electrotransfer. For primary antibodies, we used anti-ACE2 (21115-1-AP, 1:1000, Proteintech, USA), anit-AGTR1 (25343-1-AP, 1:500, Proteintech, USA), anti-TMRRSS2 (ab92323, 1:1000, Abcam, UK), anti-ISG15 (15981-1-AP, 1:500, Proteintech, USA), anti-phospho-MAPK14 (28796-1-AP, 1:1000, Proteintech, USA), anti-MAPK14 (14064-1-AP, 1:1000, Proteintech, USA), anti-phospho-RELA (ab76302, 1:1000, Abcam, UK), anti-RELA (10745-1-AP, 1:1000, Proteintech, USA), and anti-ACTB (66009-1-Ig, 1:5000, Proteintech, USA) antibodies. The membranes were then incubated with HRP-conjugated secondary antibodies. β-actin was used as the internal reference. The membrane was immersed in ECL chemiluminescent solution (K-12,045-D50, Advantesta, USA) for luminescence visualization. Protein bands were detected using a chemiluminescence imaging system (ChemiScope 6100, Clinx, China). The experiment was repeated three times.

IL-6 (KE00007, Proteintech, USA), MCP-1 (KE20009, Proteintech, USA), MDA (#A003-1, Nanjing Jiancheng Bioengineering Institute, China), and SOD (#A001-3, Nanjing Jiancheng Bioengineering Institute, China) quantitative ELISA kits were used to detect related factor levels according to the manufacturer’s recommendations. A DYNATECHM 7000 microplate reader was used to calculate each indicator concentration by forming a standard curve through the constant value. The experiment was repeated three times.

The level of ROS was assessed according to the ROS assay kit (S0033S, Beyotime, China). First, DCFH-DA (stock concentration 10 mM) was diluted 1:1000 in serum-free medium to a final concentration of 10 µM. Then, the cells treated as indicated above were removed from the cell culture medium, and DCFH-DA was added to cover the cells, followed by incubation in a 37 °C cell incubator for 20 min. The samples were collected by trypsinization. Finally, FCM (A00-1-1102, Beckman, USA) detection was performed using an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The level of ROS was analyzed with CyExpert software. The experiment was repeated three times.

GraphPad Prism 8.0 software was applied for statistical analysis. The experiments were repeated three times. Measurement data are expressed as the mean ± standard deviation. The experiments were repeated three times. The significance of three or more groups was compared by one-way ANOVA. Tukey’s test was adopted for postmortem testing. P < 0.05 indicated a statistically significant difference.