Takashi Ozaki

Natural antisense transcript stabilizes inducible nitric oxide synthase messenger RNA in rat hepatocytes

During inflammation, inducible nitric oxide synthase (iNOS) is induced to generate the important mediator nitric oxide (NO). Interleukin 1β (IL‐1β) induces iNOS messenger RNA (mRNA), iNOS protein, and NO in rat hepatocytes. We found that the stability of iNOS mRNA changed during the induction and that the antisense (AS) strand corresponding to the 3′‐untranslated region (3′UTR) of iNOS mRNA was transcribed from the iNOS gene. Expression levels of the iNOS AS transcript correlated with those of iNOS mRNA. The 1.5‐kilobase region 3′‐flanking to iNOS gene exon 27 was involved in IL‐1β induction. Knockdown experiments suggest that sense oligonucleotides to iNOS mRNA significantly reduced iNOS mRNA levels in the hepatocytes by blocking the interaction between iNOS mRNA and the AS transcript. Overexpression of iNOS AS transcript stabilized the reporter luciferase mRNA through the fused iNOS mRNA 3′UTR. These results together with the data in a yeast RNA‐hybrid assay suggested that the iNOS AS transcript interacted with iNOS mRNA and stabilized iNOS mRNA. The iNOS mRNA colocalized with the AU‐rich element‐binding protein HuR, a human homolog of embryonic lethal‐abnormal visual protein, and heterogeneous nuclear ribonucleoprotein L (hnRNP L) in the cytoplasm of rat hepatocytes. Interaction assays further revealed that the iNOS AS transcript interacted with HuR, which interacted with hnRNP L, suggesting that iNOS mRNA, the AS transcript, and the RNA‐binding proteins may mutually interact. Conclusion: The natural AS transcript of the iNOS gene interacts with iNOS mRNA and may play an important role in the stability of iNOS mRNA. This RNA‐RNA interaction may be a new therapeutic target for NO‐mediating inflammatory diseases. (HEPATOLOGY 2008.)

Dexamethasone inhibits the induction of iNOS gene expression through destabilization of its mRNA in proinflammatory cytokine-stimulated hepatocytes.

In the inflamed liver, proinflammatory cytokines including TNF-&agr;, IL-1&bgr;, and IFN-&ggr; stimulate the induction of iNOS gene expression, leading to excess production of NO and resulting in liver injury. The induction of iNOS is regulated by transactivation of the iNOS promoter with transcription factors such as nuclear factor &kgr;B and by posttranscriptional modifications such as mRNA stabilization. The synthetic glucocorticoid dexamethasone has been reported to inhibit iNOS induction, which may contribute to its inflammation-reducing effects. The objective was to investigate the mechanisms involved in the down-regulation of iNOS gene expression by dexamethasone. Primary cultured rat hepatocytes were treated with IL-1&bgr; (1 nM) in the presence or absence of dexamethasone. The induction of iNOS and its signal were analyzed. Dexamethasone (10-250 nM) inhibited the expression of iNOS mRNA and protein dose and time dependently, resulting in decreases in NO production. However, dexamethasone did not inhibit the up-regulation of type I IL-1 receptor stimulated by IL-1&bgr;. Dexamethasone also had no effect on the degradation of I&kgr;B proteins and on the activation of nuclear factor &kgr;B. Transfection experiments with iNOS promoter-luciferase constructs revealed that dexamethasone had no effect on the transactivation of the iNOS promoter but decreased the stabilization of iNOS mRNA. In support of the latter observation, dexamethasone inhibited the expression of an iNOS gene antisense transcript, which stabilizes iNOS mRNA by interacting with its 3&vprime;-untranslated region and 3&vprime;-untranslated region-binding proteins. Dexamethasone may inhibit the induction of iNOS gene expression at the step of mRNA stabilization rather than promoter activation and may provide useful therapeutic effects in iNOS induction involved in liver injuries.ABBREVIATIONS-iNOS-inducible nitric oxide synthase; IL-1&bgr;-interleukin 1&bgr;; NF-&kgr;B-nuclear factor &kgr;B; IL-1RI-type I interleukin 1 receptor; 3&vprime;-UTR-3&vprime;-untranslated region

Protective effect of FR183998, a Na+/H+ exchanger inhibitor, and its inhibition of iNOS induction in hepatic ischemia-reperfusion injury in rats.

Recent evidence indicates that inhibition of the Na+/H+ exchanger improves heart and brain injuries induced by I/R. Studies were performed to investigate whether FR183998, a Na+/H+ exchanger inhibitor, has protective effects on hepatic I/R injury in rats. Male Sprague-Dawley rats were subjected to 70% hepatic ischemia by occluding the hepatic artery, portal vein, and bile duct associated with the left and median liver lobes with a microvascular clip for 2 h. FR183998 (1 mg/kg) was administered i.v. 10 min before the hepatic ischemia. Hepatic I/R increased the serum levels of aspartate transaminase, alanine transaminase, and lactate dehydrogenase, which peaked at 9 h after reperfusion. FR183998 reduced these injury markers and recovered liver functions. Histopathologic analysis revealed that FR183998 prevented the incidences of hepatic necrosis, apoptosis, and neutrophil infiltration at 6 and 9 h (P < 0.05). FR183998 reduced the increases in proinflammatory cytokines such as TNF-&agr; (1 - 6 h), IL-6 (1 - 12 h), interferon-&ggr; (6 - 12 h), IL-1&bgr; (1 - 3 h), and cytokine-induced neutrophil chemoattractant 1 (1-3 h), but enhanced the anti-inflammatory cytokine IL-10 (1 h). FR183998 inhibited the hepatic I/R-induced activation of the transcription factor nuclear factor-&kgr;B at 1 to 6 h and reduced the induction of iNOS at 6 to 12 h, followed by inhibition of nitric oxide production. Furthermore, FR183998 decreased the expression of the iNOS gene antisense transcript, which is involved in the stability of iNOS messenger RNA, at 9 to12 h in the liver of hepatic I/R rats. These results demonstrate that FR183998 reduces the induction of proinflammatory cytokines and iNOS at least in part through inhibition of nuclear factor-&kgr;B activation and iNOS antisense transcript expression, thereby preventing hepatic I/R injury.ABBREVIATIONS - NHE-Na+/H+ exchanger; I/R-ischemia/reperfusion; NO-nitric oxide; iNOS-inducible nitric oxide synthase; TNF-&agr;-tumor necrosis factor-&agr;; CINC-1-cytokine-induced neutrophil chemoattractant 1; IFN-&ggr;-interferon-&ggr;; IL-interleukin; NF-&kgr;B-nuclear factor-&kgr;B

Active hexose correlated compound inhibits the expression of proinflammatory biomarker iNOS in hepatocytes.

Background/Aims: Excess production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated as proinflammatory biomarker in liver injury. The application of active hexose correlated compound (AHCC) as a functional food in complementary and alternative medicine has increased. The possibility that AHCC might inhibit iNOS induction was investigated as a potential liver-protective effect. Methods: Hepatocytes were isolated from rats by collagenase perfusion and cultured. Primary cultured hepatocytes were treated with interleukin-1β in the presence or absence of AHCC-sugar fraction (AHCC-SF). Results and Conclusion: AHCC-SF inhibited the production of NO and reduced expressions of iNOS mRNA and its protein. AHCC-SF had no effects on either IĸB degradation or nuclear factor-ĸB (NF-ĸB) activation. In contrast, AHCC-SF inhibited the upregulation of type I interleukin-1 receptor (IL-1RI) through the inhibition of Akt phosphorylation. Transfection experiments with iNOS promoter-luciferase constructs revealed that AHCC-SF reduced the levels of iNOS mRNA at both promoter transactivation and mRNA stabilization steps. AHCC-SF inhibited the expression of iNOS gene antisense transcript, which is involved in iNOS mRNA stabilization. These findings demonstrate that AHCC-SF suppresses iNOS gene expression through a IĸB/NF-ĸB-independent but Akt/IL-1RI-dependent pathway, resulting in the reduction of NO production. AHCC-SF may have therapeutic potential for various liver injuries.

Effect of Thiol-Containing Molecule Cysteamine on the Induction of Inducible Nitric Oxide Synthase in Hepatocytes

BACKGROUND Cysteamine, which is a known antioxidant and anti-inflammatory agent, is believed to be a key regulator of essential metabolic pathways in organisms. Cysteamine has beneficial effects in liver damaged by a variety of insults. During liver injury, inducible nitric oxide synthase (iNOS) is induced by lipopolysaccharide or proinflammatory cytokines, leading to excessive nitric oxide (NO) production. Accumulated evidence indicates that NO is an important factor associated with hepatic dysfunction. We examined whether cysteamine influences the induction of iNOS in hepatocytes. METHODS Primary cultured rat hepatocytes were treated with interleukin (IL)-1beta in the presence and absence of cysteamine. NO production, iNOS induction, and iNOS signal were analyzed. RESULTS IL-1beta stimulated the inhibitory protein kappaB (IkappaB)/nuclear factor kappaB (NFkappaB) pathway, resulting in the activation of NFkappaB (nuclear translocation and DNA binding), which was followed by the induction of iNOS and NO production. The addition of IL-1beta and cysteamine (1-4 mmol/L) markedly inhibited NO production, with a maximal effect at 4 mmol/L (80%-90% inhibition). Cysteamine also decreased the levels of iNOS protein and mRNA. Transfection experiments revealed that cysteamine decreased the transactivation activity of the iNOS promoter. An electrophoretic mobility shift assay demonstrated that cysteamine inhibited the activation of NFkappaB. Furthermore, cysteamine decreased the mRNA levels of the NFkappaB subunit p65 but increased those of the inhibitory protein IkappaB. CONCLUSIONS These findings suggest that cysteamine inhibits iNOS induction at the step of NFkappaB activation. Further study is necessary to define the molecular basis of this effect of cysteamine on the regulation of NFkappaB and its pharmacologic implications.

Infrarenal abdominal aortic aneurysm complicated by persistent endotension after endovascular repair: report of a case.

Endoleak and endotension may prevent the successful exclusion of an aneurysm after endovascular aortic aneurysm repair (EVAR). The pressurization in the excluded aneurysm sac caused by endotension may lead to rupture of the aneurysm; however, the cause of endotension and its underlying mechanisms remain unclear. We report a case of infrarenal abdominal aortic aneurysm (AAA) complicated by persistent endotension after EVAR. Although no endoleaks were found on conventional double-phase computed tomographic scans, a thrombosed endoleak existed in the side branch and attachment site of the endograft. After treating the undetectable thrombosed endoleaks, physical examination revealed that the pressure of the excluded aneurysm had diminished, with shrinkage of the aneurysm. This case report suggests that a high-pressure undetectable type I or type II endoleak could be a major cause of endotension. Thus, postoperative evaluation of the attachment site of an endograft is important after EVAR.

Surgical Control of a Primary Hepatic Carcinoid Tumor: A Case Report.

We report a primary hepatic carcinoid tumor occurring in a 47-year-old man. The patient consulted our hospital complaining of epigastralgia. Abdominal ultrasonography, computed tomography scanning, and magnetic resonance imaging showed a large mass in the right lobe of the liver. FDG-PET revealed 18F-FDG uptake by the right hepatic lobe. The tumor was a solid mass with cystic components, approximately 15 cm in diameter. We conducted an extended right lobectomy of the liver. The resected specimen was a solid tumor with cystic components and hemorrhagic lesion. Microscopic findings showed that the tumor cells had round nuclei and formed trabecular patterns. Immunohistologically, tumor cells were stained positive for chromogranin A, neuron specific enolase, CD56, and S-100. Careful examinations before and after the operation revealed no other possible origin of the tumor. Based on these findings, the tumor was diagnosed as a primary hepatic carcinoid. This is a report of a rare case of a primary hepatic carcinoid tumor with a discussion of several other relevant reports.

Preoperative weight loss program involving a 20-day very low-calorie diet for obesity before laparoscopic gastrectomy for gastric cancer: Preoperative weight loss program

The increased visceral fat in patients with obesity can increase the technical difficulty of surgery. This study was performed to evaluate a preoperative 20‐day very low‐calorie diet for obesity before laparoscopic gastrectomy for gastric cancer.