Kento Imajo

Hyperresponsivity to Low-Dose Endotoxin during Progression to Nonalcoholic Steatohepatitis Is Regulated by Leptin-Mediated Signaling

Although bacterial endotoxin, such as lipopolysaccharide (LPS), plays a key role in the pathogenesis of nonalcoholic steatohepatitis (NASH), detailed mechanisms of this pathogenesis remain unclear. Here, we demonstrate that upregulation of CD14 by leptin-mediated signaling is critical to hyperreactivity against endotoxin during NASH progression. Upregulation of CD14 in Kupffer cells and hyperreactivity against low-dose LPS were observed in high-fat diet (HFD)-induced steatosis mice, but not chow-fed-control mice. Hyperresponsivity against low-dose LPS led to accelerated NASH progression, including liver inflammation and fibrosis. Administering leptin in chow-fed mice caused increased hepatic expression of CD14 via STAT3 signaling, resulting in hyperreactivity against low-dose LPS without steatosis. In contrast, a marked decrease in hepatic CD14 expression was observed in leptin-deficient ob/ob mice, despite severe steatosis. Our results indicate that obesity-induced leptin plays a crucial role in NASH progression via enhanced responsivity to endotoxin, and we propose a mechanism of bacteria-mediated progression of NASH.

Involvement of a periodontal pathogen, Porphyromonas gingivalis on the pathogenesis of non-alcoholic fatty liver disease

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome that is closely associated with multiple factors such as obesity, hyperlipidemia and type 2 diabetes mellitus. However, other risk factors for the development of NAFLD are unclear. With the association between periodontal disease and the development of systemic diseases receiving increasing attention recently, we conducted this study to investigate the relationship between NAFLD and infection with Porphyromonas gingivalis (P. gingivalis), a major causative agent of periodontitis.MethodsThe detection frequencies of periodontal bacteria in oral samples collected from 150 biopsy-proven NAFLD patients (102 with non-alcoholic steatohepatitis (NASH) and 48 with non-alcoholic fatty liver (NAFL) patients) and 60 non-NAFLD control subjects were determined. Detection of P. gingivalis and other periodontopathic bacteria were detected by PCR assay. In addition, effect of P. gingivalis-infection on mouse NAFLD model was investigated. To clarify the exact contribution of P. gingivalis-induced periodontitis, non-surgical periodontal treatments were also undertaken for 3 months in 10 NAFLD patients with periodontitis.ResultsThe detection frequency of P. gingivalis in NAFLD patients was significantly higher than that in the non-NAFLD control subjects (46.7% vs. 21.7%, odds ratio: 3.16). In addition, the detection frequency of P. gingivalis in NASH patients was markedly higher than that in the non-NAFLD subjects (52.0%, odds ratio: 3.91). Most of the P. gingivalis fimbria detected in the NAFLD patients was of invasive genotypes, especially type II (50.0%). Infection of type II P. gingivalis on NAFLD model of mice accelerated the NAFLD progression. The non-surgical periodontal treatments on NAFLD patients carried out for 3 months ameliorated the liver function parameters, such as the serum levels of AST and ALT.ConclusionsInfection with high-virulence P. gingivalis might be an additional risk factor for the development/progression of NAFLD/NASH.

Outcomes and factors influencing survival in cirrhotic cases with spontaneous rupture of hepatocellular carcinoma: a multicenter study

BackgroundSpontaneous rupture is rare complication of hepatocellular carcinoma (HCC) with high mortality rate in cirrhotic cases. The aim of this study was to determine the factors influencing prognosis in cases of spontaneously ruptured HCC and to investigate the outcomes of the treatments employed, especially transcatheter arterial embolization (TAE).MethodsA retrospective multicenter study was conducted in 48 cirrhotic patients with spontaneous rupture of HCC. Conservative treatment was employed in 32 patients (ConT group) and TAE was performed in 16 patients (TAE group).ResultsThe median survival time (MST) in the ConT group was only 13.1 days and the survival rate was extremely poor: 59.4% at 7 days, 37.5% at 14 days, and 6.3% at 30 days. On the other hand, the MST in the TAE group was 244.8 days and the survival rate was 87.5% at 1 month, 56.3% at 3 months, 23.4% at 12 months, and 15.6% at 24 months. According to the results of univariate analyses, factors associated with poor hepatic function and poor suitability for TAE was important determinants of short-term death (less than 3 weeks) among the patients (p < 0.05). On the other hand, among the patients in whom initial TAE was successfully performed (n = 15), a multivariate analysis showed that a maximum tumor size not exceeding 7 cm was the only independent factor determining long-term survival (p = 0.0130).ConclusionDespite the inherent limitations of this retrospective study, TAE appears to be a useful treatment strategy for cirrhotic patients with spontaneous HCC rupture, as it yielded a longer survival period compared with conservative treatment in patients with ruptured HCC. Among the patients with ruptured HCC in whom initial TAE was successfully performed, the maximum tumor size was an important factor influencing survival.

Rodent models of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis.

Research in nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis (NASH), has been limited by the availability of suitable models for this disease. A number of rodent models have been described in which the relevant liver pathology develops in an appropriate metabolic context. These models are promising tools for researchers investigating one of the key issues of NASH: not so much why steatosis occurs, but what causes the transition from simple steatosis to the inflammatory, progressive fibrosing condition of steatohepatitis. The different rodent models can be classified into two large groups. The first includes models in which the disease is acquired after dietary or pharmacological manipulation, and the second, genetically modified models in which liver disease develops spontaneously. To date, no single rodent model has encompassed the full spectrum of human disease progression, but individual models can imitate particular characteristics of human disease. Therefore, it is important that researchers choose the appropriate rodent models. The purpose of the present review is to discuss the metabolic abnormalities present in the currently available rodent models of NAFLD, summarizing the strengths and weaknesses of the established models and the key findings that have furthered our understanding of the disease’s pathogenesis.

The Selective SGLT2 Inhibitor Ipragliflozin Has a Therapeutic Effect on Nonalcoholic Steatohepatitis in Mice

Background & Aims In recent years, nonalcoholic steatohepatitis (NASH) has become a considerable healthcare burden worldwide. Pathogenesis of NASH is associated with type 2 diabetes mellitus (T2DM) and insulin resistance. However, a specific drug to treat NASH is lacking. We investigated the effect of the selective sodium glucose cotransporter 2 inhibitor (SGLT2I) ipragliflozin on NASH in mice. Methods We used the Amylin liver NASH model (AMLN), which is a diet-induced model of NASH that results in obesity and T2DM. AMLN mice were fed an AMLN diet for 20 weeks. SGLT2I mice were fed an AMLN diet for 12 weeks and an AMLN diet with 40 mg ipragliflozin/kg for 8 weeks. Results AMLN mice showed steatosis, inflammation, and fibrosis in the liver as well as obesity and insulin resistance, features that are recognized in human NASH. Ipragliflozin improved insulin resistance and liver injury. Ipragliflozin decreased serum levels of free fatty acids, hepatic lipid content, the number of apoptotic cells, and areas of fibrosis; it also increased lipid outflow from the liver. Conclusions Ipragliflozin improved the pathogenesis of NASH by reducing insulin resistance and lipotoxicity in NASH-model mice. Our results suggest that ipragliflozin has a therapeutic effect on NASH with T2DM.

Unfolded protein response pathways regulate Hepatitis C virus replication via modulation of autophagy

Abstract Background Hepatitis C virus (HCV) induces endoplasmic reticulum (ER) stress which, in turn, activates the unfolding protein response (UPR). UPR activates three distinct signalling pathways. Additionally, UPR induces autophagy (UPR-autophagy pathways). On the other hand, it has become clear that some positive-single-strand RNA viruses utilize autophagy. Some groups have used the siRNA silencing approach to show that autophagy is required for HCV RNA replication. However, the mechanism of induction of the UPR-autophagy pathways remain unclear in the cells with HCV. Method and results: we used a genome-length HCV RNA (strain O of genotype 1b) replication system (OR6) in hepatoma cells (HuH-7-derived OR6 cells). As control, we used OR6c cells from which the HCV genome had been removed by treatment with interferon-α. The UPR-autophagy pathways were activated to a greater degree in the OR6 cells as compared to the OR6c cells. Rapamycin, mTOR-independent autophagy inducer, activated HCV replication in the OR6 cells. On the other hand, HCV replication in the cells was inhibited by 3-methyladenine (3-MA), which is an inhibitor of autophagy. Salubrinal (Eukaryotic Initiation Factor 2(eIF2)-alpha phosphatase inhibitor), 3-ethoxy-5, 6-dibromosalicylaldehyde (X-box binding protein-1 (XBP-1) splicing inhibitor) and sp600125 (c-Jun N-terminal kinases (JNK) inhibitor) inhibited HCV replication and autophagy. Additionally, HCV replication and autophagy were inhibited more strongly by combination of these inhibitors. Conclusion Our results suggest that UPR-autophagy pathways exert an influence on HCV replication. Therefore, control these pathways may serve as a novel therapeutic strategy against replication of HCV.

Liver abscess caused by periodontal bacterial infection with Fusobacterium necrophorum

Liver abscess is recognized as a life‐threatening disease. However, even in recent years, approximately 50% of liver abscess cases are considered to be cryptogenic. Here, we report a case of liver abscess associated with periodontal bacterial infection by Fusobacterium necrophorum, which is commonly found in the oropharyngeal flora. A 36‐year‐old man presented with fever and contrast‐enhanced abdominal computed tomography revealed multiple liver abscesses. F.necrophorum was isolated from oral smears, liver aspirates and blood samples. Liver abscesses caused by periodontal bacterial infection are rare, however, the incidence is expected to increase in the future, as periodontitis is extremely common and is on the rise as one of the most common chronic infections in the world. A systemic survey including periodontitis may be required for the exact diagnosis of the source of infection.

Microbiota and nonalcoholic steatohepatitis

The recent rise in obesity-related diseases, such as nonalcoholic fatty liver disease and its strong association with microbiota, has elicited interest in the underlying mechanisms of these pathologies. Experimental models have highlighted several mechanisms connecting microbiota to the development of liver dysfunction in nonalcoholic steatohepatitis (NASH) such as increased energy harvesting from the diet, small intestine bacterial overgrowth, modulation of the intestinal barrier by glucagon-like peptide-2 secretions, activation of innate immunity through the lipopolysaccharide–CD14 axis caused by obesity-induced leptin, periodontitis, and sterile inflammation. The manipulation of microbiota through probiotics, prebiotics, antibiotics, and periodontitis treatment yields encouraging results for the treatment of obesity, diabetes, and NASH, but data in humans is scarce.

Soluble CD14 Levels Reflect Liver Inflammation in Patients with Nonalcoholic Steatohepatitis

Background & Aims Liver inflammation is a risk factor for the progression of nonalcoholic fatty liver disease (NAFLD). However, the diagnosis of liver inflammation is very difficult and invasive liver biopsy is still the only method to reliably detect liver inflammation. We previously reported that overexpression of CD14 in Kupffer cells may trigger the progression to nonalcoholic steatohepatitis (NASH) via liver inflammation following hyper-reactivity to low-dose lipopolysaccharide. Therefore, the aim of this study was to investigate the relationship between soluble type of CD14 (sCD14) and histological features in patients with NAFLD. Methods Our cohort consisted of 113 patients with liver biopsy-confirmed NAFLD and 21 age-matched healthy controls. Serum sCD14 levels were measured by an enzyme-linked immunosorbent assay. Results Serum sCD14 levels were significantly associated with diagnosis of NASH and the area under the receiver operator characteristic curve (AUROC) to distinguish between not NASH and NASH was 0.802. Moreover, serum sCD14 levels were significantly associated with the disease activity based on NAFLD activity score and hepatic CD14 mRNA expression, which is correlated with membrane CD14 (mCD14) expression, in patients with NAFLD. In multiple regression analysis, the serum sCD14 levels were independently associated with liver inflammation. The AUROC to distinguish between mild and severe liver inflammation in patients with NAFLD was 0.752. Conclusions We found that serum sCD14 levels increased significantly with increasing liver inflammation grade in patients with NAFLD, reflecting increased hepatic CD14 expression. Serum sCD14 is a promising tool to predict the worsening of liver inflammation, and may offer a potential biomarker for evaluation of therapeutic effects in NAFLD.

Targeted-bisulfite sequence analysis of the methylation of CpG islands in genes encoding PNPLA3, SAMM50, and PARVB of patients with non-alcoholic fatty liver disease

BACKGROUND & AIMS The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is affected by epigenetic factors as well as by genetic variation. METHODS We performed targeted-bisulfite sequencing to determine the levels of DNA methylation of 4 CpG islands (CpG99, CpG71, CpG26, and CpG101) in the regulatory regions of PNPLA3, SAMM50, PARVB variant 1, and PARVB variant 2, respectively. We compared the levels of methylation of DNA in the livers of the first and second sets of patients with mild (fibrosis stages 0 and 1) or advanced (fibrosis stages 2 to 4) NAFLD and in those of patients with mild (F0 to F2) or advanced (F3 and F4) chronic hepatitis C infection. The hepatic mRNA levels of PNPLA3, SAMM50, and PARVB were measured using qPCR. RESULTS CpG26, which resides in the regulatory region of PARVB variant 1, was markedly hypomethylated in the livers of patients with advanced NAFLD. Conversely, CpG99 in the regulatory region of PNPLA3 was substantially hypermethylated in these patients. These differences in DNA methylation were replicated in a second set of patients with NAFLD or chronic hepatitis C. PNPLA3 mRNA levels in the liver of the same section of a biopsy specimen used for genomic DNA preparation were lower in patients with advanced NAFLD compared with those with mild NAFLD and correlated inversely with CpG99 methylation in liver DNA. Moreover, the levels of CpG99 methylation and PNPLA3 mRNA were affected by the rs738409 genotype. CONCLUSIONS Hypomethylation of CpG26 and hypermethylation of CpG99 may contribute to the severity of fibrosis in patients with NAFLD or chronic hepatitis C infection.