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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Background Hepatic fibrosis is a dynamic process which ultimately leads to cirrhosis in almost patients with chronic hepatic injury. However, progressive fibrosis is a reversible scarring response. Activation of hepatic stellate cells (HSCs) is the prevailing process during hepatic fibrosis. Osthole is an active component majorly contained in the fruit of Cnidium monnieri (L.) Cusson. This present study investigated the therapeutic effects of osthole on rat liver fibrosis and HSC activation. Results We established the thioacetamide (TAA)-model of Sprague–Dawley (SD) rats to induce hepatic fibrosis. Rats were divided into three groups: control, TAA, and TAA + osthole (10 mg/kg). In vivo, osthole significantly reduced liver injury by diminishing levels of plasma AST and ALT, improving histological architecture, decreasing collagen and α-SMA accumulation, and improving hepatic fibrosis scores. Additionally, osthole reduced the expression of fibrosis-related genes significantly. Osthole also suppressed the production of fibrosis-related cytokines and chemokines. Moreover, nuclear translocation of p65 was significantly suppressed in osthole-treated liver. Osthole also ameliorated TAA-induced injury through reducing cellular oxidation. Osthole showed inhibitory effects in inflammation-related genes and chemokines production as well. In vitro, we assessed osthole effects in activated HSCs (HSC-T6 and LX-2). Osthole attenuated TGF-β1-induced migration and invasion in HSCs. Furthermore, osthole decreased TNF-α-triggered NF-κB activities significantly. Besides, osthole alleviated TGF-β1- or ET-1-induced HSCs contractility. Conclusions Our study demonstrated that osthole improved TAA-caused liver injury, fibrogenesis and inflammation in rats. In addition, osthole suppressed HSCs activation in vitro significantly. Electronic supplementary material The online version of this article (doi:10.1186/s12929-015-0168-5) contains supplementary material, which is available to authorized users.