Use of a drug with antioxidant effect for preparing a drug for treating polycystic ovary syndrome

Abstract

The present invention belongs to the field of biomedicine, and in particular to the use of a drug with an antioxidant effect for preparing a drug for treating polycystic ovary syndrome. Our research found that PCOS hyperandrogenism induces hyperactivity of the polyol pathway, and the metabolites of this pathway lead to excessive oxidative stress response of cells, affecting the microenvironment of follicular development, increasing follicular atresia, and hindering follicular maturation, which is an important pathological mechanism of PCOS ovarian dysfunction. The use of drugs with an antioxidant effect can improve the microenvironment of follicular growth, help the maturation and ovulation of follicles, and thus can be used for the treatment of PCOS.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and the core of the invention is the use of a drug with antioxidant effect in treating polycystic ovary syndrome. Background Art

[0002] The main function of the ovary is to grow follicles, discharge mature eggs and synthesize and secrete sex hormones. Follicular growth is a long process. Only during the luteal-follicular transformation period of each menstrual cycle after puberty, a group of follicles that have developed to the early antral stage can enter the final rapid growth stage that is highly dependent on gonadotropin and eventually become mature follicles. When the follicles are close to maturity, under the action of luteinizing hormone, the oocytes complete the first meiosis to become mature oocytes and ovulate.

[0003] Polycystic ovary syndrome (PCOS) is the most common reproductive endocrine disorder in women of childbearing age, with an incidence rate of up to 5%-10%. The high-fat and high-sugar diet in modern society not only increases the incidence of cardiovascular diseases, but also significantly increases the incidence of PCOS. The main pathological characteristics of PCOS are hyperandrogenemia, polycystic ovarian changes, rare ovulation or anovulation. According to incomplete statistics, about 76% of PCOS patients in China have hyperandrogenemia. A large number of animal and cell experimental studies have found that the excessive response of PCOS follicular cells to luteinizing hormone (LH) and the inactivation of aromatase lead to excessive synthesis and secretion of androgens, and high androgens can prevent the further growth and maturation of mid- and late-stage follicles.

[0004] So far, hyperandrogenemia has been recognized as the core pathological feature of PCOS. Androgen is an important reproductive hormone that plays an important regulatory role in the reproductive function of both men and women, and also has a wide range of effects on other functional activities outside the human reproductive system. In the male reproductive system, androgen-induced ADR regulation of the polyol pathway has important physiological significance for male reproductive function. Clinically, androgen-lowering treatment is often used to promote follicular maturation and ovulation. Obviously, the effect of high androgen on ovarian follicle growth plays an extremely important role in the pathological process of PCOS. But to date, the mechanism by which high androgen hinders follicle growth and ovulation is still unclear. Summary of the invention

[0005] The purpose of the present invention is to overcome the problem of lack of effective drugs for polycystic ovary syndrome in the prior art, and to provide a drug with antioxidant effect for use in treating polycystic ovary syndrome.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention discloses a drug with an antioxidant effect for preparing a drug for treating polycystic ovary syndrome.

[0008] Use of drugs with antioxidant effects for the treatment of polycystic ovary syndrome.

[0009] Androgens can directly regulate the expression of aldose reductase, thereby activating the polyol pathway. Excessive glucose in follicular cells is metabolized through the polyol pathway, resulting in excessive oxidative stress, which has a negative impact on oocyte maturation.

[0010] The high androgen level in PCOS can stimulate the abnormally high expression of ADR in ovarian tissue, leading to the hyperactivity of the polyol pathway. The accumulation of sorbitol and fructose, the metabolites of this pathway, and the resulting excessive oxidative stress in cells affect the microenvironment of follicular development, ultimately leading to excessive local oxidative stress in the ovaries, causing follicular atresia, hindering follicular maturation and ovulation, and corpus luteum formation, and affecting ovarian function.

[0011] The use of drugs with antioxidant effects can eliminate the hyperactivity of the polyol pathway caused by high androgen levels, correct the microenvironment changed by excessive oxidative stress response of cells, eliminate the resulting follicle maturation and ovulation disorders, and achieve the effect of treating polycystic ovary syndrome.

[0012] Furthermore, the drug with antioxidant effect is N-acetyl-L-cysteine ​​(NAC), flutamide, and epalrestat.

[0013] Treatment of PCOS mice with the antioxidant N-acetyl-L-cysteine ​​(NAC) can inhibit oxidative stress in the ovaries, improve oxidative stress-related indicators in the mouse ovaries, and optimize ovarian function.

[0014] The antioxidant NAC was used to treat PCOS and compared with the effect of epalrestat. Epalrestat and NAC had the same efficacy in improving the content of oxidized and reduced substances in the ovaries of PCOS mice, as well as in improving ovarian hormone levels, ovarian morphology, follicle ratios at all levels, and ovulation. This suggests that epalrestat reduces the degree of oxidative stress while reducing the flux of the ovarian polyol pathway in PCOS mice, thereby improving ovarian function. Obviously, the increase in the flux of the polyol pathway in PCOS affects ovarian function through oxidative stress.

[0015] The content of oxidative substances in the ovaries increased, the content of reductive substances decreased, and the serum P4 level of mice was reduced, the ratio of atretic follicles increased, and the ratio of corpus luteum decreased. However, all these changes were restored by NAC treatment. This indicates that hyperactivity of the polyol pathway leads to oxidative stress in ovarian tissue and affects ovarian function by increasing follicle apoptosis and hindering follicle maturation.

[0016] Furthermore, the drug with antioxidant effect and other drugs are used as active ingredients of the drug. The other drugs are at least one of epalrestat and flutamide.

[0017] Excessive androgen in PCOS can stimulate over-activation of early follicles, leading to the appearance of a large number of antral follicles and even cystic follicles. The hyperactivity of the polyol pathway induced by high androgen leads to follicular maturation and ovulation disorders, thus affecting ovarian function. Flutamide and epalrestat can improve some metabolic abnormalities caused by excessive androgen, and then cooperate with antioxidants to achieve the effect of improving the microenvironment of follicle maturation.

[0018] Furthermore, drugs with antioxidant effects act by reducing polyol pathway flux.

[0019] Furthermore, drugs with antioxidant effects work by eliminating or alleviating the level of oxidative stress.

[0020] Furthermore, drugs with antioxidant effects exerted their effects by correcting ovarian Akr1b3 overexpression.

[0021] Studies have found that PCOS hyperandrogenism induces hyperactivity of the polyol pathway, and the metabolites of this pathway lead to excessive oxidative stress response in cells, affecting the microenvironment of follicular development, increasing follicular atresia, and hindering follicular maturation, which is an important pathological mechanism of ovarian dysfunction in PCOS. The use of drugs with antioxidant effects can improve the microenvironment of follicular growth, help follicle maturation and ovulation, and can therefore be used to treat PCOS.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention provides an application of an antioxidant in a drug for treating PCOS. Hyperandrogenism in PCOS mice causes overactivation of early ovarian follicle growth, and the hyperactivity of the polyol pathway induced by the antioxidant increases follicle atresia and hinders follicle maturation, thus causing ovarian dysfunction. By using an antioxidant as an active ingredient of the drug, the microenvironment can be adjusted to achieve the purpose of treating PCOS.

[0024] 2. In PCOS patients with hyperandrogenemia, a large amount of oxidative substances accumulate in the ovarian granulosa cells, and reducing substances are consumed, which indicates that there is oxidative stress in the ovarian tissue. NAC antioxidants are used to treat the microenvironment of follicular development and achieve normal follicular growth and ovulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Serum sex hormone levels in mice.

[0026] Figure 2 It is an indicator related to oxidative stress in mouse ovary.

[0027] Figure 3 Counting mouse ovarian follicles.

[0028] Figure 4 Counting of artificial superovulation in mice.

[0029] Figure 5 Serum sex hormone levels in Akr1b3 overexpressing mice.

[0030] Figure 6 It is an indicator related to oxidative stress in the ovaries of Akr1b3-overexpressing mice.

[0031] Figure 7 The ovarian follicles in Akr1b3-overexpressing mice were counted. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with test examples and specific implementation methods. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0033] Example 1

[0034] Mice at 21 days of age were randomly divided into three groups: (1) PCOS model group (PCOS), subcutaneous injection of 6 mg / 100 g DHEA and feeding with 60% high-fat diet for 21 days to establish PCOS mouse model; (2) ADR inhibition group (PCOS+Epalrestat), PCOS mice were modeled and gavaged with 20 mg / 100 g ADR inhibitor Epalrestat daily for 21 days; (3) antioxidant group (PCOS+NAC), PCOS mice were modeled and gavaged with 5 mg / 100 g antioxidant NAC daily for 21 days. After modeling, some mice underwent artificial superovulation experiment to detect ovarian ovulation. The remaining mice were killed and blood and ovarian tissues were collected. Serum sex hormone levels, ovarian oxidative stress status and follicle growth of mice were detected.

[0035] The 21-day-old mice were randomly divided into three groups: (1) control group (Control), 1011 control empty virus were injected orally into the ovaries, and the mice were fed normally for 21 days; (2) overexpression group (Akr1b3), 1011 Akr1b3 overexpression virus were injected orally into the ovaries, and the mice were fed normally for 21 days; (3) antioxidant group (Akr1b3+NAC), 1011 Akr1b3 overexpression virus were injected orally into the ovaries, and 5 mg / 100 g antioxidant NAC was gavaged daily during feeding for 21 days. After the modeling was completed, the mice were killed, and the blood and ovarian tissues of the mice were collected. The serum sex hormone levels, ovarian oxidative stress status and follicle growth of the mice were detected.

[0036] 1. Both epalrestat and NAC can improve serum reproductive hormone levels in PCOS mice

[0037] Serological test results showed that epalrestat and NAC had significant therapeutic effects on serum P4, LH, LH / FSH and AMH levels in PCOS mice, but had no effect on serum T, E2 and FSH levels. Among them, epalrestat and NAC showed comparable therapeutic effects.

[0038] The results are as follows Figure 1 As shown, the serum sex hormone levels of mice. Figure 1 (A) T level; Figure 1 (B) E2 level; Figure 1 (C) P4 level; Figure 1 (D) FSH level; Figure 1 (E) LH levels; Figure 1 (F) LH / FSH; Figure 1 (G) AMH levels. All data are expressed as Mean±SD; *, p<0.05 vs. PCOS mice, **, p<0.01 vs. PCOS mice, ***, p<0.001 vs. PCOS mice; sample size = 30.

[0039] 2. Epalrestat and NAC improve ovarian oxidative stress in PCOS mice

[0040] The levels of oxidative substances ROS and MDA in the ovaries of PCOS mice treated with epalrestat and NAC were significantly lower than those of untreated PCOS mice. At the same time, the levels of reducing substances NADPH and GSH in the ovaries of mice in the two treatment groups increased significantly, and the activity of antioxidant enzyme GSH-Px in the ovaries was also enhanced compared with that of untreated PCOS mice. Among them, the therapeutic effects of epalrestat and NAC were comparable.

[0041] The results are as follows Figure 2 As shown, the indicators related to oxidative stress in mouse ovaries. Figure 2 (A) ROS content; Figure 2 (B) MDA content; Figure 2 (C) NADPH content; Figure 2 (D) GSH content; Figure 2 (E) GSH-Px activity. All data are expressed as Mean±SD; *, p<0.05 vs. PCOS mice, **, p<0.01 vs. PCOS mice, ***, p<0.001 vs. PCOS mice; sample size = 30.

[0042] 3. Epalrestat and NAC partially improve follicular growth and ovulation disorders in PCOS mice

[0043] Like epalrestat, NAC could not reverse the increased antral follicle ratio in PCOS mice, but it reduced the ratio of cystic follicles to atretic follicles, increased the ratio of preantral follicles to corpora lutea, and increased the total number of ovarian follicles in PCOS mice. The therapeutic effects of epalrestat and NAC were comparable.

[0044] The results are as follows Figure 3 As shown, the mouse ovarian follicle counts. Figure 3 (A) HE staining of mouse ovarian sections; Figure 3 (B) Total number of single ovarian follicles in mice; Figure 3 (C) Follicle ratio counts at each level in a single ovary of mice. All data are expressed as Mean±SD; all scale bars are 200 μm; *, p<0.05 vs. PCOS mice, **, p<0.01 vs. PCOS mice, ***, p<0.001 vs. PCOS mice; sample size = 30.

[0045] The results of artificial superovulation experiments showed that epalrestat and NAC treatment increased the number of ovulations in PCOS mice, and the effects of the two treatments were comparable.

[0046] The results are as follows Figure 4 As shown, the artificial superovulation counts of mice. Figure 4 (A) Ovulation counts, each group of data refers to the number of ovulations of a single mouse; Figure 4 (B) Oocyte morphology under 40x microscope, each figure shows the number of ovulations of 3 mice. All data are expressed as Mean±SD; all scale bars are 100μm; **, p<0.01 vs. PCOS mice; sample size = 9.

[0047] 4. NAC improves abnormal serum reproductive hormone levels in mice with ovarian Akr1b3 overexpression

[0048] Serological results showed that the serum T, E2, FSH and AMH levels of Akr1b3-overexpressing mice and mice treated with NAC were no different from those of control mice. Treatment with NAC increased the serum P4, LH and LH / FSH levels of Akr1b3-overexpressing mice.

[0049] The results are as follows Figure 5 As shown, the serum sex hormone levels of Akr1b3 overexpressing mice. Figure 5 (A) T level; Figure 5 (B) E2 level; Figure 5 (C) P4 level; Figure 5 (D) FSH level; Figure 5 (E) LH levels; Figure 5 (F) LH / FSH; Figure 5 (G) AMH levels. All data are expressed as Mean+SD; *, p<0.05 vs. control mice, **, p<0.01 vs. control mice, ##, p<0.01 vs. Akr1b3 overexpressing mice; sample size = 24.

[0050] 5. NAC reduces ovarian oxidative stress in mice with Akr1b3 overexpression

[0051] Compared with the control group, the levels of oxidative substances ROS and MDA in the ovaries of Akr1b3-overexpressing mice were significantly increased, while the levels of reducing substances NADPH and GSH were significantly decreased. After NAC treatment of Akr1b3-overexpressing mice, all of these oxidative stress-related indicators returned to normal levels. There was no difference in the activity of the antioxidant enzyme GSH-Px in the ovaries among the three groups of mice.

[0052] The results are as follows Figure 6 As shown, Akr1b3 overexpression mice ovarian oxidative stress related indicators. Figure 6 (A) ROS content; Figure 6 (B) MDA content; Figure 6 (C) NADPH content; Figure 6 (D) GSH content; Figure 6 (E) GSH-Px activity. All data are expressed as Mean+SD; *, p<0.05 vs. control mice, **, p<0.01 vs. control mice, #, p<0.05 vs. Akr1b3 overexpressing mice, ##, p<0.01 vs. Akr1b3 overexpressing mice; sample size = 24.

[0053] 6. NAC improves follicular growth and ovulation abnormalities in mice with ovarian Akr1b3 overexpression

[0054] Follicle counting results showed that NAC treatment of Akr1b3-overexpressing mice could restore the ratio of atretic follicles and corpora lutea to normal levels.

[0055] The results are as follows Figure 7 As shown, the ovarian follicle counts of Akr1b3 overexpressing mice. Figure 7 (A) HE staining of mouse ovarian sections; Figure 7 (B) Total number of single ovarian follicles in mice; Figure 7 (C) Follicle ratio counts at each level in a single mouse ovary. All data are expressed as Mean+SD; all scale bars are 200 μm; **, p<0.01 vs. control mice, ##, p<0.01 vs. Akr1b3 overexpressing mice, ###, p<0.001 vs. Akr1b3 overexpressing mice; sample size = 24.

[0056] in conclusion:

[0057] Excessive oxidative stress damage in the ovaries caused by hyperactivity of the ovarian polyol pathway in PCOS mice is a direct cause of follicle and oocyte maturation disorders.

[0058] High expression of ADR, the rate-limiting enzyme of the polyol pathway, and sorbitol, the product of the pathway, in the ovaries of PCOS women. The polyol pathway in the ovaries of PCOS patients with hyperandrogenemia is hyperactive and positively correlated with serum T levels. Serum LH and LH / FSH are significantly elevated in PCOS patients with hyperandrogenemia, indicating that their pituitary-gonad axis is disordered. AMH in women is mainly secreted by early developing follicles. It can inhibit the recruitment of primordial follicles and the development of antral follicles. It is mainly used clinically to evaluate women's ovarian reserve. AMH is significantly elevated in patients with hyperandrogenemia in PCOS, indicating that their early follicles are overactivated and a large number of preantral follicles begin to develop. Ovarian function mainly includes the production and discharge of oocytes, as well as the synthesis and secretion of sex hormones. A large amount of clinical data shows that hyperandrogenemia in PCOS patients can lead to increased secretion of hypothalamic LH, thereby increasing the LH / FSH ratio. At the same time, because androgens stimulate the growth of early follicles, they will lead to an increase in AMH in PCOS patients.

[0059] A large amount of oxidative substances accumulate and reducing substances are consumed in the ovarian granulosa cells of PCOS patients with hyperandrogenemia, which indicates that there is oxidative stress in their ovarian tissue. The expression of ADR in the ovaries of PCOS patients without hyperandrogenemia also seems to be increased, but it does not cause the accumulation of enzyme products and obvious tissue oxidative stress. In addition to androgens, there are many other factors that induce the expression of tissue ADR, such as certain metabolites (glucose, uric acid, etc.) and many nuclear transcription factors produced in physiological activities (KappaB, Nrf2, TonEBP and c-jun, etc.). For PCOS patients with hyperandrogenemia, androgens may be the main factor leading to high expression of ADR.

[0060] In animal experiments, a PCOS mouse model was established using high androgen combined with high fat, and the hyperactivity of the ovarian polyol pathway was verified. Consistent with the clinical results, the PCOS model mice had abnormal ovarian function, and their ovarian polyol pathway flux increased, and was significantly positively correlated with the serum androgen levels of the mice. For PCOS model mice, high androgen is the main factor inducing the hyperactivity of their ovarian polyol pathway, not a high-fat diet.

[0061] PCOS mice were treated with the androgen antagonist flutamide and the results showed that the polyol pathway flux in the mouse ovaries was reduced as serum T levels decreased.

[0062] In order to study the effect of high androgen on the ovarian function of mice, we tried to detect the ovarian function of mice in each treatment group. We found that whether it was mice treated with high androgen or PCOS model mice, their serum T levels increased, while LH and LH / FSH levels increased, and P4 and AMH levels decreased. At the same time, the total number of ovarian follicles in mice decreased, the proportion of preantral follicles and corpus luteum decreased, the proportion of antral follicles and atretic follicles increased, and pathological cystic follicles appeared. This shows that high androgen causes damage to the pituitary-gonad axis of mice, stimulates a large number of early follicles to activate, but these follicles cannot mature smoothly and go to apoptosis, thereby accelerating the depletion of the follicle pool. For mice treated with high fat, their ovaries did not show obvious morphological and functional abnormalities, that is, high fat mainly causes lipid metabolism disorders in mice, while high androgen mainly causes ovarian morphological and functional disorders in mice.

[0063] In mice treated with high androgen or PCOS mice, the number of corpus luteum in the ovaries is significantly reduced, and therefore the serum progesterone level is reduced. Mouse studies have shown that AMH is an intraovarian growth factor that regulates the FSH sensitivity of primordial follicle recruitment and growing follicles in an inhibitory manner. AMH is mainly secreted by early growing follicles and is therefore related to the number of growing follicles. It can be used as a marker for quantification of ovarian reserve and diagnosis of PCOS. Research on the function of AMH has mainly focused on mice.

[0064] In the high androgen-treated mice or PCOS mice we studied, the total number of ovarian follicles and the proportion of preantral follicles decreased, and the number of ovulations in the artificial superovulation experiment decreased, all of which showed that the mice had follicle pool failure. Therefore, the decrease in serum AMH in the experimental group mice is a manifestation of ovarian failure.

[0065] The androgen antagonist flutamide is mainly used clinically to treat androgen-dependent tumors such as prostate cancer, and is also used to treat PCOS patients with hyperandrogenemia. After PCOS mice were treated with flutamide, almost all ovarian dysfunction induced by high androgen was reversed. However, although the serum P4 and AMH levels of mice in the treatment group improved, they were still lower than those in the control group, and their antral follicle ratio was lower than that in the control group. This may be because flutamide inhibited the effects of androgens and reduced the activation of early follicles. Similarly, in the artificial superovulation experiment, although the number of ovulations in PCOS mice treated with flutamide increased compared with PCOS mice, it was still lower than that in the control group. This may be due to the antagonism of androgen, and the number of follicles that responded to PMSG decreased accordingly.

[0066] To further demonstrate the effect of hyperandrogen-induced hyperactivity of the polyol pathway on the ovarian function of PCOS mice, PCOS mice were treated with the ADR inhibitor epalrestat. Epalrestat is a non-competitive, reversible ADR inhibitor used to treat diabetic neuropathy. Epalrestat can significantly reduce the expression of ADR and the accumulation of sorbitol, but does not have the effect of inhibiting SDH. The study found that epalrestat significantly reduced the expression of ADR and the accumulation of sorbitol in the ovaries of PCOS mice, but did not affect the expression of SDH and the content of fructose. Although epalrestat could not correct the hyperandrogenemia and overactivation of preantral follicles and antral follicles in PCOS mice, it could also increase the serum P4 and AMH levels of mice, reduce the ratio of cystic follicles and atretic follicles, and increase the ratio of corpus luteum. These facts indicate that epalrestat improves ovarian function mainly by inhibiting follicular apoptosis, promoting follicular maturation and ovulation. This can be verified by the reduced expression of ADR in follicles during estrus (which is also the ovulation period of follicular maturation).

[0067] Comparing the effects of flutamide and epalrestat, we believe that the excessive androgen in PCOS will stimulate the over-activation of early follicles, leading to the appearance of a large number of antral follicles and even cystic follicles. The hyperactivity of the polyol pathway induced by high androgen leads to follicle maturation and ovulation disorders, thus affecting ovarian function.

[0068] Finally, we investigated the relationship between hyperactivity of the ovarian polyol pathway and oxidative stress in PCOS mice. The antioxidant NAC was used to treat PCOS mice to compare the efficacy of epalrestat. Epalrestat and NAC were equally effective in improving the content of oxidized and reduced substances in the ovaries of PCOS mice, as well as in improving ovarian hormone levels, ovarian morphology, follicle ratios at different levels, and ovulation. This suggests that epalrestat reduces the degree of oxidative stress while reducing the ovarian polyol pathway flux in PCOS mice, thereby improving ovarian function. Clearly, the increased polyol pathway flux in PCOS is likely to affect ovarian function through oxidative stress.

[0069] Hyperactivity of the polyol pathway can lead to oxidative stress in tissues, and studies have confirmed that epalrestat can indeed alleviate oxidative stress in these tissues. That is, it shows that there is an inevitable connection between hyperactivity of the polyol pathway and oxidative stress. ADR was overexpressed in the ovaries of normal mice to observe the direct effect of hyperactivity of the polyol pathway. The results showed that overexpression of ovarian Akr1b3 did not affect the growth of early follicles, nor did it lead to the appearance of cystic follicles. This shows that overexpression of ovarian Akr1b3 is not the cause of cystic changes in mouse ovaries, but is caused by high androgen. However, overexpression of ovarian Akr1b3 significantly increased the content of oxidative substances in the ovaries, decreased the content of reducing substances, and reduced the level of serum P4 in mice, increased the ratio of atretic follicles, and reduced the ratio of corpus luteum. However, all of these changes were restored by NAC treatment. This shows that hyperactivity of the polyol pathway leads to oxidative stress in ovarian tissues, leading to apoptosis of granulosa cells and oocytes, interfering with the communication between granulosa cells and oocytes, affecting the quality of oocytes, thereby causing follicle apoptosis and affecting ovarian function.

[0070] Therefore, the antioxidant NAC has the potential to be used as a drug for treating PCOS. Also, the antioxidant NAC has the potential to be used to prepare drugs for treating PCOS.

Claims

1. Use of a drug with antioxidant effect for preparing a drug for treating polycystic ovary syndrome; the drug with antioxidant effect is epalrestat.

Images