Noscapine acts as a protease inhibitor of in vitro elastase-induced collagen deposition in equine endometrium

Research Areas: Biochemistry & Molecular Biology ; ChemistryABSTRACT - Endometrosis is a reproductive pathology that is responsible for mare infertility. Our recent studies have focused on the involvement of neutrophil extracellular traps enzymes, such as elastase (ELA), in the development of equine endometrosis. Noscapine (NOSC) is an alkaloid derived from poppy opium with anticough, antistroke, anticancer, and antifibrotic properties. The present work investigates the putative inhibitory in vitro effect of NOSC on collagen type I alpha 2 chain (COL1A2) mRNA and COL1 protein relative abundance induced by ELA in endometrial explants of mares in the follicular or mid-luteal phases at 24 or 48 h of treatment. The COL1A2 mRNA was evaluated by qPCR and COL1 protein relative abundance by Western blot. In equine endometrial explants, ELA increased COL 1 expression, while NOSC inhibited it at both estrous cycle phases and treatment times. These findings contribute to the future development of new endometrosis treatment approaches. Noscapine could be a drug capable of preventing collagen synthesis in mare’s endometrium and facilitate the therapeutic approach.info:eu-repo/semantics/publishedVersio

The effects of prostaglandin E-2 treatment on the secretory function of mare corpus luteum depends on the site of application : an in vivo study

Research Areas: Veterinary SciencesWe examined the effect of prostaglandin (PG) E2 on the secretory function of equine corpus luteum (CL), according to the application site: intra-CL injection vs. an intrauterine (intra-U) administration. Moreover, the effect of intra-CL injection vs. intra-U administration of both luteotropic factors: PGE2 and human chorionic gonadotropin (hCG) as a positive control, on CL function was additionally compared. Mares were assigned to the groups (n = 6 per group): (1) an intra-CL saline injection (control); (2) an intra-CL injection of PGE2 (5 mg/ml); (3) an intra-CL injection of hCG (1,500 IU/ml); (4) an intra-U saline administration (control); (5) an intra-U administration of PGE2 (5 mg/5 ml); (6) an intra-U administration of hCG (1,500 IU/5 ml). Progesterone (P4) and PGE2 concentrations were measured in blood plasma samples collected at −2, −1, and 0 (pre-treatment), and at 1, 2, 3, 4, 6, 8, 10, 12, and 24 h after treatments. Moreover, effects of different doses of PGE2 application on the concentration of total PGF2α (PGF2α and its main metabolite 13,14-dihydro-15-keto-prostaglandin F2α– PGFM) was determined. The time point of PGE2, hCG, or saline administration was defined as hour “0” of the experiment. An intra-CL injection of PGE2 increased P4 and PGE2 concentrations between 3 and 4 h or at 3 and 12 h, respectively (p < 0.05). While intra-U administration of PGE2 elevated P4 concentrations between 8 and 24 h, PGE2 was upregulated at 1 h and between 3 and 4 h (p < 0.05). An intra-CL injection of hCG increased P4 concentrations at 1, 6, and 12 h (p < 0.05), while its intra-U administration enhanced P4 and PGE2 concentrations between 1 and 12 h or at 3 h and between 6 and 10 h, respectively (p < 0.05). An application of PGE2, dependently on the dose, supports equine CL function, regardless of the application site, consequently leading to differences in both P4 and PGE2 concentrations in blood plasmainfo:eu-repo/semantics/publishedVersio

The inhibitory effect of noscapine on the in vitro cathepsin G-induced collagen expression in equine endometrium

Research Areas: Life Sciences & Biomedicine ; MicrobiologyABSTRACT - Cathepsin G (CAT) is a protease released by neutrophils when forming neutrophil extracellular traps that was already associated with inducing type I collagen (COL1) in equine endometrium in vitro. Endometrosis is a fibrotic condition mainly characterized by COL1 deposition in the equine endometrium. The objective was to evaluate if noscapine (an alkaloid for cough treatment with anti-neoplastic and anti-fibrotic properties) would reduce COL1A2 transcription (evaluated by qPCR) and COL1 protein relative abundance (evaluated by western blot) induced by CAT in equine endometrial explants from follicular and mid-luteal phases treated for 24 or 48 h. The explants treated with CAT increased COL1 expression. Noscapine decreased COL1A2 transcription at both estrous cycle phases, but COL1 relative protein only at the follicular phase, both induced by CAT. Additionally, the noscapine anti-fibrotic action was found to be more effective in the follicular phase. The CAT treatment caused more fibrosis at the longest period of treatment, while noscapine acted better at the shortest time of treatment. Our results showed that noscapine could act as an anti-fibrotic drug in equine endometrosis by inhibiting CAT in vitro. Noscapine offers a new promising therapeutic tool for treating fibrosis as a single non-selective agent to be considered in the future.info:eu-repo/semantics/publishedVersio

Temporal isolation between two strongly differentiated stocks of the Greenland halibut (Reinhardtius hippoglossoides Walbaum, 1792) from the Western Barents Sea

Identification of discrete stocks of Greenland halibut is an important aspect of proper fisheries exploitation. Available literature data indicated a lack of significant inter‑area differences between Greenland halibut populations from the Northeast Atlantic. To define the population diversity, two aspects were taken into account: enzyme‑genetic diversity and the concentration of heavy metals in tissues. Seven allozyme loci variations were used to characterize the genetic structure of four populations of Greenland halibut from the Western Barents Sea region. The samples were collected from the spawning area in the period when this species took migration to spawn. The sample RH4 was significantly different from the other samples collected from the same location (RH2 and RH3) and depth for over two days. Another sample (RH8), collected from the nearby area 6 days later was similar to the samples RH2, RH3. We noticed a significant divergence between the sample RH4 and the three remaining samples, where the value of the index FST fluctuated about 0.40 and approximately 0.01 between three similar populations. This genetic fluctuation negates the thesis of a panmictic character of the Western Atlantic population. Feeding groups of Greenland halibut are moving along the Barents Sea shelf and they are exposed to different heavy metals concentrations according to the food preferences or the exact place of feeding. We identified similar concentrations of heavy metals, i.e., Zn, Cu, Cd, and Pb in all samples. Trace metal analysis of aquatic organisms from the Barents Sea can provide important information on the degree of environmental contamination, and the potential impact of seafood consumption

The In vitro inhibitory effect of sivelestat on elastase induced collagen and metallopeptidase expression in equine endometrium

Research Areas: Agriculture ; Veterinary Sciences ; ZoologyAbstract: Neutrophil extracellular traps (NETs) fight endometritis, and elastase (ELA), a protease found in NETs, might induce collagen type I (COL1) accumulation in equine endometrium. Metallopeptidases (MMPs) are involved in extracellular matrix balance. The aim was to evaluate the e ects of ELA and sivelestat (selective elastase inhibitor) on MMP-2 and MMP-9 expression and gelatinolytic activity, as well as the potential inhibitory e ect of sivelestat on ELA-induced COL1 in equine endometrium. Endometrial explants from follicular (FP) and mid-luteal (MLP) phases were treated for 24 or 48 h with ELA, sivelestat, and their combination. Transcripts of COL1A2, MMP2, and MMP9 were evaluated by qPCR; COL1 protein relative abundance by Western blot, and MMP-2 and MMP-9 gelatinolytic activity by zymography. In response to ELA treatment, there was an increase in MMP2 mRNA transcription (24 h) in active MMP-2 (48 h), both in FP, and in MMP9 transcripts in FP (48 h) and MLP (24 h) (p < 0.05). Sivelestat inhibited ELA-induced COL1A2 transcripts in FP (24 h) and MLP (24 h, 48 h) (p < 0.05). The sivelestat inhibitory e ect was detected in MMP9 transcripts in FP at 48 h (p < 0.05), but proteases activity was unchanged. Thus, MMP-2 and MMP-9 might be implicated in endometrium fibrotic response to ELA. In mare endometrium, sivelestat may decrease ELA-induced COL1 deposition and hinder endometrosis development.info:eu-repo/semantics/publishedVersio

The inhibition of Cathepsin G on endometrial explants with endometrosis in the mare

Research Areas: Veterinary SciencesAlthough proteases found in neutrophil extracellular traps (NETs) have antimicrobial properties, they also stimulate collagen type 1 (COL1) production by the mare endometrium, contributing for the development of endometrosis. Cathepsin G (CAT), a protease present in NETs, is inhibited by specific inhibitors, such as cathepsin G inhibitor I (INH; beta-keto-phosphonic acid). Matrix metallopeptidases (MMPs) are proteases involved in the equilibrium of the extracellular matrix. The objective of this study was to investigate the effect of CAT and INH (a selective CAT inhibitor) on the expression of MMP-2 and MMP-9 and on gelatinolytic activity. In addition, the putative inhibitory effect of INH on CAT-induced COL1 production in mare endometrium was assessed. Endometrial explants retrieved from mares in follicular phase or midluteal phase were treated for 24 or 48 h with CAT, inhibitor alone, or both treatments. In explants, transcripts (quantitative polymerase chain reaction) of COL1A2, MMP2, and MMP9, as well as the relative abundance of COL1 protein (Western blot), and activity of MMP-2 and MMP-9 (zymography) were evaluated. The protease CAT induced COL1 expression in explants, at both estrous cycle phases and treatment times. The inhibitory effect of INH was observed on COL1A2 transcripts in follicular phase at 24-h treatment, and in midluteal phase at 48 h (P < 0.05), and on the relative abundance of COL protein in follicular phase and midluteal phase explants, at 48 h (P < 0.001). Our study suggests that MMP-2 might also be involved in an earlier response to CAT, and MMP-9 in a later response, mainly in the follicular phase. While the use of INH reduced CAT-induced COL1 endometrial expression, MMPs might be involved in the fibrogenic response to CAT. Therefore, in mare endometrium, the use of INH may be a future potential therapeutic means to reduce CAT-induced COL1 formation and to hamper endometrosis establishment.info:eu-repo/semantics/publishedVersio

Myeloperoxidase inhibition decreases the expression of collagen and metallopeptidase in mare endometria under in vitro conditions

Research Areas: Agriculture ; Veterinary Sciences ; ZoologyABSTRACT - Neutrophils can originate neutrophil extracellular traps (NETs). Myeloperoxidase (MPO) is a peroxidase found in NETs associated to equine endometrosis and can be inhibited by 4-aminobenzoic acid hydrazide (ABAH). Metallopeptidases (MMPs) participate in extracellular matrix stability and fibrosis development. The objectives of this in vitro work were to investigate, in explants of mare’s endometrium, (i) the ABAH capacity to inhibit MPO-induced collagen type I (COL1) expression; and (ii) the action of MPO and ABAH on the expression and gelatinolytic activity of MMP-2/-9. Explants retrieved from the endometrium of mares in follicular or mid-luteal phases were treated with MPO, ABAH, or their combination, for 24 or 48 h. The qPCR analysis measured the transcription of COL1A2, MMP2, and MMP9. Western blot and zymography were performed to evaluate COL1 protein relative abundance and gelatinolytic activity of MMP-2/-9, respectively. Myeloperoxidase elevated COL1 relative protein abundance at both treatment times in follicular phase (p < 0.05). The capacity of ABAH to inhibit MPO-induced COL1 was detected in follicular phase at 48 h (p < 0.05). The gelatinolytic activity of activated MMP-2 augmented in mid-luteal phase at 24 h after MPO treatment, but it was reduced with MPO+ABAH treatment. The activity of MMP-9 active form augmented in MPO-treated explants. However, this effect was inhibited by ABAH in the follicular phase at 48 h (p < 0.05). By inhibiting the pro-fibrotic effects of MPO, it might be possible to reduce the development of endometrosis. Metallopeptidase-2 might be involved in an acute response to MPO in the mid-luteal phase, while MMP-9 might be implicated in a prolonged exposition to MPO in the follicular phase.info:eu-repo/semantics/publishedVersio