Atherosclerosis 231 (2013) 384e391
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Coptisine protects rat heart against myocardial ischemia/reperfusion injury by suppressing myocardial apoptosis and inflammation Jing Guo a, b, Shou-Bao Wang a, b, Tian-Yi Yuan a, c, Yu-Jie Wu a, c, Yu Yan a, c, Li Li a, b, Xiao-Na Xu a, c, Li-li Gong d, Hai-lin Qin a, b, Lian-Hua Fang a, b, *, Guan-Hua Du a, b, * a
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, China Beijing Key Laboratory of Drug Targets Identification and Drug Screening, China d Beijing Chao-Yang Hospital Affiliated with Beijing Capital Medical University, Beijing 100020, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 January 2013 Received in revised form 12 September 2013 Accepted 2 October 2013 Available online 16 October 2013
Objective: Protecting the heart from myocardial ischemia and reperfusion (I/R) damage is the focus of intense research. Coptisine is an isoquinoline alkaloid isolated from Coptidis Rhizoma. The present study investigated the potential effect of coptisine on myocardial I/R damage in rats and the underlying mechanisms. Methods and results: Electrocardiogram examination showed that the administration of coptisine 10 min before ischemia significantly decreased I/R-induced arrhythmia after 30 min ischemia followed by 3 h reperfusion. The release of cardiac markers was also limited. Echocardiography was performed before ischemia and 24 h post-I/R, separately. The M-mode records showed that the reductions of ejection fraction (EF) and fractional shortening (FS) were attenuated in coptisine-treated rats compared with the I/R rats. Similar results were obtained with Evans Blue/triphenyl tetrazolium chloride (TTC) staining, in which coptisine notably reduced infarct size. Moreover, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay demonstrated coptisine suppressed myocardial apoptosis, which may be related to the upregulation of Bcl-2 protein and inhibition of caspase-3 activation. Coptisine treatment also attenuated the proinflammatory cytokines including interleukin (IL)-1b, IL-6, and tumor necrosis factor-a in heart tissue. Additionally, Western blot and immunohistochemical analysis showed that coptisine markedly reduced Rho, Rho-kinase 1 (ROCK1), and ROCK2 expression and attenuated the phosphorylation of myosin phosphatase targeting subunit-1, a downstream target of ROCK. Conclusions: Coptisine exerts pronounced cardioprotection in rats subjected to myocardial I/R likely through suppressing myocardial apoptosis and inflammation by inhibiting the Rho/ROCK pathway. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Coptisine Ischemia/reperfusion (I/R) Apoptosis Inflammation Rho-kinase (ROCK)
1. Introduction As the morbidity and mortality associated with ischemic heart disease have continued to increase and are gaining increased attention. Despite early reperfusion and improvements in antiplatelet and anti-thrombotic therapy, the mortality of acute myocardial infarction (AMI) patients remains significant even if undergoing primary percutaneous coronary intervention. One major contributing factor is the inability to protect the heart against
* Corresponding authors. Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xiannongtan Street, Beijing 100050, China. Tel.: þ86 10 63165313; fax: þ86 10 63165184. E-mail addresses: [email protected] (J. Guo), [email protected] (L.-H. Fang), [email protected] (G.-H. Du). 0021-9150/$ e see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2013.10.003
the detrimental effects of lethal myocardial reperfusion injury, which occur on restoring blood flow to the acutely ischemic myocardium. Therefore, fully understanding the mechanisms of ischemia/reperfusion (I/R) injury and seeking for novel therapeutic strategies is still the focus of intense research. The cardiomyocyte apoptosis and inflammatory reaction have been recognized as hallmarks of myocardial reperfusion injury. Recent evidences suggest that myocardial apoptosis is initiated shortly after ischemia, is amplified by reperfusion, and partially contributes to overall cardiomyocyte death [1]. Blocking the apoptotic process may prevent the loss of contractile cells, minimize cardiac injury induced by I/R, and slow the occurrence of myocardial stunning and heart failure [2]. Likewise, reducing inflammatory responses during reperfusion after ischemic insult has been shown to be beneficial in numerous studies [3,4]. In addition,
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Rho-kinase (ROCK), the best-characterized effector of the small Gprotein Rho, has recently been suggested to be potential therapeutic targets in the treatment of cardiovascular diseases [5e7]. Accumulating researches have demonstrated that ROCKs play a considerable role in mediating the myocardium apoptosis and inflammatory response to I/R injury [3,8e10]. The beneficial effects of ROCK inhibition against I/R injury using the ROCK inhibitors fasudil and Y-27632 have been well established [3,8,11,12]. Coptisine (Cop) is an isoquinoline-type alkaloid isolated from Coptidis Rhizoma, the rhizome of a traditional Chinese herb, Coptis chinensis Franch. Recently, several biological activities of coptisine have been reported, including anti-diabetic [13], antimicrobial [14], antiviral [15], anti-hepatoma, and anti-leukemia effects [16]. Moreover, we previously found that coptisine exerted cardioprotective effects on isoproterenol-induced myocardiac infarction (MI) in rats, and Rho/ROCK pathway may contribute to the effects [17]. The potential effects of coptisine on MI prompted us to investigate whether it is capable of exerting protection effects during myocardial I/R injury and the underlying mechanism responsible for its actions. In the present study, we employed a model of left anterior descending (LAD) coronary artery occlusion for 30 min followed by reperfusion in rats [18], which simulates the most common clinical manifestations of I/R (i.e., AMI followed by reperfusion therapy) to determine the potential effects and mechanisms of coptisine on I/R injury.
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due to anesthesia or failed surgery, the number of rat in each group was as follows: sham (n ¼ 18), I/R (n ¼ 18), I/R þ Cop 3 mg/kg (n ¼ 21), I/R þ Cop 10 mg/kg (n ¼ 18), I/R þ Cop 30 mg/kg (n ¼ 21). 2.4. Electrocardiographic recording and arrhythmia analysis The ECG was monitored throughout the 30 min ischemia and 3 h reperfusion process using the BL-420S Biologic Function Experiment system (Chengdu, China). Ventricular tachycardia (VT), ventricular fibrillation (VF), and premature ventricular complexes (PVCs) were evaluated according to the Lambeth Convention [20]. The arrhythmia score was graded as previously described [20,21] to evaluate the severity of arrhythmias. Detailed arrhythmias scoring criteria is enclosed in Supplementary material. 2.5. Echocardiography Cardiac function was determined by transthoracic echocardiography as previously described [22,23]. The rats were initially anesthetized with 3% isoflurane in oxygen using a nose cone. Transthoracic 2-D and M-mode images of the left ventricle (LV) were acquired using the Vevo 770 high-resolution echocardiographic system (Visual Sonics Inc., Toronto, ON, Canada). The ejection fraction (EF) and fractional shortening (FS) were calculated from digital images using Vevo 770 software. For each rat, two measurements were performed: 1 day before LAD occlusion (baseline) and after 24 h of reperfusion [19].
2. Materials and methods 2.6. Measurement of myocardial infarct size 2.1. Animals All animal care and experimental procedures were performed in accordance with institutional animal ethical committee guidelines, which conform to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. All of the studies were performed using male Spraguee Dawley rats weighing 280 20 g (Vital River Laboratory Animal Center, Beijing, China). The animals were fed a normal rodent chow diet, with tap water available ad libitum. The rats were housed at a constant temperature and relative humidity under a regular 12 h/ 12 h light/dark schedule. 2.2. Reagents Coptisine (Fig. S1 in Supplementary) was extracted by the Department of Medicinal Chemistry at our institute. Its structure was confirmed based on its physicochemical properties and spectral evidence. Antibodies and major reagents were enclosed in Supplementary material. 2.3. Experimental protocol and drug administration The I/R model was generated by LAD occlusion for 30 min followed by 3 h reperfusion. The surgical procedures were performed as previously described [18,19]. The success of the coronary occlusion and reperfusion was verified by regional cyanosis of the myocardium and typical electrocardiogram (ECG) changes [3]. Fig. S2 describes the experimental protocol. Coptisine (3, 10, and 30 mg/kg, dissolved in saline) or vehicle was orally administered 10 min before occlusion. To examine cardiac function and myocardial infarct size, reperfusion was prolonged to 24 h, during which coptisine was administered in one more doses: 4 h after reperfusion. The experiments are performed on non-diseased hearts which have no abnormal ECG. Except for accidental deaths
At the end of 24 h reperfusion, all of the rats were sacrificed with overdose sodium pentobarbital, and the area at risk (AAR), infarct size, and viable area were evaluated by Evans Blue/TTC staining as previously described [19]. Briefly, Evans blue stained areas indicated non-ischemia area. White parts in the heart indicated the infarct size. Red parts, which were stained by TTC, represented for ischemic but viable tissue. White plus red part was AAR. 2.7. Determination of release of AST, LDH, CK-MB into serum Myocardial cellular damage was evaluated by measuring serum AST, LDH, and CK-MB levels. Three hours after reperfusion, serum AST and LDH activities were measured spectrophotometrically, and serum CK-MB was quantified using a commercial ELISA kit according to the manufacturer’s instructions. 2.8. Cytokine measurement by ELISA Heart tissues from AAR were homogenized and then centrifuged. The levels of IL-1b, IL-6, and TNF-a in heart homogenates were measured using ELISA kits according to the manufacturer’s instructions. The concentrations of the cytokines were quantified by reference to standard curves. 2.9. Determination of myocardial apoptosis A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed using an In Situ Cell Death Detection kit according to the manufacturer’s instructions. Both positive (DNase-treated) and negative (no addition of terminal transferase) control tissue sections were incorporated into each assay. Individual nuclei were visualized at 400 magnification, and the percentage of TUNEL-positive nuclei (positive nuclei/total nuclei) was calculated using ImagePro Plus 5.1 (Media Cybernetics, Bethesda, MD, USA) from at least eight randomly chosen fields per slide for
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statistical analysis. All of these measurements were performed in a blinded manner.
were analyzed using the KruskaleWallis rank-sum test. Values of P < 0.05 were considered statistically significant. SPSS 16.0 software (SPSS, Chicago, IL, USA) was used for the data analyses.
2.10. Agarose gel electrophoresis of DNA 3. Results Detailed method is enclosed in Supplementary. 3.1. Coptisine alleviated I/R-induced arrhythmias in rats 2.11. Immunohistochemical (IHC) analysis of ROCK1 in heart tissue Detailed method is enclosed in Supplementary. 2.12. Western blot analysis After 3 h reperfusion, the hearts were quickly removed, and the AAR from LV was immediately snap-frozen in liquid nitrogen. Western blotting was performed as described in Supplementary material. 2.13. Statistical analysis The data are expressed as mean SEM. Quantitative data were analyzed using analysis of variance (ANOVA) followed by Dunnett’s Multiple Comparison post hoc test. The incidence of arrhythmias (%) was analyzed using Fisher’s exact probability test. Arrhythmia scores
I/R injury increased the arrhythmia score (Fig. S3A) by inducing frequent attacks of PVCs, VT, and VF (Fig. S3B). Coptisine at doses of 10 and 30 mg/kg significantly decreased the arrhythmia score (Fig. S3A) by reducing the incidence of VT and PVCs compared with the I/R group. The incidence of VF did not differ between coptisinetreated rats and I/R rats (Fig. S3B). 3.2. Coptisine limited infarct size and reduced the release of biochemical marker enzymes into serum in I/R rats Representative images of heart sections stained with Evans Blue/TTC are shown in Fig. 1A. AAR/LV (%) (Fig. 1B) was similar between all hearts exposed to I/R treatments, thus demonstrating that all of the I/R treatments had statistically similar ischemic and perfused tissue ratios. As an index of post-I/R tissue necrosis, infarcted regions were determined and normalized to AAR. Both
Fig. 1. Coptisine decreased infarct size and serum biochemical marker enzymes in I/R rats. (A) Representative illustrations of heart sections stained with Evans Blue/TTC. The area at risk (AAR) and infarct regions were determined at 24 h after reperfusion. Dark blue staineviable area; White staineinfarct region; White plus red staineAAR. (B) The ratio of AAR/LV area (%) was similar between all hearts exposed to I/R treatments. (C) The infarct size was normalized to the AAR (%), both 10 and 30 mg/kg coptisine administration significantly decreased infarct size (% of AAR) after 30 min ischemia followed by 24 h reperfusion compared with I/R group. (DeF) Coptisine dose-dependently decreased serum level of AST, LDH, and CK-MB in rats subjected to 30 min ischemia and 3 h reperfusion. Data are expressed as mean SEM. n ¼ 6e8. ##P < 0.01, vs. Sham group; *P < 0.05, **P < 0.01, vs. I/R group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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the 10 and 30 mg/kg doses of coptisine significantly decreased infarct size (% of AAR) after 30 min ischemia followed by 24 h reperfusion compared with the I/R rats (Fig. 1C). As shown in Fig. 1DeF, I/R injury induced the necrotic cell death of myocytes, reflected by a significant increase in the release of biochemical markers, including AST, LDH, and CK-MB, into serum. Coptisine at doses of 10 and 30 mg/kg decreased serum AST, LDH and CK-MB levels compared with the I/R group. Moreover, as shown in Fig. S5, coptisine at 3e30 mg/kg had no effect on the systemic blood pressure, including SBP, DBP and MBP, from 0 to 6 h after oral administration in normal rats. 3.3. Coptisine attenuated the reduction of left ventricular function after I/R in rats At 24 h post-I/R, the rats exhibited a pronounced decline in LV function, reflected by an attenuation of LV wall motion and reduction of EF and FS (i.e., EF and FS at 24 h post-I/R minus their baseline values; Fig. 2). The representative echocardiographic Mmode records (Fig. 2A) showed that LV wall motion was wellpreserved in coptisine-treated rats. Further analysis of these records demonstrated that the reductions of EF and FS in rats treated with coptisine at doses of 10 and 30 mg/kg were significantly decreased compared with the I/R group (Fig. 2B, C). 3.4. Coptisine decreased cardiomyocytes apoptosis and cleaved caspase-3 and increased Bcl-2 expression in I/R rats A representative photomicrograph showed that TUNELpositive cardiomyocyte nuclei (i.e., dark brown cell nuclei) were more frequently observed in the I/R rats. In contrast, almost no TUNEL-positive cells were observed in sections from sham
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rats (Fig. 3A). Treatment with coptisine at doses of 10 and 30 mg/ kg significantly and dose-dependently decreased the percentage of apoptotic nuclei compared with the I/R group. The anti-apoptosis effect of coptisine was confirmed by agarose gel electrophoresis of DNA (Fig. S4). DNA ladder, indicative of apoptotic internucleosomal DNA fragmentation, were clearly visible in agarose gels of DNA from the AAR zone of hearts subjected to I/R injury. Coptisine (30 mg/kg) treatment reduced the appearance of DNA fragmentation. To further investigate the anti-apoptotic effect of coptisine, the levels of cleaved caspase-3 and Bcl-2 expression in heart were determined by Western blot analysis. Consistent with the TUNEL assay result, coptisine dose-dependently reduced the expression of the cleaved, activated forms of caspase-3 (Fig. 3B) and upregulated the level of the pro-survival protein Bcl-2 (Fig. 3C). 3.5. Coptisine reduced proinflammatory cytokine levels and NF-kB activity in heart of I/R rats The ELISA results showed that 30 min ischemia and 3 h reperfusion resulted in a noticeable increase in the levels of IL-1b, IL-6, and TNF-a. Coptisine at doses of 10 and 30 mg/kg decreased the levels of IL-1b, IL-6, and TNF-a in heart tissue compared with the I/R rats (Fig. 4AeC). We further studied the effects of coptisine on nuclear factorkappa B (NF-kB) activation by detecting the translocation of NFkB p65 subunit from cytoplasm to nucleus. As shown in Fig 4D, E, I/R injury strongly decreased NF-kB p65 level in the cytosolic, but increased the NF-kB p65 level in the nucleus. Administration of coptisine markedly inhibited the degradation of NF-kB p65 in the cytosolic, as well as the increase in the nucleus in a dose-dependent manner.
Fig. 2. Coptisine attenuated the reduction of left ventricular function in rats. (A) Representative echocardiographic M-mode records obtained at the papillary muscle level at baseline and 24 h post-I/R in rats treated with saline or coptisine. The solid arrows indicates end diastolic diameter; The dotted arrows indicates the end systolic diameter. (B, C) Coptisine at 3 and 10 mg/kg exhibit a significant less loss in percentage EF and FS relative to their respective baseline level after 24 h of reperfusion compared to I/R rats. Data are expressed as mean SEM. n ¼ 6. *P < 0.05, **P < 0.01, vs. I/R group. EF, ejection fraction; FS, fractional shortening.
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Fig. 3. Effects of coptisine on I/R-induced apoptosis and the expression of cleaved caspase-3 and Bcl-2. (A, aee) Apoptotic cardiomyocytes were identified by TUNEL analysis. TUNEL-positive cells were manifested as a marked appearance of dark brown apoptotic cell nuclei (bar ¼ 50 mm). (A, f) Quantitative analysis of percentage of apoptotic cardiomyocytes (n ¼ 6). (B, C) Representative immunoblotting bands (upper lane) of cleaved caspase-3 and Bcl-2. The densitometric analysis of bands (lower histograms) were normalized to GAPDH (n ¼ 5). Data are expressed as mean SEM. #P < 0.05, ##P < 0.01, vs. Sham group; *P < 0.05, **P < 0.01, vs. I/R group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.6. Coptisine reduced Rho, ROCK1, and ROCK2 expression and attenuated MYPT-1 phosphorylation To explore the molecular mechanisms involved in coptisinemediated cardioprotection, we further investigated the effects of coptisine on the Rho/ROCK pathway, which plays an important role in I/R injury. As shown in Fig. 5, Western blot indicated that I/R injury increased Rho, ROCK1, and ROCK2 expression compared with the sham operation. Administration of coptisine at doses of 10 and 30 mg/kg noticeably reduced Rho, ROCK1, and ROCK2 expression (Fig. 5A, B). ROCK1 immunohistological staining was also determined in the heart tissues (Fig. 5D). In sham-operated hearts, minimal ROCK1 immunoreactivity was noted. In contrast, positive ROCK1 staining clearly increased in hearts subjected to 30 min ischemia and 3 h reperfusion. In rats treated with 10 and 30 mg/kg coptisine, positive ROCK1 staining was significantly reduced. To further determine whether coptisine affects the activity of ROCKs, the phosphorylation level of the downstream target MYPT1 was measured by Western blot. As shown in Fig. 5C, myocardial I/
R resulted in an increased amount of phospho-MYPT-1 compared with sham operation. In contrast, coptisine at doses of 10 and 30 mg/kg significantly and dose-dependently decreased the amount of phospho-MYPT-1. 4. Discussion In the present study, coptisine has been proven to reduce the ischemic/reperfusion damage to the rat heart using a model of LAD coronary artery occlusion. Rats treated with coptisine had a lower arrhythmia score and a smaller infarct size compared with the I/R rats. Biochemical parameters of damage detection (AST, LDH and CK-MB) demonstrated the same protective effects. Myocardial functions were also improved by administration of coptisine. Suppression of cardiomyocyte apoptosis, attenuation of inflammation and significant inhibition of the Rho/ROCK pathway may contribute to the cardioprotective effects of coptisine. Our previous study [17] tested the effects of coptisine at doses of 25, 50 and 100 mg/kg on isoproterenol-induced MI in rats. Coptisine had an apparent protective effect, even at the lowest dose
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Fig. 4. Coptisine reduced proinflammatory cytokine levels and NF-kB activity in heart tissue of rats subjected to 30 min ischemia followed by 3 h reperfusion. (AeC) are measurement of IL-1b, IL-6, TNF-a in heart tissue by ELISA (n ¼ 6e8). D and E are measurement of cytosolic and nucleic NF-kB p65 expression by western blot. The bands for cytosolic NF-kB were normalized to GAPDH, and nucleic NF-kB p65 were normalized to Histone H3 (n ¼ 4). Data are expressed as mean SEM. #P < 0.05, ##P < 0.01, vs. Sham group; * P < 0.05, **P < 0.01, vs. I/R group.
(25 mg/kg). Based on the preliminary experiment, which showed potent protection against myocardial I/R injury in rat at lower doses (data not shown), three lower doses (3, 10, and 30 mg/kg) were used in the present study. These results suggested that coptisine had potent cardioprotective effects against I/R injury at doses of 10 and 30 mg/kg, and the beneficial effect of 30 mg/kg on the heart was more pronounced than that of the 10 mg/kg dose. Various disturbances in cardiac rhythm (including PVCs, VT, and the potentially lethal condition of VF) are an important consequence of both myocardial ischemia and reperfusion. The present study showed that coptisine decreased the arrhythmia score and incidence of PVCs and VT in I/R-injured rats, suggesting that coptisine protects cardiomyocytes from ischemia and reperfusioninduced membrane damage, thereby attenuating myocardial vulnerability to arrhythmia. Additionally, the release of AST, LDH, and CK-MB, biochemical markers of necrotic cell death and clinically used diagnostic markers of myocardium damage, was significantly decreased in the coptisine treatment groups. Echocardiography is a versatile noninvasive tool for measuring cardiac function and structure in animal as well as in clinic [19,22,23]. In the present study, we used echocardiography to estimate LV function as reflected by EF and FS. The echocardiography records showed that coptisine at doses of 10 and 30 mg/kg significantly diminished the decline in EF and FS compared with the I/R group 24 h post-I/R. Furthermore, Evans Blue/TTC staining of heart tissue was consistent with the cardiac function results. A robust limitation of infarct size was observed at coptisine doses of 10 and 30 mg/kg.
Apoptosis is a mainstay of tissue damage secondary to reperfusion injury after short ischemia. In the present study, TUNEL assay was used to examine myocardial apoptosis. The percentage of apoptotic cardiomyocytes after 3 h reperfusion was 24.56 0.90%, similar to the data reported previously in rat hearts subjected to I/R [4]. Treatment with coptisine at doses of 10 and 30 mg/kg resulted in significant reductions in cardiomyocyte apoptosis rate, consistent with the infarct-limiting effect of coptisine, indicating that the anti-apoptotic effect of coptisine in early phase of reperfusion could contribute to the attenuation of later myocardial necrosis (myocardial infarction). The anti-apoptosis effect of coptisine was confirmed by reduced appearance of DNA fragmentation. Caspase3 plays a central role in the execution phase of cell apoptosis. The activation of caspase-3 is governed by a series of signal transduction cascades, among which the interaction between antiapoptotic Bcl-2 and pro-apoptotic Bax proteins plays a critical role. Bcl-2 is capable of forming a heterodimer with Bax, thereby preventing Bax homodimerization and the sequential activation of caspase-3 [24]. The present study revealed that I/R markedly downregulated expression of Bcl-2 in the myocardium of I/R rats, consistent with the previous researches [3,4]. Meanwhile, treatment with coptisine at doses of 10 and 30 mg/kg clearly attenuated I/R-induced decrease of Bcl-2 expression in myocardium, suggesting that coptisine may exert its anti-apoptotic effects by regulating Bcl-2, thus depressing the activation of caspase-3. Myocardial necrosis after AMI induces complement activation and free radical generation, triggering TNF-a release from the
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Fig. 5. Effects of coptisine on the levels of Rho, ROCK1, and ROCK2 expression and phospho-MYPT-1 in heart tissue in rats subjected to 30 min ischemia followed by 3 h reperfusion. (A, B) Representative immunoblotting bands of Rho, ROCK1, and ROCK2. (C) Phosphorylation levels of MYPT-1, a downstream target of ROCKs. n ¼ 5. (D) Representative immunohistochemical staining for ROCK1 in rat myocardium in the different groups (bar ¼ 100 mm). (D, f) Quantitative measurement of ROCK1 expression (n ¼ 6). Data are expressed as mean SEM. #P < 0.05, ##P < 0.01, vs. Sham group; *P < 0.05, **P < 0.01, vs. I/R group. IOD, integrated optical density.
infarcted myocardium. The secreted TNF-a further stimulates the release of proinflammatory cytokines, like IL-1b and IL-6, chemokines, and adhesion molecules from infiltrating leukocytes and endothelial cells, initiating the cytokine cascade. Pro-inflammatory cytokines, such as IL-1b, IL-6, and TNF-a, have emerged as significant contributors to myocardial dysfunction [3,4,25,26]. 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 [27]. In the 30 mg/kg coptisine-treated group, the heart tissue levels of IL-1b, IL-6, and TNF-a were significantly decreased compared with the I/R group. Therefore, suppressing the inflammatory reaction may be one of the mechanisms by which coptisine protects the heart against I/R injury. NF-kB pathway plays a pivotal role in myocardial I/R injury and induces many pro-inflammatory cytokines and chemokines which will greatly contribute to myocardial I/R injury. Cytokines can selfamplify through a positive feed-back loop targeting NF-kB. On the one hand, NF-kB activation mediates the transactivation of proinflammatory genes, including TNF-a, IL-1b and IL-6. On the other hand, TNF-a and IL-1b directly activate NF-kB to increase the initial inflammatory response [28]. Here, we measured the inhibitory effect of coptisine on the translocation of NF-kB p65 from cytoplasm
to nucleus, which is representative of the activation of NF-kB, in I/R injured myocardium of rats. The results showed that coptisine significantly inhibited myocardial I/R induced activation of NF-kB pathway, which may be a mechanism of suppression of IL-1/6 and TNF-a. ROCKs play a considerable role in I/R-induced myocardial damage. Upregulated expression of Rho in ischemic myocardium and subsequent activation of ROCKs occurs during reperfusion [3,8e10]. Two highly homologous isoforms of ROCK, ROCK1 and ROCK2 are both expressed in rat heart [29], but their differential effects have not yet been well characterized. We previously showed that coptisine protected the myocardium against isoproterenolinduced MI injury in rats by inhibiting Rho/ROCK1 [17]. In the current study, a consistent result was found in a rat model of I/R. We observed a distinct increase in Rho and ROCK1 in heart tissues 3 h post-I/R, which was significantly reduced by coptisine administration. The expression of ROCK2 in the heart in I/R rats was further detected in the present study. Similar to the upregulation of ROCK1 protein, ROCK2 expression also increased in the heart in I/R rats. Coptisine at doses of 10 and 30 mg/kg also attenuated the expression of ROCK2. Moreover, ROCK activity was estimated by the phosphorylation of specific downstream target proteins MYPT-1. Consistent with previous studies [29,30], a marked elevation of phospho-MYPT-1 was observed in heart tissue in I/R rats, which
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was dose-dependently attenuated by coptisine. These results and earlier studies [3,9] together suggest that Rho/ROCK activation occurred as an early feature of reperfusion, and the upregulation of both ROCK1 and ROCK2 was involved in I/R injury in rats. Several recent studies have provided evidence that ROCKinhibition reduces cardiomyocyte apoptosis and inflammation in heart ischemia/reperfusion through PI3K/Akt and JAK2/STAT3 signaling pathways and JNK-mediated AIF translocation [3,8e11]. Thus, in the present study, the anti-apoptosis and anti-inflammation effects of coptisine were associated with, at least in part, the inhibition of the Rho/ROCK pathway, but a further study of coptisine on the crosstalk between ROCK and these signaling pathways is necessary to interpret the relationship among ROCK, apoptosis and inflammation. In conclusion, the salient finding of the present study was that coptisine treatment reduced infarct size and improved cardiac function after I/R injury in rats. The cardiac protection afforded by coptisine may contribute to its ability to suppress myocardial apoptosis and inflammation, which may at least partially occur through inhibition of the Rho/ROCK pathway. Therefore, the possible therapeutic application of such a potent effect of coptisine in patients at risk for myocardial I/R damage is very promising and justifies further research. Acknowledgments This study was supported by the National Scientific & Technological major special project “significant creation of new drugs” (2013ZX09103-001-008, 2012ZX09103-101-078) and Special Foundation on Scientific and Technological basic work (No. 2007FY130100) and the National Natural Science Foundation of China (No. 81102444, 81202538, 81373269). Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.atherosclerosis.2013.10.003. References [1] Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Circ Res 1996;79(5):949e56. [2] Anselmi A, Abbate A, Girola F, et al. Myocardial ischemia, stunning, inflammation, and apoptosis during cardiac surgery: a review of evidence. Eur J Cardiothorac Surg 2004;25(3):304e11. [3] Bao W, Hu E, Tao L, et al. Inhibition of Rho-kinase protects the heart against ischemia/reperfusion injury. Cardiovasc Res 2004;61(3):548e58. [4] Sun D, Huang J, Zhang Z, et al. Luteolin limits infarct size and improves cardiac function after myocardium ischemia/reperfusion injury in diabetic rats. PLoS One 2012;7(3):e33491. [5] Loirand G, Guerin P, Pacaud P. Rho kinases in cardiovascular physiology and pathophysiology. Circ Res 2006;98(3):322e34. [6] Shimokawa H. Rho-kinase as a novel therapeutic target in treatment of cardiovascular diseases. J Cardiovasc Pharmacol 2002;39(3):319e27. [7] Dong M, Yan BP, Liao JK, Lam YY, Yip GW, Yu CM. Rho-kinase inhibition: a novel therapeutic target for the treatment of cardiovascular diseases. Drug Discov Today 2010;15(15e16):622e9. [8] Zhang J, Li XX, Bian HJ, Liu XB, Ji XP, Zhang Y. Inhibition of the activity of Rhokinase reduces cardiomyocyte apoptosis in heart ischemia/reperfusion via suppressing JNK-mediated AIF translocation. Clin Chim Acta 2009;401(1e2): 76e80. [9] Hamid SA, Bower HS, Baxter GF. Rho kinase activation plays a major role as a mediator of irreversible injury in reperfused myocardium. Am J Physiol Heart Circ Physiol 2007;292(6):H2598e606. [10] Wolfrum S, Dendorfer A, Rikitake Y, et al. Inhibition of Rho-kinase leads to rapid activation of phosphatidylinositol 3-kinase/protein kinase Akt and cardiovascular protection. Arterioscler Thromb Vasc Biol 2004;24(10):1842e7. [11] Li Y, Zhu W, Tao J, et al. Fasudil protects the heart against ischemiareperfusion injury by attenuating endoplasmic reticulum stress and modulating SERCA activity: the differential role for PI3K/Akt and JAK2/STAT3 signaling pathways. PLoS One 2012;7(10):e48115. [12] Demiryurek S, Kara AF, Celik A, Babul A, Tarakcioglu M, Demiryurek AT. Effects of fasudil, a Rho-kinase inhibitor, on myocardial preconditioning in anesthetized rats. Eur J Pharmacol 2005;527(1e3):129e40.
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[13] Jung HA, Yoon NY, Bae HJ, Min BS, Choi JS. Inhibitory activities of the alkaloids from Coptidis Rhizoma against aldose reductase. Arch Pharm Res 2008;31(11):1405e12. [14] Yan D, Jin C, Xiao XH, Dong XP. Antimicrobial properties of berberines alkaloids in Coptis chinensis Franch by microcalorimetry. J Biochem Biophys Methods 2008;70(6):845e9. [15] Li HL, Han T, Liu RH, Zhang C, Chen HS, Zhang WD. Alkaloids from Corydalis saxicola and their anti-hepatitis B virus activity. Chem Biodivers 2008;5(5):777e83. [16] Lin CC, Ng LT, Hsu FF, Shieh DE, Chiang LC. Cytotoxic effects of Coptis chinensis and Epimedium sagittatum extracts and their major constituents (berberine, coptisine and icariin) on hepatoma and leukaemia cell growth. Clin Exp Pharmacol Physiol 2004;31(1e2):65e9. [17] Gong LL, Fang LH, Wang SB, et al. Coptisine exert cardioprotective effect through anti-oxidative and inhibition of RhoA/Rho kinase pathway on isoproterenol-induced myocardial infarction in rats. Atherosclerosis 2012;222(1):50e8. [18] Bhindi R, Witting PK, McMahon AC, Khachigian LM, Lowe HC. Rat models of myocardial infarction. Pathogenetic insights and clinical relevance. Thromb Haemost 2006;96(5):602e10. [19] Ertracht O, Liani E, Bachner-Hinenzon N, et al. The cardioprotective efficacy of TVP1022 in a rat model of ischaemia/reperfusion. Br J Pharmacol 2011;163(4): 755e69. [20] Walker MJ, Curtis MJ, Hearse DJ, et al. The Lambeth conventions: guidelines for the study of arrhythmias in ischaemia infarction, and reperfusion. Cardiovasc Res 1988;22(7):447e55. [21] Feng M, Xiang JZ, Ming ZY, et al. Activation of epidermal growth factor receptor mediates reperfusion arrhythmias in anaesthetized rats. Cardiovasc Res 2012;93(1):60e8. [22] Haubner BJ, Neely GG, Voelkl JG, et al. PI3Kgamma protects from myocardial ischemia and reperfusion injury through a kinase-independent pathway. PLoS One 2010;5(2):e9350. [23] Elsherif L, Wang X, Grachoff M, Wolska BM, Geenen DL, O’Bryan JP. Cardiacspecific expression of the tetracycline transactivator confers increased heart function and survival following ischemia reperfusion injury. PLoS One 2012;7(1):e30129. [24] Dong JW, Zhu HF, Zhu WZ, Ding HL, Ma TM, Zhou ZN. Intermittent hypoxia attenuates ischemia/reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2/Bax expression. Cell Res 2003;13(5):385e91. [25] Ahn J, Kim J. Mechanisms and consequences of inflammatory signaling in the myocardium. Curr Hypertens Rep 2012;14(6):510e6. [26] Woldbaek PR, Tonnessen T, Henriksen UL, et al. Increased cardiac IL-18 mRNA, pro-IL-18 and plasma IL-18 after myocardial infarction in the mouse; a potential role in cardiac dysfunction. Cardiovasc Res 2003;59(1):122e31. [27] Liu X, Shen J, Jin Y, Duan M, Xu J. Recombinant human erythropoietin (rhEPO) preconditioning on nuclear factor-kappa B (NF-kB) activation & proinflammatory cytokines induced by myocardial ischaemia-reperfusion. Indian J Med Res 2006;124(3):343e54. [28] Zhang T, Sun L, Liu R, et al. A novel naturally occurring salicylic acid analogue acts as an anti-inflammatory agent by inhibiting nuclear factor-kappaB activity in RAW264.7 macrophages. Mol Pharm 2012;9:671e7. [29] Wibberley A, Chen Z, Hu E, Hieble JP, Westfall TD. Expression and functional role of Rho-kinase in rat urinary bladder smooth muscle. Br J Pharmacol 2003;138(5):757e66. [30] Zhang J, Bian HJ, Li XX, et al. ERK-MAPK signaling opposes rho-kinase to reduce cardiomyocyte apoptosis in heart ischemic preconditioning. Mol Med 2010;16(7e8):307e15.
Abbreviations AAR: area at risk AMI: acute myocardial infarction AST: aspartate transaminase CK-MB: creative kinase-MB Cop: coptisine ECG: electrocardiogram EF: ejection fraction ELISA: enzyme-linked immunosorbent assay FS: fractional shortening HRP: horseradish peroxidase IL: interleukin I/R: ischemia/reperfusion ISO: isoproterenol LAD: left anterior descending LDH: lactate dehydrogenase LV: left ventricle MI: myocardial infarction MYPT-1: myosin phosphatase targeting subunit-1 PVCs: premature ventricular complexes ROCK: Rho kinase TNF-a: tumor necrosis factor-alpha TTC: triphenyl tetrazolium chloride TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling VF: ventricular fibrillation VT: ventricular tachycardia
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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Coptisine protects rat heart against myocardial ischemia/reperfusion injury by suppressing myocardial apoptosis and inflammation Jing Guo a, b, Shou-Bao Wang a, b, Tian-Yi Yuan a, c, Yu-Jie Wu a, c, Yu Yan a, c, Li Li a, b, Xiao-Na Xu a, c, Li-li Gong d, Hai-lin Qin a, b, Lian-Hua Fang a, b, *, Guan-Hua Du a, b, * a
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, China Beijing Key Laboratory of Drug Targets Identification and Drug Screening, China d Beijing Chao-Yang Hospital Affiliated with Beijing Capital Medical University, Beijing 100020, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 January 2013 Received in revised form 12 September 2013 Accepted 2 October 2013 Available online 16 October 2013
Objective: Protecting the heart from myocardial ischemia and reperfusion (I/R) damage is the focus of intense research. Coptisine is an isoquinoline alkaloid isolated from Coptidis Rhizoma. The present study investigated the potential effect of coptisine on myocardial I/R damage in rats and the underlying mechanisms. Methods and results: Electrocardiogram examination showed that the administration of coptisine 10 min before ischemia significantly decreased I/R-induced arrhythmia after 30 min ischemia followed by 3 h reperfusion. The release of cardiac markers was also limited. Echocardiography was performed before ischemia and 24 h post-I/R, separately. The M-mode records showed that the reductions of ejection fraction (EF) and fractional shortening (FS) were attenuated in coptisine-treated rats compared with the I/R rats. Similar results were obtained with Evans Blue/triphenyl tetrazolium chloride (TTC) staining, in which coptisine notably reduced infarct size. Moreover, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay demonstrated coptisine suppressed myocardial apoptosis, which may be related to the upregulation of Bcl-2 protein and inhibition of caspase-3 activation. Coptisine treatment also attenuated the proinflammatory cytokines including interleukin (IL)-1b, IL-6, and tumor necrosis factor-a in heart tissue. Additionally, Western blot and immunohistochemical analysis showed that coptisine markedly reduced Rho, Rho-kinase 1 (ROCK1), and ROCK2 expression and attenuated the phosphorylation of myosin phosphatase targeting subunit-1, a downstream target of ROCK. Conclusions: Coptisine exerts pronounced cardioprotection in rats subjected to myocardial I/R likely through suppressing myocardial apoptosis and inflammation by inhibiting the Rho/ROCK pathway. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Coptisine Ischemia/reperfusion (I/R) Apoptosis Inflammation Rho-kinase (ROCK)
1. Introduction As the morbidity and mortality associated with ischemic heart disease have continued to increase and are gaining increased attention. Despite early reperfusion and improvements in antiplatelet and anti-thrombotic therapy, the mortality of acute myocardial infarction (AMI) patients remains significant even if undergoing primary percutaneous coronary intervention. One major contributing factor is the inability to protect the heart against
* Corresponding authors. Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xiannongtan Street, Beijing 100050, China. Tel.: þ86 10 63165313; fax: þ86 10 63165184. E-mail addresses: [email protected] (J. Guo), [email protected] (L.-H. Fang), [email protected] (G.-H. Du). 0021-9150/$ e see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2013.10.003
the detrimental effects of lethal myocardial reperfusion injury, which occur on restoring blood flow to the acutely ischemic myocardium. Therefore, fully understanding the mechanisms of ischemia/reperfusion (I/R) injury and seeking for novel therapeutic strategies is still the focus of intense research. The cardiomyocyte apoptosis and inflammatory reaction have been recognized as hallmarks of myocardial reperfusion injury. Recent evidences suggest that myocardial apoptosis is initiated shortly after ischemia, is amplified by reperfusion, and partially contributes to overall cardiomyocyte death [1]. Blocking the apoptotic process may prevent the loss of contractile cells, minimize cardiac injury induced by I/R, and slow the occurrence of myocardial stunning and heart failure [2]. Likewise, reducing inflammatory responses during reperfusion after ischemic insult has been shown to be beneficial in numerous studies [3,4]. In addition,
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Rho-kinase (ROCK), the best-characterized effector of the small Gprotein Rho, has recently been suggested to be potential therapeutic targets in the treatment of cardiovascular diseases [5e7]. Accumulating researches have demonstrated that ROCKs play a considerable role in mediating the myocardium apoptosis and inflammatory response to I/R injury [3,8e10]. The beneficial effects of ROCK inhibition against I/R injury using the ROCK inhibitors fasudil and Y-27632 have been well established [3,8,11,12]. Coptisine (Cop) is an isoquinoline-type alkaloid isolated from Coptidis Rhizoma, the rhizome of a traditional Chinese herb, Coptis chinensis Franch. Recently, several biological activities of coptisine have been reported, including anti-diabetic [13], antimicrobial [14], antiviral [15], anti-hepatoma, and anti-leukemia effects [16]. Moreover, we previously found that coptisine exerted cardioprotective effects on isoproterenol-induced myocardiac infarction (MI) in rats, and Rho/ROCK pathway may contribute to the effects [17]. The potential effects of coptisine on MI prompted us to investigate whether it is capable of exerting protection effects during myocardial I/R injury and the underlying mechanism responsible for its actions. In the present study, we employed a model of left anterior descending (LAD) coronary artery occlusion for 30 min followed by reperfusion in rats [18], which simulates the most common clinical manifestations of I/R (i.e., AMI followed by reperfusion therapy) to determine the potential effects and mechanisms of coptisine on I/R injury.
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due to anesthesia or failed surgery, the number of rat in each group was as follows: sham (n ¼ 18), I/R (n ¼ 18), I/R þ Cop 3 mg/kg (n ¼ 21), I/R þ Cop 10 mg/kg (n ¼ 18), I/R þ Cop 30 mg/kg (n ¼ 21). 2.4. Electrocardiographic recording and arrhythmia analysis The ECG was monitored throughout the 30 min ischemia and 3 h reperfusion process using the BL-420S Biologic Function Experiment system (Chengdu, China). Ventricular tachycardia (VT), ventricular fibrillation (VF), and premature ventricular complexes (PVCs) were evaluated according to the Lambeth Convention [20]. The arrhythmia score was graded as previously described [20,21] to evaluate the severity of arrhythmias. Detailed arrhythmias scoring criteria is enclosed in Supplementary material. 2.5. Echocardiography Cardiac function was determined by transthoracic echocardiography as previously described [22,23]. The rats were initially anesthetized with 3% isoflurane in oxygen using a nose cone. Transthoracic 2-D and M-mode images of the left ventricle (LV) were acquired using the Vevo 770 high-resolution echocardiographic system (Visual Sonics Inc., Toronto, ON, Canada). The ejection fraction (EF) and fractional shortening (FS) were calculated from digital images using Vevo 770 software. For each rat, two measurements were performed: 1 day before LAD occlusion (baseline) and after 24 h of reperfusion [19].
2. Materials and methods 2.6. Measurement of myocardial infarct size 2.1. Animals All animal care and experimental procedures were performed in accordance with institutional animal ethical committee guidelines, which conform to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. All of the studies were performed using male Spraguee Dawley rats weighing 280 20 g (Vital River Laboratory Animal Center, Beijing, China). The animals were fed a normal rodent chow diet, with tap water available ad libitum. The rats were housed at a constant temperature and relative humidity under a regular 12 h/ 12 h light/dark schedule. 2.2. Reagents Coptisine (Fig. S1 in Supplementary) was extracted by the Department of Medicinal Chemistry at our institute. Its structure was confirmed based on its physicochemical properties and spectral evidence. Antibodies and major reagents were enclosed in Supplementary material. 2.3. Experimental protocol and drug administration The I/R model was generated by LAD occlusion for 30 min followed by 3 h reperfusion. The surgical procedures were performed as previously described [18,19]. The success of the coronary occlusion and reperfusion was verified by regional cyanosis of the myocardium and typical electrocardiogram (ECG) changes [3]. Fig. S2 describes the experimental protocol. Coptisine (3, 10, and 30 mg/kg, dissolved in saline) or vehicle was orally administered 10 min before occlusion. To examine cardiac function and myocardial infarct size, reperfusion was prolonged to 24 h, during which coptisine was administered in one more doses: 4 h after reperfusion. The experiments are performed on non-diseased hearts which have no abnormal ECG. Except for accidental deaths
At the end of 24 h reperfusion, all of the rats were sacrificed with overdose sodium pentobarbital, and the area at risk (AAR), infarct size, and viable area were evaluated by Evans Blue/TTC staining as previously described [19]. Briefly, Evans blue stained areas indicated non-ischemia area. White parts in the heart indicated the infarct size. Red parts, which were stained by TTC, represented for ischemic but viable tissue. White plus red part was AAR. 2.7. Determination of release of AST, LDH, CK-MB into serum Myocardial cellular damage was evaluated by measuring serum AST, LDH, and CK-MB levels. Three hours after reperfusion, serum AST and LDH activities were measured spectrophotometrically, and serum CK-MB was quantified using a commercial ELISA kit according to the manufacturer’s instructions. 2.8. Cytokine measurement by ELISA Heart tissues from AAR were homogenized and then centrifuged. The levels of IL-1b, IL-6, and TNF-a in heart homogenates were measured using ELISA kits according to the manufacturer’s instructions. The concentrations of the cytokines were quantified by reference to standard curves. 2.9. Determination of myocardial apoptosis A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed using an In Situ Cell Death Detection kit according to the manufacturer’s instructions. Both positive (DNase-treated) and negative (no addition of terminal transferase) control tissue sections were incorporated into each assay. Individual nuclei were visualized at 400 magnification, and the percentage of TUNEL-positive nuclei (positive nuclei/total nuclei) was calculated using ImagePro Plus 5.1 (Media Cybernetics, Bethesda, MD, USA) from at least eight randomly chosen fields per slide for
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statistical analysis. All of these measurements were performed in a blinded manner.
were analyzed using the KruskaleWallis rank-sum test. Values of P < 0.05 were considered statistically significant. SPSS 16.0 software (SPSS, Chicago, IL, USA) was used for the data analyses.
2.10. Agarose gel electrophoresis of DNA 3. Results Detailed method is enclosed in Supplementary. 3.1. Coptisine alleviated I/R-induced arrhythmias in rats 2.11. Immunohistochemical (IHC) analysis of ROCK1 in heart tissue Detailed method is enclosed in Supplementary. 2.12. Western blot analysis After 3 h reperfusion, the hearts were quickly removed, and the AAR from LV was immediately snap-frozen in liquid nitrogen. Western blotting was performed as described in Supplementary material. 2.13. Statistical analysis The data are expressed as mean SEM. Quantitative data were analyzed using analysis of variance (ANOVA) followed by Dunnett’s Multiple Comparison post hoc test. The incidence of arrhythmias (%) was analyzed using Fisher’s exact probability test. Arrhythmia scores
I/R injury increased the arrhythmia score (Fig. S3A) by inducing frequent attacks of PVCs, VT, and VF (Fig. S3B). Coptisine at doses of 10 and 30 mg/kg significantly decreased the arrhythmia score (Fig. S3A) by reducing the incidence of VT and PVCs compared with the I/R group. The incidence of VF did not differ between coptisinetreated rats and I/R rats (Fig. S3B). 3.2. Coptisine limited infarct size and reduced the release of biochemical marker enzymes into serum in I/R rats Representative images of heart sections stained with Evans Blue/TTC are shown in Fig. 1A. AAR/LV (%) (Fig. 1B) was similar between all hearts exposed to I/R treatments, thus demonstrating that all of the I/R treatments had statistically similar ischemic and perfused tissue ratios. As an index of post-I/R tissue necrosis, infarcted regions were determined and normalized to AAR. Both
Fig. 1. Coptisine decreased infarct size and serum biochemical marker enzymes in I/R rats. (A) Representative illustrations of heart sections stained with Evans Blue/TTC. The area at risk (AAR) and infarct regions were determined at 24 h after reperfusion. Dark blue staineviable area; White staineinfarct region; White plus red staineAAR. (B) The ratio of AAR/LV area (%) was similar between all hearts exposed to I/R treatments. (C) The infarct size was normalized to the AAR (%), both 10 and 30 mg/kg coptisine administration significantly decreased infarct size (% of AAR) after 30 min ischemia followed by 24 h reperfusion compared with I/R group. (DeF) Coptisine dose-dependently decreased serum level of AST, LDH, and CK-MB in rats subjected to 30 min ischemia and 3 h reperfusion. Data are expressed as mean SEM. n ¼ 6e8. ##P < 0.01, vs. Sham group; *P < 0.05, **P < 0.01, vs. I/R group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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the 10 and 30 mg/kg doses of coptisine significantly decreased infarct size (% of AAR) after 30 min ischemia followed by 24 h reperfusion compared with the I/R rats (Fig. 1C). As shown in Fig. 1DeF, I/R injury induced the necrotic cell death of myocytes, reflected by a significant increase in the release of biochemical markers, including AST, LDH, and CK-MB, into serum. Coptisine at doses of 10 and 30 mg/kg decreased serum AST, LDH and CK-MB levels compared with the I/R group. Moreover, as shown in Fig. S5, coptisine at 3e30 mg/kg had no effect on the systemic blood pressure, including SBP, DBP and MBP, from 0 to 6 h after oral administration in normal rats. 3.3. Coptisine attenuated the reduction of left ventricular function after I/R in rats At 24 h post-I/R, the rats exhibited a pronounced decline in LV function, reflected by an attenuation of LV wall motion and reduction of EF and FS (i.e., EF and FS at 24 h post-I/R minus their baseline values; Fig. 2). The representative echocardiographic Mmode records (Fig. 2A) showed that LV wall motion was wellpreserved in coptisine-treated rats. Further analysis of these records demonstrated that the reductions of EF and FS in rats treated with coptisine at doses of 10 and 30 mg/kg were significantly decreased compared with the I/R group (Fig. 2B, C). 3.4. Coptisine decreased cardiomyocytes apoptosis and cleaved caspase-3 and increased Bcl-2 expression in I/R rats A representative photomicrograph showed that TUNELpositive cardiomyocyte nuclei (i.e., dark brown cell nuclei) were more frequently observed in the I/R rats. In contrast, almost no TUNEL-positive cells were observed in sections from sham
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rats (Fig. 3A). Treatment with coptisine at doses of 10 and 30 mg/ kg significantly and dose-dependently decreased the percentage of apoptotic nuclei compared with the I/R group. The anti-apoptosis effect of coptisine was confirmed by agarose gel electrophoresis of DNA (Fig. S4). DNA ladder, indicative of apoptotic internucleosomal DNA fragmentation, were clearly visible in agarose gels of DNA from the AAR zone of hearts subjected to I/R injury. Coptisine (30 mg/kg) treatment reduced the appearance of DNA fragmentation. To further investigate the anti-apoptotic effect of coptisine, the levels of cleaved caspase-3 and Bcl-2 expression in heart were determined by Western blot analysis. Consistent with the TUNEL assay result, coptisine dose-dependently reduced the expression of the cleaved, activated forms of caspase-3 (Fig. 3B) and upregulated the level of the pro-survival protein Bcl-2 (Fig. 3C). 3.5. Coptisine reduced proinflammatory cytokine levels and NF-kB activity in heart of I/R rats The ELISA results showed that 30 min ischemia and 3 h reperfusion resulted in a noticeable increase in the levels of IL-1b, IL-6, and TNF-a. Coptisine at doses of 10 and 30 mg/kg decreased the levels of IL-1b, IL-6, and TNF-a in heart tissue compared with the I/R rats (Fig. 4AeC). We further studied the effects of coptisine on nuclear factorkappa B (NF-kB) activation by detecting the translocation of NFkB p65 subunit from cytoplasm to nucleus. As shown in Fig 4D, E, I/R injury strongly decreased NF-kB p65 level in the cytosolic, but increased the NF-kB p65 level in the nucleus. Administration of coptisine markedly inhibited the degradation of NF-kB p65 in the cytosolic, as well as the increase in the nucleus in a dose-dependent manner.
Fig. 2. Coptisine attenuated the reduction of left ventricular function in rats. (A) Representative echocardiographic M-mode records obtained at the papillary muscle level at baseline and 24 h post-I/R in rats treated with saline or coptisine. The solid arrows indicates end diastolic diameter; The dotted arrows indicates the end systolic diameter. (B, C) Coptisine at 3 and 10 mg/kg exhibit a significant less loss in percentage EF and FS relative to their respective baseline level after 24 h of reperfusion compared to I/R rats. Data are expressed as mean SEM. n ¼ 6. *P < 0.05, **P < 0.01, vs. I/R group. EF, ejection fraction; FS, fractional shortening.
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Fig. 3. Effects of coptisine on I/R-induced apoptosis and the expression of cleaved caspase-3 and Bcl-2. (A, aee) Apoptotic cardiomyocytes were identified by TUNEL analysis. TUNEL-positive cells were manifested as a marked appearance of dark brown apoptotic cell nuclei (bar ¼ 50 mm). (A, f) Quantitative analysis of percentage of apoptotic cardiomyocytes (n ¼ 6). (B, C) Representative immunoblotting bands (upper lane) of cleaved caspase-3 and Bcl-2. The densitometric analysis of bands (lower histograms) were normalized to GAPDH (n ¼ 5). Data are expressed as mean SEM. #P < 0.05, ##P < 0.01, vs. Sham group; *P < 0.05, **P < 0.01, vs. I/R group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.6. Coptisine reduced Rho, ROCK1, and ROCK2 expression and attenuated MYPT-1 phosphorylation To explore the molecular mechanisms involved in coptisinemediated cardioprotection, we further investigated the effects of coptisine on the Rho/ROCK pathway, which plays an important role in I/R injury. As shown in Fig. 5, Western blot indicated that I/R injury increased Rho, ROCK1, and ROCK2 expression compared with the sham operation. Administration of coptisine at doses of 10 and 30 mg/kg noticeably reduced Rho, ROCK1, and ROCK2 expression (Fig. 5A, B). ROCK1 immunohistological staining was also determined in the heart tissues (Fig. 5D). In sham-operated hearts, minimal ROCK1 immunoreactivity was noted. In contrast, positive ROCK1 staining clearly increased in hearts subjected to 30 min ischemia and 3 h reperfusion. In rats treated with 10 and 30 mg/kg coptisine, positive ROCK1 staining was significantly reduced. To further determine whether coptisine affects the activity of ROCKs, the phosphorylation level of the downstream target MYPT1 was measured by Western blot. As shown in Fig. 5C, myocardial I/
R resulted in an increased amount of phospho-MYPT-1 compared with sham operation. In contrast, coptisine at doses of 10 and 30 mg/kg significantly and dose-dependently decreased the amount of phospho-MYPT-1. 4. Discussion In the present study, coptisine has been proven to reduce the ischemic/reperfusion damage to the rat heart using a model of LAD coronary artery occlusion. Rats treated with coptisine had a lower arrhythmia score and a smaller infarct size compared with the I/R rats. Biochemical parameters of damage detection (AST, LDH and CK-MB) demonstrated the same protective effects. Myocardial functions were also improved by administration of coptisine. Suppression of cardiomyocyte apoptosis, attenuation of inflammation and significant inhibition of the Rho/ROCK pathway may contribute to the cardioprotective effects of coptisine. Our previous study [17] tested the effects of coptisine at doses of 25, 50 and 100 mg/kg on isoproterenol-induced MI in rats. Coptisine had an apparent protective effect, even at the lowest dose
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Fig. 4. Coptisine reduced proinflammatory cytokine levels and NF-kB activity in heart tissue of rats subjected to 30 min ischemia followed by 3 h reperfusion. (AeC) are measurement of IL-1b, IL-6, TNF-a in heart tissue by ELISA (n ¼ 6e8). D and E are measurement of cytosolic and nucleic NF-kB p65 expression by western blot. The bands for cytosolic NF-kB were normalized to GAPDH, and nucleic NF-kB p65 were normalized to Histone H3 (n ¼ 4). Data are expressed as mean SEM. #P < 0.05, ##P < 0.01, vs. Sham group; * P < 0.05, **P < 0.01, vs. I/R group.
(25 mg/kg). Based on the preliminary experiment, which showed potent protection against myocardial I/R injury in rat at lower doses (data not shown), three lower doses (3, 10, and 30 mg/kg) were used in the present study. These results suggested that coptisine had potent cardioprotective effects against I/R injury at doses of 10 and 30 mg/kg, and the beneficial effect of 30 mg/kg on the heart was more pronounced than that of the 10 mg/kg dose. Various disturbances in cardiac rhythm (including PVCs, VT, and the potentially lethal condition of VF) are an important consequence of both myocardial ischemia and reperfusion. The present study showed that coptisine decreased the arrhythmia score and incidence of PVCs and VT in I/R-injured rats, suggesting that coptisine protects cardiomyocytes from ischemia and reperfusioninduced membrane damage, thereby attenuating myocardial vulnerability to arrhythmia. Additionally, the release of AST, LDH, and CK-MB, biochemical markers of necrotic cell death and clinically used diagnostic markers of myocardium damage, was significantly decreased in the coptisine treatment groups. Echocardiography is a versatile noninvasive tool for measuring cardiac function and structure in animal as well as in clinic [19,22,23]. In the present study, we used echocardiography to estimate LV function as reflected by EF and FS. The echocardiography records showed that coptisine at doses of 10 and 30 mg/kg significantly diminished the decline in EF and FS compared with the I/R group 24 h post-I/R. Furthermore, Evans Blue/TTC staining of heart tissue was consistent with the cardiac function results. A robust limitation of infarct size was observed at coptisine doses of 10 and 30 mg/kg.
Apoptosis is a mainstay of tissue damage secondary to reperfusion injury after short ischemia. In the present study, TUNEL assay was used to examine myocardial apoptosis. The percentage of apoptotic cardiomyocytes after 3 h reperfusion was 24.56 0.90%, similar to the data reported previously in rat hearts subjected to I/R [4]. Treatment with coptisine at doses of 10 and 30 mg/kg resulted in significant reductions in cardiomyocyte apoptosis rate, consistent with the infarct-limiting effect of coptisine, indicating that the anti-apoptotic effect of coptisine in early phase of reperfusion could contribute to the attenuation of later myocardial necrosis (myocardial infarction). The anti-apoptosis effect of coptisine was confirmed by reduced appearance of DNA fragmentation. Caspase3 plays a central role in the execution phase of cell apoptosis. The activation of caspase-3 is governed by a series of signal transduction cascades, among which the interaction between antiapoptotic Bcl-2 and pro-apoptotic Bax proteins plays a critical role. Bcl-2 is capable of forming a heterodimer with Bax, thereby preventing Bax homodimerization and the sequential activation of caspase-3 [24]. The present study revealed that I/R markedly downregulated expression of Bcl-2 in the myocardium of I/R rats, consistent with the previous researches [3,4]. Meanwhile, treatment with coptisine at doses of 10 and 30 mg/kg clearly attenuated I/R-induced decrease of Bcl-2 expression in myocardium, suggesting that coptisine may exert its anti-apoptotic effects by regulating Bcl-2, thus depressing the activation of caspase-3. Myocardial necrosis after AMI induces complement activation and free radical generation, triggering TNF-a release from the
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Fig. 5. Effects of coptisine on the levels of Rho, ROCK1, and ROCK2 expression and phospho-MYPT-1 in heart tissue in rats subjected to 30 min ischemia followed by 3 h reperfusion. (A, B) Representative immunoblotting bands of Rho, ROCK1, and ROCK2. (C) Phosphorylation levels of MYPT-1, a downstream target of ROCKs. n ¼ 5. (D) Representative immunohistochemical staining for ROCK1 in rat myocardium in the different groups (bar ¼ 100 mm). (D, f) Quantitative measurement of ROCK1 expression (n ¼ 6). Data are expressed as mean SEM. #P < 0.05, ##P < 0.01, vs. Sham group; *P < 0.05, **P < 0.01, vs. I/R group. IOD, integrated optical density.
infarcted myocardium. The secreted TNF-a further stimulates the release of proinflammatory cytokines, like IL-1b and IL-6, chemokines, and adhesion molecules from infiltrating leukocytes and endothelial cells, initiating the cytokine cascade. Pro-inflammatory cytokines, such as IL-1b, IL-6, and TNF-a, have emerged as significant contributors to myocardial dysfunction [3,4,25,26]. 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 [27]. In the 30 mg/kg coptisine-treated group, the heart tissue levels of IL-1b, IL-6, and TNF-a were significantly decreased compared with the I/R group. Therefore, suppressing the inflammatory reaction may be one of the mechanisms by which coptisine protects the heart against I/R injury. NF-kB pathway plays a pivotal role in myocardial I/R injury and induces many pro-inflammatory cytokines and chemokines which will greatly contribute to myocardial I/R injury. Cytokines can selfamplify through a positive feed-back loop targeting NF-kB. On the one hand, NF-kB activation mediates the transactivation of proinflammatory genes, including TNF-a, IL-1b and IL-6. On the other hand, TNF-a and IL-1b directly activate NF-kB to increase the initial inflammatory response [28]. Here, we measured the inhibitory effect of coptisine on the translocation of NF-kB p65 from cytoplasm
to nucleus, which is representative of the activation of NF-kB, in I/R injured myocardium of rats. The results showed that coptisine significantly inhibited myocardial I/R induced activation of NF-kB pathway, which may be a mechanism of suppression of IL-1/6 and TNF-a. ROCKs play a considerable role in I/R-induced myocardial damage. Upregulated expression of Rho in ischemic myocardium and subsequent activation of ROCKs occurs during reperfusion [3,8e10]. Two highly homologous isoforms of ROCK, ROCK1 and ROCK2 are both expressed in rat heart [29], but their differential effects have not yet been well characterized. We previously showed that coptisine protected the myocardium against isoproterenolinduced MI injury in rats by inhibiting Rho/ROCK1 [17]. In the current study, a consistent result was found in a rat model of I/R. We observed a distinct increase in Rho and ROCK1 in heart tissues 3 h post-I/R, which was significantly reduced by coptisine administration. The expression of ROCK2 in the heart in I/R rats was further detected in the present study. Similar to the upregulation of ROCK1 protein, ROCK2 expression also increased in the heart in I/R rats. Coptisine at doses of 10 and 30 mg/kg also attenuated the expression of ROCK2. Moreover, ROCK activity was estimated by the phosphorylation of specific downstream target proteins MYPT-1. Consistent with previous studies [29,30], a marked elevation of phospho-MYPT-1 was observed in heart tissue in I/R rats, which
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was dose-dependently attenuated by coptisine. These results and earlier studies [3,9] together suggest that Rho/ROCK activation occurred as an early feature of reperfusion, and the upregulation of both ROCK1 and ROCK2 was involved in I/R injury in rats. Several recent studies have provided evidence that ROCKinhibition reduces cardiomyocyte apoptosis and inflammation in heart ischemia/reperfusion through PI3K/Akt and JAK2/STAT3 signaling pathways and JNK-mediated AIF translocation [3,8e11]. Thus, in the present study, the anti-apoptosis and anti-inflammation effects of coptisine were associated with, at least in part, the inhibition of the Rho/ROCK pathway, but a further study of coptisine on the crosstalk between ROCK and these signaling pathways is necessary to interpret the relationship among ROCK, apoptosis and inflammation. In conclusion, the salient finding of the present study was that coptisine treatment reduced infarct size and improved cardiac function after I/R injury in rats. The cardiac protection afforded by coptisine may contribute to its ability to suppress myocardial apoptosis and inflammation, which may at least partially occur through inhibition of the Rho/ROCK pathway. Therefore, the possible therapeutic application of such a potent effect of coptisine in patients at risk for myocardial I/R damage is very promising and justifies further research. Acknowledgments This study was supported by the National Scientific & Technological major special project “significant creation of new drugs” (2013ZX09103-001-008, 2012ZX09103-101-078) and Special Foundation on Scientific and Technological basic work (No. 2007FY130100) and the National Natural Science Foundation of China (No. 81102444, 81202538, 81373269). Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.atherosclerosis.2013.10.003. References [1] Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Circ Res 1996;79(5):949e56. [2] Anselmi A, Abbate A, Girola F, et al. Myocardial ischemia, stunning, inflammation, and apoptosis during cardiac surgery: a review of evidence. Eur J Cardiothorac Surg 2004;25(3):304e11. [3] Bao W, Hu E, Tao L, et al. Inhibition of Rho-kinase protects the heart against ischemia/reperfusion injury. Cardiovasc Res 2004;61(3):548e58. [4] Sun D, Huang J, Zhang Z, et al. Luteolin limits infarct size and improves cardiac function after myocardium ischemia/reperfusion injury in diabetic rats. PLoS One 2012;7(3):e33491. [5] Loirand G, Guerin P, Pacaud P. Rho kinases in cardiovascular physiology and pathophysiology. Circ Res 2006;98(3):322e34. [6] Shimokawa H. Rho-kinase as a novel therapeutic target in treatment of cardiovascular diseases. J Cardiovasc Pharmacol 2002;39(3):319e27. [7] Dong M, Yan BP, Liao JK, Lam YY, Yip GW, Yu CM. Rho-kinase inhibition: a novel therapeutic target for the treatment of cardiovascular diseases. Drug Discov Today 2010;15(15e16):622e9. [8] Zhang J, Li XX, Bian HJ, Liu XB, Ji XP, Zhang Y. Inhibition of the activity of Rhokinase reduces cardiomyocyte apoptosis in heart ischemia/reperfusion via suppressing JNK-mediated AIF translocation. Clin Chim Acta 2009;401(1e2): 76e80. [9] Hamid SA, Bower HS, Baxter GF. Rho kinase activation plays a major role as a mediator of irreversible injury in reperfused myocardium. Am J Physiol Heart Circ Physiol 2007;292(6):H2598e606. [10] Wolfrum S, Dendorfer A, Rikitake Y, et al. Inhibition of Rho-kinase leads to rapid activation of phosphatidylinositol 3-kinase/protein kinase Akt and cardiovascular protection. Arterioscler Thromb Vasc Biol 2004;24(10):1842e7. [11] Li Y, Zhu W, Tao J, et al. Fasudil protects the heart against ischemiareperfusion injury by attenuating endoplasmic reticulum stress and modulating SERCA activity: the differential role for PI3K/Akt and JAK2/STAT3 signaling pathways. PLoS One 2012;7(10):e48115. [12] Demiryurek S, Kara AF, Celik A, Babul A, Tarakcioglu M, Demiryurek AT. Effects of fasudil, a Rho-kinase inhibitor, on myocardial preconditioning in anesthetized rats. Eur J Pharmacol 2005;527(1e3):129e40.
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Abbreviations AAR: area at risk AMI: acute myocardial infarction AST: aspartate transaminase CK-MB: creative kinase-MB Cop: coptisine ECG: electrocardiogram EF: ejection fraction ELISA: enzyme-linked immunosorbent assay FS: fractional shortening HRP: horseradish peroxidase IL: interleukin I/R: ischemia/reperfusion ISO: isoproterenol LAD: left anterior descending LDH: lactate dehydrogenase LV: left ventricle MI: myocardial infarction MYPT-1: myosin phosphatase targeting subunit-1 PVCs: premature ventricular complexes ROCK: Rho kinase TNF-a: tumor necrosis factor-alpha TTC: triphenyl tetrazolium chloride TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling VF: ventricular fibrillation VT: ventricular tachycardia
