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Ameliorating Oxidative Stress and Inflammation by Hesperidin And Turk J Biochem 2019; 44(2): 207–217 Research Article Thoria Donia*, Samar Eldaly and Ehab M.M. Ali Ameliorating oxidative stress and inflammation by Hesperidin and vitamin E in doxorubicin induced cardiomyopathy Doxorubicin ile İndüklenmiş Kardiyomiyopatide Hesperidin ve E Vitamini ile Oksidatif Stres ve İnflamasyonun İyileştirilmesi https://doi.org/10.1515/tjb-2018-0156 Conclusion: HSP and VIT.E possess a protective effect Received May 7, 2018; accepted July 3, 2018; previously published against DOX-induced cardiomyopathy via inhibiting oxi- online September 5, 2018 dative stress, inflammation, and apoptosis. Abstract Keywords: Cardiomyopathy; Doxorubicin; Hesperidin; Vitamin E; Oxidative stress. Background: Doxorubicin (DOX) is a common chemother- apeutic drug. However, it causes cardiomyopathy which reduces its clinical use in human cancer therapy. Öz Objective: The purpose of our study was to assess the cardioprotective effect of hesperidin (HSP) and vitamin E Giriş: Doksorubisin (DOX) yaygın bir kemoterapötik ilaçtır. (VIT.E) against DOX-induced cardiomyopathy. Bununla birlikte, kardiyomiyopatiye neden olduğu için bu Material and methods: Seventy rats were allocated into durum ilaçın insan kanser tedavisinde klinik kullanımını seven groups: control, HSP (50 mg/kg, orally), VIT.E azaltır. (100 mg/kg orally), DOX [4 mg/kg, intraperitoneally (i.p.)], Amaç: Çalışmamızın amacı, DOX ile indüklenen kardiyo- DOX + HSP, DOX + VIT.E and DOX + HSP + VIT.E. miyopatiye karşı hesperidin (HSP) ve vitamin E’nin (VIT.E) Results: Our findings showed that serum lactate dehy- kardiyoprotektif etkisini değerlendirmektir. drogenase (LDH), creatine kinase (CK), myeloperoxidase Gereç ve Yöntemler: Yetmiş sıçan yedi gruba ayrıldı: (MPO), cardiac catalase and caspase activities as well kontrol, HSP (50 mg/kg, oral), VIT.E (100 mg/kg oral), DOX as cardiac malondialdehyde (MDA) and serum nitric [4 mg/kg, intraperitoneal (ip)], DOX + HSP, DOX + VIT.E ve oxide (NO) concentrations were reduced DOX + HSP or DOX + HSP + VIT.E. DOX + VIT.E or DOX + VIT.E + HSP groups compared to DOX Bulgular: Bulgularımız, DOX + HSP veya DOX + VIT.E veya group. Whereas, cardiac reduced glutathione (GSH) level, DOX + VIT.E + HSP grupları DOX grubu ile karşılaştırıl- serum arylesterase, and paraoxonase activities were higher dığında serum laktat dehidrojenaz (LDH), kreatin kinaz in rats injected with DOX and administrated with HSP and (CK), miyeloperoksidaz (MPO), kardiyak katalaz ve kaspaz VIT.E than that of rats injected with DOX only. Cardiac his- aktivitelerinin yanı sıra kardiyak malondialdehid (MDA) topathology of DOX group showed some changes that were ve serum nitrik oksit (NO) konsantrasyonlarının azaldığını improved during administration with HSP and VIT.E. gösterdi. Oysa kardiyak redükte glutatyon (GSH) düzeyi, serum arilesteraz ve paraoksonaz aktiviteleri DOX ile HSP *Corresponding author: Thoria Donia, Biochemistry Division, ve VIT.E enjekte edilen sıçanlarda sadece DOX enjekte Chemistry Department, Faculty of Science, Tanta University, Tanta, edilen sıçanlara göre daha yüksekti. DOX grubunun kar- Egypt, e-mail: [email protected]. diyak histopatolojisi HSP ve VIT.E ile uygulama sırasında https://orcid.org/0000-0002-1629-1290 düzelen bazı değişiklikler gösterdi. Samar Eldaly and Ehab M.M. Ali: Biochemistry Division, Chemistry Department, Faculty of Science, Tanta University, Tanta, Egypt, Sonuç: HSP ve VIT.E, oksidatif stres, inflamasyon ve e-mail: [email protected] (S. Eldaly); [email protected] apoptozu inhibe ederek DOX ile indüklenen kardiyomiyo- (E.M.M. Ali) patiye karşı koruyucu bir etkiye sahiptir. 208 Thoria Donia et al.: Ameliorating oxidative stress and inflammation by Hesperidin and vitamin E Anahtar Kelimeler: Doxorubicin; Kardiyomiyopati; Hes- Experimental design peridin; Vitamin E; Oksidatif Stres. Seventy adult males of Sprague Dawley rats, weighing 130–150 g, were purchased from the Organization for Bio- Introduction logical Products and Vaccines, Helwan, Cairo, Egypt. Rats were maintained under standard animal house conditions Doxorubicin (DOX) is a widely used chemotherapeutic under 20–22°C. Rat experimentation was consistent with drug for the treatment of several hematogenous and solid the guidelines of Ethics by the Guide for the Use and Care human malignancies [1]. However, irreversible myocardial of Laboratory Animals in accordance with animal care damage, resulting in cardiomyopathy and subsequent committee of the Faculty of Science, Tanta University, and congestive heart failure reduces its clinical use in human Tanta, Egypt. cancer therapy [2]. Also, 41% of cancer patients who Rats were randomly allocated into seven groups each received DOX therapy are affected by various cardiac prob- with 10 rats: (1) in control group, rats received normal lems and liver function abnormalities [3]. DOX-induced rat food; (2) in HSP group, rats were intra-gastric admin- cardiomyopathy has been suggested to involve free istrated with 50 mg hesperidin/kg body weight three radical generation, inhibition of nucleic acid and protein times per week for 3 weeks, [10]; (3) in VIT.E rats were synthesis, myofibrillar degeneration, and mitochondrial intra-gastric administrated with 100 mg VIT.E/kg body abnormalities [4, 5]. Moreover, DOX administration could weight, [11] two times per week for 3 weeks; (4) DOX decrease the endogenous antioxidants that scavenge the group was intra-peritoneal injected with 4 mg doxoru- free radicals so supplementation of exogenous antioxi- bicin/kg body weight three times per week for 2 weeks, dants may improve antioxidant defense system [6]. [12]; (5) DOX + HSP group was intra-gastric administrated Hesperidin (HSP) a flavanone glycoside found in with hesperidin and intra-peritoneal injected with doxo- citrus fruits (i.e. lemon and sweet orange) has wide phar- rubicin; (6) in DOX + VIT.E group, VIT.E was intra-gastric macological and biological effects. It could act as an administrated and DOX was intra-peritoneal injected; antioxidant, anti-inflammatory, anticarcinogenic, and (7) in DOX + HSP + VIT.E, HSP and VIT.E were intra-gastric antimicrobial agent, [7] as well as its effects on the vascu- administrated and DOX was intra-peritoneal injected. lar system [8]. The administration of HSP or/and VIT.E started 1 week The fat-soluble Vitamin E (VIT.E) is one of the body’s before DOX injection and continued for the next 2 weeks main free radical scavengers that protect cell membranes with DOX injection. from lipid peroxidation. Also, it maintains the health of At the end of the experiment, rats were sacrificed by red blood cells enhances blood circulation and supports puncture under diethyl ether anesthesia. Blood was col- healthy muscle and nerve function. Moreover, VIT.E has lected, allowed to clot at room temperature and centri- antioxidant effects in other cell organelles [9]. However, fuged at 3000 rpm for 10 min to separate serum. Serum no data regarding the effectiveness of VIT.E in combi- samples were kept at −20°C for various biochemical nation with HSP in a doxorubicin-induced cardiomyopa- analyses. thy model have been reported. In addition, hearts from different rats were removed, In the current study, we explored the cardioprotective washed in ice-cold saline (0.9%) and divided into two effect of VIT.E alone or with HSP in DOX-induced cardiomy- parts; one of them was fixed in 10% formalin for histo- opathy in rats by assessment of their effects on oxidative pathological analysis the other part was homogenized stress and inflammatory as well as apoptotic parameters. (10% w/v) in 0.05 M phosphate buffer pH 7.4 then centri- fuged at 3000 rpm for 15 min. The supernatant was used for determination of the biochemical parameters. Materials and methods Biochemical analyses Drugs and chemicals Estimation of serum LDH and CK activities and lipid DOX hydrochloride was purchased from Pfizer (Bentley, profile biomarkers Australia). HSP, and VIT.Ecapsules, as well as all other chemicals, were purchased from Sigma (St. Louis, MO, LDH and CK activities were assayed by commercial kit USA). supplied by Salucea (Germany). Serum triglycerides (TG), Thoria Donia et al.: Ameliorating oxidative stress and inflammation by Hesperidin and vitamin E 209 HDL-cholesterol and total cholesterol (TC) levels were Heart tissue catalase activity was estimated according assayed by commercial kit supplied by Biodiagnostics to Luck [18] by determining of the rate for H2O2 decomposi- (Cairo, Egypt). Serum LDL-cholesterol was calculated tion at 240 nm. using the following formula: LDL = TC-HDL – TG/5. Determination of serum arylesterase (AE) and paraoxonase (PON) activities Determination of serum nitric oxide (NO) level and myeloperoxidase (MPO) activity Serum AE was measured using p-nitrophenyl acetateas a substrate as described previously [19]. Briefly, 50 mM Serum nitric oxide was evaluated by measuring nitrite phosphate buffer, pH 7.5, 5 μL serum and 0.5 μmol (NO2) and nitrate (NO3) metabolites [13]. The concentration p-nitrophenyl acetate (acetone 0.3%) were mixed in a of oxidative end products is estimated by reduction of NO3 total volume of 1 mL, incubated at 37°C for 2 min and the to NO2 using cadmium. Briefly, Samples were treated with change in absorbance at 450 nm was recorded. AE activ- cadmium after deproteinization with 30% zinc sulfate. ity was calculated as nmol of p-nitrophenol produced per Then Griess reagent reacts with NO2 producing pink color minute under assay conditions using extension coeffi- −1 −1 that measured at 540 nm against reagent blank. The NO2 cient for p-nitrophenol (ε = 17.000 M cm ). standard curve was constructed with a set of serial con- PON was assayed using paraoxon as substrate accord- centrations ranging from 5–100 μM. ing to Gi et al. [20]. Briefly, calcium chloride 1 mM, 0.1 M Serum MPO oxidized hydrogen peroxide in the pres- glycine – NaOH buffer pH 10, sodium chloride 0.3 M, 50 μL ence of o-diasnsidine as substrate to produce a brown of serum and paraoxon 2 mM were mixed in a total volume color which is spectrophotometrically measured at of 1 mL. Then, the mixture was incubated at 37°C for 5 min 405 nm [14].
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