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.