Chung-Hsi Chou

Hepatoprotection of silymarin against thioacetamide-induced chronic liver fibrosis

BACKGROUND Liver fibrosis is chronic liver damage usually caused by alcohol, viruses or other toxins and is characterised by an excessive accumulation of extracellular matrix proteins such as collagen. The aim of this study was to establish an animal model of chronic liver damage and investigate molecular mechanisms of silymarin hepatoprotective effects. RESULTS Thioacetamide (TAA; 100 mg kg(-1) intraperitoneal (i.p.) injection three times weekly) effectively induced chronic liver fibrosis in male ICR mice. Then 24 ICR mice were randomly divided into four groups: (1) saline (i.p.) + water (gavage); (2) saline (i.p.) + 150 mg kg(-1) silymarin (gavage); (3) 100 mg kg(-1) TAA (i.p.) + water (gavage); (4) 100 mg kg(-1) TAA (i.p.) + 150 mg kg(-1) silymarin (gavage). Eight weeks of TAA treatment resulted in lower body weight, serum cholesterol and triglycerides as well as increased liver size, ALT, AST and LDH values (P < 0.05). These TAA-induced effects were attenuated by silymarin (P < 0.05); therefore silymarin also ameliorated TAA-induced liver lesions. Effects of silymarin on TAA-induced chronic liver damage may be attributed to down-regulation of hepatic MMP-2, MMP-13, TIMP-1, TIMP-2, AP-1, KLF6, TGF-β1, α-SMA and COL-α1. CONCLUSION A mouse model of chronic liver fibrosis was successfully established by injecting 100 mg kg(-1) TAA three times weekly in male ICR mice. Meanwhile, silymarin showed hepatoprotection against TAA-induced damage.

Salmonellae and campylobacters in household and stray dogs in northern Taiwan.

Rectal swabs were collected from 437 household and 491 stray dogs in northern Taiwan from May 2003 to June 2005 to investigate the prevalence and antimicrobial susceptibilities of salmonellae and campylobacters. The results revealed that 2.1% of household dogs and 6.3% of stray dogs were positive for salmonellae, with Salmonella Duesseldorf being the most dominant serotype in both. Additionally, 2.7% of the household dogs and 23.8% of the stray dogs were positive for campylobacters. Campylobacter jejuni was the most prevalent species (86.8%), followed by C. upsaliensis (9.3%) and C. coli (3.9%). Both salmonella and campylobacter isolation rates from the stray dogs were significantly higher than those from the household dogs (p < 0.01). The susceptibility of 33 C. jejuni isolates to eight antimicrobials was studied by the E-test. A high rate of resistance was observed to azithromycin (93.9%), clindamycin (87.9%), erythromycin (81.8%), tetracycline (78.8%), chloramphenicol (69.7%), nalidixic acid (51.5%), gentamicin (33.3%), and ciprofloxacin (18.2%). The susceptibility of 40 Salmonella isolates to 15 antimicrobials was also studied by the disc-diffusion method. All the Salmonella isolates were susceptible to ciprofloxacin and ceftriaxone. Resistance was observed most frequently to tetracycline (77.5%), chloramphenicol (52.5%), and ampicillin (50%).

Prevalence and molecular characterization of chloramphenicol resistance in Riemerella anatipestifer isolated from ducks and geese in Taiwan

Riemerella anatipestifer is a Gram-negative bacterium that can cause disease in a wide range of wild and domesticated birds, especially waterfowl. The presence of an antibiotic-resistance gene in R. anatipestifer has not yet been reported, indicating the need for investigation. In the present study, 40.5% of R. anatipestifer isolates were found to exhibit resistance to chloramphenicol, while 45.9% showed intermediate resistance and 13.5% were susceptible to chloramphenicol, an antibiotic that has been prohibited for use in food animals in Taiwan since 2003. The resistance gene was identified as the cat gene and cloned by library sequencing. The prevalence of the cat gene in Taiwanese R. anatipestifer isolates was 78.4%. The position of the cat gene was then determined within the novel plasmid, designated pRA0511. pRA0511 was sequenced and shown to be 11,435 bp in size with 10 open reading frames (ORFs). Proteins putatively encoded by these 10 ORFs included four drug-resistance-associated proteins. Two proteins designed as chloramphenicol acetyltransferases (CATs) were encoded by two non-adjacent ORFs, and the other two were TetX2 and a multi-drug ABC transporter permease/ATPase. The putative CAT protein had 62.9 to 79.5% homology to a known type B CAT. The pRA0511 plasmid is the first identified drug-resistance plasmid in R. anatipestifer, more specifically associated with chloramphenicol resistance.

Amino acid, mineral, and polyphenolic profiles of black vinegar, and its lipid lowering and antioxidant effects in vivo

Black vinegar (BV) contains abundant essential and hydrophobic amino acids, and polyphenolic contents, especially catechin and chlorogenic acid via chemical analyses. K and Mg are the major minerals in BV, and Ca, Fe, Mn, and Se are also measured. After a 9-week experiment, high-fat/cholesterol-diet (HFCD) fed hamsters had higher (p<0.05) weight gains, relative visceral-fat sizes, serum/liver lipids, and serum cardiac indices than low-fat/cholesterol diet (LFCD) fed ones, but BV supplementation decreased (p<0.05) them which may resulted from the higher (p<0.05) faecal TAG and TC contents. Serum ALT value, and hepatic thiobarbituric acid reactive substances (TBARS), and hepatic TNF-α and IL-1β contents in HFCD-fed hamsters were reduced (p<0.05) by supplementing BV due to increased (p<0.05) hepatic glutathione (GSH) and trolox equivalent antioxidant capacity (TEAC) levels, and catalase (CAT) and glutathione peroxidase (GPx) activities. Taken together, the component profiles of BV contributed the lipid lowering and antioxidant effects on HFCD fed hamsters.

MMP-9 from sublethally irradiated tumor promotes Lewis lung carcinoma cell invasiveness and pulmonary metastasis

Matrix metalloproteinases (MMPs) associate with tumor progression and metastasis. We sought to investigate the role of MMP-9 from sublethally irradiated tumor in accelerated pulmonary metastasis of Lewis lung carcinoma (LLC-LM) and the corresponding anti-metastasis strategies in C57BL/6 mice. We used Matrigel-coated Boyden chamber assays and chicken chorioallantoic membrane assays to evaluate the invasion capability of irradiated LLC-LM cells (7.5 Gy), reverse transcription–polymerase chain reaction and the western blot assay to investigate the expression of MMPs by irradiated cells, and small interfering RNA duplexes to inhibit MMP-9 expression. LLC-LM cells differing in MMP-2 or -9 expression were subcutaneously injected into right thighs and the resulting tumors were irradiated (10 Gy × 5) to induce pulmonary metastasis. Radiation significantly enhanced MMP-9 at both the transcriptional and translational levels. MMP-9 siRNA significantly inhibited in vitro radiation-enhanced invasiveness. The number of radiation-accelerated pulmonary metastases was significantly reduced by MMP-9 knockdown and MMP-2/9 knockdown. Reverse transcription–polymerase chain reaction of LLC-LM cells in the blood and lung tissue revealed MMP-9 involvement in radiation-enhanced intravasation. Either higher-dose irradiation (30 Gy × 2) or pretreatment with prototypical MMP-9 inhibitor, zoledronic acid, significantly reduced the number of pulmonary metastases. The viability of irradiated tumor was seen on both positron emission tomography and magnetic resonance imaging, and tumor/serum MMP-9 levels suggested the association of local control of primary tumor and inhibition of time-dependent MMP-9 activities. Our results demonstrate that MMP-9 is crucially involved in radiation-enhanced LLC-LM cell invasiveness in vitro and in pulmonary metastasis from inadequately irradiated primary tumor in vivo.

Detection of florfenicol resistance genes in Riemerella anatipestifer isolated from ducks and geese

The cat gene, coding for chloramphenicol acetyltransferase has been reported for conferring the chloramphenicol resistance for Riemerella anatipestifer. Chloramphenicol acetyltransferases, however, are unable to inactivate florfenicol. In this study, 66 R. anatipestifer isolates were investigated for their susceptibility to chloramphenicol and florfenicol and the presence of floR gene. Results showed nine florfenicol intermediate or resistant R. anatipestifer isolates were all floR positive. The expression of floR gene in E. coli and inhibition studies with PAβN indicated that the floR gene was as an efflux pump conferring resistance to both chloramphenicol and florfenicol. Southern hybridization revealed the floR was located in the plasmid DNA of five isolates and in the chromosomal DNA of four isolates. Furthermore, two novel floR-carrying plasmids designated pRA0726 and pRA0846 were sequenced completely. pRA0726 was 11,704 bp in size with 10 putative open reading frames which included the floR, catB and bla(OXA-209) resistance genes. The most differences between sequences of pRA0846 and pRA0726 were the absence of a bla(OXA-209) gene and the deletion of 321 nucleotides of orf1 in pRA0846. Plasmid curing tests demonstrated that pRA0726 carried functional coding proteins for resistance to phenicol and β-lactam antimicrobials. To the best of our knowledge, this is the first report of presence of the floR and bla(OXA-209) resistance genes in R. anatipestifer.

Prevalence and typing of Listeria monocytogenes in raw catfish fillets.

Raw channel catfish fillets collected from three processing plants during four time periods were tested for the presence of Listeria species. Listeria monocytogenes was the predominant Listeria species found in these catfish fillets, with 25 to 47% prevalence. Other Listeria species, such as L. welshimeri, L. innocua, L. ivanovii, L. grayi, and L. seeligeri, were also found. L. monocytogenes isolates were further fingerprinted by a repetitive element PCR. Forty distinctive electrophoretic types (ETs) and three genetic clusters were determined by Dice coefficient analysis and UPGMA (unweighted pair group method using arithmetic averages). Twenty of 40 ETs were represented by a single isolate, and the other 20 ETs were represented by 2 to 11 isolates. Thirty-five ETs, represented by 76 isolates, were found in processing plant A, B, or C and designated plant-specific types. The remaining five ETs, represented by 21 isolates, were found in multiple plants and designated nonplant-specific types. In addition, 10 ETs from 52 isolates were found repeatedly during different seasons. Plant-specific and nonplant-specific L. monocytogenes coexisted in processed catfish fillets. Some isolates were persistently found in processed fillets, suggesting that either the current sanitation procedures used by these plants are inadequate or that these isolates originated from the natural habitats of the catfish. The results also suggest that the repetitive element PCR is a useful tool for differentiating L. monocytogenes subtypes and can be used for tracing the source of a contamination.

Prebiotic effect of diosgenin, an immunoactive steroidal sapogenin of the Chinese yam.

This study investigated the effect of diosgenin, a yam-derived phytochemical, on the growth of enteric lactic acid bacteria (LAB). The in vivo effect of diosgenin on the density of intestinal flora was examined in a murine model of food allergy. Oral administration with diosgenin markedly restored the diminished density of faecal LAB associated with allergic reactions. The direct effect of diosgenin and several structure-related steroidal compounds on the growth of faecal anaerobes isolated from diosgenin-administered mice was also investigated. The presence of diosgenin significantly enhanced the growth of Lactobacillus murinus and Lactobacillus reuteri, but not enterococci. Structure-activity relationship analysis showed that the prebiotic activity of steroidal sapogenins might require structural elements of the C5-C6 double bond and intact E- and F-rings. Collectively, these results indicate that steroidal sapogenins may be a novel class of prebiotics to LAB.

A time-course study of gene responses of chicken granulosa cells to Salmonella Enteritidis infection

Consumption of eggs contaminated with Salmonella Enteritidis (SE) has been recognized as one of the important causes of human foodborne salmonellosis. Chicken granulosa cells (cGCs) comprise the last tissue layer surrounding the yolk in preovulatory follicles and are a preferred site for SE invasion. To understand the cGC response to SE infection, we conducted an in vitro time-course study to identify cGC transcriptional changes using chicken whole genome microarrays. The expression of 135 (4h postinfection) and 120 cGC genes (48 h postinfection) were altered (P<.01) compared to uninfected cells. Many of the altered genes were related to immune response, physiological processes, signal transduction, and transcription. Furthermore, we also found that the Jak-STAT pathway, which is essential in the regulation of cellular cytokines and growth factors, was highly active in this study. Among the genes identified by microarray, the mRNA levels of TLR15, IL-6, CXCLi1, CXCLi2, and K203 were shown to be upregulated by real-time RT-PCR (qRT-PCR). In contrast, the mRNA levels of RASD1 and HB-EGF decreased according to both microarray and qRT-PCR analyses. These results suggest that during the SE infection, cGCs recruit cells of the innate immune responses; the infection may also induce suppression of cGC cell proliferation, which alters follicular development and ovulation.

Expression of GALNT2 in human extravillous trophoblasts and its suppressive role in trophoblast invasion

Extravillus trophoblast (EVT) invasion plays a critical role in placental development. Integrins bind to extracellular matrix (ECM) proteins to mediate EVT cell adhesion, migration, and invasion. Changes in O-glycans on β1-integrin have been found to regulate cancer cell behavior. We hypothesize that O-glycosyltransferases can regulate EVT invasion through modulating the glycosylation and function of β1-integrin. Here, we found that the GALNT1 and GALNT2 mRNA were highly expressed in HTR8/SVneo and first trimester EVT cells. Immunohistochemstry and immunofluorescence staining showed that GALNT2 was expressed in subpopulations of EVT cells in deciduas, but not in syncytiotrophoblasts and cytotrophoblasts of placental villi. The percentage of GALNT2-positive EVT cells increased with gestational ages. Overexpression of GALNT2 in HTR8/SVneo cells significantly enhanced cell-collagen IV adhesion, but suppressed cell migration and invasion. Notably, we found that GALNT2 increased the expression of Tn antigen (GalNAc-Ser/Thr) on β1-integrin as revealed by Vicia Villosa agglutinin (VVA) binding. Furthermore, GALNT2 suppressed the phosphorylation of focal adhesion kinase (FAK), a crucial downstream signaling molecule of β1-integrin. Our findings suggest that GALNT2 is a critical initiating enzyme of O-glycosylation for regulating EVT invasion.