Tatyana Simeonova1, Krasimira Stefanova2, Ivelina Himcheva1, Pavlina Yordanova-Laleva3, Boryana Ruseva1, Aneliya Dimitrova1 1) Department of Physiology and Pathophysiology, Medical University Pleven, 2) Diagnostic Consulting Center II-Pleven, 3) Faculty of Pharmacy, Medical University-Pleven, Bulgaria. Journal of IMAB Annual Proceeding (Scientific Papers). 2020 Jan-Mar;26(1) Journal of IMAB ISSN: 1312-773X https://www.journal-imab-bg.org

Introduction: Melatonin, the principal secretory product of the pineal gland, has antioxidant functions as a potent antioxidant and free radical scavenger. Objectives of the present study were to investigate the effect of melatonin against inflammatory response, burn-induced oxidative damage and apoptotic changes of rat liver. Methods: Melatonin (10 mg /kg, i.p.) was applied immediately after 30% of total body surface area (TBSA) burns on male Wistar rats. The level of malondialdehyde (MDA) as a marker of an oxidative stress was quantified by thiobarbituric method. Hepatic TNFα and IL-10 as inflammatory markers were assayed by ELISA. Using light immunоchistochemistry the expression Ki67 proliferative marker was investigated. Results: Hepatic MDA and TNF-α levels increased significantly following burns without any change in IL-10 level. Intracellular vacuolization, hepatic cell degeneration and apoptosis occurred in rats after burns. The number of apoptotic cells was increased whereas no significant increase in Ki67 proliferative marker. Melatonin decreased the MDA and TNF-α content and increased the IL-10 level. It also limited the degenerative changes and formation of apoptotic cells in rat liver but did not increase expression of the marker of proliferation. In conclusion, our data show that melatonin relieves burn-induced hepatic damage associated with modulation of the proinflammatory/anti-inflammatory balance, mitigation of lipid peroxidation and hepatic apoptosis.

Unacylated ghrelin (UAG) amounts to 80-90% of the circulating orexigenic hormone ghrelin. Studies suggest that both acyl ghrelin (AG) and UAG may mediate peripheral biological actions and UAG can act as a potent functional inhibitor of ghrelin. The aim of this study was to track changes in unacylated ghrelin in plasma in a control and three experimental (fructose, sucrose and aspartame) groups of rats and to compare them with some morphological and metabolic parameters. An 8-week burdening of 28 male Wistar rats with 15% fructose (Group F, n = 7), 10% sucrose (Group S, n = 7) and 0.3% aspartame solutions (Group A, n = 7) was carried out. An increase in average body weight was found in the following order: sucrose group > fructose group > aspartame group > controls. Significant difference was found in the mesenteric fat depot weight of Group F vs. Group C. An increase in unacylated ghrelin levels and in general metabolic parameters (glucose, triglycerides, total and LDL-cholesterol, AST, ALT) of the fructose and sucrose groups compared to controls was registered. Moreover, changes in some metabolic markers in the aspartame group were also seen. In conclusion, the results of the current study suggest that elevated unacylated ghrelin might trigger changes in the regulation of food intake and further development of obesity, metabolic disorders and chronic noninfectious diseases.

Introduction. Osteoporosis is a disease characterized by decreased bone density and destruction of the microarchitectonics of the bone structure. This leads to increased bone fragility and risk of fracture, particularly of the hip, spine, wrist and shoulder. Osteoporosis is known as „The Silent Epidemic of the Century“ because bone loss occurs without symptoms. An altered ovarian function is one of the most common causes of osteoporosis. Indicators for altered bone homeostasis are the changes in serum levels of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Objective. The aim of current study was to determine the activity of alkaline phosphatase (ALP) and serum concentrations of MMP-9, MMP-13 and TIMP-1 in the ovariectomized rats. Materials and Methods. An experiment was performed on 35 female Wistar rats at reproductive age – 2 months divided into 2 groups: group 1 (G1)-20 animals were sham-operated (sham) and group 2 (G2)-15 were ovariectomized (ovx). Results. The concentrations of ALP, MMP-9, MMP-13 and TIMP-1 in G2 were significantly increased compared to G1 (p<0.05). Conclusion. Our study confirmed that the serum activity of ALP, which is a marker of bone formation, was elevated in rats with OVX-induced osteoporosis. Although the level of TIMP-1 is increased, the level of MMP 9 in G2 is also increased, that confirms the thesis that MMP-9 may be a marker for osteoclast activity.

During the last years several studies have been performed to develop new materials for stimulation of bone healing and regeneration. Whilst it is known that osteoporotic bone is generally not cancellous in nature and has thin walls, the essential design paradigm of implants does not reflect this application. We have proposed a simple system for assisted bone repair by in bone injection of “intelligent” polymers combined with nanodiamonds and progenitor cells. A Safe-by-Design approach was undertaken. It utilizes autologous stem cells transplantation in combination with supportive bioresorbable matrices and bioactive molecules for enhancing growth and repair. The endothelial progenitor cells obtained from peripheral blood of the same patient may be cultured in vitro in the presence of different stimuli and/or nanoparticles to undergo osteoblasts differentiation, prior to autologous transplantation. This injectable therapy could also be used for: (1) modifying the bone interior morphology, porosity and interconnectivity, which are extremely important for cell adhesion, proliferation and differentiation; (2) prophylactic treatment for high risk patients to prevent fractures, especially the hip and vertebrae; (3) providing exceptional repair of the osteoporotic bone by releasing pharmaceuticals to the specific sites with the purpose of accelerating healing, promoting angiogenesis, reducing the risk of infection, etc. ‡ §,| ‡ | © Grigoryan A et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Endothelin-1 (ET-1) is one of the most potent vasoconstrictors known to date. While its plasma or serum concentrations are elevated in some forms of experimental and human hypertension, this is not a consistent finding in all forms of hypertension. Matrix metalloproteinases -2 and -9 (MMP-2 and MMP-9), which degrade collagen type IV of the vascular basement membrane, are responsible for vascular remodeling, inflammation, and atherosclerotic complications, including in type 2 diabetes (T2D). In our study, we compared concentrations of ET-1, MMP-2, and MMP-9 in pre-hypertensive (PHTN) and hypertensive (HTN) T2D patients with those of healthy normotensive controls (N). ET-1, MMP-2, and MMP-9 were measured by ELISA. Concentrations of ET-1 in PHTN and N were very similar, while those in HTN were significantly higher. Concentrations of MMP-2 and MMP-9 in PHTN and HTN were also significantly higher compared to N. An interesting result in our study is that concentrations of MMP-2 and MMP-9 in HTN were lower compared to PHTN. In conclusion, we showed that increased production of ET-1 in patients with T2D can lead to long-lasting increases in blood pressure (BP) and clinical manifestation of hypertension. We also demonstrated that increased levels of MMP-2 and MMP-9 in pre-hypertensive and hypertensive patients with T2D mainly reflect the early vascular changes in extracellular matrix (ECM) turnover.

Website: www.phcog.com DOI: 10.4103/0973-1296.131024 Quick Response Code: P H C O G M A G . O R I G I N A L A R T I C L E Valcheva-Kuzmanova, et al.: Effect of AMFJ in amiodarone-induced pneumotoxicity Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 133 of the release of pro-infl ammatory cytokines such as tumor necrosis factor alpha and interleukins (interleukin IL-1 beta, IL-6, IL-8).[19,20] There are numerous pathologic similarities between human and rodent lungs and as such, rodent model presents an excellent tool to investigate pathologic changes in vivo.[21] The aim of the study was to investigate the effect of A. melanocarpa fruit juice (AMFJ) in a rat model of AD-induced pulmonary toxicity. MATERIALS AND METHODS Experimental substances AD hydrochloride and all other chemicals and reagents wereof analytical grade and were purchased from Sigma-Aldrich Company (Germany). The Quantikine Rat IL-6 and IL-10 immunoassay kits were from R and D Systems (USA). AMFJ was produced from A. melanocarpa Elliot fruits grown in the Balkan Mountains, Bulgaria. They were handpicked in September, crushed and squeezed. The juice was fi ltered, pasteurized at 80°C for 10 min and stored at 0°C until the experiment. The contents of phenolic substances in 100 mL AMFJ were: Total phenolics, 709.3 ± 28.1 mg as gallic acid equivalents, determined spectrophotometrically according to the Folin-Ciocalteu procedure;[22] total fl avonoids, 189.4 ± 8.6 mg as catechin equivalents, measured by a colorimetric assay developed by Zhishen et al.;[23] total anthocyanins, 106.8 ± 6.2 mg as cyanidin-3-glucoside equivalents, determined by a pH-differential spectrophotometry at pH 1.0 and pH 4.5;[24] quercetin, 11.8 mg, measured by a high-performance liquid chromatography method.[25] The values were the mean of duplicate determinations of three samples. Animals and experimental treatments The study was carried out on 96 male Wistar rats (weight 220-250 g, age 4 months). The animals were obtained from the Research and Laboratory Animal Breeding Center of Slivnitsa (Bulgaria) and were housed in the university animal quarters for 1 month at a temperature of 22°C ± 2°C and humidity of 50 ± 10%, given normal pelleted diet and water AD libitum. All procedures concerning animal treatment and experimentation were conducted in compliance with the national laws and policies, in conformity with the international guidelines (European Economic Community EEC Council Directive 86/609, IL 358, 1, December 12, 1987). The animals were divided into four groups of 24 rats. Each group was subdivided into four subgroups of six rats. One subgroup of each group was sacrifi ced on days 3, 5, 10, and 28 under thiopental anesthesia (50 mg/kg) after receiving the respective treatment in the course of 2, 4, 9, and 10 days, respectively. Group 1 (Control) received two intratracheal (i.t.) instillations of sterile distilled water (2 mL/kg) on days 0 and 2. The four subgroups of Group 1 received distilled water (10 mL/kg) orally through an orogastric cannula from day 1 to days 2, 4, 9, and 10, respectively. Group 2 (AD) received two i.t. instillations of AD (6.25 mg/kg, as a 3.125 mg/mL water solution) on days 0 and 2.[26] The subgroups of Group 2 received distilled water (10 mL/kg) orally through an orogastric cannula from day 1 to days 2, 4, 9, and 10, respectively. Group 3(AD + AMFJ5) was treated with AD i.t. on days 0 and 2, and from day 1 to days 2, 4, 9, and 10 the respective subgroups received AMFJ orally at a dose of 5 mL/kg diluted with distilled water to a total volume of 10 mL/kg. Group 4 (AD + AMFJ10) was treated with AD i.t. on days 0 and 2, and from day 1 to days 2, 4, 9, and 10 the respective subgroups received AMFJ orally at a dose of 10 mL/kg. AD was dissolved in distilled water at 60°C and allowed to cool to room temperature before the i.t. instillation. Lung weight coeffi cient The body weight and lung weight were measured for each animal. The lung weight coeffi cient (organ weight in mg/100 g body weight) was calculated. Bronchoalveolar lavage Bronchoalveolar lavage fl uid (BALF) was obtained on days 3, 5, and 10. Rats were sacrifi ced by exsanguination through cutting v. renalis. The chest was opened, and the lungs were perfused in situ via the right heart ventricle with saline (30 mL). Triple lavage of the left lung through the trachea with a total of 5 mL of saline was performed. The volume of fl uid recovered ranged from 80% to 90% of the fl uid introduced. Cytological assays of BALF One aliquot of the BAL F was used for total cell count ×105/L. The cells were removed by centrifugation at 300 × g for 10 min. The cell pellet was resuspended in 1 mL of saline. The cell differentials were analyzed using the procedure of Danos and Keebler, modifi ed by Saltini et al.[27] Cells were collected on nitrocellulose fi lters 25 mm in diameter with 5 μm pores (SWP-025-00 Catalogue number SMWP02500; Millipore Corp.) and were stained with H and E. Biochemical assays of BALF After centrifugation of BALF, the supernatant was used for the measurement of enzyme activities such as lactate Valcheva-Kuzmanova, et al.: Effect of AMFJ in amiodarone-induced pneumotoxicity 134 Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 dehydrogenase (LDH), acid phosphatase (AcP), and alkaline phosphatase (AlP) by the methods of Bergmeyer et al.[28] Total protein content was measured by the method of Lowry et al.[29] The enzyme activities are represented in U/L, and total protein in mg/mL of BALF. Biochemical assays of lung homogenate Lung homogenate was obtained from the right lung. The tissue was homogenized with potassium chloride KCl (1.15%) in 1:10 ratio. The homogenate was centrifuged (9000 × g, 30 min), and the supernatant was stored on ice. Malondialdehyde (MDA) content i n nmol/g tissue was measured on days 3, 5, and 10 by the method of Ohkaw a et al.[30] Hydroxyproline (HP) levels (in μg/mL) were measured in lung homogenate of rats sacrifi ced on day 28 as described by Bergman and Loxley.[31] The method is based on the release of free HP from collagen by acid hydrolysis. Immunological assays of rat serum The serum of the experimental animals was used for the measurement of IL-6 and IL-10 in pg/mL on days 3, 5, and 10 by the ELISA method in accordance with the immunoassay kits manufacturer’s instructions. Statistical analysis Results are presented as mean ± SEM. The data were tested by one-way ANOVA, followed by Dunnett’s multiple comparison post-test to identify signifi cant difference. A level of P < 0.05 was considered signifi cant. All analyses were performed using GraphPad Prism Statistical Software.

Purpose: Hemodynamic stress in arterial hypertension leading to increased production of endothelin-1 (ET-1). Changes in the extracellular matrix are controlled largely by methalloproteinase-2 (MMP-2) and methalloproteinase-9 (MMP-9) which play an important role in vascular remodeling of hypertension. C-reactive protein (CRP) is an acute phase protein which is synthesized by hepatocytes under the effect of interleukin-6 (IL-6) in inflammation. The purpose of the study was to investigate the relationship of ET-1, MMP-2, MMP-9 and CRP with the degree of arterial hypertension and the systemic and vascular inflammatory response. Methods: Three groups were formed: group I - 31 patients with mild hypertension (MH); group II - 29 patients with severe hypertension (SH); group III - 15 persons in a control group (CG). ET-1 was determined by ELISA kit of "Biomedika", MMP-2 and MMP-9 by ELISA kit of the "R&D Systems", and the CRP- through immunoturbidimetric method with monoclonal anti-CRP antibodies. The analysis used the statistical program STATGRAPHICS. Results: It was found that there was a statistically significant difference between serum concentration of ET-1 (pg/ml) at MH when compared to CG (p = 0.0189) and SH compared to CG (p = 0.0181). There is a statistically significant difference between concentration of MMP-9 (ng/ml) of SH when compared to CG (p = 0.0010) and between MH when compared to SH (p = 0.0006). There is no statistically significant difference of MMP-2 (ng/ml) between the three groups (p> 0.05). There is also a statistically significant difference between the concentrations of CRP (mg/l) at MH when compared with CG (p = 0.0170) and SH when compared with CG (p = 0.0366). Conclusions: Serum levels of ET-1 are increased in the groups with arterial hypertension, which demonstrates its role in the pathogenesis of the disease. The levels of MMP-9 are greatly reduced in patients with severe hypertension which can be used as an indicator of increased cardiovascular risk in these patients. Serum levels of MMP-2 do not show specific changes at the different degrees of hypertension. Based on our experimental data can be concluded that the levels of MMP-9 in arterial hypertension are defined by complex causes - the degree of hypertension, drug therapy and accompanying cardiovascular diseases and complications. The average levels of CRP at MH and SH are significantly increased which confirms the pathogenetic relationship of arterial hypertension with systemic and vascular inflammation.

Background: The fruits of Aronia melanocarpa (Michx.) Elliot is extremely rich in biologically active polyphenols. Objective: We studied the protective effect of A. melanocarpa fruit juice (AMFJ) in a model of amiodarone (AD)-induced pneumotoxicity in rats. Materials and Methods: AD was instilled intratracheally on days 0 and 2 (6.25 mg/kg). AMFJ (5 mL/kg and 10 mL/kg) was given orally from day 1 to days 2, 4, 9, and 10 to rats, which were sacrificed respectively on days 3, 5, 10, and 28 when biochemical, cytological, and immunological assays were performed. Results: AMFJ antagonized AD-induced increase of the lung weight coefficient. In bronchoalveolar lavage fluid, AD increased significantly the protein content, total cell count, polymorphonuclear cells, lymphocytes and the activity of lactate dehydrogenase, acid phosphatase and alkaline phosphatase on days 3 and 5. In AMFJ-treated rats these indices of direct toxic damage did not differ significantly from the control values. In lung tissue, AD induced oxidative stress measured by malondialdehyde content and fibrosis assessed by the hydroxyproline level. AMFJ prevented these effects of AD. In rat serum, AD caused a significant elevation of interleukin IL-6 on days 3 and 5, and a decrease of IL-10 on day 3. In AMFJ-treated rats, these indices of inflammation had values that did not differ significantly from the control ones. Conclusion: AMFJ could have a protective effect against AD-induced pulmonary toxicity as evidenced by the reduced signs of AD-induced direct toxic damage, oxidative stress, inflammation, and fibrosis.

Haemodynamic stress in arterial hypertension leads to increased production of endothelin-1 (ET-1). Changes in the extracellular matrix are controlled largely by methalloproteinase-2 (MMP-2) and methalloproteinase-9 (MMP-9) which play an important role in vascular remodelling of hypertension. Creactive protein (CRP) is an acute phase protein which is synthesized by hepatocytes under the effect of interleukin-6 (IL-6) in inflammation. The purpose of the study was to investigate the relationship of ET-1, MMP-2, MMP-9 and CRP with the degree of arterial hypertension and the systemic and vascular inflammatory response. Three groups were formed: group I 31 patients with mild hypertension (MH); group II 29 patients with severe hypertension (SH); group III 15 persons in a control group (CG). ET-1 was determined by ELISA kit of "Biomedika", MMP-2 and MMP-9 by ELISA kit of the "R&D Systems", and the CRP through immunoturbidimetric method with monoclonal anti-CRP antibodies. The analysis used the statistical program STATGRAPHICS. The results obtained show that the serum levels of ET-1 are increased in the arterial hypertension (p < 0.05) which demonstrates its role in the pathogenesis of the disease. Levels of MMP-9 in severe hypertension were reduced almost twice (p = 0.001) which is probably a mechanism that supports the stability of the vessel wall at increased haemodynamic stress. Levels of MMP2 did not show a significant change (p > 0.05). Mean values of CRP were higher in patients with mild hypertension in comparison with the control group (p < 0.05) and those with severe hypertension which indicates that the inflammatory process is predominant from an early stage of hypertension.

Purpose: Matrix metalloproteinases -2 and -9 (MMP-2 and MMP-9) are representatives of the group of gelatinases that have similar effect, resulting in the degradation of collagen type IV. MMP-2 and MMP-9 have a key role in the pathogenesis of atherosclerosis. The aim of the present study was to determine the extent of the immunohistochemical expression of MMP-2 and MMP-9 in the vascular wall of the abdominal aorta in patients with atherosclerotic changes. Methods: We investigated by immunohistochemistry (polyclonal antibodies,class IgG, Santa Cruz Biotechnology, USA) the expressional changes of MMP-2 and MMP-9 in abdominal aorta obtained from autopsied cases. We used 5 nonatherosclerotic (control group - G1) and 20 atherosclerotic segments (group - G2) from human abdominal aorta. These arteries were colected at autopsy from 25 cases within 5 hours of death. Results: In the nonatherosclerotic aorta MMP-2 and MMP-9 were expressed mainly in the endothelium and the smooth muscle cells (VSMC) of the vascular wall. The expression of MMP-2 and MMP-9 was significantly increased (p<0.05) in the atheromatous plaques of atherosclerotic abdominal aorta in comparison with nonatherosclerotic. Intense expression of MMPs-2 in media

Experimental data suggest that oxidative stress is involved in hypertension. The aim of this study was to evaluate and compare the effect of chronic systematic treatment with AT1 receptor antagonist losartan on arterial blood pressure and the production of oxidative damage in the brain of normotensive Wistar and spontaneously hypertensive rats (SHRs). Drug administration was conducted via subcutaneous osmotic minipumps for 14 days (10 mg/kg per day). Spontaneously hypertensive rats showed an increase in the arterial blood pressure compared to normotensive Wistar rats. Long-term losartan exposure attenuated hypertension in SHRs. The level of lipid peroxidation was higher in both the frontal cortex and the hippocampus of SHRs compared to Wistar rats. However, chronic block of AT1 receptors decreased the level of lipid peroxidation of the above mentioned brain structures in Wistar and SHRs. Losartan influenced positively the citosolic superoxide dismutase (SOD/CuZn) activity in both the frontal cortex and the hippocampus of SHRs while it enhanced the activity of this antioxidant enzyme only in the hyppocampus of Wistar rats. No changes in the mitochondrial SOD/Mn activity in both the frontal cortex and the hippocampus were detected after losartan treatment in Wistar and SHRs. These data suggest that the strain differences of the level of oxidative stress in the frontal cortex and the hippocampus as well as arterial blood pressure determine different responses after long-term infusion with AT1 receptor antagonist.

Our aim was to investigate the effects of MnTE-2-PyP on some markers of inflammation and lipid peroxidation in mouse asthma model. 24 female mice were divided into four groups: group 1, controls; group 2, injected with ovalbumin (OVA); group 3, treated with MnTE-2-PyP; and group 4, treated with ovalbumin and MnTE-2-PyP. The mice from groups 2 and 4 were injected with 10 μg OVA and 1 mg Imject Alum? in 100 μL phosphate buffered saline (PBS) on days 0 and 14. The animals from groups 1 and 3 were injected with 100 μL PBS + Imject Alum? (1:1). The animals from groups 2 and 4 were subjected to a 30 min aerosol challenge of 1% ovalbumin on days 24, 25 and 26 and those from groups 1 and 3 were subjected to aerosol challenge of PBS at the same time and duration. One hour before inhalation, and 12 hours later the animals from groups 3 and 4 were injected with 100 μL MnTE-2-PyP solution in PBS containing 5 mg/kg. The total cell number, total protein content and 8-isoprostane, IL-4 and IL-5 levels in the bronchialveolar lavage fluid increased in group 2 as compared to the control group. Malone dialdehyde content in the lung homogenate and IgE levels in the serum also increased in this group. The total cell number, total protein content, and levels of 8-isoprostane, IL-4, IL-5 and IgE decreased significantly in group 4 as compared to the OVA group. The parameters set out above in group 3 did not differ significantly from those of the control group. MnTE-2-PyP administered intraperitoneally, 48 hours after the last nebulization, reduced the inflammation and lipid peroxidation in mouse asthma model.

Advanced glycation end products (AGEs) have been implicated in the pathogenesis of many disorders. Excessive accumulation of AGEs has been shown to lead to tissue damage through a variety of mechanisms including alteration of tissue protein structure and function, or increased generation of reactive oxygen species. On the other hand, oxidative stress accelerates AGEs formation. Selenium (Se) is an exogenous antioxidant which performs its biological role via selenoprotein expression. The aim of this study was to investigate the effect of selenium (Se) on the serum level of antibodies against AGEs (anti-AGEs abs) and lipid peroxidation in young normotensive Wistar (WKY) and spontaneously hypertensive (SHR) rats. Fifteen male WKY and twenty male SHR, 16 weeks old, divided into 4 groups, were tested after being on a Se-adequate diet (NSe) or a Se-supplemented diet (HSe) for eight weeks. The Se nutritional status was assessed by measuring Glutathione peroxidase (GPx-1) activity in whole blood, using "Ransel" kit of Randox Laboratories LTD. The serum anti-AGEs abs level was determined by the method of indirect ELISA. The serum lipid hydroperoxide concentration (ROOH) was evaluated by the method of Yagi. The results showed that Se supplementation increased GPx-1 activity of whole blood of the rats (p < 0.05). The serum anti-AGEs abs and ROOH levels of SHR NSe were higher than those detected in WKY NSe, and they were significantly reduced in the SHR HSe (p < 0.05). In conclusion, selenium supplementation reduces oxidative stress and serum anti-AGEs abs level in SHR.

Dimitrova, A.1; Tisheva, S.2; Atanasova, M.3; Georgieva, M.4; Russeva, A.5 Author Information