Sakuranetin downregulates inducible nitric oxide synthase expression by affecting interleukin-1 receptor and CCAAT/enhancer-binding protein β

Pruni Cortex is a herbal drug from the bark of the Japanese flowering cherries, Prunus jamasakura or Prunus verecunda , and is included in the traditional Japanese herbal (Kampo) formula Jumihaidokuto , which is administered orally to patients suffering from inflammatory skin diseases. The flavanones contained in Pruni Cortex (e.g., sakuranetin and naringenin) have potent anti-inflammatory, anti-allergic, and anti-microbial activities. Although the effects of Pruni Cortex on skin disease have been well studied, reports regarding its pharmacological effects on the liver are limited. In this study, we extracted the bark of Prunus jamasakura and purified it to isolate the pharmacologically active constituents by monitoring nitric oxide (NO) production in rat hepatocytes that were treated with the pro-inflammatory cytokine, interleukin (IL)-1β. Sakuranetin and (−)-naringenin, which were present in an ethyl acetate-soluble fraction of the bark extract, significantly inhibited NO induction and inducible nitric oxide synthase (iNOS) expression. These two flavanones decreased the expression of type 1 IL-1 receptor gene and phosphorylation of Akt, also known as protein kinase B, which is regulated by phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). Furthermore, sakuranetin decreased the phosphorylation of the activator isoforms of CCAAT/enhancer-binding protein β (C/EBPβ), which synergistically activates the transcription of the iNOS gene with nuclear factor κB (NF-κB). Therefore, sakuranetin inhibited the co-activating activity of C/EBPβ with NF-κB, leading to the suppression of iNOS gene expression in hepatocytes. Taken together, sakuranetin in Pruni Cortex downregulated the iNOS gene by inhibiting PI3K/Akt signal transduction and the phosphorylation of C/EBPβ. These results imply that sakuranetin may be primarily responsible for the anti-inflammatory effects of Pruni Cortex in the liver.