Ginger extract and zingerone ameliorated trinitrobenzene sulphonic acid-induced colitis in mice via modulation of nuclear factor-κB activity and interleukin-1ß signalling pathway.

Ginger is a commonly used spice with anti-inflammatory potential. Colitis is the common pathological lesion of inflammatory bowel diseases. In this study, we investigated the therapeutic effects of ginger and its component zingerone in mice with 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis. Ginger and zingerone ameliorated TNBS-induced colonic injury in a dose-dependent manner. Pathway analysis of ginger- and zingerone-regulated gene expression profiles showed that ginger and zingerone significantly regulated cytokine-related pathways. Network analysis showed that nuclear factor-κB (NF-κB) and interleukin-1ß (IL-1ß) were key molecules involved in the expression of ginger- and zingerone-affected genes. Ex vivo imaging and immunohistochemical staining further verified that ginger and zingerone suppressed TNBS-induced NF-κB activation and IL-1ß protein level in the colon. In conclusion, ginger improved TNBS-induced colitis via modulation of NF-κB activity and IL-1ß signalling pathway. Moreover, zingerone might be the active component of ginger responsible for the amelioration of colitis induced by TNBS.

Identification of novel mechanisms of silymarin on the carbon tetrachloride-induced liver fibrosis in mice by nuclear factor-κB bioluminescent imaging-guided transcriptomic analysis.

In this study, we applied bioluminescent imaging-guided transcriptomic analysis to evaluate and identify the therapeutic potentials and novel mechanisms of silymarin on carbon tetrachloride (CCl(4))-induced liver fibrosis. Transgenic mice, carrying the luciferase genes driven by nuclear factor-κB (NF-κB), were given with CCl(4) and/or silymarin. In vivo NF-κB activity was evaluated by bioluminescent imaging, liver fibrosis was judged by Sirius red staining and immunohistochemistry, and gene expression profiles of silymarin-treated livers were analyzed by DNA microarray. CCl(4) enhanced the NF-κB-dependent hepatic luminescence and induced hepatic fibrosis, while silymarin reduced the CCl(4)-induced hepatic luminescence and improved CCl(4)-induced liver fibrosis. Microarray analysis showed that silymarin altered the transforming growth factor-ß-mediated pathways, which play pivotal roles in the progression of liver fibrosis. Moreover, we newly identified that silymarin downregulated the expression levels of cytoskeleton organization genes and mitochondrion electron-transfer chain genes, such as cytochrome c oxidase Cox6a2, Cox7a1, and Cox8b genes. In conclusion, the correlation of NF-κB-dependent luminescence and liver fibrosis suggested the feasibility of NF-κB bioluminescent imaging for the evaluation of liver fibrosis progression and therapeutic potentials. Moreover, our findings suggested that silymarin might exhibit anti-fibrotic effects in vivo via altering the expression of genes involved in cytoskeleton organization and mitochondrion electron-transfer chain.

Inhibitory effects of Zuo-Jin-Wan and its alkaloidal ingredients on activator protein 1, nuclear factor-κB, and cellular transformation in HepG2 cells.

Zuo-Jin-Wan (ZJW) has been used to treat hepatocellular carcinoma in Asia. This study was to determine whether ZJW and its components blocked activator protein 1 (AP-1) and nuclear factor-κB (NF-κB) activities as well as tumor promotion in hepatoblastoma HepG2 cells. ZJW and its components, Coptis chinensis and Evodia rutaecarpa, inhibited AP-1 and NF-κB activities, and suppressed anchorage-independent growth of HepG2 cells. The major alkaloidal ingredients, berberine and evodiamine, inhibited AP-1 activities and/or NF-κB activation, and further suppressed hepatocellular transformation. In conclusion, ZJW and its constituents, berberine and evodiamine, suppressed tumor promotion primarily through AP-1 and/or NF-κB pathways in HepG2 cells.

Evodiamine inhibits 12-O-tetradecanoylphorbol-13-acetate-induced activator protein 1 transactivation and cell transformation in human hepatocytes.

Evodia rutaecarpa has been used to treat inflammatory digestive disorders in Asian countries. However, little is known about the antitumor activities of E. rutaecarpa and its bioactive constituent evodiamine (EVO). The aim of this study was to characterize the antitumor mechanisms of E. rutaecarpa and EVO in human hepatocytes. Human Chang liver cells were transfected with activator protein 1 (AP-1)-luciferase reporter gene and designated as Chang/AP-1 cells. The Chang/AP-1 cells were treated with E. rutaecarpa and its bioactive constituents, and challenged with the AP-1 stimulator 12-O-tetradecanoylphorbol-13- acetate (TPA). The present study showed that the methanol extract of E. rutaecarpa decreased the TPA-induced AP-1 transactivation in Chang/AP-1 cells, with an EC50 value of 24.72 µg/mL. EVO inhibited the TPA-induced AP-1 transactivation and colony formation, with EC50 values of 82 µM and 8.2 µM, respectively. Moreover, EVO significantly diminished the TPA-induced phosphorylation of extracellular signal-regulated kinases (ERKs). These results suggested that EVO treatment suppressed the TPA-induced AP-1 activity via the ERKs pathway. In conclusion, EVO inhibited the AP-1 activity and cellular transformation in human hepatocytes, suggesting that EVO was a potential agent for antitumor therapy.

Application of bioactivity database of Chinese herbal medicine on the therapeutic prediction, drug development, and safety evaluation.

AIM OF THE STUDY: Chinese herbal medicine has been used for the treatments of various diseases for years. However, it is often difficult to analyze their biological activities and molecule mechanisms because of their complex nature. In this study, we applied DNA microarray to analyze the biological events induced by herbal formulae, predict the therapeutic potentials of formulae, and evaluate the safety of formulae. MATERIALS AND METHODS: Mice were administrated orally with 15 formulae for 7 consecutive days, and the gene expression profiles in liver or kidney were further analyzed by transcriptomic tools. RESULTS: Our data showed that most formulae altered the metabolic pathways, such as glutathione metabolism and oxidative phosphorylation, and regulatory pathways, such as antigen processing and presentation and insulin-like growth factor signaling pathway. By comparing the gene expression signatures of formulae with those of disease states or drugs, we found that mice responsive to formula treatments might be related to disease states, especially metabolic and cardiovascular diseases, and drugs, which exhibit anti-cancer, anti-inflammatory, and anti-oxidative effects. Moreover, most formulae altered the expression levels of cytochrome p450, glutathione S-transferase, and UDP glycosyltransferase genes, suggesting that caution should be paid to possible drug interaction of these formulae. Furthermore, the similarities of gene expression profiles between formulae and toxic chemicals were low in kidney, suggesting that these formulae might not induce nephrotoxicities in mice. CONCLUSIONS: This report applied transcriptomic tools as a novel platform of translational medicine for Chinese herbal medicine. This platform will not only for understanding the therapeutic mechanisms involving herbal formulae and gene interactions, but also for the new theories in drug discovery.

Transcriptomic analysis of EGb 761-regulated neuroactive receptor pathway in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: EGb 761, a well-defined extract from Ginkgo biloba, has been widely used in patients with cerebral disorders. AIM OF THE STUDY: Although EGb 761 exhibits neuroprotective effects and exerts beneficial effects on many neurological disorders, its mechanism on the neuronal functions is unclear so far. MATERIALS AND METHODS: In this study, we used oligonucleotide microarray technique to investigate the effect of EGb 761 on the transcriptional profile of mouse genes. RNA samples were obtained from frontal cortex, straitum, and kidneys after the oral administration of EGb 761 for seven consecutive days. RESULTS: Our data showed that EGb 761 significantly altered the neuroactive ligand-receptor interaction pathway in frontal cortex but not in straitum and kidney. Then we analyzed 26 receptor genes that were significantly altered by EGb 761 in this pathway and found that EGb 761 treatment highly up-regulated the subgroup of dopamine receptors, especially dopamine receptor 1a (Drd1a), in frontal cortex. Quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemical staining confirmed the increased level of Drd1a expression after EGb 761 treatment. CONCLUSIONS: In summary, we investigated for the first time the overall effects of EGb 761 on the gene expression in brain using a powerful systemic biological technique. Our results suggested that EGb 761 altered unique pathways and regulated the expressions of some specific neuronal receptor genes exclusively in frontal cortex.

Identification of Escherichia coli enterotoxin inhibitors from traditional medicinal herbs by in silico, in vitro, and in vivo analyses.

ETHNOPHARMACOLOGICAL RELEVANCE: Glycyrrhiza uralensis has been used for the treatment of gastrointestinal disorders, such as diarrhea, in several ancient cultures. Glycyrrhizin is the principal component of liquorice and lots of pharmacological effects have been demonstrated. AIM OF THE STUDY: Heat-labile enterotoxin (LT), the virulence factor of enterotoxigenic Escherichia coli, induces diarrhea by initially binding to the GM1 on the surfaces of intestinal epithelial cells and consequently leading to the massive loss of fluid and ions from cells. Therefore, we evaluated the inhibitory effects of traditional medicinal herbs (TMH) on the B subunit of LT (LTB) and GM1 interaction. MATERIALS AND METHODS: The inhibitory effects of TMH on LTB-GM1 interaction were evaluated by GM1-enzyme-linked immunosorbent assay (ELISA). The likely active phytochemicals of these TMH were then predicted by in silico model (docking) and analyzed by in vitro (GM1-ELISA) and in vivo (patent mouse gut assay) models. RESULTS: We found that various TMH, which have been ethnomedically used for the treatment of diarrhea, inhibited the LTB-GM1 interaction. Docking data showed that triterpenoids were the most active phytochemicals and the oleanane-type triterpenoids presented better LTB-binding abilities than other types of triterpenoids. Moreover, by in vitro and in vivo models, we demonstrated that glycyrrhizin was the most effective oleanane-type triterpenoid that significantly suppressed both the LTB-binding ability (IC50=3.26+/-0.17 mM) and the LT-induced fluid accumulation in mice. CONCLUSIONS: We found an LT inhibitor, glycyrrhizin, from TMH by in silico, in vitro, and in vivo analyses.

Eriobotrya japonica leaf and its triterpenes inhibited lipopolysaccharide-induced cytokines and inducible enzyme production via the nuclear factor-kappaB signaling pathway in lung epithelial cells.

Pulmonary inflammation is a characteristic of many lung diseases. Increased levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory cytokines, such as interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and IL-8, have been correlated with lung inflammation. In this study, we used lipopolysaccharide (LPS) to induce iNOS, COX-2, and cytokines (TNF-alpha, IL-1beta, and IL-8) productions in human lung epithelial cells (A-549). Leaf of Eriobotrya japonica (Pi-Pa-Ye, PPY), a traditional Chinese medicine for the treatment of pulmonary inflammatory diseases, was capable of suppressing LPS-induced cytokine productions in a dose-dependent manner. Moreover, the suppression of PPY on the cytokine productions resulted from the inhibition of inhibitory kappaB-alpha phosphorylation and nuclear factor-kappaB (NF-kappaB) activation. Analysis of the anti-inflammatory effects of ursolic acid and oleanolic acid, the triterpene compounds present in PPY, showed that ursolic acid significantly inhibited LPS-induced IL-8 production, NF-kappaB activation, and iNOS mRNA expression, whereas oleanolic acid did not have these effects. In conclusion, our findings suggested the potential mechanisms of PPY and its active component, ursolic acid, in the treatment of pulmonary inflammation.

Inhibition of lipopolysaccharide-induced interleukin-1beta and tumor necrosis factor-alpha production by menthone through nuclear factor-kappaB signaling pathway in HaCat cells.

Menthone, the Chinese old remedy extracted from genus Mentha, has been widely used as a cooling agent, a counterirritant for pain relief, and for the treatment of pruritus. However, its detail mechanisms for interfering inflammatory reaction remain unknown. In this study, we found that menthone can suppress the lipopolysaccharide (LPS)-induced proinflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), as well as nuclear factor kappaB (NF-kappaB) activity induced by LPS and other inflammatory agents, including 12-O-tetradecanoylphorbol-13-acetate, hydrogen peroxide, okadaic acid, and ceramide. Furthermore, our data also demonstrated that the translocation of NF-kappaB activated by LPS into the nucleus was suppressed by menthone, and I-kappaB and beta-transducin repeat containing protein (beta-TrCP) were both involved in this suppression. To sum up, this study has provided molecular evidence for menthone effect on the LPS-induced cytokine production, NF-kappaB activation, and the involvement of I-kappaB and beta-TrCP.

Relationship Between San-Huang-Xie-Xin-Tang and its herbal components on the gene expression profiles in HepG2 cells.

Traditional Chinese medicine (TCM) has been used for thousands of years. Most Chinese herbal formulae consist of several herbal components and have been used to treat various diseases. However, the mechanisms of most formulae and the relationship between formulae and their components remain to be elucidated. Here we analyzed the putative mechanism of San-Huang-Xie-Xin-Tang (SHXXT) and defined the relationship between SHXXT and its herbal components by microarray technique. HepG2 cells were treated with SHXXT or its components and the gene expression profiles were analyzed by DNA microarray. Gene set enrichment analysis indicated that SHXXT and its components displayed a unique anti-proliferation pattern via p53 signaling, p53 activated, and DNA damage signaling pathways in HepG2 cells. Network analysis showed that most genes were regulated by one molecule, p53. In addition, hierarchical clustering analysis showed that Rhizoma Coptis shared a similar gene expression profile with SHXXT. These findings may explain why Rhizoma Coptis is the principle herb that exerts the major effect in the herbal formula, SHXXT. Moreover, this is the first report to reveal the relationship between formulae and their herbal components in TCM by microarray and bioinformatics tools.

Ginger and its bioactive component inhibit enterotoxigenic Escherichia coli heat-labile enterotoxin-induced diarrhea in mice.

Ginger is one of the most commonly used fresh herbs and spices. Enterotoxigenic Escherichia coli heat-labile enterotoxin (LT)-induced diarrhea is the leading cause of infant death in developing countries. In this study, we demonstrated that ginger significantly blocked the binding of LT to cell-surface receptor G M1, resulting in the inhibition of fluid accumulation in the closed ileal loops of mice. Biological-activity-guided searching for active components showed that zingerone (vanillylacetone) was the likely active constituent responsible for the antidiarrheal efficacy of ginger. Further analysis of chemically synthesized zingerone derivatives revealed that compound 31 (2-[(4-methoxybenzyl)oxy]benzoic acid) significantly suppressed LT-induced diarrhea in mice via an excellent surface complementarity with the B subunits of LT. In conclusion, our findings provide evidence that ginger and its derivatives may be effective herbal supplements for the clinical treatment of enterotoxigenic Escherichia coli diarrhea.

Inhibition of Escherichia coli heat-labile enterotoxin-induced diarrhea by Chaenomeles speciosa.

Enterotoxigenic Escherichia coli (ETEC) is responsible for millions of deaths in developing countries. Heat-labile enterotoxin (LT), the virulence factor of ETEC, induces diarrhea by initially binding to the G(M1) on the surface of intestinal epithelial cells and consequently leading to the massive loss of fluid and ions from cells. Fruit of Chaenomeles (FC), the dried fruit of Chaenomeles speciosa, has been used for diarrhea in China. However, the anti-diarrheal mechanism of FC is still unclear. In this study, we demonstrated that FC extract (FCE) inhibited the LT-induced diarrhea in mice by blocking the binding of the B subunit of LT (LTB) to G(M1). The ethyl acetate (EA) soluble fraction was the most active fraction of FC that significantly abolished the LTB and G(M1) interaction. Furthermore, the oleanolic acid, ursolic acid, and betulinic acid from EA fraction, blocked the toxin binding effects, resulting in the suppression of LT-induced diarrhea. Moreover, by docking techniques, these compounds fitted LTB well via hydrogen bonds and hydrophobic contacts with amino acid residues of LTB. In conclusion, our findings suggested that oleanolic acid, ursolic acid, and betulinic acid were the active constituents from FC and might be considered as lead therapeutic agents in the treatment of LT-induced diarrhea.

Emodin blocks the SARS coronavirus spike protein and angiotensin-converting enzyme 2 interaction.

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel coronavirus (SARS-CoV). SARS-CoV spike (S) protein, a type I membrane-bound protein, is essential for the viral attachment to the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening 312 controlled Chinese medicinal herbs supervised by Committee on Chinese Medicine and Pharmacy at Taiwan, we identified that three widely used Chinese medicinal herbs of the family Polygonaceae inhibited the interaction of SARS-CoV S protein and ACE2. The IC(50) values for Radix et Rhizoma Rhei (the root tubers of Rheum officinale Baill.), Radix Polygoni multiflori (the root tubers of Polygonum multiflorum Thunb.), and Caulis Polygoni multiflori (the vines of P. multiflorum Thunb.) ranged from 1 to 10 microg/ml. Emodin, an anthraquinone compound derived from genus Rheum and Polygonum, significantly blocked the S protein and ACE2 interaction in a dose-dependent manner. It also inhibited the infectivity of S protein-pseudotyped retrovirus to Vero E6 cells. These findings suggested that emodin may be considered as a potential lead therapeutic agent in the treatment of SARS.

Anti-diarrheal effect of Galla Chinensis on the Escherichia coli heat-labile enterotoxin and ganglioside interaction.

Enterotoxigenic Escherichia coli (ETEC) is the most frequently isolated enteropathogen, accounting for approximately 210 million diarrhea episodes annually. ETEC-induced diarrhea is initiated by the binding of B subunit of heat-labile enterotoxin (LTB) to the ganglioside G(M1) on the surface of intestinal epithelial cell. Therefore, we evaluated the inhibitory effects of 297 Chinese medicinal herbs on the LTB and G(M1) interaction by G(M1)-enzyme-linked immunosorbent assay. Galla Chinensis extract (GCE) exhibited anti-LT-induced diarrheal effect in the patent mouse gut assay, with IC50 value of 4.7+/-1.3 mg/ml. GCE also inhibited the binding of LTB to G(M1), suggesting that GCE suppressed the LT-induced fluid accumulation by blocking the binding of LTB to G(M1). Furthermore, the ethyl acetate (EA) soluble fraction was the most active fraction of Galla Chinensis that inhibiting the binding of LTB to G(M1) with an IC50 value of 153.6+/-3.4 microg/ml. The major components of the EA fraction should be phenolic derivatives according to a thin-layer chromatography analysis. Gallic acid, the major component of EA fraction, blocked the binding of LTB to G(M1), resulting in the suppression of LT-induced diarrhea. In conclusion, these data suggested that Galla Chinensis and gallic acid might be potent drugs for the treatment of LT-induced diarrhea.

Acetaldehyde-induced interleukin-1beta and tumor necrosis factor-alpha production is inhibited by berberine through nuclear factor-kappaB signaling pathway in HepG2 cells.

Alcoholic liver disease (ALD) is one of the most common liver diseases in the world. Increased levels of proinflammatory cytokines, including interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), have been correlated with the patients affected by ALD. However, the direct effect of alcohol in the induction of IL-1beta and TNF-alpha has not been clarified. In this study, we demonstrated that acetaldehyde, the metabolic product of ethanol, was able to induce IL-1beta and TNF-alpha production in HepG2 cells. Nuclear factor-kappaB (NF-kappaB), the transcription factor involved in the regulation of cytokine production, was also activated by acetaldehyde through inhibitory kappaB-alpha (IkappaB-alpha) phosphorylation and degradation. However, the NF-kappaB inhibitors, such as aspirin, cyclosporin A and dexamethasone, inhibited both the acetaldehyde-induced NF-kappaB activity and the induced cytokine production. Therefore, these data suggested that acetaldehyde stimulated IL-1beta and TNF-alpha production via the regulation of NF-kappaB signaling pathway. By screening 297 controlled Chinese medicinal herbs supervised by Committee on Chinese Medicine and Pharmacy at Taiwan, we found that Coptis chinensis (Huang-Lien) and Phellodendron amurense (Huang-Po) were capable of inhibiting acetaldehyde-induced NF-kappaB activity. Berberine, the major ingredient of these herbs, abolished acetaldehyde-induced NF-kappaB activity and cytokine production in a dose-dependent manner. Moreover, its inhibitory ability was through the inhibition of induced IkappaB-alpha phosphorylation and degradation. In conclusion, we first linked the acetaldehyde-induced NF-kappaB activity to the induced proinflammatory cytokine production in HepG2 cells. Our findings also suggested the potential role of berberine in the treatment of ALD.

An in vitro system combined with an in-house quantitation assay for screening hepatitis C virus inhibitors.

Hepatitis C virus (HCV) infection is a serious global health problem. Interferon-alpha (IFN-alpha) and ribavirin have demonstrated efficacy in the treatment of HCV infection; however, these therapies display many side effects. To screen the anti-HCV compounds from plants, we established an in vitro model for inoculation of HCV by centrifugation-facilitated method. The HCV RNA molecules were then quantitated by nested competitive reverse transcription-polymerase chain reaction (cRT-PCR) using fluorescein-labeled primers, and analyzed by ABI Prism 310. The positive and negative strands of HCV RNA were detectable in Vero cells on Day 7 post-infection, suggesting that the HCV RNA was present in the cell model system. The cell culture system was further used to screen the anti-HCV activities of 4 Chinese herbal formulas and 15 formula components. IFN-alpha showed an antiviral effect. The formulas exhibited no anti-HCV activities, while Arnebia euchroma, Thlaspi arvense, and Poncirus trifoliata displayed anti-HCV activities. Therefore, these results pointed out the possibility by using the cell culture system established in this study to screen the herb extracts for their anti-HCV activities.

Activation of activator protein-1 in cells exposed to electrical stimulation of various parameters used in electroacupuncture therapy.

The aim of this study was to investigate the effect of electrical stimulation on activator protein-1 (AP-1) in recombinant liver cells. In order to elucidate the molecular effects of electrical stimulation on cells, AP-1 expression was detected by a luciferase assay. The parameters used were taken from clinical electroacupuncture (EA) therapy as follows: biphasic rectangular symmetrical pulses (frequency: 2, 10 and 100 Hz; pulse width: 50, 80, 130 and 250 microsec; intensity: 1, 2, 5 and 10 mA; time: 5, 10, 20 and 30 minutes). S (10% fetal bovine serum in medium), SF (serum free medium) and 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment represented the experimental, negative and positive groups, respectively. We found that electrical stimulation with 10 Hz, a pulse width of 130 microsec, and a duration of 30 minutes gave a significant increase in AP-1 activity. In contrast, the intensity of the stimulation had no significant effect on AP-1 activity. In conclusion, our data suggest that electrical stimulation with an appropriate frequency, duration and pulse width could cause an increase in AP-1 activity in cells.