Inhibition of Myeloperoxidase Pro-Fibrotic Effect by Noscapine in Equine Endometrium

Abstract

Myeloperoxidase is an enzyme released by neutrophils when neutrophil extracellular traps (NETs) are formed. Besides myeloperoxidase activity against pathogens, it was also linked to many diseases, including inflammatory and fibrotic ones. Endometrosis is a fibrotic disease of the mare endometrium, with a large impact on their fertility, where myeloperoxidase was shown to induce fibrosis. Noscapine is an alkaloid with a low toxicity, that has been studied as an anti-cancer drug, and more recently as an anti-fibrotic molecule. This work aims to evaluate noscapine inhibition of collagen type 1 (COL1) induced by myeloperoxidase in equine endometrial explants from follicular and mid-luteal phases, at 24 and 48 h of treatment. The transcription of collagen type 1 alpha 2 chain (COL1A2), and COL1 protein relative abundance were evaluated by qPCR and Western blot, respectively. The treatment with myeloperoxidase increased COL1A2 mRNA transcription and COL1 protein, whereas noscapine was able to reduce this effect with respect to COL1A2 mRNA transcription, in a time/estrous cycle phase-dependent manner (in explants from the follicular phase, at 24 h of treatment). Our study indicates that noscapine is a promising drug to be considered as an anti-fibrotic molecule to prevent endometrosis development, making noscapine a strong candidate to be applied in future endometrosis therapies.

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

Figure 1

Effect of time of treatment (24 or 48 h) and estrous cycle phase (follicular phase—FP; mid-luteal phase—MLP) on relative collagen type I alpha chain (COL1A2) mRNA transcription (A), and COL1 relative protein abundance (B) in equine endometrial explants. The results are independent of MPO and NOSC treatment, shown as the least square means ± SEM and considered significant at p < 0.05. Asterisks above the connecting line represent significant differences between treatments (** p < 0.01; *** p < 0.001).

Figure 2

Effect of myeloperoxidase (MPO; 0.1–0.5 μg/mL), noscapine (NOSC; 45 μg/mL), or MPO (0.1–0.5 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 h or 48 h, regardless of the estrous cycle phase. Results are shown as the least square means ± SEM and considered significant at p < 0.05. Asterisks represent significant differences relative to the respective control group. Asterisks above the connecting line represent significant differences between treatments (* p < 0.05; ** p < 0.01).

Figure 3

Effect of myeloperoxidase (MPO; 0.1–0.5 μg/mL), noscapine (NOSC; 45 μg/mL), or MPO (0.1–0.5 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 from follicular (FP) or mid-luteal (MLP) phases, regardless of treatment time. Results are shown as the least square means ± SEM and considered significant at p < 0.05. Asterisks represent significant differences relative to the respective control group. Asterisks above the connecting line represent significant differences between treatments (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 4

Effect of myeloperoxidase (MPO; 0.1–0.5 µg/mL), noscapine (NOSC; 45 µg/mL), or MPO (0.1–0.5 µg/mL) + NOSC (45 µg/mL) treatments in explants of mare endometrium from the follicular phase (FP; n = 7), treated for 24 or 48 h, on relative collagen type I alpha 2 chain (COL1A2) mRNA transcription (A) and collagen type I (COL1) protein relative abundance (B). Results were considered significant at p < 0.05 and shown in a scattered dot plot, as the mean ± SEM. Asterisks represent significant differences relative to the respective control and asterisks above connecting lines indicate significant differences of MPO + NOSC treatment relative to the respective MPO-treated group (* p < 0.05; ** p < 0.01; *** p < 0.001).