Jietacin Derivative Inhibits TNF-α-Mediated Inflammatory Cytokines Production via Suppression of the NF-κB Pathway in Synovial Cells

Synovial inflammation plays a central role in joint destruction and pain in osteoarthritis (OA). The NF-κB pathway plays an important role in the inflammatory process and is activated in OA. A previous study reported that a jietacin derivative (JD), (Z)-2-(8-oxodec-9-yn-1-yl)-1-vinyldiazene 1-oxide, suppressed the nuclear translocation of NF-κB in a range of cancer cell lines. However, the effect of JD in synovial cells and the exact mechanism of JD as an NF-κB inhibitor remain to be determined. We investigated the effect of JD on TNF-α-induced inflammatory reaction in a synovial cell line, SW982 and human primary synovial fibroblasts (hPSFs). Additionally, we examined phosphorylated levels of p65 and p38 and expression of importin α3 and β1 using Western blotting. RNA-Seq analysis revealed that JD suppressed TNF-α-induced differential expression: among 204 genes significantly differentially expressed between vehicle and TNF-α-stimulated SW982 (183 upregulated and 21 downregulated) (FC ≥ 2, Q < 0.05), expression of 130 upregulated genes, including inflammatory cytokines (IL1A, IL1B, IL6, IL8) and chemokines (CCL2, CCL3, CCL5, CCL20, CXCL9, 10, 11), was decreased by JD treatment and that of 14 downregulated genes was increased. KEGG pathway analysis showed that DEGs were increased in the cytokine–cytokine receptor interaction, TNF signaling pathway, NF-κB signaling pathway, and rheumatoid arthritis. JD inhibited IL1B, IL6 and IL8 mRNA expression and IL-6 and IL-8 protein production in both SW982 and hPSFs. JD also suppressed p65 phosphorylation in both SW982 and hPSFs. In contrast, JD did not alter p38 phosphorylation. JD may inhibit TNF-α-mediated inflammatory cytokine production via suppression of p65 phosphorylation in both SW982 and hPSFs. Our results suggest that JD may have therapeutic potential for OA due to its anti-inflammatory action through selective suppression of the NF-κB pathway on synovial cells.


Introduction
Osteoarthritis (OA) is the most common type of arthritis, and an important cause of disability that degrades quality of life and causes substantial economic loss [1]. A number of guidelines for management of osteoarthritis (OA) pain in patients presenting with severe pain and musculoskeletal pain universally recommend oral nonsteroidal anti-inflammatory drugs (NSAIDs) [2][3][4][5]. However, NSAIDs are restricted to pain management rather than prevention or cure, leaving surgery as typically the last resort for knee OA. Therefore, the development of disease-modifying drugs that confer both structural and symptomatic benefits is needed for OA treatment.
Synovial inflammation plays a pivotal role in joint destruction and pain in osteoarthritis. The inducible transcription factor NF-κB has a central role in immune and inflammatory responses, and in cellular differentiation. In particular, expression of NF-κB is upregulated in synovial tissues in early and late OA [6,7]. Inflammatory cytokines, including IL-6 and IL-8 derived from the OA synovium, are well-known causes of catabolic gene induction operating via mechanisms which involve NF-κB activation [8,9]. In addition, these cytokines contribute to osteoarthritic pain [10,11]. Moreover, NF-κB regulates chemokine ligands to recruit immune cells during synovial inflammation [12,13]. Therefore, the development of NF-κB inhibitors may provide a potential therapeutic agent for OA.
Previous studies reported the potential of natural products in the treatment of OA via the inhibition of inflammatory processes [14,15]. Jietacins, a class of azoxy antibiotics, were originally isolated from culture broth of Streptomyces sp. KP-197 [16,17]. We previously reported that a jietacin derivative (JD), (Z)-2-(8-oxodec-9-yn-1-yl)-1-vinyldiazene 1-oxide ( Figure S1), suppressed the nuclear translocation of NF-κB in a range of cancer cell lines having a strong constitutive NF-κB activation [18]. However, the effect of JD on inflammatory reaction in synovial cells and the exact mechanism of JD as an NF-κB inhibitor remain to determined.
Here, we examined the effect of JD on inflammatory cytokine production and phosphorylation of NF-κB in synovial cells.

Figure 2.
Effect of jietacin derivative on inflammatory cytokine expression and production in SW982. IL1B mRNA by qPCR (A) and IL-1β protein concentration by ELISA (B). IL6 mRNA by qPCR (C) and IL-6 protein concentration by ELISA (D). IL8 mRNA by qPCR (E) and IL-8 protein concentration by ELISA (F). Synovial cell line SW982 stimulated with DMEM (vehicle), TNF-α, or TNF-α + jietacin derivative. a p < 0.05 compared to vehicle, b p < 0.05 compared to TNF-α.  Activated NF-κB increases the expression of inflammation-related cytokines and chemokines in synovial fibroblasts [12,13,20], and these expressions are associated with OA pathology [10,11,[20][21][22][23][24][25]. CCL2 and CCL3 are elevated in peripheral blood in OA patients [23], and CXCL9 and CXCL11 are increased in synovial fluids (SF) and serum of OA patients [24]. CCL20 concentrations in SF correlate with disease severity in knee OA [25]. IL-1β concentration in SF is dependent on OA grade [26]. Serum IL-6 levels are increased in patients with OA, and are associated with radiological OA grade [20,27]. In addition, level of IL-8 in SF is associated with OA severity [21]. In addition, several clinical studies in OA patients have demonstrated a correlation between pain score and concentrations of IL-6 and IL-8 in SF [10,11]. Our results suggest that JD may have therapeutic potential for OA due to its anti-inflammatory action.

Effect of JD on NF-κB Pathway
KEGG pathway analysis indicated that JD regulated the NF-κB pathway. In addition, given that the JD suppressed TNF-α-mediated IL-1β, IL-6, and IL-8, we subsequently examined the effect of JD on the NF-κB pathway. TNF-α induced the phosphorylation of p65 (p < 0.0001), which was suppressed in the presence of 2.5 µg/mL (p < 0.001) and 1.25 µg/mL (p = 0.002) JD ( Figure 4A,B). There was no difference in p65 expression among the groups ( Figure 4A,C). Consistent with the SW982 results, TNF-α induced the phosphorylation of p65 (p < 0.0001), which was suppressed in the presence of 2.5 µg/mL JD (p < 0.001) in hPSFs ( Figure 5A,B).      The mammalian NF-κB subfamily comprises five proteins, namely, p50 (NF-κB), p52 (NF-κB2), p65 (RelA), RelB, and c-Rel [22]. NF-κB is retained in the cytosol in an inactive state under normal conditions, bound to the inhibitory protein IκB [28,29]. Any or all of proinflammatory cytokines, matrix degradation enzymes, and excessive mechanical stress can induce a cascade of reactions which lead to the phosphorylation of IκB and resulting proteasome-system-mediated degradation via the ubiquitin proteasome system. Further, IκB degradation then leads to the release of active NF-κB, which is translocated to the nucleus where it induces gene transcription [28]. We previously reported that JD inhibits this translocation to the nuclei, and that its inhibitory effect is lost in cells having mutant Pharmaceuticals 2023, 16, 5 7 of 12 p65 protein [18]. Phosphorylated p65 at this position plays the pivotal role of terminating the transcriptional activity of NF-κB in nuclei [7]. JD, therefore, prevents the transcriptional activity of NF-κB via the inhibition of p65 phosphorylation.
TNF-α activates a number of intracellular signaling pathways, among which are the NF-κB and p38 pathways [41][42][43]. A previous study reported that p38 plays a role in TNFα-induced production of inflammatory cytokines independently of the NF-κB pathway in synovial fibroblasts [43]. TNF-α induced phosphorylation of p38 in both the presence and absence of JD in SW982 (p < 0.001; Figure 6A,B). However, no change was seen in phosphorylation of p38 expression in the presence of JD ( Figure 6A,C). Similarly, TNF-αstimulated phosphorylation of p38 ( Figure 7A,B, p < 0.001) was not suppressed by JD in hPSFs ( Figure 7A,C). JD suppresses production of inflammatory cytokines by selectively inhibiting p65 phosphorylation.

Cell Culture
Human synovial cell line SW982 was purchased from American Type Culture Collection (Rockville, MD, USA). hPSFs were purchased from Sigma Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured in Dulbecco's Modified Eagle Medium with supplementation with 10% fetal bovine serum, 250 ng/mL amphotericin B, 100 ng/mL streptomycin, and 100 U/mL penicillin at 37 • C in 5% CO 2 .

RNA-Seq
RNA-Seq assay was used to screen the effect of JD on TNF-α stimulated SW982 cells. SW982 cells were seeded in 2 mL at a concentration of 1 × 105 cells/mL in a 6-well plate and incubated for 72 h. The cells were then treated with either control medium (vehicle) or TNF-α in the presence of 2.5 µg/mL JD for 6 h.
The total RNA was obtained using the Trizol protocol (Invitrogen, Carlsbad, CA, USA) and spin column (Direct-zol MicroPrep kit, Zymo Research, Orange, CA, USA). RNA quantity was determined by spectrophotometer (Denovix, DX, USA) and quality was assessed on an Agilent 2100 BioAnalyzer (Agilent) with an RNA 6000 Nano Chip. RNA-Seq was conducted on extracted RNA. RNA sequencing was performed using an MGI DNBSEQ-G400 sequencer (BGI, Shenzhen, China). Two replicate samples were taken from the vehicle, TNF-α, and TNF-α + JD groups for RNA-Seq.

Quantitative PCR (qPCR)
To validate the results of the RNA-Seq analysis and to investigate the effect of JD concentrations, qPCR was used to evaluate expression levels of IL1B, IL6 and IL8. SW982 cells and hPSFs were seeded in 2 mL at a concentration of 1 × 10 5 cells/mL in a 6-well plate and incubated for 72 h. SW982 cells were then treated with either control medium (vehicle) or TNF-α in the presence of various concentration of JD (0, 0.625, 1.25, and 2.5 µg/mL) for 6 h (n = 8). hPSFs were then treated with either control medium (vehicle) or TNF-α in the presence of 2.5 µg/mL JD for 6 h (n = 8). Following RNA extraction as described above, first-strand cDNA was synthesized from purified total RNA using the SuperScript ® III First-Strand Synthesis System (Invitrogen). qPCR was conducted using the SYBR green method using the CFX connect real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Table 2 lists the primer sequences used in this study. We evaluated GAPDH as a housekeeping gene. Gene expression (Gene/GAPDH) was measured by the delta-delta CT method, and relative expression was determined when the average level of gene expression (Gene/GAPDH) in vehicle was 1.

Enzyme-Linked Immunosorbent Assay (ELISA)
SW982 cells and hPSFs were seeded at a concentration of 1 × 10 5 cells/mL in 100 µL in 96-well plates followed by incubation for 72 h. SW982 cells were then treated with TNF-α in the presence of various concentrations of JD (0, 0.625, 1.25 and 2.5 µg/mL) for 24 h (n = 8). hPSFs were then treated with TNF-α in the presence of 2.5 µg/mL JD for 24 h (n = 8). The supernatants were collected and IL-1β, IL-6 and IL-8 concentrations in the supernatants were measured using commercial ELISA kits (BioLegend, San Diego, CA, USA) according to the manufacturer's protocol.

Statistical Analysis
Differences among vehicle-, TNF-α-, and TNF-α and JD-treated cells were compared using Tukey's multiple comparisons test on SPSS (version 25.0, IBM, Armonk, NY, USA). All statistical analyses were two-sided. Statistical significance was indicated by a p value < 0.05.

Conclusions
JD inhibited TNF-α-mediated inflammatory reaction via the suppression of p65 phosphorylation in the synovial cell line SW982 and hPSFs. Our results suggest that JD may have therapeutic potential for OA due to its anti-inflammatory action through selective suppression of the NF-κB pathway on synovial cells. Further investigation using OA animal models may reveal therapeutic potential for OA.