| Since steroids and cyclooxygenase inhibitors may cause serious side effects, the IκB kinase (IKK)β/nuclear factor-KB (NF-κB) system becomes an intriguing candidate anti-inflammatory target. Rhein, the active metabolite of diacerein, possesses anti-inflammatory ability with gastrointestinal protective effect. However, in our preliminary study, we accidentally found that rhein showed both anti-and pro-inflammatory activities in lipopolysaccharide (LPS)-activated macrophages:rhein reduces proinflammatory mediators nitric oxide (NO) and interleukin-6(IL-6), but enhances tumor necrosis factor-a (TNF-a), interleukin-1β (IL-1β) and high mobility group box1(HMGB1) release. To exclude the effect of cell specificity, we carried out parallel experiments in Balb/c mice peritoneal macrophages. Similar results were also observed in mice peritoneal macrophages. Thus, in this study, we explored the underlying molecular mechanisms of the dual effect of rhein.In LPS-activated macrophages, the supernatant NO was determined by Griess method, other proinflammatory mediators IL-6, TNF-a, IL-1β and HMGB1were assayed by ELISA kits. The transcriptional levels of proinflammatory cytokines were determined by Western blot and qPCR, respectively. All protein expressions were determined by Western blot. NF-κB and AP-1activation were investigated by luciferase assay. IKKP kinase activity assay was performed using IκBa as a substrate. IKKβ (-) cells were obtained by using IKKP shRNA plasmid.The obtained results indicated that rhein inhibits LPS-induced NF-κB activation and sequentially suppresses its downstream inducible nitric oxide synthase (iNOS), IL-6, TNF-a and IL-1β transcriptions and supernatant NO and IL-6levels by inhibiting IKKP (IC50≈11.79μM). To verify whether the pro-and anti-inflammatory actions of rhein attribute to the IKKP inhibition and exclude the off-target effects by IKKβ inhibitors, we conditionally knocked down the IKKβ gene in RAW264.7cells using KKβ shRNA plasmid and obtained stable IKKP (-) cells. Its responses to LPS were similar to rhein-treated macrophages, suggesting that rhein exerted anti-and pro-inflammatory activities by virtue of targeting IKKβ inhibition, In the meantime, rhein enhances the activity of caspase-1through inhibiting intracellular (in situ) IKβ, in turn increasing the IL-1β and HMGB1release, which can be amplified by rhein’s reductive effect on intracellular superoxide anion (O2-). Unexpectedly, it is because of IKKβ inhibition that rhein significantly enhances TNF-a secretion and phagocytosis in macrophages with or without LPS. In summary, the above results indicate that rhein exerts anti-and pro-inflammatory activities by targeting IKKβ inhibition, providing a molecular mechanism for the unanticipated role of rhein in macrophages for the first time. Furthermore, our study also highlights the potential complications of IKKβ inhibitors (e.g. rhein and diacerein, etc.) application in inflammation disorders, for the overall effects of IKKβ inhibition in different organ systems and disease processes are not easily predictable under all circumstances. Considering diacerein having been widely used clinically and its apparent safety in patients, we conclude that direct inhibition IKKβ is a hitherto unrecognized property of rhein or diacerein, which may lead to the development of previously undescribed therapeutic repositioning strategies for the treatment of various human inflammatory diseases, as well as OA. Although IKKβ inhibitors are likely to be potent anti-inflammatory agents, the fact that IKKβ displays opposite functions in macrophages raises the question whether IKKP inhibitors are excellent anti-inflammatory agents in various tissues or different stages of inflammatory diseases. Answering the question would be good for the rational use of rhein or diacerein clinically. |
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