Cells and Drugs
Human normal lung epithelial cell line BEAS-2B was purchased from Shanghai Cell Bank. BEAS-2B cells were cultured in specific medium for BEAS-2B cells (CM-0496(ProCell)), with 10% fetal bovine serum (FBS) and 5% CO2 at 37℃.The triptolide (Provided by Fujian Academy of Medical Sciences) was dissolved in 0.1% dimethyl sulfoxide (DMSO, DMSO had little effect on subsequent experiments, so the data of DMSO in the experiment were not listed, and the final concentration of DMSO in the medium was adjusted to less than 0.1%). The concentrations of triptolide L,M and H were 2.5ng/ml,5ng/mL and 10ng/mL, respectively.
Building the severe COVID-19-pseudovirus cell model
The pseudovirus system was purchased from Wuhan Shumi Brain Science and Technology Co., Ltd., which included PV-SARS-CoV-2-S-VSV-△g-EGFP (V04001,1.30E+07IFU/mL); RLV-CMV-SARS-CoV-2-S-McHerry-WPRE(LV-0614,1.00e+06TU/mL). RLV-CMV-ACE2-PGK-PURO-WPRE (LV-0707,1.00E+06 TU/mL), and the infection complex number (MOI) is 1. Referring to previous studies [17], cells were inoculated with 6-well plates at 1x106, and the complete medium was replaced 24 hours later. Pseudoviral system was firstly added according to MOI=1, and then the virus supernatant was removed at 6 hours, and the fresh complete medium was replaced in the meantime. The experimental group was added with 100 μg/mL LPS (Sigma) for 24 h, and the intervention time of triptolide was 72h.
Cell Viability Assay
Take BEAS-2B cells (1×104 cells/well) and add 2.5ng/mL,5ng/mL,10ng/mL of triptolide, respectively. The effects of triptolide on the cell viability of BEAS-2B at 24, 48 and 72 hours were analyzed with the VI-CELL XRVI-CELL (Beckman Coulter). Data are expressed as mean standard deviations with three duplicates, and the results are tested by analysis of variance and Duncan multipolar test (P<0.05).
Measurement of cytokines
Cell supernatant was collected and centrifugated at 3500rpm for 10mins. Levels of IL-6, IL-10 and TNF-a in the supernatant were analyzed using ELISA kits, and optical density (OD) values were determined using a multifunctional enzyme plate analyzer (Synergy 2, USA, Bio-Tek, Inc.).
RNA extraction and detection
Total RNA was extracted by Trizol (Beijing Tiangen Biochemical Co., Ltd.), and then RNA quality was detected. The purity of RNA was detected by Nanodrop spectrophotometer (Implen, CA, USA). Agilent 2100 (Agilent Technologies, CA, USA) assesses RNA integrity.
Differential expression analysis
Deseq (1.10.1) was used for differential expression analysis. The P-values of the results of differential expression analysis were controlled for false discovery rate (FDR) with Benjamini and Hochberg methods. The standard of differential gene screening is generally Q < 0.05.
Gene Ontology(GO) enrichment analysis
GO enrichment analysis of the DEGs was implemented by the GOseq R packages based on Wallenius non-central hyper-geometric distribution [18], which can adjust for gene length bias in DEGs.
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis
KEGG [199]is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from the perspective of molecular level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies. We used KOBAS [20] software to test the statistical enrichment of differential expression genes in KEGG pathways.
Statistical Analysis
GraphPad Prism 8 was used to perform data analysis. Data for the cell viability assay were calculated from three independent experiments with each experiment containing six replicates. Results were presented as mean ± SD. Data comparison was performed using the analysis of variance (ANOVA, one-way or two-way), followed by Dunnett’s or Tukey’s post hoc tests. Results were considered statistically significant (P < 0.05).