Erzincan Binali Yildirim University Faculty of Medicine, Department of Obstetrics and Gynaecology, Erzincan, Turkey

2Erzincan Binali Yildirim University Faculty of Medicine, Department of Pharmacology, Erzincan, Turkey

3Erzincan Binali Yildirim University Faculty of Medicine, Department of Histology and Embryology, Erzincan, Turkey

4Ataturk University Faculty of Pharmacy, Department of Biochemistry, Erzurum, Turkey

5Selcuk University Faculty of Medicine, Department of Pharmacology, Konya, Turkey

ABSTRACT

KEYWORDS:Antioxidants; Ischemia-reperfusion; Ovarian damage; Rat; Taxifolin; Oxidative stress; Ovarian torsion

1. Introduction

Ovarian torsion, which accounts for 3% of gynecologic emergencies, is one of the common causes of gynecologic acute pelvic pain[1,2]. Reperfusion, increased blood flow to the ovary with reversal of the torsion, causes ovarian ischemia-reperfusion (I/R)injury[3].Ischemia-reperfusion-induced inflammatory response can lead to vascular endothelial cell damage and microcirculation disorders[4].The production of excessive reactive oxygen species (ROS)depends on the exposure of elevated levels molecular oxygen in the reperfusion process. We know that ROS are the mediators of reperfusion injury[5]. These ROS cause cellular damage through peroxidation of polyunsaturated fattyacids in cell membranes[6]. The exacerbation of cell damage is mediated by malondialdehyde(MDA), which is the toxic endproduct of lipid-peroxidation[7]and antioxidative activity can be assessed by MDA levels[8].It has been determined that ischemia-reperfusion cause an increase in the amount of MDA and a reduction in the amount of reduced-glutathione (tGSH), which is known as an endogenous antioxidant[9]. Ischemia-reperfusion induced oxidative stress causes damage in cellular DNA, protein, and lipids in ovarian cells[10]. Base change in nucleic acid and chain breaks in DNA are caused by free radical reactions. If this change cannot be repaired, mutagenic DNA is formed.

Another mechanism of ischemia-reperfusion injury is expressed as activation of phospholipase-A2 due to intracellular calcium increase during ischemia, increased production of arachidonic acid from membrane phospholipids, the release of pro-inflammatory activity of prostaglandins and free oxygen radicals from the arachidonic acid via the cyclooxygenase-2 (COX-2) enzyme[11].

图5b所示为45个拉丁超立方样本构建的Kriging近似。图中： “X”指样本位置，“+”指初始设计点和LHS最优解的位置。可以看出，LHS的最优解和实际最优解差别较大，这是因为拉丁超立方样本均匀分布于整个设计空间，重要区域(如极限状态约束边界附近)样本数较少，进而构建的Kriging近似对功能函数拟合较差，最终导致可靠性设计优化最优解不准确。

A recent study has shown a direct link between oxidant/antioxidant balance and COX-1/COX-2 activity in I/R-injury[12]. The I/Rinjury is a complex histopathological process. After insufficient oxygenation of the tissue and production of free oxygen radicals, the inflammatory response occurs and expands[13].

根据以上思考，我打算从两个方面来开展中职语文综合活动“走进家乡文化”课堂实践：一是高淳的民间故事；二是高淳的历史遗迹。并开设这两个主题的综合实践活动课。结合地域特色，我觉得高淳的民间故事丰富多彩，可以通过民间故事来挖掘家乡文化内涵;而高淳历史悠久，通过了解现存的和文献中的历史遗迹可让学生深层次了解家乡，热爱家乡文化。

Taxifolin (3,3’,4’,5,7-pentahydroxiflavanone) is naturally present in onion, milk thistle, French maritime and Douglas fir bark[14].Taxifolin has antioxidant and anti-inflammatory effects as well as removing free radicals from the environment[15]. Taxifolin has been reported to suppress oxidative-stress mediators and COX-2 production in ovaries, which are responsible for inflammation[16].Based on the data, taxifolin may be useful in ameliorating ischemiareperfusion injury in tissues. Although there are many experimental studies investigating the anti-oxidative and anti-inflammatory effects of taxifolin in many organs such as brain, liver, skin, heart,kidney and thyroid[17-22], we could not find any study examining the effects of taxifolin on I/R-induced ovarian damage. Therefore in this study we aimed to investigate the preventive effects of taxifolin on ischemia-reperfusion-induced ovarian damage.

2. Materials and methods

2.1. Animals

In this study, 18 female Wistar albino rats (about 6-7 months old) weighing 260-273 g were obtained from Atatürk University Laboratory Animals Breeding and Experimental Research Center and the study was conducted in the same center. Rats were housed in groups in cages at (21-22) ℃, 55%-60% humidity and a 12 h light: 12 h dark cycle (lights on at 07:00 a.m.). Rats were allowed free access to food and water.

2.2. Experiment procedure

The statistical analyses were performed by using IBM SPSS Statistics version 21(IBM Co.Armonk, NY, USA). A statistical evaluation of the results was carried out by using one-way ANOVA.The Tukey multiple comparison test was used to determine the differences between groups. Data were expressed as mean±standard deviation (mean±SD). P＜0.05 was considered as statistically significant.

During the experiment, every morning between 8:00-9:00 a.m. each animal cage was carried to the experimental room. Vaginal smear was collected with a plastic pipette filled with 10 µL of normal saline(NaCl 0.9%) by inserting the tip superficially into the rat vagina.Vaginal smear samples taken from each rat were put on different slides and examined under a light microscope at 400× magnification.Three types of cells could be recognized: round and nucleated ones are epithelial cells; irregular ones without nucleus are the cornified cells; and the little round ones are the leukocytes. The proportion among them was used for the determination of the estrous-cycle phases[23,24].

2.3. Surgical and pharmacological procedures

2.4. Biochemical analysis of MDA and tGSH

The samples of ovarian tissue were dissected out from each rat.The tissues were rinsed immediately with physiological saline,blotted, and placed on petri dishes. Ovarian tissues were stored at-80 ℃ until use. The frozen tissues were grinded to a fine powder in liquid nitrogen with a mortar and pestle. For determination of tGSH, MDA and protein concentration, the tissue samples were homogenized. For the tGSH and protein assay, tissue samples were homogenized with 50 mM cold phosphate buffer, pH 6-7, containing 1 mM ethylenediaminetetraacetic acid, and centrifuged at 10 000×g for 15 min at 4 ℃. Then the supernatant was stored on ice. For the MDA assay, 250 µL RIPA buffer was used to sonicate tissue samples. After sonication, the homogenate was centrifuged at 1 600×g for 10 min at 4 ℃. The supernatant was stored on ice.The supernatants were used to determine tGSH, MDA, and protein levels. The protein concentration of the supernatant was measured by using the method described by Bradford[27]. MDA and tGSH concentrations were measured by commercial kits (Glutathion Assay Kit Item No: 703002 and TBARS Assay Kit Item No:10009055,Cayman Chemical Company, USA).

2.5. Analysis of COX activity

We used a COX activity assay kit (Cayman, Ann Arbor, MI, USA;Item No.760151) for measuring the activity of COX in rat’s ovaries.Removed ovarian tissues were washed thoroughly with ice-cold Tris buffer, pH 7.4, containing 0.16 mg/mL of heparin, to remove any red blood cells and clots, and then stored at -80 ℃. For each rat, a sample of ovarian tissue was homogenized in 5 mL of cold buffer (0.1 M Tris-HCl, pH 7.8, containing 1 mM EDTA) per gram of tissue and centrifuged at 10 000 ×g for 15 min at 4 ℃. Removed supernatant was kept on ice. The peroxidase activity of COX was measured by the COX activity assay kit. This was assayed colorimetrically by monitoring the appearance of oxidized N,N,N,N’-tetramethylp-phenylenediamine at 590 nm. COX-2 activity was measured by using the COX-1-specific inhibitor[28]. Results for COX activities were given as units per milligram of protein. The activity of COX was expressed as nmol/min/mg protein (U/ mg protein).

2.6. Histopathological examination

2.7. Statistical analysis

这些问题我们是解答不了的，因为除了按书本章节背了一些理论外，其他就不甚了了。巴克夏被问得支支吾吾，满头冒汗，求援似的望着我。我灵机一动，连忙遮掩：“大家问的都是关于农作物的疾病与虫害部分，将来会讲到的，现在暂不涉及——”

2.8. Ethics statement

The experimental procedure was approved by the Committee for Animal Research of Atatürk University (Ethics Committee Number:30.03.2018/81). This study was carried out in accordance with international guidelines on ethical use of animals.

3. Results

3.1. MDA analysis results

3.2. tGSH analysis results

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