Liver disease is often times associated with increased intestinal permeability. A disruption of the gut barrier allows microbial products and viable bacteria to translocate from the intestinal lumen to extraintestinal organs. The majority of the venous blood from the intestinal tract is drained into the portal circulation, which is part of the dual hepatic blood supply. The liver is therefore the first organ in the body to encounter not only absorbed nutrients, but also gut-derived bacteria and pathogen associated molecular patterns (PAMPs). Chronic exposure to increased levels of PAMPs has been linked to disease progression during early stages and to infectious complications during late stages of liver disease (cirrhosis). It is therefore important to assess and monitor gut barrier dysfunction during hepatic disease. We review methods to assess intestinal barrier disruption and discuss advantages and disadvantages. We will in particular focus on methods that we have used to measure increased intestinal permeability and bacterial translocation during experimental liver disease models.

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Nrf2 plays a critical role in defending against oxidative stress and inflammation. We previously reported that Nrf2 confers protection against ultraviolet-B (UVB)-induced inflammation, sunburn reaction, and is involved in sulforaphane-mediated photo-protective effects in the skin. In this study, we aimed to demonstrate the protective role of Nrf2 against inflammation-mediated extracellular matrix (ECM) damage induced by UVB irradiation. Ear biopsy weights were significantly increased in both Nrf2 wild-type (Nrf2 WT) and knockout (Nrf2 KO) mice one week after UVB irradiation. However, these weights increased more significantly in KO mice compared to WT mice, suggesting a greater inflammatory response in KO mice. In addition, we analyzed the protein expression of numerous markers, including macrophage inflammatory protein-2 (MIP-2), pro-matrix metalloproteinase-9 (MMP-9), and p53. p53, a regulator of DNA repair, was overexpressed in Nrf2 KO mice, indicating that the absence of Nrf2 led to more sustained DNA damage. There was also more substantial ECM degradation and increased inflammation in UVB-irradiated Nrf2 KO mice compared to UVB-irradiated WT mice. Furthermore, the protective effects of Nrf2 in response to UVB irradiation were mediated by increased HO-1 protein expression. Collectively, our results show that Nrf2 plays a key role in protecting against UVB irradiation and that the photo-protective effect of Nrf2 is closely related to the inhibition of ECM degradation and inflammation.

Abstract. Increasing numbers of natural products have been found to possess anticancer effects. Nuclear factor erythroid-2-related factor-2 (Nrf2) is a master regulator of the antioxidative stress response, and our previous studies found that epigenetic modification of the Nrf2 gene appears to be a critical mechanism. Salvia miltiorrhiza, a Chinese herbal medicine widely used in Asian countries, has been shown to possess anticancer and antioxidant effects. Tanshinone IIA (TIIA), an active component in S. miltiorrhiza, has been reported to activate Nrf2 pathway. The objective of this study was to investigate the epigenetic regulation of Nrf2 by TIIA in mouse skin epidermal JB6 cells and the functional consequences for cell transformation. TIIA was found to induce antioxidant response element-luciferase and upregulate the mRNA and protein levels of Nrf2 and Nrf2 downstream target genes HO-1 and NQO-1. TIIA decreased the colony formation of JB6 cells by approximately 80%. TIIA decreased the protein levels of DNMT1, DNMT3a, DNMT3b, and HDAC3 and inhibited the enzymatic activity of HDACs. Bisulfite genomic sequencing indicated that TIIA demethylated the first five CpGs in the promoter region of the Nrf2 gene. Chromatin immunoprecipitation assays showed that TIIA treatment increased the recruitment of RNA polymerase II at Nrf2 transcription start site but had limited effects on enrichment of Ac-H3 in Nrf2 promoter. Taken together, our results show that TIIA activates the Nrf2 signaling pathway and induces epigenetic demethylation of the CpGs of Nrf2. The epigenetic reactivation of the Nrf2 signaling pathway by TIIA could potentially contribute to the attenuation of JB6 cellular transformation and anticancer effects.

Ginseng has long been used in the Asian Countries for more than 2,000 years. Currently, in the “Western World or Western Medicines”, many reports have indicated that they have used herbal medicines, and Ginseng is one of the most popular herbs. Several recent reports have indicated that the antioxidant / anti-oxidative stress activities of ginseng play a role in the benefits of ginseng, however the precise mechanism is lacking. The antioxidant response element (ARE) is a critical regulatory element for the expression of many anti-oxidant enzymes and phase II/III drug metabolizing/transporter genes, mediated by the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2). The aim of this study was to examine the potential activation and synergism of Nrf2-ARE-mediated transcriptional activity between three common ginsenosides present in ginseng, ginsenoside Rb1 (Rb1), ginsenoside Rg1 (Rg1) and ginsenoside 20(S)-protopanaxytriol (20S). We tested whether these ginsenosides and their combinations, could induce Nrf2-ARE activities in HepG2-C8 cells with stably transfected ARE luciferase reporter gene. Cell proliferation, antioxidant and ARE activities, western blotting of Nrf2 protein and qPCR of mRNA of Nrf2 were conducted for Rb1, Rg1 and 20S as well as the combinations of 20S with Rb1 or Rg1. To determine the combination effects, the combination index (CI) was calculated. Rb1 and Rg1 are relatively non-toxic to the cells, while 20S at 50 μM or above significantly inhibited the cell proliferation. Rb1, Rg1 or 20S induced total antioxidant activity and ARE activity in a concentration-dependent manner. Furthermore, combinations of 20S with either Rb1 or Rg1 induced total antioxidant and ARE activity synergistically. Induction of Nrf2 protein and mRNA were also found to be synergistic with the combination treatments. In summary, in this study, we show that ginsenosides Rb1, Rg1 and 20S possess antioxidant activity, transcriptionally activating ARE as well as potential of synergistic activities. The Nrf2-ARE-mediated antioxidant pathway could play a role for the overall anti-oxidative stress activities, which could be important for ginseng's health beneficial effects such as cancer chemopreventive activities.

Epigenetic control of NRF2, a master regulator of many critical anti-oxidative stress defense genes in human prostate cancer (CaP) is unknown. Our previous animal study found decreased Nrf2 expression through promoter CpG methylation/histone modifications during CaP progression in TRAMP mice. In this study, we evaluated CpG methylation of human NRF2 promoter in 27 clinical CaP samples and in LNCaP cells using MAQMA analysis and bisulfite genomic DNA sequencing. Prostate cancer tissue microarray (TMA) containing normal and prostate cancer tissues were studied by immunohistochemistry. Luciferase reporter assay using specific human NRF2 DNA promoter segments and ChIP assay against histone modifying proteins were performed in LNCaP cells. Three specific CpG sites in the NRF2 promoter were found to be hypermethylated in clinical CaP samples (BPH

Exposure to ultraviolet B (UVB) irradiation results in multitude of cellular responses including generation of reactive oxygen species and DNA damage and is responsible for non-melanoma skin cancers (NMSCs). Although genetic mutation is well documented, the epi-mutation, the alteration in epigenetics, remains elusive. In this study, we utilized CpG Methyl-seq to identify a genomewide DNA CpG methylation, to profile the DNA methylation in UVB-irradiated SKH-1 mouse skin epidermis and non-melanoma skin papillomas at various stages. Methyl-seq and RNA-seq were performed to examine the methylation and corresponding transcriptome alterations. The methylation profiles in mouse epidermis were altered by UVB-irradiation as time progresses. Ingenuity Pathways Analysis (IPA) identified many cancer related pathways including PTEN, p53, Nrf2 and inflammatory signaling in UVB-irradiation induced carcinogenesis. Additionally, some novel genes involved in skin carcinogenesis that were not previously reported were differentially methylated, including Enf2, Mgst2, Vegfa, and Cdk4. Taken together, the current study provides novel profiles and insights of methylation and transcriptomic changes at different stages of