Benzylideneacetophenone derivatives attenuate IFN-γ-induced IP-10/CXCL10 production in orbital fibroblasts of patients with thyroid-associated ophthalmopathy through STAT-1 inhibition

Abstract

The aim of the present study was to identify a new candidate anti-inflammatory compound for use in the active stage of thyroid-associated ophthalmopathy (TAO). Benzylideneacetophenone compound JC3 [(2E)-3-(4-hydroxy-3-methoxyphenyl)phenylpro-2-en-l-one] was synthesized based on a structural modification of yakuchinone B, a constituent of the seeds of Alpinia oxyphylla, which belongs to the ginger family (Zingiberaceae), has been widely used in folk medicine as an anti-inflammatory phytochemical. Orbital fibroblasts were primarily cultured from patients with TAO, and the potential of JC3 to suppress the interferon (IFN)-γ-induced protein (IP)-10/CXCL10 production in these cells was determined. IFN-γ strongly increased the level of IP-10/CXCL10 in orbital fibroblasts from patients with TAO. JC3 exerted a significant inhibitory effect on the IFN-γ-induced increase in IP-10/CXCL10 in a dose-dependent manner; its potency was greater than that of an identical concentration of yakuchinone B with no toxicity to cells at the concentration range used. Moreover, the constructed dimer and trimer polystructures of JC3, showed greater potency than JC3 in suppressing the IFN-γ-induced production of IP-10/CXCL10. JC3 significantly attenuated the IP-10/CXCL10 mRNA expression induced by IFN-γ, and a gel-shift assay showed that JC3 suppressed IFN-γ-induced DNA binding of signal transducer and activator of transcription-1 (STAT-1) in TAO orbital fibroblasts. Our results provide initial evidence that the JC3 compound reduces the levels of IP-10/CXCL10 protein and mRNA induced by IFN-γ in orbital fibroblasts of TAO patients. Therefore, JC3 might be considered as a future candidate for therapeutic application in TAO that exerts its effects by modulating the pathogenic mechanisms in orbital fibroblasts.

Figure 1

Structures of yakuchinone B; JC3, which was synthesized based on a structural modification of yakuchinone B; and constructed polystructures of JC3 (JC3 dimer and JC3 trimer).

Figure 2

IFN-γ increases IP-10/CXCL10 levels in orbital fibroblasts. Primary cultures of orbital fibroblasts (5 × 10⁴) of patients with TAO (n=2) and a subject without TAO (n=1) were incubated with IFN-γ (10 ng ml⁻¹), LPS (100 ng ml⁻¹) and IL-1β (10 ng ml⁻¹) for 24 h and H₂O₂ (100 μM) for 30 min. After treatment, supernatants were collected and IP-10/CXCL10 protein levels in each culture supernatant were measured by ELISA. Data are presented as the fold increases in relative IP-10/CXCL10 levels from orbital fibroblasts compared with each control. The graph shows the mean±s.d. of three independent replicates (*P<0.05 vs each untreated control).

Figure 3

The IFN-γ-induced IP-10/CXCL10 level was attenuated by the benzylideneacetophenone derivative compound, JC3, in a dose-dependent manner without an influence on the viability of orbital fibroblasts from patients with TAO. (a) TAO and non-TAO cells were pretreated with JC3 at 5, 10 and 20 μM for 30 min and then treated with IFN-γ (10 ng ml⁻¹) for 24 h; the supernatant was then collected to measure IP-10/CXCL10 protein by ELISA. The IP-10/CXCL10 expression level in IFN-γ-treated group was arbitrarily assigned a value of 100, and the data are presented as percentage reductions (*P<0.05 vs each IFN-γ group; BD, below detection limit). (b) TAO cells were pretreated with either yakuchinone B or JC3 at 10 μM for 30 min. Following treatment with IFN-γ (10 ng ml⁻¹) for 24 h, IP-10/CXCL10 production was measured and analyzed as described in A (*P<0.05 vs each IFN-γ-treated group; **P<0.05 in JC3-treated group vs yakuchinone B-treated group). (c) Orbital fibroblasts of patients with TAO patients were treated with IFN-γ (10 ng ml⁻¹) in the presence or absence of 5, 10 of 20 μM JC3 for 24 h. After treatment, cell viability was evaluated as described in the Materials and Methods section. Results are expressed as mean±s.d. in relative O.D. units (compared with control); normalized values are shown.

Figure 4

Polystructures of JC3 reveal an enhanced inhibitory effect of IFN-γ-induced IP-10/CXCL10 expression in orbital fibroblasts from patients with TAO patients. (a, b) Orbital fibroblasts from patients with TAO were treated with IFN-γ (10 ng ml⁻¹) in the presence of incremental doses of JC3 dimer (JC3-D, 0.1–10 μM) or JC3 trimer (JC3-T, 0.1–10 μM) as described in Figure 3. At the end of incubation, the supernatant was collected and analyzed by ELISA. (c) Cells were pretreated with 10 μM of JC3, JC3-D, or JC3-T, and the IP-10/CXCL10 level was assessed by ELISA. The IP-10/CXCL10 expression level of the IFN-γ-treated group was arbitrarily assigned a value of 100, and data are presented as percentage reductions (*P<0.05 vs IFN-γ-treated group; BD, below detection limit).

Figure 5

JC3 reduced IFN-γ-induced IP-10/CXCL10 mRNA expression in TAO orbital fibroblasts. Total RNA was extracted after 24 h of IFN-γ (10 ng ml⁻¹) treatment in the presence or absence of JC3. IP-10/CXCL10 mRNA levels were analyzed by RT–PCR. The control level of IP-10/CXCL10 expression was arbitrarily assigned a value of 1. Data are means of three independent experiments. *P<0.05 and **P<0.05 compared with the untreated control and IFN-γ-treated group, respectively.

Figure 6

JC3 inhibited DNA binding of STAT-1, but not NFκB, in IFN-γ-stimulated TAO orbital fibroblasts. Cells were treated with IFN-γ (10 ng ml⁻¹) in the presence or absence of JC3 (10 μM) for 30 min, and nuclear extracts were prepared and analyzed by EMSA as described in the Materials and Methods section. The levels of (a) STAT-1 DNA binding and (b) p50/p65 NF-κB DNA binding were evaluated by EMSA. A cold probe was used for competition control. Data shown are representative of at least three independent experiments.