Activation of transcription factor HIF inhibits IL-1ß-induced NO production in primary cultured rat hepatocytes.

Roxadustat and other hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs) have recently been approved for the treatment of chronic renal anemia. In macrophages and monocytes, the activation of HIF-1 by pro-inflammatory cytokines induces iNOS expression and activity through the NF-κB pathway to produce nitric oxide (NO), which causes liver injury when excessively produced. Few studies have reported a relationship between HIF activity and iNOS induction in hepatocytes. We investigated the effect of drug- and hypoxia-induced HIF activations on NO production in primary cultured rat hepatocytes. Roxadustat treatment and hypoxic conditions activated HIF. Contrary to expectations, HIF-PHI treatment and hypoxia inhibited IL-1ß-induced NO production. RNA-Seq analysis of mRNA expression in rat hepatocytes showed that roxadustat treatment decreased the expression of genes related to inflammation, and genes in the NF-κB signaling pathway were induced by IL-1ß. Moreover, roxadustat suppressed IL-1ß-activated signaling pathways in an HIF-dependent manner. GalN/LPS-treated rats were used as in vivo models of hepatic injury, and roxadustat treatment showed a tendency to suppress the death of rats. Therefore, exogenous HIF-1 activation, including HIF-PHI and hypoxia exposures, suppressed IL-1ß-induced iNOS mRNA expression and subsequent NO production in hepatocytes, by suppressing the NF-κB signaling pathway. Roxadustat treatment suppresses the expression of pro-inflammatory genes by activating HIF, and thus may exhibit hepatoprotective effects.

Comparative Analysis of Anti-inflammatory Activity of the Constituents of the Rhizome of Cnidium officinale Using Rat Hepatocytes.

The rhizome of Cnidium officinale (Umbelliferae) (known as Senkyu in Japan; COR) has been used as a crude drug in Japanese Kampo formulas, such as Jumihaidokuto (to treat eczema and urticaria) and Kakkontokasenkyushin'i (to treat rhinitis). COR contains phthalides, which are thought to be potent principal constituents. Few studies have been reported about the comparison of anti-inflammatory activity of COR constituents. We aimed to identify the constituents in COR and compare their anti-inflammatory activity. COR was extracted with methanol and fractionated into ethyl acetate (EtOAc)-soluble, n-butanol-soluble, and water-soluble fractions. Primary cultured rat hepatocytes were used to assess anti-inflammatory activity by monitoring the interleukin (IL)-1ß-induced production of nitric oxide (NO), an inflammatory mediator. The EtOAc-soluble fraction significantly suppressed NO production without showing cytotoxicity in IL-1ß-treated hepatocytes, whereas the n-butanol-soluble fraction showed less potency, and the water-soluble fraction did not significantly affect the NO levels. Four constituents were isolated from the EtOAc-soluble fraction and identified as senkyunolide A, (3S)-butylphthalide, neocnidilide, and cnidilide. Among these phthalides and (Z)-ligustilide, senkyunolide A and (Z)-ligustilide efficiently suppressed NO production in hepatocytes, whereas the others showed less potency in the suppression of NO production. Furthermore, senkyunolide A decreased the levels of the inducible nitric oxide synthase (iNOS) protein and mRNA, as well as the levels of mRNAs encoding proinflammatory cytokines (e.g., tumor necrosis factor α) and chemokine C-C motif ligand 20. These results suggest that senkyunolide A may cause the anti-inflammatory and hepatoprotective effects of COR by suppressing the genes involved in inflammation.

Active Hexose Correlated Compound Has Protective Effects in Ischemia-Reperfusion Injury of the Rat Small Intestine.

BACKGROUND: Ischemia-reperfusion (IR) injury of the small intestine is a serious problem in abdominal aortic aneurysm surgery or small intestine transplantation. Active hexose correlated compound (AHCC) is a popular anti-inflammatory drug in complementary and alternative medicine. The aim of this study was to examine whether pretreatment with AHCC reduces intestinal IR injury. METHODS: Rats were given a normal diet (IR group) or normal diet supplemented with 2% AHCC (IR + AHCC group) ad libitum for 10 d. After 1 d of fasting, the superior mesenteric artery was occluded by clipping for 45 min. Intestinal and blood samples were collected for 1-6 h after reperfusion. The messenger RNA (mRNA) and protein levels of inflammatory factors were analyzed. RESULTS: The IR + AHCC group had reduced mucosal abrasion and significantly increased mucosal thickness of the intestinal tissues 6 h after reperfusion, compared with the IR group. AHCC decreased mRNA expression of inducible nitric oxide synthase (iNOS), cytokine-induced neutrophil chemoattractant 1 and interleukin 6 in the mucosa of the small intestine. AHCC also decreased expression of iNOS protein. Serum levels of cytokine-induced neutrophil chemoattractant 1 and tumor necrosis factor α were decreased in the IR + AHCC group compared with the IR group. Electrophoretic mobility shift assay of mucosal nuclear extracts revealed that AHCC inhibited the activation of nuclear factor kappa B. AHCC also inhibited the expression of iNOS antisense transcript, which stabilizes iNOS mRNA. CONCLUSIONS: Our findings suggest that AHCC reduces expression of inflammatory mediators, in part, by inhibiting nuclear factor kappa B activation. AHCC may have anti-inflammatory effect in patients with intestinal IR injury.

The Proton Pump Inhibitor Lansoprazole Has Hepatoprotective Effects in In Vitro and In Vivo Rat Models of Acute Liver Injury.

BACKGROUND/AIMS: The proton pump inhibitor lansoprazole (LPZ) is clinically used to reduce gastric acid secretion, but little is known about its possible hepatoprotective effects. This study aimed to investigate the hepatoprotective effects of LPZ and its potential mechanisms using in vitro and in vivo rat models of liver injury. METHODS: For the in vitro model of liver injury, primary cultured rat hepatocytes were treated with interleukin-1ß in the presence or absence of LPZ. The influence of LPZ on inducible nitric oxide synthase (iNOS) induction and nitric oxide (NO) production and on the associated signaling pathways was analyzed. For the in vivo model, rats were treated with D-galactosamine (GalN) and lipopolysaccharide (LPS). The effects of LPZ on survival and proinflammatory mediator expression (including iNOS and tumor necrosis factor-α) in these rats were examined. RESULTS: LPZ inhibited iNOS induction partially through suppression of the nuclear factor-kappa B signaling pathway in hepatocytes, thereby reducing potential liver injury from excessive NO levels. Additionally, LPZ increased survival by 50% and decreased iNOS, tumor necrosis factor-α, and cytokine-induced neutrophil chemoattractant-1 mRNA expression in the livers of GalN/LPS-treated rats. LPZ also inhibited nuclear factor-kappa B activation by GalN/LPS. CONCLUSIONS: LPZ inhibits the induction of several inflammatory mediators (including cytokines, chemokines, and NO) partially through suppression of nuclear factor-kappa B, resulting in the prevention of fulminant liver failure. The therapeutic potential of LPZ for liver injuries warrants further investigation.

Third-generation oncolytic herpes simplex virus inhibits the growth of liver tumors in mice.

Multimodality therapies are used to manage patients with hepatocellular carcinoma (HCC), although advanced HCC is incurable. Oncolytic virus therapy is probably the next major breakthrough in cancer treatment. The third-generation oncolytic herpes simplex virus type 1 (HSV-1) T-01 kills tumor cells without damaging the surrounding normal tissues. Here we investigated the antitumor effects of T-01 on HCC and the host's immune response to HCC cells. The cytopathic activities of T-01 were tested in 14 human and 1 murine hepatoma cell line in vitro. In various mouse xenograft models, HuH-7, KYN-2, PLC/PRF/5 and HepG2 human cells and Hepa1-6 murine cells were used to investigate the in vivo efficacy of T-01. T-01 was cytotoxic to 13 cell lines (in vitro). In mouse xenograft models of subcutaneous, orthotopic and peritoneal tumor metastasis in athymic mice (BALB/c nu/nu), the growth of tumors formed by the human HCC cell lines and hepatoblastoma cell line was inhibited by T-01 compared with that of mock-inoculated tumors. In a bilateral Hepa1-6 subcutaneous tumor model in C57BL/6 mice, the growth of tumors inoculated with T-01 was inhibited, as was the case for contralateral tumors. T-01 also significantly reduced tumor growth. T-01 infection significantly enhanced antitumor efficacy via T cell-mediated immune responses. Results demonstrate that a third-generation oncolytic HSV-1 may serve as a novel treatment for patients with HCC.

A sense oligonucleotide to inducible nitric oxide synthase mRNA increases the survival rate of rats in septic shock.

Natural antisense transcripts (asRNAs) that do not encode proteins are transcribed from rat, mouse, and human genes, encoding inducible nitric oxide synthase (iNOS), which catalyzes the production of the inflammatory mediator nitric oxide (NO). In septic shock, NO is excessively produced in hepatocytes and macrophages. The iNOS asRNA interacts with and stabilizes iNOS mRNA. We found that single-stranded 'sense' oligonucleotides corresponding to the iNOS mRNA sequence reduced iNOS mRNA levels by interfering with the mRNA-asRNA interactions in rat hepatocytes. The iNOS sense oligonucleotides that were substituted with phosphorothioate bonds and locked nucleic acids efficiently decreased the levels of iNOS mRNA and iNOS protein. In this study, the gene expression patterns in the livers of two endotoxemia model rats with acute liver failure were compared. Next, we optimized the sequence and modification of the iNOS sense oligonucleotides in interleukin 1ß-treated rat hepatocytes. When a sense oligonucleotide was simultaneously administered with d-galactosamine and bacterial lipopolysaccharide (LPS) to rats, their survival rate significantly increased compared to the rats administered d-galactosamine and LPS alone. In the livers of the sense oligonucleotide-administered rats, apoptosis in the hepatocytes markedly decreased. These results suggest that natural antisense transcript-targeted regulation technology using iNOS sense oligonucleotides may be used to treat human inflammatory diseases, such as sepsis and septic shock.

pyroGlu-Leu inhibits the induction of inducible nitric oxide synthase in interleukin-1ß-stimulated primary cultured rat hepatocytes.

Pyroglutamyl leucine (pyroGlu-Leu), which is a peptide isolated from wheat gluten hydrolysate, has been reported to be a hepatoprotective compound in acute liver failure. In inflamed liver, proinflammatory cytokines including interleukin (IL)-1ß and tumor necrosis factor (TNF)-α stimulate the induction of inducible nitric oxide synthase (iNOS). Excess production of nitric oxide (NO) by iNOS is an inflammatory biomarker in liver injury. We examined proinflammatory cytokine-stimulated hepatocytes as a simple "in vitro inflammation model" to determine liver protective effects of pyroGlu-Leu and its mechanisms of action. We hypothesized that pyroGlu-Leu inhibits the induction of iNOS gene expression, resulting in the attenuation of hepatic inflammation. Hepatocytes were isolated from rats by collagenase perfusion and cultured. Primary cultured cells were treated with IL-1ß in the presence or absence of pyroGlu-Leu. The induction of iNOS and its signaling pathway were analyzed. IL-1ß stimulated the enhancement of NO production in hepatocytes and this effect was inhibited by pyroGlu-Leu. pyroGlu-Leu decreased the expression of iNOS protein and its mRNA. Transfection experiments with iNOS-luciferase constructs revealed that pyroGlu-Leu inhibited both of iNOS promoter transactivation and its mRNA stabilization. pyroGlu-Leu also decreased the expression of an iNOS gene antisense transcript, which is involved in iNOS mRNA stability. However, pyroGlu-Leu had no effects on IκB degradation and NF-κB activation. Results demonstrate that pyroGlu-Leu inhibited the induction of iNOS gene expression at transcriptional and post-transcriptional steps through IκB/NF-κB-independent pathway, leading to the prevention of NO production. pyroGlu-Leu may have therapeutic potential for liver injury through the suppression of iNOS.

Alpha-lipoic acid exerts a liver-protective effect in acute liver injury rats.

BACKGROUND: Recent evidence indicates that alpha-lipoic acid (α-LA) has a variety of liver-protective effects through the suppression of inflammatory mediators including tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS). However, there are few reports that α-LA markedly enhanced the survival rate in animal models of liver injury with more than 90% death. The aim of this study was to investigate the beneficial effects of α-LA in a rat model of acute liver injury and to clarify the mechanisms of α-LA action. METHODS: Rats were treated with d-galactosamine and lipopolysaccharide (GalN and LPS) to induce acute liver injury. α-LA (100 mg/kg) was administered intraperitoneally 1 h before GalN and LPS injection. Inflammatory mediators including TNF-α and iNOS were analyzed. RESULTS: A single injection of α-LA improved the survival rate by more than 80%. α-LA prevented serum transaminase increases, histopathologic changes, and apoptosis in the liver. In the serum, α-LA decreased TNF-α production and increased interleukin (IL)-10 production. In the liver, α-LA reduced TNF-α and IL-6 messenger RNA (mRNA) but enhanced IL-10 mRNA. α-LA decreased the expression of iNOS mRNA and its antisense transcript, leading to the reduction of iNOS protein expression and resulting in the inhibition of nitric oxide production. An electrophoretic mobility shift assay revealed that α-LA reduced the activation of nuclear factor-kappa B induced by GalN and LPS. CONCLUSIONS: α-LA inhibited the induction of inflammatory mediators, such as TNF-α and iNOS, in part through the inhibition of nuclear factor-kappa B activation and enhanced the induction of IL-10. α-LA may have therapeutic potential for use in the prevention of acute liver injury.

Influence of Rictor and Raptor Expression of mTOR Signaling on Long-Term Outcomes of Patients with Hepatocellular Carcinoma.

BACKGROUND: Aberrant signaling mediated by the mammalian target of rapamycin (mTOR) occurs at high frequency in hepatocellular carcinoma (HCC), indicating that mTOR is a candidate for targeted therapy. mTOR forms two complexes called mTORC1 (mTOR complexed with raptor) and mTORC2 (mTOR complexed with rictor). There are minor studies of the expression kinetics of mTORC1 and mTORC2 in HCC. METHODS: We studied 62 patients with HCC who underwent curative resection. We used univariate and multivariate analyses to identify factors that potentially influence disease and overall survival after hepatectomy. The mRNA and protein levels of mTOR, rictor and raptor in cancer and non-cancer tissues were analyzed using quantitative RT-PCR, immunohistochemistry and Western blotting. RESULTS/CONCLUSION: High ratio of the levels of rictor and raptor mRNAs in tumors was identified as independent prognostic indicators for disease-free survival. Low and high levels of preoperative serum albumin and mTOR mRNA in the tumor, respectively, were identified as independent indicators of overall survival. HCC is likely to recur early after hepatic resection in patients with high levels of mTOR and rictor mRNAs and high rictor/raptor ratios in cancer tissues. We conclude that analysis of mTOR expression in cancer tissues represents an essential strategy to predict HCC recurrence after curative treatment.

A new flavanone and other flavonoids from green perilla leaf extract inhibit nitric oxide production in interleukin 1ß-treated hepatocytes.

A new flavanone, shisoflavanone A (1), and several flavonoids were purified from the ethyl acetate-soluble fraction of green perilla leaves (Perilla frutescens Britton var. crispa form viridis), and their structures were identified. Shisoflavanone A was elucidated as 8-hydroxy-6,7-dimethoxyflavanone based on its spectral data. Other constituents of the ethyl acetate-soluble fraction, i.e. 5,8-dihydroxy-7-methoxyflavanone (2), negletein (5,6-dihydroxy-7-methoxyflavone) (3), luteolin (4), apigenin (5), esculetin (6), and protocatechuic acid (7), were identified. This is the first time that constituents 2, 3, and 6 have been found in green perilla. Shisoflavanone A and the other constituents (except 7) significantly inhibited nitric oxide production in interleukin 1ß-stimulated rat hepatocytes, which have been used to monitor the anti-inflammatory effects of herbal constituents. The present findings suggest that these constituents, including shisoflavanone A, may be involved in the anti-inflammatory effects of green perilla leaves.

Adenosine, a hepato-protective component in active hexose correlated compound: its identification and iNOS suppression mechanism.

Supplementation of active hexose correlated compound (AHCC) improved the prognosis of postoperative hepatocellular carcinoma patients. Excess production of nitric oxide (NO) by inducible NO synthase (iNOS) is an inflammatory biomarker in liver injury. AHCC suppressed iNOS induction in hepatocytes, suggesting that AHCC has a potential liver-protective effect. However, the active component in AHCC responsible for NO suppressive activities has not been identified. The objective of this study was to identify this NO suppressive component and to investigate its mechanisms of action. AHCC was subjected to fractionation by cation exchanger, size exclusion chromatography, and normal- and reversed-phase HPLC. Aliquots of the fractions were added to primary cultured rat hepatocytes stimulated with interleukin (IL)-1ß, and NO production was assayed. By activity-guided fractionation and electron spray ionization mass spectrometry analysis, adenosine was identified as one of the NO suppressive components in AHCC. Adenosine inhibited NO production, and reduced the expression of iNOS protein and mRNA. It had no effects on IκB degradation, but it inhibited NF-κB activation. Adenosine also inhibited the upregulation of type I IL-1 receptor (IL-1RI). Experiments with iNOS promoter-luciferase constructs revealed that adenosine decreased the levels of iNOS mRNA at the promoter transactivation and mRNA stabilization steps. Adenosine decreased the expression of the iNOS gene antisense transcript, which is involved in iNOS mRNA stability. Adenosine in AHCC suppressed iNOS induction by blocking NF-κB activation and the upregulation of the IL-1RI pathways, resulting in the inhibition of NO production.

Interleukin-1ß induces tumor necrosis factor-α secretion from rat hepatocytes.

AIM: 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). METHODS: Primary cultured rat hepatocytes were used to analyze TNF-α expression in response to the pro-inflammatory cytokine, interleukin-1ß (IL-1ß). Livers of rats subjected to LPS-induced endotoxemia were analyzed. RESULTS: Immunocytochemistry and enzyme-linked immunosorbent assays demonstrated that IL-1ß-treated rat hepatocytes secreted TNF-α, and RNA analyses indicated that TNF-α mRNA was induced specifically by IL-1ß. Northern blot analysis showed that not only mRNA, but also a natural antisense transcript (asRNA), was transcribed from the rat Tnf gene in IL-1ß-treated hepatocytes. TNF-α was detected in the hepatocytes of LPS-treated rats. Both TNF-α mRNA and asRNA were expressed in the hepatocytes of LPS-treated rats, human hepatocellular carcinoma and human monocyte/macrophage cells. To disrupt the interaction between TNF-α asRNA and TNF-α mRNA, sense oligonucleotides corresponding to TNF-α mRNA were introduced into rat hepatocytes resulting in significantly increased levels of TNF-α mRNA. One of these sense oligonucleotides increased a half-life of TNF-α mRNA, suggesting that the TNF-α asRNA may reduce the stability of TNF-α mRNA. CONCLUSION: IL-1ß-stimulated rat hepatocytes are a newly identified source of TNF-α in the liver. TNF-α mRNA and asRNA are expressed in rats and humans, and the TNF-α asRNA reduces the stability of the TNF-α mRNA. Hepatocytes and TNF-α asRNA may be therapeutic targets to regulate levels of TNF-α mRNA.

Bone marrow cells enhance liver regeneration after massive hepatectomy in mice.

BACKGROUND: Recent evidence indicates that transplanted autologous bone marrow cells (BMCs) can be converted into functional liver cells. BMC therapy can improve hepatic function and increase the potential for liver regeneration in patients with serious liver damage. We investigated whether BMC therapy influenced liver regeneration after massive hepatectomy in mice. METHODS: Male C57/BL6 mice underwent 70 % hepatectomy, followed by injection of BMCs via the portal vein (PV group), BMCs via the tail vein (IV group), or saline via the portal vein (control group). Analysis of serum enzyme levels and liver histology was performed on postoperative days (POD) 1, 3, and 5. RESULTS: Compared with the control group, the rate of liver regeneration on POD 3 and 5 was significantly higher in the PV group, but not in the IV group. Examination of the mitotic index and Ki-67 labeling index revealed that the increased liver regeneration resulted from stimulation of DNA synthesis. On POD 3, the serum levels of interleukin (IL)-6 and hepatocyte growth factor (HGF) were significantly higher and the expression of IL-6 and HGF mRNA in the remnant liver tended to be higher in the PV group than in the control group. Histological examination showed BMCs in the liver of the PV group, as well as conversion of BMCs into liver cells. CONCLUSIONS: Our findings indicate that the injection of BMCs via the portal vein, but not the injection of BMCs via the tail, enhances liver regeneration after massive hepatectomy in mice.

Temporal and spatial dependence of inflammatory biomarkers and suppression by fluvastatin in dextran sodium sulfate-induced rat colitis model.

BACKGROUND: Dextran sodium sulfate (DSS)-induced colitis in rats is widely used as an experimental model for elucidating the etiology of ulcerative colitis (UC) and developing its novel remedy. We investigated the temporal and spatial changes in inflammatory mediators such as tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) in the regions of rectum and distal colon and examined whether statins, which were designed to lower plasma cholesterol levels, influenced those mediators. METHODS: Colitis was induced in rats by oral administration of 5 % DSS for 5 days, followed by 2 % DSS for 10 days. 5 % DSS rats were treated with fluvastatin (20 mg/kg) concomitantly for 5 days. The expression of inflammatory mediators of a sequence of four regions in rectum (R) and distal colon (D0, D1, and D2) was determined by quantitative RT-PCR. RESULTS: The peak of colitic damage, which was confirmed clinically and histopathologically, was found on days 4-6. The expression of TNF-α, iNOS, cytokine-induced neutrophil chemoattractant-1, interleukin (IL)-1ß, and IL-6 mRNA increased in R time dependently, showing the peak on days 4-6, and then decreased thereafter. The levels of mRNAs reduced from R to D0, D1, and D2 region dependently. Fluvastatin decreased the expression of these markers in addition to the prevention of DSS-induced damage. CONCLUSIONS: Results demonstrated that the expression of inflammatory biomarkers had time and region specificity and was markedly inhibited by fluvastatin. To obtain a precise drug effect for UC, it is important to elucidate the temporal and spatial dependence of inflammatory biomarkers in DSS colitis model.

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.

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

BACKGROUND: Nobiletin 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. METHODS: A C. unshiu peel (CUP) extract was prepared. Primary cultured rat hepatocytes were treated with the CUP extract or nobiletin in the presence of interleukin 1ß (IL-1ß), which induces iNOS expression. NO production and iNOS gene expression were analyzed. RESULTS: High-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. CONCLUSIONS: The 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.

Gomisin N in the herbal drug gomishi (Schisandra chinensis) suppresses inducible nitric oxide synthase gene via C/EBPß 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 1ß in primary cultured rat hepatocytes. The hydrophobic fraction markedly suppressed NO induction and reduced the expression of inducible nitric oxide syntheses (iNOS) in interleukin 1ß-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 1ß and inflammatory chemokines. The overexpression of the p65 subunit of nuclear factor κB or CCAAT/enhancer-binding protein ß 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.

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 (3'UTR) 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 3'UTR. 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 3'UTR. 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.

Fluvastatin inhibits the induction of inducible nitric oxide synthase, an inflammatory biomarker, in hepatocytes.

AIM: Statins (3-hydroxy-3-methylglutaryl coenzyme A [HMG-CoA] reductase inhibitors), which were originally designed to lower plasma cholesterol levels, are increasingly recognized as anti-inflammatory agents. In the inflamed liver, pro-inflammatory cytokines stimulate the induction of inducible nitric oxide synthase (iNOS). Overproduction of NO by iNOS has been implicated as a factor in liver injury. We examined pro-inflammatory cytokine-stimulated hepatocytes as a simple in vitro injury model to determine liver-protective effects of statins. We hypothesized that statins are involved in the downregulation of iNOS, resulting in decreased hepatic inflammation. METHODS: Hepatocytes were isolated from rats by collagenase perfusion and centrifugation. Primary cultured hepatocytes were treated with interleukin (IL)-1ß in the presence or absence of fluvastatin. The induction of iNOS and its signaling pathway were analyzed. RESULTS: IL-1ß produced increased levels of NO. This effect was inhibited by fluvastatin, which exerted its maximal effects at 100 µM. Fluvastatin decreased the levels of iNOS protein and its mRNA expression. Fluvastatin had no effects on IκB degradation and nuclear factor-κB activation. However, fluvastatin inhibited the upregulation of type I IL-1 receptor mRNA and protein expression. Transfection experiments demonstrated that fluvastatin suppressed iNOS induction by the inhibition of promoter transactivation and mRNA stabilization. Fluvastatin reduced the expression of an iNOS gene antisense-transcript, which is involved in iNOS mRNA stability. CONCLUSION: Results indicate that fluvastatin inhibits the induction of iNOS at both transcriptional and post-transcriptional steps, leading to the prevention of NO production. Fluvastatin may provide therapeutic potential in iNOS induction involved in various liver injuries.

alpha-lipoic acid prevents the induction of iNOS gene expression through destabilization of its mRNA in proinflammatory cytokine-stimulated hepatocytes.

BACKGROUND/AIMS: α-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. METHODS: Primary cultured rat hepatocytes were treated with interleukin (IL)-1ß in the presence or absence of α-LA. The induction of iNOS and NO production and its signal were analyzed. RESULTS: α-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-1ß, 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. CONCLUSIONS: Results 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.