CerS5 deficiency promotes liver fibrosis development in non-alcoholic fatty liver disease.

BACKGROUND: Hepatocyte lipotoxicity mediated by sphingolipids was considered one of important factors in NAFLD development. Knocking out key enzymes for sphingolipids synthesis, such as DES-1, SPHK1 and CerS6, could reduce hepatocyte lipotoxicity and improve NAFLD progression. Previous studies showed that roles of CerS5 and CerS6 in sphingolipids metabolism were similar, but the role of CerS5 was controversial in NAFLD development. This study aimed to clarify the role and mechanism of CerS5 in NAFLD development. METHODS: Hepatocyte conditional CerS5 knockout (CerS5 CKO) and wild type (WT) mice were fed with standard control diet (SC) and choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD) and then divided into four groups: CerS5 CKO-SC, CerS5 CKO-CDAHFD, WT-SC and WT-CDAHFD. RT-PCR, IHC and WB were used to analyze the expression of inflammatory, fibrosis and bile acids (BA) metabolism factors. RNA-seq was used to analyze differences of transcriptional levels of liver molecules among the four groups. Metabolomics was used to measured differences of hepatic BAs among the four groups. RESULTS: Hepatocyte specific knockout of CerS5 did not increase or reduce the severity of 8-weeks CDAHFD induced hepatic steatosis and inflammation, but significantly worsened the progression of liver fibrosis in these mice. At the molecular level, hepatocyte specific knockout of CerS5 did not increase or reduce expression of hepatic inflammatory factors: CD68, F4/80 and MCP-1, but increased expression of hepatic fibrosis factors: α-SMA, COL1α and TGF-ß in mice fed with CDAHFD. Transcriptome analysis showed that hepatocyte specific knockout of CerS5 significantly decreased the expression of hepatic cyp27a1, and decreased expression of cyp27a1 was further validated by RT-PCR and WB. Considering that cyp27a1 was a key enzyme in the alternative pathway of BA synthesis, we further found that hepatic BA pools in CerS5 CKO mice were more conducive to the progression of liver fibrosis, which were characterized by elevated hydrophobic 12α-OH BAs and decreased hydrophilic non-12α-OH BAs. CONCLUSION: CerS5 played an important role in the progression of NAFLD related fibrosis, and hepatocyte specific knockout of CerS5 accelerated the progression of NAFLD related fibrosis, which was possibly due to the inhibition of BA synthesis alternative pathway by knocking out hepatocyte CerS5.

Association of metabolic traits with occurrence of nonalcoholic fatty liver disease-related hepatocellular carcinoma: A systematic review and meta-analysis of longitudinal cohort studies.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) has become one of the leading etiologies of hepatocellular carcinoma (HCC), but risk factors for NAFLD-related HCC occurrence have not been defined. NAFLD is often complicated by metabolic abnormalities, and there is a bidirectional association of metabolic abnormalities with NAFLD progression. This study aimed to systematically evaluate the relationship between metabolic traits and HCC occurrence in patients with NAFLD. METHOD: This study reviewed eight eligible studies that included 297,956 participants, to determine the relationship between metabolic traits and the occurrence of HCC in patients with NAFLD. RESULTS: Presence of diabetes mellitus (DM) was associated with increased risk of HCC (HR: 2.65, 95%CI: 2.02 ~ 3.49, Pheterogeneity = 0.589, I2 = 0.0%). Stratified analysis revealed that this risk was higher in NAFLD patients with advanced fibrosis/cirrhosis (HR: 4.55, 95%CI: 2.34 ~ 8.87, Pheterogeneity = 0.870, I2 = 0.0%). Nonetheless even in patients without cirrhosis, DM remained a high risk factor for HCC incidence (HR: 1.80, 95%CI: 1.05 ~ 3.06, Pheterogeneity = 0.291, I2 = 10.4%). Overweight/obesity had a slight correlation with increased risk of HCC occurrence in NAFLD patients (HR: 1.31, 95%CI: 1.00 ~ 1.71, Pheterogeneity = 0.888, I2 = 0.0%), while presence of hypertension and dyslipidemia had no correlation. CONCLUSION: DM and overweight/obesity are high risk factors for NAFLD-related HCC. In particular, DM increases 4-fold the risk of HCC incidence in NAFLD patients with advanced fibrosis/cirrhosis. There is a need to strengthen surveillance for HCC in NAFLD patients with DM, especially in those with advanced fibrosis/cirrhosis.

Knockout of neutrophil elastase protects against western diet induced nonalcoholic steatohepatitis in mice by regulating hepatic ceramides metabolism.

Previous studies reported increased expression and activity of neutrophil elastase (NE) in NASH. However, the role of NE in nonalcoholic steatohepatitis (NASH) remained unclear. Wild type (WT) and NE knockout (NE KO) mice were fed with western diet (WD) for 24 weeks to establish NASH model. The severity of liver injury in NASH mice was assessed by biochemical analysis, liver triglyceride (TG) quantitation and histological scoring. The gene and protein expression was detected by quantitative real-time PCR, immunohistochemical and immunofluorescence staining (IHC/IF) and western blot analysis. After 24 weeks, WD induced WT (WD-WT) mice had significantly up-regulated NE protein in the liver. Moreover, body weight, liver/body weight, serum and hepatic TG, liver histological score, and hepatic inflammatory factors expression were significantly higher in WD-WT mice than those in low fat diet (LFD) induced WT mice, and these effects were markedly improved by NE KO. In addition, we found incereased expression of ceramides and serine palmitoyltransferase subunit 2 (SPT2) in WD-WT mice could be reversed by NE KO. Up-regulating expression of ceramides and SPT2 by active NE treatment was also found in mice primary hepatocytes. Collectively, these findings indicated that NE KO ameliorated WD induced NASH, and this beneficial effect was due, at least in part, to the potential of NE in regulating hepatic ceramides metabolism.

The association between nonalcoholic fatty liver disease and risk of colorectal adenoma and cancer incident and recurrence: a meta-analysis of observational studies.

BACKGROUND & AIM: Lifestyle modification plays a key role in nonalcoholic fatty liver disease (NAFLD) and colorectal adenoma and/or cancer (CRA/CRC) development. However, the association between NAFLD and the risk of CRA/CRC has not been carefully evaluated. METHODS: In this meta-analysis, we assessed 21 eligible studies including 124,206 participants to determine the association between NAFLD and the risk of incident and recurrent CRA/CRC. RESULTS: NAFLD presence was associated with an increased risk of any incident CRA (aOR: 1.30, 95% CI: 1.19-1.43) and advanced incident CRA/CRC (aOR: 1.57, 95% CI: 1.21-2.04). The severity of NAFLD affected this correlation: compared to mild and/or moderate NAFLD, severe NAFLD was associated with an increased risk of incident CRA/CRC (aOR: 2.19, 95% CI: 1.33-3.60). Although pooled cOR revealed that NAFLD was associated with an increased risk of recurrent CRA/CRC (cOR = 1.73; 95% CI: 1.12-2.68), after adjustment for confounding factors, NAFLD had less correlation with the risk of recurrent CRA/CRC (aOR: 1.81, 95% CI: 0.70-4.65). CONCLUSIONS: The presence and severity of NAFLD are associated with an increased risk of incident CRA/CRC. However, there is insufficient evidence to indicate that NAFLD is associated with an increased risk of recurrent CRA/CRC.

Increased ratio of neutrophil elastase to α1-antitrypsin is closely associated with liver inflammation in patients with nonalcoholic steatohepatitis.

An imbalance between neutrophil elastase (NE) and its inhibitor α1-antitrypsin (A1 AT) is known to contribute to the development of obesity-related inflammation. This study aimed to investigate the role of the NE-A1 AT system in the histological progression of non-alcoholic fatty liver disease (NAFLD), and to evaluate the ability of it to predict nonalcoholic steatohepatitis (NASH). A total of 252 adults (NAFLD group, n = 202; healthy group, n = 50) were recruited. Clinical biochemical characteristics, NE and A1 AT concentrations were measured in all subjects. Among the NAFLD group, 86 patients had previously undergone liver biopsy and information on histological characteristics was consequently available. The area under the receiver operating characteristic curve (AUC) was used to determine the predictive accuracy of the NE-A1 AT system for NASH. NAFLD patients had an elevated serum NE concentration and a reduced A1 AT level with consequent NE/A1 AT imbalance. NE increased in the early stage of steatosis, preceding the decline in A1 AT, dating from the onset of NASH (NAS 3-4), and subsequently NE/A1 AT increased in the presence of NASH. Nonetheless, this increase began to resolve as the disease state progressed to advanced fibrosis. A1 AT had a sensitivity (SEN) of 83.8% and a specificity (SP) of 83.3% with the optimal cut-off of -1459.43, NE/A1 AT had a SEN of 88.8% and a SP of 83.3% with cut-off of 0.363 to predict NASH. An increased NE: A1 AT ratio is closely associated with liver Inflammation in patients with NASH and could serve as a novel marker to predict NASH in humans.

[Development and evaluation of a high-fat/high-fructose diet-induced nonalcoholic steatohepatitis mouse model].

OBJECTIVE: To develop and evaluate a mouse model of nonalcoholic steatohepatitis (NASH) induced by a high-fat and high-fructose (HFHFr) diet. METHODS: Six-week-old C3H mice were randomly divided into groups for HFHFr diet experimental modeling, high fat-only (HF) diet controls, high fructose-only (HFr) diet controls, and standard chow (SC) diet controls. The standard HFHFr diet was modified so that it consisted of 76.5% standard chow, 12% lard, 1% cholesterol, 5% egg yolk powder, 5% whole milk powder, and 0.5% sodium cholate, along with 20% fructose drinking water. At the end of experimental weeks 4, 8, and 16, measurements were taken for the NASH-related parameters of body mass, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), lipid profile, and wet liver weight (upon sacrifice). In addition, histological changes in the liver were evaluated by hematoxylin-eosin (HE) and oil red O staining. The significance of differences between groups was assessed by statistical analysis, using the METHODS: of t-test, Wilcoxon rank sum test, x2 test, F test or Fisher's test as appropriate. RESULTS: As compared to the mice in the SC group at the corresponding time points, the mice in the HFHFr and HF groups showed significantly higher body mass and wet liver weight, as well as more extensive and robust lipid disposition in hepatic tissues as evidenced by oil red O staining. However, HE staining indicated that the HFHFr and HF groups had different degrees of macrosteatosis accompanied with intralobular inflammatory foci, with the former showing more remarkable NASH-related histological changes. Analysis at the end of week 16 showed that about 80% of the mice in the HFHFr group had developed NASH [nonalcoholic fatty liver disease (NAFLD) activity score (NAS): less than 5]. The levels of low-and high-density lipoprotein (LDL and HDL) cholesterol, as well as the levels of ALT and AST, were increased from the end of week 4 to the end of week 8 for the HFHFr and HF groups. At the end of week 16, the two groups differed in the extent of increase in total cholesterol and LDL and HDL cholesterol, with only the HFHFr group showing statistically significant changes. Specifically, at the end of week 16, the HFHFr group showed ALT levels of 108.5 +/- 93.34 U/L (F=5.099, P =0.005 vs. HF group: 44.30 +/- 35.71 U/L, HFr group: 46.70 +/- 17.95 U/L, SC group: 24.70 +/- 6.57 U/L), AST levels of 316.30 +/- 208.98 U/L (F=6.654, P=0.001 vs. HF: 132.12 +/- 75.43 U/L, HFr: 143.30 +/- 38.53 U/L, SC: 122.60 +/- 12.76 U/L), total cholesterol levels of 5.18 +/- 0.58 mmol/L (F=72: 470, P =0.000 vs. HF: 3.94 +/- 0.75 mmol/L, HFr: 2.30 +/- 0.50 mmol/L, SC: 2.02 +/- 0.24 mmol/L), HDL cholesterol levels of 3.05 +/- 0.49 mmol/L (F=25.413, P =0.000 vs. HF: 2.65 +/- 0.54 mmol/L HFr: 1.77 +/- 0.47 mmol/L, SC: 1.58 +/- 0.16 mmol/L), LDL cholesterol levels of 1.11 +/- 0.23 mmol/L (F =83.297, P =0.000 vs. HF: 0.72 +/- 0.17 mmol/L, HFr: 0.27 +/- 0.04 mmol/L, SC: 0.20 +/- 0.05 mmol/ L). CONCLUSION: The present study suggests that a mouse model of NASH can be successfully induced by a 16-week modified HFHFr diet.

Non-alcoholic fatty liver disease (NAFLD) fibrosis score predicts 6.6-year overall mortality of Chinese patients with NAFLD.

The non-alcoholic fatty liver disease (NAFLD) fibrosis score (NFS) has emerged as a useful predictor of long-term outcome in NAFLD patients. We evaluated the predictive performance of the NFS for overall mortality in a Chinese population with NAFLD. All NAFLD patients diagnosed ultrasonographically at Xixi Hospital of Hangzhou between 1996 and 2011 were retrospectively recruited to the study. Outcome was determined by interview and causes of death were confirmed by medical records. The area under the receiver operating characteristic curve (AUCROC ) was used to determine the predictive accuracy of the NFS, BARD (body mass index, aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio, diabetes) score, FIB-4 index and the AST/platelet ratio index (APRI) for mortality. Data from a total of 180 eligible patients (median age 39 years; 96 men) were analysed, with 12 deaths over a median follow-up period of 6.6 years (range 0.5-14.8 years). Using Cox model analysis, the NFS as a continuous variable was identified as the only predictor for all-cause mortality (hazard ratio 2.743, 95% confidence interval (CI) 1.670-4.504). The NFS yielded the highest AUCROC of 0.828 (95% CI 0.728-0.928, P < 0.05), followed by the FIB-4 index, APRI and BARD score (AUCROC 0.806 (P < 0.05), 0.732 (P < 0.05) and 0.632, respectively). The data indicated that the NFS is a useful predictor of 6.6-year all-cause mortality for Chinese patients with NAFLD.

Toll-like receptor-4 signalling in the progression of non-alcoholic fatty liver disease induced by high-fat and high-fructose diet in mice.

The aim of the present study was to investigate Toll-like receptor-4 (TLR4) signalling at different stages of non-alcoholic fatty liver disease (NAFLD) induced by a high-fat, high-fructose (HFHFr) diet in mice. Both TLR4 wild-type (WT) and mutant (TLR4(mut) ) mice were fed either standard chow (SC) or the HFHFr diet for different periods of time from 4 to 16 weeks. Pathological characteristics and function of the liver were assessed. Simple steatosis, steatohepatitis and hepatic fibrosis occurred sequentially in Week 4, 8 and 16 in WT mice fed with the HFHFr. Expression of TLR4, myeloid differentiation factor 88 (MyD88), interferon regulatory factor (IRF) 3 and IRF7 started to increase at Week 4, peaked at Week 8 and then declined to basal levels at Week 16. This pattern was consistent with changes in inflammation in the liver revealed by haematoxylin and eosin staining. However, lipid accumulation, inflammation and fibrosis in livers of TLR4(mut) mice fed the HFHFr diet were significantly alleviated. In addition, the expression of activin A in WT mice fed the HFHFr diet increased at Week 16. The data suggest that TLR4 signalling mediates non-alcoholic steatohepatitis before fibrosis and that activin A is subsequently involved in NAFLD.

[The effect of RNA interfering TLR4 signal pathway on phagocytosis of Kupffer cells].

OBJECTIVE: To investigate the effect of RNA interfering TLR4 signal pathway on phagocytosis of Kupffer cells. METHODS: RAW2647 mice mononuclear macrophage leukemia cells were observed. The tested group was interfered by Tlr4-mus-1567 RNA which had the best result confirmed by QPCR, cells interfered by Negative Control RNA as NC group, and normal cell as control. We perform the phagocytosis test on each group. RESULTS: The tested group has lower phagocytes percentage than control (17.67% +/- 3.51% vs 32.00% +/- 3.00%, P < 0.01), and lower phagocytic index (46.33% +/- 7.51% vs 82.00% +/- 6.08%, P < 0.01). CONCLUSIONS: Decreased phagocytic activity was observed on Kupffer cells by RNA interference.

[Activation of Kupffer cell and related signal pathway proteins in the liver of high fat and high fructose diet induced NAFLD mice].

OBJECTIVE: To investigate the expression of F4/80, NF-kappaB, p-AKT, AKT in the liver of nonalcoholic fatty liver disease (NAFLD) mice. To determine the role of Kupffer cells (KCs) in the development of NASH (non-alcoholic steatohepatitis), and understand the pathogenic mechanism of NASH. METHODS: Five C3H/HeN mice fed with normal diet were served as controls, while fifteen fed with high fat, high fructose, high fat combined fructose diet respectively for 16 weeks were as NAFLD mice models. The liver inflammation and hepatic damage were examined, and the expression of F4/80, NF-Kb, p-AKT, AKT and the content of lipid in the liver were also detected. RESULTS: Chronic intake of high fat and 30% fructose solution caused a significant increase in hepatic steatosis in animals in comparison to water controls. Liver F4/80 and NF-kappaB were significantly higher in high fat and high fat combined fructose diet fed mice than that in controls (P < 0.01, P < 0.01), F4/80 protein were higher in high fat diet treated mice than those in fructose and high fat combined fructose groups (P < 0.01, P < 0.01). Markers of insulin resistance (e. g, hepatic phospho-AKT, AKT) were only altered in fructose-fed or high fat combined fructose animals (P < 0.01, P < 0.01). CONCLUSION: High fat and fructose diet may induce NAFLD in C3H/HeN mice. Kupffer cells and signal pathway proteins were activated, and they may play key roles in the initiation and progression of NASH.

[Model building-up and observation on the mouse carried chronic hepatitis B and nonalcoholic fatty liver disease].

OBJECTIVE: Establish the model of mouse with chronic hepatitis B virus (HBV) and nonalcoholic fatty liver disease (NAFLD). METHODS: Take 100 HBV transgenic, BALB/c mice of 4 weeks old, with each gender half. Then pick out 70 mice in one group to feed high-fat feed and the rest to feed normal feed. At the end of week 16, random kill 10 mice of high-fat, then liver tissue and serological detection target identification model is established in this paper. After that, divide the mice into model group and comparison group with 30 mice in each group. Feed model group with high-fat feed, comparison group with normal feed and normal group with normal feed till week 72 (including previous 16 weeks). Kill 10 mice of each group at the end of week 24, 48 and 72 respectively, fully automatic biochemical instrument detection of serum ALT, AST, TC, TG, FBG, fluorescence quantitative PCR method to detect HBV-DNA, chemiluminescence detection of HBsAg, liver biopsy after HE staining to evaluate histology change, observe mice model of dynamic evolution. RESULTS: (1) Feed high fat feed after 16 weeks, mice's weight, serum ALT, AST, TC, TG, FBG and blood biochemical indicators increased, HBV-DNA positive, liver HE staining obviously big blister fatty degeneration of liver cells and within the lobule lymphocytes infiltration, NAFLD activity score (NAS) getting close to NASH, the model of chronic HBV carries with NAFLD mouse built successfully. (2) The TC and TG values of model group in each period were higher than that of comparison group and normal group. (3) In week 24 and 72, HBV-DNA values of each group are obvious different from the other two groups and the difference can be applied to statistical significance (P < 0.05). (4) In week 48 and 72, NAS of each group are obvious different from the other two groups and the difference can be applied to statistical significance (P < 0.05). CONCLUSIONS: (1) Chronic HBV carries with NAFLD mice model can be established by HBV transgenic mice fed by high fat feed. (2) NAFLD accelerates the liver disease of the mice carrying HBV to some extent.

[Protective effects of rhein on hepatic progression in HBV-transgenic mice with nonalcoholic steatohepatitis induced by a high-fat diet].

OBJECTIVE: To investigate the beneficial effects of Rhein (RH) on hepatic progression in hepatitis B virus (HBV)-transgenic mice with nonalcoholic steatohepatitis induced by a high-fat (HF) diet. METHODS: A mice model of HBV chronic infection concomitant with liver steatosis was induced by a HF diet in 4-week old HBV-transgenic mice for 16 weeks (n = 130). Thereafter, the mice were divided randomly into control group (back to normal chow), model group (continuing HF diet), RH group [continuing HF diet and administering with 120 mg/(kg x d) RH by gavage] and Essentiale group [continuing HF diet and administering with 69.2 mg/(kg x d) Essentiale by gavage] with 30 mice in each, and were sacrificed at the end of 24-week and 48-week respectively. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG) and fasting plasma glucose (FPG) were measured by an automatic biochemical analyzer, and serum HBV-DNA was determined with qPCR. Hepatic histology was evaluated by HE staining with a light microscope. RESULTS: (1) An histological change composed of steatosis, lymphocytes intralobular infiltration and ballooning was observed after 48 weeks feeding of HF diet, in part mimicking that of NASH patients as evidenced by a NAFLD activity score (NAS) of 3.58 +/- 1.44 points. (2) Histologically, the NAS of model group was higher than that of control group at both time points. RH failed to lessen NAS whereas Essentiale improved the NAS at 48-week. (3) Serum levels of TC, TG and FPG were significantly different between 4 groups at 24-week, with a comparable low value in both RH and Essentiale group. A similar change was evident at 48-week. (4) In terms of HBV viral load, a significantly lower level in Essentiale group than the others was observed at both time points. CONCLUSION: HF diet feeding is able to induce a mouse model of HBV chronic infection concomitant with NASH. RH is effective in alleviating the glucose and lipid metabolism but ineffective in improving the hepatic histology in this model, in contrast, backing to normal chow achieved a better effect in this aspect.