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Marta Szandruk-Bender,1 Anna Merwid-Ląd,1 Benita Wiatrak,1 Maciej Danielewski,1 Stanisław Dzimira,2 Danuta Szkudlarek,3 Łukasz Szczukowski,4 Piotr Świątek,4 Adam Szeląg1 1Department of Pharmacology, Wroclaw Medical University, Wrocław, Poland; 2Department of Pathology, Wroclaw University of Environmental and Life Sciences, Wrocław, Poland; 3Foundation of the Wroclaw Medical University, Wroclaw Medical University, Wrocław, Poland; 4Department of Chemistry of Drugs, Wroclaw Medical University, Wrocław, PolandCorrespondence: Marta Szandruk-BenderDepartment of Pharmacology, Wroclaw Medical University, Mikulicza-Radeckiego 2, Wrocław, 50-345, PolandTel +48717841438Fax +48717840094Email marta.szandruk@umed.wroc.plBackground and Purpose: Due to the risk of gastrointestinal damage and various tissue toxicity associated with non-steroidal anti-inflammatory drugs (NSAIDs) use, investigating new anti-inflammatory agents with efficacy comparable to that of NSAIDs but reduced toxicity is still a major challenge and a clinical need. Based on our previous study, new 1,3,4-oxadiazole derivatives of pyrrolo[3,4-d]pyridazinone, especially 6-butyl-3,5,7-trimethyl-1-[[4-[[4-(4-nitrophenyl)piperazin-1-yl]methyl]-5-thioxo-1,3,4-oxadiazol-2-yl]methoxy]pyrrolo[3,4-d]pyridazin-4-one and 6-butyl-1-[[4-[[4-(4-chlorophenyl)-4-hydroxy-1-piperidyl]methyl]-2-thioxo-1,3,4-oxadiazol-5-yl]methoxy]-3,5,7-trimethyl-pyrrolo[3,4-d]pyridazin-4-one (hereafter referred to as the compounds 10b and 13b, respectively) seem to be promising anti-inflammatory agents. This study aimed to elucidate the effects of these two new derivatives on the course of experimental rat inflammation, liver and kidney function, and gastric mucosa.Methods: The anti-inflammatory effect of compounds 10b and 13b was evaluated using the carrageenan-induced paw edema test in rats. The increase in paw volume (paw edema), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α) and myeloperoxidase (MPO) levels, histological alterations, and inflammatory cell infiltration in paw tissue were determined. Serum alanine transaminase (ALT) and aspartate transaminase (AST) activities, serum urea and creatinine levels, as well as changes in gastric mucosa, were measured as indicators of hepatic, renal, and gastric toxicity.Results: Pretreatment with both novel derivatives at 10 mg/kg and 20 mg/kg doses reduced paw edema, counteracted the increased PGE2 and TNF-α levels, reduced the influx of inflammatory cells, and decreased histopathological alterations in paw tissue. Compound 13b at a dose of 20 mg/kg was more effective than indomethacin in reversing the increased TNF-α levels and reducing the influx of inflammatory cells. Only compound 13b at all studied doses (5, 10, or 20 mg/kg) counteracted the increased MPO level in paw tissue. Both compounds neither caused alterations in ALT, AST, urea, creatinine parameters nor gastric mucosal lesions.Conclusion: New compounds exert an anti-inflammatory effect, presumably via inhibiting inflammatory mediators release and inflammatory cell infiltration. Moreover, both possess a more favorable benefit–risk profile than indomethacin, especially compound 13b.Keywords: carrageenan, anti-inflammatory agents, pyrrolo[3,4-d]pyridazinone, 1,3,4-oxadiazole, inflammatory mediators, toxicity

Alpha-linolenic acid (ALA) is widely regarded as the main beneficial component of flax for the prevention of cardiovascular disease. We evaluated the effect of the transgenic flaxseed W86—which is rich in ALA—on the lipid profile, atherosclerosis progression, and vascular reactivity in hypercholesterolemic rabbits compared to the parental cultivar Linola with a very low ALA content. Rabbits were fed a basal diet (control) or a basal diet supplemented with 1% cholesterol, 1% cholesterol and 10% flaxseed W86, or 1% cholesterol and 10% Linola flaxseed. A high-cholesterol diet resulted in an elevated plasma cholesterol and triglyceride levels compared to the control animals. Aortic sections from rabbits fed Linola had lower deposits of foamy cells than those from rabbits fed W86. A potassium-induced and phenylephrine-induced contractile response was enhanced by a high-cholesterol diet and not influenced by the W86 or Linola flaxseed. Pretreatment of the aortic rings with nitro-L-arginine methyl ester resulted in a concentration-dependent tendency to increase the reaction amplitude in the control and high-cholesterol diet groups but not the flaxseed groups. Linola flaxseed with a low ALA content more effectively reduced the atherosclerosis progression compared with the W86 flaxseed with a high concentration of stable ALA. Aorta contractility studies suggested that flaxseed ameliorated an increased contractility in hypercholesterolemia but had little or no impact on NO synthesis in the vascular wall.

Cornelian cherry (Cornus mas L.) fruits possess potential cardiovascular, lipid-lowering and hypoglycemic bioactivities. The aim of this study is to evaluate the influence of resin-purified cornelian cherry extract rich in iridoids and anthocyanins on several transcription factors, intima/media ratio in aorta and serum parameters, which determine or are valuable indicators of the adverse changes observed in the course of atherosclerosis, cardiovascular disease, and metabolic syndrome. For this purpose, male New Zealand rabbits were fed a diet enriched in 1% cholesterol for 60 days. Additionally, one group received 10 mg/kg b.w. of cornelian cherry extract and the second group 50 mg/kg b.w. of cornelian cherry extract. PPAR-α and PPAR-γ expression in the aorta, LXR-α expression in the liver, cholesterol, triglycerides, adipokines, apolipoproteins, glucose and insulin levels in serum, the intima and media diameter in the thoracic and abdominal aorta were determined. Administration of cornelian cherry extract resulted in an enhancement in the expression of all tested transcription factors, a decrease in triglycerides, leptin and resistin, and an increase in adiponectin levels. In addition, a significant reduction in the I/M ratio was observed for both the thoracic and abdominal aorta. The results we have obtained confirm the potential contribution of cornelian cherry extract to mitigation of the risk of developing and the intensity of symptoms of obesity-related cardiovascular diseases and metabolic disorders such as atherosclerosis or metabolic syndrome.

In our previous publication we showed that the oral administration of cornelian cherry fruits prevented feed-induced atherosclerosis. In this study we have examined the effect of cornelian cherry lyophilisate on proteins responsible for regulation of NO synthesis: asymmetric and symmetric dimethylarginine (ADMA, SDMA) and l -arginine in plasma, and dimethylarginine dimethylaminohydrolase (DDAH) in the liver. We have also assessed the systemic and local redox status in the blood and the aorta, and the thickness of the thoracic aorta. We have shown that 60-days of administering lyophilisate to rabbits fed 1% cholesterol increased the l -arginine and l -arginine/ADMA ratio, decreased ADMA and SDMA, increased DDAH activity, and had a positive impact on the redox state in the aorta but not in the blood, measured as decreased MDA, and increased glutathione, GPx, and SOD. Moreover, lyophilisate significantly decreased intima thickness and the intima/media ratio in the thoracic aorta. Concluding, the l -arginine-ADMA-DDAH pathway may contribute to the beneficial effects of the cornelian cherry in feed-induced atherosclerosis.

Disorders of sex development (DSD) are rare in cats. They can be caused by chromosomal aberrations, gene mutations or other undefined factors. The aim of the present study was to compare the histological structure and immunohistochemical reactivity of testes in cats with DSD and in healthy cats. The research material consisted of the gonads of four cats - phenotypic males with an incorrect structure of the reproductive system. The control group consisted of the testes of four healthy cats - routinely castrated phenotypical males. The material was fixed with formalin and embedded in paraffin; the sections were stained with hematoxylin and eosin. The immunohistochemical investigation were performed using monoclonal and polyclonal antibodies directed against desmin, vimentin, actin of smooth muscles, S100 protein and MCM3 protein. The results obtained allow concluding that the testes of cats with DSD differed in certain respects, mainly in the number of blood vessels, from the normal testes. Moreover, the results of immunohistochemical examination indicate that in the testes of cats with DSD the number of supporting cells is lower, the amount of interstitial cells is comparable and spermatogenesis is correct es compared to those determined in the control gonads. The number of blood vessels in cats with DSD is reduced by about 30%. It confirms the recommendations for castration of these animals in order to eliminate the potential inheritance of sex development disorders.

Despite the availability of the current drug arsenal for pain management, there is still a clinical need to identify new, more effective, and safer analgesics. Based on our earlier study, newly synthesized 1,3,4-oxadiazole derivatives of pyrrolo[3,4-d]pyridazinone, especially 10b and 13b, seem to be promising as potential analgesics. The current study was designed to investigate whether novel derivatives attenuate nociceptive response in animals subjected to thermal or chemical noxious stimulus, and to compare this effect to reference drugs. The antinociceptive effect of novel compounds was studied using the tail-flick and formalin test. Pretreatment with novel compounds at all studied doses increased the latency time in the tail-flick test and decreased the licking time during the early phase of the formalin test. New derivatives given at the medium and high doses also reduced the late phase of the formalin test. The achieved results indicate that new derivatives dose-dependently attenuate nociceptive response in both models of pain and exert a lack of gastrotoxicity. Both studied compounds act more efficiently than indomethacin, but not morphine. Compound 13b at the high dose exerts the greatest antinociceptive effect. It may be due to the reduction of nociceptor sensitization via prostaglandin E2 and myeloperoxidase levels decrease.

We established a new B-cell leukaemia cell line CLB70 from a dog with chronic lymphocytic leukaemia. This cell line is positive for CD20, CD45, CD79a, MHC class II, IgG, IgM; weakly positive for CD21; and negative for CD3, CD4, CD5, CD8, CD14, CD34, CD117. PCR for antigen receptor gene rearrangement (PARR) analysis revealed a biclonal immunoglobulin heavy chain (IgH) gene rearrangement and negative result for TCRγ. Western blot analysis of anti- and pro-apoptotic proteins showed increased expression of Bcl-2, Mcl-1, NF-kB, and Ras, and decreased expression of p53. CLB70 cells grow rapidly in vitro and are tumourigenic in nude mice. The CLB70 line is highly sensitive to doxorubicin, less sensitive to etoposide and imatinib, and resistant to piroxicam, celecoxib and dexamethasone. Our results indicate that CLB70 cells are derived from mature B-cells and they may be a useful tool for the development of new therapeutic strategies for both dogs and humans.

Background Cornelian cherry (Cornus mas L.) is a plant growing in southeast Europe, in the past used in folk medicine. There are many previous publications showing the preventive effects of (poly)phenolic compounds, especially anthocyanins, on cardiovascular diseases, but there is a lack of studies comparing the effects of (poly)phenolics and other constituents of fruits. Objectives We have attempted to determine if iridoids and anthocyanins from cornelian cherry fruits may affect the formation of atherosclerotic plaques in the aorta as well as lipid peroxidation and oxidative stress in the livers of cholesterol-fed rabbits. Material and methods Fractions of iridoids and anthocyanins were analyzed using the high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) methods. Loganic acid (20 mg/kg b.w.) and a mixture of anthocyanins (10 mg/kg b.w.) were administered orally for 60 days to rabbits fed with 1% cholesterol. Histopathological samples of the aortas and the livers were stained with hematoxylin and eosin. Lipid peroxidation (malondialdehyde - MDA) and redox status (glutathione - GSH, glutathione peroxidase - Gpx and superoxide dismutase - SOD) were analyzed using spectrophotometrical methods. Results Both loganic acid (an iridoid) and a mixture of anthocyanins diminished the formation of atherosclerotic plaques in the aorta. Both substances also diminished lipid peroxidation, measured as a decrease of MDA, and attenuated oxidative stress, measured as an increase of GSH in the livers depleted by cholesterol feeding. Unexpectedly, cholesterol feeding decreased the Gpx activity in the liver, which was reversed by both investigated substances. Conclusions We have shown that both iridoids and anthocyanins help prevent fed-induced atherosclerosis, and the consumption of fruits rich in these substances may elicit beneficial effects on the cardiovascular system.

Cornelian cherry (Cornus mas L.) fruits have been used in traditional cuisine and in folk medicine in various countries. This study was conducted to evaluate the constituents and impact of cornelian cherry (C. mas L.) fruits lyophilisate on lipid levels, PPARα protein expression, atheromatous changes in the aorta, oxido-redox state, and proinflammatory cytokines in hypercholesterolemic rabbits. The HPLC-MS method was used for determining active constituents in cornelian cherry. In a subsequent in vivo study the protective effect of the cornelian cherry on diet-induced hyperlipidemia was studied using a rabbit model fed 1% cholesterol. Cornelian cherry (100mg/kg b.w.) or simvastatin (5mg/kg b.w.) were administered orally for 60 days. Two iridoids - loganic acid and cornuside - and five anthocyanins were identified as the main constituents of the cornelian cherry. The administering of the cornelian cherry led to a 44% significant decrease in serum triglyceride levels, as well as prevented development of atheromatous changes in the thoracic aorta. Cornelian cherry significantly increased PPARα protein expression in the liver, indicating that its hypolipidemic effect may stem from enhanced fatty acid catabolism. Simvastatin treatment did not affect PPAR-α expression. Moreover, the cornelian cherry had a significant protective effect on diet-induced oxidative stress in the liver, as well as restored upregulated proinflammatory cytokines serum levels. In conclusion, we have shown loganic acid to be the main iridoid constituent in the European cultivar of the cornelian cherry, and proven that the cornelian cherry could have protective effects on diet-induced hypertriglicerydemia and atherosclerosis through enhanced PPARα protein expression and via regulating oxidative stress and inflammation.

Present study aimed to evaluae the elasticity of tumorous testicular lesions and usefulness of the elastographic examination for diagnosing lesions in the testes. The study was carried out on nine dogs in which tumorous testicular lesions had been found in the ultrasonographic examination. In all the animals examined, the elastographic examination of the lesions was performed and then castration and the histopathological examination of specimens in order to determine the type of the changes. On the basis of the results of the histopathological examination the dogs were divided into two groups: group I - consisted of three dogs in which nonneoplastic testicular lesions were found and group II comprised six dogs in which neoplasic lesions that began in testicular interstitial cells (Leydigoma) were detected. The lesions observed in dogs of group I showed low stiffness (average 11.25 kPa, range 6.1 to 16.4 kPa), whereas the lesions found in dogs of group II were characterized by high stiffness (average 91.85 kPa, range 52.3 to 131.4 kPa). On the basis of a scale proposed by Goodie et al. (2012), the lesions in group I were in the range of SC1,and in turn, the lesions in group II were in the range of SC 3 inverted. Based on the results obtained, it can be stated that the sonoelastographic examination is useful method for the screening diagnostics of testicular lesions.

1. Acetylsalicylic acid (ASA) and sodium salicylate (SS) are considered safe for poultry and often used in avian medicine. However, information on tolerance and specific side effects of these drugs in birds is lacking. 2. The aim of this study was to determine the effects of 14 d administration of high doses (200 or 400 mg/kg) of either ASA or SS on body weight gain, blood biochemistry, white and red blood cell counts and pathology in broilers. In addition, minimal plasma salicylate concentrations were determined on the 1st, 5th, 10th and 14th d of treatment. 3. The results showed that the dose of 400 mg/kg of either ASA or SS decreased weight gain and induced gizzard ulceration. Kidney to body weight ratio was increased in a dose-dependent manner, but serum concentrations of creatinine and uric acid were not affected. A time-dependent decrease in the minimal plasma salicylate concentration was evident.

The attempt to limit the negative effects of polyester implants on the articular cavity by using preparations containing growth factors.Polyester implants used for the reconstruction of a rabbit's cranial cruciate ligament (CCL) were saturated with autogenic platelet-rich plasma (PRP), antlerogenic stem cells MIC-1 and their homogenate prior to the surgery. Six months after CCL reconstruction, morphological, and biochemical blood tests were carried out, including proteinogram and acute phase proteins. The knee joints were also examined macro- and microscopically.The results, compared to the control group, showed a favorable effect of the PRP and homogenate of antlerogenic cells on limiting the inflammation caused by the presence of polyester implant in the knee joint. The addition of growth factors caused covering the implant faster with the recipient's connective tissue, thus contributing to reducing the inflammatory reaction of the articular capsule to the presence of polyester. At the same time, no enhanced local or general reaction of the rabbit organism was observed to the presence of xenogenic antlerogenic stem cells MIC-1 homogenate which, like the PRP, may provide an easily available source of growth factors, increasingly often used in regenerative medicine.Applying antlerogenic stem cells, their homogenate or PRP increases the volume of connective tissue that surrounds and intertwines polyester CCL implant, separating it from synovial cavity environment.

Background and aims Cardiovascular benefits of fruits are attributed mainly to their (poly)phenolic constituents, especially anthocyanins. The main aim of our study is to compare effects of iridoids and anthocyanins from one fruit on diet-induced atherosclerosis. The cornelian cherry is a native or cultivated plant that grows in many European countries, used in cuisine and folk medicine. In our previous study, we showed its constituents and proved that oral administration of lyophilized fruits to hypercholesterolemic rabbits had preventive effects on atherosclerosis through the activation of PPARα expression. In this study, we have compared the effects of the main constituents of the cornelian cherry:iridoid loganic acid and anthocyanins. Methods Our experiment followed the model used in our previous study, in which rabbits were fed 1% cholesterol. Results We showed that both loganic acid (20 mg/kg b.w.) and a mixture of anthocyanins (10 mg/kg b.w.) administered orally for 60 days had a positive impact on dyslipidemia caused by cholesterol-rich diet, although the effects of anthocyanins were more pronounced. Anthocyanins decreased total and LDL-cholesterol and triglycerides and increased HDL-cholesterol. Loganic acid showed similar effects, but only the triglycerides and HDL-cholesterol changes achieved statistical significance. Anthocyanins, and to a lesser extent loganic acid, significantly decreased intima thickness and intima/media ratio in the thoracic aorta. Both substances decrease ox-LDL in the plasma. Anthocyanins significantly increased expression of PPARγ and α in the liver. Loganic acid also increased their expression, but to a lesser extent. Conversely, loganic acid showed pronounced anti-inflammatory effects, decreasing TNF-α and IL-6 activity. Conclusions Our results imply that both substances have a positive effect on factors contributing to the development of diet-induced atherosclerosis. Our results also indicate the potential health benefits of fruits containing anthocyanins and iridoids, and support the idea of creating composed phytopharmaceuticals containing both groups of substances.

The study aimed at immunohistochemical analysis of various markers of cell proliferation and comparison of the results with canine mast cell tumours grading systems according to the Patnaik and Kiupel. Tissue sections were stained using classical technique with haematoxylin and eosin, and immunohistochemical studies were performed with Ki-67, PCNA and MCM-3 antibodies. Additionally the mitotic index was assessed. Statistical analysis including rank correlation Spearman’s and ANOVA Friedman analysis was performed. The significance was set at p

Morphological and immunohistochemical analysis of 45 canine mast cell tumours was performed to determine whether the proteins examined are useful for a more precise description of tumour morphology and a more reliable determination of the prognosis in patients. Tissue sections were stained according to the standard haematoxylin and eosin (HE) technique and with toluidine blue to demonstrate cytoplasmic granules. Immunohistochemical studies were performed, using the cell markers CD117 (c-kit), p16 and von Willebrand factor (FVIII). In CD117 three different staining patterns were observed: (1) membranous reaction, (2) intense staining of cytoplasm, and (3) a diffuse, delicate cytoplasmic reaction. Von Willebrand antibody was evaluated on the basis of the number of blood vessels stained. p16 expression was evaluated by scoring positive nuclear reaction. Positive expression was demonstrated for all examined antigens, but their level of expression differed depending on the grades of tumour malignancy. Statistical analysis of the results documented a pronounced positive correlation between the markers studied and the grade of tumour malignancy (P < 0.001). It was shown that each of the cell markers examined represents a useful prognostic indicator for patients with mast cell tumours. The calculated correlation coefficients demonstrate a strong association between the expressions of CD117, FVIII and p16, and the histological malignancy of a tumour.

The results of several experimental and epidemiological studies have shown an inverse correlation between Mg status and the risk of some cancers. However, relationship between magnesium and cancer is complex. The aim of our work was to examine the precise effect of Mg deficiency on transplantable mouse tumor growth and metastasis.The results obtained indicate a significant retardation of primary tumor growth (up to 70%) in mice receiving Mg-deficient diet. However, Mg repletion caused in these mice significant increase of primary tumor burden. Analysis of cell cycle distribution showed a reduced percentage of cells in the S phase and an increase of cells in the G0/G1 phase of the cell cycle in LLC tumors caused by Mg deficiency. This is in agreement with the effect of low Mg level on cell growth observed in vitro. Interestingly, in mice inoculated with LLC cells and receiving low-magnesium diet, a higher metastatic potential was observed as compared to control mice.In conclusion, our results demonstrate a direct role of magnesium in tumor growth and also point at deleterious effect of low magnesium status on tumor metastasis.

Many efforts have been made to obtain active and less toxic Vitamin D analogs for new clinical applications. The results of previous studies demonstrated the efficacy and safety of topical treatment of psoriasis with one of these analogs, 1,24-dihydroxyvitamin D(3), tacalcitol (1,24-(OH)(2)D(3)). In the present study, we evaluated the toxicity and antitumor effect of this analog. Lethal toxicity of 1,24-(OH)(2)D(3) after s.c. injection was significantly lower than that of calcitriol. No significant differences were observed in the toxicity of the analogs when administered p.o. Calcium levels in the serum of mice treated with calcitriol were significantly higher (111%) than those in mice treated with 1,24-(OH)(2)D(3) (89%) at 5 day after the first s.c. (10 microg/kg/day) administration in comparison to the control (healthy, untreated animals). Oral administration increased the calcium level by 78% for calcitriol and only to 47% over the control for 1,24-(OH)(2)D(3). Parallel administration of clodronate prevented the calcitriol- and 1,24-(OH)(2)D(3)-induced lethal toxicity and also prevented increase in calcium levels. Single therapy with calcitriol did not affect tumor growth in the 16/C mouse mammary cancer model. In contrary, 1,24-(OH)(2)D(3) alone reduced tumor volume to 41% of control. Cisplatin alone did not affect growth of 16/C tumor in these conditions. The growth of tumors in the presence of cisplatin was inhibited by 1,24-(OH)(2)D(3) but not by calcitriol. Interestingly, the inhibition of tumor growth in cisplatin-treated mice by 1,24-(OH)(2)D(3) was greater, than that observed in mice treated with this analog alone. In conclusion, 1,24-(OH)(2)D(3) revealed higher antitumor and lower calcemic activity and toxicity than calcitriol. Application of biphosphonates along with Vitamin D analogs is sufficient to overcome the calcemic and toxic side effects of the proposed treatment.

International audience; BACKGROUND & AIMS: The aim of this experiment was to evaluate the potential beneficial effect of supplementation with different inulin-type fructan fractions against common features of the metabolic syndrome in a rat model of this syndrome (fructose-fed rat). METHODS: Forty Wistar rats were randomly divided into five groups and the animals received for 4 weeks either a semi-purified starch or fructose-based diet, or diets in which fructose was partially substituted with various fructans: 10 g/100 g of long-chain inulin or oligofructose, or an oligofructose-enriched inulin. After this period, blood pressure was measured and samples of blood and tissues were collected for selected biochemical analyses. RESULTS: As compared to the starch-fed group, the fructose-fed rats presented: hypertriglyceridemia, hypertension, increased susceptibility to heart peroxidation and renal damages. Long-chain inulin and oligofructose-enriched inulin supplementation prevented fructose induced elevated blood pressure, susceptibility to heart peroxidation and renal damages. All inulin-type fructans containing diets prevented fructose induced hypertriglyceridemia. CONCLUSIONS: These results suggest that supplementation with inulin-type fructans is efficient against fructose induced hypertension and that effects are most pronounced for long-chain inulin and oligofructose-enriched inulin. We hypothesize that the anti-hypertensive effect of inulin could be explained by the reduction of the high fructose induced oxidative stress.