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Endophytic fungi are symbiotic with plants and possess multienzyme systems showing promising metabolite potency with region selectivity and stereoselectivity. The aim of this study was to use these special microorganisms as an in vitro model to mimic the potential mammalian metabolites of a natural iridoid gentiopicroside (GPS, compound 1). The fungi isolated from a medicinal plant, Dendrobium candidum Wall. ex Lindl., were screened for their biotransformation abilities with GPS as the substrate, and one strain with high converting potency was identified as Penicillium crustosum 2T01Y01 on the basis of the sequence of the internal transcribed spacer of the ribosomal DNA region. Upon the optimized incubation of P. crustosum 2T01Y01 with the substrate, seven deglycosylated metabolites were detected by ultraperformance liquid chromatography/quadrupole time of flight mass spectrometry (UPLC/Q-TOF MS). Preparative-scale biotransformation with whole cells of the endophytic fungus resulted in the production of five metabolites, including three novel ones, 5α-(hydroxymethyl)-6β-methyl-3,4,5,6-tetrahydropyrano[3,4- c ]pyran-1(8 H )-one (compound 2), ( Z )-4-(1-hydroxybut-3-en-2-yl)-5,6-dihydropyran-2-one (compound 3), and ( E )-4-(1-hydroxybut-3-en-2-yl)-5,6-dihydropyran-2-one (compound 4), along with two known ones, 5α-(hydroxymethyl)-6β-methyl-1 H ,3 H -5,6-dihydropyrano[3,4- c ]pyran-1(3 H )-one (compound 5) and 5α-(hydroxymethyl)-6α-methyl-5,6-dihydropyrano[3,4- c ]pyran-1(3 H )-one (compound 6), aided by nuclear magnetic resonance and high-resolution mass spectral analyses. The other two metabolites were tentatively identified by online UPLC/Q-TOF MS as 5-hydroxymethyl-5,6-dihydroisochromen-1-one (compound 7) and 5-hydroxymethyl-3,4,5,6-tetrahydroisochromen-1-one (compound 8), and compound 8 is a new metabolite. To test the metabolic mechanism, the β-glucosidase activity of the fungus P. crustosum 2T01Y01 was assayed with ρ-nitrophenyl-β- d -glucopyranoside as a probe substrate, and the pathway of GPS biotransformation by strain 2T01Y01 is proposed. In addition, the hepatoprotective activities of GPS and metabolite compounds 2, 5, and 6 against human hepatocyte line HL-7702 injury induced by hydrogen peroxide were evaluated.

Hepatic fibrosis is a common pathological process of chronic liver diseases and would lead to cirrhosis, and Fuzheng Huayu (FZHY) is an effective Chinese herbal product against liver fibrosis. This study observes FZHY influence on proteome of fibrotic liver with differential proteomic approach and aims to understand FZHY multiple action mechanisms on liver fibrosis. The liver fibrosis models were induced with intraperitoneal injection of dimethylnitrosamine for 4 weeks in rats and divided into model control (model) and FZHY-treated (FZHY) groups, while normal rats were used as normal control (normal). After model establishment, rats in FZHY groups were administered 4 g/kg wt of FZHY for 4 weeks, and normal and model groups were given the same volume of saline. The liver proteins in the above 3 groups were separated by two-dimensional gel electrophoresis (2-DE), the differentially expressed spots were analyzed and compared between normal and model or model and FZHY groups, and then the proteins were identified with mass spectrum analysis and validated partially with western blot and real-time PCR. 1000~1200 spots were displayed on each 2D gel, and a total of 61 protein spots were found with significant intensity difference between normal control or FZHY and model control. 23 most obviously differential spots were excised, and in-gel digestion and 21 peptide mass fingerprints (PMF) were obtained with MALDI-TOF MS analysis, and 14 proteins were identified through protein database searching. Among 14 differentially expressed proteins, 8 proteins in normal and FZHY groups had the same tendency of differential expression compared with the ones in model group. And one of them, vimentin, was validated by western blot and real-time PCR analyses. Our study reveals 12 proteins responsible for fibrogenesis induced by DMN in rats, and among them, 8 proteins in fibrotic liver were regulated by FZHY, including aldehyde dehydrogenase, vimentin isoform (CRA_b), gamma-actin, vimentin, fructose-bisphosphate aldolase B, aldo-keto reductase, S-adenosylhomocysteine hydrolase isoform, and HSP90. It indicates that the action mechanism of FZHY antiliver fibrosis may be associated with modulation of proteins associated with metabolism and stress response, as well as myofibroblast activation. The study provides new insights and data for exploring the liver fibrogenesis pathophysiology and FZHY action mechanism against liver fibrosis.

Considerable research have described important regulatory mechanisms that provide immune homeostasis by limiting excessive immune responses and preventing loss of tolerance (1, 2). Naturally occurring regulatory T cells (Tregs) specifically express the transcription factor known as forkhead box 3 (Foxp3), a member of the forkhead/winged-helix family of transcription factors that is essential for the development, maintenance and function of Tregs. Importantly, Foxp3 expression is sufficient to confer suppressive activity to conventional non-Tregs and critical for TCR+ T cells to differentiate to Tregs in the thymus (3). Thus, functional deficiency of Tregs, an abnormality in Foxp3 expression and/or mutations in the Foxp3 gene locus results in increased susceptibility to several autoimmune diseases. Humans that have mutations in the Foxp3 gene develop IPEX (immune dysregulation, polyendocrinopathy and enteropathy, X-linked syndrome), a severe autoimmune disease, resulting in fatal lymphoproliferation at a very early age. A significantly reduced suppressive function of Tregs has been found in patients with polyglandular syndrome type II (APS-II) (4), rheumatoid arthritis (RA) (5), type-I diabetes (6), myasthenia graves (7, 8) and autoimmune hepatitis (9). Patients with multiple sclerosis (MS) not only lose functional suppression (10) but also exhibit decreases in Foxp3 mRNA and protein expressions (11). Primary biliary cirrhosis (PBC) is a progressive autoimmune liver disease characterized by immune-mediated destruction of intrahepatic small bile ducts (12). It has been suggested that the loss of Tregs plays a key role in susceptibility to PBC (13). For example, we have demonstrated a significant decrease in the frequency of Tregs in patients with PBC as well as daughters and sisters of PBC patients (14). We also reported a male child who developed liver dysfunction in association with serum anti-PDC-E2 antibodies at the age of five, who had a complete deficiency of the alpha subunit of the IL-2 receptor (IL-2Rα, CD25) in peripheral blood lymphocytes (15). CD25 is a critical component for functional Treg development and expansion. Mice deficient in IL-2 receptor also demonstrated PDC-like lesions in the liver (16). In agreement with our findings, Ae2-deficient mice that develop features resembling PBC, the proportion of CD4+CD25+FoxP3+ Tregs was reduced (17). Based on the above, we hypothesized that genetic components which are essential for the development and homeostasis of Foxp3+ Tregs is responsible for the loss of self-tolerance in PBC. Hence, we took advantage of Scurfy (Sf) mice which have a Foxp3 gene mutation that results in a deficiency of functional Tregs (18, 19) and examined levels of autoantibodies, cytokines and liver histology. We report herein that Sf mice have serologic, histologic and cytokine features characteristic of autoimmune cholangitis, similar to patients with PBC.

Multiple reports have suggested that reactive oxygen species (ROS) are implicated in hepatic fibrosis and that they are capable of causing hepatocyte apoptosis in hepatic fibrosis by causing oxidative damage to the liver. Thus, the study of antioxidant compounds may shed light on the treatment of hepatic fibrosis. The aim of the current study was to investigate the protective effects of Cordyceps polysaccharide (CPS), a major antioxidative component of Cordyceps militaris, on hydrogen peroxide (H2O2)-induced cell apoptosis. The data showed that CPS markedly inhibited H2O2-induced mitochondrial dysfunction, lowered cell viability, increased the apoptotic rate, boosted ROS production, decreased mitochondrial membrane potential (MMP), reduced the intracellular adenosine triphosphate (ATP) level, increased the Bax/Bcl-2 ratio and promoted cytochrome C (Cyt C) release. These results indicated that CPS protected HL-7702 cells, which are used as the main model of hepatic fibrosis, against H2O2-induced mitochondrial dysfunction by decreasing ROS production and regulating mitochondrial apoptotic signaling through the Cyt C, Bax and Bcl-2 apoptosis-related proteins.

In this study, we investigated the anti-angiogenic effect of the Chinese herbal decoction Danggui Buxue Tang (DBT;Radix AstragaliandRadix Angelicae sinensisin 5 : 1 ratio) in a rat model of liver fibrosis, in order to elucidate its mechanisms of action against liver fibrosis. Liver fibrosis was induced with CCl4and high-fat food for 6 weeks, and the rats were treated with oral doses of DBT (6 g raw herbs/kg/d) and N-Acetyl-L-cysteine (NAC; 0.1 g/kg/d). The results showed that both DBT and NAC attenuated liver fibrosis and neo-angiogenesis. Furthermore, DBT and NAC improved SOD activity but decreased MDA content and 8-OH-dG in fibrotic livers, with DBT being more effective than NAC. DBT decreased the expression of VEGF, Ang1 and TGF-β1 and their signaling mediators, whereas NAC had no effect on VEGF and VEGFR2 expression. Both DBT and NAC reduced HIF-1αgene and protein expression in fibrotic livers, with DBT being more effective. These data clearly demonstrate that the anti-fibrotic properties of DBT are related to its ability to inhibit angiogenesis and its anti-angiogenic mechanisms are associated with improving oxidative stress, regulating the expression and signaling of angiogenic factors, and especially modulating HIF-1αin fibrotic livers.

The fragmentation behaviors of the angular- and linear-type coumarins from Peucedanum praeruptorum Dunn and P. decursivum (Miq.) Maxim were simultaneously investigated by HPLC-DAD/ESI-MSn. For more structural identification, the fragment ions were analyzed and some possible fragmentation pathways were proposed. Different positions and numbers of the substituent also led to different fragment behaviors. Two types of coumarins from P. praeruptorum and P. decursivum were structurally elucidated by these techniques. In addition, UV spectra were applied to support the MS analysis. This is the first time that the two types of coumarins from herbal extracts have been differentiated by HPLC-DAD/ESI-MSn. The method further illustrated the importance of the ESI-MSn technique in the identification of different types of coumarins and was applied for the rapid differentiation of the two herbs.