Heat stress (HS) adversely affects survival(Reference Bogin, Avidar and Pech-Waffenschmidt1), performance(Reference Yalcin, Ozkan and Turkmut2, Reference Sandercock, Hunter and Nute3) and product quality(Reference Sandercock, Hunter and Nute3) in poultry. Moreover, HS causes oxidative stress, reflected by an increased production of reactive oxygen molecules(Reference Halliwell and Gutteridge4) and decreased concentrations of serum vitamins(Reference Sahin and Kucuk5) and minerals(Reference Sahin, Sahin and Kucuk6) that play a role in the defence system. Oxidative stress impairs cell membrane and mitochondrial integrity(Reference Meng, Velalar and Ruan7) and causes cell damage through lipid peroxidation(Reference Halliwell and Gutteridge4), which can be minimised by supplementation of antioxidant vitamins(Reference Puthpongsiriporn, Scheideler and Sell8, Reference Franchini, Sirri and Tallarico9) or natural substances that possess antioxidant potential(Reference Sahin, Orhan and Tuzcu10, Reference Tuzcu, Sahin and Karatepe11).
Berberis vulgaris L. (Barberry L. family Berberidaceae) is grown in Europe and Asia and its various parts (i.e. root, bark, leaf and fruit) are used in traditional medicine(Reference Imanshahidi and Hosseinzadeh12). B. vulgaris extract exerts numerous biological effects that are pertinent to human medicine, which include antioxidant(Reference Zovko-Koncić, Kremer and Karlović13), anti-inflammatory(Reference Ivanovska and Philipov14, Reference Souto, Tavares and da Silva15), antimicrobial(Reference Kosalec, Gregurek and Kremer16), antipyretic(Reference Imanshahidi and Hosseinzadeh12), antipruritic(Reference Imanshahidi and Hosseinzadeh12), antiurolithic(Reference Bashir, Gilani and Siddiqui17), anticonvulsant(Reference Bhutada, Mundhada and Bansod18) and antiarrhythmic(Reference Fatehi, Saleh and Fatehi-Hassanabad19) activities. The antioxidant effect of B. vulgaris extract is attributed to possessing high reductive powers to quench singlet molecular oxygen and peroxyl radicals(Reference Zovko-Koncić, Kremer and Karlović13). The root of B. vulgaris is usually used for treating a variety of ailments such as diabetes, stomach, liver and kidney discomfort(Reference Kosalec, Gregurek and Kremer16, Reference Blumenthal20). The predominant active compound in the plant is berberine, one of isoquinoline alkaloids(Reference Imanshahidi and Hosseinzadeh12, Reference Arayne, Sultana and Bahadur21).
Transcription factors are key cellular components that control gene expression in response to biological and environmental stimuli(Reference Vaquerizas, Kummerfeld and Teichmann22). NF-κB is a pleiotropic transcription factor present in almost all cell types, responsible for controlling DNA transcription and involved in cellular responses to a number of stimuli including free radicals(Reference Gilmore23). This complex protein acts as the first responder because it is normally present in the cytosol in an inactive form and enters the nucleus in response to a stimulus in order to activate the expression of specific genes(Reference Gilmore23, Reference Nelson, Ihekwaba and Elliott24). Heat shock proteins (HSP), known as stress proteins, are a group of proteins that are present in all cells and expressed at high levels when cells are exposed to high or low temperature or other stressors(Reference Mahmoud, Edens and Eisen25, Reference Figueiredo, Gertler and Cabello26). HSP play roles in protein folding and unfolding, protein assembling and disassembling and protein translocation(Reference Murakami, Pain and Blobel27, Reference Wang and Edens28).
Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), another transcription factor, is known as a master regulator of the antioxidant response. It is bound to another protein called Kelch-like ECH (eroyl CoA hydratase)-associated protein 1 in the cytosol(Reference Itoh, Wakabayashi and Katoh29). Disruption of cysteine residues in Kelch-like ECH-associated protein 1 due to oxidative stress results in accumulation of Nrf2 in the cytosol(Reference Yamamoto, Suzuki and Kobayashi30). Unbound Nrf2 is then translocated into the nucleus, where its binds to the antioxidant response element in the promoter region of many antioxidative genes to initiate their transcriptions(Reference Itoh, Chiba and Takahashi31).
Recently, active components from herbal plants have been explored as possible antioxidants in poultry(Reference Sahin, Orhan and Tuzcu32). In a previous experiment, Kermanshahi & Riasi(Reference Kermanshahi and Riasi33) demonstrated that the addition of dried B. vulgaris fruit to the diet of laying hens improved some blood and egg quality parameters that may have merit for the animal's well-being. Antioxidants are shown to modulate the transcription system(Reference Farombi, Shrotriya and Na34, Reference Jung, Hong and Zheng35). However, the role of B. vulgaris root extract as an antioxidant against HS in poultry has not been investigated. The present experiment was performed to elucidate the mechanism by which supplemental B. vulgaris root extract alleviates performance and oxidative stress in Japanese quails (Coturnix coturnix japonica) exposed to HS.
Materials and methods
Plant material and extraction
The plant samples (root of B. vulgaris) were collected during the spring season of 2009 from a forest near Białystok, Poland. After crushing and drying, the samples (50 g) were subjected to extraction with water and methanol in Soxhlet apparatus for 32 h. The extract was concentrated to dryness under vacuum at 40°C. The extraction efficiency was 14·9 (sd 0·6) and 10·5 (sd 0·7) % (w/w), respectively, with water and methanol.
GC/MS analysis conditions
B. vulgaris root extract was assayed using a Perkin Elmer Clarus 680 gas chromatograph (Perkin Elmer Inc.) with a Clarus 600S mass spectrometer (Perkin Elmer Inc.). The separation of the analyte was performed on an Elite 5MS 30 m/0·25 mm column (Perkin Elmer Inc.) at the programmed thermostat temperature of − 80°C at start-up, followed by increments of 5°C/min up to 320°C and isothermally for 22 min. The total time of analysis was 70 min. The injector temperature was 220°C and the flow of the carrier gas 1 ml/min. A split of 20 ml/min was applied. Electron ionisation with 70 eV was used in the spectrometer. The temperatures of the transfer line and the ion source were 240 and 200°C, respectively. The retrieved signals were identified using the National Institute of Standards and Technology mass spectra library. Signals with height three times larger than the background noises were assessed. Due to low content, berberine was identified using the selected ion research method for ions characteristic with masses of 339–164 and 337–321 m/s.
Evaluation of antioxidant activity and total phenolic content
Antioxidant activity of B. vulgaris root extract was determined as described by Velázquez et al. (Reference Velázquez, Tournier and Mordujovich de Buschiazzo36). Briefly, 1·5 ml of a solution of 2,2-diphenyl-1-picrylhydrazyl were added to 0·75 ml of various concentrations of each sample solution ranging from 3·9 to 500 μg/ml. The solution of 2,2-diphenyl-1-picrylhydrazyl in methanol (20 mg/l) was prepared daily before UV measurements. The mixtures were kept in the dark for 15 min at room temperature and the decrease in absorbance was measured at 517 nm against a blank consisting of a 1·5 ml of methanol and 0·75 ml of extract solution. Quercetin and gallic acid were used as positive controls. These were converted to percentage 2,2-diphenyl-1-picrylhydrazyl radical scavenging(Reference Motalleb, Hanachi and Kua37). The half-maximal inhibitory concentration value of each extract was determined graphically and all tests were performed in triplicate. A lower half-maximal inhibitory concentration value indicates stronger antioxidant activity.
Total phenolic content was determined using the Folin–Ciocalteu colorimetric method; the results were expressed as mg of gallic acid equivalents per g of extract(Reference Djeridane, Yousfi and Nadjemi38).
Animals, diets and experimental design
A total of 180, 5-week-old female Japanese quails (C. coturnix japonica) were used in accordance with animal welfare regulations at the Veterinary Control and Research Institute of Elazig, Turkey. All procedures involving birds were approved by the Institutional Animal Care and Use Committee at the Institute of Veterinary Research, Elazig, Turkey (approval code: 2011/5-1). The quails were kept in temperature-controlled rooms at either 22°C for 24 h/d (thermoneutral, TN) or 34°C for 8 h (09.00 and 17.00 hours) followed by 22°C for 16 h/d (HS) during the experimental period. The quails were then fed one of three diets: basal diet and basal diet supplemented with 100 or 200 mg B. vulgaris root extract per kg diet. Chemical composition of B. vulgaris root extract is shown in Table 1. The diets (Table 2) were mixed weekly in batches and stored in black plastic containers at 4°C to avoid photo-oxidation in airtight containers.
RT, retention time (min).
* Total area was 1 099 497 784 and 716 735 822 in extraction with water and methanol, respectively.
* Berberis vulgaris root extract (0, 200 or 400 mg B. vulgaris per kg diet) was added to the basal diet at the expense of maize.
† Per kg contained: vitamin A, 8000 IU; vitamin D3, 3000 IU; vitamin E, 25 IU; menadione, 1·5 mg; vitamin B12, 0·02 mg; biotin, 0·1 mg; folacin, 1 mg; niacin, 50 mg; pantothenic acid, 15 mg; pyridoxine, 4 mg; riboflavin, 10 mg; and thiamin, 3 mg. Cu (copper sulphate), 10·00 mg; iodine (ethylenediamine dihydriodide), 1·00 mg; Fe (ferrous sulphate monohydrate), 50·00 mg; Mn (manganese sulphate monohydrate), 60·00 mg; and Zn (zinc sulphate monohydrate), 60·00 mg; Se (sodium selenite), 0·42 mg.
‡ Calculated value according to tabular values listed for the feed ingredients(Reference Jurgens44).
All quails were hatched from a large group of the parent stock that were of identical age. Each of the 2 × 3 factorially arranged groups was replicated in ten cages (20 × 20 cm2 dimension), each consisting of three quails. During the experimental period (12 weeks), the birds were subjected to a 16 h light–8 h dark cycle and offered feed and water ad libitum.
Sample and data collection
Feed intake was measured weekly and egg production was recorded daily during the experimental period. At the end of the experiment, one bird from each cage (ten birds per group) was killed by cervical dislocation. The liver was removed and chopped into small pieces on ice for determination of the oxidative stress biomarkers, including levels of malondialdehyde (MDA) and antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px)) and expressions of hepatic nuclear transcription proteins (NF-κB, HSP70, Nrf2 and haeme-oxygenase 1 (HO-1)).
Lipid peroxidation and antioxidant enzymes
A 10 % (w/v) liver homogenate was prepared in 10 mm-phosphate buffer (pH 7·4). The homogenate was centrifuged at 13 000 g for 10 min at 4°C. The supernatant was collected and stored at − 80°C. The concentration of MDA, an index of lipid peroxidation and oxidative stress, was measured(Reference Barim and Karatepe39) using the fully automatic HPLC (Shimadzu) equipped with a pump (LC-20AD), an ultraviolet–visible detector (SPD-20A), an inertsil ODS-3 C18 column (250 × 4·6 mm, 5 m), a column oven (CTO-10ASVP), an autosampler (SIL-20A), a degasser unit (DGU-20A5) and a computer system with LC solution Software (Shimadzu). Total SOD activity was attained based on the amount of enzyme required to inhibit the rate of formazan dye formation by 50 %(Reference Spitz and Oberley40). CAT activity was estimated based on the amount of enzyme required to decompose 1 mmol of H2O2 to water and oxygen(Reference Cohen, Dembiec and Marcus41). GSH-Px activity assessment was based on the amount of enzyme required to oxidise 1 mmol NADPH(Reference Lawrence and Burk42). The enzyme activities were expressed as U/mg protein.
Western blot analyses
Accurately weighed liver tissue was homogenised in 1:10 (w/v) ratio in 10 mm-Tris–HCl buffer (pH 7·4) containing 0·1 mm-NaCl, 0·1 mm-phenylmethylsulphonyl fluoride and 5 μm-soyabean (soluble powder; Sigma) as trypsin inhibitor. Tissue homogenate was centrifuged at 15 000 g at 4°C for 30 min and the supernatant was transferred into fresh tubes. SDS-PAGE sample buffer containing 2 % β-mercaptoethanol was added to the supernatant. Equal amounts of protein (20 μg) were electrophoresed and subsequently transferred to nitrocellulose membrane (Schleicher and Schuell, Inc.). Nitrocellulose blots were washed twice for 5 min in PBS and blocked with 1 % bovine serum albumin in PBS for 1 h prior to the application of primary antibody. Chicken antibodies against NF-κB, HSP70, Nrf2 and HO-1 (Abcam) were diluted (1:1000) in the same buffer containing 0·05 % Tween 20. The nitrocellulose membrane was incubated overnight at 4°C with protein antibody. The blots were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Abcam). Specific binding was detected using diaminobenzidine and H2O2 as substrates. Protein loading was controlled using a monoclonal mouse antibody against β-actin antibody (Sigma). Samples were analysed in quadruplicates and protein levels were determined densitometrically using an image analysis system (Image J; National Institutes of Health).
The diets were analysed for crude protein (no. 988·05), Ca (no. 968·08) and P (no. 965·17) according to the procedures described by the Association of Official Analytical Chemists(43). Energy and amino acid (methionine and lysine) contents were calculated from tabular values listed for the feedstuffs(Reference Jurgens44).
Data on performance parameters, oxidative stress biomarkers and proteins were analysed by two-way ANOVA using the PROC GLM procedure(45). The linear model to test the effect of treatments on response variables was as follows: y ijk= μ+Ei+S j+(E× S)ij+e ijk, where y= response variable, μ = population mean, E= environmental temperature, S= B. vulgaris root extract supplementation and e= residual error (N (σ, μ; 0, 1)). The model also included polynomial contrast to determine changes in response variables as supplemental B. vulgaris root extract level was increased. Correlations among performance parameters, oxidative stress biomarkers and nuclear proteins were determined using the CORR procedure(45). Statistical significance was considered at P≤ 0·05.
Composition of Berberis vulgaris and antioxidant activity
Table 1 lists twenty-three compounds detected in B. vulgaris root extract (>0·05 % of total ion current). B. vulgaris root extract antioxidant activity value was 60·24 (sd 6·27) %. Total phenolic content was 8·92 (sd 0·55) mg gallic acid equivalents per g extract.
Table 3 summarises performance variables. Quails exposed to HS consumed less feed (27·6 v. 30·2 g/d) and produced less egg (81·6 v. 92·8 %) than those reared under the TN environment (P< 0·0001 for both). Overall, there were 3·1 and 4·9 % increases in feed intake and egg production in response to increasing dietary B. vulgaris root extract supplementation (linear effect; P< 0·0001 for both). These increases were greater in the HS environment than in the TN environment (environmental temperature × B. vulgaris root extract supplementation level interaction effect, P< 0·001 for both).
ET, environmental temperature; TN, thermoneutral environment (temperature-controlled rooms at 22°C for 24 h/d); HS, heat stress environment (34°C for 8 h between 09.00 and 17.00 hours followed by 22°C for 16 h/d).
Hepatic malondialdehyde and antioxidant enzymes
Heat-stressed quails had higher hepatic MDA level (2·70 v. 1·36 nmol/g) and lower hepatic SOD (126 v. 175), CAT (28 v. 49) and GSH-Px (10 v. 18) activities (U/mg protein) than control quails (P< 0·001 for all). There was a linear decrease in level of MDA (by − 25·5 %) and increases in activities of SOD, CAT and GSH-Px (by 23·5, 35·4 and 55·7 %, respectively) as supplemental B. vulgaris root extract level in the diet increased (P< 0·001 for all). Decrease in hepatic MDA level and increases in hepatic SOD, CAT and GHS-Px activities in response to increasing B. vulgaris root extract supplementation were more notable in the HS environment than in the TN environment (environmental temperature × B. vulgaris root extract level interaction effects; P< 0·001 for all; Table 4).
ET, environment temperature; TN, thermoneutral environment (temperature-controlled rooms at 22°C for 24 h/d); HS, heat stress environment (34°C for 8 h between 09.00 and 17.00 hours followed by 22°C for 16 h/d); MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase; HSP70, heat shock protein 70; Nrf2, nuclear factor (erythroid-derived 2)-like 2; HO-1, haeme oxygenase-1.
* Expressions of nuclear transcription factors are percentage of control (quails reared under the TN condition and not received B. vulgaris root extract).
Hepatic nuclear transcription factors
Expressions of hepatic NF-κB (161·6 v. 94·1 %) and HSP70 (161·2 v. 91·4 %) were greater, whereas that of hepatic Nrf2 (72·1 v. 113·1 %) and HO-1 (69·8 v. 112·8 %) were lower in the heat-stressed quails than the control quails (P< 0·001 for all). With increasing dietary B. vulgaris root extract supplementation, the mean expressions of hepatic NF-κB (by − 22·7 %) and HSP70 (by − 26·6 %) decreased linearly (P< 0·001 for both), whereas the mean expressions of hepatic Nrf2 (by 56·0 %) and HO-1 (by 38·0 %) increased linearly (P< 0·001 for both). Decreases in hepatic NF-κB and HSP70 levels (P< 0·001 for both) and increases in hepatic Nrf2 and HO-1 levels (P< 0·001 for both) in response to increasing supplemental B. vulgaris root extract level were greater in the TN environment than in the HS environment (environmental temperature × B. vulgaris root extract level interaction effects; Table 4).
Correlations among oxidative stress biomarkers and protein levels
All response variables were autocorrelated (P< 0·001; Table 5). There were positive correlations between egg production and activities of antioxidant enzymes and negative correlation between egg production and hepatic MDA level. Hepatic MDA level was negatively correlated with activities of antioxidant enzymes and expressions of Nrf2 and HO-1 and positively correlated with expressions of NF-κB and HSP70. Moreover, activities of antioxidant enzymes were negatively correlated with expressions of NF-κB and HSP70 and positively correlated with expressions of Nrf2 and HO-1.
FI, feed intake; EP, egg production; MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase; HSP70, heat shock protein 70; Nrf2, nuclear factor (erythroid-derived 2)-like 2; HO-1, haeme oxygenase-1.
* P< 0·001 for correlation coefficients for all paired variables.
HS compromises performance and productivity through reducing feed intake, while decreasing growth rate, egg production, egg quality and feed efficiency, which lead to economic losses in poultry. HS also leads to oxidative stress associated with a reduced antioxidant status in the bird in vivo, as reflected by increased oxidative damage and lowered plasma concentrations of antioxidants. In agreement with the literature, HS caused depressions in feed intake and egg production (Table 3). Moreover, supplemental B. vulgaris extract increased feed intake and egg production at a greater extent in heat-stressed quails than control quails (Table 3). These positive impacts of B. vulgaris root supplementation on performance are in agreement with a previous study(Reference Mujahid, Pumford and Bottje47, Reference Mujahid, Pumford and Bottje47). Moreover, Kermanshahi & Riasi(Reference Kermanshahi and Riasi33) reported that dried B. vulgaris fruit significantly improved haematocrit value and HDL-cholesterol in laying hens.
Cell damage occurring due to oxidative stress results in lipid peroxidation(Reference Mujahid, Pumford and Bottje47, Reference Tan, Yang and Fu48). This is reflected by elevated hepatic MDA level (Table 4). B. vulgaris root extract is a free radical scavenger and protects membrane stability(Reference Zovko-Koncić, Kremer and Karlović13, Reference Tomosaka, Chin and Salim49). Several studies reported that B. vulgaris root extract possesses antioxidant activity at different degrees in different extraction solvents(Reference Motalleb, Hanachi and Kua37, Reference Hanachi and Golkho50). Hanachi & Golkho(Reference Hanachi and Golkho50) reported that the antioxidant activity of ethanol extract was the highest (27·26 %), followed by extraction with butylated hydroxytoluene (20·29 %), methanol extract (16·80 %), vitamin E (6·68 %) and water (6·53 %). Moreover, because the extract is a pool of many named and unnamed compounds, it is always difficult to conclude the responsibility of each chemical constituent.
Living organisms facilitate the antioxidant defence system through increasing activities of detoxifying enzymes (SOD, CAT and GSH-Px (Table 4)) in order to reduce harmful effects of reactive oxygen molecules produced due to HS(Reference Tan, Yang and Fu48). B. vulgaris root extract supplementation elevates the activity of SOD, CAT and GSH-Px (Table 4), probably due to its antioxidant nature. Due to limitation of literature regarding the effects of B. vulgaris root extract on the antioxidant enzymes, the present enzyme data are not comparable. However, its antioxidant effects were postulated to be linked to cytotoxicity in human liver cell lines(Reference Hanachi, Kua and Asmah51). In the present study, B. vulgaris root extract's antioxidant activity was 60·24 (sd 6·27) %, which is in agreement with a report by Zovko-Koncić et al. (Reference Zovko-Koncić, Kremer and Karlović13). It was shown that total phenolic content of B. vulgaris increases the antioxidant activity(Reference Motalleb, Hanachi and Kua37). In the present study, total phenolic content of B. vulgaris root extract was 8·92 (sd 0·55) %. Despite not determined in the present study, other studies showed that antioxidant effects of B. vulgaris root extract could be related to its vitamin C, malic acid and tannin contents(Reference Hanachi and Golkho50). In oxidative stress induced in mice by CCl4 intoxication, Domitrović et al. (Reference Domitrović, Jakovac and Blagojević52) reported an increase in MDA level and a decrease in SOD activity, which were ameliorated by B. vulgaris root extract. This was attributed to berberine, an isoquinoline alkaloid found in B. vulgaris root extract. Similarly, the present study demonstrated aggravated lipid peroxidation and suppressed activities of oxidative enzymes in heat-stressed quails, which were alleviated by B. vulgaris root extract supplementation in a dose–response manner (Table 4).
NF-κB and Nrf2 are redox-sensitive transcription factors(Reference Nair, Li and Kong53) and play a role in induction of phase II detoxifying/antioxidant defence mechanisms to cope with oxidative stress through enhancing the expression of a number of enzymes, such as NAD(P)H quinone oxidoreductase 1, glutamate cysteine ligase, HO-1, glutathione S-transferase and uricline diphosphate (UDP)-glucuronosyltransferase(Reference Na and Surh54). In addition, NF-κB is induced by various cell stress-associated stimuli, including oxidative stress. HS-induced oxidative stress increases production of reactive oxygen molecules that lead to the activation of various redox-sensitive cell signalling molecules such as NF-κB(Reference Surh, Kundu and Na55, Reference Rahman, Biswas and Kirkham56). The literature on the NF-κB and Nrf2 pathway activity in response to dietary supplementation of B. vulgaris root extract in poultry is limited. In the present study, increased expression of NF-κB (Table 4) and decreased expression of Nrf2 (Table 4) in heat-stressed quails could be related to their activation(Reference Gilmore23, Reference Nelson, Ihekwaba and Elliott24) and translocation(Reference Yamamoto, Suzuki and Kobayashi30, Reference Nguyen, Nioi and Pickett57), respectively, to overcome oxidative stress induced by HS. In the present study, the levels of berberine were 0·03 %. Among several alkaloids of B. vulgaris, berberine was considered to be responsible for most of the biological activity, such as abolished acetaldehyde-induced NF-κB activity and cytokine production in a dose-dependent manner(Reference Hsiang, Wu and Cheng58). Liu et al. (Reference Liu, Zhang and Liu59) also reported that the immunostaining of NF-κB was decreased and the reduced degradation of inhibitor of κB level was partially restored after berberine treatment in alloxan-induced diabetic mice. Increased berberine-increased HO-1 expression is mediated by Nrf2 activation(Reference Chen, Huang and Tan60), suggesting that B. vulgaris root extract acts as modifier of signal transduction pathways to elicit cytoprotective responses.
Several studies have shown that HS triggers expression of HSP70 induction(Reference Mahmoud, Edens and Eisen25, Reference Sahin, Tuzcu and Orhan61). To our knowledge, the present study is the first to evaluate the effect of the B. vulgaris root extract supplementation on HSP in the liver of heat-stressed Japanese quail. In the present study, the HSP70 expression was much lower in quails supplemented with B. vulgaris root extract than control quails (Table 4). Other studies have ascertained the protective role of antioxidants by suppressing HSP expressions in stressed birds(Reference Sahin, Tuzcu and Orhan61, Reference Mahmoud and Edens62).
In conclusion, B. vulgaris root extract supplementation compensated depression in performance variables (feed intake and egg production) in heat-stressed quails and alleviated oxidative stress, as reflected by lipid peroxidation and activity of antioxidant enzymes. Moreover, B. vulgaris root extract exerted antioxidant effects through inhibiting NF-κB and HSP70 expressions and activating Nrf2 and HO-1 expressions, which were activated and suppressed in the HS environment, respectively. Future studies should focus on elucidation of the effect of chemicals on the modulation of oxidative stress biomarkers and nuclear transcription proteins.
The authors thank the Veterinary Control and Research, Institute of Ministry of Agriculture, Elazig, Turkey, for providing the research facility. This work was supported by Grant N N405 625438 from the National Research Committee, Warsaw, Poland. The authors declare no conflicts of interest. The contribution of the authors is as follows: K. S., M. H. B. and N. S. were involved in the study design. N. S., C. O., M. T., J. J., A. H. and O. G. performed animal experimentation, sampling, laboratory analyses and data collection/analyses. A. H., M. H. B. and K. S. wrote the first draft of the manuscript. All authors contributed to the final version of the manuscript. K. S. had primary responsibility for the final content. This work was also supported in part by the Turkish Academy of Sciences.