Application of melatonin in preparation of medicine for improving endocrine dyscrasia caused by circadian rhythm disorder

Abstract

The invention discloses an application of melatonin in preparation of a medicine for improving endocrine dyscrasia caused by circadian rhythm disorder, according to a circadian rhythm disorder animal model experiment, the melatonin is determined to be capable of improving the symptoms of disordered mood cycle, reduced female and progestational hormone levels, abnormal increase of anti-mullerian hormone (AMH) level, abnormal increase of endocrine dyscrasia caused by circadian rhythm disorder and the like caused by circadian rhythm disorder. The pulse type secretion of luteinizing hormone (LH) is enhanced, and the quantity of luteum in ovaries is reduced, so that the effect of improving female reproductive endocrine dyscrasia caused by circadian rhythm disorder is achieved. The increase of the LH basic value and the increase of the LH pulse frequency and amplitude are one of the typical characteristics of the polycystic ovarian syndrome, so that the melatonin can become a new medicine for preventing and / or treating the polycystic ovarian syndrome, and has a wide application prospect clinically.

Description

Technical Field

[0001] The present invention specifically relates to the use of melatonin in the preparation of a drug for improving endocrine disorders caused by circadian rhythm disorders. Background Art

[0002] With the rapid development of modern society, people are exposed to artificial light sources at night for a long time, and combined with irregular lifestyles such as staying up late and shift work for a long time, the proportion of women of childbearing age who have delayed sleep or irregular work and rest has increased significantly, seriously disrupting their normal biological rhythms. Biological rhythms play an important role in the regulation of female reproductive endocrinology, among which the circadian rhythm is the most common. The circadian rhythm refers to the regular changes in physiology, behavior, etc. of organisms that occur in a cycle of about 24 hours in order to adapt to the rotation of the earth, including human sleep, wakefulness, feeding, excretion, neuroendocrine, body temperature, blood pressure, etc., all of which are synchronized with the periodic changes in the external light / dark environment.

[0003] Nowadays, more and more evidence shows that chronic circadian rhythm disorders caused by shift work, transmeridian flight, long-term light pollution or unhealthy sleep habits are closely related to irregular menstruation, dysmenorrhea, polycystic ovary syndrome, infertility and even poor pregnancy and childbirth outcomes in women. A meta-analysis in 2014 that included 15 articles with a total of 123,403 women's pregnancy outcomes showed that shift work can increase the incidence of menstrual disorders in women. The results of an epidemiological survey on flight attendants in 2017 showed that the high miscarriage rate of flight attendants may be related to the circadian rhythm disorders caused by shift work.

[0004] Melatonin is a neuroendocrine hormone secreted by the pineal gland and is regulated by external environmental light / dark stimuli. Melatonin helps to normalize the sleep / wake cycle, and short-term use of melatonin is generally considered safe. At present, there are studies showing that melatonin is related to female reproduction, but there is no research on melatonin in improving female reproductive endocrine disorders caused by circadian rhythm disorders. Summary of the Invention

[0005] To solve the above problems, the present invention provides the use of melatonin in the preparation of a drug for improving endocrine disorders caused by circadian rhythm disorders.

[0006] Furthermore, the drug is a drug for improving female endocrine disorders caused by circadian rhythm disorders.

[0007] Furthermore, the drug is a drug for improving female reproductive endocrine disorders caused by circadian rhythm disorders.

[0008] Even further, the drug has the effect of improving the estrous cycle disorder caused by circadian rhythm disorder.

[0009] Furthermore, the drug has the effect of improving the reproductive endocrine hormone disorder caused by circadian rhythm disorder.

[0010] Furthermore, the drug has the effect of inhibiting the increased pulsatile secretion of LH caused by circadian rhythm disorder.

[0011] Furthermore, the drug has the effect of increasing the number of corpora lutea in the ovary.

[0012] Furthermore, the drug is a drug for preventing and / or treating polycystic ovary syndrome.

[0013] The use of melatonin of the present invention in the preparation of a drug for improving endocrine disorders caused by circadian rhythm disorder. An animal model of circadian rhythm disorder was constructed by switching between a normal light / dark cycle and an advanced 8-hour light / dark cycle every 3 days. The experiment determined that melatonin can improve the estrous cycle disorder, reduced levels of estradiol and progesterone, abnormal elevation of AMH level, enhanced pulsatile secretion of LH, and reduced number of corpora lutea caused by circadian rhythm disorder, thereby playing a role in improving female reproductive endocrine disorders caused by circadian rhythm disorder. Since the elevated basal value of luteinizing hormone (LH) and the increased frequency and amplitude of LH pulses are one of the typical characteristics of polycystic ovary syndrome, melatonin may become a new drug for preventing and / or treating polycystic ovary syndrome and has broad application prospects in clinical practice.

[0014] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modification, substitution or change can be made.

[0015] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagrams of animal model establishment for (A) normal light cycle and (B) circadian rhythm disorder light cycle in the examples;

[0017] Figure 2 Schematic diagram of repeated tail tip blood sampling of mice to detect LH pulsatile secretion in the examples;

[0018] Figure 3 Ratio of regular / irregular estrous cycles of mice in (A) CON group and CRD group, (B) CRD+MEL group and CRD+VEH group in the examples;

[0019] Figure 4The levels of reproductive endocrine hormones in mice of (A) the CON group and the CRD group, and (B) the CRD+MEL group and the CRD+VEH group in the examples;

[0020] Figure 5 The LH pulse parameters of (A) the CON group and the CRD group, and (B) the CRD+MEL group and the CRD+VEH group in the examples, including pulse frequency, pulse interval, pulse amplitude, basal LH concentration, and average LH concentration;

[0021] Figure 6 The FSH levels of (A) the CON group and the CRD group, and (B) the CRD+MEL group and the CRD+VEH group in the examples;

[0022] Figure 7 The number of corpora lutea and small antral follicles in the ovaries of (A) the CON group and the CRD group, and (B) the CRD+MEL group and the CRD+VEH group in the examples. Detailed implementation manners

[0023] The raw materials, equipment, and reagents used in the detailed implementation manners of the present invention are all known products and are obtained by purchasing commercially available products.

[0024] Example 1 Research on the application of melatonin of the present invention in improving female reproductive endocrine abnormalities caused by circadian rhythm disorders

[0025] 1 Experimental materials

[0026] (1) Experimental animals: In this experiment, 6-8-week-old CBA / CaJ female mice were selected and purchased from Shanghai Bikaiyi Biotechnology Co., Ltd. After the mice were delivered to the Key Laboratory of Chronobiology, National Health Commission (Sichuan University), they were adaptively raised for 1 week. During this period, the light cycle was 12 h light and 12 h darkness. During all experiments, the mice could freely access food and water, and the room temperature was maintained at 22±2 °C.

[0027] (2) Experimental instruments: CLOCKLAB chronobiology research system (Wuhan Pubaike Technology Co., Ltd., model: ACT-556C).

[0028] (3) Experimental drugs: Melatonin was purchased from Sigma-Aldrich, CAS number: 73-31-4.

[0029] 2. Experimental methods

[0030] (1) Experimental grouping: 60 female mice were divided into 4 groups, namely the control group (CON), the circadian rhythm disorder group (CRD), the circadian rhythm disorder + melatonin treatment group (CRD+MEL), and the circadian rhythm disorder + placebo treatment group (CRD+VEH), with 15 mice in each group.

[0031] (2) Modeling of experimental animals

[0032] The schematic diagram of the light cycle for animal modeling is as Figure 1 shown. In the CON group, mice were continuously in a normal circadian rhythm of 12:12 hours (lights on at 8:00 am and lights off at 8:00 pm). In the CRD group, mice switched between normal light conditions (lights on at 8:00 am and lights off at 8:00 pm) and 8 hours in advance (lights on at 12:00 pm and lights off at 0:00 am) every 3 days. In the CRD+MEL group, mice received intraperitoneal injection of melatonin (10 mg / kg / day) 1 hour before lights out every day while undergoing light-induced circadian rhythm disorder. Melatonin was dissolved in absolute ethanol and further diluted in physiological saline, and the final concentration of ethanol was 2.5%. In the CRD+VEH group, mice received an equal volume of physiological saline containing the same concentration of ethanol at the same time while undergoing light-induced circadian rhythm disorder. After 8 weeks of modeling, the mice returned to the starting light cycle conditions and subsequent experiments and analyses were performed 1 day after the last time shift.

[0033] (3) Detection of estrous cycle: The estrous cycle of each group was determined by microscopic analysis of the presence or absence of leukocytes, keratinized epithelial cells, and nucleated epithelial cells in vaginal exfoliated cells. Vaginal smears were taken from the 5th week of light modeling in mice until the end of the experiment, and a complete assessment was made for 21 days and statistical analysis was performed.

[0034] (4) Detection and analysis of LH pulse secretion in mice: Whole blood samples were collected from mice using a modified repeated tail tip blood sampling technique. Before the experiment, mice were trained for blood sampling adaptation for 3 weeks every day to reduce the stress response of mice on the day of formal blood sampling and avoid affecting the experimental results. On the day of the experiment (from 10:00 am to 12:00 pm), whole blood samples (5 μL) were collected from the tail tip of mice in the diestrus phase every 10 minutes, and the whole blood sampling process lasted for 120 minutes in total, as Figure 2 shown. The collected whole blood was immediately diluted and mixed in 55 μL of PBS containing 0.05% Tween 20 in 0.1 M and quickly frozen on dry ice. The samples were stored in a -80 °C refrigerator for subsequent Ultra-Sensitive Luteinizing Hormone ELISA (AL-188, AnshLabs) detection. The animals were awake throughout the process. The results of the detection were confirmed for the number, amplitude, etc. of LH pulses using DynPeak software.

[0035] (5) Specimen collection: After the modeling was completed, the mice were anesthetized before euthanasia, and their blood and ovarian specimens were collected for subsequent experiments. The blood was stored in the form of serum, and a part of the ovarian tissue was stored in 4% paraformaldehyde solution in a 4°C refrigerator; the remaining ovarian tissue was stored in an -80°C refrigerator for subsequent RNA and protein extraction.

[0036] (6) Hormone level detection: ELISA method was used to detect the levels of anti-Müllerian hormone (AMH), estradiol (E2), progesterone (P), testosterone (T), FSH, and cortisol in mice (all the above ELISA kits were purchased from Shanghai Zhuocai Biotechnology Co., Ltd.).

[0037] (7) Follicle counting: Mouse ovarian tissue was used to make paraffin specimens, and the specimens were subjected to follicle counting to evaluate the changes in the number of follicles at all levels.

[0038] 3. Statistical analysis

[0039] All data analyses were performed using GraphPad Prism statistical software. Continuous variables were expressed as means ± standard error of the mean (Means ± SEM). The data were evaluated for normality (Shapiro-Wilk test). For normally distributed data, unpaired t-tests or one-way ANOVA were used. For non-normally distributed data, the Kruskal-Wallis test was used. Percentage data analysis was performed using the chi-square test. A P value < 0.05 was considered to be statistically significantly different.

[0040] 4. Experimental results:

[0041] (1) Melatonin can effectively improve the estrous cycle disorder in mice caused by circadian rhythm disorder

[0042] As Figure 3 shown, compared with the CON group, the proportion of mice with estrous cycle disorder in the CRD group was significantly increased (66.67% in the CRD group vs 20% in the CON group, P < 0.05). Melatonin can effectively improve the estrous cycle disorder in mice caused by circadian rhythm disorder (14.29% in the CRD + MEL group vs 64.29% in the CRD + VEH group, P < 0.05).

[0043] (2) Melatonin can effectively improve the reproductive endocrine hormone disorder in mice caused by circadian rhythm disorder

[0044] As Figure 4As shown in , the serum AMH level of the mice in the CRD group increased significantly, while the levels of E2 and P showed a downward trend. There was no significant difference in T between the two groups. To investigate the effect of chronic stress on the reproductive endocrine system of mice, we measured the serum corticosterone level and found that the corticosterone level of the mice in the CRD group was higher than that in the CON group. As Figure 4 shown in , we found that the AMH level in the CRD+MEL group was lower than that in the CRD+VEH group. There was no significant difference in the levels of E2, P, T, and corticosterone between the two groups.

[0045] (3) Melatonin can effectively improve the enhanced LH pulse in mice caused by circadian rhythm disorder

[0046] As Figure 5 shown in , the LH level in the CON group was relatively low, with occasional pulses. In contrast, the CRD group showed a stronger LH secretion pattern. The LH pulse frequency (the number of LH pulses within a 120-min sampling period) in the CRD group was significantly higher than that in the CON group (P<0.0001). The pulse interval in the CRD group was correspondingly shorter than that in the CON group (P<0.001). Compared with the CON group, the average pulse amplitude (1.7-fold), basal LH concentration (2.1-fold), and average LH concentration (2.4-fold) in the CRD group also increased significantly. As Figure 5 shown in , melatonin can significantly inhibit the LH pulsatile secretion induced by CRD. As Figure 6 shown, there was no significant difference in the FSH level among the four groups.

[0047] (5) Melatonin can effectively improve the abnormal follicle count in mice caused by circadian rhythm disorder

[0048] As Figure 7 shown in , compared with the CON group, the number of corpora lutea in the ovaries of female mice in the CRD group decreased significantly, and the number of small antral follicles increased significantly (28.20±1.29 vs 24.00±1.13, P<0.05). Therefore, the CRD model had a negative impact on the reproductive function of female mice. After melatonin treatment, as Figure 7 shown in , the number of corpora lutea in the CRD+MEL group increased significantly (5.9±0.31 vs 2.80±0.36, P<0.0001), but the number of small antral follicles in the two groups was similar.

[0049] From the above results, it can be seen that melatonin can improve the estrous cycle disorder, reproductive endocrine hormone disorder, enhanced LH pulsatile secretion, and decreased number of corpora lutea caused by circadian rhythm disorder, thus playing a role in improving the reproductive endocrine disorder in women caused by circadian rhythm disorder.

[0050] Numerous current studies have shown that elevated basal luteinizing hormone (LH) levels, as well as increased LH pulse frequency and amplitude, have been observed in patients with polycystic ovary syndrome (PCOS) and PCOS animal models. The elevated LH can promote androgen synthesis in ovarian theca cells, leading to hyperandrogenemia and impaired follicular development; it can affect the synthesis of estrogen and follicle-stimulating hormone (FSH), inhibiting follicular growth and ovulation; it can also promote the ovarian secretion of insulin-like growth factor-1, facilitating the binding of LH to theca cells to promote androgen synthesis and accelerating the occurrence and progression of polycystic ovaries in PCOS patients. Therefore, melatonin may play a role in preventing and / or treating polycystic ovary syndrome by inhibiting pulsatile LH secretion and reducing basal luteinizing hormone (LH) levels, showing broad application prospects in clinical practice.

Claims

1. Use of melatonin in the preparation of a drug for improving endocrine disorders caused by circadian rhythm disorders.

2. The use according to claim 1, characterized in that: The drug is a drug for improving female endocrine disorders caused by circadian rhythm disorders.

3. The use according to claim 2, wherein: The drug is a drug for improving female reproductive endocrine disorders caused by circadian rhythm disorders.

4. The use according to claim 3, characterized in that: The drug has the effect of improving estrous cycle disorders caused by circadian rhythm disorders.

5. The use according to claim 3, characterized in that: The drug has the effect of improving reproductive endocrine hormone disorders caused by circadian rhythm disorders.

6. The use according to claim 3, wherein: The drug has the effect of inhibiting the increased pulsatile secretion of LH caused by circadian rhythm disorders.

7. The use according to claim 3, characterized in that: The drug has the effect of increasing the number of corpora lutea in the ovary.

8. The use according to any one of claims 1 to 7, characterized in that: The drug is a drug for preventing and / or treating polycystic ovary syndrome.

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