Noscapine Acts as a Protease Inhibitor of In Vitro Elastase-Induced Collagen Deposition in Equine Endometrium

Abstract

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.

Keywords: collagen; elastase; endometrosis; equine; fibrosis; inhibition; noscapine.

Figure 1

Equine endometrial explant secretion of prostaglandin (PG)F_2α treated with oxytocin (OXT; 10−7 M) for 24 or 48 h. Secretion of PGF_2α (ng/mL) is presented as mean ± SEM. Results were considered significant at p < 0.05. Asterisks represent statistical differences between OXT treatment and the respective control within the treatment time (** p < 0.01; *** p < 0.001).

Figure 2

Effect of elastase (ELA; 0.5 or 1 µg/mL), noscapine (NOSC; 45 µg/mL), or ELA (0.5 or 1 µg/mL) + NOSC (45 µg/mL) treatments during 24 or 48 h in explants of mare endometrium from follicular phase (FP) or mid-luteal phase (MLP) on relative collagen type I alpha 2 chain (COL1A2) mRNA transcription (A,B) and collagen type I (COL1) protein relative abundance (C,D). Results were considered significant at p < 0.05 and shown as least square mean ± SEM. Asterisks alone represent significant differences relative to the respective control and asterisks above connecting lines indicate significant differences of ELA + NOSC treatment relative to the respective ELA-treated group (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 3

Effect of elastase (ELA; 0.5 and 1 µg/mL) (A), noscapine (NOSC; 45 µg/mL) (B) and ELA (0.5 and 1 µg/mL) + NOSC (45 µg/mL) (C) on relative collagen type I alpha 2 chain (COL1A2) mRNA transcription and collagen type I (COL1) protein relative abundance in mare endometrial explants. The results are independent of ELA concentration, estrous cycle phase and treatment time. Results are displayed as least square means ± SEM, and considered significant at p < 0.05. Asterisks represent significant differences relative to the respective control (*** p < 0.001).

Figure 4

Noscapine (NOSC; 45 µg/mL) inhibition of the effects of elastase (ELA; 0.5 or 1 µg/mL) treatments on relative collagen type I alpha 2 chain (COL1A2) mRNA transcription and collagen type I (COL1) protein relative abundance in explants of mare endometrium, irrespective of estrous cycle phase and time of treatment. Results are shown at least square means ± SEM, and considered significant at p < 0.05. Different superscript letters indicate significant differences between ELA concentrations (a, b: ELA 0.5 µg/mL ≠ ELA 1 µg/mL; p < 0.01). Asterisks alone represent significant differences relative to the respective control and asterisks above the connecting lines indicate significant differences between treatments (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 5

Effect of elastase (ELA; 0.5 or 1 µg/mL), noscapine (NOSC; 45 µg/mL), or ELA (0.5 or 1 µg/mL) + NOSC (45 µg/mL) treatments on relative collagen type I alpha 2 chain (COL1A2) mRNA transcription (A) and collagen type I (COL1) protein relative abundance (B) in explants of mare endometrium from follicular (FP) or mid-luteal (MLP) phases, regardless of treatment time. Results are shown at least square means ± SEM and considered significant at p < 0.05. Asterisks above connecting lines indicate significant differences of the same treatment between estrous cycle phase (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 6

Effect of elastase (ELA; 0.5 or 1 µg/mL), noscapine (NOSC; 45 µg/mL), or ELA (0.5 or 1 µg/mL) + NOSC (45 µg/mL) treatments on relative collagen type I alpha 2 chain (COL1A2) mRNA transcription (A) and collagen type I (COL1) protein relative abundance (B) in equine endometrial explants treated for 24 or 48 h, regardless of estrous cycle phase. Results are shown as least square means ± SEM and considered significant at p < 0.05. Asterisks above connecting lines indicate significant differences of the same treatment between time of treatment (** p < 0.01; *** p < 0.001).

Figure 7

Representative equine endometrial biopsies classified as category IIA (A,B) and IIB (C,D) according to Kenney and Doig (1986) classification. Endometrial glands (EG), glandular nests (GN), collagen fibers (C), stromal cells (S), surface epithelium (SE). Staining with hematoxylin and eosin. Scale bar = 20 μm.