Tadayoshi Okumura

Gomisin N in the herbal drug gomishi (Schisandra chinensis) suppresses inducible nitric oxide synthase gene via C/EBPβ and NF-κB in rat hepatocytes

Gomishi is the dried fruit of Schisandra chinensis Baillon (Fructus Schisandrae chinensis, FSC) and has been used in Japanese Kampo medicine to treat inflammatory and liver diseases. However, it is unclear which constituent of FSC is primarily responsible for its pharmacological effects. FSC was extracted with methanol, fractionated by hydrophobicity, and further purified. We measured the effects of each fraction or constituent thereof on the induction of the inflammatory mediator nitric oxide (NO), which was induced by interleukin 1β in primary cultured rat hepatocytes. The hydrophobic fraction markedly suppressed NO induction and reduced the expression of inducible nitric oxide syntheses (iNOS) in interleukin 1β-treated hepatocytes. Gomisin N and γ-schizandrin, two major constituents of the hydrophobic fraction, significantly reduced NO production and the levels of the iNOS protein, mRNA, and antisense transcript. Gomisin N and γ-schizandrin also decreased the transcription of interleukin 1β and inflammatory chemokines. The overexpression of the p65 subunit of nuclear factor κB or CCAAT/enhancer-binding protein β increased the promoter activity of the iNOS gene in the firefly luciferase assay, whereas gomisin N decreased the promoter activity. The anti-inflammatory activity of FSC and its constituents were analysed, and we demonstrated that gomisin N and γ-schizandrin are involved in the hepatoprotective effect of the FSC extract, which has therapeutic potential for liver disease.

Characterization of natural antisense transcripts expressed from interleukin 1β-inducible genes in rat hepatocytes

Abstract nBackground: Natural antisense transcripts (asRNAs) are transcribed from many genes in various species. Recently, we found that asRNAs were transcribed from the rat and mouse genes encoding inducible nitric

The Anti-Inflammatory Effects of Flavanol-Rich Lychee Fruit Extract in Rat Hepatocytes

Flavanol (flavan-3-ol)-rich lychee fruit extract (FRLFE) is a mixture of oligomerized polyphenols primarily derived from lychee fruit and is rich in flavanol monomers, dimers, and trimers. Supplementation with this functional food has been shown to suppress inflammation and tissue damage caused by high-intensity exercise training. However, it is unclear whether FRLFE has in vitro anti-inflammatory effects, such as suppressing the production of the proinflammatory cytokine tumor necrosis factor α (TNF-α) and the proinflammatory mediator nitric oxide (NO), which is synthesized by inducible nitric oxide synthase (iNOS). Here, we analyzed the effects of FRLFE and its constituents on the expression of inflammatory genes in interleukin 1β (IL-1β)-treated rat hepatocytes. FRLFE decreased the mRNA and protein expression of the iNOS gene, leading to the suppression of IL-1β-induced NO production. FRLFE also decreased the levels of the iNOS antisense transcript, which stabilizes iNOS mRNA. By contrast, unprocessed lychee fruit extract, which is rich in flavanol polymers, and flavanol monomers had little effect on NO production. When a construct harboring the iNOS promoter fused to the firefly luciferase gene was used, FRLFE decreased the luciferase activity in the presence of IL-1β, suggesting that FRLFE suppresses the promoter activity of the iNOS gene at the transcriptional level. Electrophoretic mobility shift assays indicated that FRLFE reduced the nuclear transport of a key regulator, nuclear factor κB (NF-κB). Furthermore, FRLFE inhibited the phosphorylation of NF-κB inhibitor α (IκB-α). FRLFE also reduced the mRNA levels of NF-κB target genes encoding cytokines and chemokines, such as TNF-α. Therefore, FRLFE inhibited NF-κB activation and nuclear translocation to suppress the expression of these inflammatory genes. Our results suggest that flavanols may be responsible for the anti-inflammatory and hepatoprotective effects of FRLFE and may be used to treat inflammatory diseases.

Citrus nobiletin suppresses inducible nitric oxide synthase gene expression in interleukin-1β-treated hepatocytes

BACKGROUNDnNobiletin is a polymethoxylated flavone that is abundant in the peels of citrus fruits, such as Citrus unshiu (Satsuma mandarin) and Citrus sinensis. The dried peels of C. unshiu (chinpi) have been included in several formulae of Japanese Kampo medicines. Nobiletin may suppress the induction of inducible nitric oxide synthase (iNOS), which synthesizes the inflammatory mediator nitric oxide (NO) in hepatocytes.nnnMETHODSnA C. unshiu peel (CUP) extract was prepared. Primary cultured rat hepatocytes were treated with the CUP extract or nobiletin in the presence of interleukin 1β (IL-1β), which induces iNOS expression. NO production and iNOS gene expression were analyzed.nnnRESULTSnHigh-performance liquid chromatography analyses revealed that the nobiletin content in the CUP extract was 0.14%. Nobiletin dose-dependently reduced the NO levels and decreased iNOS expression at the protein, mRNA and antisense transcript levels. Flavone, which does not contain any methoxy groups, also suppressed iNOS induction. Nobiletin reduced the transcriptional activity of iNOS promoter-luciferase constructs and the DNA-binding activity of nuclear factor κB (NF-κB) in the nuclei.nnnCONCLUSIONSnThe suppression of iNOS induction by nobiletin suggests that nobiletin may be responsible for the anti-inflammatory effects of citrus peels and have a therapeutic potential for liver diseases.

Supplementation with a flavanol-rich lychee fruit extract influences the inflammatory status of young athletes.

Flavanol‐rich lychee fruit extract (FRLFE) is a processed lychee fruit extract that is higher in flavanols (monomers, dimers and trimers) than its unprocessed counterpart. FRLFE exerts antioxidant activities in vitro and is expected to protect against inflammation and tissue damage. However, the physiological effects of FRLFE intake have not been explored in vivo. The aim of this study was to examine the effects of FRLFE supplementation on inflammation and tissue damage in young athletes during intense physical training. Twenty healthy male long‐distance runners at a university were randomly assigned to receive FRLFE or placebo in a double‐blind manner. Blood and serum parameters associated with inflammation, tissue damage and oxidative stress were evaluated before (pre‐training), during (mid‐training) and after (post‐training) a 2‐month training period. Some parameters, including the white blood cell count, were significantly modified by FRLFE supplementation. Compared with the placebo group, the change in the serum interleukin‐6 level between pre‐ and mid‐training were significantly lower in the FRLFE group, while the change in the transforming growth factor‐β level between pre‐ and post‐training was significantly greater in the FRLFE group. These findings suggest that FRLFE supplementation may suppress inflammation or tissue damage caused by high‐intensity exercise training. Copyright

α-Lipoic Acid Prevents the Induction of iNOS Gene Expression Through Destabilization of Its mRNA in Proinflammatory Cytokine-Stimulated Hepatocytes

Background/Aimsα-Lipoic acid (α-LA) has been reported to reduce ischemia–reperfusion injury (IRI). Proinflammatory cytokines stimulate the induction of inducible nitric oxide synthase (iNOS) gene expression, leading to excess production of NO and resulting in liver injury including IRI. We hypothesized that inhibition of iNOS induction underlies the protective effects of α-LA on the liver. The objective was to investigate whether α-LA directly influences iNOS induction in cultured hepatocytes, which is used as a simple in vitro injury model, and the mechanism involved.MethodsPrimary cultured rat hepatocytes were treated with interleukin (IL)-1β in the presence or absence of α-LA. The induction of iNOS and NO production and its signal were analyzed.Resultsα-LA inhibited the expression of iNOS mRNA and protein dose- and time-dependently, resulting in decreases in NO production. α-LA had no effects on the degradation of IκB proteins and activation of NF-κB. In contrast, α-LA inhibited the upregulation of type I IL-1 receptor stimulated by IL-1β, although α-LA had no effect on Akt activation. Transfection experiments with iNOS promoter-luciferase constructs revealed that α-LA had no effect on the transactivation of the iNOS promoter, but decreased the stabilization of iNOS mRNA. Further, α-LA inhibited the expression of an iNOS gene antisense-transcript, which is involved in iNOS mRNA stability.ConclusionsResults indicate that α-LA inhibits the induction of iNOS gene expression at a posttranscriptional step via iNOS mRNA stabilization, rather than promoter activation. It may provide useful therapeutic effects through the suppression of iNOS induction involved in liver injury.

Interleukin‐1β induces tumor necrosis factor‐α secretion from rat hepatocytes

Tumor necrosis factor‐α (TNF‐α) is a pleiotropic cytokine involved in various inflammatory diseases. The only production of TNF‐α in the liver is thought to be from hepatic macrophages known as Kupffer cells, predominantly in response to bacterial lipopolysaccharide (LPS).

Natural antisense transcript-targeted regulation of inducible nitric oxide synthase mRNA levels.

Natural antisense transcripts (asRNAs) are frequently transcribed from mammalian genes. Recently, we found that non-coding asRNAs are transcribed from the 3 untranslated region (3UTR) of the rat and mouse genes encoding inducible nitric oxide synthase (iNOS), which catalyzes the production of the inflammatory mediator nitric oxide. The iNOS asRNA stabilizes iNOS mRNA by interacting with the mRNA 3UTR. Furthermore, single-stranded sense oligonucleotides corresponding to the iNOS mRNA sequence were found to reduce iNOS mRNA levels by interfering with mRNA-asRNA interactions in rat hepatocytes. This method was named natural antisense transcript-targeted regulation (NATRE) technology. In this study, we detected human iNOS asRNA expressed in hepatocarcinoma and colon carcinoma tissues. The human iNOS asRNA harbored a sequence complementary to an evolutionarily conserved region of the iNOS mRNA 3UTR. When introduced into hepatocytes, iNOS sense oligonucleotides that were modified by substitution with partial phosphorothioate bonds and locked nucleic acids or 2-O-methyl nucleic acids greatly reduced levels of iNOS mRNA and iNOS protein. Moreover, sense oligonucleotides and short interfering RNAs decreased iNOS mRNA to comparable levels. These results suggest that NATRE technology using iNOS sense oligonucleotides could potentially be used to treat human inflammatory diseases and cancers by reducing iNOS mRNA levels.

Stabilization of human interferon-α1 mRNA by its antisense RNA.

Antisense transcription is a widespread phenomenon in the mammalian genome and is believed to play a role in regulating gene expression. However, the exact functional significance of antisense transcription is largely unknown. Here, we show that natural antisense (AS) RNA is an important modulator of interferon-α1 (IFN-α1) mRNA levels. A ~4-kb, spliced IFN-α1 AS RNA targets a single-stranded region within a conserved secondary structure element of the IFN-α1 mRNA, an element which was previously reported to function as the nuclear export element. Following infection of human Namalwa lymphocytes with Sendai virus or infection of guinea pig 104C1 fetal fibroblasts with influenza virus A/PR/8/34, expression of IFN-α1 AS RNA becomes elevated. This elevated expression results in increased IFN-α1 mRNA stability because of the cytoplasmic (but not nuclear) interaction of the AS RNA with the mRNA at the single-stranded region. This results in increased IFN-α protein production. The silencing of IFN-α1 AS RNA by sense oligonucleotides or over-expression of antisense oligoribonucleotides, which were both designed from the target region, confirmed the critical role of the AS RNA in the post-transcriptional regulation of IFN-α1 mRNA levels. This AS RNA stabilization effect is caused by the prevention of the microRNA (miRNA)-induced destabilization of IFN-α1 mRNA due to masking of the miR-1270 binding site. This discovery not only reveals a regulatory pathway for controlling IFN-α1 gene expression during the host innate immune response against virus infection but also suggests a reason for the large number of overlapping complementary transcripts with previously unknown function.

Japanese herbal medicine, inchinkoto, inhibits inducible nitric oxide synthase induction in interleukin-1β-stimulated hepatocytes.

Aim:u2002 A herbal medicine, kampo inchinkoto (TJ‐135), is used to treat jaundice and liver fibrosis in patients with cirrhosis. In the inflamed liver, proinflammatory cytokines stimulate the induction of inducible nitric oxide synthase (iNOS) gene expression. Over‐production of nitric oxide (NO) by iNOS has been implicated as a factor in liver injury. We examined interleukin (IL)‐1β‐stimulated hepatocytes as a simple in vitro injury model to determine liver‐protective effects of TJ‐135. The objective was to investigate whether TJ‐135 influences iNOS induction and to determine its mechanism.