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Original Research Article

DTT 2022; 1(1): 27-32

Published online July 31, 2022 https://doi.org/10.58502/DTT.22.006

Copyright © The Pharmaceutical Society of Korea.

Inhibitory Effect of Icariin on Agonist-Induced Modulation of Vascular Contractility via Calcium Desensitization-Related Pathways

Jina Kim1*, Joon Seok Bang2*, Young Sil Min3, Hyeong-Dong Kim4, Hyun Dong Je1

1Department of Pharmacology, College of Pharmacy, Daegu Catholic University, Gyeongsan, Korea
2College of Pharmacy, Sookmyung Women's University, Seoul, Korea
3Department of Pharmaceutical Science, Jungwon University, Goesan, Korea
4School of Health and Environmental Science, College of Health Science, Korea University, Seoul, Korea

Correspondence to:Hyun Dong Je, hyundong@cu.ac.kr
*The first two authors contributed equally to this work.

Received: March 29, 2022; Revised: May 27, 2022; Accepted: June 3, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Icariin, a prenylated flavonol glycoside, is found in several plants of the genus Epimedium such as horny goat weed and Yin Yang Huo and has many biological functions. This study investigated the effect of icariin on vascular contractility and the mechanism involved. Data on the isometric contractions of denuded aortas of male rats were determined and combined with molecular experiments. Icariin alleviated fluoride-, thromboxane mimetic- and phorbol ester-elicited vascular contraction, suggesting its antihypertensive effect in reducing agonist-elicited vascular contraction regardless of endothelial nitric oxide synthesis. Furthermore, icariin alleviated increases elicited by fluoride in pMYPT1 levels and those by phorbol ester in pERK1/2 levels, indicating the mechanism involved in the restriction of Rho-kinase and MEK activity and the subsequent phosphorylation of MYPT1 and ERK1/2. This study presents evidence regarding the mechanism and explains the relaxation effect of icariin on agonist-stimulated vascular contraction regardless of endothelial function.

KeywordsERK1/2, icariin, fluoride, MYPT1, phorbol ester, Rho-kinase

Icariin (7-(β-D-glucopyranosyloxy)-5-hydroxy-4’-methoxy-8-(3-methylbut-2-en-1-yl)-3-(α-L-rhamnopyranosyloxy)flavone) (Fig. 1) is a prenylated flavonol glycoside monomer isolated from Herba Epimedii (Yin Yang Huo), a kidney-tonifying medicinal herb and also used as a dietary supplement in the USA (Tesch 2003). Icariin exerts various pharmacological effects such as immunity regulation, the proliferation and development of osteoblasts (Wang et al. 2018), and protection against diseases associated with estrogen deficiency (Xiao et al. 2014), with excellent medicinal and health benefits. Although the effect of endothelial nitric oxide synthesis is well established (Li et al. 2016), we evaluated the possible influence and related mechanisms of the anti-inflammatory action of icariin on vascular contractility to develop a better antihypertensive. Isometric contractions of denuded aortas of male Sprague-Dawley rats were recorded using a computerized physiograph for use with molecular experiments.

Figure 1.Chemical structure of icariin (7-(β-D-glucopyranosyloxy)-5-hydroxy-4′-methoxy-8-(3-methylbut-2-en-1-yl)-3-(α-L-rhamnopyranosyloxy)	flavone).

Alterations in the arterial tone are associated with cardiovascular disorders that may result in mortality and morbidity, including hypertension, which is complicated by diverse mechanisms involving endothelial dysfunction and risk factors for circulatory disorders. Besides endothelial dysfunction, vascular smooth muscle contractility is predominantly controlled by Ca2+ signaling involving Ca2+ influx, release or sensitization and regulation of a Ca2+-dependent increase in the phosphorylation of a 20 kDa myosin light chain (MLC20) (Somlyo and Somlyo 1994). The extent of MLC20 phosphorylation or force of contraction induced by agonist stimulation is usually higher than that caused by an increase in the cytosolic Ca2+ concentration referred to as Ca2+ sensitization (Somlyo and Somlyo 1994). Subsequent studies have suggested that the inhibition of MLC phosphatase by Rho-kinase (Kitazawa et al. 1991; Uehata et al. 1997; Somlyo and Somlyo 1998; Sakurada et al. 2003) or thin filament regulation including the activation of protein kinase C (PKC), mitogen-activated protein kinase kinases (MEK), and extracellular signal-regulated kinase (ERK) 1/2 and phosphorylation of the actin-binding protein caldesmon (Wier and Morgan 2003) may be major components of the pathway that facilitates Ca2+ sensitization.

Activation of ERK1/2 not only regulates vascular contractility but is also associated with pathologic hypertrophy, hyperplasia, hypertension, and atherosclerosis (Xu et al. 1996; Touyz et al. 1999). ERK1/2 is stimulated by threonine/tyrosine phosphorylation by the specific kinase MEK stimulated by Raf. In diverse smooth muscles, fluoride, phorbol ester, or thromboxane mimetic elicits contractions, primarily owing to enhanced calcium sensitivity or partially enhanced calcium concentration only for thromboxane mimetic. ERK1/2 activation is induced by the phorbol ester, phorbol 12,13-dibutyrate (PDBu). PDBu triggers ERK1/2-dependent cytoskeletal remodeling and formation of podosomes, inducing ERK1/2 activation (Gu et al. 2007). By contrast, contractions induced by fluoride or thromboxane mimetic may involve the RhoA/Rho-kinase pathway (Jeon et al. 2006). Whether this route is alleviated during icariin-elicited vascular relaxation in aortas contracted with MEK activator phorbol ester or Rho-kinase activator fluoride except endothelial nitric oxide synthesis is unknown. Therefore, to fill in the gap, this study examined the feasible roles of Rho-kinase or MEK inhibition on calcium desensitization during this relaxation.

Vessel preparation

All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee at our institutions such as Jungwon University and Daegu Catholic University (IACUC-2013-042). Sprague-Dawley male rats weighing 200-240 g were anesthetized with etomidate (0.3 mg/kg i.p.) as subjected to animal euthanasia. Thoracic aortas were swiftly extracted and soaked in oxygenated (5% CO2/95% O2) physiological saline solution composed of (mM): 115.0 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 KH2PO4, 25.0 NaHCO3 and 10.0 dextrose (pH 7.4). Then adherent connective tissue was wiped out, and aortic endothelia were eliminated by gentle abrasion using pipette tips or cell scrapers.

Contraction assessments

Two triangles were inserted through each aortic ring, and each ring was dangled in a water-jacketed organ bath (10 mL) maintained at 37℃ and aerated with a mixture of 5% CO2 and 95% O2. One triangle was fixed to an immovable support and the other to an isometric force transducer (Grass FT03C, Quincy, MA, USA). The rings were extended passively by applying a suitable resting tension of 2.0 g, which was sustained throughout the experiment. Each ring was stabilized in the organ bath for 1 h before contractile responses to 50 mM KCl were assessed. Isometric contractions were stored using a data acquisition system (PowerLab/8SP, AD Instruments, Castle Hill, NSW, Australia).

The direct effect of icariin application was assessed after KCl- (50 mM), thromboxane mimetic- (U46619, 0.1 µM), fluoride- (6 mM), or phorbol ester (1 µM)-elicited contractions had stabilized in normal Krebs solution.

Western blot assay

Muscle strips were swift frozen by soaking in a dry ice/acetone slurry of 10 mM dithiothreitol (DTT) and 10% trichloroacetic acid (TCA). Muscles were stored at –20℃ until use. The frozen tissues were allowed to thaw to room temperature in a dry ice/acetone/DTT/TCA mixture and homogenized in a buffer containing 20 mM MOPS, 10% glycerol, 4% SDS, 10 mM DTT, 20 mM β-glycerophosphate, 5.5 μM leupeptin, 5.5 μM pepstatin, 2 mM Na3VO4, 20 kIU aprotinin, 1 mM NaF, 100 μM ZnCl2, 5 mM EGTA, and 20 μM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF). Protein-accorded samples (revised Lowry protein assay, DC Protein Assay Kit, Bio-Rad) were electrophoresed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Protogel, National Diagnostics), transferred to polyvinylidene fluoride (PVDF) membranes, and subjected to immunostaining and densitometry using suitable antibodies. The success of protein matching was verified by Naphthol Blue Black staining of the membrane and densitometry of the actin band. Lane loading discrepancies were rectified by normalization versus β-actin.

Chemicals and antibodies

Chemicals and drugs were obtained from the following sources: potassium chloride, sodium fluoride, acetylcholine, icariin, U46619, and PDBu from Sigma (St. Louis, MO, USA); DTT, TCA, and acetone from Fisher Scientific (Hampton, NH, USA); enhanced chemiluminescence (ECL) kits from Pierce (Rockford, IL, USA). Antibodies against phospho-myosin phosphatase targeting subunit protein 1 (phospho-MYPT1) at Thr855 (1:5,000), MYPT1, ERK or phosphoERK at Thr202/Tyr204 were purchased from Cell Signaling Technology (Danvers, MA, USA) or Upstate Biotechnology (Lake Placid, NY, USA) to determine levels of RhoA/Rho-kinase activity (Wooldridge et al. 2004; Wilson et al. 2005) or MEK activity; anti-rabbit IgG (goat) and anti-mouse IgM (goat) conjugated with horseradish peroxidase, used as secondary antibodies (1:2,000, respectively), from Upstate (Lake Placid, NY, USA). Icariin was prepared in dimethyl sulfoxide (DMSO) as a 100 mM stock solution and frozen at –20℃ for later use. DMSO alone had no perceptible effect at the concentrations used (data not shown).

Statistical analysis

Data were represented as mean ± standard error of the mean. Student’s unpaired t test or analysis of variance was used to judge the statistical significance of the means between two groups using SPSS 12.0 (SPSS Inc., Chicago, IL, USA). p values < 0.05 were considered as statistically significant.

Effect of icariin on a full RhoA/Rho-kinase activator fluoride or thromboxane mimetic-elicited contractions of endothelium-denuded aortas

Endothelium was removed by gentle abrasion with a pipette tip to identify the direct effect of icariin on vascular smooth muscle. The absence of endothelium was verified by a scarcity of relaxation after treating contracted vessel segments with acetylcholine (1 µM). Icariin showed no perceptible effect on basal tension (data not shown), and remarkably alleviated the contraction elicited by a Rho-kinase activator fluoride regardless of endothelial nitric oxide synthesis (Fig. 2A). This suggests that the relaxation mechanism of icariin might involve the alleviation of Rho-kinase activity in addition to endothelial nitric oxide synthesis and the ensuing stimulation of guanylyl cyclase. By contrast, icariin at the same concentration significantly alleviated thromboxane A2 mimetic U46619- elicited contraction in denuded muscles (Fig. 3) suggesting that thromboxane mimetic acts similarly with fluoride where Rho-kinase stimulation was the main pathway.

Figure 2.Effect of icariin on fluoride-elicited vasoconstriction in denuded (A) or intact (B) muscles. Each vessel was stabilized in the organ bath for 30-60 min before relaxation responses to icariin were assessed. Data are represented as the means of three to five experiments with vertical lines constituting SEMs.

Figure 3.Effect of icariin on thromboxane mimetic-elicited vasoconstriction in denuded muscles. Each ring was stabilized in the organ bath for 30-60 min before relaxation responses to icariin were assessed. Data are represented as the means of three to five experiments with vertical lines constituting SEMs.

Effect of icariin on an MEK activator phorbol ester-elicited contractions of denuded aortas

Phorbol esters are primarily MEK activators and partial RhoA/Rho-kinase activators (data not shown). Interestingly, PDBu-elicited contraction was significantly alleviated by icariin regardless of endothelial nitric oxide synthesis (Fig. 4), which suggested that thin or actin filament modulation including MEK/ERK activation was significantly alleviated.

Figure 4.Effect of icariin on phorbol ester-elicited vasoconstriction in denuded muscles. Each ring was stabilized in the organ bath for 30-60 min before relaxation responses to icariin were assessed. Data are represented as the means of three to five experiments with vertical lines constituting SEMs.

Effect of icariin on the level of MYPT1 phosphorylation at Thr-855

The role of icariin on the thick filament modulation of smooth muscle contractility was verified by assessing the myosin phosphatase targeting subunit 1 (MYPT1) and phospho-MYPT1 levels in vessels swift frozen after 60 min exposure to icariin for equilibration. Each relaxing rat aorta in the absence of endothelium was contracted with 6 mM fluoride using swift frozen icariin (0.1 mM) treatment, and the levels were compared to those of vehicle-treated rat aortas (Fig. 5). Interestingly, icariin led to reduction in fluoride-elicited MYPT1 phosphorylation at Thr855 (Fig. 5). Thus, modulation of thick or myosin filament containing myosin phosphatase activation via RhoA/Rho-kinase inactivation is involved in the alleviated contractility of icariin-treated rat aorta.

Figure 5.Effect of icariin on fluoride-elicited augmentations in phospho-MYPT1 levels. Phospho-MYPT1 protein levels were significantly alleviated in quick frozen icariin-treated aorta in the absence of endothelium compared to vehicle-treated aorta precontracted with fluoride. Lower panel indicates average densitometric results for relative levels of phospho-MYPT1. Data are represented as the means of three to five experiments with vertical lines constituting SEMs. *p < 0.05, ##p < 0.01, versus control or normal group, respectively. Icariin: 0.1 mM icariin; fluoride: 6 mM sodium fluoride.

Effect of icariin on the levels of ERK1/2 phosphorylation at Thr-202/Tyr-204

The role of icariin on thin filament modulation of smooth muscle contractility was verified by assessing the phospho-ERK1/2 and ERK1/2 levels in vessels swift frozen after 60 min exposure to icariin for equilibration. Each relaxing ring was precontracted with 1 μM phorbol ester (PDBu). Compared with vehicle-treated rat aortas, in rat aortas without endothelium, the reduction in ERK 1/2 phosphorylation at Thr202/Tyr204 was elicited by icariin (0.1 mM) treatment (Fig. 6), showing substantial relaxation (Fig. 4) and thin filament modulation. These findings show the importance of thin or actin filament modulation including ERK1/2 phosphorylation via MEK stimulation in the alleviated contractility elicited by icariin.

Figure 6.Effect of icariin on phorbol ester-elicited augmentations in phospho-ERK1/2 levels (closed bar: pERK1; open bar: pERK2). Phospho-ERK1/2 protein levels were alleviated in quick frozen icariin-treated rat aortas in the absence of endothelium compared to vehicle-treated rat aortas contracted with phorbol ester. Lower panel indicates average densitometric results for relative levels of phospho-ERK1/2. Data are represented as the means of three to five experiments with vertical lines constituting SEMs. *p < 0.05, ##p < 0.01, versus control or normal group respectively. Icariin: 0.1 mM icariin; PDBu: phorbol 12,13-dibutyrate (1 μM).

The present study demonstrates that icariin alleviates vascular contractility in an agonist-dependent manner and that the mechanism is not only endothelium dependent but also accommodates the nonspecific inhibition of MEK and Rho-kinase activity. Considering the anti-inflammatory and antioxidant activity and endothelial vasorelaxation of icariin (Li et al. 2016), icariin-elicited relaxation by the restriction of MEK or RhoA/Rho-kinase activity was estimated in rat aortas denuded and contracted by an MEK activator phorbol ester or RhoA/Rho-kinase activator fluoride.

Although the mechanism by which phorbol ester activates MEK/ERK has been established (Kordowska et al. 2006; Gu et al. 2007), variable findings are indicated in those underlying arterial contractions elicited by fluoride or thromboxaneB mimetic in the contraction triggered by calcium entry and Rho-kinase activation (Wilson et al. 2005; Tsai and Jiang 2006). These findings are consistent with the notion that icariin can restrict phorbol ester or fluoride-elicited contraction by inhibiting MEK or Rho-kinase activity.

Several possibilities exist in the mechanism by which MEK activation promotes vasoconstriction, warranting intensive study. The phosphorylation of caldesmon by MEK/ERK regulates smooth muscle contractility (Kordowska et al. 2006), in the process stimulating MEK/ERK by PKC, which in turn is excited by phorbol esters or GPCR receptor agonists.

The present study demonstrates that icariin alleviates the maximal contraction elicited by vasoconstrictor fluorideB or phorbol ester, independently of the endothelium (Fig. 2, 4), by the MEK/ERK and RhoA/Rho-kinase pathway. Previously, vasodilation was believed to be caused by endothelial nitric oxide synthesis and the subsequent activation of guanylyl cyclase (Li et al. 2016). Li et al. (2016) reported icariin-induced endothelium-dependent relaxation and related pathway involving cGMP production, while in this study, icariin substantially alleviated phorbol ester- or fluorideB-elicited contraction regardless of endothelial function (Fig. 2, 4). Furthermore, icariin alleviated the phosphorylation of phorbol ester-evoked extracellular signal-regulated kinase (ERK) 1/2 (Fig. 6) and substantially that of fluoride-elicited MYPT1 at Thr855 (Fig. 5), with the relaxation (Fig. 2) suggesting the alleviation of MEK or Rho-kinase activity as a major mechanism.

MLC20 is known to be phosphorylated by both MLCK and Rho-kinase (Somlyo and Somlyo 1998). The activation of Rho-kinase by U46619 or fluoride inhibits the activity of myosin light chain phosphatase through the phosphorylation of MYPT1, leading to an enhanced MLC20 phosphorylation as well as contractions (Sakurada et al. 2003; Wilson et al. 2005).

Icariin substantially alleviates the contractions elicited by an MEK activator phorbol ester regardless of endothelial function and those by a Rho-kinase activator fluoride at this concentration. Thus, the mechanism underlying these relaxations involve the nonspecific restriction of MEK and Rho-kinase activities. Interestingly, during fluoride-elicited contraction, the alleviation of Rho-kinase activity and subsequent MYPT1 phosphorylation elicited by icariin suggest that Rho-kinase inactivation is needed for relaxation. In conclusion, in addition to endothelial nitric oxide synthesis (Li et al. 2016), both MEK and Rho-kinase inhibition are major contributors to the icariin-elicited vasorelaxation mechanism in the denuded muscle, and the reduction of intracellular calcium level, which should be elucidated directly, may be involved as well (Liu and Huang 2016).

No potential conflict of interest relevant to this article was reported.

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Article

Original Research Article

DTT 2022; 1(1): 27-32

Published online July 31, 2022 https://doi.org/10.58502/DTT.22.006

Copyright © The Pharmaceutical Society of Korea.

Inhibitory Effect of Icariin on Agonist-Induced Modulation of Vascular Contractility via Calcium Desensitization-Related Pathways

Jina Kim1*, Joon Seok Bang2*, Young Sil Min3, Hyeong-Dong Kim4, Hyun Dong Je1

1Department of Pharmacology, College of Pharmacy, Daegu Catholic University, Gyeongsan, Korea
2College of Pharmacy, Sookmyung Women's University, Seoul, Korea
3Department of Pharmaceutical Science, Jungwon University, Goesan, Korea
4School of Health and Environmental Science, College of Health Science, Korea University, Seoul, Korea

Correspondence to:Hyun Dong Je, hyundong@cu.ac.kr
*The first two authors contributed equally to this work.

Received: March 29, 2022; Revised: May 27, 2022; Accepted: June 3, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Icariin, a prenylated flavonol glycoside, is found in several plants of the genus Epimedium such as horny goat weed and Yin Yang Huo and has many biological functions. This study investigated the effect of icariin on vascular contractility and the mechanism involved. Data on the isometric contractions of denuded aortas of male rats were determined and combined with molecular experiments. Icariin alleviated fluoride-, thromboxane mimetic- and phorbol ester-elicited vascular contraction, suggesting its antihypertensive effect in reducing agonist-elicited vascular contraction regardless of endothelial nitric oxide synthesis. Furthermore, icariin alleviated increases elicited by fluoride in pMYPT1 levels and those by phorbol ester in pERK1/2 levels, indicating the mechanism involved in the restriction of Rho-kinase and MEK activity and the subsequent phosphorylation of MYPT1 and ERK1/2. This study presents evidence regarding the mechanism and explains the relaxation effect of icariin on agonist-stimulated vascular contraction regardless of endothelial function.

Keywords: ERK1/2, icariin, fluoride, MYPT1, phorbol ester, Rho-kinase

Introduction

Icariin (7-(β-D-glucopyranosyloxy)-5-hydroxy-4’-methoxy-8-(3-methylbut-2-en-1-yl)-3-(α-L-rhamnopyranosyloxy)flavone) (Fig. 1) is a prenylated flavonol glycoside monomer isolated from Herba Epimedii (Yin Yang Huo), a kidney-tonifying medicinal herb and also used as a dietary supplement in the USA (Tesch 2003). Icariin exerts various pharmacological effects such as immunity regulation, the proliferation and development of osteoblasts (Wang et al. 2018), and protection against diseases associated with estrogen deficiency (Xiao et al. 2014), with excellent medicinal and health benefits. Although the effect of endothelial nitric oxide synthesis is well established (Li et al. 2016), we evaluated the possible influence and related mechanisms of the anti-inflammatory action of icariin on vascular contractility to develop a better antihypertensive. Isometric contractions of denuded aortas of male Sprague-Dawley rats were recorded using a computerized physiograph for use with molecular experiments.

Figure 1. Chemical structure of icariin (7-(β-D-glucopyranosyloxy)-5-hydroxy-4′-methoxy-8-(3-methylbut-2-en-1-yl)-3-(α-L-rhamnopyranosyloxy)&#9;flavone).

Alterations in the arterial tone are associated with cardiovascular disorders that may result in mortality and morbidity, including hypertension, which is complicated by diverse mechanisms involving endothelial dysfunction and risk factors for circulatory disorders. Besides endothelial dysfunction, vascular smooth muscle contractility is predominantly controlled by Ca2+ signaling involving Ca2+ influx, release or sensitization and regulation of a Ca2+-dependent increase in the phosphorylation of a 20 kDa myosin light chain (MLC20) (Somlyo and Somlyo 1994). The extent of MLC20 phosphorylation or force of contraction induced by agonist stimulation is usually higher than that caused by an increase in the cytosolic Ca2+ concentration referred to as Ca2+ sensitization (Somlyo and Somlyo 1994). Subsequent studies have suggested that the inhibition of MLC phosphatase by Rho-kinase (Kitazawa et al. 1991; Uehata et al. 1997; Somlyo and Somlyo 1998; Sakurada et al. 2003) or thin filament regulation including the activation of protein kinase C (PKC), mitogen-activated protein kinase kinases (MEK), and extracellular signal-regulated kinase (ERK) 1/2 and phosphorylation of the actin-binding protein caldesmon (Wier and Morgan 2003) may be major components of the pathway that facilitates Ca2+ sensitization.

Activation of ERK1/2 not only regulates vascular contractility but is also associated with pathologic hypertrophy, hyperplasia, hypertension, and atherosclerosis (Xu et al. 1996; Touyz et al. 1999). ERK1/2 is stimulated by threonine/tyrosine phosphorylation by the specific kinase MEK stimulated by Raf. In diverse smooth muscles, fluoride, phorbol ester, or thromboxane mimetic elicits contractions, primarily owing to enhanced calcium sensitivity or partially enhanced calcium concentration only for thromboxane mimetic. ERK1/2 activation is induced by the phorbol ester, phorbol 12,13-dibutyrate (PDBu). PDBu triggers ERK1/2-dependent cytoskeletal remodeling and formation of podosomes, inducing ERK1/2 activation (Gu et al. 2007). By contrast, contractions induced by fluoride or thromboxane mimetic may involve the RhoA/Rho-kinase pathway (Jeon et al. 2006). Whether this route is alleviated during icariin-elicited vascular relaxation in aortas contracted with MEK activator phorbol ester or Rho-kinase activator fluoride except endothelial nitric oxide synthesis is unknown. Therefore, to fill in the gap, this study examined the feasible roles of Rho-kinase or MEK inhibition on calcium desensitization during this relaxation.

MATERIALS AND METHODS

Vessel preparation

All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee at our institutions such as Jungwon University and Daegu Catholic University (IACUC-2013-042). Sprague-Dawley male rats weighing 200-240 g were anesthetized with etomidate (0.3 mg/kg i.p.) as subjected to animal euthanasia. Thoracic aortas were swiftly extracted and soaked in oxygenated (5% CO2/95% O2) physiological saline solution composed of (mM): 115.0 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 KH2PO4, 25.0 NaHCO3 and 10.0 dextrose (pH 7.4). Then adherent connective tissue was wiped out, and aortic endothelia were eliminated by gentle abrasion using pipette tips or cell scrapers.

Contraction assessments

Two triangles were inserted through each aortic ring, and each ring was dangled in a water-jacketed organ bath (10 mL) maintained at 37℃ and aerated with a mixture of 5% CO2 and 95% O2. One triangle was fixed to an immovable support and the other to an isometric force transducer (Grass FT03C, Quincy, MA, USA). The rings were extended passively by applying a suitable resting tension of 2.0 g, which was sustained throughout the experiment. Each ring was stabilized in the organ bath for 1 h before contractile responses to 50 mM KCl were assessed. Isometric contractions were stored using a data acquisition system (PowerLab/8SP, AD Instruments, Castle Hill, NSW, Australia).

The direct effect of icariin application was assessed after KCl- (50 mM), thromboxane mimetic- (U46619, 0.1 µM), fluoride- (6 mM), or phorbol ester (1 µM)-elicited contractions had stabilized in normal Krebs solution.

Western blot assay

Muscle strips were swift frozen by soaking in a dry ice/acetone slurry of 10 mM dithiothreitol (DTT) and 10% trichloroacetic acid (TCA). Muscles were stored at –20℃ until use. The frozen tissues were allowed to thaw to room temperature in a dry ice/acetone/DTT/TCA mixture and homogenized in a buffer containing 20 mM MOPS, 10% glycerol, 4% SDS, 10 mM DTT, 20 mM β-glycerophosphate, 5.5 μM leupeptin, 5.5 μM pepstatin, 2 mM Na3VO4, 20 kIU aprotinin, 1 mM NaF, 100 μM ZnCl2, 5 mM EGTA, and 20 μM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF). Protein-accorded samples (revised Lowry protein assay, DC Protein Assay Kit, Bio-Rad) were electrophoresed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Protogel, National Diagnostics), transferred to polyvinylidene fluoride (PVDF) membranes, and subjected to immunostaining and densitometry using suitable antibodies. The success of protein matching was verified by Naphthol Blue Black staining of the membrane and densitometry of the actin band. Lane loading discrepancies were rectified by normalization versus β-actin.

Chemicals and antibodies

Chemicals and drugs were obtained from the following sources: potassium chloride, sodium fluoride, acetylcholine, icariin, U46619, and PDBu from Sigma (St. Louis, MO, USA); DTT, TCA, and acetone from Fisher Scientific (Hampton, NH, USA); enhanced chemiluminescence (ECL) kits from Pierce (Rockford, IL, USA). Antibodies against phospho-myosin phosphatase targeting subunit protein 1 (phospho-MYPT1) at Thr855 (1:5,000), MYPT1, ERK or phosphoERK at Thr202/Tyr204 were purchased from Cell Signaling Technology (Danvers, MA, USA) or Upstate Biotechnology (Lake Placid, NY, USA) to determine levels of RhoA/Rho-kinase activity (Wooldridge et al. 2004; Wilson et al. 2005) or MEK activity; anti-rabbit IgG (goat) and anti-mouse IgM (goat) conjugated with horseradish peroxidase, used as secondary antibodies (1:2,000, respectively), from Upstate (Lake Placid, NY, USA). Icariin was prepared in dimethyl sulfoxide (DMSO) as a 100 mM stock solution and frozen at –20℃ for later use. DMSO alone had no perceptible effect at the concentrations used (data not shown).

Statistical analysis

Data were represented as mean ± standard error of the mean. Student’s unpaired t test or analysis of variance was used to judge the statistical significance of the means between two groups using SPSS 12.0 (SPSS Inc., Chicago, IL, USA). p values < 0.05 were considered as statistically significant.

RESULTS

Effect of icariin on a full RhoA/Rho-kinase activator fluoride or thromboxane mimetic-elicited contractions of endothelium-denuded aortas

Endothelium was removed by gentle abrasion with a pipette tip to identify the direct effect of icariin on vascular smooth muscle. The absence of endothelium was verified by a scarcity of relaxation after treating contracted vessel segments with acetylcholine (1 µM). Icariin showed no perceptible effect on basal tension (data not shown), and remarkably alleviated the contraction elicited by a Rho-kinase activator fluoride regardless of endothelial nitric oxide synthesis (Fig. 2A). This suggests that the relaxation mechanism of icariin might involve the alleviation of Rho-kinase activity in addition to endothelial nitric oxide synthesis and the ensuing stimulation of guanylyl cyclase. By contrast, icariin at the same concentration significantly alleviated thromboxane A2 mimetic U46619- elicited contraction in denuded muscles (Fig. 3) suggesting that thromboxane mimetic acts similarly with fluoride where Rho-kinase stimulation was the main pathway.

Figure 2. Effect of icariin on fluoride-elicited vasoconstriction in denuded (A) or intact (B) muscles. Each vessel was stabilized in the organ bath for 30-60 min before relaxation responses to icariin were assessed. Data are represented as the means of three to five experiments with vertical lines constituting SEMs.

Figure 3. Effect of icariin on thromboxane mimetic-elicited vasoconstriction in denuded muscles. Each ring was stabilized in the organ bath for 30-60 min before relaxation responses to icariin were assessed. Data are represented as the means of three to five experiments with vertical lines constituting SEMs.

Effect of icariin on an MEK activator phorbol ester-elicited contractions of denuded aortas

Phorbol esters are primarily MEK activators and partial RhoA/Rho-kinase activators (data not shown). Interestingly, PDBu-elicited contraction was significantly alleviated by icariin regardless of endothelial nitric oxide synthesis (Fig. 4), which suggested that thin or actin filament modulation including MEK/ERK activation was significantly alleviated.

Figure 4. Effect of icariin on phorbol ester-elicited vasoconstriction in denuded muscles. Each ring was stabilized in the organ bath for 30-60 min before relaxation responses to icariin were assessed. Data are represented as the means of three to five experiments with vertical lines constituting SEMs.

Effect of icariin on the level of MYPT1 phosphorylation at Thr-855

The role of icariin on the thick filament modulation of smooth muscle contractility was verified by assessing the myosin phosphatase targeting subunit 1 (MYPT1) and phospho-MYPT1 levels in vessels swift frozen after 60 min exposure to icariin for equilibration. Each relaxing rat aorta in the absence of endothelium was contracted with 6 mM fluoride using swift frozen icariin (0.1 mM) treatment, and the levels were compared to those of vehicle-treated rat aortas (Fig. 5). Interestingly, icariin led to reduction in fluoride-elicited MYPT1 phosphorylation at Thr855 (Fig. 5). Thus, modulation of thick or myosin filament containing myosin phosphatase activation via RhoA/Rho-kinase inactivation is involved in the alleviated contractility of icariin-treated rat aorta.

Figure 5. Effect of icariin on fluoride-elicited augmentations in phospho-MYPT1 levels. Phospho-MYPT1 protein levels were significantly alleviated in quick frozen icariin-treated aorta in the absence of endothelium compared to vehicle-treated aorta precontracted with fluoride. Lower panel indicates average densitometric results for relative levels of phospho-MYPT1. Data are represented as the means of three to five experiments with vertical lines constituting SEMs. *p < 0.05, ##p < 0.01, versus control or normal group, respectively. Icariin: 0.1 mM icariin; fluoride: 6 mM sodium fluoride.

Effect of icariin on the levels of ERK1/2 phosphorylation at Thr-202/Tyr-204

The role of icariin on thin filament modulation of smooth muscle contractility was verified by assessing the phospho-ERK1/2 and ERK1/2 levels in vessels swift frozen after 60 min exposure to icariin for equilibration. Each relaxing ring was precontracted with 1 μM phorbol ester (PDBu). Compared with vehicle-treated rat aortas, in rat aortas without endothelium, the reduction in ERK 1/2 phosphorylation at Thr202/Tyr204 was elicited by icariin (0.1 mM) treatment (Fig. 6), showing substantial relaxation (Fig. 4) and thin filament modulation. These findings show the importance of thin or actin filament modulation including ERK1/2 phosphorylation via MEK stimulation in the alleviated contractility elicited by icariin.

Figure 6. Effect of icariin on phorbol ester-elicited augmentations in phospho-ERK1/2 levels (closed bar: pERK1; open bar: pERK2). Phospho-ERK1/2 protein levels were alleviated in quick frozen icariin-treated rat aortas in the absence of endothelium compared to vehicle-treated rat aortas contracted with phorbol ester. Lower panel indicates average densitometric results for relative levels of phospho-ERK1/2. Data are represented as the means of three to five experiments with vertical lines constituting SEMs. *p < 0.05, ##p < 0.01, versus control or normal group respectively. Icariin: 0.1 mM icariin; PDBu: phorbol 12,13-dibutyrate (1 μM).

DISCUSSION

The present study demonstrates that icariin alleviates vascular contractility in an agonist-dependent manner and that the mechanism is not only endothelium dependent but also accommodates the nonspecific inhibition of MEK and Rho-kinase activity. Considering the anti-inflammatory and antioxidant activity and endothelial vasorelaxation of icariin (Li et al. 2016), icariin-elicited relaxation by the restriction of MEK or RhoA/Rho-kinase activity was estimated in rat aortas denuded and contracted by an MEK activator phorbol ester or RhoA/Rho-kinase activator fluoride.

Although the mechanism by which phorbol ester activates MEK/ERK has been established (Kordowska et al. 2006; Gu et al. 2007), variable findings are indicated in those underlying arterial contractions elicited by fluoride or thromboxaneB mimetic in the contraction triggered by calcium entry and Rho-kinase activation (Wilson et al. 2005; Tsai and Jiang 2006). These findings are consistent with the notion that icariin can restrict phorbol ester or fluoride-elicited contraction by inhibiting MEK or Rho-kinase activity.

Several possibilities exist in the mechanism by which MEK activation promotes vasoconstriction, warranting intensive study. The phosphorylation of caldesmon by MEK/ERK regulates smooth muscle contractility (Kordowska et al. 2006), in the process stimulating MEK/ERK by PKC, which in turn is excited by phorbol esters or GPCR receptor agonists.

The present study demonstrates that icariin alleviates the maximal contraction elicited by vasoconstrictor fluorideB or phorbol ester, independently of the endothelium (Fig. 2, 4), by the MEK/ERK and RhoA/Rho-kinase pathway. Previously, vasodilation was believed to be caused by endothelial nitric oxide synthesis and the subsequent activation of guanylyl cyclase (Li et al. 2016). Li et al. (2016) reported icariin-induced endothelium-dependent relaxation and related pathway involving cGMP production, while in this study, icariin substantially alleviated phorbol ester- or fluorideB-elicited contraction regardless of endothelial function (Fig. 2, 4). Furthermore, icariin alleviated the phosphorylation of phorbol ester-evoked extracellular signal-regulated kinase (ERK) 1/2 (Fig. 6) and substantially that of fluoride-elicited MYPT1 at Thr855 (Fig. 5), with the relaxation (Fig. 2) suggesting the alleviation of MEK or Rho-kinase activity as a major mechanism.

MLC20 is known to be phosphorylated by both MLCK and Rho-kinase (Somlyo and Somlyo 1998). The activation of Rho-kinase by U46619 or fluoride inhibits the activity of myosin light chain phosphatase through the phosphorylation of MYPT1, leading to an enhanced MLC20 phosphorylation as well as contractions (Sakurada et al. 2003; Wilson et al. 2005).

Icariin substantially alleviates the contractions elicited by an MEK activator phorbol ester regardless of endothelial function and those by a Rho-kinase activator fluoride at this concentration. Thus, the mechanism underlying these relaxations involve the nonspecific restriction of MEK and Rho-kinase activities. Interestingly, during fluoride-elicited contraction, the alleviation of Rho-kinase activity and subsequent MYPT1 phosphorylation elicited by icariin suggest that Rho-kinase inactivation is needed for relaxation. In conclusion, in addition to endothelial nitric oxide synthesis (Li et al. 2016), both MEK and Rho-kinase inhibition are major contributors to the icariin-elicited vasorelaxation mechanism in the denuded muscle, and the reduction of intracellular calcium level, which should be elucidated directly, may be involved as well (Liu and Huang 2016).

Conflict of interest

No potential conflict of interest relevant to this article was reported.

Fig 1.

Figure 1.Chemical structure of icariin (7-(β-D-glucopyranosyloxy)-5-hydroxy-4′-methoxy-8-(3-methylbut-2-en-1-yl)-3-(α-L-rhamnopyranosyloxy)&#9;flavone).
Drug Targets and Therapeutics 2022; 1: 27-32https://doi.org/10.58502/DTT.22.006

Fig 2.

Figure 2.Effect of icariin on fluoride-elicited vasoconstriction in denuded (A) or intact (B) muscles. Each vessel was stabilized in the organ bath for 30-60 min before relaxation responses to icariin were assessed. Data are represented as the means of three to five experiments with vertical lines constituting SEMs.
Drug Targets and Therapeutics 2022; 1: 27-32https://doi.org/10.58502/DTT.22.006

Fig 3.

Figure 3.Effect of icariin on thromboxane mimetic-elicited vasoconstriction in denuded muscles. Each ring was stabilized in the organ bath for 30-60 min before relaxation responses to icariin were assessed. Data are represented as the means of three to five experiments with vertical lines constituting SEMs.
Drug Targets and Therapeutics 2022; 1: 27-32https://doi.org/10.58502/DTT.22.006

Fig 4.

Figure 4.Effect of icariin on phorbol ester-elicited vasoconstriction in denuded muscles. Each ring was stabilized in the organ bath for 30-60 min before relaxation responses to icariin were assessed. Data are represented as the means of three to five experiments with vertical lines constituting SEMs.
Drug Targets and Therapeutics 2022; 1: 27-32https://doi.org/10.58502/DTT.22.006

Fig 5.

Figure 5.Effect of icariin on fluoride-elicited augmentations in phospho-MYPT1 levels. Phospho-MYPT1 protein levels were significantly alleviated in quick frozen icariin-treated aorta in the absence of endothelium compared to vehicle-treated aorta precontracted with fluoride. Lower panel indicates average densitometric results for relative levels of phospho-MYPT1. Data are represented as the means of three to five experiments with vertical lines constituting SEMs. *p < 0.05, ##p < 0.01, versus control or normal group, respectively. Icariin: 0.1 mM icariin; fluoride: 6 mM sodium fluoride.
Drug Targets and Therapeutics 2022; 1: 27-32https://doi.org/10.58502/DTT.22.006

Fig 6.

Figure 6.Effect of icariin on phorbol ester-elicited augmentations in phospho-ERK1/2 levels (closed bar: pERK1; open bar: pERK2). Phospho-ERK1/2 protein levels were alleviated in quick frozen icariin-treated rat aortas in the absence of endothelium compared to vehicle-treated rat aortas contracted with phorbol ester. Lower panel indicates average densitometric results for relative levels of phospho-ERK1/2. Data are represented as the means of three to five experiments with vertical lines constituting SEMs. *p < 0.05, ##p < 0.01, versus control or normal group respectively. Icariin: 0.1 mM icariin; PDBu: phorbol 12,13-dibutyrate (1 μM).
Drug Targets and Therapeutics 2022; 1: 27-32https://doi.org/10.58502/DTT.22.006

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