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Histopathological and Biochemical Effects of Thyme Essential Oil on H2O2 Stress in Heart Tissues
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Histopathological and Biochemical Effects of Thyme Essential Oil on H2O2 Stress in Heart Tissues [TD$FIRSNAME]Fatma[TD$FIRSNAME.] [TD$SURNAME]Guesmi[TD$SURNAME.] a*, [TD$FIRSNAME]Khantouche[TD$FIRSNAME.] [TD$SURNAME]Linda[TD$SURNAME.], MD b[2_TD$IF], [TD$FIRSNAME]Houda[TD$FIRSNAME.] [TD$SURNAME]Bellamine[TD$SURNAME.], MD c, [TD$FIRSNAME]Ahmed[TD$FIRSNAME.] [TD$SURNAME]Landoulsi[TD$SURNAME.] a, [TD$FIRSNAME]Amel[TD$FIRSNAME.] [TD$SURNAME]Mehrez[TD$SURNAME.], PhD a a
Faculty of Sciences of Bizerte, Bizerte, Tunisia Preparatory Institute for Scientific and Technical Studies, La Marsa, 2075, Tunisia Service of Anatomo-Pathology of Menzel Bourguiba, Bizerte, Tunisia
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Received 26 May 2018; received in revised form 4 December 2018; accepted 14 December 2018; online published-ahead-of-print xxx
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ORIGINAL ARTICLE
Background
Thymus algeriensis (T. algeriensis) is traditionally used in Tunisia to treat many human diseases. The aim of the present study was to investigate whether terpenes extracted from the aerial parts of T. algeriensis are potent cardioprotective agents for hydrogen peroxide (H2O2)-induced cardiotoxicity in rats.
Method
Thirty rats were divided into six groups as per the experimental design: control (n = 6); 0.1 mmol/L H2O2 (LD H2O2) (n = 6); 1 mmol/L H2O2 (HD H2O2) (n = 6); oily fraction of T. algeriensis (OFTS) (180 mg/kg b.wt) (n = 6); OFTS + 0.1 mmol/L H2O2 (n = 6); and OFTS + 1 mmol/L H2O2 (n = 6).
Results
The H2O2 demonstrated concentration-dependent cardiotoxic effects in vitro. While, exposure of rats to OFTS significantly depleted H2O2-induced protein oxidation and lipid peroxidation, it raised antioxidant defence enzymes, and protected against H2O2-induced histopathological alterations. The antioxidant potential of the Thyme essence was assessed by both enzymatic and non-enzymatic antioxidants.
Conclusion
In conclusion, OFTS may be a potential compound for the therapy of oxidative stress-induced heart disease.
Keywords
Hydrogen peroxide (H2O2) Thymus algeriensis Cardiotoxicity Thiobarbituric acid reactive substances Antioxidant defence enzymes
Introduction Q11
Free radicals and oxidative stress play a crucial role in the pathophysiology of a broad spectrum of cardiovascular diseases, including: congestive heart failure, valvular heart disease, cardiomyopathy, hypertrophy, atherosclerosis, and ischaemic heart disease [1]. Cardiovascular diseases have a high prevalence in developing and developed countries, and myocardial infarction accounts for the majority of deaths and disabilities [2]. Novel antioxidants may offer an effective and safe means of counteracting some of these problems and bolstering the
body’s defences against free radicals and cardiovascular disease [3]. There is a growing interest in using natural antioxidants as a protective strategy against cardiovascular-related problems [4]. Nutraceuticals include bioactive compounds such as polyphenols with anti-inflammatory activities, thus these products have the potential to treat chronic inflammatory diseases [5]. Several in vitro and in vivo studies have demonstrated the ability of polyphenols to counteract oxidative stress-induced cardiomyocytes damage and death [6]. The importance of natural products in modern medicine are well recognised and they continue to be of interest as
*Corresponding author. Emails: [email protected], [email protected] © 2019 Published by Elsevier B.V. on behalf of Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ).
Please cite this article in press as: Guesmi F, et al. Histopathological and Biochemical Effects of Thyme Essential Oil on H2O2 Stress in Heart Tissues. Heart, Lung and Circulation (2019), https://doi.org/10.1016/j.hlc.2018.12.008
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sources of novel lead compounds. Natural products targeting components of unfolded protein response and reducing endoplasmic reticulum stress offer an innovative strategic approach to treat cardiovascular diseases [7]. These days, oils from Thymus plants and extracts, as well as thymol and carvacrol, are used for several purposes such as medical antiseptics, wound healing agents, food preservatives, and flavourings [8]. Thyme has been used as an antihypertensive agent in Mexican traditional medicine [9] and has a generally recognised-as-safe status [8]. The current study was undertaken to assess the effects of hydrogen peroxide (H2O2) on heart toxicity and to investigate the protective effects of oily fraction of Thymus algeriensis (OFTS) against H2O2-induced cardiotoxicity in rats. Biochemical analysis was carried out. Histological studies were also conducted on H2O2-induced heart pathophysiology and its protection by OFTS.
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Materials and Methods
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Chemicals
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Bovine serum albumin (BSA); 5, 5’-dithiobis-2-nitrobenzoic acid (DTNB); Tris-HCl buffer, thiobarbituric acid (TBA); and 2,4-dinitrochlorobenzene (CDNB) were purchased from Merck (Nottingham, UK), Sigma Chemical Co. (St. Louis, MO, USA) and Fluka Chemie (Buchs, Switzerland).
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Plant Material
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For plant material collection, methods described earlier were used (when studying the effect of this oil on liver and kidney in a rat model) [10]. The aerial portions of wild Thymus algeriensis plants were collected during the flowering stage in March 2014 from Orbata mountain (Tunisia), which is located on the middle west of Tunisia at a latitude and longitude of 34.39641 and 9.12914, respectively. Plant specimens (Voucher #1188) were botanically authenticated by Dr Hamdi Lazhar, Bouhedma Natural Park, Tunisia, and deposited in the herbarium of medicinal plants at the National Institute of Agronomic in Tunisia. The essential oil of OFTS was extracted with Clevenger apparatus and dried over sodium sulphate. Purified OFTS was stored in sealed dark vials at 4 C.
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Chemical Composition of Oily Fraction of Thymus algeriensis To profile OFTS essential oil, gas chromatography-mass spectrometry (GC/MS) analysis was carried out according to the methods described earlier (when studying the effect of this oil on gastric ulcer in a rat model) [11]. One microliter of Thymus essential oil diluted in 10% hexane was subjected to GC/MS. Gas chromatography analysis was performed on a model 7890 A (series II) gas chromatograph, with a flame ionisation detector (FID) and a split ratio of 1:50 using a fused silica capillary column, HP5-MS (30 m250 mm i.d., 0.25 mm film thickness). Injector or detector temperature for each analysis was about 250 C, and the carrier gas was helium,
with a flow rate of 0.8 mL/minute. Peak areas were measured by electronic integration, and relative amounts of the individual components were based on the peak areas and expressed in %. The GC/MS was carried out on an Agilent model 5975 C mass spectrometer operating at ionising energy mode at 70 eV, combined with the GC described above.
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Animals
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Thirty male Sprague Dawley rats (6-8 weeks old) were kept in polypropylene cages and received a nutritionally standard diet and tap water. Rats were handled under room temperature and 12-hour light and dark cycles. The Medical Ethical Committee for the Care and Use of Laboratory Animals of Pasteur Institute of Tunis, Tunisia, approved these experiments (Ethic#LNFP/Pro 152012).
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Experimental Design
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Experimental animals were divided into six groups (X1-X6) consisting of six rats each: Group X1: Control (Ctrl); Group X2: Rats were given low-dose (0.1 mmol/L) H2O2 (LD) with an oral dose daily for 15 days; Group X3: Rats were given high-dose (1 mmol/L) H2O2 (HD) with an oral dose daily for 15 days; Group X4: Rats were given OFTS with an oral dose (180 mg/kg per day dissolved in normal saline) for 15 days; Group X5: Rats were treated with OFTS and low-dose H2O2 with an oral dose (180 mg/kg per day and 0.1 mmol/L, respectively) for 15 days; Group X6: Rats were treated with OFTS and high-dose H2O2 with an oral dose (180 mg/kg per day and 1 mmol/ L, respectively) for 15 days. Rats in the latter two groups receiving both agents were treated with OFTS 1 hour prior to H2O2 administration to the animals. Body weight was recorded daily for 15 days.
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Experimental Process
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After 2 weeks, rats were killed by cervical dislocation and the hearts collected from experimental rats was dissected. Cardiac tissues were fixed in formalin for histopathological analysis. Portions of the hearts were extracted in phosphate buffer using a mechanical rotary homogeniser. The supernatant obtained after centrifugation at 8000 g for 15 minutes was used as a source of enzymes for biochemical assays.
Biochemical Assays 1 Protein assessment Protein levels of heart tissue were estimated, using crystalline bovine serum albumin (BSA) as a standard graph, by the method of Lowry et al. [12]. 2 Antioxidant status
Please cite this article in press as: Guesmi F, et al. Histopathological and Biochemical Effects of Thyme Essential Oil on H2O2 Stress in Heart Tissues. Heart, Lung and Circulation (2019), https://doi.org/10.1016/j.hlc.2018.12.008
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To delineate the effect of H2O2 in heart tissues, this study evaluated its mechanism of action on the antioxidant defence enzymes following exposure in the treated rats. Assessment of cellular antioxidant enzymes of the heart tissues were determined for catalase (CAT) [13] superoxide dismutase (SOD) [14], glutathione-s-transferase (GST) [15], and glutathione peroxidase (GPX) [16].
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3 Cardiac levels of thiobarbituric acid relative substances
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The level of lipid peroxidation (LPO) in the heart was estimated by the thiobarbituric acid reactive substances method of Ohkawa et al. [17]. 4 Reduced glutathione estimation The reduced glutathione (GSH) content was evaluated by the method of Sedlak and Lindsay [18].
Effect of Hydrogen Peroxide on Animal Weight
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Body weight of control and treated groups was recorded daily for 15 days.
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Histopathologic Examination
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Heart tissues from all the groups were fixed in formalin solution (10%). Sections, prepared and stained with haematoxylin and eosin (H&E), were evaluated and photographed with light microscopy. A damage scoring system was used to quantify heart tissue damage and was carried out as follows: 0, no damage; 3, mild damage; 5, moderate damage; and 10, severe damage.
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Statistical Evaluation
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Data were normally distributed and reported as mean SEM, and one-way analysis of variance (ANOVA) as a parametric method that compared the means among the groups with such small amounts of samples. This was followed by Bonferroni’s test for multiple comparison or Dunnett’s multiple range test. Statistical significance was set at p < 0.05.
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Results Chemical Composition of Thymus Essential Oil
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The present study used OFTS, which is mainly composed of Campher, 1,8-Cineol, Linalol, 4-Carvomenthenol, Terpinen4-ol, Bornyl acetate, Viridiflorol and p-cymen. Each substance was expressed in %.
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Body Weight Gain
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At the end of H2O2 treatment period, a significant (p < 0.05) reduction in body weight was observed as compared to control (Figure 1). During the experimental procedure, no death was observed.
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Figure 1 Effect of HFTS on body weight in rats. Abbreviations: CtrlControl; OFTSOily fraction ofThymus algeriensis; LDlow dose; HDhigh dose; HDL. Mean values of three independent experiments have been plotted. **Significant value p < 0.05.
Effect of Oily Fraction of Thymus Algeriensis on Cellular Enzymatic Antioxidants
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The H2O2 increased the oxidative damage in cardiotoxic rats. The levels of lipid peroxides were significantly elevated. The antioxidant enzymes SOD, CAT, GST, and glutathione peroxidase (GPx) (Figure 2D-2 G) levels were depleted in cardiotoxic rats. In the OFTS (600, 900 mg/kg) pre-treated rats, the malondialdehyde levels (Figure 2A) were significantly decreased, and all the antioxidant enzymes were increased in cardiotoxic rats when compared to H2O2. The OFTS co-treatment decreased H2O2-induced lipid peroxidation (p < 0.05) when compared with the H2O2-treated group.
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Non-enzymatic Antioxidant Levels
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The investigation of cardiac marker levels by monitoring non-enzymatic antioxidant GSH showed that it significantly increased when rats were treated with essential oil of Thyme alone when compared to H2O2 (with significance p < 0.05). Oral treatment of rats with H2O2 showed lower levels of reduced GSH (Figure 2C) when compared with the control rats (p < 0.05). Co-treatment of rats with OFTS and H2O2 showed higher levels of GSH content.
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Histopathological Examination
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Histopathological examination of the heart tissues of the control animals and the groups treated with OFTS alone showed normal architecture (Figure 3A), whereas myofibre loss and necrosis were detected in the H2O2 group. Photographs of the heart tissues showed that OFTS pre-treatment decreased the tissue damage in heart injury conditions induced by H2O2 (Figure 3B).
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Please cite this article in press as: Guesmi F, et al. Histopathological and Biochemical Effects of Thyme Essential Oil on H2O2 Stress in Heart Tissues. Heart, Lung and Circulation (2019), https://doi.org/10.1016/j.hlc.2018.12.008
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Figure 2 A. Photomicrograph of the cardiac muscle tissues of normal control (A) and experimental group (B, C, D, E) of rats. B. Semi-quantitative analyses of Haematoxylin & Eosin staining results. *p < 0.05. Abbreviations: H, haemorrhage; DMF, degeneration of myocardial fibres; E, oedema; NP, nuclear pyknosis; ICI, inflammatory cell infiltration; IS, intracellular space; CN, condensed nuclei. Magnification: A, C, D, and E, 40; B, 20.
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Discussion
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In the present study, essential oil extracted from the aerial parts of Thymus algeriensis collected in the mountains of Orbata, Gafsa province of Tunisia, was investigated for its influence on H2O2-induced heart toxicity. The OFTS protects H2O2-induced biochemical and histopathological changes in the heart tissue of animals by restoring the activities of endogenous antioxidant enzymes. Hydrogen peroxide is a weak oxidising agent and can directly inactivate a few enzymes, usually by oxidation of essential thiol (-SH) groups. When adding H2O2 to cells, it is important to realise that H2O2 can permeate through plasma membranes at reasonably rapid rates, with permeability coefficients ranging 0.010.7 cm/minute [19]. It can rapidly cross cell membranes. Inside the cell, H2O2 probably reacts with Fe2+[3_TD$IF] or other redox metals, possibly Cu2+ ions, to form a hydroxyl radical, which may be the origin of many of its toxic effects [20].
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Combating oxidative stress through plant-derived products for strengthening the cellular defence mechanisms has proven beneficial in various therapeutic strategies [21]. Several studies have reported the importance of bioactive compounds for cardioprotection. As noted before by Gautam et al. [22], cells pre-treated with compounds like linalool, myrcene, and eucalyptol were studied for repair activity by their recovery on normal media. It was found that these can reduce the damage caused by hydrogen peroxide (H2O2), a potential genotoxin, but their coadministration is not that beneficial. In another studies, quercetin pre-treatment (20 mM) of H9c2 cardiomyocytes significantly attenuated trauma-induced apoptosis, TNF-a levels, reactive oxygen species (ROS) production, and Ca2+ levels [23]. In addition, ( )-epigallocatechin-3-O-gallate (50 mM) was also able to protect H9c2 cardiomyocytes from H2O2-induced changes in the expression of b-catenin, N-cadherin, and the gap junction protein Cx43, and to
Please cite this article in press as: Guesmi F, et al. Histopathological and Biochemical Effects of Thyme Essential Oil on H2O2 Stress in Heart Tissues. Heart, Lung and Circulation (2019), https://doi.org/10.1016/j.hlc.2018.12.008
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Figure 3 Variations of lipid peroxidation (A), CAT (C), SOD (D), GSH (E), GST (F), and GPX (G) activities and protein levels (B) in heart of rat after injection of different drugs. Data are showed as mean standard deviation (SD). **Significant value was p < 0.05.
activate caveolin-1 by modulating Akt/GSK-3b signalling [24]. Hydrogen peroxide (H2O2) induces membrane oxidation and leads to haemolysis, since it causes heme degradation in the presence of haemoglobin with the release of iron ions, which catalytically activate lipid peroxidation [25,26]. H2O2 also appears to play a more general role in the phosphorylation and dephosphorylation of serine groups, resulting in modification of protein activity [27]. Lacy et al. [28] observed that systemic plasma H2O2 levels increased in patients with cardiovascular disease. Heart tissue is susceptible to free radical damage because of its highly oxidative metabolism and because its antioxidant defences – like superoxide dismutase, GSH and Catalase – are less than those of other organs such as the liver [29]. Antioxidant enzymes are activated during oxidative stress and after activation of cytokines. However, these enzymes are down-regulated during end-stage heart failure [30]. Depletion of GSH results in enhanced lipid peroxidation, and excessive lipid peroxidation can cause increased GSH consumption [31]. Current data show that the GSH level was also significantly lowered in the H2O2-treated rats, while OFTS treatment showed a significant increase in GSH level (Table 1). It is noteworthy that the hearts contained very high levels of glutathione in the OFTS-treated groups rather than the H2O2-treated groups. Experimental and clinical studies on heart failure have shown that there is increased generation of reactive oxygen species such as superoxide anion (O2-) and hydroxyl radicals (OH-), which are involved in the formation of lipid peroxides, cell membrane damage, and destruction of the antioxidative defence
system [32]. In the current study, control and OFTS treatment alone did not induce any decrease in antioxidant status. Biochemical estimations of antioxidant enzymes showed significantly decreased activities in the H2O2treated rats. The decrease in the SOD and GPx activities in the H2O2-treated rats may have been due to excessive ROS production. Glutathione peroxidase (GSH peroxidase) metabolises H2O2 in both the cytosolic and mitochondrial compartments. In this context, GPx converts H2O2 into water before it can produce damaging ROS [33]. Thymol can increase the endogenous antioxidant enzyme activities – such as SOD, CAT, GPx, GST – and the level of other nonenzymatic antioxidants such as Vitamin C, Vitamin E, and reduced glutathione [34]. Pre-treatment of rats with OFTS followed by H2O2 administration can block its free radical-mediated toxicity, and prevent heart tissue from damage and oxidative stress. Most probably, the promising effect of OFTS, so the minimal histological changes, may be due to the terpenic compounds of Thyme. In fact, Thymus species essential oil has been found to be comparatively nontoxic to the normal fibroblast cells than MCF-7 and LNCaP human cancer cell lines [35]. Polyphenols can reduce the risk of cardiovascular disease by interacting with signalling cascades and epigenetic factors [36]. Ramprasath et al. [37] investigated the effects of naringenin (50 mM) on the protection of H9c2 cardiomyocytes from H2O2-mediated cells death, reduction in lipid peroxidation, and increases in the activity of antioxidant enzymes. Previous reports have shown that thymol suppressed the progression of hyperlipidaemia in high-fat-diet rabbits and atherosclerosis by reducing aortic intimal lipid lesion,
Please cite this article in press as: Guesmi F, et al. Histopathological and Biochemical Effects of Thyme Essential Oil on H2O2 Stress in Heart Tissues. Heart, Lung and Circulation (2019), https://doi.org/10.1016/j.hlc.2018.12.008
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lowering serum lipids, as well as inhibiting oxidative stress and inflammation [38]. Upon H2O2 administration, histoarchitecture of the heart tissues showed fibre necrosis. These data coincide with previous observations that administration of 400 mM H2O2 was used to initiate the progressive apoptotic cell death. Previous studies have shown that in the body, transient exposure of cells to H2O2 is a frequently encountered situation, as in the case of acute myocardial ischaemia and reperfusion [39]. Other reports have shown increased vascular smooth muscle cell O2 levels with H2O2 exposure [40]. The current study observed that OFTS treatment significantly alleviated oxidative stress by reducing malondialdehyde levels concomitantly with marked elevation in superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, and reduced glutathione activity in H2O2-induced heart toxicity. There were several limitations to this report, which are in accordance with the therapeutic effects of Thyme essential oil. Because the effect of OFTS against H2O2-induced heart toxicity was evaluated in rats, further clinical trials are needed to justify the effect of this essence as a human cardiac preventive drug. Natural compounds including Thyme essential oil are considered safe and do not have sideeffects, but further scientific studies are needed. Furthermore, the experimental procedure using high doses of essential oil used for heart prevention need to be repeated with low doses.
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Conclusions
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This study’s findings, validated through antioxidant status and histological sections, showed that a hydrophobic fraction of Thymus algeriensis has a new application as a cardioprotective agent. Inhibitory effect of OFTS, as an antioxidant, on H2O2-induced cardiotoxicity through decline of lipid peroxidation and restoration of antioxidant enzyme activity were found.
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Competing Interests
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Please cite this article in press as: Guesmi F, et al. Histopathological and Biochemical Effects of Thyme Essential Oil on H2O2 Stress in Heart Tissues. Heart, Lung and Circulation (2019), https://doi.org/10.1016/j.hlc.2018.12.008
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[27] Coyle CH, Martinez LJ, Coleman MC, Spitz DR, Weintraub NL, Kader KN. Mechanisms of H(2)O(2)-induced oxidative stress in endothelial cells. Free Radic Biol Med 2006;40:2206–13. http://dx.doi.org/ 10.1016/j.freeradbiomed.2006.02.017. [28] Lacy F, O’Connor DT, Schmid-Schonbein GW. Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertension. J Hypertens 1998;16:291–303. [29] Seval Y, Ahmet A, Engin S, Izzet K, Songul O. Protective effect of lycopene on adriamycin-induced cardiotoxicity and nephrotoxicity. Toxicol 2006;218:164–71. http://dx.doi.org/10.1016/j.tox.2005.10.015. [30] Sabri A, Hughie HH, Lucchesi PA. Regulation of hypertrophic and apoptotic signalling pathways by reactive oxygen species in cardiac myocytes. Antioxid Redox Signal 2003;5:731–40. http://dx.doi.org/ 10.1089/152308603770380034. [31] Ganesan B, Anandan R. Protective effect of betaine on changes in the levels of lysosomal enzyme activities in heart tissue in isoprenalineinduced myocardial infarction in Wistar rats. J Cell Stress Chap 2009;14:661–7. http://dx.doi.org/10.1007/s12192-009-0111-3. [32] Lalitha G, Poornima P, Archanah A, Padma VV. Protective effect of nefrine against Isoproterenol-induced cardiac toxicity. Cardiovasc Toxicol 2013;13:168–79. http://dx.doi.org/10.1007/s12012-012-9196-5. [33] El-Desoky GE, Bashandy SA, Alhazza M, Al-Othman ZA, Aboul-Soud MA, Yusuf K. Improvement of mercuric chloride-induced testis injuries and sperm quality deteriorations by Spirulina platensis in rats. PLoS One 2013;8(3):e59177. http://dx.doi.org/10.1371/journal.pone.0059177. [34] Meeran MFN, Prince PSM. Protective effects of thymol on altered plasma lipid peroxidation and nonenzymatic antioxidants in isoproterenol-
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induced myocardial infarcted rats. J Biochem Mol Toxicol 2012;26 (9):368–73. Hussain AI, Anwar F, Chatha SAS, Latif S, Sherazi STH, Ahmad A, et al. Chemical composition and bioactivity studies of the essential oils from two Thymus species from the Pakistani flora. LWT-Food Sci Technol 2013;50(1):185–92. Blade´ C, Baselga-Escudero L, Salvado´ MJ, Arola-Arnal A. miRNAs, polyphenols, and chronic disease. Mol Nutr Food Res 2013;57:58–70. http://dx.doi.org/10.1002/mnfr.201200454. Ramprasath T, Senthamizharasi M, Vasudevan V, Sasikumar S, Yuvaraj S, Selvam GS. Naringenin confers protection against oxidative stress through upregulation of Nrf2 target genes in cardiomyoblast cells. J Physiol Biochem 2014;70:407–15. http://dx.doi.org/10.1007/s13105014-0318-3. Yu YM Chao TY, Chang WC, Chang MJ, Lee MF. Thymol reduces oxidative stress, aortic intimal thickening, and inflammation-related gene expression in hyperlipidemic rabbits. J Food Drug Analy 2016;24 (3):556–63. http://dx.doi.org/10.1016/j.jfda.2016.02.004. Han H, Long H, Wang H, Wang J, Zhang Y, Wang Z. Progressive apoptotic cell death triggered by transient oxidative insult in H9c2 rat ventricular cells: a novel pattern of apoptosis and the mechanisms. Am J Physiol Heart Circ Physiol 2004;286:H2169–82. http://dx.doi.org/ 10.1152/ajpheart.00199.2003. PMID: 14739138. Li WG, Miller Jr FJ, Brown MR, Chatterjee P, Aylsworth GR, Shao J, et al. Enhanced H(2)O(2)-induced cytotoxicity in ‘‘epithelioid” smooth muscle cells: implications for neointimal regression. Arterioscler Thromb Vasc Biol 2000;20(6):1473–9. PMID: 10845860.
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Histopathological and Biochemical Effects of Thyme Essential Oil on H2O2 Stress in Heart Tissues [TD$FIRSNAME]Fatma[TD$FIRSNAME.] [TD$SURNAME]Guesmi[TD$SURNAME.] a*, [TD$FIRSNAME]Khantouche[TD$FIRSNAME.] [TD$SURNAME]Linda[TD$SURNAME.], MD b[2_TD$IF], [TD$FIRSNAME]Houda[TD$FIRSNAME.] [TD$SURNAME]Bellamine[TD$SURNAME.], MD c, [TD$FIRSNAME]Ahmed[TD$FIRSNAME.] [TD$SURNAME]Landoulsi[TD$SURNAME.] a, [TD$FIRSNAME]Amel[TD$FIRSNAME.] [TD$SURNAME]Mehrez[TD$SURNAME.], PhD a a
Faculty of Sciences of Bizerte, Bizerte, Tunisia Preparatory Institute for Scientific and Technical Studies, La Marsa, 2075, Tunisia Service of Anatomo-Pathology of Menzel Bourguiba, Bizerte, Tunisia
b c
Received 26 May 2018; received in revised form 4 December 2018; accepted 14 December 2018; online published-ahead-of-print xxx
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ORIGINAL ARTICLE
Background
Thymus algeriensis (T. algeriensis) is traditionally used in Tunisia to treat many human diseases. The aim of the present study was to investigate whether terpenes extracted from the aerial parts of T. algeriensis are potent cardioprotective agents for hydrogen peroxide (H2O2)-induced cardiotoxicity in rats.
Method
Thirty rats were divided into six groups as per the experimental design: control (n = 6); 0.1 mmol/L H2O2 (LD H2O2) (n = 6); 1 mmol/L H2O2 (HD H2O2) (n = 6); oily fraction of T. algeriensis (OFTS) (180 mg/kg b.wt) (n = 6); OFTS + 0.1 mmol/L H2O2 (n = 6); and OFTS + 1 mmol/L H2O2 (n = 6).
Results
The H2O2 demonstrated concentration-dependent cardiotoxic effects in vitro. While, exposure of rats to OFTS significantly depleted H2O2-induced protein oxidation and lipid peroxidation, it raised antioxidant defence enzymes, and protected against H2O2-induced histopathological alterations. The antioxidant potential of the Thyme essence was assessed by both enzymatic and non-enzymatic antioxidants.
Conclusion
In conclusion, OFTS may be a potential compound for the therapy of oxidative stress-induced heart disease.
Keywords
Hydrogen peroxide (H2O2) Thymus algeriensis Cardiotoxicity Thiobarbituric acid reactive substances Antioxidant defence enzymes
Introduction Q11
Free radicals and oxidative stress play a crucial role in the pathophysiology of a broad spectrum of cardiovascular diseases, including: congestive heart failure, valvular heart disease, cardiomyopathy, hypertrophy, atherosclerosis, and ischaemic heart disease [1]. Cardiovascular diseases have a high prevalence in developing and developed countries, and myocardial infarction accounts for the majority of deaths and disabilities [2]. Novel antioxidants may offer an effective and safe means of counteracting some of these problems and bolstering the
body’s defences against free radicals and cardiovascular disease [3]. There is a growing interest in using natural antioxidants as a protective strategy against cardiovascular-related problems [4]. Nutraceuticals include bioactive compounds such as polyphenols with anti-inflammatory activities, thus these products have the potential to treat chronic inflammatory diseases [5]. Several in vitro and in vivo studies have demonstrated the ability of polyphenols to counteract oxidative stress-induced cardiomyocytes damage and death [6]. The importance of natural products in modern medicine are well recognised and they continue to be of interest as
*Corresponding author. Emails: [email protected], [email protected] © 2019 Published by Elsevier B.V. on behalf of Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ).
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sources of novel lead compounds. Natural products targeting components of unfolded protein response and reducing endoplasmic reticulum stress offer an innovative strategic approach to treat cardiovascular diseases [7]. These days, oils from Thymus plants and extracts, as well as thymol and carvacrol, are used for several purposes such as medical antiseptics, wound healing agents, food preservatives, and flavourings [8]. Thyme has been used as an antihypertensive agent in Mexican traditional medicine [9] and has a generally recognised-as-safe status [8]. The current study was undertaken to assess the effects of hydrogen peroxide (H2O2) on heart toxicity and to investigate the protective effects of oily fraction of Thymus algeriensis (OFTS) against H2O2-induced cardiotoxicity in rats. Biochemical analysis was carried out. Histological studies were also conducted on H2O2-induced heart pathophysiology and its protection by OFTS.
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Materials and Methods
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Chemicals
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Bovine serum albumin (BSA); 5, 5’-dithiobis-2-nitrobenzoic acid (DTNB); Tris-HCl buffer, thiobarbituric acid (TBA); and 2,4-dinitrochlorobenzene (CDNB) were purchased from Merck (Nottingham, UK), Sigma Chemical Co. (St. Louis, MO, USA) and Fluka Chemie (Buchs, Switzerland).
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Plant Material
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For plant material collection, methods described earlier were used (when studying the effect of this oil on liver and kidney in a rat model) [10]. The aerial portions of wild Thymus algeriensis plants were collected during the flowering stage in March 2014 from Orbata mountain (Tunisia), which is located on the middle west of Tunisia at a latitude and longitude of 34.39641 and 9.12914, respectively. Plant specimens (Voucher #1188) were botanically authenticated by Dr Hamdi Lazhar, Bouhedma Natural Park, Tunisia, and deposited in the herbarium of medicinal plants at the National Institute of Agronomic in Tunisia. The essential oil of OFTS was extracted with Clevenger apparatus and dried over sodium sulphate. Purified OFTS was stored in sealed dark vials at 4 C.
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Chemical Composition of Oily Fraction of Thymus algeriensis To profile OFTS essential oil, gas chromatography-mass spectrometry (GC/MS) analysis was carried out according to the methods described earlier (when studying the effect of this oil on gastric ulcer in a rat model) [11]. One microliter of Thymus essential oil diluted in 10% hexane was subjected to GC/MS. Gas chromatography analysis was performed on a model 7890 A (series II) gas chromatograph, with a flame ionisation detector (FID) and a split ratio of 1:50 using a fused silica capillary column, HP5-MS (30 m250 mm i.d., 0.25 mm film thickness). Injector or detector temperature for each analysis was about 250 C, and the carrier gas was helium,
with a flow rate of 0.8 mL/minute. Peak areas were measured by electronic integration, and relative amounts of the individual components were based on the peak areas and expressed in %. The GC/MS was carried out on an Agilent model 5975 C mass spectrometer operating at ionising energy mode at 70 eV, combined with the GC described above.
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Animals
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Thirty male Sprague Dawley rats (6-8 weeks old) were kept in polypropylene cages and received a nutritionally standard diet and tap water. Rats were handled under room temperature and 12-hour light and dark cycles. The Medical Ethical Committee for the Care and Use of Laboratory Animals of Pasteur Institute of Tunis, Tunisia, approved these experiments (Ethic#LNFP/Pro 152012).
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Experimental Design
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Experimental animals were divided into six groups (X1-X6) consisting of six rats each: Group X1: Control (Ctrl); Group X2: Rats were given low-dose (0.1 mmol/L) H2O2 (LD) with an oral dose daily for 15 days; Group X3: Rats were given high-dose (1 mmol/L) H2O2 (HD) with an oral dose daily for 15 days; Group X4: Rats were given OFTS with an oral dose (180 mg/kg per day dissolved in normal saline) for 15 days; Group X5: Rats were treated with OFTS and low-dose H2O2 with an oral dose (180 mg/kg per day and 0.1 mmol/L, respectively) for 15 days; Group X6: Rats were treated with OFTS and high-dose H2O2 with an oral dose (180 mg/kg per day and 1 mmol/ L, respectively) for 15 days. Rats in the latter two groups receiving both agents were treated with OFTS 1 hour prior to H2O2 administration to the animals. Body weight was recorded daily for 15 days.
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Experimental Process
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After 2 weeks, rats were killed by cervical dislocation and the hearts collected from experimental rats was dissected. Cardiac tissues were fixed in formalin for histopathological analysis. Portions of the hearts were extracted in phosphate buffer using a mechanical rotary homogeniser. The supernatant obtained after centrifugation at 8000 g for 15 minutes was used as a source of enzymes for biochemical assays.
Biochemical Assays 1 Protein assessment Protein levels of heart tissue were estimated, using crystalline bovine serum albumin (BSA) as a standard graph, by the method of Lowry et al. [12]. 2 Antioxidant status
Please cite this article in press as: Guesmi F, et al. Histopathological and Biochemical Effects of Thyme Essential Oil on H2O2 Stress in Heart Tissues. Heart, Lung and Circulation (2019), https://doi.org/10.1016/j.hlc.2018.12.008
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To delineate the effect of H2O2 in heart tissues, this study evaluated its mechanism of action on the antioxidant defence enzymes following exposure in the treated rats. Assessment of cellular antioxidant enzymes of the heart tissues were determined for catalase (CAT) [13] superoxide dismutase (SOD) [14], glutathione-s-transferase (GST) [15], and glutathione peroxidase (GPX) [16].
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3 Cardiac levels of thiobarbituric acid relative substances
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The level of lipid peroxidation (LPO) in the heart was estimated by the thiobarbituric acid reactive substances method of Ohkawa et al. [17]. 4 Reduced glutathione estimation The reduced glutathione (GSH) content was evaluated by the method of Sedlak and Lindsay [18].
Effect of Hydrogen Peroxide on Animal Weight
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Body weight of control and treated groups was recorded daily for 15 days.
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Histopathologic Examination
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Heart tissues from all the groups were fixed in formalin solution (10%). Sections, prepared and stained with haematoxylin and eosin (H&E), were evaluated and photographed with light microscopy. A damage scoring system was used to quantify heart tissue damage and was carried out as follows: 0, no damage; 3, mild damage; 5, moderate damage; and 10, severe damage.
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Statistical Evaluation
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Data were normally distributed and reported as mean SEM, and one-way analysis of variance (ANOVA) as a parametric method that compared the means among the groups with such small amounts of samples. This was followed by Bonferroni’s test for multiple comparison or Dunnett’s multiple range test. Statistical significance was set at p < 0.05.
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Results Chemical Composition of Thymus Essential Oil
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The present study used OFTS, which is mainly composed of Campher, 1,8-Cineol, Linalol, 4-Carvomenthenol, Terpinen4-ol, Bornyl acetate, Viridiflorol and p-cymen. Each substance was expressed in %.
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Body Weight Gain
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At the end of H2O2 treatment period, a significant (p < 0.05) reduction in body weight was observed as compared to control (Figure 1). During the experimental procedure, no death was observed.
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Figure 1 Effect of HFTS on body weight in rats. Abbreviations: CtrlControl; OFTSOily fraction ofThymus algeriensis; LDlow dose; HDhigh dose; HDL. Mean values of three independent experiments have been plotted. **Significant value p < 0.05.
Effect of Oily Fraction of Thymus Algeriensis on Cellular Enzymatic Antioxidants
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The H2O2 increased the oxidative damage in cardiotoxic rats. The levels of lipid peroxides were significantly elevated. The antioxidant enzymes SOD, CAT, GST, and glutathione peroxidase (GPx) (Figure 2D-2 G) levels were depleted in cardiotoxic rats. In the OFTS (600, 900 mg/kg) pre-treated rats, the malondialdehyde levels (Figure 2A) were significantly decreased, and all the antioxidant enzymes were increased in cardiotoxic rats when compared to H2O2. The OFTS co-treatment decreased H2O2-induced lipid peroxidation (p < 0.05) when compared with the H2O2-treated group.
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Non-enzymatic Antioxidant Levels
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The investigation of cardiac marker levels by monitoring non-enzymatic antioxidant GSH showed that it significantly increased when rats were treated with essential oil of Thyme alone when compared to H2O2 (with significance p < 0.05). Oral treatment of rats with H2O2 showed lower levels of reduced GSH (Figure 2C) when compared with the control rats (p < 0.05). Co-treatment of rats with OFTS and H2O2 showed higher levels of GSH content.
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Histopathological Examination
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Histopathological examination of the heart tissues of the control animals and the groups treated with OFTS alone showed normal architecture (Figure 3A), whereas myofibre loss and necrosis were detected in the H2O2 group. Photographs of the heart tissues showed that OFTS pre-treatment decreased the tissue damage in heart injury conditions induced by H2O2 (Figure 3B).
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Figure 2 A. Photomicrograph of the cardiac muscle tissues of normal control (A) and experimental group (B, C, D, E) of rats. B. Semi-quantitative analyses of Haematoxylin & Eosin staining results. *p < 0.05. Abbreviations: H, haemorrhage; DMF, degeneration of myocardial fibres; E, oedema; NP, nuclear pyknosis; ICI, inflammatory cell infiltration; IS, intracellular space; CN, condensed nuclei. Magnification: A, C, D, and E, 40; B, 20.
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Discussion
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In the present study, essential oil extracted from the aerial parts of Thymus algeriensis collected in the mountains of Orbata, Gafsa province of Tunisia, was investigated for its influence on H2O2-induced heart toxicity. The OFTS protects H2O2-induced biochemical and histopathological changes in the heart tissue of animals by restoring the activities of endogenous antioxidant enzymes. Hydrogen peroxide is a weak oxidising agent and can directly inactivate a few enzymes, usually by oxidation of essential thiol (-SH) groups. When adding H2O2 to cells, it is important to realise that H2O2 can permeate through plasma membranes at reasonably rapid rates, with permeability coefficients ranging 0.010.7 cm/minute [19]. It can rapidly cross cell membranes. Inside the cell, H2O2 probably reacts with Fe2+[3_TD$IF] or other redox metals, possibly Cu2+ ions, to form a hydroxyl radical, which may be the origin of many of its toxic effects [20].
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Combating oxidative stress through plant-derived products for strengthening the cellular defence mechanisms has proven beneficial in various therapeutic strategies [21]. Several studies have reported the importance of bioactive compounds for cardioprotection. As noted before by Gautam et al. [22], cells pre-treated with compounds like linalool, myrcene, and eucalyptol were studied for repair activity by their recovery on normal media. It was found that these can reduce the damage caused by hydrogen peroxide (H2O2), a potential genotoxin, but their coadministration is not that beneficial. In another studies, quercetin pre-treatment (20 mM) of H9c2 cardiomyocytes significantly attenuated trauma-induced apoptosis, TNF-a levels, reactive oxygen species (ROS) production, and Ca2+ levels [23]. In addition, ( )-epigallocatechin-3-O-gallate (50 mM) was also able to protect H9c2 cardiomyocytes from H2O2-induced changes in the expression of b-catenin, N-cadherin, and the gap junction protein Cx43, and to
Please cite this article in press as: Guesmi F, et al. Histopathological and Biochemical Effects of Thyme Essential Oil on H2O2 Stress in Heart Tissues. Heart, Lung and Circulation (2019), https://doi.org/10.1016/j.hlc.2018.12.008
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Figure 3 Variations of lipid peroxidation (A), CAT (C), SOD (D), GSH (E), GST (F), and GPX (G) activities and protein levels (B) in heart of rat after injection of different drugs. Data are showed as mean standard deviation (SD). **Significant value was p < 0.05.
activate caveolin-1 by modulating Akt/GSK-3b signalling [24]. Hydrogen peroxide (H2O2) induces membrane oxidation and leads to haemolysis, since it causes heme degradation in the presence of haemoglobin with the release of iron ions, which catalytically activate lipid peroxidation [25,26]. H2O2 also appears to play a more general role in the phosphorylation and dephosphorylation of serine groups, resulting in modification of protein activity [27]. Lacy et al. [28] observed that systemic plasma H2O2 levels increased in patients with cardiovascular disease. Heart tissue is susceptible to free radical damage because of its highly oxidative metabolism and because its antioxidant defences – like superoxide dismutase, GSH and Catalase – are less than those of other organs such as the liver [29]. Antioxidant enzymes are activated during oxidative stress and after activation of cytokines. However, these enzymes are down-regulated during end-stage heart failure [30]. Depletion of GSH results in enhanced lipid peroxidation, and excessive lipid peroxidation can cause increased GSH consumption [31]. Current data show that the GSH level was also significantly lowered in the H2O2-treated rats, while OFTS treatment showed a significant increase in GSH level (Table 1). It is noteworthy that the hearts contained very high levels of glutathione in the OFTS-treated groups rather than the H2O2-treated groups. Experimental and clinical studies on heart failure have shown that there is increased generation of reactive oxygen species such as superoxide anion (O2-) and hydroxyl radicals (OH-), which are involved in the formation of lipid peroxides, cell membrane damage, and destruction of the antioxidative defence
system [32]. In the current study, control and OFTS treatment alone did not induce any decrease in antioxidant status. Biochemical estimations of antioxidant enzymes showed significantly decreased activities in the H2O2treated rats. The decrease in the SOD and GPx activities in the H2O2-treated rats may have been due to excessive ROS production. Glutathione peroxidase (GSH peroxidase) metabolises H2O2 in both the cytosolic and mitochondrial compartments. In this context, GPx converts H2O2 into water before it can produce damaging ROS [33]. Thymol can increase the endogenous antioxidant enzyme activities – such as SOD, CAT, GPx, GST – and the level of other nonenzymatic antioxidants such as Vitamin C, Vitamin E, and reduced glutathione [34]. Pre-treatment of rats with OFTS followed by H2O2 administration can block its free radical-mediated toxicity, and prevent heart tissue from damage and oxidative stress. Most probably, the promising effect of OFTS, so the minimal histological changes, may be due to the terpenic compounds of Thyme. In fact, Thymus species essential oil has been found to be comparatively nontoxic to the normal fibroblast cells than MCF-7 and LNCaP human cancer cell lines [35]. Polyphenols can reduce the risk of cardiovascular disease by interacting with signalling cascades and epigenetic factors [36]. Ramprasath et al. [37] investigated the effects of naringenin (50 mM) on the protection of H9c2 cardiomyocytes from H2O2-mediated cells death, reduction in lipid peroxidation, and increases in the activity of antioxidant enzymes. Previous reports have shown that thymol suppressed the progression of hyperlipidaemia in high-fat-diet rabbits and atherosclerosis by reducing aortic intimal lipid lesion,
Please cite this article in press as: Guesmi F, et al. Histopathological and Biochemical Effects of Thyme Essential Oil on H2O2 Stress in Heart Tissues. Heart, Lung and Circulation (2019), https://doi.org/10.1016/j.hlc.2018.12.008
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lowering serum lipids, as well as inhibiting oxidative stress and inflammation [38]. Upon H2O2 administration, histoarchitecture of the heart tissues showed fibre necrosis. These data coincide with previous observations that administration of 400 mM H2O2 was used to initiate the progressive apoptotic cell death. Previous studies have shown that in the body, transient exposure of cells to H2O2 is a frequently encountered situation, as in the case of acute myocardial ischaemia and reperfusion [39]. Other reports have shown increased vascular smooth muscle cell O2 levels with H2O2 exposure [40]. The current study observed that OFTS treatment significantly alleviated oxidative stress by reducing malondialdehyde levels concomitantly with marked elevation in superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, and reduced glutathione activity in H2O2-induced heart toxicity. There were several limitations to this report, which are in accordance with the therapeutic effects of Thyme essential oil. Because the effect of OFTS against H2O2-induced heart toxicity was evaluated in rats, further clinical trials are needed to justify the effect of this essence as a human cardiac preventive drug. Natural compounds including Thyme essential oil are considered safe and do not have sideeffects, but further scientific studies are needed. Furthermore, the experimental procedure using high doses of essential oil used for heart prevention need to be repeated with low doses.
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Conclusions
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This study’s findings, validated through antioxidant status and histological sections, showed that a hydrophobic fraction of Thymus algeriensis has a new application as a cardioprotective agent. Inhibitory effect of OFTS, as an antioxidant, on H2O2-induced cardiotoxicity through decline of lipid peroxidation and restoration of antioxidant enzyme activity were found.
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Competing Interests
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346 Q14 The authors declare that they have no competing interests.
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Please cite this article in press as: Guesmi F, et al. Histopathological and Biochemical Effects of Thyme Essential Oil on H2O2 Stress in Heart Tissues. Heart, Lung and Circulation (2019), https://doi.org/10.1016/j.hlc.2018.12.008
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HLC 2824 1–7
7
Histopathological and Biochemical Effects of Thyme
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Please cite this article in press as: Guesmi F, et al. Histopathological and Biochemical Effects of Thyme Essential Oil on H2O2 Stress in Heart Tissues. Heart, Lung and Circulation (2019), https://doi.org/10.1016/j.hlc.2018.12.008
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