Chien-Yun Hsiang

Vanillin Inhibits Matrix Metalloproteinase-9 Expression through Down-Regulation of Nuclear Factor-κB Signaling Pathway in Human Hepatocellular Carcinoma Cells

Vanillin has been reported to exhibit anti-invasive and antimetastatic activities by suppressing the enzymatic activity of matrix metalloproteinase-9 (MMP-9). However, the underlying mechanism of anti-invasive activity remains unclear so far. Herein we demonstrate that vanillin reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 gelatinolytic activity and suppressed cell invasion through the down-regulation of MMP-9 gene transcription in HepG2 cells. Vanillin significantly reduced the 6.6-fold invasive capacity of HepG2 cells in noncytotoxic concentrations, and this anti-invasive effect was concentration-dependent in the Matrigel invasion assay. Moreover, vanillin significantly suppressed the TPA-induced enzymatic activity of MMP-9 and decreased the induced mRNA level of MMP-9. Analysis of the transcriptional regulation indicated that vanillin suppressed MMP-9 transcription by inhibiting nuclear factor-κB (NF-κB) activity. Western blot further confirmed that vanillin inhibited NF-κB activity through the inhibition of IκB-α phosphorylation and degradation. In conclusion, vanillin might be a potent antiinvasive agent that suppresses the MMP-9 enzymatic activity via NF-κB signaling pathway.

Toona sinensis and its major bioactive compound gallic acid inhibit LPS-induced inflammation in nuclear factor-κB transgenic mice as evaluated by in vivo bioluminescence imaging.

In the present study, we investigated the anti-inflammatory effects of a nutritious vegetable Toona sinensis (leaf extracts, TS) and its major bioactive compound gallic acid (GA) by analysing LPS-induced NF-κB activation in transgenic mice, using bioluminescence imaging. Mice were challenged intraperitoneally with LPS (1mg/kg) and treated orally with TS or GA (100 or 5mg/kg, respectively). In vivo and ex vivo imaging showed that LPS increased NF-κB luminescence in the abdominal region, which was significantly inhibited by TS or GA. Immunohistochemical and ELISA analyses confirmed that TS and GA inhibited LPS-induced NF-κB, interleukin-1β, and tumour necrosis factor-α expression. Microarray analysis revealed that biological pathways associated with metabolism and the immune responses were affected by TS or GA. Particularly, LPS-induced thioredoxin-like 4B (TXNL4B) 2 expression in the small intestine, and TXNL4B, iNOS, and COX-2 expression in RAW 264.7 cells were significantly inhibited by TS or GA. Thus, the anti-inflammatory potential of TS was mediated by the downregulation of NF-κB pathway.

5-Fluorouracil Induced Intestinal Mucositis via Nuclear Factor-κB Activation by Transcriptomic Analysis and In Vivo Bioluminescence Imaging

5-Fluorouracil (5-FU) is a commonly used drug for the treatment of malignant cancers. However, approximately 80% of patients undergoing 5-FU treatment suffer from gastrointestinal mucositis. The aim of this report was to identify the drug target for the 5-FU-induced intestinal mucositis. 5-FU-induced intestinal mucositis was established by intraperitoneally administering mice with 100 mg/kg 5-FU. Network analysis of gene expression profile and bioluminescent imaging were applied to identify the critical molecule associated with 5-FU-induced mucositis. Our data showed that 5-FU induced inflammation in the small intestine, characterized by the increased intestinal wall thickness and crypt length, the decreased villus height, and the increased myeloperoxidase activity in tissues and proinflammatory cytokine production in sera. Network analysis of 5-FU-affected genes by transcriptomic tool showed that the expression of genes was regulated by nuclear factor-κB (NF-κB), and NF-κB was the central molecule in the 5-FU-regulated biological network. NF-κB activity was activated by 5-FU in the intestine, which was judged by in vivo bioluminescence imaging and immunohistochemical staining. However, 5-aminosalicylic acid (5-ASA) inhibited 5-FU-induced NF-κB activation and proinflammatory cytokine production. Moreover, 5-FU-induced histological changes were improved by 5-ASA. In conclusion, our findings suggested that NF-κB was the critical molecule associated with the pathogenesis of 5-FU-induced mucositis, and inhibition of NF-κB activity ameliorated the mucosal damage caused by 5-FU.

Vanillin Improves and Prevents Trinitrobenzene Sulfonic Acid-Induced Colitis in Mice

Inflammatory bowel disease (IBD) is chronic inflammatory and relapsing disease of the gut. It has been known that activation of nuclear factor-κB (NF-κB) and production of proinflammatory cytokines play important roles in the pathogenesis of IBD. In this study, the effect of vanillin (4-hydroxy-3-methoxybenzaldehyde), a potent nuclear factor-κB (NF-κB) inhibitor, was evaluated in mice with trinitrobenzene sulfonic acid (TNBS)-induced colitis. Oral administration of vanillin improved macroscopic and histological features of TNBS-induced colitis in a dose-dependent manner. Vanillin not only prevented TNBS-induced colitis but also ameliorated the established colitis. By in vivo NF-κB bioluminescence imaging, electrophoretic mobility shift assay, and Western blot, we found that vanillin suppressed in vivo NF-κB activities through the inhibition of p65 translocation, inhibitor of nuclear factor-κB(IκB)-α phosphorylation, and IκB kinase activation. Furthermore, vanillin reduced the expressions of proinflammatory cytokines [interleukin (IL)-1β, IL-6, interferon-γ, and tumor necrosis factor-α] and stimulated the expression of anti-inflammatory cytokine (IL-4) in colonic tissues. In conclusion, this work identified vanillin as an anti-inflammatory compound with the capacity to prevent and ameliorate TNBS-induced colitis. Due to its safety, vanillin could be a potent candidate for the treatment of IBD.

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 extract and zingerone ameliorated trinitrobenzene sulphonic acid-induced colitis in mice via modulation of nuclear factor-κB activity and interleukin-1β 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-1β (IL-1β) 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-1β protein level in the colon. In conclusion, ginger improved TNBS-induced colitis via modulation of NF-κB activity and IL-1β signalling pathway. Moreover, zingerone might be the active component of ginger responsible for the amelioration of colitis induced by TNBS.

Differential regulation of activator protein 1 activity by glycyrrhizin.

Glycyrrhizin, a major component of Glycyrrhiza uralensis (licorice) root, is a saponin and exhibits a number of pharmacological effects, including anti-inflammation, anti-ulcer, anti-allergy, and anticarcinogenesis. Activator protein I (AP-1), a nuclear transcription factor, consists of Jun/Fos heterodimers or Jun/Jun homodimers, and blocking of tumor promoter-induced AP-1 activity could inhibit induced cellular transformation. In order to elucidate the molecular mechanism of glycyrrhizin-induced anticarcinogenesis, effect of glycyrrhizin on the AP-1 activity in untreated and tumor promoter-12-O-tetradecanoylphorbol-13-acetate (TPA)-treated conditions was analyzed in this study. Glycyrrhizin induced the AP-1/TATA reporter activity in a dose-dependent fashion, which was judged by chloramphenicol acetyltransferase assay and electrophoretic mobility-shift assay. Similar results were observed in HepG2 and Vero cells, suggested that glycyrrhizin effect was cell type-independent. In addition, the cis element responsible for glycyrrhizin activity was AP-1 responsive element. Further analysis indicated that glycyrrhizin exhibited a different regulation on the AP-1 activity in untreated and TPA-treated cells. Glycyrrhizin induced the AP-1 activity in untreated cells, while it inhibited the TPA-induced AP-1 activation in TPA-treated cells. These results provide insight into the biological actions of glycyrrhizin and the molecular basis for the development of new chemoprotective agents for cancer.

Inhibitory effect of anti-pyretic and anti-inflammatory herbs on herpes simplex virus replication.

The increasing clinical use of acyclovir, ganciclovir, and foscarnet against herpes simplex virus (HSV), varicella-zoster virus, and cytomegalovirus has been associated with the emergence of drug-resistant herpesvirus strains. To develop anti-HSV compounds from plants, 31 herbs used as antipyretic and anti-inflammatory agents in Chinese medicine were screened. Five different preparations (cold aqueous, hot aqueous, ethanolic, acid ethanolic, and methanolic) from 31 herbs were analyzed by plaque reduction assay, and 7 extracts. which showed significant antiviral activities, were further elucidated for their antiviral mechanisms. Our results showed that ethanolic extract of Rheum officinale and methanolic extract of Paeonia suffruticosa prevented the process of virus attachment and penetration. Aqueous extract of P. suffruticosa and ethanolic extract of Melia toosendan inhibited virus attachment to cell surface. Aqueous extract of Sophora flavescens and methanolic extract of M. toosendan showed no effect on virus attachment and penetration. These data indicated that these 4 herbs have a potential value as a source of new powerful anti-HSV compounds.

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

Abstract 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 IC50 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 10μg/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.

Antigenicity and receptor-binding ability of recombinant SARS coronavirus spike protein.

Abstract Severe acute respiratory syndrome (SARS) is an emerging infectious disease associated with a novel coronavirus and causing worldwide outbreaks. SARS coronavirus (SARS-CoV) is an enveloped RNA virus, which contains several structural proteins. Among these proteins, spike (S) protein is responsible for binding to specific cellular receptors and is a major antigenic determinant, which induces neutralizing antibody. In order to analyze the antigenicity and receptor-binding ability of SARS-CoV S protein, we expressed the S protein in Escherichia coli using a pET expression vector. After the isopropyl-β-d-thiogalactoside induction, S protein was expressed in the soluble form and purified by nickel-affinity chromatography to homogeneity. The amount of S protein recovered was 0.2–0.3mg/100ml bacterial culture. The S protein was recognized by sera from SARS patients by ELISA and Western blot, which indicated that recombinant S protein retained its antigenicity. By biotinylated ELISA and Western blot using biotin-labeled S protein as the probe, we identified 130-kDa and 140-kDa proteins in Vero cells that might be the cellular receptors responsible for SARS-CoV infection. Taken together, these results suggested that recombinant S protein exhibited the antigenicity and receptor-binding ability, and it could be a good candidate for further developing SARS vaccine and anti-SARS therapy.