Hepatoprotective effects of baicalein against liver ischemia-reperfusion injury and partial hepatectomy in a rat model.

BACKGROUND: Baicalein is the main active flavonoid in Scutellariae Radix and is included in shosaikoto, a Kampo formula used for treating hepatitis and jaundice. However, little is known about its hepatoprotective effects against hepatic ischemia-reperfusion injury (HIRI), a severe clinical condition directly caused by interventional procedures. We aimed to investigate the hepatoprotective effects of baicalein against HIRI and partial hepatectomy (HIRI + PH) and its potential underlying mechanisms. METHODS AND RESULTS: Male Sprague-Dawley rats received either baicalein (5 mg/kg) or saline intraperitoneally and underwent a 70% hepatectomy 15 min after hepatic ischemia. After reperfusion, liver and blood samples were collected. Survival was monitored 30 min after hepatic ischemia and hepatectomy. In interleukin 1ß (IL-1ß)-treated primary cultured rat hepatocytes, the influence of baicalein on inflammatory mediator production and the associated signaling pathway was analyzed. Baicalein suppressed apoptosis and neutrophil infiltration, which are the features of HIRI + PH treatment-induced histological injury. Baicalein also reduced the mRNA expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α). In addition, HIRI + PH treatment induced liver enzyme deviations in the serum and hypertrophy of the remnant liver, which were suppressed by baicalein. In the lethal HIRI + PH treatment group, baicalein significantly reduced mortality. In IL-1ß-treated rat hepatocytes, baicalein suppressed TNF-α and chemokine mRNA expression as well as the activation of nuclear factor-kappa B (NF-κB) and Akt. CONCLUSIONS: Baicalein treatment attenuates HIRI + PH-induced liver injury and may promote survival. This potential hepatoprotection may be partly related to suppressing inflammatory gene induction through the inhibition of NF-κB activity and Akt signaling in hepatocytes.

Hydrophobic constituents of Polygonum multiflorum roots promote renal erythropoietin expression in healthy mice.

The roots of Polygonum multiflorum Thunberg (Polygonaceae) are used as a crude drug Kashu that is considered to improve blood deficiency based on a Kampo concept. Kashu has been included in Kampo formulas, such as Tokiinshi, which is used to treat eczema and dermatitis with itchiness by inhibiting inflammation and facilitating blood circulation in the skin. However, the effects of P. multiflorum roots on erythropoiesis are unclear. Previously, we isolated six phenolic constituents from an ethyl acetate (EtOAc)-soluble fraction of P. multiflorum root extract and identified them as (E)-2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside [(E)-THSG], emodin, emodin-8-O-ß-D-glucopyranoside, physcion, physcion-8-O-ß-D-glucopyranoside, and catechin. To examine whether P. multiflorum roots facilitate erythropoiesis, the EtOAc-soluble fraction was orally administered to healthy ICR mice. When compared with mice fed a standard diet alone (Controls), the mice fed a diet including the EtOAc-soluble fraction exhibited significantly higher serum erythropoietin (Epo) levels. The renal Epo mRNA levels in EtOAc-soluble fraction-administered mice were significantly higher than those in the control mice. Then, we administered roxadustat, which is a drug to treat the patient suffering with renal anemia by specifically inhibiting hypoxia-inducible factor prolyl hydroxylases. Roxadustat slightly increased renal Epo mRNA levels in healthy mice. Administration of (E)-THSG, a major constituent, significantly increased serum Epo levels. It is likely that (E)-THSG may facilitate the process to convert inactive renal Epo-producing cells to active Epo-producing cells. Collectively, it is implied that (E)-THSG in the EtOAc-soluble fraction of P. multiflorum roots may primarily improve blood deficiency of Kampo concept by promoting erythropoiesis.

Identification of Anti-Inflammatory Compounds from Peucedanum praeruptorum Roots by Using Nitric Oxide-Producing Rat Hepatocytes Stimulated by Interleukin 1ß.

The roots of Peucedanum praeruptorum Dunn and Angelica decursiva Franchet et Savatier are designated Zenko, which is a crude drug defined by the Japanese Pharmacopoeia. This crude drug is used as an antitussive and an expectorant and is included in the Kampo formula Jinsoin, which improves cough, fever, and headache. Although the anti-inflammatory effects of this crude drug have been determined, the constituents responsible for this effect remain unknown. To investigate biologically active compounds, rat hepatocytes were used, which produce proinflammatory mediator nitric oxide (NO) in response to proinflammatory cytokine interleukin 1ß (IL-1ß). A methanol extract of P. praeruptorum roots, which suppressed IL-1ß-induced NO production, was fractionated into three crude fractions (ethyl acetate (EtOAc)-soluble, n-butanol-soluble, and water-soluble fractions) based on hydrophobicity. The EtOAc-soluble fraction markedly inhibited NO production. After this fraction was purified, three biologically active compounds were identified as praeruptorins A, B, and E, the contents of which were high. A comparison of their activities indicated that praeruptorin B exhibited the highest potency to inhibit NO production by decreasing inducible NO synthase expression and suppressed the expression of mRNAs encoding proinflammatory cytokines. Collectively, the three praeruptorins may primarily contribute to the anti-inflammatory effects of P. praeruptorum roots.

Detection of Decarboxylated Amino Acids after in Vitro Protease Digestion of the Hydrophilic Fraction of Crude Drug Extracts.

Many constituents of crude drugs in Japanese Kampo formulas are thought to function as pro-drugs, whose pharmacological activity is manifested after oral administration. Proteins and peptides in crude drugs may be digested and metabolized in the digestive tract and liver. However, few studies have reported the pharmacological activity of peptides in crude drugs. Here, we applied an analysis using LC-tandem mass spectrometry (LC-MS/MS) to identify the compounds derived from six crude drugs that are assumed to have anti-inflammatory effects. To simulate in vivo protease digestion, each water-soluble fraction of the crude drug extracts was treated with proteases, including endoproteinases and exopeptidases. Amines in the resultant digests were modified by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and analyzed using LC-MS/MS, which demonstrated the presence of four decarboxylated amino acids (primary amines). In the digest of the hydrophilic fraction of the fruit of Ziziphus jujuba Miller var. inermis Rehder (Taiso), isobutylamine, isoamylamine, and 2-methylbutylamine were identified, which may be derived from valinyl, leucinyl, and isoleucinyl residues, respectively. Additionally, tyramine possibly derived from tyrosyl residues was identified in the digests of all the crude drugs. In primary cultured rat hepatocytes treated with interleukin-1ß, all these decarboxylated amino acids suppressed the production of nitric oxide, a proinflammatory mediator. Our approach, i.e., in vitro protease digestion and LC-MS/MS analysis, suggests that decarboxylated amino acids may be formed in vivo from peptides and may be responsible for the anti-inflammatory effect of crude drugs included in Kampo medicine.

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.

Correction to: Antiinflammatory constituents of Atractylodes chinensis rhizome improve glomerular lesions in immunoglobulin A nephropathy model mice.

The article Antiinflammatory constituents of Atractylodes chinensis rhizome improve glomerular lesions in immunoglobulin A nephropathy model mice, written by Toshinari Ishii, Tetsuya Okuyama, Nao Noguchi, Yuto Nishidono, Tadayoshi Okumura, Masaki Kaibori, Ken Tanaka, Susumu Terabayashi, Yukinobu Ikeya and Mikio Nishizawa was originally published Online First without Open Access. After publication in volume 74 issue 1, page 51-64 the author decided to opt for Open Choice and to make the article an Open Access publication. Therefore, the copyright of the article has been changed to © The Author(s) 2020 and the article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice.

Qing Dai/Indigo Naturalis (QD) has been shown to ameliorate ulcerative colitis (UC) in clinical trials; however, its mechanism remains elusive. This study investigates the effects of QD on murine dextran sulfate sodium salt-induced colitis. Oral administration of QD protected the animals from colitis as manifested by weight loss, diarrhea, and rectal bleeding. QD was distinguishingly more effective than 5-aminosalicylate. Focused microarray analysis of genes expressed in the distal colon suggested that QD influences the inflammatory pathway. Anti-inflammatory activity of QD was confirmed by the suppression of nitric oxide (NO) production in response to interleukin-1ß in cultured hepatocytes. Some of the constituents in QD, such as tryptanthrin (TRYP) and indigo, suppressed NO production. TRYP maintained body weight but did not inhibit bleeding. Indigo, on the other hand, partially ameliorated bleeding, but did not maintain body weight. The combination of TRYP and indigo did not show additive ameliorating activity. The methanol extract of QD showed an anti-colitis activity like that of TRYP. In contrast, the methanol-insoluble QD fraction moderately ameliorated diarrhea and bleeding. Combining these two fractions resulted in full anti-colitis activity. Further clarification of the active constituents will help in the discovery of a safe and potent prescription for UC.

Antiinflammatory constituents of Atractylodes chinensis rhizome improve glomerular lesions in immunoglobulin A nephropathy model mice.

The crude drug Sojutsu, as defined by the Japanese Pharmacopoeia, is the rhizome of Atractylodes lancea De Candolle, Atractylodes chinensis Koidzumi, or their interspecific hybrids (Asteraceae). Sojutsu is one of the traditional Kampo formulas, which are administered to patients suffering from stomach disorders, edema, and nephrotic syndrome. Although antiinflammatory effects of Sojutsu have been reported, its effects on the liver and kidney have not been extensively investigated. Here, we used a Sojutsu sample identified as A. chinensis rhizome and isolated several constituents from its ethyl acetate (EtOAc)-soluble fraction that decreased production of the proinflammatory mediator nitric oxide (NO) in interleukin 1ß-treated rat hepatocytes. Among the constituents in this fraction, atractylodin showed the highest activity to suppress NO production, whereas hinesol, ß-eudesmol, and α-bisabolol showed low activity. Atractylodin decreased the levels of inducible nitric oxide synthase, tumor necrosis factor α, and lipocalin 2 messenger RNAs (mRNAs). The EtOAc-soluble fraction of the A. chinensis rhizome extract was administered daily for 20 weeks to high immunoglobulin A (HIGA) mice, whose pathological findings resemble human immunoglobulin A nephropathy. This fraction decreased the weight of white adipose tissue and decreased mesangial proliferation and immunoglobulin A deposition in glomeruli. These results indicate that the EtOAc-soluble fraction, which included antiinflammatory constituents, may be responsible for improvement of the mesangial lesions in HIGA mice.

Bitter melon fruit extract has a hypoglycemic effect and reduces hepatic lipid accumulation in ob/ob mice.

Bitter melon (Momordica charantia L.) is a vegetable and has been used as traditional medicine. Recently, we reported that bitter melon fruit extracts and its ethyl acetate (EtOAc)-soluble fraction markedly suppressed the expression of proinflammatory genes, including the inducible nitric oxide synthase gene. However, it is unclear whether bitter melon exhibits antidiabetic effects. In this study, we showed that cucurbitacin B, a cucurbitane-type triterpenoid, was present in an EtOAc-soluble fraction and suppressed nitric oxide production in hepatocytes. When the EtOAc-soluble fraction was administered for 7 days to ob/ob mice, a type 2 diabetes mellitus model, the mice fed with this fraction exhibited a significant decrease in body weight and blood glucose concentrations compared with the mice fed without the fraction. The administration of the fraction resulted in significant increases in serum insulin concentrations and the levels of both insulin receptor mRNA and protein in the ob/ob mouse liver. The EtOAc-soluble fraction decreased the interleukin-1ß mRNA expression, as well as hepatic lipid accumulation in hepatocytes. Taken together, these results indicate that administration of an EtOAc-soluble fraction improved hyperglycemia and hepatic steatosis, suggesting that this fraction may be responsible for both the antidiabetic and anti-inflammatory effects of bitter melon fruit.

Yokukansan, a Kampo medicine, enhances the level of neuronal lineage markers in differentiated P19 embryonic carcinoma cells.

Yokukansan (YKS), a traditional Japanese Kampo medicine, affects neurological and psychiatric disorders. It ameliorates hippocampal neurogenesis in animals. However, its effect on neuronal cell differentiation remains unclear. Therefore, we investigated the effects of YKS on pluripotent P19 embryonic carcinoma cells as neuronal differentiation model cells. Western blotting and immunocytochemistry revealed that 10 µg/mL YKS treatment during embryoid body formation or neuronal differentiation increased the expression of the neuronal stem cell marker, Nestin, by 1.9-fold and 1.7-fold, respectively, and of the mature neuron marker, NeuN, by 1.5-fold and 1.4-fold, respectively. We examined the effect of YKS on intracellular signaling pathways in P19 cells and found significant elevation in phospho-PDK1 and phospho-mTOR expression (1.1-fold and 1.2-fold, respectively). Therefore, we investigated the effect of PDK1 and mTOR inhibitors on the level of neuronal lineage markers. We found that the mTOR inhibitor significantly abolished the YKS effect on the level of neuronal lineage markers. Moreover, to identify the target(s) of YKS, antibody array analysis that simultaneously detects 16 phosphorylated proteins was performed. YKS significantly upregulated 10 phosphorylated proteins including PDK1, Akt, AMPK, PRAS40, mTOR, p70 S6 kinase, GSK-3α, Bad and ERK1/2 under cell proliferation conditions. These results suggest that YKS simultaneously activates multiple signaling pathways. Thus, we concluded that YKS enhances the level of neuronal lineage markers in differentiated P19 cells, however it does not induce neuronal differentiation. Furthermore, mTOR is the predominant mediator of the YKS effect on these cells.

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

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)-1ß. 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 ß (C/EBPß), which synergistically activates the transcription of the iNOS gene with nuclear factor κB (NF-κB). Therefore, sakuranetin inhibited the co-activating activity of C/EBPß 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/EBPß. These results imply that sakuranetin may be primarily responsible for the anti-inflammatory effects of Pruni Cortex in the liver.

Identification of anti-inflammatory constituents in Phellodendri Cortex and Coptidis Rhizoma by monitoring the suppression of nitric oxide production.

Phellodendri Cortex (Obaku in Japanese) and Coptidis Rhizoma (Oren), both of which contain berberine, have been used to prepare the kampo formula orengedokuto to treat inflammatory diseases, including dermatitis, gastric ulcers, and gastritis. These drugs are blended differently in other formulas, such as the use of Phellodendri Cortex in shichimotsukokato to treat hypertension and Coptidis Rhizoma in hangeshashinto to treat diarrhea and stomatitis. However, the differences in their medicinal properties are not well characterized. We prepared extracts from Phellodendron amurense bark (PAB) and Coptis chinensis rhizome (CCR) and separated them into alkaloid and non-alkaloid fractions. Anti-inflammatory effects were examined by monitoring the production of nitric oxide (NO), which is a pro-inflammatory mediator. A non-alkaloid fraction of the PAB extract suppressed NO production in hepatocytes more efficiently than that of the CCR extract. When each non-alkaloid fraction of the PAB and CCR extracts was administered to mice, the fractions of both extracts decreased the levels of mRNAs encoding inducible NO synthase and molecules in the interleukin-1ß signaling pathway. Limonin and obakunone identified in the PAB non-alkaloid fraction suppressed NO production, exhibiting IC50 values of 16 and 2.6 µM, respectively, whereas berberine and coptisine displayed IC50 values of 12 and 14 µM, respectively. Limonin and obakunone reduced the expression of the iNOS gene, probably through the transcription factor nuclear factor-κB. Therefore, both limonoids and alkaloids may be responsible for the anti-inflammatory effects of the PAB extract, whereas alkaloids may be primarily responsible for those of the CCR extract. The different composition of the constituents may modulate the anti-inflammatory effects of Phellodendri Cortex and Coptidis Rhizoma.

Lignans from guaiac resin decrease nitric oxide production in interleukin 1ß-treated hepatocytes.

Guaiac resin, extracted from the heartwood of Guaiacum officinale L. or G. sanctum L., is speculated to have anti-inflammatory effects. Lignans were purified from guaiac resin (also known as gum guaiacum) by monitoring the nitric oxide (NO) production in rat hepatocytes treated with an inflammatory cytokine interleukin-1ß (IL-1ß). Six lignans were purified from guaiac resin and identified as: dehydroguaiaretic acid (1), (+)-trans-1,2-dihydrodehydroguaiaretic acid (2), furoguaiaoxidin (3), meso-dihydroguaiaretic acid (4), furoguaiacin (i.e., α-guaiaconic acid) (5), and nectandrin B (6). To our knowledge, this is the first time that 1 has been isolated from guaiac resin as a non-derivative. Compounds 2 and 6 were first found in guaiac resin. Compound 3 was first isolated from a natural source as a non-derivative. Furthermore, 1-6 significantly suppressed NO production in IL-1ß-treated hepatocytes. Because anti-inflammatory compounds suppress NO production, this system is often used to measure the anti-inflammatory effects of Kampo drugs and herbal constituents. The NO-suppressing activity of the six lignans isolated in this study indicates that guaiac resin has anti-inflammatory effects and that these lignans may be responsible for the anti-inflammatory effects of guaiac resin.

Post-transcriptional inducible gene regulation by natural antisense RNA.

Accumulating data indicate the existence of natural antisense transcripts (asRNAs), frequently transcribed from eukaryotic genes and do not encode proteins in many cases. However, their importance has been overlooked due to their heterogeneity, low expression level, and unknown function. Genes induced in responses to various stimuli are transcriptionally regulated by the activation of a gene promoter and post-transcriptionally regulated by controlling mRNA stability and translatability. A low-copy-number asRNA may post-transcriptionally regulate gene expression with cis-controlling elements on the mRNA. The asRNA itself may act as regulatory RNA in concert with trans-acting factors, including various RNA-binding proteins that bind to cis-controlling elements, microRNAs, and drugs. A novel mechanism that regulates mRNA stability includes the interaction of asRNA with mRNA by hybridization to loops in secondary structures. Furthermore, recent studies have shown that the functional network of mRNAs, asRNAs, and microRNAs finely tunes the levels of mRNA expression. The post-transcriptional mechanisms via these RNA-RNA interactions may play pivotal roles to regulate inducible gene expression and present the possibility of the involvement of asRNAs in various diseases.

The constituents of licorice (Glycyrrhiza uralensis) differentially suppress nitric oxide production in interleukin-1ß-treated hepatocytes.

Licorice (Glycyrrhizae radix) is the roots and stolons of Glycyrrhiza uralensis Fischer or Glycyrrhiza glabra Linnaeus in the Japanese Pharmacopoeia. Glycyrrhizae radix has been widely used as a sweetener and a traditional medicine. A Glycyrrhizae radix extract contains many constituents and has antispasmodic, antitussive, anti-ulcer, and anti-inflammatory effects. However, reports comparing the anti-inflammatory effects of these constituents are very few. Here, we purified several constituents from the roots and stolons of G. uralensis and examined and compared their anti-inflammatory effects by monitoring the levels of the inflammatory mediator, nitric oxide (NO), in interleukin (IL)-1ß-treated rat hepatocytes. From the G. uralensis extract, we purified the main constituent glycyrrhizin and the constituents that are characteristic of G. uralensis (chalcones and flavanones). These constituents suppressed NO production in IL-1ß-treated rat hepatocytes, and isoliquiritigenin showed the greatest suppression activity. Isoliquiritigenin, isoliquiritin, and liquiritigenin significantly decreased both protein and mRNA for the inducible nitric oxide synthase. These constituents reduced the levels of mRNAs encoding tumor necrosis factor α and IL-6. In contrast, although glycyrrhizin is abundant, it showed a 100-fold lower potency in NO suppression. Therefore, both glycyrrhizin and the minor constituents (isoliquiritigenin, isoliquiritin, and liquiritigenin) may be responsible for the anti-inflammatory effects of G. uralensis. It is also implied that these constituents may have a therapeutic potential for inflammatory hepatic disorders.

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.

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.

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

AIM: 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)-1ß-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. METHODS: Primary cultured rat hepatocytes were treated with IL-1ß in the presence or absence of TJ-135. The induction of iNOS and its signaling pathway were analyzed. RESULTS: IL-1ß produced increased levels of NO. This effect was inhibited by TJ-135, which exerted its maximal effects at 3 mg/mL. TJ-135 decreased the levels of iNOS protein and its mRNA expression. Experiments with nuclear extracts revealed that TJ-135 inhibited the translocation of nuclear factor-κB (NF-κB) to the nucleus and its DNA binding. TJ-135 also inhibited the activation of Akt, resulting in the reduction of type I IL-1 receptor mRNA and protein expression. Transfection experiments with iNOS promoter-luciferase constructs demonstrated that TJ-135 suppressed iNOS induction by inhibition of promoter transactivation and mRNA stabilization. TJ-135 reduced the expression of an iNOS gene antisense-transcript. Delayed administration or withdrawal of TJ-135 after IL-1ß addition also inhibited iNOS induction. CONCLUSIONS: RESULTS indicate that TJ-135 inhibits the induction of iNOS at both transcriptional and post-transcriptional steps, leading to the prevention of NO production. TJ-135 may have therapeutic potential for various liver injuries through the suppression of iNOS induction.

Regulation of inducible gene expression by natural antisense transcripts.

Natural antisense transcripts are frequently transcribed from many genes in eukaryotes. Although natural antisense transcripts have been recognized for a long time, their importance has been overlooked due to their heterogeneity, low expression level, and unknown function. Genes induced in responses to various external stimuli are transcriptionally regulated by the activation of a gene promoter and post-transcriptionally regulated by controlling mRNA stability and translatability. Recent studies have shed light on the functions of natural antisense transcripts at the post-transcriptional level. An antisense transcript may regulate gene expression with cis-controlling elements on the mRNA, and the antisense transcript itself may act in concert with trans-acting factors, including various proteins that bind to cis-controlling elements, drugs, and microRNAs. A novel mechanism recently reported to regulate mRNA stability includes the interaction of the antisense transcript with mRNA by hybridization to single-stranded loops in secondary structures. This antisense transcript-mediated post-transcriptional regulation may be one of the general mechanisms for the regulation of inducible gene expression and presents the possibility of the involvement of natural antisense transcripts in disease.