Taxifolin attenuates ischemia-reperfusion induced oxidative ovarian damage in rats

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].

圖5b所示為45個拉丁超立方樣本構建的Kriging近似。圖中： “X”指樣本位置，“+”指初始設計點和LHS最優解的位置?？梢钥闯觯琇HS的最優解和實際最優解差別較大，這是因為拉丁超立方樣本均勻分布于整個設計空間，重要區域(如極限狀態約束邊界附近)樣本數較少，進而構建的Kriging近似對功能函數擬合較差，最終導致可靠性設計優化最優解不準確。

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].

根據以上思考，我打算從兩個方面來開展中職語文綜合活動“走進家鄉文化”課堂實踐：一是高淳的民間故事；二是高淳的歷史遺跡。并開設這兩個主題的綜合實踐活動課。結合地域特色，我覺得高淳的民間故事豐富多彩，可以通過民間故事來挖掘家鄉文化內涵;而高淳歷史悠久，通過了解現存的和文獻中的歷史遺跡可讓學生深層次了解家鄉，熱愛家鄉文化。

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

這些問題我們是解答不了的，因為除了按書本章節背了一些理論外，其他就不甚了了。巴克夏被問得支支吾吾，滿頭冒汗，求援似的望著我。我靈機一動，連忙遮掩：“大家問的都是關于農作物的疾病與蟲害部分，將來會講到的，現在暫不涉及——”

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

每當我們遇到同行的時候，大家往往都會先想他們是不是我的競爭者，其實我們的老祖宗早就告訴我們“物之不齊，物之情也”這個道理，我們不能因為別人的優點而讓自己意志消沉，也不能因為同行而讓自己走進競爭的死胡同。在行業發展的大潮中，企業與個人要想尋求長久、輝煌的發展，要有大格局和奉獻精神。擁抱一個行業，沒有保留地投入，當回頭的那一刻，你會發現，你已經走在了行業的前端。

Figure 1. The effect of taxifolin on malondialdehyde (MDA) level(A) and reduced-glutathione (tGSH) level (B). Values are expressed as mean±SD. ##: Compared with the sham group, P＜0.01; **: Compared with the ovarian ischemia reperfusion (OIR) group, P＜0.01. TOIR: the taxifolinovarian ischemia reperfusion group.

Figure 2.The effect of taxifolin on the levels of cyclooxygenase (COX)-1 and COX-2. Values are expressed as mean±SD. ##: Compared with the sham group, P＜0.01; **: Compared with the ovarian ischemia reperfusion (OIR)group, P＜0.01. TOIR:the taxifolinovarian ischemia reperfusion group.

3.3. COX-1 and COX-2 activity analysis results

3.4. Histopathological assessment results

Figure 3. Histological appearance of the ovary in the experimental groups (hematoxylin-eosin sections in rat ovaries). A: Sections from ovarian tissue show normal histological structure of developing follicules (DF), interstitium (Int), corpus luteum (CL), and blood vessel (asterisk) in the sham group (100×). B-C:Degenerated developing follicules with fluid filled cavity (DF), severe edema in intersititial area (Int), corpus luteum (CL), congested blood vessel (asterisk)and hemorrhage (arrow) in the ovarian ischemia reperfusion group (200×) are observed. D-E: Mostly normal developing follicules (DF), mild to moderate edema in the interstitium (Int), corpus luteum (CL), and mild congestion in blood vessel (asterisk) in the taxifolinovarian ischemia reperfusion group (200×)are seen.

沒等我們張口，?？拷o編輯送禮而發點兒豆腐干新聞的通訊干事李文，搶先說道：“來了，剛進屋，我看見了——是一個妙齡少女，標致得很——真有沉魚落雁之容，閉月羞花之貌！”

Histopathological evaluation was performed in six rats for each group. One central and five peripheral areas of each sample were histopathologically examined. The degeneration intensity was graded semiquantitatively as 1, 2, or 3 (mild, moderate or severe), as seen in Table 1.

Table 1. Histopathological scoring resuts of ovarian tissues (score).

Figure 4. The effect of taxifolin on the mean number of primoidal follicules,developing follicules, atretic follicules, and corpus luteum. Values are expressed as mean±SD. #: Compared with the sham group, P＜0.05;*: Campared with the ovarian ischemia reperfusion (OIR) group, P＜0.05.TOIR:the taxifolinovarian ischemia reperfusion group.

4. Discussion

Previous studies have reported that multiple mechanisms participate ischemia reperfusion-induced tissue damage such as increased production of ROS, elevation of inflammatory mediators, and the initiation of apoptotic factors in different tissues[29]. Normally in the human body, the formation and elimination of ROS are in balance[30]. Oxidative stress occurs when some pathological status produce excessive amount of ROS and human body would not be capable of eliminating this excessive amount of ROS. The elevated levels of ROS could cause damage to significant macromolecules including DNA, protein, lipids and they play a major role in the development of some pathologies such as I/R-induced ovarian damage[31].

Antioxidants hinder radical formation before injury, repair oxidative damage, remove damaged molecules and prevent mutations[32].Different antioxidants have been examined for the prevention of ROS overproduction and oxidative stress-induced damage of many tissues. Anti-inflammatory and anti-oxidative effects of taxifolin were demonstrated in many organs[18-22]. Herein we investigated the preventive effects of taxifolin on I/R-induced ovarian injury in rats biochemically and histopathologically.

硬件在回路仿真 （Hardware-in-the-Loop Simulation）主要是在上一階段已經結束后，并已完成產品，而應用的仿真。由于真實環境中測試條件有限不能對產品進行多種情況的測試。這一階段就能應用dSPACE實時仿真系統的硬件在回路仿真來實現。圖2是對汽車防抱死裝置（ABS）控制器的測試原理圖。

In this study, the effects of taxifolin in rat ovarian tissues following two hour ischemia and two hour reperfusion have been investigated.In this study, we showed that taxifolin can prevent oxidative stress in rat ovaries exposed to ischemia reperfusion. Taxifolin at 50 mg/kg protected ovaries from an oxidant parameter, MDA and prevented a decrease in antioxidant tGSH. MDA is the main product of lipidperoxidation and is also a good marker to evaluate oxidative damage in tissue[19,33]. MDA is formed as a result of the significant increase in lipid peroxidation after overproduction of ROS[34].

As is known, MDA aggravates tissue injury by causing polymerization and production of cross links between membrane compounds[35]. tGSH is one of the most important antioxidant capacity indicator, also tGSH can protect body from damage of oxidative-stress[36]. Literature has shown that ischemia reperfusioninduced oxidative stress may lead to the deleterious effects in ovarian tissues such as an increase in the levels of MDA and a decrease in the levels of tGSH[37].

物質的量濃度溶液配制常結合其他儀器進行考查,物質的量濃度計算常通過溶液的pH、電離平衡、溶解沉淀平衡等知識進行考查。

Ovarian tissue samples were first embedded into 10% formaldehyde solution for fixation. After the fixation process, tissue samples were washed under tap water in cassettes for 24 h. Then samples were treated with conventional grade of alcohol (70%, 80%, 90%, 100%)to remove the water within tissues. Tissues were then passed through xylol and embedded in paraffin. Four-to-five micron sections were cut from the paraffin blocks and hematoxylin-eosin staining was performed. Images were acquired by using an Olympus DP2-SAL software program (OlympusInc.Tokyo, Japan). Histopathological assessment was carried out by a pathologist, who was blinded to the all experimental data. Ovarian degeneration criteria were graded between 0-3. While the normal histological structure appearance was scored as grade 0, the degenerative changes were scored as mild (grade 1), moderate (grade 2) and severe (grade 3).While histochemical scoring was performed, scoring was done by evaluating one central and five peripheral areas.

阮小棉又來到樓頂。她折了九十九只紙飛機，逐一飛下去。這一次她選擇了彩色的紙，赤橙黃綠青藍紫，五顏六色的，煞是好看。樓下有幾個年幼的孩子歡快地追逐起來，邊撿邊仰起臉龐向樓頂看。

美國國立衛生研究院項目管理程序透明，全流程管理內容集中成文，及時更新《美國國立衛生研究院資助政策聲明》。美國國立衛生研究院管理信息是公開的，例如戰略規劃、顧問委員會會議紀要、績效報告、項目立項及進展情況等均在網絡公布，部分會議向公眾開放。而美國國立衛生研究院各研究所不僅公布有關項目管理人員名單、聯系方式，而且在指南意向性建議、公布指南、申請、管理等階段均可直接溝通聯系。但是有些信息僅為公開，例如科學評審小組專家名單在網站公開，并重點備注說明申請人及其單位在評審前后均不得聯系專家，一旦發現將以違反科研誠信處理。

However, there are some limitations of this study. In this study, we examined only a single dose of taxifolin. Besides, we decided the dose based on the previous studies. Therefore, we need more studies for different comparative doses to find the exact dose. Additionally,total oxidant antioxidant, proinflammatory cytokine measurements and cell apoptosis should be investigated to clarify the mechanism of action of taxifolin.

In conclusion, we show that ischemia reperfusion-procedure leads to ovarian injury related to oxidative stress. Suppression of ROS will provide a treatment for the ischemia reperfusion injury. As shown in this study, taxifolin significantly improves the biochemical and histopathological findings and it may prevent the ovarian follicules from negative effects of ischemia reperfusion damage.As a result, taxifolin may be useful in prevention of ovarian injury related to ischemia reperfusion caused by torsion-detorsion. There is also a need for other studies in the future that can reveal taxifolin and its protective properties and elucidate their mechanism of action on this subject.

隨著我國經濟的飛速發展，工業生產所排放的溫室氣體越來越多，碳污染問題日益嚴峻。因此，我們應盡快建立標準的碳會計披露制度，統一考核標準，根據利益主體的訴求，全方位研究碳信息，為使用者提供全面、真實、科學的信息，促進企業可持續發展。披露制度要吸收國外優秀經驗制度，還要蘊含中國特色，符合我國具體國情。

Conflict of interest statement

There is no conflict of interest associated with this work.

Acknowledgements

We thank Professor Halis Suleyman, Erzincan Binali Yildirim University Faculty of Medicine, Department of Pharmacology, for his technical support and suggestions.

Authors’ contributions

This work was carried out in collaboration between all authors.Author Sevil Kiremitli designed the study and contributed the statistical analysis. She also contributed literature search. She equally definated of intellectual content and wrote the first draft of the manuscript. Author Tunay Kiremitli wrote the first draft of the manuscript. He reviewed the paper and created the paper final state.Author Renad Mammadov contributed conception and design. And he performed paper’s initial editing with author Can Turkler. Author Nihal ?etin carried out most of the literature searches. Authors Umit Arslan Nayki, Nesrin Yilmaz performed the experiment, handling the tissue processing for histology and biochemistry. Sevil Kiremitli,Tunay Kiremitli, Renad Mamadov and Nihal Cetin also assisted Umit Nayki and Nesrin Yilmaz in conducting the experiment.Authors Mine Gulaboglu and Gülce Naz Yazici performed the histological and biochemical analysis. Author Kemal Dinc performed the statistical analysis. Before the article was submitted, the article was read and approved by all authors. All responsibilities that may arise regarding the content are accepted by the authors.