ReN VM spheroids in matrix: A neural progenitor three-dimensional in vitro model reveals DYRK1A inhibitors as potential regulators of radio-sensitivity.

INTRODUCTION: Pre-clinical testing of small molecules for therapeutic development across many pathologies relies on the use of in-vitro and in-vivo models. When designed and implemented well, these models serve to predict the clinical outcome as well as the toxicity of the evaluated therapies. The two-dimensional (2D) reductionist approach where cells are incubated in a mono-layer on hard plastic microtiter plates is relatively inexpensive but not physiologically relevant. In contrast, well developed and applied three dimensional (3D) in vitro models could be employed to bridge the gap between 2D in vitro primary screening and expensive in vivo rodent models by incorporating key features of the tissue microenvironment to explore differentiation, cortical development, cancers and various neuronal dysfunctions. These features include an extracellular matrix, co-culture, tension and perfusion and could replace several hundred rodents in the drug screening validation cascade. METHODS: Human neural progenitor cells from middle brain (ReN VM, Merck Millipore, UK) were expanded as instructed by the supplier (Merck Millipore, UK), and then seeded in 96-well low-attachment plates (Corning, UK) to form multicellular spheroids followed by adding a Matrigel layer to mimic extracellular matrix around neural stem cell niche. ReN VM cells were then differentiated via EGF and bFGF deprivation for 7 days and were imaged at day 7. Radiotherapy was mimicked via gamma-radiation at 2Gy in the absence and presence of selected DYRK1A inhibitors Harmine, INDY and Leucettine 41 (L41). Cell viability was measured by AlamarBlue assay. Immunofluorescence staining was used to assess cell pluripotency marker SOX2 and differentiation marker GFAP. RESULTS: After 7 days of differentiation, neuron early differentiation marker (GFAP, red) started to be expressed among the cells expressing neural stem cell marker SOX2 (green). Radiation treatment caused significant morphology change including the reduced viability of the spheroids. These spheroids also revealed sensitizing potential of DYRK1A inhibitors tested in this study, including Harmine, INDY and L41. DISCUSSION & CONCLUSIONS: Combined with the benefit of greatly reducing the issues associated with in vivo rodent models, including reducing numbers of animals used in a drug screening cascade, cost, ethics, and potential animal welfare burden, we feel the well-developed and applied 3D neural spheroid model presented in this study will provide a crucial tool to evaluate combinatorial therapies, optimal drug concentrations and treatment dosages.

Isofraxidin ameliorated influenza viral inflammation in rodents via inhibiting platelet aggregation.

Platelets have been proved to exacerbate influenza infection and its complications. Inhibition of platelet activation may be a feasible method for preventing severe infection and secondary acute lung injury (ALI). Isofraxidin (IFD) is a natural coumarin isolated from the plants Sarcandra glabra and Siberian ginseng, and exerts anticancer, antioxidant and antiinflammatory effects. In the present study, we examined the therapeutic effects of IFD in ADP- or arachidonic acid (AA)-induced platelet aggregation model and in influenza A virus (IAV)-induced ALI mouse model. The results showed that IFD significantly inhibited platelet aggregation induced by ADP and AA in vitro in a concentration-dependent manner as well as the release of soluble P-selectin and platelet factor 4. Moreover, IFD significantly relieved IAV-induced lung inflammation, reduced the expressions of platelet activation biomarkers (P-selectin and CD61), decreased the serum levels of TNF-α, IL-1ß, IL-6 and MIP-2, suppressed peripheral platelet aggregation and prolonged the survival time of infected mice. The western blotting results also demonstrated that IFD reduced the phosphorylation levels of PI3K, AKT and p38 in the activated platelets stimulated by ADP and IAV infection. But IFD did not have any effects on IAV replication. It indicated that IFD ameliorated IAV-induced severe lung damage and lethal infection by suppressing platelet aggregation via regulating PI3K/AKT and MAPK pathways.

Mosla scabra flavonoids ameliorate the influenza A virus-induced lung injury and water transport abnormality via the inhibition of PRR and AQP signaling pathways in mice.

ETHNOPHARMACROLOGICAL RELEVANCE: Mosla scabra (Thunb.) C.Y. Wu and H.W. Li has been used as a traditional medicinal herb for centuries in East Asian countries. It has antibacterial, antiviral, antioxidant, anti-inflammatory and immunomodulatory effects. In folk medicine, it is used as a remedy for the treatment of pulmonary diseases, such as fever, cold, cough, pulmonary edema and emphysema. AIM OF THE STUDY: This study was to investigate the protective mechanism of total flavonoids from M. scabra (MF) in influenza A virus (IAV)-infected mice. MATERIALS AND METHODS: The mice were infected with IAV and then were treated daily with MF for five days. At the end of the experiment, the levels of inflammatory-related cytokines (IFN-α, IL-6, TNF-α and IL-1ß) were determined by ELISA. Pathological changes of lung tissue were examined by H&E staining. The protein expressions of AQP5, p-p38, caspase-3 and NF-κB p65 were detected by western blot analysis while the gene expressions of key effectors in AQP5 and PRRs signaling pathways were detected by real-time Fluorescence Quantitative Polymerase Chain Reaction (RFQ-PCR) analysis. RESULTS: The results showed that treatment with MF at doses of 120-360mg/kg for five days to IAV-infected mice significantly attenuated IAV-induced pulmonary injury and decreased the serum levels of IL-6, TNF-α and IL-1ß, but increased IFN-α levels. MF treatment could up-regulate the mRNA expressions of TLR-7, RIG-1, TRAF6, Bcl-2, Bax, VIPR1, PKCα and AQP5 and down-regulate caspase-3 and NF-κB p65 protein expression. CONCLUSION: Treatment with MF could significantly alleviate IAV-induced pulmonary inflammation, apoptosis and water transport abnormality, which was probably through the regulation of TLR7, RIG-1 and AQP5 signaling pathway.

Protective effect of a polyphenolic rich extract from Magnolia officinalis bark on influenza virus-induced pneumonia in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Magnolia officinalis bark is used in traditional Chinese medicine for the treatment of cough, colds, fever, chronic bronchitis and stomach ailments. AIM OF THE STUDY: To investigate therapeutic effects of polyphenol rich extract from M. officinalis bark (MPE) in influenza virus A-infected mice, and to provide evidence for the inflammation response and immunomodulatory potential during infection. MATERIALS AND METHODS: Mice were infected with influenza virus A (IVA) and MPE at doses of 10 and 20mg/kg were orally administrated daily for 5 days after challenge. The levels of serum L-6 and TNF-α were determined by ELISA while protein expressions of NF-κB and TLR3 were detected by western blotting analysis. RESULTS: MPE exhibited significant therapeutical effects on reducing levels of serum NO, IL-6 and TNF-α, inhibiting pneumonia, decreasing lung viral titers and sensitizing IVA-induced apoptosis through down-regulation of NF-κB and TLR3 protein expression in the lung tissue of IVA-infected mice. CONCLUSIONS: MPE could exhibit preventive and therapeutical effects on IVA-infected mice as a suppressor of the production of inflammatory mediators, NO and pro-inflammatory cytokines, TNF-α and IL-6. These effects appeared to be mediated, at least in part, by an inhibition of TLR3 and NF-κB activation. Therefore, MPE could provide a safe and effective therapeutic approach for influenza and its subsequent viral pneumonia.

Anti-influenza virus effects of the aqueous extract from Mosla scabra.

ETHNOPHARMACOLOGICAL RELEVANCE: Mosla scabra (Thunb.) C. Y. Wu is a broadly used species in the southeastern China as an antipyretic and antiviral drug for the treatment of colds, fever, inflammation and chronic bronchitis. AIM OF THE STUDY: To investigate the anti-influenza virus activities of the aqueous extract from Mosla scabra (AEMS), and to provide evidence for the implication of its immunomodulatory potential for the overall protective effect in lethal murine experimental influenza A infection. MATERIALS AND METHODS: The anti-influenza virus activities of AEMS or ribavirin were evaluated in embryonated eggs and in a mouse infection model and the effects of AEMS on early immune responses during influenza virus infection were evaluated in a mouse infection model. RESULTS: At the concentration of 0.3-30.0mg/ml, AEMS exhibited both preventive and therapeutical effect on embryonated egg. Oral administration of AEMS to mice infected with influenza virus A (IVA) was highly effective in preventing death, inhibiting pneumonia and reducing lung viral titers. It also significantly enhanced IFN-gamma, IL-2, IL-4 and IL-5 cytokine-producing splenocytes, increased T-cell subsets like CD4(+) and CD4(+)/CD8(+), decreased levels of IL-6 after infection, and provided protective immunity. At a dosage of 4800 mg/kg, almost all treated mice survived, suggesting that AEMS is of low toxicity. However, ribavirin has weaker efficacy compared to AEMS. CONCLUSIONS: These results showed, for the first time, while both AEMS and ribavirin appeared to have similar efficacy against IVA, AEMS playing a role as an immunomodulator and antiviral inhibitor during influenza virus infection, was considered to be less toxic and may warrant further evaluation as a possible agent for the treatment of influenza.

Expression of extracellular matrix genes in cultured hepatic oval cells: an origin of hepatic stellate cells through transforming growth factor beta?

BACKGROUND: Hepatic oval cells, progenitor cells in the liver, can differentiate into hepatocytes and bile duct cells both in vitro and in vivo. Although hepatic stellate cells are another important cell component in the liver, less attention has been focused on the relationship between hepatic oval cells and hepatic stellate cells. METHODS: Hepatic oval cells were isolated from rats fed a choline-deficient diet supplemented with 0.1% ethionine for 6 weeks and characterized by electron microscopy, flow cytometry, reverse transcription polymerase chain reaction, Western blot and bi-direction differentiation. After treatment with transforming growth factor-beta1 (TGF-beta1), changes in cell viability, morphology, extracellular matrix (ECM) expression and immune phenotype were analysed in these cultured and adherent hepatic oval cells. RESULTS: The primary cultured hepatic oval cells were positive for the oval cell-specific markers OV-6, BD-1/BD-2 and M2PK as well as the hepatocyte markers albumin and alpha-foetoprotein. These hepatic oval cells differentiated bipotentially into hepatocytes or bile duct-like cells under appropriate conditions. It is noteworthy that these bipotential hepatic oval cells expressed ECM genes stably, including collagens, matrix metalloproteinases and tissue inhibitor of mellatoproteinase. Furthermore, except for growth inhibition and morphological changes in the hepatic oval cells after exposure to TGF-beta1, there was an increased expression of ECM genes, the onset expression of snail and loss expression of E-cadherin. During this process, TGF-beta1 treatment induced an upregulation of marker genes for hepatic stellate cells in hepatic oval cells, such as desmin and GFAP. CONCLUSION: Except for the expression of ECM, the cultured hepatic oval cells could induce an increased expression of hepatic stellate cell markers by TGF-beta1 through an epithelial-mesenchymal transition process, which might indicate the contribution of hepatic oval cells to liver fibrosis.