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Protective effects of Astragalus polysaccharides against endothelial dysfunction in hypertrophic rats induced by isoproterenol
International Immunopharmacology 38 (2016) 306–312
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Protective effects of Astragalus polysaccharides against endothelial dysfunction in hypertrophic rats induced by isoproterenol☆ Ronghui Han 1, Futian Tang 1, Meili Lu, Chonghua Xu, Jin Hu, Meng Mei, Hongxin Wang ⁎ Key Laboratory of Cardiovascular and Cerebrovascular Drug Research of Liaoning Province, Drug Research Institute, Jinzhou Medical University, No. 40, Section 3, Songpo Road, Jinzhou 121001, PR China
a r t i c l e
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Article history: Received 8 February 2016 Received in revised form 22 May 2016 Accepted 17 June 2016 Available online xxxx Keywords: Astragalus polysaccharide Cardiac hypertrophy Endothelial dysfunction cGMP Isoproterenol
a b s t r a c t Astragalus polysaccharide (APS) is an important bioactive component extracted from Chinese herb Astragalus membranaceus. It has been widely used in treatment of cardiovascular diseases. We have previously reported that APS could inhibit isoproterenol-induced cardiac hypertrophy. The present study was designed to evaluate the protective effect of APS on vascular endothelia in cardiac hypertrophy rats induced by isoproterenol (ISO). ISO (10 mg × kg−1) was intraperitoneally injected once daily for 2 weeks to induce cardiac hypertrophy. APS (400 and 800 mg × kg−1) was intragastrically injected once daily along with ISO. The results showed that combination with APS significantly ameliorates the endothelial dysfunction while attenuates cardiac hypertrophy induced by ISO. We found that administration with APS could attenuate the increase in number of circulating endothelial cell (CEC). APS also decreases the superoxide anion generation and the protein expression of p65 and the levels of TNF-α and IL-6; while increases the cGMP levels, an activity marker for nitric oxide (NO) in aortas. In addition, APS improves the relaxation dysfunction in isolated aortic rings and increases the protein expression of IκBα and Cu/Zn-SOD in aortas. In conclusion, our results suggested that APS had a protective effect against endothelial dysfunction in hypertrophic rats induced by ISO. The underlining mechanisms may be contributed to the anti-inflammatory effects and the improvement of the imbalance between reactive oxygen species (ROS) and NO. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cardiac hypertrophy is considered as a compensatory response to various kinds of physiological and pathological conditions. It has been recognized as an independent predictor to a variety of cardiovascular diseases such as hypertension [1], diabetes [2], atherosclerosis [3], chronic kidney disease [4], as well as in general population [5]. It has been reported that endothelial dysfunction of conduit or resistance arteries is often concurrent with myocardial hypertrophy in hypertensive model rats [6–8], angiotensin II-infused model rats [9] and model of swines with aortic banding [10]. The association between cardiac hypertrophy and impaired endothelial function has also been reported in humans of chronic kidney disease [11], atherosclerosis [3] and nevertreated hypertensive patients [12]. Abbreviations: APS, Astragalus polysaccharide; ROS, reactive oxygen species; ISO, isoproterenol; CEC, circulating endothelial cell. ☆ This work was supported by National Natural Science Foundation of China (81374008). ⁎ Corresponding author at: Department of Pharmacology, Jinzhou Medical University, No. 40, Section 3, Songpo Road, Jinzhou City, Liaoning 121001, PR China. E-mail addresses: [email protected] (R. Han), [email protected] (H. Wang). 1 Both authors are equally contributed to this work.
http://dx.doi.org/10.1016/j.intimp.2016.06.014 1567-5769/© 2016 Elsevier B.V. All rights reserved.
Persistent stimulation of catecholamines to adrenoceptors induced by an increased sympathetic tone in a variety of pathological conditions has been reported to be associated with many cardiovascular diseases, such as essential hypertension, cardiac hypertrophy and heart failure [13,14]. Although the cardiac hypertrophy induced by ISO is a model without hypertension [15] or significantly changes of arterial and ventricular hemodynamic parameters [16], it has been reported that sustained stimulation of ISO could also result in injury to the endothelial function [17]. It is known that myocardial hypertrophy is often accompanied by inflammatory responses [18]. Although the association between inflammation, endothelial dysfunction, and cardiac hypertrophy has not been examined systematically in rats induced by ISO, the association between systemic inflammation and endothelial dysfunction has been reported [19]. And the inflammation was also reported to be one of the most potential trigger to endothelial dysfunction [20]. It was reported that oxidative stress and inflammation seems to play key roles in endothelial dysfunction in the rats model induced by ISO [16]. Astragalus polysaccharide (APS) is an important bioactive ingredient obtained from traditional Chinese herb Astragalus membranaceus that has a range of activities in many pharmacological effects, including the increase of cGMP and cAMP levels in plasma and tissues, promotion of
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immune responses, anti-inflammation, protection of vessels, antioxidant effects, anti-insulin resistance and anti-tumor [21–25]. We have previously reported that APS could inhibit isoproterenol-induced cardiac hypertrophy [26,27]. However, the effects of Astragalus polysaccharide to endothelial dysfunction in hypertrophic rats induced by isoproterenol remains unclear.
2. Materials Astragalus polysaccharide(APS)was purchased from Nanjing Jing Zhu Biological Technology Co.(purity N98%; Nanjing, China). Isoproterenol, ADP were purchased from Sigma(St. Louis, USA). Hydroethidine was obtained from Shanghai Hao Ran Biological Technology Co., Ltd. (Shanghai, China). The Enzyme-linked immunosorbent assay (ELISA) kits for rat TNF-α and IL-6 were obtained from R&D Systems (Minneapolis, MN, USA). cGMP assay kit was supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Rabbit anti-rat factor VIII serum, antibody of IκBα, Cu/Zn-SOD, were purchased from Beijing Boosen Biological Technology Co., Ltd. (Beijing, China). FITC-conjugated goat anti-rabbit IgG fluorescent antibody and antibody of p65 (Proteintech Group, USA). Healthy male Sprague-Dawley rats aged at 4–6 weeks (200 ± 20 g) were supplied by the experimental animal center of Liaoning Medical University, certificate number: SCXK (Liao) 2009-0004), for the current study. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Liaoning Medical University (Permit Number: LMU-2014-118), China. 3. Methods 3.1. Experimental design Forty SD rats were randomly divided into the following four groups (n = 10) A: the control group; B: the ISO model group, rats received ISO injections (10 mg × kg−1 × day−1,i.p.); C: ISO + APS 400 mg × kg−1 ×day−1 group, rats received ISO injections and APS (400 mg × kg−1 ×day− 1, i.g); D: ISO + APS800 mg × kg−1 × day− 1 group, rats received ISO injections and APS (800 mg × kg−1 × day−1, i.g). All administered rats in C and D groups were pre-treated with APS for 1 day before Iso injection. After this, the rats were intraperitoneally injected with ISO for 2 weeks. The rats of control group were treated with the same volume of physiological saline (i.p.). The criteria for selection of the doses of APS and ISO in rats were based on the combination of our preliminary experiment and previous report [26,28,29]. Rats were housed in a room with a constant room temperature (25.0 °C ± 0.2 °C) in a Specific Pathogen Free laboratory, with a 12-h/ 12-h light/dark light cycle and 50% humidity. The rats were allowed free access to food and water ad libitum. 3.2. Parameters of heart weight assay At the end of the treatment, all animals were anaesthetized with a 20% urethane (0.5 ml/100 g, i.p.). Then all animals were killed by complete collection of the blood through right carotid cannula and the heart were immediately harvested, rinsed in ice-cold phosphate buffer solution (PBS). The blood were collected to anticoagulant tube which was ice-cold and added with heparin for further experiments and plasma was obtained by centrifugation, aliquoted and stored at −20 °C. The isolated heart was used for ventricular weight measurement. The heart weight index (HMI = HW/BW), the left ventricle weight index (LVMI = LVW/BW) were calculated separately. The aorta was isolated for vascular function experiments, ethidium fluorescence analysis and protein expression. Except for vascular reactivity experiments, the aorta was kept frozen (−80 °C) for further experiments.
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3.3. Circulating endothelial cell counting The circulating endothelial cell (CEC) counting assessment was made as previously described [30]. In brief, the blood samples were centrifuged at 4 °C for 20 min, at 395 ×g, and then the supernatants were collected. After this, the ADP (Sigma; 1 mg/ml solution) was added at a ratio of 5:1. After centrifuging the mixture at 4 °C for 20 min at 395 ×g, the platelet aggregates were removed. The circulating vascular endothelial cell precipitates were obtained by centrifuging the supernatants at 4 °C for 20 min at 2100 ×g. Then, 0.1 ml of 0.9% NaCl was added to prepare cell suspension. The numbers of cell were counted in a hemocytometer under a microscope (Olympus). A fraction suspension was placed on glass slides and equilibrated for 30 min at 37 °C with rabbit anti-rat factor VIII serum and FITC-conjugated goat anti-rabbit IgG fluorescent antibody after fixed with methanol to identify the CEC [31].
3.4. Assessment of vascular function According to the method described in reference [16,17], the vascular function of control group, ISO-treated model group and ISO + APS 800 mg kg−1 day−1 group were assessed in isolated aortic rings. Briefly, the aortas were dissected out and cleaned of connective tissue. The relaxations of ring preparations (4 mm length) of isolated aorta to acetylcholine were measured in organ baths in a modified Krebs solution (in mM: NaCl 122, KCl 5.9, NaHCO3 15, glucose 11, CaCl2 1.25, MgCl2 1.2 and ethylenediamine-tetraacetic acid (EDTA) 0.01). Relaxations to acetylcholine were measured in norepinephrine-precontracted (10−6 mol/l) aortic rings. The acetylcholine were added cumulatively to final concentrations of 10−10 to 10−5 mol/l.
3.5. The ROS production measurement in aorta by ethidium fluorescence analysis The production of ROS in aorta was measured by using dihydroethidium (DHE) staining as described previously [10,17]. Hydroethidine, an oxidative fluorescent dye, can be oxidized to ethidium bromidein and trapped by intercalation into DNA in the presence of superoxide anions. The aortic sections (14 mm) were obtained on a cryostat with freezing microtome from previously frozen aorta and collected on glass slides. After this, the tissue sections were equilibrated for 30 min at 37 °C in phosphate buffered saline (PBS) buffer. Then the sections were incubated with 2 mM hydroethidine for 30 min at 37 °C in a light-protected, humidified chamber. The images were obtained and analyzed by inverted fluorescent microscopy.
3.6. Enzyme-linked immunosorbent assay (ELISA) The plasma was used to measure the levels of TNF-α and IL-6 with commercially available ELISA kits according to the manufacturer's protocol.
3.7. Measurement of the intracellular cGMP The cGMP levels was detected with an assay kit according to the manufacturer's protocol. The Frozen rat aortas were ultrasonic grinded. The homogenate was prepared in PBS and centrifuged at 1000 × g for 5 min at 4 °C. cGMP level in the supernatant was determined according to the guidelines of the kit. Values of cGMP level were expressed as micromole per grams of protein. Protein concentration was measured with BCA protein assay kit (Beijing Dingguo biotechnology limited company) and BSA as standards. cGMP is a second messenger of NO, the level of which reflects the level of NO in organisms [32].
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3.8. Western blot analysis Frozen rat aortas were homogenized with ice-cold RIPA extraction buffer for protein extraction. Then, the protein concentration was quantified using a bicinchoninic acid protein assay (BCA). Protein extracts (30 μg) of aortas were electrophoretically separated by 12% or 10% SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane. The membranes were blocked with 5% nonfat dry milk and then incubated overnight at 4 °C with the following primary antibodies: antiIκBα (1:300 dilution), anti-p65(1:500 dilution), anti-Cu/Zn-SOD (1:400 dilution). After being washed with TBST(10 mM Tris, 100 mM NaCl, and 0.1% Tween 20), membranes were incubated horseradishperoxidase (HRP)-conjugated secondary antibodies (1:2000 dilution) for 2 h at room temperature. Then the membranes were thoroughly washed, and immunocomplexes were visualized using an ECL system. In the same membrane, the expression of β-actin was determined and used as an internal control for the experiments. 3.9. Statistical analysis All data were expressed as the mean ± SD. Each experiment was performed at least 3 times. Statistical analysis was performed using Student's t-test or one-way ANOVA with SPSS software. P b 0.05 was considered statistically significant. 4. Results 4.1. APS attenuates ISO induced myocardial hypertrophy in rats We first measured the parameters of heart weight to investigate whether the hypertrophic model was successfully made. As shown in Fig. 1, compared with the control group, in ISO model group HMI, LVMI increased by 38.41%, 44.91% (P b 0.01) which indicated that the hypertrophic model was successfully made. The results also showed that in the APS administrated groups HMI and LVMI decreased significantly compared with ISO group, which indicated that the APS had cardiac protective effects. The result is consistent with the previous studies [26]. 4.2. APS reduced the number of CEC in hypertrophy rats induced by ISO The increasing number of CEC has been reported to be an biomarker of endothelial dysfunction. As shown in Fig. 2, the number of CEC in ISO model group increased significantly compared with the control group which indicated that there were obvious endothelial injury in the cardiac hypertrophy rats induced by ISO. However, the CEC number of APS treated groups decreased compared with the ISO model group which
indicated that APS had protective effects to the endothelial function of cardiac hypertrophy rats induced by ISO. 4.3. APS improved the endothelium-dependent relaxation function in isolated aortic rings We also evaluated the endothelium-dependent relaxation function in isolated aortic rings. It has been reported that the vascular endothelial integrity could be tested by measuring the diastolic level with 10−6 M acetylcholine to norepinephrine-precontracted (10−6 M) aortic rings. The endothelial function was regarded to be injured when the diastolic level was lower than 10%. As shown in Fig. 3, the endothelial function of ISO model group was impaired obviously in comparison with that of control group. And the impairments were reduced in APS administrated groups compared with that of ISO model group. 4.4. APS decreased the superoxide anion production in aortas To investigate the potential mechanisms of APS, we measured the production of ROS by using dihydroethidium (DHE) staining. As shown in Fig. 4, an increase of production of superoxide anion was observed in aortas from ISO-treated rats as compared with control rats which indicated an increased superoxide anion generation. This increase could be significantly reduced in APS administrated groups. 4.5. APS reduced the levels of TNF-α and IL-6 in plasma It is known that inflammation is one of the most potential trigger to both myocardial hypertrophy and endothelial dysfunction. We measured the levels of TNF-α and IL-6 with ELISA kits. As shown in Fig. 5, The inflammatory cytokines levels of TNF-α and IL-6 in ISO model group were much higher than that of the control. Compared with the ISO model group, the levels of TNF-α and IL-6 in APS administrated groups were significantly decreased. 4.6. APS increased the cGMP levels in aortics To further investigate the protective effects of APS, we measured the influence of APS on cGMP accumulation. As shown in Fig. 6, in the ISO model group an decrease of the cGMP accumulation was observed in aortas which indicated that the bioavailability of NO was reduced. However, the levels of cGMP in APS administrated groups could be significantly increased compared with that of ISO model group. 4.7. APS regulated the expressions of IκBα, p65 and Cu/Zn-SOD in aortas The expression of IκBα, p65 and Cu/Zn-SOD were examined to explore the potential molecular mechanisms contributing to the protective effects of APS. As shown in Fig. 7, the expression of IκBα and Cu/ Zn-SOD in the Iso model group were decreased and the protein expression of p65 were increased in comparison with that of control group. However, the APS administrated could significantly attenuate the decrease of IκBα and Cu/Zn-SOD expression and the increase of p65 protein expression which may contribute to the APS-mediated protective effects of HUVECs. 5. Discussion
Fig. 1. Effects of Astragalus polysaccharide (APS) on heart mass index (HMI) and left ventricular mass index (LVMI) in myocardial hypertrophy rats induced by ISO. The data were expressed as the mean ± SD, n = 10, **P b 0.01 vs. control. ##P b 0.01 vs. ISO model group.
The aim of this study was to assess the protective effects of APS against endothelial dysfunction in hypertrophy rats induced by ISO. Our results suggests that APS could ameliorate the endothelial function while attenuates cardiac hypertrophy induced by ISO. Endothelial function plays an important role in vascular pathophysiology and is considered to be a biomarker/mediator of cardiovascular risk factors [33,34]. Endothelial dysfunction has also been known to be a predictor of outcomes in different settings of patients [35–38]. The
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Fig. 2. Representative images of CEC (×400): (A): control group; (B): ISO model group; (C): ISO + APS 400 mg × kg−1 × day−1group; (D): ISO + APS 800 mg × kg−1 × day−1 group; (E): Statistical results of APS on the number of CEC in hypertrophy rats induced by ISO. Data were expressed as the mean ± SD, n = 5, **P b 0.01 vs. control. ##P b 0.01 vs. ISO model group.
association between endothelial dysfunction and cardiac hypertrophy has been examined in many previously published experimental data [1,11]. Although the association between endothelial dysfunction and cardiac hypertrophy has not been examined systematically in animal
Fig. 3. Effects of APS on the endothelium-dependent relaxation function of isolated aortic rings in hypertrophy rats induced by ISO. The vascular tension were expressed as percentage of precontraction induced by norepinephrine(10−6 M) (n = 5).
model induced by ISO, the injury of endothelial function has been found in myocardial hypertrophy rats induced by ISO as examined by circulating endothelial cell counting and assessment of vascular relaxation function in this study. This result is also in agreement with the previous study [17]. Astragalus polysaccharide (APS) is an important bioactive ingredient obtained from Astragalus membranaceus which has a range of activities in many pharmacological effects. It has been widely used in treatment of cardiovascular diseases. APS is a polysaccharide component. It consists of D-glucose, D-galactose, rhamnose, arabinose and L-arabinose and there are also reports indicating that APS was safe without any distinct toxicity and side effects, the safety dosage range is 5.7–39.9 g/kg for rats [39]. APS was orally administered to animals in many studies and found to be effective in treating many diseases [25,40–42]. We have also previously reported that APS could inhibit ISO-induced cardiac hypertrophy [26,27]. It has also been reported that Astragalus membranaceus and its main components including APS could potently ameliorate endothelial dysfunction induced by homocysteine in which anti-oxidation played a key role [43]. Nevertheless, the effects of APS on endothelial injury in hypertrophy rats induced by ISO are limited and mechanism has not yet been elucidated. Hence, we conducted this experiment to
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Fig. 4. Effects of APS on the ROS production in aortas of hypertrophy rats induced by ISO. Representative ethidium fluorescence images (×400):(A): control group; (B): ISO model group; (C): ISO + APS 400 mg × kg−1 × day−1 group; (D): ISO + APS 800 mg × kg−1 × day−1 group; (E): Statistical results of APS on the ROS production represented as relative fluorescence intensity. Data were expressed as the mean ± SD, n = 5, **P b 0.01 vs. control. ##P b 0.01 vs. ISO model group.
investigate the protective effects of APS against endothelial dysfunction in hypertrophy rats induced by ISO. It is known that myocardial hypertrophy is often accompanied by inflammatory responses. In this study we measured the inflammatory cytokines levels of TNF-α and IL-6 with ELISA and examined the inflammation associated protein expression of IκBα and p65. As shown in Figs. 5 and 7, the levels of TNF-α and IL-6 in APS administrated groups could be significantly decreased in comparison with that of ISO model group. APS could also attenuate the decrease of IκBα and the increase of p65 protein expression. It has also been reported that the associations have been made between endothelial dysfunction and systemic inflammation [44]. Hence, the protective effects of APS to endothelial function while attenuates cardiac hypertrophy induced by ISO may be related to its anti-inflammatory effects.
Nitric oxide (NO), an endothelium-dependent vasodilator, is an important mediator in the regulation of endothelial cell functions. Reactive oxygen species (ROS) are free radicals found in all vascular cells which can inactivate NO and decrease NO bioavailability in blood vessels and lead to the injury of endothelial functions. It has also been reported that the reduced formation of NO or impairment of nitric oxide effect may be associated with aortic sclerosis [45]. Studies also suggest that the imbalance between ROS and NO levels, rather than the individual levels, can be a major cause of endothelial dysfunction in numerous cardiovascular diseases. In this study we detected the intracellular cGMP levels to evaluate the bioavailability of NO which is the second messenger of NO. Our results suggest that APS could potently increase the levels
Fig. 5. Effects of APS on the levels of TNF-α and IL-6in hypertrophy rats induced by ISO. Data were expressed as the mean ± SD, n = 4, **P b 0.01 vs. control. ##P b 0.01 vs. ISO model group.
Fig. 6. APS restored the decrease of cGMP contents in aortas of hypertrophy rats induced by ISO. Data were expressed as the mean ± SD, n = 4, **P b 0.01 vs. control. ##P b 0.01 vs. ISO model group.
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Fig. 7. Effect of APS on expressions of IκBα, p65 and Cu/Zn-SOD in aortas of hypertrophy rats induced by ISO. (a): control group; (b): ISO model group; (c): ISO + APS 800 mg × kg−1 × day−1 group; (d): ISO + APS 400 mg × kg−1 × day−1 group; (B):Statistical results of protein expression are represented as ratio to β-actin. Data were expressed as the mean ± SD, n = 5, **P b 0.01 vs. control. ##P b 0.01 vs. ISO model group.
of cGMP as shown in Fig. 6, which may contribute to the APS-mediated protective effects to endothelial functions. It was reported that oxidative stress and inflammation seems to play key roles in endothelial dysfunction in the rats model induced by ISO [16]. Numerous observations also suggest that ROS plays an essential role in vascular remodeling and endothelial dysfunction [46]. There are researches suggest that the endothelial cells express virtually a variety of enzymes from which ROS can be generated. Thus, the endothelial cells are regarded as an important source of vascular ROS production [47]. In this study we measured the ROS production with DHE staining and evaluated the antioxidant capacity with examined the protein expression of Cu/Zn-SOD. As shown in Figs. 4 and 7, APS could significantly reduced the production of ROS and increase the Cu/Zn-SOD expression. In conclusion, our studies showed that APS had a protective effect against endothelial dysfunction in hypertrophy rats induced by ISO. The underlining mechanisms of the protection are attributed to the anti-inflammatory effects and the improvement of the imbalance between ROS and NO levels through increasing the cell antioxidant capacity and NO bioavailability. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by National Natural Science Foundation of China (81374008). References [1] F. Perticone, R. Maio, M. Perticone, S. Miceli, A. Sciacqua, E.J. Tassone, et al., Endothelial dysfunction predicts regression of hypertensive cardiac mass, Int. J. Cardiol. 167 (2013) 1188–1192. [2] A. Ilercil, R.B. Devereux, M.J. Roman, M. Paranicas, M.J. O'Grady, T.K. Welty, et al., Relationship of impaired glucose tolerance to left ventricular structure and function: the Strong Heart Study, Am. Heart J. 141 (2001) 992–998. [3] J. Yeboah, J.R. Crouse, D.A. Bluemke, J.A. Lima, J.F. Polak, G.L. Burke, et al., Endothelial dysfunction is associated with left ventricular mass (assessed using MRI) in an adult population (MESA), J. Hum. Hypertens. 25 (2011) 25–31.
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Protective effects of Astragalus polysaccharides against endothelial dysfunction in hypertrophic rats induced by isoproterenol☆ Ronghui Han 1, Futian Tang 1, Meili Lu, Chonghua Xu, Jin Hu, Meng Mei, Hongxin Wang ⁎ Key Laboratory of Cardiovascular and Cerebrovascular Drug Research of Liaoning Province, Drug Research Institute, Jinzhou Medical University, No. 40, Section 3, Songpo Road, Jinzhou 121001, PR China
a r t i c l e
i n f o
Article history: Received 8 February 2016 Received in revised form 22 May 2016 Accepted 17 June 2016 Available online xxxx Keywords: Astragalus polysaccharide Cardiac hypertrophy Endothelial dysfunction cGMP Isoproterenol
a b s t r a c t Astragalus polysaccharide (APS) is an important bioactive component extracted from Chinese herb Astragalus membranaceus. It has been widely used in treatment of cardiovascular diseases. We have previously reported that APS could inhibit isoproterenol-induced cardiac hypertrophy. The present study was designed to evaluate the protective effect of APS on vascular endothelia in cardiac hypertrophy rats induced by isoproterenol (ISO). ISO (10 mg × kg−1) was intraperitoneally injected once daily for 2 weeks to induce cardiac hypertrophy. APS (400 and 800 mg × kg−1) was intragastrically injected once daily along with ISO. The results showed that combination with APS significantly ameliorates the endothelial dysfunction while attenuates cardiac hypertrophy induced by ISO. We found that administration with APS could attenuate the increase in number of circulating endothelial cell (CEC). APS also decreases the superoxide anion generation and the protein expression of p65 and the levels of TNF-α and IL-6; while increases the cGMP levels, an activity marker for nitric oxide (NO) in aortas. In addition, APS improves the relaxation dysfunction in isolated aortic rings and increases the protein expression of IκBα and Cu/Zn-SOD in aortas. In conclusion, our results suggested that APS had a protective effect against endothelial dysfunction in hypertrophic rats induced by ISO. The underlining mechanisms may be contributed to the anti-inflammatory effects and the improvement of the imbalance between reactive oxygen species (ROS) and NO. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cardiac hypertrophy is considered as a compensatory response to various kinds of physiological and pathological conditions. It has been recognized as an independent predictor to a variety of cardiovascular diseases such as hypertension [1], diabetes [2], atherosclerosis [3], chronic kidney disease [4], as well as in general population [5]. It has been reported that endothelial dysfunction of conduit or resistance arteries is often concurrent with myocardial hypertrophy in hypertensive model rats [6–8], angiotensin II-infused model rats [9] and model of swines with aortic banding [10]. The association between cardiac hypertrophy and impaired endothelial function has also been reported in humans of chronic kidney disease [11], atherosclerosis [3] and nevertreated hypertensive patients [12]. Abbreviations: APS, Astragalus polysaccharide; ROS, reactive oxygen species; ISO, isoproterenol; CEC, circulating endothelial cell. ☆ This work was supported by National Natural Science Foundation of China (81374008). ⁎ Corresponding author at: Department of Pharmacology, Jinzhou Medical University, No. 40, Section 3, Songpo Road, Jinzhou City, Liaoning 121001, PR China. E-mail addresses: [email protected] (R. Han), [email protected] (H. Wang). 1 Both authors are equally contributed to this work.
http://dx.doi.org/10.1016/j.intimp.2016.06.014 1567-5769/© 2016 Elsevier B.V. All rights reserved.
Persistent stimulation of catecholamines to adrenoceptors induced by an increased sympathetic tone in a variety of pathological conditions has been reported to be associated with many cardiovascular diseases, such as essential hypertension, cardiac hypertrophy and heart failure [13,14]. Although the cardiac hypertrophy induced by ISO is a model without hypertension [15] or significantly changes of arterial and ventricular hemodynamic parameters [16], it has been reported that sustained stimulation of ISO could also result in injury to the endothelial function [17]. It is known that myocardial hypertrophy is often accompanied by inflammatory responses [18]. Although the association between inflammation, endothelial dysfunction, and cardiac hypertrophy has not been examined systematically in rats induced by ISO, the association between systemic inflammation and endothelial dysfunction has been reported [19]. And the inflammation was also reported to be one of the most potential trigger to endothelial dysfunction [20]. It was reported that oxidative stress and inflammation seems to play key roles in endothelial dysfunction in the rats model induced by ISO [16]. Astragalus polysaccharide (APS) is an important bioactive ingredient obtained from traditional Chinese herb Astragalus membranaceus that has a range of activities in many pharmacological effects, including the increase of cGMP and cAMP levels in plasma and tissues, promotion of
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immune responses, anti-inflammation, protection of vessels, antioxidant effects, anti-insulin resistance and anti-tumor [21–25]. We have previously reported that APS could inhibit isoproterenol-induced cardiac hypertrophy [26,27]. However, the effects of Astragalus polysaccharide to endothelial dysfunction in hypertrophic rats induced by isoproterenol remains unclear.
2. Materials Astragalus polysaccharide(APS)was purchased from Nanjing Jing Zhu Biological Technology Co.(purity N98%; Nanjing, China). Isoproterenol, ADP were purchased from Sigma(St. Louis, USA). Hydroethidine was obtained from Shanghai Hao Ran Biological Technology Co., Ltd. (Shanghai, China). The Enzyme-linked immunosorbent assay (ELISA) kits for rat TNF-α and IL-6 were obtained from R&D Systems (Minneapolis, MN, USA). cGMP assay kit was supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Rabbit anti-rat factor VIII serum, antibody of IκBα, Cu/Zn-SOD, were purchased from Beijing Boosen Biological Technology Co., Ltd. (Beijing, China). FITC-conjugated goat anti-rabbit IgG fluorescent antibody and antibody of p65 (Proteintech Group, USA). Healthy male Sprague-Dawley rats aged at 4–6 weeks (200 ± 20 g) were supplied by the experimental animal center of Liaoning Medical University, certificate number: SCXK (Liao) 2009-0004), for the current study. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Liaoning Medical University (Permit Number: LMU-2014-118), China. 3. Methods 3.1. Experimental design Forty SD rats were randomly divided into the following four groups (n = 10) A: the control group; B: the ISO model group, rats received ISO injections (10 mg × kg−1 × day−1,i.p.); C: ISO + APS 400 mg × kg−1 ×day−1 group, rats received ISO injections and APS (400 mg × kg−1 ×day− 1, i.g); D: ISO + APS800 mg × kg−1 × day− 1 group, rats received ISO injections and APS (800 mg × kg−1 × day−1, i.g). All administered rats in C and D groups were pre-treated with APS for 1 day before Iso injection. After this, the rats were intraperitoneally injected with ISO for 2 weeks. The rats of control group were treated with the same volume of physiological saline (i.p.). The criteria for selection of the doses of APS and ISO in rats were based on the combination of our preliminary experiment and previous report [26,28,29]. Rats were housed in a room with a constant room temperature (25.0 °C ± 0.2 °C) in a Specific Pathogen Free laboratory, with a 12-h/ 12-h light/dark light cycle and 50% humidity. The rats were allowed free access to food and water ad libitum. 3.2. Parameters of heart weight assay At the end of the treatment, all animals were anaesthetized with a 20% urethane (0.5 ml/100 g, i.p.). Then all animals were killed by complete collection of the blood through right carotid cannula and the heart were immediately harvested, rinsed in ice-cold phosphate buffer solution (PBS). The blood were collected to anticoagulant tube which was ice-cold and added with heparin for further experiments and plasma was obtained by centrifugation, aliquoted and stored at −20 °C. The isolated heart was used for ventricular weight measurement. The heart weight index (HMI = HW/BW), the left ventricle weight index (LVMI = LVW/BW) were calculated separately. The aorta was isolated for vascular function experiments, ethidium fluorescence analysis and protein expression. Except for vascular reactivity experiments, the aorta was kept frozen (−80 °C) for further experiments.
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3.3. Circulating endothelial cell counting The circulating endothelial cell (CEC) counting assessment was made as previously described [30]. In brief, the blood samples were centrifuged at 4 °C for 20 min, at 395 ×g, and then the supernatants were collected. After this, the ADP (Sigma; 1 mg/ml solution) was added at a ratio of 5:1. After centrifuging the mixture at 4 °C for 20 min at 395 ×g, the platelet aggregates were removed. The circulating vascular endothelial cell precipitates were obtained by centrifuging the supernatants at 4 °C for 20 min at 2100 ×g. Then, 0.1 ml of 0.9% NaCl was added to prepare cell suspension. The numbers of cell were counted in a hemocytometer under a microscope (Olympus). A fraction suspension was placed on glass slides and equilibrated for 30 min at 37 °C with rabbit anti-rat factor VIII serum and FITC-conjugated goat anti-rabbit IgG fluorescent antibody after fixed with methanol to identify the CEC [31].
3.4. Assessment of vascular function According to the method described in reference [16,17], the vascular function of control group, ISO-treated model group and ISO + APS 800 mg kg−1 day−1 group were assessed in isolated aortic rings. Briefly, the aortas were dissected out and cleaned of connective tissue. The relaxations of ring preparations (4 mm length) of isolated aorta to acetylcholine were measured in organ baths in a modified Krebs solution (in mM: NaCl 122, KCl 5.9, NaHCO3 15, glucose 11, CaCl2 1.25, MgCl2 1.2 and ethylenediamine-tetraacetic acid (EDTA) 0.01). Relaxations to acetylcholine were measured in norepinephrine-precontracted (10−6 mol/l) aortic rings. The acetylcholine were added cumulatively to final concentrations of 10−10 to 10−5 mol/l.
3.5. The ROS production measurement in aorta by ethidium fluorescence analysis The production of ROS in aorta was measured by using dihydroethidium (DHE) staining as described previously [10,17]. Hydroethidine, an oxidative fluorescent dye, can be oxidized to ethidium bromidein and trapped by intercalation into DNA in the presence of superoxide anions. The aortic sections (14 mm) were obtained on a cryostat with freezing microtome from previously frozen aorta and collected on glass slides. After this, the tissue sections were equilibrated for 30 min at 37 °C in phosphate buffered saline (PBS) buffer. Then the sections were incubated with 2 mM hydroethidine for 30 min at 37 °C in a light-protected, humidified chamber. The images were obtained and analyzed by inverted fluorescent microscopy.
3.6. Enzyme-linked immunosorbent assay (ELISA) The plasma was used to measure the levels of TNF-α and IL-6 with commercially available ELISA kits according to the manufacturer's protocol.
3.7. Measurement of the intracellular cGMP The cGMP levels was detected with an assay kit according to the manufacturer's protocol. The Frozen rat aortas were ultrasonic grinded. The homogenate was prepared in PBS and centrifuged at 1000 × g for 5 min at 4 °C. cGMP level in the supernatant was determined according to the guidelines of the kit. Values of cGMP level were expressed as micromole per grams of protein. Protein concentration was measured with BCA protein assay kit (Beijing Dingguo biotechnology limited company) and BSA as standards. cGMP is a second messenger of NO, the level of which reflects the level of NO in organisms [32].
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3.8. Western blot analysis Frozen rat aortas were homogenized with ice-cold RIPA extraction buffer for protein extraction. Then, the protein concentration was quantified using a bicinchoninic acid protein assay (BCA). Protein extracts (30 μg) of aortas were electrophoretically separated by 12% or 10% SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane. The membranes were blocked with 5% nonfat dry milk and then incubated overnight at 4 °C with the following primary antibodies: antiIκBα (1:300 dilution), anti-p65(1:500 dilution), anti-Cu/Zn-SOD (1:400 dilution). After being washed with TBST(10 mM Tris, 100 mM NaCl, and 0.1% Tween 20), membranes were incubated horseradishperoxidase (HRP)-conjugated secondary antibodies (1:2000 dilution) for 2 h at room temperature. Then the membranes were thoroughly washed, and immunocomplexes were visualized using an ECL system. In the same membrane, the expression of β-actin was determined and used as an internal control for the experiments. 3.9. Statistical analysis All data were expressed as the mean ± SD. Each experiment was performed at least 3 times. Statistical analysis was performed using Student's t-test or one-way ANOVA with SPSS software. P b 0.05 was considered statistically significant. 4. Results 4.1. APS attenuates ISO induced myocardial hypertrophy in rats We first measured the parameters of heart weight to investigate whether the hypertrophic model was successfully made. As shown in Fig. 1, compared with the control group, in ISO model group HMI, LVMI increased by 38.41%, 44.91% (P b 0.01) which indicated that the hypertrophic model was successfully made. The results also showed that in the APS administrated groups HMI and LVMI decreased significantly compared with ISO group, which indicated that the APS had cardiac protective effects. The result is consistent with the previous studies [26]. 4.2. APS reduced the number of CEC in hypertrophy rats induced by ISO The increasing number of CEC has been reported to be an biomarker of endothelial dysfunction. As shown in Fig. 2, the number of CEC in ISO model group increased significantly compared with the control group which indicated that there were obvious endothelial injury in the cardiac hypertrophy rats induced by ISO. However, the CEC number of APS treated groups decreased compared with the ISO model group which
indicated that APS had protective effects to the endothelial function of cardiac hypertrophy rats induced by ISO. 4.3. APS improved the endothelium-dependent relaxation function in isolated aortic rings We also evaluated the endothelium-dependent relaxation function in isolated aortic rings. It has been reported that the vascular endothelial integrity could be tested by measuring the diastolic level with 10−6 M acetylcholine to norepinephrine-precontracted (10−6 M) aortic rings. The endothelial function was regarded to be injured when the diastolic level was lower than 10%. As shown in Fig. 3, the endothelial function of ISO model group was impaired obviously in comparison with that of control group. And the impairments were reduced in APS administrated groups compared with that of ISO model group. 4.4. APS decreased the superoxide anion production in aortas To investigate the potential mechanisms of APS, we measured the production of ROS by using dihydroethidium (DHE) staining. As shown in Fig. 4, an increase of production of superoxide anion was observed in aortas from ISO-treated rats as compared with control rats which indicated an increased superoxide anion generation. This increase could be significantly reduced in APS administrated groups. 4.5. APS reduced the levels of TNF-α and IL-6 in plasma It is known that inflammation is one of the most potential trigger to both myocardial hypertrophy and endothelial dysfunction. We measured the levels of TNF-α and IL-6 with ELISA kits. As shown in Fig. 5, The inflammatory cytokines levels of TNF-α and IL-6 in ISO model group were much higher than that of the control. Compared with the ISO model group, the levels of TNF-α and IL-6 in APS administrated groups were significantly decreased. 4.6. APS increased the cGMP levels in aortics To further investigate the protective effects of APS, we measured the influence of APS on cGMP accumulation. As shown in Fig. 6, in the ISO model group an decrease of the cGMP accumulation was observed in aortas which indicated that the bioavailability of NO was reduced. However, the levels of cGMP in APS administrated groups could be significantly increased compared with that of ISO model group. 4.7. APS regulated the expressions of IκBα, p65 and Cu/Zn-SOD in aortas The expression of IκBα, p65 and Cu/Zn-SOD were examined to explore the potential molecular mechanisms contributing to the protective effects of APS. As shown in Fig. 7, the expression of IκBα and Cu/ Zn-SOD in the Iso model group were decreased and the protein expression of p65 were increased in comparison with that of control group. However, the APS administrated could significantly attenuate the decrease of IκBα and Cu/Zn-SOD expression and the increase of p65 protein expression which may contribute to the APS-mediated protective effects of HUVECs. 5. Discussion
Fig. 1. Effects of Astragalus polysaccharide (APS) on heart mass index (HMI) and left ventricular mass index (LVMI) in myocardial hypertrophy rats induced by ISO. The data were expressed as the mean ± SD, n = 10, **P b 0.01 vs. control. ##P b 0.01 vs. ISO model group.
The aim of this study was to assess the protective effects of APS against endothelial dysfunction in hypertrophy rats induced by ISO. Our results suggests that APS could ameliorate the endothelial function while attenuates cardiac hypertrophy induced by ISO. Endothelial function plays an important role in vascular pathophysiology and is considered to be a biomarker/mediator of cardiovascular risk factors [33,34]. Endothelial dysfunction has also been known to be a predictor of outcomes in different settings of patients [35–38]. The
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Fig. 2. Representative images of CEC (×400): (A): control group; (B): ISO model group; (C): ISO + APS 400 mg × kg−1 × day−1group; (D): ISO + APS 800 mg × kg−1 × day−1 group; (E): Statistical results of APS on the number of CEC in hypertrophy rats induced by ISO. Data were expressed as the mean ± SD, n = 5, **P b 0.01 vs. control. ##P b 0.01 vs. ISO model group.
association between endothelial dysfunction and cardiac hypertrophy has been examined in many previously published experimental data [1,11]. Although the association between endothelial dysfunction and cardiac hypertrophy has not been examined systematically in animal
Fig. 3. Effects of APS on the endothelium-dependent relaxation function of isolated aortic rings in hypertrophy rats induced by ISO. The vascular tension were expressed as percentage of precontraction induced by norepinephrine(10−6 M) (n = 5).
model induced by ISO, the injury of endothelial function has been found in myocardial hypertrophy rats induced by ISO as examined by circulating endothelial cell counting and assessment of vascular relaxation function in this study. This result is also in agreement with the previous study [17]. Astragalus polysaccharide (APS) is an important bioactive ingredient obtained from Astragalus membranaceus which has a range of activities in many pharmacological effects. It has been widely used in treatment of cardiovascular diseases. APS is a polysaccharide component. It consists of D-glucose, D-galactose, rhamnose, arabinose and L-arabinose and there are also reports indicating that APS was safe without any distinct toxicity and side effects, the safety dosage range is 5.7–39.9 g/kg for rats [39]. APS was orally administered to animals in many studies and found to be effective in treating many diseases [25,40–42]. We have also previously reported that APS could inhibit ISO-induced cardiac hypertrophy [26,27]. It has also been reported that Astragalus membranaceus and its main components including APS could potently ameliorate endothelial dysfunction induced by homocysteine in which anti-oxidation played a key role [43]. Nevertheless, the effects of APS on endothelial injury in hypertrophy rats induced by ISO are limited and mechanism has not yet been elucidated. Hence, we conducted this experiment to
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Fig. 4. Effects of APS on the ROS production in aortas of hypertrophy rats induced by ISO. Representative ethidium fluorescence images (×400):(A): control group; (B): ISO model group; (C): ISO + APS 400 mg × kg−1 × day−1 group; (D): ISO + APS 800 mg × kg−1 × day−1 group; (E): Statistical results of APS on the ROS production represented as relative fluorescence intensity. Data were expressed as the mean ± SD, n = 5, **P b 0.01 vs. control. ##P b 0.01 vs. ISO model group.
investigate the protective effects of APS against endothelial dysfunction in hypertrophy rats induced by ISO. It is known that myocardial hypertrophy is often accompanied by inflammatory responses. In this study we measured the inflammatory cytokines levels of TNF-α and IL-6 with ELISA and examined the inflammation associated protein expression of IκBα and p65. As shown in Figs. 5 and 7, the levels of TNF-α and IL-6 in APS administrated groups could be significantly decreased in comparison with that of ISO model group. APS could also attenuate the decrease of IκBα and the increase of p65 protein expression. It has also been reported that the associations have been made between endothelial dysfunction and systemic inflammation [44]. Hence, the protective effects of APS to endothelial function while attenuates cardiac hypertrophy induced by ISO may be related to its anti-inflammatory effects.
Nitric oxide (NO), an endothelium-dependent vasodilator, is an important mediator in the regulation of endothelial cell functions. Reactive oxygen species (ROS) are free radicals found in all vascular cells which can inactivate NO and decrease NO bioavailability in blood vessels and lead to the injury of endothelial functions. It has also been reported that the reduced formation of NO or impairment of nitric oxide effect may be associated with aortic sclerosis [45]. Studies also suggest that the imbalance between ROS and NO levels, rather than the individual levels, can be a major cause of endothelial dysfunction in numerous cardiovascular diseases. In this study we detected the intracellular cGMP levels to evaluate the bioavailability of NO which is the second messenger of NO. Our results suggest that APS could potently increase the levels
Fig. 5. Effects of APS on the levels of TNF-α and IL-6in hypertrophy rats induced by ISO. Data were expressed as the mean ± SD, n = 4, **P b 0.01 vs. control. ##P b 0.01 vs. ISO model group.
Fig. 6. APS restored the decrease of cGMP contents in aortas of hypertrophy rats induced by ISO. Data were expressed as the mean ± SD, n = 4, **P b 0.01 vs. control. ##P b 0.01 vs. ISO model group.
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Fig. 7. Effect of APS on expressions of IκBα, p65 and Cu/Zn-SOD in aortas of hypertrophy rats induced by ISO. (a): control group; (b): ISO model group; (c): ISO + APS 800 mg × kg−1 × day−1 group; (d): ISO + APS 400 mg × kg−1 × day−1 group; (B):Statistical results of protein expression are represented as ratio to β-actin. Data were expressed as the mean ± SD, n = 5, **P b 0.01 vs. control. ##P b 0.01 vs. ISO model group.
of cGMP as shown in Fig. 6, which may contribute to the APS-mediated protective effects to endothelial functions. It was reported that oxidative stress and inflammation seems to play key roles in endothelial dysfunction in the rats model induced by ISO [16]. Numerous observations also suggest that ROS plays an essential role in vascular remodeling and endothelial dysfunction [46]. There are researches suggest that the endothelial cells express virtually a variety of enzymes from which ROS can be generated. Thus, the endothelial cells are regarded as an important source of vascular ROS production [47]. In this study we measured the ROS production with DHE staining and evaluated the antioxidant capacity with examined the protein expression of Cu/Zn-SOD. As shown in Figs. 4 and 7, APS could significantly reduced the production of ROS and increase the Cu/Zn-SOD expression. In conclusion, our studies showed that APS had a protective effect against endothelial dysfunction in hypertrophy rats induced by ISO. The underlining mechanisms of the protection are attributed to the anti-inflammatory effects and the improvement of the imbalance between ROS and NO levels through increasing the cell antioxidant capacity and NO bioavailability. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by National Natural Science Foundation of China (81374008). References [1] F. Perticone, R. Maio, M. Perticone, S. Miceli, A. Sciacqua, E.J. Tassone, et al., Endothelial dysfunction predicts regression of hypertensive cardiac mass, Int. J. Cardiol. 167 (2013) 1188–1192. [2] A. Ilercil, R.B. Devereux, M.J. Roman, M. Paranicas, M.J. O'Grady, T.K. Welty, et al., Relationship of impaired glucose tolerance to left ventricular structure and function: the Strong Heart Study, Am. Heart J. 141 (2001) 992–998. [3] J. Yeboah, J.R. Crouse, D.A. Bluemke, J.A. Lima, J.F. Polak, G.L. Burke, et al., Endothelial dysfunction is associated with left ventricular mass (assessed using MRI) in an adult population (MESA), J. Hum. Hypertens. 25 (2011) 25–31.
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