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The objective was to investigate endocrine-disrupting effects of polar compounds from oxidized frying oil. Estrogenicity of polar compounds was tested with a rat uterotrophic bioassay. Dietary oxidized frying oil (containing 51% polar compounds) or polar compounds isolated from it were incorporated into feed (in lieu of fresh soybean oil) and fed to ovariectomized rats, with or without treatment with exogenous ethynyl estradiol. Exogenous estrogen restored uterine weight, and caused histological abnormalities (stratified epithelia and conglomerate glands) as well as proliferation of uterine epithelial cells. However, tamoxifen or polar compounds reduced these effects. Furthermore, tamoxifen or polar compounds down-regulated uterine mRNA expression of estrogen receptor (ER)-target genes, implicating reduced ER activity in this hypo-uterotrophic effect. Inhibition of ER signaling and mitosis by polar compounds were attributed to reduced MAPK and AKT activation, as well as a reduced ligand binding domain-transactivity of ERα/β. We concluded polar compounds from frying oil are potential endocrine-disrupting chemicals, with implications for food and environmental safety.

The present study aimed to establish embryonic stem (ES) cell lines, i.e., ntES cells, using rabbit blastocyst stage embryos cloned by somatic cell nuclear transfer. First, we investigated the development of cloned rabbit embryos reconstructed with normal fibroblasts and fibroblasts transfected with enhanced green fluorescence protein (eGFP). Blastocyst rates were 27.4% and 23.9%, respectively, for the embryos reconstructed with normal fibroblasts and fibroblasts transfected with eGFP compared with that from the parthenogenetic group (43.1%). One ntES cell line was established from embryos reconstructed with eGFP-transfected fibroblasts (1 of 17, 5.9%), and three ntES cell lines were derived from those with normal fibroblasts (3 of 17, 17.6%). All the ntES cell lines retained alkaline phosphatase activity and expressed ES cell-specific markers SSEA-4, Oct-4, TRA-1-60, and TRA-1-81. The pluripotency was further confirmed by reverse transcription-polymerase chain reaction analyses of Oct-4, Nanog, and Sox-2 expressions in ntES cell lines. The differentiation capacity of ntES cells was also examined in vitro and in vivo, by which these ntES cell lines were able to differentiate into all three germ layers through embryoid bodies and teratomas. In conclusion, it is apparent that the efficiency of ntES cells derived using eGFP-transfected donor cells is lower than that with nontransfected, normal fibroblasts donor cells. Similar to those from parthenogenetic embryos, all ntES cell lines derived from cloned rabbit embryos are able to express pluripotency markers and retain their capability to differentiate into various cell lineages both in vitro and in vivo.

Background Few studies have evaluated the effects and precise molecular mechanism of mycophenolate mofetil (MMF) in the treatment of human cutaneous lupus erythematosus (CLE). Our findings shed light on the therapeutic effects of MMF in a UVB-induced NZB × NZW (NZBW) F1 CLE mouse model. Methods Continuous MMF treatment (60 mg/kg/day) was administered up to Day 50 from the beginning of UVB induction (Day 0; 20 weeks old), as the pathologic features of CLE are present after 50 days. The therapeutic effects of MMF treatment in NZBW lupus mice were examined by comparing histopathological changes, lupus band test (deposition of immune complexes at the dermal–epidermal junction) and colocalization of autoantibodies with a dermal autoantigen Dsg3, and by evaluating the associations of local matrix metalloprotease activities. Results MMF improved survival in the NZBW lupus mice from 35.7% to 81.8%. The proteinuria, blood urea nitrogen, and interleukin 6 levels were significantly reduced after MMF treatment. The dermal lymphocytic infiltration, deposition of immune complexes at the dermal–epidermal junction, colocalized autoantibodies with Dsg3, and epidermal matrix metalloprotease activity were also attenuated in MMF-treated NZBW F1 mice. Conclusion The results confirmed that MMF could substantially attenuate skin damage due to CLE in the NZBW F1 mouse model.

Trypanosoma (subgenus Megatrypanum) theileri was first identified over one hundred years ago, and is a widespread parasite in cattle. Its life cycle within the mammalian host has rarely been reported. Whether there is an intracellular stage in tissues is unknown and such a stage has not been demonstrated experimentally. Intriguingly, using Giemsa staining with light microscopy and transmission electron microscopy examination, we found that the parasite was able not only to attach to cells but also to invade several phagocytic and non-phagocytic mammalian cells. Based on these findings, we conducted further investigations using a special antibody in immunofluorescence confocal images. Moreover, we examined a series of possible events of cell invasion in T. theileri. The results revealed that GM1, a marker of membrane rafts, was implicated in the mechanism of entry by this parasite. After incubation with tissue culture trypomastigotes, the gelatinolytic activity was significantly increased and accumulated at the attachment sites. Using ultrastructural localization detection by CytoTracker live imaging and confocal immunofluorescence microscopy, we found that lysosome fusion and the autophagy pathway were engaged in invaginating processes. T. theileri amastigotes also invaded cells and were enclosed by the lysosomes. Furthermore, tissue-cultured trypomastigotes were found to be capable of triggering intracellular free Ca2+ transients and TGF-β-signaling. Our findings that intracellular amastigote stages exist in mammalian cells infected with T. theileri and that the invasion processes involved various host cell components and cell signalings were extremely surprising and warrant further investigation.

Background This study aimed to investigate the relationship between oxidized regenerated cellulose (ORC) and mucosa healing in an experimental animal model. Methods Fifteen adult Sprague-Dawley rats were randomly divided into three groups that underwent different wound treatments. In Group 1, no pharyngeal wound was created. In Group 2, the pharyngeal wound was sutured with Prolene only. In Group 3, the pharyngeal wound was sutured with Prolene, and covered with one layer of ORC before closure of the skin wound. The animals were euthanized either 5 or 10 days after operation, and wound conditions were inspected and recorded. Specimens including sections of larynx and pharynx/upper esophagus were taken for microscopic and molecular biological examination. Results The pharyngotomy/esophagotomy wounds achieved good healing outcomes 10 days after operation. Wounds treated with ORC had significantly diminished inflammatory cell infiltration in microscopic examination when compared with that of those without ORC 5 days after operation. The matrix metalloproteinases (MMP) expression level was higher in wounds of Group 2 and Group 3, when compared with that of group 1. In addition, the MMP expression level was lower in the ORC-treated wounds when compared with that of those without ORC. There was no significant difference in fibroblast proliferation, collagen deposition, endothelin-1, alpha-smooth muscle actin, and transforming growth factor beta 1 expression level between wounds treated with ORC and those without ORC. Conclusion Reduced inflammatory response and decreased MMP expression level was observed in ORC-treated wounds. Whether ORC facilitates mucosa healing requires further investigation.

Tumor necrosis factor-alpha (TNF-α) plays a central role in cellular necrosis, apoptosis, organ failure, tissue damage, inflammation and fibrosis. These processes, occurring in liver injury, may lead to cirrhosis. Thalidomide, α-N-phthalidoglutarimide, (C13H10N2)4, has been shown to have immunomodulatory and anti-inflammatory properties, possibly mediated through its anti-TNF-α effect. In this study, we investigated the in vitro and in vivo effects of thalidomide on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-β1 (TGF-β1) or TNF-α. The inhibitory effects of thalidomide on the NFκB signaling cascade and fibrosis markers including α-smooth muscle actin (α-SMA) and collagen, were assessed. An in vivo therapeutic study was conducted in dimethylnitrosamine (DMN)-treated rats, which were randomly assigned to 1 of 4 groups: vehicle (0.7% carboxyl methyl cellulose, CMC), thalidomide (40 mg/kg), thalidomide (200 mg/kg), or silymarin (50 mg/kg), each given by gavage twice daily for 3 weeks starting after 1 week of DMN administration. Thalidomide (100–800 nM) concentration-dependently inhibited NFκB transcriptional activity induced by TNF-α, including IKKα expression and IκBα phosphorylation in HSC-T6 cells. In addition, thalidomide also suppressed TGF-β1-induced α-SMA expression and collagen deposition in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats receiving high dose of thalidomide (0.89±0.20) were significantly reduced in comparison with those of DMN-treated rats receiving vehicle (1.56±0.18). Hepatic collagen contents of DMN rats were also significantly reduced by either thalidomide or silymarin treatment. Immunohistochemical double staining results showed that α-SMA- and NFκB-positive cells were decreased in the livers from DMN rats receiving either thalidomide or silymarin treatment. In addition, real-time PCR analysis indicated that hepatic mRNA expressions of TGF-β1, α-SMA, collagen 1α2, TNF-α and iNOS genes were attenuated by thalidomide treatment. In conclusion, our results showed that thalidomide inhibited activation of HSC-T6 cells by TNF-α and ameliorated liver fibrosis in DMN-intoxicated rats.

Bronchiolitis obliterans organizing pneumonia (BOOP) is a chronic respiratory disease. Although the pathogenesis of BOOP is still incompletely understood, BOOP is responsive to steroids and has a good prognosis. In our five pigs with chronic postweaning multisystemic wasting syndrome (PMWS), typical BOOP lesions were revealed. All five porcine lungs showed typical intraluminal plugs, and porcine circovirus type 2 (PCV2) was identified. They also exhibited similar pathologic findings such as proliferation of type II pneumocytes and myofibroblasts (MFBs), extracellular collagen matrix (ECM) deposition, and fragmentation of elastic fibers. MFBs migration correlative molecules, for instance, gelatinase A, B and osteopontin, appeared strongly in the progressing marginal area of polypoid intraluminal plugs of fibrotic lesion. These molecules colocalized with the active MFBs. Both gelatinase activity and intercellular level of active MFBs were significantly increased (). Porcine chronic bronchopneumonia leads to BOOP and it is associated with PCV2 persistent infection. Swine BOOP demonstrates similar cellular constituents with human BOOP. Perhaps their molecular mechanisms of pathogenesis operate in a similar way. Thus we infer that the swine BOOP can be considered as a potential animal model for human BOOP associated with natural viral infection. Moreover, it is more convenient to obtain samples.

Baculovirus is an enveloped virus that infects insects in nature and has emerged as a novel vaccine vector. We previously constructed a recombinant baculovirus displaying the hemagglutinin protein (HA) of avian influenza virus (AIV) on the viral envelope (Bac-HA64), and demonstrated the induction of humoral responses in immunized mice. To improve the vector design and explore how the vector forms influence the vaccine efficacy, we constructed two more baculoviruses Bac-CHA and Bac-CHA/HA64. Bac-CHA expressed HA after transducing the host cells while Bac-CHA/HA64 not only expressed HA but also displayed HA on the envelope. After administration into BALB/c mice, all three vectors elicited HA-specific humoral (IgG1, IgG2a and hemagglutination inhibition titers), mucosal (IgA titers) and cellular (interferon (IFN)-γ and IL-4 producing T cells and IFN-γ(+)/CD8(+) T cells) immune responses. Intriguingly, the magnitudes and types of responses hinged on the vaccine form and administration route. Via intranasal (i.n.) and subcutaneous (s.c.) inoculation, the HA-displaying vectors Bac-HA64 and Bac-CHA/HA64 triggered stronger humoral and mucosal responses than Bac-CHA, but upon intramuscular (i.m.) injection the HA-expressing vectors (Bac-CHA and Bac-CHA/2HA64) elicited more robust humoral and cellular responses than Bac-HA64. Via either administration route, the dual form vaccine Bac-CHA/HA64 gave rise to superior or at least comparable HA-specific immune responses than the other two vaccine forms, implicating the potential of Bac-CHA/HA64 as a vaccine candidate against AIV infection.

Although Trypanosoma (Megatrypanum) theileri, a blood parasite of bovid species, is spread widely throughout the world, it has never been reported in Taiwan. When an anti-coagulated blood sample from febrile dairy cattle was directly smeared, no parasite was observed. However, a highly distinctive morphological feature of trypanosome appeared in baby hamster kidney (BHK) cell culture inoculated with non-thrown blood buffy coat. The different stages and typical ultrastructures of trypanosome were observed in our isolate. The isolate was subsequently identified as T. theileri by species-specific PCR assay (Tth625), 18S rDNA sequencing alignment and internal transcribed spacer of ribosomal genes (ITS) as a marker for molecular phylogenetic analysis. The first T. theileri isolate in Taiwan (TWTth1) could be periodically passaged in BHK cell culture for more than one year and retained good re-cryopreservation viability. The BHK culture method would be excellent for diagnostic isolation and maintenance long-term development of this parasite.

Detection of the myocardial damage in the early stage of subarachnoid hemorrhage (SAH) is impossible without a good animal model. We investigated the feasibility of using immunohistochemical cardiac troponin I (cTnI) staining to detect the early myocardial damage in rats subject to SAH by endovascular perforation. SAH induced increases in mean blood pressure, heart rate, intracranial pressure, plasma norepinephrine and cTnI accompanied with electrocardiogram abnormalities. The heart was dilated. The myocardium revealed coagulative myocytolysis and remarkable loss of myocardial tissue cTnI in myocytolytic areas within 3 h after SAH. Thus, we create a rat SAH model that can be applied in studying the mechanisms of the early myocardial damage following SAH and for evaluating therapeutic strategies in SAH patients.

Liver fibrosis has been characterized as chronic inflammatory processes involving multiple molecular pathogenetic pathways. This therapeutic study investigated whether a combination regimen of Salvia miltiorrhiza (S), Ligusticum chuanxiong (L) and Glycyrrhiza glabra (G) exerted in vivo antifibrotic effects on rats with hepatic fibrosis. Fibrosis was induced in rats by dimethylnitrosamine (DMN) administration for 4 weeks. Fibrotic rats were randomly assigned to one of the three groups: control, SLG (50 mg/kg) or silymarin (50 mg/kg), each given by gavage twice daily for 3 weeks starting 1 week after DMN injection. The results showed that fibrosis scores of livers from DMN-treated rats with SLG (1.13 ± 0.13) were significantly reduced in comparison with DMN-treated rats receiving vehicle (1.63 ± 0.18). Moreover, the hepatic collagen content of DMN rats was significantly reduced by either SLG or silymarin treatment. The double immunohistochemical staining results also showed that α-SMA positive cells with NFκB nuclear translocation were decreased in the fibrotic livers by SLG and silymarin treatments. The mRNA expression levels of TGF-β1, α-SMA, collagen1α2, iNOS and ICAM-1 genes were attenuated by SLG and silymarin treatment. The results showed that SLG exerted antifibrotic effects in rats with DMN-induced hepatic fibrosis. Copyright © 2007 John Wiley & Sons, Ltd.

The formation of pharyngo-cutaneous fistula (PCF) is controversial and various predisposing factors have been proposed. This study aimed to compare different suture materials in the closure of pharyngeal wounds in experimental animals. Fifty-two Sprague-Dawley rats were divided into three groups. Artificial pharyngotomy was performed and was then repaired with Vicryl, polypropylene, and Vicryl plus fibrin glue, respectively. Outcome measurements included gross wound inspection and histological examination. There was a significant difference in the rates of PCF formation between group I and group II (Fisher's exact test, p = 0.046). In addition, minimal inflammatory response was found in the polypropylene group when compared to the other groups. The fibrin glue-treated group had the highest fibroblast activity and collagen deposition. Polypropylene produced minimal tissue reaction, which facilitated the healing process. Therefore, proper selection of suture material can probably reduce the rate of PCF but should not be substituted for proper aseptic and meticulous surgical techniques.

Glutamate stimulation of the dorsal facial area, an area located dorsal to the facial nucleus, increases common carotid arterial blood flow. Nitrergic neurons are important in cardiovascular regulatory areas. We investigated whether the nitrergic neurons might be present and play a role in the dorsal facial area to regulate the arterial blood flow. Injections of l -arginine (an NO precursor) and sodium nitroprusside (an NO donor) into the area caused dose-dependent increases in the arterial blood flow. Injection of NG-nitro-arginine methyl ester ( l -NAME, an NO synthase inhibitor) or methylene blue (a guanylate cyclase inhibitor) decreased the arterial blood flow. Nitrergic neurons and fibers were found in the dorsal facial area by histochemical staining of nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase, a maker of NO synthase. In conclusion, nitrergic neurons are present in the dorsal facial area and appear to release NO tonically in stimulating the area to cause increase in common carotid arterial blood flow.

Acetaminophen (APAP) overdose causes severe liver and kidney damage. APAP-induced liver injury (AILI) represents the most frequent cause of drug-induced liver failure. APAP is relatively insoluble and can only be taken orally; however, its prodrug, propacetamol, is water soluble and usually injected directly. In this study, we examined the time-dependent effects of AILI after propacetamol injection in mice. After analyses of alanine aminotransferase and aspartate aminotransferase activities and liver histopathology, we demonstrated that a novel AILI mouse model can be established by single propacetamol injection. Furthermore, we compared the protective and therapeutic effects of galangin with a known liver protective extract, silymarin, and the only clinical agent for treating APAP toxicity, N-acetylcysteine (NAC), at the same dose in the model mice. We observed that galangin and silymarin were more effective than NAC for protecting against AILI. However, only NAC greatly improved both the survival time and rate consequent to a lethal dose of propacetamol. To decipher the hepatic protective mechanism(s) of galangin, galangin pretreatment significantly decreased the hepatic oxidative stress, increased hepatic glutathione level, and decreased hepatic microsomal CYP2E1 levels induced by propacetamol injection. In addition, propacetamol injection also reproduced the probability of APAP-induced kidney injury (AIKI), appearing similar to a clinical APAP overdose. Only galangin pretreatment showed the protective effect of AIKI. Thus, we have established a novel mouse model for AILI and AIKI using a single propacetamol injection. We also demonstrated that galangin provides significant protection against AILI and AIKI in this mouse model.

UNLABELLED Increased ECM in canine PVs. INTRODUCTION Cell uncoupling due to fibrosis or increased extracellular collagen matrix (ECM) affects the formation of ectopic focal activity. Whether or not the increase of ECM also exists in the pulmonary veins (PVs) with rapid atrial pacing is unknown. We sought to test the hypothesis that in chronic atrial pacing dogs with sustained atrial fibrillation (AF), the amount of ECM was increased in both atria and the PVs. METHODS AND RESULTS We induced sustained AF in dogs by rapid atrial pacing. Computerized mapping techniques were used to map both atria and the PVs. We also used histological assessment to quantify the amount of ECM. After 118+/-24 days of rapid atrial pacing, sustained AF was induced in 7 dogs. Repetitive rapid activities (RRAs) either continuously or intermittently arose from the PVs during sustained AF. Histological study shows that there was no fibrosis in both atrial free walls and the PVs. However, the amount of ECM was increased in these regions. The mean ECM surface area fraction at each region in the dogs with sustained AF was all significantly higher compared to the corresponding region in normal dogs. Similarly, the heterogeneity of the ECM surface area fraction at each region in the dogs with sustained AF was also all significantly higher compared to normal dogs. CONCLUSIONS In chronic atrial pacing-induced sustained AF, structural remodeling (i.e., inhomogeneous increase of ECM) also involves the PVs. Reduced coupling of the myocytes in the PV due to histological changes may provide an additional mechanism facilitating RRAs.

Fibrosis-related changes in livers of cirrhotic rats induced by dimethylnitrosamine (DMN) have not yet been fully clarified. The aim of this study was to investigate changes in molecular and biochemical markers in DMN-intoxicated rats. DMN was administered to Sprague-Dawley rats for 2 and 5 weeks to induce different degrees of hepatic fibrosis. Liver tissues were assessed for the degree of fibrosis and gene expression. Histological examination of the liver showed a progressive increase in fibrosis scores (1.33 +/- 0.21 and 3.03 +/- 0.29, respectively) and expansion of fibrous septa with collagen-staining fibers in rats after 2 and 5 weeks of DMN administration. Hepatic protein contents of alpha-smooth muscle actin (alpha-SMA) and total collagen were significantly higher in rats administered DMN for both 2 and 5 weeks compared with those in control rats. Hepatic mRNA expressions of alpha-SMA, transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor, tissue inhibitor of metalloproteinase-1, and procollagen I and III were increased in DMN rats after 2 and 5 weeks. Abnormal increases in plasma alanine transaminase (ALT) and aspartate transaminase (AST) levels, plasma and mitochondrial MDA levels, and portal venous pressure were also noted in DMN rats. DMN administration to rats for 2 and 5 weeks induced progressive increases in hepatic fibrosis scores, hepatic mRNA expressions of TGF-beta1 and procollagen I and III genes, plasma levels of ALT and AST, and portal venous pressure, as well as progressive decreases in both liver and body weights. Our results suggest that DMN administration in rats induces biochemical and molecular changes related to fibrogenesis in the liver.

Objectives : Heart failure is associated with increased plasma norepinephrine (NE) and endothelial apoptosis. Recent reports have suggested that endothelial dysfunction is an important target for future therapies of heart failure. However, whether NE can induce endothelial apoptosis and its mechanism remains unknown. Methods : Endothelial cells from neonatal rat heart were treated with various concentrations of NE for different durations. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) and DNA fragmentation assays. Caspase activity was measured using specific fluorogenic substrates. Proteins of Bcl-2 family and cytochrome c were assayed by Western blotting. Results : NE induced endothelial apoptosis in a dose- and time-dependent manner. After treatment for 48 h, increasing NE concentration from 5, 10, 50, 100 to 200 μM resulted in 6±3%, 14±5%, 43±4%, 66±5%, and 89±6% apoptotic cells, respectively. The apoptosis was accompanied by down-regulation of Bcl-2 protein synthesis but not by cytosolic cytochrome c translocation. Caspase-2, -3, -6 and -9 were activated during apoptosis and caspase-2 inhibitor (Z-VDVAD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) significantly attenuated the apoptosis. Overexpression of Bcl-2 inhibited caspase activity and decreased the apoptosis. Moreover, the NE-mediated apoptotic effect was attenuated by β- (β2>β>β1) adrenergic antagonists (ICI-118,551>propranolol>atenolol) but was not affected by α1- or α2-adrenergic antagonists (prazosin or yohimbine). Conclusion : Our study is the first report documenting that NE induces apoptosis in neonatal rat endothelial cells mainly through down-regulation of Bcl-2 protein and activation of the β-adrenergic (β2>β1) and caspase-2 pathways. β-Adrenergic antagonists and caspases inhibitors may be useful in the prevention and management of NE-mediated endothelial apoptosis during heart failure.

The role of serotonin and glutamate release in dorsal medulla (DM) for regulation of systemic arterial pressure (SAP) was examined with microdialysis and high performance liquid chromatograph in anesthetized cats. KCl-perfusion in DM increased serotonin and glutamate concentrations in DM. Perfusion of serotonin resulted in decreases in glutamate concentration and SAP. Perfusion of alaproclate, a serotonin reuptake inhibitor that produced an increase in serotonin concentration in DM, had the same results as perfusion of serotonin. In conclusion, serotonin and glutamate appeared to be tonically and endogenously released from nerve terminals in DM, and the decrease in SAP could be attributed to the decreased glutamate release resulting from inhibitory action of serotonin in DM. The putative roles of serotonin and glutamate in DM may be important in SAP regulation.

Non-alcoholic steatohepatitis (NASH) is associated with hepatic fibrogenesis. Despite well-known cholesterol-lowering action of statins, their mechanisms against NASH-mediated fibrogenesis remain unclear. This study aimed at investigating the in vitro and in vivo anti-fibrotic properties of fluvastatin (Flu). Palmitate (PA)-induced changes in intracellular hydrogen peroxide levels in primary rat hepatocytes (PRHs) and human hepatoma cell line (HepG2) were quantified by dichlorofluorescein diacetate (DCF-DA) dye assay, whereas changes in expressions of NADPH oxidase gp91 phox subunit, α-smooth muscle actin (α-SMA), and NFκB p65 nuclear translocation were quantified with Western blotting. Quantitative real-time polymerase chain reaction (q-PCR) was used to investigate mRNA expressions of pro-inflammatory genes (ICAM-1, IL-6, TNF-α). Conditioned medium (CM) from PA-treated PRHs was applied to cultured rat hepatic stellate cell line, HSC-T6, with or without Flu-pretreatment for 2 h. Pro-fibrogenic gene expressions (COL1, TIMP-1, TGF-β1, α-SMA) and protein expression of α-SMA were analyzed. In vivo study using choline-deficient L-amino acid defined (CDAA) diet-induced rat NASH model was performed by randomly assigning Wistar rats (n = 28) to normal controls (n = 4), CDAA diet with vehicles, and CDAA diet with Flu (5 mg/kg or 10 mg/kg) (n = 8 each) through gavage for 4 or 8 weeks. Livers were harvested for histological, Western blot (α-SMA), and q-PCR analyses for expressions of pro-inflammatory (IL-6, iNOS, ICAM-1) and pro-fibrogenic (Col1, α-SMA, TIMP-1) genes. In vitro, Flu (1–20 μM) inhibited PA-induced free-radical production, gp91 phox expression, and NFκB p65 translocation in HepG2 and PRHs, while CM-induced α-SMA protein expression and pro-fibrogenic gene expressions in HSC-T6 were suppressed in Flu-pretreated cells compared to those without pretreatment. Moreover, α-SMA protein expression was significantly decreased in HSC-T6 cultured with CM from PA-Flu-treated PRHs compared to those cultured with CM from PA-treated PRHs. Flu also reduced steatosis and fibrosis scores, α-SMA protein expression, mRNA expression of pro-inflammatory and pro-fibrogenic genes in livers of CDAA rats. We demonstrated PA-induced HSC activation through paracrine effect of hepatocyte in vitro that was significantly suppressed by pre-treating HSC with Flu. In vivo, Flu alleviated steatosis-induced HSC activation and hepatic fibrogenesis through mitigating inflammation and oxidative stress, suggesting possible therapeutic role of Flu against NASH.

Lupus nephritis (LN) is usually associated with widespread effacement of the podocytes’ foot processes leading to proteinuria. Induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via promoting podocytes’ motility and kidney permeability in the glomerulus. Very little is known about uPAR signaling in LN. Mycophenolate mofetil (MMF), an immunosuppressive agent, efficiently modulates the development of LN in humans and mice, but there are no data concerning the direct uPAR involvement on podocytes in LN. The MMF efficiency and uPAR involvement signaling in NZB×NZW F1 lupus-prone mice were examined by proteinuria, renal function and pathology, immune complex deposits, and uPAR expression of podocytes by immunofluorescence staining and quantitative RT-PCR. After MMF treatment, the proteinuria ( p

The purpose of this study was to investigate the effects of 50% red beans ethanol natto extracts fermented by Bacillus subtilis on hyperlipidemia in hamsters. Forty male hamsters were randomly assigned to normal control, high-fat control, and high-fat with RBNE (red beans natto extracts) (200 and 1000 mg/kg BW) groups. RBNE treatment for 8 weeks significantly decreased the body and visceral fat weight in hamsters. Compared to the high-fat diet group, the plasma total cholesterol (TC), total triacylglycerol (TAG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and oxidized LDL (oxLDL) concentrations as well as atherogenic index of plasma AIP [Log(TAG/HDL-C)] were significantly lower in the RBNE-fed group. The consumption of RBNE significantly reduced the liver contents of TC, esterified cholesterol (CE) and alanine aminotransferase (ALT), and increased a regulator of lipid metabolism: peroxisome proliferator-activated receptor alpha (PPAR-alpha). Furthermore, the circulating level of irisin was decreased. Practical Application The oral administration of Bacillus subtilis-fermented red bean natto extract (RBNE), a popular Asian dessert, effectively moderated the metabolism of lipids and positively affected the blood and liver profiles of hamsters fed with a high-cholesterol and high-fat diet. Importantly, RBNE significantly suppressed the increase in visceral fat mass. Additionally, RBNE appears to protect the liver and alleviate the magnitude of fatty liver development. RBNE also upregulated PPAR-alpha protein expression in the liver, suggesting that supplementation may improve lipid metabolism in part through the beta-oxidation of lipids and promotion of lipid secretion. Furthermore, RBNE may ameliorate hyperlipidemia involving irisin, a newly identified hormone, in hamsters fed a cholesterol- and lipid-enriched diet. In conclusion, RBNE with high hypocholesterolemic activities may be helpful for patients with atherosclerosis.

Background Matrix metalloproteinases (MMPs) are involved in acute myocardial dysfunction by degrading several intracellular contractile proteins, including cardiac troponin I (cTnI). Here, we examined the temporal profiles of MMPs and cTnI in plasma and myocardial tissue in the acute stage of subarachnoid hemorrhage (SAH). Materials and methods SAH was induced by the endovascular suture method in rats. Intracranial pressure and left ventricular (LV) function were recorded. Plasma cTnI and MMPs were measured at 0, 5, 15, 30, 60, 120, and 180 minutes after SAH. Myocardial cTnI and MMP activities were quantified at 30, 60 and 180 min after SAH from homogenized hearts. Results SAH-induced rats showed a marked decline in -LV dP/dt max (index of LV diastolic function). Plasma samples revealed a noticeable increase in cTnI and pro-MMP-9 activities over the course of 180 minutes. In myocardial tissue, there was a marked increase in pro-MMP-9, pro-MMP-2 activities and expression of activated MMP-2. Western blot analysis revealed a striking decrease in cTnI content and increase in cTnI degradation in myocardium. Simultaneous cTnI depletion and MMP-2 expression in myocardium was detected by immunohistochemistry as early as 30 minutes after SAH. MMPs correlated with -LV dP/dt max (% of baseline) both in plasma and in myocardial tissue. Furthermore, activated MMP-2 activity correlated positively with cTnI degradation in myocardium. Conclusions Early activation of MMPs was observed in myocardium and plasma following SAH. Activated MMP-2 may regulate proteolytic cTnI and contribute to myocardium stunning injury in SAH rats.

Shugan-Huayu powder (SHP) has been administered to outpatients with chronic liver disease without clear anti-fibrosis mechanism. To investigate the anti-fibrotic effects of SHP on liver fibrosis in a rat model and in hepatic stellate cells (HSCs) in vitro, rats were gavaged with CCl4 at 1.0 g/kg body weight twice a week for 8 weeks to induce liver fibrosis and the rats were randomly assigned to one of the three groups: -CCl4 alone, low-dose SHP and high-dose SHP. SHP was given by gavages 5 times a week for 8 weeks. Serum, livers and HSCs were assayed for serology, pathology, western blot, zymography and quantitative RT-PCR. Hepatic function improved as decreased serum aspartate aminotransferase and alanine aminotransferase, and collagen deposition and active HSCs were significantly reduced in CCl4-induced liver by SHP treatment. The expression of matrix metalloproteinase-2 (MMP-2) and transforming growth factor-beta1 (TGF-beta1) mRNA in fibrotic liver showed significant downregulation after SHP treatment. In vitro, inhibition of alpha-smooth muscle actin (alpha-SMA) expression and MMP-2 secretion of active HSCs were also noticed by SHP treatment. SHP has an antifibrotic effect on CCl4-induced liver fibrosis in rats. Anti-fibrotic mechanisms were probably inhibiting activation of HSCs and decreased expression of MMP-2 and TGF-beta1.

The aim of this study was to investigate if armepavine (Arm, C19H23O3N) could exert inhibitory effects against hepatic fibrosis in rats. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with tumour necrosis factor-α (TNF-α) to evaluate the inhibitory effects of Arm. Rats were injected with thioacetamide (TAA; 300 mg/kg, intraperitoneally) thrice a week for 4 weeks to induce hepatic fibrosis, with Arm (3 or 10 mg/kg) given by gavage twice a day. Liver sections were taken for western blotting, fibrosis scoring and immunofluorescence staining. Arm (1–10 µm) concentration-dependently attenuated TNF-α-stimulated: (i) protein expressions of α-smooth muscle actin (α-SMA), collagen type I and angiopoietin-1; (ii) H2O2 production; and (iii) NF-κB, JunD and C/EBPs (cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT)/enhancer binding protein-s (EBPs)) nuclear translocations in HSC-T6 cells. In vivo Arm treatment significantly reduced plasma aspartate transaminase and alanine transaminase levels, hepatic α-SMA expression and collagen contents, and fibrosis scores of TAA-injected rats. Moreover, Arm treatment decreased α-SMA- and NF-κB-positive cells in immunohistochemical staining, and mRNA expression levels of IL-6, TGF-s1, TIMP-1, col1α2, iNOS and ICAM-1 genes, but up-regulated the metallothionein gene in the livers of TAA-injected rats. Our results indicated that Arm exerted both in vitro and in vivo antifibrotic effects in rats, with inhibition of NF-κB, JunD and C/EBPs pathways. Copyright © 2011 John Wiley & Sons, Ltd.

We investigated the individual and combined effects of leukemia inhibitory factor (LIF) and basic fibroblast growth factor 2 (bFGF2) on the derivation and maintenance of rabbit embryonic stem cell lines isolated from parthenogenetic activated embryos (p-rES). First, we demonstrated that p-rES cell lines can be prevented from differentiation via LIF (STAT3) and bFGF2 (MEK-ERK1/2 and PI3K-AKT) signaling on MEF feeders. High levels of ERK1/2 and AKT activities were crucial for maintaining p-rES cells in an undifferentiated state. Although the p-rES cells under the influence of LIF (500, 1000, and 2000 U/mL) or bFGF2 (5, 10, and 20 ng/mL) alone showed enhanced expression in the pluripotency markers, the highest levels of marker expressions coincided with the simultaneous presence of LIF (1000 U/mL) and bFGF2 (10 ng/mL). The phosphorylation status of LIF and bFGF2 downstream signaling molecules including STAT3, ERK, and AKT was also intensively involved in the maintenance of p-rES cell proliferation and self-renewal. Induced dephosphorylation of STAT3, ERK1/2, and AKT by specific inhibitors caused remarkable losses of self-renewal capacity of p-rES cells. We conclude that bFGF2 and LIF by itself are self-sufficient in maintaining the state of undifferentiation and self-renewal of rabbit p-ES cells, yet are most effective when acting concomitantly.

Triptolide (C₃₈H₄₂O₆N₂, TP, a diterpene triepoxide derived from Tripterygium wilfordii Hook F.), is a potent immunosuppresive and antiinflammatory agent. The present study investigated whether TP exerted antihepatofibrotic effects in vitro and in vivo. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with tumor necrosis factor-α (TNF-α) or transforming growth factor (TGF)-β1. The inhibitory effects of TP on the nuclear factor-κB (NFκB) signaling cascade and fibrosis markers, including α-smooth muscle actin (α-SMA) and collagen, were assessed. An in vivo therapeutic study was conducted in dimethylnitrosamine (DMN)-treated rats. The rats were randomly assigned to one of three groups: control rats, DMN rats receiving vehicle only and DMN rats receiving TP (20 μg/kg). Treatment was given by gavage twice daily for 3 weeks starting 1 week after the start of DMN administration. TP (5-100 nM) concentration-dependently inhibited the NFκB transcriptional activity induced by TNF-α, lipopolysaccharide and phorbol 12-myristate 13-acetate in HSC-T6 cells. In addition, TP also suppressed TNF-α and TGF-β1-induced collagen deposition and α-SMA secretion in HSC-T6 cells. In vivo, TP treatment significantly reduced hepatic fibrosis scores, collagen contents, IL-6 and TNF-α levels, and the number of α-SMA and NFκB-positive cells in DMN rats. The results showed that TP exerted antifibrotic effects in both HSC-T6 cells and DMN rats.

The aim of this study was to investigate if armepavine (Arm, C₁₉H₂₃O₃N) could exert inhibitory effects against hepatic fibrosis in rats. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with tumour necrosis factor-α (TNF-α) to evaluate the inhibitory effects of Arm. Rats were injected with thioacetamide (TAA; 300 mg/kg, intraperitoneally) thrice a week for 4 weeks to induce hepatic fibrosis, with Arm (3 or 10 mg/kg) given by gavage twice a day. Liver sections were taken for western blotting, fibrosis scoring and immunofluorescence staining. Arm (1-10 µm) concentration-dependently attenuated TNF-α-stimulated: (i) protein expressions of α-smooth muscle actin (α-SMA), collagen type I and angiopoietin-1; (ii) H₂O₂ production; and (iii) NF-κB, JunD and C/EBPß (cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT)/enhancer binding protein-ß (EBPß)) nuclear translocations in HSC-T6 cells. In vivo Arm treatment significantly reduced plasma aspartate transaminase and alanine transaminase levels, hepatic α-SMA expression and collagen contents, and fibrosis scores of TAA-injected rats. Moreover, Arm treatment decreased α-SMA- and NF-κB-positive cells in immunohistochemical staining, and mRNA expression levels of IL-6, TGF-ß1, TIMP-1, col1α2, iNOS and ICAM-1 genes, but up-regulated the metallothionein gene in the livers of TAA-injected rats. Our results indicated that Arm exerted both in vitro and in vivo antifibrotic effects in rats, with inhibition of NF-κB, JunD and C/EBPß pathways.

The protective effect of combination therapy with valsartan and aliskiren against renal fibrosis remains to be defined. This study was undertaken to examine the protective effects of the combination of valsartan and aliskiren against renal fibrosis induced by unilateral ureteral obstruction (UUO). Combination therapy with valsartan (15 mg·kg−1·day−1) and aliskiren (10 mg·kg−1·day−1), valsartan monotherapy (30 mg·kg−1·day−1), and aliskiren monotherapy (20 mg·kg−1·day−1) all significantly ameliorated the increase in blood urea nitrogen and the degree of hydronephrosis determined by the increase in weight and length of the obstructed kidney. The dose titration study and blood pressure measurement confirmed that the combination therapy provided a greater benefit independent of the vasodilatory effect. There were no significant changes in serum levels of creatinine, sodium, and potassium in UUO rats and any treatment groups. Combination therapy also attenuated UUO-related increases in the scores of tubular dilatation, interstitial volume, interstitial collagen deposition, α-smooth muscle actin, the activation of ERK 1/2, the infiltration of monocytes/macrophages, the mRNA expression of snail-1, and transforming growth factor-β1 to a greater extent compared with aliskiren or valsartan used alone. The mRNA expression of renin and the (pro)renin receptor significantly increased after UUO. Combination therapy and monotherapy of valsartan and aliskiren had a comparable enhancing effect on the mRNA expression of renin, whereas all these treatments did not affect the expression of the (pro)renin receptor. In conclusion, a direct renin inhibitor in conjunction with an angiotensin II receptor blocker exerts increased renal protection against renal fibrosis and inflammation during obstruction over either agent alone.

We previously reported a very high cumulative incidence of urothelial carcinoma in Taiwanese kidney transplant recipients. Rapamycin, the inhibitor of mTOR Complex 1, provides alternative immunosuppressive therapy after kidney transplantation with less neoplastic potential. We examined the in vivo and in vitro effects of rapamycin on urothelial carcinoma.The rat model of urothelial carcinoma was induced by 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in Fischer F344 rats. The anti-tumor effect of rapamycin was assessed grossly, microscopically, and by Western blot analysis. The mechanism of rapamycin's attenuation of urothelial carcinoma was also evaluated by T24 cells.Rapamycin significantly reduced urinary bladder tumor growth in the rat model of 0.05% BBN-induced urothelial carcinoma (P0.001). The blood trough levels of rapamycin were correlated with the occurrence of urothelial carcinoma. In vitro, rapamycin also inhibited the cell proliferation, migration, and invasion, as well as the protein expression of vascular endothelial growth factor-A of T24 urothelial carcinoma cells, whereas rapamycin did not induce significant apoptosis in T24 cells. Rapamycin decreased the expression of phospho-mTOR, phospho-S6K, cyclin D1, and VEGF-A. Rapamycin also activated AKT in T24 cells in the rat model of urothelial carcinoma. The rapamycin-associated activation of AKT was inhibited by rictor siRNA, but not raptor siRNA.This study provides in vitro and in vivo evidence that rapamycin may inhibit the development of urothelial carcinoma. The present findings also suggest rictor-dependent AKT activation as a consequence of mTORC1 inhibition.

Background: Ventricular fibrillation (VF) during prolonged (>5 min) global ischemia (GI) could be due to repetitive endocardial focal discharges (REFDs). This hypothesis was tested in isolated rabbit hearts. Methods and Results: With optical mapping, simultaneous endocardial (left ventricle, LV) and epicardial (both ventricles) activations during VF with prolonged GI were studied (protocol I, 8 hearts). Lugol solution was applied to the LV endocardium in additional 5 hearts after 5-min GI (protocol II). During prolonged GI, sustained VF (>30 s) was successfully induced in 7 protocol I hearts. The dominant frequency of summed optical signals at the LV endocardium was higher than at the epicardium (P 90% for recording time. There were 18 windows of optical recording showing spontaneous VF termination. In 10, once REFDs ceased, the VF episode terminated immediately. Electrical defibrillation was also performed on 3 hearts. Eight shocks showed early VF recurrence after successful defibrillation. REFDs were consistently involved in the initiation period of recurrence. In protocol II, Lugol subendocardial ablation diminished REFD genesis during re-induced VF. These VF episodes were all non-sustained. Conclusions: REFDs at the LV endocardium were important for both VF maintenance and post-shock recurrence during prolonged GI in this model. (Circ J 2009; 73: 1803-1811)

Activation of hepatic stellate cells (HSCs) plays a crucial role in liver fibrogenesis. armepavine (Arm, C19H23O3N), an active compound from Nelumbo nucifera, has been shown to exert immunosuppressive effects on T lymphocytes and on lupus nephritic mice. The aim of this study was to investigate whether Arm could exert anti-hepatic fibrogenic effects in vitro and in vivo. A cell line of rat HSCs (HSC-T6) was stimulated with tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS) to evaluate the inhibitory effects of Arm. An in vivo therapeutic study was conducted in bile duct-ligated (BDL) rats. BDL rats were given Arm (3 or 10 mg/kg) by gavage twice daily for 3 weeks starting from the onset of BDL. Liver sections were taken for fibrosis scoring, immuno-fluorescence staining and quantitative real-time mRNA measurements. In vitro, Arm (1-10 μM) concentration-dependently attenuated TNF-α- and LPS-stimulated α-SMA protein expression and AP-1 activation by HSC-T6 cells without adverse cytotoxicity. Arm also suppressed TNF-α-induced collagen collagen deposition, NFκB activation and MAPK (p38, ERK1/2, and JNK) phosphorylations. In vivo, Arm treatment significantly reduced plasma AST and ALT levels, hepatic α-SMA expression and collagen contents, and fibrosis scores of BDL rats as compared with vehicle treatment. Moreover, Arm attenuated the mRNA expression levels of col 1α2, TGF-β1, TIMP-1, ICAM-1, iNOS, and IL-6 genes, but up-regulated metallothionein genes. Our study results showed that Arm exerted both in vitro and in vivo antifibrotic effects in rats, possibly through anti-NF-κB activation pathways.

Interactions of glutamatergic and purinergic actions in the medulla regulate important cardiovascular functions. The glutamatergic action in dorsal facial area (DFA) of the medulla increases blood flow of common carotid artery (CCA) in cats. We hypothesized that interactions of glutamatergic and purinergic actions in the DFA may regulate the CCA blood flow. Purinergic and glutamatergic agonists and antagonists were microinjected into the DFA through a four-barrel tubing in anesthetized cats. Drug effects were evaluated by changes in the CCA blood flow. Microinjection with 20 nmol ATP or alpha,beta-methyleneATP (alpha,beta-MeATP, a P2 purinergic receptor agonist) induced an increase of the CCA blood flow. This increase was dose-dependently reduced by prior administration with 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX, a specific P1 purinergic receptor antagonist), or pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, a selective P2 purinergic receptor antagonist) as well as with MK-801 (a non-competitive NMDA receptor antagonist) or glutamate diethyl ester (GDEE, a competitive AMPA/kainate receptor antagonist). It was almost completely blocked by administrations with combined maximal doses of P1 and P2 receptor antagonists as well as NMDA and AMPA receptor antagonists. Nevertheless, P1 receptor agonist induced only mild and poorly reproducible increase in the CCA blood flow. In conclusion, prominent P2 and minor P1 purinergic receptors appear to be present in the DFA; the purinergic activation can mediate a release of glutamate that stimulates NMDA and AMPA to induce the increase of the CCA blood flows. These findings may provide important information for developing therapeutic strategy for diseases involving the CCA blood flow, such as hypertensive disease and cerebral ischemia.

The purposes of this study were to examine technical details in deriving and maintaining rabbit embryonic stem (rES) cell lines and to analyze their characteristics. When STO cells were used as feeder cells, no rES cell lines were established using either intact blastocysts or inner cell masses (ICMs). On the mouse embryonic fibroblasts (MEF) feeder, rES cell lines were efficiently (24%) derived. Addition of leukemia inhibitory factor (LIF) to the cells cultured on the MEF feeders further increased the derivation efficiency (57%) of rES cells. The fact that LIF induced serine-phosphorylation of STAT3 suggested LIF-dependent maintenance of rES cells. Most of the rES cell lines expressed AP, SSEA-4, Oct4, TRA-1-60, and TRA-1-81. Western blot or RT-PCR analysis also confirmed the expression of Oct4, Nanog, and Sox2. When induced to form EBs in vitro or injected to the severe combined immunodeficiency (SCID) mice, the rES cells generated embryoid bodies (EBs) and teratomas with three germ layers expressing the marker genes including MAP2, Desmin, and GATA4, respectively. In conclusion, rabbit ES cell lines can be efficiently established using our current protocols with LIF supplement. These ES cells express pluripotent stem cell markers and retain their capability to differentiate into different tissue cells. Furthermore, rES cells depend on LIF for self-renewal, likely via the JAK-STAT pathway.

Sympathetic hyperactivation in many kinds of neurocardiogenic injury can result in obvious heart failure. We generated a vagotomized feline model in which sympathetic hyperactivation was induced by electrical stimulation of dorsal medulla (ESDM) of brain stem to investigate the relationship between disruption of extracellular collagen matrix (ECM) and activation of matrix metalloproteinases (MMPs) in myocardium in the sympathetic hyperactivity. Mean blood pressure, heart rate and plasma norepinephrine were all significantly increased from baseline to a peak at 5 min after ESDM. Echocardiographic study showed significant left ventricular dilatation and hypokinesia (ejection fraction: from 87.7 +/- 6.3% to 39.4 +/- 7.8%) from baseline to 180 mm after ESDM. Histopathological finding revealed significant overstretching or spring-like disappearance and disruption of ECM. MMP-2 expression was significantly increased in left ventricular myocardium as compared to sham. These results suggest that ESDM-induced sympathetic hyperactivity causes the expression of MMP-2 that disrupts myocardial ECM, contributing to the development of cardiac dysfunction.

Nitric oxide (NO) or glutamate stimulation of dorsal facial area (DFA) increases blood flow in the common carotid artery (CCA), which supplies intra-and extra-cranial tissues. Nitrergic fibers and neurons as well as preganglionic cholinergic neurons are present in the DFA. We hypothesized the presence of nitrergic–glutamatergic fibers and preganglionic nitrergic–cholinergic neurons in the DFA that are involved in the regulation of CCA blood flow. In microdialysis studies, perfusion of the DFA with S-nitroso-N-acetylpenicillamine (SNAP, an NO donor) increased the glutamate concentration in the dialysate. This effect was abolished by co-perfusion of methylene blue (a guanylyl cyclase inhibitor). Intra-DFA injection of l-arginine (an NO precursor) or glutamate increased CCA blood flow. The l-arginine-induced flow increase was reduced by prior administration of NG-nitro-arginine methyl ester (l-NAME, a non-specific NO synthase inhibitor), 7-nitroindazole (7-NI, a relatively selective neuronal NO synthase inhibitor), d-2-amino-5-phosphonopentanoate (d-AP5, a competitive NMDA receptor antagonist), or glutamate diethylester (GDEE, a competitive AMPA receptor antagonist). The glutamate-induced blood flow increase was reduced by prior administration of l-NAME, 7-NI, or methylene blue. The induced increase in CCA blood flow, however, was not affected by endothelial NO synthase inhibitor. The findings indicate that NO-signal transduction within the DFA might cause glutamate release from presynaptic nitrergic-glutamatergic fibres and that the released glutamate activates NMDA/AMPA receptors on preganglionic nitrergic-cholinergic neurons in the nucleus to activate neuronal NO synthase and guanylyl cyclase in the neurons, leading to an increase in CCA blood flow. These findings may be important for developing therapeutic strategies for the diseases associated with CCA blood flow.

Despite implementation of practical guidelines, continuing education programs in our hospital, the percentage of inappropriate prophylactic antibiotic usage remains high. The aim of this study was to investigate whether information technology can reduce the misuse of surgical prophylactic antibiotic. We started physician continuing education programs in January 2005 and initiated feedback system in July 2005. The computerized reminder system was implemented in April 2006. Relevant data about the surgical prophylactic antibiotic usage were collected and was separated into three groups. Group 1 consisted of data from January 2005 to June 2005, while group 2 and group 3 consisted of data from July 2005 to March 2006 and April 2006 to December 2006, respectively. The percentage of no prophylactic antibiotic in clean procedures and the duration of prophylactic antibiotic in clean-contaminated procedures were recorded and analyzed. Furthermore, the surgical wound infection rates were also collected. In clean procedures, the percentage of no prophylactic antibiotic after surgery decreased in the long run. In parotidectomy, submandibular gland surgery and thyroidectomy patients, the percentage even reached 100% at the end of this study. In clean-contaminated procedures, the duration of prophylactic antibiotic after surgery was also reduced except in laryngectomized patients at the end of this study. Information technology such as feedback and reminder systems is an effective method to reduce inappropriate usage of surgical prophylactic antibiotic.

Liver fibrosis has been characterized as chronic inflammatory processes involving multiple molecular pathogenetic pathways. This therapeutic study investigated whether a combination regimen of Salvia miltiorrhiza (S), Ligusticum chuanxiong (L) and Glycyrrhiza glabra (G) exerted in vivo antifibrotic effects on rats with hepatic fibrosis. Fibrosis was induced in rats by dimethylnitrosamine (DMN) administration for 4 weeks. Fibrotic rats were randomly assigned to one of the three groups: control, SLG (50 mg/kg) or silymarin (50 mg/kg), each given by gavage twice daily for 3 weeks starting 1 week after DMN injection. The results showed that fibrosis scores of livers from DMN-treated rats with SLG (1.13 +/- 0.13) were significantly reduced in comparison with DMN-treated rats receiving vehicle (1.63 +/- 0.18). Moreover, the hepatic collagen content of DMN rats was significantly reduced by either SLG or silymarin treatment. The double immunohistochemical staining results also showed that alpha-SMA positive cells with NF kappa B nuclear translocation were decreased in the fibrotic livers by SLG and silymarin treatments. The mRNA expression levels of TGF-beta1, alpha-SMA, collagen1 alpha 2, iNOS and ICAM-1 genes were attenuated by SLG and silymarin treatment. The results showed that SLG exerted antifibrotic effects in rats with DMN-induced hepatic fibrosis.

Background Anti-inflammation strategies are one of the proposed therapeutic approaches to hepatic fibrosis. Tetrandrine (C(38)H(42)O(8)N(2), molecular weight: 622; Tet), an alkaloid isolated from the Chinese medicinal herb Stephania tetrandra, has been shown to exert anti-inflammatory activity in pulmonary diseases. The purpose of the present study was to investigate the in vitro and in vivo effects of Tet on hepatic fibrosis. Methods A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-beta1 (TGF-beta1) or tumor necrosis factor-alpha (TNF-alpha). The inhibitory effects of Tet on the nuclear factor kappaB (NFkappaB) signaling cascade and molecular markers including intercellular adhesion molecule-1 (ICAM-1) and alpha-smooth muscle actin (alpha-SMA) secretion were assessed. Fibrosis was induced by dimethylnitrosamine (DMN) administration in rats for 4 weeks. Fibrotic rats were randomly assigned to one of the four groups: vehicle (0.7% carboxyl methyl cellulose, CMC), Tet (1 mg/kg), Tet (5 mg/kg), or silymarin (50 mg/kg), each given by gavage twice daily for 3 weeks starting after 1 week of DMN administration. At the end of the study, liver tissues were scored for fibrosis and analyzed for molecular markers of fibrosis. Results Tetrandrine (0.5-5.0 micromol/L) concentration-dependently inhibited NFkappaB transcriptional activity induced by TNF-alpha, including IkappaBalpha phosphorylation and mRNA expressions of ICAM-1 in HSC-T6 cells. In addition, Tet also inhibited TGF-beta1-induced alpha-SMA secretion and collagen deposition in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats with high-dose Tet (1.3 +/- 0.3) were significantly reduced in comparison with DMN-treated rats receiving saline (2.0 +/- 0.2). Hepatic collagen content of DMN rats was significantly reduced by either Tet or silymarin treatment. Double-staining results showed that alpha-SMA- and NFkappaB-positive cells were decreased in the fibrotic livers by Tet and silymarin treatment. In addition, mRNA expression of ICAM-1, alpha-SMA, and TGF-beta1 was attenuated by Tet treatment. Moreover, levels of plasma aspartate aminotransferase and alanine aminotransferase activities were reduced by Tet and silymarin treatment. Conclusion Tetrandrine exerts antifibrotic effects in both HSC-T6 cells and in rats with DMN-induced fibrosis.

The homeobox gene families play important roles in the transcriptional regulation of gene expression prior to and during embryo development. To identify novel homeobox genes expressed in early embryonic development, we conducted a degenerated oligonucleotide polymerase chain reaction (PCR) to screen a mouse embryonic stem (ES) cell cDNA library. A novel homeobox-containing gene, Eso-1, which is preferentially expressed in ES cells and ovaries, was identified. The full-length Eso-1 cDNA was found to be 1,710 bp with a predicted homeodomain that has no significant homology to previously reported home- odomain proteins. Eso-1 was mapped to chromosome 14A3. Reverse transcription-polymerase chain reac- tion (RT-PCR) analyses showed that Eso-1 was expressed through oogenesis and continuing to be expressed through to the blastocyst stage. De novo expression of Eso-1 started at 13.5 days postcoitum in the ovaries, which coincides with the initiation of oogenesis. Northern blot analyses demonstrated that Eso-1 is preferentially expressed in both ovaries and ES cells as a 1.7-kb transcript. Results from whole mount in situ hybridization revealed that Eso-1 in oocytes showed increased expression from primordial to antral follicles. The 3 0 -untranslated region of Eso-1 transcripts contained cytoplasmic polyadenylation sequences while the length of poly (A) tails changed during oocyte maturation, indicating that Eso-1 expression is con- trolled by time-dependent translational activation. We suggest that the novel homeodomain protein, Eso-1, plays a role during oocyte maturation and early embryonic development. Mol. Reprod. Dev. 73: 825-833, 2006. 2006 Wiley-Liss, Inc.

Spinal muscular atrophy (SMA) is the most common genetic motoneuron degenerative disorder, but the mechanism(s) of motoneuron death is unclear. Previously, a direct interaction between tumor-suppressive TP53 protein and the SMA determinant gene product, survival motor neuron protein, was identified and therefore it has been suggested that a mechanism of TP53-dependent apoptosis plays an important role in motoneuron degeneration in SMA. We used our SMA model mice, generated by a combination of knockout and transgenic techniques, to decipher the role of TP53 protein in the motoneuron degeneration in SMA. We detected a significant increase of Trp53 expression in the spinal cord of SMA-like mice compared to their normal littermates. After crossing SMA-like mice with Trp53 knockout mice, the progeny Trp53-deficient SMA-like mice did not show milder disease severity or longer lifespan compared to SMA littermates with wild-type Trp53 genes. Our studies provide in vivo evidence indicating that Trp53-dependent apoptosis does not play a crucial role in motoneuron degeneration in SMA-like mice. European Journal of Human Genetics (2006) 14, 372-375. doi:10.1038/sj.ejhg.5201556; published online 4 January 2006.

Tumor necrosis factor-alpha (TNF-alpha) plays a central role in cellular necrosis, apoptosis, organ failure, tissue damage, inflammation and fibrosis. These processes, occurring in liver injury, may lead to cirrhosis. Thalidomide, alpha-N-phthalidoglutarimide, (C(13)H(10)N(2))(4), has been shown to have immunomodulatory and anti-inflammatory properties, possibly mediated through its anti-TNF-alpha effect. In this study, we investigated the in vitro and in vivo effects of thalidomide on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-beta1 (TGF-beta1) or TNF-alpha. The inhibitory effects of thalidomide on the NFkappaB signaling cascade and fibrosis markers including alpha-smooth muscle actin (alpha-SMA) and collagen, were assessed. An in vivo therapeutic study was conducted in dimethylnitrosamine (DMN)-treated rats, which were randomly assigned to 1 of 4 groups: vehicle (0.7% carboxyl methyl cellulose, CMC), thalidomide (40 mg/kg), thalidomide (200 mg/kg), or silymarin (50 mg/kg), each given by gavage twice daily for 3 weeks starting after 1 week of DMN administration. Thalidomide (100-800 nM) concentration-dependently inhibited NFkappaB transcriptional activity induced by TNF-alpha, including IKKalpha expression and IkappaBalpha phosphorylation in HSC-T6 cells. In addition, thalidomide also suppressed TGF-beta1-induced alpha-SMA expression and collagen deposition in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats receiving high dose of thalidomide (0.89 +/- 0.20) were significantly reduced in comparison with those of DMN-treated rats receiving vehicle (1.56 +/- 0.18). Hepatic collagen contents of DMN rats were also significantly reduced by either thalidomide or silymarin treatment. Immunohistochemical double staining results showed that alpha-SMA- and NFkappaB-positive cells were decreased in the livers from DMN rats receiving either thalidomide or silymarin treatment. In addition, real-time PCR analysis indicated that hepatic mRNA expressions of TGF-beta1, alpha-SMA, collagen 1alpha2, TNF-alpha and iNOS genes were attenuated by thalidomide treatment. In conclusion, our results showed that thalidomide inhibited activation of HSC-T6 cells by TNF-alpha and ameliorated liver fibrosis in DMN-intoxicated rats.

Objective Amniotic fluid mesenchymal stem cells (MSCs) have the potential to differentiate into neuronal stem cells in vitro. We evaluated using amniotic fluid MSCs to support or enhance the ability of the injured sciatic nerve to cross a nerve gap. Materials and methods We created a 5 mm nerve defect in Sprague Dawley rats. One group received therapy with MSCs embedded into woven oxidised regenerated cellulose gauze (Surgical; Ethicon, Somerville, NJ) and fibrin glue, while a control group received woven Surgicel and fibrin glue only. Evaluation methods included behavioural, electrophysiological and immunohistochemical studies. Results In gait analysis, the angle of the ankles in the treatment and control group were 46.4° (standard deviation [SD] = 15°) and 36° (SD = 8.2°), respectively, which was statistically significant ( p = 0.045). Five of 10 treated rats (50%) demonstrated partial foot movement, while none of the control group had any movement. The percentage amplitude of muscle compound action potential in the experimental group was 43% (SD = 12.5%) compared to 29% (SD = 8.8%) in the control group ( p = 0.038). The conduction latencies in the control and experimental groups was 2.5 ms (SD = 0.45) and 1.7 ms (SD = 0.47), respectively ( p = 0.005). Histological examination demonstrated that 70% of the treatment group achieved a maximum axon diameter percentage across the nerve gap of greater than 50%, compared with 0% in the control group. There were no differences in direction of fibre growth and fibrotic reaction between the two groups. Conclusion Amniotic fluid MSC can augment growth of injured nerve across a nerve gap. This effect may be due to neurotrophic or induction effects of the MSC interacting with Schwann cells. Further study is required to determine the underlying mechanism of this effect.

Although serum leptin concentrations are reported by several studies to increase in patients with liver cirrhosis, the mechanisms underpinning this increase remain unclear. Circulating tumor necrosis factor alpha (TNF-alpha) concentrations are also recognized to increase in liver cirrhosis. Furthermore, TNF-alpha administration to rodents results in increased expression and secretion of leptin from adipose tissue in a manner dependent on type 1 TNF-alpha receptor (TNF-RI). The present study was undertaken to examine adipose leptin expression and to explore potential relationships between leptin expression and TNF-alpha in subjects with liver cirrhosis. Liver cirrhosis was induced in male Sprague-Dawley rats by dimethylnitrosamine (DMN) administration or by common bile duct ligation (BDL). Ad libitum and pair-fed animals constituted controls. Serum leptin and TNF-alpha concentrations were determined by immunoassay. Gene expression was determined by the reverse transcription-polymerase chain reaction, and protein levels were measured by Western blotting. Serum leptin values after adjustment of body fat mass in DMN-treated rats were significantly higher than in pair-fed or ad libitum groups. Leptin mRNA and protein levels in epididymal fat in DMN rats increased by 1.8-fold and 2.3-fold, respectively, as compared with ad libitum controls, and by 4-fold and 6-fold, respectively, as compared with the pair-fed group. Epididymal TNF-alpha and membranous TNF-RI (mTNF-RI) concentrations were both 2.3 times higher in DMN rats than in ad libitum controls but did not differ between ad libitum and pair-fed groups. Adipose leptin protein levels correlated directly with TNF-alpha and mTNF-RI concentrations in combined DMN, ad libitum, and pair-fed rats (r=0.64 and r=0.49, respectively; P

Excessive oxidative stress is implicated in hepatic fibrogenesis. Extracts of Salvia miltiorrhiza (Sm) have been shown to protect cells against oxidative stress. In this study we investigated the in vitro and in vivo effects of Sm on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-beta1 (TGF-beta1). The inhibitory effects of Sm (50-400 microg/ml) on TGF-beta1-induced alpha-smooth muscle actin (alpha-SMA) secretion and the mRNA expressions of fibrosis-related genes, including alpha-SMA, connective tissue growth factor (CTGF), and tissue inhibitor of metalloproteinase-1 (TIMP-1), were assessed. Fibrosis was induced by dimethylnitrosamine (DMN) administration in rats. DMN-treated rats were randomly assigned to 1 of 4 groups: saline, Sm (20 mg/kg), Sm (100 mg/kg), or silymarin (100 mg/kg), each given by gavage twice daily for 5 weeks starting from the onset of DMN administration. Sm (200 and 400 microg/ml) significantly inhibited TGF-beta1-stimulated alpha-SMA secretion and the mRNA expressions of alpha-SMA, CTGF, and TIMP-1 in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats with either a low (1.8 +/- 0.2) or high (1.8 +/- 0.1) dose of Sm, or silymarin (1.4 +/- 0.2) were significantly reduced in comparison with DMN-treated rats receiving saline (3.1 +/- 0.1). Hepatic collagen contents were also significantly reduced by either Sm or silymarin treatment. The mRNA expression levels of alpha-SMA, TGF-beta1, and procollagen I were all attenuated in Sm- and silymarin-treated rats. Moreover, levels of plasma aspartate transaminase activities were reduced by Sm and silymarin treatment. In conclusion, our results show that Sm exerted antifibrotic effects in both HSC-T6 cells and in rats with DMN-induced fibrosis.

Many kinds of brain lesions cause sympathetic hyperexcitation and myocardial damage. A novel animal model was developed for the correlation of sympathetic excitation with ventricular dysfunction and myocardial damage. Six cats (3.23+/-0.26 kg) under intraperitoneal urethane-chloralose anesthesia were artificially ventilated and bilaterally vagotomized. They underwent an electrical stimulation of unilateral dorsal medulla for 180 min (monopolar square-wave pulses, 10 Hz, 10 V, 0.5 ms). Mean blood pressure, heart rate plasma concentration of norepinephrine and left ventricular size and ejection fraction were measured at 0, 5, 15, 30, 60, 90, 120 and 180 min. Mean blood pressure (mm Hg), heart rate (beats/min) and norepinephrine (pg/ml) increased abruptly from 128+/-15, 203+/-22 and 353+/-123 to 234+/-26, 240+/-13 and 4727+/-2159 at 5 min after electrical stimulation (all p0.01). The left ventricles showed significant dilatation (end-diastolic diameter: from 1.35+/-0.13 to 1.84+/-0.21 cm, p=0.006; end-systolic diameter: from 0.65+/-0.20 to 1.54+/-0.24 cm, p=0.002) and hypokinesia (ejection fraction: from 88.9+/-6.4% to 37.3+/-8.7%, p0.001). Cardiac pathology revealed myocardial hemorrhage, cardiomyocyte apoptosis and coagulative myocytolysis (contraction band necrosis), characterized by sarcoplasmic coagulation, granulation and disruption. In conclusions, the present experiment develops a novel animal model in which stimulation of the pressor area in the dorsal medulla in vagotomized cats produces sympathetic hyperexcitation accompanied with myocardial dysfunction and damage. This model may be applicable for studying protective effect of drugs on myocardial dysfunction and damage caused by sympathetic hyperexcitation occurring in brain diseases.

Objective: Norepinephrine (NE)-induced apoptosis in cardiomyocytes is an important cause of heart failure. Previous studies revealed that reactive oxygen species (ROS) are involved in apoptosis. Tumor necrosis factor-α (TNF), a well-known mediator that stimulates apoptosis, is not only produced by macrophages but also by cardiomyocytes. Until now, the role of TNF and its relationship to ROS in NE-induced apoptosis of cardiomyocytes have never been investigated. Methods: Neonatal rat cardiomyocytes were treated with various concentrations of NE. Apoptosis of cardiomyocytes was determined using the TUNEL assay. The level of secreted TNF was measured by ELISA and TNF mRNA expression was determined by semiquantitative reverse transcriptional polymerase chain reaction. Caspase activity was measured by a fluorogenic protease assay kit. Anti-TNF antibodies, caspase inhibitors and antioxidants ( N -acetyl-l-cysteine or vitamin C) were added to determine if they could inhibit the apoptotic effect of NE. Results: NE induced apoptosis of cardiomyocytes in a dose- and time-dependent manner. NE up-regulated TNF mRNA expression and increased TNF secretion and caspase-2,-3,-6, and -9 activities. A neutralizing anti-TNF antibody and caspase-2 and -3 inhibitors significantly attenuated NE-induced apoptosis. Antioxidants completely abrogated NE-induced TNF secretion, caspase activation, and apoptotic death. Conclusion: NE induced apoptosis in neonatal rat cardiomyocytes through a ROS–TNF–caspase signaling pathway.