Lippia origanoides essential oil induces tocolytic effect in virgin rat uterus and inhibits writhing in a dysmenorrhea mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: The species Lippia origanoides Kunth, popularly known as "salva-de-marajó", is used in Brazilian traditional "quilombola" communities to treat menstrual cramps and uterine inflammation. AIM OF THE STUDY: Evaluate the spasmolytic activity of Lippia origanoides essential oil (LOO) on experimental models of uterine conditions related to menstrual cramps and investigate its mechanism of action. MATERIALS AND METHODS: Virgin rat-isolated uterus was mounted in the organ bath apparatus to evaluate the spasmolytic effect of LOO on basal tonus and contractions induced by carbachol, KCl, or oxytocin. We used pharmacological agents to verify the relaxation mechanism of LOO. The evaluation of uterine contractility in virgin rats, after treatment with LOO for three consecutive days, was carried out by the construction of a concentration-response curve with oxytocin or carbachol. The primary dysmenorrhea animal model was replicated with an injection of estradiol cypionate in female mice for three consecutive days, followed by intraperitoneal application of oxytocin. RESULTS: LOO relaxed the rat uterus precontracted with 10-2 IU/mL oxytocin (logEC50 = 1.98 ± 0.07), 1 µM carbachol (logEC50 = 1.42 ± 0.07) or 60 mM KCl (logEC50 = 1.53 ± 0.05). It was also able relax uterus on spontaneous contractions (logEC50 = 0.41 ± 0.05). Preincubation with glibenclamide, propranolol, phentolamine or L-NAME in contractions induced by carbachol did not alter significantly the relaxing effect of LOO. However, in the presence of 4-aminopyridine, CsCl or tetraethylammonium there was a reduction of LOO potency, whereas the blockers methylene blue, ODQ, aminophylline and heparin potentiated the LOO relaxing effect. Preincubation with LOO in a Ca2+ free medium at concentrations of 27 µg/mL or 81 µg/mL reduced the contraction induced by carbachol. The administration of LOO for 3 days did not alter uterus contractility. The treatment with LOO at 30 or 100 mg/kg intraperitoneally, or 100 mg/kg orally, inhibited writhing in female mice. The association of LOO at 10 mg/kg with nifedipine or mefenamic acid potentiated writhing inhibition in mice. CONCLUSIONS: The essential oil of L. origanoides has tocolytic activity in rat isolated uterus pre-contracted with KCl, oxytocin, or carbachol. This effect is possibly related to the opening of potassium channels (Kir, KV, and KCa), cAMP increase, and diminution of intracellular Ca2+. This relaxant effect, probably, contributed to reduce the number of writhings in an animal model of dysmenorrhea being potentiated by nifedipine or mefenamic acid. Taken together, the results here presented indicate that this species has a pharmacological potential for the treatment of primary dysmenorrhea, supporting its use in folk medicine.

Ethanolic Extracts of Adlay Testa and Hull and Their Active Biomolecules Exert Relaxing Effect on Uterine Muscle Contraction through Blocking Extracellular Calcium Influx in Ex Vivo and In Vivo Studies.

Dysmenorrhea is one of the most prevalent disorders in gynecology. Historically, adlay (Coix lachryma-jobi L. var. Ma-yuen Stapf.) has been explored for its anti-tumor, pain relief, anti-inflammatory, and analgesic effects. The aim of this study was to evaluate the effects of adlay seeds on the inhibition of uterine contraction and thus dysmenorrhea relief, in vitro and in vivo. HPLC-MS and GC were used to elucidate the ethyl acetate fraction of adlay testa ethanolic extract (ATE-EA) and ethyl acetate fraction of adlay hull ethanolic extract (AHE-EA). Elucidation yielded flavonoids, phytosterols, and fatty acids. Uterine leiomyomas and normal adjacent myometrial tissue were evaluated by oxytocin- and PG-induced uterine contractility. ATE-EA and AHE-EA suppressed uterine contraction induced by prostaglandin F2 alpha (PGF2α), oxytocin, carbachol, and high-KCl solution ex vivo. In addition, the external calcium (Ca2+) influx induced contraction, and increased Ca2+ concentration was inhibited by ATE-EA and AHE-EA on the uterine smooth muscle of rats. Furthermore, ATE-EA and AHE-EA effectively attenuated the contraction of normal human myometrium tissues more than adjacent uterine leiomyoma in response to PGF2α. 3,5,6,7,8,3',4'-Heptamethoxyflavone and chrysoeriol produced a remarkable inhibition with values of IC50 = 24.91 and 25.59 µM, respectively. The experimental results showed that treatment with ATE-EA at 30 mg/day effectively decreased the writhing frequency both on the oxytocin-induced writhing test and acetic acid writhing test of the ICR mouse.

Evaluating aminophylline and progesterone combination treatment to modulate contractility and labor-related proteins in pregnant human myometrial tissues.

Progesterone (P4) and cyclic adenosine monophosphate (cAMP) are regarded as pro-quiescent factors that suppress uterine contractions during pregnancy. We previously used human primary cells in vitro and mice in vivo to demonstrate that simultaneously enhancing myometrial P4 and cAMP levels may reduce inflammation-associated preterm labor. Here, we assessed whether aminophylline (Ami; phosphodiesterase inhibitor) and P4 can reduce myometrial contractility and contraction-associated proteins (CAPs) better together than individually; both agents are clinically used drugs. Myometrial tissues from pregnant non-laboring women were treated ex vivo with Ami acutely (while spontaneous contracting) or throughout 24-h tissue culture (±P4); isometric tension measurements, PKA assays, and Western blotting were used to assess tissue contractility, cAMP action, and inflammation. Acute (1 h) treatment with 250 and 750 µM Ami reduced contractions by 50% and 84%, respectively, which was not associated with a directly proportional increase in whole tissue PKA activity. Sustained myometrial relaxation was observed during 24-h tissue culture with 750 µM Ami, which did not require P4 nor reduce CAPs. COX-2 protein can be reduced by 300 nM P4 but this did not equate to myometrial relaxation. Ami (250 µM) and P4 (100 and 300 nM) co-treatment did not prevent oxytocin-augmented contractions nor reduce CAPs during interleukin-1ß stimulation. Overall, Ami and P4 co-treatment did not suppress myometrial contractions more than either agent alone, which may be attributed to low specificity and efficacy of Ami; cAMP and P4 action at in utero neighboring reproductive tissues during pregnancy should also be considered.

Quercetin affects uterine smooth muscle contractile activity in gilts.

Quercetin is a polyphenolic flavonoid occurring in leaves, stems, flowers and fruits of many plants. In traditional Chinese medicine, it is used as a natural therapeutic agent with a broad spectrum of activities (antioxidant, neuroprotective, anti-inflammatory, anticancer, antibacterial and antiviral). Moreover, quercetin affects function of the reproductive tract, however the knowledge of this activity is still fragmentary. Therefore, this study aimed to determine the influence of quercetin on the contractile activity of the porcine myometrium collected from immature (n = 6), cyclic (n = 6) and early pregnant (n = 6) gilts. Strips of the myometrium (comprising longitudinal and circular layer) were resected from the middle part of the uterine horns and the isometric contractions were recorded. After 60-90 min of preincubation, the strips were stimulated with quercetin in increasing (10-13-10-1 M) concentrations and the changes in the tension amplitude and frequency of contractions were measured. Quercetin decreased (P<0.01-0.001) the amplitude of contractions at concentrations 10-11-10-1 M and 10-10-10-1 M in cyclic and early pregnant groups, respectively. The frequency of contractions decreased in all groups but was the highest (at concentrations 10-11-10-1 M; P<0.05-0.001) in the cyclic group and the lowest (at concentrations 10-5-10-1 M; P<0.01) in the immature group. The tension decreased only in the cyclic group after quercetin administration in high concentrations (10-6-10-1 M; P<0.05-0.01). The results indicate that quercetin causes relaxation of the porcine uterine smooth muscle but this activity is strongly related to the physiological status of the gilts.

The mechanism of action of oxytocin antagonist nolasiban in ART in healthy female volunteers.

RESEARCH QUESTION: What are the effects of the oxytocin receptor (OTR) antagonist nolasiban on uterine contractions, endometrial perfusion and endometrial mRNA expression? DESIGN: Randomized, double-blind, parallel-group, mechanism-of-action study with nolasiban. Forty-five healthy, pre-menopausal women were treated with placebo, 900 mg or 1800 mg nolasiban on the day corresponding to blastocyst transfer. Ultrasonographic uterine contraction frequency and endometrial perfusion were assessed, and endometrial biopsies analysed by next-generation sequencing. RESULTS: Both doses of nolasiban showed decreased contraction frequency and increased endometrial perfusion depending on the time point assessed. At 1800 mg, 10 endometrial genes (DPP4, CNTNAP3, CNTN4, CXCL12, TNXB, CTSE, OLFM4, KRT5, KRT6A, IDO2) were significantly differentially expressed (adjusted P < 0.05). Of these, OLFM4, DPP4 and CXCL12 were regulated in the same direction as genes involved in implantation during the window of implantation. In addition, three genes (DPP4, CXCL12 and IDO2) were associated with decidualization and endometrial receptivity. CONCLUSIONS: These data expand our knowledge of the mechanism of action of nolasiban in increasing pregnancy rates after embryo transfer. The results suggest more marked effects of nolasiban 1800 mg compared with the 900 mg dose, supporting testing at higher doses in IVF patients.

Using shear wave elastography to assess uterine tonicity after vaginal delivery.

This study aims to evaluate the feasibility and clinical interest of shear wave elastography, by quantitatively estimating the baseline stiffness of the myometrium before and after placental expulsion. We conducted a prospective cohort study of women at term, without known risk factors for postpartum hemorrhage, who gave birth via spontaneous labor in our tertiary center. Myometrium tonicity was evaluated based on measurements of shear wave speed (SWS) in the anterior uterine corpus. All data points were collected by a single operator. Measurements were carried out at three different time points: after fetal delivery (T1), after placental delivery (T2) and 30 min after placental delivery (T3). Our primary objective was to assess the feasibility of this new imaging technique. Ten valid SWS measurements obtained at each of the three different time points were considered as a positive primary outcome. Our secondary objectives were to evaluate the difference in median myometrial shear wave velocity between each time point, as well as to determine the correlation between myometrial shear wave velocity and patients' characteristics. 38 women were recruited during the study period, of whom 34 met the study criteria. 1017 SWS measurements were obtained. The median time to perform measurements was 16 s for one value, and 2 min 56 s for ten. For 11 women (32%) it was not possible to achieve ten SWS at T1 as placental expulsion immediately followed the birth of the newborn. One patient experienced placental retention and only measurements at T1 were performed. For all other patients, we were successfully able to obtain all measures as intended. There was no difference in the mean shear wave speed between the three time points. After adjustments for confounders, we observed a significant correlation for total blood loss (correlation coefficient = - 0.26, p < 0.001, units of oxytocin (correlation coefficient = - 0.34, p = 0.03), and newborn weight (correlation coefficient = - 0.08, p = 0.001). It is feasible to assess uterine tonicity by shear wave imaging, after placental expulsion. We did not observe a variance in uterine tonicity between the three time points. Women who had higher blood loss, received more units of oxytocin and/or those with newborns of a higher weight exhibited lower shear wave speed measures.

Conflicting Nongenomic Effects of Progesterone in the Myometrium of Pregnant Rats.

Recently, it has been suggested that progesterone affects the contractile activity of pregnant myometrium via nongenomic pathways; therefore, we aimed to clarify whether progesterone causes and/or inhibits pregnant myometrial contractions via nongenomic pathways. Our in vitro experiments using myometrial strips obtained from rats at 20 days of gestation revealed that progesterone caused myometrial contractions in a concentration- and time-dependent manner at concentrations up to 5 × 10-7 M; however, this effect decreased at concentrations higher than 5 × 10-5 M. Similarly, progesterone enhanced oxytocin-induced contractions up to 5 × 10-7 M and inhibited contractions at concentrations higher than 5 × 10-5 M. Conversely, progesterone did not enhance high-KCl-induced contractions but inhibited contractions in a concentration- and time-dependent manner at concentrations higher than 5 × 10-7 M. We also found that RU486 did not affect progesterone-induced contractions or the progesterone-induced inhibition of high-KCl-induced contractions; however, progesterone-induced contractions were blocked by calcium-free phosphate saline solution, verapamil, and nifedipine. In addition, FPL64176, an activator of L-type voltage-dependent calcium channels, enhanced high-KCl-induced contractions and rescued the decrease in high-KCl-induced contractions caused by progesterone. Together, these results suggest that progesterone exerts conflicting nongenomic effects on the contractions of pregnant myometrium via putative L-type voltage-dependent calcium channels.

Myometrial Responses to Beta-Adrenoceptor Antagonists in Gynecological Malignancies.

OBJECTIVE: The aim of the study was to determine the influence of beta-adrenoceptor (ADRB) antagonists on contractile activity of the nonpregnant human uterus in patients affected by gynecological malignancies. DESIGN: This was a controlled and prospective ex vivo study. SETTING: The work was conducted as a collaboration between 4 academic departments. MATERIALS AND METHODS: Myometrial specimens were obtained from women undergoing hysterectomy for benign gynecological disorders (reference group; N = 15), and ovarian (N = 15), endometrial (N = 15), synchronous ovarian-endometrial (N = 3), and cervical cancer (N = 10). Contractions of myometrial strips in an organ bath before and after applications of ADRB antagonists (propranolol, bupranolol, SR 59230A, and butoxamine) were studied under isometric conditions. RESULTS: Propranolol and bupranolol attenuated contractions in the endometrial and cervical cancer groups similar to that in the reference group (all p < 0.05), whereas opposite effects were observed in the ovarian and synchronous ovarian-endometrial cancer groups. SR 59230A and butoxamine significantly increased contractions in the ovarian cancer group (both p < 0.001). LIMITATIONS: These results require now to be placed into a firm clinical context. CONCLUSIONS: Our study indicates that ovarian cancer considerably alters contractile activity of the nonpregnant human uterus in response to ADRB antagonists. This suggests a pathogenetic role of beta-adrenergic pathways in this malignancy. Furthermore, propranolol and bupranolol substantially influence spontaneous uterine contractility.

The ontogeny of kisspeptin receptor in the uterine contractions in rats: Its possible role in the quiescence of non-pregnant and pregnant uteri.

The objectives of this study were to investigate the effects of KISS1 94-121 fragment on the contractility of non-pregnant and pregnant rat uteri, and to determine the uterine and myometrial expressions of Kiss1r. Uterine muscle strips were obtained from non-pregnant Sprague-Dawley rats in oestrous phase and from pregnant rats on gestational days 5, 15, 18, 20 or 22. The in vitro contractility measurements were carried out in an isolated organ bath in the presence of KISS1 94-121. Experiments with 5-day pregnant tissues were also performed in the presence of kisspeptin-234 trifluoroacetate. The mRNA and protein expressions of Kiss1r were measured by RT-PCR and Western blot analysis, while localizations of receptors were defined by fluorescent immunohistochemistry. KISS1 94-121 induced a dose-dependent relaxation both in non-pregnant and pregnant intact and endometrium-denuded uteri. A gradual decrease was found in the uterine expressions of Kiss1r mRNA and protein towards the end of the gestational period, and it was further confirmed by the immunohistochemical results. The significant majority of Kiss1r is found in the myometrium, however the few endometrial Kiss1r also influences the uterine contractions. The relaxing effect of kisspeptin is continuously reduced towards the end of gestational period in parallel with the reduction of Kiss1r expression. Our results suggest a putative role of kisspeptin in the maintenance of uterine quiescence that may have significance in miscarriage or preterm contractions.

Uterotonic Effects of Aqueous and Methanolic Extracts of Lannea acida in Wistar Rats: An In Vitro Study.

Lannea acida (Anacardiaceae), commonly called Kikié in the Noun division (West-Cameroon), is a tree whose bark is used locally to facilitate delivery. This study was aimed at evaluating the in vitro uterotonic effects of aqueous and methanol extracts of L. acida in Wistar rats. Uterine strips isolated from rats pretreated with 5 µg estradiol (48 h) were mounted in a single-organ bath containing aerated and thermostated De Jalon solution (37 °C). After equilibration, non-cumulative effects of L. acida extracts were recorded after application. The effect of the methanol extract (the most active extract) was monitored in the presence of atosiban (a competitive antagonist of oxytocin receptors), atropine (a specific type 3 muscarinic receptor antagonist), nifedipine (an L-type calcium channel antagonist), and 2-Aminoethoxydiphenyl borate (2-ADB, a specific antagonist of inositol 1,4,5-triphosphate receptors type 1), and in calcium-free medium containing EGTA to elucidate its mechanism of action. L. acida induced uterine contraction in a concentration-dependent manner with the methanol extract (1.506 ± 0.032 gf) being the most effective. Administration of atosiban (2 µmol/L) and atropine (1 µmol/L) reduced the contractile effect of L. acida. Complete inhibition was observed with nifedipine, 2-APB, and calcium-free medium containing EGTA. These results suggest that L. acida possesses uterotonic effects mediated through oxytocin receptors with mobilization of extracellular calcium.

Essential oil from Lippia microphylla Cham. modulates nitric oxide pathway and calcium influx to exert a tocolytic effect in rat uterus.

The essential oil of Lippia microphylla (LM-OE) presents several pharmacological activities. This work evaluates the tocolytic effect of LM-OE on rats. LM-OE inhibited phasic contractions and relaxed tonic contractions on rat uterus. Considering that nitric oxide (NO) pathway regulates uterine contraction, LM-OE potency was attenuated in the presence of NO synthase (NOS) inhibitor and this reduction was reversed in the presence of a NOS substrate. Similarly, the relaxant potency of LM-OE was reduced in the presence of soluble guanylyl cyclase (sGC) and protein kinase G (PKG) inhibitors. LM-OE also demonstrates a positive modulation of large and small conductance calcium-activated, voltage-gated and adenosine triphosphate-sensitive potassium channels and inhibited curves to CaCl2 as well as relaxed the uterus pre-contracted by S-(-)-Bay K8644, suggesting voltage-gated calcium channels type-1 (CaV1) blockade. Thus, the tocolytic effect of LM-OE on rat involves positive modulation of NO/NOS/sGC/PKG/K+-channels pathway and Ca2+ influx blockade through CaV1.[Formula: see text].

The role of somatostatin and its receptors (sstr2, sstr5) in the contractility of gilt inflamed uterus.

We studied the inflammation effect on somatostatin receptors subtypes 2 (sstr2) and 5 (sstr5) expression in myometrium and somatostatin influence alone or with sstr2 and sstr5 antagonists on the contractility of gilt inflamed uterus. On day 3 of the estrous cycle, either E.coli suspension (E.coli group) or saline (SAL group) were injected into uterine horns. In the control pigs (CON group), only laparotomy was performed. Eight days later, in the E.coli group developed severe acute endometritis. In this group, myometrial sstr2 mRNA expression lowered and protein expression increased compared to other groups. Compared to period before somatostatin administration, somatostatin did not change tension in myometrium and endometrium/myometrium of three groups, reduced amplitude and frequency in the CON and SAL groups, and increased amplitude and decreased frequency in the E.coli group. In this group, amplitude was increased by somatostatin compared to other groups. In the CON and SAL groups, sstr2 eliminated inhibitory somatostatin effect on amplitude, while sstr5 antagonist reversed inhibitory somatostatin effect on amplitude. In the E.coli group, sstr2 antagonist reversed stimulatory somatostatin effect on amplitude, while in sstr5 antagonist presence stimulatory somatostatin effect was more deepened compared to somatostatin action alone. After using sstr2 antagonist more deepened inhibitory somatostatin effect on frequency in the CON and E.coli groups was found. Sstr5 antagonist partly eliminated inhibitory somatostatin effect on frequency in the SAL group. Summarizing, the uterine inflammation increases the myometrial sstr2 protein expression; somatostatin raises amplitude of the inflamed uterus acting by sstr2, while drops this parameter by sstr5.

The Inhibitory Effect of Extra Virgin Olive Oil and Its Active Compound Oleocanthal on Prostaglandin-Induced Uterine Hypercontraction and Pain-Ex Vivo and In Vivo Study.

Primary dysmenorrhea is a common occurrence in adolescent women and is a type of chronic inflammation. Dysmenorrhea is due to an increase in oxidative stress, which increases cyclooxygenase-2 (COX-2) expression, increases the concentration of prostaglandin F2α (PGF2α), and increases the calcium concentration in uterine smooth muscle, causing excessive uterine contractions and pain. The polyphenolic compound oleocanthal (OC) in extra virgin olive oil (EVOO) has been shown to have an anti-inflammatory and antioxidant effect. This study aimed to investigate the inhibitory effect of extra virgin olive oil and its active ingredient oleocanthal (OC) on prostaglandin-induced uterine hyper-contraction, its antioxidant ability, and related mechanisms. We used force-displacement transducers to calculate uterine contraction in an ex vivo study. To analyze the analgesic effect, in an in vivo study, we used an acetic acid/oxytocin-induced mice writhing model and determined uterus contraction-related signaling protein expression. The active compound OC inhibited calcium/PGF2α-induced uterine hyper-contraction. In the acetic acid and oxytocin-induced mice writhing model, the intervention of the EVOO acetonitrile layer extraction inhibited pain by inhibiting oxidative stress and the phosphorylation of the protein kinase C (PKC)/extracellular signal-regulated kinases (ERK)/ myosin light chain (MLC) signaling pathway. These findings supported the idea that EVOO and its active ingredient, OC, can effectively decrease oxidative stress and PGF2α-induced uterine hyper-contraction, representing a further treatment for dysmenorrhea.

Non-genomic actions of sex hormones on pregnant uterine contractility in rats: An in vitro study at term.

AIMS: The non-genomic (prompt) actions of sex steroids on pregnant uterine contractility are not fully explored yet, the aim of our study was to clarify such effects of 17-ß estradiol (E2), progesterone (P4) and testosterone (T) on late (22-day) pregnant uterine contractions together with the signaling pathways in rats in vitro. METHODS: The uterine effects of sex steroids on KCl-stimulated contractions were examined in the presence of genomic pathway blocker actinomycin D and cycloheximide, sex hormone receptor antagonists (flutamide, fulvestrant, mifepristone) and also after removing the endometrium. The modifications in uterine G-protein activation and cAMP levels were also detected. RESULTS: T and E2 both relaxed the uterine contractions in the concentration range of 10-8-10-3 M with an increase in the activated G-protein and cAMP levels of the uterus, while P4 was ineffective. Cycloheximide, actinomycin D, antagonist for T and E2 were not able to modify the responses along with the endothelium removal. Mifepristone blocked the relaxing effects of T and E2 and reduced the activation of G-protein and the formation of cAMP. SIGNIFICANCE: T and E2 can inhibit KCl-stimulated contractions in the late pregnant uterus in high concentrations and in a non-genomic manner. Their actions are mediated by a G-protein coupled receptor that can be blocked by mifepristone. A single and high dose of T or E2 might be considered in premature contractions, however, further preclinical and clinical studies are required for the approval of such a therapeutic intervention.

Smooth muscle 22α deficiency impairs oxytocin-induced uterine contractility in mice at full-term pregnancy.

Smooth muscle 22α (SM22α, namely Transgelin), as an actin-binding protein, regulates the contractility of vascular smooth muscle cells (VSMCs) by modulation of the stress fiber formation. However, little is known about the roles of SM22α in the regulation of uterine contraction during parturition. Here, we showed that contraction in response to oxytocin (OT) was significantly decreased in the uterine muscle strips from SM22α knockout (Sm22α-KO) mice, especially at full-term pregnancy, which may be resulted from impaired formation of stress fibers. Furthermore, serious mitochondrial damage such as the mitochondrial swelling, cristae disruption and even disappearance were observed in the myometrium of Sm22α-KO mice at full-term pregnancy, eventually resulting in the collapse of mitochondrial membrane potential and impairment in ATP synthesis. Our data indicate that SM22α is necessary to maintain uterine contractility at delivery in mice, and acts as a novel target for preventive or therapeutic manipulation of uterine atony during parturition.

Mechanisms of thrombin-Induced myometrial contractions: Potential targets of progesterone.

Intrauterine bleeding during pregnancy is a major risk factor for preterm birth. Thrombin, the most abundant coagulation factor in blood, is associated with uterine myometrial contraction. Here, we investigated the molecular mechanism and signaling of thrombin-induced myometrial contraction. First, histologic studies of placental abruption, as a representative intrauterine bleeding, revealed that thrombin was expressed within the infiltrating hemorrhage and that thrombin receptor (protease-activated receptor 1, PAR1) was highly expressed in myometrial cells surrounding the hemorrhage. Treatment of human myometrial cells with thrombin resulted in augmented contraction via PAR1. Thrombin-induced signaling to myosin was then mediated by activation of myosin light chain kinase- and Rho-induced phosphorylation of myosin light chain-2. In addition, thrombin increased prostaglandin-endoperoxidase synthase-2 (PTGS2 or COX2) mRNA and prostaglandin E2 and F2α synthesis in human myometrial cells. Thrombin significantly increased the mRNA level of interleukine-1ß, whereas it decreased the expressions of prostaglandin EP3 and F2α receptors. Progesterone partially blocked thrombin-induced myometrial contractions, which was accompanied by suppression of the thrombin-induced increase of PTGS2 and IL1B mRNA expressions as well as suppression of PAR1 expression. Collectively, thrombin induces myometrial contractions by two mechanisms, including direct activation of myosin and indirect increases in prostaglandin synthesis. The results suggest a therapeutic potential of progesterone for preterm labor complicated by intrauterine bleeding.

Chlamydia muridarum infection differentially alters smooth muscle function in mouse uterine horn and cervix.

Chlamydia trachomatis infection is a primary cause of reproductive tract diseases including infertility. Previous studies showed that this infection alters physiological activities in mouse oviducts. Whether this occurs in the uterus and cervix has never been investigated. This study characterized the physiological activities of the uterine horn and the cervix in a Chlamydia muridarum (Cmu)-infected mouse model at three infection time points of 7, 14, and 21 days postinfection (dpi). Cmu infection significantly decreased contractile force of spontaneous contraction in the cervix (7 and 14 dpi; P < 0.001 and P < 0.05, respectively), but this effect was not observed in the uterine horn. The responses of the uterine horn and cervix to oxytocin were significantly altered by Cmu infection at 7 dpi (P < 0.0001), but such responses were attenuated at 14 and 21 dpi. Cmu infection increased contractile force to prostaglandin (PGF2α) by 53-83% in the uterine horn. This corresponded with the increased messenger ribonucleic acid (mRNA) expression of Ptgfr that encodes for its receptor. However, Cmu infection did not affect contractions of the uterine horn and cervix to PGE2 and histamine. The mRNA expression of Otr and Ptger4 was inversely correlated with the mRNA expression of Il1b, Il6 in the uterine horn of Cmu-inoculated mice (P < 0.01 to P < 0.001), suggesting that the changes in the Otr and Ptger4 mRNA expression might be linked to the changes in inflammatory cytokines. Lastly, this study also showed a novel physiological finding of the differential response to PGE2 in mouse uterine horn and cervix.

Macrophage-induced reactive oxygen species promote myometrial contraction and labor-associated mechanisms†.

At labor, the myometrium is infiltrated by a massive influx of macrophages that secrete high levels of pro-inflammatory cytokines inducing the expression of specific labor-associated markers. However, the interactions between myocytes and macrophages and the role of macrophages in the myometrium at labor remain to be elucidated. In this work, we studied the role of myometrium-infiltrated macrophages and their interaction with myocytes in lipopolysaccharide-induced preterm labor. A co-culture model of human primary myometrial cells and macrophages was developed and validated. Collagen lattices were used to evaluate myocyte contraction. Differentiation steps were assessed by (i) phalloidin and vinculin staining for cytoskeleton reorganization, (ii) gap junction protein alpha 1 expression and scrape loading/dye transfer with Lucifer Yellow for gap junction intercellular communication, and (iii) calcium imaging for cell excitability. We demonstrated that macrophages favored lipopolysaccharide-induced contraction and early differentiation of myometrial cells. Transwell assays showed that previous activation of macrophages by lipopolysaccharide was essential for this differentiation and that macrophage/myocyte interactions involved macrophage release of reactive oxygen species (ROS). The effects of macrophage-released ROS in myometrial cell transactivation were mimicked by H2O2, suggesting that superoxide anion is a major intermediate messenger in macrophage/myocyte crosstalk during labor. These novel findings provide the foundation for innovative approaches to managing preterm labor, specifically the use of antioxidants to inhibit the initial stages of labor before the contractile phenotype has been acquired. In addition, the co-culture model developed by our team could be used in future research to decipher pathophysiological signaling pathways or screen/develop new tocolytics.

Pimpinella anisum extract attenuates spontaneous and agonist-induced uterine contraction in term-pregnant rats.

ETHNOPHARMACOLOGY RELEVANCE: Pimpinella anisum is a well-known traditional medicinal herb which has been used in folk medicine as an antiulcer, anticancer, antibacterial and as a muscle relaxant. AIM OF THE STUDY: This study was performed to explore the modulatory effects of Pimpinella anisum on term-pregnant rat uterine contractility and to investigate its possible underlying mechanisms. MATERIAL AND METHODS: Intact uterine strips without endometrial layer were isolated from female term-pregnant Wistar rats (22 days of gestation) and mounted in a tissue bath apparatus for in vitro isometric force recording. The effects of different concentrations of Pimpinella anisum extract (PAE) (1, 3, 5, and 7 mg/mL) were examined on uterine contractions generated spontaneously or induced with oxytocin (5 nmol/L), Bay K8644 (1 µmol/L), and carbachol (10 µmol/L). In some experiments, PAE was applied on depolarized myometrium in the presence of high-KCl solution (60 mmol/L). The effect on Ca2+ release was also examined. RESULTS: Application of PAE significantly reduced uterine contractions generated spontaneously or induced with oxytocin, Bay K8644, and carbachol in a concentration-dependent manner (n = 7; P < 0.01). In depolarized myometrium, PAE significantly reduced the tonic force induced by high-KCl solution (n = 7; P < 0.01). PAE prevented oxytocin-induced transient contraction in the entire absence of external calcium (n = 7; P < 0.01). CONCLUSION: The present findings demonstrate the potentials of PAE to relax pregnant uterine contractions possibly by blocking Ca2+ entry via L-type calcium channels and inhibiting Ca2+ release from the internal store. The tocolytic effects of PAE may be a potential adjuvant against strong premature uterine contractions which threaten early pregnancy although clinical studies are required.

Paeonol alleviates primary dysmenorrhea in mice via activating CB2R in the uterus.

BACKGROUND AND PURPOSE: Primary dysmenorrhea is the most common gynaecologic problem in menstruating women and is characterized by spasmodic uterine contraction and pain symptoms associated with inflammatory disturbances. Paeonol is an active phytochemical component that has shown anti-inflammatory and analgesic effects in several animal models. The aim of this study was to explore whether paeonol is effective against dysmenorrhea and to investigate the potential mechanism of cannabinoid receptor signalling. EXPERIMENTAL APPROACH: Dysmenorrhea was established by injecting oestradiol benzoate into female mice. The effects of paeonol on writhing time and latency, uterine pathology and inflammatory mediators were explored. Isolated uterine smooth muscle was used to evaluate the direct effect of paeonol on uterine contraction. KEY RESULTS: The oral administration of paeonol reduced dysmenorrhea pain and PGE2 and TNF-α expression in the uterine tissues of mice, and paeonol was found to be distributed in lesions of the uterus. Paeonol almost completely inhibited oxytocin-, high potassium- and Ca2+-induced contractions in isolated uteri. Antagonists of CB2R (AM630) and the MAPK pathway (U0126), but not of CB1R (AM251), reversed the inhibitory effect of paeonol on uterine contraction. Paeonol significantly blocked L-type Ca2+ channels and calcium influx in uterine smooth muscle cells via CB2R. Molecular docking results showed that paeonol fits well with the binding site of CB2R. CONCLUSIONS AND IMPLICATIONS: Paeonol partially acts through CB2R to restrain calcium influx and uterine contraction to alleviate dysmenorrhea in mice. These results suggest that paeonol has therapeutic potential for the treatment of dysmenorrhea.