Circulating miRNAs in maternal plasma as potential biomarkers of early pregnancy in sheep.

MicroRNA (miRNA) plays an important role in the control of gene expression and is implied in many biological functions, including embryo implantation and development. The aim was to assess plasma miRNA profiles during the peri-implantation and ascertain potential candidate miRNA markers for early pregnancy diagnosis in ovine plasma. The plasma samples were obtained from a total of 24 ewes on days 12 (pre-implantation; P12, n = 4), 16 (implantation; P16, n = 4) and 22 (post-implantation; P22, n = 4) after mating, and on their corresponding days of 12 (Pre-C; C12, n = 4), 16 (Imp-C; C16, n = 4) and 22 (Post-C; C22, n = 4) of the estrous cycle. The miRNA profiles in plasma were assessed by microarray technology. We detected the presence of 60 ovine-specific miRNAs in plasma samples. Of these miRNAs, 22 demonstrated a differential expression pattern, especially between the estrous cycle and early pregnancy, and targeted 521 genes. Two miRNAs (oar-miR-218a and oar-miR-1185-3p) were confirmed using RT-qPCR in the ovine plasma samples. Protein-protein interaction (PPI) network of target genes established six functional modules, of which modules 1 and 3 were enriched in the common GO terms, such as inflammatory response, defense response, and regulation of immune response. In contrast, module 2 was enriched in the developmental process involved in reproduction, embryo development, embryonic morphogenesis, and regulation of the developmental process. The results indicate that miRNAs profiles of plasma seemed to be modulated during the peri-implantation stage of pregnancy in ewes. Circulating miRNAs could be promising candidates for diagnosis in early ovine pregnancy.

Expression pattern of microRNAs in ovine endometrium during the peri-implantation.

MicroRNA (miRNA), acting as the transcriptional regulator of gene expression, has been widely demonstrated to be involved in many biological functions, including embryo implantation and development. The objective of the current study was to illuminate the expression pattern of microRNAs (miRNAs) in the endometrium during the peri-implantation in ewes. Intercaruncular endometrial samples was obtained from a total of 24 ewes on days of 12 (pre-implantation, n = 4), 16 (implantation, n = 4) and 22 (post-implantation, n = 4) of pregnancy following mating, and on their corresponding days of 12 (n = 4), 16 (n = 4) and 22 (n = 4) of the estrous cycle. The miRNA profiles were examined in the endometrium by microarray technology. We detected 116 ovine specifics miRNAs in the endometrium. Of these, nineteen were differentially expressed in early pregnancy. Four miRNAs (oar-miR-370-3p, oar-miR-411b-5p, oar-miR-379-3p and oar-miR-411a-3p) that had the most differential fold change were confirmed by RT-qPCR in ovine endometrium. The differentially expressed miRNAs targeted a total of 315 genes, resulting in 39 GO terms in molecular function, 353 in biological process, and 17 in the cellular component. The construction of the PPI network of target genes established two functional modules mostly enriched in the innate immune system, toll receptor cascades in module 1, whereas genes in module 2 were associated with GMCSF-mediated signaling events, insulin pathway, and mTOR signaling pathway. Based on the results, we may imply that miRNAs modulate ovine endometrium during the peri-implantation.

Protective effects of silymarin on methotrexate-induced damages in rat testes

Abstract The present study aimed to investigate the protective effects of silymarin (SMN), an antioxidant, on methotrexate (MTX)-induced damage in rat testes. Thirty-two Wistar albino rats were divided into four groups (n = 8): control, MTX (20 mg/kg, i.p. on days 1 and 5), SMN (200 mg/kg, orally), and MTX + SMN (20 mg/kg, i.p. on days 1 and 5 and SMN 200 mg/kg orally) groups. At the end of the 6-week trial period, histopathological, immunohistochemical, biochemical, and spermatological analyses were performed on testes tissues. Histopathologically, MTX-induced damage, including depletion of germ cell and loos of spermatozoa, was significantly improved with SMN treatment. Immunohistochemically, the immunoreactivity of glutathione peroxidase 1 (GPx1) and manganese superoxide dismutase 2 (SOD2) were detected more intensely in the MTX + SMN group than in the MTX group. Biochemical examinations revealed that SMN supplementation decreased the lipid peroxidation and increased enzymatic antioxidants in the SMN-treated rats. Spermatologically, significant differences were found in the density, motility, dead-to-live sperm ratio, and abnormal sperm rate in the MTX + SMN group compared to the MTX group. In conclusion, SMN seems to have protective effects as an antioxidant against MTX-induced damage in rat testes.

Early pregnancy-related changes in toll-like receptors expression in ovine trophoblasts and peripheral blood leukocytes.

In the present study, we aimed to 1) demonstrate the presence of all 10 toll-like receptors (TLRs) in ovine trophoblasts, and 2) investigate the expression profiles of TLR1-10 mRNAs in peripheral blood leukocytes (PBLs) in ewes during early pregnancy. For those purposes, ovine trophoblasts (n = 6) were collected from pregnant ewes on day 13. PBLs were collected from non-pregnant (n = 6) and pregnant ewes (n = 17) on days of mating (d) 0 and 18. TLR mRNAs in ovine trophoblasts were visualized by free-floating in situ hybridization (ISH). To assess the expression profiles of TLR1-10 in PBLs, total RNA was isolated and transcribed to cDNA. TLR1-10 mRNA levels were determined by real-time PCR in triplicate. The Relative Expression Software Tool (REST 2009) was used for statistical analysis. We detected mRNAs for TLR2, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR10 but not for TLR1, TLR3, and TLR9 in trophoblasts. TLR2, TLR5, TLR6, TLR7, TLR8, and TLR10 mRNAs were expressed by all trophoblasts, whereas TLR4 mRNA and protein in trophoblasts were more limited. In PBLs, TLR expression did not differ between day 0 and day 18 in non-pregnant ewes; however, ewes in early pregnancy exhibited significantly upregulated expression of TLR2 (2.3-fold), TLR4 (3.1-fold), TLR6 (1.7-fold), and TLR8 (2.2-fold) on day 18 compared with day 0. In contrast, TLR10 was downregulated (2-fold) on day 18 by pregnancy. Similar results were also obtained for TLR2, TLR4, TLR6, TLR8 and TLR10 from the comparison between day 18 non -pregnant and day 18 pregnant groups. According to these results, the presence of TLRs in early ovine trophoblasts suggests that these cells play an immunological role at the maternal-fetal interface. The results also suggest that tight regulation of some components of TLRs in PBLs due to embryo- and/or pregnancy-related factors is necessary for successful establishment of early pregnancy in ewes.

Antidiabetic properties of lyophilized extract of acorn (Quercus brantii Lindl.) on experimentally STZ-induced diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Acorn obtained from the Quercus brantii Lindl. (QB) tree has been used in Turkish folk medicine. Some studies have reported as an antioxidant, antimicrobial, anti-inflammatory, gastroprotective, antitumoural of QB properties previously. however, its effect on the management of type diabetic 2 and oxidative stress complications is still unexplored. The aims of our study were the evaluation of the protective effect and antioxidant role of acorn lyophilized seed plant extract against STZ-induced diabetic complications as oxidative stress, hepatotoxicity and nephropathy, lipidemia and serum biomarkers of diabetes (SBD). MATERIALS AND METHODS: In-vivo studies were performed on STZ-induced diabetic rats Experiment was designed as I [Normal Control (NC)], II [Diabetes mellitus (DM)], III [DM+Acarbose (20mg/kg b.w) (DM+AC-20)], IV [DM+QB (100mg/kg b.w) (DM+QB-100)], V [DM+QB (250 mg/kg b.w) (DM+QB-250)] and VI [DM+QB (500 mg/kg b.w) (DM+QB-500)] groups. RESULTS: This study showed that the biochemical analysis showed a considerable increase in the HRDB, DB, LP, MDA and fluctuated ADSC in the II group as compared to that of control group whereas, AC and the plant lyophilized seed plant extract supplementations diet restored the STZ-induced diabetic complications towards the control. α-glycosidase activity in DM group showed statistically significant increase with respect to control group in small intestine. Moreover, in accordance with the effects of seed extract; in diabetic rat groups to whom acorn seed extract and acarbose were given, the levels of almost all the concerned parameters were reached to the ones measured at control group. As a result, it was concluded that acorn seed extract had certain healing effects on many complications caused by diabetes.

In case of obesity, longevity-related mechanisms lead to anti-inflammation.

The exact mechanisms which contribute to longevity have not been figured out yet. Our aim was to find out a common way for prompting longevity by bringing together the well-known applications such as food restriction, exercise, and probiotic supplementing in an experimental obesity model. Experimental obesity was promoted in a total of 32 young (2 months old) and 32 aged (16 months old) male Wistar albino rats through 8-week cafeteria diet (salami, chocolate, chips, and biscuits). Old and young animals were divided into groups each consisting of eight animals and also divided into four subgroups as obese control, obese food restriction, obese probiotic-fed and obese exercise groups. Probiotic group diet contained 0.05 %w/total diet inactive and lyophilized Lactobacillus casei str. Shirota. The exercise group was subjected to treadmill running 1 h/day, at 21 m/min and at an uphill incline of 15 % for 5 days a week. Food restriction group was formed by giving 40 % less food than the others. The control group was fed regular pellet feed ad libitum. This program was continued for 16 weeks. Blood samples from all the groups were analyzed for fasting glucose, insulin, IGF-1, insulin-like growth factor binding protein 3 (IGFBP-3), interleukin (IL)-6, IL-12, malondialdehyde (MDA), fT3, TT3, fT4, TT4, and liver tissue MDA levels were measured. All applications showed anti-inflammatory effects through the observed changes in the levels of IGFBP-3, IL-6, and IL-12 in the young and old obese rats. While the interventions normally contribute to longevity by recruiting different action mechanisms, anti-inflammatory effect is the only mode of action for all the applications in the obesity model.

The antioxidant role of Sideritis caesarea infusion against TCA toxicity in rats.

Sideritis caesarea (SC) Duman, Aytac & Baser is a member of the Lamiaceae family. The present study was designed to investigate the antioxidant properties of the aerial parts of island green tea SC against TCA effects in rats. Biomarkers selected for monitoring antioxidant capacity were the activities of glutathione reductase (GR), superoxide dismutase (SOD), glutathione-S-transferase, catalase (CAT), GSH level and malondialdehyde (MDA) content in various organs of rats. Three experimental groups, A (untreated = control), B (only TCA-treated) and C (TCA+SC-treated), were studied. At the end of the 50 d experiment, the MDA content in tissues increased significantly in group B, whereas no significant changes were observed in group C as compared with that of the control group. Antioxidant enzyme activities such as SOD and CAT increased significantly in the brain, liver and kidneys of group B but decreased significantly in group C as compared with group B. The GSH level and GR activity increased significantly in the brain and liver of group C as compared with the control and TCA-exposed rats. Hence, the study reveals that the constituents present in SC impart protection against chemical-induced oxidative injury that may result in the development of cancer.

Evaluation of neurotoxic and immunotoxic effects of trichloroacetic acid on rats.

This study was carried out to investigate the neurotoxic and immunotoxic effects of trichloroacetic acid (TCA) on rats at subchronic exposure. The neurotoxic effects of TCA were evaluated by measuring the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Biomarkers selected for immunotoxic monitoring were the activities of adenosine deaminase (ADA) and myeloperoxidase (MPO) in various tissues of rats exposed to 2000 parts per million (ppm) dosage of TCA for 52 days. Results showed that the administrations of TCA decreased BChE activities in heart and lungs tissue of the rats treated with TCA. With regard to the immunotoxic effects, ADA activity significantly decreased in the heart, lungs and spleen whereas MPO activity increased after subchronic exposure with 2000 ppm dosage in all of the tissues except for heart tissue of rats compared with controls. The observations presented led us to conclude that the administration of TCA at subchronic was decreased BChE and ADA activities whereas increased MPO activity in various tissues of rats. This may reflect the potential role of these parameters as useful biomarkers for toxicity of TCA.