Progress in Nutrition 2019; Vol.21, N. 4: 849-857 DOI: 10.23751/pn.v21i4.7751 © Mattioli 1885 Original articles The impact of astaxanthin on adverse effects of hyperglycemia induced by STZ in retinal tissue of rat Shahnaz Mojarrab1,2, Farideh Bahrami1,2, Ali Khoshbaten2, Ahmad Shojaei3, Narsis Daftarian4, Fatemeh Salem5, Mohammad Taghi Mohammadi2 1Neuroscience Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran; 2Department of Physiology and Medical physics, School of Medicine, Baqiyatallah University of Medical Sciences, Tehran, Iran - E-Mail: [email protected]; 3Department of Ophthalmology, Faculty of Medicine, Baqiyatallah University of Medical Sciences, Tehran, Iran; 4Ocular Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran; 5Department of Pharmacology, School of Pharmacy, Baqiyatallah University of Medical Sciences, Tehran, Iran Summary. Astaxanthin (ATX) is a powerful natural antioxidant belongs to xanthophylls and the aim of this study is to investigate its protective roles on adverse effects of hyperglycemia in retinal tissue. Sixty rats were randomly divided into Controls, and hyperglycemic groups. ATX (20 mg/kg) was administrated over 47 days. After 47 days the final blood glucose concentration and body weight also the expression of vascular endo- thelial growth factor (VEGF), tumor necrosis factor α (TNF-α) proteins, antioxidant capacity and vessels dimension in ganglionic cell layer (RGC) layer in retinal tissue were measured as well as immunohistochemis- try and histopathological assessments. Hyperglycemia-induced decrement in Catalase (CAT) (0.096 ± 0.026) and Glutathione (GSH) (133.80 ± 65.10) activity in retinal tissue but increase Superoxide dismutase (SOD) (15.52 ± 1.36 mU/mg) and Malondialdehyde (MDA) (2.64 ± 0.12) content. Administration of ATX in- creased the antioxidant capacity in the treated group (p<0.05). An increment in the expression of VEGF and TNF-α and vasodilation were shown in the hyperglycemic groups (p<0.01). Immune and histopathological assessments indicated that the ATX treatment could repair vasodilation in RGC layer vessels and also reduce the TNF-α content in retinal tissue (p<0.05). ATX could repair vasodilation and inflammation presumably because of removal of oxidizing and inflammatory agents in retinal tissue but during 47- day treatment it could not significantly decrease expression of VEGF in retinal tissue of hyperglycemic group. Keywords: Astaxanthin; Oxidative stress; Hyperglycemia; Retinal tissue; RGC layer; VEGF Introduction stress (4). Research has shown that advanced glyca- tion end products increase vascular endothelial growth Diabetes is a main public health problem. Several factor (VEGF) and inflammatory factors such as tu- factors such as genetic susceptibility, environmental mor necrosis factor α (TNF-α) (5, 6). Inflammation factor, poor control of blood sugar, lifestyles and co- plays an important role in the pathology of hypergly- morbidities have an impact on the prognosis of dia- cemic retinopathy (6). Overexpression of VEGF and betes complication (1, 2). Hyperglycemic retinopathy TNFαinduces the adverse effect on tight junction pro- is one of the prevalent complications of diabetes (3). teins in vascular endothelial cells in the retina which During diabetes, hyperglycemia increases free radicals they cause blood-retinal barrier break down. This event through different metabolism pathways (4). Oxidative increased vascular permeability and edema in retinal stress is the lack of balance between overproduction tissue in the hyperglycemic condition (7). and eliminating ROS in the body. During diabetes, the Any organism has enzymatic and non-enzymatic retinal cells and their capillaries experience oxidative defense mechanisms that remove harmful free radicals. 850 S. Mojarrab, F. Bahrami, A. Khoshbaten, et al. The enzymatic defense mechanisms include superoxide Animals dismutase (SOD) and catalase(CAT) (8). The non-en- In this study, 48 male Wistar rats weighing 200- zymatic defense mechanisms consist of glutathione, acid 225 were used. The rats were kept in standard cages ascorbic, vitamin E and etc. Some studies confirm that with free access to food and water in temperature of antioxidant capacity decreases in diabetes and some other 22 ± 2°C, humidity of 40% - 60%, and 12 hours light/ demonstrate increment of antioxidant agents in saliva and dark cycle condition. serum along with the oxidants enhancement in diabetes. Also, it was found that during hyperglycemia vitamin E, C Diabetes induction and Beta-carotene have been decreased (9). Diabetes was induced by a single tail vein injection Some of the carotenoids are used as a nutritional of Streptozotocin (Sigma UK) (45 mg/kg) dissolved supplement to prevent oxidative lesions in the retinal tis- in 0.1 M citrate buffer. Blood glucose was measured sue. Carotenoids are the phytochemical substance which in the first day before streptozotocin (STZ) injection has hydrocarbon chain with carbon-carbon double bonds, and days 5th and 47th after injection of streptozotocin therefore, they can scavenge free radicals and inhibit lipid by Accu-Chek Blood Glucose Meter. Animals with a peroxidation by the unique structure (10). In human one blood glucose of more than 350 mg/dl were selected as of the significant role of carotenoids are in the macula. Lu- hyperglycemic animals. tein and Zexanthin are the macular pigment carotenoids which exist in the eye in different amounts (11). Research Retina Sampling results have shown that during the past two decades the The animals were euthanized by a lethal dose of antioxidant action of macular carotenoids, as a filter, is re- Ketamine and Diazepam. The eyeballs were removed moving optical radiation damage in the retina (11). and washed with cold PBS (phosphate buffer saline, Astaxanthin (AXT) is a carotenoid from xantho- PH 7.4). The retinas were detached from the retinal phylls category that shows pharmaceutical potential pigmented epithelium cell layer and submerged in liq- (12, 13) and also some of the investigators attribute to it uid nitrogen to freeze then stored at -80° C for protein therapeutic role in neurodegeneration (14). The unique extraction or enzyme assays. For the histopathological feature of AXT is the existence of oxygen with a dou- and immunohistochemical studies, the whole eye caps ble bond in the ionic ring at the end of the hydrocar- were fixed in formalin or paraformaldehyde. bon chain. This property and the carbon-carbon double bonds contribute to the powerful antioxidant AXT (12). Protocols and Groups of Experiment However, the interaction of this potent antioxidant Animals in the control group were randomly with other cellular and molecular targets and their exact divided into two groups of (n=12) control and AXT mechanisms are not clear in the hyperglycemic condition treated rats. Treated- control rats group was fed with in retinal tissue. The aim of this study is to investigate the AXT (1-800-921-8482 manufactured for Viva Labs effect of AXT on antioxidant capacity, vascular variations Inc, made in the USA), 20 mg/ kg orally once a day and expression of VEGF and TNF ά in retinal tissue in by gavages during six weeks. Hyperglycemic animals an uncontrolled hyperglycemia condition. In were randomly divided into two groups (n=12) hyper- glycemic and treated-hyperglycemic rats. The treated- hyperglycemic rats group were treated with AXT, 20 Methods mg/kg orally once a day by gavages over six weeks. Af- ter six weeks the final blood glucose concentration and Ethics statement body weight were checked. The experimental methods of this study were ap- proved by bioethics committee of the animal house Enzyme Assays and Protein measurement in Baqiyatallah University of Medical Science and Isolated retinas were homogenized and sonicated have followed the NIH guidelines for use and care of on ice-cold in PBS solution (pH=7.4) incubated at animals(Approval code: IR.bmsu.Rec.1396.620). 4°C for 30 minutes then centrifuged (15,000 RPM The impact of astaxanthin on adverse effects of hyperglycemia induced by STZ in retinal tissue of rat 851 at 4°C for 30 minutes). The supernatants were used It was stored at -80º temperature for cutting. Each for protein (Bradford method) (15) and enzyme as- slide had 10 µm diameters. The slides were defrosted says. All measures were based on the spectro- and blocked in a goat serum for one hour. The retinal photometric assay. The method of Tietz was used to vasculature and adherent leukocytes were imaged by determine the GSH content of retinal tissues (16). DPI or VEGF antibody (Santa Cruze) which was di- Briefly interaction of cell lysate with Na2HPO4 and luted in a goat serum 3% incubated overnight in 4°C. 5, 5’-dithiobis-(2-nitrobenzoic acid) (DTNB) and Finally, the secondary antibody conjugated to FITC the absorbance of DTNB was monitored at 412 nm was added. After dehydrating and contrasting, the im- for 5 minutes. The SOD activity of the retina tissues ages were observed under fluorescent microscope. The was measured according to Winterbourne method vascular dimension and area of vessels were measured (17). In this method, potassium phosphate buffer was by Olysia software. added to the EDTA solution containing sodium cya- nide, NBT and homogenized sample. In presence of Protein extraction and western blot analysis the riboflavin, the reaction initiates. The absorbance of Isolated retinas were homogenized and sonicated samples was measured at 560 nm. For Catalase activity in a lysis buffer [0.5 M Tris-HCl (pH 7.4), 150 mM assay the Abei method was used (18). The principle NaCl, 0.5% deoxycholic acid, 1% Triton X100, and of this method was based on measuring the decrease protease inhibitors(1 tablet/50 ml Tris Buffer (pH in absorbance of the test sample by the induced de- 7.2),0.1% SDS] incubated at 4°C for 30 minutes and composition of H2O2 in the presence of the analytic centrifuged with 14,000 rpm at 4°C for 20 minutes. enzyme at 240 nm wavelength. Protein concentration was determined by the Brad- MDA content of the retinal tissues was measured by ford assay, each sample (50 µg of protein) were mixed the method of Satoh (19).