Microbial products linked to steatohepatitis are reduced by deletion of nuclear hormone receptor SHP in mice.

Deletion of the nuclear hormone receptor small heterodimer partner (Shp) ameliorates the development of obesity and nonalcoholic steatohepatitis (NASH) in mice. Liver-specific SHP plays a significant role in this amelioration. The gut microbiota has been associated with these metabolic disorders, and the interplay between bile acids (BAs) and gut microbiota contributes to various metabolic disorders. Since hepatic SHP is recognized as a critical regulator in BA synthesis, we assessed the involvement of gut microbiota in the antiobesity and anti-NASH phenotype of Shp-/- mice. Shp deletion significantly altered the levels of a few conjugated BAs. Sequencing the 16S rRNA gene in fecal samples collected from separately housed mice revealed apparent dysbiosis in Shp-/- mice. Cohousing Shp-/- mice with WT mice during a Western diet regimen impaired their metabolic improvement and effectively disrupted their distinctive microbiome structure, which became indistinguishable from that of WT mice. While the Western diet challenge significantly increased lipopolysaccharide and phenylacetic acid (PAA) levels in the blood of WT mice, their levels were not increased in Shp-/- mice. PAA was strongly associated with hepatic peroxisome proliferator-activated receptor gamma isoform 2 (Pparg2) activation in mice, which may represent the basis of the molecular mechanism underlying the association of gut bacteria and hepatic steatosis. Shp deletion reshapes the gut microbiota possibly by altering BAs. While lipopolysaccharide and PAA are the major driving forces derived from gut microbiota for NASH development, Shp deletion decreases these signaling molecules via dysbiosis, thereby partially protecting mice from diet-induced metabolic disorders.

Glycan biomarkers of autoimmunity and bile acid-associated alterations of the human glycome: Primary biliary cirrhosis and primary sclerosing cholangitis-specific glycans.

We have recently introduced multiple reaction monitoring (MRM) mass spectrometry as a novel tool for glycan biomarker research and discovery. Herein, we employ this technique to characterize the site-specific glycan alterations associated with primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). Glycopeptides associated with disease severity were also identified. Multinomial regression modelling was employed to construct and validate multi-analyte diagnostic models capable of accurately distinguishing PBC, PSC, and healthy controls from one another (AUC = 0.93 ± 0.03). Finally, to investigate how disease-relevant environmental factors can influence glycosylation, we characterized the ability of bile acids known to be differentially expressed in PBC to alter glycosylation. We hypothesize that this could be a mechanism by which altered self-antigens are generated and become targets for immune attack. This work demonstrates the utility of the MRM method to identify diagnostic site-specific glycan classifiers capable of distinguishing even related autoimmune diseases from one another.

Epigenetic changes of the thioredoxin system in the tx-j mouse model and in patients with Wilson disease.

Wilson disease (WD) is caused by mutations in the copper transporter ATP7B, leading to copper accumulation in the liver and brain. Excess copper inhibits S-adenosyl-L-homocysteine hydrolase, leading to variable WD phenotypes from widespread alterations in DNA methylation and gene expression. Previously, we demonstrated that maternal choline supplementation in the Jackson toxic milk (tx-j) mouse model of WD corrected higher thioredoxin 1 (TNX1) transcript levels in fetal liver. Here, we investigated the effect of maternal choline supplementation on genome-wide DNA methylation patterns in tx-j fetal liver by whole-genome bisulfite sequencing (WGBS). Tx-j Atp7b genotype-dependent differences in DNA methylation were corrected by choline for genes including, but not exclusive to, oxidative stress pathways. To examine phenotypic effects of postnatal choline supplementation, tx-j mice were randomized to one of six treatment groups: with or without maternal and/or continued choline supplementation, and with or without copper chelation with penicillamine (PCA) treatment. Hepatic transcript levels of TXN1 and peroxiredoxin 1 (Prdx1) were significantly higher in mice receiving maternal and continued choline with or without PCA treatment compared to untreated mice. A WGBS comparison of human WD liver and tx-j mouse liver demonstrated a significant overlap of differentially methylated genes associated with ATP7B deficiency. Further, eight genes in the thioredoxin (TXN) pathway were differentially methylated in human WD liver samples. In summary, Atp7b deficiency and choline supplementation have a genome-wide impact, including on TXN system-related genes, in tx-j mice. These findings could explain the variability of WD phenotype and suggest new complementary treatment options for WD.

RARß acts as both an upstream regulator and downstream effector of miR-22, which epigenetically regulates NUR77 to induce apoptosis of colon cancer cells.

This study investigates the mechanism and consequences of microRNA-22 ( miR-22) induction. Our data revealed for the first time that retinoic acid (RA) and histone deacetylase (HDAC) inhibitors, including short-chain fatty acids and suberanilohydroxamic acid (SAHA), could individually or in combination induce miR-22. This induction was mediated via RA receptor ß (RARß) binding to a direct repeat 5 (DR5) motif. In addition, we uncovered HDAC1 as a novel miR-22 target. In an miR-22-dependent manner, HDAC inhibitors and RA reduced HDAC1, HDAC4, and sirtuin 1 (SIRT1), which were involved in chromatin remodeling of the RARß and nerve growth factor IB ( NUR77). Thus, HDAC inhibitors and RA-induced miR-22 resulted in simultaneous induction of cytoplasmic RARß and NUR77, leading to apoptosis of colon cancer cells. In mice, miR-22 and its inducers inhibited the growth of xenograft colon cancer. Moreover, tumor size reduction was accompanied by elevated miR-22, NUR77, and RARß and by reduced HDACs. In human colon polyps and adenocarcinomas, miR-22 and RARß were consistently reduced, which was associated with elevated HDAC1, HDAC4, and SIRT1 in colon adenocarcinomas. Results from this study revealed a novel anticancer mechanism of RARß via miR-22 induction to epigenetically regulate itself and NUR77, providing a promising cancer treatment modality using miR-22 and its inducers.-Hu, Y., French, S. W., Chau, T., Liu, H.-X., Sheng, L., Wei, F., Stondell, J., Garcia, J. C., Du, Y., Bowlus, C. L., Wan, Y.-J. Y. RARß acts as both an upstream regulator and downstream effector of miR-22, which epigenetically regulates NUR77 to induce apoptosis of colon cancer cells.

Dysregulated bile acid synthesis and dysbiosis are implicated in Western diet-induced systemic inflammation, microglial activation, and reduced neuroplasticity.

The goal of this study was to identify the intrinsic links that explain the effect of a Western diet (WD) on cognitive dysfunction. Specific pathogen-free, wild-type mice were fed either a control diet (CD) or a high-fat, high-sucrose WD after weaning and were euthanized at 10 mo of age to study the pathways that affect cognitive health. The results showed that long-term WD intake reduced hippocampal synaptic plasticity and the level of brain-derived neurotrophic factor mRNA in the brain and isolated microglia. A WD also activated ERK1/2 and reduced postsynaptic density-95 in the brain, suggesting postsynaptic damage. Moreover, WD-fed mice had increased inflammatory signaling in the brain, ileum, liver, adipose tissue, and spleen, which was accompanied by microglia activation. In the brain, as well as in the digestive tract, a WD reduced signaling regulated by retinoic acid and bile acids (BAs), whose receptors form heterodimers to control metabolism and inflammation. Furthermore, a WD intake caused dysbiosis and dysregulated BA synthesis with reduced endogenous ligands for BA receptors, i.e., farnesoid X receptor and G-protein-coupled bile acid receptor in the liver and brain. Together, dysregulated BA synthesis and dysbiosis were accompanied by systemic inflammation, microglial activation, and reduced neuroplasticity induced by WD.-Jena, P. K., Sheng, L., Di Lucente, J., Jin, L.-W., Maezawa, I., Wan, Y.-J. Y. Dysregulated bile acid synthesis and dysbiosis are implicated in Western diet-induced systemic inflammation, microglial activation, and reduced neuroplasticity.

Obesity treatment by epigallocatechin-3-gallate-regulated bile acid signaling and its enriched Akkermansia muciniphila.

Dysregulated bile acid (BA) synthesis is accompanied by dysbiosis, leading to compromised metabolism. This study analyzes the effect of epigallocatechin-3-gallate (EGCG) on diet-induced obesity through regulation of BA signaling and gut microbiota. The data revealed that EGCG effectively reduced diet-increased obesity, visceral fat, and insulin resistance. Gene profiling data showed that EGCG had a significant impact on regulating genes implicated in fatty acid uptake, adipogenesis, and metabolism in the adipose tissue. In addition, metabolomics analysis revealed that EGCG altered the lipid and sugar metabolic pathways. In the intestine, EGCG reduced the FXR agonist chenodeoxycholic acid, as well as the FXR-regulated pathway, suggesting intestinal FXR deactivation. However, in the liver, EGCG increased the concentration of FXR and TGR-5 agonists and their regulated signaling. Furthermore, our data suggested that EGCG activated Takeda G protein receptor (TGR)-5 based on increased GLP-1 release and elevated serum PYY level. EGCG and antibiotics had distinct antibacterial effects. They also differentially altered body weight and BA composition. EGCG, but not antibiotics, increased Verrucomicrobiaceae, under which EGCG promoted intestinal bloom of Akkermansia muciniphila. Excitingly, A. muciniphila was as effective as EGCG in treating diet-induced obesity. Together, EGCG shifts gut microbiota and regulates BA signaling thereby having a metabolic beneficial effect.-Sheng, L., Jena, P. K., Liu, H.-X., Hu, Y., Nagar, N., Bronner, D. N., Settles, M. L., Bäumler, A. J. Wan, Y.-J. Y. Obesity treatment by epigallocatechin-3-gallate-regulated bile acid signaling and its enriched Akkermansia muciniphila.

Western Diet-Induced Dysbiosis in Farnesoid X Receptor Knockout Mice Causes Persistent Hepatic Inflammation after Antibiotic Treatment.

Patients who have liver cirrhosis and liver cancer also have reduced farnesoid X receptor (FXR). The current study analyzes the effect of diet through microbiota that affect hepatic inflammation in FXR knockout (KO) mice. Wild-type and FXR KO mice were on a control (CD) or Western diet (WD) for 10 months. In addition, both CD- and WD-fed FXR KO male mice, which had hepatic lymphocyte and neutrophil infiltration, were treated by vancomycin, polymyxin B, and Abx (ampicillin, neomycin, metronidazole, and vancomycin). Mice were subjected to morphological analysis as well as gut microbiota and bile acid profiling. Male WD-fed FXR KO mice had the most severe steatohepatitis. FXR KO also had reduced Firmicutes and increased Proteobacteria, which could be reversed by Abx. In addition, Abx eliminated hepatic neutrophils and lymphocytes in CD-fed, but not WD-fed, FXR KO mice. Proteobacteria and Bacteroidetes persisted in WD-fed FXR KO mice even after Abx treatment. Only polymyxin B could reduce hepatic lymphocytes in WD-fed FXR KO mice. The reduced hepatic inflammation by antibiotics was accompanied by decreased free and conjugated secondary bile acids as well as changes in gut microbiota. Our data revealed that Lactococcus, Lactobacillus, and Coprococcus protect the liver from inflammation.

Hepatic inflammation caused by dysregulated bile acid synthesis is reversible by butyrate supplementation.

Dysregulated bile acid (BA) synthesis or reduced farnesoid X receptor (FXR) levels are found in patients having metabolic diseases, autoimmune hepatitis, and liver cirrhosis or cancer. The objective of this study was to establish the relationship between butyrate and dysregulated BA synthesis-induced hepatitis as well as the effect of butyrate in reversing the liver pathology. Wild-type (WT) and FXR knockout (KO) male mice were placed on a control (CD) or western diet (WD) for 15 months. In the presence or absence of butyrate supplementation, feces obtained from 15-month-old WD-fed FXR KO mice, which had severe hepatitis and liver tumors, were transplanted to 7-month-old WD-fed FXR KO for 3 months. Hepatic phenotypes, microbiota profile, and BA composition were analyzed. Butyrate-generating bacteria and colonic butyrate concentration were reduced due to FXR inactivation and further reduced by WD intake. In addition, WD-fed FXR KO male mice had the highest concentration of hepatic ß-muricholic acid (ß-MCA) and bacteria-generated deoxycholic acid (DCA) accompanied by serious hepatitis. Moreover, dysregulated BA and reduced SCFA signaling co-existed in both human liver cancers and WD-fed FXR KO mice. Microbiota transplantation using butyrate-deficient feces derived from 15-month-old WD-fed FXR KO mice increased hepatic lymphocyte numbers as well as hepatic ß-MCA and DCA concentrations. Furthermore, butyrate supplementation reduced hepatic ß-MCA as well as DCA and eliminated hepatic lymphocyte infiltration. In conclusion, reduced butyrate contributes to the development of hepatitis in the FXR KO mouse model. In addition, butyrate reverses dysregulated BA synthesis and its associated hepatitis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

MiR-22-silenced cyclin A expression in colon and liver cancer cells is regulated by bile acid receptor.

Because of the significant tumor-suppressive role of microRNA-22 (miR-22), the current study was designed to understand the regulation of miR-22 and to identify additional downstream miR-22 targets in liver and colon cells. The data showed that miR-22 was transcriptionally regulated by bile acid receptor farnesoid X receptor (FXR) through direct binding to an invert repeat 1 motif located at -1012 to -1025 bp upstream from miR-22. Among the studied primary and secondary bile acids, chenodeoxycholic acid, which has the highest binding affinity to FXR, induced miR-22 level in both Huh7 liver and HCT116 colon cells in a dose- and time-dependent manner. In addition, cyclin A2 (CCNA2) was identified as a miR-22 novel target in liver and colon cancer cells. The sequence of miR-22, which is conserved in mice, rats, humans, and other mammalians, aligns with the sequence of 3'-UTR of CCNA2. Chenodeoxycholic acid treatment and miR-22 mimics reduced CCNA2 protein and increased the number of G0/G1 Huh7 and HCT116 cells. In FXR KO mice, reduction of miR-22 was accompanied by elevated hepatic and ileal CCNA2 protein, as well as an increased number of hepatic and colonic Ki-67-positive cells. In humans, the expression levels of miR-22 and CCNA2 are inversely correlated in liver and colon cancers. Taken together, our data showed that bile acid-activated FXR stimulates miR-22-silenced CCNA2, a novel pathway for FXR to exert its protective effect in the gastrointestinal tract.

Functional analysis of the relationship between intestinal microbiota and the expression of hepatic genes and pathways during the course of liver regeneration.

BACKGROUND & AIMS: The pathways regulating liver regeneration have been extensively studied within the liver. However, the signaling contribution derived from the gut microbiota to liver regeneration is poorly understood. METHODS: Microbiota and expression of hepatic genes in regenerating livers obtained from mice at 0h to 9days post 2/3 partial hepatectomy were temporally profiled to establish their interactive relationships. RESULTS: Partial hepatectomy led to rapid changes in gut microbiota that was reflected in an increased abundance of Bacteroidetes S24-7 and Rikenellaceae and decreased abundance of Firmicutes Clostridiales, Lachnospiraceae, and Ruminococcaceae. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was used to infer biological functional changes of the shifted microbiota. RNA-sequencing data revealed 6125 genes with more than a 2-fold difference in their expression levels during regeneration. By analyzing their expression pattern, six uniquely expressed patterns were observed. In addition, there were significant correlations between hepatic gene expression profiles and shifted bacterial populations during regeneration. Moreover, hepatic metabolism and immune function were closely associated with the abundance of Ruminococcacea, Lachnospiraceae, and S24-7. Bile acid profile was analyzed because bacterial enzymes produce bile acids that significantly impact hepatocyte proliferation. The data revealed that specific bacteria were closely associated with the concentration of certain bile acids and expression of hepatic genes. CONCLUSIONS: The presented data established, for the first time, an intimate relationship between intestinal microbiota and the expression of hepatic genes in regenerating livers.

Implications of microbiota and bile acid in liver injury and regeneration.

Studies examining the mechanisms by which the liver incurs injury and then regenerates usually focus on factors and pathways directly within the liver, neglecting the signaling derived from the gut-liver axis. The intestinal content is rich in microorganisms as well as metabolites generated from both the host and colonizing bacteria. Through the gut-liver axis, this complex "soup" exerts an immense impact on liver integrity and function. This review article summarizes data published in the past 30 years demonstrating the signaling derived from the gut-liver axis in relation to liver injury and regeneration. Due to the intricate networks of implicated pathways as well as scarcity of available mechanistic data, it seems that nutrigenomic, metabolomics, and microbiota profiling approaches are warranted to provide a better understanding regarding the interplay and impact between nutrition, bacteria, and host response in influencing liver function and healing. Therefore elucidating the possible molecular mechanisms that link microbiota alteration to host physiological response and vice versa.

Donor PNPLA3 rs738409 genotype affects fibrosis progression in liver transplantation for hepatitis C.

UNLABELLED: The rs738409 G>C single nucleotide polymorphism occurring in the patatin-like phospholipase 3 gene has been identified as a novel genetic marker for hepatic steatosis. Recent studies also associated rs738409 with fibrosis in hepatitis C (HCV). Therefore, we sought to determine the impact of donor and recipient rs738409 genotype on the progression of fibrosis after liver transplantation for HCV. This cohort study included 101 patients infected with HCV who underwent liver transplantation between January 2008, and June 2011. Donor and recipient rs738409 genotypes were determined from donor wedge biopsies and recipient explants. The time to Ishak stage 3 fibrosis, or HCV-related mortality/graft loss was analyzed by the Cox model adjusting for HCV-Donor Risk Index, warm ischemic time, pretransplant Model for Endstage Liver Disease (MELD) and viral load. The rs738409 CC variant was present in 56% of donors and 57% of recipients. The median follow-up period was 620 days. A total of 39 patients developed the primary outcome of ≥stage 3 fibrosis or HCV-related mortality/graft loss, the time to which differed by donor (P = 0.019) but not recipient (P = 0.89) genotype. In the multivariate model, donor GC or GG variants had 2.53 times the risk (95% confidence interval [CI] 1.25-5.02, P = 0.008) compared to CC variants. In the alternative endpoint: stage 3 fibrosis or all-cause mortality/graft loss, the effect of donor genotype was attenuated but remained significant at 1.98 (95% CI 1.11-3.53). CONCLUSIONS: The rs738409 genotype is an important predictor of posttransplant outcome in HCV. Liver, and not adipocytes, is the site at which this effect occurs. Our finding may be useful in donor selection for liver transplantation with HCV, and may guide decisions regarding early antiviral treatment.

The effect of miRNA-122 in regulating fat deposition in a cell line model.

Accumulating evidence supports the role of miR-122 in fatty liver disease. We investigated miR-122 expression in a steatotic hepatocyte model, the effect of miR-122 over-expression and inhibition in the pathogenesis. Human hepatic cell line L02 was induced with oleic acid to establish the steatotic hepatocyte model. Intracellular lipid content was observed with laser scanning confocal microscope (LSCM), and triglyceride content was determined with kits. Total RNA was extracted and reversely transcribed into cDNA. miR-122 expression was measured using qRT-PCR. Subsequently, miR-122 mimic and miR-122 inhibitor were transfected into steatotic hepatocytes to observe their effect on intracellular lipid content. The lipid fluorescence intensity and triglyceride content within the steatotic hepatocytes were significantly higher than those in normal control (860.01 ± 26.52 vs. 257.77 ± 29.69 and 3.47 ± 0.12 vs. 1.85 ± 0.02 at 24 h) (P < 0.01). miR-122 expression in steatotic hepatocytes was down-regulated compared with that in control (2-ΔCt value: 0.0286 ± 0.0078 vs. 0.0075 ± 0.0012) (P ≪ 0.01). After transfection, miR-122 expression (2-ΔCt value) in the miR-122 mimic group increased 2.96-fold compared with that in control, and its lipid fluorescence intensity was significantly lower than that in control (790.92 ± 46.72 vs. 1,022.16 ± 49.66) (P < 0.01). Nevertheless, miR-122 expression decreased 3.45-fold in the miR-122 inhibitor group compared with that in control, and its fluorescence intensity was significantly higher than that in control (1,386.49 ± 40.34 vs 1,022.16 ± 49.66)(P ≪ 0.01). We concluded that miR-122 was down-regulated in steatotic hepatocytes model. The pathogenesis of hepatocyte steatosis was enhanced by miR-122 mimic and reduced with miR-122 inhibitor.

Biological functional annotation of retinoic acid alpha and beta in mouse liver based on genome-wide binding.

Retinoic acid (RA) has diverse biological effects. The liver stores vitamin A, generates RA, and expresses receptors for RA. The current study examines the hepatic binding profile of two RA receptor isoforms, RARA (RARα) and RARB (RARß), in response to RA treatment in mouse livers. Our data uncovered 35,521, and 14,968 genomic bindings for RARA and RARB, respectively. Each expressed unique and common bindings, implying their redundant and specific roles. RARB has higher RA responsiveness than RARB. RA treatment generated 18,821 novel RARB bindings but only 14,798 of RARA bindings, compared with the control group. RAR frequently bound the consensus hormone response element [HRE; (A/G)G(G/T)TCA], which often contained the motifs assigned to SP1, GABPA, and FOXA2, suggesting potential interactions between those transcriptional factors. Functional annotation coupled with principle component analysis revealed that the function of RAR target genes were motif dependent. Taken together, the cistrome of RARA and RARB revealed their extensive biological roles in the mouse liver. RAR target genes are enriched in various biological processes. The hepatic RAR genome-wide binding data can help us understand the global molecular mechanisms underlying RAR and RA-mediated gene and pathway regulation.

NURBS: a database of experimental and predicted nuclear receptor binding sites of mouse.

SUMMARY: Nuclear receptors (NRs) are a class of transcription factors playing important roles in various biological processes. An NR often impacts numerous genes and different NRs share overlapped target networks. To fulfil the need for a database incorporating binding sites of different NRs at various conditions for easy comparison and visualization to improve our understanding of NR binding mechanisms, we have developed NURBS, a database for experimental and predicted nuclear receptor binding sites of mouse (NURBS). NURBS currently contains binding sites across the whole-mouse genome of 8 NRs identified in 40 chromatin immunoprecipitation with massively parallel DNA sequencing experiments. All datasets are processed using a widely used procedure and same statistical criteria to ensure the binding sites derived from different datasets are comparable. NURBS also provides predicted binding sites using NR-HMM, a Hidden Markov Model (HMM) model. AVAILABILITY: The GBrowse-based user interface of NURBS is freely accessible at http://shark.abl.ku.edu/nurbs/. NR-HMM and all results can be downloaded for free at the website. CONTACT: jwfang@ku.edu

Metabolomic and genomic evidence for compromised bile acid homeostasis by senecionine, a hepatotoxic pyrrolizidine alkaloid.

Pyrrolizidine alkaloids (PAs) are among the most hepatotoxic natural products that produce irreversible injury to humans via the consumption of herbal medicine and honey, and through tea preparation. Toxicity and death caused by PA exposure have been reported worldwide. Metabolomics and genomics provide scientific and systematic views of a living organism and have become powerful techniques for toxicology research. In this study, senecionine hepatotoxicity on rats was determined via a combination of metabolomic and genomic analyses. From the global analysis generated from two omics data, the compromised bile acid homeostasis in vivo was innovatively demonstrated and confirmed. Serum profiling of bile acids was altered with significantly elevated conjugated bile acids after senecionine exposure, which was in accordance with toxicity. Similarly, the hepatic mRNA levels of several key genes associated with bile acid metabolism were significantly changed. This process included cholesterol 7-α hydroxylase, bile acid CoA-amino acid N-acetyltransferase, sodium taurocholate cotransporting polypeptide, organic anion-transporting polypeptides, and multidrug-resistance-associated protein 3. In conclusion, a cross-omics study provides a comprehensive analysis method for studying the toxicity caused by senecionine, which is a hepatotoxic PA. Moreover, the change in bile acid metabolism and the respective transporters may provide a new PA toxicity mechanism.

BCG as an Innovative Option for HCC Treatment: Repurposing and Mechanistic Insights.

This study investigates Bacillus Calmette-Guérin (BCG) as a potential treatment for hepatocellular carcinoma (HCC), a condition often associated with unfavorable treatment outcomes. Exploiting BCG's recognized immune-boosting properties, preclinical trials are conducted using HCC mice, with a single subcutaneous dose of BCG administered post-tumor formation. Results indicate that BCG treatment effectively diminishes tumor burden and extends survival in both male and female HCC mice. Positive influences on hepatic fibrosis and metabolism are observed, leading to a reduction in lipid levels. Spatial analysis underscores BCG's tumor-specific effects, inducing the enrichment of metabolic pathways and inhibiting various cancer-related pathways. Furthermore, BCG promotes immune cell infiltration, including CD4+, CD8+ T cells, and M1 macrophages, in both v-akt murine thymoma viral oncogene homolog 1(AKT)/neutoblastoma RAS viral oncogene homolog (RAS) and ß-catenin positive HCC models. Interestingly, blocking T cells, trained immunity, and Interferon-γ (IFN-γ) function reverses BCG's anti-HCC effects. In conclusion, BCG emerges as a promising treatment option for HCC, characterized by a favorable safety profile and efficacy in inhibiting fibrosis, improving metabolism, and engaging both trained immunity and T cells in therapeutic mechanisms.

The role of retinoic acid in hepatic lipid homeostasis defined by genomic binding and transcriptome profiling.

BACKGROUND: The eyes and skin are obvious retinoid target organs. Vitamin A deficiency causes night blindness and retinoids are widely used to treat acne and psoriasis. However, more than 90% of total body retinol is stored in liver stellate cells. In addition, hepatocytes produce the largest amount of retinol binding protein and cellular retinoic acid binding protein to mobilize retinol from the hepatic storage pool and deliver retinol to its receptors, respectively. Furthermore, hepatocytes express the highest amount of retinoid x receptor alpha (RXRα) among all the cell types. Surprisingly, the function of endogenous retinoids in the liver has received very little attention. RESULTS: Based on the data generated from chromatin immunoprecipitation followed by sequencing, the global DNA binding of transcription factors including retinoid x receptor α (RXRα) along with its partners i.e. retinoic acid receptor α (RARα), pregnane x receptor (PXR), liver x receptor (LXR), farnesoid x receptor (FXR), and peroxisome proliferator-activated receptor α (PPARα) has been established. Based on the binding, functional annotation illustrated the role of those receptors in regulating hepatic lipid homeostasis. To correlate the DNA binding data with gene expression data, the expression patterns of 576 genes that regulate lipid homeostasis were studied in wild type and liver RXRα-null mice treated with and without RA. The data showed that RA treatment and RXRα-deficiency had opposite effects in regulating lipid homeostasis. A subset of genes (114), which could clearly differentiate the effect of ligand treatment and receptor deficiency, were selected for further functional analysis. The expression data suggested that RA treatment could produce unsaturated fatty acids and induce triglyceride breakdown, bile acid secretion, lipolysis, and retinoids elimination. In contrast, RXRα deficiency might induce the synthesis of saturated fatty acids, triglyceride, cholesterol, bile acids, and retinoids. In addition, DNA binding data indicated extensive cross-talk among RARα, PXR, LXR, FXR, and PPARα in regulating those RA/RXRα-dependent gene expression levels. Moreover, RA reduced serum cholesterol, triglyceride, and bile acid levels in mice. CONCLUSIONS: We have characterized the role of hepatic RA for the first time. Hepatic RA mediated through RXRα and its partners regulates lipid homeostasis.

Protein expression profiling of nuclear membrane protein reveals potential biomarker of human hepatocellular carcinoma.

BACKGROUND: Complex molecular events lead to development and progression of liver cirrhosis to HCC. Differentially expressed nuclear membrane associated proteins are responsible for the functional and structural alteration during the progression from cirrhosis to carcinoma. Although alterations/ post translational modifications in protein expression have been extensively quantified, complementary analysis of nuclear membrane proteome changes have been limited. Deciphering the molecular mechanism that differentiate between normal and disease state may lead to identification of biomarkers for carcinoma. RESULTS: Many proteins displayed differential expression when nuclear membrane proteome of hepatocellular carcinoma (HCC), fibrotic liver, and HepG2 cell line were assessed using 2-DE and ESI-Q-TOF MS/MS. From the down regulated set in HCC, we have identified for the first time a 15 KDa cytochrome b5A (CYB5A), ATP synthase subunit delta (ATPD) and Hemoglobin subunit beta (HBB) with 11, 5 and 22 peptide matches respectively. Furthermore, nitrosylation studies with S-nitrosocysteine followed by immunoblotting with anti SNO-cysteine demonstrated a novel and biologically relevant post translational modification of thiols of CYB5A in HCC specimens only. Immunofluorescence images demonstrated increased protein S-nitrosylation signals in the tumor cells and fibrotic region of HCC tissues. The two other nuclear membrane proteins which were only found to be nitrosylated in case of HCC were up regulated ATP synthase subunit beta (ATPB) and down regulated HBB. The decrease in expression of CYB5A in HCC suggests their possible role in disease progression. Further insight of the functional association of the identified proteins was obtained through KEGG/ REACTOME pathway analysis databases. String 8.3 interaction network shows strong interactions with proteins at high confidence score, which is helpful in characterization of functional abnormalities that may be a causative factor of liver pathology. CONCLUSION: These findings may have broader implications for understanding the mechanism of development of carcinoma. However, large scale studies will be required for further verification of their critical role in development and progression of HCC.

The contributions of bacteria metabolites to the development of hepatic encephalopathy.

Over 20% of mortality during acute liver failure is associated with the development of hepatic encephalopathy (HE). Thus, HE is a complication of acute liver failure with a broad spectrum of neuropsychiatric abnormalities ranging from subclinical alterations to coma. HE is caused by the diversion of portal blood into systemic circulation through portosystemic collateral vessels. Thus, the brain is exposed to intestinal-derived toxic substances. Moreover, the strategies to prevent advancement and improve the prognosis of such a liver-brain disease rely on intestinal microbial modulation. This is supported by the findings that antibiotics such as rifaximin and laxative lactulose can alleviate hepatic cirrhosis and/or prevent HE. Together, the significance of the gut-liver-brain axis in human health warrants attention. This review paper focuses on the roles of bacteria metabolites, mainly ammonia and bile acids (BAs) as well as BA receptors in HE. The literature search conducted for this review included searches for phrases such as BA receptors, BAs, ammonia, farnesoid X receptor (FXR), G protein-coupled bile acid receptor 1 (GPBAR1 or TGR5), sphingosine-1-phosphate receptor 2 (S1PR2), and cirrhosis in conjunction with the phrase hepatic encephalopathy and portosystemic encephalopathy. PubMed, as well as Google Scholar, was the search engines used to find relevant publications.