Oxidative Stress and Natural Antioxidants in Osteoporosis: Novel Preventive and Therapeutic Approaches.

This review reports in detail the cellular and molecular mechanisms which regulate the bone remodeling process in relation to oxidative stress (OS), inflammatory factors, and estrogen deficiency. OS is considered an important pathogenic factor of osteoporosis, inducing osteocyte apoptosis and varying levels of specific factors, such as receptor activator κB ligand (RANKL), sclerostin, and, according to recent evidence, fibroblast growth factor 23, with consequent impairment of bone remodeling and high bone resorption. Bone loss increases the risk of fragility fractures, and the most commonly used treatments are antiresorptive drugs, followed by anabolic drugs or those with a double effect. In addition, recent data show that natural antioxidants contained in the diet are efficient in preventing and reducing the negative effects of OS on bone remodeling and osteocytes through the involvement of sirtuin type 1 enzyme. Indeed, osteocytes and some of their molecular factors are considered potential biological targets on which antioxidants can act to prevent and reduce bone loss, as well as to promote bone anabolic and regenerative processes by restoring physiological bone remodeling. Several data suggest including antioxidants in novel therapeutic approaches to develop better management strategies for the prevention and treatment of osteoporosis and OS-related bone diseases. In particular, anthocyanins, as well as resveratrol, lycopene, oleuropein, some vitamins, and thiol antioxidants, could have protective and therapeutic anti-osteoporotic effects.

Effect of Oxidative Stress-Induced Apoptosis on Active FGF23 Levels in MLO-Y4 Cells: The Protective Role of 17-ß-Estradiol.

The discovery that osteocytes secrete phosphaturic fibroblast growth factor 23 (FGF23) has defined bone as an endocrine organ. However, the autocrine and paracrine functions of FGF23 are still unknown. The present study focuses on the cellular and molecular mechanisms involved in the complex control of FGF23 production and local bone remodeling functions. FGF23 was assayed using ELISA kit in the presence or absence of 17ß-estradiol in starved MLO-Y4 osteocytes. In these cells, a relationship between oxidative stress-induced apoptosis and up-regulation of active FGF23 levels due to MAP Kinases activation with involvement of the transcriptional factor (NF-kB) has been demonstrated. The active FGF23 increase can be due to up-regulation of its expression and post-transcriptional modifications. 17ß-estradiol prevents the increase of FGF23 by inhibiting JNK and NF-kB activation, osteocyte apoptosis and by the down-regulation of osteoclastogenic factors, such as sclerostin. No alteration in the levels of dentin matrix protein 1, a FGF23 negative regulator, has been determined. The results of this study identify biological targets on which drugs and estrogen may act to control active FGF23 levels in oxidative stress-related bone and non-bone inflammatory diseases.

Protective Role of Natural and Semi-Synthetic Tocopherols on TNFα-Induced ROS Production and ICAM-1 and Cl-2 Expression in HT29 Intestinal Epithelial Cells.

Vitamin E, a fat-soluble compound, possesses both antioxidant and non-antioxidant properties. In this study we evaluated, in intestinal HT29 cells, the role of natural tocopherols, α-Toc and δ-Toc, and two semi-synthetic derivatives, namely bis-δ-Toc sulfide (δ-Toc)2S and bis-δ-Toc disulfide (δ-Toc)2S2, on TNFα-induced oxidative stress, and intercellular adhesion molecule-1 (ICAM-1) and claudin-2 (Cl-2) expression. The role of tocopherols was compared to that of N-acetylcysteine (NAC), an antioxidant precursor of glutathione synthesis. The results show that all tocopherol containing derivatives used, prevented TNFα-induced oxidative stress and the increase of ICAM-1 and Cl-2 expression, and that (δ-Toc)2S and (δ-Toc)2S2 are more effective than δ-Toc and α-Toc. The beneficial effects demonstrated were due to tocopherol antioxidant properties, but suppression of TNFα-induced Cl-2 expression seems not only to be related with antioxidant ability. Indeed, while ICAM-1 expression is strongly related to the intracellular redox state, Cl-2 expression is TNFα-up-regulated by both redox and non-redox dependent mechanisms. Since ICAM-1 and Cl-2 increase intestinal bowel diseases, and cause excessive recruitment of immune cells and alteration of the intestinal barrier, natural and, above all, semi-synthetic tocopherols may have a potential role as a therapeutic support against intestinal chronic inflammation, in which TNFα represents an important proinflammatory mediator.

Current Therapies and New Targets to Fight Melanoma: A Promising Role for the ß3-Adrenoreceptor.

Melanoma is one of the most aggressive types of cancer and the most deadly skin cancer. According to World Health Organization, about 132,000 melanoma skin cancers occur globally each year. Thanks to the efficacy of new therapies, life expectation has been improved over the last years. However, some malignant melanomas still remain unresponsive to these therapies. The ß-adrenergic system, among its many physiological roles, has been recognized as the main mediator of stress-related tumorigenic events. In particular, catecholamine activation of ß-adrenergic receptors (ß-ARs) affects several processes that sustain cancer progression. Among the ß-AR subtypes, the ß3-AR is emerging as an important regulator of tumorigenesis. In this review, we summarize data of different experimental studies focused on ß3-AR involvement in tumor development in various types of cancer and, particularly, in melanoma. Taken together, the preclinical evidences reported in this review demonstrate the crucial role of ß3-AR in regulating the complex signaling network driving melanoma progression. Therefore, a need exists to further disseminate this new concept and to investigate more deeply the role of ß3-AR as a possible therapeutic target for counteracting melanoma progression at clinical level.

Vitamin D regulates claudin-2 and claudin-4 expression in active ulcerative colitis by p-Stat-6 and Smad-7 signaling.

PURPOSE: The tight junctions (TJ) responsible for the integrity of the intestinal barrier are altered in patients with inflammatory bowel disease (IBD), but the physiopathological mechanisms that lead to this alteration are not yet clear. The aim of this study was to determine whether vitamin D, which regulates the integrity of the epithelial barrier by expressing TJ proteins, reduces claudin-2 (Cl-2) levels by inhibiting Stat-6 phosphorylation and whether it increases claudin-4 (Cl-4) levels by blocking Smad-7 activity. METHODS: Biopsies were obtained from inflamed and non-inflamed tracts of the right side colon (caecum or ascending colon) from the same patient with active UC. All the patients were affected by a recent flare-up of ulcerative rectocolitis (RCU), with no previous biologic or immunosuppressive therapy, and all the biopsies were obtained before any treatments. The biopsies were cultured in the presence or not of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). We also used T84 cells as an in vitro model to perform transfection experiments with Stat-6 and Smad-7. RESULTS: Our results indicate that 1,25(OH)2D3 is able to regulate CL-2 and CL-4 protein levels, which are increased and reduced in the intestinal mucosa of UC patients, respectively. In the biopsies obtained from UC patients 1,25(OH)2D3 reduces Cl-2 levels by blocking Stat-6 phosphorylation and increases Cl-4 levels by blocking Smad-7 activity. T84 cells, transfected with siRNA of Stat-6 and Smad-7, showed reduced Cl-2 levels and increased Cl-4 levels, confirming that 1,25(OH)2D3 regulates Cl-2 and Cl-4 by decreasing p-Stat-6 and Smad-7 levels. CONCLUSIONS: Our results indicate that the effects of vitamin D on Cl-2 and Cl-4 are mediated by p-Stat-6 and Smad-7 signal, respectively. The study suggests that vitamin D administration to UC patients could be a useful therapeutic intervention, given that vitamin D deficiency is found in these patients.

Blueberry Juice Antioxidants Protect Osteogenic Activity against Oxidative Stress and Improve Long-Term Activation of the Mineralization Process in Human Osteoblast-Like SaOS-2 Cells: Involvement of SIRT1.

Diets rich in fruits and vegetables with many antioxidants can be very important in the prevention and treatment of osteoporosis. Studies show that oxidative stress, often due to lack of antioxidants, is involved in alteration of bone remodeling and reduction in bone density. This study demonstrates in human osteoblast-like SaOS-2 cells that blueberry juice (BJ), containing 7.5 or 15 µg∙mL-1 total soluble polyphenols (TSP), is able to prevent the inhibition of osteogenic differentiation and the mineralization process due to oxidative stress induced by glutathione depletion. This situation mimics a metabolic condition of oxidative stress that may occur during estrogen deficiency. The effect of BJ phytochemicals occurs through redox- and non-redox-regulated mechanisms. BJ protects from oxidative damage factors related to bone remodeling and bone formation, such as alkaline phosphatase and Runt-related transcription factor 2. It upregulates these factors by activation of sirtuin type 1 deacetylase expression, a possible molecular target for anti-osteoporotic drugs. Quantitative analysis of TSP in BJ shows high levels of anthocyanins with high antioxidant capacity and bioavailability. These novel data may be important to elucidate the molecular and cellular beneficial effects of blueberry polyphenols on bone regeneration, and they suggest their use as a dietary supplement for osteoporosis prevention and therapies.

Resveratrol decreases TNFα-induced ICAM-1 expression and release by Sirt-1-independent mechanism in intestinal myofibroblasts.

Up-regulation of intercellular adhesion molecule-1 (ICAM-1) and its soluble form are involved in the chronic inflammation. For the first time, we demonstrated that resveratrol (RE), a natural polyphenol with antioxidant and anti-inflammatory properties, reduces the increase of expression and release of ICAM-1, due to TNFα-induced oxidative stress, in a myofibroblast cell line derived from human colonic (18Co cells). RE is scavenger of radical oxygen species (ROS) and modulates signaling pathways in which Sirt-1 and NF-κB are involved. Effectively, in TNFα-stimulated 18Co cells RE decreases ROS production and increases Sirt-1 expression and activity, but it reduces TNFα-induced ICAM-1 up-regulation by a Sirt-1-independent mechanism, as demonstrated by EX527 and Sirt-1 siRNA treatments. RE inhibits TNFα-induced activation of NF-κB by reducing both ROS and the degradation of IκB-α, an endogenous inhibitor of NF-κB, with consequent decrease of NF-κB nuclear translocation. This study also shows that NF-κB is not the only factor involved in the TNFα-induced ICAM-1 up-regulation and confirms our previous evidence according to which TNFα increases ICAM-1 levels by redox- and non-redox-regulated mechanisms. RE can represent good and useful support in therapies for intestinal inflammatory diseases in which TNFα plays a crucial role in the increase of adhesion molecule expression.

Blueberry juice protects osteocytes and bone precursor cells against oxidative stress partly through SIRT1.

Oxidative stress and abnormal osteocyte apoptosis are often related to dysregulation of bone turnover and chronic bone loss, and so fruit and vegetables with high antioxidant potential may play an important role in the prevention and/or management of osteoporosis. Osteocytes are the main regulators of bone remodelling. For the first time, we demonstrate here that blueberry juice (BJ), obtained from Vaccinium myrtillus, rich in polyphenols, shows antioxidant and antiosteoclastogenic properties in MLO-Y4 osteocytes. We report that BJ prevents oxidative stress-induced apoptosis and reverses the increase in receptor activator of nuclear factor κB ligand and sclerostin expression, crucial factors for osteoclast activation and bone resorption. BJ is also able to prevent oxidative stress-induced cell cytotoxicity in bone marrow mesenchymal stromal cells (MSCs), which are considered to be an important tool for cell therapy in bone disorders. No significant difference in preventing these events was observed between BJ and blueberry dry extract containing equal amounts of total soluble polyphenols. We have also shown that blueberry acts as both an antioxidant and an activator of sirtuin type 1, a class III histone deacetylase involved in cell death regulation and considered a molecular target for blocking bone resorption without affecting osteoclast survival. Overall, these novel data obtained in osteocytes and MSCs may help us clarify the mechanisms by which blueberry counteracts oxidative stress-induced damage in bone remodelling and osteogenesis at the cellular and molecular level. Our findings are consistent with the reported beneficial effects of blueberry on bone tissue reported in animal studies, which suggest that blueberry may be a useful supplement for the prevention and/or management of osteoporosis and osteogenic process.

Oxidative stress in bone remodeling: role of antioxidants.

ROS are highly reactive molecules which consist of a number of diverse chemical species, including radical and non-radical oxygen species. Oxidative stress occurs as a result of an overproduction of ROS not balanced by an adequate level of antioxidants. The natural antioxidants are: thiol compounds among which GSH is the most representative, and non-thiol compounds such as polyphenols, vitamins and also various enzymes. Many diseases have been linked to oxidative stress including bone diseases among which one of the most important is the osteoporosis. The redox state changes are also related to the bone remodeling process which allows the continuous bone regeneration through the coordinated action of bone cells: osteoclasts, osteoblasts and osteocytes. Changes in ROS and/or antioxidant systems seem to be involved in the pathogenesis of bone loss. ROS induce the apoptosis of osteoblasts and osteocytes, and this favours osteoclastogenesis and inhibits the mineralization and osteogenesis. Excessive osteocyte apoptosis correlates with oxidative stress causing an imbalance in favor of osteoclastogenesis which leads to increased turnover of bone remodeling and bone loss. Antioxidants either directly or by counteracting the action of oxidants contribute to activate the differentiation of osteoblasts, mineralization process and the reduction of osteoclast activity. In fact, a marked decrease in plasma antioxidants was found in aged or osteoporotic women. Some evidence shows a link among nutrients, antioxidant intake and bone health. Recent data demonstrate the antioxidant properties of various nutrients and their influence on bone metabolism. Polyphenols and anthocyanins are the most abundant antioxidants in the diet, and nutritional approaches to antioxidant strategies, in animals or selected groups of patients with osteoporosis or inflammatory bone diseases, suggest the antioxidant use in anti-resorptive therapies for the treatment and prevention of bone loss.

Protective role of benzoselenophene derivatives of resveratrol on the induced oxidative stress in intestinal myofibroblasts and osteocytes.

Resveratrol (RE), a polyphenolic compound present in some food and plants, is characterized by anti-inflammatory and antioxidant properties. However, it is quickly metabolized with consequent loss of its efficacy. In this study, the antioxidant effect of 2-phenyl-benzoselenophene derivatives (VD0, VD1 and VD2) was detected in intestinal myofibroblast and osteocyte cell lines in which the oxidative stress was induced by GSH depletion or starvation, respectively. In fact, the oxidative stress is involved in pathogenesis of inflammatory bowel diseases (IBD) and in increased osteoclastogenesis in osteoporosis. Our results show that these derivatives have major antioxidant power in reducing and/or restoring radical oxygen species to control values than RE itself in both cell types. Moreover, derivatives have different antioxidant capacity in myofibroblasts and in osteocytes and this can be due to different degree of oxidative stress and structural characteristics of these compounds. Some of the synthesized RE analogs have shown anti-bacterial role in IBD and anti-resorptive activity in bone pathologies related to inflammatory and osteoporotic processes. Thus, we suggest benzoselenophene derivatives as good candidates for alternative therapy and/or therapeutic support in these pathologies.

Estrogen inhibits starvation-induced apoptosis in osteocytes by a redox-independent process involving association of JNK and glutathione S-transferase P1-1.

Estrogen deficiency causes bone loss as a result of microdamage, oxidative stress, and osteocyte apoptosis. A relationship between oxidative stress-induced apoptosis, c-Jun N-terminal kinase (JNK) activation, and expression of factors involved in bone remodeling has been demonstrated in osteocytes. However, the molecular regulation of these events in osteocytes treated with 17ß-estradiol (17ß-E2) remains unexplored. The MLO-Y4 murine osteocyte-like cell line was used as a model to study starvation-induced apoptosis and ROS production during 17ß-E2 treatment. Expression of glutathione S-transferase P1-1 (GSTP1-1), receptor activator kB ligand (RANKL), osteoprotegerin (OPG), sclerostin, and kinases activation were measured by western blot. In addition, the GSTP1-1/JNK association was assessed by immunoprecipitation, and GSTP1-1 involvement in the osteocyte response to 17ß-E2 was detected by specific siRNA transfection. 17ß-E2 prevents starvation-induced apoptosis (DNA fragmentation and caspase activation), the increase in sclerostin expression and the RANKL/OPG ratio, which are all related to JNK activation due to oxidative stress in osteocytes. This occurs through GSTP1-1 overexpression, which can inhibit JNK activation by formation of a GSTP1-1/JNK complex. No early antioxidant action of 17ß-E2 has been found but the estrogen effect is similar to N-acetylcysteine which, by increasing the intracellular redox state, maintains JNK bound to GSTP1-1. Thus, the antiapoptotic and osteogenic effect of 17ß-E2 in MLO-Y4 occurs by a redox-independent process involving GSTP1-1/JNK association. This study clarifies at molecular level the effect of 17ß-E2 on osteocyte activity and identifies a possible role of GSTP1-1 and JNK activity in bone remodeling and repair mechanisms.

Tumor Necrosis Factor-Alpha Up-Regulates ICAM-1 Expression and Release in Intestinal Myofibroblasts by Redox-Dependent and -Independent Mechanisms.

Intercellular adhesion molecule-1 (ICAM-1) is distributed and expressed on cell surface and is present in circulation as soluble form (sICAM-1). Tumor necrosis factor-alpha (TNFα) and radical oxygen species (ROS) up-regulate the expression of ICAM-1. This study demonstrates for the first time in 18 Co cells, a myofibroblast cell line derived from human colonic mucosa, an up-regulation of ICAM-1 expression and sICAM-1 release induced by oxidative stress and TNFα stimulation. The intracellular redox state was modulated by L-buthionine-S,R-sulfoximine (BSO) or N-acetylcysteine (NAC), inhibitor and precursor respectively of GSH synthesis. ROS production increases in cells treated with BSO or TNFα, and this has been related to an up-regulation of ICAM-1 expression and sICAM-1 release. The involvement of metalloproteinases in ICAM-1 release has been demonstrated. Moreover, also expression and activation of A disintegrin and metalloproteinase 17, a membrane-bound enzyme known as TNFα-converting enzyme (TACE), have been related to ROS levels. This suggests the possible involvement of TACE in the cleavage of ICAM-1 on cell surface in condition of oxidative stress. NAC down-regulates the expression and release of ICAM-1 as well as the expression and activation of TACE. However, in TNFα stimulated cells NAC treatment reduces only in part ICAM-1 expression and sICAM-1 release. Given this TNFα may also act on these events by a redox-independent mechanism.