XTT Test Research Articles

Investigation of the Anti-Leishmanial Effects of Prangos ferulacea and Ferula orientalis Extracts Collected from Şırnak Province Against Leishmania tropica Isolated in Turkey

Leishmaniasis is a vector-borne disease that is caused by the protozoa of Leishmania genus. Leishmaniasis is endemic in tropical, subtropical, and large areas of the Mediterranean basin, and covers a total of 98 countries worldwide. It is estimated, according to the World Health Organization (WHO) data, that approximately 350 million people are at risk in these areas, and approximately 12 million people are infected. Increased drug resistance has been documented lately, in the treatment of leishmaniasis which causes almost 1.2 million new cases annually. Thus, interest in plant-derived active substances has increased in recent years, and new anti-leishmanial agents are investigated with in vitro studies. The aim of the present study was to investigate the anti-leishmanial effects of Prangos ferulacea and Ferula orientalis plant extracts collected from the rural areas of Şırnak province against Leishmania tropica. The water, chloroform, and ethanol extracts of the roots, stems, and fruits of P.ferulaceae and F.orientalis plants were obtained, and the cytotoxic activity tests of the extracts were performed. L.tropica isolate obtained from the Parasite Bank in Manisa Celal Bayar University in Turkey (MHOM/TR/2012/CBCL-LT) was grown on NNN and RPMI 1640 broth medium. The cytotoxicity of each extract on the L.tropica isolate was evaluated with the XTT test. Amphotericin B (AmpB) was used as the positive control, and the IC50 values were determined. The lowest IC50 values of the plant extracts were found to be as follows: P.ferulaceae root chloroform extract 36 µg/ml and fruit chloroform extract 20 µg/ml, F.orientalis root ethanol extract 2.5 µg/ml, and fruit ethanol extract 48 µg/ml, stem chloroform extract 24 µg/ml, and fruit chloroform extract 3.1 µg/ml. It was also determined in our study that only P.ferulaceae root ethanol extract showed cytotoxic activity on the WI-38 fetal lung fibroblast cell line at 65.19 µg/ml at 72 hours. This is the first study that assessed the anti-leishmanial activities of P.ferulaceae and F.orientalis plants that grow in high altitude areas of our country. It was determined that P.ferulaceae root ethanol extract and fruit chloroform extract had the lowest IC50 values among the 18 plant extracts that we examined for their anti-leishmanial activities. The outcomes of this study will be useful in further studies for the determination of active compounds in P.ferulaceae and F.orientalis plant extracts.

Effects of Adipose-Derived Stem Cells and Their Conditioned Medium in a Human Ex Vivo Wound Model.

Adult stem cells have been extensively investigated for tissue repair therapies. Adipose-derived stem cells (ASCs) were shown to improve wound healing by promoting re-epithelialization and vascularization as well as modulating the inflammatory immune response. In this study, we used ex vivo human skin cultured in a six-well plate with trans-well inserts as a model for superficial wounds. Standardized wounds were created and treated with allogeneic ASCs, ASCs conditioned medium (ASC-CM), or cell culture medium (DMEM) supplemented with fetal calf serum (FCS). Skin viability (XTT test), histology (hematoxylin and eosin, H and E), β-catenin expression as well as inflammatory mediators and growth factors were monitored over 12 days of skin culture. We observed only a moderate time-dependent decrease in skin metabolic activity while skin morphology was preserved, and re-epithelialization occurred at the wound edges. An increase in β-catenin expression was observed in the newly formed epithelia, especially in the samples treated with ASC-CM. In general, increased growth factors and inflammatory mediators, e.g., hepatocytes growth factor (HGF), platelet-derived growth factor subunit AA (PDGF-AA), IL-1α, IL-7, TNF-α, and IL-10, were observed over the incubation time. Interestingly, different expression profiles were observed for the different treatments. Samples treated with ASC-CM significantly increased the levels of inflammatory cytokines and PDGF-AA with respect to control, whereas the treatment with ASCs in DMEM with 10% FCS resulted in significantly increased levels of fibroblast growth factor-basic (FGF-basic) and moderate increases of immunomodulatory cytokines. These results confirm that the wound microenvironment can influence the type of mediators secreted by ASCs and the mode as to how they improve the wound healing process. Comparative investigations with pre-activated ASCs will elucidate further aspects of the wound healing mechanism and improve the protocols of ACS application.

Influence of Different Nanometals Implemented in PMMA Bone Cement on Biological and Mechanical Properties.

Cemented arthroplasty is a common process to fix prostheses when a patient becomes older and his/her bone quality deteriorates. The applied cements are biocompatible, can transfer loads, and dampen vibrations, but do not provide antibacterial protection. The present work is aimed at the development of cement with antibacterial effectivity achieved with the implementation of nanoparticles of different metals. The powders of Ag, Cu with particles size in a range of 10–30 nm (Cu10) and 70–100 nm (Cu70), AgCu, and Ni were added to PMMA cement. Their influence on compression strength, wettability, and antibacterial properties of cement was assessed. The surface topography of samples was examined with biological and scanning electron microscopy. The mechanical properties were determined by compression tests. A contact angle was observed with a goniometer. The biological tests included an assessment of cytotoxicity (XTT test on human cells Saos-2 line) and bacteria viability exposure (6 months). The cements with Ag and Cu nanopowders were free of bacteria. For AgCu and Ni nanoparticles, the bacterial solution became denser over time and, after 6 months, the bacteria clustered into conglomerates, creating a biofilm. All metal powders in their native form in direct contact reduce the number of eukaryotic cells. Cell viability is the least limited by Ag and Cu particles of smaller size. All samples demonstrated hydrophobic nature in the wettability test. The mechanical strength was not significantly affected by the additions of metal powders. The nanometal particles incorporated in PMMA-based bone cement can introduce long-term resistance against bacteria, not resulting in any serious deterioration of compression strength.

Synthesis, structural characterization and anticancer activity of 3-(3,5-dinitrobenzoyl)-1H-imidazolidine-2-thione

The present study describes the synthesis, characterization and in vitro anticancer potential evaluation of a novel 3-(3,5-dinitrobenzoyl)-1 H -imidazolidine-2-thione compound. In the first step, structure analysis of the compound has been elucidated by NMR and FT-IR techniques. Theoretical NMR, FT-IR spectra, HOMO and LUMO orbital energies and MEP analyses have been used to determine the activity of the molecule by Gaussian 09 package program using DFT techniques. Furthermore, docking calculations have been performed for the BRCA2 (PDB Code: 3EU7) active side to foresee the possible mechanism of action of the synthesized compound. In the second step of the study, the synthesized compound has been screened for its potential in vitro anticancer activity against MCF-7 human breast cancer cell line using the cell proliferation (XTT), apoptosis, cell cycle arrest and intracellular ROS production assays. The results of XTT test revealed significant dereases in MCF-7 cell viability with the 24h IC 50 value of 7.57 µM. It has been also observed that treatment with the IC 50 concentration of the compound can significantly induce apoptosis, intracellular ROS production and G2/M phase arrest in MCF-7 cells.

Antimicrobial potential of plant extracts from the Brazilian Cerrado.

Bacteria are related do different oral diseases, such as dental caries and periodontal disease. Therefore, the control or/and eradication of microorganisms and their by-products is primordial for the success of their treatment. An alternative for decrease bacterial load is the use of plant extracts used in popular medicine. The cytotoxicity and antimicrobial action of extracts of Cariniana rubra Gardiner ex Miers, Senna martiniana, Anadenanthera colubrina (Vell.) Brenan and Spiranthera odoratissima St. Hil. against strains of Streptococcus mutans, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Aggregatibacter actinomyces- tencomitans and Candida albicans were investigated. Cytotoxicity was assessed at concentrations of 1, 10, 40, 80, 100 and 1000 μg/mL by means of the MTT test and compared to a control group with untreated cells. Those with acceptable cytotoxicity had the antimicrobial action measured by the XTT test. As a positive control, sodium hypochlorite was used. Cariniana rubra Gardiner ex Miers had the highest citototoxicity results while Spiranthera odoratissima St. Hil. had the best results, but all extracts showed acceptable cytotoxicity at different concentrations. The plant extracts showed higher activity against A. actinomycetencomitans: Anadenanthera columbrina (Vell.) Brenan (80.52%) at 40 μg/mL, Spiranthera odoratissima St. Hil (78.48%) in 1 μg/mL, Senna martiniana (73.28%) in the concentration of 40 μg/mL and Cariniana rubra Gardiner ex Miers (70.50%) in 10 μg/mL. All extracts analyzed showed acceptable cytotoxicity at different concentrations and were promising for inhibition of the pathogenic microorganisms studied.

Anti-Inflammatory Effect of an O-2-Substituted (1-3)-β-D-Glucan Produced by Pediococcus parvulus 2.6 in a Caco-2 PMA-THP-1 Co-Culture Model.

Bacterial β-glucans are exopolysaccharides (EPSs), which can protect bacteria or cooperate in biofilm formation or in bacterial cell adhesion. Pediococcus parvulus 2.6 is a lactic acid bacterium that produces an O-2-substituted (1-3)-β-D-glucan. The structural similarity of this EPS to active compounds such as laminarin, together with its ability to modulate the immune system and to adhere in vitro to human enterocytes, led us to investigate, in comparison with laminarin, its potential as an immunomodulator of in vitro co-cultured Caco-2 and PMA-THP-1 cells. O-2-substituted (1-3)-β-D-glucan synthesized by the GTF glycosyl transferase of Pediococcus parvulus 2.6 or that by Lactococcus lactis NZ9000[pGTF] were purified and used in this study. The XTT tests revealed that all β-glucans were non-toxic for both cell lines and activated PMA-THP-1 cells’ metabolisms. The O-2-substituted (1-3)-β-D-glucan modulated production and expression of IL-8 and the IL-10 in Caco-2 and PMA-THP-1 cells. Laminarin also modulated cytokine production by diminishing TNF-α in Caco-2 cells and IL-8 in PMA-THP-1. All these features could be considered with the aim to produce function foods, supplemented with laminarin or with another novel β-glucan-producing strain, in order to ameliorate an individual’s immune system response toward pathogens or to control mild side effects in remission patients affected by inflammatory bowel diseases.

Synthesis, structural characterization and evaluation of anticancer activity of polymeric silver(I) complexes based on niflumic acid/naproxen and picoline derivatives

Two polymeric mixed-ligand silver(I) complexes, [Ag(µ-nif)(4-pic)]n (1) and [Ag4(µ-nap)4(2-pic)2]n (2), where Hnif: niflumic acid, Hnap: naproxen, 4-pic: 4-picoline and 2-pic: 2-picoline, were synthesized and characterized by single-crystal X-ray diffraction, FT-IR and 1H NMR spectroscopies, elemental and thermal analysis techniques. In 1, nif ligand behaves as a µ3-N,O,O' bridge connecting three silver(I) ions and each silver(I) ion exhibits a seesaw geometry. In 2, nap ligands act as a µ-carboxylato-(O,O') bridge between two silver(I) ions and µ3-carboxylato-(O,O,O') bridge linking three silver(I) ions. Each silver(I) ion occurs in trigonal geometry. To uncover the anticancer properties of the complexes, first, their cytotoxic abilities were tested against three cancer cell lines, HT-29 (human colorectal adenocarcinoma), A-549 (human lung carcinoma), and MDA-MB-453 (human breast adenocarcinoma), and normal 3T3-L1 (mouse fibroblast) cell line by XTT tests. Afterwards, the anticancer mechanism of the complexes was tried to be elucidated through the flow cytometry analyzes. XTT assay results showed that the complexes exhibited the highest cytotoxic activity and selectivity on HT-29 cells among all the cell lines. Flow cytometry analyses revealed that both complexes induced the apoptosis through the mitochondrial membrane depolarization and activation of the caspase cascade in HT-29 cells.

Intranuclear cell uptake and toxicity of titanium dioxide and zirconia particles as well as bacterial adhesion on dental titanium- and zirconia-implants

ObjectivePrevious studies have shown that particles can be released from dental titanium (Ti)- and zirconia (ZrO2)-implants. Titanium dioxide (TiO2)- and ZrO2-particles were compared regarding their toxicity and intranuclear cell uptake as well as the adhesion of various anaerobic bacteria on Ti- and ZrO2-implants. MethodsCyto- and genotoxicity of TiO2-microparticles (TiO2-MPs) and TiO2-nanoparticles (TiO2-NPs) in periodontal ligament (PDL)-hTERT cells were determined with XTT test and DNA damage with comet assay. Particle sizes of TiO2- and ZrO2-particles were measured with scanning electron microscope. Intranuclear uptake in PDL-hTERT cells was determined with laser scanning confocal microscopy. Adhesions of relevant anaerobic mouth bacteria Porphyromonas gingivalis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans on Ti- and ZrO2-implants were investigated by cultivation and counting bacterial colonies. ResultsParticle size measurements revealed that 99% of the TiO2-NPs had a size below 100 nm and 88% of the TiO2-MPs sizes were between 50 and 200 nm. Following EC50 values were found for particles (mg/l): 92 (TiO2-MPs) and 15 (TiO2-NPs). A significant increase in olive tail moment (OTM) was found for TiO2-NPs at a concentration of 1/10 EC50. TiO2- and ZrO2-NPs had a higher intranuclear cell uptake efficiency, compared to corresponding TiO2- and ZrO2-MPs. All investigated particles could be detected in cell nucleus. Adhesion of all investigated bacterial species was significantly higher on Ti-implants, compared to ZrO2-implants. ConclusionTi usually develops an oxide layer (TiO2). Particles released from Ti-implants should be TiO2-particles or Ti-particles coated with a TiO2-layer. Toxicity of released Ti-particles depends on their oxidation state and on their size (NP or MP). Particularly, NPs were more cyto- and genotoxic compared to the corresponding MPs. TiO2- and ZrO2-NPs showed a significant increase in the intranuclear cell uptake ratio at higher exposure concentration, compared to lower concentrations and consequently might lead to a higher potential of DNA damage. Adhesion of bacteria to ZrO2-implants is reduced, compared to Ti-implants. Therefore, ZrO2-implants might contribute to reduced biological complications (e.g. periimplantitis).

Erianin, a promising agent in the treatment of glioblastoma multiforme triggers apoptosis in U373 and A172 glioblastoma cells

Glioblastoma is an aggressive, common and deadly primary intracranial brain tumor in adults. The antitumor activity of erianin, a dibenzyl compound found in Dendrobium chrysotoxum Lindl. extract, has not been previously demonstrated in glioblastoma. We investigated the anticancer activity and underlying mechanisms of erianin in human U373 and A172 glioma cells. The effects of erianin on cell viability, apoptosis, migration and invasion were estimated by the XTT test, the reverse transcription-polymerase chain reaction (RT-PCR), annexin V staining assay protocol for apoptosis, wound healing assay, and Matrigel? invasion chamber, respectively. The effective amounts of erianin in U373 and A172 cells were 16 and 64 ?M at 48 h, respectively. Erianin also significantly induced apoptosis by inhibiting B-cell lymphoma 2 (Bcl-2), caspase-8, caspase-9 and tumor necrosis factor receptor type 1-associated DEATH domain protein (TRADD), and activation of caspase-3 and BH3 interacting domain death agonist (BID) gene expression. In addition, erianin significantly increased the number of apoptotic cells in U373 and A172 cells and significantly decreased invasion and migration in U373 and A172 cells. Taken together, our results suggest that erianin may be a new therapeutic anticancer drug component with a potent apoptotic effect and a potential for treating glioblastoma.

Mevastatin in colon cancer by spectroscopic and microscopic methods – Raman imaging and AFM studies

One of the most important areas of medical science is oncology, which is responsible for both the diagnostics and treatment of cancer diseases. Simultaneously one of the main challenges of oncology is the development of modern drugs effective in the fight against cancer. Statins are a group of biologically active compounds with the activity of 3-hydroxy-3-methyl glutaryl-CoA reductase inhibitors, an enzyme catalyzing the reduction of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) to mevalonic acid. By acting on this enzyme, statins inhibit the endogenous cholesterol synthesis which in turn causes the reduction of its systemic concentrations. However, in vitro and in vivo studies confirm also the cytostatic and cytotoxic effects of statins against various types of cancer cells including colon cancer. In the presented studies the influence of mevastatin on cancerous colon cells CaCo-2 by Raman spectroscopy and imaging is discussed and compared with biochemistry characteristic for normal colon cells CCD-18Co. Based on vibrational features of colon cells: normal cells CCD-18Co, cancerous cells CaCo-2 and cancerous cells CaCo-2 treated by mevastatin in different concentrations and incubation times we have confirmed the influence of this statin on biochemistry composition of cancerous human colon cells. Moreover, the spectroscopic results for colon normal cells and cancerous cells based on data typical for nucleic acids, proteins, lipids have been compared. The cytotoxisity of mevastatin was determined by using XTT tests.

Lamivudine-conjugated and efavirenz-loaded G2 dendrimers: Novel anti-retroviral nano drug delivery systems.

Infection with human immunodeficiency virus (HIV)‐1 causes immunological disorders and death worldwide which needs to be further assisted by novel anti‐retroviral drug delivery systems. Consequently, finding newer anti‐retroviral pharmaceuticals by using biocompatible, biodegradable nanomaterials comprising a nanoparticle as core and a therapeutic agent is of high global interest. In this experiment, a second generation of a negatively charged nano‐biopolymer linear globular G2 dendrimer was carefully conjugated and loaded with well‐known anti‐HIV drugs lamivudine and efavirenz, respectively. They were characterised by a variety of analytical methods such as Zetasizer, Fourier‐transform infrared spectroscopy, elemental analysis and liquid chromatography‐mass spectroscopy. Additionally, conjugated lamivudine and loaded efazirenz with globular PEGylated G2 dendrimer were tested on an HEK293 T cell infected by single‐cycle replicable HIV‐1 virion and evaluated using XTT test and HIV‐1 P24 protein load. The results showed that lamivudine‐conjugated G2 significantly decreased retroviral activity without any cell toxicity. This effect was more or less observed by efavirenz‐loaded G2. These nano‐constructs are strongly suggested for further in vivo anti‐HIV assays.

Cytotoxicity and genotoxicity of salicylate- and calcium silicate-based root canal sealers on primer human periodontal ligament fibroblasts.

The aim of this study was to evaluate the biocompatibility of epoxy-resin-based AHPlus, salicylate-based MTA-Fillapex and calcium silicate-based iRootSP root canal sealers. Cytotoxicity was assessed by XTT test. The extracts from sealers of different setting times were serially diluted. Cell viability was calculated as the percentage of the control group (100%). The optimal concentration of each sealer was used at genotoxicity test, and micronuclei formations were detected. Statistical analyses were done by using Kruskal-Wallis and Dunn post hoc test with Bonferroni correction. AHPlus and MTA-Fillapex showed the lowest percentage of cell viability at higher concentrations (1:1, 1:2, 1:4), especially at first 12 h. iRootSP showed higher viability at all concentrations and times than AHPlus and MTA-Fillapex. At genotoxicity assay, AHPlus increased the number of micronuclei. MTA-Fillapex slightly induced micronucleus formation (not significant) and iRootSP was not increased. In conclusion, calcium silicate-based iRootSP had lowest cytotoxic and genotoxic potential and can be considered as a highly biocompatible material.

Evaluation the Effects of Helichrysum plicatum Subsp. pseudoplicatum on an In-Vitro Wound Model Using Human Dermal Fibroblast Cells.

Wound is tissue damage that occurs in the skin. Helichrysum species (Altınotu) are rich in phenolic compounds used in traditional medicine for wound healing. The main component in their flower head (capitulum) is phenolic compounds. The present study investigates the proliferative, oxidative stress, and wound healing properties of the methanolic extract of Helichrysum plicatum subsp. pseudoplicatum capitulum on a human dermal fibroblast (HDF) cell line in this study. H plicatum subsp. pseudoplicatum capitulums were collected in Erzurum, Turkey (altitude 1950 m), dried, pulverized, and extracted with methanol. Firstly, total phenolic contents were determined and secondly, the proliferative effect, oxidative stress activities, and wound healing effects on HDF cells were evaluated by the cell proliferation kit (XTT) test, total antioxidant status (TAS), and total oxidant status (TOS) commercial kits, and the scratch experiment by taking microscopic images of the cells at 0, 12, 18, and 24 h, respectively. Total phenolic content was found to be 142.00 ± 0.73 mg gallic acid equivalent per gram (GAE/g) extract. The capitulum extract has a proliferative effect at 0.5 to 10 µg/mL concentrations according to the XTT test results. It was observed that TAS levels significantly increased in the plant extract at the concentration ranges 1 to 10 µg/mL (P < .01). About 1 to 5 µg/mL plant extract started to increase cell migration at the 12 h and significantly closed the wound area at the 24 h. At the doses between 1 to 5 μg/mL, it has the most substantial effect on both cell viability and antioxidant effect, and wound healing was found to be in this concentration range. These findings suggested that the H plicatum subsp. pseudoplicatum capitulum is a valuable source of phenolic content with important antioxidant activity at wound healing and it was concluded that the capitulum extract accelerates wound healing by increasing cell migration in low doses.

Pharmaceutical Drug Metformin and MCL1 Inhibitor S63845 Exhibit Anticancer Activity in Myeloid Leukemia Cells via Redox Remodeling.

Metabolic landscape and sensitivity to apoptosis induction play a crucial role in acute myeloid leukemia (AML) resistance. Therefore, we investigated the effect of metformin, a medication that also acts as an inhibitor of oxidative phosphorylation (OXPHOS), and MCL-1 inhibitor S63845 in AML cell lines NB4, KG1 and chemoresistant KG1A cells. The impact of compounds was evaluated using fluorescence-based metabolic flux analysis, assessment of mitochondrial Δψ and cellular ROS, trypan blue exclusion, Annexin V-PI and XTT tests for cell death and cytotoxicity estimations, also RT-qPCR and Western blot for gene and protein expression. Treatment with metformin resulted in significant downregulation of OXPHOS; however, increase in glycolysis was observed in NB4 and KG1A cells. In contrast, treatment with S63845 slightly increased the rate of OXPHOS in KG1 and KG1A cells, although it profoundly diminished the rate of glycolysis. Generally, combined treatment had stronger inhibitory effects on cellular metabolism and ATP levels. Furthermore, results revealed that treatment with metformin, S63845 and their combinations induced apoptosis in AML cells. In addition, level of apoptotic cell death correlated with cellular ROS induction, as well as with downregulation of tumor suppressor protein MYC. In summary, we show that modulation of redox-stress could have a potential anticancer activity in AML cells.

Induced Biological Response in Contact with Ag-and Cu-Doped Carbon Coatings for Potential Orthopedic Applications.

Silver and copper as additives of various biomaterials have been reported as the potential solutions for biomedicine applications, mostly because of inducing bactericidal effects. The application of those admixtures in diamond-like carbon (DLC) coatings may be desirable for orthopedic implants. In the present manuscript, the biological effect of coatings with up to about 7 at.% and 14 at.% of, respectively, Cu and Ag is compared. The morphology, chemical structure, and composition of films deposited on AISI 316LVM and Ti6Al7Nb is characterized. The live/dead analysis conducted with Escherichia coli shows a higher bactericidal potential of silver than copper. Although the Cu-doped coatings can positively affect the proliferation of Saos-2 and EA.hy926 cell lines, the results of XTT test are on the verge of 70% of viability. Biological effect of silver on EA.hy926 cell lines is negative but that admixture ensures high proliferation of osteoblasts in contact with coatings deposited on titanium alloy (over 20% better than for substrate material). In that case, the viability is reaching about 85% for Ag-doped coatings on AISI 316LVM and 75% on Ti6Al7Nb. The results indicate that for the sake of bactericidal coatings that may promote osteointegration, the candidates are DLC with silver content no higher than 10 at.%.

Effect of Elaeagnus umbellata (Thunb.) fruit extract on H2O2-induced oxidative and inflammatory responses in normal fibroblast cells.

BackgroundElaeagnus umbellata is a plant commonly used in traditional Asian medicine for its many health benefits and strong antioxidative activity. Its therapeutic potential is believed to be connected to its effect on fibroblasts. This study aimed to investigate E. umbellata methanol-acetone extract’s (EUE) defense against hydrogen peroxide (H2O2)-induced fibroblast damage.MethodsBecause the main biologically active compounds of E. umbellata are water-insoluble, we evaluated the effects of methanol-acetone fruit extracts using liquid chromatography (for ascorbic acid and beta-carotene) and spectrophotometry (for lycopene and total phenolics). The extract’s antioxidative activity was measured using DPPH radical inhibition, and EUE’s effect on human fibroblasts was also evaluated. We assessed the metabolic activity and apoptosis of HFFF-2 fibroblasts exposed to EUE and/or H2O2using the XTT test and flow cytometry, respectively. Superoxide dismutase activity and reactive oxygen species (ROS) production were evaluated using colorimetric and fluorometric assays, respectively. We measured pro-inflammatory cytokine (MIF, fractalkine, MCP-4, BLC, GCP-2, NAP-2, Eotaxin-2, and Eotaxin-3) expression in HFFF-2 cells using immunocytochemistry.ResultThe extract increased HFFF-2 cell proliferation and reduced cell death caused by H2O2-induced stress. H2O2-treated fibroblasts had greater ROS production than cells treated with both H2O2 and EUE. Additionally, the group treated with H2O2 alone showed higher pro-inflammatory cytokine (MIF, MCP-4, NAP-2, Eotaxin-2, and Eotaxin-3) expression.ConclusionEUE protected human fibroblasts from H2O2-induced oxidative stress and reduced the fibroblast-mediated inflammatory response triggered by ROS.

Toxicity assessment of &lt;em&gt;Gentiana lutea&lt;/em&gt; L. root extract and its monoterpene compounds

Root of Gentiana lutea commercially available as gentian root, a natural antidote for different types of poisons, possess antioxidative, immunomodulatory, cytoprotective and anti-inflammatory, and adverse, genotoxic and mutagenic effects. It has monoterpenes loganic acid, swertiamarin, gentiopicroside and sweroside as most abundant constituents. In this study, we assessed the toxicity of monoterpenes’ reactive molecular fragments using in silico prediction by VEGA-QSAR platform. Further, we compared the data obtained with in vitro geno- and cyto- toxicity testing of the above monoterpenes and the G. lutea root extract (GE), on human primary unstimulated and mitogen-stimulated peripheral blood mononuclear cells (PBMCs). Viability was assessed by TB and XTT tests after 48 h treatmen. DNA damage was evaluated by alkaline comet assay on unstimulated cells, whereas cytokinesis-block micronucleus assay was employed on mitogen-stimulated PBMCs. Stability of compounds throughout treatment was monitored by UPLC. The observed in vitro results had highest compliance with in silico IRFMN/ISSCAN-CGX prediction model. Compounds showed high stability during experiment while treatment with single compounds reduced number of viable cells and increased DNA damage. GE treatment had toxic impact on unstimulated PBMCs but no significant genotoxic influence on mitogen-stimulated PBMCs. In summary, the mild GE effect suggests that the complexity of crude GE extract chemical composition may attenuate the toxicity of the tested monoterpenes loganic acid, swertiamarin, gentiopicroside and sweroside.

INTERLEUKIN -22 MEDIATED INHIBITION OF H2O2-INDUCED APOPTOSIS IN HK-2 CELLS THROUGH SUPPRESSION OF ER STRESS

Introduction: Oxidative stress plays a key role in ischemic-reperfusion (IR) injury. Recent studies have shown that endoplasmic reticulum (ER) stress in the tubular cells of kidneys is associated with IR injury. However, there is little information about the anti-apoptotic cytokines that help overcome ER stress in HK-2 cells. In this study, we investigated the effect of interleukin-22 on H2O2-induced apoptosis and ER stress in HK-2 cells. Methods: For this study, we used the HK-2 cell line to examine interleukin-22 toxicity and its therapeutic concentrations using XTT test. H2O2-induced injury is well known as an oxidative stress model for acute kidney injury. We stimulated HK-2 cells with H2O2, which induced apoptosis. The expression of ER stress marker proteins, such as glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), phosphorylated eukaryotic translation initiation factor 2 alpha (p-eIF2α), and C/EBP homologous protein (CHOP), were evaluated by western blot analysis. Results: In HK-2 cells, H2O2 increased the levels of apoptotic proteins that cleaved PARP in a dose-dependent manner at 4 h. ATF4, p-eIF2α, and C/EBP homologous protein (CHOP) were also increased by H2O2 stimulation. Interleukin-22 pre-treatment prevented H2O2-induced apoptosis and decreased expression of these ER stress proteins in HK-2 cells as observed through western blot analysis. Interleukin-22 blocking antibody abolished interleukin-22 induced protective effects on HK-2 cells. Conclusions: Our results suggest that interleukin-22 prevents H2O2-induced apoptosis in HK-2 cells through suppression of ER stress. Therefore, interleukin-22 can potentially be an anti-apoptotic cytokine for IR injury.

Techniques for obtaining dermal extracellular matrix scaffold

Despite advancements in modern surgery in the treatment of cutaneous injuries, the search for new methods of ensuring faster and more effective wound healing appears especially urgent today. Tissue engineering is undoubtedly of interest when it comes to developing such technologies. Objective : to determine the optimal protocol for obtaining a decellularized dermal matrix scaffold for subsequent development of tissue-engineered skin. Materials and methods . One Landrace piglet was used as the experimental animal. After preliminary skin treatment with dermatome, 0.3 cm thick samples were taken. Two decellularization protocols were considered: protocol No. 1 was based on the use of Triton X-100 and deoxycholate, protocol No. 2 was only based on deoxycholate. There were 5 processing cycles in total for the 2 protocols. After treatment, acellular matrix scaffolds were examined through histological analysis and quantitative determination of DNA concentration. Next, static recellarization of the matrix scaffolds was carried out with porcine dermal fibroblasts. After that, the matrix scaffolds were tested for cytotoxicity using XTT test and differential staining test to differentiate between live and dead cells. Results . Comparative analysis of the two protocols for porcine dermis decellularization showed that both protocols effectively remove cells and nuclear material, while maintaining the architectonics of the intercellular substance intact, since fibrous structures are not destroyed. But when assessing the biocompatibility of matrix scaffolds based on analysis of cell viability according to data obtained from XTT test and cell–matrix adhesion, the matrix scaffold processed under protocol No. 1, shows advantages. Conclusion . In this study, a decellularization protocol based on Triton X-100 and deoxycholate was noted. The results obtained mark the first stage towards developing a tissue-engineered skin.

The effects of endosulfan on the expression levels of DNA damage and apoptotic genes in HT22 cells: First preliminary study

Aim: The aim of this study was to determine the IC50 dose of endosulfan in the hippocampal HT22 cell line, and also to elucidate the effect of expression of DNA-PK, Bax, Bcl2 and Casp-3 genes involved in DNA repair and apoptotic pathway.Material and Methods: Cytotoxic effect of endosulfan and IC50 dose were determined by using XTT method after 24 hours of culture by applying endosulfan at 5 different concentrations (10, 25, 50, 75 and 100 μM) to HT22 cell lines.HT22 cells were then seeded into 6 sterile plates and treated with Endosulfan at IC50 for 12 hours. The expression changes of DNA-Pk, Bax, Bcl2 and Casp-3 genes, after total RNA isolation and cDNA formation, were determined by RT-PCR. Expression levels were calculated using the comparative 2-ΔΔCt method.Results: After 24 hours of endosulfan treatment at different doses in HT22 cell lines, a significant loss of viability was observed in all endosulfan treated groups. It was determined by XTT test, that the IC50 dose of endosulfan was 50 μM in 24 hours treatment. After 12 hours administration of IC50 endosulfan dose in HT22 cells following the examinations of DNA-PK and some apoptotic genes we observed different amounts of increases in expression as follows; 5-fold for DNA-PK, 18-fold for Bax, and 4-fold for Casp-3. On the other hand, approximately 2-fold decrease was detected in Bcl-2 gene.Conclusion: The IC50 dose of 24-hour endosulfan administration in HT22 cell lines was found to be 50 μM. Expression changes in the proapoptotic and antiapoptotic genes have shown that apoptosis is induced in endosulfan-administrated cells. In addition, the increase in DNA-PK gene expression suggests that endosulfan causes DNA damage in cells and triggers DNA repair mechanisms