Quercetin, a Main Flavonoid in Onion, Inhibits the PGF2α-Induced Uterine Contraction in Vitro and in Vivo
Chi-Hao Wu1*, Tzong-Ming Shieh2, Kai-Lee Wang1, Tsui-Chin Huang3, Shih-Min Hsia1*
1. School of Nutrition and Health Science, Taipei Medical University, Taipei, Taiwan
2. Department of Dental Hygiene, College of Health Care, China Medical University, Taichung, Taiwan
3. PhD Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei, Taiwan
*Corresponding authors: Chi-Hao Wu Ph.D.
School of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan Telephone: 886-2-7361661-6554
E-mail address: wch@tmu.edu.tw
Shih-Min Hsia Ph.D.
School of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan Telephone: 886-2-7361661-6558
Abstract
Dysmenorrhea is considered to be caused by excessive levels of prostaglandins, which stimulate abnormal uterine contractions. This study aimed to investigate the modulatory effects of quercetin that is a main flavonoid in onion on uterine contractility and its possible underlying mechanisms. In vitro and in vivo contractile activities of the uteri were determined using uterine horns isolated from adult rats. The results indicated that (1) for contractions induced by PGF2α-, oxytocin-, and carbachol, quercetin showed the most potent suppressing effect among the tested flavonoids; (2) Ca2+-dependent uterine contractions were inhibited by quercetin; (3) quercetin reduced the PGF2α-elicited Ca2+ responses in human uterine smooth muscle cells; (4) quercetin inhibited uterine contractions stimulated by Ca2+ channel activator and depolarization in response to high K+; (5) quercetin was able to block Ca2+ influx through voltage-operated Ca2+ channels in plasma membrane, and (6) quercetin effectively reduce PGF2α-induced contractions through the administration of increasing doses of 10, 25, and 50 mg/kg in rats. The present findings suggest that onion’s major active compound, quercetin, may be a potential adjuvant for treating uterine disorders.
Keywords: Onion, Calcium, Dysmenorrhea, flavonoids, PGF2α, Quercetin, Uterine contractions
1. Introduction
Flavonoids are widely occurring polyphenols that are foundin vegetables, fruits, wine, and tea. They are recognized for their diverse physiological activity including antioxidation, anticancer, anti-inflammatory, anticarcinogenesis, antidiabetic, antiallergic, acrylamide reduction and anti-vascular smooth muscle contractions (Cheng, Chen, Zhao, & Zhang, 2015; Fu, Chen, Li, Zheng, & Li, 2013; Park et al., 2012; Plaza et al., 2014; Xu et al., 2013). Flavonoids based on different structures have been identified as: flavones, flavanones, flavanols, flavonols, anthocyanidins, and isoflavonoids (Agati, Azzarello, Pollastri, & Tattini, 2012; Friedman, 2007; Terahara, 2015). The relaxant activity of flavonoids has been reported in different organs showing spontaneous rhythmicity, including bladder (Dambros et al., 2005), and intestine (Amira, Rotondo, & Mule, 2008).
Onion is a common vegetable and ingredient in the traditional medicine and dietary culture all over the world. Research in modern medicine has confirmed that onion possesses many positive health effects, including tumor, anti-inflammatory, and prevention and treatment of cardiovascular diseases (Albishi, John, Al-Khalifa, & Shahidi, 2013a; Jakubowski, 2003; Terahara, 2015). Onions are known to contain a large amount of flavonoids including free forms (quercetin and keampferol) and glucose derivatives forms; the majority is glucose derivative of
quercetin and keampferol (Albishi, John, Al-Khalifa, & Shahidi, 2013b). Quercetin is a flavonoid found in vegetables, nut, fruits and onion (Albishi et al., 2013a, 2013b; John & Shahidi, 2010). This compound possesses physiological activity including the ability to induce glucose uptake in L6 myotubes under oxidative stress (Dhanya et al., 2014). In a previous study, we provided evidence that flavonoids (quercetin and naringenin) have the most potent inhibitory effect on uterine contractions in rats (Hsia, Kuo, Chiang, & Wang, 2008). However, phenolic acids (ferulic acid, vanillic acid, caffic acid, gallic acid, syringic acid, and p-coumaric acid) did not exhibit inhibitory effect on uterine contractions. Our previous study also showed that resveratrol had spasmolytic activity on uterine contractions in rats (Hsia, Wang, & Wang, 2011). This results demonstrated that flavonoids could be considered as a therapeutic agent for dysmenorrhea.
Dysmenorrhea or painful periods is a medical term for cramps during menstruation (Latthe & Champaneria, 2014). The prevalence of dysmenorrhea is about 25% and it usually decreases with age. According to a survey done on adolescent females, the prevalence of varies from 67.2 to 90% (Al-Jefout et al., 2015; Ju, Jones, & Mishra, 2014; Kazama, Maruyama, & Nakamura, 2015). In clinical practice, there are two types of dysmenorrheal, primary and secondary. Primary dysmenorrhea is a more common gynecological disorder than secondary
dysmenorrhea (Dawood, 2006; Latthe & Champaneria, 2014). Secondary dysmenorrhea is mainly due to pelvic lesions such as endometriosis. Primary dysmenorrhea has been reported to cause elevation in prostaglandin production such as prostaglandin F2α (PGF2α), which may result in the contraction of blood vessels and myometrium and insufficient blood flow to the endometrium and cause pain in women (Bottcher et al., 2014). Many treatments are available to alleviate the symptoms, such as the use of non-steroidal anti-inflammatory drugs (NSAIDs). Most women choose to take NSAIDs as the first therapeutic option for dysmenorrhea (Rainsford, 2006). Although NSAIDs are effective and rapid in relieving menstrual pain, however, it has many side effects that can affect the hepatic, digestive, cardiac and renal systems (Hayes & Rock, 2002). Therefore, traditional Chinese medicine may be a feasible alternative to improve dysmenorrhea (Mirabi, Alamolhoda, Esmaeilzadeh, & Mojab, 2014). In this study, we investigated the effect of quercetin on PGF2α-induced uterine smooth muscle contraction both in vitro and in vivo.
2. Materials and Methods
2.1 Materials and chemicals
The analytic standards used in this research were purchased from Sigma Chemical (St. Louis, MO, USA) except for luteolin (ChromaDex, Irvine, CA, USA). Onion extract (#7-178A) contain 2% quercetin were purchased from herbal company in Taiwan (BIOMED HERBAL RESEARCH CO., LTD., Taichung, Taiwan)
2.2 Uterine preparations and measurement of uterine contraction
Female Sprague-Dawley (SD) rats weighing 200~300g were housed in a temperature-controlled room (22±1°C) with 14 h/day artificial illumination (0600~2000), and food and water provided ad libitum. The use of the animals was approved by the Institutional Animal Care and Use Committee of the Taipei Medical University (No. LAC-101-0236). All animals received humane care in compliance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals, published by the National Science Council, Taiwan. In the experiment, the rats at estrus stage, which was confirmed by microscopic examination of a vaginal smear, were decapitated; both uterine horns were surgically removed and placed in a petri dish containing Krebs’s solution (113 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 18 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM glucose, 30
mM mannitol, and pH adjusted to 7.4). After the adherent fat and mesenteric attachments were removed, each uterine horn was cut into segments of equal-length (10 mm); these were used for measuring uterine oscillatory contraction. The segments were placed in isolated organ baths containing physical solution at 37°C with 95% O2~5% CO2 supply. Each station was equilibrated using 1 g preload for at least 60 min. Contractions were recorded with force displacement transducers (PowerLab recorder ML785; Castle Hill, NSW, Australia) by using Chart 5 software (Castle Hill, NSW, Australia) (Fig 2A).
2.3 Human uterine smooth muscle cell (HutSMCs) culture
Human uterine smooth muscle cells (HutSMCs) were purchased from PromoCell Co. (PromoCell, Heidelberg, Germany). HutSMCs were cultured in smooth muscle cell growth medium-2 containing 5% (v/v) fetal calf serum, 0.5 ng/ml epidermal growth factor, 2 ng/ml basic fibroblast growth factor, and 5 μg/mL insulin (PromoCell); seeded in a 24-well plate; and incubated at 37°C in Dulbecco’s modified Eagle’s medium (DMEM)-F12 containing 100 U/mL penicillin and 100 μg/mL streptomycin. After incubation for approximately 24 h, the uterine smooth muscle cells were collected and intracellular calcium mobilization was measured.
2.4 Measurement of [Ca2+]i
HutSMCs were treated with 10, 25, 50, 75, or 100 μM quercetin and 200 nM PGF2α for 24 h. They were then harvested using the culture medium and washed twice with the same medium. A cell suspension (1 × 106 cells/mL) was loaded with 5 mg of fura-2/acetoxymethyl ester
(Fura 2-AM; Fluka Chemical Corporation, Milwaukee, WI, USA) dissolved in 5 mL of DMSO. A fluorescent probe was used for monitoring the intracellular calcium concentrations ([Ca2+]i). The cells were incubated in the dark for 30 min at 37°C. After extensive washing, 1
× 106 cells were resuspended in 2.5 mL loading buffer (NaCl, 152 mM; MgCl2, 1.2 mM;
CaCl2, 2.2 mM; KCl, 4.98 mM; and HEPES, 10 mM). Fluorescence emission at 505 nm was monitored at 37°C by a dual-wavelength spectrometer system, with excitation at 340 and 380 nm. Free [Ca2+]i was calculated using the method developed by Grynkiewicz (Grynkiewicz,
Poenie, & Tsien, 1985) by using the ratio of fluorescence intensities obtained every second with a dissociation constant (Kd) of 135 nM. The dye was considered saturated after lysis with digitonin at the final concentration of 0.16 mM. Minimum fluorescence was determined by adding 0.5 mL ethylene glycol bis(3-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA; Sigma Chemical Co.) to obtain a final concentration of 8 mM.
2.5 Western blotting for MLC20 and p-ser19-MLC20
quercetin for 20 min at 37 °C, The treatment uterine strips were collected and frozen immediately in liquid nitrogen. The strips were homogenized in homogenization buffer (pH 8.0) containing 1.5% sodium-lauroylsarcosine, 1 x 10-3 M ethylenediaminetetraacetic acid
(EDTA), 2.5 x 10-3 M Tris-base, 0.68% phenylmethylsulfonyl fluoride (PMSF), and 2%
proteinase inhibitor cocktail, and then disrupted by ground-glass homogenizer in ice-cold buffer. Tissue extracts were centrifuged at 13,500 × g for 10 min. The supernatant fluid was collected and the protein concentration was determined. Extracted proteins were denatured by boiling for 5 min in SDS buffer (0.125 M Tris-base, 4% SDS, 0.001% bromophenol blue, 12% sucrose, and 0.15 M dithiothreitol). The proteins (20 μg) in the samples were separated on 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 50 V for 30 min and then at 90 V for 90 min using a running buffer. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (NEN Life Science Products, Inc., Boston, MA, USA) using a Trans-Blot SD semi-dry transfer cell (170-3940, Bio-Rad, Hercules, CA, USA) at 64 mA (for 8 mm ×10 mm membrane) for 45 min in a blotting solution. ECL detection reagent (PerkinElmer Life Sciences Boston, MA, USA) was used to visualize the immunoreactive proteins on PVDF membranes after transfer. The quantification software was Multi-Gauge V3.0.
In the experiment, the rats that were confirmed to be in the estrous stage by microscopic examination of a vaginal smear were used. They were anesthetized with pentobarbitone (18 mg in 0.3 mL, i.p). A small mid-portion of a uterine horn with associated mesometrium was obtained through a ventral incision made in the skin and body wall. A 1- to 2-mm-long incision was made at the distal end of the exposed uterus, and a thin, finger-shaped latex balloon was attached to a polyethylene catheter (Becton-Dickinson, Franklin Lakes, NJ, USA), which was a modification of a method described in a previous study (Hsia et al., 2011; Hsia, Yeh, Kuo, Wang, & Chiang, 2007). The catheter was connected to a transducer (PowerLab recorder ML785; Castle Hill). Then, the rats were catheterized via the right jugular vein and injected with (PGF2α 0.2 mg/kg) or PGF2α plus quercetin (10, 25, or 50 mg/·kg) via the jugular catheter, and the contractions were recorded by this transducer with Chart 5.1 software (Castle Hill, NSW, Australia) (Fig 5A).
2.7 Statistical analysis
Data were presented as the mean ± standard deviation (SD). Differences between the means were analyzed by one-way analysis of variance (ANOVA) using the SPSS system, version 11.0 (SPSS, Chicago, IL, USA). Group means were compared using a one-way ANOVA and Duncan’s multiple-range test. For comparison of two groups,
Student’s t-test was used. The difference between two means was considered statistically significant when p<0.05 and highly significant when p<0.01.
3. Results and Discussion
3.1 Effect of onion extract on PGF2α - induced contraction
An increase in the [Ca2+]i in the uterine smooth muscles induces muscles to contract. Previous studies have demonstrated that [Ca2+]i is regulated via two different Ca2+ channels: receptor- and voltage-operated channels (ROCs and VOCs) in the membrane of uterine smooth muscle cells (Berridge, 2008; Bolton, 1979). For ROCs, when uterotonic compounds (PGF2α, oxytocin or carbachol) bind to uterine smooth muscle cell membrane G-protein coupled receptors to elicit uterine smooth muscle contractions, the [Ca2+]i increases via both the influx of extracellular Ca2+ through Ca2+ channels and by release of intracellular stored Ca2+. To investigate the potential inhibition of PGF2-induced uterine contraction by onion extract in rats, we first examined the effect of flavonoids on PGF2-induced uterine contraction. PGF2α is a major factor for inducing uterine contractions during dysmenorrhea (Bottcher et al., 2014). PGF2� connects with PGF2� receptor (FP) on the spiral arterioles to increase uterine contractility, causing ischemia pain. PGF2α increases the intracellular calcium concentration by stimulating the release of stored calcium, which produces a phasic contraction (Rosenwaks et al., 1981). When PGF2� was added to spontaneously contracting uteri, a significant increase in the amplitude was observed. (Dawood, 2006; Rosenwaks et al., 1981) In this study, onion extract was effective in inhibiting PGF2-induced
uterine contraction (Fig 1A-B). We also assessed tissue viability using a recovery study by removing the inhibitory substance and washing the tissue with Krebs’s solution for 20 min, then re-analyzing the tissue in the presence of a stimulus (PGF2�, 10-6 M). From our results, we found that PGF2� could reverse the inhibition after removing the resveratrol (Fig 1C). Our previous study showed that adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) hull extract could inhibit PGF2α-induced uterine smooth muscle contraction both in vitro and in vivo (Hsia et al., 2008). Thus, adlay hull may be considered as a potential alternative therapeutic agent for dysmenorrhea. Adlay is a plant used both as a medicine and food. Research has confirmed that adlay possesses many positive health effects, including the ability to regulate blood sugar, blood lipids, and blood pressure; to improve gastrointestinal physiology; to regulate immune and having anti-inflammatory ,antitumor effects and regulating reproductive endocrine hormones (Hsia, Chiang, Kuo, & Wang, 2006; Hsia et al., 2008; Hsia et al., 2009; Hsia et al., 2007; Hsu, Lin, Lin, Kuo, & Chiang, 2003; Huang, Chung, Kuo, Lin, & Chiang, 2009; Huang et al., 2014; Kuo et al., 2002; Wu et al., 2014). We also provided evidence that flavonoids (quercetin and naringenin) have the most potent inhibitory effect on uterine contractions in rats (Hsia et al., 2008). However, phenolic acid (ferulic acid, vanillic acid, caffic acid, gallic acid, syringic acid, and p-coumaric acid) did not exhibit inhibitory effect on uterine contractions. Our previous study also
showed that resveratrol had spasmolytic activity on uterine contractions in the rats (Hsia et al., 2011). Thus, this flavonoids could be considered as a therapeutic agent for dysmenorrhea.
3.2 Effect of flavonoids on PGF2α-, Oxytocin-, and Carbachol- induced contraction
To investigate the potential inhibition of PGF2-induced uterine contraction by flavonoids (apigenin, naringenin, luteolin, kaempferol and quercetin) in rats, we first examined the effect of flavonoids on PGF2-induced uterine contraction. In this study, quercetin was more effective in inhibiting PGF2-induced uterine contraction compared to other flavonoids (apigenin, naringenin, luteolin and kaempferol) (Fig 2B). Oxytocin is a nonapeptide hormone responsible for uterine and mammary gland contractions. Carbachol also has the ability to increase [Ca2+]i in the uterine smooth muscles to induce uterine contractions. In our study, PGF2α (10-6 M), oxytocin (10–7 M) and carbachol (10–6 M) caused rhythmic contractions of the isolated rat uterus. Quercetin (10-100 μM) showed a significant inhibitory effect on the amplitude of PGF2α-, oxytocin-, and carbachol-induced contraction in a concentration-dependent manner (Fig 2C-D). All contractions were ceased when the highest concentration of quercetin (100 μM) was added. Quercetin and kaempferol as natural flavonoids and well-known antioxidative agents (Albishi et al., 2013b). Also, quercetin is a major
active compound in onion skin extract. A previous study has shown that quercetin could interfere with vascular smooth muscle contraction (Hou, Liu, Niu, Cui, & Zhang, 2014). In the present study, quercetin was found to block ROCs and being able to cause uterine contraction. These results suggest that quercetin acts on the downstream receptors for the muscle relaxation effect.
O
wing to quercetin is a major flavonoid component present in onions and the different components present in onions probably have an additive action, the c ommercial onion extracts which contain 2% quercetin were used in this study. We estimated that 50, 125, 250, 500, 1000, and 2000 μg/mL onion extracts contain 1, 2.5, 5, 10, 20, and 40 μg/mL quercetin, respectively. Therefore, quercetin and the same quercetin content of onion extracts were used to examine the anti-contraction capacity. The present results show that quercetin incorporated either as a purified compound or as component in onion extracts at the same concentrations could similarly attenuate PGF2α-induced uterine contraction in SD rats, suggesting no observable matrix effects and the anti-contraction activity of onion is likely attributable to its higher levels of quercetin (Supplemental Fig. 1) .
3.2 Effect of quercetin on on Ca2+-dependent contractions
external Ca2+ influx into cell through calcium channels and induce uterine contraction. To investigate whether the inhibition of uterine contractions by quercetin is due to blocking the external Ca2+ influx, we performed the following experiments in a Ca2+ -free Krebs’s solution. In the absence of external Ca2+, the spontaneous contractions were abolished. Further, when the Ca2+-free Krebs’s solution was supplied with increasing concentrations of Ca2+ from 0.05 to 5 mM, the spontaneous contractions were restored. However, when 100 μg/mL quercetin was added to the Ca2+-containing Krebs’s solution, the Ca2+-induced uterine contractions were not observed (Fig 3A). Thus, this inhibitory effect of quercetin was due to the blockage of external Ca2+ influx. In our previous study, we also found that resveratrol and adlay extract could block external Ca2+ influx into smooth muscle cell.
3.3 Effects of quercetin on [Ca2+]i and Myocine light chain (MLC20) phosphorylation
In order to study whether quercetin inhibits [Ca2+]i and affects muscular contraction, the HutSMCs were treated with different concentrations of quercetin (10, 25, 50, and 100 μM) along with PGF2α (200 nM). Quercetin (25 to 100 μmM) significantly reduced the PGF2α-induced [Ca2+]i (Fig 3B). In contrast, administration of quercetin (10~100 μM) had no effect on HutSMCs number (data not shown). This result implies that the decrease in [Ca2+]i was not attributed to the cytotoxicity of
quercetin on smooth muscle cells. In addition, in order to study whether quercetin inhibits the MLC20 phosphorylation and leads to smooth muscle relaxation, the rat uterine smooth muscle was treated with quercetin (10, 25, 50, and 100 μM) along with PGF2α (10-6 M). Quercetin treatment (50 to 100 μM) significantly reduced the PGF2α-induced MLC20 phosphorylation (Fig 3 C-D). Myosin light chain (MLC20) which is phosphorylated at Thr18 and Ser19 by myosine light chain kinase (MLCK) in a Ca2+/camodulin-dependent manner is one of the proteins involved in uterine smooth muscle contraction. The phosphorylation of MLC20 could interact with α-actin filaments, resulting in uterine smooth muscle contraction. Conversely, when MLC20 is dephosphorylated by myosine light chain phosphatase (MLCP), it leads to relaxation (Ikebe & Hartshorne, 1985; Ito, Nakano, Erdodi, & Hartshorne, 2004). Here, we demonstrated that quercetin inhibited smooth muscle contraction by affecting MLC20 phosphorylation.
3.4 Effect of quercetin on KCl- and BayK8644-induced contraction
Addition of KCl (50 mM) caused tonic contraction and maximum contraction of the uterine strips. Bay K8644 also caused rhythmic contractions of the isolated rat uterus. However, in the presence of quercetin (10-100 μM), the amplitude of KCl-and Bay K8644-induced contraction was significantly decreased in a
concentration-dependent manner (Fig 4 A-B). High K+ (> 30mM) is known to cause smooth muscle contraction through the opening of voltage-dependent L-type Ca2+ channels, thus allowing an influx of extracellular Ca2+ causing a contractile effect (Bolton, 1979). In the present study, quercetin was able to reduce the contraction produced by KCl on isolated uterus tissues, showing its ability in blocking Ca2+ influx. Bay K8644 is a L-type Ca2+-channel activator that can increase intracellular calcium [Ca2+]i, and activate the phosphatidylinsitol-signaling pathway and cytosolic calcium oscillation-like phenomena, thereby resulting in the generation of phasic myometrial contractions (Chien, Saunders, & Phillippe, 1996). Both Bay K 8644 and high K+ induced contraction were also abolished by quercetin administration. Moreover, the spontaneous contractions stimulated by elevated extracellular Ca2+ were also abolished by quercetin treatment. In addition, PGF2α increased [Ca2+]i concentration which was also reduced by quercetin treatment in HutSMCs. This result demonstrated that quercetin is capable of inhibiting Ca2+ influx. Therefore, the effect of quercetin on uterine contractions could be due to its interference with ROCs or/and VOCs. Our present study demonstrated that quercetin could inhibit [Ca2+]i induced by PGF
2α,
oxytocin, carbachol, KCl, and Bay K 8644, and block the Ca2+ influx through ROCs and VOCs.
3.5 Effects of quercetin on isolated uterus in rat (in vivo).
Flavonoids are widely found in plants and food and exhibit numerous pharmacological and biological effects including vascular protection. Previous studies revealed that flavonoids could inhibit vascular smooth muscle contraction (Sun et al., 2013; Wang et al., 2014). Results from this study showed that quercetin at pharmacologically relevant concentration (100 μM) was able to inhibit PGF2α-induced uterine contraction in vivo (Fig 5 B-C). To the best of our knowledge, this study is the first to investigate the effect of quercetin in inhibiting PGF2α-induced uterine contraction in rats.
4. Conclusions
The present study demonstrated that quercetin could inhibit PGF2α-induced uterine smooth muscle contraction both in vitro and in vivo. The inhibition of uterine smooth muscle contraction is, in part, due to the blockage of ROCs and VOCs in rats. Therefore, quercetin may potentially be used in the treatment of dysmenorrhea. However, additional clinical experiments are required to confirm this finding.
Acknowledgement
This study was supported by the grants [MOST103-2313-B-038-003-MY3, MOST103-2313-B-038-001-MY3, NSC102-2313-B-038-001-,] from the Ministry of Science and Technology, Taiwan, Republic of China.
Figure Legends
Fig. 1-Effect of onion extract on PGF2α-induced uterine contractions in the rats. (A)
Representative recordings of PGF2 (10–6 M)-induced contractions treated with vehicle (PBS)
only, and the effects of cumulative additions of onion extract (0.1-1 mg/ml) are shown. (B) Rat uterine segments were treated PGF2 (10–6 M), and exposure of rat uterine smooth muscles
to vehicle or onion extract (0.1, 0.125, 0.25, 0.5 and 1 mg/ml). Recovery test about onion extract on induced uterine smooth muscle contractions in the rats. 1st: First PGF2α-treated; 2nd: Second PGF2α-treated after wash. PGF2α-induced contractions before the addition of quercetin were considered as the control (100%, quercetin: 0 mg/ml group). Data represent mean ± SEM from six independent experiments. *p < 0.05, **p < 0.01 when compared with control at stimulated with PGF2α.
Fig. 2-Effect of flavonoids on PGF2α-induced uterine contractions in the rats. (A) The scheme
of rat uterine preparations and measurement of uterine contraction in vitro. (B) Rat uterine segments were treated PGF2 (10–6 M), and exposure of rat uterine smooth muscles to
vehicle or flavonoids (apigenin, naringenin, luteolin, quercetin and kaempferol) (10, 25, 50, 75 and 100 μM). (C) Representative recordings of PGF2 (10–6 M), oxytocin (10–6 M) or
carbachol (10–5 M)-induced contractions treated with vehicle (DMSO) only, and the effects of
cumulative additions of quercetin (10-100 μM) are shown. (D) Dose-dependent effects of quercetin on the mean peak amplitude. PGF2α-, oxytocin or carbachol induced contractions before the addition of quercetin were considered as the control (100%, quercetin: 0 μM group). Data represent mean ± SEM from six independent experiments. *p < 0.05, **p < 0.01 when compared with control at stimulated with PGF2α-, oxytocin or carbachol.
Fig. 3-Inhibitory actions of quercetin on Ca2+-dependent contractile responses. (A) Muscle
segments were initially pretreated in a Ca2+-free medium containing the vehicle (0.2%
cumulatively applied to trigger muscle contraction. (B) Inhibition of PGF2α-induced increases in [Ca2+]i by quercetin in HutSMCs. HutSMCs were treated with vehicle (0.2% DMSO) or quercetin (10, 25, 50 and 100 μg/mL) along with PGF2α (200 nM). (C) Inhibition of PGF2α-induced increases in MLC20 phosphation by quercetin in rat uterine smooth muscle. Rat uterine segments were treated with vehicle (DMSO) or quercetin (10, 25, 50 and 100 μg/mL) along with PGF2α (10-6 M). **P < 0.01 vs. PGF2α-treated group, assessed by Duncan’s
multiple-range test. #P < 0.05 vs. vehicle-treated group, assessed by Student’s t test. Each column represents the mean SEM.
Fig. 4-Effects of quercetin on high K+ (KCl) or Bay K 8644-induced uterine contractions in
the rats. (A) Representative recordings of high K+ (KCl 50 mM) or Bay K 8644 (10–6
M)-induced contractions and the effects of cumulative additions of quercetin (10-100 μM) are shown. (B) Rat uterine segments were treated high K+ (KCl 50 mM) or Bay K 8644 (10–6 M),
and exposure of rat uterine smooth muscles to quercetin (10, 25, 50, 75 and 100 μM). High K+
(KCl) or Bay K 8644-induced contractions before the addition of quercetin were considered as the control (100%, quercetin: 0 μM group).
Fig. 5-Effects of quercetin on PGF2α-induced uterine contractions in vivo. (A) The scheme of rat uterine preparations and measurement of uterine contraction in vivo. The rats were catheterized via the right jugular vein and injected with (PGF2α 0.2 mg/kg) or PGF2α plus quercetin (10, 25, or 50 mg/kg) via the jugular catheter, and the contractions were recorded. Representative recordings of PGF2α-induced contractions and the effects of cumulative additions of quercetin (10-100 μM) are shown. (B) Dose-dependent effects of quercetin on the mean peak amplitude. These results are representative of the records of 6 rats (n=6). **P < 0.01 vs. PGF2α-treated group, assessed by Duncan’s multiple-range test. #P < 0.05 vs. vehicle-treated group, assessed by Student’s t test. Each column represents the mean SEM.
Fig. 6-The mechanism of quercetin inhibit PGF2α-induced uterine contractions. (1) Ca2+ -dependent uterine contractions were inhibited by quercetin; (2) quercetin reduced the PGF2α-elicited Ca2+ responses in human uterine smooth muscle cells; (3) quercetin inhibited uterine contractions stimulated by Ca2+ channel activator and depolarization in response to high K+; (4) quercetin was able to block Ca2+ influx through voltage-operated Ca2+ channels in plasma membrane.
Fig.
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