Effect of Inducible BMP-7 Expression on the Osteogenic Differentiation of Human Dental Pulp Stem Cells.

BMP-7 has shown inductive potential for in vitro osteogenic differentiation of mesenchymal stem cells, which are an ideal resource for regenerative medicine. Externally applied, recombinant BMP-7 was able to induce the osteogenic differentiation of DPSCs but based on our previous results with BMP-2, we aimed to study the effect of the tetracyclin-inducible BMP-7 expression on these cells. DPSC, mock, and DPSC-BMP-7 cell lines were cultured in the presence or absence of doxycycline, then alkaline phosphatase (ALP) activity, mineralization, and mRNA levels of different osteogenic marker genes were measured. In the DPSC-BMP-7 cell line, the level of BMP-7 mRNA significantly increased in the media supplemented with doxycycline, however, the expression of Runx2 and noggin genes was upregulated only after 21 days of incubation in the osteogenic medium with doxycycline. Moreover, while the examination of ALP activity showed reduced activity in the control medium containing doxycycline, the accumulation of minerals remained unchanged in the cultures. We have found that the induced BMP-7 expression failed to induce osteogenic differentiation of DPSCs. We propose three different mechanisms that may worth investigating for the engineering of expression systems that can be used for the induction of differentiation of mesenchymal stem cells.

SIRT1 Activation by Equisetum Arvense L. (Horsetail) Modulates Insulin Sensitivity in Streptozotocin Induced Diabetic Rats.

BACKGROUND: Equisetum arvense L., commonly known as field horsetail is a perennial fern of which extracts are rich sources of phenolic compounds, flavonoids, and phenolic acids. Activation of SIRT1 that was shown to be involved in well-known signal pathways of diabetic cardiomyopathy has a protective effect against oxidative stress, inflammatory processes, and apoptosis that are the basis of diseases such as obesity, diabetes mellitus, or cardiovascular diseases. The aim of our study was to evaluate the antidiabetic and cardioprotective effects of horsetail extract in streptozotocin induced diabetic rats. METHODS: Diabetes was induced by a single intraperitoneal injection of 45 mg/kg streptozotocin. In the control groups (healthy and diabetic), rats were administered with vehicle, whilst in the treated groups, animals were administered with 50, 100, or 200 mg/kg horsetail extract, respectively, for six weeks. Blood glucose levels, glucose tolerance, and insulin sensitivity were determined, and SIRT1 levels were measured from the cardiac muscle. RESULTS: The horsetail extract showed moderate beneficial changes in blood glucose levels and exhibited a tendency to elevate SIRT1 levels in cardiomyocytes, furthermore a 100 mg/kg dose also improved insulin sensitivity. CONCLUSIONS: Altogether our results suggest that horsetail extract might have potential in ameliorating manifested cardiomyopathy acting on SIRT1.

Anthocyanin-Rich Sour Cherry Extract Attenuates the Lipopolysaccharide-Induced Endothelial Inflammatory Response.

The anthocyanin content of Hungarian sour cherry is remarkable based on our preliminary investigations. Nutraceutical and pharmaceutical effects of anthocyanins have been extensively studied. The objective of this work was to investigate the the effect of purified sour cherry extract using human umbilical cord vein endothelial cells (HUVECs) as the inflammatory model. HUVECs were isolated by enzymatic digestion and characterized by flow cytometry. The optimal concentration range of sour cherry extract was selected based on MTT, apoptosis, and necrosis assays. Cells were divided into three groups, incubating with M199 medium as control, or with lipopolysaccharide (LPS) or with LPS plus anthocyanin extract (ACE). The effect of sour cherry extract on oxidative stress, pro-inflammatory factors, and arachidonic pathway was investigated. An amount of 50 µg/mL ACE (ACE50) was able to increase the level of glutathione and decrease the ROS, thereby improving the unbalanced redox status in inflammation. ACE50 lowered pro-inflammatory cytokine levels including Interleukin-6 (IL-6), regulated on activation, normal T cell expressed and secreted (RANTES), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-α). ACE50 affected the arachidonic acid pathway by reducing the LPS-induced enzyme expression (cyclooxygenase-1, cyclooxygenase-2, and prostacyclin synthase). The extract under investigation seems to have a pleiotropic effect including anti-oxidative, anti-inflammatory, hemostatic, and vasoactive effects. Our results indicate that purified sour cherry extract could reduce the LPS-induced inflammatory response, thereby improving endothelial dysfunction.

Spilanthol Inhibits Inflammatory Transcription Factors and iNOS Expression in Macrophages and Exerts Anti-inflammatory Effects in Dermatitis and Pancreatitis.

Activated macrophages upregulate inducible nitric oxide synthase (iNOS) leading to the profuse production of nitric oxide (NO) and, eventually, tissue damage. Using macrophage NO production as a biochemical marker of inflammation, we tested different parts (flower, leaf, and stem) of the medicinal plant, Spilanthes acmella. We found that extracts prepared from all three parts, especially the flowers, suppressed NO production in RAW macrophages in response to interferon-γ and lipopolysaccharide. Follow up experiments with selected bioactive molecules from the plant (α-amyrin, ß-caryophylline, scopoletin, vanillic acid, trans-ferulic acid, and spilanthol) indicated that the N-alkamide, spilanthol, is responsible for the NO-suppressive effects and provides protection from NO-dependent cell death. Spilanthol reduced the expression of iNOS mRNA and protein and, as a possible underlying mechanism, inhibited the activation of several transcription factors (NFκB, ATF4, FOXO1, IRF1, ETS, and AP1) and sensitized cells to downregulation of Smad (TF array experiments). The iNOS inhibitory effect translated into an anti-inflammatory effect, as demonstrated in a phorbol 12-myristate 13-acetate-induced dermatitis and, to a smaller extent, in cerulein-induced pancreatitis. In summary, we demonstrate that spilanthol inhibits iNOS expression, NO production and suppresses inflammatory TFs. These events likely contribute to the observed anti-inflammatory actions of spilanthol in dermatitis and pancreatitis.

Effect of Anthocyanin-Rich Tart Cherry Extract on Inflammatory Mediators and Adipokines Involved in Type 2 Diabetes in a High Fat Diet Induced Obesity Mouse Model.

Male C57BL/6J mice were used to determine the possible therapeutic effects of our previously described tart cherry extract in a chronic obesity mouse model on metabolic parameters, glucose tolerance, inflammatory mediators, and antioxidant capacity. The control group received standard mouse chow, and the high fat control group was switched to a high fat diet and tap water supplemented with 5% sucrose. The high fat + anthocyanin group received the high fat and sucrose diet, but received the anthocyanin-rich tart cherry extract dissolved in their drinking water. After six weeks, an oral glucose tolerance test was performed, and the water-soluble antioxidant capacity (ACW), superoxide dismutase (SOD) activity, and the plasma levels of insulin, C-peptide, leptin, IL-6, MCP-1, adiponectin and resistin were measured. The high fat diet increased body weight, reduced glucose tolerance, and caused an elevation in leptin, IL-6, MCP-1, and resistin levels. Furthermore, antioxidant capacity was decreased with a significant elevation of SOD activity. Anthocyanin treatment failed to reverse the effects of the high fat diet on body weight and glucose tolerance, but significantly reduced the leptin and IL-6 levels. The tart cherry extract also made a significant enhancement in antioxidant capacity and SOD activity. Our results show that chronic anthocyanin intake has a potential to enhance redox status and alleviate inflammation associated with obesity.

Silymarin: Friend or Foe of UV Exposed Keratinocytes?

The application of natural plant extracts in UV-protection is popular and intensively studied. Silymarin (from Silibum marianum), a naturally occurring polyphenol, has recently received attention due to its antioxidant, anti-inflammatory and anti-apoptotic effects. However, its role in the UV-mediated keratinocyte cell response is still controversial. In this study, we investigated the effects of Silibum marianum extracts with different origins and formulations on UVA-exposed HaCaT keratinocytes in vitro. Our results show, that silymarin treatment caused an inverse dose-dependent photosensitivity relationship (at higher doses, a decrease in cell viability and ROS production) after UVA exposure. The attenuation of the UVA-induced ROS generation after silymarin treatment was also observed. Moreover, silymarin pre-treatment increased the cyclobutane pyrimidine dimer photolesions in keratinocytes after UVA exposure. These results indicated the dual role of silymarin in UVA-exposed keratinocytes. It scavenges ROS but still induces phototoxicity. Based on our results dermatological applications of silymarin and related compounds should be considered very carefully.

Diosgenin and Its Fenugreek Based Biological Matrix Affect Insulin Resistance and Anabolic Hormones in a Rat Based Insulin Resistance Model.

Fenugreek is known since ancient times as a traditional herbal medicine of its multiple beneficial effects. Fenugreek's most studied and employed effect is its hypoglycemic property, but it can also be useful for the treatment of certain thyroid disorders or for the treatment of anorexia. The regulation of glucose homeostasis is a complex mechanism, dependent on the interaction of different types of hormones and neurotransmitters or other compounds. For the study of how diosgenin and fenugreek seeds modify insulin sensitivity, we used a rat insulin resistance model induced by high-fat diet. Diosgenin in three different doses (1mg/bwkg, 10mg/bwkg, and 50 mg/bwkg, respectively) and fenugreek seed (0.2 g/bwkg) were administered orally for 6 weeks. Insulin sensitivity was determined by hyperinsulinemic euglycemic glucose clamp method. Our research group found that although glucose infusion rate was not significantly modified in either group, the increased insulin sensitivity index and high metabolic clearance rate of insulin found in the 1 mg/kg diosgenin and the fenugreek seed treated group suggested an improved peripheral insulin sensitivity. Results from the 10 mg/kg diosgenin group, however, suggest a marked insulin resistance. Fenugreek seed therapy results on the investigated anabolic hormones support the theory that, besides insulin and gastrointestinal peptides, the hypothalamic-hypopituitary axis regulated hormones synchronized action with IGF-1 also play an important role in the maintaining of normal glucose levels. Both diosgenin and fenugreek seeds are capable of interacting with substrates of the above-mentioned regulatory mechanisms, inducing serious hormonal disorders. Moreover, fenugreek seeds showed the ability to reduce the thyroid hormone levels at the periphery and to modify the T4/T3 ratio. It means that in healthy people this effect could be considered a severe side effect; however, in hypothyroidism this effect represents a possibility of alternative natural therapy.

Diverse effect of BMP-2 homodimer on mesenchymal progenitors of different origin.

Bone morphogenetic protein-2 (BMP-2), is a potential factor to enhance osseointegration of dental implants. However, the appropriate cellular system to investigate the osteogenic effect of BMP-2 in vitro in a standardized manner still needs to be defined. The aim of this study was to examine the effect of BMP-2 on the cell proliferation and osteogenic differentiation of human osteogenic progenitors of various origins: dental pulp stem cells (DPSC), human osteosarcoma cell line (Saos-2) and human embryonic palatal mesenchymal cell line (HEPM). For induction of osteogenic differentiation, cell culture medium was supplemented with BMP-2 homodimer alone or in combination with conventionally used differentiation inducing agents. Differentiation was monitored for 6-18 days. To assess differentiation, proliferation rate, alkaline phosphatase activity, calcium deposition and the expression level of osteogenic differentiation marker genes (Runx2, BMP-2) were measured. BMP-2 inhibited cell proliferation in a concentration and time-dependent manner. In a concentration which caused maximal cell proliferation, BMP-2 did not induce osteogenic differentiation in any of the tested systems. However, it had a synergistic effect with the osteoinductive medium in both DPSC and Saos-2, but not in HEPM cells. We also found that the differentiation process was faster in Saos-2 than in DPSCs. Osteogenic differentiation could not be induced in the osteoblast progenitor HEPM cells. Our data suggest that in a concentration that inhibits proliferation the differentiation inducing effect of BMP-2 is evident only in the presence of permissive osteoinductive components. ß-glycerophosphate, was identified interacting with BMP-2 in a synergistic manner.

Effect of long-term olanzapine treatment on meal-induced insulin sensitization and on gastrointestinal peptides in female Sprague-Dawley rats.

Meal-induced insulin sensitization (MIS), an endogenous adaptive mechanism is activated post-prandially. Reduced MIS leads to diabetes, but its activation improves insulin sensitivity. MIS is preserved to single olanzapine administration, therefore we aimed to investigate the chronic effect of olanzapine on fasted-state insulin sensitivity and on MIS in female Sprague-Dawley rats. Daily food and water intake, stool and urine production and body weight were determined. The MIS was characterized by a rapid insulin sensitivity test. Fasting hepatic and peripheral insulin sensitivity were determined by a hyperinsulinaemic euglycaemic glucose clamping supplemented with radiotracer technique. Fasted and post-prandial blood samples were obtained for plasma insulin, leptin, ghrelin, amylin, GLP-1, GIP, PYY and PP determination. Adiposity was characterized by weighing intra-abdominal and inguinal fat pads. Olanzapine caused hepatic insulin resistance and a reduced metabolic clearance rate of insulin, but the MIS retained its function. Body weight and adiposity were enhanced, but olanzapine failed to increase food intake. Fasting insulin and leptin were elevated and the post-prandial reduction in ghrelin level was inhibited by olanzapine.The MIS remained functionally intact after long-term olanzapine treatment. Altered insulin, leptin and ghrelin levels indicate olanzapine-induced metabolic derangements. Pharmacological activation of MIS could potentially be exploited to treat or prevent olanzapine-induced insulin resistance.

Effects of non-toxic zinc exposure on human epidermal keratinocytes.

Zinc is an essential microelement; its importance to the skin is highlighted by the severe skin symptoms in hereditary or acquired zinc deficiency, by the improvement of several skin conditions using systemic or topical zinc preparations and by the induced intracellular zinc release upon UVB exposure, which is the main harmful environmental factor to the skin. Understanding the molecular background of the role of zinc in skin may help gain insight into the pathology of skin disorders and provide evidence for the therapeutic usefulness of zinc supplementation. Herein, we studied the effects of zinc chloride (ZnCl2) exposure on the function of HaCaT keratinocytes, and the results showed that a non-toxic elevation in the concentration of extracellular zinc (100 µM) facilitated cell proliferation and induced significant alterations in the mRNA expression of NOTCH1, IL8, and cyclooxygenase-2. In addition, increased heme oxygenase-1 (HMOX1) expression and non-toxic generation of superoxide were detected in the first 4 h. Regarding the effects on the UVB-induced toxicity, although the level of cyclobutane pyrimidine dimers in the keratinocytes pre-treated with zinc for 24 h was reduced 3 h after UVB irradiation, significantly enhanced superoxide generation was observed 10 h after UVB exposure in the zinc pre-exposed cells. The overall survival was unaffected; however, there was a decrease in the percentage of early apoptotic cells and an increase in the percentage of late apoptotic plus necrotic cells. These results suggest that the exposure of human keratinocytes to non-toxic concentrations of ZnCl2 impacts gene expression, cell proliferation and the responses to environmental stress in the skin. It would be important to further examine the role of zinc in skin and further clarify whether this issue can affect our thinking regarding the pathogenesis of skin diseases.