RU2776131C1 - Method for treatment of polycystic ovarian syndrome based on experimental model of wistar rats - Google Patents

Abstract

FIELD: experimental medicine.

SUBSTANCE: invention relates to experimental medicine, namely to obstetrics and gynecology, and can be used for the treatment of polycystic ovary syndrome (PCOS) in an experimental model of Wistar rats. The method consists in the fact that on the model of drug-induced polycystic ovary syndrome using letrozole at a dosage of 800 mcg/day or 400 mcg /day for 60 days, oral administration of natural phytoalexin resveratrol is performed at a dosage of 30 mg/kg/day or 20 mg/day. kg/day for 30 days.

EFFECT: invention helps to restore the estrous cycle, reduce body weight, reduce cystic changes, increase the layer of granular cells, as well as the appearance of oocytes.

1 cl, 1 ex, 3 dwg

Description

The invention relates to experimental medicine, namely to obstetrics and gynecology, and can be used for the treatment of polycystic ovary syndrome (PCOS) based on an experimental model of Wistar rats.

Polycystic ovary syndrome (PCOS) is a common endocrine pathology among women of reproductive age, with an incidence of 8-21% [Neven ACH, Laven J, Teede HJ, Boyle JA. A Summary on Polycystic Ovary Syndrome: Diagnostic Criteria, Prevalence, Clinical Manifestations, and Management According to the Latest International Guidelines. Semin. reproduction. Med. 2018; 36(1): 5-12]. It is known that PCOS is associated with metabolic disorders, insulin resistance (IR), impaired glucose tolerance (IGT), diabetes mellitus (DM) and a significant increase in the risk of developing cardiovascular diseases [Murri M, Luque-ram'irez M, Insensor MM, Ojeda-Ojeda M, Escobar-Morreale HF. Circulating markers of oxidative stress and polycystic ovary syndrome (PCOS): a systematic review and meta-analysis. Hum Reprod Update. 2013; 19(3): 268-88; Zhang J, Fan P, Liu H, Bai H, Wang Y, Zhang F. Apolipoprotein A-I and B levels, dyslipidemia and metabolic syndrome in south-west Chinese women with PCOS. Hum reproduction. 2012; 27(8): 2484-93]. The pathogenesis of the damaging effect of carbohydrate metabolism disorders (hyperglycemia, insulin resistance) is associated with the initiation of the polyol pathway of glucose metabolism, the accumulation of early and end glycation products (AGEs) in tissues, which include products of non-enzymatic glycation and protein oxidation. AGEs are a chemically heterogeneous group of compounds - glycotoxins. Pathological glycation processes lead to irreversible cross-linking of proteins, loss of protein structure and function, followed by cell apoptosis. The damaging effect of AGE manifests itself both independently (receptor-independent) and through binding to receptors for advanced glycation end products (RAGE - receptor for advanced glycation end products). Recent studies have shown that advanced glycation end products are involved in the pathogenesis of metabolic disorders in PCOS [Diamanti-Kandarakis E, Chatzigeorgiou A, Papageorgiou E, Koundouras D, Koutsilieris M. Advanced glycation end-products and insulin signaling in granulosa cells. Exp Biol Med (Maywood). 2016 Jul; 241(13): 1438-45. doi: 10.1177/1535370215584937. Epub 2015 May 7. PMID: 25956684; PMCID: PMC4994916].

Well-known treatments for PCOS are lifestyle modification, drug therapy, which is considered as the first line - combined oral contraceptives (COC), metformin, and laparoscopic drilling [Teede HJ, Misso ML, Costello MF, Dokras A, Laven J, Moran L , Piltonen T, Norman RJ; International PCOS Network. Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome. Fertil Steril. 2018 Aug; 110(3): 364-379]. In women with PCOS who are not interested in pregnancy, the first line of therapy is COCs with an antiandrogenic effect, taking into account the acceptability of prescribing contraception. WHO [World Health Organization. 2015 Quick Reference Chart for the WHO Medical Eligibility Criteria for Contraceptive Use. Adapted from Medical Eligibility Criteria for Contraceptive Use, 5th Edition 2015]. However, it should also be taken into account that PCOS is associated with a number of concomitant conditions: obesity, hyperlipidemia, arterial hypertension, in which COC administration may be contraindicated due to the possible development of complications. In this case, the indication for laparoscopic drilling is the presence of infertility or resistance to drugs used to stimulate ovulation [Bordewijk EM, Ng KYB, Rakic L, et al. Laparoscopic ovarian drilling for ovulation induction in women with anovulatory polycystic ovary syndrome. Cochrane Database Syst Rev 2020; 2: CD001122], but it is not recommended to resort to surgical treatment to regulate the menstrual cycle and eliminate hyperandrogenemia in the absence of the above indications [Lepine S, Jo J, Metwally M, Cheong YC. Ovarian surgery for symptom relief in women with polycystic ovary syndrome. Cochrane Database Syst Rev. 2017; 11(11): CD009526].

Currently, there is no single approach and universal method for the treatment of PCOS, given the extremely polymorphic clinical picture and the identification of four disease phenotypes. It is promising to study the effectiveness of the use of various drugs on the course of the disease in the framework of preclinical trials using experimental models on rats.

Most experimental models of PCOS are created in rats using androgens. Modeling of PCOS in rats, in our opinion, is the most appropriate, since these laboratory animals have a similar course of physiological and pathological processes with humans, which allows using the results of animal studies to create new methods of treating this disease in humans. The use of androgens to initiate PCOS is due to the fact that hyperandrogenemia is one of the main criteria for this disease [Ota H., Fukushima M., Maki M. Endocrinological and Histological Aspects of the Process of Polycystic Ovary Formation in the Rat Treated with Testosterone Propionate. Tohoku J. exp. Med., 1983, 140, 121-131; Wu X.Y., Li Z.L., Wu C.Y., Liu Y.M., Lin H., Wang S.H., Xiao W.F. Endocrine Traits of Polycystic Ovary Syndrome in Prenatally Androgenized Female Sprague-Dawley Rats. Endocrine Journal 2010, 57(3), 201-209; Tyndall V., Broyde M., Sharpe R., Welsh M., Drake A.J. and McNeilly A.S. Effect of androgen treatment during foetal and/or neonatal life on ovarian function in prepubertal and adult rats. Reproduction, 2012, 143 21-33].

There is a known method for the treatment of PCOS syndrome based on an experimental model in which metformin was used on a rat model as the only therapeutic drug, as well as in combination with resveratrol [Rencber S.F., Sema Kurnaz Ozbek S.K, Eraldemir C, Sezer Z., Kum T. , Ceylan S., Guzel E. Effect of resveratrol and metformin on ovarian reserve and ultrastructure in PCOS: an experimental study. Furat Rencber et al. Journal of Ovarian Research (2018) 11:55 https://doi.org/l0.1186/s 13048-018-0427-7]. Metformin (1,1-dimethylbiguanide hydrochloride) is an insulin sensitizer used to treat type 2 diabetes mellitus [Kai Y., Kawano Y., Yamamoto H., Narahara H. A possible role for AMP - activated protein kinase activated by metformin and AICAR in human granulosa cells. Reprod Biol Endocrinol. 2015; 13(1): 1] and polycystic ovary syndrome [Patel R., Shah G. Effect of metformin on clinical, metabolic and endocrine outcomes in women with polycystic ovary syndrome: a metaanalysis of randomized controlled trials. Curr Med Res Opin. 2017: 1-13]. During the use of metformin as the main drug, or in combination with resveratrol, a decrease in body weight of rats was noted, as well as normalization of ovarian morphology. Against the background of the use of only resveratrol, the authors did not reveal any significant changes. The use of metformin in the experimental model was taken by us as a prototype.

However, in clinical practice, when using metformin, one should be aware of the presence of common side effects in the form of dyspeptic phenomena.

Therefore, it is necessary to further study the pathogenesis of PCOS with the development of alternative methods of pathogenetically substantiated therapy.

The technical result of the invention is the expansion of the arsenal of drugs used for the treatment of PCOS, the possibility of using natural phytoalexin resveratrol, restoration of the estrous cycle, weight loss, normalization of the morphological structure of the ovaries - a significant decrease in cystic changes, an increase in the layer of granular cells, as well as the appearance of oocytes (mainly when using a dosage of resveratrol 30 mg / kg / day).

The specified technical result is achieved in a method for the treatment of PCOS based on an experimental model of Wistar rats, in which oral administration is performed on a histologically confirmed experimental model of drug-induced PCOS formed using letrozole at a dosage of 400 μg/day or 800 μg/day for 60 days. natural phytoalexin resveratrol at a dosage of 30 mg/kg/day or 20 mg/kg/day, respectively, for 30 days.

The method is illustrated in Fig. 1-3 where:

in fig. 1 - morphological changes in the ovary of the Wistar rat against the background of the use of letrozole: cystic changes in the follicles, flattening of the layer of granular cells, absence of oocytes, H-E, ×500 μm;

in fig. 2 - morphology of the ovaries of rats of the control group, G-E ×500 µm;

in fig. 3 - increase in the layer of granular cells, the appearance of oocytes after resveratrol therapy in rats with polycystic ovary syndrome, G-E ×500 μm.

For histological confirmation of the formation of the PCOS model, a unilateral oophorectomy was performed. At the same time, after the surgical intervention, in the first 4 days from the moment of the operation, in order to prevent postoperative complications, an antibacterial drug - ceftriaxone 100 mg/kg twice a day and a nonsteroidal anti-inflammatory drug - ketoprofen 40 mg/kg twice a day were administered intramuscularly. In addition, before the start of the experiment, and also 60 days after the start of daily use of the non-selective aromatase inhibitor letrozole, all rats underwent blood sampling from the peripheral part of the tail to determine the level of soluble receptors for advanced glycation end products (sRAGE).

After receiving histological confirmation of the formation of the PCOS model, oral administration of a solution of natural phytoalexin - resveratrol at a dosage of 20 mg/kg/day and 30 mg/kg/day is performed for 30 days. Then, after 30 days, a second surgical intervention, ovariectomy, is performed.

The isoflavone resveratrol (trans-3,4',5-trihydroxystilbene) can be isolated from the skin of grapes and a number of other products, including mulberries and flowers of some plants [I B, Shi C, Meng J, et al. Resveratrol alleviates ethanol-induced neuroinflammation in vivo and in vitro: involvement of TLR2-MyD88-NF-jB pathway. Int J Biochem Cell Biol. 2018; 103:56-64; Gao Y, Kang L, Li C, et al. Resveratrol ameliorates diabetes-induced cardiac dysfunction through AT1R-ERK/p38 MAPK signaling pathway. Cardiovasc. Toxicol. 2016; 16(2): 130-137]. According to the literature, resveratrol has anti-inflammatory, antidiabetic activity [Guo S, Yao Q, Ke Z, et al. Resveratrol attenuates high glucoseinduced oxidative stress and cardiomyocyte apoptosis through AMPK. Mol Cell Endocrinol. 2015; 412: 85-94] and is also able to induce [Harati K, Slodnik P, Chromik AM, et al. Resveratrol induces apoptosis and alters gene expression in human fibrosarcoma cells. Anticancer Res. 2015; 35(2): 767-774; Wu XP, Xiong M, Xu CS, et al. Resveratrol induces apoptosis of human chronic myelogenous leukemia cells in vitro through p38 and JNK-regulated H2AX phosphorylation. Acta Pharmacol Sin. 2015; 36(3): 353-361], or inhibit [Singh CK, Kumar A, Hitchcock DB, et al. Resveratrol prevents embryonic oxidative stress and apoptosis associated with diabetic embryopathy and improves glucose and lipid profile of diabetic dam. Mol Nutr Food Res. 2011; 55(8): 1186-1196; MacCarrone M, Lorenzon T, Guerrieri P, et al. Resveratrol prevents apoptosis in K562 cells by inhibiting lipoxygenase and cyclooxygenase activity. Eur J Biochem. 1999; 265(1): 27-34] apoptosis.

The claimed method is based on experimental studies on 24 female Wistar rats. All laboratory animals were bred at the Rappolovo Laboratory Animal Nursery and kept under regulated vivarium conditions in compliance with all rules for keeping laboratory animals (time and procedure for quarantine, marking of all individuals, constant sanitary control, standard diet, free access to water and food, automatic lighting mode "day / night"). Animal care and experiments were carried out in accordance with the basic moral and ethical principles for conducting biomedical experiments on animals, formulated in the following documents: "Rules of laboratory practice in the Russian Federation" approved by order of the Ministry of Health of the Russian Federation No. 199n of 04/01/2016 practice” and the European Convention of 22.09.2010

The study included 2 stages.

Randomization of rats was carried out, 3 groups were formed: group 1, receiving non-steroidal aromatase inhibitor letrozole at a dosage of 800 mcg/day (n=11); the 2nd, receiving the non-steroidal aromatase inhibitor letrozole at a dosage of 400 mcg/day (n=9); 3rd - control, receiving physiological saline orally (n=4).

At the first stage of the experiment, a model of polycystic ovary syndrome was formed in Wistar rats with an average weight of 203.67 ± 12.3 grams, with oral administration of a non-steroidal aromatase inhibitor - letrozole at a dosage of 400 and 800 μg / day for 60 days. The inclusion of female rats in the experiment was carried out in the estrus phase, then, for 2 months, the vaginal cytology of rat smears was studied, which showed irregular estrous cycling against the background of the use of aromatase inhibitors, thereby confirming the successful modeling of polycystic ovary syndrome. The appearance of irregular estrous cycling is considered as a factor indicating the formation of PCOS based on the literature [Furat Rencber, S., Kurnaz Ozbek, S., Eraldemir, C, Sezer, Z., Kum, T., Ceylan, S., & Guzel , E. (2018). Effect of resveratrol and metformin on ovarian reserve and ultrastructure in PCOS: an experimental study. Journal of Ovarian Research, 11(1). doi:10.1186/sl3048-018-0427-7]. In addition, a significant increase in body weight of rats receiving letrozole was noted in comparison with the control group (287.24 ± 12.12 grams - when taking 800 μg / day; 268.75 ± 32.27 grams - when taking 400 μg / day and 210 .00±15.81 grams, p<0.05, respectively), also the weight of the rats was significantly higher than at the stage before the start of letrozole intake (287.24±12.12 grams - when taking 800 mcg/day; 268.75 ±32.27 grams - when taking 400 mcg/day and 203.45±11.35 grams, p<0.05). Also, we studied the levels of soluble glycation end product receptors (sRAGE) in the peripheral blood of rats by enzyme immunoassay using the RayBio Rat RAGE ELISA Kit test system, USA. Blood sampling was carried out from the peripheral part of the tail before the start of the experiment, as well as after the end of the administration of the non-steroidal aromatase inhibitor letrozole. The level of sRAGE in the peripheral blood of rats when taking letrozole was significantly lower (464.17±14.96 pg/ml when taking 800 µg/day and 462.27±12.92 pg/ml when taking 400 µg/day) than in control group (897.5±98.45 pg/ml), as well as than in the group before taking the aromatase inhibitor (733.8±198.35 pg/ml), p<0.05. These results also indicate the successful formation of a model of polycystic ovary syndrome.

After 60 days from the start of the aromatase inhibitor, surgery was performed to confirm the formation of the PCOS model. Operations were performed under general anesthesia of animals using zolazepam hydrochloride 100 at a dosage of 30 mg/kg intramuscularly. After the site of the surgical access was freed from hair with the help of a chemical depilatory agent (“Veet”), a laparotomy followed by a left-sided oophorectomy was performed. In the first 4 days after the operation, in order to prevent postoperative complications, an antibacterial drug - ceftriaxone 100 mg/kg once a day and a non-steroidal anti-inflammatory drug - ketoprofen 40 mg/kg twice a day were administered intramuscularly.

The resulting material was fixed in 10% neutral buffered formalin. The material was stained with hematoxylin and eosin, then, using light microscopy, the change in the morphology of the ovaries was assessed in the form of cystic-modified follicles and a decrease in the layer of granular cells. Light microscopy was performed on an Olympus СХ31 microscope (Olympus, Japan) at ×200, ×500 magnification.

Samples of rat ovaries after the use of aromatase inhibitors, as well as the control group, were examined. Morphological changes in the structure of the ovaries were detected in 18 (90%) ovarian samples from letrozole-treated rats. In 2 samples of ovaries from rats receiving a dosage of letrozole 400 μg/day, morphological changes in the structure were not detected (Fig. 1). In the studied samples of the control group, the presence of oocytes, an increase in the layer of granular cells, and the absence of cystic changes in the follicles were observed (Fig. 2).

The second stage consisted in the treatment of PCOS in an experimental model of Wistar rats using natural phytoalexin-resveratrol.

After confirming the formation of the model and assessing the morphological changes in the structure of the ovaries (n=18), rats were randomized. During randomization, 3 groups were formed: the 1st group included animals with PCOS receiving a solution of natural phytoalexin resveratrol at a dosage of 30 mg/kg/day (n=8); in the 2nd group - rats with PCOS formed, receiving a solution of natural phytoalexin resveratrol at a dosage of 20 mg/kg/day (n=6); in the 3rd group - comparisons, animals with PCOS were included, receiving saline orally (n=4). Resveratrol therapy was carried out for 30 days, after which the animals were withdrawn from the experiment. After opening, a visual assessment of the morphological changes in the ovaries was performed, and a right-sided oophorectomy was performed. The material was fixed in a neutral 10% formalin solution for subsequent histological examination.

A histological study of ovarian samples of rats that received resveratrol, as well as animals from the comparison group, was carried out.

In the study of samples of the ovaries of rats of the comparison group, a flattening of the layer of granular cells was observed, as well as the presence of cystic-modified follicles.

In studies of ovarian samples of animal groups (after resveratrol therapy), a significant decrease in cystic changes, an increase in the layer of granular cells, the appearance of oocytes in 87.5% of cases in female rats (n = 7) receiving resveratrol at a dosage of 30 mg / kg / day, and in 66.7% of cases in female rats (n=5) receiving the drug at a dosage of 20 mg/kg/day (Fig. 3).

The results obtained demonstrated the dose-dependent efficacy of natural phytoalexin resveratrol in the treatment of drug-induced polycystic ovary syndrome based on an experimental model in rats. The presented data are the basis for continuing further experimental studies in order to evaluate the most effective minimum doses, duration of therapy, as well as the possibility of using resveratrol in clinical practice in patients with PCOS.

An example of the implementation of the method.

When taking smears in rats No. 5 and No. 12 weighing 200 and 196 grams, the estrus phase was confirmed. Blood was taken from the peripheral part of the tail to determine the level of soluble glycation end products receptors (610 pg / ml in rat No. 5 and 500 pg / ml in rat No. 12), then, for 60 days, rat No. 5 received 800 mcg / day non-selective aromatase inhibitor letrozole, rat No. 12 received 400 μg/day of the above drug. Within 60 days, smears were also taken from rats to determine changes in the astral cycle - an irregular estrous cycle was observed, weight was measured once a week - a significant increase in weight was noted in rats (285 grams - rat No. 5 and 290 grams - rat No. 12) against the background of use of the non-selective aromatase inhibitor letrozole. After 60 days, blood was taken from the peripheral part of the tail to determine the level of soluble receptors for advanced glycation end products (rat No. 5 - 470 pg / ml; rat No. 12 - 438 pg / ml), then, after anesthesia of the animals and preparation of the surgical field, performed laparotomy using a median vertical incision. Next, a visual assessment of the morphology of the ovaries was performed, and a left-sided ovariectomy was performed. The next 4 days after the operation, to prevent complications, ceftriaxone 9 IU was administered using an insulin syringe for 100 IU-1 ml, ketoprofen 40 IU twice a day. Then, after receiving histological confirmation of the creation of a model of polycystic ovary syndrome, for 30 days the animals were injected with a solution of natural phytoalexin restveratrol at a dosage of: rat No. 5-30 mg/kg/day, rat No. 12-20 mg/kg/day. Further, within 30 days of drug use in rats, vaginal cytology was examined for the regularity of the estrous cycle, and the body weight of rats was measured once a week. After 30 days, the rats were taken out of the experiment, to evaluate the effectiveness of the use of resveratrol in relation to changes in the morphology of the ovaries, a laparotomy, right-sided ovariectomy was performed. According to the results of histological examination, a significant decrease in cystic changes, an increase in the layer of granular cells, and the appearance of oocytes were noted.

The claimed method expands the arsenal of drugs for the treatment of polycystic ovary syndrome, allows you to study the effectiveness of the use of resveratrol as a drug for the treatment of this syndrome, improve the personification of the treatment of the disease, conduct research on the assessment of the most effective minimum doses, duration of therapy, as well as the possibility of using resveratrol in clinical practice in patients with polycystic ovary syndrome.

Claims (1)

A method for the treatment of polycystic ovary syndrome based on an experimental model in Wistar rats, which consists in the fact that in the model of drug-induced polycystic ovary syndrome using letrozole at a dosage of 800 μg/day or 400 μg/day for 60 days, oral administration of natural phytoalexin resveratrol at a dosage of 30 mg/kg/day or 20 mg/kg/day for 30 days.

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