Our study results revealed that the pretreatment of RAW 264.7 cells with various concentrations of DEX could significantly reduce the LPS-induced mRNA expression of IL-6 in a dose-dependent manner. A protective role of synthetic DEX was indicated that affects LPS-induced inflammation. Our results are consistent with the findings of Yamazaki et al. who reported that GCs inhibit the synthesis of IL-1, TNF-α, IL-1β, IL-6, MMP-I, and COX-2 mRNA expression in SW 982 cells .
The molecular mechanism of GCs, wherein they interact with their GRs to regulate inflammatory responses, has been well-studied. GCs bind to GRs and are transported to the nucleus subsequently to form a dimer and combinations of transcriptional complexes, such as AP-1 and NF-κB, either in transrepression or transactivation, which are responsible for the anti-inflammatory action of GCs [21, 22]. The results of previous studies have suggested that the treatment of naïve monocytes with fluticasone or DEX did not cause global suppression of activated monocytic functions instead induced a cellular differentiation with an anti-inflammatory phenotype [23, 24]. The recent evidence revealed that GCs could efficiently inhibit these processes by downregulating pro-inflammatory mediators from macrophages and monocytes and their migration toward inflammatory stimuli. In addition, GCs could remove endo- and exogenous danger signals by increasing phagocytic capacity and limiting T-cell activation .
Our results revealed that LPS stimulation resulted in the upregulated expressions of inflammatory transcription factors, such as NF-κB , AP-1 , and Sp1-2 . In addition, LPS increased IL-6 and its promoter activity. The increase of LPS-stimulated IL-6 promoter activities could be suppressed using DEX, especially in NF-κB, AP-1, and Sp1-2, but not in Sp1-1. These results are consistent with those of previous studies [29, 30]. A previous study revealed two Sp1 sites on the IL-6 promoter and Sp1 bind to this region in human monocytes . However, no reports were available to determine which Sp1 site was crucial for IL-6 expression. Our study is the first to report that only the Sp1-2 site was involved in IL-6 expression. Conclusively, the transcription factors NF-κB, AP-1, and Sp1-2 bind to the IL-6 promoter and play crucial roles in the basal and inducible expression of murine IL-6 gene.
Our study determined five putative GR binding sites on the IL-6 promoter, and DEX could reduce the promoter activities of IL-6 GR binding sites. After LPS stimulation and sequence construct, the promoter activity of IL-6 exhibited a significant increase in LPS-induced promoter activity. Notably, GR2 and GR3 sites appeared to play a crucial role in both basal and inducible promoter activities in LPS-induced inflammation. By contrast, the GR1, GR4, and GR5 sites did not exhibit changes in LPS-induced or basal promoter activities. Although a previous study revealed that the recombinant GR binds to IL-6 promoter regions, namely the GR1–5 elements, in vitro , no significant shifted bands were noted using the probes of GR1, GR4, and GR5 during EMSA, and no changes of promoter activities were observed after the mutation of these three sites (data not shown). Furthermore, GRs may interact with transcription factors, such as AP-1 and NF-κB, to mediate downstream proinflammatory genes, including IL-6 . Nevertheless, the precise targeted regulation of GR2 and GR3 on IL-6 and the possible mechanism related to transcriptional synergism with the GC response element (GRE) remain unknown. Nonetheless, additional studies are warranted to determine whether GR2 and GR3 represent a novel negative GRE , directly binding through GRα—the pivotal subunit of GR—that could facilitate interactions between GRα and AP-1 and NF-κB.
Our results provide possible insights into the mechanism of controversial steroids that would help with the decision-making of their dosages—low or high—in hemodynamically unstable patients with sepsis. Moreover, in the future, knowledge regarding GR2 and GR3 binding sites could prove crucial in facilitating the selection of individual-specific therapeutic agents in vulnerable patients with sepsis.