| Fungi as an important source of natural active compounds have rich diversity of metabolites with unique chemical structure and biological activity.In the present researches, it was focused on the isolation of active compounds from Fungi and their activities of antioxidant, anti-HIV-1and immunoregulation as well as the relative mechanism.Extracts from64different strains of Fungi were investigated for their antioxidant capacities in two different assays, namely inhibition of erythrocyte hemolysis and lipid peroxidation assay, from which Pholiota adiposa was screened for further study. The active compound PB3, PC3-3and HEB were isolated and identified from P. adiposa for the first time by a combination of chromatography techniques, including NKA macroporous resin, Sephadex G-15, Sephadex LH-20and HPLC, after extraction with suitable concentration of ethanol or/and ethyl acetate. Comparing to the retention time of standards by HPLC, PC3-3was identified to be steroidal saponins, PB3was identified to be adenosine with molecular formula of C10H13N5O4, and HEB was identified to be methyl gallate with molecular formula of C8H8O5, which were verified again by mass spectrum (MS) and Fourier transform infrared spectroscopy (FT-IR) analysis separately. The inhibition rate of PC3-3and HEB were26.9%and82.4%on erythrocyte hemolysis, while88.2%and17.3%on lipid peroxidation at concentration250μg/ml. They exhibited strong antioxidative activities in different assays. Furthermore, HEB could eliminate superoxide anion and DPPH radical with the clearance rate of71.4%and85.6%at the same concentration.In the present studies, the gene of redox-sensitive green fluorescent protein which could characterize intracellular redox level by fluorescence intensity of GFP was recombinant expressed in E. coli BL21and TZM-BL cell line with HIV-LTR driven luciferase in genomes separately. As a result, two novel intracellular detection models named E. coli-croGFP model and TZM-BL-croGFP model were established for antioxidant and anti-HIV active compounds screening. In E. coli-croGFP model, only six kinds of fungal strains with antioxidant activity in vitro could display intracellular activity partly detected by fluorescence spectrophotometer. It showed that not all antioxidants were able to enter the cell to exert its activity. In TZM-BL-croGFP model, it showed that intracellular fluorescence intensity increased while the expression of HIV-LTR driven luciferase enhanced under H2O2stimulation detected by flow cytometry and luciferase activity assay. Thus, it directly confirmed the close relationship between oxidative stress and HIV-LTR activation. Moreover, antioxidant HEB could inhibit the oxidative stress induced by H2O2effectively with an IC50value of26.0μM and HIV-1replication in TZM-BL cell infected by pseudovirus with an IC50value of11.9μM, as well as restrain the HIV-LTR activation induced by200μM H2O2from18.2%to about2%. These two intracellular models with advantages of cheap, simple and stable were suitable for high-throughput screening of active ingredients.To reveal the relationship between oxidative stress and HIV-LTR activation, the transformation of nuclear transcription factor kappa B (NF-κB) signal transduction pathway was detected by Western blot because there was κB target sequence in HIV-LTR gene. It showed that NF-κB p65was increasing in nucleus while its repressor IκB degrading in cytoplasm which meant that NF-κB pathway could be activated by H2O2. Antioxidant HEB could prevent the activation of NF-κB pathway by inhibiting the increasing of p65in nucleus and the degradation of IκB in cytoplasm in order to inhibit HIV-1replication and transcription mediated by HIV-LTR with κB target sequence.Antioxidant HEB inhibited HIV-1replication through multiple targets which were important for HIV-1life cycle, including virus-cell fusion/entry, reverse transcription, integration and proteins processing. Compared to medication2h after virus infection, HEB pre-protection showed better inhibition activity to viral replication, which proved that the inhibitory effect of HEB did not limited to virus-cell fusion/entry process. Moreover, HEB inhibited the activity of key enzymes which were essential for HIV-1replication. HEB showed higher activity than the positive control pepstatin A in anti-HIV-1protease assay with the inhibition rate of17.1%. It also inhibited HIV-1reverse transcriptase and integrase activities efficiently with the IC50values of80.1μM and228.5μM.Cytokines were critical regulatory factors and played important roles in immune system. Mice were intraperitoneally injected with purified fraction HEB and PB3every24hours, and the expressive level of cytokines was detected in spleen by real-time quantitative PCR seven days later. Antioxidant HEB significantly improved the expression of IFN-y more than twice, while decreased the expression of Th2type cytokines IL-4and IL-10to about50%. It was conducive to regulate the Thl/Th2type cytokines balance in AIDS patients. Fraction PB3displayed anti-inflammatory potency by up regulating the expression of cytokine synthesis inhibitory factor IL10to150%, while down regulating the expression of IL-2, IL-6and IFN-y to49.0%,56.9%and73.4%respectively.In a word, this study was focused on the isolation of natural active compounds from P. adiposa and detection of their activities of antioxidant, anti-HIV-1and immunoregulation as well as searching on their mechanism and interaction. What is more, two novel intracellular detection models named E. coli-croGFP model and TZM-BL-croGFP model were established for high-throughput screening of ingredients with antioxidantive and anti-HIV activity. Natural products with better biosecurity and pleiotropic effects could overcome the disadvantage of virus resistance caused by single chemically synthetic drug. Therefore, this study provides a new direction for anti HIV-1drug research, and it is also significant for application of natural products in HIV-1therapy. |
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