Avaliação dos efeitos do inibidor multiquinase sorafenibe sobre as células musculares lisas arteriais de Rattus norvegicus: Estudo in vitro / Evaluation of the effects of the multikinase inhibitor sorafenib on the arterial smooth muscle cells of Rattus norvegicus: An in vitro study

A reestenose arterial é um processo inflamatório que pode ocorrer após colocação de stent por cateterismo. Os stents farmacológicos surgiram para reduzir esse problema e o inibidor multiquinase sorafenibe demonstrou ser um composto com ação efetiva. Este estudo in vitro avaliou os efeitos do sorafenibe sobre a citotoxicidade, migração celular e distribuição das células nas fases do ciclo celular. A linhagem celular de músculo liso de rato A7r5 foi tratada com sorafenibe em concentrações que variaram de 0 a 5 μM. Os efeitos citotóxicos foram avaliados por dois ensaios colorimétricos, MTT e SRB após 24 horas de tratamento. A distribuição das células nas fases do ciclo celular foi avaliada por citometria de fluxo e a capacidade de cicatrização/migração celular pelo ensaio scratch wound assay. Comparado com o controle positivo paclitaxel, o sorafenibe demonstrou um efeito 1,6 vezes maior na redução da proliferação celular. Na avaliação do ciclo celular, o sorafenibe mostrou um bloqueio na fase G0/G1. Além disso, o sorafenibe aumentou o número de A7r5 células na fase sub-G1, sugerindo morte celular. No entanto, no estudo de cicatrização/migração celular, não foi observado efeito quando comparado ao controle negativo. Assim, esses resultados in vitro sugerem que o sorafenibe é eficaz para uso em stents farmacológicos, sugerindo uma continuidade na investigação desse fármaco.

Synthesis and evaluation of an alginate-methacrylate xerogel for insulin delivery towards wound healing applications

Background: Alginate is one of the most widely used biopolymer for wound healing. But poor mechanical strength and degradability limits its application especially as a drug-delivery matrix. The aim of this study was to develop stable alginate based scaffold for insulin delivery toward wound care. Materials & methods: The xerogel alginate-g-poly (methacrylic acid; AGM2S) was characterized by various analytical techniques. Results: AGM2S xerogel showed improved physical stability, low degradation, good swelling and water vapour transmission rate (WVTR). About 70% of insulin was released from loaded xerogel over a period of 48 h and favorably modulated the healing response in in vitro scratch wound assay. Conclusion: Grafting improved the strength and stability of alginate xerogel and the results suggest the application of insulin loaded AGM2S xerogels as a potential wound healing material.

Abnormal Cannabidiol Affects Production of Pro-Inflammatory Mediators and Astrocyte Wound Closure in Primary Astrocytic-Microglial Cocultures

Abnormal cannabidiol (abn-CBD) exerts neuroprotective effects in vivo and in vitro. In the present study, we investigated the impact of abn-CBD on the glial production of proinflammatory mediators and scar formation within in vitro models. Primary astrocytic-microglial cocultures and astrocytic cultures from neonatal C57BL/6 mice and CB2 receptor knockout mice were stimulated with lipopolysaccharide (LPS), and the concentrations of tumor necrosis factor α (TNFα), interleukin-6 (IL-6) and nitrite were determined. Furthermore, we performed a live cell microscopy-based scratch-wound assay. After LPS stimulation, TNFα, IL-6 and nitrite production was more strongly increased in cocultures than in isolated astrocytes. Abn-CBD treatment attenuated the LPS-induced production of TNFα and nitrite in cocultures, while IL-6 production remained unaltered. In isolated astrocytes, only LPS-induced TNFα production was reduced by abn-CBD. Similar effects were observed after abn-CBD application in cocultures of CB2 knockout mice. Interestingly, LPS-induced TNFα and nitrite levels were far lower in CB2 knockout cultures compared to wildtypes, while IL-6 levels did not differ. In the scratch-wound assay, treatment with abn-CBD decelerated wound closure when microglial cells were present. Our data shows a differential role of abn-CBD for modulation of glial inflammation and astrocytic scar formation. These findings provide new explanations for mechanisms behind the neuroprotective potential of abn-CBD.

Dissecting the invasive potential of esophageal cancer by siRNA library screening.

86 Background: The low survival for esophageal cancer is in part attributed to its high invasive potential and distant metastasis. In cancer, abnormal cell migration is an essential component of metastasis, and it is reasonable to consider that the conversion between different forms of morphology permits tumor cells to metastasise. Discovering regulators of esophageal cancer morphogenesis may aid in the development of novel targeted therapies that limit metastatic potential. Methods: GOhTRT cells were seeded and treated with siRNA (Human Druggable Genome, Dharmacon) by reverse transfection. Cells were fixed, immunostained for DNA, tubulin and actin and imaged with automated microscope. Cell images were processed using the InCell Analyzer software and the R statistical software systems CellHTS2 and RNAither. The effect of RNAi knockdown on cell viability and cell motility were assessed using MTT cell proliferation assay and scratch wound assay. Results: 127 gene candidates greatly exhibited effects on F-actin and α-tubulin area staining. This list was refined to six high quality candidates (RRM2, ITGB8, GPS1, SPRY1, NOL1 and MYO9B). Silencing of GPS1, MYO9B and SPRY1 increased the rate of migration in a scratch wound assay, with 86.98% ± 3.097%, 75.78% ±5.454% and 72.97% ± 5.463% (p =0.0022) respectively. There was no significant difference in cell viability absorbance values for siGPS1 (0.9037 ± 0.06575; p =0.1905) and siSPRY1 (0.9088 ± 0.09849; p =0.2985), which suggests that the increased rate of wound closure previously seen is by virtue of migratory signalling as oppose to an increase in cell proliferation. Cell viability was decreased considerably in siRRM2 cells (0.2492 ± 0.02798; p <0.0001) and siMYO9B (0.4048 ± 0.04663; p<0.0001) in comparison to siNT cells (1.046 ± 0.07712). Conclusions: In summary, this screen successfully identified high confidence hits associated with cytoskeletal remodelling, some of which are already associated with metastasis in literature and database searches. Further mechanistic studies and gene pathway analysis of candidate genes will provide novel therapeutic targets which can be utilised to block the spread of cancer in patients.

Auswirkungen Konservierungsmittel-freier Fluorchinolon-Augenlösungen auf menschliche Hornhaut-Epithelzellen in vitro

Ziele: Die Beurteilung der biologischen Wirkungen Konservierungsmittel-freier Fluorchinolon-Augenlösungen auf Zellkulturen menschlichen Hornhaut-Epithels in vitro.Methoden: Wir untersuchten die Auswirkung von topischen Fluorchinolonen verschiedener Generationen, wie Ofloxacin 0,3%, Levofloxacin 0,5%, Tosufloxacin 0,3%, Moxifloxacin 0,5% und Gatifloxacin 0,3%, auf gezüchtete menschliche Hornhaut-Epithelzellen. Die Untersuchung erfolgte mittels MTT-basiertem kalorimetrischem Assay, Laktatdehydrogenase(LDH)-Leakage-Assay und Scratch-Wound-Assay. Die Morphologie der Hornhaut-Epithelzellen wurde mittels inverser Lichtmikroskopie und Transmissionselektronenmikroskopie untersucht.Ergebnisse: Bei allen topischen Fluorchinolonen ging die metabolische Aktivität der Hornhaut-Epithelzellen zeitabhängig zurück, und der LDH-Titer stieg mit zunehmender Dauer der Wirkstoffexposition. Insbesondere nach einer Exposition gegenüber Moxifloxacin 0,5% und Gatifloxacin 0,3% war ein signifikanter Anstieg der LDH-Titer im Vergleich zu den Kontrollen festzustellen. Die Migrationsraten der Hornhaut-Epithelzellen waren bei Ofloxacin 0,3% und Levofloxacin 0,5% höher als bei den anderen Fluorchinolonen. Nach einer Exposition gegenüber Moxifloxacin 0,5% und Gatifloxacin 0,3% waren schwere morphologische Schäden an den Zellen zu beobachten.Schlussfolgerung: Da Moxifloxacin 0,5% und Gatifloxacin 0,3% eine stärkere toxische Wirkung auf die Hornhaut-Epithelzellen ausübten als die anderen Fluorchinolone, sind diese Fluorchinolon-Augenlösungen der 4. Generation nur nach sorgfältiger Abwägung im Hinblick auf die mögliche Schädigung des Hornhaut-Epithels bei langer Behandlungsdauer oder zu hoher Dosierung anzuwenden.Übersetzung aus Ophthalmic Res 2014;51:216-223 (DOI: 10.1159/000357976)