{"paper_id":"2861fb3a-a14d-49db-93f1-c5118fc6b6b4","body_text":"Participation of neuropeptide Y and its receptors in leukotriene generation in the pig inflamed endometrium | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Participation of neuropeptide Y and its receptors in leukotriene generation in the pig inflamed endometrium Barbara Jana, Jarosław Całka, Michał Bulc This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7738675/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract The aim of the study was to determine neuropeptide Y (NPY) receptor subtypes 1 (Y1R) and 2 (Y2R) mRNA and protein abundances in the inflamed porcine endometrium and NPY influence alone and with Y1R or Y2R antagonists on 5-lipoxygenase (5-LOX), LTA4 hydrolase (LTAH) and LTC4 synthase (LTCS) protein abundances and LTB4 and LTC4 release from this tissue. Either saline solution (CON group) or Escherichia coli (E. coli) suspension (E. coli group) were injected into uterine horns. After eight days, in E. coli group severe acute endometritis was diagnosed, as well as decreased Y1R mRNA and protein abundances and Y2R mRNA abundance and increased Y2R protein abundance compared to CON group. NPY increased 5-LOX, LTAH and LTCS protein abundances and LTB4 and LTC4 release from the endometrial stripes of both groups, however in the E. coli group above parameters were higher compared to CON group. In both groups, Y1R antagonist reduced NPY-induced 5-LOX and LTCS protein abundances in reference to NPY influence alone. This effect was also exerted by Y1R antagonist combined with NPY on LTB4 release in the CON group and on LTAH protein abundance and LTC4 release in E. coli group. As compared to NPY action alone, Y2R antagonist with NPY caused a decrease in LTAH protein abundance and LTB4 and LTC4 release in both CON and E. coli groups. Summarizing, in the inflamed porcine endometrium changes Y1R and Y2R mRNA and protein abundances. NPY by interaction with Y1Rs and Y2Rs stimulates LTAH protein abundance and LTC4 release as well as acting through Y1Rs increases 5-LOX and LTCS protein abundances and by Y2Rs release LTB4. NPY, in an indirect manner, can affect the LT-regulated processes in an inflamed endometrium. Health sciences/Diseases Biological sciences/Microbiology Biological sciences/Physiology endometrium inflammation neuropeptide Y and its receptors leukotriene B4 and C4 formation pig Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Uterine inflammation (endometritis, metritis) very often occurs in domestic animals and women developing mainly during puerperal period. In pigs this disorder is very often connected with the mammary gland inflammatory process, creating a postpartum dysgalactia syndrome [1, 2]. Severe kinds of uterine inflammation in cows and pigs cause the reproductive problems and bringing substantial economic losses on farm [3, 4]. The pathology develops as a result of disturbances in the endometrial immune defense mechanisms and/or in contractility of myometrial layer [5]. Most often, uterine inflammation is caused by bacteria, including Escherichia coli ( E. coli ). Streptococcus spp., Staphylococcus spp., Trueperella pyogenes , and Klebsiella pneumoniae [6, 7]. Leukotrienes (LTs) are a family of pro-inflammatory lipid mediators playing a significant role in immune-mediated diseases. In response to inflammatory, allergic and immune signals, activated cytosolic phospholipase A2 converts phospholipids from cellular membrane to arachidonic acid. This acid is serially converted to 5-hydroperoxyeicosatetraenoic acid and then to LTA4 by 5-lipoxygenase (5-LOX, also called 5-LO) in the presence of 5-LOX activating peptide (FLAP). LTA4 can be either hydrolyzed by LTA4 hydrolase (LTAH) to LTB4 or conjugated with glutathione by LTC4 synthase (LTCS) to yield LTC4. LTC4, first of three cysteinyl-LTs (cys-LTs) may be converted to LTD4 by glutamyl transpeptidase and then to LTD4 by dipeptidase [8, 9]. CysLTs are ligands for cysLT1, cysLT2 and cysLT3 receptors, while LTB4 exerts its actions by binding to BLT1 and BLT2 receptors [10,11]. Neuropeptide Y (NPY), a highly conserved 36-amino acid peptide, is the most widely distributed neuropeptide, performing a wide spectrum of physiological functions in both the central and peripheral nervous systems. Moreover, NPY was also identified in non-neuronal cells, for example, platelets, endothelial and immune cells. Biological effects of NPY are mediated by six subtypes of G protein-coupled Y-family receptors (YRs), termed from Y1 to Y6 [12], however, this peptide preferentially acts via Y1R, Y2R and Y5R subtypes [ 13 ]. Immunoreactivity for NPY is present in the cholinergic and noradrenergic neurons in the pig paracervical ganglion (PCG) [14]. Moreover, NPY was determined in the pig uterine noradrenergic perikarya in the caudal mesenteric ganglion (CaMG) [15, 16] and PCG [17, 18]. In human [19] and pigs [20] uteri NPY-positive nerve fibers occur in the vicinity of blood vessels, glands and myocytes of the myometrium. It is known that under physiological conditions NPY increases the contractile activity in the rat myometrium through Y1Rs [21]. NPY acting by Y1Rs and Y2Rs reduces the contractility of the porcine uterus [22]. This peptide is also a potent modulator of the immune response during inflammation [23]. Its role as well as Y1Rs and Y2Rs was reported, for example, in the inflammatory bowel disease [24] and asthma [25]. In relation to the inflamed uterus, it is known that during severe acute endometritis in pigs increased the populations of uterine sympathetic perikarya expressing NPY in the CaMG [16] and PCG [18]. Inflammation changes also the Y1R and Y2R expression in the pig myometrium and decreases the NPY-evoked contractility of inflamed uterus [22]. In relation to the 5-LOX pathway, it was reported that uterine inflammation significantly increased the LTB4 and LTC4 formation and secretion [26-28]. Up to now, NPY receptors have not been determined in the inflamed (also healthy) endometrium of any species, and the action of NPY on arachidonic acid metabolites formation, including LTs, in this tissue is not known. In relation to LTs, it was only reported that NPY enhanced platelet activating factor (PAF)-stimulated LTD4 level in the rat lungs [29]. It is hypothesized that inflammation alters the abundance of YRs in endometrium and that the NPY action on LT biosynthesis pathway in the inflamed endometrium. Defining the connection between NPY and LTs will contribute to further understanding of neuro-immune interactions. Therefore, we studied the effect of inflammation on Y1R and Y2R mRNA and protein abundances in pig endometrium and the NPY influence on 5-LOX, LTAH and LTCS protein abundances and LTB4 and LTC4 release by this tissue. Results Messenger RNA abundances of Y1Rs and Y2Rs In the E. coli group, the Y1R (P<0.001) and Y2R (P<0.05) mRNA abundances in the endometrium were lower than in the CON group (Fig. 1A, B). Protein abundances of Y1Rs and Y2Rs In the E. coli group endometrium, the Y1R protein abundance was reduced (P<0.001) compared to the CON group (Fig. 2A). The Y2R protein abundance in the endometrium of E. coli group was increased (P<0.05) in relation to the CON group (Fig. 2B). Distribution of Y1Rs and Y2Rs Immunofluorescent method showed the presence of Y1Rs and Y2Rs in the luminal and glandular epithelium and blood vessels (endothelium, muscle layer) of the endometrium of the CON (Fig. 3A, B, C, G, H, I) and E. coli (Fig. 3D, E, F, J, K, L) groups. No staining of Y1Rs (Fig. 3M) and Y2Rs (Fig. 3N) was observed after replacement of primary antibodies by normal rabbit IgG. Effect of NPY and/or antagonists of Y1Rs and Y2Rs on 5-LOX protein abundance in endometrium Antagonists of Y1Rs and Y2Rs alone: In the CON and E. coli groups, the 5-LOX protein abundances under the NPY influence were higher (P<0.001) compared to influences of Y1R and Y2Rantagonists (Table 1). The exposition of the E. coli group endometrium to the antagonists increased (P<0.001) the enzyme abundances versus the CON group. NPY alone: In both groups, NPY augmented (P<0.01) the 5-LOX abundances versus the control values (obtained from an endometrium of the CON or E. coli groups that had not undergone any in vitro treatment) (Fig. 4). In the E. coli group, the control value of protein abundance and that in response to NPY were higher (P<0.001) than in the CON group. Y1R antagonist or Y2R antagonist with NPY : In the CON (P<0.01) and E. coli (P<0.001) groups, Y1R antagonist with NPY decreased the 5-LOX abundances in reference to the NPY action(Fig. 4). In both groups, the enzyme abundances did not significantly change by Y2R antagonist with NPY versus the NPY action. The protein abundance in response to Y1R antagonist with NPY did not differ significantly between both groups. The enzyme abundance in response to Y2R antagonist with NPY in the E. coli group was higher (P<0.001) than in the CON group. Effect of NPY and/or antagonists of Y1Rs and Y2Rs on LTAH protein abundance and LTB4 secretion from endometrium LTAH protein abundance Antagonists of Y1Rs and Y2Rs alone: The LTAH abundances in the CON and E. coli groups were lowered (P<0.001) after using Y1R and Y2R antagonists compared to the NPY action (Table 1). In the E. coli group, the enzyme abundance after using Y1R antagonist was increased (P<0.05) versus the CON group. NPY alone: NPY led to a rise (P<0.01) in the LTAH protein abundances in the CON and E. coli groups compared to the control values (Fig. 5A). In the E. coli group, the control value and that after exposition to NPY were higher (P<0.05) than in the CON group. Y1R antagonist or Y2R antagonist with NPY: In the CON group, Y1R antagonist with NPY did not significantly change the LTAH protein abundance versus the NPY action (Fig. 5A). In E. coli group, these substances reduce (P<0.001) the enzyme abundance versus the NPY influence. In the CON (P<0.01) and E. coli (P<0.001) groups, the LTAH protein abundances in response to Y2R antagonist with NPY were lowered compared to the NPY effect. The enzyme abundances in response to Y1R antagonist or Y2R antagonist with NPY did not differ significantly between both groups. LTB4 secretion Antagonists of Y1Rs and Y2Rs alone: In the CON group, the endometrial LTB4 secretion in response to Y1R (P<0.05) and Y2R (P<0.01) antagonists was reduced versus the NPY action (Table 2). The LT release in the E. coli group lowered after exposition to antagonists for Y1Rs (P<0.05) and Y2Rs (P<0.001) compared to the NPY influence. Y1R antagonist evoked greater (P<0.01) secretion of LT in the E. coli group than in the CON group. NPY alone: The LTB4 release by NPY in the CON and E. coli groups was higher (P<0.001) versus the control values (Fig. 5B). Compared to the CON group, the control value and that after using NPY were increased (P<0.01) in the E. coli group. Y1R antagonist or Y2R antagonist with NPY: In the CON group, Y1R antagonist with NPY reduced (P<0.001) the LTB4 release in relation to the NPY influence (Fig. 5B). In E. coli group, Y1R antagonist with NPY did not significantly change the LTB4 secretion versus the NPY action. In the CON (P<0.01) and E. coli (P<0.001) groups, the LTB4 release under the action of Y2R antagonist with NPY was lower than the NPY effect. The LTB4 release by endometrium of the E. coli group in response to Y1R antagonist with NPY was higher (P<0.001) than in the CON group. Effect of NPY and/or antagonists of Y1Rs and Y2Rs on LTCS protein abundance and LTC4 secretion from endometrium LTCS protein abundance Antagonists of Y1Rs and Y2Rs alone: The LTCS abundances in the CON and E. coli groups were decreased by Y1R (P<0.01) and Y2R (P<0.05) versus the NPY influence (Table 1). In the E. coli group, this enzyme abundances after exposition to Y1R and Y2R antagonists were higher (P<0.05) than in the CON group. NPY alone: In the CON and E. coli groups, the LTCS abundances were elevated (P<0.05) in versus the control values (Fig. 6A). In the E. coli group, the control value (P<0.01) and that in response to NPY (P<0.05) were increased versus the CON group. Y1R antagonist or Y1R antagonist with NPY: In the CON(P<0.05)and E. coli (P<0.01) groups, the enzyme abundances were dropped by Y1R antagonist with NPY versus the NPY action (Fig. 6A). In both groups, the LTCS abundances after using Y2R antagonist with NPY did not differ significantly in relation to the NPY action. After using Y2R antagonist with NPY, the LTCS abundance in the E. coli group was higher (P<0.001) than in the CON group. LTC4 secretion Antagonists of Y1Rs and Y2Rs alone: In the CON and E. coli groups, the LTC4 secretion after using Y1R and Y2R antagonists was lowered (P<0.001) versus the NPY influence (Table 2). The LT release in the E. coli group by Y1R and Y2R antagonists was higher (P<0.001) than in the CON group. NPY alone: The LTC4 release by NPY in the CON and E. coli groups was higher (P<0.001) versus the control values (Fig. 6B). In the E. coli group, the control value of LTC4 (P<0.01) and that in response to NPY (P<0.001) were increased in relation to the CON group. Y1R antagonist or Y2R antagonist with NPY: In the CON group, the LTC4 release by Y1R antagonist with NPY did not significantly change in relation to the effect of NPY (Fig. 6B). In the E. coli group, Y1R antagonist with NPYreduced (P<0.05) the LTC4 release compared to the NPY effect. The LTC4 secretion in the CON (P<0.05) and E. coli (P<0.001) groups was decreased by Y2R antagonist with NPY compared to the NPY influence (Fig. 6B). After using Y1R antagonist or Y2R antagonist with NPY, the LTC4 release did not differ significantly between both groups. Discussion The current study shows the Y1Rs and Y2Rs abundances in the porcine inflamed endometrium and the contribution of NPY, Y1Rs and Y2Rs in the biosynthesis pathway for LTs in this tissue. Histopathological assessment ofthe E. coli -injected uteri revealed the presence of a severe acute endometritis. In the endometrium following changes were determined: edema, hyperemia, the increased number of neutrophils and the damage of luminal and/or glandular epithelium [30]. To date, it has only been reported that the paired transcript NPY-Y1Rs is one of many paired transcripts mediating the dialogue extraembryonic membrane and endometrium during pre- and peri-implantation in sheep under physiological conditions [31]. Here, we demonstrated, for the first time, the Y2R mRNA abundance and Y1R and Y2R protein abundances in the healthy endometrial tissue. Completely new findings of the current study concern also the abundances of Y1Rs and Y2Rs in the endometrium under inflammatory conditions. In the inflamed endometrium we revealed a drop in the Y1R mRNA and protein abundances and Y2R mRNA abundance and a rise in the Y2R protein abundance versus the healthy endometrium. Similar situation was found in the porcine myometrium following intrauterine E. coli injection, with exception of the lack of significant change in the Y2R mRNA abundance [22]. It is also known that in the heart tissue of diabetic patients, the level of the Y2R protein increased and the expression of Y1R and Y2R mRNA was unchanged [32], as well as that the expression of Y1R mRNA in the uterosacral ligaments was increased in women with pelvic organ prolapse [33]. Using immunofluorescent method we found that Y1Rs and Y2Rs are present in the luminal and glandular epithelium and endometrial blood vessel cells in the gilts of both CON and E. coli groups.Thus, a severe acute inflammation did not change the localization of Y1Rs and Y2Rs in the endometrium. It allows to suppose that epithelium and blood vessels are the sites of NPY impact under physiological and inflammatory conditions. The changes in endometrial Y1R and Y2R mRNA and protein abundances shown in the present study, may be a consequence of bacterial infection and the development of inflammation. However, there is a lack of data in the available literature on the effect of inflammatory mediators on the expression pattern of YRs. We suppose that the biological signals leading to the changes in the endometrial Y1R and Y2R abundances might include lipopolysaccharide (LPS) originated from bacteria [34] or pro- and anti-inflammatory cytokines generated in large amounts in the course of endometritis [35-37]. The ability of steroid hormones to modulate the endometrial abundance of Y1R and Y2R cannot be ruled out. Dihydrotestosterone implanted in rats to stimulate polycystic ovarian syndrome increases the Y2R and Y5R protein contents in the ovarian granulosa cells [38] and estrogen stimulates the Y1R mRNA expression the in human breast cancer cell line [39]. Decreased the levels of estrogens and progesterone and increased the level of androstenedione in the peripheral blood of pigs with endometritis were previously reported [40]. However, above suppositions needs further studies. On the basis of changes in the Y1R and Y2R abundances in the inflamed endometrium it is suggested that Y1R and Y2R participate in the function of this tissue. The presence of both Y1Rs and Y2Rs in the muscle layer of blood vessels of the E. coli group (also CON group) allows to assume that these receptors mediate the NPY-controlled blood flow in the endometrium. NPY is able to modulate the contractile activity of the guinea pig uterine arteries acting mainly via Y1Rs [41, 42] and of the rabbit ovarian artery [19] and the guinea pig intestine arterioles [43] via Y1Rs and Y2Rs. It is also known that NPY regulates the influence of noradrenaline [44] and vasoactive intestinal peptide [45] on uterine blood flow. Revealed in our study the immunoreaction for Y1Rs and Y2Rs in the luminal and glandular epithelium of both studied groups suggests that these receptors may be important for secretory activity of endometrium. It is known that the endometrium is the first line of defense against reproductive tract infections after delivery [46] and endometrial cells play an important role in innate immune defense [47]. Given that the inflammation-provoking factors in the endometrium increase the production of inflammatory mediators derived from arachidonic acid, this study was focused on the participation of NPY in LT generation and release by the inflamed endometrium. To our knowledge, there are so far no reports of the participation of NPY in the uterine synthesis and secretion of the LTs under physiological and pathological conditions. This report for the first time shows that in the CON and E. coli groups endometrial stripes under the influence of NPY increased the 5-LOX, LTAH and LTCS protein abundances and the LTB4 and LTC4 secretion. It should be added that in the E. coli group NPY led to greater rise of above enzyme abundances and both LT secretion than in the CON group. Up to now in relation to the interaction between NPY and LT production it was only presented that NPY augmented the PAF-induced LTD4 content in the rat lungs and had not effect on LTC4 and LTE4 levels [29], as well as that NPY produced relaxation of the cod celiac arteries without LT involvement [48]. It is also known that NPY stimulated PGI2 production in the porcine aortic endothelial cells [49] and the isolated rat kidney (also PGE2) [50], and inhibited the bradykinin-induced PGI2 release (also thromboxane B2) from the guinea pig perfused lung [51]. There is also a lack of any information on the receptor mechanism of NPY influence on LT synthesis and secretion from the healthy and pathologically-changed uterus. Our results show that the engagement of Y1Rs in the generation and release of LT partially differs between the studied groups. In the endometrial strips of the CON and E. coli groups, this receptor subtype mediated in the NPY influence on 5-LOX and LTCS protein abundances. Moreover, Y1Rs participate in the NPY action on LTAH protein abundance and LTC4 release only in the E. coli group, as well as were important for LTB4 release only in the CON group. In both groups, we indicate the role of Y2Rs in the NPY-induced LTAH protein abundance and LTB4 and LTC4 release, and the lack of significance of this receptor subtype in the NPY influence on 5-LOX and LTCS protein abundances. It should be mentioned that the involvement of the studied YRs in the NPY excitatory effect on the LTB4 and LTC4 synthesis and secretion by the inflamed endometrial stripes was consistent with the reduction of Y1R and Y2R mRNA and Y1R protein abundance and with a rise in Y2R protein abundance in the endometrium. Available literature show that the pro-inflammatory (LPS, tumor necrosis factor-α, interleukin /IL)/-1β) and anti-inflammatory (IL-4, IL-10) mediators [52], as well as acetylcholine [53] regulate LT formation and release from the endometrium under inflammatory conditions. Above-mentioned inflammatory mediators act also the biosynthesis pathway for LTs in the pig endometrial epithelial [54], endothelial [55] and stromal [56] cells. Thus, the current results by demonstrating the NPY action on the 5-LO, LTAH and LTCS protein abundances and LTB4 and LTC4 secretion by the endometrial stripes complement the data about the immune-neuronal control of LT production in the inflamed uterus. Obtained findings allow to suppose that NPY may indirectly influence processes modulated by LTs in endometrium, including inflammatory state and secretory function. It is possible that NPY through Y1Rs and/or Y2Rs stimulates the LTB4 and LTC4 production and secretion in the course of endometritis, and contributes to the development and maintenance of the inflammatory process. LTB4 is an active chemoreceptor especially for granulocytes and phagocytes. It is related to many functions, including: stimulation and activation of neutrophils and increased interleukin-6 production which causes early gene transcription in mononuclear cells. LTC4 (also the rest cys-LTs) are present in many groups of cells such as mast cells and macrophages. Cys-LTs cause vasodilation (relevant in the recruitment of leukocytes) which initiates the inflammatory response, as well as play role in the maintenance and regeneration of damage tissues [57-59]. Both LTB4 and LTC4 are able to a rise in the PGE2, PGF2α and IL-6 release from E. coli -stimulated bovine uterine stripes [60] and the contents of these PGs in the bovine healthy endometrium [61]. The involvement of Y1Rs and Y2Rs in the NPY influence on the formation and release of LTs during endometritis suggests new potential application of antagonists and/or agonists of individual NPY receptors to modulate the inflammatory response and uterine function. Conclusion This study revealed that in the porcine endometrium with a severe acute endometritis, the Y1R and Y2R mRNA and protein abundances change. In this tissue, NPY stimulates the LTAH protein abundance and LTC4 release acting through Y1Rs and Y2Rs. The NPY effect mediated by Y1Rs increases 5-LOX and LTCS protein abundances, while by Y2Rs the release of LTB4. On this basis, it is conjecturable that NPY together with these receptors may be at least partly responsible for the elevated LT generation in the endometrium in the course of spontaneous inflammatory process. Thus, NPY, in an indirect manner, can affect the LT-modulated processes in the endometrium with inflammation. The mechanisms underlying changes in the YR abundances as well as their significance in an inflamed endometrium function need be further investigated. We postulate that pharmacological modulation of Y1Rs and Y2Rs may be important for control of secretory activity of an inflamed endometrium. Materials and Methods Animals All study procedures were approved by The Local Ethics Committee for Experiments on Animals (University of Warmia and Mazury in Olsztyn, Poland, Consent no. 65/2015). The guidelines in EU Directive 2010/63/EU for animal studies were also followed. Ten crossbred gilts (Large White x Landrace) of similar age (7-8 months) and weighing between 90 and 120 kg were used in this experiment. Behavioral estrus was detected using a tester boar. The animals were characterized by no disturbances in reproductive processes (vaginal discharges were not observed, the second estrous cycle occurred regularly). For acclimatization, three days before the start of the study, the gilts were transported from the commercial farm (Agro-Wronie Sp. z o.o., Wronie, Wąbrzeźno, Poland) to the local animal house (University of Warmia and Mazury, Olsztyn, Poland). Pigs were maintained in individual pens (with an area of about 5 m 2 ), under natural light (14.5±1.5 h - day and 9.5±1.5 h - night) and temperature (18±2°C) conditions. They were fed typically for this species and animal age and had access to water. During the experiment, the gilts were not exposed to any treatment. Study design At the end of the acclimatization period (day 3 of the second estrous cycle - day 0 of the study), the gilts were randomly divided into the control group (CON, the gilts with saline injections into uterine horns, n=5) and E. coli group ( E. coli, the gilts with E. coli injections into uterine horns, n=5). Study procedures have been reported earlier [62]. Briefly, to induce premedication atropine (Atropinum sulf. WZF, Warszawskie Zakłady Farmaceutyczne Polfa S.A., Poland), azaperone (Stresnil, Janssen Pharmaceutica, Beerse, Belgium) and ketamine hydrochloride (Ketamina, Biowet, Puławy, Poland) were used. General anesthesia was induced by ketamine hydrochloride. After median laparotomy was done, in the E. coli group, E. coli suspension (50 ml content: 10 9 colony forming units/ml, strain with a serotype O25:K23/a/:H1; Department of Microbiology, National Veterinary Research Institute, Puławy, Poland) was injected into each uterine horn. The quantity of injected bacteria was based on previous report which revealed that the injections into the pig uterine horns of the same strain of E. coli in the same amount as in the current experiment, evoked the severe (mostly) or moderate acute endometritis after eight days [62]. In the CON group, saline solution (50 ml) was injected into each uterine horn. Bacterial suspension and saline solution were injected into the horn of the uterus at 5 sites at a similar distance from each other. Ten ml of bacterial suspension/saline was administered into each site. The animals from both groups were untreated in the period from surgery until euthanasia. The euthanasia of gilts was performed by an overdose of sodium pentobarbital on day 8 of the study (the expected day 11 of the estrous cycle) and uteri were harvested. Immediately following this, pieces of the uterine horn were collected from the paraoviductal, middle and paracervical parts of the organ and intended for real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) and Western blot analyses. For this purpose, endometrial and myometrial layers were separated using a scalpel blade. The separation of these layers was evaluated using a dissecting microscope. The endometrial pieces were immediately shock-frozen in liquid nitrogen and stored at -80 °C for real-time RT and Western blot analysis. The collection of uterine horn fragments, separation of both uterine layers and their freezing were carried out under sterile conditions to avoid contamination. Pieces of the uterine horns were collected also from three parts of the organ for immunofluorescent study. First, they were divided into smaller pieces and fixed in 4% paraformaldehyde solution (pH 7.4) After 24 h, pieces were washed in 0.1 M phosphate buffered-saline (PBS, pH 7.4). In order to perform immunofluorescent staining, tissues were cryoprotected in 18% sucrose until sectioning. For examination of the secretory activity of endometrium, fragments of the uterine horns collected from the middle part of the horns were placed on ice and transported to the laboratory within 20 min of collection. Preparation and treatment of endometrial explants The uterine fragments were washed twice using sterile PBS. Next, uterine wall was divided into endometrium and myometrium with the help of a scalpel blade. The endometrium fragments were cut in slices weighing 60-70 mg and then rinsed in Medium (cat. no. M2520, Sigma). Single endometrial explants were put into glass vials with 2 ml of Medium 199 which contained: 0.1% bovine serum albumin (BSA; cat. no. A2058, Sigma) and antibiotics (500 μl/500 ml gentamicin, 100 μl/500 ml neomycin, cat. no. G1272, N1142, respectively, both from Sigma). Preincubation and incubation of slices were performed in a shaking water bath (temperature: 37 °C, a humidified atmosphere: 95% air and 5% CO 2 ). Preincubation was carried out for 1.5 h. Next, the endometrial explants were treated, for 16 h, with fresh (control value) medium or with the addition of NPY (10 -7 M, cat. no. H-4430.0500, Bachem) alone or YR antagonists alone (each at a dose of 10 -6 M) for: Y1Rs (BIBO 3304 trifluoroacetate, no cat. 2412, Tocris Biotechnology) and Y2Rs (CYM 9484, no cat. 4606, Tocris Biotechnology). The endometrial explants were also incubated with NPY (10 -7 M) in combination with particular antagonists (each at a dose of 10 -6 M). Initial dilutions of NPY and the antagonists were done in accordance with the manufacturer’s instructions (NPY was diluted in deionized water, antagonists for Y1Rs and Y2Rs were diluted in dimethyl sulfoxide /cat. no. W387509, Sigma/), and then stored at -20 °C. The final antagonists solutions and NPY solution were prepared using the same medium as for preincubation and incubation of explants. Each treatment was performed in duplicate (five separate studies for particular groups, n=5). The PGF2α secretion after exposure to a nitric oxide (NO) donor (NONOate; at a dose of 10 -4 M, cat. no. 82150, Cayman Chemical Co.) was applied to control the reactivity of endometrial explants. The doses of NPY, antagonists and NONOate and the time of incubation, were selected based on the findings from preliminary studies or according to the authors’ previous experiments. After the end of incubation, the endometrial explants were blotted with a paper filter, weighed and placed at -80 °C until estimation of 5-LOX, LTAH and LTCS protein abundances by Western blot analysis. The medium was placed into tubes with 5% EDTA (cat. no. 118798103, Chempur, Piekary Śląskie, Poland), 1% acetylsalicylic acid (cat. no. 107140422, POCH Gliwice, Poland) solution (pH 7.4), and stored at -20 °C until determination of LTB4 and LTC4 concentrations by ELISA method. RNA extraction, and real-time RT-PCR Total RNA was isolated from endometrial layers. Tissues were homogenized in TRI Reagent solution (Invitrogen, Thermo Fisher Scientific, USA) using a FastPrep 24 homogenizer (MP Biomedicals, LCC, USA). For phase separation, a BCP reagent (Molecular Research Center Inc., USA) was used, and the RNA was then purified by using an RNeasy Mini Kit (QIAGEN, USA), according to the manufacturer's instructions. RNA was stored until further use at -80 °C in RNase-free water with the addition of RNAse Inhibitor (Applied Biosystems, Thermo Fisher Scientific, USA). The quality and quantity of extracted RNA was estimated using NanoDrop 1000 (Thermo Fisher Scientific, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA). RNA with an RNA Integrity number ranging 7.0-9.6 was used in real-time RT-PCR. Real-time RT-PCR was performed using TaqMan assays and a one-step PCR Master mix (Applied Biosystems). TaqMan assays for porcine Y1Rs (NPY1R), Y2Rs (NPY2R), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (ACTB) and hypoxanthine guanine phosphoribosyl transferase (HPRT) were reported previously [22]. Each reaction (10 μl) contained: 15 ng of total RNA in a volume of 3 µl, 5 μl 2 x TaqMan RT-PCR Mix, 0.25 μl 40 x TaqMan RT Enzyme Mix, 0.5 μl 20 x TaqManGene Expression Assays and 1.25 μl RNase-free water (Applied Biosystems). The real-time RT-PCR reaction was performed in duplicates in 384-well plates using the following conditions: reverse transcription for 15 min at 48 °C, initial denaturation for 10 min at 95 °C, followed by 45 cycles of 15 s of denaturation at 95 °C and then 1 min of annealing at 60 °C, in an ABI Prism 7900HT system (Applied Biosystems). The negative control was prepared by replacing the RNA template with RNase free water. Data obtained by real-time PCR were analyzed using the Miner method [63]. In order to select the most stable housekeeping gene among ACTB, HPRT and GAPDH, the NormFinder algorithm was applied 64]. The analysis indicated the best stability value for the combination of ACTB and GAPDH genes (0.171). Therefore, abundances levels for each target gene were normalized relatively to the geometric mean of ACTB and GAPDH gene abundance. Western blotting Endometrial pieces for Y1R, Y2R, 5-LOX, LTAH and LTCS protein abundance estimation were minced and placed on ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.4; 50 mM EDTA, 150 mM NaCl, 1% Triton X100, all from Sigma) supplemented with a proteinase inhibitor (Proteinase Inhibitor Cocktail Tablets EDTA-Free, cat. no. S8830-2Tab, Sigma ). Tissue samples were sonicated and centrifuged (15 min, 13500 g , 4 °C). Obtained supernatants were frozen at -80 °C until determination. The total protein concentration was estimated by Bradford method [65]. Equal amounts of protein isolate (120 μg) were dissolved in sodium dodecyl sulphate (SDS, cat. no. L3771, Sigma), a gel-loading buffer, heated (4 min, 95 °C) and separated by 12% SDS-polyacrylamide gel electrophoresis. The separated proteins were then electroblotted onto 0.45 µm pore size Immobilon®-P PVDF membranes (cat. no. IOVH00010, Sigma) in a transfer buffer. In order to block nonspecific binding sites, the incubation was carried out with 5% fat-free dry milk (Spółdzielnia Mleczarska, Gostyń, Poland) in Tris (cat. no. T1503, Sigma) -buffered saline Tween 20 (cat. no. P1379, Sigma) buffer (1.5 h, 21 °C). The membranes were incubated (18 h, 4°C) with polyclonal rabbit antibodies for Y1Rs (diluted 1:500, cat. no. AP01221PU-N, Acris an OriGene Co.), Y2Rs (diluted 1:1000, cat. no. TA314282, Acris an OriGene Co.), 5-LOX (diluted 1:200, cat. no. 160402, Cayman Chemical Co.), LTAH (diluted 1:200, cat. no. 1600250, Cayman Chemical Co.) and for LTCS (diluted 1:1000, cat. no. DF14129, Affinity Biosciences). Subsequently, the membranes were incubated (1.5 h, 21 °C) with secondary antibodies alkaline phosphatase-conjugated goat anti-rabbit for all determined factors (diluted 1:10000, cat. no. 111-055-003, Jackson Immunoresearch). Protein immune complexes were visualized by applied a standard alkaline phosphatase method (NBT-BCIP; cat. no. 72091, Sigma). Analysis was made three times. Specificity of antibodies for Y1Rs and Y2Rs [22] as well as 5-LOX, LTAH and LTCS [28, 53] was earlier presented for pig uterus. As internal control for protein loading, polyclonal rabbit GAPDH (diluted 1:5000, cat. no. G9545, Sigma) was used. Images were acquired and quantified using a CHEMIDOC Touch Imaging System (Image Lab 5.2, Bio-Rad Laboratories, Hercules, CA, USA). Immunofluorescence To determine the tissue distribution of Y1Rs and Y2Rs, fragments of uterine horns were cut in cryostat (Leica CM 1950). Next, 10-µm-thick sections were subjected to the routine single-immunofluorescence technique. After air-drying (30 min, 21 °C), sections were then washed in 0.1 M PBS (pH 7.4; 3 x 15 min) and then incubated (1 h, 21 °C) in blocking buffer, containing 0.1 M PBS, 10% normal goat serum (MP Biomedicals), 0.1% BSA (Sigma), 0.05% Thimerosal (Sigma), 1% Triton X-100 (Sigma) and 0.01% NaN3. After further rinsing in PBS (3 x 15 min), tissues were incubated (18 h, 21 °C) in a humidity chamber, with primary antibodies, the same as for Western blot analysis against Y1Rs and Y2Rs (both diluted 1:100). Subsequently, after following washing in PBS (3 x 15 min), the sections were incubated (1 h, 21 °C) with biotinylated anti-rabbit IgG (diluted 1:1000, cat. no AP132B, Chemicon International). Next, the sections were incubated (1 h, 21 °C) with carbocyanine 3 (CY3)-conjugated streptavidin (diluted 1:9000, cat. no. 016160084, Jackson Immunoresearch). Negative control staining was accomplished by replacing the primary antibodies with the same concentration of rabbit normal IgG. Endometrial cells immunoreactive for the used antibody were estimated under an Olympus BX51 microscope (Olympus Consilio sp. z.o.o., Warsaw, Poland) equipped with epi-fluorescence and an appropriate filter. ELISA procedure Concentrations of LTB4 and LTC4 in the incubation medium were determined by the use of ELISA kits (cat. no. 502390 and 501070, respectively, Cayman Chemical Co.) according to the manufacturer’s instructions. The standard curve for LTB4 ranged from 1.96 to 1000 pg/ml, and the effective dose for 50% inhibition (ID50) of assay was 2.5 pg/ml. The intra- and interassay coefficients of variation were 4.5% and 7.2%, respectively. The standard curve for LTC4 ranged from 0.98 to 500 pg/ml, and the effective dose for ID50 of the assay was 1.85 pg/ml. The intra-assay and interassay coefficients of variation were 4.7% and 6.2%, respectively. Statistical analysis The normal distribution (P>0.05) of all data and residuals were determined using the Shapiro–Wilk test. The significant differences in mRNA and protein receptor abundances between both groups were analyzed via Student`s test. The results from the incubation of endometrium for which the PGF2α secretion in response to NONOate was statistically significant, were only taken into account. A two-way ANOVA (group, treatment) followed by the Bonferroni test was applied to compare the mean (± sem) values. Differences were accepted as statistically significant at P<0.05. For above analyses InStat Graph Pad (San Diego, CA) was used. Ethical approval The studies presented in the manuscript were carried out in accordance with the ARRIVE guidelines. The all study procedures were approved by the Local Ethics Committee for Experiments on Animals (University of Warmia and Mazury in Olsztyn, Poland; Consent no. 65/2015). The guidelines in EU Directive 2010/63/EU for animals experiments were included. Declarations Acknowledgments This work was supported by the State Committee for Scientific Research (grant No. 2014/15/B/N25/03572). The cost of the publication was covered by a statutory research grant from the Department of Clinical Physiology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn. We thank Joanna Kalinowska and Andrzej Pobiedziński for their assistance. Author contributions B.J.: contributed to the conception and design of the study, performed surgical procedures, participated in the laboratory analyses, analyzed and interpreted data, written draft, reviewed and edited the manuscript. J.C.: analyzed and interpreted data, and reviewed and edited the manuscript. M.B.: participated in the laboratory analyses, and reviewed and edited the manuscript the manuscript. Data availability The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. 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Enzyme abundances were determined by Western blotting and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein used as loading control. Data are expressed as the mean ± sem. CV: control value (gained for an endometrial stripes from the CON or E. coli groups that had not undergone any treatment in vitro conditions); anta.: antagonist Mean values with various letters (a, b) differ significantly (P<0.05-0.001) within the CON or E. coli groups for the same enzyme between CV, action of NPY and actions of particular antagonists; * P<0.05, ** P<0.01,*** P<0.001 - represent statistical differences between groups for the same enzyme and treatment. Treatment Group Protein abundance (arbitrary units) 5-LOX LTAH LTCS CV CON 0.63 ± 0.08 a 1.84 ± 0.63 a 0.62 ± 0.03 a E. coli 1.94 ± 0.18 a,*** 3.78 ± 0.22 a,* 1.74 ± 0.22 a,** NPY CON 1.75 ± 0.15 b 4.65 ± 0.41 b 1.67 ± 0.26 b E. coli 3.18 ± 0.22 b,*** 6.57 ± 0.61 b,* 2.75 ± 0.44 b,* Y1R anta. CON 0.81 ± 0.07 a 1.42 ± 0.35 a 0.53 ± 0.04 a E. coli 1.76 ± 0.19 a,*** 3.23 ± 0.19 a* 1.53 ± 0.19 a,* Y2R anta. CON 0.59 ± 0.09 a 1.75 ± 0.22 a 0.61 ± 0.04 a E. coli 1.67 ± 0.21 a,*** 2.89 ± 0.17 a 1.66 ± 0.21 a,* Table 2. Influence of neuropeptide Y (NPY, 10 -7 M) alone or antagonists of NPY receptors subtype 1 (Y1R) and NPY receptors subtype 2 (Y2R) (10 -6 M) alone on the leukotriene (LT)B4 and LTC4 secretion from the endometrium of gilts from the CON and E. coli groups (n=5 in each group). Treatments were performed in duplicate for each gilt in the CON and E. coli groups. LTs contents were determined by ELISA. Data are expressed as the mean ± sem. CV: control value (gained for an endometrial stripes from the CON or E. coli groups that had not undergone any treatment in vitro conditions); anta.: antagonist. Treatment Group Concentrations (pg/ml of medium) LTB4 LTC4 CV CON 140.4 ± 8.6 a 28.8 ± 3.9 a E. coli 234.8 ± 13.7 a,** 56.1 ± 3.1 a,** NPY CON 258.3 ± 24.3 b 65.4 ± 5.3 b E. coli 358.5 ± 28.1 b,** 98.2 ± 7.3 b,*** Y1R anta. CON 179.5 ± 9.8 a 31.8 ± 2.9 a E. coli 277.2 ± 15.6 a,** 60.3 ± 4.8 a,*** Y2R anta. CON 167.9 ± 8.9 a 29.8 ± 3.1 a E. coli 236.4 ± 18.1 a 57.2 ± 5.2 a,*** Mean values with various letters (a, b) differ significantly (P<0.05-0.001) within the CON or E. coli groups for the same LT between CV, action of NPY and actions of particular antagonists; ** P<0.01, *** P<0.001 - represent statistical differences between groups for the same LT and treatment. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-7738675\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":595818653,\"identity\":\"e313cb0d-3b9d-4d1d-867d-46e00e3dcc1a\",\"order_by\":0,\"name\":\"Barbara Jana\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYBACAwaGBCBlA+E9gAk/wKYWVUsahAdkSsAY+LSAwGEStJgzMDz8XFBzPo9/dgPjg4Q/dXUGtxvYHuDTYtnAkCw949jtYok7B5gNEtvYJAzuHGA3wOuwAwwJ0jxstxMbbiSwSSQ28EgYgBgEtCT/5vl3LnE+WOUfCaK0pEnzth1I3ABWyWZAWItlM0OaNW9fcrHhjcRmoF8SJGfeSGzH6xdz9p7k2zzf7PLkbiQffPDhTx0/343kYw8+4NHCwMwDNhFIMDaABRQOMLbh0wAE7AegWqBAvoGBjYCWUTAKRsEoGGEAAOi3TqZuvq6YAAAAAElFTkSuQmCC\",\"orcid\":\"\",\"institution\":\"Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences in Olsztyn, 18 Trylińskiego St., 10-683 Olsztyn, Poland\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Barbara\",\"middleName\":\"\",\"lastName\":\"Jana\",\"suffix\":\"\"},{\"id\":595818654,\"identity\":\"b719feb9-ca2f-4679-9f9e-8443f50b4213\",\"order_by\":1,\"name\":\"Jarosław Całka\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Warmia and Mazury\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jarosław\",\"middleName\":\"\",\"lastName\":\"Całka\",\"suffix\":\"\"},{\"id\":595818655,\"identity\":\"b9722357-3cfb-4411-9c0b-40d3d59437ed\",\"order_by\":2,\"name\":\"Michał Bulc\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Warmia and Mazury\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Michał\",\"middleName\":\"\",\"lastName\":\"Bulc\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-09-29 06:38:26\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-7738675/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-7738675/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":103415485,\"identity\":\"c3779a46-30ca-48b5-8aba-e500686e06b1\",\"added_by\":\"auto\",\"created_at\":\"2026-02-25 11:57:30\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":34416,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMessenger RNA abundances of neuropeptide Y (NPY) receptor subtype 1 (Y1R, A) and NPY receptor subtype 2 (Y2R, B) in the endometrium of gilts from the control (CON) and \\u003cem\\u003eE. coli\\u003c/em\\u003e(\\u003cem\\u003eE. coli\\u003c/em\\u003e) groups (n=5 for each group). Results of quantification by real-time PCR are expressed as the mean ± sem. Receptor abundances were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA used as loading control. * P\\u0026lt;0.05, *** P\\u0026lt;0.001 - represent statistical differences between groups for the same receptor.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7738675/v1/1360519dfa2aa2ca8a138216.jpg\"},{\"id\":103415478,\"identity\":\"2a306488-576e-411d-a3a7-435f6cda5c85\",\"added_by\":\"auto\",\"created_at\":\"2026-02-25 11:57:25\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":34287,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eProtein abundances\\u003cstrong\\u003e \\u003c/strong\\u003eof neuropeptide Y (NPY) receptor subtype 1 (Y1R, A) and NPY receptor subtype 2 (Y2R, B) in the endometrium of gilts from the control (CON) and \\u003cem\\u003eE. coli\\u003c/em\\u003e (\\u003cem\\u003eE. coli\\u003c/em\\u003e) groups (n=5 for each group). Results of quantification by Western blotting are expressed as the mean ± sem. Receptor abundances were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein used as loading control. Representative blots with protein bands for each receptors are presented on Supplementary Fig. 1A and B. * P\\u0026lt;0.05, *** P\\u0026lt;0.001 - represent statistical differences between groups for the same receptor.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7738675/v1/f055ae1b29814eb7b6cdede0.jpg\"},{\"id\":103415476,\"identity\":\"cf5e94af-38fc-42d9-b1a4-3edd026b8343\",\"added_by\":\"auto\",\"created_at\":\"2026-02-25 11:57:25\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":112435,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eImages of neuropeptide Y (NPY) receptor subtype 1 (Y1R, A-F) and NPY receptor subtype 2 (Y2R, G-L) in the endometrium of gilts from the control (CON) and \\u003cem\\u003eE. coli (E. coli) \\u003c/em\\u003egroups. Positive reaction to Y1Rs is visible in luminal epithelium (LE), glands (G) and artery (A) of the CON (A-C) and \\u003cem\\u003eE. coli\\u003c/em\\u003e (D-F) groups. Immunoreactivity for Y2Rs displays above-mentioned structures in the CON (G-I) and \\u003cem\\u003eE. coli\\u003c/em\\u003e (J-L) groups. Negative control (NC) for Y1Rs (M) and Y2Rs (O) after replacing primary antibodies with rabbit normal IgG.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7738675/v1/bffde0df9a9f5975159a46d6.jpg\"},{\"id\":103415470,\"identity\":\"45b798bc-8494-4cff-93d1-f5bf65fbc9df\",\"added_by\":\"auto\",\"created_at\":\"2026-02-25 11:57:25\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":33866,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eInfluence of neuropeptide Y (NPY, 10\\u003csup\\u003e-7\\u003c/sup\\u003e M) alone or antagonists of NPY receptor subtype 1 (Y1R) and NPY receptor subtype 2 (Y2R) (10\\u003csup\\u003e-6\\u003c/sup\\u003e M) with NPY (10\\u003csup\\u003e-7\\u003c/sup\\u003e M) on 5-lipoxygenase (5-LOX) protein abundances in the endometrium of gilts from the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups (n=5 for each group). Treatments were performed in duplicate for each gilt in the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups. Enzyme abundances were determined by Western blotting and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein used as loading control. Data are expressed as the mean ± sem. Representative blot with protein bands for each treatment is presented on Supplementary Fig. 2A. Mean values with various letters (a, b) differ significantly (P\\u0026lt;0.01, P\\u0026lt;0.001) within the CON or \\u003cem\\u003eE. coli\\u003c/em\\u003e groups; *** P\\u0026lt;0.001 - represents statistical differences between groups for the same treatment; CV: control value (gained for an endometrial stripes from the CON or \\u003cem\\u003eE. coli\\u003c/em\\u003e groups that had not undergone any treatment\\u003cem\\u003e in vitro \\u003c/em\\u003econditions); anta.: antagonist.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7738675/v1/72284284f1614fcf289b2a16.jpg\"},{\"id\":103415477,\"identity\":\"92e1b97d-400a-40ed-a521-6b6b1f054ffa\",\"added_by\":\"auto\",\"created_at\":\"2026-02-25 11:57:25\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":34471,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eInfluence of neuropeptide Y (NPY, 10\\u003csup\\u003e-7\\u003c/sup\\u003e M) alone or antagonists of NPY receptor subtype 1 (Y1R) and NPY receptor subtype 2 (Y2R) (10\\u003csup\\u003e-6\\u003c/sup\\u003e M) with NPY (10\\u003csup\\u003e-7\\u003c/sup\\u003e M) on leukotriene A4 hydrolase (LTAH) protein abundance (A) and on leukotriene (LT)B4 (B) secretion by the endometrium of gilts from the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups (n=5 for each group). Treatments were performed in duplicate for each gilt in the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups. Enzyme abundances were determined by Western blotting and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein used as loading control. LT contents were determined by ELISA. Data are expressed as the mean ± sem. Representative blot with protein bands for each treatment is presented on Supplementary Fig. 2B. Mean values with various letters (a, b, c) differ significantly (P\\u0026lt;0.01, P\\u0026lt;0.001) within the CON or \\u003cem\\u003eE. coli\\u003c/em\\u003e groups; * P\\u0026lt;0.05, ** P\\u0026lt;0.01,\\u003csup\\u003e \\u003c/sup\\u003e*** P\\u0026lt;0.001 - represent statistical differences between groups for the same treatment; CV: control value (gained for an endometrial stripes from the CON or \\u003cem\\u003eE. coli\\u003c/em\\u003e groups that had not undergone any treatment\\u003cem\\u003e in vitro \\u003c/em\\u003econditions); anta.: antagonist.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7738675/v1/adcba449df4ca458aabadce3.jpg\"},{\"id\":103415468,\"identity\":\"6fd8b677-ed98-44dd-8b3d-1c2b8e20d7c5\",\"added_by\":\"auto\",\"created_at\":\"2026-02-25 11:57:25\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":55553,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eInfluence of neuropeptide Y (NPY, 10\\u003csup\\u003e-7\\u003c/sup\\u003e M) alone or antagonists of NPY receptor subtype 1 (Y1R) and NPY receptor subtype 2 (Y2R) (10\\u003csup\\u003e-6\\u003c/sup\\u003e M) with NPY (10\\u003csup\\u003e-7\\u003c/sup\\u003e M) on leukotriene C4 synthase (LTCS) protein abundance (A) and on leukotriene (LT)C4 (B) secretion by the endometrium of gilts from the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups (n=5 for each group). Treatments were performed in duplicate for each gilt in the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups. Enzyme abundances were determined by Western blotting and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein used as loading control. LT contents were determined by ELISA. Data are expressed as the mean ± sem. Representative blot with protein bands for each treatment is presented on Supplementary Fig. 2C. Mean values with various letters (a, b, c) differ significantly (P\\u0026lt;0.05-0.001) within the CON or \\u003cem\\u003eE. coli\\u003c/em\\u003e groups; * P\\u0026lt;0.05,\\u003csup\\u003e \\u003c/sup\\u003e** P\\u0026lt;0.01,\\u003csup\\u003e \\u003c/sup\\u003e*** P\\u0026lt;0.001 - represent statistical differences between groups for the same treatment; CV: control value (gained for an endometrial stripes from the CON or \\u003cem\\u003eE. coli\\u003c/em\\u003e groups that had not undergone any treatment\\u003cem\\u003e in vitro \\u003c/em\\u003econditions); anta.: antagonist.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7738675/v1/7f85dffc64e4e7c2086d7448.jpg\"},{\"id\":103415487,\"identity\":\"e425c385-e855-4ca4-96ba-a22b551f8344\",\"added_by\":\"auto\",\"created_at\":\"2026-02-25 11:57:36\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1563131,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7738675/v1/3064ad01-bea4-4288-a1ff-fc7373371924.pdf\"},{\"id\":103415475,\"identity\":\"f1fec8f6-0bb5-4bb3-832f-15a2b3a4f3c8\",\"added_by\":\"auto\",\"created_at\":\"2026-02-25 11:57:25\",\"extension\":\"docx\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":335150,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Suppl.materialsBJ.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7738675/v1/e97e668b3ba0656515f8c7bf.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Participation of neuropeptide Y and its receptors in leukotriene generation in the pig inflamed endometrium\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eUterine inflammation (endometritis, metritis) very often occurs in domestic animals and women developing mainly during puerperal period.\\u0026nbsp;In pigs this disorder is\\u0026nbsp;very often connected with the mammary gland inflammatory process, creating a postpartum dysgalactia syndrome\\u0026nbsp;[1, 2]. Severe kinds of uterine inflammation in cows and pigs cause the reproductive problems and bringing substantial economic losses on farm [3, 4]. The pathology develops as a result of disturbances in the endometrial immune defense mechanisms and/or in contractility of myometrial layer [5].\\u0026nbsp;Most often, uterine inflammation is caused by bacteria, including \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e (\\u003cem\\u003eE. coli\\u003c/em\\u003e).\\u0026nbsp;\\u003cem\\u003eStreptococcus\\u0026nbsp;\\u003c/em\\u003espp., \\u003cem\\u003eStaphylococcus\\u0026nbsp;\\u003c/em\\u003espp., \\u003cem\\u003eTrueperella\\u0026nbsp;pyogenes\\u003c/em\\u003e, and \\u003cem\\u003eKlebsiella pneumoniae\\u0026nbsp;\\u003c/em\\u003e[6, 7].\\u003c/p\\u003e\\n\\u003cp\\u003eLeukotrienes (LTs) are a family of pro-inflammatory lipid mediators playing a significant role in immune-mediated diseases. In response to inflammatory, allergic and immune signals, activated cytosolic phospholipase A2 converts phospholipids from cellular membrane to arachidonic acid. This acid is serially converted to 5-hydroperoxyeicosatetraenoic acid and then to LTA4 by 5-lipoxygenase\\u0026nbsp;(5-LOX, also called 5-LO)\\u0026nbsp;in the presence of 5-LOX activating peptide (FLAP). LTA4 can be either hydrolyzed by LTA4 hydrolase (LTAH) to LTB4 or conjugated with glutathione by LTC4 synthase (LTCS) to yield LTC4. LTC4, first of three cysteinyl-LTs (cys-LTs) may be converted to LTD4 by glutamyl transpeptidase and then to LTD4 by dipeptidase [8, 9]. CysLTs are ligands for cysLT1, cysLT2 and cysLT3 receptors, while LTB4 exerts its actions by binding to BLT1 and BLT2 receptors [10,11]. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eNeuropeptide Y (NPY), a highly conserved 36-amino acid peptide, is the most widely distributed neuropeptide, performing a wide spectrum of physiological functions in both the central and peripheral nervous systems.\\u0026nbsp;Moreover, NPY was also identified in non-neuronal cells, for example, platelets, endothelial and immune cells. Biological effects of NPY are mediated by six subtypes of G protein-coupled Y-family receptors (YRs), termed from Y1 to Y6 [12], however, this peptide preferentially acts \\u003cem\\u003evia\\u003c/em\\u003e Y1R, Y2R and Y5R subtypes [\\u003ca href=\\\"https://www.sciencedirect.com/science/article/pii/S0143417918300969#bb0100\\\"\\u003e13\\u003c/a\\u003e]. Immunoreactivity for NPY is present in the cholinergic and noradrenergic neurons in the pig paracervical ganglion (PCG) [14]. Moreover, NPY was determined in the pig uterine noradrenergic perikarya in the caudal mesenteric ganglion (CaMG) [15, 16] and PCG [17, 18]. In human [19] and pigs [20] uteri NPY-positive nerve fibers occur in the vicinity of blood vessels, glands and myocytes of the myometrium. It is known that under physiological conditions NPY increases the contractile activity in the rat myometrium through Y1Rs [21]. NPY acting by Y1Rs and Y2Rs reduces the contractility of the porcine uterus [22]. This peptide is also a potent modulator of the immune response during inflammation [23]. Its role as well as Y1Rs and Y2Rs was reported, for example, in the inflammatory bowel disease [24] and asthma [25].\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eIn relation to the inflamed uterus, it is known that during severe acute endometritis in pigs increased the populations of uterine sympathetic perikarya expressing NPY in the CaMG [16] and PCG [18]. Inflammation changes also the Y1R and Y2R expression in the pig myometrium and decreases the NPY-evoked contractility of inflamed uterus [22]. In relation to the 5-LOX pathway, it was reported that uterine inflammation significantly increased the LTB4 and LTC4 formation and secretion [26-28]. Up to now, NPY receptors have not been determined in the inflamed (also healthy) endometrium of any species, and the action of NPY on arachidonic acid metabolites formation, including LTs, in this tissue is not known. In relation to LTs, it was only reported that NPY enhanced platelet activating factor (PAF)-stimulated LTD4 level in the rat lungs [29]. It is hypothesized that inflammation alters the abundance of YRs in endometrium and that the NPY action on LT biosynthesis pathway in the inflamed endometrium. Defining the connection between NPY and LTs will contribute to further understanding of neuro-immune interactions. Therefore, we studied the effect of inflammation on Y1R and Y2R mRNA and protein abundances in pig endometrium and the NPY influence on 5-LOX, LTAH and LTCS protein abundances and LTB4 and LTC4 release by this tissue.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eMessenger RNA abundances of Y1Rs and Y2Rs \\u0026nbsp;\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIn the \\u003cem\\u003eE. coli\\u003c/em\\u003e group, the Y1R (P\\u0026lt;0.001) and Y2R (P\\u0026lt;0.05) mRNA abundances in the endometrium were lower than in the CON group (Fig. 1A, B).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eProtein abundances of Y1Rs and Y2Rs\\u003c/strong\\u003e \\u003c/p\\u003e\\n\\u003cp\\u003eIn the \\u003cem\\u003eE. coli\\u003c/em\\u003e group endometrium, the Y1R protein abundance was reduced (P\\u0026lt;0.001) compared to the CON group (Fig. 2A). The Y2R protein abundance in the endometrium of \\u003cem\\u003eE. coli\\u003c/em\\u003e group was increased (P\\u0026lt;0.05) in relation to the CON group (Fig. 2B).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDistribution of Y1Rs and Y2Rs\\u003c/strong\\u003e\\u0026nbsp; \\u0026nbsp;\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eImmunofluorescent method showed the presence of Y1Rs and Y2Rs in the luminal and glandular epithelium and blood vessels (endothelium, muscle layer) of the endometrium of the CON (Fig. 3A, B, C, G, H, I) and \\u003cem\\u003eE. coli\\u003c/em\\u003e (Fig. 3D, E, F, J, K, L) groups. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eNo staining of Y1Rs (Fig. 3M) and Y2Rs (Fig. 3N) was observed after replacement of primary antibodies by normal rabbit IgG.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEffect of NPY and/or antagonists of Y1Rs and Y2Rs\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eon 5-LOX protein abundance in endometrium\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eAntagonists of\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003eY1Rs and Y2Rs\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003ealone:\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003eIn the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups, the 5-LOX protein abundances under the NPY influence were higher (P\\u0026lt;0.001) compared to influences of Y1R and Y2Rantagonists (Table 1). The exposition of the \\u003cem\\u003eE. coli\\u003c/em\\u003e group endometrium to the antagonists increased (P\\u0026lt;0.001) the enzyme abundances versus the CON group.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eNPY alone:\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003eIn both groups, NPY augmented (P\\u0026lt;0.01) the 5-LOX abundances versus the control values (obtained from an endometrium of the CON or \\u003cem\\u003eE. coli\\u003c/em\\u003e groups that had not undergone any \\u003cem\\u003ein vitro\\u003c/em\\u003e treatment) (Fig. 4). In the \\u003cem\\u003eE. coli\\u003c/em\\u003e group, the control value of protein abundance and that in response to NPY were higher (P\\u0026lt;0.001) than in the CON group.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eY1R\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003eantagonist or\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003eY2R\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003eantagonist with NPY\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003e:\\u003c/em\\u003e\\u003c/strong\\u003eIn the CON (P\\u0026lt;0.01) and \\u003cem\\u003eE. coli\\u003c/em\\u003e (P\\u0026lt;0.001) groups, Y1R antagonist with NPY decreased the 5-LOX abundances in reference to the NPY action(Fig. 4). In both groups, the enzyme abundances did not significantly change by Y2R antagonist with NPY versus the NPY action. The protein abundance in response to Y1R antagonist with NPY did not differ significantly between both groups. The enzyme abundance in response to Y2R antagonist with NPY in the \\u003cem\\u003eE. coli\\u003c/em\\u003e group was higher (P\\u0026lt;0.001) than in the CON group.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEffect of NPY and/or antagonists of Y1Rs and Y2Rs\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eon LTAH protein abundance and LTB4 secretion from endometrium\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eLTAH protein abundance\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eAntagonists of Y1Rs and Y2Rs\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003ealone:\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003eThe LTAH abundances in the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups were lowered (P\\u0026lt;0.001) after using Y1R and Y2R antagonists compared to the NPY action (Table 1). In the \\u003cem\\u003eE. coli\\u003c/em\\u003e group, the enzyme abundance after using Y1R antagonist was increased (P\\u0026lt;0.05) versus the CON group.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eNPY alone:\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003eNPY led to a rise (P\\u0026lt;0.01) in the LTAH protein abundances in the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups compared to the control values (Fig. 5A). In the \\u003cem\\u003eE. coli\\u003c/em\\u003e group, the control value and that after exposition to NPY were higher (P\\u0026lt;0.05) than in the CON group.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eY1R antagonist or\\u0026nbsp;Y2R antagonist with NPY:\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003eIn the CON group, Y1R antagonist with NPY did not significantly change the LTAH protein abundance versus the NPY action (Fig. 5A). In \\u003cem\\u003eE. coli\\u003c/em\\u003e group, these substances reduce (P\\u0026lt;0.001) the enzyme abundance versus the NPY influence. In the CON (P\\u0026lt;0.01) and \\u003cem\\u003eE. coli\\u003c/em\\u003e (P\\u0026lt;0.001) groups, the LTAH protein abundances in response to Y2R antagonist with NPY were lowered compared to the NPY effect. The enzyme abundances in response to Y1R antagonist or Y2R antagonist with NPY did not differ significantly between both groups.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eLTB4 secretion\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eAntagonists of Y1Rs and Y2Rs\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003ealone:\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003eIn the CON group, the endometrial LTB4 secretion in response to Y1R (P\\u0026lt;0.05) and Y2R (P\\u0026lt;0.01) antagonists was reduced versus the NPY action (Table 2). The LT release in the \\u003cem\\u003eE. coli\\u003c/em\\u003e group lowered after exposition to antagonists for Y1Rs (P\\u0026lt;0.05) and Y2Rs (P\\u0026lt;0.001) compared to the NPY influence. Y1R antagonist evoked greater (P\\u0026lt;0.01) secretion of LT in the \\u003cem\\u003eE. coli\\u003c/em\\u003e group than in the CON group.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eNPY alone:\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003eThe LTB4 release by NPY in the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups was higher (P\\u0026lt;0.001) versus the control values (Fig. 5B). Compared to the CON group, the control value and that after using NPY were increased (P\\u0026lt;0.01) in the \\u003cem\\u003eE. coli\\u003c/em\\u003e group.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eY1R antagonist or Y2R antagonist with NPY:\\u003c/em\\u003e\\u003c/strong\\u003eIn the CON group, Y1R antagonist with NPY reduced (P\\u0026lt;0.001) the LTB4 release in relation to the NPY influence (Fig. 5B). In \\u003cem\\u003eE. coli\\u003c/em\\u003e group, Y1R antagonist with NPY did not significantly change the LTB4 secretion versus the NPY action. In the CON (P\\u0026lt;0.01) and \\u003cem\\u003eE. coli\\u003c/em\\u003e (P\\u0026lt;0.001) groups, the LTB4 release under the action of Y2R antagonist with NPY was lower than the NPY effect. The LTB4 release by endometrium of the \\u003cem\\u003eE. coli\\u003c/em\\u003e group in response to Y1R antagonist with NPY was higher (P\\u0026lt;0.001) than in the CON group.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEffect of NPY and/or antagonists of Y1Rs and Y2Rs\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eon LTCS protein abundance and LTC4 secretion from endometrium\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eLTCS protein abundance\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eAntagonists of Y1Rs and Y2Rs\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003ealone:\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003eThe LTCS abundances in the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups were decreased by Y1R (P\\u0026lt;0.01) and Y2R (P\\u0026lt;0.05) versus the NPY influence (Table 1). In the \\u003cem\\u003eE. coli\\u003c/em\\u003e group, this enzyme abundances after exposition to Y1R and Y2R antagonists were higher (P\\u0026lt;0.05) than in the CON group.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eNPY alone:\\u003c/em\\u003e\\u003c/strong\\u003e In the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups, the LTCS abundances were elevated (P\\u0026lt;0.05) in versus the control values (Fig. 6A). In the \\u003cem\\u003eE. coli\\u003c/em\\u003e group, the control value (P\\u0026lt;0.01) and that in response to NPY (P\\u0026lt;0.05) were increased versus the CON group.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eY1R antagonist or Y1R antagonist with NPY:\\u003c/em\\u003e\\u003c/strong\\u003eIn the CON(P\\u0026lt;0.05)and\\u003cem\\u003e\\u0026nbsp;E. coli\\u003c/em\\u003e (P\\u0026lt;0.01) groups, the enzyme abundances were \\u0026nbsp;dropped by Y1R antagonist with NPY versus the NPY action (Fig. 6A). In both groups, the LTCS abundances after using Y2R antagonist with NPY did not differ significantly in relation to the NPY action. After using Y2R antagonist with NPY, the LTCS abundance in the \\u003cem\\u003eE. coli\\u003c/em\\u003e group was higher (P\\u0026lt;0.001) than in the CON group.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eLTC4 secretion\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eAntagonists of Y1Rs and Y2Rs\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003ealone:\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003eIn the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups, the LTC4 secretion after using\\u0026nbsp;Y1R and Y2R antagonists was lowered (P\\u0026lt;0.001) versus the NPY influence (Table 2). The LT release in the \\u003cem\\u003eE. coli\\u003c/em\\u003e group by Y1R and Y2R antagonists was higher (P\\u0026lt;0.001) than in the CON group.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eNPY alone:\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003eThe LTC4 release by NPY in the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups was higher (P\\u0026lt;0.001) versus the control values (Fig. 6B). In the \\u003cem\\u003eE. coli\\u003c/em\\u003e group, the control value of LTC4 (P\\u0026lt;0.01) and that in response to NPY (P\\u0026lt;0.001) were increased in relation to the CON group.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eY1R antagonist or\\u0026nbsp;Y2R antagonist with NPY:\\u003c/em\\u003e\\u003c/strong\\u003eIn the CON group, the LTC4 release by Y1R antagonist with NPY did not significantly change in relation to the effect of NPY (Fig. 6B). In the \\u003cem\\u003eE. coli\\u003c/em\\u003e group, Y1R antagonist with NPYreduced (P\\u0026lt;0.05) the LTC4 release compared to the NPY effect. The LTC4 secretion in the CON (P\\u0026lt;0.05) and \\u003cem\\u003eE. coli\\u003c/em\\u003e (P\\u0026lt;0.001) groups was decreased by Y2R antagonist with NPY compared to the NPY influence (Fig. 6B). After using Y1R antagonist or Y2R antagonist with NPY, the LTC4 release did not differ significantly between both groups.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThe current study shows the Y1Rs and Y2Rs abundances in the porcine inflamed endometrium and the contribution of NPY, Y1Rs and Y2Rs in the biosynthesis pathway for LTs in this tissue. Histopathological assessment ofthe\\u003cem\\u003e\\u0026nbsp;E. coli\\u003c/em\\u003e-injected uteri revealed the presence of a severe acute endometritis. In the endometrium following changes were determined: edema, hyperemia, the increased number of neutrophils and the damage of luminal and/or glandular epithelium [30].\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTo date, it has only been reported that the paired transcript NPY-Y1Rs is one of many paired transcripts mediating the dialogue extraembryonic membrane and endometrium during\\u0026nbsp;pre- and peri-implantation in sheep under physiological conditions [31]. Here, we demonstrated, for the first time, the Y2R mRNA abundance and Y1R and Y2R protein abundances in the healthy endometrial tissue. Completely new findings of the current study concern also the abundances of Y1Rs and Y2Rs in the endometrium under inflammatory conditions. In the inflamed endometrium we revealed a drop in the Y1R mRNA and protein abundances and Y2R mRNA abundance and a rise in the Y2R protein abundance versus the healthy endometrium. Similar situation was found in the porcine myometrium following intrauterine \\u003cem\\u003eE. coli\\u0026nbsp;\\u003c/em\\u003einjection, with exception of the lack of significant change in the Y2R mRNA abundance [22]. It is also known that in the heart tissue of diabetic patients, the level of the Y2R protein increased and the expression of Y1R and Y2R mRNA was unchanged [32], as well as that the expression of Y1R mRNA in the uterosacral ligaments was increased in women with pelvic organ prolapse [33].\\u003c/p\\u003e\\n\\u003cp\\u003eUsing immunofluorescent method we found that Y1Rs and Y2Rs are present in the luminal and glandular epithelium and endometrial blood vessel cells in the gilts of both CON and \\u003cem\\u003eE. coli\\u0026nbsp;\\u003c/em\\u003egroups.Thus, a severe acute inflammation did not change the localization of Y1Rs and Y2Rs in the endometrium. It allows to suppose that epithelium and blood vessels are the sites of NPY impact\\u0026nbsp;under physiological and inflammatory conditions.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe changes in endometrial Y1R and Y2R mRNA and protein abundances shown in the present study, may be a consequence of bacterial infection and the development of inflammation.\\u0026nbsp;However, there is a lack of data in the available literature on the effect of inflammatory mediators on the expression pattern of YRs. We suppose that the biological signals leading to the changes in the endometrial Y1R and Y2R abundances might include lipopolysaccharide (LPS) originated from bacteria [34] or pro- and anti-inflammatory cytokines generated in large amounts in the course of endometritis [35-37]. The ability of steroid hormones to modulate the endometrial abundance of Y1R and Y2R cannot be ruled out.\\u0026nbsp;Dihydrotestosterone implanted in rats to stimulate polycystic ovarian syndrome increases\\u0026nbsp;the\\u0026nbsp;Y2R and Y5R protein contents in the ovarian granulosa cells [38] and estrogen stimulates the Y1R mRNA expression the in human breast cancer cell line [39]. Decreased the levels of estrogens and progesterone and increased the level of androstenedione in the peripheral blood of pigs with endometritis were previously reported [40]. However, above suppositions\\u0026nbsp;needs further studies.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eOn the basis of changes in the\\u0026nbsp;Y1R and Y2R abundances in\\u0026nbsp;the inflamed endometrium it is suggested that\\u0026nbsp;Y1R and Y2R participate\\u0026nbsp;in the function of this tissue. The presence of both\\u0026nbsp;Y1Rs and Y2Rs in the muscle layer of blood vessels of the \\u003cem\\u003eE. coli\\u003c/em\\u003e group (also CON group) allows to assume that these receptors mediate the NPY-controlled blood flow in the endometrium. NPY is able to modulate the contractile activity of the guinea pig uterine arteries acting mainly \\u003cem\\u003evia\\u003c/em\\u003e Y1Rs [41, 42] and of the rabbit ovarian artery [19] and the guinea pig intestine arterioles [43] \\u003cem\\u003evia\\u0026nbsp;\\u003c/em\\u003eY1Rs and Y2Rs. It is also known that NPY regulates the influence of noradrenaline [44] and vasoactive intestinal peptide [45] on uterine blood flow. Revealed in our study the immunoreaction for Y1Rs and Y2Rs in the luminal and glandular epithelium of both studied groups suggests that these receptors may be important for secretory activity of endometrium. It is known that the endometrium is the first line of defense against reproductive tract infections after delivery [46] and endometrial cells play an important role in innate immune defense [47]. Given that the inflammation-provoking factors in the endometrium increase the production of inflammatory mediators derived from arachidonic acid, this study was focused on the participation of NPY in LT generation and release by the inflamed endometrium.\\u003c/p\\u003e\\n\\u003cp\\u003eTo our knowledge, there are so far no reports of the participation of NPY in the uterine synthesis and secretion of the LTs under physiological and pathological conditions. This report for the first time shows that in the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups endometrial stripes under the influence of NPY increased the 5-LOX, LTAH and LTCS protein abundances and the LTB4 and LTC4 secretion. It should be added that in the \\u003cem\\u003eE. coli\\u003c/em\\u003e group NPY led to greater rise of above enzyme abundances and both LT secretion than in the CON group. Up to now in relation to the interaction between NPY and LT production it was only presented that NPY augmented the PAF-induced LTD4 content in the rat lungs and had not effect on LTC4 and LTE4 levels [29], as well as that NPY produced relaxation of the cod celiac arteries without LT involvement [48]. It is also known that NPY stimulated PGI2 production in the porcine aortic endothelial cells [49] and the isolated rat kidney (also PGE2) [50], and inhibited the bradykinin-induced PGI2 release (also thromboxane B2) from the guinea pig perfused lung [51].\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThere is also a lack of any information on the receptor mechanism of NPY influence\\u0026nbsp;on LT synthesis and secretion from the healthy and pathologically-changed uterus. Our results show that the engagement of Y1Rs in the generation and release of LT partially differs between the studied groups.\\u0026nbsp;In the endometrial strips of the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups,\\u0026nbsp;this receptor subtype mediated in the NPY influence on\\u0026nbsp;5-LOX and LTCS protein abundances. Moreover, Y1Rs participate in the NPY action on LTAH protein abundance and LTC4 release only in the \\u003cem\\u003eE. coli\\u003c/em\\u003e group, as well as were important for LTB4 release only in the CON group. In both groups, we indicate the role of Y2Rs in the NPY-induced LTAH protein abundance and LTB4 and LTC4 release, and the lack of significance of this receptor subtype in the NPY influence on 5-LOX and LTCS protein abundances. It should be mentioned that the involvement of the studied YRs in the NPY excitatory effect on the LTB4 and LTC4 synthesis and secretion by the inflamed endometrial stripes was consistent with the reduction of Y1R and Y2R mRNA and Y1R protein abundance and with a rise in Y2R protein abundance in the endometrium.\\u003c/p\\u003e\\n\\u003cp\\u003eAvailable literature show that the pro-inflammatory (LPS, tumor necrosis factor-α, interleukin /IL)/-1β) and anti-inflammatory (IL-4, IL-10) mediators [52], as well as acetylcholine [53] regulate LT formation and release from the endometrium under inflammatory conditions. Above-mentioned inflammatory mediators act also the biosynthesis pathway for LTs in the pig endometrial epithelial [54], endothelial [55] and stromal [56] cells. Thus, the current results by demonstrating the NPY action on the 5-LO, LTAH and LTCS protein abundances and LTB4 and LTC4 secretion by the endometrial stripes complement the data about the immune-neuronal control of LT production in the inflamed uterus. Obtained findings allow to suppose that NPY may indirectly influence processes modulated by LTs in endometrium, including inflammatory state and secretory function. It is possible that NPY through Y1Rs and/or Y2Rs stimulates the LTB4 and LTC4 production and secretion in the course of endometritis, and contributes to the development and maintenance of the inflammatory process.\\u0026nbsp;LTB4 is an active chemoreceptor especially for granulocytes and phagocytes. It is related to many functions, including: stimulation and activation of neutrophils and increased interleukin-6 production which causes early gene transcription in mononuclear cells. LTC4 (also the rest cys-LTs) are present in many groups of cells such as mast cells and macrophages. Cys-LTs cause vasodilation (relevant in the recruitment of leukocytes) which initiates the inflammatory response, as well as play role in the maintenance and regeneration of damage tissues [57-59]. Both LTB4 and LTC4 are able to a rise in the PGE2, PGF2α and IL-6 release from \\u003cem\\u003eE. coli\\u003c/em\\u003e-stimulated bovine uterine stripes [60] and the contents of these PGs in the bovine healthy endometrium [61]. The involvement of Y1Rs and Y2Rs in the NPY influence on the formation and release of LTs during endometritis suggests new potential application of antagonists and/or agonists of individual NPY receptors to modulate the inflammatory response and uterine function.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eThis study revealed that in the porcine endometrium with a severe acute endometritis, the Y1R and Y2R mRNA and protein abundances change. In this tissue, NPY stimulates the LTAH protein abundance and LTC4 release acting through Y1Rs and Y2Rs. The NPY effect mediated by Y1Rs increases 5-LOX and LTCS protein abundances, while by Y2Rs the release of LTB4. On this basis, it is conjecturable that NPY together with these receptors may be at least partly responsible for the elevated LT generation in the endometrium in the course of spontaneous inflammatory process. Thus, NPY, in an indirect manner, can affect the LT-modulated processes in the endometrium with inflammation. The mechanisms underlying changes in the YR abundances as well as their significance in an inflamed endometrium function need be further investigated. We postulate that pharmacological modulation of Y1Rs and Y2Rs may be important for control of secretory activity of an inflamed endometrium.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eAnimals\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll study procedures were approved by The Local Ethics Committee for Experiments on Animals (University of Warmia and Mazury in Olsztyn, Poland, Consent no. 65/2015). The guidelines in EU Directive 2010/63/EU for animal studies were also followed.\\u0026nbsp;Ten crossbred gilts (Large White x Landrace) of similar age (7-8 months) and weighing between 90 and 120 kg were used in this experiment. Behavioral estrus was detected using a tester boar. The animals were characterized by no disturbances in reproductive processes (vaginal discharges were not observed, the second estrous cycle occurred regularly).\\u0026nbsp;For acclimatization, three days before the start of the study, the gilts were transported from the\\u0026nbsp;commercial farm (Agro-Wronie Sp. z o.o., Wronie, Wąbrzeźno, Poland)\\u0026nbsp;to the local animal house\\u0026nbsp;(University of Warmia and Mazury, Olsztyn, Poland). Pigs were maintained in\\u0026nbsp;individual pens (with an area of about 5 m\\u003csup\\u003e2\\u003c/sup\\u003e),\\u0026nbsp;under natural light (14.5\\u0026plusmn;1.5 h - day and 9.5\\u0026plusmn;1.5 h - night) and temperature (18\\u0026plusmn;2\\u0026deg;C) conditions. They were fed typically for this species and animal age and had access to water.\\u0026nbsp;During the experiment,\\u0026nbsp;the gilts were not exposed to any treatment.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eStudy design\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAt the end of the acclimatization period (day 3 of the second estrous cycle - day 0 of the study), the gilts were randomly divided into the control group (CON, the gilts with saline injections into uterine horns, n=5) and\\u0026nbsp;\\u003cem\\u003eE. coli\\u003c/em\\u003e group (\\u003cem\\u003eE. coli,\\u0026nbsp;\\u003c/em\\u003ethe gilts with\\u0026nbsp;\\u003cem\\u003eE. coli\\u0026nbsp;\\u003c/em\\u003einjections into uterine horns, n=5).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eStudy procedures have been reported earlier [62]. Briefly, to induce premedication\\u0026nbsp;atropine\\u0026nbsp;(Atropinum sulf. WZF, Warszawskie Zakłady Farmaceutyczne Polfa S.A., Poland), azaperone (Stresnil, Janssen Pharmaceutica, Beerse, Belgium)\\u0026nbsp;and ketamine hydrochloride\\u0026nbsp;(Ketamina, Biowet, Puławy, Poland) were used. General anesthesia was induced by\\u0026nbsp;ketamine hydrochloride. After median\\u0026nbsp;laparotomy was done, in the\\u0026nbsp;\\u003cem\\u003eE. coli\\u0026nbsp;\\u003c/em\\u003egroup, \\u003cem\\u003eE. coli\\u003c/em\\u003e suspension (50 ml content: 10\\u003csup\\u003e9\\u003c/sup\\u003e colony forming units/ml, strain with a serotype O25:K23/a/:H1; Department of Microbiology, National Veterinary Research Institute, Puławy, Poland) was injected\\u0026nbsp;into each uterine horn.\\u0026nbsp;The quantity of injected bacteria was based on previous report which revealed that the injections into the pig uterine horns of the same strain of \\u003cem\\u003eE. coli\\u003c/em\\u003e in the same amount as in the current experiment, evoked the severe (mostly) or moderate acute endometritis after eight days [62].\\u0026nbsp;In the CON group, saline solution (50 ml) was injected into each uterine horn. Bacterial suspension and saline solution were injected into the horn of the uterus at 5 sites at a similar distance from each other.\\u0026nbsp;Ten ml of bacterial suspension/saline was administered into each site. The animals from both groups were untreated in the period from surgery until euthanasia. The euthanasia of gilts was performed by an overdose of sodium pentobarbital on day 8 of the study (the expected day 11 of the estrous cycle) and uteri were harvested.\\u0026nbsp;Immediately following this, pieces of the uterine horn were collected from the paraoviductal, middle and paracervical parts of the organ and intended for\\u0026nbsp;real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) and Western blot analyses. For this purpose, endometrial and myometrial layers were separated\\u0026nbsp;using a scalpel blade.\\u0026nbsp;The separation of these layers was evaluated using a dissecting microscope. The endometrial pieces were immediately shock-frozen in liquid nitrogen and stored at -80\\u0026nbsp;\\u0026deg;C for real-time RT and Western blot analysis. The collection of uterine horn fragments, separation of both uterine layers and their freezing were carried out under sterile conditions to avoid contamination. Pieces of the uterine horns were collected also from three parts of the organ for immunofluorescent study. First, they were divided into smaller pieces and fixed\\u0026nbsp;in 4% paraformaldehyde solution\\u0026nbsp;(pH 7.4) After 24 h,\\u0026nbsp;pieces were washed in\\u0026nbsp;0.1 M\\u0026nbsp;phosphate buffered-saline\\u0026nbsp;(PBS, pH 7.4). In order to perform immunofluorescent staining, tissues were\\u0026nbsp;cryoprotected in 18% sucrose until sectioning.\\u0026nbsp;For examination of the secretory activity of endometrium, fragments of the uterine horns collected from the middle part of the horns were placed on ice and transported to the laboratory within 20 min of collection.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003ePreparation and treatment of endometrial explants \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe uterine\\u0026nbsp;fragments\\u0026nbsp;were washed twice using sterile\\u0026nbsp;PBS.\\u0026nbsp;Next,\\u0026nbsp;uterine wall\\u0026nbsp;was divided into endometrium\\u0026nbsp;and myometrium\\u0026nbsp;with the help of a scalpel blade. The endometrium fragments were cut in slices weighing 60-70 mg and then rinsed in Medium (cat. no. M2520, Sigma). Single endometrial explants were put into glass vials with 2 ml of Medium 199 which contained: 0.1% bovine serum albumin (BSA; cat. no. A2058, Sigma) and antibiotics (500 \\u0026mu;l/500 ml gentamicin, 100 \\u0026mu;l/500 ml neomycin, cat. no. G1272, N1142, respectively, both from Sigma). Preincubation and incubation of slices were\\u0026nbsp;performed in a shaking water bath (temperature: 37\\u0026nbsp;\\u0026deg;C, a humidified atmosphere: 95% air and 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e). Preincubation was carried out for 1.5 h.\\u0026nbsp;Next, \\u0026nbsp;the endometrial explants were treated, for 16 h, with fresh (control value) medium or with the addition of\\u0026nbsp;NPY (10\\u003csup\\u003e-7\\u003c/sup\\u003e M,\\u0026nbsp;cat. no. H-4430.0500, Bachem) alone or YR antagonists alone (each at a dose of\\u0026nbsp;10\\u003csup\\u003e-6\\u003c/sup\\u003e M)\\u0026nbsp;for: Y1Rs (BIBO 3304 trifluoroacetate, no cat. 2412, Tocris Biotechnology)\\u0026nbsp;and Y2Rs\\u0026nbsp;(CYM 9484, no cat. 4606, Tocris Biotechnology).\\u0026nbsp;The endometrial explants\\u0026nbsp;were also incubated with NPY (10\\u003csup\\u003e-7\\u003c/sup\\u003e M)\\u0026nbsp;in combination with particular antagonists (each at a dose of\\u0026nbsp;10\\u003csup\\u003e-6\\u003c/sup\\u003e M).\\u0026nbsp;Initial dilutions of NPY and the antagonists were done in accordance with the manufacturer\\u0026rsquo;s instructions (NPY was diluted in deionized water, antagonists for Y1Rs and Y2Rs were diluted in dimethyl sulfoxide /cat. no. W387509, Sigma/), and then stored at -20\\u0026nbsp;\\u0026deg;C. The final antagonists solutions and NPY solution were prepared using the same medium as for preincubation and incubation of explants.\\u0026nbsp;Each treatment was performed in\\u0026nbsp;duplicate\\u0026nbsp;(five separate studies for particular groups, n=5). The PGF2\\u0026alpha; secretion after exposure\\u0026nbsp;to a nitric oxide (NO) donor (NONOate; at a dose of 10\\u003csup\\u003e-4\\u0026nbsp;\\u003c/sup\\u003eM, cat. no. 82150, Cayman Chemical Co.) was applied to control the reactivity of endometrial explants. The doses of\\u0026nbsp;NPY, antagonists and NONOate\\u0026nbsp;and\\u0026nbsp;the time of incubation, were selected based on the\\u0026nbsp;findings\\u0026nbsp;from preliminary studies or according to the authors\\u0026rsquo; previous\\u0026nbsp;experiments.\\u0026nbsp;After the end of incubation, the endometrial explants were blotted with a paper filter, weighed and placed at -80\\u0026nbsp;\\u0026deg;C until estimation of 5-LOX, LTAH and LTCS protein abundances\\u0026nbsp;by Western blot analysis.\\u0026nbsp;The medium was placed into tubes with 5% EDTA (cat. no. 118798103, Chempur, Piekary Śląskie, Poland), 1% acetylsalicylic acid (cat. no. 107140422, POCH Gliwice, Poland) solution (pH 7.4), and stored at -20\\u0026nbsp;\\u0026deg;C until determination of LTB4 and LTC4 concentrations by ELISA method.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eRNA extraction, and real-time RT-PCR\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTotal RNA was isolated from endometrial layers. Tissues were homogenized in TRI Reagent solution (Invitrogen, Thermo Fisher Scientific, USA) using a\\u0026nbsp;FastPrep 24 homogenizer (MP Biomedicals, LCC, USA). For phase separation, a BCP reagent (Molecular Research Center Inc., USA) was used, and the RNA was then purified by using an RNeasy Mini Kit (QIAGEN, USA), according to the manufacturer\\u0026apos;s instructions. RNA was stored until further use at -80 \\u0026deg;C in RNase-free water with the addition of RNAse Inhibitor (Applied Biosystems, Thermo Fisher Scientific, USA). The quality and quantity of extracted RNA was estimated using NanoDrop 1000 (Thermo Fisher Scientific, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA). RNA with an RNA Integrity number ranging 7.0-9.6 was used in real-time RT-PCR.\\u003c/p\\u003e\\n\\u003cp\\u003eReal-time RT-PCR was performed using TaqMan assays and a one-step PCR Master mix (Applied Biosystems). TaqMan assays for porcine Y1Rs (NPY1R), Y2Rs (NPY2R), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), \\u0026beta;-actin (ACTB) and hypoxanthine guanine phosphoribosyl transferase (HPRT) were reported previously [22]. Each reaction (10 \\u0026mu;l) contained: 15 ng of total RNA in a volume of 3 \\u0026micro;l, 5 \\u0026mu;l 2 x TaqMan RT-PCR Mix, 0.25 \\u0026mu;l 40 x TaqMan RT Enzyme Mix, 0.5 \\u0026mu;l 20 x TaqManGene Expression Assays and 1.25 \\u0026mu;l RNase-free water (Applied Biosystems). The real-time RT-PCR reaction was performed in duplicates in 384-well plates using the following conditions: reverse transcription for 15 min at 48 \\u0026deg;C, initial denaturation for 10 min at 95 \\u0026deg;C, followed by 45 cycles of 15 s of denaturation at 95 \\u0026deg;C and then 1 min of annealing at 60 \\u0026deg;C, in an ABI Prism 7900HT system (Applied Biosystems). The negative control was prepared by replacing the RNA template with RNase free water. Data obtained by real-time PCR were analyzed using the Miner method [63]. In order to select the most stable housekeeping gene among ACTB, HPRT and GAPDH, the NormFinder algorithm was applied 64]. The analysis indicated the best stability value for the combination of ACTB and GAPDH genes (0.171). Therefore, abundances levels for each target gene were normalized relatively to the geometric mean of ACTB and GAPDH gene abundance.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eWestern blotting\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eEndometrial pieces for Y1R, Y2R, 5-LOX, LTAH and LTCS protein abundance estimation were minced and placed on ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.4; 50 mM EDTA, 150 mM NaCl, 1% Triton X100, all from Sigma) supplemented with a proteinase inhibitor (Proteinase Inhibitor Cocktail Tablets EDTA-Free, cat. no. S8830-2Tab, Sigma ). Tissue samples were sonicated and centrifuged (15 min, 13500 \\u003cem\\u003eg\\u003c/em\\u003e, 4\\u0026nbsp;\\u0026deg;C). Obtained supernatants were frozen at -80\\u0026nbsp;\\u0026deg;C until determination. The total protein concentration was estimated by\\u0026nbsp;Bradford method\\u0026nbsp;[65]. Equal amounts of protein isolate (120 \\u0026mu;g) were dissolved in sodium dodecyl sulphate (SDS, cat. no. L3771, Sigma), a gel-loading buffer, heated (4 min, 95\\u0026nbsp;\\u0026deg;C) and separated by 12% SDS-polyacrylamide gel electrophoresis. The separated proteins were then electroblotted onto 0.45 \\u0026micro;m pore size Immobilon\\u0026reg;-P PVDF membranes (cat. no. IOVH00010, Sigma) in a transfer buffer. In order to block nonspecific binding sites, the incubation was carried out with 5% fat-free dry milk (Sp\\u0026oacute;łdzielnia Mleczarska, Gostyń, Poland) in Tris (cat. no. T1503, Sigma) -buffered saline Tween 20 (cat. no. P1379, Sigma) buffer (1.5 h, 21\\u0026nbsp;\\u0026deg;C).\\u0026nbsp;The membranes were incubated (18 h, 4\\u0026deg;C) with polyclonal rabbit antibodies for Y1Rs (diluted 1:500, cat. no. AP01221PU-N, Acris an OriGene Co.), Y2Rs (diluted 1:1000, cat. no. TA314282, Acris an OriGene Co.),\\u0026nbsp;5-LOX (diluted 1:200, cat. no. 160402, Cayman Chemical Co.), LTAH (diluted 1:200, cat. no. 1600250, Cayman Chemical Co.) and for LTCS (diluted 1:1000, cat. no. DF14129, Affinity Biosciences). Subsequently, the membranes were incubated (1.5 h,\\u0026nbsp;21\\u0026nbsp;\\u0026deg;C) with secondary antibodies alkaline phosphatase-conjugated goat anti-rabbit for all determined factors (diluted 1:10000, cat. no. 111-055-003, Jackson Immunoresearch). Protein immune complexes were visualized by applied a standard alkaline phosphatase method (NBT-BCIP; cat. no. 72091, Sigma). Analysis was made three times. Specificity of antibodies for Y1Rs and Y2Rs [22] as well as 5-LOX, LTAH and LTCS [28, 53] was earlier presented for pig uterus. As internal control for protein loading,\\u0026nbsp;polyclonal rabbit\\u0026nbsp;GAPDH (diluted 1:5000, cat. no. G9545, Sigma) was used. Images were acquired and quantified using a CHEMIDOC Touch Imaging System (Image Lab 5.2, Bio-Rad Laboratories, Hercules, CA, USA).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eImmunofluorescence\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo determine the tissue distribution of Y1Rs and Y2Rs, fragments of uterine horns were cut in cryostat (Leica CM 1950). Next, 10-\\u0026micro;m-thick sections were subjected to the routine single-immunofluorescence technique. After air-drying (30 min,\\u0026nbsp;21\\u0026nbsp;\\u0026deg;C), sections were then washed in 0.1 M PBS (pH 7.4; 3 x 15 min) and then incubated (1 h,\\u0026nbsp;21\\u0026nbsp;\\u0026deg;C)\\u0026nbsp;in blocking buffer, containing 0.1 M PBS, 10% normal goat serum (MP Biomedicals), 0.1% BSA (Sigma), 0.05% Thimerosal (Sigma), 1% Triton X-100 (Sigma) and\\u0026nbsp;0.01% NaN3.\\u0026nbsp;After further rinsing in PBS (3 x 15 min), tissues were incubated (18 h,\\u0026nbsp;21\\u0026nbsp;\\u0026deg;C)\\u0026nbsp;in a humidity chamber, with\\u0026nbsp;primary antibodies, the same as for Western blot analysis against\\u0026nbsp;Y1Rs and Y2Rs (both diluted 1:100).\\u0026nbsp;Subsequently,\\u0026nbsp;after following washing in PBS (3 x 15 min), the sections were incubated (1 h,\\u0026nbsp;21\\u0026nbsp;\\u0026deg;C)\\u0026nbsp;with biotinylated anti-rabbit IgG (diluted 1:1000, cat. no AP132B, Chemicon International). Next, the sections were incubated (1 h,\\u0026nbsp;21\\u0026nbsp;\\u0026deg;C)\\u0026nbsp;with carbocyanine 3 (CY3)-conjugated streptavidin (diluted 1:9000, cat. no. 016160084, Jackson Immunoresearch). Negative control staining was accomplished by replacing the primary antibodies with the same concentration of rabbit normal IgG. Endometrial cells immunoreactive for the used antibody were estimated under an Olympus BX51 microscope (Olympus Consilio sp. z.o.o., Warsaw, Poland) equipped with epi-fluorescence and an appropriate filter.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eELISA procedure\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eConcentrations of LTB4 and LTC4 in the incubation medium were\\u0026nbsp;determined by the use of\\u0026nbsp;ELISA kits (cat. no. 502390\\u0026nbsp;and\\u0026nbsp;501070, respectively,\\u0026nbsp;Cayman Chemical Co.) according to the manufacturer\\u0026rsquo;s instructions. The standard curve for LTB4 ranged from 1.96 to 1000 pg/ml, and the effective dose for 50% inhibition (ID50) of assay was 2.5 pg/ml. The intra- and interassay coefficients of variation were 4.5% and 7.2%, respectively. The standard curve for LTC4 ranged from 0.98 to 500 pg/ml, and the effective dose for ID50 of the assay was 1.85 pg/ml. The intra-assay and interassay coefficients of variation were 4.7% and 6.2%, respectively.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eStatistical analysis\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe normal distribution (P\\u0026gt;0.05) of all data and residuals were determined using the Shapiro\\u0026ndash;Wilk test.\\u0026nbsp;The significant differences in mRNA and protein receptor abundances between both groups were analyzed \\u003cem\\u003evia\\u0026nbsp;\\u003c/em\\u003eStudent`s test.\\u0026nbsp;The results from the incubation of endometrium for which the PGF2\\u0026alpha; secretion in response to NONOate was statistically significant, were only taken into account. A two-way ANOVA (group, treatment) followed by the\\u0026nbsp;Bonferroni test was applied to compare the mean (\\u0026plusmn; sem) values.\\u0026nbsp;Differences were accepted as statistically significant at P\\u0026lt;0.05. For above analyses\\u0026nbsp;InStat Graph Pad (San Diego, CA) was used.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical approval\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe studies presented in the manuscript were carried out in accordance with the ARRIVE guidelines. The all study procedures were approved by the Local Ethics Committee for Experiments on Animals (University of Warmia and Mazury in Olsztyn, Poland; Consent no. 65/2015). The guidelines in EU Directive 2010/63/EU for animals experiments were included.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by the State Committee for Scientific Research (grant No. 2014/15/B/N25/03572). The cost of the publication was covered by a statutory research grant from the Department of Clinical Physiology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn. We thank Joanna Kalinowska and Andrzej Pobiedziński for their assistance.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eB.J.: contributed to the conception and design of the study, performed surgical procedures, participated in the laboratory analyses, analyzed and interpreted data, written draft, reviewed and edited the manuscript. J.C.: analyzed and interpreted data, and reviewed and edited the manuscript. M.B.: participated in the laboratory analyses, and reviewed and edited the manuscript the manuscript.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets used and/or analysed during the current study are available from the corresponding author\\u0026nbsp;upon\\u0026nbsp;reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAdditional information\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCorrespondence\\u003c/strong\\u003e and requests for materials should be addressed to B.J.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003ePendl, W. et al. 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Biochem.\\u003c/em\\u003e \\u003cstrong\\u003e72,\\u003c/strong\\u003e 248-254 (1976).\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"},{\"header\":\"Tables\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eTable 1.\\u003c/strong\\u003e Influence of neuropeptide Y (NPY, 10\\u003csup\\u003e-7\\u003c/sup\\u003e M) alone or antagonists of NPY receptors subtype 1 (Y1R) and\\u0026nbsp;NPY receptors subtype 2 (Y2R) (10\\u003csup\\u003e-6\\u003c/sup\\u003e M) alone on the 5-lipoxygenase (5-LOX), leukotriene (LT)A4 hydrolase (LTAH) and LTC4 synthase (LTCS) protein abundances in the endometrium of gilts from the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups (n=5 in each group). Treatments were performed in duplicate for each gilt in the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups.\\u0026nbsp;Enzyme abundances were determined by Western blotting and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein used as loading control. Data are expressed as the mean \\u0026plusmn; sem. CV: control value (gained for an endometrial stripes from the CON or \\u003cem\\u003eE. coli\\u003c/em\\u003e groups that had not undergone any treatment\\u003cem\\u003e\\u0026nbsp;in vitro\\u0026nbsp;\\u003c/em\\u003econditions); anta.: antagonist\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eMean values with various letters (a, b) differ significantly (P\\u0026lt;0.05-0.001) within the CON or \\u003cem\\u003eE. coli\\u003c/em\\u003e groups for the same enzyme between CV, action of NPY and actions of particular antagonists; * P\\u0026lt;0.05, ** P\\u0026lt;0.01,*** P\\u0026lt;0.001 -\\u0026nbsp;represent statistical differences between groups for the same enzyme and treatment. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eTreatment\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eGroup\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; Protein abundance \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;(arbitrary units)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e5-LOX\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eLTAH\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eLTCS\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eCV\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eCON\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e0.63 \\u0026plusmn; 0.08\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e1.84 \\u0026plusmn; 0.63\\u003csup\\u003ea\\u003c/sup\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.62 \\u0026plusmn; 0.03\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eE. coli\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; 1.94 \\u0026plusmn; 0.18\\u003csup\\u003ea,***\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp;3.78 \\u0026plusmn; 0.22\\u003csup\\u003ea,*\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp;1.74 \\u0026plusmn; 0.22\\u003csup\\u003ea,**\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eNPY\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eCON\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e1.75 \\u0026plusmn; 0.15\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u0026nbsp;4.65 \\u0026plusmn; 0.41\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.67 \\u0026plusmn; 0.26\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eE. coli\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e3.18 \\u0026plusmn; 0.22\\u003csup\\u003eb,***\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; 6.57 \\u0026plusmn; 0.61\\u003csup\\u003eb,*\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; 2.75 \\u0026plusmn; 0.44\\u003csup\\u003eb,*\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eY1R anta.\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eCON\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e0.81 \\u0026plusmn; 0.07\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e1.42 \\u0026plusmn; 0.35\\u003csup\\u003ea\\u003c/sup\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.53 \\u0026plusmn; 0.04\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eE. coli\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e1.76 \\u0026plusmn; 0.19\\u003csup\\u003ea,***\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp;3.23 \\u0026plusmn; 0.19\\u003csup\\u003ea*\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp; 1.53 \\u0026plusmn; 0.19\\u003csup\\u003ea,*\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eY2R anta.\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eCON\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e0.59 \\u0026plusmn; 0.09\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e1.75 \\u0026plusmn; 0.22\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.61 \\u0026plusmn; 0.04\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eE. coli\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e1.67 \\u0026plusmn; 0.21\\u003csup\\u003ea,***\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e2.89 \\u0026plusmn; 0.17\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp; 1.66 \\u0026plusmn; 0.21\\u003csup\\u003ea,*\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 2.\\u003c/strong\\u003e Influence of neuropeptide Y (NPY, 10\\u003csup\\u003e-7\\u003c/sup\\u003e M) alone or antagonists of NPY receptors subtype 1 (Y1R) and NPY receptors subtype 2 (Y2R) (10\\u003csup\\u003e-6\\u003c/sup\\u003e M) alone on the leukotriene (LT)B4 and LTC4 secretion from the endometrium of gilts from the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups (n=5 in each group). Treatments were performed in duplicate for each gilt in the CON and \\u003cem\\u003eE. coli\\u003c/em\\u003e groups. LTs contents were determined by ELISA. Data are expressed as the mean \\u0026plusmn; sem. CV: control value (gained for an endometrial stripes from the CON or \\u003cem\\u003eE. coli\\u003c/em\\u003e groups that had not undergone any treatment\\u003cem\\u003e\\u0026nbsp;in vitro\\u0026nbsp;\\u003c/em\\u003econditions); anta.: antagonist.\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eTreatment\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eGroup \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eConcentrations (pg/ml of medium)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eLTB4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eLTC4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eCV\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eCON\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e140.4 \\u0026plusmn; 8.6\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e28.8 \\u0026plusmn; 3.9\\u003csup\\u003ea\\u003c/sup\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eE. coli\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp;234.8 \\u0026plusmn; 13.7\\u003csup\\u003ea,**\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp;56.1 \\u0026plusmn; 3.1\\u003csup\\u003ea,**\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eNPY\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eCON\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u0026nbsp; 258.3 \\u0026plusmn; 24.3\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e65.4 \\u0026plusmn; 5.3\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eE. coli\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp;358.5 \\u0026plusmn; 28.1\\u003csup\\u003eb,**\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; 98.2 \\u0026plusmn; 7.3\\u003csup\\u003eb,***\\u0026nbsp;\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eY1R anta.\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eCON\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;179.5 \\u0026plusmn; 9.8\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e31.8 \\u0026plusmn; 2.9\\u003csup\\u003ea\\u003c/sup\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eE. coli\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp;277.2 \\u0026plusmn; 15.6\\u003csup\\u003ea,**\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; 60.3 \\u0026plusmn; 4.8\\u003csup\\u003ea,***\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eY2R anta.\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eCON\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e167.9 \\u0026plusmn; 8.9\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u0026nbsp;29.8 \\u0026plusmn; 3.1\\u003csup\\u003ea\\u003c/sup\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eE. coli\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u0026nbsp; 236.4 \\u0026plusmn; 18.1\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp;57.2 \\u0026plusmn; 5.2\\u003csup\\u003ea,***\\u003c/sup\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003eMean values with various letters (a, b) differ significantly (P\\u0026lt;0.05-0.001) within the CON or \\u003cem\\u003eE. coli\\u003c/em\\u003e groups for the same LT between CV, action of NPY and actions\\u0026nbsp;of particular antagonists; ** P\\u0026lt;0.01, *** P\\u0026lt;0.001 - represent statistical differences between groups for the same LT and \\u0026nbsp;treatment. \\u0026nbsp;\\u003c/p\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"endometrium, inflammation, neuropeptide Y and its receptors, leukotriene B4 and C4 formation, pig\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7738675/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7738675/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"The aim of the study was to determine neuropeptide Y (NPY) receptor subtypes 1 (Y1R) and 2 (Y2R) mRNA and protein abundances in the inflamed porcine endometrium and NPY influence alone and with Y1R or Y2R antagonists on 5-lipoxygenase (5-LOX), LTA4 hydrolase (LTAH) and LTC4 synthase (LTCS) protein abundances and LTB4 and LTC4 release from this tissue. Either saline solution (CON group) or Escherichia coli (E. coli) suspension (E. coli group) were injected into uterine horns. After eight days, in E. coli group severe acute endometritis was diagnosed, as well as decreased Y1R mRNA and protein abundances and Y2R mRNA abundance and increased Y2R protein abundance compared to CON group. NPY increased 5-LOX, LTAH and LTCS protein abundances and LTB4 and LTC4 release from the endometrial stripes of both groups, however in the E. coli group above parameters were higher compared to CON group. In both groups, Y1R antagonist reduced NPY-induced 5-LOX and LTCS protein abundances in reference to NPY influence alone. This effect was also exerted by Y1R antagonist combined with NPY on LTB4 release in the CON group and on LTAH protein abundance and LTC4 release in E. coli group. As compared to NPY action alone, Y2R antagonist with NPY caused a decrease in LTAH protein abundance and LTB4 and LTC4 release in both CON and E. coli groups. Summarizing, in the inflamed porcine endometrium changes Y1R and Y2R mRNA and protein abundances. NPY by interaction with Y1Rs and Y2Rs stimulates LTAH protein abundance and LTC4 release as well as acting through Y1Rs increases 5-LOX and LTCS protein abundances and by Y2Rs release LTB4. 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