Increased apoptosis, tumor necrosis factor-α, and DNA damage attenuated by 3',4'-dihydroxyflavonol in rats with brain İschemia-reperfusion,Dervis Dasdelen1, Merve Solmaz2, Esma Menevse3, Rasim Mogulkoc1, Abdulkerim Kasim Baltaci1, Ender Erdogan2, : Indian Journal of Pharmacology

Home

RESEARCH ARTICLE	[Download PDF]

Year : 2021 \| Volume : 53 \| Issue : 1 \| Page : 39--49

Increased apoptosis, tumor necrosis factor-α, and DNA damage attenuated by 3',4'-dihydroxyflavonol in rats with brain İschemia-reperfusion

Dervis Dasdelen¹, Merve Solmaz², Esma Menevse³, Rasim Mogulkoc¹, Abdulkerim Kasim Baltaci¹, Ender Erdogan²,
¹ Deparment of Physiology, Selcuk University, Medical School, Konya, Turkey
² Deparment of Histology, Selcuk University, Medical School, Konya, Turkey
³ Deparment of Biochemistry, Selcuk University, Medical School, Konya, Turkey

Correspondence Address:
Dr. Rasim Mogulkoc
Department of Physiology, Selcuk University, Medical School, 42075 Konya
Turkey

Abstract

OBJECTIVES: This research was aimed to find out the effects of 3',4'-dihydroxyflavonol (DiOHF) on apoptosis, DNA damage, and tumor necrosis factor-α (TNF-α) levels in the frontal cortex of rats with induced experimental brain ischemi reperfusion. MATERIALS AND METHODS: A total of 38 Wistar albino male rats were used. Groups were created as 1-Sham; 2-Ischemia-reperfusion (I/R); 3-I/R + DiOHF (10 mg/kg); 4-Ischemia + DiOHF + reperfusion; 5-DiOHF + I/R. I/R was performed by carotid artery ligation for 30 min in anesthesized animals. Following experimental applications, blood samples were taken from anesthetized rats to obtain erythrocyte and plasma. Later, the rats were killed by cervical dislocation, and frontal cortex samples were taken and stored at − 80^oC for the analysis. RESULTS: In the ischemic frontal cortex tissue sections degenerate neuron numbers, Terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) positive cell ratio and caspase-3 positive cell ratio increased. Malondialdehyde, TNF-α, and 8-OHdG levels were increased in both plasma and tissue in ischemia group, whereas tissue and erythrocyte glutathione levels were significantly suppressed. However, these values were significantly reversed by DiOHF treatment. CONCLUSION: The results of the study showed that I/R significantly increased apoptosis, TNF-α, and DNA damage in rats with brain I/R. However, 10 mg/kg intraperitoneal DiOHF treatment improved deterioted parameters.

How to cite this article:
Dasdelen D, Solmaz M, Menevse E, Mogulkoc R, Baltaci AK, Erdogan E. Increased apoptosis, tumor necrosis factor-α, and DNA damage attenuated by 3',4'-dihydroxyflavonol in rats with brain İschemia-reperfusion.Indian J Pharmacol 2021;53:39-49

How to cite this URL:
Dasdelen D, Solmaz M, Menevse E, Mogulkoc R, Baltaci AK, Erdogan E. Increased apoptosis, tumor necrosis factor-α, and DNA damage attenuated by 3',4'-dihydroxyflavonol in rats with brain İschemia-reperfusion. Indian J Pharmacol [serial online] 2021 [cited 2023 Mar 22 ];53:39-49
Available from: https://www.ijp-online.com/text.asp?2021/53/1/39/315086

Full Text

Introduction

Ischemia is defined as the reduction or disappearance of the blood supply to a part of the body for various reasons.[1] After ischemia, neurons cannot maintain their normal structure and functions due to they cannot be fed and apoptosis results.[2] The damaged brain region increases as the blood flow reduces.[3] Ischemia reperfusion (I/R)-induced brain damage includes mitochondrial dysregulation, increased oxidative stress, reactive oxygen species (ROS), microvascular permeability, neuronal apoptosis, and blood–brain barrier destruction.[4],[5] And also, brain I/R injury causes neuronal damage by contributing to inflammation and oxidative damage by activating microglias.[2],[6] It has been shown that malondialdehyde (MDA) is widely used as an indicator of oxidative stress which was increased significantly in hypoxic ischemic brain damage.[7] Antioxidant mechanisms that convert ROS to less harmful compounds include many antioxidant enzymes such as superoxide dismutases, catalase and glutathione (GSH) peroxidase,[8] and low-molecular-weight nonenzymatic antioxidants such as GSH.[9] GSH levels were decreased significantly in the brain I/R study.[10]

A significant increase in ROS caused by cerebral ischemia can accelerate neuronal death by triggering the signaling pathways in the neurons.[11] Oxidative stress and excitotoxicity caused by cerebral ischemia causes microglias to secrete proinflammatory cytokines such as tumour necrosis factor-α (TNF-α) and interleukin 6. Cytokines attract neutrophils, monocytes, and T-cells to the damaged area in the brain lead to increases inflammation.[2],[12] Damage-associated molecular patterns (DAMPs) released from necrotic neurons stimulate these cells by activating their receptors on microglias and astrocytes.[13] Brain ischemia causes hypoxia, decreases energy production, and disrupts the function of ion pumps, leading to the deterioration of membrane potential and activation of many apoptotic factors.[14] DNA damage, growth factor reduction, hypoxia, and cytotoxic drugs initiate the intrinsic apoptotic pathway.[15] In intrinsic apoptosis, cytochrome C, released from the pores created by the proapoptotic proteins Bax and/or Bak in the mitochondria, merged with apoptotic protease activating factor-1 and caspase-9 by combining to create apoptosis.[16] The downstream caspases of the intrinsic pathway are then activated and the effector caspases which are caspase-3 and caspase-7 break down the cell by targeting substrates that break down the homeostatic, cell skeleton, repair, metabolic, and cell signaling proteins.[17]

Flavonoids, whose simple structures are composed of an oxygenated heterocyclic and two phenolic rings were divided into different subgroups according to the oxidation state of the heterocyclic pyran ring.[18] Many flavonoid-containing grape seed proanthocyanidin extracts have been found to reduce ischemic neuronal damage by preventing DNA damage occurring during 2 h, 3 h, 4 day reperfusion periods after 5 min of transient forebrain ischaemia.[19],[20] 3',4'-dihydroxyflavonol (DiOHF) used 5 min before ischemia in the hindpaw or 5 min before the start of reperfusion at the dose of 5 mg intra-jugularly reduces the degree of deterioration of the dilated response of ACh and sodium nitroprusside that occurs in response to IR. Ach, in response to IR, significantly increases the corresponding vasodilatation. DiOHF reduces the accumulation of superoxide anions concentration under in vitro conditions.[21]

This research aimed to determine the effect of DiOHF administration on apoptotic activity, DNA damage, TNF-α levels, and oxidant and antioxidant systems in the blood and brain frontal cortex tissue at different stages of brain I/R (before, during, and after ischemia) in rats.

Materials and Methods

This study was performed at the Selcuk Experimental Medicine Research and Application Center and experimental procedures were approved by local ethics committee (20.06.2017/2017-22). Biochemical analyses of the study were performed in the Molecular Physiology Laboratory and histological analyses were performed in the Histology Laboratory of Faculty of Medicine. In this study, 38 wistar albino male rats were used with the mean weight of 300–400 g.

Surgical applications for ischemia

Surgical procedures were conducted as in previous study but with minor changes.[1] First, the animals were anesthesized by intraperitoneal (i.p.) ketamine HCl (60mg/kg) and xylazine (Rompun, Bayer) (5 mg/kg). Anesthetized rats were placed on the operation table with their backs to the floor, and the legs were fixed to the operation table with a cloth band. The right and left carotid arteries were carefully isolated from the surrounding tissues and vagus nerve after ventral incision in the midline of the animal's neck. Two-vessel occlusion model was used to induce experimental brain ischemia. Animals were only incised and then closed in sham group. In the I/R group, the ischemia was visually confirmed. Ischemia was induced for 30 min. After the ischemia, the ropes were thawed and blood flow was confirmed visually. Reperfusion was also induced for 30 min.

Groups

Sham Group (n = 6): After general anesthesia was administered, the carotid arteries were isolated and closedI/R Group (I/R, n = 8): Under general anesthesia, carotid arteries were isolated and ligated for 30 min to induce ischemia. Later, reperfusion was induced for 30 minI/R + DiOHF Group (n = 8): I/R of carotid arteries were induced in as Group 2. At the end of reperfusion, DiOHF (10 mg/kg) was administered to animals intraperitoneally, and 30 min later, the animals were killedIschaemia + DiOHF + Reperfusion (I + DiOHF + R) Group (n = 8): Carotid arteries were ligated for 30 min followed by 10 mg/kg DiOHF administered by i.p.; subsequently, animals were reperfused for 30 min and then killedDiOHF + I/R Group (n = 8): Animals were administered DiOHF at a dose of 10 mg/kg 30 min before I/R. The carotid arteries were then ligated for 30 min to produce ischaemia. Later, reperfusion was induced for 30 min [Figure 1].{Figure 1}

Animals were sacrificed under general anesthesia at the end of the experimental period. A portion of each tissue taken (frontal cortex) and blood samples were maintained at −80°C until analysis. TUNEL, caspase-3, hematoxylin-eosin (HE), and Toluidin Blue staining was performed for the histological examination in the sections of the tissues. Plasma and erythrocytes were obtained from the blood samples taken from the animals. Erythrocyte hemoglobin and GSH levels were analyzed immediately. The plasma was stored at −80°C until the study was performed. Biochemical analysis was carried out for 8-OHdG, GSH, MDA and TNF-α in the plasma and tissues.

3',4'-dihydroxyflavonol application

Ninety-nine percent purity DiOHF (Indofine Chemical Company, U.S.A.) having catalog number T-601 was dissolved in hazelnut oil and 5% DMSO and administered intraperitoneally to rats as 10 mg/kg.

Biochemical analysis of blood and brain tissue

Tissue homogenization

The frontal cortex to be analyzed was weighed and placed in glass test tubes and homogenized in Misonix's Microscan ultrasonic tissue shredder at 4°C to produce 10% homogenate in 150 mM KCl.[1] The resulting homogenates were centrifuged at 3000 rpm for 15 min. After homogenization, the samples were placed into Ependorf tubes for GSH, MDA, TNF-α, and 8-OHdG analyses.

Measurement of hemoglobin levels

Hemoglobin levels were analyzed using the Drabkin method. Levels were calculated as g/dl.[22]

Analysis of tissue malondialdehyde levels

MDA level of the frontal cortex was measured by Uchiyama and Mihara method as previous research.[23] Values were calculated as nmol/g tissue.

Analysis of plasma malondialdehyde levels

Plasma MDA was determined as in our previous research. The results were calculated in nmol/ml.[1]

Analysis of tissue glutathione levels

This analysis was performed as in previous studies to determine GSH levels.[1] The results were calculated as mg/gr tissue.

Analysis of erythrocyte glutathione levels

Erythrocyte analysis was performed as in previous studies.[1] Levels of GSH were calculated in µmol/gr Hb.

Analysis of tissue and plasma 8-OHdG levels

Tissue and plasma 8-OHdG levels were determined by using the Cayman brand rat ELISA assay (Cat. No. 589320). Values were given in pg/ml.

Analysis of tissue and plasma tumor necrosis factor-α levels

Tissue and plasma TNF-α levels were analyzed using Sunred brand rat ELISA assay (Catalog No: 201-11-0765). Values were given as ng/ml.

Histological examination

Tissues were taken into a 10% formalin solution with a fixative/tissue ratio of 10/1. Half of each tissue was taken to 4% freshly prepared paraformaldehyde solution for immunofluorescence staining. It was detected at 4°C for at least 24 h. Tissue fixation was performed in formaldehyde. Tissues were washed for one night in the running water for tissue processing and then treated with 50%, 60%, 70%, 80%, and 90% alcohols, respectively, 30 min, 96% and 100% alcohol for 45 min for dehydration. It was then treated with alcohol + xylene (1/1) for 20 min, xylene for 1 h, second time xylene for 1.5 h, xylene + paraffin (1/1) for 20 min, and paraffin in 57°C incubator for 15 min. Tissues were embedded in paraffin, and 5 µm thick sections were taken with a microtome (LEICA RM2125RT) on polylysine slides.

Hematoxylin-eosin staining

Hematoxylin-Eosin (HE) staining was performed. For HE staining, tissue preparations were left in the incubator at 57°C for one night for deperfusion. They were then kept in xylene twice for 30 min and passed through a series of reduced concentrations of alcohol. They were washed under running water for 5 min and stained with hematoxylin dye for 5 min. They were again washed under running water for 5 min. The sections were soaked in acid alcohol and removed. Subsequently, they were stained with eosin dye for 2–3 min and passed through an increased concentration of alcohol series. One drop of entellan was dropped onto the tissue and covered with a coverslip.

Toluidine blue staining

Toluidine blue (TB) staining was performed. For toluidine blue staining, 1 g of toluidine blue in 100 cc of distilled water was shaken well and filtered with filter paper. Tissue preparations were left in the incubator at 57°C for one night, kept in xylene twice for 30 min, and passed through a series of reduced concentrations of alcohol. The sections were heated on a 60°C heating tray for approximately 1 min (not to boil). Toluidine blue was added dropwise to cover the tissue. The sections were held on the heating plate until the edges of the paint gave a color halo. The sections were washed by immersing them in distilled water several times and dried on the heating plate and closed with Entellan. HE staining and Toluidine blue staining were evaluated. A 5-point index that had been used in a previous study was modified and used for evaluation.[24] Accordingly, all preparations were scored according to their indices (Score 0: normal neurons; Score 1: degenerative neurons <25%; Score 2: degenerative neurons 25%–50%; Score 3: degenerative neurons 50%–75%; and Score 4: degenerative neurons >75%). The preparations were also evaluated for inflammation, bleeding, and ischemia.

In acute ischemia, neurons become smaller and eosinophilic due to hypoxia. Nuclei become denser and lose their sharp lines. These types of neurons are called “red neurons.” Red neurons are indicators of neuron degeneration.[25] HE staining was scored by considering the amount of red neurons, which is an indicator of neurons degeneration. TB staining scoring was done by considering the degenerated and shrunken neurons.

Immunofluorescence staining

First, the tissues were fixed in paraformaldehyde for 24 h and then taken to 30% sucrose and kept for at least 24 h. Serial sections of 4 µm thickness were taken with a cryostat device (Thermo Shandon Cryostat 210160GB). They were stored at − 18°C. TUNEL and caspase-3 stainings were performed on the frozen sections. The stained tissue sections were examined by fluorescence microscope (Olympus BX51). TUNEL positive cell ratio (TUNEL positive cell number/4′,6-diamidino-2-phenylindole [DAPI] stained nucleus number) was used for the evaluation of TUNEL staining, and caspase-3 positive cell ratio (number of caspase-3 positive cells/DAPI-stained nucleus number) was used in the evaluation of caspase-3 staining.

TUNEL staining

TUNEL staining was performed using the kit (In situ Cell Death Detection Kit POD, Roche 11684817). For TUNEL staining, sections were washed with phosphate buffer saline (PBS) twice for 5 min. The sections were then incubated with proteinase K (Roche Applied Science Cat. No. 03115836001) for 40 min at the room temperature and washed twice with PBS for 5 min. They were then incubated with TUNEL reaction mixture (labeling solution + TdT [Terninal deoxynucleidyl transferase] solution) at 37°C for 2 h in a humidity chamber. Some sections were incubated with the labeling solution only for negative control and washed twice with PBS for 5 min. The tissues were closed with DAPI. Randomly, four areas were photographed from each tissue preparation. Two different observers counted cells stained with TUNEL and nuclei stained with DAPI at different times and environments.

Caspase-3 staining

Caspase-3 staining was performed using the primary antibody (anti-caspase-3 antibody, Abcam ab13847). For caspase-3, frozen sections stored at −18°C were removed and stored for 5 min at the room temperature. The area to be painted was marked with a pen and washed twice with PBS for 5 min. They were then incubated with 0.1% Triton X-100 at the room temperature for 10 min and washed twice with PBS for 5 min. The sections were incubated with block solution for 30 min at the room temperature, and the block solution was withdrawn later by a pipette. Sections were incubated in proteinase K (Roche Applied Science Cat. No. 03115836001) for 20 min at 57°C. It was then incubated with anti-caspase-3 primary antibody in the refrigerator for 1 night and washed thrice with PBS for 5 min. It was incubated with the secondary antibody (Goat anti rabbit Texas Red, ab6719) at room temperature for 3 h and washed thrice with PBS for 5 min. The tissues were subsequently sealed with DAPI closing medium (abcam ab104139). Randomly, four areas were photographed from each tissue preparation. Two different observers counted cells stained with caspase-3 and nuclei stained with DAPI at different times and environments.

Statistical evaluations

Statistical analysis of the findings was performed with SPSS22.0, (IBM, Inc, Chicago, IL, USA) and arithmetic mean and standard deviation of all parameters were calculated. In order to determine the homogeneity of the data, “Shapiro–Wilk” test was performed, and the data were found to show normal distribution. The analysis of variance test was used to determine the differences between the groups, and “Tukey” test from post hoc test was used to determine which group caused the difference.

Results

Hematoxylin-eosin and Toluidine blue staining results

The HE staining scores of the groups are given as for Sham: 0.66 ± 0.51; I/R: 3.33 ± 0.51; I/R + DiOHF: 2.42 ± 0.53; I + DiOHF + R: 2.28 ± 0.75; DiOHF + I/R: 2.14 ± 0.69. The TB staining scores of the groups are given . Sham: 0.66 ± 0.33; I/R: 3.50 ± 0.54; I/R + DiOHF: 2.42 ± 0.53; I + DiOHF + R: 2.28 ± 0.75 and DiOHF + I/R: 2.28 ± 0.75 respectively.

For general routine morphological evaluation, HE and TB were used. It is known that TB stain is better the nervous system and myelin sheath. Histological evaluation of the study showed no bleeding areas or cell infiltration in the frontal cortex. In all ischemia-induced groups, small red neurons were observed that are indicative of neuronal damage (degeneration) due to ischemia and hypoxia. The highest score was obtained in I/R group [P < 0.001; [Figure 2], [Figure 3]a and [Figure 3]b. This score was lower in the DiOHF groups than I/R group (P < 0.001).{Figure 2}{Figure 3}

TUNEL and caspase-3 staining results

The TUNEL positive cell ratio of the groups is given, respectively. Sham: 0.14 ± 0.03; I/R: 0.42 ± 0.14; I/R + DiOHF: 0.28 ± 0.06; I + DiOHF + R: 0.27 ± 0.03; DiOHF + I/R: 0.20 ± 0.01. The Caspase-3 positive cell ratio of the groups is given for Sham: 0.21 ± 0.04; I/R: 0.45 ± 0.05; I/R + DiOHF: 0.29 ± 0.02; I + DiOHF + R: 0.31 ± 0.06; DiOHF + I/R: 0.27 ± 0.04 respectively.

TUNEL staining of the relevant tissue [Figure 4] and TUNEL positive cell ratio is given in [Figure 3]c. Accordingly, the I/R group had the highest ratio, whereas sham and the DiOHF + I/R group had the lowest mean values. Group 2 has higher values than the other groups and also Groups 3 and 4 had increased values compared to Groups 1 and 5 (P < 0.001). I/R increased Caspase-3 positive cell ratio [Figure 3]d and [Figure 5] increased; however, DiOHF application decreased (P < 0.001).{Figure 4}{Figure 5}

Glutathione levels of erythrocyte and frontal cortex tissue

The erythrocyte GSH values of the groups are given, respectively. Sham: 2.15 ± 0.80; I/R: 1.53 ± 0.42; I/R + DiOHF: 2.89 ± 0.68; I + DiOHF + R: 3.90 ± 1.12; DiOHF + I/R: 3.58 ± 1.43. The results were calculated in µmol/gr Hb. The tissue GSH values of the groups are given, respectively. Sham: 22.33 ± 1.96; I/R: 26.88 ± 2.97; I/R + DiOHF: 32.92 ± 5.85; I + DiOHF + R: 32.26 ± 5.96; DiOHF + I/R: 32.40 ± 2.75. The results were calculated as mg/gr tissue.

The erythrocyte and tissue GSH levels are given in [Figure 3]e and [Figure 3]f. When the erythrocyte GSH values were compared, it was found that Groups 4 and 5 obtained the highest values [P < 0.020; [Figure 3]e]. Although the sham and I/R + DiOHF groups had higher values than the I/R group (Group 2), there was no statistical difference. When tissue GSH levels were examined, the groups treated with DiOHF had higher values than the sham and I/R groups [P < 0.001; [Figure 3]]f.

Tissue and plasma malondialdehyde levels

The plasma MDA is given as follows: Sham: 2.35 ± 0.61; I/R: 4.81 ± 1.08; I/R + DiOHF: 1.93 ± 0.93; I + DiOHF + R: 1.20 ± 0.26; DiOHF + I/R: 1.34 ± 0.21. The results were calculated in nmol/ml. The tissue MDA values of the groups are given, respectively, Sham: 16.57 ± 2.50; I/R: 41.20 ± 5.91; I/R + DiOHF: 18.61 ± 7.07; I + DiOHF + R: 14.74 ± 1.41; DiOHF + I/R: 16.38 ± 3.69. Values were calculated as nmol/gr tissue.

Plasma and tissue MDA values of experimental groups are given in [Figure 3]g and h. I/R increased MDA levels [P < 0.001; [Figure 3]g and h]. Plasma MDA levels showed a significant decrease in Groups 4 and 5 compared to the sham group (P < 0.001). However, the sham group did not show a statistically significant difference with the I/R + DiOHF group. Furthermore, tissue MDA levels were not different among the groups except the I/R group.

Tissue and plasma tumor necrosis factor-α levels

Plasma TNF-α levels of groups are given, respectively. Sham: 26.69 ± 4.58; I/R: 59.46 ± 4.35; I/R + DiOHF: 26.16 ± 5.42; I + DiOHF + R: 20.91 ± 3.71; DiOHF + I/R: 20.36 ± 3.62. Values were given as ng/ml. The tissue TNF-α values of the groups are given, respectively. Sham: 27.12 ± 4.11; I/R: 123.01 ± 16.54; I/R + DiOHF: 42.67 ± 11.33; I + DiOHF + R: 58.27 ± 8.89; DiOHF + I/R: 57.54 ± 4.41. Values were given as ng/ml.

TNF-α values are one of the indicators of inflammatory values which are given in [Figure 3]i and j. I/R group plasma and tissue TNF-α values were higher than all other groups [P < 0.001; [Figure 3]i and [Figure 3]j.

Tissue and plasma DNA damage (8-OHdG) results

The plasma 8-OHdG values of the groups are given, respectively. Sham: 17.27 ± 1.16; I/R: 34.27 ± 0.92; I/R + DiOHF: 20.36 ± 5.37; I + DiOHF + R: 24.26 ± 5.19; DiOHF + I/R: 25.22 ± 1.10. Values were given in pg/ml. The tissue 8-OHdG values of the groups are given, respectively. Sham: 0.70 ± 0.34; I/R: 3.01 ± 0.81; I/R + DiOHF: 1.39 ± 0.15; I + DiOHF + R: 1.81 ± 0.65; DiOHF + I/R: 1.57 ± 0.41. Values were given in pg/ml.

Plasma and tissue 8-OHdG values of the groups are given in [Figure 3]k and l. Comparing the groups, it was found that this parameter increased significantly in plasma and tissues of I/R group (P < 0.001; [Figure 3]k and l]. In addition, when 8-OHdG levels were compared in plasma, this parameter was found to decrease significantly in Groups 3, 4, and 5 compared to I/R group (P < 0.001).

Discussion

In this study, the affects of I/R injury on the oxidant/antioxidant system, apoptotic activity, DNA damage, and TNF-α levels in the blood and the frontal cortex tissue were determined in rats with brain I/R. Our results showed that experimental I/R led to apoptosis and DNA damage and increased TNF-α levels in the frontal cortex. Brain ischemia causes hypoxia, reducing energy production, disrupting the function of ion pumps, and consequently leading to a deterioration of membrane potential and activation of a number of apoptotic factors.[14] In the present study, histological examination of the tissue (HE, TB, TUNEL, and caspase-3) showed that I/R increased apoptotic activity significantly. However, it was determined that DiOHF administration with I/R (before and after I/R or after ischemia and during reperfusion) significantly suppressed the number of degenerated neurons in HE and TB staining as well as TUNEL and caspase-3 positive cell ratio. Previous brain-I/R studies showed significant increases in the number of TUNEL positive cells (apoptotic index) due to I/R.[26] Similar increases were observed in the number of degenerated neurons in HE-staining.[26] Komur et al.[7] showed that hypoxic ischemia significantly increased TUNEL positive and caspase-3 positive cell ratio. Similar findings have also been reported by Ghosh et al.[10] Lan et al.[6] in a study conducted on the hypoxic ischemic brain damage in the cortex and hippocampus, showed that HE staining significantly reduced the number of neurons, and neuronal order was distorted; furthermore, cellular vacuolization and karyopyknosis was found. Ghosh et al.[10] reported in the study that caspase-3 activity was significantly increased due to different reperfusion times (30 min, 24 h and 72 h). In our study, similar to the studies mentioned above, neuron degeneration in HE, TB staining, and also TUNEL positive cell ratio and caspase-3 activity increased significantly due to 30/30 min I/R. However, the use of DiOHF significantly suppressed the TUNEL positive cell number (ratio) and caspase-3 positive cell ratio and neuron degeneration performed by HE and TB staining. In a previous study, it was determined that DiOHF significantly reduced the area of infarct in the heart tissue.[27] In our study, the administration of 10 mg/kg i.p. DiOHF significantly suppressed degenerated neuron numbers in HE and TB staining (number of red neurons) and TUNEL positive cell ratio which is one of the indicators of apoptotic activity. It might be postulated that DiOHF has a protective activity against neuron degeneration and apoptosis. However, pretreatment of DiOHF before the I/R was the most affective especially on apoptosis.

Free radicals are oxygen radicals and ROS which can be present in a reduced or oxidized form if an electron is not paired; this might destroy biological systems. Maintaining normal balance between oxidant and the antioxidant system activities of the body against free radicals is important for body functions. In our study, GSH levels in erythrocyte and frontal cortex tissues were evaluated to determine antioxidant activity. Erythrocyte GSH values showed significant suppression in I/R groups. However, DiOHF administration significantly increased suppressed erythrocyte GSH values, but this increase was more pronounced in Groups 4 and 5. When tissue GSH values were examined, there was a partial increase in I/R group. This increase is thought to be indicative of the antioxidant system activity of the tissue against I/R. However, tissue GSH values were higher in I/R + DiOHF supplemented groups. In an experimental study, male Sprague–Dawley reperfusion of 30 min, 24 h and 72 h after 30 min of bilateral carotid artery clamping led to increased ROS levels in brain. GSH levels were significantly reduced in different brain regions; however, the hippocampus being the most adversely affected region.[10] In our study, similar to the previously mentioned studies, GSH values decreased especially in erythrocytes due to I/R. However, DiOHF administration significantly increased GSH levels in both erythrocytes and frontal cortex tissue. In our study, DiOHF administration can be said to significantly support the antioxidant activity by increasing GSH levels in I/R. However, I + DiOHF and DiOHF + I/R have much more affective.

MDA is one of the oxidative stresses, primarily targeting membrane lipids. In our study, MDA values were evaluated in both plasma and brain tissue as an indicator of oxidant stress and lipid peroxidation. This parameter was found to increase significantly due to I/R. However, DiOHF administration significantly suppressed the increase due to I/R. In a study, it was determined that MDA levels significantly were increased in hypoxic ischemic brain damage in neonatal rats[6] and plasma.[7] It has also been reported that MDA causes lipid peroxidation in cortex, citriatum, and hippocampus, and its levels significantly increased when 6-h reperfusion was formed after 5-min occlusion in gerbils.[28] In a previous study, it was found that brain I/R injury significantly increased MDA levels in the brain compared to the sham control group which is similar to our increased MDA level findings.[1] In our study, the increased levels of MDA in both plasma and brain cortex tissue are in line with the results obtained in the aforementioned studies. Furthermore, the application of DiOHF before and after ischemia (30 min) and at the end of ischemia significantly reduced MDA levels in the blood tissue and brain and reduced them to the sham group levels. In a study by Caliskan et al.,[1] DiOHF application significantly suppressed MDA values. This shows that the dose of DiOHF used considerbaly affects the oxidant system by suppressing MDA levels significantly. The most affective treatment to reduce MDA levels, DiOHF treatment must be given before the reperfusion.

TNF-α levels were also evaluated in the plasma and cerebral cortex tissue. This parameter increased significantly in both plasma and tissue due to I/R, whereas DiOHF given significantly reduced the levels of TNF-α to sham and control group levels. Tissue damage caused by focal ischemia in the brain increases cytokines such as TNF-α and IL-1β, leading to increased adhesion of molecules and subsequent adherence of neutrophils to the endothelium.[29] In male spontaneous hypertensive rats, TNF-α immunopositive cells started appearing in the ischemic hemisphere 30 min after permanent left MCAO. TNF-α mRNA increased maximally after 3 hours and very few TNF-α immunopositive cells could be detected 4 days after ischemia onset.[30] This suggests that TNF-α levels may vary depending on the duration of I/R. In our study, since the I/R time was 60–90 min [Figure 1], TNF-α levels were also increased during this time. Oxidative stress and excitotoxicity caused by cerebral ischemia causes microglias to secrete proinflammatory cytokines such as TNF-α and IL-6. These secreted cytokines attract neutrophils, monocytes, and T-cells to the damaged area in the brain, further enhancing inflammation.[2] In addition, during the acute stroke, the molecule called “fractalkine” is secreted from ischemic neurons and attracts NK cells to the ischemic region, releasing cytokines such as TNF-α, leading to glutamate release. This leads to excitotoxicity in ischemic neurons. NK cells activate microglia and macrophages that secrete various inflammatory mediators by releasing various cytokines.[12] DAMPs released from necrotic neurons activate microglia and astrocytes by stimulating receptors on cells.[13] Excitotoxicity and inflammation caused by microglia and NK cells contribute to neurotoxicity and apoptotic cell death.[2] Binding of TNF-α to the R2 receptor triggers cell death by activating the inflammatory cascade through the production of NF��B or p38 MAPK (Mitogen-activated protein kinase) through TNF-α receptor-associated factor 2.[31] TNF-α is one of the parameters in the extrinsic pathway in apoptosis event and induces apoptosis by caspase-8 activation after binding to TNFR1.[32] Therefore, in our study, we examined TNF-α levels and the effect of TNF-α change in extrinsic pathway in apoptosis and the effect of DiOHF on it. One of the main results of this research is that increased TNF-α levels due to I/R causes apoptosis through extrinsic pathway, which it is one of the extrinsic apoptosis mechanisms, but re-suppression of elevated TNF-α levels by DiOHF application showed that DiOHF significantly suppressed I/R-induced apoptosis by suppressing TNF-α levels. In our study, all treatments of DiOHF had similar affects on TNF-α levels.

In our study, we examined 8-OHdG levels as an indicator of DNA damage. This parameter significantly increased due to I/R, while DiOHF supplementation significantly suppressed increased DNA damage. DNA damage is divided into subgroups as endonuclease-mediated active DNA damage and ROS-induced endonuclease-independent passive DNA damage.[33] The significant ROS increase caused by cerebral ischemia triggers premortality signaling by inducing apurinic/apyrimidinic regions in addition to base modifications in neurons, single-strand breaks, or double-chain breaks.[11] Events such as DNA damage, oxidative stress, and excitotoxicity cause transcriptional activation of p53, transactivating proapoptotic target genes encoding Bax, PUMA, Noxa, and disruption of the mitochondrial membrane potential, and cytochrome-C, and apoptotic factors such as AIF.[34] The grape seed proanthocyanidin extract[19] containing many flavonoids, prevent DNA damage that occur during 2-h, 3-h, and 4-day reperfusion period after 5 min of transient forebrain ischemia. It was found to reduce neuronal damage.[20] Resveratrol, a member of the polyphenol group, has been shown to exert protective effects by inhibiting oxidative DNA damage in spontaneous hypertensive rats with a tendency to stroke.[35] MDA and 8-OHdG levels were measured to assess oxidative stress. 8-OHdG levels are small in the sham group, whereas a significant amount of 8-OHdG was detected only in the I/R group given solvent. It has been shown that 8-OHdG levels increase significantly in rats with brain I/R.[26] In our study, all treatments of DiOHF had similar affects on 8-OHdG.

We have used three different therapeutic regimens, all treatments have similar affects on neuronal degeneration by HE and TB staining, TNF-α for inflammation and 8-OHdG for DNA damage. When used 30 min before ischemia or in the beginning of the reperfusion, DiOHF treatment was more effective in reducing oxidative stress and increasing antioxidant activity. In Group 3 (I/R + DiOHF), DiOHF treatment was effective but, DiOHF treatment much more affective in Groups 4 and 5. When the anti-apoptotic effect of DiOHF is also evaluated and DiOHF pretreatment 30 min before ischemia (DiOHF + I/R group) was the best among them.

Conclusion

As shown in the aforementioned studies, various flavonoid species' effects on DNA damage were examined in different experimental I/R models. In this study, one of the important results is that DiOHF has a significant protective effect against DNA damage in brain I/R. It was seen that 30 min of ischemia and 30 min of reperfusion activate the oxidant system in brain tissue and blood and also suppress the antioxidant system in rats. TUNEL positive cell ratio and caspase-3 activity were significantly increased as an indicator of apoptosis, but DiOHF application suppressed the I/R-induced damage through both extrinsic (via TNF-α pathway) and intrinsic-extrinsic pathways (caspase-3) and reduced the levels of 8-OHdG.

Limitation of the study that different I/R durations and various DiOHF supplementations ways and doses were not used. Second, other molecular methods were not applicated to determine intrinsic and extrinsic apoptotic pathways. However, in future studies, the effect of DiOHF should be detailed at the molecular level by identifying different molecules related to apoptosis, inflammation, and DNA damage which might provide a more detailed understanding of the mechanisms.

Acknowledgment

This research has been supported by Selcuk University (ÖYP) (Project number is 2015-ÖYP-117).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1	Caliskan M, Mogulkoc R, Baltaci AK, Menevse E. The effect of 3',4'-dihydroxyflavonol on lipid peroxidation in rats with cerebral ıschemia reperfusion ınjury. Neurochem Res 2016;41:1732-40.
2	Khoshnam SE, Winlow W, Farzaneh M, Farbood Y, Moghaddam HF. Pathogenic mechanisms following ischemic stroke. Neurol Sci 2017;38:1167-86.
3	Majid A. Neuroprotection in stroke: Past, present, and future. ISRN Neurol 2014;2014:515716.
4	Esposito E, Cordaro M, Cuzzocrea S. Roles of fatty acid ethanolamides (FAE) in traumatic and ischemic brain injury. Pharmacol Res 2014;86:26-31.
5	Lin L, Wang X, Yu Z. Ischemia-reperfusion ınjury in the brain: Mechanisms and potential therapeutic strategies. Biochem Pharmacol (Los Angel) 2016;5:1-6.
6	Lan XB, Wang Q, Yang JM, Ma L, Zhang WJ, Zheng P, et al. Neuroprotective effect of Vanillin on hypoxic-ischemic brain damage in neonatal rats. Biomed Pharmacother 2019;118:109196.
7	Komur M, Okuyaz C, Celik Y, Resitoglu B, Polat A, Balci S, et al. Neuroprotective effect of levetiracetam on hypoxic ischemic brain injury in neonatal rats. Childs Nerv Syst 2014;30:1001-9.
8	Hayes JD, McLellan LI. Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic Res 1999;31:273-300.
9	Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O. Oxidative stress and antioxidant defense. World Allergy Organ J 2012;5:9-19.
10	Ghosh A, Sarkar S, Mandal AK, Das N. Neuroprotective role of nanoencapsulated quercetin in combating ischemia-reperfusion induced neuronal damage in young and aged rats. PLoS One 2013;8:e57735.
11	Li P, Stetler RA, Leak RK, Shi Y, Li Y, Yu W, et al. Oxidative stress and DNA damage after cerebral ischemia: Potential therapeutic targets to repair the genome and improve stroke recovery. Neuropharmacology 2018;134:208-17.
12	Gan Y, Liu Q, Wu W, Yin JX, Bai XF, Shen R, et al. Ischemic neurons recruit natural killer cells that accelerate brain infarction. Proc Natl Acad Sci U S A 2014;111:2704-9.
13	Anrather J, Iadecola C. Inflammation and stroke: An overview. Neurotherapeutics 2016;13:661-70.
14	Zhang WF, Jin YC, Li XM, Yang Z, Wang D, Cui JJ. Protective effects of leptin against cerebral ischemia/reperfusion injury. Exp Ther Med 2019;17:3282-90.
15	Dasgupta A, Nomura M, Shuck R, Yustein J. Cancer's achilles' heel: Apoptosis and necroptosis to the rescue. Int J Mol Sci 2016;18:23.
16	Johnston MV, Fatemi A, Wilson MA, Northington F. Treatment advances in neonatal neuroprotection and neurointensive care. Lancet Neurol 2011;10:372-82.
17	Doyle KP, Simon RP, Stenzel-Poore MP. Mechanisms of ischemic brain damage. Neuropharmacology 2008;55:310-8.
18	Tressera-Rimbau A, Arranz S, Eder M, Vallverdú-Queralt A. Dietary polyphenols in the prevention of stroke. Oxid Med Cell Longev 2017;2017:7467962.
19	Bagchi D, Bagchi M, Stohs SJ, Das DK, Ray SD, Kuszynski CA, et al. Free radicals and grape seed proanthocyanidin extract: İmportance in human health and disease prevention. Toxicology 2000;148:187-97.
20	Hwang IK, Yoo KY, Kim DS, Jeong YK, Kim JD, Shin HK, et al. Neuroprotective effects of grape seed extract on neuronal injury by inhibiting DNA damage in the gerbil hippocampus after transient forebrain ischemia. Life Sci 2004;75:1989-2001.
21	Chan EC, Drummond GR, Woodman OL. 3', 4'-dihydroxyflavonol enhances nitric oxide bioavailability and improves vascular function after ischemia and reperfusion injury in the rat. J Cardiovasc Pharmacol 2003;42:727-35.
22	Balasubramaniam P, Malathi A. 1992. Comparative study of hemoglobin estimated by drabkin's and sahli's methods. J Postgrad Med 38: 8-9.
23	Mihara M, Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1978;86:271-8.
24	Plaschke K, Grant M, Weigand MA, Züchner J, Martin E, Bardenheuer HJ. Neuromodulatory effect of propentofylline on rat brain under acute and long-term hypoperfusion. Br J Pharmacol 2001;133:107-16.
25	Ellison D, Love S, Chimelli LMC, Harding B, Lowe JS, Vinters HV, et al. Neuropathology: A Reference Text of CNS Pathology. 3rd ed. Mosby, Elsevier, Edinburg; 2013. p. 17.
26	Ya BL, Liu Q, Li HF, Cheng HJ, Yu T, Chen L, et al. Uric acid protects against focal cerebral ıschemia/reperfusion-ınduced oxidative stress via activating Nrf2 and regulating neurotrophic factor expression. Oxid Med Cell Longev 2018;2018:6069150.
27	Wang S, Dusting GJ, May CN, Woodman OL. 3',4'-Dihydroxyflavonol reduces infarct size and injury associated with myocardial ischaemia and reperfusion in sheep. Br J Pharmacol 2004;142:443-52.
28	Candelario-Jalil E, Mhadu NH, Al-Dalain SM, Martínez G, León OS. Time course of oxidative damage in different brain regions following transient cerebral ischemia in gerbils. Neurosci Res 2001;41:233-41.
29	del Zoppo G, Ginis I, Hallenbeck JM, Iadecola C, Wang X, Feuerstein GZ. Inflammation and stroke: putative role for cytokines, adhesion molecules and iNOS in brain response to ischemia. Brain Pathol 2000;10:95-112.
30	Buttini M, Appel K, Sauter A, Gebicke-Haerter PJ, Boddeke HW. Expression of tumor necrosis factor alpha after focal cerebral ischaemia in the rat. Neuroscience 1996;71:1-6.
31	O'Connor JJ. Targeting tumour necrosis factor-α in hypoxia and synaptic signalling. Ir J Med Sci 2013;182:157-62.
32	Wang L, Du F, Wang X. TNF-alpha induces two distinct caspase-8 activation pathways. Cell 2008;133:693-703.
33	Li P, Hu X, Gan Y, Gao Y, Liang W, Chen J. Mechanistic insight into DNA damage and repair in ischemic stroke: Exploiting the base excision repair pathway as a model of neuroprotection. Antioxid Redox Signal 2011;14:1905-18.
34	Culmsee C, Mattson MP. p53 in neuronal apoptosis. Biochem Biophys Res Commun 2005;331:761-77.
35	Mizutani K, Ikeda K, Kawai Y, Yamori Y. 2001. Protective effect of resveratrol on oxidative damage in male and female stroke-prone spontaneously hypertensive rats. Clin Exp Pharmacol Physiol 28: 55-59.