Brahma-Related Gene 1 Inhibition Prevents Liver Fibrosis and Cholangiocarcinoma by Attenuating Progenitor Expansion.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (iCCA) is closely correlated with hepatic progenitor cell (HPC) expansion and liver fibrosis. Brahma-related gene 1 (Brg1), an enzymatic subunit of the switch/sucrose nonfermentable complex that is critical in stem cell maintenance and tumor promotion, is prominently up-regulated in both HPCs and iCCA; however, its role in this correlation remains undefined. APPROACH AND RESULTS: A retrospective cohort study indicated that high Brg1 expression suggests poor prognosis in patients with iCCA. In chronically injured livers induced by a 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet or bile duct ligation surgery, HPCs were dramatically activated, as indicated by their enhanced expression of Brg1 and a subset of stem cell markers; however, Brg1 ablation in HPCs strongly suppressed HPC expansion and liver fibrosis. Furthermore, in a chemically induced iCCA model, inhibition of Brg1 by a specific inhibitor or inducible gene ablation markedly improved histology and suppressed iCCA growth. Mechanistically, in addition to transcriptionally promoting both Wnt receptor genes and target genes, Brg1 was found to bind to the ß-catenin/transcription factor 4 transcription complex, suggesting a possible approach for regulation of Wnt/ß-catenin signaling. CONCLUSIONS: We have demonstrated the function of Brg1 in promoting HPC expansion, liver cirrhosis, and, ultimately, iCCA development in chronically injured livers, which is largely dependent on Wnt/ß-catenin signaling. Our data suggest that therapies targeting Brg1-expressing HPCs are promising for the treatment of liver cirrhosis and iCCA.

N-n-Butyl haloperidol iodide ameliorates liver fibrosis and hepatic stellate cell activation in mice.

N-n-Butyl haloperidol iodide (F2) is a novel compound that has antiproliferative and antifibrogenic activities. In this study we investigated the therapeutic potential of F2 against liver fibrosis in mice and the underlying mechanisms. Two widely used mouse models of fibrosis was established in mice by injection of either carbon tetrachloride (CCl4) or thioacetamide (TAA). The mice received F2 (0.75, 1.5 or 3 mg·kg-1·d-1, ip) for 4 weeks of fibrosis induction. We showed that F2 administration dose-dependently ameliorated CCl4- or TAA-induced liver fibrosis, evidenced by significant decreases in collagen deposition and c-Jun, TGF-ß receptor II (TGFBR2), α-smooth muscle actin (α-SMA), and collagen I expression in the liver. In transforming growth factor beta 1 (TGF-ß1)-stimulated LX-2 cells (a human hepatic stellate cell line) and primary mouse hepatic stellate cells, treatment with F2 (0.1, 1, 10 µM) concentration-dependently inhibited the expression of α-SMA, and collagen I. In LX-2 cells, F2 inhibited TGF-ß/Smad signaling through reducing the levels of TGFBR2; pretreatment with LY2109761 (TGF-ß signaling inhibitor) or SP600125 (c-Jun signaling inhibitor) markedly inhibited TGF-ß1-induced induction of α-SMA and collagen I. Knockdown of c-Jun decreased TGF-ß signaling genes, including TGFBR2 levels. We revealed that c-Jun was bound to the TGFBR2 promoter, whereas F2 suppressed the binding of c-Jun to the TGFBR2 promoter to restrain TGF-ß signaling and inhibit α-SMA and collagen I upregulation. In conclusion, the therapeutic benefit of F2 against liver fibrosis results from inhibition of c-Jun expression to reduce TGFBR2 and concomitant reduction of the responsiveness of hepatic stellate cells to TGF-ß1. F2 may thus be a potentially new effective pharmacotherapy for human liver fibrosis.

Ketogenic Diet Enhances the Cholesterol Accumulation in Liver and Augments the Severity of CCl4 and TAA-Induced Liver Fibrosis in Mice.

Persistent chronic liver diseases increase the scar formation and extracellular matrix accumulation that further progress to liver fibrosis and cirrhosis. Nevertheless, there is no antifibrotic therapy to date. The ketogenic diet is composed of high fat, moderate to low-protein, and very low carbohydrate content. It is mainly used in epilepsy and Alzheimer's disease. However, the effects of the ketogenic diet on liver fibrosis remains unknown. Through ketogenic diet consumption, ß-hydroxybutyrate (bHB) and acetoacetate (AcAc) are two ketone bodies that are mainly produced in the liver. It is reported that bHB and AcAc treatment decreases cancer cell proliferation and promotes apoptosis. However, the influence of bHB and AcAc in hepatic stellate cell (HSC) activation and liver fibrosis are still unclear. Therefore, this study aimed to investigate the effect of the ketogenic diet and ketone bodies in affecting liver fibrosis progression. Our study revealed that feeding a high-fat ketogenic diet increased cholesterol accumulation in the liver, which further enhanced the carbon tetrachloride (CCl4)- and thioacetamide (TAA)-induced liver fibrosis. In addition, more severe liver inflammation and the loss of hepatic antioxidant and detoxification ability were also found in ketogenic diet-fed fibrotic mouse groups. However, the treatment with ketone bodies (bHB and AcAc) did not suppress transforming growth factor-ß (TGF-ß)-induced HSC activation, platelet-derived growth factor (PDGF)-BB-triggered proliferation, and the severity of CCl4-induced liver fibrosis in mice. In conclusion, our study demonstrated that feeding a high-fat ketogenic diet may trigger severe steatohepatitis and thereby promote liver fibrosis progression. Since a different ketogenic diet composition may exert different metabolic effects, more evidence is necessary to clarify the effects of a ketogenic diet on disease treatment.

Dietary α-Lactalbumin protects against thioacetamide-induced liver cirrhosis by maintaining gut-liver axis function in rats.

We tested the hypothesis that α-lactalbumin inhibits the disruption of intestinal barrier function and liver cirrhosis by restoring gut-liver axis function in thioacetamide (TAA) -treated rats. Rat diets were supplemented with α-lactalbumin replacing 50% of dietary protein. After consuming α-lactalbumin for one week, rats were intraperitoneally injected with TAA twice a week for 14 weeks. The α-lactalbumin-enriched diet significantly inhibited the elevation of plasma alanine aminotransferase, aspartate aminotransferase, and hyaluronic acids. The supplement significantly reduced plasma lipopolysaccharide levels and increased occludin mRNA level. Hepatic fibrosis and regenerative nodules was developed and intestinal villi were shortened by TAA; α-Lactalbumin attenuated these histopathological changes. These results indicated that α-lactalbumin improved intestinal barrier function, suppressing endotoxin levels. These data also suggested that α-lactalbumin ameliorated the impairment of the gut-liver axis by TAA, inhibiting the development of liver cirrhosis.

Concordance between Thioacetamide-Induced Liver Injury in Rat and Human In Vitro Gene Expression Data.

The immense resources required and the ethical concerns for animal-based toxicological studies have driven the development of in vitro and in silico approaches. Recently, we validated our approach in which the expression of a set of genes is uniquely associated with an organ-injury phenotype (injury module), by using thioacetamide, a known liver toxicant. Here, we sought to explore whether RNA-seq data obtained from human cells (in vitro) treated with thioacetamide-S-oxide (a toxic intermediate metabolite) would correlate across species with the injury responses found in rat cells (in vitro) after exposure to this metabolite as well as in rats exposed to thioacetamide (in vivo). We treated two human cell types with thioacetamide-S-oxide (primary hepatocytes with 0 (vehicle), 0.125 (low dose), or 0.25 (high dose) mM, and renal tubular epithelial cells with 0 (vehicle), 0.25 (low dose), or 1.00 (high dose) mM) and collected RNA-seq data 9 or 24 h after treatment. We found that the liver-injury modules significantly altered in human hepatocytes 24 h after high-dose treatment involved cellular infiltration and bile duct proliferation, which are linked to fibrosis. For high-dose treatments, our modular approach predicted the rat in vivo and in vitro results from human in vitro RNA-seq data with Pearson correlation coefficients of 0.60 and 0.63, respectively, which was not observed for individual genes or KEGG pathways.

Heat shock protein 47 as indispensible participant in liver fibrosis: Possible protective effect of lactoferrin.

Lactoferrin (LF) was previously suggested to have a protective effect against liver fibrosis by preventing hepatic stellate cells (HSCs) activation. The effect of LF on heat shock protein 47 (HSP47) has not yet been studied so this study was designed to investigate LF effect on HSP47 as a potential target for management of liver fibrosis and comparing it with silymarin (SM) in a thioacetamide (TAA)-induced liver fibrosis model. Rats were divided into four groups; normal control, TAA (TAA-treated), LF (LF + TAA-treated), and SM (SM + TAA-treated). After 6 weeks, both LF and SM improved the grade of cirrhosis, reduced collagen fibers deposition, inactivated HSCs, significantly decreased elevated liver enzymes, HSP47, hydroxyproline content, transforming growth factor-beta 1, matrix metalloproteinase-2, 8-hydroxydeoxyguanosine, malondialdehyde, nitric oxide levels and the percentage of alpha smooth muscle actin positive HSCs compared with TAA group. Moreover, LF significantly increased the total antioxidant capacity compared with TAA group. It could be concluded that LF is a promising antifibrotic drug and could be considered as one of the HSP47 inhibitors but SM is still more potent. © 2018 IUBMB Life, 70(8):795-805, 2018.

"Extracellular matrix remodelling in the liver of rats subjected to dietary choline deprivation and/or thioacetamide administration".

Choline deprivation is a recognized experimental approach to nonalcoholic steatohepatitis, while thioacetamide (TAA)-induced liver fibrosis resembles alcoholic liver fibrogenesis. In order to elucidate the effect of TAA on liver extracellular matrix composition under choline deprivation due to choline-deficient diet (CDD) administration, we evaluated the transcriptional and immunohistochemical (IHC) pattern of major hepatic matrix metalloproteinases (namely, MMP-2, -9) and their tissue inhibitors (TIMP-1, -2) in adult male albino Wistar rats at 30, 60 and 90 days. In the CDD+TAA group, IHC showed an early progressive increase in MMP-2 expression, while MMP-9 initially exhibited a significant increase followed by a gradual decrease; TIMP-1 and TIMP-2 IHC expressions showed gradual increase throughout the experiment. The MMPs-TIMPs regulation at the transcriptional level was found to be increased in all groups throughout the experiment. The increased MMP-2/TIMP-2 and suppressed MMP-9/TIMP-1 ratios in IHC and in real-time polymerase chain reaction (RT-PCR) seemed to correlate with the degree of liver fibrosis. These results support the important role of MMPs and TIMPs in controlling the hepatic pathogenesis and shed more light on the recently described experimental approach to liver disease (steatohepatitis) under the impact of two insults (TAA and CDD).

Oxidative stress caused by a SOD1 deficiency ameliorates thioacetamide-triggered cell death via CYP2E1 inhibition but stimulates liver steatosis.

We investigated the responses of mice that are defective in the superoxide-scavenging enzyme SOD1 to thioacetamide (TAA)-induced hepatotoxicity. When a lethal dose of TAA (500 mg/kg) was intraperitoneally injected, the wild-type (WT) mice all died within 36 h, but all of the SOD1-knockout (KO) mice survived. Treatment with an SOD1 inhibitor rendered the WT mice resistant to TAA toxicity. To elucidate the mechanism responsible for this, we examined the acute effects of a sublethal dose of TAA (200 mg/kg) on the livers of WT and KO mice. The extent of TAA-induced liver damage was less in the KO mice, but, instead, lipogenesis was further advanced in the SOD1-KO livers. The levels of proteins modified with acetyllysine, a marker for TAA-mediated injury, were lower in the KO mice than the WT mice upon the TAA treatment. The KO mice, which were under oxidative stress per se, exhibited a lower CYP2E1 activity, and this appeared to result in a decrease in the production of reactive oxygen species (ROS) during TAA metabolism. Both cleaved ATF6, a transcriptional regulator that is activated by endoplasmic reticulum (ER) stress, and CHOP, a death signal mediator, were highly elevated in the WT mice as the result of the TAA treatment and consistent with the liver damage. We conclude that elevated TAA metabolites and reactive oxygen species that are produced by CYP-mediated drug metabolism trigger lipogenesis as well as liver damage via ER stress and determine the fate of the mice.

Hepatic inflammation facilitates transcription-associated mutagenesis via AID activity and enhances liver tumorigenesis.

Chronic inflammation triggers the aberrant expression of a DNA mutator enzyme, activation-induced cytidine deaminase (AID), and contributes to tumorigenesis through the accumulation of genetic aberrations. To gain further insight into the inflammation-mediated genotoxic events required for carcinogenesis, we examined the role of chronic inflammation in the emergence of genetic aberrations in the liver with constitutive AID expression. Treatment with thioacetamide (TAA) at low-dose concentrations caused minimal hepatic inflammation in both wild-type (WT) and AID transgenic (Tg) mice. None of the WT mice with low-dose TAA administration or AID Tg mice without hepatic inflammation developed cancers in their liver tissues over the 6 month study period. In contrast, all the AID Tg mice with TAA treatment developed multiple macroscopic hepatocellular carcinomas during the same observation period. Whole exome sequencing and additional deep-sequencing analyses revealed the enhanced accumulation of somatic mutations in various genes, including dual specificity phosphatase 6 (Dusp6), early growth response 1 (Egr1) and inhibitor of DNA binding 2 (Id2), which are putative tumor suppressors, in AID-expressing liver with TAA-mediated hepatic inflammation. Microarray and quantitative reverse transcription-polymerase chain reaction analyses showed the transcriptional upregulation of various genes including Dusp6, Egr1 and Id2 under hepatic inflammatory conditions. Together, these findings suggest that inflammation-mediated transcriptional upregulation of target genes, including putative tumor suppressor genes, enhances the opportunity for inflamed cells to acquire somatic mutations and contributes to the acceleration of tumorigenesis in the inflamed liver tissues.

Increased serum bile acid concentration following low-dose chronic administration of thioacetamide in rats, as evidenced by metabolomic analysis.

A liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS)-based metabolomics approach was employed to identify endogenous metabolites as potential biomarkers for thioacetamide (TAA)-induced liver injury. TAA (10 and 30mg/kg), a well-known hepatotoxic agent, was administered daily to male Sprague-Dawley (SD) rats for 28days. We then conducted untargeted analyses of endogenous serum and liver metabolites. Partial least squares discriminant analysis (PLS-DA) was performed on serum and liver samples to evaluate metabolites associated with TAA-induced perturbation. TAA administration resulted in altered levels of bile acids, acyl carnitines, and phospholipids in serum and in the liver. We subsequently demonstrated and confirmed the occurrence of compromised bile acid homeostasis. TAA treatment significantly increased serum levels of conjugated bile acids in a dose-dependent manner, which correlated well with toxicity. However, hepatic levels of these metabolites were not substantially changed. Gene expression profiling showed that the hepatic mRNA levels of Ntcp, Bsep, and Oatp1b2 were significantly suppressed, whereas those of basolateral Mrp3 and Mrp4 were increased. Decreased levels of Ntcp, Oatp1b2, and Ostα proteins in the liver were confirmed by western blot analysis. These results suggest that serum bile acids might be increased due to the inhibition of bile acid enterohepatic circulation rather than increased endogenous bile acid synthesis. Moreover, serum bile acids are a good indicator of TAA-induced hepatotoxicity.

Induction of chemokines and cytokines before neutrophils and macrophage recruitment in different regions of rat liver after TAA administration.

Single-dose thioacetamide (TAA) administration induces inflammation and acute liver damage. The mechanism of inflammatory cell recruitment in the liver is still unclear. The aim of this study was to examine the sequence and recruitment of inflammatory cells in different liver regions in relation to CXC- and CC-chemokine and cytokine expression during acute liver injury. Single-dose TAA was administered to rats intraperitoneally, and animals were killed at different time points thereafter. Serum and liver tissue were taken and frozen immediately. Tissue was used for immunostaining cryostat sections, RNA, and protein extraction. RT-PCR and western blotting were performed for RNA and protein analysis, respectively. An early increase (3 h) in CXCL8/IL-8 levels was measured followed by a marked release in MCP1/CCL2 (24 h) serum levels after TAA administration compared with controls. Similarly, an early increase in specific RNA of hepatic chemokines CXCL1/KC and CXCL8/IL-8 was found at 3 h, followed by an upregulation of CXCL5/LIX (6 h), CXCL2/MIP-2 (12 h), and MCP1/CCL2 gene expression at 24-48 h. Further, an induction of pro-inflammatory cytokines IFN-γ and IL-1ß followed by IL-6 and TNF-α was observed with a maximum at 12 h. The magnitude of increase in gene expression of TNF-α and MCP1/CCL2 was the highest among all cytokines and chemokines, respectively. By means of immunohistochemistry, an early (12-24 h) increase in the number of only neutrophil granulocytes (NGs) attached to and around portal vessel walls was observed, followed by increased numbers of mononuclear phagocytes (24-48 h) along the sinusoids. Treatment of the human monocytic cell line U-937 with TNF-α increased the gene expression of CXCL1/KC, CXCL8/IL-8, and MCP1/CCL2. Conversely, adding of infliximab (IFX) to the culture medium inhibited this upregulation significantly. In conclusion, single-dose TAA administration induces a sequence of events with a defined upregulation of gene expression of inflammatory chemokines and cytokines and a transient accumulation of NGs within the portal area and macrophages along the sinusoids throughout the liver. Periportal inflammation seems to precede hepatocellular damage.

Promoter-region hypermethylation and expression downregulation of Yy1 (Yin yang 1) in preneoplastic liver lesions in a thioacetamide rat hepatocarcinogenesis model.

Thioacetamide (TAA) has been used to develop a rodent model for hepatocarcinogenesis. To determine the genes with epigenetic modifications in early hepatocarcinogenesis, we did a genome-wide scan for hypermethylated promoter regions using CpG island microarrays in TAA-promoted rat liver tissue. Eight genes were selected based on the microarray profile; of these, Yy1 and Wdr45b were confirmed to be hypermethylated by methylation-specific polymerase chain reaction (PCR) and pyrosequencing and downregulated by real-time reverse transcription PCR. Non-neoplastic liver cells had nuclear Yy1 immunoreactivity, while preneoplastic foci with glutathione S-transferase placental form (GST-P) immunoreactivity had decreased Yy1 immunoreactivity. The incidence of these foci was proportional to the dose of TAA administered. Co-expression analysis of gene products downstream of Yy1 revealed increased nuclear phospho-c-Myc(+) foci as well as nuclear and cytoplasmic p21(Cip1+) foci in Yy1(-) or GST-P(+) foci in response to TAA-promotion dose. Although the absolute number of cells was low, the incidence of death receptor 5(-) foci was increased in Yy1(-) foci in proportion to the TAA dose. Yy1(-)/GST-P(+) foci revealed a higher number of proliferating cell nuclear antigen (PCNA)-immunoreactive cells than Yy1(+)/GST-P(+) foci, while cleaved caspase-3(+) cells were unchanged between Yy1(-)/GST-P(+) and Yy1(+)/GST-P(+) foci. In the case of Wdr45b, most GST-P(+) foci were Wdr45b(-) and were not increased by TAA promotion. These results suggest involvement of Yy1 in the epigenetic gene regulation at the early stages of TAA promoted cell proliferation and concomitant cell cycle arrest in preneoplastic lesions.

P-glycoprotein and multidrug resistance-associated protein 2 are oppositely altered in brain of rats with thioacetamide-induced acute liver failure.

BACKGROUND: P-glycoprotein (P-GP) and multidrug resistance-associated protein 2 (MRP2) are involved in transport of many drugs across blood-brain barrier (BBB). The function and expression of P-GP and MRP2 may be modulated by different pathologies. Acute liver failure (ALF) was reported to impair BBB function, resulting in the increased BBB permeability. AIMS: We investigated whether ALF altered function and expression of P-GP and MRP2 in brain of thioacetamide-induced ALF rats. METHODS: ALF was induced by intraperitoneal injection of thioacetamide (300 mg/kg) for 2 days with a 24-h interval. The rats were used for experiments at 6, 12 and 24 h after the second administration. P-GP and MRP2 function in brain were determined using the brain-to-plasma ratios of corresponding substrates (rhodamine 123 and vincristine for P-GP; sulfobromophthalein and dinitrophenyl-S-glutathione for MRP2). Evans blue was used for examining the BBB integrity. Western blot was accomplished to determine P-GP and MRP2 protein expression. RESULTS: The brain-to-plasma ratios of rhodamine 123 and vincristine were significantly increased in ALF-6 h rats and almost returned to normal levels in ALF-24 h rats, whereas those of sulfobromophthalein and dinitrophenyl-S-glutathione were decreased in all ALF rats. Western blot results showed that ALF decreased brain P-GP levels at 6 and 12 h, whereas increased MRP2 levels at 6, 12 and 24 h. No significant difference of Evans blue concentrations in brain was found among the four groups. CONCLUSIONS: Function and expression of P-GP and MRP2 in brain of thioacetamide-induced ALF rats were oppositely altered.

Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cytoprotective agent in several organ systems but its roles in liver fibrosis are unclear. We studied the roles of HB-EGF in experimental liver fibrosis in mice and during hepatic stellate cell (HSC) activation. Thioacetamide (TAA; 100 mg/kg) was administered by intraperitoneal injection three times a week for 4 weeks to wild-type HB-EGF(+/+) or HB-EGF-null (HB-EGF(-/-)) male mice. Livers were examined for histology and expression of key fibrotic markers. Primary cultured HSCs isolated from untreated HB-EGF(+/+) or HB-EGF(-/-) mice were examined for fibrotic markers and/or cell migration either during culture-induced activation or after exogenous HB-EGF (100 ng/ml) treatment. TAA induced liver fibrosis in both HB-EGF(+/+) and HB-EGF(-/-) mice. Hepatic HB-EGF expression was decreased in TAA-treated HB-EGF(+/+) mice by 37.6% (P<0.05) as compared with animals receiving saline alone. HB-EGF(-/-) mice treated with TAA showed increased hepatic α-smooth muscle actin-positive cells and collagen deposition, and, as compared with HB-EGF(+/+) mice, TAA-stimulated hepatic mRNA levels in HB-EGF(-/-) mice were, respectively, 2.1-, 1.7-, 1.8-, 2.2-, 1.2- or 3.3-fold greater for α-smooth muscle actin, α1 chain of collagen I or III (COL1A1 or COL3A1), transforming growth factor-ß1, connective tissue growth factor or tissue inhibitor of metalloproteinase-1 (P<0.05). HB-EGF expression was detectable in primary cultured HSCs from HB-EGF(+/+) mice. Both endogenous and exogenous HB-EGF inhibited HSC activation in primary culture, and HB-EGF enhanced HSC migration. These findings suggest that HB-EGF gene knockout in mice increases susceptibility to chronic TAA-induced hepatic fibrosis and that HB-EGF expression or action is associated with suppression of fibrogenic pathways in HSCs.

Effects of armepavine against hepatic fibrosis induced by thioacetamide in 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.

Soy isoflavone delays the progression of thioacetamide-induced liver fibrosis in rats.

OBJECTIVE: Our aim was to investigate the effect of soy isoflavone (SI) on liver fibrosis in a thioacetamide (TAA)-induced rat model. MATERIALS AND METHODS: Twenty-eight rats were assigned to four groups: sham group, fibrosis group, low-dose treatment group (LDg) and high-dose treatment group (HDg). SI (90 or 270 mg/kg) was administered daily during the model development by TAA. Standard liver tests, platelet derived growth factor-BB (PDGF-BB) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were measured. The expression of collagen, α-smooth muscle actin (α-SMA) and transforming growth factor-ß1 (TGF-ß1) in liver tissue was determined. Electron microscopy was used to perform ultrastructural analysis of the livers. RESULTS: Hepatic fibrosis was induced by 8 weeks of TAA administration. However, following the administration of SI, collagen staining significantly declined as compared with the fibrosis group (p < 0.01). Less collagen fibers around the hepatic stellate cells (HSCs) were observed in HDg as compared to the fibrosis group and LDg. There was no significant difference in standard liver tests between the fibrosis group and the two treatment groups. The levels of PDGF-BB and TIMP-1 in the two SI-treated groups were significantly lower than in the fibrosis group (p < 0.01). The expression of α-SMA and TGF-ß1 in HDg was less than that in the fibrosis group and LDg (p < 0.01). CONCLUSION: Administration of a high dose of SI resulted in an obvious inhibitory effect on liver fibrosis induced by TAA in rats. One hypothesis is that the effect may be related to the inhibition of HSC activation and proliferation.

PGC-1α is downregulated in a mouse model of obstructive cholestasis but not in a model of liver fibrosis.

Several studies have indicated that cholestatic liver damage involves mitochondria dysfunction. However, the precise mechanism by which hydrophobic bile salts cause mitochondrial dysfunction is not clear. In this study, we intended to determine the pathogenesis of cholestatic liver injury associated with peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α). A mouse model of cholestatic liver disease was generated by surgical ligation of the bile duct (BDL), and a mouse model of fibrosis was developed through serial administration of thioacetamide. After obtaining liver specimens on scheduled days, we compared the expression of the antioxidant enzymes (superoxide dismutase 2 [SOD2], catalase, and glutathione peroxidase-1[GPx-1]) and PGC-1α in livers from mice with fibrosis and cholestasis using western blotting, immunohistochemistry, and immunofluorescence. We found that cholestatic livers exhibit lower expression of antioxidant enzymes, such as SOD2, catalase, and PGC-1α. In contrast, fibrotic livers exhibit higher expression of antioxidant enzymes and PGC-1α. In addition, cholestatic livers exhibited significantly lower expression of pro-apoptotic markers (Bax) as compared to fibrotic livers. It is well known that overexpression of PGC-1α increases mitochondrial antioxidant enzyme expression, and vice versa. Thus, we concluded that obstructive cholestasis decreases expression of PGC-1α, which may lead to decreased expression of mitochondrial antioxidant enzymes, thereby rendering mice with cholestatic livers vulnerable to ROS-induced cell death.

Myocardial Dysfunction in Cirrhotic Cardiomyopathy is Associated with Alterations of Phospholamban Phosphorylation and IL-6 Levels.

BACKGROUND: Decreased cardiac contractility has been observed in cirrhosis, but the mechanisms that initiate and maintain cardiac dysfunction are not entirely understood. AIM OF THE STUDY: We test the hypothesis that cirrhotic cardiomyopathy is related to deterioration of myocardial contractility due to alterations in calcium-handling proteins expression. In addition, we evaluated whether cardiac pro-inflammatory cytokine levels are associated with this process. METHODS: Cirrhosis was induced by thioacetamide (TAA, 100 mg/kg/i.p., twice weekly for eight weeks). The myocardial performance was evaluated in isolated left ventricle papillary muscles under basal conditions and after inotropic challenge. The cardiac calcium handling protein expression was detected by Western blotting. Cardiac TNF-α and IL-6 levels were measured by ELISA. RESULTS: Thioacetamide induced liver cirrhosis, which was associated with cirrhotic cardiomyopathy characterized by in vivo left ventricular diastolic and systolic dysfunction as well as cardiac hypertrophy. In vitro baseline myocardial contractility was lower in cirrhosis. Also, myocardial responsiveness to post-rest contraction stimulus was declined. Protein expression for RYR2, SERCA2, NCX, pPBL Ser16 and L-type calcium channel was quantitatively unchanged; however, pPBL Thr17 was significantly lower while IL-6 was higher. CONCLUSIONS: Our study demonstrates that cirrhotic cardiomyopathy is associated with decreased cardiac contractility with alteration of phospholamban phosphorylation in association with higher cardiac pro-inflammatory IL-6 levels. These findings provided molecular and functional insights about the effects of liver cirrhosis on cardiac function.

Brassica juncea L. (Mustard) Extract Silver NanoParticles and Knocking off Oxidative Stress, ProInflammatory Cytokine and Reverse DNA Genotoxicity.

Detoxification is one of the main vital tasks performed by the liver. The purpose of this study was to investigate whether mustard in its normal or nanoparticles could confer a protective/therapeutic effect against TAA-induced acute liver failure in experimental animal models. Mustard ethanolic extract was analyzed by HPLC/MS. To induce liver failure, male rats were injected with 350 mg/kg bw TAA IP, then treated orally with a dose of 100 mg/kg for 15 d of mustard extract and its nanoform before and following induction. The levels of serum liver functions, total cholesterol (TCHo), total glyceride (TG), total bilirubin (TBIL), hepatic malonaldhyde (MDA) and nitric oxide (NO),glutathione (GSH), sodium oxide dismutase (SOD), as well as tumor necrosis factor (TNF-α,) and interleukin 6 (IL-6), were estimated. DNA genotoxicity and hepatic pathology, and immunohistologic (IHC) changes were assayed. The antioxidant content of Phenolic acids, flavonoids in mustard ethanolic extract substantially decreased the levels of ALT, AST, ALP and rehabilitated the histopathological alterations. In addition, nanoforms of mustard ethanol extract have notably increased the levels of GSH, SOD and significantly reduced the levels of MDA. The expression levels of TNF-α and IL-6 in serum and tissue were markedly downregulated. DNA genotoxicity was significantly reversed. Mustard introduced a protective and medicinal effect against TAA in both its forms.

Participation of functionally different macrophage populations and monocyte chemoattractant protein-1 in early stages of thioacetamide-induced rat hepatic injury.

Macrophages are crucial in hepatic fibrogenesis. In acute hepatic necrosis induced in rats by a single injection of 300 mg/kg body weight (BW) of thioacetamide (TAA), macrophage properties were investigated using single or double immunohistochemistry. Macrophages reacting with anti-CD68, anti-CD163, or major histocompatibility complex (anti-MHC) class II antibody appeared in injured centrilobular areas on days 1-5 after injection. Increased expression of CD68 and CD163 reflect phagocytosis and production of pro-inflammatory factors, respectively. There were also macrophages double-positive to CD68/CD163, CD68/MHC class II, or CD163/MHC class II; of these, macrophages double-positive to CD68/MHC class II were most frequent, indicating that macrophages with enhanced phagocytic activity came to express MHC class II. The appearance of these macrophages corresponded to increased expression of mRNAs of monocyte chemoattractant protein-1 (MCP-1), a chemokine, on day 1, and TGF-beta1, a fibrogenic factor, on day 3. Some hepatic stellate cells (HSCs) in injured areas reacted with anti-MCP-1 antibody. To investigate the effects of MCP-1, we added MCP-1 to HS-P, a rat macrophage line. Addition of MCP-1 increased immunoexpression for CD68 and CD163 and up-regulated TGF-beta1 mRNA expression. Collectively, macrophages in acute hepatic necrosis may express different properties such as phagocytosis, MHC class II expression, and TGF-beta1 production; such expression may be influenced by MCP-1 produced by HSCs.