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Akt signaling pathway in myocardial ischemia injury
Biomedicine & Pharmacotherapy 98 (2018) 308–317
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Cardioprotective effects of total flavonoids from Jinhe Yangxin prescription by activating the PI3K/Akt signaling pathway in myocardial ischemia injury
T
Yangang Chenga,1, Jinyan Tana,1, Huifeng Lib, Xiangpeng Kongb, Yan Liua, Rui Guob, Guoyan Lib, ⁎ Bingyou Yanga, Miaorong Peia,b, a
Key Laboratory of Chinese Materia Medica (Ministry of Education), Heilongjiang University of Chinese Medicine, 24 Heping Road, Xiangfang District, Harbin 150040, PR China b School of Chinese Pharmacy, Shanxi University of Chinese Medicine, 121 Daxue Road, Yuci District, Jinzhong 030619, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Myocardial ischemia PI3K/Akt Flavonoids Oxidative stress Apoptosis Herb pairs
The purpose of the present study was to investigate the cardioprotective effects of total flavonoids of Jinhe yangxin prescription (JHTF) on myocardial ischemia (MI) injury rats induced by Isoproterenol (ISO) and explore the potential mechanisms underlying these effects. 128 male rats were randomized into 8 groups: Control, Model, Positive, JHTF-H (2.64 g/kg/d), JHTF-M (1.32 g/kg/day), JHTF-L (0.66 g/kg/d), LY + JHTF (JHTF-H plus LY294002, an inhibitor of PI3K/Akt) and LY groups. Electrocardiogram, histopathological examination and terminal deoxynucleotidyl transferase dUTP nickend labeling (TUNEL) assay were performed. Heart weight index, markers of cardiac marker enzymes [creatine kinase (CK), creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) and cardiac troponin I (cTnI)], oxidative stress [superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px) and nitric oxide (NO)] and inflammation [tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6)] were also measured in each group. Proteins involved in PI3K/Akt pathway were detected by Western blot. JHTF decreased the ST elevation induced by MI, decreased serum levels of CK, CK-MB, cTnI, LDH, MDA, IL-6 and TNF-α, and increased serum SOD, GSH-Px and NO activities. Furthermore, JHTF inhibited myocardial apoptosis, which may be related to downregulated caspase-3 and Bax, upregulated Bcl-2, and increased the protein levels of phosphorylated Akt, GSK-3β and endothelial nitric oxide synthase (eNOS). However, all the previously mentioned effects of JHTF were blocked when JHTF was coadministered with LY294002. In conclusion, these observations indicated that JHTF has cardioprotective effects against MI, and these effects seem to be related to the activation of PI3K/Akt signaling pathway in the myocardium.
1. Introduction
damage. Oxidative stress is regarded as excessive generation or accumulation of free radical and decrease of antioxidant enzymes [5–7]. Oxidative stress resulting from production of reactive oxygen species (ROS) play an important role in cardiac remodeling and heart failure [8]. ROS are biological molecules that play important roles in cardiovascular physiology and contribute to disease initiation, progression, and severity [9]. Increasing evidences also suggest the importance of sterile inflammatory response in MI pathogenesis, which is involved in mediating impaired myocardial function and heart failure. So, therapeutic approaches that target components of the oxidative stress and inflammatory response have been investigated as potential and useful
Myocardial ischemia (MI), a major pathogenic factor for cardiovascular diseases, is a pathological condition characterized by a restriction of blood in the heart, resulting in the heart does not receive adequate oxygen-rich blood to keep up with its metabolic requirements [1,2]. Around 40% of death are caused by cardiovascular diseases and MI is the leading reason. MI is also the leading cause of cardiac injury, including myocardial infarction and life-threatening arrhythmias [3,4]. Recently, considerable experimental evidences have been proven that oxidative stress should be a major apoptotic stimulus in MI
Abbreviations: cTnI, cardiac troponin I; CK, creatine kinase; CK-MB, creatine kinase-MB; DF, Dijincao- Fenxinmu; DX, Dijincao-Xianhecao; ECG, electrocardiogram; eNOS, endothelial nitric oxide synthase; GSH-Px, glutathione peroxidase; HWI, heart weight index; IL-6, interleukin-6; ISO, Isoproterenol; JHTF, total flavonoids of Jinhe yangxin prescription; LDH, lactate dehydrogenase; MDA, malondialdehyde; MI, myocardial ischemia; NO, nitric oxide; PI3K-Akt, phosphoinositide 3-kinases/serine/threonine protein kinase; ROS, reactive oxygen species; SOD, superoxide dismutase; TCM, traditional Chinese medicine; TNF-α, tumor necrosis factor-alpha; TUNEL, terminal deoxynucleotidyl transferase dUTP nickend labeling ⁎ Corresponding author at: Key Laboratory of Chinese Materia Medica (Ministry of Education), Heilongjiang University of Chinese Medicine, 24 Heping Road, Xiangfang District, Harbin 150040, PR China. E-mail address: [email protected] (M. Pei). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2017.12.052 Received 1 September 2017; Received in revised form 4 December 2017; Accepted 13 December 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
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height of chromatographic column was 1:6, the flow speed for adsorption was 2 BV/h, then cleaned by 1.5 BV deionized water and eluated by 3 BV 50% ethanol at the speed of 2 BV/h, respectively. Eluate was then collected, concentrated and freeze dried in FDU-1200 lyophilizer (Tokyo Rikakikai Co., Ltd., Japan), thus JHTF was obtained. With rutin as reference substance, the content of total flavonoids in JHTF was determined by ultraviolet spectrophotometry and expressed as mg rutin equivalents per gram dry weight (mg RE/g DW). The total flavonoids content of JHTF was 855.40 mg RE/g DW.
treatments for MI injury [10]. Previous studies indicate that the phosphoinositide 3-kinases/ serine/threonine protein kinase (PI3K-Akt) signaling pathway is an endogenous negative feedback regulator which limits proinflammatory and apoptotic events in response to harmful stimuli. Active Akt is a downstream effector of PI3K, which can inhibit apoptosis by regulating multiple target such as TNF-α, eNOS, and Bcl-2 family [11]. Activation of PI3K/Akt signaling pathway could suppress cardiac myocyte and prevent heart [12] . Traditional Chinese medicine (TCM) has played an important role in clinical therapy for MI, especially the flavonoids, which are a vast group of polyphenols found ubiquitously in TCM, and have received more and more attention due to their pharmacological activities such as antioxidation, scavenging free radicals, anti-inflammation, regulating blood lipid and reducing cardiovascular damage [13,14]. Herb pairs (mixture of two or more herbs) is a basic essential element of Chinese herbal formulas, which are much simpler than other complex formulas without altering the basic therapeutic features, meanwhile, the herb pair presents significantly better pharmacological efficacy than the individual plants [15,16]. Jinhe yangxin prescription is a combination of two herb pairs, namely Dijincao [dry entire grass of Euphorbia humifusa Willd]-Xianhecao [dry aerial parts of Agrimomia pilosa ledeb.] (DX) and Dijincao-Fenxinmu [wooden diaphragma of the kernel of Juglans regia L] (DF), which are recorded in Shijinmoduiyao written by Lv Jingshan, a famous TCM master in China [17–19]. DX and DF are usually used for the treatment of various heart diseases in clinically by master Lv. The present experiments were aimed to explore the cardioprotective effects of total flavonoids from jinhe yangxin prescription (JHTF) against MI injury induced by ISO in rats and investigated the underlying myocardial protective mechanisms.
2.3. Animals Male Wistar rats (200 ± 20 g) were supplied by the Experimental Animal Center of the Academy of Military Medical Sciences (Beijing, China). Rats were housed under controlled conditions with a 12 h lightdark cycle at 25 ± 2 ℃ and 45 ± 5% relative humidity. Standard food and water were provided ad libitum. All rats were acclimated to the facility for 7 days. All protocols for experiments were performed in accordance with the Animal Ethics Committee of Shanxi University of TCM (Shanxi, China) on May 22, 2016 (number 2016052202). 2.4. Induction of MI in rats 128 male Wistar rats were randomly assigned into 8 groups (n = 16 per group) named Control, Model, Positive, High-dose JHTF (JHTF-H), Middle-dose JHTF (JHTF-M), Low-dose JHTF (JHTF-L), LY294002 plus High-dose JHTF (LY + JHTF) and LY groups. JHTF-H, JHTF-M, JHTF-L and LY + JHTF groups were given 2.64, 1.32, 0.66, and 2.64 g/kg/d of JHTF, respectively, whereas Positive group was given 20 mg/kg/d of PH. Other groups (Control, Model and LY groups) were administered distilled water. All treatments were administrated intragastrically for a period of 14 days. Except for the Control group, all rats were injected subcutaneously with ISO at a dosage of 85 mg/kg/d on the 12th, 13th and 14th day to set up MI model, whereas LY + JHTF and LY groups were intraperitoneal injection (ip) with LY294002 (0.3 mg/kg/d) prior to injected subcutaneously with ISO. Control group was injected with equivalent volume of sterile saline to instead of ISO. Twenty-four hours after the third ISO injection, the rats were anesthetized by 10% chloral hydrate (ip, 3 mL/kg). The blood was collected and processed for further biochemical estimations and then heart tissue was excised immediately, washed with chilled isotonic saline.
2. Materials and methods 2.1. Materials Dijicao, Xianhecao and Fenximu were purchased from Anguo Chinese Medicine market (Hebei, China). The pharmaceutical botany of the these TCM materials were identified by Prof. Xiangping-Pei from Shanxi University of Chinese Medicine (Shanxi, China). Voucher specimens (NO. SXUTCM2016-1101; NO. SXUTCM2016- 1102 and NO. SXUTCM2016-1103, respectively) were deposited in the department of medicinal plant, Shanxi University of Chinese Medicine (Shanxi, China). The standard of rutin (100080-201202, 98.0% purity), was obtained from National Institutes for Food and Drug Control (Beijing, China). Isoproterenol (ISO), propranolol hydrochloride (PH, positive) and LY294002 (a PI3K inhibitor) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Creatine kinase (CK), creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), interleukin-6 (IL-6) and tumor necrosis factorα (TNF-α) ELISA kits were obtained from Sangon Biotech Co., Ltd. (Shanhai, China). Lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide (NO), glutathione peroxidase (GSH-Px) assay kits and situ TUNEL apoptosis detection kit were acquired from Jiancheng Bioengineering Research Institute (Nanjing, China).
2.5. Electrocardiogram detection The electrocardiogram (ECG) test (Lead II) was conducted in anesthetized rats before the first injection and after finishing the final injection of ISO. The needle electrodes were linked to the right arm, left arm and left leg of the rats, and the electrocardiographic-patterns were recorded with an ECG recording and analysis system (BL-420F, Chengdu TME Technology Company, Chengdu, China). 2.6. Heart weight index determination After rats were sacrificed, the heart tissues were excised (excluding large blood vessels, and connective tissue), and weighed after blotting with filter paper. The heart weight index (HWI) was computed as HWI = heart weight (HW)/body weight (BW).
2.2. Preparation of JHTF A mixture of Dijincao (500 g), Xianhecao (1000 g) and Fenxinmu (500 g) was refluxed twice with 55% ethanol (1:17, w/v) for 147 min, and the collected extract was filtrated and vacuum evaporated with rotary evaporation at 40 ℃ to obtain extract at a final concentration of 0.5 g/mL (w/v, expressed as the weight of raw materials). The total flavonoids were enriched by the AB-8 macroporous resin chromatography, and the purification technologies were as follows: the volume of chromatographic column was 1600 mL, the ratio of diameter to
2.7. Cardiac marker enzymes After the ECG detection, arterial blood samples were collected and centrifuged (3000 rpm for 15 min) to obtain serum, and then stored at −80 ℃. Serum samples were thawed at room temperature before analysis. Myocardial damage was evaluated by measuring the serum levels of CK, CK-MB, cTnI and LDH according to the manufacturer's 309
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Fig. 1. Effects of JHTF on ST-segment elevation. Values represent the mean ± SD,n = 16. ## p < 0.01 vs. control group; **p < 0.01 vs. model group; ++p < 0.01 vs. JHTF-H group.
instructions of kits.
2.10. Histological studies The hearts were removed immediately after the sacrifice of the rats, and fixed in 10% formalin solution. The heart tissue was processed for sectioning and staining by standard histological methods. Sections (5 mm, Leica RM 2125, Germany) from the left ventricle were stained with hematoxylin and eosin (H&E) and examined by light microscopy (Nikon, Tokyo, Japan) at 200 × magnification.
2.8. Antioxidant enzymes assay SOD, MDA, GSH-Px and NO levels in serum were measured using commercial kits according to the manufacturer's protocols.
2.9. Inflammation assay 2.11. Determination of myocardial apoptosis IL-6 and TNF-α levels of serum were determined by standard commercial kits to explore the relationship between the cardioprotective effects of JHTF and the levels of inflammatory cytokines.
Myocardial apoptosis was determined by TUNEL staining according to the manufacturer’s instructions (Jiancheng Bioengineering Research Institute, Nanjing, China). Briefly, after the sacrifice of the rats, the tissue near the cardiac apex was fixed in 10% neutral formalin. 310
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heart tissue proteins involved in PI3K/Akt pathway. The tissue proteins were obtained from the heart 24 h after the third dose of ISO administration and were mechanically homogenized in radioimmunoprecipitation assay buffer lysis buffer (25 mg/mL) with 1 mmol/L Phenylmethanesulfonyl fluoride (PMSF) on ice. The whole lysates were then centrifuged (12,000 rpm for 15 min, 4 ℃) to obtain the supernatant. Total protein was determined using bicinchoninic assay kit (Biyuntian Biotechnology, Wuhan, China). Protein sample of 50 ug was loaded per lane and separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to the poly-vinylidnene fluoride (PVDF) membranes (San Yin-tan, Beijing, China). Afterwards, these membranes were blocked with 5% non-fat skim milk in tris-buffered saline solution containing Tween-20 (TBST; 10 mmol/L tris, pH 7.5; 140 mmoL/L NaCl; 0.1% Tween-20) for 1 h at room temperature, followed by incubated overnight at 4 ℃ with the corresponding primary antibody in TBST containing 3% bovine serum albumin (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA). The primary antibodies were as follows: rabbit anti-Akt (1:1000; 4691), rabbit anti-p-Akt (1:500; 4060), rabbit anti-Caspase-3 (1:500, 9665), rabbit anti-p-GSK-3β (1:1000, 5558), rabbit anti-GSK-3β (1:1000, 12356), rabbit anti-p-eNOS (1:1000, 9570), rabbit anti-eNOS (1:1000, 9586), rabbit anti-Bcl-2 (1:1000; 3498), rabbit anti-Bax (1:1000; 5023; Cell Signaling Technology, Inc., Danvers, MA, USA). Subsequently, the membrane was washed for three times, and the membranes were further incubated with horseradish peroxidase-conjugated secondary antibody anti-rabbit IgG (1:2000) in TBST solution for 1 h. In the end, the signals of detected proteins were visualized by an enhanced chemiluminescence reaction system (Millipore, Billerica, Massachusetts, USA) and the density of each reactive band was quantified using the LabWorks Image Acquisition platform (UVP, Inc., Upland, CA, USA) and ImageJ (National Institutes of Health, Bethesda, MA, USA). β-actin (1:3000, 4970; Cell Signaling Technology, Inc., Danvers, MA, USA) was
Fig. 2. Effects of JHTF on heart weight index. Data are shown as mean ± SD,n = 16. ## p < 0.01 vs. control group; **p < 0.01 vs. model group; *p < 0.05 vs. model group; ++p < 0.01 vs. JHTF-H group.
embedded in paraffin blocks, cut into ultrathin sections (5 μm), deparaffinized, and myocardial apoptosis was examined. Cells with brown stained nuclei indicated TUNEL-positive, apoptotic cells, and the apoptotic cells and bodies were counted in five high-power fields. The apoptotic index (AI) was calculated as the percentage of positively stained cells using the following equation: AI = number of apoptotic cells/total number of nucleated cells. 2.12. Western blot analysis Western blot analysis was used to detect the expression levels of
Fig. 3. Effects of JHTF on serum level of cardiac marker enzymes; CK (A), CK-MB (B), cTnI (C) and LDH (D). Data are expressed as mean ± SD, n = 16. ##p < 0.01 vs. control group; **p < 0.01 vs. model group; *p < 0.05 vs. model group; ++p < 0.01 vs. JHTF-H group.
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Fig. 4. Effects of JHTF on serum level of cardiac oxidative stress parameters; SOD (A), MDA (B), GSH-Px (C) as well as NO (D). Data are shown as mean ± SD, n = 16. control group; **p < 0.01 vs. model group; *p < 0.05 vs. model group; ++p < 0.01 vs. JHTF-H group; +p < 0.05 vs. JHTF-H group.
##
p < 0.01 vs.
HWI became larger (p < 0.01 or p < 0.05, Fig. 2).
used for normalization of protein levels. 2.13. Statistical analysis
3.3. Effects of JHTF on CK, CK-MB, cTnI and LDH
All statistical analyses were carried out using SPSS 17.0 software including one-way ANOVA and Student's t-test. Data were expressed as mean ± standard deviation (SD). Difference between groups were considered as significant when p < 0.05.
CK, CK-MB, cTnI and LDH are identified as essential diagnostic markers of ischemia injury to the myocardial tissues and leak out from damaged myocardium to blood [21]. Current studies measured the contents of CK, CK-MB, cTnI and LDH in serum to evaluate the myocardial damage. As shown in Fig. 3, a marked increase in the levels of CK, CK-MB, cTnI and LDH were detected in MI model rats compared with those in the control group (p < 0.01). While, compared with the model group, the JHTF-H significantly decreased serum CK, CK-MB, cTnI and LDH (p < 0.01). When LY294002 was applied to eliminate the effects of JHTF, the levels of CK and CK-MB were higher in LY + JHTF compared with the JHTF-H group (p < 0.01), and the levels of cTnI, LDH also have the same trend.
3. Results 3.1. Effects of JHTF on ST-segment MI occurs when the blood flow through the heart muscle is decreased by a partial or complete blockage of heart arteries, which causes ST level changes in an ECG signal. A normal ST segment is horizontal, whereas an abnormal ST segment deviates from the horizontal [20]. As depicted in Fig. 1(A and B), ST-segment was significantly elevated in model rats compared with those in the control group (p < 0.01). In groups pre-administrated with PH (Positive) and JHTF-H, ST-segment was significantly decreased as compared with that in the rats of model group (p < 0.01). However, in the presence of LY294002, a specific inhibitor of PI3K/Akt pathway, this effect of JHTF was eliminated compared with JHTF-H (p < 0.01), while LY294002 alone had no significant effect on the ST-segment. These results reflect the positive effect of JHTF on cardiac function.
3.4. Effects of JHTF on SOD, MDA, GSH-Px and NO In this study, we evaluated the levels of SOD, MDA, GSH-Px and NO in the serum to explore the relationship between the cardioprotective effect of JHTF in MI and its antioxidant status. As shown in Fig. 4, compared with control group, the model rats exhibited a significant increase in MDA content, while the levels of SOD, GSH-Px and NO were all significantly decreased, and those changes were reversed in Positive, JHTF-H, JHTF-M and JHTF-L groups (p < 0.01 or p < 0.05). These results suggested the antioxidative stress effect of JHTF in alleviating MI injury. However, LY294002 significantly abolished the effect of JHTF on the levels of SOD, GSH-Px, NO, and it also has a similar effect on the levels of MDA, which indicated that JHTF-attenuated oxidative stress in MI injury was associated with PI3K/Akt pathway.
3.2. Effects of JHTF on heart weight indices HWI was greater in the model group rats than in the control group rats (p < 0.01). Administration with JHTF decreased the HWI compared with the model group rats. As the dose increased, the decrease in 312
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the LY + JHTF group, the reduction of myocardial structure disorder and partial rupture, and lyse of muscle fibers with moderate infiltration of lymphocytes and macrophages were eliminated. Administration of LY294002 alone before ISO-treated had no effect on myocardial tissue (Fig. 6G and H). 3.7. Effects of JHTF on the cardiomyocyte apoptosis Apoptosis is the major mechanism of cell death induced by MI. Herein, myocardial apoptosis was determined by TUNEL staining. TUNEL-positive cells were defined as the death of cells. As shown in Fig. 7, in the model group, the TUNEL-positive cells expressed as the percent of total nucleiwere significantly increased when compared with the control group (p < 0.01). However, the percentage of TUNEL-positive cardiomyocytes was significantly lower in the Positive, JHTF-H, JHTF-M groups compared with the model group (p < 0.01 or p < 0.05), and JHTF-L group also has the same trend. Furthermore, cotreated with LY294002 in the LY + JHTF group significantly attenuated the effect of JHTF on the cardiomyocyte apoptosis compared with the JHTF-H group (p < 0.01). 3.8. Effects of JHTF on the expression of proteins in PI3K/Akt pathway To clarify whether the PI3K/Akt pathway was involved in the cardioprotective effect of JHTF in MI, the expression level of proteins related to PI3K/Akt pathway in the ischemic of myocardial tissue were detected by western blots. As shown in Fig. 8, there were no detectable differences in the expression of total Akt, GSK-3β and eNOS among the six groups. JHTF treatment markedly increased the myocardial levels of p-Akt in rats compared with those in model group (p < 0.01). However, administration of LY294002 could significantly attenuate the JHTF-induced upregulation of myocardial p-Akt in rats. Moreover, JHTF treatment significantly increased the levels of p-GSK-3β, a downstream kinase of Akt, in the myocardium compared with levels in model group (p < 0.01). Administration of the PI3K inhibitor, LY294002, could significantly prevent the JHTF-induced enhancement of phosphorylation of GSK-3β in the MI myocardium (p < 0.01). Akt induced eNOS phosphorylation at serine 1177 is an important mechanism to protect heart against injury. ISO-induced MI resulted in significantly decreased expression of p-eNOS in the myocardium as compared to control group (p < 0.01). JHTF pretreatment markedly increased the expressions of p-eNOS compared with the MI model group (p < 0.01). We further determined whether administration of LY294002 could alter the effect of JHTF on the expression level of p-eNOS. The result showed that cotreatment of LY294002 blocked the eNOS activation induced by JHTF. Furthermore, to determine whether the administration of JHTF could affect the expressions of apoptosis-related proteins, we analyzed the levels of Bcl-2, Bax and caspase-3 in myocardial tissues by Western blot. Obvious increases in Bax and caspase-3 levels and decrease in Bcl2 level were detected in the model group compared with control group (p < 0.01, Fig. 9). While, JHTF treatment markedly downregulated Bax and caspase-3, but upregulated Bcl-2 expressions in comparison to the model group (p < 0.01). However, LY294002 abolished the effects of JHTF on Bcl-2, Bax and caspase-3 expression (p < 0.01).
Fig. 5. Effects of JHTF on serum level of inflammatory markers; IL-6 (A) and TNF-α (B). Data are shown as mean ± SD, n = 16. ##p < 0.01 vs. control group; **p < 0.01 vs. model group; *p < 0.05 vs. model group; ++p < 0.01 vs. JHTF-H group; +p < 0.05 vs. JHTF-H group.
3.5. Effects of JHTF on IL-6 and TNF-α To further evaluate and validate the protective function of JHTF during MI injury, we measured the levels of IL-6 and TNF-α in serum. It was noted that a marked increase in the levels of the IL-6 and TNF-α as compared with that of rats in the control group (p < 0.01, Fig. 5). Notably, the administration of PH and JHTF could inhibit the increase, which indicated that PH and JHTF exhibited markedly alleviation of pro-inflammatory cytokines. However, the serum IL-6 and TNF-α concentrations in LY + JHTF and LY groups remained higher than those in JHTF-H group. These indicated that the activation of PI3K/Akt signaling by JHTF significantly suppressed MI-induced inflammatory cytokine secretion in the serum. 3.6. Effects of JHTF on myocardial histology The representative micrographs of rat myocardial HE staining are shown in Fig. 6. The myocardial structure in the control group exhibited a regular arrangement, normal cardiac muscle fibers and no necrosis (Fig. 6A). However, compared with the control group, the model group demonstrated ruptured cardiac muscle fibers, necrosis with inflammatory cell infiltration and an enlargement of the extracellular space was also observed (Fig. 6B). The myocardial damage in the JHTF groups was less than that in the model group and there was dose-effect relationship between the three JHTF pretreated groups. Cardiac sections obtained from rats undergoing JHTF treatment demonstrated a relatively more intact and less myocardial fiber disruption, necrosis and inflammatory cell infiltration compared with those from the model group (Fig. 6D–F). When co-treated with LY294002 in
4. Discussion In the present study, we demonstrated that JHTF had beneficial effects on cardiac function in MI rats. These effects were in line with the suppressing oxidative stress-triggered damage and inflammatory cytokines, decreasing caspase-3 and Bax expressions, and up-regulating the Bcl-2 expression involving in the PI3K/Akt signaling pathway, and thereby attenuate apoptosis after MI and the ameliorative effects of JHTF presented in a dose-dependent manner. ISO is a synthetic catecholamine and β-adrenergic agonist which at 313
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Fig. 6. Representative slides of HE staining results for myocardial tissues in all groups of rats.A. Control, B. Model, C. Positive, D. JHTF-H, E. JHTF-M, F. JHTF-L, G. LY + JHTF, H. LY (×200).
Fig. 7. Effects of JHTF on myocardial apoptosis induced by ISO in rats. (A-H) representative micrographs of myocardium stained with TUNEL method observed by lightmicroscope (×400). A. Control, B. Model, C. Positive, D. JHTF-H, E. JHTF-M, F. JHTF-L, G. LY + JHTF, H. LY, I. Quantitative analysis of percentage of apoptotic cells. Data are expressed as mean ± SD, n = 6. ##p < 0.01 vs. control group; **p < 0.01 vs. model group; *p < 0.05 vs. model group; ++p < 0.01 vs. JHTF-H group; +p < 0.05 vs. JHTF-H group.
ISO-alone-induced rats, suggesting the improvement of JHTF on cardiac electrical activity. Following ISO administration, we observed that the heart weight increased significantly, and thus the HWI increased when compared to control with relatively unchanged body weight. The increased heart weight might be attributed to the increased extensive edematous intramuscular space and necrosis of cardiac muscle fibers followed by invasion of the damaged tissues by inflammatory cells [22,25,26]. Pretreatment with JHTF significantly reduced HWI as compared to ISO alone treated group, and indicative of their myocardial protection against infiltration and it also could be due to the decrease in water content of the myocardium. HE staining is an important method for evaluating myocardial
higher doses could produces necrosis, hypoxia, hyperplasia and MI [22,23]. In the current study, MI was induced in rats by intraperitoneal administration of ISO in a dose of 85 mg/kg for three successive days with an interval of 24 h between the applications. Observing alterations in the ECG pattern, particularly ST-segment elevation, is the standard method for accurately diagnosing MI injury in animals [4,24]. Our findings showed that subcutaneous injection of supramaximal doses of ISO in rats induced notable modifications on ECG pattern: a significant ST-segment elevation. It was the consequence of the cell membrane lesion in the ischemia myocardium [25]. However, the ECG patterns were marked by the disappearance of these abnormalities in MI rats pretreated with JHTF, and a complete neutralization of the ST-segment elevation was observed as compared to 314
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Fig. 8. Expression levels of p-Akt (A), p-GSK-3β (B) and p-eNOS (C) in myocardial tissues. Representative Western blots of p-Akt (A), p-GSK-3β (B) and p-eNOS (C) in the myocardial tissue of each experimental group. β-Actin was used as a loading control. Average quantification obtained by densitometric analysis of the results of western blot analysis. Data are presented as mean ± SD, n = 16. ##p < 0.01 vs. control group; **p < 0.01 vs. model group; *p < 0.05 vs. model group; ++p < 0.01 vs. JHTF-H group; +p < 0.05 vs. JHTF-H group.
Fig. 9. Expression levels of Bcl-2 (A), Bax (B) and caspase-3 (C) in myocardial tissues of each group. β-Actin was used as a loading control. Average quantification obtained by densitometric analysis of the results of western blot analysis. Data are presented as mean ± SD, n = 16. ##p < 0.01 vs. control group; **p < 0.01 vs. model group; *p < 0.05 vs. model group; ++p < 0.01 vs. JHTF-H group; +p < 0.05 vs. JHTF-H group.
muscle, and injury to the cardiac tissue results in the release of these enzymes into the blood stream [4]. The concentrations of CK, CK-MB and LDH in blood reflects the degree of cell membrane and integrity and serum levels of these enzymes are also serve as the diagnostic markers of myocardial damage [24,26]. cTnI is a low molecular weight protein constituent of the myofibrillary contractile apparatus of the cardiac muscle [29]. ISO-induced MI in rats caused a significant
injury [27,28]. In the current study, the cardioprotective effect of JHTF was further shown by the significant improvement in the HE. Treatment with JHTF restored the histopathological change and the sections from heart tissue showed fewer area of degeneration compared with those in MI model group. The results provide strong evidence to prove that JHTF provides cardioprotection against ISO-induced myocardial injury. CK, CK-MB and LDH are cytosolic enzymes present in cardiac 315
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5. Conclusion
increase in the levels of CK, CK-MB, cTnI and LDH. The leakage of these marker enzymes into the blood stream may be due to the lipid peroxide mediated membrane permeability enhance and damage [26,29,30]. Meanwhile, elevated cTnI levels can predict the risk of both cardiac death and subsequent infarction [22]. In our present study, JHTF can partially decrease the serum CK, CK-MB, cTnI and LDH which are related to cellular damage and loss functional integrity, indicating that JHTF effectively maintains cell functional integrity and restricts the leakage of these enzyme into circulation. Many reports reveal that oxidative damage accompanied by free radical progresses the pathogenesis of cardiac damage [31]. In the present study, increased level of free radicals such as MDA and decreased level of antioxidant defense system such as SOD, NO and GSHPx in ISO-induced group indicated the severe oxidative stress. The deterioration of these oxidative stress markers was consistent with the previous literature [32–34]. However, JHTF treatment decreased the level of MDA and normalized the level of SOD, NO and GSH-Px, confirming the antioxidant activity of JHTF against ISO-induced myocardial injury. Inflammatory is a key process and the inflammatory cytokines appear to be useful in the diagnosis of ischemia and myocardium lesions [22,25]. Studies have shown that excess of pro-inflammatory cytokines, such as IL-6 and TNF-α, is responsible for myocardial injury [35]. In agreement with previous studies, our results demonstrated that the levels of pro-inflammatory cytokines IL-6 and TNF-α were increased in ISO-induced group [10,36]. Whereas, treatment with JHTF markedly attenuated the cardiac inflammation by inhibiting the generations of inflammatory cytokines in MI model group rats. These results suggest an anti-inflammatory action of JHTF. LY294002, the PI3K inhibitor, was applied to confirm the role of PI3K/Akt pathway in the present study. All the findings indicated that PI3K participated in the cardioprotective effect of JHTF on MI. Furthermore, the data of Western blots proved that the expression levels of p-Akt, p-GSK-3β and p-eNOS were significantly decreased in the MI model rats, while, the phosphorylated levels of these proteins were markedly improved by JHTF treatment. More importantly, by using the PI3K inhibitor LY294002, the effects of JHTF on related phosphorylated proteins were largely reversed. According to all the comprehensive description as above, our study implicated that the potential benefits of JHTF in rats with MI were likely mediated by the activation of PI3K/ Akt pathway. Apoptosis is an essential contributor in cardiac dysfunction. In our study, TUNEL assay was performed to examine myocardial apoptosis. Pretreatment with JHTF significantly decreased myocardial apoptosis, indicating that the anti-apoptotic effect of JHTF in MI injury. Moreoner, caspase- dependent apoptosis is considered as an essential molecular mechanism and Bcl-2 family members as well as play important roles in regulating apoptotic signaling, while, Bax, belong to Bcl-2 family, is an essential pro-apoptotic molecule [37,38]. The present study we demonstrated that treatment with JHTF can effectively downregulate the levels of caspase-3 protein in the myocardium. Apoptosis following MI has been determined to be associated with increased levels of Bax and decreased levels of Bcl-2 [39]. Our research showed that JHTF treatment significantly increased Bcl-2 protein expression and decreased Bax protein expression by method of western blot. These results indicated that inhibition of apoptosis may be one of the mechanisms underlying the cardioprotective effect of JHTF treatment. But the protective effects of JHTF were largely reversed by LY294002. These observations indicated that PI3K/Akt pathway might be involved in the protection of JHTF after MI. Notably, the present study revealed that JHTF increased the expression levels of p-Akt, p-GSK3β and p-eNOS in myocardium. In addition, JHTF decreased the expression levels of the downstream proteins of the PI3K/Akt signaling pathway, Bax and caspase, and increased the expression levels of Bcl-2. Conversely, the effects of JHTF on MI and PI3K/Akt signaling were attenuated by LY294002.
The present study provided that the JHTF shows remarkable cardioprotective effect, which suppresses oxidative stress and inflammatory cytokines and thereby attenuate apoptosis after MI. The cardioprotective function may be associated with its ability to attenuate myocardial damage and apoptosis involving in the PI3K/Akt signaling pathway. Taking all our data together, it may be suggested that JHTF could be used as potential cardioprotective drug in the patient suffering from MI injury. Conflict of interest There is no any conflict of interest in this study. Acknowledgement The authors would like to acknowledge the financial support kindly provided by the program of National Natural Science Foundation (grant number No. 30472125) of China. References [1] Z.C. Ke, G. Wang, L. Yang, H.H. Qiu, H. Wu, M. Du, et al., Crude terpene glycoside component from Radix paeoniae rubra protects against isoproterenol induced myocardial ischemic injury via activation of the PI3K/AKT/mTOR signaling pathway, J. Ethnopharmacol. 206 (2017) 160–169. [2] C.G. Zhao, F.X. Meng, L.L. Geng, X. Zhao, H. Zhou, Y. Zhang, et al., Cardiac-protective effects and the possible mechanisms of alatamine during acute myocardial ischemia, Can. J. Physiol. Pharmacol. 94 (2016) 433–440. [3] K. Aras, B. Burton, D. Swenson, R. Macleod, Spatial organization of acute myocardial ischemia, J. Electrocardiol. 49 (2016) 323–336. [4] Y. Li, J. Feng, Y.Q. Mo, H.G. Liu, B. Yang, Concordance between cardio-protective effect on isoproterenol-induced acute myocardial ischemia and phenolic content of different extracts of Curcuma aromatica, Pharm. Biol. 54 (2016) 3226–3231. [5] H.B. He, J. Xu, Y.Q. Xu, C.C. Zhang, H.W. Wang, Y.M. He, et al., Cardioprotective effects of saponins from Panax japonicus on acute myocardial ischemia against oxidative stress-triggered damage and cardiac cell death in rats, J. Ethnopharmacol. 140 (2012) 73–82. [6] D.W. Wang, J. Wang, Y.T. Liu, Z. Zhao, Q. Liu, Roles of Chinese herbal medicines in ischemic heart diseases by regulating oxidative stress, Int. J. Cardiol. 220 (2016) 314–319. [7] A.N.C. Simão, M.F. Lehmann, D.F. Alfieri, M.Z. Meloni, T. Flauzino, B.M. Scavuzzi, et al., Metabolic syndrome increases oxidative stress but does not influence disability and short-time outcome in acute ischemic stroke patients, Metab. Brain Dis. 30 (2015) 1409–1416. [8] M.E. Worou, K. Belmokhtar, P. Bonnet, P. Vourc'h, M.C. Machet, G. Khamis, et al., Hemin decreases cardiac oxidative stress and fibrosis in a rat model of systemic hypertension via PI3K/Akt signalling, Cardiovasc. Res. 91 (2011) 320–329. [9] K.K. Griendling, R.M. Touyz, J.L. Zweier, S. Dikalov, W. Chilian, Y.R. Chen, et al., Measurement of reactive oxygen species, reactive nitrogen species, and redox-dependent signaling in the cardiovascular system, Circ. Res. 119 (2016) e39–e75. [10] H.Y. Li, S.S. Zhang, Li F.L, L.J. Qin, NLRX1 attenuates apoptosis and inflammatory responses in myocardial ischemia by inhibiting MAVS-dependent NLRP3 inflammasome activation, Mol. Immunol. 76 (2016) 90–97. [11] J. Jian, F.F. Xuan, F.Z. Qin, R.B. Huang, Bauhinia championii flavone inhibits apoptosis and autophagy via the Pi3K/Akt pathway in myocardial ischemia/reperfusion injury in rats, Drug Des. Dev. Ther. 9 (2015) 5933–5945. [12] Y.H. Pei, J. Chen, L. Xie, X.M. Cai, R.H. Yang, X. Wang, et al., Hydroxytyrosol protects against myocardial ischemia/reperfusion injury through a PI3K/Akt-dependent mechanism, Med. Inflamm. 2016 (2016) 9 p.. [13] X.J. Hou, J.C. Han, C.S. Yan, H.H. Ren, Y. Zhang, T. Zhang, et al., Cardioprotective effects of total flavonoids extracted from Xinjiang Sprig Rosa rugosa against acute ischemia/reperfusion-induced myocardial injury in isolated rat heart, Cardiovasc. Toxicol. 16 (2015) 54–66. [14] X.Y. Liu, L. Xu, Y. Wang, J.X. Li, Y. Zhang, C. Zhang, et al., Protective effects of total flavonoids of Astragalus against adjuvant-induced arthritis in rats by regulating OPG/RANKL/NF-κB pathway, Int. Immunopharmcol. 44 (2017) 105–114. [15] S. Song, F.L. Chen, X.F. Xing, M.Y. Ren, Q.H. Ma, Y. Xie, et al., Concurrent quantification and comparative pharmacokinetic analysis of bioactive compounds in the Herba Ephedrae-Semen Armeniacae Amarum herb pair, J. Pharm. Biomed. Anal. 109 (2015) 67–73. [16] Y. Jin, Y.P. Qu, H.Q. Pang, L.L. Liu, Z.H. Zhu, E.X. Shang, et al., Herb pairs containing Angelicae Sinensis Radix (Danggui): a review of bio-active constituents and compatibility effects, J. Ethnopharmacol. 181 (2016) 158–171. [17] D.H. Nguyen, U.M. Seo, B.T. Zhao, D.D. Le, S.H. Seong, J.S. Choi, et al., Ellagitannin and flavonoid constituents from Agrimonia pilosa Ledeb. With their protein tyrosine phosphatase and acetylcholinesterase inhibitory activities, Bioorg. Chem. 72 (2017) 293–300.
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Cardioprotective effects of total flavonoids from Jinhe Yangxin prescription by activating the PI3K/Akt signaling pathway in myocardial ischemia injury
T
Yangang Chenga,1, Jinyan Tana,1, Huifeng Lib, Xiangpeng Kongb, Yan Liua, Rui Guob, Guoyan Lib, ⁎ Bingyou Yanga, Miaorong Peia,b, a
Key Laboratory of Chinese Materia Medica (Ministry of Education), Heilongjiang University of Chinese Medicine, 24 Heping Road, Xiangfang District, Harbin 150040, PR China b School of Chinese Pharmacy, Shanxi University of Chinese Medicine, 121 Daxue Road, Yuci District, Jinzhong 030619, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Myocardial ischemia PI3K/Akt Flavonoids Oxidative stress Apoptosis Herb pairs
The purpose of the present study was to investigate the cardioprotective effects of total flavonoids of Jinhe yangxin prescription (JHTF) on myocardial ischemia (MI) injury rats induced by Isoproterenol (ISO) and explore the potential mechanisms underlying these effects. 128 male rats were randomized into 8 groups: Control, Model, Positive, JHTF-H (2.64 g/kg/d), JHTF-M (1.32 g/kg/day), JHTF-L (0.66 g/kg/d), LY + JHTF (JHTF-H plus LY294002, an inhibitor of PI3K/Akt) and LY groups. Electrocardiogram, histopathological examination and terminal deoxynucleotidyl transferase dUTP nickend labeling (TUNEL) assay were performed. Heart weight index, markers of cardiac marker enzymes [creatine kinase (CK), creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) and cardiac troponin I (cTnI)], oxidative stress [superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px) and nitric oxide (NO)] and inflammation [tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6)] were also measured in each group. Proteins involved in PI3K/Akt pathway were detected by Western blot. JHTF decreased the ST elevation induced by MI, decreased serum levels of CK, CK-MB, cTnI, LDH, MDA, IL-6 and TNF-α, and increased serum SOD, GSH-Px and NO activities. Furthermore, JHTF inhibited myocardial apoptosis, which may be related to downregulated caspase-3 and Bax, upregulated Bcl-2, and increased the protein levels of phosphorylated Akt, GSK-3β and endothelial nitric oxide synthase (eNOS). However, all the previously mentioned effects of JHTF were blocked when JHTF was coadministered with LY294002. In conclusion, these observations indicated that JHTF has cardioprotective effects against MI, and these effects seem to be related to the activation of PI3K/Akt signaling pathway in the myocardium.
1. Introduction
damage. Oxidative stress is regarded as excessive generation or accumulation of free radical and decrease of antioxidant enzymes [5–7]. Oxidative stress resulting from production of reactive oxygen species (ROS) play an important role in cardiac remodeling and heart failure [8]. ROS are biological molecules that play important roles in cardiovascular physiology and contribute to disease initiation, progression, and severity [9]. Increasing evidences also suggest the importance of sterile inflammatory response in MI pathogenesis, which is involved in mediating impaired myocardial function and heart failure. So, therapeutic approaches that target components of the oxidative stress and inflammatory response have been investigated as potential and useful
Myocardial ischemia (MI), a major pathogenic factor for cardiovascular diseases, is a pathological condition characterized by a restriction of blood in the heart, resulting in the heart does not receive adequate oxygen-rich blood to keep up with its metabolic requirements [1,2]. Around 40% of death are caused by cardiovascular diseases and MI is the leading reason. MI is also the leading cause of cardiac injury, including myocardial infarction and life-threatening arrhythmias [3,4]. Recently, considerable experimental evidences have been proven that oxidative stress should be a major apoptotic stimulus in MI
Abbreviations: cTnI, cardiac troponin I; CK, creatine kinase; CK-MB, creatine kinase-MB; DF, Dijincao- Fenxinmu; DX, Dijincao-Xianhecao; ECG, electrocardiogram; eNOS, endothelial nitric oxide synthase; GSH-Px, glutathione peroxidase; HWI, heart weight index; IL-6, interleukin-6; ISO, Isoproterenol; JHTF, total flavonoids of Jinhe yangxin prescription; LDH, lactate dehydrogenase; MDA, malondialdehyde; MI, myocardial ischemia; NO, nitric oxide; PI3K-Akt, phosphoinositide 3-kinases/serine/threonine protein kinase; ROS, reactive oxygen species; SOD, superoxide dismutase; TCM, traditional Chinese medicine; TNF-α, tumor necrosis factor-alpha; TUNEL, terminal deoxynucleotidyl transferase dUTP nickend labeling ⁎ Corresponding author at: Key Laboratory of Chinese Materia Medica (Ministry of Education), Heilongjiang University of Chinese Medicine, 24 Heping Road, Xiangfang District, Harbin 150040, PR China. E-mail address: [email protected] (M. Pei). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2017.12.052 Received 1 September 2017; Received in revised form 4 December 2017; Accepted 13 December 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
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height of chromatographic column was 1:6, the flow speed for adsorption was 2 BV/h, then cleaned by 1.5 BV deionized water and eluated by 3 BV 50% ethanol at the speed of 2 BV/h, respectively. Eluate was then collected, concentrated and freeze dried in FDU-1200 lyophilizer (Tokyo Rikakikai Co., Ltd., Japan), thus JHTF was obtained. With rutin as reference substance, the content of total flavonoids in JHTF was determined by ultraviolet spectrophotometry and expressed as mg rutin equivalents per gram dry weight (mg RE/g DW). The total flavonoids content of JHTF was 855.40 mg RE/g DW.
treatments for MI injury [10]. Previous studies indicate that the phosphoinositide 3-kinases/ serine/threonine protein kinase (PI3K-Akt) signaling pathway is an endogenous negative feedback regulator which limits proinflammatory and apoptotic events in response to harmful stimuli. Active Akt is a downstream effector of PI3K, which can inhibit apoptosis by regulating multiple target such as TNF-α, eNOS, and Bcl-2 family [11]. Activation of PI3K/Akt signaling pathway could suppress cardiac myocyte and prevent heart [12] . Traditional Chinese medicine (TCM) has played an important role in clinical therapy for MI, especially the flavonoids, which are a vast group of polyphenols found ubiquitously in TCM, and have received more and more attention due to their pharmacological activities such as antioxidation, scavenging free radicals, anti-inflammation, regulating blood lipid and reducing cardiovascular damage [13,14]. Herb pairs (mixture of two or more herbs) is a basic essential element of Chinese herbal formulas, which are much simpler than other complex formulas without altering the basic therapeutic features, meanwhile, the herb pair presents significantly better pharmacological efficacy than the individual plants [15,16]. Jinhe yangxin prescription is a combination of two herb pairs, namely Dijincao [dry entire grass of Euphorbia humifusa Willd]-Xianhecao [dry aerial parts of Agrimomia pilosa ledeb.] (DX) and Dijincao-Fenxinmu [wooden diaphragma of the kernel of Juglans regia L] (DF), which are recorded in Shijinmoduiyao written by Lv Jingshan, a famous TCM master in China [17–19]. DX and DF are usually used for the treatment of various heart diseases in clinically by master Lv. The present experiments were aimed to explore the cardioprotective effects of total flavonoids from jinhe yangxin prescription (JHTF) against MI injury induced by ISO in rats and investigated the underlying myocardial protective mechanisms.
2.3. Animals Male Wistar rats (200 ± 20 g) were supplied by the Experimental Animal Center of the Academy of Military Medical Sciences (Beijing, China). Rats were housed under controlled conditions with a 12 h lightdark cycle at 25 ± 2 ℃ and 45 ± 5% relative humidity. Standard food and water were provided ad libitum. All rats were acclimated to the facility for 7 days. All protocols for experiments were performed in accordance with the Animal Ethics Committee of Shanxi University of TCM (Shanxi, China) on May 22, 2016 (number 2016052202). 2.4. Induction of MI in rats 128 male Wistar rats were randomly assigned into 8 groups (n = 16 per group) named Control, Model, Positive, High-dose JHTF (JHTF-H), Middle-dose JHTF (JHTF-M), Low-dose JHTF (JHTF-L), LY294002 plus High-dose JHTF (LY + JHTF) and LY groups. JHTF-H, JHTF-M, JHTF-L and LY + JHTF groups were given 2.64, 1.32, 0.66, and 2.64 g/kg/d of JHTF, respectively, whereas Positive group was given 20 mg/kg/d of PH. Other groups (Control, Model and LY groups) were administered distilled water. All treatments were administrated intragastrically for a period of 14 days. Except for the Control group, all rats were injected subcutaneously with ISO at a dosage of 85 mg/kg/d on the 12th, 13th and 14th day to set up MI model, whereas LY + JHTF and LY groups were intraperitoneal injection (ip) with LY294002 (0.3 mg/kg/d) prior to injected subcutaneously with ISO. Control group was injected with equivalent volume of sterile saline to instead of ISO. Twenty-four hours after the third ISO injection, the rats were anesthetized by 10% chloral hydrate (ip, 3 mL/kg). The blood was collected and processed for further biochemical estimations and then heart tissue was excised immediately, washed with chilled isotonic saline.
2. Materials and methods 2.1. Materials Dijicao, Xianhecao and Fenximu were purchased from Anguo Chinese Medicine market (Hebei, China). The pharmaceutical botany of the these TCM materials were identified by Prof. Xiangping-Pei from Shanxi University of Chinese Medicine (Shanxi, China). Voucher specimens (NO. SXUTCM2016-1101; NO. SXUTCM2016- 1102 and NO. SXUTCM2016-1103, respectively) were deposited in the department of medicinal plant, Shanxi University of Chinese Medicine (Shanxi, China). The standard of rutin (100080-201202, 98.0% purity), was obtained from National Institutes for Food and Drug Control (Beijing, China). Isoproterenol (ISO), propranolol hydrochloride (PH, positive) and LY294002 (a PI3K inhibitor) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Creatine kinase (CK), creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), interleukin-6 (IL-6) and tumor necrosis factorα (TNF-α) ELISA kits were obtained from Sangon Biotech Co., Ltd. (Shanhai, China). Lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide (NO), glutathione peroxidase (GSH-Px) assay kits and situ TUNEL apoptosis detection kit were acquired from Jiancheng Bioengineering Research Institute (Nanjing, China).
2.5. Electrocardiogram detection The electrocardiogram (ECG) test (Lead II) was conducted in anesthetized rats before the first injection and after finishing the final injection of ISO. The needle electrodes were linked to the right arm, left arm and left leg of the rats, and the electrocardiographic-patterns were recorded with an ECG recording and analysis system (BL-420F, Chengdu TME Technology Company, Chengdu, China). 2.6. Heart weight index determination After rats were sacrificed, the heart tissues were excised (excluding large blood vessels, and connective tissue), and weighed after blotting with filter paper. The heart weight index (HWI) was computed as HWI = heart weight (HW)/body weight (BW).
2.2. Preparation of JHTF A mixture of Dijincao (500 g), Xianhecao (1000 g) and Fenxinmu (500 g) was refluxed twice with 55% ethanol (1:17, w/v) for 147 min, and the collected extract was filtrated and vacuum evaporated with rotary evaporation at 40 ℃ to obtain extract at a final concentration of 0.5 g/mL (w/v, expressed as the weight of raw materials). The total flavonoids were enriched by the AB-8 macroporous resin chromatography, and the purification technologies were as follows: the volume of chromatographic column was 1600 mL, the ratio of diameter to
2.7. Cardiac marker enzymes After the ECG detection, arterial blood samples were collected and centrifuged (3000 rpm for 15 min) to obtain serum, and then stored at −80 ℃. Serum samples were thawed at room temperature before analysis. Myocardial damage was evaluated by measuring the serum levels of CK, CK-MB, cTnI and LDH according to the manufacturer's 309
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Fig. 1. Effects of JHTF on ST-segment elevation. Values represent the mean ± SD,n = 16. ## p < 0.01 vs. control group; **p < 0.01 vs. model group; ++p < 0.01 vs. JHTF-H group.
instructions of kits.
2.10. Histological studies The hearts were removed immediately after the sacrifice of the rats, and fixed in 10% formalin solution. The heart tissue was processed for sectioning and staining by standard histological methods. Sections (5 mm, Leica RM 2125, Germany) from the left ventricle were stained with hematoxylin and eosin (H&E) and examined by light microscopy (Nikon, Tokyo, Japan) at 200 × magnification.
2.8. Antioxidant enzymes assay SOD, MDA, GSH-Px and NO levels in serum were measured using commercial kits according to the manufacturer's protocols.
2.9. Inflammation assay 2.11. Determination of myocardial apoptosis IL-6 and TNF-α levels of serum were determined by standard commercial kits to explore the relationship between the cardioprotective effects of JHTF and the levels of inflammatory cytokines.
Myocardial apoptosis was determined by TUNEL staining according to the manufacturer’s instructions (Jiancheng Bioengineering Research Institute, Nanjing, China). Briefly, after the sacrifice of the rats, the tissue near the cardiac apex was fixed in 10% neutral formalin. 310
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heart tissue proteins involved in PI3K/Akt pathway. The tissue proteins were obtained from the heart 24 h after the third dose of ISO administration and were mechanically homogenized in radioimmunoprecipitation assay buffer lysis buffer (25 mg/mL) with 1 mmol/L Phenylmethanesulfonyl fluoride (PMSF) on ice. The whole lysates were then centrifuged (12,000 rpm for 15 min, 4 ℃) to obtain the supernatant. Total protein was determined using bicinchoninic assay kit (Biyuntian Biotechnology, Wuhan, China). Protein sample of 50 ug was loaded per lane and separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to the poly-vinylidnene fluoride (PVDF) membranes (San Yin-tan, Beijing, China). Afterwards, these membranes were blocked with 5% non-fat skim milk in tris-buffered saline solution containing Tween-20 (TBST; 10 mmol/L tris, pH 7.5; 140 mmoL/L NaCl; 0.1% Tween-20) for 1 h at room temperature, followed by incubated overnight at 4 ℃ with the corresponding primary antibody in TBST containing 3% bovine serum albumin (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA). The primary antibodies were as follows: rabbit anti-Akt (1:1000; 4691), rabbit anti-p-Akt (1:500; 4060), rabbit anti-Caspase-3 (1:500, 9665), rabbit anti-p-GSK-3β (1:1000, 5558), rabbit anti-GSK-3β (1:1000, 12356), rabbit anti-p-eNOS (1:1000, 9570), rabbit anti-eNOS (1:1000, 9586), rabbit anti-Bcl-2 (1:1000; 3498), rabbit anti-Bax (1:1000; 5023; Cell Signaling Technology, Inc., Danvers, MA, USA). Subsequently, the membrane was washed for three times, and the membranes were further incubated with horseradish peroxidase-conjugated secondary antibody anti-rabbit IgG (1:2000) in TBST solution for 1 h. In the end, the signals of detected proteins were visualized by an enhanced chemiluminescence reaction system (Millipore, Billerica, Massachusetts, USA) and the density of each reactive band was quantified using the LabWorks Image Acquisition platform (UVP, Inc., Upland, CA, USA) and ImageJ (National Institutes of Health, Bethesda, MA, USA). β-actin (1:3000, 4970; Cell Signaling Technology, Inc., Danvers, MA, USA) was
Fig. 2. Effects of JHTF on heart weight index. Data are shown as mean ± SD,n = 16. ## p < 0.01 vs. control group; **p < 0.01 vs. model group; *p < 0.05 vs. model group; ++p < 0.01 vs. JHTF-H group.
embedded in paraffin blocks, cut into ultrathin sections (5 μm), deparaffinized, and myocardial apoptosis was examined. Cells with brown stained nuclei indicated TUNEL-positive, apoptotic cells, and the apoptotic cells and bodies were counted in five high-power fields. The apoptotic index (AI) was calculated as the percentage of positively stained cells using the following equation: AI = number of apoptotic cells/total number of nucleated cells. 2.12. Western blot analysis Western blot analysis was used to detect the expression levels of
Fig. 3. Effects of JHTF on serum level of cardiac marker enzymes; CK (A), CK-MB (B), cTnI (C) and LDH (D). Data are expressed as mean ± SD, n = 16. ##p < 0.01 vs. control group; **p < 0.01 vs. model group; *p < 0.05 vs. model group; ++p < 0.01 vs. JHTF-H group.
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Fig. 4. Effects of JHTF on serum level of cardiac oxidative stress parameters; SOD (A), MDA (B), GSH-Px (C) as well as NO (D). Data are shown as mean ± SD, n = 16. control group; **p < 0.01 vs. model group; *p < 0.05 vs. model group; ++p < 0.01 vs. JHTF-H group; +p < 0.05 vs. JHTF-H group.
##
p < 0.01 vs.
HWI became larger (p < 0.01 or p < 0.05, Fig. 2).
used for normalization of protein levels. 2.13. Statistical analysis
3.3. Effects of JHTF on CK, CK-MB, cTnI and LDH
All statistical analyses were carried out using SPSS 17.0 software including one-way ANOVA and Student's t-test. Data were expressed as mean ± standard deviation (SD). Difference between groups were considered as significant when p < 0.05.
CK, CK-MB, cTnI and LDH are identified as essential diagnostic markers of ischemia injury to the myocardial tissues and leak out from damaged myocardium to blood [21]. Current studies measured the contents of CK, CK-MB, cTnI and LDH in serum to evaluate the myocardial damage. As shown in Fig. 3, a marked increase in the levels of CK, CK-MB, cTnI and LDH were detected in MI model rats compared with those in the control group (p < 0.01). While, compared with the model group, the JHTF-H significantly decreased serum CK, CK-MB, cTnI and LDH (p < 0.01). When LY294002 was applied to eliminate the effects of JHTF, the levels of CK and CK-MB were higher in LY + JHTF compared with the JHTF-H group (p < 0.01), and the levels of cTnI, LDH also have the same trend.
3. Results 3.1. Effects of JHTF on ST-segment MI occurs when the blood flow through the heart muscle is decreased by a partial or complete blockage of heart arteries, which causes ST level changes in an ECG signal. A normal ST segment is horizontal, whereas an abnormal ST segment deviates from the horizontal [20]. As depicted in Fig. 1(A and B), ST-segment was significantly elevated in model rats compared with those in the control group (p < 0.01). In groups pre-administrated with PH (Positive) and JHTF-H, ST-segment was significantly decreased as compared with that in the rats of model group (p < 0.01). However, in the presence of LY294002, a specific inhibitor of PI3K/Akt pathway, this effect of JHTF was eliminated compared with JHTF-H (p < 0.01), while LY294002 alone had no significant effect on the ST-segment. These results reflect the positive effect of JHTF on cardiac function.
3.4. Effects of JHTF on SOD, MDA, GSH-Px and NO In this study, we evaluated the levels of SOD, MDA, GSH-Px and NO in the serum to explore the relationship between the cardioprotective effect of JHTF in MI and its antioxidant status. As shown in Fig. 4, compared with control group, the model rats exhibited a significant increase in MDA content, while the levels of SOD, GSH-Px and NO were all significantly decreased, and those changes were reversed in Positive, JHTF-H, JHTF-M and JHTF-L groups (p < 0.01 or p < 0.05). These results suggested the antioxidative stress effect of JHTF in alleviating MI injury. However, LY294002 significantly abolished the effect of JHTF on the levels of SOD, GSH-Px, NO, and it also has a similar effect on the levels of MDA, which indicated that JHTF-attenuated oxidative stress in MI injury was associated with PI3K/Akt pathway.
3.2. Effects of JHTF on heart weight indices HWI was greater in the model group rats than in the control group rats (p < 0.01). Administration with JHTF decreased the HWI compared with the model group rats. As the dose increased, the decrease in 312
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the LY + JHTF group, the reduction of myocardial structure disorder and partial rupture, and lyse of muscle fibers with moderate infiltration of lymphocytes and macrophages were eliminated. Administration of LY294002 alone before ISO-treated had no effect on myocardial tissue (Fig. 6G and H). 3.7. Effects of JHTF on the cardiomyocyte apoptosis Apoptosis is the major mechanism of cell death induced by MI. Herein, myocardial apoptosis was determined by TUNEL staining. TUNEL-positive cells were defined as the death of cells. As shown in Fig. 7, in the model group, the TUNEL-positive cells expressed as the percent of total nucleiwere significantly increased when compared with the control group (p < 0.01). However, the percentage of TUNEL-positive cardiomyocytes was significantly lower in the Positive, JHTF-H, JHTF-M groups compared with the model group (p < 0.01 or p < 0.05), and JHTF-L group also has the same trend. Furthermore, cotreated with LY294002 in the LY + JHTF group significantly attenuated the effect of JHTF on the cardiomyocyte apoptosis compared with the JHTF-H group (p < 0.01). 3.8. Effects of JHTF on the expression of proteins in PI3K/Akt pathway To clarify whether the PI3K/Akt pathway was involved in the cardioprotective effect of JHTF in MI, the expression level of proteins related to PI3K/Akt pathway in the ischemic of myocardial tissue were detected by western blots. As shown in Fig. 8, there were no detectable differences in the expression of total Akt, GSK-3β and eNOS among the six groups. JHTF treatment markedly increased the myocardial levels of p-Akt in rats compared with those in model group (p < 0.01). However, administration of LY294002 could significantly attenuate the JHTF-induced upregulation of myocardial p-Akt in rats. Moreover, JHTF treatment significantly increased the levels of p-GSK-3β, a downstream kinase of Akt, in the myocardium compared with levels in model group (p < 0.01). Administration of the PI3K inhibitor, LY294002, could significantly prevent the JHTF-induced enhancement of phosphorylation of GSK-3β in the MI myocardium (p < 0.01). Akt induced eNOS phosphorylation at serine 1177 is an important mechanism to protect heart against injury. ISO-induced MI resulted in significantly decreased expression of p-eNOS in the myocardium as compared to control group (p < 0.01). JHTF pretreatment markedly increased the expressions of p-eNOS compared with the MI model group (p < 0.01). We further determined whether administration of LY294002 could alter the effect of JHTF on the expression level of p-eNOS. The result showed that cotreatment of LY294002 blocked the eNOS activation induced by JHTF. Furthermore, to determine whether the administration of JHTF could affect the expressions of apoptosis-related proteins, we analyzed the levels of Bcl-2, Bax and caspase-3 in myocardial tissues by Western blot. Obvious increases in Bax and caspase-3 levels and decrease in Bcl2 level were detected in the model group compared with control group (p < 0.01, Fig. 9). While, JHTF treatment markedly downregulated Bax and caspase-3, but upregulated Bcl-2 expressions in comparison to the model group (p < 0.01). However, LY294002 abolished the effects of JHTF on Bcl-2, Bax and caspase-3 expression (p < 0.01).
Fig. 5. Effects of JHTF on serum level of inflammatory markers; IL-6 (A) and TNF-α (B). Data are shown as mean ± SD, n = 16. ##p < 0.01 vs. control group; **p < 0.01 vs. model group; *p < 0.05 vs. model group; ++p < 0.01 vs. JHTF-H group; +p < 0.05 vs. JHTF-H group.
3.5. Effects of JHTF on IL-6 and TNF-α To further evaluate and validate the protective function of JHTF during MI injury, we measured the levels of IL-6 and TNF-α in serum. It was noted that a marked increase in the levels of the IL-6 and TNF-α as compared with that of rats in the control group (p < 0.01, Fig. 5). Notably, the administration of PH and JHTF could inhibit the increase, which indicated that PH and JHTF exhibited markedly alleviation of pro-inflammatory cytokines. However, the serum IL-6 and TNF-α concentrations in LY + JHTF and LY groups remained higher than those in JHTF-H group. These indicated that the activation of PI3K/Akt signaling by JHTF significantly suppressed MI-induced inflammatory cytokine secretion in the serum. 3.6. Effects of JHTF on myocardial histology The representative micrographs of rat myocardial HE staining are shown in Fig. 6. The myocardial structure in the control group exhibited a regular arrangement, normal cardiac muscle fibers and no necrosis (Fig. 6A). However, compared with the control group, the model group demonstrated ruptured cardiac muscle fibers, necrosis with inflammatory cell infiltration and an enlargement of the extracellular space was also observed (Fig. 6B). The myocardial damage in the JHTF groups was less than that in the model group and there was dose-effect relationship between the three JHTF pretreated groups. Cardiac sections obtained from rats undergoing JHTF treatment demonstrated a relatively more intact and less myocardial fiber disruption, necrosis and inflammatory cell infiltration compared with those from the model group (Fig. 6D–F). When co-treated with LY294002 in
4. Discussion In the present study, we demonstrated that JHTF had beneficial effects on cardiac function in MI rats. These effects were in line with the suppressing oxidative stress-triggered damage and inflammatory cytokines, decreasing caspase-3 and Bax expressions, and up-regulating the Bcl-2 expression involving in the PI3K/Akt signaling pathway, and thereby attenuate apoptosis after MI and the ameliorative effects of JHTF presented in a dose-dependent manner. ISO is a synthetic catecholamine and β-adrenergic agonist which at 313
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Fig. 6. Representative slides of HE staining results for myocardial tissues in all groups of rats.A. Control, B. Model, C. Positive, D. JHTF-H, E. JHTF-M, F. JHTF-L, G. LY + JHTF, H. LY (×200).
Fig. 7. Effects of JHTF on myocardial apoptosis induced by ISO in rats. (A-H) representative micrographs of myocardium stained with TUNEL method observed by lightmicroscope (×400). A. Control, B. Model, C. Positive, D. JHTF-H, E. JHTF-M, F. JHTF-L, G. LY + JHTF, H. LY, I. Quantitative analysis of percentage of apoptotic cells. Data are expressed as mean ± SD, n = 6. ##p < 0.01 vs. control group; **p < 0.01 vs. model group; *p < 0.05 vs. model group; ++p < 0.01 vs. JHTF-H group; +p < 0.05 vs. JHTF-H group.
ISO-alone-induced rats, suggesting the improvement of JHTF on cardiac electrical activity. Following ISO administration, we observed that the heart weight increased significantly, and thus the HWI increased when compared to control with relatively unchanged body weight. The increased heart weight might be attributed to the increased extensive edematous intramuscular space and necrosis of cardiac muscle fibers followed by invasion of the damaged tissues by inflammatory cells [22,25,26]. Pretreatment with JHTF significantly reduced HWI as compared to ISO alone treated group, and indicative of their myocardial protection against infiltration and it also could be due to the decrease in water content of the myocardium. HE staining is an important method for evaluating myocardial
higher doses could produces necrosis, hypoxia, hyperplasia and MI [22,23]. In the current study, MI was induced in rats by intraperitoneal administration of ISO in a dose of 85 mg/kg for three successive days with an interval of 24 h between the applications. Observing alterations in the ECG pattern, particularly ST-segment elevation, is the standard method for accurately diagnosing MI injury in animals [4,24]. Our findings showed that subcutaneous injection of supramaximal doses of ISO in rats induced notable modifications on ECG pattern: a significant ST-segment elevation. It was the consequence of the cell membrane lesion in the ischemia myocardium [25]. However, the ECG patterns were marked by the disappearance of these abnormalities in MI rats pretreated with JHTF, and a complete neutralization of the ST-segment elevation was observed as compared to 314
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Fig. 8. Expression levels of p-Akt (A), p-GSK-3β (B) and p-eNOS (C) in myocardial tissues. Representative Western blots of p-Akt (A), p-GSK-3β (B) and p-eNOS (C) in the myocardial tissue of each experimental group. β-Actin was used as a loading control. Average quantification obtained by densitometric analysis of the results of western blot analysis. Data are presented as mean ± SD, n = 16. ##p < 0.01 vs. control group; **p < 0.01 vs. model group; *p < 0.05 vs. model group; ++p < 0.01 vs. JHTF-H group; +p < 0.05 vs. JHTF-H group.
Fig. 9. Expression levels of Bcl-2 (A), Bax (B) and caspase-3 (C) in myocardial tissues of each group. β-Actin was used as a loading control. Average quantification obtained by densitometric analysis of the results of western blot analysis. Data are presented as mean ± SD, n = 16. ##p < 0.01 vs. control group; **p < 0.01 vs. model group; *p < 0.05 vs. model group; ++p < 0.01 vs. JHTF-H group; +p < 0.05 vs. JHTF-H group.
muscle, and injury to the cardiac tissue results in the release of these enzymes into the blood stream [4]. The concentrations of CK, CK-MB and LDH in blood reflects the degree of cell membrane and integrity and serum levels of these enzymes are also serve as the diagnostic markers of myocardial damage [24,26]. cTnI is a low molecular weight protein constituent of the myofibrillary contractile apparatus of the cardiac muscle [29]. ISO-induced MI in rats caused a significant
injury [27,28]. In the current study, the cardioprotective effect of JHTF was further shown by the significant improvement in the HE. Treatment with JHTF restored the histopathological change and the sections from heart tissue showed fewer area of degeneration compared with those in MI model group. The results provide strong evidence to prove that JHTF provides cardioprotection against ISO-induced myocardial injury. CK, CK-MB and LDH are cytosolic enzymes present in cardiac 315
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5. Conclusion
increase in the levels of CK, CK-MB, cTnI and LDH. The leakage of these marker enzymes into the blood stream may be due to the lipid peroxide mediated membrane permeability enhance and damage [26,29,30]. Meanwhile, elevated cTnI levels can predict the risk of both cardiac death and subsequent infarction [22]. In our present study, JHTF can partially decrease the serum CK, CK-MB, cTnI and LDH which are related to cellular damage and loss functional integrity, indicating that JHTF effectively maintains cell functional integrity and restricts the leakage of these enzyme into circulation. Many reports reveal that oxidative damage accompanied by free radical progresses the pathogenesis of cardiac damage [31]. In the present study, increased level of free radicals such as MDA and decreased level of antioxidant defense system such as SOD, NO and GSHPx in ISO-induced group indicated the severe oxidative stress. The deterioration of these oxidative stress markers was consistent with the previous literature [32–34]. However, JHTF treatment decreased the level of MDA and normalized the level of SOD, NO and GSH-Px, confirming the antioxidant activity of JHTF against ISO-induced myocardial injury. Inflammatory is a key process and the inflammatory cytokines appear to be useful in the diagnosis of ischemia and myocardium lesions [22,25]. Studies have shown that excess of pro-inflammatory cytokines, such as IL-6 and TNF-α, is responsible for myocardial injury [35]. In agreement with previous studies, our results demonstrated that the levels of pro-inflammatory cytokines IL-6 and TNF-α were increased in ISO-induced group [10,36]. Whereas, treatment with JHTF markedly attenuated the cardiac inflammation by inhibiting the generations of inflammatory cytokines in MI model group rats. These results suggest an anti-inflammatory action of JHTF. LY294002, the PI3K inhibitor, was applied to confirm the role of PI3K/Akt pathway in the present study. All the findings indicated that PI3K participated in the cardioprotective effect of JHTF on MI. Furthermore, the data of Western blots proved that the expression levels of p-Akt, p-GSK-3β and p-eNOS were significantly decreased in the MI model rats, while, the phosphorylated levels of these proteins were markedly improved by JHTF treatment. More importantly, by using the PI3K inhibitor LY294002, the effects of JHTF on related phosphorylated proteins were largely reversed. According to all the comprehensive description as above, our study implicated that the potential benefits of JHTF in rats with MI were likely mediated by the activation of PI3K/ Akt pathway. Apoptosis is an essential contributor in cardiac dysfunction. In our study, TUNEL assay was performed to examine myocardial apoptosis. Pretreatment with JHTF significantly decreased myocardial apoptosis, indicating that the anti-apoptotic effect of JHTF in MI injury. Moreoner, caspase- dependent apoptosis is considered as an essential molecular mechanism and Bcl-2 family members as well as play important roles in regulating apoptotic signaling, while, Bax, belong to Bcl-2 family, is an essential pro-apoptotic molecule [37,38]. The present study we demonstrated that treatment with JHTF can effectively downregulate the levels of caspase-3 protein in the myocardium. Apoptosis following MI has been determined to be associated with increased levels of Bax and decreased levels of Bcl-2 [39]. Our research showed that JHTF treatment significantly increased Bcl-2 protein expression and decreased Bax protein expression by method of western blot. These results indicated that inhibition of apoptosis may be one of the mechanisms underlying the cardioprotective effect of JHTF treatment. But the protective effects of JHTF were largely reversed by LY294002. These observations indicated that PI3K/Akt pathway might be involved in the protection of JHTF after MI. Notably, the present study revealed that JHTF increased the expression levels of p-Akt, p-GSK3β and p-eNOS in myocardium. In addition, JHTF decreased the expression levels of the downstream proteins of the PI3K/Akt signaling pathway, Bax and caspase, and increased the expression levels of Bcl-2. Conversely, the effects of JHTF on MI and PI3K/Akt signaling were attenuated by LY294002.
The present study provided that the JHTF shows remarkable cardioprotective effect, which suppresses oxidative stress and inflammatory cytokines and thereby attenuate apoptosis after MI. The cardioprotective function may be associated with its ability to attenuate myocardial damage and apoptosis involving in the PI3K/Akt signaling pathway. Taking all our data together, it may be suggested that JHTF could be used as potential cardioprotective drug in the patient suffering from MI injury. Conflict of interest There is no any conflict of interest in this study. Acknowledgement The authors would like to acknowledge the financial support kindly provided by the program of National Natural Science Foundation (grant number No. 30472125) of China. References [1] Z.C. Ke, G. Wang, L. Yang, H.H. Qiu, H. Wu, M. Du, et al., Crude terpene glycoside component from Radix paeoniae rubra protects against isoproterenol induced myocardial ischemic injury via activation of the PI3K/AKT/mTOR signaling pathway, J. Ethnopharmacol. 206 (2017) 160–169. [2] C.G. Zhao, F.X. Meng, L.L. Geng, X. Zhao, H. Zhou, Y. Zhang, et al., Cardiac-protective effects and the possible mechanisms of alatamine during acute myocardial ischemia, Can. J. Physiol. Pharmacol. 94 (2016) 433–440. [3] K. Aras, B. Burton, D. Swenson, R. Macleod, Spatial organization of acute myocardial ischemia, J. Electrocardiol. 49 (2016) 323–336. [4] Y. Li, J. Feng, Y.Q. Mo, H.G. Liu, B. Yang, Concordance between cardio-protective effect on isoproterenol-induced acute myocardial ischemia and phenolic content of different extracts of Curcuma aromatica, Pharm. Biol. 54 (2016) 3226–3231. [5] H.B. He, J. Xu, Y.Q. Xu, C.C. Zhang, H.W. Wang, Y.M. He, et al., Cardioprotective effects of saponins from Panax japonicus on acute myocardial ischemia against oxidative stress-triggered damage and cardiac cell death in rats, J. Ethnopharmacol. 140 (2012) 73–82. [6] D.W. Wang, J. Wang, Y.T. Liu, Z. Zhao, Q. Liu, Roles of Chinese herbal medicines in ischemic heart diseases by regulating oxidative stress, Int. J. Cardiol. 220 (2016) 314–319. [7] A.N.C. Simão, M.F. Lehmann, D.F. Alfieri, M.Z. Meloni, T. Flauzino, B.M. Scavuzzi, et al., Metabolic syndrome increases oxidative stress but does not influence disability and short-time outcome in acute ischemic stroke patients, Metab. Brain Dis. 30 (2015) 1409–1416. [8] M.E. Worou, K. Belmokhtar, P. Bonnet, P. Vourc'h, M.C. Machet, G. Khamis, et al., Hemin decreases cardiac oxidative stress and fibrosis in a rat model of systemic hypertension via PI3K/Akt signalling, Cardiovasc. Res. 91 (2011) 320–329. [9] K.K. Griendling, R.M. Touyz, J.L. Zweier, S. Dikalov, W. Chilian, Y.R. Chen, et al., Measurement of reactive oxygen species, reactive nitrogen species, and redox-dependent signaling in the cardiovascular system, Circ. Res. 119 (2016) e39–e75. [10] H.Y. Li, S.S. Zhang, Li F.L, L.J. Qin, NLRX1 attenuates apoptosis and inflammatory responses in myocardial ischemia by inhibiting MAVS-dependent NLRP3 inflammasome activation, Mol. Immunol. 76 (2016) 90–97. [11] J. Jian, F.F. Xuan, F.Z. Qin, R.B. Huang, Bauhinia championii flavone inhibits apoptosis and autophagy via the Pi3K/Akt pathway in myocardial ischemia/reperfusion injury in rats, Drug Des. Dev. Ther. 9 (2015) 5933–5945. [12] Y.H. Pei, J. Chen, L. Xie, X.M. Cai, R.H. Yang, X. Wang, et al., Hydroxytyrosol protects against myocardial ischemia/reperfusion injury through a PI3K/Akt-dependent mechanism, Med. Inflamm. 2016 (2016) 9 p.. [13] X.J. Hou, J.C. Han, C.S. Yan, H.H. Ren, Y. Zhang, T. Zhang, et al., Cardioprotective effects of total flavonoids extracted from Xinjiang Sprig Rosa rugosa against acute ischemia/reperfusion-induced myocardial injury in isolated rat heart, Cardiovasc. Toxicol. 16 (2015) 54–66. [14] X.Y. Liu, L. Xu, Y. Wang, J.X. Li, Y. Zhang, C. Zhang, et al., Protective effects of total flavonoids of Astragalus against adjuvant-induced arthritis in rats by regulating OPG/RANKL/NF-κB pathway, Int. Immunopharmcol. 44 (2017) 105–114. [15] S. Song, F.L. Chen, X.F. Xing, M.Y. Ren, Q.H. Ma, Y. Xie, et al., Concurrent quantification and comparative pharmacokinetic analysis of bioactive compounds in the Herba Ephedrae-Semen Armeniacae Amarum herb pair, J. Pharm. Biomed. Anal. 109 (2015) 67–73. [16] Y. Jin, Y.P. Qu, H.Q. Pang, L.L. Liu, Z.H. Zhu, E.X. Shang, et al., Herb pairs containing Angelicae Sinensis Radix (Danggui): a review of bio-active constituents and compatibility effects, J. Ethnopharmacol. 181 (2016) 158–171. [17] D.H. Nguyen, U.M. Seo, B.T. Zhao, D.D. Le, S.H. Seong, J.S. Choi, et al., Ellagitannin and flavonoid constituents from Agrimonia pilosa Ledeb. With their protein tyrosine phosphatase and acetylcholinesterase inhibitory activities, Bioorg. Chem. 72 (2017) 293–300.
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