Effect of boric acid application on antioxidant enzymes activity and gene expression in Safflower (Carthamus tinctorius L) cultivars

Abstract: Boron is one of the most important micronutrients for plants. Plants may suffer from deficiency or with boron toxicity. Boron plays a role in significant physiological and biochemical events in plants such as synthesis of the cell wall, membrane integrity, antioxidation, transport of photosynthesis products to other organs of the plant. The enzyme activities of ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD) in three different safflower cultivars (Balcı, Dinçer and Remzibey) subjected to different boric acid concentrations (0, 5, 10, 15 mM) were measured spectrophotometrically, and the changes in the expression levels of the genes that encode these enzymes were obtained by quantitative RT-qPCR. When both the spectrophotometric measurements and the mRNA values were evaluated together, both the activity and mRNA values of APX and GR enzymes were found to be the highest in the Dinçer cultivar among the varieties treated with 15 mM boric acid, while the lowest values of these enzymes were determined in the Remzibey cultivar. According to the RT-qPCR results, the lowest SOD and CAT values were determined in Remzibey. The Dinçer cultivar was found to have the highest antioxidant capacity (APX, GR) to cope with oxidative stress caused by boric acid application at high concentrations. The sensitive Remzibey cultivar was found to have the lowest antioxidant capacity to cope with such oxidative stress. Balcı was found to be closer to Dinçer than to Remzibey in terms of boron tolerance. As a result, the boron-sensitive cultivar had low antioxidant activity.

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos / Uso da melatonina como inibidor do processo apoptotico para a criopreservação de embriões de zebrafish (Danio rerio)

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.

Gene expression and apoptosis response in hepatocellular carcinoma cells induced by biocompatible polymer/magnetic nanoparticles containing 5-fluorouracil

BACKGROUND: Super-paramagnetic iron oxide nanoparticles (SPION) contain a chemotherapeutic drug and are regarded as a promising technique for improving targeted delivery into cancer cells. RESULTS: In this study, the fabrication of 5-fluorouracil (5-FU) was investigated with loaded Dextran (DEXSPION) using the co-precipitation technique and conjugated by folate (FA). These nanoparticles (NPs) were employed as carriers and anticancer compounds against liver cancer cells in vitro. Structural, magnetic, morphological characterization, size, and drug loading activities of the obtained FA-DEX-5-FUSPION NPs were checked using FTIR, VSM, FESEM, TEM, DLS, and zeta potential techniques. The cellular toxicity effect of FA-DEX-5-FU-SPION NPs was evaluated using the MTT test on liver cancer (SNU-423) and healthy cells (LO2). Furthermore, the apoptosis measurement and the expression levels of NF-1, Her-2/neu, c-Raf-1, and Wnt-1 genes were evaluated post-treatment using flow cytometry and RT-PCR, respectively. The obtained NPs were spherical with a suitable dispersity without noticeable aggregation. The size of the NPs, polydispersity, and zeta were 74 ± 13 nm, 0.080 and 45 mV, respectively. The results of the encapsulation efficiency of the nano-compound showed highly colloidal stability and proper drug maintenance. The results indicated that FA-DEX-5-FU-SPION demonstrated a sustained release profile of 5-FU in both phosphate and citrate buffer solutions separately, with higher cytotoxicity against SNU-423 cells than against other cells types. These findings suggest that FA-DEX-SPION NPs exert synergistic effects for targeting intracellular delivery of 5-FU, apoptosis induction, and gene expression stimulation. CONCLUSIONS: The findings proved that FA-DEX-5-FU-SPION presented remarkable antitumor properties; no adverse subsequences were revealed against normal cells.

Effects of maternal exposure to arsenic on social behavior and related gene expression in F2 male mice

BACKGROUND@#Arsenic is a developmental neurotoxicant. It means that its neurotoxic effect could occur in offspring by maternal arsenic exposure. Our previous study showed that developmental arsenic exposure impaired social behavior and serotonergic system in C3H adult male mice. These effects might affect the next generation with no direct exposure to arsenic. This study aimed to detect the social behavior and related gene expression changes in F2 male mice born to gestationally arsenite-exposed F1 mice.@*METHODS@#Pregnant C3H/HeN mice (F0) were given free access to tap water (control mice) or tap water containing 85 ppm sodium arsenite from days 8 to 18 of gestation. Arsenite was not given to F1 or F2 mice. The F2 mice were generated by mating among control F1 males and females, and arsenite-F1 males and females at the age of 10 weeks. At 41 weeks and 74 weeks of age respectively, F2 males were used for the assessment of social behavior by a three-chamber social behavior apparatus. Histological features of the prefrontal cortex were studied by ordinary light microscope. Social behavior-related gene expressions were determined in the prefrontal cortex by real time RT-PCR method.@*RESULTS@#The arsenite-F2 male mice showed significantly poor sociability and social novelty preference in both 41-week-old group and 74-week-old group. There was no significant histological difference between the control mice and the arsenite-F2 mice. Regarding gene expression, serotonin receptor 5B (5-HT 5B) mRNA expression was significantly decreased (p < 0.05) in the arsenite-F2 male mice compared to the control F2 male mice in both groups. Brain-derived neurotrophic factor (BDNF) and dopamine receptor D1a (Drd1a) gene expressions were significantly decreased (p < 0.05) only in the arsenite-F2 male mice of the 74-week-old group. Heme oxygenase-1 (HO-1) gene expression was significantly increased (p < 0.001) in the arsenite-F2 male mice of both groups, but plasma 8-hydroxy-2'-deoxyguanosine (8-OHdG) and cyclooxygenase-2 (COX-2) gene expression were not significantly different. Interleukin-1β (IL-1β) mRNA expression was significantly increased only in 41-week-old arsenite-F2 mice.@*CONCLUSIONS@#These findings suggest that maternal arsenic exposure affects social behavior in F2 male mice via serotonergic system in the prefrontal cortex. In this study, COX-2 were not increased although oxidative stress marker (HO-1) was increased significantly in arsnite-F2 male mice.

mRNAs biomarker related to the control of proliferation and cell death in HepG2/C3A spheroid and monolayer cultures treated with piperlongumine

Background: Cell culture (spheroid and 2D monolayer cultures) is an essential tool in drug discovery. Piperlongumine (PLN), a naturally occurring alkaloid present in the long pepper (Piper longum), has been implicated in the regulation of GSTP1 activity. In vitro treatment of cancer cells with PLN increases ROS (reactive oxygen species) levels and induces cell death, but its molecular mode of action has not been entirely elucidated. Methods: In this study, we correlated the antiproliferative effects (2D and 3D cultures) of PLN (CAS 20069­09-4, Sigma-Aldrich) with morphological and molecular analyses in HepG2/C3A cell line. We performed assays for cytotoxicity (MTT), comet assays for genotoxicity, induction of apoptosis, analysis of the cell cycle phase, and analysis of the membrane integrity by flow cytometry. Relative expression of mRNA of genes related to proliferation, apoptosis, cell cycle control, metabolism of xenobiotics, and reticulum endoplasmic stress. Results: PLN reduced the cell proliferation by the cell cycle arrest in G2/M. Changes in the mRNA expression for CDKN1A (4.9x) and CCNA2 (0.5x) of cell cycle control genes were observed. Cell death occurred due to apoptosis, which may have been induced by increased expression of proapoptotic mRNAs (BAK1, 3.1x; BBC3, 2.4x), and by an increase in 9 and 3/7 active caspases. PLN induced cellular injury by ROS generation and DNA damage. DNA damage induced MDM2 signaling (3.0x) associated with the appearance of the monastral spindle in mitosis. Genes associated with ROS degradation also showed increased mRNA expression (GSR, 2.0x; SOD1, 2.1x). PLN induce endoplasmic reticulum stress with the increase in the mRNA expression of ERN1 (4.5x) and HSPA14 (2.2x). The xenobiotic metabolism showed increased mRNA expression for CYP1A2 (2.2x) and CYP3A4 (3.4x). In addition to 2D culture, PLN treatment also inhibited the growth of 3D culture (spheroids). Conclusion: Thus, the findings of our study show that several gene expression biomarkers (mRNAs) and monastral spindle formation indicated the many pathways of damage induced by PLN treatment that contributes to its antiproliferative effects

Impact of calcium aluminate cement with additives on dental pulp-derived cells

Abstract Calcium aluminate cement (CAC) has been highlighted as a promising alternative for endodontic use aiming at periapical tissue repair. However, its effects on dental pulp cells have been poorly explored. Objective: This study assessed the impact of calcium chloride (CaCl2) and bismuth oxide (Bi2O3) or zinc oxide (ZnO) additives on odontoblast cell response to CAC. Methodology: MDPC-23 cells were exposed for up to 14 d: 1) CAC with 2.8% CaCl2 and 25% ZnO (CACz); 2) CAC with 2.8% CaCl2 and 25% Bi2O3 (CACb); 3) CAC with 10% CaCl2 and 25% Bi2O3 (CACb+); or 4) mineral trioxide aggregate (MTA), placed on inserts. Non-exposed cultures served as control. Cell morphology, cell viability, gene expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), and dentin matrix protein 1 (DMP-1), ALP activity, and extracellular matrix mineralization were evaluated. Data were compared using ANOVA (α=5%). Results: Lower cell density was detected only for MTA and CACb+ compared with Control, with areas showing reduced cell spreading. Cell viability was similar among groups at days one and three (p>0.05). CACb+ and MTA showed the lowest cell viability values at day seven (p>0.05). CACb and CACb+ promoted higher ALP and BSP expression compared with CACz (p<0.05); despite that, all cements supported ALP activity. Matrix mineralization were enhanced in CACb+ and MTA. Conclusion: In conclusion, CAC with Bi2O3, but not with ZnO, supported the expression of odontoblastic phenotype, but only the composition with 10% CaCl2 promoted mineralized matrix formation, rendering it suitable for dentin-pulp complex repair.

Effect of Rolipram on in vitro maturation, gene expression and embryonic development in bovines / Efeito de rolipram na maturação in vitro, expressão gênica e desenvolvimento embrionário em bovinos

The aim of this work was to evaluate the effect of the Rolipram during the maturation of bovine oocytes and gene expression of embryos produced in vitro. Bovine ovaries were collected in slaughterhouse. The COCs were selected and divided into 5 groups: Control 0 time; Control: IVM for 24 hours; Rolipram treatments with IVM blocking for 24 hours in maturation medium containing (100, 150 and 200µM). After 24 hours all groups were reseated in IVM for another 24 hours. Subsequently COCs were subjected to the same IVM system and fertilized, being checked for cleavage post fertilization and for blastocyst. In addition, performed expression of the following genes: Mater, BMP15 and Bax. No difference was found in gene expression. Of oocytes evaluated shortly after follicular aspiration, 79.00% were in GV, GVBD, MI, while 13.40%, were in MII and 7.60%, D/NI. Significant difference was observed in different concentrations (T100, T200 and T150µM) in oocytes that have reached the MII phase compared to control treatments (P= 0.003). Differences were observed in cleavage rate (P< 0.05) between T150 and T200 when compared to the C/24 Group. A high difference was observed on blastocyst rate (P< 0.001) among treatments compared to the control group.(AU)

O objetivo deste trabalho foi avaliar o efeito do rolipram durante a maturação de oócitos bovinos, expressão gênica e embriões produzidos in vitro. Os ovários bovinos foram coletados no matadouro. Os COCs foram selecionados e divididos em cinco grupos: controle 0 tempo; controle: MIV por 24 horas; tratamentos rolipram com bloqueio MIV por 24 horas em meio de maturação contendo 100, 150 e 200µM. Após 24 horas, todos os grupos foram recolocados em MIV por mais 24 horas. Subsequentemente COCs foram submetidos ao mesmo sistema MIV e fertilizados, sendo avaliada a taxa de clivagem e de blastocisto, além da expressão dos seguintes genes: Mater, BMP15 e Bax. Nenhuma diferença foi observada na expressão gênica. Dos oócitos avaliados logo após a aspiração folicular, 79,0% estavam em GV, GVBD, MI, enquanto 13,40% estavam em MII, e 7,60% em D/NI. A diferença significativa foi observada em diferentes concentrações (T100, T200 e T150µM) em oócitos que atingiram a fase MII em comparação aos tratamentos de controle (P=0,3). Diferenças foram observadas nas taxas de clivagem (P<0,5) entre T150 e T200 quando comparadas com as taxas do grupo C/24. Uma grande diferença foi observada na taxa de blastocisto (P<0,1) entre os tratamentos em relação ao grupo controle.(AU)

Inhibición del crecimiento y modificación genética de Phytophthora capsici usando quitosano de bajo grado de polimerización / Growth inhibition and genetic modification of Phytophthora capsici using chitosan with low degree of polymerization

Phytophtora capsici es un patógeno que incide sobre cultivos de la familia de las solanáceas causando pérdidas económicas en cultivos de pimientos, tomates, berenjenas y cur-cubitáceas. En este trabajo evaluamos el efecto del quitosano de bajo grado de polimerización (QBP) sobre el crecimiento de P. capsici y sobre la regulación génica de este fitopatógeno a nivel transcripcional. A una concentración de 0,4mg/l de QBP se obtuvo un 88% de inhibición en el crecimiento; concentraciones superiores a 1,6 mg/l inhibieron el crecimiento en un 100%. Mediante ensayos de cambio en la movilidad electroforética de ácidos nucleicos se comprobó que el quitosano interactúa con el ADN y el ARN del hongo frente a concentraciones entre 2 y 4 mg/l de ADN y entre 0,5 y 3 mg/l de ARN. Además, se efectuó un análisis de despliegue diferencial de los productos de amplificación por RT-PCR de los ARN mensajeros de P. capsici obtenidos en presencia o ausencia de QBP; este mostró cambios en el perfil de expresión inducidos por el tratamiento con quitosano. El análisis bioinformático de las secuencias de los transcritos expresados diferencialmente sugiere que el QBP afectó la regulación génica de elementos involucrados en la síntesis de quitina y de proteínas de unión a hidratos de carbono.

Phytophthora blight of peppers, caused by oomycete Phytophthora capsici, currently causes economic losses in crops such as peppers, tomatoes, eggplant and cucurbits. In this work, we evaluated the effect of chitosan with low degree of polymerization (LDP) on growth and gene expression of P. capsici cultures. LDP chitosan inhibited 88% of P. capsici mycelial growth at concentrations up to 0,4 mg/l, whereas at concentrations higher than 1,6 mg/l it completely inhibit growth. Gel mobility shift assays demonstrated that chitosan interacts with DNA and RNA of the fungus at concentrations ranging from 2 to 4 mg/l for DNA and 0,5 to 3 mg/l for RNA. The differential display analysis of RT-PCR-amplification products of P. capsici messenger RNA revealed changes in gene expression profiles after the chitosan treatment. Bioinformatic analysis of sequences from selected differentially-expressed bands showed the gene regulation of elements involved in chitin synthesis and carbohydrate-binding proteins.

Effects of sub-minimum inhibitory concentrations of ciprofloxacin on biofilm formation and virulence factors of Escherichia coli

ABSTRACT Objective: To evaluate the influence of sub-minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP) on biofilm formation and virulence factors of Escherichia coli clinical isolates. Methods: Sub-MICs of CIP were determined using growth curve experiments. The biofilm-forming capacity of E. coli clinical isolates and E. coli ATCC 25922 treated or untreated with sub-MICs of CIP was assessed using a crystal violet staining assay. The biofilm structure of E. coli isolate was assessed with scanning electron microscopy (SEM). The expression levels of the virulence genes fim, usp, and iron and the biofilm formation genes of the pgaABCD locus were measured using quantification RT-PCR (qRT-PCR) in E. coli isolates and E. coli ATCC 25922. Results: Based on our results, the sub-MICs of CIP were 1/4 MICs. Sub-MICs of CIP significantly inhibited biofilm formation of E. coli clinical isolates and E. coli ATCC 25922 (p < 0.01). SEM analyses indicated that the biofilm structure of the E. coli changed significantly after treatment with sub-MICs of CIP. Expression levels of the virulence genes fim, usp, and iron and the biofilm formation genes of the pgaABCD locus were also suppressed. Conclusions: The results revealed that treatment with sub-MICs of CIP for 24 h inhibited biofilm formation and reduced the expression of virulence genes and biofilm formation genes in E. coli.

Modulatory effect of baicalein on gene expression and activity of antioxidant enzymes in streptozotocin-nicotinamide induced diabetic rats

Oxidative stress plays the central role in the pathogenesis and progression of diabetic complications. The present study aims to investigate the beneficial effect of oral administration of flavone baicalein in streptozotocin-nicotinamide (STZ-NA) induced diabetic rats by measuring oxidative stress markers, antioxidant enzyme activities and expression analysis of antioxidant genes. Experimental diabetes was induced by a single intraperitoneal (i.p.) injection of STZ (55 mg /kg b.wt), 15 min after the i.p. administration of NA. At the end of the experimental period, thiobarbituric acid reactive substances (TBARS), activities of antioxidant enzymes and expression levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione peroxidase (GPx) were measured in diabetic rats along with serum biochemical parameters namely total cholesterol (TC), total triglyceride (TG), aspartate transaminase (AST) alanine transaminase (ALT) and glycosylated hemoglobin (HbA1c). Oral administration of baicalein (40 mg/kg b.wt/day) demonstrated a significant ameliorative effect on all studied biochemical and oxidative stress parameters. Biochemical findings were corroborated by qPCR expression analysis which showed significant upregulation of antioxidant genes in diabetic rats. These results suggest that baicalein supplementation may reduce diabetes and its complications by suppressing oxidative stress and enhancing gene expression and antioxidant enzyme activities in diabetic rats.

Inhibitory effect of Astragalus polysaccharide on osteoporosis in ovariectomized rats by regulating FoxO3a /Wnt signaling pathway

Abstract Purpose: To investigate inhibitory effect of Astragalus polysaccharide (APS) on osteoporosis in ovariectomized rats by regulating FoxO3a/Wnt2 signaling pathway. Methods: Postmenopausal osteoporosis (PMOP) animal model was developed by excising the bilateral ovaries of rats. The model rats were administered with APS (200 mg/kg, 400 mg/kg, 800 mg/kg) by intragastric administration once daily for 12 weeks. Bone density, bone metabolism index and oxidative stress index were measured in all groups. Furthermore, the regulation of APS of FoxO3a / Wnt2 signaling pathway was observed. Results: APS has an estrogen-like effect, which can increase bone mass, lower serum ALP and BGP values, increase blood calcium content, and increase bone density of the femur and vertebrae in rats. At the same time, APS can increase the bone mineral content of the femur, increase the maximum stress, maximum load and elastic modulus of the ovariectomized rats, improve oxidative stress in rats by increasing the gene expression of β-catenin and Wnt2 mRNA and inhibiting the gene expression of FoxO3a mRNA. Conclusion: Astragalus polysaccharide can effectively alleviate oxidative stress-mediated osteoporosis in ovariectomized rats, which may be related to its regulation of FoxO3a/Wnt2/β-catenin pathway.

Comparing the effects of vitamin E tocotrienol-rich fraction supplementation and α-tocopherol supplementation on gene expression in healthy older adults

OBJECTIVES This study aims to compare the differential gene expression resulting from tocotrienol-rich fraction and α-tocopherol supplementation in healthy older adults. METHODS A total of 71 eligible subjects aged 50 to 55 years from Gombak and Kuala Lumpur, Malaysia, were divided into three groups and supplemented with placebo (n=23), α-tocopherol (n=24) or tocotrienol-rich fraction (n=24). Blood samples were collected at baseline and at 3 and 6 months of supplementation for microarray analysis. RESULTS The number of genes altered by α-tocopherol was higher after 6 months (1,410) than after 3 months (273) of supplementation. α-Tocopherol altered the expression of more genes in males (952) than in females (731). Similarly, tocotrienol-rich fraction modulated the expression of more genes after 6 months (1,084) than after 3 months (596) and affected more genes in males (899) than in females (781). α-Tocopherol supplementation modulated pathways involving the response to stress and stimuli, the immune response, the response to hypoxia and bacteria, the metabolism of toxins and xenobiotics, mitosis, and synaptic transmission as well as activated the mitogen-activated protein kinase and complement pathways after 6 months. However, tocotrienol-rich fraction supplementation affected pathways such as the signal transduction, apoptosis, nuclear factor kappa B kinase, cascade extracellular signal-regulated kinase-1 and extracellular signal-regulated kinase-2, immune response, response to drug, cell adhesion, multicellular organismal development and G protein signaling pathways. CONCLUSION Supplementation with either α-tocopherol or tocotrienol-rich fraction affected the immune and drug response and the cell adhesion and signal transduction pathways but modulated other pathways differently after 6 months of supplementation, with sex-specific responses.

Stanozolol promotes osteogenic gene expression and apposition of bone mineral in vitro

Abstract Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. Objective: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. Material and Methods: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. Results: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. Conclusions: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.

The role of atenolol in the modulation of the expression of genes encoding pro- (caspase-1) and anti- (Bcl2L1) apoptotic proteins in endothelial cells exposed to intestinal ischemia and reperfusion in rats

Abstract Purpose: To investigate the role of atenolol in the gene expression of caspase 1 (Casp1) and Bcl2L1 on vascular endothelium of rat intestine after ischemia and reperfusion (IR). Methods: Eighteen adult male Wistar rats were randomly divided into 3 groups (n=6): SG (Sham group): no clamping of the superior mesenteric artery; IRG: IR plus saline group: IRG+At: IR plus Atenolol group. Rats from IRG and IRG+At were subjected to 60 min of intestinal ischemia and 120 min of reperfusion. Atenolol (2mg/kg) or saline were injected in the femoral vein 5 min before ischemia, 5 min and 55 min after reperfusion. Thereafter, intestinal segments were appropriately removed and processed for Endothelial Cell Biology Rat RT2 Profiler PCR Array. Results: the anti-apoptotic Bcl2L1 gene expression was significantly down-regulated (-1.10) in the IRG and significantly up-regulated in the IRG+At (+14.15). Meanwhile, despite Casp1 gene expression was upregulated in both groups, it was significantly higher in the IRG (+35.06) than the IRG+At (+6.68). Conclusions: Atenolol presents antiapoptotic effects on rat intestine subjected to IR partly by the up-regulation of the anti-apoptotic Bcl2L1 gene expression. Moreover, atenolol can mitigate the pro-apoptotic and pro-inflammatory effects of Casp1 gene on rat intestine after IR.

The role of vitamin C in the gene expression of oxidative stress markers in fibroblasts from burn patients

Abstract Purpose: To assess the action of vitamin C on the expression of 84 oxidative stress related-genes in cultured skin fibroblasts from burn patients. Methods: Skin samples were obtained from ten burn patients. Human primary fibroblasts were isolated and cultured to be distributed into 2 groups: TF (n = 10, fibroblasts treated with vitamin C) and UF (n = 10, untreated fibroblasts). Gene expression analysis using quantitative polymerase chain reaction array was performed for comparisons between groups. Results: The comparison revealed 10 upregulated genes as follows: arachidonate 12-lipoxygenase (ALOX12), 24-dehydrocholesterol reductase (DHCR24), dual oxidase 1 (DUOX1), glutathione peroxidase 2 (GPX2), glutathione peroxidase 5 (GPX5), microsomal glutathione S-transferase 3 (MGST3), peroxiredoxin 4 (PRDX4), phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-REX1), prostaglandin-endoperoxide synthase 1 (PTGS1), and ring finger protein 7 (RNF7). Conclusion: Cultured fibroblasts obtained from burn patients and treated with vitamin C resulted in 10 differentially expressed genes, all overexpressed, with DUOX1, GPX5, GPX2 and PTGS1 being of most interest.

Graves' ophthalmopathy: low-dose dexamethasone reduces retinoic acid receptor-alpha gene expression in orbital fibroblasts

ABSTRACT Objective: Graves' ophthalmopathy (GO) is an autoimmune disease that leads to ocular proptosis caused by fat accumulation and inflammation, and the main treatment is corticosteroid therapy. Retinoid acid receptor-alpha (RARα) seems to be associated with inflammation and adipocyte differentiation. This study aimed to assess the effect of glucocorticoid treatment on orbital fibroblasts of GO patient treated or not with different glucocorticoid doses. Materials and methods: Orbital fibroblasts collected during orbital decompression of a female patient with moderately severe/severe GO were cultivated and treated with 10 nM and 100 nM dexamethasone (Dex). rRARα gene expression in the treated and untreated cells was then compared. Results: Fibroblast RARα expression was not affected by 100 nM Dex. On the other hand, RARα expression was 24% lower in cells treated with 10 nM Dex (p < 0.05). Conclusions: Orbital fibroblasts from a GO patient expressed the RARα gene, which was unaffected by higher, but decreased with lower doses of glucocorticoid.

Extracellular calcium increases fibroblast growth factor 2 gene expression via extracellular signal-regulated kinase 1/2 and protein kinase A signaling in mouse dental papilla cells

Abstract We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). Objective: The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Materials and Methods: Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Results: Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. Conclusions: These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.

Efecto de las nanopartículas industriales TiO 2 , SiO 2 y ZnO sobre la viabilidad celular y expresión génica en médula ósea roja de mus musculus / Effect of the Industrial Nanoparticles TiO 2 , SiO 2 and ZnO on Cell Viability and Gene Expression in Red Bone Marrow of Mus Musculus

RESUMEN Objetivos Evaluar el efecto de las nanopartículas de ZnO, TiO2 y SiO2 sobre la viabilidad celular y la expresión génica de las interleuquinas 7 y 3 y del factor estimulante de colonias de granulocito - macrófago (GM-CSF) en Mus musculus. Materiales y métodos Se extrajo médula ósea roja de cinco roedores (Balb/c) para el estudio de viabilidad celular mediante la prueba de MTT. Por otro lado, grupos cinco roedores fueron inoculados vía intraperitoneal con dosis de 0,5; 1; 2,5; 5 y 10 mg/kg de nanopartículas de ZnO y SiO2 y de 5; 10; 15; 20 y 25 mg/kg de nanopartículas de TiO2, 30 h después, se obtuvo el ARN a partir de la médula ósea roja para los análisis de expresión génica empleando las técnicas de PCR y RT-PCR cuantitativa. Resultados Las nanopartículas de ZnO y SiO2 redujeron la viabilidad celular de una manera dosis-dependiente en un 37 y 26%, respectivamente, a partir de una dosis de 1 mg/kg. En cuanto al efecto sobre la expresión génica, a las dosis 5 y 10 mg/kg, las nanopartículas de TiO2 redujeron en mayor porcentaje la expresión de las interleuquinas 7 y 3 (55,3 y 70,2% respectivamente), con respecto a la expresión del GM-CSF, el mayor porcentaje de reducción lo produjo las nanopartículas de SiO2 (91%). Las nanopartículas de ZnO redujeron a partir de las dosis de 20 y 25 mg/kg. Conclusiones Las nanopartículas de ZnO, SiO2 y TiO2 alteran la viabilidad celular y la expresión génica en la médula ósea de ratón.

ABSTRACT Objectives To evaluate the effect of ZnO, TiO2 and SiO2 nanoparticles on cell viability and expression of the interleukin 7, interleukin 3, and granulocyte-macrophage colony stimulating factor (GM-CSF) genes in Mus musculus. Material and methods Red bone marrow was extracted from five Balb/c mice for the analysis of cell viability using the MTT test. The mice were divided into two groups of five each: one group was inoculated intraperitoneally with 0.5, 1.0, 2.5, 5.0, and 10 mg/kg of ZnO and SiO2 nanoparticles, respectively, and the other group was inoculated with 5.0, 10.0, 15.0, 20.0, and 25 mg/kg of TiO2 nanoparticles, respectively. Thirty hours later, RNA was extracted from the red bone marrow of the mice in both groups for gene expression analysis using quantitative PCR and RT-PCR. Results ZnO and SiO2 nanoparticles reduced cell viability in a dose-dependent manner by 37% and 26%, respectively, starting at a dose of 1 mg/kg. TiO2 nanoparticles at 5 mg/kg and 10 mg/kg reduced the gene expression of interleukins 7 and 3 by 55.3% and 70.2%, respectively, and SiO2 nanoparticles caused the greatest decrease (91%) in the expression of GM-CSF. ZnO nanoparticles reduced the expression of GM-CSF starting at doses of 20 mg/kg and 25 mg/kg. Conclusions ZnO, SiO2 and TiO2 nanoparticles affect cell viability and gene expression in the mouse bone marrow.

Roles of the ERK1/2 and PI3K/PKB signaling pathways in regulating the expression of extracellular matrix genes in rat pulmonary artery smooth muscle cells

Abstract Purpose: To investigate the mechanisms by which PD98059 and LY294002 interfere with the abnormal deposition of extracellular matrix regulated by connective tissue growth factor (CTGF) of rat pulmonary artery smooth muscle cells (PASMCs). Methods: Rat PASMCs were cultured and separated into a control group. Real-time fluorescence quantitative PCR was performed to detect the expression of collagen III and fibronectin mRNA. Immunohistochemistry and western blot analyses were performed to detect the expression of collagen III protein. Results: The expression of collagen III and fibronectin mRNA was greater in PASMCs stimulated with CTGF for 48 h, than in the control group. After 72h of stimulation, the expression of collagen III protein in the PASMCs was greater than in the control. The equivalent gene and protein expression of the CPL group were much more significant. Conclusions: CTGF can stimulate the gene expression of collagen III and fibronectin in PASMCs, which may be one of the factors that promote pulmonary vascular remodeling (PVR) under the conditions of pulmonary arterial hypertension (PAH). PD98059 and LY294002 can inhibit the ERK1/2 and PI3K/PKB signaling pathways, respectively, thus interfering with the biological effects of CTGF. This may be a new way to reduce PAH-PVR.

Retinoic acid protects from experimental cerebral infarction by upregulating GAP-43 expression

The aim of this study was to investigate whether exogenous retinoic acid (RA) can upregulate the mRNA and protein expression of growth-associated protein 43 (GAP-43), thereby promoting brain functional recovery in a rat distal middle cerebral artery occlusion (MCAO) model of ischemia. A total of 216 male Sprague Dawley rats weighing 300–320 g were divided into 3 groups: sham-operated group, MCAO+vehicle group and MCAO+RA group. Focal cortical infarction was induced with a distal MCAO model. The expression of GAP-43 mRNA and protein in the ipsilateral perifocal region was assessed using qPCR and immunocytochemistry at 1, 3, 7, 14, 21, and 28 days after distal MCAO. In addition, an intraperitoneal injection of RA was given 12 h before MCAO and continued every day until the animal was sacrificed. Following ischemia, the expression of GAP-43 first increased considerably and then decreased. Administration of RA reduced infarction volume, promoted neurological functional recovery and upregulated expression of GAP-43. Administration of RA can ameliorate neuronal damage and promote nerve regeneration by upregulating the expression of GAP-43 in the perifocal region after distal MCAO.