Protective Effects of Resveratrol against Chronic Immobilization Stress on Testis

Objective. The aim of this study was to investigate protective effects of resveratrol, a strong antioxidant, against possible negative effects of chronic immobilization stress on testes of male rats histochemically, immunohistochemically, ultrastructurally, and biochemically. Material and Methods. Male Wistar rats were divided into 4 groups (n = 7). Group I, control group (C), was not exposed to stress. Group II, stress group (S), was exposed to chronic immobilization stress. In Group III, low dose resveratrol + stress group (LRS), rats were given 10 mg/kg/day resveratrol just before the stress application. In Group IV, high dose resveratrol + stress group (HRS), rats were given 20 mg/kg/day resveratrol just before the stress application. For chronic immobilization stress application animals were put in the plastic tubes (6 cm in diameter, 15 cm in length) during 32 days for 6 hours. All animals were sacrificed 18 hours after the last stress application. Results. Histochemical and ultrastructural investigations showed that in stress group there was germ cell deprivation in seminiferous tubules and increase of connective tissue on interstitial area. No significant changes were seen in low and high dose resveratrol groups. After immunohistochemical investigations, TUNEL (+) and Active Caspase-3 (+) cells were increased in seminiferous tubules of stress group compared with those control group, but they were decreased in low and high dose resveratrol groups. According to biochemically results, MDA, GSH, and testosterone levels in stress group showed no significant difference when compared with those of the other groups. Conclusion. The chronic immobilization stress increases oxidative stress and apoptosis and causes histological tissue damages; resveratrol can minimize the histological damage in testes significantly

An improved collagen zymography approach for evaluating the collagenases MMP-1, MMP-8, and MMP-13

Collagen zymography is an SDS-PAGE-based method for detecting both the proenzyme and active forms of collagenases. Although collagen zymography is used for assessment of the matrix metalloproteinases MMP-1 and MMP-13, it can be difficult to detect these collagenases due to technical issues. Moreover, it remains unclear whether the collagenase activity of MMP-8 can be detected by this method. Here, we present an improved collagen zymography method that allows quantification of the activities of MMP-1, MMP-8, and MMP-13. Activities of recombinant collagenases could be detected in collagen zymogram gels copolymerized with 0.3 mg/mL type I collagen extracted from rat tail tendon. This improved method is sensitive enough to detect the activity of as little as 1 ng of collagenase. We generated standard curves for the three collagenases to quantify the collagenolytic activity levels of unknown samples. To validate our improved method, we investigated MMP-1 activity levels in human thyroid cancer (8505C) and normal thyroid (Nthy-ori-3-1) cell lines, finding that the proenzyme and active MMP-1 levels were greater in 8505C cells than in Nthy-ori-3-1 cells. Taken together, our data show that collagen zymography can be used in both molecular and clinical investigations to evaluate collagenase activities in various pathological conditions

Is There any Relationship Between the Grade of Mucoid Degeneration of Torn Menisci and Biochemical Marker Levels in Synovial Fluid?

OBJECTIVES: Mucoid degeneration (MD) leads to nontraumatic tears of the meniscus even in the young population. The tears are often irrepairable. The purpose of this study is to find out if there is any relationship between the severity of mucoid degeneration (MD) and the biochemical environment of the knee. Our hypothesis is that meniscal tears due to more severe MD are associated with higher levels of markers in the synovial fluid. METHODS: Synovial fluid samples were taken during isolated arthroscopic meniscectomies. Samples of excised menisci were sent to the department of pathology for grading of MD. According to the Copenhaver staging classification, stage 1-2 menisci comprised group A (n:13), and grade 3 menisci group B (n:19). Cases with instability and with greater than grade 2 chondral lesions were excluded. Synovial fluid samples were also aspirated from 9 normal knees of individuals operated from other sites of lower extremities; these comprised the control group (C). The synovial fluids were examined for MMP-3,TIMP-1,COMP and proteoglycan (PG)fragment levels. Results were statistically analyzed with nonparametric Mann Whitney's U test. RESULTS: PG fragment levels were significantly higher in group B as compared to group A (p=0.044).When groups B and C were compared, the difference between PG fragment levels almost displayed significance (p=0.055).There were no significant differences between the groups A,B and C for MMP-3,TIMP-1,and COMP levels. MMP-3 levels were significantly higher for traumatic meniscal tears than nontraumatic tears (p=0.025). CONCLUSION: In this study, our hypothesis was partially confirmed. Higher levels of PG fragments were found in knees with higher grade of meniscal mucoid degeneration. MD may be associated with an insidious degenerative process in the knee

Glutathione Depletion by Buthionine Sulfoximine Induces Oxidative Damage to DNA in Organs of Rabbits in Vivo

WOS: 000266601500034PubMed ID: 19374446Glutathione (GSH) exists in mammalian tissues in vivo at high concentrations and plays an important protective role against oxidatively induced damage to biological molecules, including DNA. We investigated oxidatively Induced damage to DNA by GSH depletion in different organs of rabbits in vivo. Rabbits were treated subcutaneously with buthionine sulfoximine (BSO), an effective GSH-depleting compound. GSH levels were measured in heart, brain, liver, and kidney of animals. BSO treatment significantly reduced GSH levels in heart, brain, and liver, but not in kidney. DNA was isolated from these tissues to test whether GSH depletion causes oxidatively induced DNA damage in vivo. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry with isotope dilution methods were applied to measure typical products of oxidatively induced damage in isolated DNA samples. Several such products were identified and quantified in all organs. BSO treatment caused significant formation of 8-hydroxyguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyadenine, and (5'S)-8,5'-cyclo-2'-deoxyadenosine in DNA of organs of rabbits. Animals were fed with the semiessential amino acid 2-aminoethanesulfonic acid (taurine) during BSO treatment. Taurine significantly inhibited GSH depletion and also formation of DNA products. Depletion of GSH correlated well with formation of DNA products, indicating the role of GSH in preventing oxidatively induced DNA damage. Our findings might contribute to the understanding of pathologies associated with DNA damage, oxidative stress, and/or defective antioxidant responses and improve our understanding of the effect of BSO in increasing the efficacy of anticancer therapeutics.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [SBAG-K-70]This research was supported in part by Grant SBAG-K-70 (Z.K.) from The Scientific and Technological Research Council of Turkey (TUBITAK)

Ectasia and Severe Atherosclerosis: Relationships with Chlamydia pneumoniae, Helicobacter pylori, and Inflammatory Markers

To date, there has been no convincing evidence for an association between Chlamydia pneumoniae or Helicobacter pylori and ectasia. In this case-control study, we have investigated the association of H. pylori and C. pneumoniae seropositivity with ectasia, severe coronary atherosclerosis, and normal vessels, which were so classified by coronary angiography. We have also evaluated the influence of these infections on inflammatory markers such as high-sensitive C-reactive protein (hsCRP) and interleukin 6 (IL-6). Of the 796 patients undergoing coronary angiography for suspected ischemic heart disease, 244 patients were recruited. Of these, 91 had normal vessels, 88 had 3 or more obstructed vessels, and 65 had ectatic vessels without atherosclerosis. Eighty-seven atherosclerotic patients (98.9%) were positive for C. pneumoniae IgG, as were 64 ectatic patients (98.5%) and 76 controls (83.5%) (P < 0.001). Forty-two atherosclerotic patients (47.7%) were positive for C. pneumoniae IgM, as were 43 ectatic patients (66.2%) and 43 controls (47.3%) (P = 0.036). Seventy-two atherosclerotic patients (81.8%) were positive for H. pylori IgA, as were 26 ectatic patients (40.0%) and 44 controls (48.4%) (P < 0.001). High-sensitive CRP levels were significantly higher in ectatic patients (5.639 mg/L) than in controls (4.390 mg/L) (P = 0.032), and IL-6 levels were significantly higher in atherosclerotic patients (33.92 U/L) than in controls (14.01 U/L) (P < 0.001). Interleukin-6 levels were higher in H. pylori seropositive patients, and hsCRP levels were higher in C. pneumoniae seropositive patients, when compared with seronegatives. We suggest that, as in atherosclerosis, C. pneumoniae infection is related to ectasia, with raised CRP levels

Roles of matrix metalloproteinases in the cornea: A special focus on macular corneal dystrophy

Matrix metalloproteinases (MMPs) are endopeptidases that are responsible for the degradation of several components of the extracellular matrix (ECM) and some non-ECM proteins. MMPs are subdivided into 6 groups according to their structure and substrate specificity: collagenases, gelatinases, membrane-type MMPs, stromelysins, and matrilizines. Collagenases are important proteolytic tools during ECM remodeling, tissue regeneration, and organ development. MMPs, especially collagenases, have important roles in ocular processes such as retinal neurogenesis and corneal wound healing. MMP studies on eye research are limited, but there is growing evidence that MMP physiology is key for the ocular system, especially for the cornea. The cornea is predominantly composed of collagen fibrils, which form uniform lamellar lattices. Collagenase-driven ECM remodeling is essential for the cornea. Macular corneal dystrophy (MCD) is a rare inherited disease and characterized by progressive, insoluble accumulation of irregular substances in the corneal ECM. MCD can cause visual acuity loss up to blindness, and there is currently no treatment available. It has been recently reported that certain collagenases are downregulated in MCD disease progression. Here, we review the roles of MMPs in eye diseases and propose possible treatment strategies for MCD

In vitro reoxygenation following hypoxia increases MMP-2 and TIMP-2 secretion by human umbilical vein endothelial cells

Endothelial cells lining the inner blood vessel walls play a key role in the response to hypoxia, which is frequently encountered in clinical conditions such as myocardial infarction, renal ischemia and cerebral ischemia. In the present study we investigated the effects of hypoxia and hypoxia/reoxygenation on gelatinases (matrix metalloproteinase-2 and -9), their inhibitor (TIMP-2) and activator (MT1-MMP), in human umbilical vein endothelial (HUVE) cells. HUVE cells were subjected to 4 h of hypoxia or hypoxia followed by 4 and 24 h of reoxygenation. The pro- and active forms of MMP-2 and MMP-9 were analyzed by gelatin zymography; TIMP-2 protein level was assayed using ELISA, while MT1-MMP activity was measured using an activity assay. The secretion of MMP-2 proform increased significantly in cells subjected to 4 h of hypoxia followed by 4 or 24 h of reoxygenation, compared with the normoxic group. TIMP-2 protein level also increased significantly in the hypoxia/reoxygenation groups, compared with the normoxic group. There were no statistically significant differences in the levels of active MT1-MMP in all groups. This study indicates that MMP-2 and TIMP-2 could be regarded as important components of a mechanism in the pathophysiology of ischemic injury following reperfusion

Endothelin Receptor-Independent Correlation Between HSP47 and Collagen in Rabbit Model of Early Atherosclerosis

WOS: 000298720200003Objective: Collagen in the extracellular matrix (ECM) plays an important role in modulation of response to the vascular injury during the progression of atherosclerosis and restenosis. Collagen can regulate smooth muscle cell (SMC) proliferation, migration and matrix metalloproteinase (MMP) production. Therefore, collagen turnover in the arteries is an important determinant of intimal thickening. Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is thought to be essential for the processing and secretion of procollagen molecules. Endothelin (ET) is a strong chemoatractant and mitogen promoting SMC proliferation and migration. The aim of this study was to investigate the possible role of HSP47 and its relation to collagen synthesis, and the effects of a nonselective ETA/ETB receptor antagonist, TAK-044 in collar-induced early atherosclerosis model. Material and Methods: New Zealand white rabbits were divided into two groups. Both groups received vehicle (0.9% NaCl 0.8 ml/kg/day, s.c.) or TAK-044 (5 mg/kg/day, s.c.) for three weeks. On 8th day, a non-occlusive silicon collar was placed around the left carotid artery. The right carotid artery was sham-operated. HSP47 expression in carotid arteries were determined immunohistochemically. Furthermore, total collagen levels, collagen expression and type I procollagen expression were established. Results: HSP47 expression correlated with collagen expression did not change in collared arteries. TAK-044 treatment did not affect HSP47 and collagen levels. Conclusion: There was a correlation between HSP47 expression and collagen expression in carotid arteries. However, intimal thickening did not induce HSP47 expression and early collagen development. The ineffectivenes of TAK-044 suggests that ET-1 signaling may not be implicated in HSP47 and collagen in this model.Ege UniversityEge University [04ECZ 020]This study was supported by Ege University Research Fund (Project No: 04ECZ 020 to Z.K.)