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.

A Chemerin Peptide Analog Stimulates Tumor Growth in Two Xenograft Mouse Models of Human Colorectal Carcinoma

Background: Chemerin plasma concentration has been reported to be positively correlated with the risk of colorectal cancer. However, the potential regulation of CRC tumorigenesis and progression has not yet been investigated in an experimental setting. This study addresses this hypothesis by investigating proliferation, colony formation, and migration of CRC cell lines in vitro as well as in animal models. Methods: In vitro, microscopic assays to study proliferation, as well as a scratch-wound assay for migration monitoring, were applied using the human CRC cell lines HCT116, HT29, and SW620 under the influence of the chemerin analog CG34. The animal study investigated HCT116-luc and HT29-luc subcutaneous tumor size and bioluminescence during treatment with CG34 versus control, followed by an ex-vivo analysis of vessel density and mitotic activity. Results: While the proliferation of the three CRC cell lines in monolayers was not clearly stimulated by CG34, the chemerin analog promoted colony formation in three-dimensional aggregates. An effect on cell migration was not observed. In the treatment study, CG34 significantly stimulated both growth and bioluminescence signals of HCT116-luc and HT29-luc xenografts. Conclusions: The results of this study represent the first indication of a tumor growth-stimulating effect of chemerin signaling in CRC.

Combination of Two Kinds of Medicated Microparticles Based on Hyaluronic Acid or Chitosan for a Wound Healing Spray Patch

Olive leaves extract (OLE) has been extensively studied as antioxidant and antibiotic and these characteristics make it particularly interesting for use on wounds. For this reason, the aim of this study was to introduce OLE in microparticles (MP) of hyaluronic acid (MPHA-OLE) or chitosan (MPCs-OLE) to obtain a spray patch for the treatment of wounds in anatomical areas that are difficult to protect with traditional patches. The MP were characterized for particle size and ability to protect OLE from degradation, to absorb water from wound exudate, to control OLE release from MP. The MPHA and MPCs medicated or not and mixtures of the two types in different proportions were studied in vitro on fibroblasts by the scratch wound healing assay. The MP size was always less than 5 µm, and therefore, suitable for a spray patch. The MPCs-OLE could slow down the release of OLE therefore only about 60% of the polyphenols contained in it were released after 4 h. Both MPHA and MPCs could accelerate wound healing. A 50% MPHA-OLE-50% MPCs-OLE blend was the most suitable for accelerating wound healing. The MPHA-OLE-MPCs-OLE blends studied in this work were shown to have the characteristics suitable for a spray patch, thus giving a second life to the waste products of olive growers.

Hydroxamate-Based Histone Deacetylase Inhibitors as Potential Mediators to Induce Dentine Regeneration by Human Dental Pulp Cell

Human dental pulp cells (hDPCs) have shown their plasticity when treated with the hydroxamate-based histone deacetylase (HDAC) inhibitor members, Trichostatin A (TSA), and suberoylanilide hydroxamic acid (SAHA). However, a comparison of their potency to stimulate odontoblast-like differentiation and mineralization has not been reported. The aim of our study was to confirm and compare these TSA and SAHA effects. Primary hDPCs cultured with/without various TSA or SAHA concentrations were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), ALP activity, alizarin red staining, and scratch wound healing assays. The inhibitory effect of TSA and SAHA on inhibiting the activity of HDAC was evaluated by HDAC activity assay. Odontoblast-related gene expression was determined using RT-qPCR. The MTT assay indicated that TSA or SAHA did not affect hDPC viability. TSA or SAHA treatment-induced odontoblast-like differentiation as evidenced by a significant increase in alkaline phosphatase activity and mineral deposition after 400 nM TSA or 1 μM SAHA treatment. A significant increase in nuclear factor I C, kruppel like factor 4, dentin matrix acidic phosphoprotein 1, dentin sialophosphoprotein, collagen type I alpha 1 chain, alkaline phosphatase (ALPL), integrin-binding sialoprotein, bone gamma-carboxyglutamate protein, vascular endothelial growth factor A, and cyclin-dependent kinase inhibitor 1A gene expression analyzed by RT-qPCR, at 24, 72 h, 7, and 10 days of treatment. The activity of HDAC in hDPCs culture was significantly inhibited after 72 h TSA and SAHA treatment. The scratch wound healing assay displayed enhanced cell migration at 72 h after TSA or SAHA treatment. Our findings demonstrated that TSA and SAHA have similar stimulatory effects in inducing HDPC odontogenic differentiation and mineralization and propose another potential use of TSA and SAHA to promote dentin regeneration.

The Potential of Sky Fruit as an Anti-Aging and Wound Healing Cosmeceutical Agent

There are many extrinsic factors that can contribute to the premature aging of the skin. In recent years, the demand for natural cosmetic from the general population has noticeable grow. Therefore, this research aimed to investigate the bioproperties of sky fruit (Swietenia macrophylla) seed extract that could help to inhibit premature skin aging. Firstly, the extract and its fractions were tested on HaCaT cells for their wound healing properties. The presence of sky fruit’s extract and its fractions on scratch wound significantly improved cellular proliferation, migration, and closure of the wound. These effects were distinctly observed following the treatment with S. macrophylla hexane fraction (SMHF) and S. macrophylla water fraction (SMWF). Our continuous research study revealed that SMWF had antioxidant properties, which might be one of the factors contributing to its emerging wound healing properties because antioxidants are known to act as suppressors of the inflammatory pathway and aid the transition towards cell proliferation. In addition, all samples had critical wavelengths that indicated that they were able to absorb the whole UVB range and some parts of the UVA wavelength. This suggested that S. macrophylla might contain potential photoprotective bioactive compounds, which could be developed into anti-UVB photoprotective sunscreens. Thus, this warrants further studies focusing on isolation and identifications of the bioactive compounds responsible for both its photoprotective and wound healing properties. A deeper study on mechanisms of the pathways that were affected by these compounds should be conducted as well to better understand this natural product and develop it into a potential cosmeceutical ingredient.

Modeling and Predicting the Cell Migration Properties from Scratch Wound Healing Assay on Cisplatin-Resistant Ovarian Cancer Cell Lines Using Artificial Neural Network

The study of artificial neural networks (ANN) has undergone a tremendous revolution in recent years, boosted by deep learning tools. The presence of a greater number of learning tools and their applications, in particular, favors this revolution. However, there is a significant need to deal with the issue of implementing a systematic method during the development phase of the ANN to increase its performance. A multilayer feedforward neural network (FNN) was proposed in this paper to predict the cell migration assay on cisplatin-sensitive and cisplatin-resistant (CisR) ovarian cancer (OC) cell lines via scratch wound healing assay. An FNN training algorithm model was generated using the MATLAB fitting function in a MATLAB script to accomplish this task. The input parameters were types of cell lines, times, and wound area, and outputs were relative wound area, percentage of wound closure, and wound healing speed. In addition, we tested and compared the initial accuracy of various supervised learning classifier and support vector regression (SVR) algorithms. The proposed ANN model achieved good agreement with the experimental data and minimized error between the estimated and experimental values. The conclusions drawn demonstrate that the developed ANN model is a useful, accurate, fast, and inexpensive method to predict cancerous cell migration characteristics evaluated via scratch wound healing assay.

Internalization of Neutrophil-Derived Microvesicles Modulates TNFα-Stimulated Proinflammatory Cytokine Production in Human Fibroblast-Like Synoviocytes

Neutrophil-derived microvesicles (NDMVs) have the potential to exert anti-inflammatory effects. Our study aimed to explore the effects of NDMVs on proinflammatory cytokines expressed by tumor necrosis factor α (TNFα)-stimulated fibroblast-like synoviocytes (FLS). FLS were isolated from the synovium of knee osteoarthritis (OA) patients undergoing surgery. NDMVs, isolated from TNFα-stimulated healthy neutrophils, were characterized by electron microscopy and nanoparticle tracking analysis. MTT and scratch wound healing assays were used to measure FLS viability and migration after treatment with NDMVs, while internalization of fluorescently labeled NDMVs was appraised by flow cytometry and confocal microscopy. Levels of proinflammatory cytokines in supernatants were quantified by the Bio-Plex system. Incubation of FLS with NDMVs at a vesicle/cell ratio of 100 resulted in a time-dependent uptake, with 35% of synoviocytes containing microvesicles over a 6–24 h time period, with no significant change in cell viability. TNFα stimulated the cytokine expression in FLS, and NDMVs down-regulated TNFα-induced expression of IL-5, IL-6, IL-8, MCP-1, IFNγ and MIP-1β. However, this down-regulation was selective, as NDMVs had no significant effects on TNFα-stimulated expression of IL-2 or IL-4. NDMVs were internalized by FLS to inhibit TNFα-stimulated broad-spectrum proinflammatory cytokine secretion. NDMVs, therefore, may exhibit an anti-inflammatory role in the regulation of the FLS function.

Combination of ferulic acid, ligustrazine and tetrahydropalmatine inhibits invasion and metastasis through MMP/TIMP signaling in endometriosis

Background The design of the combination of ferulic acid, ligustrazine and tetrahydropalmatine (FLT) is inspired by the Chinese herbal prescription Foshou San. Previous work has shown that FLT inhibited endometriosis growth in rat autograft models. However, the mechanism behind this is unclear. MMP/TIMP signaling is considered as the vital pathway of metastasis and invasion in endometriosis. In this study, we aim to disclose effects of FLT on MMP/TIMP signaling in invasion and metastasis during endometrial cells and xenograft endometriosis. Methods In vivo, effect of FLT on endometriosis was evaluated in a xenogeneic mice model. In vitro, cell viability assay was performed with an IC50 measurement of FLT in hEM15A and HEC1-B cells. The effect of FLT on invasion and metastasis was analyzed in scratch wound and transwell assay. Gene and protein expression of MMP/TIMP signaling were detected by qPCR and Western blotting. Results In xenograft endometriosis, FLT reduced ectopic volume without effect on weight. FLT inhibitory effects on cell growth exhibited a dose-dependent manner in hEM15A and HEC1-B cells. IC50s of FLT in hEM15A cells were 839.30 ± 121.11 or 483.53 ±156.91 μg·ml−1 after the treatment for 24 or 48 h, respectively. In HEC1-B cells, IC50 values of 24 or 48 h were 625.20 ± 59.52 or 250.30 ± 68.12 μg·ml−1. In addition, FLT significantly inhibited invasion and metastasis in scratch wound and transwell assay. Furthermore, FLT inactivated MMP/TIMP signaling with decreasing expression of MMP-2/9, and an enhancing expression of TIMP-1. Conclusions MMP/TIMP inactivation is a reasonable explanation for the inhibition of FLT on invasion and metastasis in endometriosis. This result reveals a potential mechanism on the role of FLT in endometriosis and may benefit for its further application.

TRPM2 promotes pancreatic cancer by PKC/MAPK pathway

The mechanism of pancreatic cancer (PA) is not fully understanded. In our last report, TRPM2 plays a promising role in pancreatic cancer. However, the mechanism of TRPM2 is still unknown in this dismal disease. This study was designed to investigate the role and mechanism of TRPM2 in pancreatic cancer. TRPM2 overexpressed and siRNA plasmid were created and transfected with pancreatic cancer cell line (BxPC-3) to construct the cell model. We employed CCK-8, Transwell, scratch wound, and nude mice tumor-bearing model to investigate the role of TRPM2 in pancreatic cancer. Besides, we collected the clinical data, tumor tissue sample (TT) and para-tumor sample (TP) from the pancreatic cancer patients treated in our hospital. We analyzed the mechanism of TRPM2 in pancreatic cancer by transcriptome analysis, western blot, and PCR. We blocked the downstream PKC/MEK pathway of TRPM2 to investigate the mechanism of TRPM2 in pancreatic cancer by CCK8, scratch wound healing, and transwell assays. Overexpressed TRPM2 could promote pancreatic cancer in proliferation, migration, and invasion ability in no matter the cell model or nude mice tumor-bearing model. TRPM2 level is highly negative correlated to the overall survival and progression-free survival time in PA patients, however, it is significantly increased in PA tissue as the tumor stage increases. The transcriptome analysis, GSEA analysis, western-blot, and PCR results indicate TRPM2 is highly correlated with PKC/MAPK pathways. The experiments of PKC/MEK inhibitors added to TRPM2 overexpressed BxPC-3 cell showed that significant inhibition of PA cells happened in CCK8, transwell, and wound-healing assay. TRPM2 may directly activate PKCα by calcium or indirectly activate PKCε and PKCδ by increased DAG in PA, which promote PA by downstream MAPK/MEK pathway activation.

Characterization, Antimicrobial Effects, and Cytocompatibility of a Root Canal Sealer Produced by Pozzolan Reaction between Calcium Hydroxide and Silica

This study aimed to evaluate a newly developed pozzolan-based bioceramic sealer (PZBS) regarding setting time, radiopacity, antibacterial effect, and cytocompatibility. The PZBS was manufactured by mixing calcium hydroxide and silica. The pozzolan reaction was verified by identification of calcium silicate hydrate (C-S-H) using X-ray diffraction analysis. The initial setting time and radiopacity were measured using the ISO 6876/2012 protocol in comparison with other commercially available calcium silicate (CS) sealers. The antibacterial effect of PZBS on biofilms cultured in the bovine root canal was evaluated by measurement of colony-forming units and volume of biofilms in comparison with other calcium hydroxide pastes. The morphological features of the biofilms were observed by scanning electron microscopy (SEM). The cytocompatibility of PZBS was assessed by the viability of bone marrow–derived mesenchymal stem cells and scratch wound healing rate in comparison with other CS sealers. The morphology of the cells cultured on the tested sealers was observed by SEM. The detection of the CS peak confirmed the formation of C-S-H. The initial setting time of PZBS was around 11 h, which was twice as long as the other tested sealers. The radiopacity of PZBS was 4.3 mm/Al, which satisfied the ISO criteria. The antibacterial effect and cytocompatibility of PZBS were comparable to those of the commercially available intracanal medicaments and CS endodontic sealers, respectively. The PZBS has the potential to be used for root canal obturation, and is expected to exert a favorable antibacterial effect.