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Published in final edited form as: Pregnancy Hypertens. 2020 Oct 23;23:11–17. doi: 10.1016/j.preghy.2020.10.008

Luteolin-induced vasorelaxation in uterine arteries from normal pregnant rats

Weiwei Yang 1,3, Qinghua Li 2,3, Jeremy W Duncan 3, Bhavisha A Bakrania 3, Jessica L Bradshaw 3, Joey P Granger 3, Sarosh Rana 4, Frank T Spradley 3,5
PMCID: PMC7904643  NIHMSID: NIHMS1644185  PMID: 33161224

Abstract

Background.

The flavonoid, luteolin, promotes vasorelaxation in various arteries through endothelial-dependent and independent mechanisms. Although there is growing interest in the vasoactive effects of flavonoids on maternal vascular function during pregnancy, it is unknown whether luteolin elicits vasorelaxation in the uterine circulation. We tested the hypothesis that luteolin induces vasorelaxation via endothelial-dependent mechanisms in uterine arteries from normal pregnant rats during late gestation.

Methods.

Uterine arteries and aortas were isolated from Sprague-Dawley rats at gestational day 19 and prepared for wire myography.

Results.

The potency of luteolin-induced vasorelaxation was examined between uterine arteries and the aortas. By 50 μM of luteolin, there was complete relaxation (100.5 ± 5.2%) in uterine arteries as compared to aortas (27.5 ± 10.0%). Even the highest concentration of 100 μM luteolin produced less than half relaxation (43.6 ± 8.6%) in aortas compared to uterine arteries. We then explored if luteolin-induced vasorelaxation in uterine arteries from pregnant rats was mediated by endothelial-dependent vasorelaxation pathways, including nitric oxide synthase (NOS), cyclooxygenase (COX), or potassium (K+) channels. Blocking these pathways with N(G)-Nitro-L-arginine methyl ester hydrochloride (L-NAME), indomethacin, or tetraethylammonium (TEA)/high potassium chloride (KCl), respectively, did not alter luteolin responses in uterine arteries from pregnant rats. These findings suggested that endothelial factors may not mediate luteolin-induced vasorelaxation in uterine arteries during pregnancy. Indeed, experiments where the endothelium was removed did not alter luteolin-induced vasorelaxation in uterine arteries during pregnancy.

Conclusions.

Luteolin directly promotes vasorelaxation in the medial smooth muscle layer of uterine arteries during normal pregnancy.

Keywords: Flavonoid, Pregnancy, Uterine Artery, Vasculature, Women’s Health

INTRODUCTION

Luteolin is a naturally occurring tetrahydroxyflavone, which is a type of flavonoid. It is found in numerous edible plants, including but not limited to celery, broccoli, peppers, and citrus fruits [1–3]. Luteolin extracts are available in capsular form as a dietary supplement as well [4]. Studies in humans and experimental animal models have shown that luteolin has promise for protection in diseases like cancer and neurological deficits [5–7]. There is also increasing evidence that luteolin has protective actions in the cardiovascular system [8].

Flavonoids, like luteolin, have beneficial vasoactive properties [9, 10]. Investigations in isolated blood vessels have shown that luteolin promotes vasorelaxation via differing mechanisms depending on the vessel bed. Luteolin relaxed rat thoracic aortic rings in an endothelium-dependent fashion mostly via stimulating the nitric oxide synthase (NOS) pathway [11], but cyclooxygenase (COX) also mediated part of this response [10]. Whereas, luteolin-induced vasorelaxation in coronary arteries involves activation of potassium (K+) channels [12]. However, all of those vascular function studies were conducted in vessels isolated from male rats.

During normal pregnancy, the uterine artery is responsive to substances that promote vasorelaxation [13]. Proper uterine artery function and blood flow is important for the success of fetal-placental outcomes [14]. Whereas, pregnancies complicated by preeclampsia and fetal growth restriction have impaired uterine artery function with impaired endothelium-dependent vasorelaxation and increased vasocontractile responses [15, 16]. Thus, the identification of a potential treatment that elicits potent vasorelaxation in the uterine artery may have beneficial implications for a variety of pregnancy-related disorders resulting from poor uterine artery function. While there is growing interest about the potential impact of flavonoids on protecting vascular function during pregnancy [17, 18], there have been no studies to our knowledge examining the impact of luteolin on uterine artery function during normal pregnancy. Thus, in this study, we conducted experiments to test the hypothesis that luteolin induces vasorelaxation in uterine arteries derived from normal pregnant rats. Our first aim was to examine the potency of luteolin-induced vasorelaxation in uterine arteries and the conduit aorta. The second aim was to determine the mechanism of luteolin-induced vasorelaxation in uterine arteries from normal pregnant rats. We proposed that the vasorelaxant effect of luteolin is mediated by an endothelium-dependent mechanism. The endothelium controls vascular tone via three known pathways: NOS, COX, or EDH (endothelium-dependent hyperpolarization) [19]. Therefore, we explored each of these pathways by pharmacological inhibition of NOS, COX, or K+ channels using N(G)-Nitro-L-arginine methyl ester hydrochloride (L-NAME), indomethacin, or tetraethylammonium (TEA), respectfully. Vascular preparations also included removal of the endothelium to confirm whether it plays a role in luteolin-induced vasorelaxation in uterine arteries from normal pregnant rats.

MATERIALS AND METHODS

Reagents and drugs

Luteolin was acquired from Supersmart USA, LLC (Miami, FL, USA). Acetylcholine (ACh), dimethyl sulfoxide (DMSO), indomethacin (non-selective COX inhibitor at 10 μM), N(G)-Nitro-L-arginine methyl ester hydrochloride (L-NAME, non-selective NOS inhibitor at 100 μM), phenylephrine (Phe), tetraethylammonium (TEA, a non-selective K+ channel blocker at 10 mM), and were obtained from Sigma (St. Louis, MO, USA). Potassium chloride (KCl) was purchased from Fisher Scientific (Waltham, MA, USA).

Luteolin was solubilized in 100 % DMSO to make a 2 mM stock concentration. This stock solution was added at increasing volumes of 2.5, 10, 12.5, 50, 50, and 100 μL to the 5 mL vessel baths containing physiological saline solution (PSS), which resulted in generation of the luteolin-induced vasorelaxation curves. For the control vascular function curves, the same volume of DMSO was added to separate vessel segments isolated from the normal pregnant rats.

Experimental animals

All animal protocols were approved by The Institutional Animal Care and Use Committee of The University of Mississippi Medical Center. Experiments were carried out in accordance with NIH guidelines on the ethical use of animals. All of the dams included in this study were ~12 weeks of age and this was their first pregnancy. Timed-pregnant Sprague-Dawley rats were received from Envigo at gestational day 10–11 and housed on a 12/12 light cycle at 22 ± 2°C with free access to Envigo 8640 diet and water.

Blood vessel isolation, preparation, and functional studies

On gestational day 19, rats were euthanized with 5% isoflurane. The thoracic aorta and uterine vessel beds were harvested and immersed in ice-cold PSS (Physiological Saline Solution, [mmol/L]: 118.3 NaCI, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11.1 dextrose; Thermo Fisher Scientific, Waltham, MA, USA). Segments of thoracic aorta and the main uterine artery were cleaned of connective tissue and fat and cut into 2–3 mm-long rings for wire myography. It has been shown that thoracic aortas and main uterine arteries are 2 – 3 mm and 150 – 300 μM in diameter at around gestational day 19 in Sprague-Dawley rats [20].

Rings of the main uterine artery were suspended on chucks and thoracic aortic rings placed on pins in tissue-bath chambers (DMT-USA, Inc., Ann Arbor, MI) containing 5 mL PSS continuously bubbled with carbogen (95% O2 / 5% CO2) at 37°C. Wall tension was increased to 4 mN for uterine arteries and 28 mN in aortic rings. Preload tensions were based on the following equations: 4 mN preload/2.5 mm vessel length = 1.8 mN/mm for uterine arteries and 28 mN/2.5 mm = 4.48 mN/mm for aortic rings, which are similar to other published wire-myography studies to induce maximal vasoactive responses in these vessels isolated from pregnant rats [20, 21]. Vessel segments were allowed to equilibrate for 15 min. Arteries were constricted by the addition of Phe (1 μM). Once constriction stabilized, increasing amounts of luteolin were added to the bath to result in a 6-point curve at final concentrations of 1, 5, 10, 30, 50, and 100 μM, which is similar to curves that have been conducted by Si et al [11]. Sufficient time was allowed for force to stabilize after each dose. Force changes were recorded using a PowerLab 8/35 acquisition system (ADInstruments, CO, USA). Relaxation responses were expressed as a percent relaxation from maximal constriction.

Depending on the endogenous mediator of vasorelaxation being examined, endothelium-intact vessels were incubated for 15 min with the NOS inhibitor, L-NAME (100 μM) [22, 23]; the COX inhibitor, indomethacin (10 μM) [24, 25]; or a dose of TEA (10 mM) previously shown to non-selectively block K+ channels [26] prior to conducting luteolin relaxation curves. Another way to assess whether vasorelaxation to luteolin is mediated by activation of K+ channel-induced hyperpolarization is by conducting relaxation curves in KCl-constricted and depolarized blood vessel rings [27]. Therefore, additional vessels were constricted with 60 mM of KCl followed by assessment of luteolin-induced vasorelaxation.

In a subset of the vascular reactivity experiments, to further gauge the role of the endothelium in luteolin-induced vasorelaxation, the endothelium was removed by gently rubbing of the lumen using a coarse human hair. The presence of an intact endothelium was assessed by the ability of ACh to induce >80% relaxation in Phe-contracted artery rings. Endothelium removal was considered successful when ACh caused <10% relaxation.

Statistical analysis

All data are presented as mean ± SEM. The number of rats used in each study are detailed in the figures and corresponding legends. These numbers represent individual vessels from separate animals. The luteolin-induced vasorelaxation curves were performed as paired experiments whereby each of the curves with or without treatments or perturbations were conducted at the same time but in different vessel rings from the same rats. Data were graphed and analyzed using GraphPad Prism (8.2.0). The effects of luteolin in uterine arteries versus aortic rings or in uterine arteries in the presence or absence of pharmacological agents or endothelium denudation were analyzed by two-way ANOVA with repeated measures. Specific differences for each concentration of luteolin were revealed using Sidak’s multiple comparisons tests. The maximum effect (Emax) of luteolin was statistically assess with a parametric Student’s t-test. A P-value <0.05 was considered statistically significant. The vascular response to the highest concentration of luteolin at 100 μM did not significantly differ (P>0.05) from that of 50 μM in any of these studies. Thus, the Emax response to luteolin was graphed at 50 μM and calculated as: Emax = [(maximum constriction to Phe – relaxation at 50 μM of luteolin) ÷ (maximum constriction to Phe – baseline tension pre-Phe)]*100. The Emax responses are illustrated as scattered-dot plots and presented alongside each vasorelaxation curve.

RESULTS

Luteolin-induced vasorelaxation in uterine artery versus aorta

We initially evaluated the relaxant effect of luteolin in Phe-constricted uterine arteries versus aortas isolated from normal pregnant rats at gestational day 19. As shown in Figure 1A, upon reaching the concentration of 30 μM luteolin, there was already a significant difference (P=0.0004) for greater relaxation in uterine arteries versus aortas. By 50 μM of luteolin (Figure 1A and B), there was complete relaxation (100.5 ± 5.2%) in uterine arteries, which was significantly greater (P=0.0003) than in aortas (27.5 ± 10.0%). Even the highest concentration of 100 μM luteolin (data not shown) produced less than half relaxation (43.6 ± 8.6%) in aortas compared to the uterine artery response (101.8 ± 4.6%). The response reached at this final concentration was significantly greater (P=0.0006) in uterine arteries versus the aortas. DMSO alone did not cause vasorelaxation in aortas or uterine arteries (data not shown). These data show that luteolin is a potent inducer of vasorelaxation in uterine arteries from normal pregnant rats.

Figure 1.

Figure 1.

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A: Luteolin-induced vasorelaxation in phenylephrine (Phe)-constricted aortas (blue circles, n = 8) and uterine arteries (red circles, n=8) isolated from normal pregnant Sprague-Dawley rats at gestational day 19. B: Maximum responses to 50 μM luteolin in aortas vs. uterine arteries. Data were analyzed using a two-way ANOVA with repeated measures followed by Sidak’s post-hoc analysis in A and Student’s t-test in B. *P=0.0004 for the response to 30 μM luteolin and **P=0.0003 for 50 μM luteolin in uterine arteries vs. aortas.

NOS, COX, K+ channel inhibitors and luteolin-induced vasorelaxation

Although it has been demonstrated that luteolin induces vasorelaxation via NOS, COX, and K+ channels in other vessels, it was unknown whether these mediators contribute to luteolin-induced relaxation in uterine arteries isolated from pregnant rats. To begin this assessment, endothelium-intact uterine artery segments were incubated in the presence or absence of the non-selective NOS inhibitor, L-NAME, prior to performing luteolin-induced vasorelaxation curves in Phe-constricted vessel rings. Figure 2A and B show that L-NAME did not impact luteolin-induced relaxation in uterine arteries. These data indicate that the NOS pathway is not a mechanism by which luteolin modulates vasorelaxation in the uterine artery isolated from normal pregnant rats.

Figure 2.

Figure 2.

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Luteolin-induced vasorelaxation in phenylephrine (Phe)-constricted uterine arteries isolated from normal pregnant Sprague-Dawley rats at gestational day 19. Uterine artery rings (n = 6–8) remained untreated (solid symbols) or were treated (unfilled symbols) with nitric oxide (NO) pathway inhibitor L-NAME (A and B, unfilled circles), cyclooxygenase (COX) pathway inhibitor Indomethacin (C and D, unfilled squares), or endothelium-derived hyperpolarization (EDH) pathway inhibitor TEA (E and F, unfilled triangles). B, D, and F: Maximum responses to 50 μM luteolin in the presence and absence of respective inhibitors. G and H: A comparison was made for luteolin-induced vasorelaxation in Phe-constricted (red circles) vs. KCl-constricted (green circles) uterine arteries.

Endothelium-intact vessels were also incubated in the presence or absence of the COX inhibitor, indomethacin. As illustrated in Figure 2C and D, indomethacin failed to alter luteolin-induced vasorelaxation in uterine arteries. These data indicate that the COX pathway is not involved in luteolin-induced relaxation in the uterine artery from normal pregnant rats.

We then explored the involvement of K+ channels in mediating luteolin-induced relaxation by non-selectively blocking this pathway with TEA in endothelium-intact uterine arteries. As shown in Figure 2E and F, TEA had no effect on luteolin-induced relaxation in uterine arteries. Moreover, Figure 2G and H illustrate that luteolin was able to relax uterine arteries in KCl-constricted vessel preparations from normal pregnant rats. Overall, these data indicate that luteolin does not promote vasorelaxation in uterine arteries isolated from pregnant rats via modulating K+ channel function.

Luteolin-induced vasorelaxation following removal of the endothelium

The data above suggested that luteolin-induced vasorelaxation in uterine arteries isolated from normal pregnant rats does not require classical endothelial mediators and pathways of relaxation. To confirm this, we tested the hypothesis that removal of the endothelium would not alter the ability of luteolin to promote relaxation in uterine artery rings in normal pregnant rats. Indeed, Figure 3A and B demonstrate the response to luteolin was similar in vessel preparations with and without the endothelium. These results indicated that luteolin bypasses the endothelium to directly promote vasorelaxation in the medial smooth muscle layer of uterine arteries during normal pregnancy.

Figure 3.

Figure 3.

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A: Concentration response curves to luteolin-induced vasorelaxation in phenylephrine (Phe)-constricted uterine arteries with or without endothelium (n = 8) isolated from normal pregnant Sprague-Dawley rats at gestational day 19. B: Maximum responses to 50 μM luteolin in uterine arteries with or without endothelium. Red symbols = endothelium intact, black symbols = endothelium denuded.

DISCUSSION

This study is the first report to demonstrate that the flavonoid, luteolin, has a potent effect to promote vasorelaxation in isolated uterine arteries, as compared to aortic rings, from normal pregnant rats. Moreover, it was found that this vasorelaxant effect of luteolin was not dependent on NOS, COX, or K+ channels. These findings suggested that endothelial factors and pathways do not mediate luteolin-induced vasorelaxation in uterine arteries during pregnancy. Indeed, experiments where the endothelium was removed demonstrated that luteolin is able to directly promote vasorelaxation by acting in the medial smooth muscle layer of uterine arteries during normal pregnancy.

The impetus for the present study is the growing interest regarding the beneficial impact of natural flavonoids, like luteolin, on the maternal cardiovascular system during pregnancy [17]. During normal pregnancy, the maternal uterine arteries dilate significantly and are responsive to a variety of endogenous vasorelaxant substances that act on the endothelium or vascular smooth muscle [28, 29]. In contrast, there is a paucity of data that has assessed the vasoactive effects of agents that can be found and consumed from nature on pregnancy. Therefore, in this study, we tested the hypothesis that luteolin induces vasorelaxation in isolated uterine arteries from normal pregnant rats.

Within this overall hypothesis, our first aim was to examine the potency of luteolin-induced vasorelaxation in uterine arteries and the conduit aorta. The reason for including the aorta in this investigation is that most studies examining the vascular effects of luteolin have focused on this vessel in male animals. There have been no studies examining the effects of luteolin on aortas and uterine arteries during normal pregnancy. Our results show that luteolin has vasorelaxing actions in aortas isolated from normal pregnant rats at gestational day 19. This response seems less potent than what has been documented in the literature using non-pregnant (male) rats [10, 11]. We found that luteolin produced a relaxation effect of ~40% in Phe-constricted aortas from pregnant rats, whereas other investigators found that it led to >60% in aortas from male rats. It is unknown if this difference was due to sex differences or because our study was conducted in pregnant rats. However, what the current study solidly demonstrates is that luteolin-induced vasorelaxation is significantly more potent in uterine arteries compared to aortas from normal pregnant rats.

Because we found such a potent vasorelaxation response to luteolin in uterine arteries during normal pregnancy, our study then focused on examining if this response was dependent on endothelial mediators and pathways of relaxation. Such classical mediators include NOS-derived NO and COX-derived metabolites along with other factors that promote vascular smooth muscle hyperpolarization via stimulating K+ efflux through K+ channels, which are termed the endothelial-derived hyperpolarizing factors (EDHF). It was previously demonstrated that luteolin elicits relaxation in aortas and coronary arteries from male rats via stimulating NOS, COX, and K+ channel pathways [10–12]. When we blocked each of these pathways with L-NAME, indomethacin, and TEA/high KCl, respectively, there was no impact on the ability of luteolin to promote vasorelaxation in uterine arteries from pregnant rats. These findings suggested that endothelial factors do not mediate luteolin-induced vasorelaxation in uterine arteries during pregnancy. Indeed, in experiments where the endothelium was denuded confirmed that luteolin was able to directly promote vasorelaxation by acting in the medial smooth muscle layer of uterine arteries during normal pregnancy.

Our finding that luteolin is capable of causing vasorelaxation that is independent of the endothelium is potentially promising for treatment of hypertensive vascular disease where the endothelium is damaged and dysfunctional. This includes the maternal hypertensive disorder of preeclampsia. It has been reported that abnormal uterine artery Doppler waveforms and microvascular endothelial dysfunction precede the onset on preeclamptic symptoms [30]. Preeclampsia can result from improper uteroplacental vascular function resulting in placenta ischemia/hypoxia [31]. Certainly, surgically-mediated reductions in uteroplacental perfusion lead to placenta-ischemia induced systemic vascular reactive oxygen species (ROS) production, endothelial dysfunction, and hypertension in pregnant rats [32–35]. Other studies show that luteolin inhibits ROS production in response to the pro-oxidant molecule, platelet-derived growth factor (PDGF), in isolated rat aortic smooth muscle cells [36] as well as attenuates superoxide-induced vascular dysfunction in an ex vivo model using mesenteric arteries exposed to auto-oxidation of pyrogallol [37]. Those studies along with our present investigation suggest that luteolin could bypass endothelial dysfunction in preeclampsia and directly promote vasorelaxation in the underlying smooth muscle and protect vascular function via reducing ROS. Our current study was conducted using uterine arteries isolated from normal pregnant rats, but we found that the greatest degree of luteolin-induced vasorelaxation appeared to be between the concentrations of 10 and 30 μM. Future studies will be designed to examine whether incubation of uterine arteries within this range of doses attenuates the response to pro-hypertensive vasoconstrictor substances. These include endothelin-1 (ET-1), which is elevated in preeclampsia and is the most potent endogenous vasoconstrictor known [38], or non-receptor mediated vasoconstriction in response to KCl. It could be that this is the range of doses that would be most beneficial in treating uterine artery dysfunction in preeclampsia. Thus, it will be determined if luteolin administration in vivo, using models of preeclampsia like rats with Reduced Uterine Perfusion Pressure (RUPP) that have increased vascular ET-1 [39], is protective for maternal cardiovascular function in the face of placental ischemia-induced vascular dysfunction and hypertension.

Limitations.

We only assessed luteolin-induced vasorelaxation at one maternal age (12 weeks of age), one time point during gestation (gestational day 19), and this was their first pregnancy. Thus, future studies will determine if luteolin-induced vasorelaxation in uterine arteries differs in younger or older dams, at different time before or after gestational day 19, or after multiple pregnancies.

Although our study suggests that luteolin causes vasorelaxation in uterine arteries from normal pregnant rats via direct action on the smooth muscle, we did not determine the intracellular mechanisms whereby luteolin promotes smooth muscle relaxation in uterine vessels. Luteolin is lipophilic [40] and likely crosses the plasma membrane. A hint about its mechanism of action was provided by a study using porcine splenic arteries [41]. The sex of the pigs was not specified, but they found that luteolin-induced relaxation was associated with an inhibition of calcium (Ca2+) entry by conducting vascular reactivity experiments in (Ca2+)-free buffer. Moreover, previous work in guinea pig gallbladder strips found that luteolin-mediated blunting of KCl-induced tension was attenuated by inhibition of Protein Kinase A (PKA) and intracellular Ca2+ release [42]. It has been shown that luteolin also induces activation of PKA in other cell types [43]. Future studies will examine whether this molecular pathway mediates luteolin-induced vasorelaxation in uterine arteries during pregnancy.

Our current study focused on luteolin-induced vasorelaxation in uterine arteries from normal pregnant rats. Although investigators have previously assessed luteolin-induced vasorelaxation in various vessels, those studies were to our knowledge only conducted in male rats [10–12]. Therefore, it is unclear at this point whether there is a sex difference in the vascular responsiveness to luteolin between males and non-pregnant female rats, and secondly, whether pregnancy alters this response compared to non-pregnant females. However, we believe that the current results are important to report showing that luteolin promotes vasorelaxation in uterine arteries from pregnant rats by bypassing the endothelium to act at the level of the vascular smooth muscle.

CONCLUSIONS

We report that the flavonoid compound, luteolin, promotes vasorelaxation in small vessels like the uterine arteries in normal pregnant rats. This response was greater than the response found in the conduit aorta. The potent capability of luteolin to induce vasorelaxation in uterine arteries was not dependent on the endothelium. Thus, we postulate that the direct luteolin-induced relaxation in vascular smooth muscle in the uterine circulation could be vasoprotective in states where pregnancy is complicated by uterine and systemic vascular dysfunction, like preeclampsia.

HIGHLIGHTS.

  • Luteolin induced vasorelaxation in uterine arteries from normal pregnant rats

  • Endothelial-dependent vasorelaxing pathways do not mediate the luteolin response

  • Endothelial denudation did not alter luteolin-induced vasorelaxation

  • Luteolin directly relaxed the tunica media of uterine arteries during pregnancy

ACKNOWLEDGMENTS

The authors thank Drs. Lorena M. Amaral and B. Babbette LaMarca for providing uterine arteries and aortas. The authors also thank Dr. Ying Ge for her invaluable help with vascular reactivity.

FUNDING

This project was funded by grants from the National Heart, Lung, and Blood Institute (T32HL105324, P01HL051971, R00HL130577), the National Institute of General Medical Sciences (P20GM104357, P20GM121334, U54GM115428), the American Heart Association (18POST33990293), and the Natural Science Foundation of China (81601318).

Footnotes

CONFLICT OF INTEREST STATEMENT

No conflict to disclose.

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