Effects of Triptolide on Cell Viability, Secretion of In�ammatory Cytokines, and Gene Expression of Severe Coronavirus Disease 2019(cid:0)COVID-19(cid:0) Pseudovirus Cell Model

Background: Acute respiratory distress syndrome (ARDS), which is caused by severe immune cell response and associated alveolar in�ammation, is also a critical complication in hospitalized patients with COVID-19. Triptolide is a powerful anti-inammatory and immunosuppressive drug and is proved to help relieve the in�ammation of ARDS. However, its anti-inammatory effect on COVID-19 patients with ARDS complications remains uncertain. Methods: In this study, human normal lung epithelial cells (BEAS-2B), the pseudovirus system of syndrome coronavirus 2(SARS-CoV-2) and lipopolysaccharide (LPS) were used to construct as severe COVID-19-pseudovirus cell model to explore the effects of triptolide on cell viability, secretion of in�ammatory cytokines, and gene expression. Results: The results showed that triptolide increased cell viability, decreased the secretion levels of cytokines IL-6, TNF-a, and increased the expression of IL-10. Furthermore, transcriptome analysis in this cell models showed that the Differentially expressed genes (DEGs) were related to plasma membrane integrity, metabolic activity and mitochondrial function, and were associated with TNF, FOXO, mTOR and MAPK signaling pathways. Conclusion: Take into consideration previous studies on the functions of triptolide in BEAS-2B cells, the current study indicated that triptolide can play a critical role in protecting against in�ammatory damage and maintaining the normal physiological function of BEAS-2B cells in response to pseudovirus and LPS infection.

However, the anti-in ammatory function of triptolide in patients with severe COVID-19 accompanied by ARDS and systemic in ammation is less understood.With the help of Connectivity Map (CMap) database [11] and network pharmacology and data mining [12], recent research indicates that triptolide would provide new options for improving treatment of patients infected with SARS-CoV-2.In addition, triptolide may serve as an important function in promoting autophagy and inhibiting apoptosis [13], reducing oxidative stress [14][15] and improving antioxidant activity [16], all of which were correlated in terms of the protective effect of cell.
To get a better understanding of its functions in severe COVID-19, we explored the effects of triptolide on cell viability, secretion of in ammatory cytokines, and gene expression in severe COVID-19-pseudovirus cell model.It was observed that triptolide moderately increased cell viability in BEAS-2B and inhibited the secretion of in ammatory cytokines.More importantly, the DEGs provided convincing evidence showing that triptolide maintains normal physiological functions of the severe COVID-19-pseudovirus cell model through TNF, FOXO, mTOR and MAPK signaling pathway.

Cells and Drugs
Human normal lung epithelial cell line BEAS-2B was purchased from Shanghai Cell Bank.BEAS-2B cells were cultured in speci c 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 nal 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.

Cell Viability Assay
Take BEAS-2B cells (1×10 4 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 highthroughput 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, oneway or two-way), followed by Dunnett's or Tukey's post hoc tests.Results were considered statistically signi cant (P < 0.05).

Effect of triptolide on Cell Viability and in ammatory cytokines secretion
As is shown in Fig. S1A-C, the concentration of triptolide less than 10ng/ml had no signi cant cytotoxic effects on the BEAS-2B.Compared with the model group (Virus + LPS), the cell viability of BEAS-2B was improved after 72 h of triptolide treatment(P<0.05) (Fig. 1A-C).In addition, Fig. 1D-F shows that triptolide decrease the levels of cytokines IL-6, TNF-a and increase the levels of IL-10 (P<0.01).These results veri ed that triptolide can help reduce the in ammatory response and improve cell survival activity in severe COVID-19-pseudovirus cell model.
Effect of triptolide on Gene Expression in severe COVID-19-pseudovirus cell model.
The gene expression affected by triptolide treatment was detected by transcriptomics.DEGs were highlighted in green and red in the volcano plots (Fig. 2A).As Fig. 2C shows, 639 DEGs in model control group, and 1,236 DEGs in triptolide intervention group, were identi ed respectively.Moreover, the differential gene expression cluster analysis showed that the expression patterns of genes in the similar color cluster region were identical, indicating that these genes may have similar functions or participate in the regulation of the same metabolic pathway (Fig. 2B).The top 30 up-and downregulated DEGs are summarized in Table.S1.

Go Enrichment Analysis
A number of GO terms belonging to biological process (cellular metabolic process, nucleic acid metabolic process, localization, transport and oxidation-reduction process), cellular component (intracellular, organelle, cytoplasm, membrane-bounded organelle and endoplasmic reticulum), molecular function (nucleic acid binding, protein binding and oxidoreductase activity) were signi cantly enriched within the dataset (Fig. 3A-B).By regulating these genes, triptolide can affect cellular activity in terms of metabolic activity and plasma membrane integrity.

Kegg Enrichment Analysis
KEGG enrichment analysis provided additional information on possible functional pathways that the DEGs are involved in (Fig. 4A-B).Further bioinformatics analysis showed that the downregulated DEGs were signi cantly enriched in signaling pathways such as the TNF, FOXO, mTOR and MAPK; metabolic pathways and cellular physiological processes such as mitophagy, autophagy and endocytosis (Fig. 4A).For the upregulated DEGs, the enriched pathways were mainly involved in ribosome, oxidative phosphorylation, lysosome, ECM-receptor interaction and glycolysis/ gluconeogenesis (Fig. 4B).

Discussion
In the current study, we observe the protection effects of triptolide on cell viability of severe COVID-19pseudovirus cell model, and we found that triptolide inhibits the secretion of in ammatory cytokine IL-6 and TNF-a, and enhances the secretion of anti-in ammatory cytokine IL-10.What's more, to better understand the mechanisms underlying regulation of cell viability, multiple signaling pathways heavily involved and well-studied, including TNF, FOXO, mTOR and MAPK, mitophagy, autophagy and metabolic pathways have now been shown to be associated with triptolide.
Our observation was in line with previous studies that triptolide protects cell viability of neural cells [21][22], cardiac cells [23]and the neuroin ammation response induced by LPS [24].However, these previous studies were mainly carried out with NF-kappa B or any other single signaling pathway.There are also many reports on the role of triptolide in antiviral mechanisms.For example, triptolide inhibits human immunode ciency virus type 1 replication [25], and triptolide impaired the number of viral DNA copies and virion production [26].Whereas, whether triptolide can also protect the activity of cells infected with SARS-CoV-2 and exert its powerful anti-in ammatory effect has not been reported when our research was carried out.Besides, mitochondria are mainly related to cell bioenergy, which is an important cause of cell apoptosis and death [27], and mitochondria also impact on complex cellular processes including in the regulation of autophagy [28][29], immune response [30] and some other complex cell process.Our study found that the levels of mitochondria-related genes were regulated by triptolide (Fig. 4A, Table .1 and Fig. S2), indicating that the improvement of mitochondrial function was an important factor in the protection of cell activity of triptolide.
Existing studies have con rmed that triptolide plays an anti-in ammatory role in different diseases involving different signaling pathways.For example, triptolide induces protective autophagy in human cervical cancer cells by targeting phosphoinositol 3-kinase/Akt/mTOR, p38, MAPK, p53 and FOXO3a signaling pathways [31].And in angiocardiopathy, ErbB, Hippo and hypoxia-inducible factor-1α (HIF-1α) signaling pathways are involved [31][32][33].In addition, FOXO and MAPK pathways are involved in a variety of cellular functions and have clinical signi cance, including its effects on cell cycle arrest, cell differentiation, cell proliferation, migration, senescence and apoptosis [34][35].These signaling pathways's functions have also been con rmed in this study, and our research focuses on the TNF, FOXO, mTOR and MAPK signaling pathways and explores the effects of triptolide on cell viability and antiin ammatory effect.
In view of the fact that monitoring cell viability is a key task for basic research, such as apoptosis, necrosis and drug discovery, this study focused on maintaining cell activity with triptolide.Through our analysis, in addition to regulating the expression level of in ammatory factors, it was found that triptolide can change the expression of cell viability-related genes and proteins through a variety of signaling pathways, so as to protect cell activity by down-regulating the expression levels of mitochondrial damage, autophagy and other related genes.Taking all these factors together, triptolide can maintain the normal physiological function of BEAS-2B cells by regulating the integrity of plasma membrane, protecting mitochondrial function and balancing normal cell metabolic activity, and thus protect against pseudovirus and LPS-mediated BEAS-2B cells damage.
On the other hand, it is worth noting that large doses or long-term use of triptolide also can cause many adverse reactions, including membrane damage, mitochondrial disruption, metabolism dysfunction, oxidative stress, apoptosis and autophagy, so there is also a concern between the e cacy and toxicology of triptolide.Of course, TNF, FOXO, mTOR and MAPK signaling pathway are not independent of each other in the action of triptolide, and these signaling pathways are interrelated.Although we have selected a certain amount of related genes and signaling pathways, many of the targets and exact mechanisms involved in these events remain unknown.Therefore, the exact mechanism and function of triptolide in severe COVID-19-pseudovirus cell model is also worth further study.

Figures Figure 1
Figures

Figure 2 Effect
Figure 2
TablesTable 1 is not available with this version.