BackgroundNonalcoholic fatty liver disease begins with the aberrant accumulation of triglyceride in the liver. Its spectrum includes the earliest stage of hepatic simple steatosis (SS), nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma. Generally, hepatic SS is often self-limited; however 10%-30% of patients with hepatic SS progress to NASH. The cause(s) of the transition from SS to NASH are unclear. We aimed to test the contribution of nuclear erythroid 2-related factor 2 (Nrf2) on the progression of “benign” SS to NASH in mice fed a high fat diet. In doing so, we discovered the influence of fatty acid in that progression.MethodThe involvement of Nrf2 in defending against the development of NASH was studied in an experimental model induced by a high-fat diet. Wild-type and Nrf2-null mice were fed the diet. Their specimens were analyzed for pathology as well as for fatty acid content and ratios.ResultIn feeding the high-fat diet to the Wild-type and the Nrf2-null mice, the Wild-type mice increased hepatic fat deposition without inflammation or fibrosis (i.e., simple steatosis), while the Nrf2-null mice had significantly more hepatic steatosis and substantial inflammation, (i.e., nonalcoholic steatohepatitis). In addition, as a result of the high-fat diet, SFA (C20: 0, C22: 0) and MUFA (C18: 1, C20: 1) content in Nrf2-null mice were significantly higher than in Wild-type mice. In the Nrf2-null mice the PUFA/TFA ratio decreased; conversely, the MUFA/TFA ratio increased.ConclusionThe deletion of Nrf2 causes “benign” SS to develop into NASH in mice fed with a high-fat diet, through prompt fatty acid accumulation and disruption of hepatic fatty acid composition in the liver.

Mouse cytochrome P450 2A5 (CYP2A5) is upregulated in various liver diseases and a putative common feature for all of these conditions is altered cellular redox status. Nuclear factor erythroid 2-like 2 (Nrf2) is a transcription factor that is post-translationally regulated by oxidative stress and controls the transcription of protective target genes. In the present study, we have characterized the regulation of CYP2A5 by Nrf2 and evaluated gene expression, protein content and activity of anti-oxidant enzymes in the Nrf2+/+ and Nrf2−/− mice model of non-alcoholic fatty liver (NAFLD). After eight weeks of feeding on a high-fat diet, livers from Nrf2−/− mice showed a substantial increase in macro and microvesicular steatosis and a massive increase in the number of neutrophil polymorphs, compared to livers from wild-type mice treated similarly. Livers of Nrf2−/− mice on the high-fat diet exhibited more oxidative stress than their wild-type counterparts as assessed by a significant depletion of reduced glutathione that was coupled with increases in malondialdehyde. Furthermore, results in Nrf2-deficient mice showed that CYP2A5 expression was significantly attenuated in the absence of Nrf2, as was found with the conventional target genes of Nrf2. The treatment of wild-type mice with high-fat diet leaded to nuclear accumulation of Nrf2, and co-immunoprecipitation experiments showed that Nrf2 was bound to Cyp2a5. These findings suggest that the high-fat diet induced alteration in cellular redox status and induction of CYP2A5 was modulated through the redox-sensitive transcription Nrf2.

To investigate the role of hepatic 18-carbon fatty acids (FA) accumulation in regulating CYP2A5/2A6 and the significance of Nrf2 in the process during hepatocytes steatosis, Nrf2-null, and wild type mice fed with high-fat diet (HFD), and Nrf2 silenced or over expressed HepG2 cells administered with 18-carbon FA were used. HE and Oil Red O staining were used for mice hepatic pathological examination. The mRNA and protein expressions were measured with real-time PCR and Western blot. The results showed that hepatic CYP2A5 and Nrf2 expression levels were increased in HFD fed mice accompanied with hepatic 18-carbon FA accumulation. The Nrf2 expression was increased dose-dependently in cells administered with increasing concentrations of stearic acid, oleic acid, and alpha-linolenic acid. The Nrf2 expression was dose-dependently decreased in cells treated with increasing concentrations of linoleic acid, but the Nrf2 expression level was still found higher than the control cells. The CYP2A6 expression was increased dose-dependently in increasing 18-carbon FA treated cells. The HFD-induced up-regulation of hepatic CYP2A5 in vivo and the 18-carbon FA treatment induced up-regulation of CYP2A6 in HepG2 cells were, respectively, inhibited by Nrf2 deficiency and Nrf2 silencing. While the basal expression of mouse hepatic CYP2A5 was not impeded by Nrf2 deletion. Nrf2 over expression improved the up-regulation of CYP2A6 induced by 18-carbon FA. As the classical target gene of Nrf2, GSTA1 mRNA relative expression was increased in Nrf2 over expressed cells and was decreased in Nrf2 silenced cells. In presence or absence of 18-carbon FA treatment, the change of CYP2A6 expression level was similar to GSTA1 in Nrf2 silenced or over expressed HepG2 cells. It was concluded that HFD-induced hepatic 18-carbon FA accumulation contributes to the up-regulation of CYP2A5/2A6 via activating Nrf2. However, the CYP2A5/2A6 expression does not only depend on Nrf2.

The antimicrobial peptide fowlicidin-2 identified in chicken is a member of the cathelicidins family. The mature fowlicidin-2 possesses high antibacterial efficacy and lipopolysaccharide (LPS) neutralizing activity, and also represents an excellent candidate as an antimicrobial agent. In the present study, the recombinant fowlicidin-2 was successfully produced by Escherichia coli (E. coli) recombinant expression system. The gene encoding fowlicidin-2 with the codon preference of E. coli was designed through codon optimization and synthesized in vitro. The gene was then ligated into the plasmid pET-32a(+), which features fusion protein thioredoxin at the N-terminal. The recombinant plasmid was transformed into E. coli BL21(DE3) and cultured in Luria-Bertani (LB) medium. After isopropyl-β-D-thiogalactopyranoside (IPTG) induction, the fowlicidin-2 fusion protein was successfully expressed as inclusion bodies. The inclusion bodies were dissolved and successfully released the peptide in 70% formic acid solution containing cyanogen bromide (CNBr) in a single step. After purification by reverse-phase high-performance liquid chromatography (RP-HPLC), ∼6.0 mg of fowlicidin-2 with purity more than 97% was obtained from 1 litre of bacteria culture. The recombinant peptide exhibited high antibacterial activity against the Gram-positive and Gram-negative bacteria, and even drug-resistant strains. This system could be used to rapidly and efficiently produce milligram quantities of a battery of recombinant antimicrobial peptides as well as for large-scale production.