L-Carnitine Prevents Progression of Non-Alcoholic Steatohepatitis in a Mouse Model with Upregulation of Mitochondrial Pathway

Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease characterized by lobular inflammation, hepatocellular ballooning, and fibrosis with an inherent risk for progression to cirrhosis and hepatocellular carcinoma (HCC). Mitochondrial dysfunction appears to play a role in the progression from simple steatosis to NASH. L-carnitine (L-b-hydroxy-g-N-trimethylaminobutyric acid), an essential nutrient that converts fat into energy in mitochondria, has been shown to ameliorate liver damage. The aim of the present study was to explore the preventive and therapeutic effect of L-carnitine in NASH model mice. Eight-week-old male STAM mice, a NASH-cirrhosis-hepatocarcinogenic model, were divided into 3 experimental groups and fed as follows: 1) high-fat diet (HFD) (control group); 2) HFD mixed with 0.28% L-carnitine (L-carnitine group); and 3) HFD mixed with 0.01% alpha-tocopherol (alpha-tocopherol group). After 4 or 8 weeks, mice were sacrificed. Blood samples and livers were collected, and hepatic tumors were counted and measured. Livers were subjected to histological study, immunohistochemical staining of 4-hydroxynonenal and ferritin, determination of 8-OHdG levels, mRNA and protein expressions for multiple genes, and metabolomic analysis. The intestinal microbiome was also analyzed. L-carnitine increased hepatic expression of genes related to long-chain fatty acid transport, mitochondrial beta-oxidation, and antioxidant enzymes following suppression of hepatic oxidative stress markers and inflammatory cytokines in NASH, and mice treated with L-carnitine developed fewer liver tumors. Although alpha-tocopherol resulted in NASH improvement in the same manner as L-carnitine, it increased periodontitis-related microbiotic changes and hepatic iron transport-related gene expression and led to less effective for anti-hepatocarcinogenesis. Conclusion: L-carnitine prevents progression of non-alcoholic steatohepatitis in a mouse model by upregulating the mitochondrial beta-oxidation and redox system

Hepatitis C Virus-specific T-cell Response Correlates with Hepatitis Activity and Donor IL28B Genotype Early after Liver Transplantation

It is not known how the immune system targets hepatitis C virus (HCV)-infected HLA-mismatched hepatocytes under immune-suppressed conditions after orthotopic liver transplantation (OLT). In addition, the relationship between the HCV-specific immune response and IL28B variants as predictors of HCV clearance has not been well-characterized. We determined the IL28B polymorphisms for 57 post-OLT HCV carriers, and we assessed the HCV-specific immune responses by measuring the peripheral blood mononuclear cell-derived HCV-specific interferon-gamma (IFN-γ) response using an enzyme-linked immunospot assay. At 1-3 years after OLT, patients with no active hepatitis showed higher total spots on the immunospot assay. At＞3 years after OLT, patients with resolved HCV showed higher levels of core, NS3, NS5A, and total spots compared to the chronic hepatitis patients. The IL28B major genotype in the donors correlated with higher spot counts for NS5A and NS5B proteins at 1-3 years after OLT. In the post-OLT setting, the HCV-specific immune response could be strongly induced in patients with no active hepatitis with an IL28B major donor or sustained virological response. Strong immune responses in the patients with no active hepatitis could only be maintained for 3 years and diminished later. It may be beneficial to administer IFN treatment starting 3 years after OLT, to induce the maximum immunological effect

Quantitative real-time PCR results and Western blotting results for expression of hepatic mitochondrial pathway-related genes.

<p>(A) mRNA levels of L-carnitine transport-related gene OCTN-2 and long chain fatty acid transport-related genes Cpt1a and Cpt2 were analyzed. (B) mRNA levels of mitochondrial β-oxidation-related gene MCAD were measured. (C) mRNA levels of antioxidant system-related genes Sod2, CAT, and Gpx1 were analyzed. Data are expressed as means ± SD. (D) Western blotting was performed with the following antibodies directed to Cpt1a, Cpt2, Sod2, CAT and Gpx4. β-actin was used as a loading control. *P<0.05. OCTN, organic cation/carnitine transporter; Cpt, carnitine palmitoyltransferase; MCAD, medium chain acyl CoA dehydrogenase; Sod, superoxide dismutase; CAT, catalase; Gpx, glutathione peroxidase.</p

Intestinal microbiome analysis.

<p>(A) The 100% stacked column chart of microbiome from intestinal feces. (B) Representative percentages of bacterial species in experimental groups. *P<0.05.</p

Body weight, plasma and hepatic biochemical levels.

<p>(A) Body weight of the three experimental groups. Eight-week-old male STAM mice were divided into three experimental groups and fed for 4 weeks as follows: 1) high-fat diet (HFD) (control group); 2) HFD mixed with 0.28% L-carnitine (L-carnitine group); and 3) HFD mixed with 0.01% α-tocopherol (α-tocopherol group). After 4 weeks, mice were weighed. (B) Fasting blood glucose levels of the three experimental groups. (C) Plasma biochemical findings of experimental groups. (D) Hepatic triglyceride levels of experimental groups. Data are expressed as mean ± standard deviation (SD).</p

Liver histological findings.

<p>(A) Representative H&E-stained liver sections are shown. (B) Non-alcoholic fatty liver disease activity scores (NAS) for mouse liver specimens of the three experimental groups. Steatosis, inflammation, and hepatocyte ballooning were categorized, and the sum of these scores was designated as NAS. Data are expressed as means ± SD. *P<0.05.</p

Iron metabolism related pathway analysis.

<p>(A) Representative immunohistochemical staining for ferritin of STAM mouse liver tissue. Intensity of ferritin was calculated by computerized image analysis using Olympus cellSens imaging software. (B) mRNA levels of iron uptake-related hepcidin coding gene Hamp and iron transport-related gene DMT-1 were analyzed. Data are expressed as means ± SD. (C) Liver extracted proteins were analyzed with Western blotting with antibodies directed to DMT-1 and Hamp (hepcidine). *P<0.05. DMT, divalent metal transporter; Hamp, hepcidin coding gene.</p

Assessment of oxidative stress and inflammation in the liver.

<p>(A) Concentrations of 8-OHdG in the liver. (B) Representative immunohistochemical staining for 4-HNE in STAM mouse liver tissue. The intensity of 4-HNE was calculated by computerized image analysis with Olympus cellSens imaging software. (C) Results of quantitative real-time PCR assay to detect TNF-α and IL-1β mRNA levels are shown. (D) Western blotting analysis of the hepatic extracts was performed with each antibodies. Data are expressed as means ± SD. *P<0.05. 8-OHdG, 8-hydroxy-deoxyguanosine; 4-HNE, 4-hydroxynonenal; TNF-α, tumor necrosis factor alpha; IL-1β, interleukin-1β.</p