Ewa Ambrożewicz

Hydroxyapatite-based biomaterials are commonly used in surgery to repair bone damage. However, the introduction of biomaterials into the body can cause metabolic alterations, including redox imbalance. Because vitamins D3 and K (K1, MK-4, MK-7) have pronounced osteoinductive, anti-inflammatory, and antioxidant properties, it is suggested that they may reduce the adverse effects of biomaterials. The aim of this study was to investigate the effects of vitamins D3 and K, used alone and in combination, on the redox metabolism of human osteoblasts (hFOB 1.19 cell line) cultured in the presence of hydroxyapatite-based biomaterials (Maxgraft, Cerabone, Apatos, and Gen-Os). Culturing of the osteoblasts in the presence of hydroxyapatite-based biomaterials resulted in oxidative stress manifested by increased production of reactive oxygen species and decrease of glutathione level and glutathione peroxidase activity. Such redox imbalance leads to lipid peroxidation manifested by an increase of 4-hydroxynonenal level, which is known to influence the growth of bone cells. Vitamins D3 and K were shown to help maintain redox balance and prevent lipid peroxidation in osteoblasts cultured with hydroxyapatite-based biomaterials. The strongest effect was observed for the combination of vitamin D3 and MK-7. Moreover, vitamins promoted growth of the osteoblasts, manifested by increased DNA biosynthesis. Therefore, it is suggested that the use of vitamins D3 and K may protect redox balance and support the growth of osteoblasts affected by hydroxyapatite-based biomaterials.

Inflammatory granulocytes are characterized by an oxidative burst, which may promote oxidative stress and lipid modification both in affected tissues and on a systemic level. On the other hand, redox signaling involving lipid peroxidation products acting as second messengers of free radicals play important yet not fully understood roles in the pathophysiology of inflammation and various stress-associated disorders. Therefore, the aim of this study was to evaluate the onset of oxidative stress and alterations of enzyme-dependent lipid metabolism resulting from redox imbalance in granulocytes and plasma obtained from patients with psoriasis vulgaris or psoriatic arthritis in comparison to the healthy subjects. The results obtained revealed enhanced activity of pro-oxidant enzymes nicotinamide adenine dinucleotide phosphate (NADPH) and xanthine oxidases in granulocytes with a decrease of enzymatic and non-enzymatic antioxidants in the plasma of psoriatic patients. The nuclear factor erythroid 2–related factor 2 (Nrf2) and its regulators were increased in both forms of psoriasis while heme oxygenase 1 levels were increased only in psoriasis vulgaris. The redox imbalance was associated with decreased levels of phospholipids and of free polyunsaturated fatty acids but with enhanced activity of enzymes involved in lipid metabolism (phospholipase A2, acetylhydrolase PAF, cyclooxygenases 1 and 2) and increased lipid peroxidation products 4-hydroxynonenal, isoprostanes, and neuroprostanes. Increased endocannabinoids and G protein-coupled receptor 55 were observed in both forms of the disease while expression of the cannabinoid type 1 receptor (CB1) was increased only in patients with psoriatic arthritis, which is opposite to the cannabinoid type 2 receptor. This receptor was increased only in psoriasis vulgaris. Changes in protein expression promoted the apoptosis of granulocytes by increased caspases mainly in psoriasis vulgaris. This study indicates that inhibition of the Nrf2 pathway in psoriatic arthritis promotes a redox imbalance. In addition, increased expression of CB1 receptors leads to increased oxidative stress, lipid modifications, and inflammation, which, in turn, may promote the progression of psoriasis into the advanced, arthritic form of the disease.

Fatty acid amide hydrolase (FAAH) inhibitor [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate (URB597) may influence redox balance and blood pressure through the modulation of endocannabinoids levels. Therefore, this study aimed to compare changes in oxidative metabolism and apoptosis in the hearts of rats with spontaneous hypertension (SHR) and secondary hypertension (11-deoxycorticosterone acetate; DOCA-salt rats) treated by URB597 via intraperitoneal injection for 14 days. The results showed that URB597 decreased the activity of NADPH and xanthine oxidases in both groups of rats. Moreover, in the heart of SHR rats, URB597 led to an increase of enzymatic and nonenzymatic antioxidant activity and levels (catalase, vitamin C, glutathione/glutathione disulfide [GSH/GSSG]) and upregulation of the thioredoxin system; however, NRf2 expression was downregulated. The opposite effect in relation to Nrf2 activity and the thioredoxin system was observed in DOCA-salt rats after URB597 administration. Despite improvement in antioxidant parameters, URB597 enhanced oxidative modifications of phospholipids (4-hydroxynonenal and isoprostanes) and proteins (carbonyl groups) in SHR heart, whereas 4-hydroxynonenal and carbonyl groups levels decreased in the heart of DOCA-salt rats. Obtained results suggest that examined lipid mediators are involved in peroxisome proliferator-activated receptors (PPAR)-independent and PPAR-dependent modulation of cardiac inflammatory reactions. Furthermore, decreased expression of pro-apoptotic proteins (Bax and caspase 3 and 9) was observed after URB597 administration in the heart of both groups of hypertensive rats, whereas expression of the antiapoptotic protein (Bcl-2) increased in SHR rats. Long-term administration of URB597 altered cardiac redox status depending on the type of hypertension. URB597 enhanced oxidative metabolism and reduced pro-apoptotic factors in the heart of SHR rats, increasing the probability of heart metabolic disorders occurrence or progression.

Recent evidence suggests that endocannabinoids acting via cannabinoid CB₁receptors may modulate vascular responses of various vasoconstrictors in the rodent systemic vasculature. The aim of the study was to investigate whether endocannabinoids modulate the contractile responses evoked by a thromboxane A₂analog (U46619), angiotensin II (ANG II), serotonin (5-HT), and phenylephrine, which stimulate distinct G_q/11protein-coupled receptors (thromboxane, ANG II type 1, 5-HT₂, and α₁-adrenergic receptors) in isolated endothelium-intact human and rat pulmonary arteries (hPAs and rPAs, respectively). The CB₁receptor antagonist AM251 (1 μM) and diacylglycerol lipase (2-arachidonoylglycerol synthesis enzyme) inhibitor RHC80267 (40 μM) enhanced contractions induced by U46619 in hPAs and rPAs and by ANG II in rPAs in an endothelium-dependent manner. AM251 did not influence vasoconstrictions induced by 5-HT or phenylephrine in rPAs. The monoacylglycerol lipase (2-arachidonoylglycerol degradation enzyme) inhibitor JZL184 (1 μM), but not the fatty acid amide hydrolase (anandamide degradation enzyme) inhibitor URB597 (1 μM), attenuated contractions evoked by U46619 in hPAs and rPAs and ANG II in rPAs. 2-Arachidonoylglycerol concentration-dependently induced relaxation of hPAs, which was inhibited by endothelium denudation or AM251 and enhanced by JZL184. Expression of CB₁receptors was confirmed in hPAs and rPAs using Western blotting and immunohistochemistry. The present study shows the protective interaction between the endocannabinoid system and vasoconstriction in response to U46619 and ANG II in the human and rat pulmonary circulation. U46619 and ANG II may stimulate rapid endothelial release of endocannabinoids (mainly 2-arachidonoylglycerol), leading to CB₁receptor-dependent and/or CB₁receptor-independent vasorelaxation, which in the negative feedback mechanism reduces later agonist-induced vasoconstriction.

Disorder of cellular metabolic homeostasis resulting in the enhanced level of reactive oxygen species, glucose or reactive carbonyl compounds (products of lipid, glucose and amino acids oxidation) causes modifications of cellular components, particularly proteins. As a result of polypeptide chain direct oxidative modifications, carbonyl groups are formed. Reactions of polypeptides with glucose resulting in advanced glycation end products (AGE) generation and reaction with lipid peroxidation products giving advanced lipoxidation end products (ALE) and mechanisms of the above products generation are described. Since accumulation of modified proteins in cells can lead to the development of different diseases including diabetes and its associated diseases as well as neurodegenerative diseases their identification as well as quantitative determination is very important. Obtained data are used in diagnosis and in pharmacotherapy. However, variety of products and a wide range of their concentration in biological samples, cause that different analytic techniques must be used for their analysis. In this review, methods for their determination, with particular emphasis on proteomic approach using state of the art analytical techniques are shown.

Background. UVA is one of the most ubiquitous and penetrating stress factor to human skin and it is crucially involved in various forms of skin damages including the photoaging and cancerogenesis. Mentioned skin damages are connected with redox status imbalance and consequently with cellular metabolic disturbances (changes in phospholipid mediators, including lipid peroxidation products and endocannabinoids). To prevent the redox status changes in skin cells many natural antioxidants which reduce the amount of ROS, resulted from UV in particular, in the cells thereby protecting them from their toxic effects are used. One of them is well known plant-derived polyphenol – rutin. Therefore, the aim of the study was to exam the effect of rutin on redox balance and lipid mediators after UVA irradiation of skin fibroblasts. Methods. Fibroblasts were subjected to UVA [30 J/cm²] and were treated and pretreated with – rutin [25μM]. The redox status was estimated by xanthine and NADPH oxidases activity and ROS generation (ESR/spectrophotometry), enzymatic and non-enzymatic antioxidants (HPLC, spectrophotometry), and lipid mediators: lipid peroxidation products (LCMS; GC/MS) and endocannabinoids (LC/MS) were examined. The cannabinoids receptors, transcriptional factor Nrf2 and its activators/inhibitors, as well as pro-inflammatory, pro- and anti-apoptotic protein levels were also measured (Western blot). Results. Obtained results demonstrate that rutin significantly reduced UVA-induced xanthine and NADPH oxidase activity what was accompanied by changes in ROS generation. Rutin also enhanced antioxidants activity [SOD] and non-enzymatic antioxidant level [GSH, vitamin E and C], which decreased due to UVA exposure. It was shown that UVA-induced Nrf2 activity has been reduced by rutin. Simultaneously, rutin led to increase in Nrf2 inhibitor Bach2 in UVA irradiated cells. It was shown that UVA caused significant decrease in linoleic and arachidonic acids levels and in consequence a strong increase in lipid peroxidation products - MDA and 4-HNE level were observed. Also another lipid mediators – endocannabinoids level was changed during UV irradiation. The significantly reduced level of cytosolic AEA and 2-AG and accompanied higher expression in the level of their receptors CB1, CB2, VR1 and GPR55 were demonstrated. Rutin given after irradiation prevented the most changes caused by UV irradiation. Rutin also protect cells against inflammatory effect after UV irradiation, and decrease apoptotic fibroblasts activity, confirmed by increase in Bcl-2, and decrease in caspases levels. Discussion. Obtained results show that rutin efficiently prevents oxidative stress and inflammatory response formation as well as changes in phospholipid metabolism including endocannabinoids and lipid peroxidation products level, what may be important point to search the skin protecting method against harmful ultraviolet radiation.

Background. UVA is one of the most ubiquitous and penetrating stress factor to human skin and it is crucially involved in various forms of skin damages including the photoaging and cancerogenesis. Mentioned skin damages are connected with redox status imbalance and consequently with cellular metabolic disturbances (changes in phospholipid mediators, including lipid peroxidation products and endocannabinoids). To prevent the redox status changes in skin cells many natural antioxidants which reduce the amount of ROS, resulted from UV in particular, in the cells thereby protecting them from their toxic effects are used. One of them is well known plant-derived polyphenol – rutin. Therefore, the aim of the study was to exam the effect of rutin on redox balance and lipid mediators after UVA irradiation of skin fibroblasts. Methods. Fibroblasts were subjected to UVA [30 J/cm²] and were treated and pretreated with – rutin [25μM]. The redox status was estimated by xanthine and NADPH oxidases activity and ROS generation (ESR/spectrophotometry), enzymatic and non-enzymatic antioxidants (HPLC, spectrophotometry), and lipid mediators: lipid peroxidation products (LCMS; GC/MS) and endocannabinoids (LC/MS) were examined. The cannabinoids receptors, transcriptional factor Nrf2 and its activators/inhibitors, as well as pro-inflammatory, pro- and anti-apoptotic protein levels were also measured (Western blot). Results. Obtained results demonstrate that rutin significantly reduced UVA-induced xanthine and NADPH oxidase activity what was accompanied by changes in ROS generation. Rutin also enhanced antioxidants activity [SOD] and non-enzymatic antioxidant level [GSH, vitamin E and C], which decreased due to UVA exposure. It was shown that UVA-induced Nrf2 activity has been reduced by rutin. Simultaneously, rutin led to increase in Nrf2 inhibitor Bach2 in UVA irradiated cells. It was shown that UVA caused significant decrease in linoleic and arachidonic acids levels and in consequence a strong increase in lipid peroxidation products - MDA and 4-HNE level were observed. Also another lipid mediators – endocannabinoids level was changed during UV irradiation. The significantly reduced level of cytosolic AEA and 2-AG and accompanied higher expression in the level of their receptors CB1, CB2, VR1 and GPR55 were demonstrated. Rutin given after irradiation prevented the most changes caused by UV irradiation. Rutin also protect cells against inflammatory effect after UV irradiation, and decrease apoptotic fibroblasts activity, confirmed by increase in Bcl-2, and decrease in caspases levels. Discussion. Obtained results show that rutin efficiently prevents oxidative stress and inflammatory response formation as well as changes in phospholipid metabolism including endocannabinoids and lipid peroxidation products level, what may be important point to search the skin protecting method against harmful ultraviolet radiation.