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    Dietary supplementation of omega-3 fatty acid and circulating levels of interleukin-1 beta, osteocalcin, and C-reactive protein in rats
    (Amer Acad Periodontology, 2006) Vardar-Sengul, Saynur; Buduneli, Nurcan; Buduneli, Eralp; Kardesler, Levent; Baylas, Haluk; Atilla, Gul; Lappin, David; Kinane, Denis F.
    Background: In this study, we evaluated the effects of two different regimes of dietary supplementation of omega-3 fatty acid on serum levels of interleukin-1 beta (IL-1 beta), osteocalcin (OC), and C-reactive protein (CRP) in experimental periodontitis. Methods: Experimental periodontitis was induced by repeated injections of Escherichia coli lipopolysaccharide (LPS). Thirty-nine adult male Sprague-Dawley rats were divided into four study groups as follows: an LPS positive control group; a saline (negative) control group; and two different groups with omega-3 fatty acid dietary supplementation, one in which we gave the supplement subsequent to disease induction (TO3) and the other in which the agent was started prior to and continued subsequent to LPS injections (P + TO3). In the TO3 group, omega-3 fatty acid administration was performed for 14 days following induction of experimental periodontitis. In the P + TO3 group, omega-3 fatty acid was given for 14 days prior to the start of LPS injections and was continued for another 14 days subsequent to the induction of experimental periodontitis. On day 15 of the first LPS injection, serum samples were obtained and rats were sacrificed. Serum samples were analyzed for IL-1 beta, OC, and CRP concentrations by enzymelinked immunosorbent assay. Defleshed jaws were analyzed morphometrically for alveolar bone loss. Data were evaluated statistically by non-parametric tests. Results: LPS injection resulted in statistically significantly more bone loss compared to the saline control group (P < 0.05). None of the omega-3 fatty acid administration groups showed evidence that this fatty acid was effective in preventing LPS-induced alveolar bone loss. TO3 and P + TO3 groups revealed significantly higher IL-1 beta and OC levels than the LPS group (P < 0.05). The study groups exhibited no significant differences in the serum CRP levels. Conclusions: Omega-3 fatty acid administration does not seem to influence circulating levels of CRP. The significantly increased serum OC level observed in both omega-3 fatty acid regimes is curious and could have an effect on bone turnover, as could the further significant increase in serum IL-1 beta, which could counteract any osteoblastic induction by OC through promotion of osteoclast activity. The lack of a therapeutic benefit of omega-3 fatty acid in this study, despite the effects on OC and IL-1 beta, is difficult to explain, and further studies are required to more fully assess the potential role of this fatty acid in periodontal treatment.
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    Editorial: antimicrobial peptides, mainly Defensins in oral cavity
    (Bentham Science Publ Ltd, 2007) Vardar-Sengul, Saynur; Mercola, Dan
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    Effects of selective cyclooxygenase-2 inhibitor and omega-3 fatty acid on serum interleukin-1 beta, osteocalcin, and C-reactive protein levels in rats
    (Amer Acad Periodontology, 2006) Vardar-Sengul, Saynur; Buduneli, Nurcan; Buduneli, Eralp; Baylas, Haluk; Atilla, Gul; Lappin, David; Kinane, Denis F.
    Background: The aim of this study was to evaluate the effects of selective cyclooxygenase-2 inhibitor, celecoxib, and omega-3 fatty acid on serum levels of interleukin 1-beta (IL-1 beta), osteocalcin (OC), and C-reactive protein (CRP) in experimental periodontitis. Methods: Experimental periodontitis in rats was induced by repeated injection of purified lipopolysaccharide (LPS) derived from Escherichia coli endotoxin. Forty-seven adult male Sprague-Dawley rats were divided into five study groups as follows: saline control, LPS, LPS + celecoxib, LPS + omega-3 fatty acid, and LPS + celecoxib + omega-3 fatty acid. Celecoxib and omega-3 fatty acid were given alone or in combination during 14 days of the experimental study period. At the end of the 2-week protocol, serum samples were obtained, and the rats were sacrificed. Serum samples were analyzed for IL-1 beta, OC, and CRP concentrations by enzyme-linked immunosorbent assay. Defleshed jaws were analyzed morphometrically for alveolar bone loss. Data were evaluated statistically by non-parametric tests. Results: According to the morphometric measurements, the LPS and drug treatment groups showed significantly higher bone loss than the saline control group (P < 0.05). Omega-3 fatty acid, both alone and in combination with celecoxib, revealed significantly higher IL-1 beta levels than LPS and celecoxib groups (P < 0.05). Individual and combined administration of celecoxib and omega-3 fatty acid significantly increased OC levels compared to the LPS group (P < 0.05). There were no significant differences in serum CRP levels. Conclusions: Celecoxib and/or omega-3 fatty acid administration does not significantly influence circulating levels of CRP. The significantly increased serum OC level observed after individual and combination administration suggests that celecoxib and omega-3 fatty acid may in influence bone remodeling and thereby inhibit the progression of alveolar bone resorption. However, the failure to observe any significant inhibition of bone loss in celecoxib- and/or omega-3 fatty acid-treated rats compared to the LPS group suggests that their therapeutic effect may be reduced by other factors, such as increases in serum IL-1 beta promoting osteoclast activity.
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    Egr1 regulates the coordinated expression of numerous EGF receptor target genes as identified by ChIP on chip
    (Bmc, 2008) Arora, Shilpi; Wang, Yipeng; Jia, Zhenyu; Vardar-Sengul, Saynur; Munawar, Ayla; Doctor, Kutbuddin S.; Birrer, Michael; McClelland, Michael; Adamson, Eileen; Mercola, Dan
    Background: UV irradiation activates the EGF receptor, induces Egr1 expression and promotes apoptosis in a variety of cell types. We examined the hypothesis that Egr1 regulates genes that mediate this process by use of a chip-on-chip protocol in human tumorigenic prostate M12 cells. Results: UV irradiation led to significant binding of 288 gene promoters by Egr1. A major functional subgroup consisted of apoptosis related genes. The largest subgroup of 24 genes; belong to the EGFR-signal transduction pathway. Egr1 promoter binding had a significant impact on gene expression of target genes. Conventional ChIP and qRTPCR were used to validate promoter binding and expression changes. siRNA experiments were used to demonstrate the specific role of Egr1 in gene regulation. UV-stimulation promotes growth arrest and apoptosis of M12 cells and our data clearly show that downstream target of EGFR, namely Egr1, mediates this apoptotic response. Our study also identified numerous previously unknown targets of Egr1. These include FasL, MAX and RRAS2, which may play a role in the apoptotic response/growth arrest. Conclusion: Our results indicate that M12 cells undergo Egr1-dependent apoptotic response upon UV-stimulation and led to the identification of downstream targets of Egr1, which mediate EGFR function.
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    Expression Profile of Human Gingival Fibroblasts Induced by Interleukin-1 beta Reveals Central Role of Nuclear Factor-Kappa B Stabilizing Human Gingival Fibroblasts During Inflammation
    (Wiley, 2009) Vardar-Sengul, Saynur; Arora, Shilpi; Baylas, Haluk; Mercola, Dan
    Background: Interleukin (IL)-1 beta is a key cytokine in the pathogenesis of periodontitis, and it induces inflammatory mediators in periodontal diseases. We developed immortalized human gingival fibroblasts (HGFs), investigated the effects of IL-1 beta on the gene expression using expression arrays containing similar to 40,000 genes, and tested the role of nuclear factor-kappa B (NF-kappa B) in maintaining an activated HGF population. Methods: Total RNA was isolated from IL-1 beta-induced and mock-induced control cells. Gene expression analyses were performed using expression arrays and confirmed by quantitative real-time polymerase chain reaction. Western blot analysis to show inhibitor of kappa B-alpha (I kappa B alpha) phosphorylation and immunostaining of cells for NF-kappa B nuclear translocation were performed. Apoptosis was confirmed by assay of poly ADP-ribose polymerase (PARP) cleavage. Results: A total of 382 probe sets corresponding to 254 genes were differentially expressed in IL-1 beta-induced cells (P <0.001). A total of 215 genes were upregulated, and 39 genes were downregulated. Most notable NF-kappa B pathway members (NF kappa B1, NF kappa B2, I kappa B alpha, I kappa B epsilon, I kappa B zeta, REL, RELB, and TA-NFKBH) were upregulated. I kappa B alpha was phosphorylated, and NF-kappa B accumulated in the nucleus. An IL-1 beta-induced set of 27 genes was downregulated by an NF-kappa B inhibitor, leading to a decreased number of viable cells and suggesting an antiapoptotic role for NF-kappa B. Conclusions: IL-1 beta leads to a large number of significant expression changes consistent with a pathologic role in periodontitis, including enhancement of inflammatory cytokines, chemokines, transcription factors, matrix metalloproteinases, adhesion molecules, and especially NF-kappa B-dependent antiapoptotic genes. NF-kappa B activation blocks apoptosis, thereby stabilizing the HGF population in inflammation. J Periodontol 2009;80:833-849.
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    Matrix metalloproteinases, tissue inhibitor of matrix metalloproteinase-1, and laminin-5 gamma 2 chain immunolocalization in gingival tissue of endotoxin-induced periodontitis in rats: Effects of low-dose doxycycline and alendronate
    (Amer Acad Periodontology, 2007) Buduneli, Eralp; Vardar-Sengul, Saynur; Buduneli, Nurcan; Atilla, Gul; Wahlgren, Jaana; Sorsa, Timo
    Background: Matrix metalloproteinases (MMPs) play important roles in tissue destruction mechanisms of periodontitis. MMP-8 and -13 are the major collagenases that act in extracellular matrix degradation in periodontal tissues. MMP-14 is a membrane-type MMP, and laminin (Ln)-5 is a basal membrane component. The aim of the present study was to evaluate the effects of doxycycline and alendronate on gingival tissue expression of MMP-8, -13, and -14; tissue inhibitors of MMP (TIMP)-1; and Ln-5 gamma 2 chain in experimental periodontitis induced by Escherichia coli endotoxin (LPS) in rats. Methods: Experimental periodontitis was induced by repeated injection of LPS. Forty-four adult male Sprague-Dawley rats were divided into five study groups: saline control, LPS, LPS + doxycycline, LPS + alendronate, and LPS + doxycycline + alendronate. Doxycycline and alendronate were given as a single agent or as combination therapy during the 7 days of the experimental study period. On day 7, the rats were sacrificed, and the gingival tissues were analyzed immunohistochemically for expression of MMP-8, -13, and -14, Ln-5 gamma 2 chain, and TIMP-1. Alveolar bone loss was evaluated morphometrically under a stereomicroscope. Data were tested statistically by Kruskal-Wallis and Mann-Whitney tests and Spearman correlation analysis. Results: Alveolar bone loss was significantly higher in the LPS, doxycycline, alendronate, and combination groups than in the saline control group (all P < 0.01). MMP-8 expression was significantly higher in the LPS group than in the saline control group (P = 0.001). Individual administration of doxycycline or alendronate significantly decreased the expression of MMP-8 compared to LPS (P = 0.01). Combined drug administration reduced MMP-14 significantly compared to doxycycline (P = 0.004). No significant differences in Ln-5 gamma 2 chain expression were found between the study groups (P > 0.05). MMP-14 significantly correlated with the Ln-5 gamma 2 chain in the LPS + alendronate group (P = 0.04) and with the amount of alveolar bone loss in the LPS + doxycycline + alendronate group (P = 0.03). Conclusions: Our findings suggest that alendronate and/or doxycycline may inhibit MMP-8 expression significantly; particularly, their combined administration may provide beneficial effects in periodontal treatment. Moreover, individual administration of alendronate and doxycycline results in significant increases in TIMP-I expression in gingiva. However, these effects of combined low-dose doxycycline and alendronate on MMPs and TIMP should be verified by clinical human trials before these agents are used in dental practice.