Evaluation of the Effect of Melatonin Treatment on Telomere Length of the Retinal Pigment Epithelium in Streptozotocin-Induced Diabetic Rat Model

Evaluation of the Effect of Melatonin Treatment on Telomere Length of the Retinal Pigment Epithelium in Streptozotocin-Induced Diabetic Rat Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of the Effect of Melatonin Treatment on Telomere Length of the Retinal Pigment Epithelium in Streptozotocin-Induced Diabetic Rat Model Ayla Eren Ozdemir This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3950753/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2024 Read the published version in BMC Ophthalmology → Version 1 posted 4 You are reading this latest preprint version Abstract Objectives: We aimed to investigate the effect of diabetic retinopathy and melatonin treatment on the relative telomer lengths (RTL) in retinal pigment epithelium (RPE) cells in a streptozotocin-induced diabetic rat model. Background: TL can be used to evaluate diabetes mellitus, its complications, and the effectiveness of its treatment. However, TL assessment has not been performed in retinal cells in a diabetic retinopathy model until now. Methods: Forty Sprague-Dawley male rats were randomly divided into four groups. The experimental groups were: Control Group (C): non- diabetic rats; Diabetes Mellitus Group (DM): rats induced to diabetes without treatment; Melatonin and Diabetes Mellitus Group (Mel +DM): rats induced to diabetes and after confirmation, treated with melatonin; Melatonin Group (Mel): rats were not induced to diabetes, treated with melatonin. Diabetes was induced by intraperitoneal administration of streptozotocin solution after 12 h food fasting. For eight weeks after the diabetes was induced, melatonin was administered via subcutaneous injection at a dose of 10 mg / kg. RTLs were measured by qPCR method with modifications. The comparison of averaged data among groups was performed using least significant difference (LSD) and Kruskal – Wallis Test and One way ANOVA test. Results: RTL was significantly similar in control and melatonin group. RTL was thinnest in DM group, in addition melatonin treatment significantly prevented the RTL shortening in DM + Mel group (p=0.031). Conclusion: We demonstrated that diabetic retinopathy led to the shortening of RTL in RPE cells in rats and melatonin treatment prevents this shortening. diabetes mellitus diabetic retinopathy melatonin retinal pigment epithelium telomer length Figures Figure 1 Introduction Diabetic retinopathy is a worldwide leading cause of blindness in working age population ( 1 ). Chronic hyperglycemic environment induces oxidative stress and inflammation which impair the blood-retinal barrier and neurosensory retinal units leading to ischemia, edema, angiogenesis, and eventual gliosis of the retina ( 2 ). Treatment options in diabetes mellitus and diabetic retinopathy are limited and can only interfere with the already established and progressed disease and its complications. Therefore, alternative treatment searches which may ameliorate or decelerate the initial pathological processes in diabetic retinopathy like oxidative stress have become a necessity. Melatonin, a hormone derived from tryptophan and mainly secreted from the pineal gland, is a candidate for these treatment alternatives since it acts as a free radical scavenger and stimulates antioxidant enzymes ( 3 ). Moreover, melatonin can easily pass-through cell membranes and bio-barriers due to its biological structure. Studies demonstrated that melatonin levels are decreased in diabetic patients with complications compared to diabetic patients without complications and also healthy individuals have more melatonin levels than the diabetic patients ( 4 ). The blood sugar regulatory effect of melatonin was indicated both in human and animal research ( 5 – 6 ). In experimental diabetic animal models and cell cultures, melatonin yielded significant benefits in diabetic retinopathy in terms of attenuating the oxidative stress, inflammation, apoptosis, and regulation autophagy ( 7 – 9 ). Telomeres are tandem repeats of DNA protein structures located at the free ends of the human chromosomes which serve as a maintainer of chromosome integrity. Telomers normally shorten in every DNA replication and cell division. Therefore, telomer lengths are the indicators of the cellular life span ( 10 ). Besides, abnormal stressful metabolic conditions like hyperglycemia and oxidative stress may lead to disrupted replication and telomere shortening ( 11 – 12 ). Hence, telomer length is also a significant biomarker of oxidative stress and associated diseases ( 13 ). Research showed that telomer shortening is related with diabetes and its vascular complications ( 14 ). It has been shown that increased oxidative stress in diabetes mellitus causes shortening of telomeres in both peripheral blood leukocyte cells and insulin-producing islet beta cells ( 15 – 16 ). In addition, it was found that the telomere length in peripheral blood leukocytes was shorter in diabetic patients with complications than in patients with uncomplicated diabetes, and the shortening in telomeres increased as the number of complications increased. Based on this, it has been suggested that telomere length can be used as a marker to evaluate diabetes complications ( 17 ). Accordingly, antioxidant therapies such as melatonin may prevent the attrition of the telomeres thus contribute to the treatment of diabetes and its vascular complications. Retinal pigment epithelium (RPE) cells have multiple tasks in regular functioning of the retina including forming the outer blood-retinal barrier, transporting the essential metabolites to retina, removing metabolic wastes, and participating in fatty acid metabolism. These high metabolic activity and contiguity with the choriocapillaris makes RPE vulnerable to metabolic stressful conditions like hyperglycemia induced oxidative stress ( 18 ). It has been shown that disruption of the integrity of the RPE, which forms the outer blood-retina barrier, has a significant effect on the formation of retinal edema in diabetic retinopathy ( 19 ). Therefore, we aimed to investigate the effect of diabetic retinopathy and melatonin treatment on the relative telomer lengths (RTL) in RPE cells in a streptozotocin-induced diabetic rat model. Materials and Methods The experimental protocol was carried out in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Academy of Sciences and ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The protocol was approved by the Ethics Committee of the Faculty of Medicine (S-1105-224-15), University of Sakarya, Turkey. The study was reported in accordance with ARRIVE guidelines. Forty Sprague-Dawley male rats weighting 250–300 grams of 10–12 weeks of age were used. Rats were placed in cages under standard laboratory conditions at 22 ° C and 50–60% humidity for six weeks in light and dark cycles for 12/12 hours. All rats were given standard tap water and fed with pellet feed. The rats were randomly divided into four groups, with ten rats per group. The experimental groups were: Control Group (C): non- diabetic rats; Diabetes Mellitus Group (DM): rats induced to diabetes without treatment; Melatonin and Diabetes Mellitus Group (Mel + DM): rats induced to diabetes and after confirmation, treated with melatonin; Melatonin Group (Mel): rats were not induced to diabetes, treated with melatonin. At the end of the experiment, rats were sacrifced under general anesthesia with 65 mg/kg (i.p.) ketamine and 7 mg/kg xylazine (i.p.) injection. Diabetes induction It was induced by intraperitoneal administration of streptozotocin solution (Cayman Chemical product number, 13104, USA) after 12 h food fasting. Streptozotocin was diluted in 10 mM sodium citrate buffer and pH 4.5 at the single dosage of 60 mg/kg animal weight. Non-diabetic and diabetic animals without treatment received equivalent doses of saline. After 30 min all animals were fed normally. Diabetes was confirmed seven days after induction. Only animals that presented blood glucose above 250 mg/dL (Media Smart, Switzerland) were included in the study, except for the control group. Melatonin administration For eight weeks after the diabetes was induced, melatonin (Cayman Chemical East Ellsworth rd. Item No. 14427, USA) was prepared in isotonic NaCl solution containing 10% ethanol daily and subcutaneous injection at a dose of 10 mg / kg. RPE isolation Eyes were cut and excess tissues were removed in cold − 4 degrees phosphate buffered saline (PBS). Eyes were excised and then kept in a large petri dish at 37°C for 1 hour in 20 U/ml papain solution (Turklab). Place two eyes in the petri dish and add 1 ml of papain solution and cover the eyes. Obtained eyes were transferred to Dulbecco’s modified eagle media supplemented with 10% fetal bovine serum to inhibit papain absorption. A recess close to the lens and ora serrata was created with a fine needle and an incision was made through the ora serrata. The absorption induced by papain allowed the retina to disappear easily and led to the appearance of the choroid-sclera complex. retina-attached RPE cells were left to absorb in 1 ml of 20 U/ml papain for an average of 8 minutes at 37°C. RPE layers were separated from the retina. The plates were incubated in trypsin-PBS and then pulverized and ready to use to obtain smaller RPE cells. Telomere Length Measurement In epidemiological studies, TL analysis and chain reaction (Q-PCR) used to measure polymerase are the two most common methods for such analysis. Q- PCR Application The number of 36B4 copies from rat RPE was determined to use as a nuclear genome standard. DNA absorbance at 260 and 280 nm was examined using an ultraviolet spectrophotometer. and the DNA concentration was measured using a Nano-Drop 2000 spectrophotometer. The relative length of telomere was measured using quantitative polymerase chain reaction (qPCR), with the primers synthesized by Sango Biotech Co., Ltd. (Shanghai, China). DNA in RPE of rats was isolated from plates using a DNA extraction kit (Tiangen, Beijing, China). Relative telomere lengths were measured by qPCR method with modifications. Briefly, qPCR (36B4) was used for the master mix obtained by creating a total volume of 20 µL of Power SYBR Master mix, including DNA samples for single copy genes and telomeres. Table 1 demonstrates the detailed content of Power SYBR Master mix. Table 1 Power SYBR Master Mix Preparation. Reagents Volumes for one sample (µl) Final concentration Power SYBR Green master mix (2x) 10 1x Primer 36B4 F (2 µM)) 1 0.1 µM Primer 36B4 R (2 µM) 1 0.1 µM H 2 0 4 DNA (5 ng/µl DNA) 4 20 ng total µl: microliter; µM: micrometer; ng: nanogram Analysis of the results was performed using an integrated software to determine the threshold cycle (Ct) value with the negative control and baseline, and to determine the availability of the Ct value according to the dissolution curve. Triple reactions were performed simultaneously for each sample and the mean Ct value of the samples was calculated. The negative control reaction was also used for each round of PCR. The telomere / copy (T / S) ratio was calculated according to the formula: △Ct1 = Ct TL – Ct 36B4 , relative T/S ratio = 2 –(△ Ct telomere – △ Ct 36B4) = 2 –△△Ct . △Ct1 stands for T/S ratio, and △Ct2 stands for the T/S ratio of the reference DNA. Statistical analysis The covariance analysis of repeated measurements by the mixed model approach was used for the telomere length analysis. The comparison of averaged data among groups was performed using least significant difference (LSD) and Kruskal – Wallis Test and One way ANOVA test. Results RTL was significantly similar in controls and melatonin group. RTL was thinnest in DM group, in addition melatonin treatment significantly prevented the RTL shortening in DM + Mel group (P = 0.031) Table 2 ; Fig. 1 . Table 2 Comparison of the relative telomer lengths among the groups. Number of Rats Relative Telomere Length (mean ± StD ) P value DM 10 1139 ± 46.3 DM – DM + Mel; P < 0.001 DM – C; P < 0.001 DM + Mel 10 1285 ± 35.3 DM + Mel – C; P = 0,264 DM + Mel – DM; P < 0.001 DM + Mel – Mel; P = 0.022 C 10 1329 ± 63.7 C – Mel; P = 0.641 C – DM; P < 0.001 Mel 10 1357 ± 61.3 Mel – C; P = 0.641 Mel – DM + Mel; P = 0.022 Mel – DM; P < 0.001 DM: Diabetes mellitus group; Mel: Melatonin group; C: Control group; DM + Mel: Diabetes mellitus treated with melatonin group StD: Standard Deviation Discussion In our study, we revealed that while diabetic retinopathy led to the shortening of the RTL in RPE, melatonin yielded a protective function on the telomer structure. To the best of our knowledge, this is the first study investigating the impact of diabetic retinopathy on the telomer length of a retinal cell in vivo. We generated an experimental diabetic retinopathy model by administering streptozotocin in rats. It was demonstrated that streptozotocin induced diabetic rats could be adopted on the purpose of evaluating the pathogenesis and treatment of diabetic retinopathy in humans ( 20 ). We enrolled RPE cells for our research as they have potent antioxidant defense mechanisms and RPE TL measurements showed similarity throughout the retina ( 21 – 22 ). Previous research suggested that diabetes mellitus and oxidative stress is associated with telomer shortening and treatments including glycemic control and antioxidant therapy prevent telomer attrition ( 23 – 25 ). Additionally, diabetic microvascular complications like diabetic retinopathy which involves endothelial cell and RPE damage are caused by the hyperglycemia induced oxidative stress which is also related to chromosome insult and telomere shortening ( 26 – 29 ). Most of these studies were examined the TL in peripheral blood cells and none of them investigated the TL of a retinal cell in a diabetic retinopathy model. In our study, we revealed that melatonin had a preventive effect on TL of RPE in diabetic retinopathy. The emergence of these result may have been caused by the effect of melatonin on many steps in the pathophysiology of diabetes mellitus. When we consider the effects of melatonin systematically; melatonin levels were demonstrated to be decreased in diabetic animal models and melatonin treatment was shown to be beneficial in controlling insulin and glucose levels ( 30 – 31 ). Moreover, studies indicated its potential use for glycemic control and decreasing the insulin resistance in human ( 32 ). Thus, it is highly probable that the glycemic control provided by melatonin contributed to the control of diabetic retinopathy. Therapeutic effects of melatonin were also investigated in animal models of diabetic retinopathy. Mehrzadi et al. ( 33 ) suggested that melatonin reduced the malondialdehyde and reactive oxygen species levels in the retina and decreased the fluorescein leakage scores on fundus fluorescence angiography in diabetic rats. Ferreira de Melo et al. ( 34 ) showed that simultaneous administration of melatonin with the streptozotocin induction provided similar insulin levels with the healthy control group due to possible antioxidative protection effect of melatonin on pancreatic β islet cells. Additionally, they demonstrated that melatonin had a regulatory effect on the expression of the inflammatory cytokines, vascular endothelial growth factor and apoptosis in diabetic retinopathy in rats. In another study, it was indicated that melatonin reduces oxidative stress and inflammation of Müller cells in diabetic retinopathy induced rats ( 35 ). These studies demonstrated that melatonin treatment showed antioxidative and anti-inflammatory properties both systemically and locally, reducing apoptosis of both islet beta cells and retinal cells, and contributed positively to the progression of diabetic retinopathy. RPE cells play a major role in maintaining the retinal integrity and proper functioning via forming the outer blood-retinal barrier, absorbing photooxidative lights, transporting essential metabolites and waste, producing cytokines and growth factors. Therefore, RPE disfunction takes part in pathophysiology of diabetic retinopathy. It was demonstrated that hyperglycemia led to acceleration of ROS which induced vascular endothelial growth factor expression in RPE ( 36 ). Our study showed that melatonin can attenuate the RPE apoptosis via preventing the telomere shortening. Chang et al. ( 37 ) indicated that melatonin can protect oxidative-stress induced apoptosis and autophagy in human RPE cells. Doğanlar et al. ( 38 ) generated an in vitro diabetic macular edema model via employing human RPE cell cultures and found that melatonin maintained the outer blood-retinal-barrier integrity and decreases hyperpermeability. They also demonstrated that melatonin reduced the expression of hypoxia-angiogenesis related genes. Finally, they revealed that melatonin reduced the expression of apoptotic genes and increased the expression of antiapoptotic genes in RPE. The outcomes of these studies along with our study supporting the hypothesis that melatonin treatment might be useful in diabetic retinopathy treatment via preventing the damage and apoptosis of the retinal cells. Conclusion In conclusion, we demonstrated that diabetic retinopathy led to the shortening of RTL in RPE in rats and melatonin treatment prevents this shortening. Moreover, possibly because melatonin maintains the integrity of both the inner and outer blood retinal blood barrier, with the effect of preventing damage to retinal cells, it may reduce the vascular leakage and ameliorated the pathological vascular signs in diabetic retinopathy. Declarations Acknowledgements: Not applicable Author Contribution: AE conceptualized and designed this study. AE participated in data collection. AE performed statistical analysis. AE created the initial draft of this manuscript. AE. contributed to critical revision of the manuscript. Author approved the final version of this manuscript. Funding: There is no funding. The author has not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. Data Availability: The data presented in this study are available on request from the corresponding author. Ethics Approval: This study involves animal participants. The protocol was approved by the Ethics Committee of the Faculty of Medicine, University of Sakarya, Turkey (S-1105-224-15). The study was reported in accordance with ARRIVE guidelines. This study was Conducted in accordance with the tenets of the Declaration of Helsinki. Consent for publication: Not applicable Conflict of Interest: The authors declare no conflict of interest. Author details: Department of health laboratory techniques, Sakarya University, Serdivan Sakarya. References Cai X, McGinnis JF. Diabetic retinopathy: animal models, therapies, and perspectives. J Diabetes Res 2016; 2016: 3789217. Lechner J, O'Leary OE, Stitt AW. The pathology associated with diabetic retinopathy. Vision Res 2017; 139 :7-14. Reiter RJ, Tan DX , Rosales-Corral S, et al. The universal nature, unequal distribution and antioxidant functions of melatonin and its derivatives. Mini Rev Med Chem 2013; 13: 373–384. Hikichi T, Tateda N, Miura T. Alteration of melatonin secretion in patients with type 2 diabetes and proliferative diabetic retinopathy. Clin Ophthalmol 2011; 5: 655–660. Sun H, Wang X, Chen J, et al. Melatonin treatment improves insulin resistance and pigmentation in obese patients with acanthosis nigricans. Int J Endocrinol 2018; 2018: 2304746. Sartori C, Dessen P, Mathieu C, et al. Melatonin improves glucose homeostasis and endothelial vascular function in high-fat diet-fed insulin-resistant mice. Endocrinology 2009; 150(12) :5311–5317. Reiter RJ, Mayo JC, Tan DX, et al. Melatonin as an antioxidant: under promises but over delivers. J Pineal Res 2016; 61(3): 253–278. Gurpinar T, Ekerbicer N, Uysal N, et al. The effects of the melatonin treatment on the oxidative stress and apoptosis in diabetic eye and brain. Sci World J 2012; 2012: 498489. Ma Y, Zhao Q, Shao Y, et al. Melatonin inhibits the inflammation and apoptosis in rats with diabetic retinopathy via MAPK pathway. 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Ophthalmol Vis Sci 2016; 57: 5547–5555. Ma D, Zhu W, Hu S, et al. Association between oxidative stress and telomere length in Type 1 and Type 2 diabetic patients. J Endocrinol Invest 2013; 36(11) :1032-1037. Uziel O, Singer JA, Danicek V, et al. Telomere dynamics in arteries and mononuclear cells of diabetic patients: effect of diabetes and of glycemic control. Exp Gerontol 2007; 42: 971-978. New evidence: bariatric surgery also reverses the effects of aging. Telomeres, genetic biomarkers of aging, are found to be longer after the surgery. Duke Med Health News 2014; 20(2): 6. Sutanto, SSI, McLennan SV, Keech AC, et al. Shortening of telomere length by metabolic factors in diabetes: Protective effects of fenofibrate. J. Cell Commun Signal 2019; 13: 523–530. Sampson MJ, Hughes DA. Chromosomal telomere attrition as a mechanism for the increased risk of epithelial cancers and senescent phenotypes in T2D. Diabetologia 2006; 49(8): 1726-1731. Matsunaga H, Handa JT, Aotaki-Keen A, et al. Beta-galactosidase histochemistry and telomere loss in senescent retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 1999; 40(1): 197-202. Honda S, Weigel A, Hjelmeland LM, et al. Induction of telomere shortening and replicative senescence bycryopreservation. Biochem Biophys Res Commun 2001; 282(2): 493-498. de Oliveira AC, Andreotti S, Sertie RAL, et al. Combined treatment with melatonin and insulin improves glycemic control, white adipose tissue metabolism and reproductive axis of diabetic male rats. Life Sci 2018; 199: 158–166. R Zanuto, MA Siqueira-Filho, LC Caperuto, et al., Melatonin improves insulin sensitivity independently of weight loss in old obese rats, J. Pineal Res 2013; 55: 156–165. Doosti-Irani A, Ostadmohammadi V, Mirhosseini N, et al. Correction: the effects of melatonin supplementation on glycemic control: a systematic review and meta-analysis of randomized controlled trials. Hormone Metabol Res 2018; 50(11): e6. S. Mehrzadi, M. Motevalian, M. Rezaei Kanavi, et al. Protective effect of melatonin in the diabetic rat retina. Fundam Clin Pharmacol 2018; 32: 414–421. Ferreira de Melo IM, Martins Ferreira CG, Lima da Silva Souza EH, et al. Melatonin Regulates the Expression of Inflammatory Cytokines, VEGF and Apoptosis in Diabetic Retinopathy in Rats. Chemico-Biological Interactions 2020; 327: 109383. Tu Y, Song E, Wang Z, et al. Melatonin attenuates oxidative stress and inflammation of Muller cells in diabetic retinopathy via activating the Sirt1 pathway. Biomed Pharmacother. 2021; 137: 111274. Simão S, Bitoque DB, Calado SM, et al. Oxidative stress modulates the expression of VEGF isoforms in the diabetic retina. N Front Ophthalmol. 2016; 2(1): 77–83. Chang CC, Huang TY, Chen HY, et al. Protective Effect of Melatonin against Oxidative Stress-Induced Apoptosis and Enhanced Autophagy in Human Retinal Pigment Epithelium Cells. Oxidative Med Cell Longev 2018; 2018: 9015765. Doğanlar ZB, Doğanlar O, Kurtdere K, et al. Melatonin prevents blood-retinal barrier breakdown and mitochondrial dysfunction in high glucose and hypoxia-induced in vitro diabetic macular edema model. Toxicol In Vitro 2021; 75: 105191. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 18 Dec, 2024 Read the published version in BMC Ophthalmology → Version 1 posted Editorial decision: Revision requested 11 Mar, 2024 Editor assigned by journal 08 Mar, 2024 Submission checks completed at journal 07 Mar, 2024 First submitted to journal 12 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3950753","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":277393080,"identity":"2c8a9bff-835c-416b-b049-22d8fb5d8e5e","order_by":0,"name":"Ayla Eren Ozdemir","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYFAC5gYGhgoGBgkol7GBsBaQmjMoWpiJ0MLYRooW/hmJjR8+zquTl2w/+0ziB4ON7IYD/Mc+4NMicSOxWXLmtsOGs3nSzSR7GNKMNxxgZp6B15obiQ3SvNsOMM5jSGOT4GE4nAjSgleHPNCW33/n1NnP43/GJvmH4T9hLQY3EtukGRuYE2dLpLFJ8zAcIKzF8MzDNsueY4eTZ854xmwtY5BsPPMwszFeLXLHkw/f+FFTZzvjfBrjzTcVdrJ9xxsf49XCIJAAZ7JIMBgAKYIxyX8AzmTGGx2jYBSMglEwcgEAm8tKpL1rIKoAAAAASUVORK5CYII=","orcid":"","institution":"Sakarya University","correspondingAuthor":true,"prefix":"","firstName":"Ayla","middleName":"Eren","lastName":"Ozdemir","suffix":""}],"badges":[],"createdAt":"2024-02-12 10:30:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3950753/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3950753/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12886-024-03732-y","type":"published","date":"2024-12-18T15:57:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52404880,"identity":"fa2b3eda-555b-45c6-b132-3ecbc8d2d80d","added_by":"auto","created_at":"2024-03-11 08:30:34","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":19580,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplot graph of the telomere lengths according to the groups.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3950753/v1/e0c929e7e0c7a5ba746412c6.jpg"},{"id":72202022,"identity":"b576d2d1-18ae-4409-86ad-628af37a176c","added_by":"auto","created_at":"2024-12-23 16:13:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":397989,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3950753/v1/aeb4b6ae-584a-40c2-a369-cf4b83344a6a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEvaluation of the Effect of Melatonin Treatment on Telomere Length of the Retinal Pigment Epithelium in Streptozotocin-Induced Diabetic Rat Model\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetic retinopathy is a worldwide leading cause of blindness in working age population (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Chronic hyperglycemic environment induces oxidative stress and inflammation which impair the blood-retinal barrier and neurosensory retinal units leading to ischemia, edema, angiogenesis, and eventual gliosis of the retina (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Treatment options in diabetes mellitus and diabetic retinopathy are limited and can only interfere with the already established and progressed disease and its complications. Therefore, alternative treatment searches which may ameliorate or decelerate the initial pathological processes in diabetic retinopathy like oxidative stress have become a necessity.\u003c/p\u003e \u003cp\u003eMelatonin, a hormone derived from tryptophan and mainly secreted from the pineal gland, is a candidate for these treatment alternatives since it acts as a free radical scavenger and stimulates antioxidant enzymes (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Moreover, melatonin can easily pass-through cell membranes and bio-barriers due to its biological structure. Studies demonstrated that melatonin levels are decreased in diabetic patients with complications compared to diabetic patients without complications and also healthy individuals have more melatonin levels than the diabetic patients (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The blood sugar regulatory effect of melatonin was indicated both in human and animal research (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In experimental diabetic animal models and cell cultures, melatonin yielded significant benefits in diabetic retinopathy in terms of attenuating the oxidative stress, inflammation, apoptosis, and regulation autophagy (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTelomeres are tandem repeats of DNA protein structures located at the free ends of the human chromosomes which serve as a maintainer of chromosome integrity. Telomers normally shorten in every DNA replication and cell division. Therefore, telomer lengths are the indicators of the cellular life span (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Besides, abnormal stressful metabolic conditions like hyperglycemia and oxidative stress may lead to disrupted replication and telomere shortening (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Hence, telomer length is also a significant biomarker of oxidative stress and associated diseases (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Research showed that telomer shortening is related with diabetes and its vascular complications (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). It has been shown that increased oxidative stress in diabetes mellitus causes shortening of telomeres in both peripheral blood leukocyte cells and insulin-producing islet beta cells (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). In addition, it was found that the telomere length in peripheral blood leukocytes was shorter in diabetic patients with complications than in patients with uncomplicated diabetes, and the shortening in telomeres increased as the number of complications increased. Based on this, it has been suggested that telomere length can be used as a marker to evaluate diabetes complications (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Accordingly, antioxidant therapies such as melatonin may prevent the attrition of the telomeres thus contribute to the treatment of diabetes and its vascular complications.\u003c/p\u003e \u003cp\u003eRetinal pigment epithelium (RPE) cells have multiple tasks in regular functioning of the retina including forming the outer blood-retinal barrier, transporting the essential metabolites to retina, removing metabolic wastes, and participating in fatty acid metabolism. These high metabolic activity and contiguity with the choriocapillaris makes RPE vulnerable to metabolic stressful conditions like hyperglycemia induced oxidative stress (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). It has been shown that disruption of the integrity of the RPE, which forms the outer blood-retina barrier, has a significant effect on the formation of retinal edema in diabetic retinopathy (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Therefore, we aimed to investigate the effect of diabetic retinopathy and melatonin treatment on the relative telomer lengths (RTL) in RPE cells in a streptozotocin-induced diabetic rat model.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e The experimental protocol was carried out in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Academy of Sciences and ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The protocol was approved by the Ethics Committee of the Faculty of Medicine (S-1105-224-15), University of Sakarya, Turkey. The study was reported in accordance with ARRIVE guidelines.\u003c/p\u003e \u003cp\u003eForty Sprague-Dawley male rats weighting 250\u0026ndash;300 grams of 10\u0026ndash;12 weeks of age were used. Rats were placed in cages under standard laboratory conditions at 22 \u0026deg; C and 50\u0026ndash;60% humidity for six weeks in light and dark cycles for 12/12 hours. All rats were given standard tap water and fed with pellet feed. The rats were randomly divided into four groups, with ten rats per group. The experimental groups were: Control Group (C): non- diabetic rats; Diabetes Mellitus Group (DM): rats induced to diabetes without treatment; Melatonin and Diabetes Mellitus Group (Mel\u0026thinsp;+\u0026thinsp;DM): rats induced to diabetes and after confirmation, treated with melatonin; Melatonin Group (Mel): rats were not induced to diabetes, treated with melatonin. At the end of the experiment, rats were sacrifced under general anesthesia with 65 mg/kg (i.p.) ketamine and 7 mg/kg xylazine (i.p.) injection.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDiabetes induction\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIt was induced by intraperitoneal administration of streptozotocin solution (Cayman Chemical product number, 13104, USA) after 12 h food fasting. Streptozotocin was diluted in 10 mM sodium citrate buffer and pH 4.5 at the single dosage of 60 mg/kg animal weight. Non-diabetic and diabetic animals without treatment received equivalent doses of saline. After 30 min all animals were fed normally. Diabetes was confirmed seven days after induction. Only animals that presented blood glucose above 250 mg/dL (Media Smart, Switzerland) were included in the study, except for the control group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMelatonin administration\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor eight weeks after the diabetes was induced, melatonin (Cayman Chemical East Ellsworth rd. Item No. 14427, USA) was prepared in isotonic NaCl solution containing 10% ethanol daily and subcutaneous injection at a dose of 10 mg / kg.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRPE isolation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eEyes were cut and excess tissues were removed in cold \u0026minus;\u0026thinsp;4 degrees phosphate buffered saline (PBS). Eyes were excised and then kept in a large petri dish at 37\u0026deg;C for 1 hour in 20 U/ml papain solution (Turklab). Place two eyes in the petri dish and add 1 ml of papain solution and cover the eyes. Obtained eyes were transferred to Dulbecco\u0026rsquo;s modified eagle media supplemented with 10% fetal bovine serum to inhibit papain absorption. A recess close to the lens and ora serrata was created with a fine needle and an incision was made through the ora serrata. The absorption induced by papain allowed the retina to disappear easily and led to the appearance of the choroid-sclera complex. retina-attached RPE cells were left to absorb in 1 ml of 20 U/ml papain for an average of 8 minutes at 37\u0026deg;C. RPE layers were separated from the retina. The plates were incubated in trypsin-PBS and then pulverized and ready to use to obtain smaller RPE cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTelomere Length Measurement\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn epidemiological studies, TL analysis and chain reaction (Q-PCR) used to measure polymerase are the two most common methods for such analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQ- PCR Application\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe number of 36B4 copies from rat RPE was determined to use as a nuclear genome standard. DNA absorbance at 260 and 280 nm was examined using an ultraviolet spectrophotometer. and the DNA concentration was measured using a Nano-Drop 2000 spectrophotometer. The relative length of telomere was measured using quantitative polymerase chain reaction (qPCR), with the primers synthesized by Sango Biotech Co., Ltd. (Shanghai, China). DNA in RPE of rats was isolated from plates using a DNA extraction kit (Tiangen, Beijing, China). Relative telomere lengths were measured by qPCR method with modifications. Briefly, qPCR (36B4) was used for the master mix obtained by creating a total volume of 20 \u0026micro;L of Power SYBR Master mix, including DNA samples for single copy genes and telomeres. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e demonstrates the detailed content of Power SYBR Master mix.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePower SYBR Master Mix Preparation.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReagents\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVolumes for one sample (\u0026micro;l)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFinal concentration\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePower SYBR Green master mix (2x)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer 36B4 F (2 \u0026micro;M))\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer 36B4 R (2 \u0026micro;M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDNA (5 ng/\u0026micro;l DNA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20 ng total\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u0026micro;l: microliter; \u0026micro;M: micrometer; ng: nanogram\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAnalysis of the results was performed using an integrated software to determine the threshold cycle (Ct) value with the negative control and baseline, and to determine the availability of the Ct value according to the dissolution curve. Triple reactions were performed simultaneously for each sample and the mean Ct value of the samples was calculated. The negative control reaction was also used for each round of PCR. The telomere / copy (T / S) ratio was calculated according to the formula:\u003c/p\u003e \u003cp\u003e△Ct1\u0026thinsp;=\u0026thinsp;Ct\u003csub\u003eTL\u003c/sub\u003e \u0026ndash; Ct\u003csub\u003e36B4\u003c/sub\u003e, relative T/S ratio\u0026thinsp;=\u0026thinsp;2\u003csup\u003e\u0026ndash;(△ Ct telomere \u0026ndash; △ Ct 36B4)\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;2\u003csup\u003e\u0026ndash;△△Ct\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003e△Ct1 stands for T/S ratio, and △Ct2 stands for the T/S ratio of the reference DNA.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe covariance analysis of repeated measurements by the mixed model approach was used for the telomere length analysis. The comparison of averaged data among groups was performed using least significant difference (LSD) and Kruskal \u0026ndash; Wallis Test and One way ANOVA test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eRTL was significantly similar in controls and melatonin group. RTL was thinnest in DM group, in addition melatonin treatment significantly prevented the RTL shortening in DM\u0026thinsp;+\u0026thinsp;Mel group (P\u0026thinsp;=\u0026thinsp;0.031) Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of the relative telomer lengths among the groups.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of Rats\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRelative Telomere Length\u003c/p\u003e \u003cp\u003e(mean \u0026plusmn; StD )\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1139 \u0026plusmn; 46.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDM \u0026ndash; DM\u0026thinsp;+\u0026thinsp;Mel; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003cp\u003eDM \u0026ndash; C; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDM\u0026thinsp;+\u0026thinsp;Mel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1285 \u0026plusmn; 35.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDM\u0026thinsp;+\u0026thinsp;Mel \u0026ndash; C; P\u0026thinsp;=\u0026thinsp;0,264\u003c/p\u003e \u003cp\u003eDM\u0026thinsp;+\u0026thinsp;Mel \u0026ndash; DM; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003cp\u003eDM\u0026thinsp;+\u0026thinsp;Mel \u0026ndash; Mel; P\u0026thinsp;=\u0026thinsp;0.022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1329 \u0026plusmn; 63.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC \u0026ndash; Mel; P\u0026thinsp;=\u0026thinsp;0.641\u003c/p\u003e \u003cp\u003eC \u0026ndash; DM; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1357 \u0026plusmn; 61.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMel \u0026ndash; C; P\u0026thinsp;=\u0026thinsp;0.641\u003c/p\u003e \u003cp\u003eMel \u0026ndash; DM\u0026thinsp;+\u0026thinsp;Mel; P\u0026thinsp;=\u0026thinsp;0.022\u003c/p\u003e \u003cp\u003eMel \u0026ndash; DM; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eDM: Diabetes mellitus group;\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eMel: Melatonin group;\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eC: Control group;\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eDM\u0026thinsp;+\u0026thinsp;Mel: Diabetes mellitus treated with melatonin group\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eStD: Standard Deviation\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn our study, we revealed that while diabetic retinopathy led to the shortening of the RTL in RPE, melatonin yielded a protective function on the telomer structure. To the best of our knowledge, this is the first study investigating the impact of diabetic retinopathy on the telomer length of a retinal cell in vivo. We generated an experimental diabetic retinopathy model by administering streptozotocin in rats. It was demonstrated that streptozotocin induced diabetic rats could be adopted on the purpose of evaluating the pathogenesis and treatment of diabetic retinopathy in humans (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). We enrolled RPE cells for our research as they have potent antioxidant defense mechanisms and RPE TL measurements showed similarity throughout the retina (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrevious research suggested that diabetes mellitus and oxidative stress is associated with telomer shortening and treatments including glycemic control and antioxidant therapy prevent telomer attrition (\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Additionally, diabetic microvascular complications like diabetic retinopathy which involves endothelial cell and RPE damage are caused by the hyperglycemia induced oxidative stress which is also related to chromosome insult and telomere shortening (\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Most of these studies were examined the TL in peripheral blood cells and none of them investigated the TL of a retinal cell in a diabetic retinopathy model.\u003c/p\u003e \u003cp\u003eIn our study, we revealed that melatonin had a preventive effect on TL of RPE in diabetic retinopathy. The emergence of these result may have been caused by the effect of melatonin on many steps in the pathophysiology of diabetes mellitus. When we consider the effects of melatonin systematically; melatonin levels were demonstrated to be decreased in diabetic animal models and melatonin treatment was shown to be beneficial in controlling insulin and glucose levels (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Moreover, studies indicated its potential use for glycemic control and decreasing the insulin resistance in human (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Thus, it is highly probable that the glycemic control provided by melatonin contributed to the control of diabetic retinopathy.\u003c/p\u003e \u003cp\u003eTherapeutic effects of melatonin were also investigated in animal models of diabetic retinopathy. Mehrzadi et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) suggested that melatonin reduced the malondialdehyde and reactive oxygen species levels in the retina and decreased the fluorescein leakage scores on fundus fluorescence angiography in diabetic rats. Ferreira de Melo et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) showed that simultaneous administration of melatonin with the streptozotocin induction provided similar insulin levels with the healthy control group due to possible antioxidative protection effect of melatonin on pancreatic β islet cells. Additionally, they demonstrated that melatonin had a regulatory effect on the expression of the inflammatory cytokines, vascular endothelial growth factor and apoptosis in diabetic retinopathy in rats. In another study, it was indicated that melatonin reduces oxidative stress and inflammation of M\u0026uuml;ller cells in diabetic retinopathy induced rats (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). These studies demonstrated that melatonin treatment showed antioxidative and anti-inflammatory properties both systemically and locally, reducing apoptosis of both islet beta cells and retinal cells, and contributed positively to the progression of diabetic retinopathy.\u003c/p\u003e \u003cp\u003eRPE cells play a major role in maintaining the retinal integrity and proper functioning via forming the outer blood-retinal barrier, absorbing photooxidative lights, transporting essential metabolites and waste, producing cytokines and growth factors. Therefore, RPE disfunction takes part in pathophysiology of diabetic retinopathy. It was demonstrated that hyperglycemia led to acceleration of ROS which induced vascular endothelial growth factor expression in RPE (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Our study showed that melatonin can attenuate the RPE apoptosis via preventing the telomere shortening. Chang et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) indicated that melatonin can protect oxidative-stress induced apoptosis and autophagy in human RPE cells. Doğanlar et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) generated an in vitro diabetic macular edema model via employing human RPE cell cultures and found that melatonin maintained the outer blood-retinal-barrier integrity and decreases hyperpermeability. They also demonstrated that melatonin reduced the expression of hypoxia-angiogenesis related genes. Finally, they revealed that melatonin reduced the expression of apoptotic genes and increased the expression of antiapoptotic genes in RPE. The outcomes of these studies along with our study supporting the hypothesis that melatonin treatment might be useful in diabetic retinopathy treatment via preventing the damage and apoptosis of the retinal cells.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, we demonstrated that diabetic retinopathy led to the shortening of RTL in RPE in rats and melatonin treatment prevents this shortening. Moreover, possibly because melatonin maintains the integrity of both the inner and outer blood retinal blood barrier, with the effect of preventing damage to retinal cells, it may reduce the vascular leakage and ameliorated the pathological vascular signs in diabetic retinopathy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eContribution:\u0026nbsp;\u003c/strong\u003eAE conceptualized and designed this study. AE participated in data collection. AE performed statistical analysis. AE created the initial draft of this manuscript. AE. contributed to critical revision of the manuscript. Author approved the final version of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThere is no funding. The author has not declared a specific grant for this research from \u0026nbsp; any funding agency in the public, commercial or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eThe data presented in this study are available on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval:\u0026nbsp;\u003c/strong\u003eThis study involves animal participants.\u0026nbsp;The protocol was approved by the Ethics Committee of the Faculty of Medicine, University of Sakarya, Turkey (S-1105-224-15). The study was reported in accordance with ARRIVE guidelines. This study was Conducted in accordance with the tenets of the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details:\u0026nbsp;\u003c/strong\u003eDepartment of health laboratory techniques, Sakarya University, Serdivan Sakarya.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCai X, McGinnis JF. Diabetic retinopathy: animal models, therapies, and perspectives. J Diabetes Res 2016; 2016: 3789217.\u003c/li\u003e\n\u003cli\u003eLechner J, O\u0026apos;Leary OE, Stitt AW. The pathology associated with diabetic retinopathy. Vision Res 2017; 139 :7-14.\u003c/li\u003e\n\u003cli\u003eReiter RJ, Tan DX , Rosales-Corral S, et al. The universal nature, unequal distribution and antioxidant functions of melatonin and its derivatives. Mini Rev Med Chem 2013; 13: 373\u0026ndash;384. \u003c/li\u003e\n\u003cli\u003eHikichi T, Tateda N, Miura T. Alteration of melatonin secretion in patients with type 2 diabetes and proliferative diabetic retinopathy. Clin Ophthalmol 2011; 5: 655\u0026ndash;660.\u003c/li\u003e\n\u003cli\u003eSun H, Wang X, Chen J, et al. Melatonin treatment improves insulin resistance and pigmentation in obese patients with acanthosis nigricans. Int J Endocrinol 2018; 2018: 2304746.\u003c/li\u003e\n\u003cli\u003eSartori C, Dessen P, Mathieu C, et al. Melatonin improves glucose homeostasis and endothelial vascular function in high-fat diet-fed insulin-resistant mice. Endocrinology 2009; 150(12) :5311\u0026ndash;5317.\u003c/li\u003e\n\u003cli\u003eReiter RJ, Mayo JC, Tan DX, et al. Melatonin as an antioxidant: under promises but over delivers. 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Oxidative stress shortens telomeres. Trends Biochem Sci 2002; 27: 339\u0026ndash;344.\u003c/li\u003e\n\u003cli\u003eTamura Y, Takubo K, Aida J, Araki A, Ito H. Telomere attrition and diabetes mellitus. Geriatr. Gerontol Int 2016; 16: 66\u0026ndash;74.\u003c/li\u003e\n\u003cli\u003eMurillo-Ortiz B, Albarr\u0026aacute;n-Tamayo F, Arenas-Aranda D et al. Telomere length and type 2 diabetes in males, a premature aging syndrome. Aging Male 2012; 15: 54\u0026ndash;58.\u003c/li\u003e\n\u003cli\u003eTamura Y, Izumiyama-Shimomura N, Kimbara Y, et al. beta-Cell Telomere Attrition in Diabetes: inverse Correlation Between HbA1c and Telomere Length. J Clin Endocrinol Metab 2014; 99(8): 2771-2777\u003c/li\u003e\n\u003cli\u003eQi Nan W, Ling Z, Bing C. The infuence of the telomeretelomerase system on diabetes mellitus and its vascular complications. Expert Opin Ther Targets 2015; 19: 849\u0026ndash;864.\u003c/li\u003e\n\u003cli\u003eXie M, Hu A, Luo Y, et al. Interleukin-4 and melatonin ameliorate high glucose and interleukin-1\u0026beta; stimulated inflammatory reaction in human retinal endothelial cells and retinal pigment epithelial cells. Mol Vis 2014; 20: 921\u0026ndash;928.\u003c/li\u003e\n\u003cli\u003eXu HZ, Le YZ. Significance of Outer Blood-Retina Barrier Breakdown in Diabetes and Ischemia. Invest. Ophthalmol Vis Sci 2010; 52:2160\u0026ndash;2164.\u003c/li\u003e\n\u003cli\u003eNaderi A, Zahed R, Aghajanpour L, et al. Long term features of diabetic retinopathy in streptozotocin‑induced diabetic Wistar rats. Exp Eye Res 2019; 184: 213‑220.\u003c/li\u003e\n\u003cli\u003eHanda JT. How does the macula protect itself from oxidative stress? Mol Aspects Med 2012; 33: 418\u0026ndash;435.\u003c/li\u003e\n\u003cli\u003eDrigeard Desgarnier, MC, Zinflou C, Mallet JD, et al. Telomere Length Measurement in Different Ocular Structures: A Potential Implication in Corneal Endothelium Pathogenesis. Investig. Ophthalmol Vis Sci 2016; 57: 5547\u0026ndash;5555.\u003c/li\u003e\n\u003cli\u003eMa D, Zhu W, Hu S, et al. Association between oxidative stress and telomere length in Type 1 and Type 2 diabetic patients. J Endocrinol Invest 2013; 36(11) :1032-1037.\u003c/li\u003e\n\u003cli\u003eUziel O, Singer JA, Danicek V, et al. Telomere dynamics in arteries and mononuclear cells of diabetic patients: effect of diabetes and of glycemic control. Exp Gerontol 2007; 42: 971-978.\u003c/li\u003e\n\u003cli\u003eNew evidence: bariatric surgery also reverses the effects of aging. Telomeres, genetic biomarkers of aging, are found to be longer after the surgery. Duke Med Health News 2014; 20(2): 6.\u003c/li\u003e\n\u003cli\u003eSutanto, SSI, McLennan SV, Keech AC, et al. Shortening of telomere length by metabolic factors in diabetes: Protective effects of fenofibrate. J. Cell Commun Signal 2019; 13: 523\u0026ndash;530.\u003c/li\u003e\n\u003cli\u003eSampson MJ, Hughes DA. Chromosomal telomere attrition as a mechanism for the increased risk of epithelial cancers and senescent phenotypes in T2D. Diabetologia 2006; 49(8): 1726-1731.\u003c/li\u003e\n\u003cli\u003eMatsunaga H, Handa JT, Aotaki-Keen A, et al. Beta-galactosidase histochemistry and telomere loss in senescent retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 1999; 40(1): 197-202.\u003c/li\u003e\n\u003cli\u003eHonda S, Weigel A, Hjelmeland LM, et al. Induction of telomere shortening and replicative senescence bycryopreservation. Biochem Biophys Res Commun 2001; 282(2): 493-498.\u003c/li\u003e\n\u003cli\u003ede Oliveira AC, Andreotti S, Sertie RAL, et al. Combined treatment with melatonin and insulin improves glycemic control, white adipose tissue metabolism and reproductive axis of diabetic male rats. Life Sci 2018; 199: 158\u0026ndash;166.\u003c/li\u003e\n\u003cli\u003eR Zanuto, MA Siqueira-Filho, LC Caperuto, et al., Melatonin improves insulin sensitivity independently of weight loss in old obese rats, J. Pineal Res 2013; 55: 156\u0026ndash;165.\u003c/li\u003e\n\u003cli\u003eDoosti-Irani A, Ostadmohammadi V, Mirhosseini N, et al. Correction: the effects of melatonin supplementation on glycemic control: a systematic review and meta-analysis of randomized controlled trials. Hormone Metabol Res 2018; 50(11): e6.\u003c/li\u003e\n\u003cli\u003eS. Mehrzadi, M. Motevalian, M. Rezaei Kanavi, et al. Protective effect of melatonin in the diabetic rat retina. Fundam Clin Pharmacol 2018; 32: 414\u0026ndash;421.\u003c/li\u003e\n\u003cli\u003eFerreira de Melo IM, Martins Ferreira CG, Lima da Silva Souza EH, et al. Melatonin Regulates the Expression of Inflammatory Cytokines, VEGF and Apoptosis in Diabetic Retinopathy in Rats. Chemico-Biological Interactions 2020; 327: 109383.\u003c/li\u003e\n\u003cli\u003eTu Y, Song E, Wang Z, et al. Melatonin attenuates oxidative stress and inflammation of Muller cells in diabetic retinopathy via activating the Sirt1 pathway. Biomed Pharmacother. 2021; 137: 111274.\u003c/li\u003e\n\u003cli\u003eSim\u0026atilde;o S, Bitoque DB, Calado SM, et al. Oxidative stress modulates the expression of VEGF isoforms in the diabetic retina. N Front Ophthalmol. 2016; 2(1): 77\u0026ndash;83.\u003c/li\u003e\n\u003cli\u003eChang CC, Huang TY, Chen HY, et al. Protective Effect of Melatonin against Oxidative Stress-Induced Apoptosis and Enhanced Autophagy in Human Retinal Pigment Epithelium Cells. Oxidative Med Cell Longev 2018; 2018: 9015765.\u003c/li\u003e\n\u003cli\u003eDoğanlar ZB, Doğanlar O, Kurtdere K, et al. Melatonin prevents blood-retinal barrier breakdown and mitochondrial dysfunction in high glucose and hypoxia-induced in vitro diabetic macular edema model. Toxicol In Vitro 2021; 75: 105191.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-ophthalmology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"boph","sideBox":"Learn more about [BMC Ophthalmology](http://bmcophthalmol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/boph","title":"BMC Ophthalmology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"diabetes mellitus, diabetic retinopathy, melatonin, retinal pigment epithelium, telomer length","lastPublishedDoi":"10.21203/rs.3.rs-3950753/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3950753/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjectives: \u003c/strong\u003eWe aimed to investigate the effect of diabetic retinopathy and melatonin treatment on the relative telomer lengths (RTL) in retinal pigment epithelium (RPE) cells in a streptozotocin-induced diabetic rat model.\u003c/p\u003e\n\u003cp\u003eBackground: TL can be used to evaluate diabetes mellitus, its complications, and the effectiveness of its treatment. However, TL assessment has not been performed in retinal cells in a diabetic retinopathy model until now.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Forty Sprague-Dawley male rats were randomly divided into four groups. The experimental groups were: Control Group (C): non- diabetic rats; Diabetes Mellitus Group (DM): rats induced to diabetes without treatment; Melatonin and Diabetes Mellitus Group (Mel +DM): rats induced to diabetes and after confirmation, treated with melatonin; Melatonin Group (Mel): rats were not induced to diabetes, treated with melatonin. Diabetes was induced by intraperitoneal administration of streptozotocin solution after 12 h food fasting. For eight weeks after the diabetes was induced, melatonin was administered via subcutaneous injection at a dose of 10 mg / kg. RTLs were measured by qPCR method with modifications. The comparison of averaged data among groups was performed using least significant difference (LSD) and Kruskal – Wallis Test and One way ANOVA test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e RTL was significantly similar in control and melatonin group. RTL was thinnest in DM group, in addition melatonin treatment significantly prevented the RTL shortening in DM + Mel group (p=0.031).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eWe demonstrated that diabetic retinopathy led to the shortening of RTL in RPE cells in rats and melatonin treatment prevents this shortening.\u003c/p\u003e","manuscriptTitle":"Evaluation of the Effect of Melatonin Treatment on Telomere Length of the Retinal Pigment Epithelium in Streptozotocin-Induced Diabetic Rat Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-11 08:30:29","doi":"10.21203/rs.3.rs-3950753/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-11T08:03:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-08T12:59:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-07T12:57:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Ophthalmology","date":"2024-02-12T10:25:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-ophthalmology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"boph","sideBox":"Learn more about [BMC Ophthalmology](http://bmcophthalmol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/boph","title":"BMC Ophthalmology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"45a702c4-3f5c-456e-a26c-6f430947f8e3","owner":[],"postedDate":"March 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-23T16:07:03+00:00","versionOfRecord":{"articleIdentity":"rs-3950753","link":"https://doi.org/10.1186/s12886-024-03732-y","journal":{"identity":"bmc-ophthalmology","isVorOnly":false,"title":"BMC Ophthalmology"},"publishedOn":"2024-12-18 15:57:26","publishedOnDateReadable":"December 18th, 2024"},"versionCreatedAt":"2024-03-11 08:30:29","video":"","vorDoi":"10.1186/s12886-024-03732-y","vorDoiUrl":"https://doi.org/10.1186/s12886-024-03732-y","workflowStages":[]},"version":"v1","identity":"rs-3950753","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3950753","identity":"rs-3950753","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}