AZM is identified as an antagonist of UPR signaling and activity
The transcriptome sequencing (RNA-seq) analysis was applied to identify the differentially expressed genes (DEGs) in TNF-α or IL-1β-activated RA FLSs with AZM treatment, and 198 genes and 217 genes were significantly differentially expressed, respectively (Fig. 3A). Then, enrichment analysis of GO classification and KEGG pathway was conducted. Among the biological processes, 68 genes and 52 genes of the DEGs were enriched in the biosynthesis and metabolism of lipid, cholesterol and steroid, cell proliferation, protein exit from ER, SREBP-SCAP complex retention in ER, redox process and regulation of transcription (Fig. 3B). KEGG analysis showed that 66 genes and 64 genes of the DEGs were mainly involved in Signal transduction, Immune system, Cholesterol and Glycerolipid metabolism, PPAR, AMPK, TNF and PI3K-Akt signaling pathway (Fig. 3C). qRT-PCR was used to confirm the results of RNA-seq analysis that the expression trends of genes involved in cholesterol biosynthetic process (CYP51A1, ACAT2, PCSK9),lipid metabolic process (MSMO1, IDI1, SCD, PNPLA3, LIPG, SC5D, HSD17B7, FADS2) were increased following AZM treatment (online Supplementary Fig. 4). Several other genes, involved in steroid biosynthetic process (LDLR and ABCA1) and apoptosis (HRK and TUBA1A), were also found and validated to be increased (online Supplementary Fig. 4).
Intriguingly, in the cellular component field of TNF-α or IL-1β-activated RA FLSs with AZM treatment, 32 and 28 DEGs were distributed to ER (Fig. 3B). ER is the site of cholesterol- or lipid-induced cytotoxicity in various cell types (Yeo et al., 2014). Accumulation of free cholesterol (FC) and lipid in the ER leads to activation of the unfolded protein response (UPR) and C/EBP homologous protein (CHOP)–induced apoptosis (Erdi, Sozen, Kartal, & Ozer, 2017). Then, we tested the effect of AZM on the UPR effector, CHOP. As shown in Fig. 3D, CHOP was induced markedly 12 h post AZM exposure. Furthermore, the activity of UPR components enhanced as evidenced by the increased phosphorylation of PERK and elF2α, and the up-regulated expression of IRE1α and, ATF4 (Fig. 3E). RA FLSs are characterized by apoptotic resistance against ER stress (Rahmati et al., 2018). In this study, the expression of p-p38, CREB3L2 and p-Akt which represents pro-apoptotic markers was obviously upregulated after AZM treatment (Fig. 3F). Furthermore, in the presence of ER stress inducer, more proportion of apoptosis were found when RA FLSs were treated AZM as compared to its control (Fig. 3G). Taken together, AZM treatment may disturb the biosynthetic process of cholesterol, lipid and steroid, activate UPR and subsequently induce apoptosis of RA FLSs.