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The Protective Effect of Total Flavones from Rhododendron Simsii Planch Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2018, Article ID 6139372, 13 pages https://doi.org/10.1155/2018/6139372 Research Article The Protective Effect of Total Flavones from Rhododendron simsii Planch. on Myocardial Ischemia/Reperfusion Injury and Its Underlying Mechanism Sheng-Yong Luo ,1,2 Qing-Hua Xu,1,2 Gong Peng ,3 and Zhi-Wu Chen 1 1 Department of Pharmacology, Anhui Medical University, Hefei, Anhui 230032, China 2Anhui Academy of Medical Sciences, Hefei, Anhui 230061, China 3Department of Pharmacy, Te First Afliated Hospital of Anhui Medical University, Hefei, Anhui 230032, China Correspondence should be addressed to Gong Peng; [email protected] and Zhi-Wu Chen; [email protected] Received 28 August 2017; Accepted 25 October 2017; Published 9 January 2018 Academic Editor: Christian Lehmann Copyright © 2018 Sheng-Yong Luo et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objectives. Total favones from Rhododendron simsii Planch. (TFR) are the efective part extracted from the fowers of Rhododendron simsii Planch. and have obvious protective efects against cerebral ischemic or myocardial injuries in rabbits and rats. However, their mechanism of cardioprotection is still unrevealed. Terefore, the present study was designed to investigate the efect of TFR on myocardial I/R injury and the underlying mechanism. Methods. TFR groups were treated by gavage once a day for 3 days at a dose of 20, 40, and 80 mg/kg, respectively, and then the model of myocardial I/R injury was established. Myocardial infarction, ST-segment elevation, and the expression of UTR, ROCK1, ROCK2, and p-MLC protein in rat myocardium were determined at 90 min afer reperfusion. UTR siRNA in vivo transfection and competition binding assay method were used to study the relationship between the protective efect of TFR and UTR. Results. Te expression of UTR protein markedly decreased in myocardium of UTR siRNA transfection group rats. TFR could signifcantly reduce the infarct size and inhibit the increase of RhoA activity and ROCK1, ROCK2, and p-MLC protein expressions both in WT and UTR knockdown rats. Te reducing rate of TFR in myocardial infarction area, RhoA activity, and ROCK1, ROCK2, and p-MLC protein expressions in UTR knockdown rats decreased markedly compared with that in WT rats. In addition, TFR had no obvious efect on the increase of ΣST in UTR knockdown rats in comparison with that 125 in model group. In particular, TFR could signifcantly inhibit the combination of [ I]-hu-II and UTR, and IC50 was 0.854 mg/l. Conclusions. Te results indicate that the protective efect of TFR on I/R injury may be correlated with its blocking UTR and the subsequent inhibition of RhoA/ROCK signaling pathway. 1. Introduction and myocardial infarction [9]. Furthermore, the serum human U-II concentration was positively correlated with the Urotensin-II (U-II) was initially isolated from the goby degree of myocardium injury in a rat myocardial infarction urophysis in the caudal portion of the teleost fsh. Subsequent model [10]. It was also found that RhoA/Rho-kinase (Rho- studies have shown that homologs of U-II were present in all associated coiled-coil-containing protein kinase, ROCK) sig- mammals acting as a vasoactive peptide [1]. Both U-II and naling pathway played an important role in a variety of itsreceptor(UTR),anorphanGprotein-coupledreceptor14, cardiovascular diseases [11]. Blocking this pathway could are widely expressed throughout the body, particularly in the inhibit the vascular tone and protect myocardial I/R injury cardiovascular system [2]. Large amount of data indicated [12, 13]. Moreover, some studies have proven the involvement that the level of plasma human U-II and tissue expression of RhoA/ROCK pathway in the human U-II-induced phys- levelsofhumanU-IIandUTRwereupregulatedinvarious iological and pathological processes, such as heart contrac- cardiovascular diseases such as ischemia [3], heart failure [4], tion, arterial smooth muscle cell proliferation, and cardiac atherosclerosis [5], hypertension [6, 7], chronic hypoxia [8], diastolic dysfunction [14]. Our previous study has shown that 2 Evidence-Based Complementary and Alternative Medicine � myocardial I/R injury may upregulate the UTR expression in negative control siRNA (sense, 5 -UUCUCCGAACGU- � � myocardium of rats and UTR antagonist (SB-710411) could GUCACGUTT-3 ;antisense,5-ACGUGACACGUUCGG- � signifcantly reduce the cardiac I/R injury via the inhibition AGAATT-3 ) were supplied by Shanghai GenePharma Co., of RhoA/ROCK signaling pathway [15], which points to the Ltd. (Shanghai, China). Entranster6-in vivo RNA transfec- fact that RhoA/ROCK pathway is also engaged in the human tion reagent was gained from Engreen Biosystem Co., Ltd. 125 U-II-induced myocardial I/R injury. Terefore, the hU-II/UT (Beijing, China). Na I was purchased from PerkinElmer receptor system might be a promising pharmacological target Co., Ltd. (Shanghai, China). in the treatment of ischemic heart disease. Flavonoids, which are efective ingredients in many Chi- 2.3. Animals. Male Sprague-Dawley rats weighing 250–300 g nese herbs and are commonly found in natural plants, exert were purchased from the Anhui Medical University Animal pharmacological functions in various biological activities, Center. All rats were housed in a room temperature of 22 ∘ including vasorelaxing and anti-infammatory efect as well ± 4 C and kept on a 12 hr light/dark cycle with free access as protective efect against myocardial I/R injury [16]. Rhodo- to food and water. Animal care and experimental protocols dendron simsii Planch. is a traditional Chinese medicine were approved by the committee on the Ethics of Animal thathasbeenusedintreatmentofbronchitisinChina Experiments of Anhui Medical University, in accordance with for thousands of years. Total favones from Rhododendron theGuidefortheCareandUseofLaboratoryAnimalsofthe simsii Planch. (TFR) are the efective part extracted from the National Institutes of Health (NIH Publication, 8th edition, fowers of Rhododendron simsii Planch. consisting of hyperin, 2011). quercetin, matteucinol, and rutin [17, 18]. Our previous studieshaveshownthatTFRhasobviousprotectiveefects 2.4. UTR siRNA In Vivo Transfection. To knockdown the against cerebral ischemic or myocardial injuries in rabbits UTR expression in rat heart, UTR siRNA in vivo transfection was performed by injecting a predesigned siRNA specifcally and rats [19, 20]. However, its mechanism of cardioprotection � � targeting UTR (5 -UGGCCUCCAUGUACGUCUATT-3 ; is still unrevealed. Tus, the present study was designed to � � 5 -UAGACGUACAUGGAGGCCATT-3 ). A scrambled investigate the efect of TFR on myocardial I/R injury and the � siRNA was used as a negative control (5 -UUCUCCGAA- underlying mechanism. Specifcally, the main focus is on the � � CGUGUCACGUTT-3 ;5-ACGUGACACGUUCGGAGA- relationship between TFR and UTR/RhoA/ROCK pathway. � ATT-3 ). Te UTR siRNA or scrambled siRNA (50 ug) was dilutedin25uLofEntranster-in vivo reagent and 10% glucose 2. Materials and Methods mixture according to the manufacturer’s instructions. Each 2.1. Sample Preparation. Rhododendron simsii Planch. was rat was intravenously injected with the siRNA mix (the fnal dried and then milled. Te total favonoids were extracted dose was 250 ug/ml) in a volume of 200 uL. Afer 72 h of UTR according to a previously reported method [21]. In brief, siRNA in vivo transfection, successful knockdown of UTR in air-dried aerial parts were inserted into a Soxhlet appara- rat heart was assessed by Western blot [22]. tus. Rhododendron simsii Planch. was exhaustively extracted sequentially with 250 mL petroleum ether, dichloromethane, 2.5. Experimental Protocol. Sprague-Dawley rats (WT and acetonitrile,ethylacetate,methanol,n-butanol,andH2O. UTR knockdown rats) were randomly divided into the Te organic extracts were dried over anhydrous magnesium following 7 groups: sham, model, nifedipine 5.4 mg/kg, SB- ∘ � sulfate, and the solvent was removed in vacuo (40 C). 710411 2.0 g/kg, and TFR 20, 40, and 80 mg/kg groups. Te ∘ Te water extract was concentrated in vacuo (40 C) and rats in nifedipine and SB-710411 groups were intravenously lyophilized. Te total favonoid extracts were dissolved in injected (i.v.) daily for 3 days at a dose of 5.4 mg/kg and 2.0 �g/kg, respectively, and TFR groups were treated by MeOH prior to analysis and the content was determined by gavageonceadayfor3daysatadoseof20,40,and80mg/kg, UV-spectrophotometry. respectively. 2.2. Reagents. SB-710411 underwent custom synthesis by GL 2.6. Surgical Procedures. Te surgical protocol was carried Biochem Ltd. (Shanghai, China). G-LISA RhoA Activation �� out in accordance with the previous method with slight Assay Biochemistry Kit was purchased from Cytoskeleton modifcations [23]. In brief, Sprague-Dawley rats were anaes- (Denver, USA). UTR antibody (1 : 200) was obtained from thetized with pentobarbital (40 mg/kg) by intraperitoneal WuXi AppTec (San Diego, USA); mouse monoclonal anti- injection. Te fourth and ffh ribs on the lef side of the chest bodies against ROCK1 and ROCK2 (1 : 1000) were bought were cut to perform the thoracotomy and the pericardium from Nanjing Enogene Biological Co. (Nanjing, China). was incised. Te heart was gently exteriorized and a 5/0 Mouse monoclonal antibodies against myosin light chain silk suture was placed around
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