- Email: [email protected]
reperfusion injury in rats
Journal Pre-proof Cardioprotection of hydralazine against myocardial ischemia/reperfusion injury in rats Chengzong Li, Zhongping Su, Liqi Ge, Yuchen Chen, Xuguan Chen, Yong Li PII:
S0014-2999(19)30802-7
DOI:
https://doi.org/10.1016/j.ejphar.2019.172850
Reference:
EJP 172850
To appear in:
European Journal of Pharmacology
Received Date: 22 April 2019 Revised Date:
3 December 2019
Accepted Date: 9 December 2019
Please cite this article as: Li, C., Su, Z., Ge, L., Chen, Y., Chen, X., Li, Y., Cardioprotection of hydralazine against myocardial ischemia/reperfusion injury in rats, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/j.ejphar.2019.172850. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Cardioprotection of hydralazine against myocardial ischemia/reperfusion injury in rats Chengzong Lia,b , Zhongping Sub, Liqi Gea, Yuchen Chenc, Xuguan Chenb*, Yong Lib* a
Department of Cardiology, the Affiliated Hospital of Xuzhou Medical University,
Xuzhou, PR China. b
Department of Cardiology, the First Affiliated Hospital of Nanjing Medical
University, Nanjing, PR China. c
Cape Henry Collegiate, 1320 Mill Dam Road, Virginia Beach, Virginia, USA.
* Corresponding author. E-mail address: [email protected] (Y. Li) or [email protected] (X. Chen). Abstract This study aimed to investigate whether hydralazine could reduce cardiac ischemia/reperfusion (I/R) injury in rats. Anesthetized male Sprague–Dawley rats underwent myocardial I/R injury. Saline, hydralazine (HYD, 10-30mg/kg) was administered intraperitoneally 10 min before reperfusion. After 30 min of ischemia and 24 h of reperfusion, the myocardial infarct size was determined using TTC staining. Heart function and oxidative stress were determined through biochemical assay and DHE staining. HE staining was used for histopathological evaluation. Additionally, the cardiomyocytes apoptosis and protein expression of PI3K-Akt-eNOS pathway marker were detected by TUNEL and Western blotting. The serum levels of malonaldehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) and reactive oxygen species were significantly elevated in cardiac I/R group, but the superoxide dismutase (SOD) level was suppressed. However, intraperitoneal pretreatment with hydralazine at a dose of 10 to 30 mg/kg before cardiac I/R significantly limited the increase in CK-MB, LDH, oxidative stress, inflammatory factors, histological damage and apoptosis in the hearts.
In addition, hydralazine also increased p-PI3K, p-AKT, p-eNOS expression and decreased Cleaved Caspase-3, Cleaved Caspase-9 expression in the hearts. Our results suggest that the cardioprotective effect of hydralazine against I/R injury might be a cooperation of the inhibition of oxidative stress, inflammatory response, apoptosis with the motivation of eNOS phosphorylation via activating the PI3K/AKT signal pathway.
Key words: hydralazine; oxidative stress; inflammatory cytokines; apoptosis; heart ischemia/reperfusion.
1. Introduction Ischemic heart disease is a common cardiovascular disease and a main cause of death worldwide (Ferdinandy et al., 2007; Xia et al., 2016). It is mandatory to invigorate ischemic myocardium via reperfusion of coronary blood flow (Kennedy, 1993); however, reperfusion itself can cause cardiomyocytic death and the subsequent irreversible myocardial impairment, termed ischemia/reperfusion (I/R) injury (Yellon, 2007). In the past three decades, a number of anti-ischemic and pharmacological cardioprotective strategies have been tested in experimental animals and replicated in the clinical setting to treat acute myocardial infarction, but the outcomes were disappointing, and no therapeutic consensus has been made (Hausenloy et al., 2016; Heusch, 2017). The underlying mechanisms of cardiac I/R injury include excessive reactive oxygen species generation, inflammation, apoptosis, intracellular calcium overload, neutrophil aggregation and adhesion, endothelial cell dysfunction, etc. Great effort is needed to seek novel therapeutic strategies regulating myocardial oxidative stress and subsequent inflammation and apoptosis induced by ischemia/reperfusion injury (Heusch, 2015). Hydralazine, a non-nucleoside analogue, was invented in 1951 and has ever since been widely used as an adjunctive treatment for hypertension (Morrow et al., 1953). It is a potent arterial vasodilator approved by FDA for severe hypertension, chronic heart failure and hypertension in pregnancy (Cornacchia et al., 1988; Klein et
al., 2003; Magee et al., 2003). Hydralazine works directly on the arterioles, resulting in decreased systemic vascular resistance and blood pressure reduction (Taketomo et al., 2013). The biological mechanisms through which hydralazine dilates vessels are diverse and not fully understood. Among them, the demethylating activity of hydralazine has long been recognized (Coronel J, 2011, Zambrano P, 2005) and exploited for a tumor therapy (Coronel et al., 2011; Tampe et al., 2014; Zambrano et al., 2005). It was reported that low-dose hydralazine effectively attenuated renal fibrogenesis in murine models of unilateral ureter obstruction and folic acid–induced nephropathy (Tampe et al., 2015), and I/R induced renal injury in rats (Tampe et al., 2015; Vergona et al., 1987; Yong et al., 2019). However, the curative effects of hydralazine on myocardial infarction induced by cardiac I/R remain obscure. This study answered whether hydralazine can reduce myocardial I/R injury. 2. Materials and methods 2.1 Animals Male Sprague-Dawley rats weighing 250–280 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The rats were housed in SPF facility in Animal Core Facility of Nanjing Medical University Nanjing Medical University under standard conditions of temperature and 12 h light/dark cycle with ad libitum feeding. All experimental protocols and animal handling procedures were performed in accordance with the “Guide for the Care and Use of Laboratory Animals” (National Academic Press, USA, 1996). This experimental animal study was performed with the approval of the Institutional Animal Care and Use Committee of Nanjing Medical University. The ethical was approved by Nanjing Medical University and the approval number is 17060186.
2.2 Grouping and Experimental Protocols We performed two studies. In study 1, a total of 60 rats were randomly allocated
into 5 groups. Rats in Group 1 (Sham group) were subjected to the surgical procedure described above and intraperitoneally administered with sterile normal saline (i.g., 10 ml/kg) for 10 min, but without left anterior descending (LAD) branch occlusion or reperfusion; rats in Group 2 (I/R group) with normal saline (i.g.); rats in Group 3 (I/R+Hyd 10mg/kg group) with hydralazine (i.g., 10 mg/kg); rats in Group 4 (I/R+Hyd 20mg/kg group) with hydralazine (i.g., 20 mg/kg); rats in Group 5 (I/R+Hyd 30mg/kg group) with hydralazine (i.g., 30 mg/kg). Ten minutes after the above mentioned pretreatments, the rats in all groups except Group 1 were subjected to LAD ligation for 30 min followed by 24 h of reperfusion. All rats were killed under general anesthesia using intraperitoneal injection of sodium pentobarbital (50 mg/kg). The blood was collected from the punctured heart, and the right kidney was stored at −80 °C for mRNA and protein extraction. In study 2, H9c2 cells were assigned to 3 groups: (1) H/R: H9c2 cells were subjected to 24 h of Hypoxia followed by 12 h of reoxygenation (Busheng Zhang, 2014); (2) H/R+Hyd: H9c2 cells pretreated with hydralazine (80 µmol/ml) were subjected to 24 h of Hypoxia followed by 12 h of reoxygenation; (3) H/R+Hyd+WM: H9c2 cells pretreated with hydralazine (80 µmol/ml) and wortmannin (100 umol/l, PI3K/AKT inhibitor) were subjected to 24 h of Hypoxia followed by 12 h of reoxygenation. Cells under normoxia throughout the experimental processes were included as a control. All experiments were repeated for three times. 2.3 Animal model of myocardial ischemia/reperfusion I/R model was induced by ligating the LAD for 30 min followed by 24 h of reperfusion. Rat models of coronary ischemia/reperfusion injury were established as those in previous studies with small modification (Gao et al., 2011; Sun et al., 2014). Briefly, the rats were anesthetized with 3.5% inhalational isoflurane on a heating pad (40°C). The body temperature of each rat was maintained at 37℃ throughout the experiments. The rats were anesthetized by administering sodium pentobarbital (50 mg/kg) intraperitoneally. Following tracheal intubation, 6–0 silk ligature was used to ligate the LAD for a 30-min ischemic period. After a 30-min ischemia treatment, reperfusion was allowed for 24 h after the ligated LAD was released. Sham control
group rats were treated with the same surgical procedures except that the left coronary artery was not ligated. The rats were killed at 24 h after reperfusion or sham operation. The blood was collected and the kidneys were harvested for examination. 2.4 H9c2 cell culture The H9c2 cells (ventricular myocardiocyte, rat in origin; Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were seeded in 6-well plates (2x104 cells/cm2) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St. Louis, MO, USA) containing 10% (v/v) fetal bovine serum (FBS, Gibco, USA) in a humidified atmosphere of 95% air and 5% CO2 at 37℃. 2.5 Simulated hypoxia/reoxygenation model Hypoxia/reoxygenation (H/R) model was established according to previously described methods (Zhang et al., 2014). H/R in H9c2 Cardiomyocytes Hypoxia was induced by exposing the cells to 1% O2, 94% N2, and 5% CO2 for 24 h using a modular incubator (Model 3131, Forma Scientific, Marietta, USA). Reoxygenation (95% air, 5% CO2, 37℃) lasted for 12 h. 2.6 Biochemical assays All blood samples were allowed to clot at room temperature and centrifuged at 2,000 g for 10 min to harvest serum. The biochemical indicators including lactate dehydrogenase (LDH), creatine kinase MB (CK-MB), superoxide dismutase (SOD), malonaldehyde (MDA), tumor Necrosis Factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were measured (n=8 per group) spectrophotometrically using commercially available kits for LDH (A020-2), CK-MB (H197), SOD (A001-1), MDA (A003-1), TNF-α (H052), IL-1β (H002) and IL-6 (H007) (Jiancheng Bioengineering Institute, Nanjing, China). 2.7 Histopathological examination The hearts removed from the euthanized rat immediately at end-diastole were placed in a 10% potassium chloride solution, washed with saline solution, and then placed in 4% paraformaldehyde at 4℃ overnight. A transverse cut was made close to the heart apex to expose the left and right ventricles. The samples were then dehydrated in an ethanol gradient, rinsed in xylene, and embedded in paraffin. Finally, paraffin blocks
were cut into 4-µm sections. The paraffin sections were stained in H&E for histopathology, and then visualized by light microscopy. Heart histology was assessed using previously described methods (Zhu et al., 2015). According to the scope and severity of the heart, it was graded on a scale of 0.5–4: 0.5 = minor, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe. Five random fields on each heart slide (five slides per animal) were examined (n = 7 per group). 2.8 Estimation of infarct size Myocardial infarct size was estimated by stating of 2, 3, 5- triphenyltetrazolium hydrochloride (TTC). Animals were killed after administrating overdose of anesthetics (2–3 times of conventional anesthetic dose), then the hearts were rapidly excised and immediately frozen at −20 °C for 1 h. The slices of the heart were cut into cross sections 2 mm thick and incubated with 1% TTC in phosphate buffer (0.1 M, pH 7.4) for 15 min at room temperature. Slices were incubated with 10% formalin overnight and then scanned from both sides for myocardial necrosis. White parts indicated the infarct size, and red indicated normal. Images of the stained slices were captured using a digital camera and analyzed using Image J2x analysis software (National Institutes of Health). The severity of the myocardial infarction was indicated by the ratio of the infarct size to the total size. 2.9 Reactive oxygen species staining Total reactive oxygen species was stained with dihydroethidium (DHE, D-23107; Invitrogen, USA) on fresh frozen sections. The hearts taken out of the euthanized rats were mounted in OCT embedding compound (3801480; Leica) and frozen at - 80℃. The frozen section was cut 5 um thick using a cryostat and then thawed; the sections were mounted onto gelatin-coated histological slides. Thereafter, 5 µM of DHE dissolved in DMSO was added to fresh frozen rat heart sections (thickness 5 um) after dilution in PBS, incubated in darkness at 37℃ for exactly 30 min, and rinsed twice with cold PBS. Photos were taken immediately. The fluorescence density of arterial walls was quantified using Image J. The relative fluorescence intensity was determined by the ratio of the average intensity at ischemia area normalized to that of non-ischemia area after subtracting background intensity.
2.10 In situ TUNEL staining assay A terminal deoxynucleotidyltransferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay was performed according to the manufacturer’s instructions (11684817910, Roche, Switzerland). Heart tissues were fixed in 4% paraformaldehyde overnight, dehydrated, embedded in paraffin, sectioned into 4-µm-thick sections and placed on numbered polylysine-coated glass slides. Deparaffinized tissue sections were incubated with proteinase K (20 mg/ml, Sigma, USA) in a humidified chamber for 15 min, and endogenous peroxidase activity was blocked via treating with 3% H2O2 for 10 min. The sections were incubated with TdT labeling buffer at 37°C for 1 h in a moist chamber and then counterstained with DAPI. TUNEL-positive cells showed brown and the nuclei showed blue. Five random fields on each slide (five slides per animal, seven animals per group) were examined. The nuclei number of TUNEL-positive cells (Numerator) and the total number of cellular nuclei (denominator) were counted as previously mentioned (Tang et al., 2017). The rate of TUNEL-positive cells in each field was analyzed using Image Pro Plus 6.0 software. 2.11 Western blotting Western blotting analyses were performed according to previously described methods with slight modifications (Choi et al., 2009). Briefly, 30 mg of protein was separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in TBST buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% Tween-20) for 1 h prior to incubation with a primary antibody against PI3K (4108, Cell Signal Technology, USA), p-PI3K (sc-48341, Santa Cruz Biotechnology, USA), AKT (4685, Cell Signal Technology, USA), p-AKT (4060, Cell Signal Technology, USA), eNOS (32027, Cell Signal Technology, USA) and p-eNOS (9570, Cell Signal Technology, USA), Cleaved Caspase 3 (9665, Cell Signal Technology, USA), Cleaved Caspase 9 (7237, Cell Signal Technology, USA) and GAPDH (sc-166574, Santa Cruz Biotechnology, USA) at 4℃ overnight, followed by an incubation with a goat anti-rabbit IgG HRP-conjugated secondary antibody (sc-2004, Santa Cruz Biotechnology) or a goat anti-mouse IgG HRP-conjugated
secondary antibody (sc-2005, Santa Cruz Biotechnology). Then, the membranes were washed for three times in TBST. The blots were imaged using the ChemiDoc XRS+ Molecular Imager (Bio-Rad) with the Pierce ECL Western Blotting Substrate (32209, Thermo Scientific) and analyzed using image analysis software (ImageJ 1.42). The housekeeping protein GAPDH was used as the internal reference. The Western blotting quantification was corrected to GAPDH expression prior to normalization. 2.12 Immunofluorescence staining To explore which type of cells are undergoing apoptosis (cardiomyocytes or endothelial cells), immunofluorescence staining was performed. CD31 is the endothelial-specific
marker, and sarcomeric is the cardiomyocytes-specific marker. Heart samples were fixed with 4% paraformaldehyde and incubated with a blocking solution consisting of 10% bovine serum albumin for 1 h. The samples were incubated with antibodies of CD31 or sarcomeric (Abcam, MA, USA) at 4°C overnight, and then washed three times in PBS and incubated in secondary antibodies (Jackson ImmunoResearch Laboratories Inc., PA, USA)) for 2 h at room temperature. Then, 4',6-diamidino-2-phenylindole (DAPI, Life Technology, Carlsbad, CA, USA) was used to counterstain the nucleus. Fluorescent imaging was achieved using a microscope (ZEISS, Oberkochen, Germany). 2.13 Statistical analysis All statistical analyses were performed using Graphpad Prism version 5 software. Comparisons between two groups were finished using the two-tailed unpaired t test. One-way ANOVA followed by Bonferroni multiple comparison test was used for comparison between more than two groups. Results were expressed as mean ± S.D. P < 0.05 was considered statistically significant. 3. Results 3.1 Effects of hydralazine on myocardial tissue damage induced by I/R The effects of hydralazine on heart infarct size was assessed by the TTC assay, showed in Fig. 1A, B. The proportion of the infarcted area in the I/R group averaged 40%. The infarct size decreased significantly in I/R+Hyd(10mg/kg) and I/R+Hyd(30mg/kg) groups (P < 0.01). This proportion was 15% in I/R+Hyd(30mg/kg)
group (P < 0.01, compared with I/R group). Histopathological examination of the hearts is shown in Fig. 1C, D. Sham group showed obvious integrity of myocardial membrane without any infarction, inflammatory infiltration or cardiac necrosis, and the cardiomyocytes demonstrated well-arranged myofilaments. I/R group showed myocardial structure disorder, myocardial fibers fractured, inflammatory infiltration and cardiac necrosis. However, hydralazine (10-30 mg/kg) groups significantly alleviated the damage of myocardium. 3.2 Effect of hydralazine on heart function and inflammation induced by I/R The effects of hydralazine on heart function levels in rats are shown in Fig. 2A,B. I/R operation significantly (P < 0.01) increased CK-MB and LDH levels in both the I/R and Hyd(30mg/kg) groups compared with the sham group. There was no significant difference in CK-MB levels between the I/R and I/R+Hyd(10mg/kg) groups. Hydralazine significantly reduced CK-MB level in I/R+Hyd(20mg/kg) and I/R+Hyd(30mg/kg) groups compared with I/R group (P < 0.01); Hydralazine also significantly reduced
LDH level in I/R+Hyd(10mg/kg), I/R+Hyd(20mg/kg) and
I/R+Hyd(30mg/kg) groups compared with I/R group (P < 0.01). To examine the effects of hydralazine on inflammation in the heart, serum levels and mRNA expression levels of IL-1ß, IL-6 and TNF-α were assessed (Fig. 2C-E). The serum levels and mRNA expression levels of IL-1ß, IL-6 and TNF-α were all significantly up-regulated in I/R group compared with the sham group (P < 0.01). However, hydralazine dose-dependently decreased serum levels and mRNA expression levels of this inflammatory factors. The serum levels of IL-1ß, IL-6 and TNF-α decreased in I/R+Hyd(10mg/kg), I/R+Hyd(20mg/kg) and I/R+Hyd(30mg/kg) compared with I/R group (P < 0.01). 3.3 Effects of hydralazine on oxidative stress induced by I/R To assess the effects of hydralazine on oxidative stress, we detected serum SOD activity and MDA level (Fig 3). Serum SOD activity and MDA level decreased significantly in the I/R and I/R+Hyd(10mg/kg) groups compared with the sham group (P < 0.01) (Fig. 3A). Heart MDA levels increased significantly (P < 0.01) in the
I/R+Hyd(30mg/kg) groups compared with the sham group (P < 0.01) (Fig. 3B). Hydralazine significantly increased SOD activity and reduced MDA level in I/R+Hyd(10mg/kg), I/R+Hyd(10mg/kg) and I/R+Hyd(30mg/kg) groups compared with the I/R group (P < 0.05 or P < 0.01, respectively). In addition, to assess the effects of hydralazine on reactive oxygen species production, we detected the intracellular generation of reactive oxygen species moiety O2- with the fluoroprobe DHE (Fig. 3C). Confocal microscopy showed that all heart sections from I/R rats had a widespread and markedly intensified DHE fluorescence compared with those in the sham group (P < 0.01, Fig. 3D). Dose-dependently, hydralazine (10 mg/kg to 30 mg/kg) before I/R operation induced a significant decrease in reactive oxygen species fluorescence intensity compared with that of the sham group (P < 0.01). 3.4 Effect of hydralazine on myocardial cell apoptosis induced by I/R In the TUNEL assay, the nuclei of TUNEL-positive cells were stained green, indicating the presence of apoptotic cells (Fig. 4A). The level of apoptosis was indicated by the percentage of TUNEL-positive cells in the total cells (Fig. 4B). Few apoptotic cells were observed in the sham group, but the I/R group displayed more TUNEL-positive cells than the sham group (P < 0.01). Dose-dependently, hydralazine significantly decreased the number of TUNEL-positive cells. Fewer apoptotic cells were observed in the I/R+Hyd (10mg/kg), I/R+Hyd (20mg/kg) and I/R+Hyd (30mg/kg) groups compared with the I/R group (P < 0.01). To explore which type of cells
are
undergoing
apoptosis
(cardiomyocytes
or
endothelial
cells),
immunofluorescence staining was performed. CD31 is the endothelial-specific marker, and sarcomeric is the cardiomyocytes-specific marker. The apoptosis cells were cardiomyocytes, but not endothelial cells (Figure 4C and D).
3.5 Effect of hydralazine on related pathways in the heart tissues The expression of PI3K-Akt-eNOS pathway and apoptosis associated proteins were detected by Western blotting (Fig. 5A). No significant difference in the PI3K, Akt and eNOS protein expression appeared between groups. The expression levels of p-PI3K, p-AKT and p-eNOS were significantly lower in the I/R group than in the sham group
(P< 0.05 or P< 0.01, respectively), and were markedly up-regulated in Hyd(10mg/kg), Hyd(20mg/kg) and Hyd(30mg/kg) groups compared with I/R group (P < 0.05 or P < 0.01, respectively) (Fig. 5B). Besides, the expression of Cleaved Caspase-3 and Cleaved Caspase-9 was remarkably enhanced in I/R group compared with the sham group (P < 0.01), and was markedly down-regulated in Hyd(10mg/kg), Hyd(20mg/kg) and Hyd(30mg/kg) compared with I/R group (P < 0.05 or P < 0.01). 3.6 Effect of PI3K/Akt inhibitor and hydralazine on related pathways in the H9C2 cells subjected to H/R The PI3K and AKT expression and phosphorylation in H9C2 cells subjected to H/R were detected by Western blotting. As shown in Fig. 6, there was no difference in total PI3K and AKT expression among all groups in cultured cardiomyocytes pretreated with 80 mmol/L hydralazine and exposed to H/R, and hydralazine treatment promoted PI3K and AKT phosphorylation. PI3K/AKT inhibitor WM (100 nmol/L) discounted the effect of hydralazine on PI3K and AKT phosphorylation. Thus, co-treatment with hydralazine and WM blocked the activation of PI3K/AKT pathway induced by hydralazine. eNOS is a downstream molecule of AKT. As shown in Fig. 6, hydralazine enhanced eNOS phosphorylation in cultured cardiomyocytes exposed to H/R. PI3K/AKT inhibitor blocked eNOS phosphorylation induced by hydralazine, whereas the expression of total eNOS remained unchanged. These results indicated that hydralazine activated PI3K-Akt and eNOS in rat cardiomyocytes exposed to H/R. 4 Discussion I/R injury was first coined in 1960 to describe a phenomenon that reperfusion itself exacerbates myocardial injury (Jennings et al., 1960). The underlying mechanisms is complicated (Heusch, 2015). I/R injury may involve mitochondrial dysfunction, overproduction of reactive oxygen species, inflammation, apoptosis, endothelial cell dysfunction reduction of nitric oxide bioavailability, and intracellular calcium overload, etc. (Xia et al., 2016; Zhang et al., 2014). A potent anti-hypertensive agent and an arterial vasodilator for chronic heart failure and hypertension in pregnancy (Cornacchia et al., 1988; Klein et al., 2003; Magee et al., 2003), hydralazine can remarkably inhibit semicarbazide-sensitive amine oxidases
activity (Kinemuchi et al., 2004). With a powerful aldehyde-trapping ability (Burcham et al., 2000), hydralazine is also more specific to normalize aberrant promoter methylation of select genes than broad demethylating drug 5’-azacytidine (Tampe et al., 2015). Low-dose hydralazine reduces leukocyte migration, not blood pressure, in SHR rats (Rodrigues et al., 2008), attenuates renal fibrosis and preserves excretory renal function regardless of its blood pressure–lowering effects in C57BL/6 mice (Tampe et al., 2017). In the current study, we observed hydralazine (>10 mg/kg) protected myocardium against I/R injury by inhibiting the overproduction of reactive oxygen species and the release of inflammatory factors. Reactive oxygen species include peroxides, superoxide, hydroxyl radical, and singlet oxygen (Hayyan et al., 2016), all acting as the key mediators of myocardial I/R injury (Dhalla et al., 2000). Reactive oxygen species and redox signaling pathways are involved in myocardial ischemia-reperfusion injury and cardioprotection (Cadenas et al., 2018). During ischemia, hypoxia, even reperfusion, the produced reactive oxygen species exceeds the scavenged reactive oxygen species and the antioxidative defense may be compromised and eventually crumbled, all contributing to inflammation, apoptosis and heart dysfunction(Granger et al., 2015). High level of reactive oxygen species activates the pro-apoptotic proteins caspase-3 and caspase-9 (Ibanez et al., 2015), and initiates inflammatory response to ischemia/reperfusion injury (Granger, 2015) by increasing the inflammatory cytokines (such as IL-6, IL-1β, and TNF-α) and decreasing anti-inflammatory cytokines (such as IL-10) (Kleinbongard et al., 2011; Yang et al., 2008). In this study, hydralazine treatment increased serum SOD level and simultaneously decreased serum MDA level (a lipid peroxidation index) and reactive oxygen species level. Thus, antioxidation is one mechanism through which hydralazine protects the heart against myocardial I/R injury. Inflammatory reaction also plays a crucial role in myocardial I/R injury (Xiong et al., 2010). The myocardial I/R injury can stimulate the release of pro-inflammatory cytokines, IL-1β, IL-6 and TNF-α (Ahn et al., 2012; Ying et al. 2015). TNF-α can further activate caspases apoptosis process by the death receptor pathway (Yang et al.,
2007). In the present study, pretreatment with hydralazine significantly decreased IL-1β, IL-6, and TNF-α levels in serum compared with I/R group. Therefore, suppression of inflammatory reaction is another mechanism responsible for hydralazine-achieved protection. Apoptosis brings with infarction during myocardial I/R (Ekhterae et al., 2011). In our study, TUNEL staining revealed that apoptotic cells in the hearts of I/R injury rats treated by hydralazine were markedly decreased; Western blotting results also showed the down-regulation of Cleaved Caspase-3 and Cleaved Caspase-9 in the group treated with hydralazine compared with I/R group. In apoptosis, caspase cascade is activated to promote apoptotic body formation and cell fragmentation (Saikumar et al., 1998). In the pathway of cell apoptosis, lowering pro-apoptotic proteins caspases levels can suppress apoptosis (Atif et al., 2015; Rong et al., 2008; Chalah and Khosravi-Far, 2008). Otherwise, caspase-3 and caspase-9 are closely coupled to upstream, pro-apoptotic signals(Chalah and Khosravi-Far, 2008); Cleaved Caspase-9 and Cleaved Caspase-3, two activated caspases, are involved in a mitochondrial-dependent pathway that eventually leads to cell apoptosis (Portt et al., 2011). These data suggest that hydralazine could significantly reduce apoptosis through mitochondrial-dependent pathway in the kidney. PI3K–AKT–eNOS pathway, plays a key role in antiapoptotic action and protection of cardiomyocytes from I/R injury (Gao et al., 2002), and PI3K/AKT pathway has been identified as a key component of the protective mechanism of ischemia preconditioning (Ansley et al., 2013; Shanmuganathan et al., 2005). The activation of AKT, which is downstream of PI3K, may ameliorate I/R injury (Forster et al., 2006). PI3K/AKT-dependent signaling pathway plays a key role in anti-apoptotic activity and cardioprotection from I/R injury (Gao et al., 2002; Xing et al., 2009; Zhang et al., 2007). It was reported that the adenoviral gene transfer of activated PI3K and AKT inhibited the apoptosis of hypoxic cardiomyocytes in vitro (Matsui et al., 1999); AKT activation ameliorated cardiac function and prevented injury from transient cardiac ischemia in vivo(Matsui T, 2001). Our results demonstrated that enhanced eNOS, PI3K and AKT activation endowed hydralazine
with an anti-apoptotic and cardioprotective ability. It has been shown that phosphorylation of eNOS by AKT is a downstream effector in the survival signaling in myocardial ischemia and reperfusion (Gao et al., 2002). The present study also revealed that pretreatment with hydralazine induced the upregulation of p-eNOS, a process during which NO is generated (Salloum et al., 2003). NO exerts beneficial effects on the heart during myocardial reperfusion through guaranteeing myocardial contractility, sarcolemmal and mitochondrial KATP channel opening, antioxidation, and oxygen free radical production (Li et al., 2012; Pang et al., 2016). NO also activates guanylate cyclase, resulting in enhanced formation of cGMP, which activates PKG to open the mitochondrial KATP channels conferring cardioprotective effects against IR injury (Kukreja et al., 2005). Our study showed that pretreatment with wortmannin significantly blocked eNOS phosphorylation in H9c2 cells induced by hydralazine in I/R+Hyd groups, suggesting that PI3K–AKT–eNOS signaling may contribute to the hydralazine elicited cardioprotection and anti-apoptotic effect against I/R injury in H9c2. The present study also revealed that pretreatment with hydralazine plus wortmannin not only blocked PI3K, AKT and eNOS phosphorylation induced by cardiomyocytes H/R injury, but also markedly increased Cleaved Caspase-3 and Cleaved Caspase-9 expression compared with those in the hydralazine-treated group, suggesting that hydralazine inhibited I/R induced cardiomyocytes apoptosis via PI3K/AKT signal pathway. However, eNOS phosphorylation was not stopped completely by PI3K– AKT signal inhibitor wortmannin. It was reported that dominant negative AKT mutants were unable to block eNOS phosphorylation completely, suggesting that there may be other mechanism responsible for the activation of eNOS (Boo et al., 2002). In conclusion, hydralazine protected the myocardium against the injury induced by I/R (a 30-min ischemia and a 24-h reperfusion). Hydralazine lessened the released inflammatory factors and attenuated tissue damage and apoptosis in the hearts. Our results might provide a rationale to use hydralazine as a new therapy to prevent myocardial I/R injury induced after vascular surgery, heart surgery and heart
transplantation.
Acknowledgements This work was supported by the Natural Science Foundation of China (81627802). Conflicts of interest The authors declare no conflicts of interest. Authors' contributions Yong Li designed the study and drafted the manuscript. Chengzong Li and Xuguan Chen undertook animal and cell experiments. Zhongping Su and Liqi Ge helped to perform the experiments regarding molecular biological technique. Yuchen Chen provided much help on biochemical index detection. All authors reviewed the manuscript.
Reference Ahn J, Kim J. Mechanisms and consequences of inflammatory signaling in the myocardium. Curr Hypertens Rep 2012; 14(6): 510-6. Ansley DM, Wang B. Oxidative stress and myocardial injury in the diabetic heart. J Pathol 2013; 229(2): 232-41. Atif F, Yousuf S, Stein DG. Anti-tumor effects of progesterone in human glioblastoma multiforme: role of PI3K/Akt/mTOR signaling. The Journal of steroid biochemistry and molecular biology 2015; 146: 62-73. Boo YC, Sorescu G, Boyd N, Shiojima I, Walsh K, Du J, et al. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A. J Biol Chem 2002; 277(5): 3388-96. Burcham PC, Kerr PG, Fontaine F. The antihypertensive hydralazine is an efficient scavenger of acrolein. Redox Rep. 2000; 5(1): 47-9. Cadenas S. ROS and redox signaling in myocardial ischemia-reperfusion injury and cardioprotection. Free Radic Biol Med 2018; 117: 76-89. Chalah A, Khosravi-Far R. The mitochondrial death pathway. Advances in
experimental medicine and biology 2008; 615: 25-45. Choi DE, Jeong JY, Lim BJ, Chung S, Chang YK, Lee SJ, et al. Pretreatment of sildenafil attenuates ischemia-reperfusion renal injury in rats. Am J Physiol Renal Physiol 2009; 297(2): 362-70. Cornacchia E, Golbus J, Maybaum J, Strahler J, Hanash S, Richardson B. Hydralazine and procainamide inhibit T cell DNA methylation and induce autoreactivity. J Immunol 1988; 140(7): 2197-200. Coronel J, Cetina L, Pacheco I, Trejo-Becerril C, González-Fierro A, de la Cruz-Hernandez E, et al. A double-blind, placebo-controlled, randomized phase III trial of chemotherapy plus epigenetic therapy with hydralazine valproate for advanced cervical cancer. Preliminary results. Med Oncol 2011; 28(suppl 1): 540-6. Dhalla NS, Elmoselhi AB, Hata T, Makino N. Status of myocardial antioxidants in ischemia-reperfusion injury. Cardiovasc Res. 2000; 47(3): 446-56. Ekhterae D, Hinmon R, Matsuzaki K, Noma M, Zhu W, Xiao RP, et al. Infarction induced myocardial apoptosis and ARC activation. J Surg Res 2011; 166(1): 59-67. Ferdinandy P, Schulz R, Baxter GF. Interaction of cardiovascular risk factors with myocardial ischemia/reperfusion injury, preconditioning, and postconditioning. Pharmacol Rev 2007; 59(4): 418-58. Förster K, Paul I, Solenkova N, Staudt A, Cohen MV, Downey JM, et al. NECA at reperfusion limits infarction and inhibits formation of the mitochondrial permeability transition pore by activating p70S6 kinase. Basic Res Cardiol 2006; 101(4): 319-26. Gao F, Gao E, Yue TL, Ohlstein EH, Lopez BL, Christopher TA, et al. Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia-reperfusion: the roles of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphorylation. Circulation 2002; 105(12): 1497-502. Gao XM, Liu Y, White D, Su Y, Drew BG, Bruce CR, et al. Deletion of macrophage migration inhibitory factor protects the heart from severe ischemia-reperfusion
injury: a predominant role of anti-inflammation. J Mol Cell Cardiol. 2011; 50(6): 991-9. Granger DN, Kvietys PR. Reperfusion injury and reactive oxygen species: the evolution of a concept. Redox Biol 2015; 6: 524-51. Hausenloy DJ, Yellon DM. Ischaemic conditioning and reperfusion injury. Nat Rev Cardiol. 2016; 13: 193-209. Hayyan M, Hashim MA, AlNashef IM. Superoxide ion: generation and chemical implications. Chem Rev. 2016; 116(5): 3019-85. Heusch G. Molecular basis of cardioprotection: signal transduction in ischemic pre-, post-, and remote conditioning. Circ Res 2015; 116(4): 674-99. Heusch G. Critical issues for the translation of cardioprotection. Circ Res 2017; 120(9): 1477-86. Ibáñez B, Heusch G, Ovize M, Van de Werf F. Evolving therapies for myocardial ischemia/reperfusion injury. J Am Coll Cardiol 2015; 65(14): 1454-71. Jennings RB SH, Smyth GA, Flack HA, Linn H. Myocardial necrosis induced by temporary occlusion of a coronary artery in the dog. . Arch Pathol 1960; 70: 68-78. Kennedy JW. Limiting the size of myocardial infarction by early coronary artery reperfusion. Heart Dis Stroke 1993; 2(2): 93-7. Kinemuchi H SH, Obata T, Satoh N, Ueda S. Selective inhibitors of membranebound semicarbazide-sensitive amine oxidase (SSAO) activity in mammalian tissues. Neurotoxicology 2004; 25(1-2): 325-35. Klein L, O'Connor CM, Gattis WA, Zampino M, de Luca L, Vitarelli A, et al. Pharmacologic therapy for patients with chronic heart failure and reduced systolic function: review of trials and practical considerations. The American journal of cardiology. 2003; 91(9A): 18-40. Kleinbongard P, Schulz R, Heusch G. TNF alpha in myocardial ischemia/reperfusion, remodeling and heart failure. Heart Fail Rev 2011; 16(1): 49-69. Kukreja RC, Salloum F, Das A, Ockaili R, Yin C, Bremer YA, et al. Pharmacological preconditioning with sildenafil: basic mechanisms and clinical implications.
Vascul Pharmacol 2005; 42(5-6): 219-32. Li Y, Hou D, Chen X, Zhu J, Zhang R, Sun W, et al. Hydralazine protects against renal ischemia-reperfusion injury in rats. Eur J Pharmacol. 2019;843:199-209. Li Y, Xiong W, Yang J, Zhong J, Zhang L, Zheng J, et al. Attenuation of Inflammation by Emodin in Lipopolysaccharide-induced Acute Kidney Injury via Inhibition of Toll-like Receptor 2 Signal Pathway. Kidney Diseases 2015; 9(3): 202-8. Li XD, Yang YJ, Geng YJ, Zhao JL, Zhang HT, Cheng YT, et al. Phosphorylation of endothelial NOS contributes to simvastatin protection against myocardial no-reflow and infarction in reperfused swine hearts: partially via the PKA signaling pathway. Acta Pharmacol Sin. 2012; 33(7): 879-87. Magee LA, Cham C, Waterman EJ, Ohlsson A, von Dadelszen P. Hydralazine for treatment of severe hypertension in pregnancy: meta-analysis. BMJ 2003; 327(7421): 955-60. Matsui T, Li L, del MonteF, Fukui Y, Franke TF, Hajjar RJ, et al. Adenoviral gene transfer of activated phosphatidylinositol 30-kinase and Akt inhibits apoptosis of hypoxic cardiomyocytes in vitro. Circulation. 1999; 100(23): 2373-9. Matsui T, Tao J, del Monte F, Lee KH, Li L, Picard M, et al. Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo. Circulation. 2001; 104(3): 330-5. Morrow JD, Schroeder HA, Perry HM Jr. Studies on the control of hypertension by hyphex. II. Toxic reactions and side effects. Circulation. 1953; 8(6): 829-39. Pang L, Cai Y, Tang EH, Yan D, Kosuru R, Li H, et al. Cox-2 inhibition protects against hypoxia/reoxygenation-induced cardiomyocyte apoptosis via aktdependent enhancement of iNOS expression. Oxid Med Cell Longev. 2016; 2016: 3453059. Portt L, Norman, G, Clapp C, Greenwood M, Greenwood MT. Anti-apoptosis and cell survival: a review. Biochimica et biophysica acta 2011; 1813: 235-59. Rodrigues SF, de Oliveira MA, dos Santos RA, Soares AG, de Cássia Tostes R, Carvalho MH, et al. Hydralazine reduces leukocyte migration through different mechanisms in spontaneously hypertensive and normotensive rats. Eur J
Pharmacol. 2008; 589(1-3): 206-14. Rong Y, Distelhorst CW. Bcl-2 protein family members: versatile regulators of calcium signaling in cell survival and apoptosis. Annual review of physiology. 2008; 70: 73-91. Saikumar P, Dong Z, Patel Y, Hall K, Hopfer U, Weinberg JM, et al. Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury. Oncogene. 1998; 17(26): 3401-15. Salloum F, Yin C, Xi L, Kukreja RC. Sildenafil induces delayed preconditioning through inducible nitric oxide synthase-dependent pathway in mouse heart. Circ Res. 2003; 92(6): 595-7. Shanmuganathan S, Hausenloy DJ, Duchen MR, Yellon DM. Mitochondrial permeability transition pore as a target for cardioprotection in the human heart. Am J Physiol Heart Circ Physiol. 2005; 289(1): 237-42. Sun L, Chen C, Jiang B, Li Y, Deng Q, Sun M, et al. Grb2-associated binder 1 is essential for cardioprotection against ischemia/reperfusion injury. Basic Res Cardiol 2014; 109(4): 420. Taketomo CK HJ, Kraus DM. Pediatric & neonatal dosage handbook. American Pharmacists Association, Lexicomp 2013; 20. Tampe B, Steinle U, Tampe D, Carstens JL, Korsten P, Zeisberg EM, et al. Low-dose hydralazine prevents fibrosis in a murine model of acute kidney injury-to-chronic kidney disease progression. Kidney International 2017; 91(1): 157-176. Tampe B, Tampe D, Zeisberg EM, Müller GA, Bechtel-Walz W, Koziolek M, et al. Induction of Tet3-dependent epigenetic remodeling by low-dose hydralazine attenuates progression of chronic kidney disease. EBioMedicine 2015; 2(1): 19-36. Tampe B, Zeisberg M. Evidence for the involvement of epigenetics in the progression of renal fibrogenesis. Nephrol Dial Transplant. 2014; 29(suppl 1): 1-8. Tang CY, Lai CC, Huang PH, Yang AH, Chiang SC, Huang PC, et al. Magnolol Reduces Renal Ischemia and Reperfusion Injury via Inhibition of Apoptosis. The American Journal of Chinese Medicine. 2017; 45(7): 1-19.
Vergona RA, Agnello A, Hirkaler G, Urbano S, Rosenberger L. Protective effects of hydralazine in a renal ischemia model in the rat. Life Sci 1987; 41(5): 563-9. Xia Z, Li H, Irwin MG. Myocardial ischaemia reperfusion injury: the challenge of translating ischaemic and anaesthetic protection from animal models to humans. Br J Anaesth. 2016;117(2):44-62. Xing W, Yan W, Fu F, Jin Y, Ji L, Liu W, et al. Insulin inhibits myocardial ischemia-induced apoptosis and alleviates chronic adverse changes in post-ischemic cardiac structure and function. Apoptosis. 2009; 14(9): 1050-60. Xiong J, Xue FS, Yuan YJ, Wang Q, Liao X, Wang WL. Cholinergic anti-inflammatory pathway: a possible approach to protect against myocardial ischemia reperfusion injury. Chin Med J (Engl) 2010; 123(19): 2720-6. Yang J, Yang J, Ding JW, Chen LH, Wang YL, Li S, et al. Sequential expression of TLR4 and its effects on the myocardium of rats with myocardial ischemia-reperfusion injury. Inflammation. 2008;31(15):304-12. Yang SJ, Meng JP, Qu Y, Liu YB The progress on the signal transduction pathways of apoptosis. Chin J Comp Med. 2007;17:297-301. Yellon DM HD. Myocardial reperfusion injury. N Engl J Med 2007; 357: 1121-35. Zambrano P, Segura-Pacheco B, Perez-Cardenas E, Cetina L, Revilla-Vazquez A, Taja-Chayeb L, et al. A phase I study of hydralazine to demethylate and reactivate the expression of tumor suppressor genes. BMC Cancer 2005; 5: 44. Zhang KR, Liu HT, Zhang HF, Zhang QJ, Li QX, Yu QJ, et al. Long-term aerobic exercise protects the heart against ischemia/reperfusion injury via PI3 kinase-dependent and Aktmediated mechanism. Apoptosis. 2007; 12(9): 1579-88. Zhang X, Liang X, Lin X, Zhang S, Huang Z, Chen C, et al. Mechanism of the protective effect of Yulangsan flavonoid on myocardial ischemia/reperfusion injury in rats. Cell Physiol Biochem. 2014; 34(4): 1050-62. Zhu L, Wei T, Gao J, Chang X, He H, Luo F, et al. The cardioprotective effect of salidroside against myocardial ischemia reperfusion injury in rats by inhibiting apoptosis and inflammation. Apoptosis. 2015; 20(11): 1433-43.
Figure Legends Fig. 1 (A) Hydralazine decreased infarct size assessed by the TTC assay in I/R rats. Representative images from TTC staining are shown for Sham, I/R, Hyd 10mg/kg, Hyd 20mg/kg, Hyd 30mg/kg. (B) Infarct size of each rats were quantitatively analyzed (n=8). Red stain: viable area; White stain: infarct region; infarct size (%) = infarct region x 100 %/ (viable area + infarct region). Data were shown as mean ± S.D. * P < 0.05, ** P < 0.01 compared with sham group; # P < 0.05, ## P < 0.01 compared with I/R group. (C) Histopathological examination of heart tissue by H&E staining. Representative images from HE staining are shown for Sham, I/R, Hyd 10mg/kg, Hyd 20mg/kg, Hyd 30mg/kg. (B) Histological injury scores of the heart were shown (n=8). Fig. 2 Biochemical analysis of heart function and inflammatory markers. Biochemical analysis of CK-MB level (A), LDH level (B), IL-1β (C), IL-6 (D) and TNF-α (E) in serum was shown (n=8). All data were expressed as the mean ± S.D. Statistical significance:* P < 0.05 and ** P < 0.01 versus the Sham group, #P < 0.05 and ## P < 0.01 versus the I/R group, respectively. Fig. 3 Biochemical analysis of oxidative stress in hearts. The activity of SOD (A) and MDA (B) in serum was measured (n=8). (C) The reactive oxygen species levels in the hearts of rats from all groups were revealed by DHE staining of frozen sections. (D) The fluorescence intensity of DHE staining was analyzed using Image-Pro Plus (n=8). All data are expressed as the mean ± S.D. Statistical significance:* P < 0.05 and ** P < 0.01 versus the Sham group, #P < 0.05 and ## P < 0.01 versus the I/R group, respectively. Fig. 4 (A) Apoptosis was analyzed using in situ TUNEL fluorescence staining. The nuclei of TUNEL-positive (apoptotic) cells stained green. Five random fields per section (five sections per tissue from each mouse) were examined in each experiment (10x). (B) The numbers of TUNEL-positive myocardial cells (%) were compared among the four groups (10x, n=8). (C) TUNEL and CD31 immunofluorescence staining (10x). (D) TUNEL and sarcomeric immunofluorescence staining (10x). All data are expressed as the mean ± S.D. All data were expressed as the mean ± S.D. Statistical significance:* P < 0.05 and ** P < 0.01 versus the Sham group, # P < 0.05
and ## P < 0.01 versus the I/R group, respectively. Fig. 5 Apoptosis assay and expression of NO related signaling pathway proteins in the hearts. (A) Representative western blots depicting protein levels of p-PI3K, PI3K, p-AKT, AKT, eNOS, p-eNOS, Cleaved Caspase-3 and Cleaved Caspase-9 in heart tissue were shown. (B) The expression levels of proteins were quantitatively analyzed (n=8). All data were expressed as the mean ± S.D. Statistical significance:* P < 0.05 and ** P < 0.01 versus the Sham group, # P < 0.05 and ## P < 0.01 versus the I/R group, respectively. Fig. 6 Expression of eNO signaling pathway proteins in H/R-induced H9c2 injury pretreated with or without PI3K/Akt inhibitor WM (100 nmol/L). (A) Representative western blots depicting protein levels of p-PI3K, PI3K, p-AKT, AKT, eNOS and peNOS in heart tissue were shown. (B) The expression levels of proteins were quantitatively analyzed (n=8). WM: wortmannin. All data were expressed as the mean ± S.D. Statistical significance:* P < 0.05 and ** P < 0.01 versus the Sham group, # P < 0.05 and ## P < 0.01 versus the I/R group, respectively.
S0014-2999(19)30802-7
DOI:
https://doi.org/10.1016/j.ejphar.2019.172850
Reference:
EJP 172850
To appear in:
European Journal of Pharmacology
Received Date: 22 April 2019 Revised Date:
3 December 2019
Accepted Date: 9 December 2019
Please cite this article as: Li, C., Su, Z., Ge, L., Chen, Y., Chen, X., Li, Y., Cardioprotection of hydralazine against myocardial ischemia/reperfusion injury in rats, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/j.ejphar.2019.172850. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Cardioprotection of hydralazine against myocardial ischemia/reperfusion injury in rats Chengzong Lia,b , Zhongping Sub, Liqi Gea, Yuchen Chenc, Xuguan Chenb*, Yong Lib* a
Department of Cardiology, the Affiliated Hospital of Xuzhou Medical University,
Xuzhou, PR China. b
Department of Cardiology, the First Affiliated Hospital of Nanjing Medical
University, Nanjing, PR China. c
Cape Henry Collegiate, 1320 Mill Dam Road, Virginia Beach, Virginia, USA.
* Corresponding author. E-mail address: [email protected] (Y. Li) or [email protected] (X. Chen). Abstract This study aimed to investigate whether hydralazine could reduce cardiac ischemia/reperfusion (I/R) injury in rats. Anesthetized male Sprague–Dawley rats underwent myocardial I/R injury. Saline, hydralazine (HYD, 10-30mg/kg) was administered intraperitoneally 10 min before reperfusion. After 30 min of ischemia and 24 h of reperfusion, the myocardial infarct size was determined using TTC staining. Heart function and oxidative stress were determined through biochemical assay and DHE staining. HE staining was used for histopathological evaluation. Additionally, the cardiomyocytes apoptosis and protein expression of PI3K-Akt-eNOS pathway marker were detected by TUNEL and Western blotting. The serum levels of malonaldehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) and reactive oxygen species were significantly elevated in cardiac I/R group, but the superoxide dismutase (SOD) level was suppressed. However, intraperitoneal pretreatment with hydralazine at a dose of 10 to 30 mg/kg before cardiac I/R significantly limited the increase in CK-MB, LDH, oxidative stress, inflammatory factors, histological damage and apoptosis in the hearts.
In addition, hydralazine also increased p-PI3K, p-AKT, p-eNOS expression and decreased Cleaved Caspase-3, Cleaved Caspase-9 expression in the hearts. Our results suggest that the cardioprotective effect of hydralazine against I/R injury might be a cooperation of the inhibition of oxidative stress, inflammatory response, apoptosis with the motivation of eNOS phosphorylation via activating the PI3K/AKT signal pathway.
Key words: hydralazine; oxidative stress; inflammatory cytokines; apoptosis; heart ischemia/reperfusion.
1. Introduction Ischemic heart disease is a common cardiovascular disease and a main cause of death worldwide (Ferdinandy et al., 2007; Xia et al., 2016). It is mandatory to invigorate ischemic myocardium via reperfusion of coronary blood flow (Kennedy, 1993); however, reperfusion itself can cause cardiomyocytic death and the subsequent irreversible myocardial impairment, termed ischemia/reperfusion (I/R) injury (Yellon, 2007). In the past three decades, a number of anti-ischemic and pharmacological cardioprotective strategies have been tested in experimental animals and replicated in the clinical setting to treat acute myocardial infarction, but the outcomes were disappointing, and no therapeutic consensus has been made (Hausenloy et al., 2016; Heusch, 2017). The underlying mechanisms of cardiac I/R injury include excessive reactive oxygen species generation, inflammation, apoptosis, intracellular calcium overload, neutrophil aggregation and adhesion, endothelial cell dysfunction, etc. Great effort is needed to seek novel therapeutic strategies regulating myocardial oxidative stress and subsequent inflammation and apoptosis induced by ischemia/reperfusion injury (Heusch, 2015). Hydralazine, a non-nucleoside analogue, was invented in 1951 and has ever since been widely used as an adjunctive treatment for hypertension (Morrow et al., 1953). It is a potent arterial vasodilator approved by FDA for severe hypertension, chronic heart failure and hypertension in pregnancy (Cornacchia et al., 1988; Klein et
al., 2003; Magee et al., 2003). Hydralazine works directly on the arterioles, resulting in decreased systemic vascular resistance and blood pressure reduction (Taketomo et al., 2013). The biological mechanisms through which hydralazine dilates vessels are diverse and not fully understood. Among them, the demethylating activity of hydralazine has long been recognized (Coronel J, 2011, Zambrano P, 2005) and exploited for a tumor therapy (Coronel et al., 2011; Tampe et al., 2014; Zambrano et al., 2005). It was reported that low-dose hydralazine effectively attenuated renal fibrogenesis in murine models of unilateral ureter obstruction and folic acid–induced nephropathy (Tampe et al., 2015), and I/R induced renal injury in rats (Tampe et al., 2015; Vergona et al., 1987; Yong et al., 2019). However, the curative effects of hydralazine on myocardial infarction induced by cardiac I/R remain obscure. This study answered whether hydralazine can reduce myocardial I/R injury. 2. Materials and methods 2.1 Animals Male Sprague-Dawley rats weighing 250–280 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The rats were housed in SPF facility in Animal Core Facility of Nanjing Medical University Nanjing Medical University under standard conditions of temperature and 12 h light/dark cycle with ad libitum feeding. All experimental protocols and animal handling procedures were performed in accordance with the “Guide for the Care and Use of Laboratory Animals” (National Academic Press, USA, 1996). This experimental animal study was performed with the approval of the Institutional Animal Care and Use Committee of Nanjing Medical University. The ethical was approved by Nanjing Medical University and the approval number is 17060186.
2.2 Grouping and Experimental Protocols We performed two studies. In study 1, a total of 60 rats were randomly allocated
into 5 groups. Rats in Group 1 (Sham group) were subjected to the surgical procedure described above and intraperitoneally administered with sterile normal saline (i.g., 10 ml/kg) for 10 min, but without left anterior descending (LAD) branch occlusion or reperfusion; rats in Group 2 (I/R group) with normal saline (i.g.); rats in Group 3 (I/R+Hyd 10mg/kg group) with hydralazine (i.g., 10 mg/kg); rats in Group 4 (I/R+Hyd 20mg/kg group) with hydralazine (i.g., 20 mg/kg); rats in Group 5 (I/R+Hyd 30mg/kg group) with hydralazine (i.g., 30 mg/kg). Ten minutes after the above mentioned pretreatments, the rats in all groups except Group 1 were subjected to LAD ligation for 30 min followed by 24 h of reperfusion. All rats were killed under general anesthesia using intraperitoneal injection of sodium pentobarbital (50 mg/kg). The blood was collected from the punctured heart, and the right kidney was stored at −80 °C for mRNA and protein extraction. In study 2, H9c2 cells were assigned to 3 groups: (1) H/R: H9c2 cells were subjected to 24 h of Hypoxia followed by 12 h of reoxygenation (Busheng Zhang, 2014); (2) H/R+Hyd: H9c2 cells pretreated with hydralazine (80 µmol/ml) were subjected to 24 h of Hypoxia followed by 12 h of reoxygenation; (3) H/R+Hyd+WM: H9c2 cells pretreated with hydralazine (80 µmol/ml) and wortmannin (100 umol/l, PI3K/AKT inhibitor) were subjected to 24 h of Hypoxia followed by 12 h of reoxygenation. Cells under normoxia throughout the experimental processes were included as a control. All experiments were repeated for three times. 2.3 Animal model of myocardial ischemia/reperfusion I/R model was induced by ligating the LAD for 30 min followed by 24 h of reperfusion. Rat models of coronary ischemia/reperfusion injury were established as those in previous studies with small modification (Gao et al., 2011; Sun et al., 2014). Briefly, the rats were anesthetized with 3.5% inhalational isoflurane on a heating pad (40°C). The body temperature of each rat was maintained at 37℃ throughout the experiments. The rats were anesthetized by administering sodium pentobarbital (50 mg/kg) intraperitoneally. Following tracheal intubation, 6–0 silk ligature was used to ligate the LAD for a 30-min ischemic period. After a 30-min ischemia treatment, reperfusion was allowed for 24 h after the ligated LAD was released. Sham control
group rats were treated with the same surgical procedures except that the left coronary artery was not ligated. The rats were killed at 24 h after reperfusion or sham operation. The blood was collected and the kidneys were harvested for examination. 2.4 H9c2 cell culture The H9c2 cells (ventricular myocardiocyte, rat in origin; Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were seeded in 6-well plates (2x104 cells/cm2) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St. Louis, MO, USA) containing 10% (v/v) fetal bovine serum (FBS, Gibco, USA) in a humidified atmosphere of 95% air and 5% CO2 at 37℃. 2.5 Simulated hypoxia/reoxygenation model Hypoxia/reoxygenation (H/R) model was established according to previously described methods (Zhang et al., 2014). H/R in H9c2 Cardiomyocytes Hypoxia was induced by exposing the cells to 1% O2, 94% N2, and 5% CO2 for 24 h using a modular incubator (Model 3131, Forma Scientific, Marietta, USA). Reoxygenation (95% air, 5% CO2, 37℃) lasted for 12 h. 2.6 Biochemical assays All blood samples were allowed to clot at room temperature and centrifuged at 2,000 g for 10 min to harvest serum. The biochemical indicators including lactate dehydrogenase (LDH), creatine kinase MB (CK-MB), superoxide dismutase (SOD), malonaldehyde (MDA), tumor Necrosis Factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were measured (n=8 per group) spectrophotometrically using commercially available kits for LDH (A020-2), CK-MB (H197), SOD (A001-1), MDA (A003-1), TNF-α (H052), IL-1β (H002) and IL-6 (H007) (Jiancheng Bioengineering Institute, Nanjing, China). 2.7 Histopathological examination The hearts removed from the euthanized rat immediately at end-diastole were placed in a 10% potassium chloride solution, washed with saline solution, and then placed in 4% paraformaldehyde at 4℃ overnight. A transverse cut was made close to the heart apex to expose the left and right ventricles. The samples were then dehydrated in an ethanol gradient, rinsed in xylene, and embedded in paraffin. Finally, paraffin blocks
were cut into 4-µm sections. The paraffin sections were stained in H&E for histopathology, and then visualized by light microscopy. Heart histology was assessed using previously described methods (Zhu et al., 2015). According to the scope and severity of the heart, it was graded on a scale of 0.5–4: 0.5 = minor, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe. Five random fields on each heart slide (five slides per animal) were examined (n = 7 per group). 2.8 Estimation of infarct size Myocardial infarct size was estimated by stating of 2, 3, 5- triphenyltetrazolium hydrochloride (TTC). Animals were killed after administrating overdose of anesthetics (2–3 times of conventional anesthetic dose), then the hearts were rapidly excised and immediately frozen at −20 °C for 1 h. The slices of the heart were cut into cross sections 2 mm thick and incubated with 1% TTC in phosphate buffer (0.1 M, pH 7.4) for 15 min at room temperature. Slices were incubated with 10% formalin overnight and then scanned from both sides for myocardial necrosis. White parts indicated the infarct size, and red indicated normal. Images of the stained slices were captured using a digital camera and analyzed using Image J2x analysis software (National Institutes of Health). The severity of the myocardial infarction was indicated by the ratio of the infarct size to the total size. 2.9 Reactive oxygen species staining Total reactive oxygen species was stained with dihydroethidium (DHE, D-23107; Invitrogen, USA) on fresh frozen sections. The hearts taken out of the euthanized rats were mounted in OCT embedding compound (3801480; Leica) and frozen at - 80℃. The frozen section was cut 5 um thick using a cryostat and then thawed; the sections were mounted onto gelatin-coated histological slides. Thereafter, 5 µM of DHE dissolved in DMSO was added to fresh frozen rat heart sections (thickness 5 um) after dilution in PBS, incubated in darkness at 37℃ for exactly 30 min, and rinsed twice with cold PBS. Photos were taken immediately. The fluorescence density of arterial walls was quantified using Image J. The relative fluorescence intensity was determined by the ratio of the average intensity at ischemia area normalized to that of non-ischemia area after subtracting background intensity.
2.10 In situ TUNEL staining assay A terminal deoxynucleotidyltransferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay was performed according to the manufacturer’s instructions (11684817910, Roche, Switzerland). Heart tissues were fixed in 4% paraformaldehyde overnight, dehydrated, embedded in paraffin, sectioned into 4-µm-thick sections and placed on numbered polylysine-coated glass slides. Deparaffinized tissue sections were incubated with proteinase K (20 mg/ml, Sigma, USA) in a humidified chamber for 15 min, and endogenous peroxidase activity was blocked via treating with 3% H2O2 for 10 min. The sections were incubated with TdT labeling buffer at 37°C for 1 h in a moist chamber and then counterstained with DAPI. TUNEL-positive cells showed brown and the nuclei showed blue. Five random fields on each slide (five slides per animal, seven animals per group) were examined. The nuclei number of TUNEL-positive cells (Numerator) and the total number of cellular nuclei (denominator) were counted as previously mentioned (Tang et al., 2017). The rate of TUNEL-positive cells in each field was analyzed using Image Pro Plus 6.0 software. 2.11 Western blotting Western blotting analyses were performed according to previously described methods with slight modifications (Choi et al., 2009). Briefly, 30 mg of protein was separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in TBST buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% Tween-20) for 1 h prior to incubation with a primary antibody against PI3K (4108, Cell Signal Technology, USA), p-PI3K (sc-48341, Santa Cruz Biotechnology, USA), AKT (4685, Cell Signal Technology, USA), p-AKT (4060, Cell Signal Technology, USA), eNOS (32027, Cell Signal Technology, USA) and p-eNOS (9570, Cell Signal Technology, USA), Cleaved Caspase 3 (9665, Cell Signal Technology, USA), Cleaved Caspase 9 (7237, Cell Signal Technology, USA) and GAPDH (sc-166574, Santa Cruz Biotechnology, USA) at 4℃ overnight, followed by an incubation with a goat anti-rabbit IgG HRP-conjugated secondary antibody (sc-2004, Santa Cruz Biotechnology) or a goat anti-mouse IgG HRP-conjugated
secondary antibody (sc-2005, Santa Cruz Biotechnology). Then, the membranes were washed for three times in TBST. The blots were imaged using the ChemiDoc XRS+ Molecular Imager (Bio-Rad) with the Pierce ECL Western Blotting Substrate (32209, Thermo Scientific) and analyzed using image analysis software (ImageJ 1.42). The housekeeping protein GAPDH was used as the internal reference. The Western blotting quantification was corrected to GAPDH expression prior to normalization. 2.12 Immunofluorescence staining To explore which type of cells are undergoing apoptosis (cardiomyocytes or endothelial cells), immunofluorescence staining was performed. CD31 is the endothelial-specific
marker, and sarcomeric is the cardiomyocytes-specific marker. Heart samples were fixed with 4% paraformaldehyde and incubated with a blocking solution consisting of 10% bovine serum albumin for 1 h. The samples were incubated with antibodies of CD31 or sarcomeric (Abcam, MA, USA) at 4°C overnight, and then washed three times in PBS and incubated in secondary antibodies (Jackson ImmunoResearch Laboratories Inc., PA, USA)) for 2 h at room temperature. Then, 4',6-diamidino-2-phenylindole (DAPI, Life Technology, Carlsbad, CA, USA) was used to counterstain the nucleus. Fluorescent imaging was achieved using a microscope (ZEISS, Oberkochen, Germany). 2.13 Statistical analysis All statistical analyses were performed using Graphpad Prism version 5 software. Comparisons between two groups were finished using the two-tailed unpaired t test. One-way ANOVA followed by Bonferroni multiple comparison test was used for comparison between more than two groups. Results were expressed as mean ± S.D. P < 0.05 was considered statistically significant. 3. Results 3.1 Effects of hydralazine on myocardial tissue damage induced by I/R The effects of hydralazine on heart infarct size was assessed by the TTC assay, showed in Fig. 1A, B. The proportion of the infarcted area in the I/R group averaged 40%. The infarct size decreased significantly in I/R+Hyd(10mg/kg) and I/R+Hyd(30mg/kg) groups (P < 0.01). This proportion was 15% in I/R+Hyd(30mg/kg)
group (P < 0.01, compared with I/R group). Histopathological examination of the hearts is shown in Fig. 1C, D. Sham group showed obvious integrity of myocardial membrane without any infarction, inflammatory infiltration or cardiac necrosis, and the cardiomyocytes demonstrated well-arranged myofilaments. I/R group showed myocardial structure disorder, myocardial fibers fractured, inflammatory infiltration and cardiac necrosis. However, hydralazine (10-30 mg/kg) groups significantly alleviated the damage of myocardium. 3.2 Effect of hydralazine on heart function and inflammation induced by I/R The effects of hydralazine on heart function levels in rats are shown in Fig. 2A,B. I/R operation significantly (P < 0.01) increased CK-MB and LDH levels in both the I/R and Hyd(30mg/kg) groups compared with the sham group. There was no significant difference in CK-MB levels between the I/R and I/R+Hyd(10mg/kg) groups. Hydralazine significantly reduced CK-MB level in I/R+Hyd(20mg/kg) and I/R+Hyd(30mg/kg) groups compared with I/R group (P < 0.01); Hydralazine also significantly reduced
LDH level in I/R+Hyd(10mg/kg), I/R+Hyd(20mg/kg) and
I/R+Hyd(30mg/kg) groups compared with I/R group (P < 0.01). To examine the effects of hydralazine on inflammation in the heart, serum levels and mRNA expression levels of IL-1ß, IL-6 and TNF-α were assessed (Fig. 2C-E). The serum levels and mRNA expression levels of IL-1ß, IL-6 and TNF-α were all significantly up-regulated in I/R group compared with the sham group (P < 0.01). However, hydralazine dose-dependently decreased serum levels and mRNA expression levels of this inflammatory factors. The serum levels of IL-1ß, IL-6 and TNF-α decreased in I/R+Hyd(10mg/kg), I/R+Hyd(20mg/kg) and I/R+Hyd(30mg/kg) compared with I/R group (P < 0.01). 3.3 Effects of hydralazine on oxidative stress induced by I/R To assess the effects of hydralazine on oxidative stress, we detected serum SOD activity and MDA level (Fig 3). Serum SOD activity and MDA level decreased significantly in the I/R and I/R+Hyd(10mg/kg) groups compared with the sham group (P < 0.01) (Fig. 3A). Heart MDA levels increased significantly (P < 0.01) in the
I/R+Hyd(30mg/kg) groups compared with the sham group (P < 0.01) (Fig. 3B). Hydralazine significantly increased SOD activity and reduced MDA level in I/R+Hyd(10mg/kg), I/R+Hyd(10mg/kg) and I/R+Hyd(30mg/kg) groups compared with the I/R group (P < 0.05 or P < 0.01, respectively). In addition, to assess the effects of hydralazine on reactive oxygen species production, we detected the intracellular generation of reactive oxygen species moiety O2- with the fluoroprobe DHE (Fig. 3C). Confocal microscopy showed that all heart sections from I/R rats had a widespread and markedly intensified DHE fluorescence compared with those in the sham group (P < 0.01, Fig. 3D). Dose-dependently, hydralazine (10 mg/kg to 30 mg/kg) before I/R operation induced a significant decrease in reactive oxygen species fluorescence intensity compared with that of the sham group (P < 0.01). 3.4 Effect of hydralazine on myocardial cell apoptosis induced by I/R In the TUNEL assay, the nuclei of TUNEL-positive cells were stained green, indicating the presence of apoptotic cells (Fig. 4A). The level of apoptosis was indicated by the percentage of TUNEL-positive cells in the total cells (Fig. 4B). Few apoptotic cells were observed in the sham group, but the I/R group displayed more TUNEL-positive cells than the sham group (P < 0.01). Dose-dependently, hydralazine significantly decreased the number of TUNEL-positive cells. Fewer apoptotic cells were observed in the I/R+Hyd (10mg/kg), I/R+Hyd (20mg/kg) and I/R+Hyd (30mg/kg) groups compared with the I/R group (P < 0.01). To explore which type of cells
are
undergoing
apoptosis
(cardiomyocytes
or
endothelial
cells),
immunofluorescence staining was performed. CD31 is the endothelial-specific marker, and sarcomeric is the cardiomyocytes-specific marker. The apoptosis cells were cardiomyocytes, but not endothelial cells (Figure 4C and D).
3.5 Effect of hydralazine on related pathways in the heart tissues The expression of PI3K-Akt-eNOS pathway and apoptosis associated proteins were detected by Western blotting (Fig. 5A). No significant difference in the PI3K, Akt and eNOS protein expression appeared between groups. The expression levels of p-PI3K, p-AKT and p-eNOS were significantly lower in the I/R group than in the sham group
(P< 0.05 or P< 0.01, respectively), and were markedly up-regulated in Hyd(10mg/kg), Hyd(20mg/kg) and Hyd(30mg/kg) groups compared with I/R group (P < 0.05 or P < 0.01, respectively) (Fig. 5B). Besides, the expression of Cleaved Caspase-3 and Cleaved Caspase-9 was remarkably enhanced in I/R group compared with the sham group (P < 0.01), and was markedly down-regulated in Hyd(10mg/kg), Hyd(20mg/kg) and Hyd(30mg/kg) compared with I/R group (P < 0.05 or P < 0.01). 3.6 Effect of PI3K/Akt inhibitor and hydralazine on related pathways in the H9C2 cells subjected to H/R The PI3K and AKT expression and phosphorylation in H9C2 cells subjected to H/R were detected by Western blotting. As shown in Fig. 6, there was no difference in total PI3K and AKT expression among all groups in cultured cardiomyocytes pretreated with 80 mmol/L hydralazine and exposed to H/R, and hydralazine treatment promoted PI3K and AKT phosphorylation. PI3K/AKT inhibitor WM (100 nmol/L) discounted the effect of hydralazine on PI3K and AKT phosphorylation. Thus, co-treatment with hydralazine and WM blocked the activation of PI3K/AKT pathway induced by hydralazine. eNOS is a downstream molecule of AKT. As shown in Fig. 6, hydralazine enhanced eNOS phosphorylation in cultured cardiomyocytes exposed to H/R. PI3K/AKT inhibitor blocked eNOS phosphorylation induced by hydralazine, whereas the expression of total eNOS remained unchanged. These results indicated that hydralazine activated PI3K-Akt and eNOS in rat cardiomyocytes exposed to H/R. 4 Discussion I/R injury was first coined in 1960 to describe a phenomenon that reperfusion itself exacerbates myocardial injury (Jennings et al., 1960). The underlying mechanisms is complicated (Heusch, 2015). I/R injury may involve mitochondrial dysfunction, overproduction of reactive oxygen species, inflammation, apoptosis, endothelial cell dysfunction reduction of nitric oxide bioavailability, and intracellular calcium overload, etc. (Xia et al., 2016; Zhang et al., 2014). A potent anti-hypertensive agent and an arterial vasodilator for chronic heart failure and hypertension in pregnancy (Cornacchia et al., 1988; Klein et al., 2003; Magee et al., 2003), hydralazine can remarkably inhibit semicarbazide-sensitive amine oxidases
activity (Kinemuchi et al., 2004). With a powerful aldehyde-trapping ability (Burcham et al., 2000), hydralazine is also more specific to normalize aberrant promoter methylation of select genes than broad demethylating drug 5’-azacytidine (Tampe et al., 2015). Low-dose hydralazine reduces leukocyte migration, not blood pressure, in SHR rats (Rodrigues et al., 2008), attenuates renal fibrosis and preserves excretory renal function regardless of its blood pressure–lowering effects in C57BL/6 mice (Tampe et al., 2017). In the current study, we observed hydralazine (>10 mg/kg) protected myocardium against I/R injury by inhibiting the overproduction of reactive oxygen species and the release of inflammatory factors. Reactive oxygen species include peroxides, superoxide, hydroxyl radical, and singlet oxygen (Hayyan et al., 2016), all acting as the key mediators of myocardial I/R injury (Dhalla et al., 2000). Reactive oxygen species and redox signaling pathways are involved in myocardial ischemia-reperfusion injury and cardioprotection (Cadenas et al., 2018). During ischemia, hypoxia, even reperfusion, the produced reactive oxygen species exceeds the scavenged reactive oxygen species and the antioxidative defense may be compromised and eventually crumbled, all contributing to inflammation, apoptosis and heart dysfunction(Granger et al., 2015). High level of reactive oxygen species activates the pro-apoptotic proteins caspase-3 and caspase-9 (Ibanez et al., 2015), and initiates inflammatory response to ischemia/reperfusion injury (Granger, 2015) by increasing the inflammatory cytokines (such as IL-6, IL-1β, and TNF-α) and decreasing anti-inflammatory cytokines (such as IL-10) (Kleinbongard et al., 2011; Yang et al., 2008). In this study, hydralazine treatment increased serum SOD level and simultaneously decreased serum MDA level (a lipid peroxidation index) and reactive oxygen species level. Thus, antioxidation is one mechanism through which hydralazine protects the heart against myocardial I/R injury. Inflammatory reaction also plays a crucial role in myocardial I/R injury (Xiong et al., 2010). The myocardial I/R injury can stimulate the release of pro-inflammatory cytokines, IL-1β, IL-6 and TNF-α (Ahn et al., 2012; Ying et al. 2015). TNF-α can further activate caspases apoptosis process by the death receptor pathway (Yang et al.,
2007). In the present study, pretreatment with hydralazine significantly decreased IL-1β, IL-6, and TNF-α levels in serum compared with I/R group. Therefore, suppression of inflammatory reaction is another mechanism responsible for hydralazine-achieved protection. Apoptosis brings with infarction during myocardial I/R (Ekhterae et al., 2011). In our study, TUNEL staining revealed that apoptotic cells in the hearts of I/R injury rats treated by hydralazine were markedly decreased; Western blotting results also showed the down-regulation of Cleaved Caspase-3 and Cleaved Caspase-9 in the group treated with hydralazine compared with I/R group. In apoptosis, caspase cascade is activated to promote apoptotic body formation and cell fragmentation (Saikumar et al., 1998). In the pathway of cell apoptosis, lowering pro-apoptotic proteins caspases levels can suppress apoptosis (Atif et al., 2015; Rong et al., 2008; Chalah and Khosravi-Far, 2008). Otherwise, caspase-3 and caspase-9 are closely coupled to upstream, pro-apoptotic signals(Chalah and Khosravi-Far, 2008); Cleaved Caspase-9 and Cleaved Caspase-3, two activated caspases, are involved in a mitochondrial-dependent pathway that eventually leads to cell apoptosis (Portt et al., 2011). These data suggest that hydralazine could significantly reduce apoptosis through mitochondrial-dependent pathway in the kidney. PI3K–AKT–eNOS pathway, plays a key role in antiapoptotic action and protection of cardiomyocytes from I/R injury (Gao et al., 2002), and PI3K/AKT pathway has been identified as a key component of the protective mechanism of ischemia preconditioning (Ansley et al., 2013; Shanmuganathan et al., 2005). The activation of AKT, which is downstream of PI3K, may ameliorate I/R injury (Forster et al., 2006). PI3K/AKT-dependent signaling pathway plays a key role in anti-apoptotic activity and cardioprotection from I/R injury (Gao et al., 2002; Xing et al., 2009; Zhang et al., 2007). It was reported that the adenoviral gene transfer of activated PI3K and AKT inhibited the apoptosis of hypoxic cardiomyocytes in vitro (Matsui et al., 1999); AKT activation ameliorated cardiac function and prevented injury from transient cardiac ischemia in vivo(Matsui T, 2001). Our results demonstrated that enhanced eNOS, PI3K and AKT activation endowed hydralazine
with an anti-apoptotic and cardioprotective ability. It has been shown that phosphorylation of eNOS by AKT is a downstream effector in the survival signaling in myocardial ischemia and reperfusion (Gao et al., 2002). The present study also revealed that pretreatment with hydralazine induced the upregulation of p-eNOS, a process during which NO is generated (Salloum et al., 2003). NO exerts beneficial effects on the heart during myocardial reperfusion through guaranteeing myocardial contractility, sarcolemmal and mitochondrial KATP channel opening, antioxidation, and oxygen free radical production (Li et al., 2012; Pang et al., 2016). NO also activates guanylate cyclase, resulting in enhanced formation of cGMP, which activates PKG to open the mitochondrial KATP channels conferring cardioprotective effects against IR injury (Kukreja et al., 2005). Our study showed that pretreatment with wortmannin significantly blocked eNOS phosphorylation in H9c2 cells induced by hydralazine in I/R+Hyd groups, suggesting that PI3K–AKT–eNOS signaling may contribute to the hydralazine elicited cardioprotection and anti-apoptotic effect against I/R injury in H9c2. The present study also revealed that pretreatment with hydralazine plus wortmannin not only blocked PI3K, AKT and eNOS phosphorylation induced by cardiomyocytes H/R injury, but also markedly increased Cleaved Caspase-3 and Cleaved Caspase-9 expression compared with those in the hydralazine-treated group, suggesting that hydralazine inhibited I/R induced cardiomyocytes apoptosis via PI3K/AKT signal pathway. However, eNOS phosphorylation was not stopped completely by PI3K– AKT signal inhibitor wortmannin. It was reported that dominant negative AKT mutants were unable to block eNOS phosphorylation completely, suggesting that there may be other mechanism responsible for the activation of eNOS (Boo et al., 2002). In conclusion, hydralazine protected the myocardium against the injury induced by I/R (a 30-min ischemia and a 24-h reperfusion). Hydralazine lessened the released inflammatory factors and attenuated tissue damage and apoptosis in the hearts. Our results might provide a rationale to use hydralazine as a new therapy to prevent myocardial I/R injury induced after vascular surgery, heart surgery and heart
transplantation.
Acknowledgements This work was supported by the Natural Science Foundation of China (81627802). Conflicts of interest The authors declare no conflicts of interest. Authors' contributions Yong Li designed the study and drafted the manuscript. Chengzong Li and Xuguan Chen undertook animal and cell experiments. Zhongping Su and Liqi Ge helped to perform the experiments regarding molecular biological technique. Yuchen Chen provided much help on biochemical index detection. All authors reviewed the manuscript.
Reference Ahn J, Kim J. Mechanisms and consequences of inflammatory signaling in the myocardium. Curr Hypertens Rep 2012; 14(6): 510-6. Ansley DM, Wang B. Oxidative stress and myocardial injury in the diabetic heart. J Pathol 2013; 229(2): 232-41. Atif F, Yousuf S, Stein DG. Anti-tumor effects of progesterone in human glioblastoma multiforme: role of PI3K/Akt/mTOR signaling. The Journal of steroid biochemistry and molecular biology 2015; 146: 62-73. Boo YC, Sorescu G, Boyd N, Shiojima I, Walsh K, Du J, et al. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A. J Biol Chem 2002; 277(5): 3388-96. Burcham PC, Kerr PG, Fontaine F. The antihypertensive hydralazine is an efficient scavenger of acrolein. Redox Rep. 2000; 5(1): 47-9. Cadenas S. ROS and redox signaling in myocardial ischemia-reperfusion injury and cardioprotection. Free Radic Biol Med 2018; 117: 76-89. Chalah A, Khosravi-Far R. The mitochondrial death pathway. Advances in
experimental medicine and biology 2008; 615: 25-45. Choi DE, Jeong JY, Lim BJ, Chung S, Chang YK, Lee SJ, et al. Pretreatment of sildenafil attenuates ischemia-reperfusion renal injury in rats. Am J Physiol Renal Physiol 2009; 297(2): 362-70. Cornacchia E, Golbus J, Maybaum J, Strahler J, Hanash S, Richardson B. Hydralazine and procainamide inhibit T cell DNA methylation and induce autoreactivity. J Immunol 1988; 140(7): 2197-200. Coronel J, Cetina L, Pacheco I, Trejo-Becerril C, González-Fierro A, de la Cruz-Hernandez E, et al. A double-blind, placebo-controlled, randomized phase III trial of chemotherapy plus epigenetic therapy with hydralazine valproate for advanced cervical cancer. Preliminary results. Med Oncol 2011; 28(suppl 1): 540-6. Dhalla NS, Elmoselhi AB, Hata T, Makino N. Status of myocardial antioxidants in ischemia-reperfusion injury. Cardiovasc Res. 2000; 47(3): 446-56. Ekhterae D, Hinmon R, Matsuzaki K, Noma M, Zhu W, Xiao RP, et al. Infarction induced myocardial apoptosis and ARC activation. J Surg Res 2011; 166(1): 59-67. Ferdinandy P, Schulz R, Baxter GF. Interaction of cardiovascular risk factors with myocardial ischemia/reperfusion injury, preconditioning, and postconditioning. Pharmacol Rev 2007; 59(4): 418-58. Förster K, Paul I, Solenkova N, Staudt A, Cohen MV, Downey JM, et al. NECA at reperfusion limits infarction and inhibits formation of the mitochondrial permeability transition pore by activating p70S6 kinase. Basic Res Cardiol 2006; 101(4): 319-26. Gao F, Gao E, Yue TL, Ohlstein EH, Lopez BL, Christopher TA, et al. Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia-reperfusion: the roles of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphorylation. Circulation 2002; 105(12): 1497-502. Gao XM, Liu Y, White D, Su Y, Drew BG, Bruce CR, et al. Deletion of macrophage migration inhibitory factor protects the heart from severe ischemia-reperfusion
injury: a predominant role of anti-inflammation. J Mol Cell Cardiol. 2011; 50(6): 991-9. Granger DN, Kvietys PR. Reperfusion injury and reactive oxygen species: the evolution of a concept. Redox Biol 2015; 6: 524-51. Hausenloy DJ, Yellon DM. Ischaemic conditioning and reperfusion injury. Nat Rev Cardiol. 2016; 13: 193-209. Hayyan M, Hashim MA, AlNashef IM. Superoxide ion: generation and chemical implications. Chem Rev. 2016; 116(5): 3019-85. Heusch G. Molecular basis of cardioprotection: signal transduction in ischemic pre-, post-, and remote conditioning. Circ Res 2015; 116(4): 674-99. Heusch G. Critical issues for the translation of cardioprotection. Circ Res 2017; 120(9): 1477-86. Ibáñez B, Heusch G, Ovize M, Van de Werf F. Evolving therapies for myocardial ischemia/reperfusion injury. J Am Coll Cardiol 2015; 65(14): 1454-71. Jennings RB SH, Smyth GA, Flack HA, Linn H. Myocardial necrosis induced by temporary occlusion of a coronary artery in the dog. . Arch Pathol 1960; 70: 68-78. Kennedy JW. Limiting the size of myocardial infarction by early coronary artery reperfusion. Heart Dis Stroke 1993; 2(2): 93-7. Kinemuchi H SH, Obata T, Satoh N, Ueda S. Selective inhibitors of membranebound semicarbazide-sensitive amine oxidase (SSAO) activity in mammalian tissues. Neurotoxicology 2004; 25(1-2): 325-35. Klein L, O'Connor CM, Gattis WA, Zampino M, de Luca L, Vitarelli A, et al. Pharmacologic therapy for patients with chronic heart failure and reduced systolic function: review of trials and practical considerations. The American journal of cardiology. 2003; 91(9A): 18-40. Kleinbongard P, Schulz R, Heusch G. TNF alpha in myocardial ischemia/reperfusion, remodeling and heart failure. Heart Fail Rev 2011; 16(1): 49-69. Kukreja RC, Salloum F, Das A, Ockaili R, Yin C, Bremer YA, et al. Pharmacological preconditioning with sildenafil: basic mechanisms and clinical implications.
Vascul Pharmacol 2005; 42(5-6): 219-32. Li Y, Hou D, Chen X, Zhu J, Zhang R, Sun W, et al. Hydralazine protects against renal ischemia-reperfusion injury in rats. Eur J Pharmacol. 2019;843:199-209. Li Y, Xiong W, Yang J, Zhong J, Zhang L, Zheng J, et al. Attenuation of Inflammation by Emodin in Lipopolysaccharide-induced Acute Kidney Injury via Inhibition of Toll-like Receptor 2 Signal Pathway. Kidney Diseases 2015; 9(3): 202-8. Li XD, Yang YJ, Geng YJ, Zhao JL, Zhang HT, Cheng YT, et al. Phosphorylation of endothelial NOS contributes to simvastatin protection against myocardial no-reflow and infarction in reperfused swine hearts: partially via the PKA signaling pathway. Acta Pharmacol Sin. 2012; 33(7): 879-87. Magee LA, Cham C, Waterman EJ, Ohlsson A, von Dadelszen P. Hydralazine for treatment of severe hypertension in pregnancy: meta-analysis. BMJ 2003; 327(7421): 955-60. Matsui T, Li L, del MonteF, Fukui Y, Franke TF, Hajjar RJ, et al. Adenoviral gene transfer of activated phosphatidylinositol 30-kinase and Akt inhibits apoptosis of hypoxic cardiomyocytes in vitro. Circulation. 1999; 100(23): 2373-9. Matsui T, Tao J, del Monte F, Lee KH, Li L, Picard M, et al. Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo. Circulation. 2001; 104(3): 330-5. Morrow JD, Schroeder HA, Perry HM Jr. Studies on the control of hypertension by hyphex. II. Toxic reactions and side effects. Circulation. 1953; 8(6): 829-39. Pang L, Cai Y, Tang EH, Yan D, Kosuru R, Li H, et al. Cox-2 inhibition protects against hypoxia/reoxygenation-induced cardiomyocyte apoptosis via aktdependent enhancement of iNOS expression. Oxid Med Cell Longev. 2016; 2016: 3453059. Portt L, Norman, G, Clapp C, Greenwood M, Greenwood MT. Anti-apoptosis and cell survival: a review. Biochimica et biophysica acta 2011; 1813: 235-59. Rodrigues SF, de Oliveira MA, dos Santos RA, Soares AG, de Cássia Tostes R, Carvalho MH, et al. Hydralazine reduces leukocyte migration through different mechanisms in spontaneously hypertensive and normotensive rats. Eur J
Pharmacol. 2008; 589(1-3): 206-14. Rong Y, Distelhorst CW. Bcl-2 protein family members: versatile regulators of calcium signaling in cell survival and apoptosis. Annual review of physiology. 2008; 70: 73-91. Saikumar P, Dong Z, Patel Y, Hall K, Hopfer U, Weinberg JM, et al. Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury. Oncogene. 1998; 17(26): 3401-15. Salloum F, Yin C, Xi L, Kukreja RC. Sildenafil induces delayed preconditioning through inducible nitric oxide synthase-dependent pathway in mouse heart. Circ Res. 2003; 92(6): 595-7. Shanmuganathan S, Hausenloy DJ, Duchen MR, Yellon DM. Mitochondrial permeability transition pore as a target for cardioprotection in the human heart. Am J Physiol Heart Circ Physiol. 2005; 289(1): 237-42. Sun L, Chen C, Jiang B, Li Y, Deng Q, Sun M, et al. Grb2-associated binder 1 is essential for cardioprotection against ischemia/reperfusion injury. Basic Res Cardiol 2014; 109(4): 420. Taketomo CK HJ, Kraus DM. Pediatric & neonatal dosage handbook. American Pharmacists Association, Lexicomp 2013; 20. Tampe B, Steinle U, Tampe D, Carstens JL, Korsten P, Zeisberg EM, et al. Low-dose hydralazine prevents fibrosis in a murine model of acute kidney injury-to-chronic kidney disease progression. Kidney International 2017; 91(1): 157-176. Tampe B, Tampe D, Zeisberg EM, Müller GA, Bechtel-Walz W, Koziolek M, et al. Induction of Tet3-dependent epigenetic remodeling by low-dose hydralazine attenuates progression of chronic kidney disease. EBioMedicine 2015; 2(1): 19-36. Tampe B, Zeisberg M. Evidence for the involvement of epigenetics in the progression of renal fibrogenesis. Nephrol Dial Transplant. 2014; 29(suppl 1): 1-8. Tang CY, Lai CC, Huang PH, Yang AH, Chiang SC, Huang PC, et al. Magnolol Reduces Renal Ischemia and Reperfusion Injury via Inhibition of Apoptosis. The American Journal of Chinese Medicine. 2017; 45(7): 1-19.
Vergona RA, Agnello A, Hirkaler G, Urbano S, Rosenberger L. Protective effects of hydralazine in a renal ischemia model in the rat. Life Sci 1987; 41(5): 563-9. Xia Z, Li H, Irwin MG. Myocardial ischaemia reperfusion injury: the challenge of translating ischaemic and anaesthetic protection from animal models to humans. Br J Anaesth. 2016;117(2):44-62. Xing W, Yan W, Fu F, Jin Y, Ji L, Liu W, et al. Insulin inhibits myocardial ischemia-induced apoptosis and alleviates chronic adverse changes in post-ischemic cardiac structure and function. Apoptosis. 2009; 14(9): 1050-60. Xiong J, Xue FS, Yuan YJ, Wang Q, Liao X, Wang WL. Cholinergic anti-inflammatory pathway: a possible approach to protect against myocardial ischemia reperfusion injury. Chin Med J (Engl) 2010; 123(19): 2720-6. Yang J, Yang J, Ding JW, Chen LH, Wang YL, Li S, et al. Sequential expression of TLR4 and its effects on the myocardium of rats with myocardial ischemia-reperfusion injury. Inflammation. 2008;31(15):304-12. Yang SJ, Meng JP, Qu Y, Liu YB The progress on the signal transduction pathways of apoptosis. Chin J Comp Med. 2007;17:297-301. Yellon DM HD. Myocardial reperfusion injury. N Engl J Med 2007; 357: 1121-35. Zambrano P, Segura-Pacheco B, Perez-Cardenas E, Cetina L, Revilla-Vazquez A, Taja-Chayeb L, et al. A phase I study of hydralazine to demethylate and reactivate the expression of tumor suppressor genes. BMC Cancer 2005; 5: 44. Zhang KR, Liu HT, Zhang HF, Zhang QJ, Li QX, Yu QJ, et al. Long-term aerobic exercise protects the heart against ischemia/reperfusion injury via PI3 kinase-dependent and Aktmediated mechanism. Apoptosis. 2007; 12(9): 1579-88. Zhang X, Liang X, Lin X, Zhang S, Huang Z, Chen C, et al. Mechanism of the protective effect of Yulangsan flavonoid on myocardial ischemia/reperfusion injury in rats. Cell Physiol Biochem. 2014; 34(4): 1050-62. Zhu L, Wei T, Gao J, Chang X, He H, Luo F, et al. The cardioprotective effect of salidroside against myocardial ischemia reperfusion injury in rats by inhibiting apoptosis and inflammation. Apoptosis. 2015; 20(11): 1433-43.
Figure Legends Fig. 1 (A) Hydralazine decreased infarct size assessed by the TTC assay in I/R rats. Representative images from TTC staining are shown for Sham, I/R, Hyd 10mg/kg, Hyd 20mg/kg, Hyd 30mg/kg. (B) Infarct size of each rats were quantitatively analyzed (n=8). Red stain: viable area; White stain: infarct region; infarct size (%) = infarct region x 100 %/ (viable area + infarct region). Data were shown as mean ± S.D. * P < 0.05, ** P < 0.01 compared with sham group; # P < 0.05, ## P < 0.01 compared with I/R group. (C) Histopathological examination of heart tissue by H&E staining. Representative images from HE staining are shown for Sham, I/R, Hyd 10mg/kg, Hyd 20mg/kg, Hyd 30mg/kg. (B) Histological injury scores of the heart were shown (n=8). Fig. 2 Biochemical analysis of heart function and inflammatory markers. Biochemical analysis of CK-MB level (A), LDH level (B), IL-1β (C), IL-6 (D) and TNF-α (E) in serum was shown (n=8). All data were expressed as the mean ± S.D. Statistical significance:* P < 0.05 and ** P < 0.01 versus the Sham group, #P < 0.05 and ## P < 0.01 versus the I/R group, respectively. Fig. 3 Biochemical analysis of oxidative stress in hearts. The activity of SOD (A) and MDA (B) in serum was measured (n=8). (C) The reactive oxygen species levels in the hearts of rats from all groups were revealed by DHE staining of frozen sections. (D) The fluorescence intensity of DHE staining was analyzed using Image-Pro Plus (n=8). All data are expressed as the mean ± S.D. Statistical significance:* P < 0.05 and ** P < 0.01 versus the Sham group, #P < 0.05 and ## P < 0.01 versus the I/R group, respectively. Fig. 4 (A) Apoptosis was analyzed using in situ TUNEL fluorescence staining. The nuclei of TUNEL-positive (apoptotic) cells stained green. Five random fields per section (five sections per tissue from each mouse) were examined in each experiment (10x). (B) The numbers of TUNEL-positive myocardial cells (%) were compared among the four groups (10x, n=8). (C) TUNEL and CD31 immunofluorescence staining (10x). (D) TUNEL and sarcomeric immunofluorescence staining (10x). All data are expressed as the mean ± S.D. All data were expressed as the mean ± S.D. Statistical significance:* P < 0.05 and ** P < 0.01 versus the Sham group, # P < 0.05
and ## P < 0.01 versus the I/R group, respectively. Fig. 5 Apoptosis assay and expression of NO related signaling pathway proteins in the hearts. (A) Representative western blots depicting protein levels of p-PI3K, PI3K, p-AKT, AKT, eNOS, p-eNOS, Cleaved Caspase-3 and Cleaved Caspase-9 in heart tissue were shown. (B) The expression levels of proteins were quantitatively analyzed (n=8). All data were expressed as the mean ± S.D. Statistical significance:* P < 0.05 and ** P < 0.01 versus the Sham group, # P < 0.05 and ## P < 0.01 versus the I/R group, respectively. Fig. 6 Expression of eNO signaling pathway proteins in H/R-induced H9c2 injury pretreated with or without PI3K/Akt inhibitor WM (100 nmol/L). (A) Representative western blots depicting protein levels of p-PI3K, PI3K, p-AKT, AKT, eNOS and peNOS in heart tissue were shown. (B) The expression levels of proteins were quantitatively analyzed (n=8). WM: wortmannin. All data were expressed as the mean ± S.D. Statistical significance:* P < 0.05 and ** P < 0.01 versus the Sham group, # P < 0.05 and ## P < 0.01 versus the I/R group, respectively.