18 research outputs found

    Supplementary Material for: Verbascoside Alleviates Atopic Dermatitis-Like Symptoms in Mice via Its Potent Anti-Inflammatory Effect

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    <b><i>Background:</i></b> Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by the interplay between multiple genetic and environmental factors. The pathogenesis of AD remains incompletely understood. Treatment with topical steroids for chronic AD symptoms has severe side effects and so a new treatment is required.<b><i></i></b> Verbascoside is a hydrophilic phenylethanoid glycoside with antioxidant, anti-inflammatory properties. <b><i>Methods:</i></b> Verbascoside was evaluated in AD-like lesions induced by the repetitive and alternative application of 2,4-dinitrochlorobenzene (DNCB) in BALB/c mice. Overall symptomatic score and serological and molecular changes of the skin lesions were investigated. <b><i>Results:</i></b> Verbascoside relieved the overall AD-like symptoms such as scratching behavior and skin lesion severity. At whole-body level, verbascoside significantly reduced DNCB-induced IgE and Th2 cytokines in the peripheral blood. At the skin lesion site, verbascoside also inhibited DNCB-induced production of proinflammatory cytokine TNF-α, IL-6, and IL-4 mRNA. In a human monocyte THP-1 model, verbascoside could suppress DNCB-induced upregulation of CD86 and CD54 at the cell surface, the secretion of the proinflammatory cytokines TNF-α and IL-6, and the activation of NFκB signaling in a dose-dependent manner. <b><i>Conclusion:</i></b> Our results demonstrate that verbascoside could be a potential therapeutic agent for the treatment of AD

    Supplementary Material for: Isotetrandrine Reduces Astrocyte Cytotoxicity in Neuromyelitis Optica by Blocking the Binding of NMO-IgG to Aquaporin 4

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    <b><i>Objective:</i></b> Neuromyelitis optica (NMO) is a severe neurological demyelinating autoimmune disease that affects the optic nerves and spinal cord with no cure and no FDA-approved therapy. Research over the last decade revealed that the binding of NMO-IgG to the water channel protein astrocyte aquaporin 4 (AQP4) might be the primary cause of NMO pathogenesis. The purpose of this study was to identify potential blockers of NMO-IgG and AQP4 binding. <b><i>Methods:</i></b> We developed a two-step screening platform consisting of a reporter cell-based high-throughput screen assay and a cell viability-based assay. Purified NMO-IgG from NMO patient serum and transfected Chinese hamster lung fibroblast V79 cells stably expressing human M23-AQP4 were used for primary screening of 40,000 small molecule fractions from 500 traditional Chinese herbs. <b><i>Results:</i></b> Thirty-six positive fractions were identified, of which 3 active fractions (at 50 μg/ml) were found to be from the same Chinese traditional herb <i>Mahonia japonica </i>(Thunb.). A bioactivity-guided method based on a primary screening assay for blocking activity led to the isolation of an active single natural compound, isotetrandrine, from the 3 fractions. Our immunofluorescence staining results showed that isotetrandrine can block NMO-IgG binding to AQP4 without affecting the expression and function of AQP4. It can also inhibit NMO-IgG binding to astrocyte AQP4 in NMO patient sera and block NMO-IgG-dependent complement-mediated cytotoxicity with the IC<sub>50</sub> at ∼3 μM. <b><i>Conclusions:</i></b> The present study developed a cell-based high-throughput screen to identify small molecule inhibitors for NMO-IgG and AQP4 binding, and suggests a potential therapeutic value of isotetrandrine in NMO

    Supplementary Material for: The Regulator Gene <b><i>rnc</i></b> Is Closely Involved in Biofilm Formation in <b><i>Streptococcus mutans</i></b>

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    <i>Streptococcus mutans</i> is an important factor in the etiology and pathogenesis of dental caries, largely owing to its ability to form a stable biofilm. Previous animal studies have indicated that <i>rnc</i> could decrease the amount of sulcal caries, and that the downregulation of cariogenicity might be due to its capacity to disrupt biofilm formation. However, the biofunctions by which <i>rnc</i> is involved in biofilm formation remain to be elucidated. In this study, we further investigate the role of <i>rnc</i> based on the study of mature biofilm. Scanning electron microscopy and the crystal violet assay were used to detect the biofilm forming ability. The production and distribution of exopolysaccharides within biofilm was analyzed by exopolysaccharide staining. Gel permeation chromatography was used to perform molecular weight assessment. Its adhesion force was measured by atomic force microscopy. The expression of biofilm formation-associated genes was analyzed at the mRNA level by qPCR. Here, we found that <i>rnc</i> could occur and function in biofilm formation by assembling well-structured, exopolysaccharide-encased, stable biofilms in <i>S. mutans</i>. The weakened biofilm forming ability of <i>rnc</i>-deficient strains was associated with the reduction of exopolysaccharide production and bacterial adhesion. Over all, these data illustrate an interesting situation in which an unappreciated regulatory gene acquired for virulence, <i>rnc</i>, most likely has been coopted as a potential regulator of biofilm formation in <i>S. mutans</i>. Further characterization of <i>rnc</i> may lead to the identification of a possible pathogenic biofilm-specific treatment for dental caries

    Supplementary Material for: Desmosterol in Brain Is Elevated because <b><i>DHCR24</i></b> Needs REST for Robust Expression but <b><i>REST</i></b> Is Poorly Expressed

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    Cholesterol synthesis in the fetal brain is inhibited because activity of DHCR24 (24-dehydrocholesterol reductase) is insufficient, causing concentrations of the precursor desmosterol to increase temporarily to 15-25% of total sterols at birth. We demonstrate that failure of <i>DHCR24</i> to be adequately upregulated during periods of elevated cholesterol synthesis in the brain results from the presence in its promoter of the repressor element 1 (RE1) nucleotide sequence that binds the RE1-silencing transcription factor (REST) and that REST, generally reduced in neural tissues, uncharacteristically but not without precedent, enhances <i>DHCR24</i> transcription. <i>DHCR24</i> and <i>REST</i> mRNA levels are reduced 3- to 4-fold in fetal mouse brain compared to liver (p < 0.001). Chromatin immunoprecipitation assays suggested that REST binds to the human <i>DHCR24</i> promoter in the vicinity of the predicted human RE1 sequence. Luminescent emission from a human <i>DHCR24</i> promoter construct with a mutated RE1 sequence was reduced 2-fold compared to output from a reporter with wild-type RE1 (p < 0.005). Silencing <i>REST</i> in HeLa cells resulted in significant reductions of <i>DHCR24</i> mRNA (2-fold) and DHCR24 protein (4-fold). As expected, relative concentrations of Δ<sup>24</sup>-cholesterol precursor sterols increased 3- to 4-fold, reflecting the inhibition of DHCR24 enzyme activity. In contrast, mRNA levels of <i>DHCR7</i> (sterol 7-dehydrocholesterol reductase), a gene essential for cholesterol synthesis lacking an RE1 sequence, and concentrations of HMGR (3-hydroxy-3-methyl-glutaryl-CoA reductase) enzyme protein were both unaffected. Surprisingly, a dominant negative fragment of REST consisting of just the DNA binding domain (about 20% of the protein) and full-length REST enhanced <i>DHCR24</i> expression equally well. Furthermore, RE1 and the sterol response element (SRE), the respective binding sites for REST and the SRE binding protein (SREBP), are contiguous. These observations led us to hypothesize that REST acts because it is bound in close proximity to SREBP, thus amplifying its ability to upregulate <i>DHCR24</i>. It is likely that modulation of <i>DHCR24</i> expression by REST persisted in the mammalian genome either because it does no harm or because suppressing metabolically active DHCR24 while providing abundant quantities of the multifunctional sterol desmosterol during neural development proved useful

    Erratum: Endoplasmic Reticulum Stress Predicts Clinical Response to Cyclosporine Treatment in Primary Membranous Nephropathy

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    <b><i>Background:</i></b> Little is known about the endoplasmic reticulum stress (ERS) marker glucose regulated protein 78 (GRP78) and calcineurin in the kidney in primary membranous nephropathy (PMN) and if they could predict post-cyclosporine treatment outcome. <b><i>Methods:</i></b> This is a retrospective study using a dataset of biopsy-confirmed PMN from Peking Union Medical College Hospital from 1996 to 2014. Seventy-six adult patients treated with cyclosporine as primary immunosuppression for at least 6 months were studied. Immunohistochemistry was used to detect GRP78 and calcineurin in the kidney. Serum calcineurin was assayed by ELISA. Patients were grouped into no-remission (NR, n = 17), partial remission (PR, n = 39), or complete remission (CR, n = 20) at the end of 6 months of treatment. <b><i>Results:</i></b> There was no difference of initial dose of cyclosporine among NR, PR, and CR groups. Kidney calcineurin expression in PMN was significantly increased compared to that in controls (p < 0.0083). The glomerular GRP78 in NR PMN was higher than that in control, CR and PR patients (p < 0.0083). Kidney calcineurin expression and GRP78 expression was positively correlated. However, there were no differences in either serum calcineurin levels or kidney calcineurin expressions among NR, PR or CR groups. There was a negative correlation between serum calcineurin activity and whole kidney calcineurin expression (p = 0.034) or glomerular calcineurin expression (p = 0.007). Neither kidney calcineurin nor GRP78 expression was correlated with proteinuria. <b><i>Conclusions:</i></b> ERS marker GRP78 in the glomeruli but not serum or kidney calcineurin expression could be a useful marker in PMN to negatively predict the response to cyclosporine treatment at the sixth month
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