High Soluble Fiber Promotes Colorectal Tumorigenesis Through Modulating Gut Microbiota and Metabolites in Mice.

BACKGROUND & AIMS: Dietary fibers are mainly fermented by the gut microbiota, but their roles in colorectal cancer (CRC) are largely unclear. Here, we investigated the associations of different fibers with colorectal tumorigenesis in mice. METHODS: Apcmin/+ mice and C57BL/6 mice with azoxymethane (AOM) injection were used as CRC mouse models. Mice were fed with mixed high-fiber diet (20% soluble fiber and 20% insoluble fiber), high-inulin diet, high-guar gum diet, high-cellulose diet, or diets with different inulin dose. Germ-free mice were used for validation. Fecal microbiota and metabolites were profiled by shotgun metagenomic sequencing and liquid chromatography-mass spectrometry, respectively. RESULTS: Mixed high-fiber diet promoted colorectal tumorigenesis with increased tumor number and tumor load in AOM-treated and Apcmin/+ mice. Antibiotics use abolished the pro-tumorigenic effect of mixed high-fiber diet, while transplanting stools from mice fed with mixed high-fiber diet accelerated tumor growth in AOM-treated germ-free mice. We therefore characterized the contribution of soluble and insoluble fiber in CRC separately. Our results revealed that soluble fiber inulin or guar gum, but not insoluble fiber cellulose, promoted colorectal tumorigenesis in AOM-treated and Apcmin/+ mice. Soluble fiber induced gut dysbiosis with Bacteroides uniformis enrichment and Bifidobacterium pseudolongum depletion, accompanied by increased fecal butyrate and serum bile acids and decreased inosine. We also identified a positive correlation between inulin dosage and colorectal tumorigenesis. Moreover, transplanting stools from mice fed with high-inulin diet increased colonic cell proliferation and oncogene expressions in germ-free mice. CONCLUSION: High-dose soluble but not insoluble fiber potentiates colorectal tumorigenesis in a dose-dependent manner by dysregulating gut microbiota and metabolites in mice.

Dietary cholesterol drives fatty liver-associated liver cancer by modulating gut microbiota and metabolites.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD)-associated hepatocellular carcinoma (HCC) is an increasing healthcare burden worldwide. We examined the role of dietary cholesterol in driving NAFLD-HCC through modulating gut microbiota and its metabolites. DESIGN: High-fat/high-cholesterol (HFHC), high-fat/low-cholesterol or normal chow diet was fed to C57BL/6 male littermates for 14 months. Cholesterol-lowering drug atorvastatin was administered to HFHC-fed mice. Germ-free mice were transplanted with stools from mice fed different diets to determine the direct role of cholesterol modulated-microbiota in NAFLD-HCC. Gut microbiota was analysed by 16S rRNA sequencing and serum metabolites by liquid chromatography-mass spectrometry (LC-MS) metabolomic analysis. Faecal microbial compositions were examined in 59 hypercholesterolemia patients and 39 healthy controls. RESULTS: High dietary cholesterol led to the sequential progression of steatosis, steatohepatitis, fibrosis and eventually HCC in mice, concomitant with insulin resistance. Cholesterol-induced NAFLD-HCC formation was associated with gut microbiota dysbiosis. The microbiota composition clustered distinctly along stages of steatosis, steatohepatitis and HCC. Mucispirillum, Desulfovibrio, Anaerotruncus and Desulfovibrionaceae increased sequentially; while Bifidobacterium and Bacteroides were depleted in HFHC-fed mice, which was corroborated in human hypercholesteremia patients. Dietary cholesterol induced gut bacterial metabolites alteration including increased taurocholic acid and decreased 3-indolepropionic acid. Germ-free mice gavaged with stools from mice fed HFHC manifested hepatic lipid accumulation, inflammation and cell proliferation. Moreover, atorvastatin restored cholesterol-induced gut microbiota dysbiosis and completely prevented NAFLD-HCC development. CONCLUSIONS: Dietary cholesterol drives NAFLD-HCC formation by inducing alteration of gut microbiota and metabolites in mice. Cholesterol inhibitory therapy and gut microbiota manipulation may be effective strategies for NAFLD-HCC prevention.

Lactobacillus gallinarum modulates the gut microbiota and produces anti-cancer metabolites to protect against colorectal tumourigenesis.

OBJECTIVE: Using faecal shotgun metagenomic sequencing, we identified the depletion of Lactobacillus gallinarum in patients with colorectal cancer (CRC). We aimed to determine the potential antitumourigenic role of L. gallinarum in colorectal tumourigenesis. DESIGN: The tumor-suppressive effect of L. gallinarum was assessed in murine models of CRC. CRC cell lines and organoids derived from patients with CRC were cultured with L. gallinarum or Escherichia coli MG1655 culture-supernatant to evaluate cell proliferation, apoptosis and cell cycle distribution. Gut microbiota was assessed by 16S ribosomal DNA sequencing. Antitumour molecule produced from L. gallinarum was identified by liquid chromatography mass spectrometry (LC-MS/MS) and targeted mass spectrometry. RESULTS: L. gallinarum significantly reduced intestinal tumour number and size compared with E. coli MG1655 and phosphate-buffered saline in both male and female murine intestinal tumourigenesis models. Faecal microbial profiling revealed enrichment of probiotics and depletion of pathogenic bacteria in L. gallinarum-treated mice. Culturing CRC cells with L. gallinarum culture-supernatant (5%, 10% and 20%) concentration-dependently suppressed cell proliferation and colony formation. L. gallinarum culture-supernatant significantly promoted apoptosis in CRC cells and patient-derived CRC organoids, but not in normal colon epithelial cells. Only L. gallinarum culture-supernatant with fraction size <3 kDa suppressed proliferation in CRC cells. Using LC-MS/MS, enrichments of indole-3-lactic acid (ILA) was identified in both L. gallinarum culture-supernatant and the gut of L. gallinarum-treated mice. ILA displayed anti-CRC growth in vitro and inhibited intestinal tumourigenesis in vivo. CONCLUSION: L. gallinarum protects against intestinal tumourigenesis by producing protective metabolites that can promote apoptosis of CRC cells.

O-GlcNAcylation inhibits hepatic stellate cell activation.

BACKGROUND AND AIM: Protein O-GlcNAcylation is a critical post-translational modification regulating gene expression and fundamental cell functions. O-GlcNAc transferase (OGT) emerged as a key regulator of liver pathophysiology and disease. In this study, we aimed to evaluate the role of OGT in hepatic stellate cells (HSCs) and its consequent role in liver fibrosis. METHODS: Primary HSCs were isolated from C57/B6 mice. Cell morphology and α-SMA immunofluorescence staining were observed under scanning confocal microscope. Transcriptomic profile was evaluated by RNAseq (Illumina). Promoter activity was examined by luciferase and ß--Galactosidase reporter assays. Liver fibrosis mouse models were induced either by intraperitoneal injection of CCl4 at 3 times/week for 4 weeks or by feeding with methionine and choline deficient (MCD) diet for 4 weeks. RESULTS: OGT protein expression and protein O-GlcNAcylation were significantly decreased in CCl4 - or MCD diet-induced liver fibrosis as compared with normal liver in mice. OGT expression and protein O-GlcNAcylation were also decreased in primary HSCs isolated from liver with CCl4 -induced fibrosis compared with those from normal liver. RNA-seq showed that OGT knockdown in HSCs modulated key signaling pathways involved in HSC activation. Promoter sequence analysis of the differentially expressed genes predicted serum response factor (SRF) as a key transcription factor regulated by OGT. Luciferase reporter assay confirmed that OGT repressed activity of SRF to induce α-SMA transcription. Mutations of specific O-GlcNAcylation sites on SRF increased its transcriptional activity, validating negative regulation of SRF by OGT-mediated O-GlcNAcylation. CONCLUSIONS: Our results suggest that OGT functions as a negative regulator of HSC activation by promoting SRF O-GlcNAcylation to protect against liver fibrosis.

Fecal microbial DNA markers serve for screening colorectal neoplasm in asymptomatic subjects.

BACKGROUND AND AIM: We have previously shown that fecal microbial markers might be useful for non-invasive diagnosis of colorectal cancer (CRC) and adenoma. Here, we assessed the application of microbial DNA markers, as compared with and in combination with fecal immunochemical test (FIT), in detecting CRC and adenoma in symptomatic patients and asymptomatic subjects. METHODS: We recruited 676 subjects [210 CRC, 115 advanced adenoma (AA), 86 non-advanced adenoma, and 265 non-neoplastic controls], including 241 symptomatic and 435 asymptomatic subjects. Fecal abundances of Fusobacterium nucleatum, a Lachnoclostridium sp. m3, Bacteroides clarus, and Clostridium hathewayi were quantified by quantitative PCR. Combining score of the four microbial markers (4Bac) and diagnostic prediction were determined using our previously established scoring model and cutoff values and FIT with a cutoff of 100 ng Hb/mL. RESULTS: 4Bac detected similar percentages of CRC [85.3% (95%CI: 79.2-90.2%) vs 84.9% (68.1-94.9%)] and AA [35.7% (12.8-64.9%) vs 38.6% (29.1-48.8%)], while FIT detected more CRC [72.1% (63.7-79.4%) vs 66.7% (48.2-82.0%)] and AA [28.6% (8.4-58.1%) vs 16.8% (10.1-25.6%)], in symptomatic vs asymptomatic subjects, respectively. Focusing on the asymptomatic cohort, 4Bac was more sensitive for diagnosing CRC and AA than FIT (P < 0.001), with lower specificity [83.3% (77.6-88.0%) vs 98.6% (96.0-99.7%)]. FIT failed to detect any non-advanced adenoma [0% (0.0-4.2%)] compared with 4Bac [41.9% (31.3-53.0%), P < 0.0001]. Combining 4Bac with FIT improved sensitivities for CRC [90.9% (75.7-98.1%)] and AA [48.5% (38.4-58.7%)] detection. CONCLUSION: Quantitation of fecal microbial DNA markers may serve as a new test, stand alone, or in combination with FIT for screening colorectal neoplasm in asymptomatic subjects.

Gastric microbes associated with gastric inflammation, atrophy and intestinal metaplasia 1 year after Helicobacter pylori eradication.

OBJECTIVE: Helicobacter pylori is associated with gastric inflammation, precancerous gastric atrophy (GA) and intestinal metaplasia (IM). We aimed to identify microbes that are associated with progressive inflammation, GA and IM 1 year after H. pylori eradication. DESIGN: A total of 587 H. pylori-positive patients were randomised to receive H. pylori eradication therapy (295 patients) or placebo (292 patients). Bacterial taxonomy was analysed on 404 gastric biopsy samples comprising 102 pairs before and after 1 year H. pylori eradication and 100 pairs before and after 1 year placebo by 16S rRNA sequencing. RESULTS: Analysis of microbial sequences confirmed the eradication of H. pylori in treated group after 1 year. Principal component analysis revealed distinct microbial clusters reflected by increase in bacterial diversity (p<0.00001) after H. pylori eradication. While microbial interactions remained largely unchanged after placebo treatment, microbial co-occurrence was less in treated group. Acinetobacter lwoffii, Streptococcus anginosus and Ralstonia were enriched while Roseburia and Sphingomonas were depleted in patients with persistent inflammation 1 year after H. pylori eradication. A distinct cluster of oral bacteria comprising Peptostreptococcus, Streptococcus, Parvimonas, Prevotella, Rothia and Granulicatella were associated with emergence and persistence of GA and IM. Probiotic Faecalibacterium praustznii was depleted in subjects who developed GA following H. pylori eradication. Functional pathways including amino acid metabolism and inositol phosphate metabolism were enriched while folate biosynthesis and NOD-like receptor signalling decreased in atrophy/IM-associated gastric microbiota. CONCLUSION: This study identified that gastric microbes contribute to the progression of gastric carcinogenesis after H. pylori eradication.

A novel faecal Lachnoclostridium marker for the non-invasive diagnosis of colorectal adenoma and cancer.

OBJECTIVE: There is a need for early detection of colorectal cancer (CRC) at precancerous-stage adenoma. Here, we identified novel faecal bacterial markers for diagnosing adenoma. DESIGN: This study included 1012 subjects (274 CRC, 353 adenoma and 385 controls) from two independent Asian groups. Candidate markers were identified by metagenomics and validated by targeted quantitative PCR. RESULTS: Metagenomic analysis identified 'm3' from a Lachnoclostridium sp., Fusobacterium nucleatum (Fn) and Clostridium hathewayi (Ch) to be significantly enriched in adenoma. Faecal m3 and Fn were significantly increased from normal to adenoma to CRC (p<0.0001, linear trend by one-way ANOVA) in group I (n=698), which was further confirmed in group II (n=313; p<0.0001). Faecal m3 may perform better than Fn in distinguishing adenoma from controls (areas under the receiver operating characteristic curve (AUROCs) m3=0.675 vs Fn=0.620, p=0.09), while Fn performed better in diagnosing CRC (AUROCs Fn=0.862 vs m3=0.741, p<0.0001). At 78.5% specificity, m3 and Fn showed sensitivities of 48.3% and 33.8% for adenoma, and 62.1% and 77.8% for CRC, respectively. In a subgroup tested with faecal immunochemical test (FIT; n=642), m3 performed better than FIT in detecting adenoma (sensitivities for non-advanced and advanced adenomas of 44.2% and 50.8% by m3 (specificity=79.6%) vs 0% and 16.1% by FIT (specificity=98.5%)). Combining with FIT improved sensitivity of m3 for advanced adenoma to 56.8%. The combination of m3 with Fn, Ch, Bacteroides clarus and FIT performed best for diagnosing CRC (specificity=81.2% and sensitivity=93.8%). CONCLUSION: This study identifies a novel bacterial marker m3 for the non-invasive diagnosis of colorectal adenoma.

Bone marrow-derived macrophage contributes to fibrosing steatohepatitis through activating hepatic stellate cells.

The role of macrophages in fibrosing steatohepatitis is largely unclear. We characterized the origin and molecular mechanisms of macrophages and its targeted therapy of fibrosing steatohepatitis. Fibrosing steatohepatitis was established in Alms1 mutant (foz/foz) and C57BL/6J wildtype mice fed high-fat/high-cholesterol or methionine- and choline-deficient diet. Bone marrow transplantation was performed to track the macrophage origin in fibrosing steatohepatitis. Macrophages were depleted using liposomal clodronate. Primary macrophages were isolated from bone marrow for adoptive transfer into mice. We found that macrophage infiltration is induced in two mouse models of fibrosing steatohepatitis and human nonalcoholic steatohepatitis-fibrosis patients. Bone marrow-derived macrophages (BMMs) contribute to the hepatic macrophage accumulation in experimental fibrosing steatohepatitis. Depletion of hepatic BMMs by liposomal clodronate during liver injury attenuated fibrosing steatohepatitis, whilst BMMs depletion after liver injury delayed the regression of fibrosing steatohepatitis. The pro-fibrotic effect of macrophages was associated with reduced activation of hepatic stellate cells (HSCs), collagen deposition and hepatic expression of key pro-fibrotic factors (TIMP1, TIMP2, and TGFß1) and endoplasmic reticulum stress markers (GRP78, IRE1α, and PDI). Conversely, adoptive transfer of BMMs significantly aggravated fibrosing steatohepatitis. Moreover, macrophage-conditioned medium directly promoted the phenotypic transition of primary quiescent HSCs to activated HSCs; it enhanced activation and proliferation but decreased apoptosis of HSC cell lines (LX-2 and HSC-T6). The effect of BMMs in promoting fibrosing steatohepatitis was mediated by inducing key pro-fibrosis factors and signaling pathways including cytokine/chemokine, TGFß and complement cascade as assessed by cDNA expression array. Complement 3a receptor (C3ar1) was a predominant effector of macrophage mediated fibrosing steatohepatitis. Knockout of C3ar1 in mice blunted development of fibrosing steatohepatitis. In conclusion, BMMs promoted the progression of fibrosing steatohepatitis during injury, whereas macrophages reduced fibrosing steatohepatitis in the recovery phase of liver injury. Increasing anti-fibrotic macrophages and decreasing pro-fibrotic macrophages are promising approaches for fibrosing steatohepatitis. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Defective lysosomal clearance of autophagosomes and its clinical implications in nonalcoholic steatohepatitis.

Autophagic impairment is implicated in nonalcoholic fatty liver disease (NAFLD), but the molecular mechanism is unclear. We found that autophagic flux was significantly inhibited in 3 murine models of NAFLD. Interestingly, the number of acidic organelles and the level of mature cathepsin D were reduced, suggesting defective lysosome acidification. Asparagine synthetase (ASNS) was induced by endoplasmic reticulum stress, leading to the generation of asparagine, which inhibited lysosome acidification. Both steatotic- and asparagine-treated hepatocytes showed reduced lysosomal acidity and retention of lysosomal calcium. Knockdown of ASNS in steatotic hepatocytes restored autophagic flux. As a potential biomarker, increased serum p62/sequestosome 1 (SQSTM1) level was an independent risk factor for patients with steatosis and lobular inflammation. Impaired autophagy in NAFLD is elicited by defective lysosome acidification, which is caused by ASNS-induced asparagine synthesis under endoplasmic reticulum stress and subsequent retention of lysosomal calcium. p62/SQSTM1 could be used as a noninvasive biomarker in the diagnosis of NAFLD patients.-Wang, X., Zhang, X., Chu, E. S. H., Chen, X., Kang, W., Wu, F., To, K.-F., Wong, V. W. S., Chan, H. L. Y., Chan, M. T. V., Sung, J. J. Y., Wu, W. K. K., Yu, J. Defective lysosomal clearance of autophagosomes and its clinical implications in nonalcoholic steatohepatitis.

Peptostreptococcus anaerobius Induces Intracellular Cholesterol Biosynthesis in Colon Cells to Induce Proliferation and Causes Dysplasia in Mice.

BACKGROUND & AIMS: Stool samples from patients with colorectal cancer (CRC) have a higher abundance of Peptostreptococcus anaerobius than stool from individuals without CRC, based on metagenome sequencing. We investigated whether P anaerobius contributes to colon tumor formation in mice and its possible mechanisms of carcinogenesis. METHODS: We performed quantitative polymerase chain reaction analyses to measure P anaerobius in 112 stool samples and 255 colon biopsies from patients with CRC or advanced adenoma and from healthy individuals (controls) undergoing colonoscopy examination at hospitals in Hong Kong and Beijing. C57BL/6 mice were given broad-spectrum antibiotics, followed by a single dose of azoxymethane, to induce colon tumor formation. Three days later, mice were given P anaerobius or Esherichia coli MG1655 (control bacteria), via gavage, for 6 weeks. Some mice were also given the nicotinamide adenine dinucleotide phosphate oxidase inhibitor apocynin. Intestine tissues were collected and analyzed histologically. The colon epithelial cell line NCM460 and colon cancer cell lines HT-29 and Caco-2 were exposed to P anaerobius or control bacteria; cells were analyzed by immunoblot, proliferation, and bacterial attachment analyses and compared in gene expression profiling studies. Gene expression was knocked down in these cell lines with small interfering RNAs. RESULTS: P anaerobius was significantly enriched in stool samples from patients with CRC and in biopsies from patients with colorectal adenoma or CRC compared with controls. Mice depleted of bacteria and exposed to azoxymethane and P anaerobius had a higher incidence of intestinal dysplasia (63%) compared with mice not given the bacteria (8.3%; P < .01). P anaerobius mainly colonized the colon compared with the rest of the intestine. Colon cells exposed to P anaerobius had significantly higher levels of proliferation than control cells. We found genes that regulate cholesterol biosynthesis, Toll-like receptor (TLR) signaling, and AMP-activated protein kinase signaling to be significantly up-regulated in cells exposed to P anaerobius. Total cholesterol levels were significantly increased in colon cell lines exposed to P anaerobius via activation of sterol regulatory element-binding protein 2. P anaerobius interacted with TLR2 and TLR4 to increase intracellular levels of reactive oxidative species, which promoted cholesterol synthesis and cell proliferation. Depletion of reactive oxidative species by knockdown of TLR2 or TLR4, or incubation of cells with an antioxidant, prevented P anaerobius from inducing cholesterol biosynthesis and proliferation. CONCLUSIONS: Levels of P anaerobius are increased in human colon tumor tissues and adenomas compared with non-tumor tissues; this bacteria increases colon dysplasia in a mouse model of CRC. P anaerobius interacts with TLR2 and TLR4 on colon cells to increase levels of reactive oxidative species, which promotes cholesterol synthesis and cell proliferation.

CXC chemokine receptor 3 promotes steatohepatitis in mice through mediating inflammatory cytokines, macrophages and autophagy.

BACKGROUND & AIMS: CXC chemokine receptor 3 (CXCR3) is involved in virus-related chronic liver inflammation. However, the role of CXCR3 in non-alcoholic steatohepatitis (NASH) remains unclear. We aimed to investigate the role of CXCR3 in NASH. METHODS: Human liver tissues were obtained from 24 non-alcoholic fatty liver disease (NAFLD) patients and 20 control subjects. CXCR3 knockout (CXCR3(-/-)), obese db/db mice and their wild-type (WT) littermates were used in both methionine-and-choline-deficient (MCD) diet and high-fat high-carbohydrate high-cholesterol (HFHC) diet-induced NASH models. In addition, MCD-fed WT mice were administrated with CXCR3 specific antagonists. RESULTS: CXCR3 was significantly upregulated in liver tissues of patients with NAFLD and in dietary-induced NASH animal models. Compared with WT littermates, CXCR3(-/-) mice were more resistant to both MCD and HFHC diet-induced steatohepatitis. Induction of CXCR3 in dietary-induced steatohepatitis was associated with the increased expression of hepatic pro-inflammatory cytokines, activation of NF-κB, macrophage infiltration and T lymphocytes accumulation (Th1 and Th17 immune response). CXCR3 was also linked to steatosis through inducing hepatic lipogenic genes. Moreover, CXCR3 is associated with autophagosome-lysosome impairment and endoplasmic reticulum (ER) stress in steatohepatitis as evidenced by LC3-II and p62/SQSTM1 accumulation and the induction of GRP78, phospho-PERK and phospho-eIF2α. Inhibition of CXCR3 using CXCR3 antagonist significantly suppressed MCD-induced steatosis and hepatocytes injury in AML-12 hepatocytes. Blockade of CXCR3 using CXCR3 antagonists in mice reversed the established steatohepatitis. CONCLUSIONS: CXCR3 plays a pivotal role in NASH development by inducing production of cytokines, macrophage infiltration, fatty acid synthesis and causing autophagy deficiency and ER stress.

Acute atorvastatin is hepatoprotective against ischaemia-reperfusion injury in mice by modulating eNOS and microparticle formation.

BACKGROUND & AIMS: Steatosis accentuates the severity of hepatic ischaemia-reperfusion injury (IRI); 'statins' (HMG-CoA reductase inhibitors) protect the heart and brain against post-ischaemic injury. We tested whether short-term administration of atorvastatin protects fatty livers in obese mice against IRI. METHODS: Mice with dietary or genetic simple steatosis (SS) or non-alcoholic steatohepatitis (NASH) were subjected to 60 min partial hepatic ischaemia/24 h reperfusion. Atorvastatin was injected intravenously (5 mg/kg) 1 h before IRI. Liver injury, Toll-like receptor-4 (TLR4), cytokines/chemokines, iNOS/eNOS expression, eNOS activity and thromboxane B2 (TXB2) production were determined. RESULTS: Ischaemia-reperfusion injury was exaggerated by two- to five-fold in SS and NASH compared with lean liver. Atorvastatin pretreatment conferred 70-90% hepatic protection in all animals. Atorvastatin increased post-ischaemic eNOS mRNA/protein and strikingly enhanced eNOS activity (by phospho-eNOS). It also attenuated microparticle (MP) production, NF-κB activation, significantly dampened post-ischaemic thromboxane B2 production, induction of TNF-α, IL-6, MIP-1a, MCP-1, GM-CSF and vascular cell adhesion molecule-1 (VCAM), with a resultant reduction on macrophage and polymorphonuclear neutrophil recruitment. Up-regulation of HMGB1 and TLR4 after IRI was marked in fatty livers; 1 h pretreatment with atorvastatin reduced HMGB1 and TLR4 expression in all livers. CONCLUSIONS: Acute (1 h) atorvastatin administration is highly hepatoprotective against IRI in NASH, fatty and lean livers. Key mechanisms include suppression of inflammation by prevention of NF-κB activation, microvascular protection via eNOS activation and suppression of TXB2 and MP release. Short-term intravenous statin treatment is a readily available and effective preventive agent against hepatic IRI, irrespective of obesity and fatty liver disease, and merits clinical trials in at-risk patients.

CXCL10 plays a key role as an inflammatory mediator and a non-invasive biomarker of non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Perpetuate liver inflammation is crucial in the pathogenesis of non-alcoholic steatohepatitis (NASH). Expression of CXCL10, a pro-inflammatory cytokine, correlates positively with obesity and type 2 diabetes. Whether CXCL10 plays a role in NASH was unknown. We aimed to investigate the functional and clinical impact of CXCL10 in NASH. METHODS: Cxcl10 gene-deleted (Cxcl10(-/-)) and C57BL/6 wild type (WT) mice were fed a methionine- and choline-deficient (MCD) diet for 4 or 8 weeks. In other experiments, we injected neutralizing anti-CXCL10 mAb into MCD-fed WT mice. Human serum was obtained from 147 patients with biopsy-proven non-alcoholic fatty liver disease and 73 control subjects. RESULTS: WT mice, fed the MCD diet, developed steatohepatitis with higher hepatic CXCL10 expression. Cxcl10(-/-) mice were refractory to MCD-induced steatohepatitis. We further revealed that CXCL10 was associated with the induction of important pro-inflammatory cytokines (TNF-α, IL-1ß, and MCP-1) and activation of the NF-κB pathway. CXCL10 was linked to steatosis through upregulation of the lipogenic factors SREBP-1c and LXR, and also to oxidative stress (upregulation of CYP2E1 and C/EBPß). Blockade of CXCL10 protected against hepatocyte injury in vitro and against steatohepatitis development in mice. We further investigated the clinical impact of CXCL10 and found circulating and hepatic CXCL10 levels were significantly higher in human NASH. Importantly, the circulating CXCL10 level was correlated with the degree of lobular inflammation and was an independent risk factor for NASH patients. CONCLUSIONS: We demonstrate for the first time that CXCL10 plays a pivotal role in the pathogenesis of experimental steatohepatitis. CXCL10 maybe a potential non-invasive biomarker for NASH patients.

Inhibitory role of Smad7 in hepatocarcinogenesis in mice and in vitro.

Smad7 is a principal inhibitor of the TGFß-Smad signalling pathway. We have investigated the functional significance of Smad7 in hepatocellular carcinoma (HCC). Smad7 knockout (KO) and wild-type (WT) mice were injected with diethylnitrosamine (DEN) to induce HCC. The effects of Smad7 on cellular features were examined in HCC cells, using a Smad7 over-expression or deletion approach. Signalling pathway components modulated by Smad7 in HCC were evaluated using luciferase reporter assay and co-immunoprecipitation. Smad7 was down-regulated in human HCCs compared with the adjacent normal tissues (p < 0.001). Smad7 KO mice were more susceptible to DEN-induced HCC than WT mice (78% versus 22%, p < 0.05). HCCs from KO mice displayed a greater proliferation activity (p < 0.05) and a reduced apoptotic index compared with WT littermates (p < 0.05). Deletion of Smad7 promoted cell proliferation in primary cultured HCC cells. In addition, over-expression of Smad7 in HCC cell lines markedly suppressed cell growth (p < 0.0001) and colony formation (p < 0.01). Cell cycle analysis revealed an increase in the G1 phase and a reduction in the S-phase populations, accompanied by up-regulation of p27(Kip1) and down-regulation of cyclin D1. Smad7 increased cell apoptosis (p < 0.01) by mediating an intrinsic [caspase-9, caspase-3 and poly(ADP-ribose) polymerase] apoptotic pathway. Moreover, Smad7 inhibited NF-κB signalling by interacting with TAB2, an upstream activator of NF-κB, and inhibited TGFß signalling by suppressing phosphorylation of Smad3. In conclusion, loss of Smad7 enhances susceptibility to HCC. Smad7 suppresses HCC cell growth by inhibiting proliferation and G1 -S phase transition and inducing apoptosis through attenuation of NF-κB and TGFß signalling. Smad7 acts as a potential tumour suppressor in liver.

Lactobacillus acidophilus suppresses non-alcoholic fatty liver disease-associated hepatocellular carcinoma through producing valeric acid.

BACKGROUND: Gut probiotic depletion is associated with non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC). Here, we investigated the prophylactic potential of Lactobacillus acidophilus against NAFLD-HCC. METHODS: NAFLD-HCC conventional and germ-free mice were established by diethylnitrosamine (DEN) injection with feeding of high-fat high-cholesterol (HFHC) or choline-deficient high-fat (CDHF) diet. Orthotopic NAFLD-HCC allografts were established by intrahepatic injection of murine HCC cells with HFHC feeding. Metabolomic profiling was performed using liquid chromatography-mass spectrometry. Biological functions of L. acidophilus conditional medium (L.a CM) and metabolites were determined in NAFLD-HCC human cells and mouse organoids. FINDINGS: L. acidophilus supplementation suppressed NAFLD-HCC formation in HFHC-fed DEN-treated mice. This was confirmed in orthotopic allografts and germ-free tumourigenesis mice. L.a CM inhibited the growth of NAFLD-HCC human cells and mouse organoids. The protective function of L. acidophilus was attributed to its non-protein small molecules. By metabolomic profiling, valeric acid was the top enriched metabolite in L.a CM and its upregulation was verified in liver and portal vein of L. acidophilus-treated mice. The protective function of valeric acid was demonstrated in NAFLD-HCC human cells and mouse organoids. Valeric acid significantly suppressed NAFLD-HCC formation in HFHC-fed DEN-treated mice, accompanied by improved intestinal barrier integrity. This was confirmed in another NAFLD-HCC mouse model induced by CDHF diet and DEN. Mechanistically, valeric acid bound to hepatocytic surface receptor GPR41/43 to inhibit Rho-GTPase pathway, thereby ablating NAFLD-HCC. INTERPRETATION: L. acidophilus exhibits anti-tumourigenic effect in mice by secreting valeric acid. Probiotic supplementation is a potential prophylactic of NAFLD-HCC. FUNDING: Shown in Acknowledgments.

Epigenetic inactivation of BCL6B, a novel functional tumour suppressor for gastric cancer, is associated with poor survival.

OBJECTIVE: Using genome-wide promoter methylation assay, B cell CLL/lymphoma 6 member B (BCL6B) was found to be preferentially methylated in cancer. A study was undertaken to examine the epigenetic regulation, biological function and clinical significance of BCL6B in gastric cancer (GC). METHODS: BCL6B promoter methylation was evaluated by combined bisulfite restriction analysis and sequencing. The biological functions of BCL6B were determined by cell viability, colony formation, flow cytometry and in vivo tumorigenicity assays. The molecular targets of BCL6B were identified by cDNA expression array. RESULTS: BCL6B was silenced or downregulated in all nine GC cell lines and readily expressed in normal gastric tissues. Loss of BCL6B expression was regulated by promoter hypermethylation. Re-expression of BCL6B in GC cell lines inhibited colony formation, suppressed cell viability, induced apoptosis and restrained the tumorigenecity in nude mice. These effects were associated with upregulation of the pro-apoptosis genes tumour necrosis factor receptor superfamily member 1A, caspase-8, caspase-9, caspase-3 and caspase-7 and nuclear enzyme poly (ADP-ribose) polymerase, downregulation of the pro-proliferation genes S100 calcium binding protein A4 and vascular endothelial growth factor A, and induction of the tumour suppressor genes ataxia telangiectasia mutated homologue and p53. BCL6B hypermethylation was detected in 49.0% (102/208) and 66.3% (67/101) of two independent cohorts of patients with GC, respectively. BCL6B methylation was an independent factor for the survival of patients with GC (p=0.001 for cohort I, p=0.02 for cohort II). CONCLUSIONS: BCL6B plays a pivotal role as a potential tumour suppressor in GC. Detection of methylated BCL6B may serve as an independent biomarker for the prognosis of GC.

Parabacteroides distasonis uses dietary inulin to suppress NASH via its metabolite pentadecanoic acid.

Non-alcoholic steatohepatitis (NASH) is the severe form of non-alcoholic fatty liver disease, and is characterized by liver inflammation and fat accumulation. Dietary interventions, such as fibre, have been shown to alleviate this metabolic disorder in mice via the gut microbiota. Here, we investigated the mechanistic role of the gut microbiota in ameliorating NASH via dietary fibre in mice. Soluble fibre inulin was found to be more effective than insoluble fibre cellulose to suppress NASH progression in mice, as shown by reduced hepatic steatosis, necro-inflammation, ballooning and fibrosis. We employed stable isotope probing to trace the incorporation of 13C-inulin into gut bacterial genomes and metabolites during NASH progression. Shotgun metagenome sequencing revealed that the commensal Parabacteroides distasonis was enriched by 13C-inulin. Integration of 13C-inulin metagenomes and metabolomes suggested that P. distasonis used inulin to produce pentadecanoic acid, an odd-chain fatty acid, which was confirmed in vitro and in germ-free mice. P. distasonis or pentadecanoic acid was protective against NASH in mice. Mechanistically, inulin, P. distasonis or pentadecanoic acid restored gut barrier function in NASH models, which reduced serum lipopolysaccharide and liver pro-inflammatory cytokine expression. Overall this shows that gut microbiota members can use dietary fibre to generate beneficial metabolites to suppress metabolic disease.

Altered gut microbiome composition by appendectomy contributes to colorectal cancer.

Appendectomy impacts the homeostasis of gut microbiome in patients. We aimed to study the role of appendectomy in colorectal cancer (CRC) risk through causing gut microbial dysbiosis. Population-based longitudinal study (cohort 1, n = 129,155) showed a 73.0% increase in CRC risk among appendectomy cases throughout 20 years follow-up (Adjusted sub-distribution hazard ratio (SHR) 1.73, 95% CI 1.49-2.01, P < 0.001). Shotgun metagenomic sequencing was performed on fecal samples from cohort 2 (n = 314). Gut microbial dysbiosis in appendectomy subjects was observed with significant enrichment of 7 CRC-promoting bacteria (Bacteroides vulgatus, Bacteroides fragilis, Veillonella dispar, Prevotella ruminicola, Prevotella fucsa, Prevotella dentalis, Prevotella denticola) and depletion of 5 beneficial commensals (Blautia sp YL58, Enterococcus hirae, Lachnospiraceae bacterium Choco86, Collinsella aerofaciens, Blautia sp SC05B48). Microbial network analysis showed increased correlation strengths among enriched bacteria and their enriched oncogenic pathways in appendectomy subjects compared to controls. Of which, B. fragilis was the centrality in the network of the enriched bacteria. We further confirmed that appendectomy promoted colorectal tumorigenesis in mice by causing gut microbial dysbiosis and impaired intestinal barrier function. Collectively, this study revealed appendectomy-induced microbial dysbiosis characterized by enriched CRC-promoting bacteria and depleted beneficial commensals, signifying that the gut microbiome may play a crucial role in CRC development induced by appendectomy.

Paired box gene 5 is a novel tumor suppressor in hepatocellular carcinoma through interaction with p53 signaling pathway.

UNLABELLED: The paired box 5 (PAX5) is a member of PAX transcription factors family involved in the regulation of embryonic development. However, the role of PAX5 in carcinogenesis is largely unclear. We identified that PAX5 is involved in human cancer by methylation-sensitive representational difference analysis. We examined the biological functions and related molecular mechanisms of PAX5 in hepatocellular carcinoma (HCC). Promoter methylation of PAX5 was evaluated by methylation-specific polymerase chain reaction (PCR) and bisulfite genomic sequencing (BGS). The functions of ectopic PAX5 expression were determined by viability assay, colony formation, and cell cycle analyses, along with in vivo tumorigenicity assays. The PAX5 target signal pathway was identified by promoter luciferase assay, chromosome immunoprecipitation (ChIP), and pathway PCR array. PAX5 is expressed in normal human liver tissue, but silenced or down-regulated in 83% (10/12) of HCC cell lines. The mean expression level of PAX5 was significantly lower in primary HCCs as compared to their adjacent normal tissues (P < 0.0001). The promoter methylation contributes to the inactivation of PAX5. Restoring PAX5 expression in silenced HCC cell lines suppressed cell proliferation, induced apoptosis in vitro, and inhibited tumor growth in nude mice (P < 0.0001). The pathway luciferase reporter assay indicated that PAX5 activated p53 and p21 signaling. ChIP analysis demonstrated that PAX5 directly bound to the p53 promoter. The antitumorigenic function of PAX5 was at least up-regulated by p53 and its downstream targets including tumor necrosis factor, Fas ligand, leucine-rich repeats, and death domain-containing, poly(rC) binding protein 4, p21, and growth arrest and DNA-damage-inducible alpha. CONCLUSION: PAX5 is frequently inactivated by promoter methylation in HCC. PAX5 appears to be a functional tumor suppressor involved in liver carcinogenesis through direct regulation of the p53 signaling pathway.

MR T1ρ as an imaging biomarker for monitoring liver injury progression and regression: an experimental study in rats with carbon tetrachloride intoxication.

OBJECTIVES: Recently it was shown that the magnetic resonance imaging (MRI) T1ρ value increased with the severity of liver fibrosis in rats with bile duct ligation. Using a rat carbon tetrachloride (CCl(4)) liver injury model, this study further investigated the merit of T1ρ relaxation for liver fibrosis evaluation. METHODS: Male Sprague-Dawley rats received intraperitoneal injection of 2 ml/kg CCl(4) twice weekly for up to 6 weeks. Then CCl(4) was withdrawn and the animals were allowed to recover. Liver T1ρ MRI and conventional T2-weighted images were acquired. Animals underwent MRI at baseline and at 2 days, 2 weeks, 4 weeks and 6 weeks post CCl(4) injection, and they were also examined at 1 week and 4 weeks post CCl(4) withdrawal. Liver histology was also sampled at these time points. RESULTS: Liver T1ρ values increased slightly, though significantly, on day 2, and then increased further and were highest at week 6 post CCl(4) insults. The relative liver signal intensity change on T2-weighted images followed a different time course compared with that of T1ρ. Liver T1ρ values decreased upon the withdrawal of the CCl(4) insult. Histology confirmed the animals had typical CCl(4) liver injury and fibrosis progression and regression processes. CONCLUSIONS: MR T1ρ imaging can monitor CCl(4)-induced liver injury and fibrosis. KEY POINTS: • MR T1ρ is a valuable imaging biomarker for liver injury/fibrosis. • Liver T1ρ was only mildly affected by oedema and acute inflammation. • Liver MR T1ρ decreased when liver fibrosis and injury regressed.