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Abstract

This study aimed to investigate the antihyperlipidemic and anti-inflammatory effect of zingiberene (ZBN) on isoproterenol-(ISO) induced myocardial infarction in rats. ZBN (10 mg/kg b.wt.) was orally administered to rats for 21 days and ISO (85 mg/kg b.wt.) was subcutaneously injected into the rats at 24 h intervals for the last 2 consecutive days. We observed increased serum creatine kinase, creatine kinase-MB, cardiac troponin T, and I levels in ISO-treated MI rats. Conversely, ZBN oral administration significantly prevented in cardiac marker enzyme activities in ISO-mediated rats. We also noticed that ZBN oral administration prevented ISO-induced expression of lipid peroxidative markers, total cholesterol, triglycerides, phospholipids, free fatty acids, very-low-density lipoprotein cholesterol (VLDL-C), low-density lipoprotein cholesterol (LDL-C) to the normal basal level. Furthermore, ZBN restored ISO-mediated antioxidant status, increased level of high-density lipoprotein cholesterol (HDL-C), and tissue phospholipids to the near-normal levels. Besides, ZBN pre-treatment significantly reduced the level of inflammatory markers (TNF-α, IL-6, NF-κB, and IL-1β) in ISO-induced MI in rats. We noticed that ZBN pretreatment inhibited the pro-apoptotic proteins Bax and cytochrome c and increased the Bcl-2 expression in ISO induced rats. The gene expression profiling by qRT-PCR array illustrates that ZBN treatment prevents the ISO mediated activation of cardiac markers, inflammatory, and fibrosis-related genes in the heart tissue. Taken together, pre-treatment with ZBN attenuated ISO-induced MI resolved exhibits the anti-inflammatory and antiapoptotic effect.

Keywords

Zingiberene isoproterenol cardiac marker enzymes anti-apoptotic anti-inflammatory

Introduction

Myocardial infarction (MI) is acute necrotic damage to the heart muscles which might be the major reason for mortality in the developed countries.¹ Ischemic heart disease is currently at the top of the mortality list. As per 2016 statistics, about 2,303 deaths occur every day due to cardiovascular diseases.² Sudden blockage of coronary arteries minimizes a portion of myocardial tissue from the constant blood supply. A most common phenomenon of MI is the development of coronary atherosclerosis that has strongly been associated with hyperlipidemia, oxidative changes, and inflammatory signaling.³ The lifestyle factors mainly nutrition plays an important role in the development of MI. Experimental studies illustrate the enlarged infarct size during ischemia in rat models.^4,5 Epidemiological studies indicate that an increased risk of coronary heart disease (CHD) is associated with high levels of serum total cholesterol, triglyceride, and decreased levels of high-density lipoprotein (HDL).^6
–8

Cardiac markers are enzymes or factors released to the bloodstream due to the necrotic death of cardiac myocardial tissue. Myoglobin, creatine kinase-MB, troponin I/T are the major gold standard cardiac markers, and analyzing these markers in the serum or plasma might be useful during MI.⁹ Isoproterenol hydrochloride (ISO) is a β-adrenergic agonist that causes necrotic damage in the heart tissue.¹⁰ ISO generates free radicals which may be an underlying factor for irreversible damage to the myocardial membrane.¹¹ Reactive oxygen species (ROS) induces several biochemical events associated with atherogenesis. ISO has been reported to increases lipids such as total cholesterol, triglycerides (TGs), free fatty acids (FFAs), and phospholipids (PLs) in circulation.¹² It also increases the levels of low-density lipoprotein cholesterol (LDL-C) in the blood which causes the buildup of harmful lipid deposits in the blood vessels.¹³

MI-mediated cellular damages are implemented by manly signaling molecules. Activation NF-κB has been reported during ISO-induced MI.¹⁴ NF-κB has been considered as a key regulator in the condition of inflammation and fibrosis. The ISO administration induces phosphorylation of NF-κB which initiates the intracellular signaling cascade that resulted in the overexpression of pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β as well as cardiac-specific markers.¹⁵ The matrix metalloproteinases specifically MMP-2 and MMP-9 play a major role in the degradation of myocardial fibrillar collagen.¹⁶ The ISO treatment induces acute proteolytic activity thereby induces myocyte loss and finally causes MI and heart failure. The hypoxia-induced angiogenesis takes place in ISO-treated cardiac tissues. Inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) play an important role in reducing the infarct size during ISO mediate MI.¹⁷ The iNOS-derived NO and VEGF were reported to promote survival of ischemic tissue by stimulating angiogenesis.¹⁸ Apoptotic cell death is a major contributor for MI and acute heart failure. The ISO administration activates proapoptotic protein such as Bax and Bad resulted in the instability of outer mitochondrial membrane which causes the release of cytochrome-c into the cytosol. In the cytosol, cytochrome c activates caspase-9, which cleaves and activates caspase-3, causing the induction of apoptosis.¹⁹

The regular consumption of antioxidant phytochemicals is associated with the prevention of atherogenesis and ischemia. The bioactive secondary metabolites present in the medicinal plants were endowed with potential pharmacological properties. It has been postulated that the antioxidant nature of the phytochemicals scavenges ISO-induced free radicals formation thereby terminates subsequent heart injury.²⁰ Treatment with phytochemical formulations shows attenuated inflammation-related (TNF-α, IL-6, MMP-9) and oxidative stress-related (ox-LDL, MDA, SOD) indicators in CHD patients.²¹ Sesquiterpenes are a group of terpenoids modulates ISO-induced lipid peroxidation, mitochondrial dysfunction and histological alterations.^22,23 Zingiberene (ZBN) is a monocyclic sesquiterpene, mainly present in ginger with oil content (Zingiber officinale). It can contribute up to 30% of the essential oils in ginger rhizomes.²⁴ ZBN has several pharmacological properties such as antioxidant,²⁵ anticancer,²⁶ antiulcer,²⁷ antibacterial,²⁸ and antiviral²⁹ effect. ZBN possess the ability to modulate the inflammatory PI3K/AKT/mTOR pathway in human cells.³⁰ Although the ginger and its active compound ZBN have well been investigated for various pharmacological properties the anti-inflammatory and antiapoptotic effect of ZBN in ISO-induced myocardial infarction has not yet been revealed. Therefore, we assessed the preventive effect of ZBN on ISO-induced cardiac serum markers, lipid peroxidative markers, antioxidant status, lipid profile, inflammatory markers, and apoptotic markers in MI rats.

Materials and methods

Chemicals

ISO (catalog No. 15627) was obtained from Sigma Aldrich (St Louis, Missouri, USA). ZBN (Catalog No. 495-60-3) was purchased from Guidechem Chemical Network (China). All the primary antibodies such as Bax (SC-20067), Bcl-2 (SC-7382), Cytochrome c (SC-13156), Caspase-3 (SC-56053) and β-actin (SC-8432) were purchased from Santa Cruz Biotechnology, USA. TNF-α (ab236712), IL-6 (ab234570), NF-κB (ab176647), and IL-1β (ab255730) ELISA kits were procured from Abcam Scientific Company, USA. All the other chemicals of analytical grade used in the study.

Myocardial infarction and treatment plan

Wistar albino rats (males) with the bodyweight of 160–180 g were used for MI experiments. The MI induction and treatment protocol was approved by the Institutional Animal Ethical Committee. The ZBN was dissolved in 0.05% DMSO and ISO-hydrochloride were dissolved in physiological saline. ZBN was given oral administration for 21 consecutive days. The experimental dose of ZBN was selected by acute toxicity studies (Table 1). MI was induced by injection of 85 mg.kg bw (s.c.) ISO on the 20th and 21st days. The dose of ISO used for MI induction was selected as per the previously published literature.^19,31 The rats randomly divided into four experimental groups with six rats (n = 6) in each group. The animals were given food and water ad libidum during the experimental period. The animals were maintained normal dark and light cycle.

Group I: Control rats (received 0.5% DMSO s.c., for 21 days).

Group II: Control+ ZBN (10 mg/kg BW, s.c., for 21 days).

Group III: ISO control (85 mg/kg BW, s.c., for 20th and 21st day).

Group IV: ZBN (10 mg/kg BW, s.c., for 21 days) + ISO (85 mg/kg BW, s.c., for 20th and 21st days).

Table 1.

Effect of various concentrations of ZBN on the acute toxicity study of male albino Wistar rats.

	Mortality on different days at drug treatment
ZBN (mg/kg b.wt)	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	% Mortality	Survivors/total
5																						0	6/6
10																						0	6/6
20																						0	6/6
50																						0	6/6
80																						0	6/6
100																						0	6/6
200																						0	6/6
300																						0	6/6
400																						0	6/6
500															1							16.66	1/6
800				1														1				33.33	2/6
1000		1								1											1	50	3/6

Different doses of ZBN (5–1000 mg/kg. b.wt.) were administered to rats and mortality for 21 consecutive days.

After 24 h of the last ISO and/or ZBN treatment, the rats were anesthetized using ketamine hydrochloride (24 mg/kg b.wt.) and were sacrificed by cervical dislocation.

Assessment of acute toxicity of ZBN in male albino Wistar rats

The acute toxicity study of ZBN was determined according to Begum et al.³² Briefly, the animals were allowed to fast by withdrawing the food and water for 18 hours. The fasted animals were divided into differenet groups of six each. Each group of animals were given different doses (5, 10, 20, 50, 80, 100, 200, 300, 400, 500, 800 and 1000 mg/kg b. wt.) of freshly prepared ZBN. The animals were provided with food and water immediately after the drug administration. The animals were observed continuously for 21 days, for any signs of morbidity, mortality and behavioral toxicity.

Analysis of cardiac markers in serum

The measurement of cardiac markers in serum plays a major role in the early diagnosis of MI. In this study, we measured the activity of creatine kinase-MB (CK-MB) and Creatine kinase (CK) during ISO and/or ZBN by the methods described by Okinaka et al.³³ Troponins are released to the bloodstream during MI. We measured the serum levels of cTn T and cTn I using commercial ELISA kit.^34,35 We strictly followed the methods and calculations as per the manufacturer’s protocol.

Measurement of lipid peroxidative markers in plasma and heart tissue

The ISO-treatment induces lipid peroxidation and elevates the levels of peroxidative products like TBARS and lipid hydroperoxides (LHP) in the bloodstream. We measured the plasma and heart TBARS levels during ISO and/or ZBN treatment the method of Niehaus and Samuelsson.³⁶ We measured plasma and cardiac tissue lipid hydroperoxides (LHPs) were carried out as per the method of Jiang et al.³⁷

Estimation of intracellular antioxidant defense system

The ISO administration severely hampers the intracellular antioxidant status. We measured the levels of reduced glutathione (GSH) and activities of catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) during ISO and/or ZBN was estimated by the following methods^38

–41 in plasma, erythrocytes, and heart tissue.

Estimation of lipids in plasma and heart tissue

We extracted myocardial tissue lipids and plasma lipids as per the protocol described by Folch et al.⁴² after the ISO and/or ZBN treatment. Then, we analyzed the levels of TC, TG, FFA, and PL by the protocols of Allain et al.,⁴³ McGowan et al.,⁴⁴ Falholt et al.,⁴⁵ and Zilversmit and Davis⁴⁶ respectively. Plasma high-density lipoprotein-cholesterol (HDL-C) was estimated by the method of Izzo et al.⁴⁷ and low-density lipoprotein-cholesterol (LDL-C), very low-density lipoprotein cholesterol (VLDL-C) in the plasma was calculated by Friedwald’s⁴⁸ formula. Then, we calculated LDL-C and VLDL-C fractions as per the following formula: VLDL-C = TG/5 and LDL-C = TC − (HDL-C + VLDL-C).

Determination of inflammatory markers

The inflammatory proteins were the prominent markers to understand the ISO-induced MI. We measured the serum level of TNF-α, IL-6, NF-κB, and IL-1β during ISO and/or ZBN treatment by ELISA kits procured from Abcam Scientific Company. We followed the methodology and calculations as per the manufacturer’s protocol. Briefly, 50 µL of standard or experimental sample was added to designated wells. Then, 50 µL of antibody cocktail was added to all the wells. Then, the plates were incubated at room temperature for 1 hour. After washing with wash buffer 100 µL of TMB substrates were added to each well and incubated for 10 minutes. After the addition of a 100 µL stop solution the color developed was read OD at 450 nm using Tecan Multimode Reader.

Protein expression by western blot analysis

Protein expression studies were done by the western blotting technique by standard protocol.¹⁹ Pro and anti-apoptotic markers such as Bax, Bcl-2, cytochrome c, and caspase-3 were examined by western blotting. The proteins were extracted from heart tissue by RIPA buffer contained cocktail protease enzyme. The proteins were located to SDS-PAGE (10%) and the gel was reallocated to the membrane (PVDF). Then the membrane was blocked with bovine serum albumin (5%) for 2 hrs at room temperature. Then, membranes were incubated with primary antibodies (Bax, Bcl-2, cytochrome c and caspase-3) luminescence underlying substance, and band illumination were analyzed by software image J.

Analysis of inflammatory, angiogenesis and fibrotic related gene expression by a qRT-PCR array

The total RNA content was isolated from control, ISO control, ISO plus ZBN treated heart tissues using an RNeasy mini kit, and used for qRT-PCR arrays. The purity of the isolated RNA was measured by Nanodrop spectrophotometer. The cDNA was reverse transcribed using a First Strand cDNA Synthesis Kit and used for PCR amplification by SYBR green chemistry using the Qiagen kit. Briefly, the total of 25 µl of reaction mixture contains 100 ng of RNA (100 ng), primers (2.5 μl), PCR master mix (12.5 μlof SYBR Green) and reverse transcriptase enzyme (0.25 μl). The relative expression pattern of cardiac markers (CK, CKMB, cTnT, cTnI), transcription factors (NF-κB), proangiogenic and fibrotic related molecules (TNF-α, IL-6, IL-1β, p-eNOS, iNOS, MMP-2, MMP9), growth factors (VEGF) and signaling molecules (AKT) were analyzed by a qRT-PCR array. The fold changes of gene expression were calculated as per 2^{^(−ΔΔCt)} and plotted as cluster grams. The heat map was developed by clustering the normalized signal values. The heatmap was produced using Cluster/Treeview software. The GAPDH was used as internal control.

Statistical analysis

The experimental data was generally expressed as mean ± standard deviation (SD) unless otherwise stated. We used SPSS® software platform for the comparison of mean values between experimental groups. We employed One Way ANOVA followed by DMRT was used to analyze the statistical significance between control, ZBN, ISO, and ISO plus ZBN groups. The level of significance was considered as p < 0.05.

Results

Effect of various concentrations of ZBN on the acute toxicity study of male albino Wistar rats

The administration of different concentration of ZBN (5, 10 20, 50, 80, 100, 200, 300 and 400 mg/kg b.wt.) has not induced mortality during the 21 days observation period. However, 16.6% and 33.33% animals died when the ZBN dose was raised to 500 and 800 mg/kg b.wt. A further increase in the dose ZBN (1000 mg/kg.b.wt.) resulted in 50% mortality. The concentration between 5 to 400 mg/kg b.wt of ZBN produce no mortality. In this study, we used just 10 mg/kg b.wt. ZBN for myocardial infarctions study (Table 1).

Effect of ZBN on ISO-induced serum cardiac marker enzymes

Figure 1(a) and (b) show the effect of ZBN treatment on the activities of CK, CK-MB, cTn T, and cTn I. ISO treatment significantly increased the activities of these cardiac marker enzymes. Conversely, a significant decrease in the activities of CK, CK-MB cTn T, and cTn I was observed in ZBN plus ISO-treated rats. The ZBN alone treatment does not alter the activities of cardiac markers when compared to the sham control p-value ≤ 0.05 (DMRT).

Figure 1.

Effect of ZBN on ISO-induced serum cardiac marker enzymes. (a) Effect of ZBN on CK and CK-MB in the serum of control and ISO-induced rats. (b) Effect of ZBN on cTn T and cTn I in the serum of control and ISO-induced rats. Values are given as means ± S.D (n = 6). Values not sharing a common marking superscript (a, b, c) are different significantly at p value ≤ 0.05 (DMRT).

Effects of ZBN on ISO-induced lipid peroxidation in plasma and heart tissue

Rats induced with ISO showed increased levels of lipid peroxidation products such as TBARS and LOOH levels when compared to the sham control group. Both, the heart tissue and rat plasma showed increased lipid peroxidative products during ISO-administration. However, ZBN administration significantly reduced ISO-induced TBARS and LOOH as compared to MI alone induced group. The ZBN alone treatment does not alter the levels of these lipid peroxidative markers when compared to the sham control (Table 2).

Table 2.

Effect of ZBN on the levels of TBARS and LOOH in the plasma and heart of experimental rats.

Groups	Plasma (mg/dL)		Heart (mmol/100 g wet tissue)
Groups	TBARS	LOOH	TBARS	LOOH
Control	0.17 ± 0.02^a	7.24 ± 0.92^a	0.61 ± 0.05^a	50.39 ± 6.95^a
ZBN control (10 mg/kg b.wt.)	0.16 ± 0.03^a	7.19 ± 1.02^a	0.60 ± 0.04^a	51.61 ± 4.04^a
ISO (85 mg/kg b.wt.)	0.38 ± 0.04^b	15.65 ± 1.92^b	1.50 ± 0.18^b	98.50 ± 7.72^b
ZBN (10 mg/kg b.wt.) + ISO (85 mg/kg b.wt.)	0.21 ± 0.02^a	8.78 ± 0.09^c	0.74 ± 0.09^c	64.08 ± 8.03^c

Values are expressed as means ± S.D for six rats in each group. Values not sharing a common superscript (a, b, c) differ significantly at p ≤ 0.05 (DMRT).

Effects of ZBN on ISO induced antioxidant status

We measured the antioxidants enzyme status in the blood erythrocytes and the reduced GSH levels in the plasma. Further, we also analyzed these antioxidants status in the heart muscles of experimental rats (Tables 3 and 4). Animals treated with ISO had drastically reduced in the activity of enzymatic antioxidants such as SOD, catalase, and GPx and decreased the GSH levels when compared to the untreated control group. Whereas, ZBN pretreatment prevented the ISO-induced loss of antioxidant enzymes. The ZBN treatment alone does not alter the antioxidants status in the heart tissue as well as in the rat plasma when compared to the sham control p-value ≤ 0.05 (DMRT).

Table 3.

Effect of ZBN on the activities of SOD, catalase, GPx in erythrocytes and GSH levels in the plasma of experimental rats.

Parameters	Control	ZBN control (10 mg/kg b.wt.)	ISO (85 mg/kg b.wt.)	ZBN (10 mg/kg b.wt.) + ISO (85 mg/kg b.wt.)
SOD (U^$/mg of Hb)	7.35 ± 0.80^a	7.30 ± 0.68^a	5.08 ± 0.42^b	7.20 ± 0.73^a
Catalase (U^$$/mg of Hb)	178.26 ± 12.59^a	176.67 ± 16.62^a	131.89 ± 11.09^b	168.32 ± 18.02^a
GPx (U*/mg of Hb)	12.95 ± 1.31^a	12.79 ± 1.04^a	09.28 ± 0.70^b	12.68 ± 1.35^a
GSH (mg/dL)	39.14 ± 4.22^a	38.95 ± 4.25^a	20.39 ± 2.18^b	35.17 ± 4.11^c

Values are expressed as means ± S.D for six rats in each group. Values not sharing a common marking superscript (a, b, c) are different significantly at p value ≤ 0.05 (DMRT). U^$ = enzyme required to scavenge the chromogen formed by 50% in 1 min. U^$$ = µmole of hydrogen peroxide decayed per min. U* = µmole of GSH required/min.

Table 4.

Effect of ZBN on the activities of SOD, catalase, GPx and the levels of reduced GSH in the heart of experimental rats.

Parameters	Control	ZBN control (10 mg/kg b.wt.)	ISO (85 mg/kg b.wt.)	ZBN (10 mg/kg b.wt.) + ISO (85 mg/kg b.wt.)
SOD (U^$/ mg protein)	6.98 ± 0.78^a	6.95 ± 0.82^a	4.26 ± 0.48^b	6.38 ± 0.56^c
Catalase (U^$$/mg protein)	43.17 ± 3.17^a	44.50 ± 6.07^a	28.46 ± 2.09^b	39.59 ± 3.93^c
GPx (U*/mg protein)	6.61 ± 2.31^a	6.58 ± 1.04^a	4.53 ± 0.53^b	6.19 ± 0.46^c
GSH (µg /mg protein)	5.04 ± 0.54^a	5.10 ± 0.62^a	3.08 ± 0.25^b	4.95 ± 0.39^c

Effect of ZBN on ISO-induced lipid profile and lipoproteins in plasma and heart tissue

We measured the lipid profile and lipoprotein status in the plasma of experimental rats. We observed that the ISO-administration elevated the levels of TC, TG, FFA, PL, LDL-C, and VLDL-C and decreased the levels of HDL-C in the blood plasma. Conversely, the ZBN pretreatment prevented ISO-mediated circulating lipoproteins levels and significantly (p-value ≤ 0.05 (DMRT) brought back to near-normal levels (Figure 2(a) and (b)).

Figure 2.

Effect of ZBN on ISO-induced lipid profile and lipoproteins. (a) Effect of ZBN on the levels of TC, TG, FFA and PL in the plasma of control and ISO-induced rats. (b) Effect of ZBN on the levels of lipoproteins in the plasma of control and ISO-induced rats. Values are given as means ± S.D (n = 6). Values not sharing a common marking superscript (a, b, c) are different significantly at p-value ≤ 0.05 (DMRT).

We also measured the levels of lipids in control and ISO-induced rats (Figure 3). The ISO-treatment elevated TC, TG, FFA levels accompanied by a decrease in PL levels. Conversely, ZBN pre-treatment prevented ISO-induced TC, TG, and FFA levels to near normal (p-value ≤ 0.05 (DMRT). The ZBN alone treatment does not alter the lipids and lipid peroxidation parameters in the plasma and heart tissue.

Figure 3.

Effect of ZBN on the levels of TC, TG, FFA and PL in the heart tissue of control and ISO-induced rats. Values are given as means ± S.D (n = 6). Values not sharing a common marking superscript (a, b, c) are different significantly at p-value ≤ 0.05 (DMRT).

Effect of ZBN on inflammatory markers level in the serum

Inflammation is the primary event during ISO-mediated MI development. We measured the serum levels of inflammatory markers such as TNF-α, IL-6, NF-κB, and IL-1β in the experimental rats. The ISO administration significantly increased the levels of these inflammatory markers when compared to control rats (p-value ≤ 0.05 (DMRT). Whereas, ZBN pre-treatment significantly prevented ISO-induced activation of the TNF-α, IL-6, NF-κB, and IL-1β levels in the rat serum (Figure 4). The ZBN treatment alone does not alter the expression of the inflammatory markers in the serum.

Figure 4.

Effect of ZBN on inflammatory markers level in the serum of control and ISO-induced rats. Values are given as means ± S.D (n = 6). Values not sharing a common marking superscript (*, **) are different significantly at p-value ≤ 0.05 (DMRT).

Effect of ZBN on ISO-induced apoptotic markers expression by western blot analysis

We isolated total proteins from the heart tissue upon completion of the experimental period. The ISO treatment significantly upregulated the expression of pro-apoptotic proteins such as Bax and cytochrome c in the heart tissue when compared to the sham control group. Conversely, the anti-apoptotic protein Bcl-2 was found to be down-regulated in ISO-administered rats. The pretreatment with ZBN significantly reversed the ISO-mediated apoptotic markers expression in heart tissue. No changes in the expression pattern of apoptosis markers were observed in sham control and ZBN alone treated rats (Figure 5(a) and (b)).

Figure 5.

Effect of ZBN on Bax, Bcl-2, cytochrome c and caspase-3 protein expressions in the heart tissue of experimental rats. (a) Bax, Bcl-2, cytochrome c and caspase-3 protein expressions by western blot analysis. Lane 1: control; 2: ZBN control; 3: ISO control; 4: ZBN + ISO. (b) Band intensity scanned by the densitometer. The graph depicts quantitation of three independent experiments (means ± S.D), with data normalized by the control group with Bax, Bcl-2, cytochrome c and caspase-3 as unit 1. Values not sharing a common superscript (*, **) differ significantly at p ≤ 0.05 (DMRT).

Effect of ZBN on ISO-induced gene expression by PCR array

We analyzed the transcriptional profile of target genes in the heart tissue of experimental animals. The relative mRNA expression pattern (RQ) of cardiac markers (CK, CKMB, cTnT, cTnI), transcription factors (NF-κB), proangiogenic and fibrotic related molecules (TNF-α, IL-6, IL-1β, p-eNOS, iNOS, MMP-2, MMP-9), growth factors (VEGF) and signaling molecules (AKT) were found to be overexpressed in the ISO-administered heart tissue when compared to the sham control. Conversely, the ZBN treatment prevented the ISO-mediated overexpression of inflammation, angiogenesis, and fibrosis-related gene expression toward a normal level (Figure 6(a) and (b)). The ZBN treatment alone does not alter the transcriptome pattern of inflammatory markers when compared to sham control.

Figure 6.

Effect of ZBN on ISO-induced inflammatory, angiogenesis and fibrotic related gene expression by qRT-PCR array. (a) Hierarchical clustergram analysis of PCR array results for inflammatory, angiogenesis and fibrotic related gene expression in ZBN and ISO-induced rats. Bright red indicates the highest normalized signal values, bright bluerepresents the lowest signal values and white and light blue represents median signal values. The total mRNA was isolated from heart tissues and were detected using custom PCR array following the manufacturer’s instructions. (b) Relative gene expression levels of inflammatory, angiogenesis and fibrotic related gene expression in ISO and ZBN + ISO treated rats. The fold changes of gene expression were calculated as per 2^{^(−ΔΔCt)}.

Discussion

In this study, we noticed that ZBN prevented ISO-induced myocardial infarction in experimental rats. The subcutaneous ISO treatment conveniently induces myocardial injury and cardiac tissue dysfunction in the mice heart. Therefore, researchers widely employ ISO-treated animals for testing preventive effects of therapeutics.⁴⁹ The ISO administration induces the release of cardiac-specific inflammatory biomarkers into the bloodstream. The creatine kinases are generally present in the cellular partition and outflow into circulation in cardiac damage leads to degeneration of contractile component.⁵⁰ Further, the cardiac-specific markers like troponins T and I are highly specific and sensitive markers for cardiac diseases and also the desired markers for the diagnosis of MI.⁵¹ Measurement of serum concentration of cardiac markers during ISO treatment might be a useful approach for pharmacological studies as well as for drug discovery purposes.⁵² In this study, we found that there were elevated levels and activities of CK, CK-MB, cTnT, and I in MI rats. Conversely, pretreatment with ZBN significantly attenuated the actions of CK and CK-MB and decreased the levels of cTn T and I when compared to control rats. These results might be due to its potent efficacy in regulating membrane reliability by regulating the leakage of the cardiac enzymes into the flow. Ansari et al. reported that the ethanolic extract of Zingiber officinale pretreatment had a significant cardioprotective effect and maintains myocardial membrane integrity in ISO-induced in rats.⁵³

Lipid peroxidation has been considered as a major pathogenic process in cardiac injury.¹⁹ The ISO administration in the cellular milieu generates quinone intermediates which subsequently generates highly reactive oxidative free radicals.⁵⁴ In this current study, we found augmented the levels of lipid peroxidation products (TBARS and LOOH) in ISO caused cardiotoxicity in rats due to increased free radical-mediated membrane destruction. Whereas, pretreated with ZBN reduced the levels of lipid peroxidation products in ISO induced MI rats due to its potent antioxidant activities. Thus, these results suggested that ZBN scavenges excessive free radical production by inductions of ISO rats as compared to untreated control rats. Similarly, the ZBN scavenges H₂O₂ induced oxidative changes in the neuronal cells through its antioxidative nature.²⁶ This protective nature of ginger phytochemicals against oxidative damages was further validated by maintaining normal levels of antioxidant enzymes.⁵⁵

The enzymatic antioxidants like SOD converted into superoxide to H₂O₂, Catalase and GPx are accountable for converting hydrogen peroxide to H₂O.⁵⁶ The levels of GSH is an essential non-enzymatic antioxidant, which has straightly countered the free radicals and also it donates electron for the reduction of peroxides to the enzymatic reaction catalyzed by GPx.⁵⁷ Furthermore, our findings suggested that the declined the activities such as SOD, Catalase, GPx and GSH level indicating cardiac injury by ISO-induced rats of heart, erythrocytes, and plasma. These results attained that the ZBN acts as a strong antioxidant mediator when induced with ISO in MI rats. Further, ZBN possesses the potency to restores the activities of SOD, Catalase, GPx, and the reduced GSH levels toward normalcy. It might be due to the ability of ZBN to interact with hydroxyl and superoxide radicals thereby consequently having the protection of free radical scavenging activity.⁵⁸ The main source of ZBN from Zingiber officinale (ZO) gingerol⁵⁹ and zingerone and zingiberene.^60,61 The ZO compounds scavenge free radicals mediated thereby inhibits lipid peroxidation and maintain cellar homeostasis.^62
–64

Lipid parameters are useful in determining risk factors for cardiovascular diseases. Hyperlipidemia and hypercholesterolemia are risk factors for the development of heart failure.⁶⁵ In the present study, ISO-administration elevated TC, TG, and FFA in both the heart and plasma. Alterations of lipid profile during ISO in the heart tissue have already been reported.⁶⁶ The ISO-administration activates adenylate cyclase activity thereby increasing the formation cAMP. This mechanism has widely been accepted for lipolytic behavior of ISO⁶⁷ Then cAMP induces lipolytic activity resulting in the hydrolysis of stored triacylglycerol thereby caused hyperlipidemia.⁶⁸ ZBN pre-treatment significantly restored ISO-mediated hyperlipidemia and maintain membrane architecture in the myocardial tissue. ISO-induced rats showed an increase in the level of LDL-C, VLDL-C, and decreased in HDL-C. In the present study, it was evident that ZBN significantly reduced the lipid profile and elevate the HDL level. Bhandari et al. reported that the preventive effect of ZO extract against altered lipid metabolism in high cholesterol-fed rabbits.⁶⁹

Previous reports found that oxidative stress and inflammatory responses play an important role in the MI.^70,71 In the current study, the major inflammatory cytokines like TNF-α, IL-6, and IL-1β were significantly increased in the ISO-treated rats. The ZBN pretreatment prevented ISO-induced overexpression of TNF-α, IL-6, IL-1β probably by inhibiting the activation of the NF-κB signaling pathway. The ZBN pretreatment might inhibit free radical reactions and thus suppress the activation of NF-κB that resulted in the downregulation of various proinflammatory cytokines. Further, we observed, the ISO treatment induces several genes including VEGF and iNOS, and eNOS which thought to be potentially involved in angiogenesis. Conversely, ZBN treatment attenuated ISO-induced mRNA expression pattern of angiogenesis factors in the heart tissue. Similarly, rosuvastatin reverses isoproterenol-induced acute myocardial infarction through modulating iNOS and VEGF.¹⁷ In this study, the ISO-treatment also induces the overexpression of MMPs. These MMPs induces cardiac myocyte loss through apoptosis and ultimately leads to heart failure. Radhiga et al. reported that ursolic acid attenuated MMPs expression in isoproterenol-induced MI in experimental rats.⁷² AKT signal transduction pathway plays an important role in apoptotic cell death through modulating several anti-apoptotic proteins expression. Li et al. reported that ulinastatin against ISO-induced chronic heart failure through the PI3 K Akt, p38 MAPK, and NF-κB pathways.⁷³ In this study, the ZBN treatment prevented the mRNA expression pattern of AKT signaling molecules. Similarly, the ZBN treatment modulates mTOR/PI3K/AKT signaling events in colon cancer cells.³⁰ Overall, transcriptome analysis revealed that ZBN treatment modulated ISO-mediated the fibrotic and inflammatory signal transduction pathways in the heart tissue of rats.

Apoptosis plays an important role in the regulation of MI in experimental animals. Bcl-2 family members like Bcl-2 and Bcl-xL are a group of important factors which function as apoptotic inhibitors and Bid proapoptotic proteins like Bax, Bak and Bad known to promote apoptosis.⁷⁴ In the current study, we found that upregulated expression of pro-apoptotic expression of Bax, cytochrome c and caspase-3, while, down-regulated expression of anti-apoptotic protein Bcl-2 in ISO-induced rats indicating myocardial apoptosis. The ISO-induced ROS activates Bax to permeabilize the mitochondrial membrane which resulted in the release of cytochrome c. This released cytochrome then activates apoptotic protease-activating factor-1 (Apaf-1). The Apaf-1 forms apoptosome with caspase-9 which triggers the caspase-3 activation which leads to myocardial apoptotic cell death.⁷⁵ The western blot results clearly illustrate the ability of ZBN treatment against the ISO mediated activation of apoptotic markers expression. Similar to our present findings, several studies report the preventive potential of phytochemicals against ISO-induced apoptosis in the cardiac tissue.^19,76,77

Conclusion

In conclusion, the ZBN pretreatment significantly attenuated the ISO-induced cardiac markers expression, oxidative damages, alterations in the lipids profiles, inflammatory and apoptotic markers expression in the heart tissue. Therefore, ZBN may be considered as a potential cardioprotective agent and deserves further scientific validation.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Jianxia Wei

References

Bhushan

Kulshreshtha

. A scientific update on myocardial infarction: a life-threatening issue. Cardiol Plus 2019; 4(3): 71.

Benjamin

Muntner

Alonso

, et al. Heart disease and stroke statistics-2019 update: a report from the American Heart Association. Circulation 2019; 139: e56–e528.

Rafieian-Kopaei

Setorki

Doudi

, et al. Atherosclerosis: process, indicators, risk factors, and new hopes. Int J Prev Med 2014; 5(8): 927.

Zheng

Jin

, et al. Cardioprotective effects of genistein in rat myocardial ischemia-reperfusion injury studies by regulation of the P2X7/NF-κB pathway. Evid Based Complement Alternat Med 2016; 2016: 5381290.

Radhiga

Rajamanickam

Senthil

, et al. Effect of ursolic acid on cardiac marker enzymes, lipid profile and macroscopic enzyme mapping assay in isoproterenol-induced myocardial ischemic rats. Food Chem Toxicol 2012; 50(11): 3971–3977.

Ascaso

Fernández-Cruz

Santos

, et al. Significance of high density lipoprotein-cholesterol in cardiovascular risk prevention. Am J Cardiovasc Drug 2004; 4(5): 299–314.

Wilkins

Ning

Stone

, et al. Coronary heart disease risks associated with high levels of HDL cholesterol. J Am Heart Assoc 2014; 3(2): e000519.

Ahmed

Miller

Nasir

, et al. Primary low level of high-density lipoprotein cholesterol and risks of coronary heart disease, cardiovascular disease, and death: results from the multi-ethnic study of atherosclerosis. Am J Epidemiol 2016; 183(10): 875–883.

Mythili

Malathi

. Diagnostic markers of acute myocardial infarction. Biomed Rep 2015; 3(6): 743–748.

10.

Khalil

Ahmmed

Ahmed

, et al. Amelioration of isoproterenol-induced oxidative damage in rat myocardium by Withania somnifera leaf extract. Biomed Res Int 2015; 2015: 624159.

11.

Kumar

Anandan

Devaki

, et al. Cardioprotective effects of Picrorrhiza kurroa against isoproterenol-induced myocardial stress in rats. Fitoterapia 2001; 72(4): 402–405.

12.

Tavori

Rosenblat

Vaya

, et al. Paraoxonase 1 interactions with atherosclerotic lesions and arterial macrophages protect against foam cell formation and atherosclerosis development. Clin Lipidol 2010; 5(5): 685–697.

13.

Hemalatha

Stanely Mainzen Prince

. Antihyperlipidaemic, antihypertrophic, and reducing effects of zingerone on experimentally induced myocardial infarcted rats. J Biochem Mol Toxic 2015; 29(4): 182–188.

14.

Yuan

Chen

, et al. Cardioprotective effects of puerarin-V on isoproterenol-induced myocardial infarction mice is associated with regulation of PPAR-Υ/NF-κB pathway. Molecules 2018; 23(12): 3322.

15.

Ojha

Azimullah

Mohanraj

, et al. Thymoquinone protects against myocardial ischemic injury by mitigating oxidative stress and inflammation. Evid Based Complement Alternat Med 2015; 2015: 143629.

16.

Bencsik

Pálóczi

Kocsis

, et al. Moderate inhibition of myocardial matrix metalloproteinase-2 by ilomastat is cardioprotective. Pharmacol Res 2014; 80: 36–42.

17.

Zaitone

Abo-Gresha

. Rosuvastatin promotes angiogenesis and reverses isoproterenol-induced acute myocardial infarction in rats: role of iNOS and VEGF. Eur J Pharmacol 2012; 691(1–3): 134–142.

18.

Lee

Wolf

Escudero

, et al. Early expression of angiogenesis factors in acute myocardial ischemia and infarction. N Engl J Med 2000; 342: 626–633.

19.

Radhiga

Rajamanickam

Sundaresan

, et al. Effect of ursolic acid treatment on apoptosis and DNA damage in isoproterenol-induced myocardial infarction. Biochimie 2012; 94(5): 1135–1142.

20.

Eilat-Adar

Sinai

Yosefy

, et al. Nutritional recommendations for cardiovascular disease prevention. Nutrients 2013; 5(9): 3646–3683.

21.

Sanchis-Gomar

Perez-Quilis

Leischik

, et al. Epidemiology of coronary heart disease and acute coronary syndrome. Ann Transl Med 2016; 4(13): 256.

22.

Meeran

Laham

Azimullah

, et al. α-Bisabolol abrogates isoproterenol-induced myocardial infarction by inhibiting mitochondrial dysfunction and intrinsic pathway of apoptosis in rats. Mol Cell Biochem 2019; 453(1–2): 89–102.

23.

Meeran

Laham

Al-Taee

, et al. Protective effects of α-bisabolol on altered hemodynamics, lipid peroxidation, and nonenzymatic antioxidants in isoproterenol-induced myocardial infarction: in vivo and in vitro evidences. J Biochem Mol Toxic 2018; 32(10): e22200.

24.

Misbah

Haq Nawaz

Zafar

. Chemical analysis of essential oil of ginger (Zingiber officinale). Pakistan J Biol Sci 2005; 8: 1576–1578.

25.

El-Ghorab

Nauman

Anjum

, et al. A comparative study on chemical composition and antioxidant activity of ginger (Zingiber officinale) and cumin (Cuminum cyminum). J Agric Food Chem 2010; 58: 8231–8237.

26.

Togar

. The cytological, biochemical and genetic effects of selected sesquiterpenes on healthy neuron and N2a neuroblastoma cell cultures. PhD Thesis, Department of Biology, Graduate School of Natural and Applied Sciences, Atatürk University, Erzurum, Turkey, 2013.

27.

Singh

Kaur

. Synbiotic (probiotic and ginger extract) loaded floating beads: a novel therapeutic option in an experimental paradigm of gastric ulcer. J Pharm Pharmacol 2012; 64: 207–217.

28.

Peña

Rojas

Aparicio

, et al. Chemical composition and antibacterial activity of the essential oil of Espeletia nana . Nat Prod Commun 2012; 7(5): 661–662.

29.

Han

, et al. In vitro and in vivo anti-tobacco mosaic virus activities of essential oils and individual compounds. J Microbiol Biotechnol 2013; 23: 771–778.

30.

Chen

Tang

Liu

, et al. Zingiberene inhibits in vitro and in vivo human colon cancer cell growth via autophagy induction, suppression of PI3K/AKT/mTOR pathway and caspase 2 deactivation. J BUON 2019; 24(4): 1470–1475.

31.

Song

Duan

, et al. Paeonol and danshensu combination attenuates apoptosis in myocardial infarcted rats by inhibiting oxidative stress: roles of Nrf2/HO-1 and PI3K/Akt pathway. Sci Rep 2016; 6: 23693.

32.

Begum

Prasad

Thayalan

. Apigenin protects gamma-radiation induced oxidative stress, hematological changes and animal survival in whole body irradiated Swiss albino mice. Int J Nutr Pharm Neurol Dis 2012; 2(1): 45.

33.

Okinaka

Kumagai

Ebashi

, et al. Serum creatine phosphokinase: activity in progressive muscular dystrophy and neuromuscular diseases. Arch Neurol 1961; 4(5): 520–525.

34.

Baum

Braun

Gerhardt

, et al. Multicenter evaluation of a second-generation assay for cardiac troponin T. Clin Chem 1997; 43(10): 1877–1884.

35.

Larue

Calzolari

Bertinchant

, et al. Cardiac-specific immunoenzymometric assay of troponin I in the early phase of acute myocardial infarction. Clin Chem 1993; 39(6): 972–979.

36.

Niehaus

Jr Samuelsson

. Formation of malonaldehyde from phospholipid arachidonate during microsomal lipid peroxidation. Eur J Biochem 1968; 6(1): 126–130.

37.

Jiang

Hunt

Wolff

. Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein. Anal Biochem 1992; 202(2): 384–389.

38.

Ellman

. Tissue sulfhydryl groups. Arch Biochem Biophys 1959; 82(1): 70–77.

39.

Sinha

. Colorimetric assay of catalase. Anal Biochem 1972; 47(2): 389–394.

40.

Rotruck

Pope

Ganther

, et al. Selenium: biochemical role as a component of glutathione peroxidase. Science 1973; 179(4073): 588–590.

41.

Kakkar

Das

Viswanathan

. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 1984; 21(2): 130–132.

42.

Folch

Lees

Stanley

. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957; 226(1): 497–509.

43.

Allain

Poon

Chan

, et al. Enzymatic determination of total serum cholesterol. Clin Chem 1974; 20(4): 40–45.

44.

McGowan

Artiss

Strandbergh

, et al. A peroxidase-coupled method for the colorimetric determination of serum triglycerides. Clin Chem 1983; 29(3): 538–542.

45.

Falholt

Lund

Falholt

. An easy colorimetric micro method for routine determination of free fatty acids in plasma. Clinica Chimica Acta 1973; 46(2): 105–111.

46.

Zilversmit

Davis

. Micro determination of plasma phospholipids by trichloroacetic acid precipitation. J Lab Clin Med 1950; 35(1): 155–160.

47.

Izzo

Grillo

Murador

Improved method for determination of high-density-lipoprotein cholesterol I. Isolation of high-density lipoproteins by use of polyethylene glycol 6000. Clin Chem 1981; 27(3): 371–374.

48.

Friedwald

Levy

Fredricken

. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972; 18(6): 499–502.

49.

Brooks

Conrad

. Isoproterenol-induced myocardial injury and diastolic dysfunction in mice: structural and functional correlates. Comparative Med 2009; 59(4): 339–343.

50.

Baird

Graham

Baker

, et al. Creatine-kinase- and exercise-related muscle damage implications for muscle performance and recovery. J Nutr Metab 2012; 2012: 960363.

51.

Panteghini

. Role and importance of biochemical markers in clinical cardiology. Eur Heart J 2004; 25(14): 1187–1196.

52.

Engle

Jordan

Pritt

, et al. Qualification of cardiac troponin I concentration in mouse serum using isoproterenol and implementation in pharmacology studies to accelerate drug development. Toxicol Pathol 2009; 37(5): 617–628.

53.

Ansari

Bhandari

Pillai

. Ethanolic Zingiber officinale R. extract pretreatment alleviates isoproterenol-induced oxidative myocardial necrosis in rats. Indian J Exp Biol 2006; 44: 892–897.

54.

Ahmed

Tanvir

Hossen

, et al. Antioxidant properties and cardioprotective mechanism of Malaysian propolis in rats. Evid Based Complement Alternat Med 2017; 2017: 1–11.

55.

Shanmugam

Mallikarjuna

Nishanth

, et al. Protective effect of dietary ginger on antioxidant enzymes and oxidative damage in experimental diabetic rat tissues. Food Chem 2011; 124: 1436–1442.

56.

Sinha

Kumar Dabla

. Oxidative stress and antioxidants in hypertension—a current review. Curr Hypertens Rev 2015; 11(2): 132–142.

57.

Han

Wang

, et al. Cardioprotection against ischemia/reperfusion by licochalcone B in isolated rat hearts. Oxidative Med Cell Longevity 2014; 1: 1–11.

58.

Kusano

Ferrari

. Total antioxidant capacity: a biomarker in biomedical and nutritional studies. J Cell Mol Biol 2008; 7(1): 1–5.

59.

Aeschbach

Löliger

Scott

, et al. Antioxidant actions of thymol, carvacrol, 6-gingerol, zingerone and hydroxytyrosol. Food Chem Toxicol 1994; 32: 31.

60.

Krishnakanth

Lokesh

. Scavenging of super oxide anions by spice principles. Indian J Biochem Biophys 1993; 30: 133.

61.

Reddy

Lokesh

. Studies on spice principles as an antioxidant in the inhibition of lipid peroxide in rat liver microsomes. Mol Cell Biochem 1992; 111: 117.

62.

Cao

Chen

Guo

, et al. Scavenging effects of ginger on superoxide anion and hydroxyl radical. Zhongguo Zhong Yao Za Zhi 1993; 18(12): 750–751.

63.

Liu

Huo

Zhang

, et al. Effect of Zingiber officinale Rosc on lipid peroxidation in hyperlipidemia rats. Wei Sheng Yan Jiu 2003; 32(1): 22–23.

64.

Ahmed

Seth

Banerjee

. Influence of dietary ginger (Zingiber officinale Rosc) on antioxidant defense system in rat: comparison with ascorbic acid. Indian J Exp Biol 2000; 38(6): 604–606.

65.

Meeran

Prince

Basha

. Preventive effects of N-acetyl cysteine on lipids, lipoproteins and myocardial infarct size in isoproterenol induced myocardial infarcted rats: an in vivo and in vitro study. Eur J Pharmacol 2012; 677(1–3): 116–122.

66.

Upaganlawar

Balaraman

. Effects of Lagenaria sicessaria fruit juice on lipid profile and glycoprotein contents in cardiotoxicity induced by isoproterenol in rats. Toxicol Int 2012; 19(1): 15.

67.

Morimoto

Kiyama

Kameda

, et al. Mechanism of the stimulatory action of okadaic acid on lipolysis in rat fat cells. J Lipid Res 2000; 41(2): 199–204.

68.

Onyeneke

Oluba

Ojeaburu

, et al. Effect of soy protein on serum lipid profile and some lipid-metabolizing enzymes in cholesterol fed rats. Afr J Biotech 2007; 6(19): 2267–2273.

69.

Bhandari

Sharma

Zafar

. The protective action of ethanolic ginger (Zingiber officinale) extract in cholesterol fed rabbits. J Ethnopharmacol 1998; 61(2): 167–171.

70.

Kumar

Kasala

Bodduluru

, et al. Baicalein protects isoproterenol induced myocardial ischemic injury in male Wistar rats by mitigating oxidative stress and inflammation. Inflamm Res 2016; 65(8): 613–622.

71.

Reed

Rossi

Cannon

. Acute myocardial infarction. Lancet 2017; 389(10065): 197–210.

72.

Radhiga

Senthil

Sundaresan

, et al. Ursolic acid modulates MMPs, collagen-I, α-SMA, and TGF-β expression in isoproterenol-induced myocardial infarction in rats. Hum Exp Toxicol 2019; 38(7): 785–793.

73.

Hao

Jiang

, et al. Cardioprotective effects of ulinastatin against isoproterenol-induced chronic heart failure through the PI3K Akt, p38 MAPK and NF-κB pathways. Mol Med Rep 2018; 17(1): 1354–1360.

74.

Baskaran

Poornima

Huang

, et al. Neferine prevents NF-κB translocation and protects muscle cells from oxidative stress and apoptosis induced by hypoxia. Biofactors 2016; 42(4): 407–417.

75.

Xie

Yang

, et al. Cardioprotective effect of paeonol and danshensu combination on isoproterenol-induced myocardial injury in rats. PloS One 2012; 7(11): e48872.

76.

Saranya

Baskaran

Poornima

, et al. Berbamine ameliorates isoproterenol-induced myocardial infarction by inhibiting mitochondrial dysfunction and apoptosis in rats. J Cellular Biochem 2019; 120(3): 3101–3113.

77.

Sun

. Catalpol pretreatment attenuates cardiac dysfunction following myocardial infarction in rats. Anatol J Cardiol 2018; 19(5): 296.

Anti-inflammatory and anti-apoptotic effect of zingiberene on isoproterenol-induced myocardial infarction in experimental animals

Abstract

Keywords

Introduction

Materials and methods

Chemicals

Myocardial infarction and treatment plan

Assessment of acute toxicity of ZBN in male albino Wistar rats

Analysis of cardiac markers in serum

Measurement of lipid peroxidative markers in plasma and heart tissue

Estimation of intracellular antioxidant defense system

Estimation of lipids in plasma and heart tissue

Determination of inflammatory markers

Protein expression by western blot analysis

Analysis of inflammatory, angiogenesis and fibrotic related gene expression by a qRT-PCR array

Statistical analysis

Results

Effect of various concentrations of ZBN on the acute toxicity study of male albino Wistar rats

Effect of ZBN on ISO-induced serum cardiac marker enzymes

Effects of ZBN on ISO-induced lipid peroxidation in plasma and heart tissue

Effects of ZBN on ISO induced antioxidant status

Effect of ZBN on ISO-induced lipid profile and lipoproteins in plasma and heart tissue

Effect of ZBN on inflammatory markers level in the serum

Effect of ZBN on ISO-induced apoptotic markers expression by western blot analysis

Effect of ZBN on ISO-induced gene expression by PCR array

Discussion

Conclusion

Footnotes

Declaration of conflicting interests

Funding

ORCID iD

References