GENE-39671; No. of pages: 7; 4C: Gene xxx (2014) xxx–xxx

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Gene journal homepage: www.elsevier.com/locate/gene

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Guanghao Ge, Qiong Zhang, Jiangwei Ma ⁎, Zengyong Qiao ⁎, Jianhua Huang, Wenbo Cheng, Hongwei Wang

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Department of Cardiology, Fengxian Branch of Shanghai 6th People's Hospital, Shanghai 201400, China

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a r t i c l e

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Article history: Received 20 March 2014 Received in revised form 23 April 2014 Accepted 10 May 2014 Available online xxxx

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Keywords: JAK2/STAT3 signaling pathway Salvia miltiorrhiza aqueous extract Myocardium ischemia reperfusion Antioxidant

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Salvia miltiorrhiza has strong antioxidative activity. They may have a strong potential as cardioprotective agents in ischemic–reperfusion injury. Experiments were carried out in Sprague–Dawley rats with myocardium ischemia reperfusion (IR). Myocardial injuries during IR were determined by changes in electrocardiogram analysis of arrhythmias, antioxidant enzyme activities, AST, CK-MB, lactate dehydrogenase (LDH) levels, and myocyte apoptosis. Results showed that S. miltiorrhiza aqueous extract (SAME) pre-treatment significantly decreased the ST-segment (ΣST120) and myocardium MDA, AST, CK-MB, lactate dehydrogenase (LDH) levels, increased myocardium antioxidant enzyme activities, and inhibit myocardium cell apoptosis. Furthermore, the SAME pre-treatment significantly upregulated p-JAK2 and p-STAT3 protein expression, decreased myocardium TNF-α and IL-6 concentrations in IR rats. The levels of TNF-α and IL-6 were positively correlated with the changes in myocardium p-JAK2 and p-STAT3 protein expression levels in IR rats. It can be concluded that the SAME pre-treatment has anti-ischemic and anti-apoptosis activity in heart IR rats. SAME pre-treatment protects heart against IR injury, at least in part, through its stimulating effects on injury-induced deactivation of JAK2/STAT3 signaling pathway. © 2014 Published by Elsevier B.V.

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1. Introduction

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Danshen, the dried root of Salvia miltiorrhiza, is used in Chinese medicine to treat vascular disease. According to Chinese medicine theory, Danshen promotes blood flow and resolves blood stasis. Among stroke patients, 80% suffer from cerebral infarction and 20% cerebral hemorrhage (Dietl et al., 2009). It is widely used to treat coronary heart disease, cerebrovascular disease, hepatitis, cirrhosis and chronic renal failure (Chan et al., 2004). This plant has received much interest due to its ability to accumulate large amounts of active natural products such as tanshinones (Chen et al., 1997) and phenolic compounds (Zupko et al., 2001). Seven phenolic compounds isolated from S. miltiorrhiza as active components have a strong protective action against oxidative damage to liver microsomes, hepatocytes, and erythrocytes (Liu et al., 1992). Cardiac muscle is a heterogeneous system and many parameters such as blood flow and perfusion (Groeneveld et al., 2001; Muehling et al., 2004; Stanley et al., 2005), patterns of ion channel activation (Gaborit et al., 2007; Näbauer et al., 1996; Qian et al., 2001) differ in distinct heart regions. As gene expression is concerned, spatial

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Protective effect of Salvia miltiorrhiza aqueous extract on myocardium oxidative injury in ischemic–reperfusion rats

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Abbreviations: IR, myocardium ischemia reperfusion; (LDH) levels, lactate dehydrogenase; SAME, Salvia miltiorrhiza aqueous extract; ROS, reactive oxygen species; MDA, malondialdehyde; TNFα, tumor necrosis factor alpha; (IL)-6, interleukin; GSH-Px, glutathione peroxidase; GSH, glutathione. ⁎ Corresponding authors. E-mail addresses: [email protected] (J. Ma), [email protected] (Z. Qiao).

heterogeneity between cardiac chambers as well as between the left and right ventricles has long been recognized (Barth et al., 2005; Chugh et al., 2003). However, mounting evidences suggest that also conduction velocity, repolarization heterogeneities, and arrhythmia susceptibility in different left ventricle (LV) regions can be attributable to regional differences in their protein expression pattern and function (Barth et al., 2009; Strom et al., 2010). The spatial, functional and temporal heterogeneity that is distinctive becomes especially relevant in the injured heart (LaFramboise et al., 2005; Mirotsou et al., 2003; Stanton et al., 2000). The oxygen free-radical system has been implicated in the pathogenesis of myocardial ischemia/reperfusion (I/R) injury (Maxwell and Lip, 1997; Park and Lucchesi, 1998). The oxygen radicals are generated by injured myocytes and endothelial cells in the ischemic zone, as well as neutrophils that enter the ischemic zone, and become activated on reperfusion. These oxygen radicals exacerbate membrane damage, which leads to calcium loading. The mechanism of tissue damage due to ischemia reperfusion is common to other organs such as the brain, myocardium and kidneys. Neutrophil infiltration and the generation of reactive oxygen species (ROS) can cause tissue damage through cell membrane lipid peroxidation, protein denaturation and DNA damage (Bozlu et al., 2003). Recently, the serum malondialdehyde (MDA) concentration in patients with testis torsion has been identified as a reliable marker of lipid peroxidation and tissue damage. For the above reasons different therapeutic strategies (Palmer et al., 1998; Savas et al., 2002) have been investigated with the aim of reducing short- and long-term testis reperfusion damages. The inflammation response to hepatic I/R

http://dx.doi.org/10.1016/j.gene.2014.05.021 0378-1119/© 2014 Published by Elsevier B.V.

Please cite this article as: Ge, G., et al., Protective effect of Salvia miltiorrhiza aqueous extract on myocardium oxidative injury in ischemic–reperfusion rats, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.021

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2.1. Preparation of S. miltiorrhiza aqueous extract

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S. miltiorrhiza was purchased from a local herb shop in Shanghai City, China. The plant material was dried at ambient temperature and stored in a dry place prior to use. The plant was washed well with water, dried at room temperature in the dark, and then ground in an electric grinder to give a coarse powder. A 150 g of the powdered aerial parts was suspended in 2000 mL distilled water, heated and boiled under reflux for 60 min. The decoction obtained was filtered, and the filtrate frozen at −20 °C and then lyophilised. The average yield of the lyophilised material (Za-extract) was approximately 21.4%. It was stored at ambient temperature until further use.

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2.2. Surgical procedure and experimental design in rat in vivo

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Male Sprague–Dawley rats aged 8 weeks, body weight 200–230 g, were purchased from experimental animal center and were anesthetized by intraperitoneal injection of ketamine hydrochloride (90 mg/kg) and xylazine hydrochloride (10 mg/kg), and ventilated with room air using a rodent ventilator with the body temperature maintained between 36 and 37 °C using a heating pad. Rats were divided into five groups: (I) sham control group; (II) ischemic reperfusion (IR) group; (III) IR + AG490 group; (IV) IR + SAME group; and (V) IR + SAME + AG490 group. In sham and IR control groups, rats (10 rats in each group) were given saline by oral gavage for 15 days before IR operation; In IR + SAME group, rats were administered with SAME (400 mg/kg) by oral gavage for 15 days before IR operation; In IR + SAME + AG490 group, rats were administered with SAME (400 mg/kg) and AG490 (an inhibitor of JAK2, 0.3 mg/kg, s.c.) by oral gavage for 15 days before IR operation; In IR + AG490 group, rats were administered with AG490 (an inhibitor of JAK2, 0.3 mg/kg, s.c.) by oral gavage for 15 days before IR operation. Rats were subjected to a 60 min coronary artery occlusion followed by 3 h of reperfusion. The snare encircling the coronary artery was used for occlusion by pulling up on the suture and clamping it with a plastic tube. Coronary artery reperfusion was restored by releasing the clamp. The sham groups were not exposed to

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2.3. Analysis of antioxidant enzyme levels

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Lipid peroxidation was determined indirectly by measuring the production of MDA in the renal extract following the method of Buege and Aust (1978) based on TBA reactivity. Briefly, 125 mL of supernatant was mixed with 50 mL of saline buffer (TBS, pH 7.4) and 125 mL of 20% trichloroacetic acid containing 1% butylhydroxytoluene (BHT), and centrifuged (1000 g, 10 min, 4 °C). Then, 200 mL of supernatant was mixed with 40 mL of HCl (0.6 M) and 160 mL of Tris–thiobarbituric acid (120 mM) and the mixture was heated at 80 °C for 10 min. The absorbance was measured at 530 nm. The amount of TBARS was calculated using an extinction coefficient of 1.56 × 10−5 M−1 cm−1 and expressed in nmol of MDA/mg protein. The activity of SOD in tissue homogenate was assayed by the inhibition of autoxidation of pyrogallol as described by Marklund and Marklund (1974). Tissue supernatant was mixed with equal volume of Triton X-100 (1%) on ice for 30 min. After centrifuged at 9300 g for 5 min (4 °C), the supernatant was collected for SOD activity analysis. A final 3.017 mL volume of the reaction systems contained 10 μL sample, 3 mL of 50 mM sodium phosphate buffer containing 0.1 mM ethylenediamine tetraacetic acid (EDTA, pH 8.0) and 50 mM pyrogallol (0.7 μL), and the absorbance was recorded every 15 s for 5 min at 420 nm. One unit of SOD activity was defined as the amount of enzyme required for producing half maximal inhibition of autoxidation. Glutathione peroxidase (GSH-Px) activity was analyzed by a spectrophotometric assay. A reaction mixture consisting of 1 mL of 0.4 M phosphate buffer (pH 7.0) containing 0.4 mM EDTA, 1 mL of 5 mM NaN3, 1 mL of 4 mM GSH, and 0.2 mL of supernatant was preincubated at 37 °C for 5 min. Then 1 mL of 4 mM H2O2 was added and incubated at 37 °C for further 5 min. The excess amount of GSH was quantified by the DTNB method as described by Sharma and Gupta (2002). One unit of GSH-Px is defined as the amount of enzyme required to oxidize 1 nmol GSH/min. Catalase activity was assayed following the method of Sinha (1972). The reaction mixture consisted of 150 μL phosphate buffer (0.01 M, pH 7.0), 100 μL supernatant. Reaction was started by adding 250 μL H2O2 0.16 M, incubated at 37 °C for 1 min and the reaction was stopped by the addition of 1.0 mL of dichromate:acetic acid reagent. The tubes were immediately kept in a boiling water bath for 15 min and the green color developed during the reaction was read at 570 nm on a spectrophotometer. Control tubes, devoid of enzyme, were also processed in parallel. The enzyme activity is expressed as μmol of H2O2 consumed/min/mg protein. Glutathione (GSH) was measured following the method of Fukuzawa and Tokumura (1976). 200 μL of supernatant was added to 1.1 mL of 0.25 M sodium phosphate buffer (pH 7.4) followed by the addition of 130 μL DTNB 0.04%. Finally, the mixture was brought to a final volume of 1.5 mL with distilled water and the absorbance was read in a spectrophotometer at 412 nm and the results were expressed as μg GSH/μg protein. GR activity was assayed at 25 °C according to the method of Carlberg and Mannervik (1975) with slight modifications. The assay mixture contained 250 mM phosphate buffer (pH 7.4), 1 mM EDTA, 2.2 mM GSSG, 0.17 mM NADPH and an appropriate amount of the enzyme. The action was initiated by the addition of GSSG, and NADPH oxidation was monitored spectrophotometrically at 340 nm. One unit of enzyme activity was defined as the amount that oxidizes 1 μmol NADPH/min under the assay conditions.

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ischemia reperfusion, but to a time-matched normal perfusion. Echocardiography was performed as previously described, with a Hewlett–Packard echocardiography system SONOS 2000 with a 3.5/2.7 MHz transducer, and recorded. The experiment was approved by the Institutional Committee on Ethics of Animal Experimentation of Shanghai JiaoTong University.

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injury continues with the releasing of proinflammatory cytokines as tumor necrosis factor alpha (TNFα), interleukin (IL)-6, reactive oxygen species (ROS), and neutrophil infiltration. A great evidence has shown that reperfusion injury in the myocardium is an acute inflammatory reaction, which involves multiple cytokines. There is substantial evidence that the production of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 is increased during ischemia–reperfusion in the myocardium (Chandrasekar et al., 1997; Gurevitch et al., 1996; Wei et al., 2013; Yang et al., 2009), and that anti-TNF-α and anti-IL-6 treatment improve the recovery of post-ischemic myocardial function (Cain et al., 1999b; Gurevitch et al., 1997; Meldrum et al., 1997). The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways are responsive to a large number of cytokines, growth factors and hormones and play a vital role in mediating cardioprotection from IR injuries (Boengler et al., 2008b; Fuglesteg et al., 2008; Heusch et al., 2012). Experimental evidence indicates that the JAK2/STAT3 signaling pathway is specifically involved in preventing myocardial IR injury (Heusch et al., 2011; Kelly et al., 2010). However, whether the JAK2/STAT3 pathway plays an important role in the cardioprotection that is induced by S. miltiorrhiza aqueous extract treatment requires further investigation. In the present study, we for the first time investigated the role of the JAK2/STAT3 pathway in the cardioprotective effect of S. miltiorrhiza aqueous extract by examining the effect of S. miltiorrhiza aqueous extract on myocardial apoptosis in an in vivo model in rats.

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Please cite this article as: Ge, G., et al., Protective effect of Salvia miltiorrhiza aqueous extract on myocardium oxidative injury in ischemic–reperfusion rats, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.021

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G. Ge et al. / Gene xxx (2014) xxx–xxx

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2.4. TUNEL assay

2.8. Statistical analysis

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Programmed cell death was determined through Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, as described earlier (Roy et al., 2012). In brief, after 4 h of treatment mature proglottids of plant extracts treated (10 mg/mL) and control cestodes were subjected to cryomicrotomy (Leica CM 1850, Germany) at a thickness of 7–10 μm. The sections were first fixed in 4% paraformaldehyde and digested by proteinase K (20 μg/mL) (Sigma, St. Louis, USA) for 15 min at room temperature. Sections were than incubated in elongation buffer (Ultrapure water, 10 × TdT buffer, 25 mM CoCl2, 1 mM Bio-16-dUTP and 25 U/μL TdT). The fragmented DNA was labeled by biotinylated nucleotide mix (Enzo Life Sciences, UK) in the presence of deoxynucleotidyl transferase (TdT) (Enzo Life Sciences, UK) for 60 min in a humidified chamber and stopped the reaction by transferring the slides to termination buffer (300 mM NaCl, 30 mM sodium citrate) for 15 min at room temperature. Finally, the labeled fragments were incubated with avidin–peroxidase (Enzo Life Sciences, UK) and then with color developer AEC (3-amino-9-ethylcarbazole) for 30 min at 37 °C. Leitz Dialux 20 fluorescence microscope was used for photography.

All data are presented as means ± standard error of the mean (SEM). Statistically significant differences among the groups were determined by one-way analysis of variance (ANOVA) followed by the Tukey–Kramer test and CISE-treated groups were compared with the each CON group, and significant differences between the groups were analyzed by Student's t-test. A p-value of less than 0.05 was considered statistically significant.

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3. Results

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In our experiments, acute myocardial I/R injury causes ST-segment (ΣST120) elevation in rats. Pretreatment with SAME at 400 mg/kg significantly inhibited the ST-segment elevation in group IV compared to group II (p b 0.01, Table 1). However, the ST-segment (ΣST120) in group V was significantly higher than that in group IV. As shown in Table 2, myocardium MDA level was significantly higher, whereas GSH level was markedly lower in group II compared to those in group I. Pretreatment with SAME at 400 mg/kg significantly decreased the myocardium MDA and increased GSH level in group IV compared to group II (p b 0.01, Table 1). However, AG490 (0.3 mg/kg, s.c.) pre-treatment significantly (p b 0.01) suppressed the reduction of myocardium MDA and the elevation of myocardium GSH induced by SAME. As a consequence of I/R injury, the activities of myocardium SOD, CAT, GSH-Px and GR in group II rats were significantly lower than animals in group I rats (p b 0.01). Compared with group II, SAME pretreatment significantly inhibited the reduction of myocardium SOD, CAT, GSH-Px and GR (p b 0.05 and p b 0.01) activities induced by I/R injury. However, AG490 (0.3 mg/kg, s.c.) pre-treatment significantly (p b 0.01) suppressed the elevation of myocardium SOD, CAT, GSH-Px and GR activities induced by SAME. As a consequence of I/R injury, the myocardium activities of Na+– + K -ATPase and Ca2+–Mg2+-ATPase in group II rats were significantly higher than the animals in group I rats (p b 0.01). Compared with group II, SAME pre-treatment significantly inhibited the elevation of Na+–K+-ATPase and Ca2+–Mg2+-ATPase (p b 0.05 and p b 0.01) activities induced by I/R injury. However, AG490 (0.3 mg/kg, s.c.) pretreatment significantly (p b 0.01) suppressed the reduction of myocardium activities of Na+–K+-ATPase and Ca2+–Mg2+-ATPase induced by SAME (see Tables 3 and 4). In the current studies, we examined whether the cardioprotective effect of SAME is related to the activation of JAK2/STAT3 pathways. As shown in Fig. 1A,B,C,D, myocardium p-JAK2 and p-STAT3 protein expression levels were significantly greater in group II relative to group I (p b 0.01). In contrast, myocardium p-JAK2 and p-STAT3 protein expression levels were significantly lower in the group treated with SAME relative to group II (p b 0.01). When giving SAME to each rat, we simultaneously administered inhibitor (AG490) of the pathway to rats to determine whether the cardioprotective effect of SAME can be abolished by the inhibitor. We found that AG490 (0.3 mg/kg, s.c.) pretreatment significantly (p b 0.01) suppressed the reduction of expression

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2.5. Na+/K+-ATPase assay

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For the Na+/K+-ATPase assay, 20 μL of the enzyme preparation (8–12 μg of protein) was added to 200 μL of the reaction mixture as described by Tsakiris and Deliconstantinos (1984), pre-incubated for 10 min at 37 °C. The reaction was initiated by the addition of ATP to a final concentration of 3.0 mM. The enzyme assays were stopped by the addition of 200 μL of 10% TCA to a final concentration of 5%. Na+/K+ATPase activity was calculated by the difference between the two assays. Released inorganic phosphate (Pi) was measured using the method of Chan et al. (1986).

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2.6. Analysis of Ca2 +–Mg2 +-ATPase, TNF-α, IL-6, AST, LDH and CK-MB levels

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2.7. Western blot analysis

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Myocardium cellular lysates were prepared with RIPA lysis buffer (Sunshine Biotechnology (Nanjing) Co. Ltd., China) and protein concentrations were measured using BCA Protein Assay Kit (Beijing Cellchip Biotechnology Co. Ltd., China) following the kit's instruction. Protein extracts were heat denatured at 95 °C for 5 min, electrophoretically separated on 12% SDS–PAGE, and transferred to PVDF membranes. The membranes were subjected to the reaction with a 1:1000 dilution of goat anti-JAK2, anti-STAT3, anti-p-JAK2, anti-p-STAT3 polyclonal antibody in TBS buffer at 4 °C overnight, followed by a reaction with a 1:2000 dilution of mouse anti-goat antibodies conjugated with horseradish peroxidase (HRP) at room temperature for 2 h. After the membrane was washed, the HRP activity was detected using an ECL kit. The image was scanned with a GS800 Densitometer Scanner (BioRad), and the data were analyzed using PDQuest 7.2.0 software (BioRad). β-actin (1:300) was used as an internal control.

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Ca2 +–Mg2 +-ATPase, TNF-α and IL-6 levels were measured with commercially available kits. Biochemical examinations were performed using blood collected into plain tubes. Blood samples were centrifuged for 5 min at 3000 rpm. The following biochemical assays were performed using Selectra Junior Version 04 autoanalyzer for biochemical assays (Vital Scientific BV, Netherlands). Aspartate amino transferase (AST), lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) were assayed.

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Table 1 ST-segment (ΣST120) change in groups. Group

ΣST120 (mV)

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I II III IV V

0.61 3.4 3.9 1.4 2.4

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± ± ± ± ±

0.22 0.5a 0.6 0.4b 0.5b,c

ΣST120: The sum of ST segment elevation in 120 min. a p b 0.01, compared with group I. b p b 0.01, compared with group II. c p b 0.01, compared with group IV.

t1:9

Q2t1:10 t1:11 t1:12

Please cite this article as: Ge, G., et al., Protective effect of Salvia miltiorrhiza aqueous extract on myocardium oxidative injury in ischemic–reperfusion rats, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.021

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4 Table 2 Myocardium MDA and GSH levels in groups.

Table 4 Myocardium activities of Na+–K+-ATPase and Ca2+–Mg2+-ATPase in groups.

t2:3

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MDA (nmol/mg)

GSH (μg/mg)

t2:4 t2:5 t2:6 t2:7 t2:8

I II III IV V

6.98 22.61 29.73 12.59 18.05

125.38 53.99 36.02 118.59 81.05

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13.57 6.05a 4.11c 10.49c 7.47c,d

p b 0.01, compared with group I. p b 0.01. p b 0.01, compared with group II. p b 0.01, compared with group IV.

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Table 3 Activities of myocardium SOD, CAT, GSH-Px and GR in groups.

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SOD

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285.7 121.9 93.5 277.9 184.3

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0.13 0.06a 0.05b 0.08b 0.08b,c

p b 0.01, compared with group I. p b 0.01, compared with group II. p b 0.01, compared with group IV.

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It is well-known that various arrhythmias caused by myocardial infarction are life-threatening; so, it is critical for clinicians to reverse the arrhythmias to normal rhythm along with the treatment with the initial heart disease. In this study, the sum of ST segment elevation in 120 min in group IR is significantly higher than that in sham group, showing an obvious ischemia injury. Pretreatment with SAME at 400 mg/kg significantly inhibited the ST-segment elevation in group IV compared to group II. This indicates that pretreatment with SAME improves heart rhythm in myocardium IR rats. Understanding the pathological mechanisms of MI is necessary for the development of therapeutic strategies and agents against cardiac ischemia. Oxidative stress and inflammation are the two keystones in the pathogenesis of MI (Kaminski et al., 2002). Oxidative stress occurred during myocardial ischemia and induced injuries directly by ROS itself, but also indirectly through an amplification of inflammatory cascade (Braunersreuther et al., 2012; Zhang et al., 2010); inversely, ROS can stimulate the production of inflammatory cytokines to cause myocardium damage (Hori and Nishida, 2009). Therefore, ROS and inflammatory response jointly lead to irreversible damage by cell necrosis and apoptosis which contribute to MI and cardiac dysfunction (Ferrari et al., 2004).

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0.17 0.07a 0.04b 0.16b 0.12b,c

Considering this, reducing ROS generation and inflammation cascade to break the vicious circle between each other might be useful therapeutic strategies as protection against myocardial ischemia injuries. Ischemia causes antioxidant deficit as well as increased oxidative stress. A significant elevation of MDA and reduction in the activities of GSH, SOD, CAT, GR and GSH-Px were observed after myocardium IR in our study. However, pretreatment with SAME significantly restrained the formation of MDA while increasing GSH, CAT, GSH-Px, GR, and SOD activities. The combination of AG490 and SAME significantly decreases the antiischemic effect of SAME which may result from enhancing activity of endogenous antioxidants. There are plenty of data suggesting that blocking pathological ROS production is likely to have beneficial clinical effects (Zhang et al., 2010). Suppressing the generation of ROS could limit infarct size and attenuate cardiac dysfunction after MI (Huang et al., 2010). Inhibition of Na+–K+-ATPase and Ca2+–Mg2+-ATPase activities has been reported to be one of the major mechanisms responsible for hypertension (Blaustein, 1996). Ca2+–Mg2+-ATPase activity of the vascular smooth muscle has been demonstrated to decrease in vascular disease. Altered cellular handling of Na+, Ca2 +, and Mg2 + has been shown in human and experimental hypertension to be associated with cardiac cell damage and death (Chinopoulos et al., 2007; Torres et al., 2009). In the present study, SAME pre-treatment significantly increased myocardium Na+–K+-ATPase and Ca2+–Mg2+-ATPase activities in IR rats, indicating that SAME can inhibit Ca2+ influx, reduce the content of Ca2+ in myocardial cells and mitochondria, and alleviate calcium overload. In the present study, we investigated the signaling pathways by which SAME triggers anti-apoptosis effect following IR injury in mouse heart. Specifically, we focused on the potential role of JAK2– STAT3 pathway in SAME-induced protection. This is because inactivation of STAT3 or deletion of STAT3 appears to be a key event in the diminution of cardioprotection in response to various physiological stresses including IR (Boengler et al., 2008b; Barry et al., 2009; Bolli et al., 2011). Moreover, it has been shown that mice with a cardiomyocyte-restricted deletion of STAT3 develop spontaneous heart failure in response to stress (Boengler et al., 2008a; Hilfiker-Kleiner et al., 2004; Jacoby et al., 2003). Our results demonstrate that SAME caused significant reduction in myocardium apoptosis rate, oxidative injury and inflammatory reaction in IR rats. Such cardioprotective effect of SAME was associated with significant increase in phosphorylation of STAT3 and JAK2. Pharmacological inhibition of JAK2–STAT3 and targeted in vivo blocking of STAT3 and JAK2 using AG490 abolished the heart protective effect of SAME against IR injury. The cardiac damage due to ischemia reperfusion was monitored by the presence of cardiac marker enzymes in the cardiac perfusate and the level of these enzymes in myocardium. The presence of aspartate aminotransferase, lactate dehydrogenase and creatine kinase in coronary perfusate of isolated rat heart indicated myocardial necrosis (Ishikawa et al., 1995). In this study, the levels of these enzymes in IR rats significantly increased and a subsequently decreased level was found in the blood of heart IR rat pre-treated with SAME.

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4. Discussion

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1.11 0.54 0.41 0.98 0.72

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1.62 0.61 0.36 1.48 1.05

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of p-JAK2 and p-STAT3 proteins induced by SAME. In addition, the results (Fig. 1D) still demonstrated that the administration of AG490 abolished the apoptosis reduction effect of SAME, as evidenced by an apoptosis rate comparable to that of group IV (Fig. 2). AG490 (0.3 mg/kg, s.c.) pretreatment significantly (p b 0.01) suppressed the reduction of serum activities of AST, CK-MB and LDH induced by SAME (Table 5). There was no significant difference in JAK2, STAT3 protein expression levels of the myocardium tissue among the rats between groups. Compared with those of group I, myocardium TNF-α and IL-6 concentrations were increased significantly in group II (Table 6). SAME pre-treatment significantly decreased serum TNF-α and IL-6 concentrations in group IV compared to group II. AG490 (0.3 mg/kg, s.c.) pretreatment significantly (p b 0.01) suppressed the reduction of myocardium TNF-α and IL-6 concentrations induced by SAME. As shown in Table 7, we found that the levels of TNF-α and IL-6 were positively correlated with the changes in myocardium p-JAK2 and pSTAT3 protein expression levels in IR rats.

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t4:3

I II III IV V

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Ca2+–Mg2+-ATPase (μmol·mg protein−1·h−1)

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0.61 1.98a 3.08b 1.42c 1.93c,d

Na+–K+-ATPase (μmol·mg protein−1·h−1)

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b

± ± ± ± ±

CAT ± ± ± ± ±

31.64 13.61a 10.42b 29.85b 20.47b,c

59.74 24.15 16.82 52.96 43.11

p b 0.01, compared with group I. p b 0.01, compared with group II. p b 0.01, compared with group IV.

GSH-Px ± ± ± ± ±

6.31 2.51a 1.77b 5.82b 4.59b,c

83.16 35.09 22.63 79.42 53.49

± ± ± ± ±

GR 9.76 4.22a 1.95b 6.66b 4.52b,c

77.47 35.42 21.72 69.61 55.26

± ± ± ± ±

6.93 3.29a 2.41b 5.93b 5.05b,c

t4:1 t4:2

Group

E

t2:1 t2:2

G. Ge et al. / Gene xxx (2014) xxx–xxx

Please cite this article as: Ge, G., et al., Protective effect of Salvia miltiorrhiza aqueous extract on myocardium oxidative injury in ischemic–reperfusion rats, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.021

347 348 349 Q7 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398

G. Ge et al. / Gene xxx (2014) xxx–xxx

I

II

III

IV

V

5

I

II

III

IV

V STAT3

JAK2

β-actin

β-actin 1.4

0.1

1.2

0.09 0.08 0.07

0.8 0.6 0.4

0.06 0.05 0.04

F

STAT3/β-actin

0.03 0.02

0.2

0.01

0

0

I

I

II

II

III group

III

IV

IV

C

V

I

V

I

II

d

1.4

d,f b d

C

0.8

T

1.6

p-STAT3/β-actin

E

1.8

1 0.6

E

0.4 0.2 0 II

III group

IV

V

R

I

B

III

IV

IV

V

V p-STAT3

β-actin

D

β-actin

1.2

III group

P

p-JAK2

2

II

R O

A

p-JAK2/β-actin

O

JAK2/β-actin

1

d

1

d,f 0.8 0.6

b d

0.4 0.2 0

D

I

II

III group

IV

V

N C O

403 404

40

U

401 402

Cytokines are a heterogeneous group of proteins which orchestrate the inflammatory response in ischemia/reperfusion injury (Cutler and Brombacher, 2005). It has been shown that ischemia/reperfusion (IR) increase cytokine levels including TNF-α and IL-6 in the myocardium (Cain et al., 1999a). Myocardial necrosis after AMI induces complement activation and free radical generation, triggering TNF-α release from the

35 30

Apoptosis rate (%)

399 400

R

Fig. 1. Myocardium JAK2 (A), p-JAK2 (B), STAT3 (C), p-STAT3 (D) protein expression in group rats. bp b 0.01, compared with group I; dp b 0.01, compared with group II; fp b 0.01, compared with group IV.

d b

405 406 407 408 409 410 411 412 413 414

d,f

25

t5:1 t5:2

Table 5 Serum AST, CK-MB and LDH activities in group rats.

20 15

d

Group

AST (U/L)

IV

I II III IV V

451.52 802.71 931.34 519.7 682.79

10 5 0

infarcted myocardium. The secreted TNF-α further stimulates the release of proinflammatory cytokines, like IL-6, chemokines, and adhesion molecules from infiltrating leukocytes and endothelial cells, initiating the cytokine cascade. Pro-inflammatory cytokines, such as IL-6, and TNF-α, have emerged as significant contributors to myocardial dysfunction (Lu et al., 2011; Noyola-Martínez et al., 2013). Proinflammatory cytokines promote further inflammatory cell adhesion and infiltration into the myocardium and cause acute tissue injury by obstructing capillary vessels, the production of vasoactive substances, and the release of cytotoxic agents (Moreland and Curtis, 2009). In the SAME-treated

I

II

III group

V

a b

Fig. 2. Myocardium apoptosis rate in groups rats.

c

± ± ± ± ±

CK-MB (U/L) 48.59 83.69a 99.42 b 57.01b 72.48b,c

5106.51 12,285.93 15,215.38 6452.61 8926.53

± ± ± ± ±

t5:3

LDH (U/L) 532.73 1362.47a 1639.52b 707.47b 931.62b,c

1429.59 3027.05 3829.37 1761.44 2418.5

± ± ± ± ±

154.73 327.59a 400.51b 183.06b 261.75b,c

p b 0.01, compared with group I. p b 0.01, compared with group II. p b 0.01, compared with group IV.

Please cite this article as: Ge, G., et al., Protective effect of Salvia miltiorrhiza aqueous extract on myocardium oxidative injury in ischemic–reperfusion rats, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.021

t5:4 t5:5 t5:6 t5:7 t5:8 t5:9 t5:10 t5:11

IL-6 (pg/mg)

16.83 36.02 43.52 22.71 29.63

1.1 1.48 1.61 1.22 1.36

t6:9 t6:10 t6:11 t6:12

a

t7:1 t7:2 t7:3

b c d

± ± ± ± ±

1.07 2.16a 1.73b 1.33c 1.95c,d

± ± ± ± ±

0.09 0.1a 0.13b 0.08c 0.09c,d

p b 0.01, compared with group I. p b 0.01. p b 0.01, compared with group II. p b 0.01, compared with group IV.

Table 7 Correlation analysis of p-JAK2, p-STAT3 protein expression and TNF-α, IL-6 in myocardium in IR rats.

t7:4

Index

TNF-α

IL-6

t7:5 t7:6

p-JAK2 p-STAT3

r = 0.929; P = 0.031 r = 0.918; P = 0.039

r = 0.962; P = 0.022 r = 0.905; P = 0.043

415

444

Acknowledgments

432 433 434 435 436 437 438 439 440 441 442

445

C

E

430 431

R

428 429

R

426 427

O

424 425

C

422 423

N

420 421

U

418 419

T

443 Q8

group, the heart tissue levels of IL-6, and TNF-α were significantly decreased compared with the IR group. Therefore, suppressing the inflammatory reaction may be one of the mechanisms by which SAME protects the heart against IR injury. In this study, we found that the levels of TNF-α and IL-6 were positively correlated with the changes in myocardium p-JAK2 and p-STAT3 protein expression levels in IR rats. TUNEL examination showed that the rat myocardial apoptosis was significantly higher in group IV than in group I. This supports the hypothesis that the production and secretion of inflammatory cytokines, including TNF-α and IL-6, are dependent on the expression of p-JAK2 and p-STAT3 protein expression, and that these elevated cytokines might mediate subsequent myocardial injury. AG490 (0.3 mg/kg, s.c.) pre-treatment significantly (p b 0.01) suppressed the reduction of serum TNF-α and IL-6 concentrations induced by SAME. Based on the results generated in the present study, we speculate that blocking p-JAK2 and p-STAT3 or related signaling pathways may represent a novel method to alleviate TNF-α, IL-6-mediated inflammation in myocardium IR injury. In conclusion, we have provided direct evidence for the essential role of JAK2–STAT3 signaling in rapamycin-induced protection against I/R injury. Our results show that SAME pretreatment significantly decreased myocardium oxidative damage and improve immunity and heart function in IR rats. Our results still show that SAME induced phosphorylation of JAK2 and STAT3. The cardioprotection mechanisms of SAME may be attributed to decreasing oxidative damage and improving immunity function, which subsequently decreases myocardium apoptosis via activating the JAK2/STAT3 pathways. These findings indicate that SAME may be an effective and promising medicine for treating ischemic stroke.

416 417

F

TNF-α (pg/mg)

I II III IV V

O

Group

t6:4 t6:5 t6:6 t6:7 t6:8

R O

t6:3

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P

Table 6 Myocardium TNF-α and IL-6 concentrations in group rats.

D

t6:1 t6:2

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The authors thank Professor Wang Li for his useful help.

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C

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7

Please cite this article as: Ge, G., et al., Protective effect of Salvia miltiorrhiza aqueous extract on myocardium oxidative injury in ischemic–reperfusion rats, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.021

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