PHYTOTHERAPY RESEARCH Phytother. Res. (2014) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5184
Modulating Effects of Pycnogenol® on Oxidative Stress and DNA Damage Induced by Sepsis in Rats Gökçe Taner,1* Sevtap Aydın,2 Merve Bacanlı,2 Zehra Sarıgöl,2 Tolga Şahin,3 A. Ahmet Başaran4 and Nurşen Başaran2 1
Department of Department of Department of 4 Department of 2 3
Biology, Faculty of Science, Gazi University, Ankara, Turkey Pharmaceutical Toxicology, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey Surgery, Faculty of Medicine, Kastamonu University, Ankara, Turkey Pharmacognosy, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey
The aim of this study was to evaluate the protective effects of Pycnogenol® (Pyc), a complex plant extract from the bark of French maritime pine, on oxidative stress parameters (superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities and total glutathione (GSH) and malondialdehyde (MDA) levels), an inflammatory cytokine (tumor necrosis factor alpha (TNF-α) level) and also DNA damage in Wistar albino rats. Rats were treated with 100 mg/kg intraperitonally Pyc following the induction of sepsis by cecal ligation and puncture. The decreases in MDA levels and increases in GSH levels, and SOD and GPx activities were observed in the livers and kidneys of Pyc-treated septic rats. Plasma TNF-α level was found to be decreased in the Pyc-treated septic rats. In the lymphocytes, kidney, and liver tissue cells of the sepsis-induced rats, Pyc treatment significantly decreased the DNA damage and oxidative base damage using standard alkaline assay and formamidopyrimidine DNA glycosylase-modified comet assay, respectively. In conclusion, Pyc treatment might have a role in the prevention of sepsis-induced oxidative damage not only by decreasing DNA damage but also increasing the antioxidant status and DNA repair capacity in rats. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Sepsis; Pycnogenol; DNA damage; Alkaline comet assay; Fpg-modified comet assay.
INTRODUCTION Sepsis is a complex multiple organ dysfunction syndrome characterized by an imbalance between pro- and antiinflammatory response to pathogens (Hotchkiss and Karl, 2003). It is known as a common cause of morbidity and mortality in the intensive care units (Dare et al., 2009). Oxidative damage is suggested to be one of the major factors that lead to cell damage, multiorgan dysfunction and death in sepsis (Messaris et al., 2004). Since the involvement of free radicals in the pathogenesis of sepsis and its degradation by cellular antioxidants is well documented (Andrades et al., 2009; Prauchner et al., 2011), the use of antioxidants as complementary tools for the treatment of the disease has been evaluated in both experimental and clinical models (Zimmermann, 1995; Fox et al., 1997; Messaris et al., 2004; Prauchner et al., 2011). Pycnogenol® (Pyc), a registered trademark of Horphag Research Ltd., is a water-soluble extract of French maritime pine bark consisting of several types of phenolic compounds (catechin, epichatechin and taxofolin), condensed flavonoids (procyanidines and proanthocyanidines) and phenolic acids (caffeic, ferulic and p-hydroxybenzoid acid) (Packer et al., 1999; Moini et al., 2000). Between 65% and 75% of Pyc are procyanidins comprising catechin and epicatechin * Correspondence to: Gökçe Taner, Department of Biology, Faculty of Science, Gazi University, 06500, Teknikokullar, Ankara, Turkey. E-mail:
[email protected] Copyright © 2014 John Wiley & Sons, Ltd.
subunits with varying chain lengths (Packer et al., 1999; D’Andrea, 2010). Pyc has been also suggested to have the ability to enhance the status of the antioxidants, vitamin E, total glutathione (GSH) and antioxidant enzymes (Packer et al., 1999; Siler-Marsiglio et al., 2004; Wei et al., 1997; Gulati, 2014). It has been reported to have diverse beneficial pharmacological effects on a wide range of medical conditions, inflammation and immune diseases (Parveen et al., 2010; Gulati, 2014). It is suggested that Pyc due to its free radical scavenging and antioxidant properties ameliorated oxidative organ injury and also DNA damage. The comet assay (SCGE; single cell gel electrophoresis) is a rapid, simple and sensitive technique for the measurement of DNA damage at the single cell level (Faust et al., 2003; Olive, 1999). The comet assay detects single and double-stranded breaks at the level of DNA molecule, sites of incomplete repair, alkali labile sites, DNA–DNA and DNA–protein cross-links. In addition, lesion specific repair enzymes such as formamidopyrimidine glycosilase (Fpg) can be used for detection of oxidative damage at the level of the DNA molecule caused by reactive oxygen species (ROS) (Collins et al., 1993; Krokan et al., 1997; Speit et al., 2004). The previous studies have proven that when Fpg is used in the comet assay, in addition to oxidized purines (products of DNA oxidation), apurinic sites and various ring-opened purine adducts are also detected (Speit et al., 2004). Fpg initiates the repair of oxidized bases by excising them and cutting the sugar-phosphate backbone of the DNA molecule. Thus, additional strand breaks are induced at the location of oxidized base, Received 21 February 2014 Revised 12 May 2014 Accepted 12 May 2014
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causing DNA relaxation and migration. The detection of Fpg-sensitive DNA lesions revealed the presence of oxidized purine bases (Collins et al., 1993; Collins et al., 1996). In the present study, the aim was to investigate the in vivo modulating effects of Pyc on sepsis-induced DNA damage in the lymphocytes and the kidney and liver cells of Wistar albino rats using the standard alkaline and Fpg-modified comet assays. The oxidative stress parameters such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and GSH and malondialdehyde (MDA) levels in the liver and kidney tissues of rats were also evaluated.
MATERIALS AND METHODS Chemicals. The chemicals used in the experiments were purchased from the following suppliers: Pyc, a registered trade mark of Horphag Research Ltd., Geneva, Switzerland), was provided by Henkel Corporation (La Grange, IL, U.S.A.); SOD assay kit, GPx assay kit and GSH assay kit from Cayman Chemicals Co. (Ann Arbor, MI, USA); normal melting point agarose (NMA) and low melting point agarose (LMA) from Boehringer Manheim (Mannheim, Germany); sodium chloride (NaCl), sodium hydroxide (NaOH), potassium chloride (KCl) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) from Merck Chemicals (Darmstadt, Germany); formamidopyridine DNA glycosylase (Fpg), bovine serum albumin, dimethyl sulfoxide (DMSO), ethidium bromide (EtBr), Triton X-100, phosphatebuffered saline (PBS) tablets, trichloroacetic acid, thiobarbituric acid and n-butanol from Sigma-Aldrich Chemicals (St. Louis, Missouri, USA); and etheylenediamine tetraacetic acid disodium salt dehydrate (EDTA-Na2), natrium lauroyl sarcosinate and Tris from ICN Biochemicals Inc. (Aurora, Ohio, USA).
Animals. A total of 32 Wistar albino rats (3 months old, 200–300 g), from Refik Saydam National Public Health Agency in Ankara, were used in this study. Animals were housed in plastic cages with stainless-steel grid tops. Rats were subjected to a controlled environment regarding temperature (23 °C), humidity (50%) and a 12-h light– dark cycle. Animals were fed with standard laboratory chow and allowed to access ad libitum feed and drinking water before and after operation. The animals were treated humanely and with regard for alleviation of suffering, and the study was approved by Hacettepe University Animal Ethical Committee (2011/45-14).
Experimental groups and procedures. The 32 rats were divided into four experimental groups Group I Group II Group III Group IV
: Sham group (n = 8) : Sepsis-induced group (n = 8) : 100 mg/kg i.p. Pyc-treated group (n = 8) : 100 mg/kg i.p. Pyc-treated + sepsis-induced group (n = 8)
All surgical procedures were performed under anesthesia by intraperitonal (i.p.) injection of 90 mg/kg ketamine hydrochloride (Ketalar, Eczacıbaşı WarnerCopyright © 2014 John Wiley & Sons, Ltd.
Lambert, Istanbul, Turkey). Sepsis was induced by cecal ligation and puncture (CLP). Under the anesthesia, a midline laparatomy was made using minimal dissection, and cecum was ligated just below the ileocecal valve with 3-0 silk ligatures so that intestinal continuity was maintained. On the antimesentric surface of the cecum, using an 18-gauge needle, the cecum was perforated at two locations 1 cm apart, and the cecum was gently compressed until the feces were extruded. The bowel was then returned to the abdomen, and the incision was closed. At the end of the operation, all rats were resuscitated with saline (5 mL/100 g bw) subcutaneously. The rats were deprived of food but had free access of water after the operation. The sham-operated control group underwent laparotomy; the cecum was manipulated but was not ligated or perforated. For the experimental groups, the sham group consisted of animals treated i.p. with 0.5 mL of saline alone following laparatomy. Abdominal layers were closed with appropriate suture materials. All animals were maintained under the same conditions after surgery. The sepsis group consisted of animals in which only CLP was performed, and the animals were treated with 0.5-mL saline i.p. following the induction of CLP. The Pyc-treated group consisted of animals immediately treated with a dose of 100 mg/kg i.p. Pyc in 0.5-mL saline following laparatomy. The Pyc treatment dose was selected according to the study of Ince et al. (2009). They studied 50 mg/kg, 75 mg/kg and 100 mg/kg Pyc i.p. in a rat model of carrageenan-induced inflammation. The Pyc-treated and sepsis-induced group consisted of animals immediately treated with 0.5 mL of a solution containing 100 mg/kg i.p. Pyc following the induction of CLP. After 24 h following the treatment, all rats were decapitated under an anesthesia. The liver and kidney tissues were carefully dissected from its attachment and totally excised, and cardiac blood was collected into preservative-free heparin tubes for DNA damage analysis. The samples were kept in the dark at 4 °C and processed within 4 h.
Determination of oxidative stress parameters in the liver and kidney tissues. The liver and kidney tissues were weighted and extracted following the homogenization and sonication procedure (Sier et al., 1996). The homogenates of the tissue samples were kept 80 °C until the time of analysis. The determination of SOD and GPx enzyme activities in the liver and kidney tissues was performed with a SOD and GPx assay kit (Cayman Chemicals Co., Ann Arbor, MI, USA) at 440 and 340 nm, respectively. Results were expressed as mmol/min/mg tissue. GSH levels were determined spectrophotometrically with a GSH assay kit (Cayman Chemicals Co., Ann Arbor, MI, USA) at 405 nm according to the manufacturer’s introduction. Results were expressed as nmol/mg tissue. The levels of MDA, a biomarker of lipid peroxidation, were determined spectrophotometrically by measuring thiobarbituric acid-reactive substances (Uchiyama and Mihara, 1978). To 0.5 mL of the 10% homogenates of the tissue samples, 2.5 mL of 20% trichloroacetic acid and 1.0 mL of 0.67% thiobarbituric acid were added. Phytother. Res. (2014)
PROTECTİVE EFFECTS OF PYCNOGENOL® ON SEPSİS-İNDUCED DNA DAMAGE
The mixtures were incubated at 100 °C for 30 min. n-Butanol (4 mL) was added after cooling and mixed vigorously. After centrifugation, absorbance of the butanol layer was measured at 535 nm. For the standard curve, 1,1,3,3-tetraethoxypropane was used. The results were expressed as nmol/g tissue.
Detemination of plasma tumor necrosis factor alpha (TNF-α) levels. Whole blood samples were obtained via the intracardiac method. The plasma was immediately separated by centrifugation at 4000 rpm for 10 min at +4 °C and stored at 80 °C until being assayed. TNF-α from each sample was measured at 30 min in duplicate according to the manufacturer’s instructions with highly sensitive enzyme-linked immunosorbent assay (ELISA) kits containing Invitrogen KRC0011 (USA) at 450 nm. The results were expressed as pg/mL.
Single cell gel electrophoresis (comet assay) and Fpg-modified comet assay. The basic alkaline technique of Singh et al. (1988) as further described by Anderson et al. (1994) and Collins et al. (1997) was followed. Lymphocytes from whole heparinized blood were separated by Ficoll-Hypaque density gradient and centrifugation (Boyum, 1976), and then the cells were washed with PBS buffer. A small piece of liver and kidney tissues was placed in 1 mL of cold Hanks Balanced Salt Solution containing 20 mM EDTA/10% DMSO and is minced into fine pieces. After it was settled, the supernatant was used. The concentration of the lymphocytes and renal and hepatic tissue cells was adjusted to approximately 2 × 106 cells/mL in PBS buffer. The cells were suspended in 75 μL of 0.5% LMA. The suspensions were then embedded on slides precoated with a layer of 1% NMA. Slides were allowed to solidify on ice for 5 min. Coverslips were then removed. The slides were immersed in cold lysing solution (2.5 M NaCl, 100 mM EDTA, 100 mM Tris, 1% sodium sarcosinate, and pH: 10), with 1% Triton X-100 and 10% DMSO added just before use for a minimum of 1 h at 4 °C. Then they were removed from the lysing solution, drained and were left in the electrophoresis solution (1 mM sodium EDTA and 300 mM NaOH, pH:13) for 20 min at 4 °C to allow unwinding of the DNA and expression of alkali-labile damage. The alkaline comet assay using Fpg, lesion-specific enzyme was used to detect oxidized pyrimidines as a result of oxidative stress-induced DNA damage as described with some modifications (Collins et al., 1993). The cell–agarose suspension slides were prepared as described earlier for the standard comet assay. After lysing, the slides were washed three times for 5 min with the enzyme buffer (40 mM HEPES, 100 mM KCl, 0.5 mM EDTA and 0.2 mg/mL bovine serumalbumin) at room temperature and were incubated at 37 °C for 30 min with Fpg (1:500) and with enzyme buffer (control). Then, they were left in the electrophoresis solution (1 mM sodium EDTA and 300 mM NaOH, pH: 13) for 20 min at 4 °C to allow unwinding of the DNA and expression of alkali-labile damage. Electrophoresis was also conducted at a low temperature (4 °C) for 20 min using 24 V and adjusting the current to 300 mA by raising or lowering the buffer level. The slides were Copyright © 2014 John Wiley & Sons, Ltd.
neutralized by washing three times in 0.4 M Tris–HCL (pH: 7.5) for 5 min at room temperature. After neutralization, the slides were incubated in 50%, 75% and 98% of alcohol for 5 min each. The dried microscopic slides were stained with EtBr (20 mg/mL in distilled water, 60 mL/slide), covered with a cover glass prior to analysis with a Leica® fluorescence microscope under green light. The microscope was connected to a charge-coupled device camera and a personal computer-based analysis system (Comet Analysis Software, version 3.0, Kinetic Imaging Ltd, Liverpool, UK) to determine the extent of DNA damage after electrophoretic migration of the DNA fragments in the agarose gel. In order to visualize DNA damage, slides were examined at 40× magnification. Results were expressed as tail length, tail intensity and tail moment. From each of the two replicate slides, 100 cells were assayed.
Statistical analysis. Statistical analysis was performed by SPSS for Windows 15.0 computer program. The results were expressed as mean ± standard deviation. The differences among the groups for oxidative stress parameters were evaluated with Kruskal–Wallis test, followed by Mann–Whitney U test. For comet assay, differences between the means of data were compared by the oneway analysis of variance test, and the post hoc analysis of group differences was performed by least significant difference test. p values of less than 0.05 were considered as statistically significant.
RESULTS Oxidative stress parameters in the liver and kidney tissues The SOD and GPx enzyme activities and GSH and MDA levels in the liver and kidney tissues are shown in Tables 1 and 2, respectively. Hepatic and renal SOD enzyme activities were found to significantly decrease in the sepsis group compared to the sham group ( p < 0.05). Although SOD enzyme activities in both kidney and liver in the sepsis + Pyc group were found to be significantly lower than the sham group ( p < 0.05), SOD enzyme activities in both kidney and liver increased significantly in the sepsis + Pyc group compared to the sepsis group ( p < 0.05). Hepatic and renal GPx enzyme activities were found to significantly decrease in the sepsis group compared to the sham group (p < 0.05). GPx enzyme activities in both kidney and liver in the sepsis + Pyc group were significantly lower than the sham group (p < 0.05). However, GPx enzyme activities increased significantly in the sepsis + Pyc group compared to the sepsis group ( p < 0.05). Hepatic GSH levels were found to significantly decrease in both sepsis and sepsis + Pyc groups compared to the sham group (p < 0.05). But the levels were found to significantly increase in the sepsis + Pyc group compared to the sepsis group ( p < 0.05). Renal GSH levels were found to significantly decrease in the sepsis group compared to the sham group (p < 0.05). The levels were found to significantly increase in the sepsis + Pyc group compared to the sepsis group ( p < 0.05). Phytother. Res. (2014)
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Table 1. Liver antioxidant enzyme activities and total glutathione and malondialdehyde levels SOD activity (mmol/min/mg tissue) Sham group Sepsis group Sepsis + Pyc group Pyc group
GPx activity (nmol/min/mL)
a
GSH levels (nmol/mg tissue)
a
164.6 ± 6.9 b 131.6 ± 5.9 c 149.2 ± 7.29 a 164.0 ± 8.6
a
86.58 ± 4.69 b 20.34 ± 3.31 c 58.84 ± 7.80 a 78.72 ± 5.03
3.52 ± 0.40 b 1.05 ± 0.30 c 2.79 ± 0.27 a 3.92 ± 0.41
MDA levels (nmol/g tissue) a
11.32 ± 2.73 b 24.80 ± 4.36 c 16.52 ± 3.16 a 11.32 ± 1.16
The results were given as mean ± standard deviation for eight rats in each group. SOD, superoxide dismutase; GPx, glutathione peroxide; GSH, total glutathione; and MDA, malondialdehyde. Superscripts of different letters differ significantly (p < 0.05) from each other within the same column.
Table 2. Renal antioxidant enzyme activities and total glutathione and malondialdehyde levels SOD activity (mmol/min/mg tissue) Sham group Sepsis group Sepsis + Pyc group Pyc group
a
148.0 ± 15.6 b 102.2 ± 12.1 c 136.5 ± 8.7 a 149.6 ± 9.3
GPx activity (nmol/min/mL)
GSH levels (nmol/mg tissue) a
123.78 ± 10.99 b 46.94 ± 6.42 c 78.84 ± 11.13 a 123.02 ± 5.88
a
2.13 ± 0.47 b 0.47 ± 0.24 c 1.61 ± 0.37 a 2.26 ± 0.41
MDA levels (nmol/g tissue) a
13.81 ± 0.80 b 32.20 ± 4.35 c 18.16 ± 1.72 a 12.63 ± 1.62
The results were given as mean ± standard deviation for eight rats in each group. SOD, superoxide dismutase; GPx, glutathione peroxide; GSH, total glutathione; and MDA, malondialdehyde. Superscripts of different letters differ significantly (p < 0.05) from each other within the same column.
Hepatic MDA levels were found to significantly increase in both sepsis and sepsis + Pyc groups compared to the sham group (p < 0.05). But the levels were found to significantly decrease in the sepsis + Pyc group compared to the sepsis group ( p < 0.05). Renal MDA levels were found to significantly increase in the sepsis group compared to the sham group ( p < 0.05). The levels were found to significantly decrease in the sepsis + Pyc group compared to the sepsis group (p < 0.05). In all oxidative stress parameters studied, Pyc alone did not cause significant changes compared to the sham group.
Detemination of plasma TNF-α levels Plasma TNF-α levels were found to be significantly increased in both sepsis and sepsis + Pyc groups compared to the sham group ( p < 0.05). But the level was found to be significantly decreased in the sepsis + Pyc group compared to the sepsis group (p < 0.05) (Fig. 1).
Single cell gel electrophoresis (comet assay) and Fpg-modified comet assay The DNA damage expressed as tail length, tail intensity, and tail moment in the lymphocytes and the liver and kidney cells of Wistar albino rats in alkaline and Fpg modified comet assay were given in Figs. 2, 3 and 4, respectively. In the standard comet assay, there were no statistically significant differences in terms of tail length, tail intensity and tail moment for lymphocytes and liver cells between the sham group and the Pyc-treated group ( p < 0.05). Even in the kidney cells, DNA damage of the Pyc-treated group was lower than the sham group. The parameters of tail length, tail intensity and tail Copyright © 2014 John Wiley & Sons, Ltd.
Figure 1. Plasma TNF-α levels in study group. The results were given as mean ± standard deviation for eight rats in each group. TNF-α, tumor necrosis factor alpha; Pyc, pycnogenol. Bars that do not share same letters (superscripts) are significantly different from each other (p < 0.05).
moment were found to be significantly higher in the sepsis-induced group in comparison with the sham group ( p < 0.05). Pyc treatment in the sepsis-induced group was also found to decrease the DNA damage significantly ( p < 0.05). In the Fpg-modified comet assay, no statistically significant differences in terms of tail length, tail intensity and tail moment for the lymphocytes, liver and kidney tissue cells were also found in sham and the Pyc-treated groups ( p < 0.05). The parameters of tail length, tail intensity and tail moment were all found to be significantly higher in the sepsis-induced group than the sham group ( p < 0.05). Pyc treatment in the sepsis-induced group was found to decrease the DNA damage significantly ( p < 0.05). Furthermore, the enzyme-sensitive sites are more prone for the standard breaks, which showed significantly higher DNA damage in the Fpg-modified comet assay than the standard comet assay. Phytother. Res. (2014)
PROTECTİVE EFFECTS OF PYCNOGENOL® ON SEPSİS-İNDUCED DNA DAMAGE
Figure 2. DNA damage in lymphocytes of the experimental groups expressed as (A) DNA tail length; (B) DNA tail intensity; and (C) DNA tail moment. The values are expressed mean ± standard a deviation (n = 8). Pyc, pycnogenol. p < 0.05, compared with b p < 0.05, the sham group for the standard comet assay; compared with the sepsis group for the standard comet assay; c p < 0.05, standard comet assay was compared to Fpg-modified d comet assay; p < 0.05, compared with the sham group for the e Fpg-modified comet assay; and p < 0.05, compared with sepsis group for the Fpg-modified comet assay.
For the lymphocytes, the tail length obtained from the Fpg-modified comet assay was found to be 1.2-, 2.0-, 1.2and 1.6-fold higher than that of standard comet assay for the sham, sepsis, Pyc and sepsis + Pyc groups, respectively. The tail intensity values obtained from the Fpg-modified comet assay were found to be 1.2-, 3.1-, 1.4- and 3.3-fold higher than that of standard comet assay for the sham, sepsis, Pyc and sepsis + Pyc groups, respectively. The tail moment values obtained from the Fpg-modified comet assay were found to be 1.5-, 5.9-, 1.6- and 5.2-fold higher than that of standard comet assay for the sham, sepsis, Pyc and sepsis + Pyc groups, respectively. For the liver cells, the tail length obtained from the Fpg-modified comet assay was found to be 1.3-, 1.8-, 1.2and 1.6-fold higher than that of standard comet assay for the sham, sepsis, Pyc and sepsis + Pyc groups, respectively. The tail intensity values obtained from the Fpg-modified comet assay were found to be 1.3-, 2.5-, 1.3- and 2.1-fold higher than that of standard comet assay for the sham, sepsis, Pyc and sepsis + Pyc groups, respectively. The tail moment values obtained from the Fpg-modified comet Copyright © 2014 John Wiley & Sons, Ltd.
Figure 3. DNA damage liver cells of the experimental groups expressed as (A) DNA tail length; (B) DNA tail intensity; and (C) DNA tail moment. The values are expressed mean ± standard a deviation (n = 8). Pyc, pycnogenol. p < 0.05, compared with b the sham group for the standard comet assay; p < 0.05, compared with the sepsis group for the standard comet assay; c p < 0.05, standard comet assay was compared to Fpg-modified d comet assay; p < 0.05, compared with the sham group for the e Fpg-modified comet assay; and p < 0.05, compared with the sepsis group for the Fpg-modified comet assay.
assay were found to be 1.7-, 6.9-, 1.8- and 3.9-fold higher than that of standard comet assay for the sham, sepsis, Pyc and sepsis + Pyc groups, respectively. For the kidney cells, the tail length obtained from the Fpg-modified comet assay was found to be 1.4-, 2.4-, 1.4and 2.1-fold higher than that of standard comet assay for the sham, sepsis, Pyc and sepsis + Pyc groups, respectively. The tail intensity values obtained from the Fpg-modified comet assay were found to be 1.4-, 3.6-, 1.6- and 3.4-fold higher than that of standard comet assay for the sham, sepsis, Pyc and sepsis + Pyc groups, respectively. The tail moment values obtained from the Fpg-modified comet assay were found to be 1.8-, 8.0-, 1.8- and 5.1-fold higher than that of standard comet assay for the sham, sepsis, Pyc and sepsis + Pyc groups, respectively.
DISCUSSION Phenolic compounds such as flavonoids found in daily diets have various appreciable effects on diseases Phytother. Res. (2014)
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Figure 4. DNA damage kidney cells of the experimental groups expressed as (A) DNA tail length; (B) DNA tail intensity; and (C) DNA tail moment. The values are expressed mean ± standard devia ation (n = 8). Pyc, pycnogenol. p < 0.05, compared with the b sham group for the standard comet assay; p < 0.05, compared c with the sepsis group for the standard comet assay; p < 0.05, standard comet assay was compared to Fpg-modified comet d assay; p < 0.05, compared with the sham group for the Fpge modified comet assay; and p < 0.05, compared with the sepsis group for the Fpg-modified comet assay.
such as cancer, neurodegenerative diseases, diabetes, obesity, cardiovascular and respiratory diseases (Gulati, 2014), and experimental data are accumulating regarding phenolic compounds as natural phytochemical antioxidants important for human health (Romano et al., 2013). Biochemical and histopathologic indices of inflammation, oxidative and nitrosative stress, apoptosis and necrosis were lowered by polyphenols as were markers for all liver, kidney and pancreatic injury and dysfunction (Shapiro et al., 2009). Research on antioxidant substances has focused on the potential benefits of both purified phytochemicals and plant extracts such as the pine bark extract (PBE) Pyc. The studies indicate that Pyc components are highly bioavailable. Pyc is a procyanidin-enriched extract of Pinus pinaster bark, and it consists of a variety of flavonoids, which are known as potent antioxidants (Lee et al., 2010). Uniquely, Pyc displays greater biologic effects as a mixture than its purified components do individually, indicating that the components Copyright © 2014 John Wiley & Sons, Ltd.
interact synergistically (Masquelier et al., 1979; Packer et al., 1999). Pyc has been extensively used in Europe as a dietary food supplement. It has been suggested to have free radical scavenging and antioxidant properties (Parveen et al., 2010; D’Andrea, 2010), to protect the biomolecules (proteins) against oxidative damage (Voss et al., 2006), and to ameliorate oxidative organ injury and also DNA damage. A number of different studies have addressed the antioxidant capacity of Pyc using either simplified assay systems in vitro or by testing its activity in cultured cell models and in perfused organs. Pyc has been reported to have diverse beneficial effects in many in vitro and in vivo studies (Taner et al., 2013; Shapiro et al., 2009). Despite the growing evidence that ROS is important during sepsis development and its potential as therapeutic target, there is a lack of evidence supporting the actual role and source of ROS in organ dysfunction and the advantage of antioxidant administration over usual sepsis therapies (Andrades et al., 2011). In this study, we determined the modulating effects of Pyc treatment on oxidative stress parameters and DNA damage induced by sepsis. In our study, the oxidative stress parameters such as MDA and GSH levels, and SOD and GPx activities in the liver and kidney tissues were evaluated. Among the various markers, MDA is a lipid peroxidation product formed by free radical attack of lipids (Del Rio et al., 2005), and a very common and simple biomarker of oxidative stress remains the most useful in clinical settings. On the other hand, intracellular antioxidant mechanisms against the inflammatory stresses involve antioxidant enzymes like SOD and molecules like GSH in tissue (Dotan et al., 2004). In our experiments, we observed that hepatic and renal MDA levels were significantly increased and hepatic and renal GPX and SOD enzyme activities and GSH levels were significantly decreased in sepsis induced rats. The description of oxidative stress in sepsis is related to the increased production of ROSs and reduction in antioxidant levels (Himmelfarb et al., 2004). The increase in MDA levels and the concomitant decrease in GSH levels demonstrate the role of oxidative mechanisms in sepsisinduced tissue damage (Şener et al., 2005). Our findings are consistent with the data of some recent studies showing the increase in oxidative stress parameters in sepsis. Increased MDA as a biomarker of lipid peroxidation and decreased antioxidant enzymes such as GSH are the oxidative stress signals observed in sepsis (Galley et al., 1996; Crimi et al., 2006; Berger and Chioléro, 2007). It was recently suggested that antioxidants might counteract the toxicity of oxygen radicals, and this antioxidant remedy for the treatment of sepsis could be useful in the clinical setting of sepsis-induced multiple organ failure (Heller et al., 2001). It has been proposed that antioxidants may restore the cellular defense mechanisms and block lipid peroxidation, thereby protecting against oxidative tissue damage. Glutathione depletion, which is often associated with sepsis, was suggested to be detrimental, impairing the host response to infection (Villa et al., 2002). In accordance with our findings, Şener et al. reported that the melatonin by its free radical scavenging and antioxidant properties ameliorated oxidative damage as demonstrated Phytother. Res. (2014)
PROTECTİVE EFFECTS OF PYCNOGENOL® ON SEPSİS-İNDUCED DNA DAMAGE
by the increased lipid peroxidation and decreased GSH levels in sepsis. Shapiro et al. (2009) reported the effects of different polyphenols in the prevention and treatment of sepsis. Li et al. (2013) investigated the mitochondria-induced oxidative stress during cardiac apoptosis in septic rats, and both SOD and GPx activities decreased, while mitochondrial lipid and protein oxidation increased between 6 and 24 h after lipopolysaccharide challenge. Janka et al. (2013) also found the increases in GPx activities in the septic patients treated with antioxidant selenium compared to negative correlation in placebo subgroups. Consistent with the studies, we found that Pyc treatment clearly reversed all the alterations induced by sepsis. Pyc treatment significantly decreased the MDA levels and also significantly increased the GSH levels and GPx and SOD enzyme activities. In another study, the protective effects of PBE against CCl4-induced oxidative stress and hepatotoxicity in rats were investigated. It was indicated that PBE probably acted by inhibiting lipid peroxidation and increasing antioxidant activity (Yang et al., 2008). The efficacy of PBE in reducing [Cr(VI)]-induced toxicity had also been assessed (Parveen et al., 2009). Nephro toxicity of hexavalent chromium [Cr(VI)] in humans and in animals, correlated with over production of free radicals, has been suggested to be responsible for oxidative damage. Pre-treatment with PBE was effective in the prevention of K2Cr2O7-induced oxidative mediated nephrotoxicity. In vivo studies support the protective effects of PBE against oxidative damage. Sehirli and Sener (2009) reported the protective effect of PBE in the ischemia/ reperfusion-induced renal damage. Their results revealed that ischemia/reperfusion caused a significant decrease in tissue GSH level and Na+ and K +-ATPase activity, while significant increases in the renal MDA level and MPO activity were noted. PBE treatment showed reversion of all the biochemical indices as serum creatinine and BUN, as well as LDH and IL-1β, IL- 6, and TNF-α levels, as well as histopathological alterations that were induced by ischemia and reperfusion. Rocha et al. (2012) reported the importance of antioxidant therapy in sepsis. Sepsis syndrome is caused by an excessive defensive and inflammatory response characterized by massive increases of ROS, nitric oxide (NO) and inflammatory cytokines. The septic response of the organism comprises complex relationships between microbial signalling molecules, leukocytes, humoral mediators and the vascular endothelium. Inflammatory cytokines strengthen and diversify the host response. TNF-α stimulates leukocytes and vascular endothelial cells to release other cytokines, express cell surface adhesion molecules and increase arachidonic acid turnover. Serum TNF-α levels are elevated during severe sepsis or septic shock (Reiter et al., 2001). Experiments with various rodent animal models supported the observation of anti-inflammatory effects of the herbal extract (Grimm et al., 2004). The release of ROS was inhibited by pretreatment of cells with PBE before challenge with TNF-α (Peng et al., 2000). In our study, we demonstrated that Pyc treatment decreased plasma TNF-α levels in the septic rats. It has been demonstrated that PYC significantly affects inducible nitric oxide synthase (iNOS) activity both in vivo (Kobuchi et al., 1999) and in murine Copyright © 2014 John Wiley & Sons, Ltd.
macrophages (RAW 264.7 cell line) activated by LPS and IFN-g (Virgili et al., 1998). Purified catechin and epicatechin, which are the basic blocks of procyanidin structure, were found to inhibit iNOS activity to a significantly lesser extent (Kobuchi et al., 1999). In vitro, Packer et al. (1999) reported that PYC had a remarkable modulatory effect on iNOS enzyme activity, producing a slight stimulatory effect at low concentrations (10 μg/mL) while acting as a powerful inhibitor of iNOS activity at higher, though still physiologically achievable, concentrations (50–100 μg/mL). They suggested that PYC modulated NO metabolism in activated macrophages by quenching the NO radical and inhibiting both iNOS mRNA expression and iNOS activity. We found that the DNA damage was significantly higher in the lymphocytes, liver and kidney cells of sepsis-induced rats compared with the control rats. On the other hand the parameters were significantly decreased in the Pyc-treated sepsis-induced group when compared with the sepsis-induced group. Pyc treatment seemed to prevent sepsis induced DNA damage and also increased the DNA repair capacity in the lymphocytes, liver and kidneys of the rats. To date very few data is existing relating to the role of Pyc in the prevention of DNA damage. Consistent with our data, in the study of Jeong et al., 2009, the protective effects of water extracts from pine needle (WEPN) against DNA damage and apoptosis induced by hydroxyl radical were also investigated in non-cellular and cellular system. WEPN exhibited strong scavenging action on hydroxyl radical and intracellular ROS, and chelating action of Fe2+ ion. WEPN inhibited oxidative DNA damage induced by hydroxyl radical. Also, WEPN prevented the cells from oxidative damage. These data indicate that WEPN possesses a spectrum of antioxidant and DNA-protective properties common to cancer chemopreventive agents. Owing to the basic chemical structure of its components, the most obvious feature of Pyc is its strong antioxidant activity. Phenolic acids, polyphenols and in particular flavonoids are composed of one (or more) aromatic rings bearing one or more hydroxyl groups and are therefore potentially able to quench free radicals by forming resonance-stabilized phenoxyl radicals (RiceEvans et al., 1996). Several key markers of oxidative stress and inflammation were measured for Pyc’s beneficial effects. In an in vitro study, results indicate Pyc’s antioxidative and anti-inflammatory efficacy in suppressing lipid peroxidation, total reactive species generation by using the dichlorofluorescein assay, superoxide (·O2), nitric oxide (NO·), peroxynitrite(ONOO ), pro-inflammatory iNOS and cyclooxygenase-2, and nuclear factor-kappa B nuclear translocation were shown (Kim et al., 2011). In conclusion, the results of this study have demonstrated the protective effects of Pyc on the sepsis-induced oxidative DNA damage. Pyc treatment significantly decreased the MDA levels and significantly increased the GSH levels, SOD and GPx enzyme activities. Pyc treatment also significantly decreased the TNF-α level. The DNA damage parameters in all the lymphocytes, kidney and liver tissue cells of the Pyctreated sepsis-induced rats were found to decrease compared to the sepsis-induced rats. These results implied that oxidative stress may be a triggering factor for the DNA damage in sepsis and also suggest that Pyc might have a role not only in the prevention of sepsis-induced oxidative DNA damage but also in the increase of the Phytother. Res. (2014)
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antioxidant status and DNA repair. Pyc can be suggested as a useful natural remedy for the treatment of disorders related to oxidative stress.
Conflict of Interest The authors have declared that there is no conflict of interest.
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