Nerolidol, a Sesquiterpene Alcohol, Attenuates Acute Myocardial Infarction in Rats

Cardiovascular diseases have a high morbidity and mortality rate and their treatment is not effective in reducing the damage caused by tissue reperfusion during an acute myocardial infarction (MI). This study aimed to investigate whether nerolidol (NRD), a sesquiterpene alcohol, would attenuate the MI in isoproterenol-treated rat model. MI was induced by the administration of two doses of ISO (100 mg/kg, i.p.) in an interval of 24 h. The animals were divided into 4 groups: control (CTR) (vehicle – saline NaCl 0.9% + TWEEN 80 0.2%), ISO (ISO + vehicle), ISO + NRD (NRD 50 or 100 mg/kg). Electrocardiogram, contractile parameters, cardiac enzymes, infarction size and antioxidant parameters in the heart were measured. ISO group showed a signicant rise in ST-segment, QTc and heart rate associated to a reduction of left ventricular developed pressure (LVDP), +dP/dt and –dP/dt. Increase in content of creatine kinase (CK), CK-MB, lactate dehydrogenase (LDH), TBARS and infarction size as well as fall in activities of superoxide dismutase (SOD) and catalase (CAT) were observed. NRD signicantly prevented almost all the parameters of ISO-induced MI mentioned above. Our results suggests that nerolidol has a signicant effect on the protection of heart through maintaining endogenous antioxidant enzyme activities.

. MI is a coronary arterial disease which has high level of morbimortality. It is characterized by partial or total necrosis of the heart caused by the death of myocardial cells. MI, or heart attack, begins with the occlusion of a coronary artery, and by consequence, the area which that artery supplies is deprived of oxygen leading to the death of those cells if the blood ow does not return soon (Piegas et al. 2015;Hu et al. 2017).
Nevertheless, the reestablishment of the blood ow after a period of ischemia can cause greater damage to the cardiac tissue, leading to a reperfusion lesion. This lesion result in several dysfunctional alterations, such as cellular edema, increased in ammatory process and oxidative stress (Berne et al. 2008; Yellon and Hausenloy 2009). Therefore, MI treatment consists in the restoration of the blood ow on the affected areas, and this restoration may lead even more complications, which is one of the major challenges to physicians and researchers (Yang 2018).
Natural products (NP) have been stood out for their wide pharmacological effects. Among the NP, the terpenes is one of the most studied classes of secondary metabolites produced by plants (Bakkali et al. 2008; Dewick 2009). They have been studied in several therapeutic areas, especially in the treatment of cardiovascular disorders (Santos et al. 2011;Silva et al. 2019). In this sense, nerolidol (3,7,11-trimethyl-1,6,10-dodecatrien-3-ol) (NRD), is a sesquiterpene alcohol that can be found in two isomers cis-and transnerolidol (Fig. 1). It can be found in several plants such as lavender, lemon grass, and ginger (Chan et al. 2016 Given the above, the aim of the present study was to evaluate the cardiovascular effects of NRD on the treatment of acute myocardial infarction (AMI) induced by isoproterenol in rats, its effects on cardiac lesion biomarkers and in the oxidative stress.

Animals
Adult male Wistar normotensive rats (250-300 g) were used for all the experiments. The animals were randomly housed in appropriate cages (258 cm 2 base and 17,8 cm high) at a controlled temperature (23 ± 2 °C) on a 12-h light/dark cycle (6:00 a.m. to 6:00 p.m.) with free access to food (Purina®, Sao Paulo, Brazil) and tap water. In addition, all efforts were made to minimize the number of animals used and any discomfort.
Myocardial infarction (MI) protocol MI was induced by two doses of (-)-isoproterenol (ISO) (100 mg/kg; i.p.) with a 24h-interval between each dose. After 30 min of the second dose of ISO, the animals were treated with NRD (50 or 100 mg/kg; i.p.).

Acquisition of the Electrocardiographic pro le
After MI induction (3º day), ECG was recorded in anesthetized animals (ketamine 80 mg/kg and xylazine 10 mg/kg; i.p.) by subdermal electrodes placed in the DII derivation (negative pole placed in the right and left superior thoracic region, and positive pole in the left inguinal region) connected to a cardioscope (TEB Electronics, São Paulo, Brazil). The electrical signals were ampli ed and digitalized (PowerLab 4/35 ADInstrument, EUA). The ECG signals were registered for 5 minutes and the LabChart 8 program (ADInstruments) was used for analysis. Measurements of heart rate (HR) corrected QT interval (QTc), QRS complex were used in all experimental groups. The QT interval was corrected normalized for rodents by HR using Bazett's modi ed formula (QTc = QT / √RR /f). Any abnormalities in the ST segment morphology for MI were also analysed (Preda and Burlacu 2010).

Determination of myocardial infarct size
After MI was induced, the animals were anesthetized with ketamine (80 mg/kg; i.p.) and xylazine (10 mg/kg; i.p.), and decapitated. The hearts were quickly removed, and the myocardial infarct size was measured using the 2,3,5-triphenyl tetrazolium tetrachloride (TTC) staining method. All hearts were sectioned in a transversal fashion in the median region to obtain a better exposure of the left ventricle. The cardiac apex region was placed in a Falcon tube with TTC 1% diluted in Krebs−Henseleit solution (composition in mM: NaCl 120, KCl 5.4, MgCl 2 1.2, NaHCO 3 27, CaCl 2 1.25, glucose 11, NaH 2 PO 4 2.0, pH 7.4) for 15 min at a 37 ºC water bath. Then, after staining, the area was scanned in high resolution and analysed with ImageJ software (free software ImageJ bundled with 64-bit Java 1.8.0_112, NIH, Bethesda, MD, USA).

Measurement of biochemical markers of tissue damage -Lactate Dehydrogenase (LDH) and Creatine Kinase Total and Myocardial Band (CK-TOTAL and CK-MB)
After experiments, the animals were euthanatized and decapitated, and the blood was collected. For serum obtainment, the blood was centrifuged at 3.500 rpm (Neofuge 15R, Heal Force, Shanghai, China) for 15 min at 4 ºC. The enzymatic essays were performed in triplicate using Labtest commercial dosing kit. All following sample analysis were performed on the LABMAX 240 Premium apparatus.

Measurement of Lipoperoxidation by the Thiobarbituric Acid (TBARS) Method
Malondialdehyde (MDA) is a known stress oxidative biomarker, one of the oxidation products of hydroperoxides of polyunsaturated fatty acids that are formed during lipoperoxidation process (Amara et al. 1995).
To perform TBARS experiments, the hearts were weighted and homogenized at the ration of 100 mg of tissue/mL of phosphate buffer (PBS 0.1 mol/L, pH 7.4). Next, the homogenates were incubated for 45 min at 90 ºC with a solution containing thiobarbituric acid (TBA 0.37%) in acid solution (15% trichloroacetic acid and 0.25 N hydrochloric acid). Samples were centrifuged at 14.000 rpm (Neofuge 15R, Heal Force) for 30 min at 4 °C, and the supernatant was mixed with n-butanol and saturated NaCl solution. The mixture was mixed in a vortex for 30 s and again centrifuged at 14.000 rpm for 2 min. Aliquots of the supernatant were pipetted into 96-well plates to read absorbance on a microplate reader (Biotek, ELx800 absorbance microplate reader, VT, USA) at 535 nm, correcting for absorbance values at 572 nm. The amount of MDA was expressed in nanomoles per grams of tissue (nmol/g) and was interpreted as a marker of lipid peroxidation formed by the reaction with TBARS (Esterbauer and Cheeseman 1990).

Measurement of Measurement of Protein Carbonylation
To measure the possible oxidative damage to proteins carbonylation was used, which is the formation of carbonyl groups in proteins by the reaction with 2,4-dinitrophenylhydrazine (DNPH) with reactive ketones or aldehydes to form hydrazones, which are formed from protein oxidation. The total carbonyl group concentration was expressed as nmol/mg protein (Levine et al. 1990).
Cardiac tissues were weighted and homogenized. Initially, 2 microtubes were prepared with DNPH (experimental) and control (white), 1 mg of supernatant protein was added in both microtubes and completed to 200 μl with PBS. Next, 200 μl of trichloroacetic acid (TCA) 20% was added and homogenized for 5 min, after was centrifuged at 4.000 rpm for 5 min. The supernatant was discarded, and the pellet was resuspended in 100 μl NaOH 0,2 mol/L.
Next was added 100 μl of hydrochloric acid (HCl) 2 M in the white tube and 100 μl of DNPH 10 mM in the sample following 100 μl of TCA 20 %, the tubes were centrifuged at 16.000 rpm for 5 min in triplicate. Thereafter, was added 500 μl of ethanol/ethyl acetate (1:1). The pellet was resuspended with 1 mL of urea 8 M (pH 2.3), centrifuged again at 16.000 rpm for 3 min. Finally, 200 μl was pipetted into 96-well ELISA plate to read at an absorbance from 360 to 385 nm.

Endogenous Antioxidant Enzyme Activities
Superoxide Dismutase Activity (SOD) SOD activity was measured as previously described (Madesh and Balasubramanian 1998). Cardiac tissue was homogenized in phosphate buffer (PBS, 50 mmol/L, pH 7.4) and centrifuged at 14.000 rpm for 30 min at 4 °C. The supernatant, PBS, tetrazolium (1.25 mmol), and adrenaline (60 mmol) were transferred to a microplate and shaken for 5 min. Thereafter, DMSO was added to the mixture, and read in a spectrophotometer (ELx 800, Biotek Instruments, VT, USA) was measured at 570 nm absorbance. SOD activity was expressed as U/μg protein.
Catalase Activity (CAT) CAT activity was measured following a protocol previously described (Nelson and Kiesow 1972)

Results
Nerolidol decrease ECG changes in a rat model of acute myocardial infarction ST-elevation and T-wave inversion are typical electrocardiographic changes observed in acute myocardial infarction (MI). Fig. 3a shows representative ECG records in the 4 experimental groups. It can be noted that in the IM group there was a ST-elevation characteristic of transmural ischemia. Elevation of ST was observed with less frequency in the MI groups IM + NRD 50 (50%) or IM + NRD 100 (33%) compared to the MI group (66%) (Fig. 3b). There was no change in the PR interval and duration of the QRS complex in analysed groups ( Fig. 3c and d). However, a signi cant increase in QTc and heart rate was observed in the MI group, which was prevented by treatment with NRD (100 mg/kg, p < 0.05) but not with NRD (50 mg/kg) ( Fig. 3e and f). Fig. 4a shows representative traces of left ventricular developed pressure (LVDP) in different experimental groups. As can be seen, isoproterenol administrated for two consecutive days promoted a marked reduction in LVDP in animals in the MI group that were fully prevented by treatment with the two doses of NRD evaluated (Fig 4a and b). Administration of NRD (50 or 100 mg/kg) alleviated the reductions in +dp/dt and -dp/dt induced by MI (p < 0.05, Fig. 2c and d). These ndings suggested that NRD could improve the cardiac function in rats submitted to MI.

Nerolidol ameliorates the level of CK, CK-MB and LDH in a rat model of acute myocardial infarction
The measurement level of CK, CK-MB and LDH (Fig. 5) were tested in this study to investigate whether the effects of NRD ameliorates cardiac function in MI. In MI group, the level of CK, CK-MB and LDH were signi cantly increased compared with the control group. Interesting, treatment with NRD at the dose of 50 mg/kg and 100 mg/kg signi cantly reduced the levels of these biochemical markers of tissue injury, compared to that in the isoproterenol-induced acute myocardial infarction group.

Nerolidol reduces infarct size in a rat model of acute myocardial infarction
In order to explore whether NRD ameliorates the infarct size in acute myocardial infarction rat model, infarct size was measurement in our study. Fig. 6a shows representative illustrations of infarction size as stained TTC in all experimental groups. While MI group indicated a large unstained area, the heart slice of MI + NRD exhibited a major portion stained positively showing tissue viability. As shown in Fig. 6b isoproterenol induced increase of infarct size (27.76 ± 1.51%, p < 0.05) in compared to control group (4.12 ± 0.54%). On the other hand, treatment with NRD (50 mg/kg or 100 mg/kg) declined the infarct size to 13.57 ± 1.09% and 3.57 ± 0.53%, respectively, compared to MI group.

Nerolidol ameliorates oxidative stress in a rat model of acute myocardial infarction
To evaluate whether the cardioprotection mediated by NRD on the MI was associated with the improvement of oxidative stress, it was veri ed in heart samples in all experimental groups the levels of TBARS, CAT and SOD activity, SH and carbonyl groups. MI signi cantly increased the levels of MDA (Fig.  7a) and suppressed the levels of SOD (Fig. 7b). Notably, NRD treatment signi cantly reversed these changes. CAT activity was reduced in MI group but the NRD treatment was not able to reverse this change (Fig. 7c). The results showed that there was no change in the SH and carbonyl group induced by MI ( Fig.   7d and e). However, the MI groups treated with 50 and 100 NRD showed a reduction in carbonyl group compared to MI group. One of the criteria used to diagnosis of MI is ECG-abnormalities such as ST-segment elevation and Twaves inversion (Coppola et al. 2013). ST-segment elevation is generally a sign of transmural infarct and occurs in the majority of acute MI that are caused by complete blockage of a coronary artery or in isoproterenol-induced myocardial infarction in rat (Klabunde 2017). T-waves inversion occurs in myocardial ischemia that shortens the action potential duration of cells, which results in repolarization occurring earlier than normal. Treatment with NRD (100 mg/kg) markedly reduced isoproterenol-induced ST-segment elevation. QTc prolongation on ECG is a marker of delayed electrical repolarization and is risk factor for occurrence of ventricular arrhythmias and all-cause mortality. Another nding was that animals treated with isoproterenol showed an increase in heart rate by activation of β-adrenergic receptors and is responsible for augmented oxygen consumption contributing for myocardial necrosis (Rona 1985;Soraya et al. 2012). However, this increase in QTc and heart rate were not observed in the animals treated with NRD at 100 mg/kg. Short-term administration of NRD strongly prevented electrocardiographic alterations in the ECG, indicating its protective effects on cell membrane function.

Discussion
Isoproterenol administration induced signi cantly decreased of LVDP, +dP/dt and -dP/dt. Interestingly, treatment with the two doses of NRD improved all the evaluated parameters preventing the left ventricular dysfunction induced by isoproterenol. It was reported that nerolidol showed ability to reduce the contractile response of guinea pig left atrium probably by reduction calcium in ux in cardiomyocyte (Vasconcelos et al. 2018). ISO-induced myocardium dysfunctions are mediated by an increased level of intracellular Ca 2+ , through L-type calcium channels (Singh et al. 2001). Thus, drugs that promote negative inotropism, such as L-type calcium channel blocker, has signi cantly diminished isoproterenol cytotoxicity and preserved the ultrastructural architecture of the ventricle tissue of rats (Hassan et al. 2016). These results suggest that NRD may be acting as a calcium blocker to induce its protective effects on the heart.
Cardiomyocyte contains a high concentration of marker enzymes (CK, CK-MB and LDH) and in the face of MI these enzymes are released into the extracellular uid and were observed in the plasma of ISOinduced rats. Notably, in our study, administration of NRD at doses of 50 and 100 mg/kg signi cantly lowered the levels of marker enzymes in ISO-induced rats. Another study reported that prolonged pretreatment with NRD at different doses (100 and 200 mg/kg) for 21 days prevented the increase of cardiac and hepatic marker enzymes induced by ISO (Asaikumar et al. 2019).
Furthermore, another important nding of this study is that the NRD administration resulted in a remarkable cardioprotection against the early complications of myocardial infarction such as observed by reduction of infarction size.
It is reported that increases in the ROS production and/or depletion of the antioxidants in the defence system result in necrotic lesions in the heart of ISO-induced rats. Free radicals produced can react with polyunsaturated fatty acids in cell membrane leading to lipid peroxidation increasing the free radical production. In ISO-induced rats was reported that the concentrations of TBARS was increased in the plasma and heart. Furthermore, activities of enzymatic antioxidants such as superoxide dismutase (SOD) and catalase (CAT) in heart tissue were decreased in ISO-induced rats. However, NRD was able to signi cantly increase the SOD activity. Decreasing in concentrations of TBARS and increase of SOD activity in animal treated with NRD showed that it exert antioxidative effects important to minimizes the myocardial injury induced by isoproterenol.
Taken together, the ndings of our study demonstrate that nerolidol exert antioxidative effects by suppressing myocardial tissue oxidative stress and enhancing the antioxidative defence system in a rat model of acute myocardial infarction. Other cardioprotective effects offered by nerolidol are observed by prevention of contractile and ECG alterations on the isoproterenol-induced myocardial infarction. This study provides a novel strategy of cardioprotection mediated by nerolidol, which may be helpful in     Nerolidol (NRD) ameliorates the level of CK-total, CK-MB and lactate dehydrogenase (LDH) induced by isoproterenol-induced myocardial infarction (MI) in rats. a CK-total, b CK-MB and c LDH (n = 3-5). *p < 0.05 vs. Control; #p < 0.05 vs. MI. One-way ANOVA followed by Tukey's post-test. Figure 6 Nerolidol (NRD) reduces the infarct size induced by isoproterenol-induced myocardial infarction (MI) in rats. a Representative images of heart slices stained with TTC in control (a), MI (b), MI + 50 mg/kg NRD (c) and MI + 100 mg/kg NRD, b Infarct size (n = 3-5). *p < 0.05 vs. Control; #p < 0.05 vs. MI; €p < 0.05 NRD 50 vs. NRD 100. One-way ANOVA followed by Tukey's post-test. Figure 7