Figure 5

Immunohistochemical localization and Western blot analysis of TRAF6, TRIF, and IRF3 in serous papillary ovarian carcinoma. The immunoreactivity for TRAF6 was moderate in the OC group (A) and weak in the OC + Mel group (B). The OC + EtOH (C) and OC + EtOH + Mel groups (D) displayed weak immunostaining for TRAF6 (arrow). The immunoreactivity for TRIF was enhanced in the epithelium of the OC (E) and OC + EtOH groups (G), but mel therapy resulted in weak reaction for TRIF in the OC + Mel (F) and OC + EtOH + Mel groups (H). Strong immunoreactivity for IRF3 was observed in the animals from the OC (I) and OC + EtOH groups (K), in contrast to the moderate to high reactions in the surface epithelium of the OC + Mel (J) and OC + EtOH + Mel groups (L) (arrow). Bar = 20 μm. Negative controls were used. (M) Representative TRAF6, TRIF, and IRF3 expression profiles in extracts (70 μg protein) pooled from 10 samples/group (upper panel). (N–P) Extracts obtained from individual rats were used for densitometric analysis of the TRAF6, TRIF, and IRF3 levels following normalization to a loading control (β-actin). All results are expressed as the means ± SD (n = 10 animals/group). ^aP < 0.05 vs. OC; ^cP < 0.05 vs. OC + EtOH. (Q) Schematic representation of TLR signaling. MyD88 acts as an essential TIR domain-containing adaptor for the induction of inflammatory cytokines in all TLR subtypes (canonical pathway). In the TLR4-mediated signaling pathway, the TIR domain-containing adaptor TRIF stimulates a MyD88-independent pathway (non-canonical pathway). This signal leads to the activation of IRF3 via TBK1 and IKKƐ/IKKi, facilitating the nuclear translocation of IRF3, which activates the expression of inflammatory factor-related target genes (e.g., IFN-β). Treatment with mel induces the downregulation of TRAF6, TRIF, and IRF3. mel: melatonin; TRAF6: tumor necrosis factor receptor-associated factor 6; TRIF: TIR domain-containing adaptor; TBK-1: TANK-binding kinase 1; IRF3: interferon regulatory factor 3; IFN-β: interferon β.