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Cardioprotective effects of a Fructus Aurantii polysaccharide in isoproterenol-induced myocardial ischemic rats
BIOMAC-13844; No of Pages 8 International Journal of Biological Macromolecules xxx (xxxx) xxx
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Cardioprotective effects of a Fructus Aurantii polysaccharide in isoproterenol-induced myocardial ischemic rats Yanni Yang a,b,1, Zihe Ding a,b,1, Renxing Zhong a,b, Tianyi Xia a,b, Wujing Wang c, Hong Zhao d, Yi Wang a, Zunpeng Shu a,b,⁎ a
Research Centre for Good Practice in TCM Processing Technology, Guangdong Pharmaceutical University, 510006, Guangzhou, PR China The College of Traditional Chinese Medicine, Guangdong Pharmaceutical University, 510006, Guangzhou, PR China School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, 510006 Guangzhou, PR China d Pharmacy College, Jiamusi University, 154002 Jiamusi, PR China b c
a r t i c l e
i n f o
Article history: Received 23 August 2019 Received in revised form 28 October 2019 Accepted 7 November 2019 Available online xxxx Keywords: Myocardial injury Fructus aurantii Polysaccharide CALB-3 Oxidative stress
a b s t r a c t CALB-3, a purified acidic hetero-polysaccharide isolated from Fructus aurantii, has been shown to exert cardioprotective effects in vitro. Recently, we investigated the protective effects of CALB-3 on myocardial injury and its possible mechanisms of action using a rat model of myocardial ischemia. In this study, a myocardial ischemia model was established via intragastric administration of 2 mg/kg isoproterenol (ISO) to male Sprague– Dawley rats (200–220 g) daily for 3 days. We found that pretreatment with CALB-3 (50, 100, and 200 mg/kg, i. g.) daily for 21 days prevented ISO-induced myocardial damage, including improvement in electrocardiographic parameters, and decrease in serum cardiac enzymes, heart vacuolation, and TUNEL-positive cells. We used western blotting to identify the underlying mechanisms and determine the possible signal pathways involved. We found that CALB-3 pretreatment prevented apoptosis, increased the expression of antioxidant enzymes, and enhanced the binding of Nrf2 to the antioxidant response element. In addition, CALB-3 activated the phosphorylation of PI3K/Akt and ERK to increase the cytoprotective effect. Overall, our results show that CALB-3 is a promising polysaccharide for protecting against myocardial injury induced by ISO. © 2019 Published by Elsevier B.V.
1. Introduction Ischemic heart disease (IHD), characterized by ischemia of the heart tissue, is a leading cause of disability and mortality worldwide [1]. Myocyte apoptosis plays an important role in IHD [2]. Evidence suggests that oxidative stress induced by reactive oxygen species (ROS) is one of the main factors triggering myocyte apoptosis [3]. Excessive ROS production detrimentally alters vital intracellular macromolecules such as DNA, proteins, and lipids, resulting in cellular apoptotic death [4]. Hence, the modulation of ROS levels and regulation of the apoptotic cascade are considered crucial therapeutic strategies for treating IHD. Fructus aurantii (FA) is dried immature fruit of Citrus aurantium L. (Rutaceae) and its cultivated variety [5]. FA is a well-known medicinal and edible plant frequently used for treating cardiovascular diseases [6]. In our previous studies, we showed that CALB-3, a purified acidic hetero-polysaccharide of FA, possessed significant antioxidant activity, ⁎ Corresponding author at: Guangdong Pharmaceutical University, No. 280 Guangzhou Higher Education Mega Center, Guangzhou 510006, Guangdong Province, PR China. E-mail address: [email protected] (Z. Shu). 1 These authors contributed equally to this manuscript and worked as co-first authors.
determined using four free radical scavenging tests in vitro [7]. In further experiments, we found that CALB-3 exhibits anti-myocardial cell injury effect induced by hypoxia-reoxygenation in H9c2 myocardial cells. We suspected that this effect is partly due to the function of CALB-3 in anti-apoptosis and anti-oxidation. Myocardial ischemia-induced oxidative stress influences signaling pathways that contribute to apoptosis and altered cell proliferation [8]. The PI3K/Akt signaling pathway is known to play pivotal roles in controlling the survival and apoptosis of cardiomyocytes. Various studies have demonstrated that the activation of PI3K and its downstream Akt molecules can inhibit the apoptosis of cardiomyocytes in IHD [9]. In addition, some studies have showed that the PI3K/Akt signaling pathway can activate Nrf2, and subsequently induce antioxidant enzymes including HO-1, NQO-1, GCLM, and γ-GCS [10]. Nrf2, a redox-sensitive transcription factor capable of interacting with anti-oxidant response elements (AREs), plays a key role in the transcriptional activation of antioxidant enzyme gene expression [11] Recent evidence has revealed that the induction of antioxidant enzymes through Nrf2 activation protects against oxidative stress in the heart [12,13]. In addition, mitogenactivated protein kinases (MAPK), including three major subgroups ERK1/2, JNK, and P38, are also shown to be responsible for ISO-
https://doi.org/10.1016/j.ijbiomac.2019.11.063 0141-8130/© 2019 Published by Elsevier B.V.
Please cite this article as: Y. Yang, Z. Ding, R. Zhong, et al., Cardioprotective effects of a Fructus Aurantii polysaccharide in isoproterenol-induced myocardial ischemic rats..., , https://doi.org/10.1016/j.ijbiomac.2019.11.063
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mediated cardiomyocyte apoptosis. For example, it has been shown that the protein level of phosphorylated ERK1/2, JNK, and P38 was inhibited in hypoxia-reoxygenation H9c2 cardiomyocytes [14]. Therefore, an intriguing question is whether CALB-3 activates Nrf2 and the subsequent up-regulation of antioxidant enzyme expression to suppress cardiomyocyte apoptosis by affecting the phosphorylation of the PI3K/Akt and MAPK signaling pathway. In the present study, we aimed to further identify the protective effects of CALB-3 and investigate its potential molecular action mechanisms against isoproterenol (ISO)-mediated myocardial injury in vivo, focusing on the PI3K/Akt/Nrf2 signal transduction pathway. 2. Materials and methods
distilled water, whereas Groups 3, 4, and 5 were dosed intragastrically with CALB-3 (50, 100, and 200 mg/kg, respectively) for 21 day. Within 2 h of CALB-3 administration on days 19, 20, and 21, rats in the groups 2 to 5 were injected with ISO (2 mg/kg, i.p.), whereas Groups 1 was injected with saline. At the end of the study period, all rats were weighed, and then anesthetized with 20% ethyl urethane by i.p. After sacrificing the rats, their heart was quickly and meticulously harvested for histopathological and biochemical analyses 24 h after the final injection. 2.3.2. Heart rate and electrocardiography The measurements of heart rate (HR) and electrocardiography (ECG) were carried out on day 21 using the 16-Channel Advanced Research Workstation (MP150; BIOPAC Systems, Inc., CA, USA) [15].
2.1. Plant materials and preparation of CALB-3 2.1.1. Plant materials Dry immature fruits of Citrus aurantium L. were collected in May 2014 from Xingan in Jiangxi Province, China and identified by Prof. Zhenyue Wang, a pharmacognosist from the Department of Pharmacognosy, School of Pharmacy, Heilongjiang University of Chinese Medicine (Harbin China), where the voucher specimen (20140018) has been deposited. 2.1.2. Preparation of CALB-3 CALB-3, a purified acidic hetero-polysaccharide isolated from Fructus aurantii, was extracted and purified according to previously reported procedures [7]. Using the phenol-sulfuric acid colorimetric method and HPLC analysis, a single and symmetrical peak was shown for CALB-3, which indicated that CALB-3 is a homogeneously pure polysaccharide based on the distribution of molecular weight. 2.2. Materials The kits for determining the content of malondialdehyde and activity of creatine kinase (CK), catalase (CAT), aspartate aminotransferase (AST), lactate dehydrogenase, glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), and superoxide dismutase (SOD) were acquired from Jiancheng Bioengineering Institute (Nanjing, China). ROS and caspase-3 ELISA kits were purchased from RapidBio Lab (Calabasas, CA, USA). The terminal deoxynucleotidyl transferasemediated dUTP nick end-labeling (TUNEL) assay kit was purchased from Roche Diagnostics GmbH (Mannheim, Germany). Protein extraction kits were purchased from CoWin Bioscience Co., Ltd. (Beijing, China). A BCA protein assay kit was purchased from Pierce Corporation (Rockford, USA). Primary antibodies against PI3K, p-PI3K, Akt, p-Akt, ERK, p-ERK, JNK, p-JNK, P38, p-P38, Nrf2, Bcl-2, Bax, HO-1, NQO-1, GCLM, γ-GCS, lamin B, and β-actin were obtained from Santa Cruz Biotechnology (USA) and other chemicals were purchased from SigmaAldrich (St. Louis, MO, USA). 2.3. Methods 2.3.1. Animals and experimental protocols Healthy Sprague-Dawley (SD) male rats (200 ± 20 g) were used in the experiments. The animals were housed under the following standard conditions: temperature, 24 ± 2 °C; relative humidity, 60% ± 10%; 12-h light/dark cycle; standard rodent chow; and free access to water. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of the Guangdong Pharmaceutical University and approved by the Animal Ethics Committee of Guangdong Pharmaceutical University. One hundred SD rats were randomly divided into the following five groups: Group 1, Control; Group 2, ISO treatment; Groups 3, 4, and 5, ISO combined with CALB-3 (50, 100, and 200 mg/kg, respectively). Groups 1 and 2 were dosed intragastrically with the same volume of
2.3.3. Preparation of samples and measurement of biochemical variables After ECG, blood was collected from the abdominal aorta, and then centrifuged to collect the serum. Serum samples were assayed to determine myocardial enzymes (LDH, CK, and AST) using kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) on an automated chemical analyzer (7060; Hitachi, Tokyo, Japan). Subsequently, the heart was immediately removed after perfusion with saline. The left ventricle (LVs) was excised for histopathological examination, and cardiac homogenates were used to determine the content of MDA, and activity of T-AOC, SOD, CAT, and GSH-Px using the corresponding kits, according to the manufacturers' instructions. ROS were detected using the corresponding ELISA kits (RapidBio Lab, Calabasas, CA, USA). 2.3.4. Heart histopathological examination (HE) The LV was harvested and fixed with 4% paraformaldehyde for 48–52 h. The heart was then dissected and embedded in paraffin blocks, sectioned, stained with hematoxylin and eosin (H&E), and examined under a light microscope (CKX41; 170 Olympus, Tokyo, Japan) by a pathologist, who was blinded to the study groups [16]. 2.3.5. TUNEL assay DNA fragmentation was detected using the TUNEL assay, according to the apoptosis kit instructions. After dewaxing and rehydration, the heart sections were incubated with proteinase K (20 mg/mL) for 20 min at 22 °C. The slices were washed with PBS, and then incubated with working-strength terminal deoxynucleotidyl transferase enzyme in a humidor for 60 min at 37 °C. The slices were again washed with PBS and incubated with working-strength anti-digoxigenin conjugate for 30 min at room temperature. The slices were stained with 4,5diamino-2-phenylindole (DAPI) and observed under a fluorescence microscope (Leica, Heidelberg, Germany) [16]. 2.3.6. Western blot analysis The cardiac muscle tissues were harvested, washed with PBS, and lysed with protein extraction reagent containing 1% phenylmethylsulfonyl fluoride on ice. The supernatant was collected after centrifuging for 15 min at 12,000 rpm and 4 °C, and the protein concentration was measured using a BCA protein assay kit. Equal amounts of protein from each sample were separated by 10% SDSPAGE, and transferred onto nitrocellulose membranes (Millipore Corporation, Bedford, MD, USA) in Tris-glycine buffer at 100 V for 55 min in an ice box. The membranes were blocked with 5% (w/v) non-fat milk powder in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBST) for 2 h at room temperature. The membranes were then incubated overnight at 4 °C with the primary antibodies (PI3K, p-PI3K, Akt, p-Akt, ERK, p-ERK, JNK, p-JNK, P38, p-P38, Nrf2, Bcl-2, Bax, HO-1, NQO-1, GCLM, γ-GCS, lamin B, and β-actin; all ratios were 1:500; Santa Cruz Biotechnology). Subsequently, the membranes were washed three times with TBST, incubated with appropriate secondary HRPconjugated antibodies (1:1000) for 2 h on a shaking table, and washed with TBST three times for 45 min. Enhanced chemiluminescence
Please cite this article as: Y. Yang, Z. Ding, R. Zhong, et al., Cardioprotective effects of a Fructus Aurantii polysaccharide in isoproterenol-induced myocardial ischemic rats..., , https://doi.org/10.1016/j.ijbiomac.2019.11.063
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solution was used to develop the blots for 5 min, and then analyzed using Image Lab software (Bio-Rad, USA). The different protein levels are expressed as a percentage of the control, calculated using Gel-Pro Analyzer software [17]. 2.4. Statistical analysis All experiments were repeated a minimum of three times. The experimental results are expressed as mean ± SD. The analysis of oneway analysis of variance (ANOVA) followed by Student–Newman– Keuls test for pairwise comparisons was used for the intergroup analysis. The results with P b 0.05 were considered statistically significant. 3. Results 3.1. Body weight and heart weight There were no significant differences in the body weight among the groups (Table 1). There was a statistically significant increase in the heart weight of the ISO-treated rats compared with that of the other group rats (P b 0.05). CALB-3 treatment decreased the heart weight significantly in a dose-dependent manner (P b 0.05). 3.2. Heart rate The HR values were significantly decreased in the ISO-treated rats compared to those of the control group; whereas the HR values were significantly increased in the three CALB-3 groups compared with those of the ISO group (P b 0.05; Table 1). 3.3. ECG analysis To evaluate the protective effects of CALB-3 on myocardial injury, we measured ECG changes in each group. As shown in Fig. 1, the ISO treatment significantly impaired the rat heart. The ST segment elevation was significantly prolonged (3.22-fold that of the control; P b 0.01) in the ISO group compared with that in the control group. However, the elevation of the ST segment was significantly reduced when the rats received pretreatment of 50, 100, and 200 mg/kg CALB-3, compared with that of the control (0.779 ± 0.088, 0.975 ± 0.103, and 1.137 ± 0.131 vs. 1.452 ± 0.134, respectively; P b 0.01); the effect was dose dependent. 3.4. CALB-3 prevents ISO-induced myocardial injury To analyze the cardioprotective effect of CALB-3, we measured the expression of serum cardiac enzymes (LDH, CK, and AST) using the ELISA methods in different groups. As shown in Fig. 2A, ISO exposure induced myocardial damage, resulting in significantly increased levels of the myocardial enzymes LDH, CK, and AST. Nevertheless, the pretreatment with different doses of CALB-3 before ISO exposure significantly ameliorated these effects (P b 0.05 or P b 0.01) in a dose-dependent manner. In addition, using H&E staining, the pathological changes in t1:1 t1:2
Table 1 Effects of CALB-3 on the levels of body, heart weight, and heart rate (HR).
t1:3 t1:4
Groups (n = 10)
Body weight (g)
Heart weight (g)
Heart rate (beats/min)
t1:5 t1:6 t1:7 t1:8 t1:9
Control ISO CA-high CA-middle CA-low
293.71 ± 22.4 279.32 ± 18.5 288.96 ± 17.5 284.47 ± 16.2 283.71 ± 16.6
0.91 ± 0.05 1.24 ± 0.12# 0.96 ± 0.08⁎⁎ 0.98 ± 0.07⁎⁎ 0.98 ± 0.09⁎⁎
437.61 ± 30.76 350.47 ± 36.01# 428.72 ± 21.77⁎⁎ 418.09 ± 26.28⁎⁎
t1:10 t1:11 t1:12 t1:13 t1:14
373.21 ± 18.90
ISO, isoproterenol; CA-high, high dose of CALB-3; CA-middle, middle dose of CALB-3; CAlow, low dose of CALB-3. Data are expressed as mean ± SD. # P b 0.01 vs the control group. ⁎⁎ P b 0.05 vs the ISO group.
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myocardial tissue were detected by light microscopy. As shown in Fig. 2B, no obvious abnormalities were observed in the control group, whereas severe myocardial damage was observed in the ISO group, characterized by myocardial fiber fracture, interstitial widening, local wavy cytoplasm dissolution, and membrane rupture. Pretreatment with CALB-3 at three different doses significantly improved the above pathological changes. This result was consistent with above results, suggesting that CALB-3 has protective effect against myocardial injury induced by ISO in rats. 3.5. CALB-3 inhibits the ISO-induced cardiomyocyte apoptosis The caspase enzymes regulate several events leading to cellular and biochemical changes associated with apoptosis [14]. The activation of caspase-3, a typical pro-apoptotic protein, was evaluated. As shown in Fig. 3A, the caspase-3 activity was significantly increased by ISO (5.4fold that of the control; P b 0.01); however, pretreatment with CALB-3 inhibited this activity in a dose-dependent manner (P b 0.01). Similarly, the caspase-8 activity was also upregulated in the heart tissue treated with ISO, and its activity was downregulated with CALB-3 pretreatment (P b 0.01). Bcl-2 and Bax are two well-known proteins in the Bcl-2 family that regulates mitochondrial outer membrane permeability. The ISOinduced apoptosis was accompanied by decreased expression of the anti-apoptotic protein Bcl-2, but an increased expression of the proapoptotic protein Bax. As shown in Fig. 3B, CALB-3 preconditioning reversed the effects of ISO by decreasing Bax (2.89, 2.40, and 1.94 vs. 3.58, respectively; P b 0.05 or P b 0.01) and increasing Bcl-2 (0.849 and 0.907 vs. 0.682, respectively; P b 0.05 or P b 0.01) expression level in rat myocardial tissue in a dose-dependent manner. Additionally, the TUNEL assay was performed to investigate the effects of CALB-3 on cardiomyocyte apoptosis. The administration of ISO caused DNA fragmentation in nucleosomes. As shown in Fig. 4, the number of TUNEL-positive cells increased considerably in the ISO group compared with that in the control group, and CALB-3 pre-administration significantly reduced the number of TUNEL-positive cells compared with that in the ISO group in a dose-dependent manner. The above results suggested that CALB-3 can inhibit ISO-mediated apoptosis via the mitochondria-dependent apoptotic pathway. 3.6. CALB-3 enhances antioxidant capacity in cardiac tissues To determine whether the protective effects of CALB-3 against ISOinduced myocardial injury were due to its antioxidative activity, we measured oxidative stress-related parameters of the heart. As shown in Fig. 5A, we found that ISO significantly reduced the SOD, CAT, and GSH-Px activities and T-AOC level in the heart compared with those of the control group. Pretreatment with different doses of CALB-3 before ISO administration significantly ameliorated these effects. Furthermore, ISO induced an increase in MDA production and intracellular ROS levels, and pretreatment with CALB-3 decreased the levels of MDA and ROS in a dose-dependent manner (P b 0.05 or P b 0.01). However, with western blotting, we detected the other four important antioxidant enzymes, HO-1, NQO-1, GCLM, and γ-GCS. In agreement with our ELISA results, the activity of the four enzymes was considerably inhibited in ISOtreated rats compared with that in the control. This was reversed by CALB-3 treatment in a dose-dependent manner (P b 0.05 or P b 0.01; Fig. 5B). These results suggest that CALB-3 can considerably enhance the antioxidative capacity to scavenge ROS against ISO-induced oxidative stress. 3.7. CALB-3-induced nuclear accumulation of the Nrf2 protein Given that activated Nrf2 is the major transcription factor that enhances the expression of antioxidative enzyme genes [18,19], we attempted to measure the nuclear accumulation of Nrf2 in ISOinduced myocardial tissue. Western blotting showed that ISO
Please cite this article as: Y. Yang, Z. Ding, R. Zhong, et al., Cardioprotective effects of a Fructus Aurantii polysaccharide in isoproterenol-induced myocardial ischemic rats..., , https://doi.org/10.1016/j.ijbiomac.2019.11.063
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Fig. 1. CALB-3 improved cardiac function in ISO-induced rats, ST depression, n = 8; The values represent the means ± SEMs. P b 0.01 vs the control group; *P b 0.05, **P b 0.01 vs the model group.
significantly downregulated the expression of nuclear Nrf2 and inhibited Nrf2 translocation from the cytosol to the nucleus. However, this could be reversed by the pretreatment with CALB-3 in a dosedependent manner (P b 0.05 or P b 0.01; Fig. 5B). 3.8. CALB-3 inhibits ISO-induced activation of ERK1/2 MAPK in cardiac tissues Previous studies have shown that ISO can result in the apoptosis of cardiomyocytes via the activation of MAPK, including ERK, JNK, and p38 MAPK [20]. Therefore, we investigated the effect of CALB-3 on the phosphorylation level of MAPK in ISO-induced rats by western blotting. As shown in Fig. 6, the protein level of total ERK1/2, JNK1/2, and P38 MAPK was almost unchanged in the ISO and ISO + CALB-3 (50, 100,
and 200 mg/kg) groups compared with that in the control group. However, the protein level of p-JNK1/2 and p-P38 was increased, whereas that of p-ERK1/2 was decreased in the ISO groups, demonstrating the activation of MAPK by ISO. Interestingly, the reduced phosphorylation level of ERK1/2 was increased, whereas that of JNK1/2 and P38 remained unchanged in the ISO + CALB-3 group compared with that in the ISO groups, suggesting the involvement of ERK in the protective effect of CALB-3 against ISO-induced myocardial injury. 3.9. CALB-3 enhances antioxidant capacity via PI3K/Akt signaling The PI3K/Akt signaling pathway plays key roles in ISO-induced oxidative stress and apoptosis. Numerous studies have indicated that they are the signal upstream of Nrf2 [21–23]. As shown in
Fig. 2. CALB-3 prevented the ISO-induced myocardial injury. (A) The levels of myocardial enzymes (LDH, CK, and AST) were determined, n = 9 or 10 per group; (B) representative images of HE staining of heart tissue, n = 3. The values represent the means ± SEMs. #P b 0.01 vs the control group; *P b 0.05, **P b 0.01 vs the model group.
Please cite this article as: Y. Yang, Z. Ding, R. Zhong, et al., Cardioprotective effects of a Fructus Aurantii polysaccharide in isoproterenol-induced myocardial ischemic rats..., , https://doi.org/10.1016/j.ijbiomac.2019.11.063
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Fig. 3. CALB-3 protected against ISO-induced myocardial injury through by improving the internal mitochondrial apoptotic pathway. (A) The activities of caspase-3 and -8 were measured using a fluorometric assay, n = 8; (B) the western blot analysis of Bcl-2, Bax and β-actin, n = 4; the values represent the means ± SEMs. #P b 0.01 vs the control group; *P b 0.05, **P b 0.01 vs the model group.
Fig. 7, western blotting showed that ISO significantly reduced the ratio of PI3K/Akt phosphorylation in the heart (P b 0.05 or P b 0.01); however, the effect was neutralized by treatment with CALB-3 in a dose-dependent manner. The results showed that CALB-3 exerted an anti-oxidative effect by activating the phosphorylation of PI3K/Akt to protect against ISO.
4. Discussion Fructus aurantii has been used as a medicinal herb in Traditional Chinese Medicine for thousands of years, particularly to treat cardiovascular diseases. Both flavonoid glycosides and polymethoxylated flavones, including naringin, hesperidin, tangeratin, and neohesperidin, are
Fig. 4. TUNEL staining, n = 4. The values represent the means ± SEMs. Arrows indicate apoptotic cells. #P b 0.01 vs the control group; *P b 0.05, **P b 0.01 vs the model group.
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Fig. 5. CALB-3 promotes the expression of Nrf2-mediated anti-oxidative enzymes. (A) The effects of CALB-3 on T-AOC, SOD, CAT, and GSH-Px activities as well as MDA and ROS level, n = 9 or 10 per group. (B) The relative expression levels of HO-1, NQO-1, GCLM, γ-GCS, Nucl-Nrf2, and Cyto-Nrf2 relative to β-actin and Lamin B and the bar graphs, n = 4. The values represent the means ± SEMs. #P b 0.01 vs the control group; *P b 0.05, **P b 0.01 vs the model group.
consistently regarded as the main active ingredients in the prevention and treatment of ischemic heart disease [24,25]. Interestingly, in our previous studies, polysaccharides, another main active ingredient of FA, also exhibited strong antioxidant effects in vitro and in vivo. Among the four purified polysaccharides, CALB-3 had the strongest antioxidant activity and its structure was elucidated by FTIR, SEM, AFM, partial acid hydrolysis, periodate oxidation, Smith degradation, methylation analysis, GC–MS, and NMR [7]. In the present study, we aimed to further investigate the protective effects of CALB-3 against ISOinduced myocardial ischemia in vivo. It is important to note that myocardial infarction continues to be the major cause of cardiovascular diseases and occurs when the blood supply from the coronary artery to the heart is blocked, resulting in irreversible damage or death of heart tissue [26–28]. Therefore, the pathogenesis and therapeutic targets of acute myocardial injury are currently popular research topics. Apoptosis of cardiac myocytes induced by oxidative stress plays a pivotal role in the pathogenesis of IHD. Therefore, the elimination of excessive intracellular ROS and prevention of oxidative stress-induced apoptosis may serve as a beneficial intervention for the treatment of these diseases. In the present study, the ISO treatment group showed a considerable increase in heart weight compared with that of the control group. One possible explanation for these findings is that ISO may lead to hypertrophy of the heart tissue and reduction of the heart rate. Similarly, Kalkan showed that ISO treatment causes a significant increase in heart weight
and decrease in heart rate [29]. This study definitively demonstrates that ISO treatment induces morphologic abnormalities and that CALB3 treatment reverses the ECG changes associated with cardiocytes and cell damage. In addition, the LDH, CK, and AST levels were increased in blood during myocardial infarction. These enzymes are released into the blood stream from cells when the cell membrane ruptures and serve as a diagnostic tool. Unlike those in the experiment model, CALB-3 administration reduced the level of serum cardiac enzymes and ameliorated histological damage in the heart, indicating that CALB-3 exerted a pronounced protective effect against myocardial injury. Apoptosis may be initiated either by the intrinsic (mitochondrial) or extrinsic (death receptor-dependent) pathways [30]. Caspases are a family of aspartate-specific cysteine proteases that play key roles in regulating the two pathways of apoptosis induced by different stimuli, including oxidative stress [31]. Caspase-3 and -8 are the key executioners of apoptosis and regulating factors involved in DNA degradation, chromatin condensation, and nuclear fragmentation [32]. Caspase-3 and -8 were activated by ISO treatment, and administration of CALB-3 inhibited the activation. Moreover, CALB-3 pretreatment inhibited TUNEL-positive cells and modulated the protein expression of the Bcl-2 family of proteins. CALB-3 pretreatment increased the expression of Bcl-2, as it decreased the expression of pro-apoptotic Bax. Therefore, CALB-3 may exert protective effects by maintaining mitochondrial function and modulating the balance between anti-
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on the heart tissue [35]. The results revealed that ISO treatment led to a decrease in antioxidant enzyme activities. Similarly, Ilhan et al. showed that the SOD, GSH-Px, and CAT enzyme activities decreased in ISO model rats when compared with those in the control group [36]. Our study demonstrated that ISO is a powerful oxidizing agent that caused the release of ROS and led to significant oxidative damage in rat heart tissue. However, CALB-3 exerted its beneficial effects on the heart tissue and protected against the toxic effects of ISO via its strong antioxidant properties. Serine/threonine kinase Akt is an important component of the intracellular signaling pathways tightly linked with apoptosis and oxidative stress [37]. Akt signaling can affect communication with other signaling pathways including Nrf2. Furthermore, our previous studies have confirmed that phosphorylation of Akt regulates Nrf2 activation and HO-1, NQO-1, GLLM, and γ-GCS expression. As we hypothesized, not only phospho-Akt was inhibited, but also phospho-ERK was inhibited in the ISO-Induced myocardial ischemia rats; however, both processes were reversed by CALB-3 in a dose-dependent manner. 5. Conclusions
Fig. 6. Effect of CALB-3 on phosphorylation levels of ERK, JNK, and p38 MAPK in ISOinduced rats. The ERK, JNK, and p38 MAPK phosphorylation were determined by western blotting and densitometric quantification is shown in the bar diagrams, n = 4. The values represent the means ± SEMs. #P b 0.01 vs the control group; *P b 0.05, **P b 0.01 vs the model group.
apoptotic protein Bcl-2 and pro-apoptotic protein Bax, thus inhibiting the release of pro-apoptotic molecules from the mitochondrial intermembrane space into the cytoplasm. A growing body of evidence indicates that the MAPK signaling pathway is involved in the Bcl-2 family-mediated mitochondria-dependent apoptotic pathway in ISO-induced rats [33,34] For example, Verma et al. reported that ISO could induce apoptosis via the upregulation of JNK and p38 MAPK in the heart of rats, while ERK is usually downregulated in acute myocardial ischemia [20]. The results reported are agreement with our findings. In addition, we found that CALB-3 only affected ERK phosphorylation, and had no effect on p-JNK and p-P38. Therefore, we surmise that CALB-3, an acidic hetero-polysaccharide, may differentially regulate three cascades of MAPK in ISO-induced rat model. To test whether a close association existed between oxidative stress and cell apoptosis induced by ISO, biochemical analysis was performed
In summary, this is the first study to show that an acidic FA polysaccharide exerts cardioprotective effect in an ISO-induced rat model. This occurred via the activation of Akt and ERK signaling as well as the suppression of cardiac oxidative stress and apoptosis. Accordingly, we believe that CALB-3 should be considered as a potential therapeutic medicine to prevent myocardial ischemia. Data availability The data used to support the findings of this study are included within the article. Declaration of competing interest The authors declare that they have no conflicts of interest. Acknowledgments This study was supported by the National Natural Science Foundation of China (Grant Nos. 81603366), the Natural Science Foundation of Heilongjiang Province (Grant Nos. H2015041), the China Postdoctoral Science Foundation (Grant Nos. 2017M610816), and the Foundation of Postdoctoral Science of Chinese Academy of Medical Sciences & Peking Union Medical College (2016). References
Fig. 7. CALB-3 enhances antioxidant capacity by the activity of PI3K/Akt signaling. The PI3K and Akt phosphorylation were determined by western blotting and densitometric quantification is shown in the bar diagrams, n = 4. The values represent the means ± SEMs. #P b 0.01 vs the control group; *P b 0.05, **P b 0.01 vs the model group.
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Cardioprotective effects of a Fructus Aurantii polysaccharide in isoproterenol-induced myocardial ischemic rats Yanni Yang a,b,1, Zihe Ding a,b,1, Renxing Zhong a,b, Tianyi Xia a,b, Wujing Wang c, Hong Zhao d, Yi Wang a, Zunpeng Shu a,b,⁎ a
Research Centre for Good Practice in TCM Processing Technology, Guangdong Pharmaceutical University, 510006, Guangzhou, PR China The College of Traditional Chinese Medicine, Guangdong Pharmaceutical University, 510006, Guangzhou, PR China School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, 510006 Guangzhou, PR China d Pharmacy College, Jiamusi University, 154002 Jiamusi, PR China b c
a r t i c l e
i n f o
Article history: Received 23 August 2019 Received in revised form 28 October 2019 Accepted 7 November 2019 Available online xxxx Keywords: Myocardial injury Fructus aurantii Polysaccharide CALB-3 Oxidative stress
a b s t r a c t CALB-3, a purified acidic hetero-polysaccharide isolated from Fructus aurantii, has been shown to exert cardioprotective effects in vitro. Recently, we investigated the protective effects of CALB-3 on myocardial injury and its possible mechanisms of action using a rat model of myocardial ischemia. In this study, a myocardial ischemia model was established via intragastric administration of 2 mg/kg isoproterenol (ISO) to male Sprague– Dawley rats (200–220 g) daily for 3 days. We found that pretreatment with CALB-3 (50, 100, and 200 mg/kg, i. g.) daily for 21 days prevented ISO-induced myocardial damage, including improvement in electrocardiographic parameters, and decrease in serum cardiac enzymes, heart vacuolation, and TUNEL-positive cells. We used western blotting to identify the underlying mechanisms and determine the possible signal pathways involved. We found that CALB-3 pretreatment prevented apoptosis, increased the expression of antioxidant enzymes, and enhanced the binding of Nrf2 to the antioxidant response element. In addition, CALB-3 activated the phosphorylation of PI3K/Akt and ERK to increase the cytoprotective effect. Overall, our results show that CALB-3 is a promising polysaccharide for protecting against myocardial injury induced by ISO. © 2019 Published by Elsevier B.V.
1. Introduction Ischemic heart disease (IHD), characterized by ischemia of the heart tissue, is a leading cause of disability and mortality worldwide [1]. Myocyte apoptosis plays an important role in IHD [2]. Evidence suggests that oxidative stress induced by reactive oxygen species (ROS) is one of the main factors triggering myocyte apoptosis [3]. Excessive ROS production detrimentally alters vital intracellular macromolecules such as DNA, proteins, and lipids, resulting in cellular apoptotic death [4]. Hence, the modulation of ROS levels and regulation of the apoptotic cascade are considered crucial therapeutic strategies for treating IHD. Fructus aurantii (FA) is dried immature fruit of Citrus aurantium L. (Rutaceae) and its cultivated variety [5]. FA is a well-known medicinal and edible plant frequently used for treating cardiovascular diseases [6]. In our previous studies, we showed that CALB-3, a purified acidic hetero-polysaccharide of FA, possessed significant antioxidant activity, ⁎ Corresponding author at: Guangdong Pharmaceutical University, No. 280 Guangzhou Higher Education Mega Center, Guangzhou 510006, Guangdong Province, PR China. E-mail address: [email protected] (Z. Shu). 1 These authors contributed equally to this manuscript and worked as co-first authors.
determined using four free radical scavenging tests in vitro [7]. In further experiments, we found that CALB-3 exhibits anti-myocardial cell injury effect induced by hypoxia-reoxygenation in H9c2 myocardial cells. We suspected that this effect is partly due to the function of CALB-3 in anti-apoptosis and anti-oxidation. Myocardial ischemia-induced oxidative stress influences signaling pathways that contribute to apoptosis and altered cell proliferation [8]. The PI3K/Akt signaling pathway is known to play pivotal roles in controlling the survival and apoptosis of cardiomyocytes. Various studies have demonstrated that the activation of PI3K and its downstream Akt molecules can inhibit the apoptosis of cardiomyocytes in IHD [9]. In addition, some studies have showed that the PI3K/Akt signaling pathway can activate Nrf2, and subsequently induce antioxidant enzymes including HO-1, NQO-1, GCLM, and γ-GCS [10]. Nrf2, a redox-sensitive transcription factor capable of interacting with anti-oxidant response elements (AREs), plays a key role in the transcriptional activation of antioxidant enzyme gene expression [11] Recent evidence has revealed that the induction of antioxidant enzymes through Nrf2 activation protects against oxidative stress in the heart [12,13]. In addition, mitogenactivated protein kinases (MAPK), including three major subgroups ERK1/2, JNK, and P38, are also shown to be responsible for ISO-
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mediated cardiomyocyte apoptosis. For example, it has been shown that the protein level of phosphorylated ERK1/2, JNK, and P38 was inhibited in hypoxia-reoxygenation H9c2 cardiomyocytes [14]. Therefore, an intriguing question is whether CALB-3 activates Nrf2 and the subsequent up-regulation of antioxidant enzyme expression to suppress cardiomyocyte apoptosis by affecting the phosphorylation of the PI3K/Akt and MAPK signaling pathway. In the present study, we aimed to further identify the protective effects of CALB-3 and investigate its potential molecular action mechanisms against isoproterenol (ISO)-mediated myocardial injury in vivo, focusing on the PI3K/Akt/Nrf2 signal transduction pathway. 2. Materials and methods
distilled water, whereas Groups 3, 4, and 5 were dosed intragastrically with CALB-3 (50, 100, and 200 mg/kg, respectively) for 21 day. Within 2 h of CALB-3 administration on days 19, 20, and 21, rats in the groups 2 to 5 were injected with ISO (2 mg/kg, i.p.), whereas Groups 1 was injected with saline. At the end of the study period, all rats were weighed, and then anesthetized with 20% ethyl urethane by i.p. After sacrificing the rats, their heart was quickly and meticulously harvested for histopathological and biochemical analyses 24 h after the final injection. 2.3.2. Heart rate and electrocardiography The measurements of heart rate (HR) and electrocardiography (ECG) were carried out on day 21 using the 16-Channel Advanced Research Workstation (MP150; BIOPAC Systems, Inc., CA, USA) [15].
2.1. Plant materials and preparation of CALB-3 2.1.1. Plant materials Dry immature fruits of Citrus aurantium L. were collected in May 2014 from Xingan in Jiangxi Province, China and identified by Prof. Zhenyue Wang, a pharmacognosist from the Department of Pharmacognosy, School of Pharmacy, Heilongjiang University of Chinese Medicine (Harbin China), where the voucher specimen (20140018) has been deposited. 2.1.2. Preparation of CALB-3 CALB-3, a purified acidic hetero-polysaccharide isolated from Fructus aurantii, was extracted and purified according to previously reported procedures [7]. Using the phenol-sulfuric acid colorimetric method and HPLC analysis, a single and symmetrical peak was shown for CALB-3, which indicated that CALB-3 is a homogeneously pure polysaccharide based on the distribution of molecular weight. 2.2. Materials The kits for determining the content of malondialdehyde and activity of creatine kinase (CK), catalase (CAT), aspartate aminotransferase (AST), lactate dehydrogenase, glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), and superoxide dismutase (SOD) were acquired from Jiancheng Bioengineering Institute (Nanjing, China). ROS and caspase-3 ELISA kits were purchased from RapidBio Lab (Calabasas, CA, USA). The terminal deoxynucleotidyl transferasemediated dUTP nick end-labeling (TUNEL) assay kit was purchased from Roche Diagnostics GmbH (Mannheim, Germany). Protein extraction kits were purchased from CoWin Bioscience Co., Ltd. (Beijing, China). A BCA protein assay kit was purchased from Pierce Corporation (Rockford, USA). Primary antibodies against PI3K, p-PI3K, Akt, p-Akt, ERK, p-ERK, JNK, p-JNK, P38, p-P38, Nrf2, Bcl-2, Bax, HO-1, NQO-1, GCLM, γ-GCS, lamin B, and β-actin were obtained from Santa Cruz Biotechnology (USA) and other chemicals were purchased from SigmaAldrich (St. Louis, MO, USA). 2.3. Methods 2.3.1. Animals and experimental protocols Healthy Sprague-Dawley (SD) male rats (200 ± 20 g) were used in the experiments. The animals were housed under the following standard conditions: temperature, 24 ± 2 °C; relative humidity, 60% ± 10%; 12-h light/dark cycle; standard rodent chow; and free access to water. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of the Guangdong Pharmaceutical University and approved by the Animal Ethics Committee of Guangdong Pharmaceutical University. One hundred SD rats were randomly divided into the following five groups: Group 1, Control; Group 2, ISO treatment; Groups 3, 4, and 5, ISO combined with CALB-3 (50, 100, and 200 mg/kg, respectively). Groups 1 and 2 were dosed intragastrically with the same volume of
2.3.3. Preparation of samples and measurement of biochemical variables After ECG, blood was collected from the abdominal aorta, and then centrifuged to collect the serum. Serum samples were assayed to determine myocardial enzymes (LDH, CK, and AST) using kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) on an automated chemical analyzer (7060; Hitachi, Tokyo, Japan). Subsequently, the heart was immediately removed after perfusion with saline. The left ventricle (LVs) was excised for histopathological examination, and cardiac homogenates were used to determine the content of MDA, and activity of T-AOC, SOD, CAT, and GSH-Px using the corresponding kits, according to the manufacturers' instructions. ROS were detected using the corresponding ELISA kits (RapidBio Lab, Calabasas, CA, USA). 2.3.4. Heart histopathological examination (HE) The LV was harvested and fixed with 4% paraformaldehyde for 48–52 h. The heart was then dissected and embedded in paraffin blocks, sectioned, stained with hematoxylin and eosin (H&E), and examined under a light microscope (CKX41; 170 Olympus, Tokyo, Japan) by a pathologist, who was blinded to the study groups [16]. 2.3.5. TUNEL assay DNA fragmentation was detected using the TUNEL assay, according to the apoptosis kit instructions. After dewaxing and rehydration, the heart sections were incubated with proteinase K (20 mg/mL) for 20 min at 22 °C. The slices were washed with PBS, and then incubated with working-strength terminal deoxynucleotidyl transferase enzyme in a humidor for 60 min at 37 °C. The slices were again washed with PBS and incubated with working-strength anti-digoxigenin conjugate for 30 min at room temperature. The slices were stained with 4,5diamino-2-phenylindole (DAPI) and observed under a fluorescence microscope (Leica, Heidelberg, Germany) [16]. 2.3.6. Western blot analysis The cardiac muscle tissues were harvested, washed with PBS, and lysed with protein extraction reagent containing 1% phenylmethylsulfonyl fluoride on ice. The supernatant was collected after centrifuging for 15 min at 12,000 rpm and 4 °C, and the protein concentration was measured using a BCA protein assay kit. Equal amounts of protein from each sample were separated by 10% SDSPAGE, and transferred onto nitrocellulose membranes (Millipore Corporation, Bedford, MD, USA) in Tris-glycine buffer at 100 V for 55 min in an ice box. The membranes were blocked with 5% (w/v) non-fat milk powder in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBST) for 2 h at room temperature. The membranes were then incubated overnight at 4 °C with the primary antibodies (PI3K, p-PI3K, Akt, p-Akt, ERK, p-ERK, JNK, p-JNK, P38, p-P38, Nrf2, Bcl-2, Bax, HO-1, NQO-1, GCLM, γ-GCS, lamin B, and β-actin; all ratios were 1:500; Santa Cruz Biotechnology). Subsequently, the membranes were washed three times with TBST, incubated with appropriate secondary HRPconjugated antibodies (1:1000) for 2 h on a shaking table, and washed with TBST three times for 45 min. Enhanced chemiluminescence
Please cite this article as: Y. Yang, Z. Ding, R. Zhong, et al., Cardioprotective effects of a Fructus Aurantii polysaccharide in isoproterenol-induced myocardial ischemic rats..., , https://doi.org/10.1016/j.ijbiomac.2019.11.063
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solution was used to develop the blots for 5 min, and then analyzed using Image Lab software (Bio-Rad, USA). The different protein levels are expressed as a percentage of the control, calculated using Gel-Pro Analyzer software [17]. 2.4. Statistical analysis All experiments were repeated a minimum of three times. The experimental results are expressed as mean ± SD. The analysis of oneway analysis of variance (ANOVA) followed by Student–Newman– Keuls test for pairwise comparisons was used for the intergroup analysis. The results with P b 0.05 were considered statistically significant. 3. Results 3.1. Body weight and heart weight There were no significant differences in the body weight among the groups (Table 1). There was a statistically significant increase in the heart weight of the ISO-treated rats compared with that of the other group rats (P b 0.05). CALB-3 treatment decreased the heart weight significantly in a dose-dependent manner (P b 0.05). 3.2. Heart rate The HR values were significantly decreased in the ISO-treated rats compared to those of the control group; whereas the HR values were significantly increased in the three CALB-3 groups compared with those of the ISO group (P b 0.05; Table 1). 3.3. ECG analysis To evaluate the protective effects of CALB-3 on myocardial injury, we measured ECG changes in each group. As shown in Fig. 1, the ISO treatment significantly impaired the rat heart. The ST segment elevation was significantly prolonged (3.22-fold that of the control; P b 0.01) in the ISO group compared with that in the control group. However, the elevation of the ST segment was significantly reduced when the rats received pretreatment of 50, 100, and 200 mg/kg CALB-3, compared with that of the control (0.779 ± 0.088, 0.975 ± 0.103, and 1.137 ± 0.131 vs. 1.452 ± 0.134, respectively; P b 0.01); the effect was dose dependent. 3.4. CALB-3 prevents ISO-induced myocardial injury To analyze the cardioprotective effect of CALB-3, we measured the expression of serum cardiac enzymes (LDH, CK, and AST) using the ELISA methods in different groups. As shown in Fig. 2A, ISO exposure induced myocardial damage, resulting in significantly increased levels of the myocardial enzymes LDH, CK, and AST. Nevertheless, the pretreatment with different doses of CALB-3 before ISO exposure significantly ameliorated these effects (P b 0.05 or P b 0.01) in a dose-dependent manner. In addition, using H&E staining, the pathological changes in t1:1 t1:2
Table 1 Effects of CALB-3 on the levels of body, heart weight, and heart rate (HR).
t1:3 t1:4
Groups (n = 10)
Body weight (g)
Heart weight (g)
Heart rate (beats/min)
t1:5 t1:6 t1:7 t1:8 t1:9
Control ISO CA-high CA-middle CA-low
293.71 ± 22.4 279.32 ± 18.5 288.96 ± 17.5 284.47 ± 16.2 283.71 ± 16.6
0.91 ± 0.05 1.24 ± 0.12# 0.96 ± 0.08⁎⁎ 0.98 ± 0.07⁎⁎ 0.98 ± 0.09⁎⁎
437.61 ± 30.76 350.47 ± 36.01# 428.72 ± 21.77⁎⁎ 418.09 ± 26.28⁎⁎
t1:10 t1:11 t1:12 t1:13 t1:14
373.21 ± 18.90
ISO, isoproterenol; CA-high, high dose of CALB-3; CA-middle, middle dose of CALB-3; CAlow, low dose of CALB-3. Data are expressed as mean ± SD. # P b 0.01 vs the control group. ⁎⁎ P b 0.05 vs the ISO group.
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myocardial tissue were detected by light microscopy. As shown in Fig. 2B, no obvious abnormalities were observed in the control group, whereas severe myocardial damage was observed in the ISO group, characterized by myocardial fiber fracture, interstitial widening, local wavy cytoplasm dissolution, and membrane rupture. Pretreatment with CALB-3 at three different doses significantly improved the above pathological changes. This result was consistent with above results, suggesting that CALB-3 has protective effect against myocardial injury induced by ISO in rats. 3.5. CALB-3 inhibits the ISO-induced cardiomyocyte apoptosis The caspase enzymes regulate several events leading to cellular and biochemical changes associated with apoptosis [14]. The activation of caspase-3, a typical pro-apoptotic protein, was evaluated. As shown in Fig. 3A, the caspase-3 activity was significantly increased by ISO (5.4fold that of the control; P b 0.01); however, pretreatment with CALB-3 inhibited this activity in a dose-dependent manner (P b 0.01). Similarly, the caspase-8 activity was also upregulated in the heart tissue treated with ISO, and its activity was downregulated with CALB-3 pretreatment (P b 0.01). Bcl-2 and Bax are two well-known proteins in the Bcl-2 family that regulates mitochondrial outer membrane permeability. The ISOinduced apoptosis was accompanied by decreased expression of the anti-apoptotic protein Bcl-2, but an increased expression of the proapoptotic protein Bax. As shown in Fig. 3B, CALB-3 preconditioning reversed the effects of ISO by decreasing Bax (2.89, 2.40, and 1.94 vs. 3.58, respectively; P b 0.05 or P b 0.01) and increasing Bcl-2 (0.849 and 0.907 vs. 0.682, respectively; P b 0.05 or P b 0.01) expression level in rat myocardial tissue in a dose-dependent manner. Additionally, the TUNEL assay was performed to investigate the effects of CALB-3 on cardiomyocyte apoptosis. The administration of ISO caused DNA fragmentation in nucleosomes. As shown in Fig. 4, the number of TUNEL-positive cells increased considerably in the ISO group compared with that in the control group, and CALB-3 pre-administration significantly reduced the number of TUNEL-positive cells compared with that in the ISO group in a dose-dependent manner. The above results suggested that CALB-3 can inhibit ISO-mediated apoptosis via the mitochondria-dependent apoptotic pathway. 3.6. CALB-3 enhances antioxidant capacity in cardiac tissues To determine whether the protective effects of CALB-3 against ISOinduced myocardial injury were due to its antioxidative activity, we measured oxidative stress-related parameters of the heart. As shown in Fig. 5A, we found that ISO significantly reduced the SOD, CAT, and GSH-Px activities and T-AOC level in the heart compared with those of the control group. Pretreatment with different doses of CALB-3 before ISO administration significantly ameliorated these effects. Furthermore, ISO induced an increase in MDA production and intracellular ROS levels, and pretreatment with CALB-3 decreased the levels of MDA and ROS in a dose-dependent manner (P b 0.05 or P b 0.01). However, with western blotting, we detected the other four important antioxidant enzymes, HO-1, NQO-1, GCLM, and γ-GCS. In agreement with our ELISA results, the activity of the four enzymes was considerably inhibited in ISOtreated rats compared with that in the control. This was reversed by CALB-3 treatment in a dose-dependent manner (P b 0.05 or P b 0.01; Fig. 5B). These results suggest that CALB-3 can considerably enhance the antioxidative capacity to scavenge ROS against ISO-induced oxidative stress. 3.7. CALB-3-induced nuclear accumulation of the Nrf2 protein Given that activated Nrf2 is the major transcription factor that enhances the expression of antioxidative enzyme genes [18,19], we attempted to measure the nuclear accumulation of Nrf2 in ISOinduced myocardial tissue. Western blotting showed that ISO
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Fig. 1. CALB-3 improved cardiac function in ISO-induced rats, ST depression, n = 8; The values represent the means ± SEMs. P b 0.01 vs the control group; *P b 0.05, **P b 0.01 vs the model group.
significantly downregulated the expression of nuclear Nrf2 and inhibited Nrf2 translocation from the cytosol to the nucleus. However, this could be reversed by the pretreatment with CALB-3 in a dosedependent manner (P b 0.05 or P b 0.01; Fig. 5B). 3.8. CALB-3 inhibits ISO-induced activation of ERK1/2 MAPK in cardiac tissues Previous studies have shown that ISO can result in the apoptosis of cardiomyocytes via the activation of MAPK, including ERK, JNK, and p38 MAPK [20]. Therefore, we investigated the effect of CALB-3 on the phosphorylation level of MAPK in ISO-induced rats by western blotting. As shown in Fig. 6, the protein level of total ERK1/2, JNK1/2, and P38 MAPK was almost unchanged in the ISO and ISO + CALB-3 (50, 100,
and 200 mg/kg) groups compared with that in the control group. However, the protein level of p-JNK1/2 and p-P38 was increased, whereas that of p-ERK1/2 was decreased in the ISO groups, demonstrating the activation of MAPK by ISO. Interestingly, the reduced phosphorylation level of ERK1/2 was increased, whereas that of JNK1/2 and P38 remained unchanged in the ISO + CALB-3 group compared with that in the ISO groups, suggesting the involvement of ERK in the protective effect of CALB-3 against ISO-induced myocardial injury. 3.9. CALB-3 enhances antioxidant capacity via PI3K/Akt signaling The PI3K/Akt signaling pathway plays key roles in ISO-induced oxidative stress and apoptosis. Numerous studies have indicated that they are the signal upstream of Nrf2 [21–23]. As shown in
Fig. 2. CALB-3 prevented the ISO-induced myocardial injury. (A) The levels of myocardial enzymes (LDH, CK, and AST) were determined, n = 9 or 10 per group; (B) representative images of HE staining of heart tissue, n = 3. The values represent the means ± SEMs. #P b 0.01 vs the control group; *P b 0.05, **P b 0.01 vs the model group.
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Fig. 3. CALB-3 protected against ISO-induced myocardial injury through by improving the internal mitochondrial apoptotic pathway. (A) The activities of caspase-3 and -8 were measured using a fluorometric assay, n = 8; (B) the western blot analysis of Bcl-2, Bax and β-actin, n = 4; the values represent the means ± SEMs. #P b 0.01 vs the control group; *P b 0.05, **P b 0.01 vs the model group.
Fig. 7, western blotting showed that ISO significantly reduced the ratio of PI3K/Akt phosphorylation in the heart (P b 0.05 or P b 0.01); however, the effect was neutralized by treatment with CALB-3 in a dose-dependent manner. The results showed that CALB-3 exerted an anti-oxidative effect by activating the phosphorylation of PI3K/Akt to protect against ISO.
4. Discussion Fructus aurantii has been used as a medicinal herb in Traditional Chinese Medicine for thousands of years, particularly to treat cardiovascular diseases. Both flavonoid glycosides and polymethoxylated flavones, including naringin, hesperidin, tangeratin, and neohesperidin, are
Fig. 4. TUNEL staining, n = 4. The values represent the means ± SEMs. Arrows indicate apoptotic cells. #P b 0.01 vs the control group; *P b 0.05, **P b 0.01 vs the model group.
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Fig. 5. CALB-3 promotes the expression of Nrf2-mediated anti-oxidative enzymes. (A) The effects of CALB-3 on T-AOC, SOD, CAT, and GSH-Px activities as well as MDA and ROS level, n = 9 or 10 per group. (B) The relative expression levels of HO-1, NQO-1, GCLM, γ-GCS, Nucl-Nrf2, and Cyto-Nrf2 relative to β-actin and Lamin B and the bar graphs, n = 4. The values represent the means ± SEMs. #P b 0.01 vs the control group; *P b 0.05, **P b 0.01 vs the model group.
consistently regarded as the main active ingredients in the prevention and treatment of ischemic heart disease [24,25]. Interestingly, in our previous studies, polysaccharides, another main active ingredient of FA, also exhibited strong antioxidant effects in vitro and in vivo. Among the four purified polysaccharides, CALB-3 had the strongest antioxidant activity and its structure was elucidated by FTIR, SEM, AFM, partial acid hydrolysis, periodate oxidation, Smith degradation, methylation analysis, GC–MS, and NMR [7]. In the present study, we aimed to further investigate the protective effects of CALB-3 against ISOinduced myocardial ischemia in vivo. It is important to note that myocardial infarction continues to be the major cause of cardiovascular diseases and occurs when the blood supply from the coronary artery to the heart is blocked, resulting in irreversible damage or death of heart tissue [26–28]. Therefore, the pathogenesis and therapeutic targets of acute myocardial injury are currently popular research topics. Apoptosis of cardiac myocytes induced by oxidative stress plays a pivotal role in the pathogenesis of IHD. Therefore, the elimination of excessive intracellular ROS and prevention of oxidative stress-induced apoptosis may serve as a beneficial intervention for the treatment of these diseases. In the present study, the ISO treatment group showed a considerable increase in heart weight compared with that of the control group. One possible explanation for these findings is that ISO may lead to hypertrophy of the heart tissue and reduction of the heart rate. Similarly, Kalkan showed that ISO treatment causes a significant increase in heart weight
and decrease in heart rate [29]. This study definitively demonstrates that ISO treatment induces morphologic abnormalities and that CALB3 treatment reverses the ECG changes associated with cardiocytes and cell damage. In addition, the LDH, CK, and AST levels were increased in blood during myocardial infarction. These enzymes are released into the blood stream from cells when the cell membrane ruptures and serve as a diagnostic tool. Unlike those in the experiment model, CALB-3 administration reduced the level of serum cardiac enzymes and ameliorated histological damage in the heart, indicating that CALB-3 exerted a pronounced protective effect against myocardial injury. Apoptosis may be initiated either by the intrinsic (mitochondrial) or extrinsic (death receptor-dependent) pathways [30]. Caspases are a family of aspartate-specific cysteine proteases that play key roles in regulating the two pathways of apoptosis induced by different stimuli, including oxidative stress [31]. Caspase-3 and -8 are the key executioners of apoptosis and regulating factors involved in DNA degradation, chromatin condensation, and nuclear fragmentation [32]. Caspase-3 and -8 were activated by ISO treatment, and administration of CALB-3 inhibited the activation. Moreover, CALB-3 pretreatment inhibited TUNEL-positive cells and modulated the protein expression of the Bcl-2 family of proteins. CALB-3 pretreatment increased the expression of Bcl-2, as it decreased the expression of pro-apoptotic Bax. Therefore, CALB-3 may exert protective effects by maintaining mitochondrial function and modulating the balance between anti-
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on the heart tissue [35]. The results revealed that ISO treatment led to a decrease in antioxidant enzyme activities. Similarly, Ilhan et al. showed that the SOD, GSH-Px, and CAT enzyme activities decreased in ISO model rats when compared with those in the control group [36]. Our study demonstrated that ISO is a powerful oxidizing agent that caused the release of ROS and led to significant oxidative damage in rat heart tissue. However, CALB-3 exerted its beneficial effects on the heart tissue and protected against the toxic effects of ISO via its strong antioxidant properties. Serine/threonine kinase Akt is an important component of the intracellular signaling pathways tightly linked with apoptosis and oxidative stress [37]. Akt signaling can affect communication with other signaling pathways including Nrf2. Furthermore, our previous studies have confirmed that phosphorylation of Akt regulates Nrf2 activation and HO-1, NQO-1, GLLM, and γ-GCS expression. As we hypothesized, not only phospho-Akt was inhibited, but also phospho-ERK was inhibited in the ISO-Induced myocardial ischemia rats; however, both processes were reversed by CALB-3 in a dose-dependent manner. 5. Conclusions
Fig. 6. Effect of CALB-3 on phosphorylation levels of ERK, JNK, and p38 MAPK in ISOinduced rats. The ERK, JNK, and p38 MAPK phosphorylation were determined by western blotting and densitometric quantification is shown in the bar diagrams, n = 4. The values represent the means ± SEMs. #P b 0.01 vs the control group; *P b 0.05, **P b 0.01 vs the model group.
apoptotic protein Bcl-2 and pro-apoptotic protein Bax, thus inhibiting the release of pro-apoptotic molecules from the mitochondrial intermembrane space into the cytoplasm. A growing body of evidence indicates that the MAPK signaling pathway is involved in the Bcl-2 family-mediated mitochondria-dependent apoptotic pathway in ISO-induced rats [33,34] For example, Verma et al. reported that ISO could induce apoptosis via the upregulation of JNK and p38 MAPK in the heart of rats, while ERK is usually downregulated in acute myocardial ischemia [20]. The results reported are agreement with our findings. In addition, we found that CALB-3 only affected ERK phosphorylation, and had no effect on p-JNK and p-P38. Therefore, we surmise that CALB-3, an acidic hetero-polysaccharide, may differentially regulate three cascades of MAPK in ISO-induced rat model. To test whether a close association existed between oxidative stress and cell apoptosis induced by ISO, biochemical analysis was performed
In summary, this is the first study to show that an acidic FA polysaccharide exerts cardioprotective effect in an ISO-induced rat model. This occurred via the activation of Akt and ERK signaling as well as the suppression of cardiac oxidative stress and apoptosis. Accordingly, we believe that CALB-3 should be considered as a potential therapeutic medicine to prevent myocardial ischemia. Data availability The data used to support the findings of this study are included within the article. Declaration of competing interest The authors declare that they have no conflicts of interest. Acknowledgments This study was supported by the National Natural Science Foundation of China (Grant Nos. 81603366), the Natural Science Foundation of Heilongjiang Province (Grant Nos. H2015041), the China Postdoctoral Science Foundation (Grant Nos. 2017M610816), and the Foundation of Postdoctoral Science of Chinese Academy of Medical Sciences & Peking Union Medical College (2016). References
Fig. 7. CALB-3 enhances antioxidant capacity by the activity of PI3K/Akt signaling. The PI3K and Akt phosphorylation were determined by western blotting and densitometric quantification is shown in the bar diagrams, n = 4. The values represent the means ± SEMs. #P b 0.01 vs the control group; *P b 0.05, **P b 0.01 vs the model group.
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Please cite this article as: Y. Yang, Z. Ding, R. Zhong, et al., Cardioprotective effects of a Fructus Aurantii polysaccharide in isoproterenol-induced myocardial ischemic rats..., , https://doi.org/10.1016/j.ijbiomac.2019.11.063