Application of dyclonine hydrochloride in prevention of hypothalamic-pituitary-ovarian axis injury in female mice

Abstract

The application relates to the field of biological medicines, in particular to application of dyclonine hydrochloride in preventing damage of a hypothalamus-pituitary-ovary axis of a female mouse. 2+ The application discloses a potential mechanism of combined procymidone damaging a hypothalamus-pituitary-ovary axis of a mouse, and provides a small-molecule drug, dyclonine hydrochloride, for preventing damage based on the mechanism. The configuration method of the small-molecule drug is as follows: dissolving dyclonine hydrochloride in a phosphate buffer, and then adjusting the pH value to about 7.4; the concentration for intraperitoneal injection is 10 mg / kg.bw / d. The small-molecule drug preparation method is simple, can effectively inhibit an RA signal path and an NF-kB inflammation path, is relatively safe, and can effectively protect the hypothalamus-pituitary-ovary axis of the female mouse.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to a medicine for preventing and treating damage of environmental endocrine disruptors to hypothalamus-pituitary-ovary axis, in particular a medicine based on low-level Cd 2+ The present application relates to the field of biological medicine, and particularly relates to a medicine for preventing and treating damage of environmental endocrine disruptors to hypothalamus-pituitary-ovary axis, in particular a medicine based on low-level Cd BACKGROUND

[0002] The hypothalamus-pituitary-ovary axis (HPOA) is the main biological axis for regulating female neuroendocrine and reproduction. The hypothalamus plays a core role in the hormone regulation of the female reproductive system, and the hormones released by the hypothalamus-pituitary system can cause a series of activities corresponding to the menstrual cycle. Studies have shown that some environmental pollutants can directly act on the HPOA in the body, interfere with the synthesis and release of normal hormones, and affect the normal reproductive function of the ovary.

[0003] Cadmium (Cd) is easily released into the environment in the form of Cd 2+ through environmental media such as wastewater and waste gas, and is a highly toxic environmental pollutant to humans and animals. Cadmium in the environment mainly enters the human body through the respiratory tract and digestive tract, and is easily accumulated in organs in the body. Studies have found that prepubertal exposure to Cd 2+ can reduce the number of neuronal cells and cause damage to the hypothalamus and pituitary tissue. In addition, Cd 2+ has estrogen-like effects and can affect the function of estrogen by interfering with the transcriptional activity of estrogen receptors, is a potential pathogenic factor for estrogen-dependent diseases such as breast cancer and endometrial cancer, and can also affect ovarian function.

[0004] Procymidone (PCM) is an environmental endocrine disruptor (EEDs) commonly used to prevent and treat sclerotinia, botrytis and alternaria diseases of fruits and crops. Studies by the European Food Safety Authority have shown that the dietary intake of PCM by residents in some areas exceeds the acceptable daily intake, and countries / regions such as China, the United States, the European Union, Japan and Canada have set maximum residue limits for PCM in agricultural products. As an EEDs, PCM exhibits anti-androgen properties in vivo and in vitro, can prevent androgen from binding to the corresponding receptors, thereby inhibiting the expression of androgen-regulated genes, and ultimately causing abnormal development of male HPOA. In addition, PCM has a certain liposolubility and can cause brain tissue damage by passing through the blood-brain barrier.

[0005] PCM and Cd 2+Cd and PCM are two common environmental pollutants with EEDs properties, and the two toxicants are simultaneously ingested by human beings through drinking water and food in daily life, and higher doses of Cd 2+ Cd and PCM can both pass through the blood-brain barrier and cause damage to the nervous system, especially the hypothalamus and pituitary gland. Therefore, it is of great practical significance to provide a method for preventing damage to the hypothalamus-pituitary-ovary axis caused by environmental endocrine disruptors. SUMMARY

[0006] Therefore, the application provides the use of dyclonine hydrochloride in preventing damage to the hypothalamus-pituitary-ovary axis of female mice. The application discloses low-dose Cd 2+ The application discloses a potential mechanism of combined procymidone in damaging the hypothalamus-pituitary-ovary axis of mice, and provides a small-molecule drug, dyclonine hydrochloride, for preventing damage based on the mechanism. The preparation method of the small-molecule drug comprises the following steps: dissolving dyclonine hydrochloride in a phosphate buffer, and adjusting the pH value to about 7.4. The concentration of the small-molecule drug for intraperitoneal injection is 10 mg / kg·bw / d. The small-molecule drug has the advantages of simple preparation method, effective inhibition of the RA signal pathway and the NF-kB inflammatory pathway, high safety, and effective protection of the hypothalamus-pituitary-ovary axis of female mice.

[0007] To achieve the above-mentioned application purposes, the application provides the following technical solutions.

[0008] The application provides the use of dyclonine hydrochloride in any one of the following:

[0009] i) preparing a drug for restoring or improving reproductive capacity or fertility;

[0010] ii) preparing a drug for preventing or treating damage to the hypothalamus-pituitary-ovary axis.

[0011] In some specific embodiments of the application, the preparation method of the drug in the above-mentioned use comprises the following steps: dissolving dyclonine hydrochloride in a phosphate buffer, and adjusting the pH value to neutral to obtain the drug.

[0012] In some specific embodiments of the application, the neutral in the above-mentioned use is 7.0-7.6.

[0013] In some specific embodiments of the application, the concentration of dyclonine hydrochloride in the phosphate buffer in the above-mentioned use is 10 mg / mL.

[0014] In some specific embodiments of the application, the intraperitoneal injection dose of the drug in the above-mentioned use is 5 mg / kg·bw / d, 6 mg / kg·bw / d, 7 mg / kg·bw / d, 8 mg / kg·bw / d, 9 mg / kg·bw / d or 10 mg / kg·bw / d.

[0015] In some embodiments of the present application, the above application of the hypothalamic-pituitary-ovarian axis damage is caused by Cd 2+ combined with procymidone.

[0016] In some embodiments of the present application, the above application of the recovery or improvement of reproductive capacity or fertility includes increasing the number of births.

[0017] In some embodiments of the present application, the above application of the prevention or treatment of hypothalamic-pituitary-ovarian axis damage is achieved by inhibiting the RA signaling pathway and the NF-kB inflammatory pathway.

[0018] The present application also provides a method for constructing a hypothalamic-pituitary-ovarian axis damage model, based on Cd 2+ combined with procymidone.

[0019] In some embodiments of the present application, the above construction method of the hypothalamic-pituitary-ovarian axis damage model is a hypothalamic-pituitary-ovarian axis damage model in female mice or a hypothalamic-pituitary-ovarian axis damage model in vitro in female mice;

[0020] The method for constructing a hypothalamic-pituitary-ovarian axis damage model in female mice comprises administering 0.0015 mg / L, 0.002 mg / L, 0.003 mg / L, 0.004 mg / L, 0.005 mg / L, 0.006 mg / L, 0.007 mg / L, 0.008 mg / L, 0.009 mg / L, 0.01 mg / L, 0.012 mg / L or 0.0135 mg / L Cd 2+ combined with 30 mg / kg / d, 40 mg / kg / d, 50 mg / kg / d, 60 mg / kg / d or 70 mg / kg / d procymidone;

[0021] The method for constructing a hypothalamic-pituitary-ovarian axis damage model in vitro in female mice comprises contacting the target organ with 2.8 μg / L, 3 μg / L, 4 μg / L, 5 μg / L, 6 μg / L, 7 μg / L, 8 μg / L, 9 μg / L, 10 μg / L, 11 μg / L, 12 μg / L, 13 μg / L, 14 μg / L, 15 μg / L, 16 μg / L, 17 μg / L, 18 μg / L, 19 μg / L, 20 μg / L, 21 μg / L, 22 μg / L, 23 μg / L, 24 μg / L or 25.2 μg / L Cd 2+ and 0.01 mg / L, 0.02 mg / L, 0.03 mg / L, 0.04 mg / L, 0.05 mg / L, 0.63 mg / L, 0.07 mg / L, 0.08 mg / L, 0.09 mg / L or 0.1 mg / L procymidone;

[0022] The target organ is at least one of hypothalamus, pituitary and ovary.

[0023] The application also provides the application of dyclonine hydrochloride in any one of the following:

[0024] i) restoring or improving the reproductive ability or fertility;

[0025] ii) preventing or treating hypothalamus-pituitary-ovary axis damage.

[0026] The application provides the application of a DYC small molecule in preventing HPOA damage of female mice. 2+ The DYC small molecule has a preventive effect on HPOA damage of female mice caused by low-level Cd

[0027] To achieve the purpose of the application, the application provides the application of a DYC small molecule in preventing HPOA damage of female mice.

[0028] Further, the HPOA damage of the female mice is caused by low-level Cd 2+ and 1 / 2 NOAEL (No observed adverse effect level) dose of PCM.

[0029] Further, the application uses the DYC to prepare a small molecule drug for preventing HPOA damage of female mice caused by EEDs.

[0030] Further, the configuration method of the small molecule drug is as follows: the DYC is dissolved in a phosphate buffer, and then the pH value is adjusted to about 7.4.

[0031] Further, the concentration of the small molecule drug for intraperitoneal injection is 5-10 mg / kg·bw / d.

[0032] The application protects the HPOA of female mice by using the small molecule drug to antagonize low-level Cd 2+ combined with PCM. 2+ The application protects the HPOA of female mice by using the small molecule drug to antagonize low-level Cd 2+ combined with PCM. The model for protecting the HPOA of female mice by using the small molecule drug to antagonize low-level Cd

[0033] The application also provides a method for preparing an EEDs damaged HPOA animal model, that is, a mouse is intragastrically administered with a PCM solution mixed with corn oil at a concentration of 1 / 2 NOAEL, and the mouse is simultaneously allowed to freely drink water or ultrapure water containing Cd 2+ at a concentration of 1 / 2 NOAEL.

[0034] The application also provides a method for verifying the mechanism of EEDs damaged HPOA, that is, the Cd 2+ concentration regulated in the Chinese Standards for Drinking Water Quality is combined with the PCM at a concentration of 1 / 2 NOAEL to damage the HPOA of a female mouse at puberty, and the expression of follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), progesterone (P), Adh1b, Rdh 10, Aldh2, Cyp26a1, Nf-kb, Nlrp3, IL-1β, IL-18, and Caspase-1 proteins can be used to qualitatively (or semi-quantitatively) evaluate the damage degree of HPOA.

[0035] The application also provides an in-vitro culture method of HPOA of a female mouse.

[0036] The in-vitro culture method of the hypothalamus, pituitary and ovary of a female mouse: the hypothalamus, pituitary and bilateral ovary of a mouse are taken out in a sterile environment, immediately washed with sterile phosphate buffer solution (PBS, pH 7.4) for three times, and then the hypothalamus, pituitary and ovary are cut into 1-2 mm 3 tissue blocks under a stereomicroscope using a razor blade, and the hypothalamus, pituitary and ovary tissue blocks are randomly inoculated into the culture medium in each hole at about 2-3 blocks per hole. The culture is placed in a 5% CO2, 95% air incubator, and the culture temperature is 37℃.

[0037] The main components of the culture medium are: DMEM / F12 culture medium, 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% ascorbic acid.

[0038] In the application, the in-vitro culture of HPOA of a female mouse preferentially drops low-melting-point agarose gel in the holes of a 24-hole culture plate to solidify into "columns", and then each group is cultured and added into the culture holes, respectively, and the culture model processing is shown in the schematic diagram of the in-vitro culture model of HPOA. Figure 1 In addition, in the application, the culture medium containing DYC is adjusted to a pH value of 7.35-7.45 by using sodium hydroxide or sodium carbonate to make the solution alkaline, so as to balance the damage of acidic substances to the tissue.

[0039] The application also provides a method for collecting and detecting HPOA of a puberty mouse. That is, blood of the mouse is taken from the abdominal aorta for detection after anesthesia, and then the mouse is killed by cervical dislocation, and brain tissue and ovary are quickly taken after death; the supernatant of the mouse blood (or organ culture medium) is taken for sex hormone detection after centrifugation; the HPOA of the mouse is observed by histopathology, and the key protein in the HPOA tissue is detected by western blot.

[0040] The application has the following effects:

[0041] 1. The application constructs an EEDs damaged female mouse HPOA model to preliminarily explore the possible mechanism of EEDs damaging the HPOA of the mouse, and finds that low-level Cd 2+ The combination of PCM can cause damage to the HPOA of the puberty mouse, and therefore an intervention drug is developed to achieve the best prevention and treatment effect; a small molecule drug is applied to inhibit the RA signal pathway and the NLRP3 / NF-kB pathway, and plays a preventive and therapeutic role, and finally produces a HPOA protection or damage repair effect;

[0042] 2. The application is evidenced by basic experimental data, and the drug has high effectiveness and safety; the application not only verifies the effect of the small molecule drug for relieving HPOA damage, but also further explores the specific effect of different drug doses on the HPOA of the damaged female mouse, so that the dose-response relationship of the drug to play an effect can be determined, which is beneficial to guide practical application. BRIEF DESCRIPTION OF DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description.

[0044] Figure 1 An in vitro culture model of HPOA is shown;

[0045] Figure 2 Low-level Cd 2+ Pathological section of HPOA tissue of a female puberty mouse damaged by low-level Cd

[0046] Figure 3 Low-level Cd 2+ Nissl staining of HPOA tissue of a female puberty mouse damaged by low-level Cd

[0047] Figure 4 Low-level Cd 2+ Electrophoresis strip of differential expression of Adh1b, Rdh10, Aldh2 and Cyp26a1 related proteins in HPOA tissue of a female puberty mouse damaged by low-level Cd

[0048] Figure 5 Low-level Cd2+ Electrophoretic banding chart of differential expression of Nf-kb, Nlrp3, IL-1β, IL-18, Caspase-1 related proteins in hypothalamus of female pubertal mice induced by combined PCM;

[0049] Figure 6 Low level Cd 2+ Pathological section chart of pituitary tissue damage of female pubertal mice induced by combined PCM;

[0050] Figure 7 Low level Cd 2+ Nissl staining chart of pituitary tissue damage of female pubertal mice induced by combined PCM;

[0051] Figure 8 Low level Cd 2+ Electrophoretic banding chart of differential expression of Adh1b, Rdh10, Aldh2, Cyp26a1 related proteins in pituitary of female pubertal mice induced by combined PCM;

[0052] Figure 9 Low level Cd 2+ Electrophoretic banding chart of differential expression of Nf-kb, Nlrp3, IL-1β, IL-18, Caspase-1 related proteins in pituitary of female pubertal mice induced by combined PCM;

[0053] Figure 10 Low level Cd 2+ Pathological section chart of ovarian tissue damage of female pubertal mice induced by combined PCM;

[0054] Figure 11 Low level Cd 2+ Immunofluorescence chart of ovarian tissue damage of female pubertal mice induced by combined PCM;

[0055] Figure 12 Low level Cd 2+ Electrophoretic banding chart of differential expression of Adh1b, Rdh10, Aldh2, Cyp26a1 related proteins in ovary of female pubertal mice induced by combined PCM;

[0056] Figure 13 Low level Cd 2+ Electrophoretic banding chart of differential expression of Nf-kb, Nlrp3, IL-1β, IL-18, Caspase-1 related proteins in ovary of female pubertal mice induced by combined PCM;

[0057] Figure 14 Low level Cd 2+ Pathological section chart of hypothalamus damage of female mice in vitro induced by combined PCM;

[0058] Figure 15 Low level of Cd 2+ The figure shows the result of the Nissl staining of the hypothalamus tissue of the female mice in vitro cultured with PCM;

[0059] Figure 16 Low level of Cd 2+ The figure shows the result of the electrophoretic strip of the differential expression of the Adh1b, Rdh10, Aldh2, Cyp26a1 related proteins of the hypothalamus tissue of the female mice in vitro cultured with PCM;

[0060] Figure 17 Low level of Cd 2+ The figure shows the result of the electrophoretic strip of the differential expression of the Nf-kb, Nlrp3, IL-1β, IL-18, Caspase-1 related proteins of the hypothalamus tissue of the female mice in vitro cultured with PCM;

[0061] Figure 18 Low level of Cd 2+ The figure shows the result of the pathological section of the pituitary tissue of the female mice in vitro cultured with PCM;

[0062] Figure 19 Low level of Cd 2+ The figure shows the result of the Nissl staining of the pituitary tissue of the female mice in vitro cultured with PCM;

[0063] Figure 20 Low level of Cd 2+ The figure shows the result of the electrophoretic strip of the differential expression of the Adh1b, Rdh10, Aldh2, Cyp26a1 related proteins of the pituitary tissue of the female mice in vitro cultured with PCM;

[0064] Figure 21 Low level of Cd 2+ The figure shows the result of the electrophoretic strip of the differential expression of the Nf-kb, Nlrp3, IL-1β, IL-18, Caspase-1 related proteins of the pituitary tissue of the female mice in vitro cultured with PCM;

[0065] Figure 22 Low level of Cd 2+ The figure shows the result of the pathological section of the ovary tissue of the female mice in vitro cultured with PCM;

[0066] Figure 23 Low level of Cd 2+ The figure shows the result of the electrophoretic strip of the differential expression of the Adh1b, Rdh10, Aldh2, Cyp26a1 related proteins of the ovary tissue of the female mice in vitro cultured with PCM;

[0067] Figure 24 Low level of Cd 2+Electrophoretic banding chart of differential expression of Nf-kb, Nlrp3, IL-1β, IL-18, Caspase-1 related proteins in female mouse ovary in vitro culture combined with PCM;

[0068] Figure 25 DYC antagonizes low level Cd 2+ Pathological section chart of hypothalamus of female puberty mice protected by combined PCM;

[0069] Figure 26 DYC antagonizes low level Cd 2+ Nissl staining chart of hypothalamus of female puberty mice protected by combined PCM;

[0070] Figure 27 DYC antagonizes low level Cd 2+ Column chart of differential expression of Adh1b, Rdh10, Aldh2, Cyp26a1 related genes in hypothalamus of female puberty mice protected by combined PCM;

[0071] Figure 28 DYC antagonizes low level Cd 2+ Column chart of differential expression of Nf-kb, Nlrp3, IL-1β, IL-18, Caspase-1 related genes in hypothalamus of female puberty mice protected by combined PCM;

[0072] Figure 29 DYC antagonizes low level Cd 2+ Pathological section chart of pituitary of female puberty mice protected by combined PCM;

[0073] Figure 30 DYC antagonizes low level Cd 2+ Nissl staining chart of pituitary of female puberty mice protected by combined PCM;

[0074] Figure 31 DYC antagonizes low level Cd 2+ Column chart of differential expression of Adh1b, Rdh10, Aldh2, Cyp26a1 related genes in pituitary of female puberty mice protected by combined PCM;

[0075] Figure 32 DYC antagonizes low level Cd 2+ Column chart of differential expression of Nf-kb, Nlrp3, IL-1β, IL-18, Caspase-1 related genes in pituitary of female puberty mice protected by combined PCM;

[0076] Figure 33 DYC antagonizes low level Cd 2+ Pathological section chart of ovary of female puberty mice protected by combined PCM;

[0077] Figure 34 DYC antagonizes low level Cd 2+ Column chart of differential expression of Adh1b, Rdh10, Aldh2, Cyp26a1 related genes in the ovary of female puberty mice combined with PCM;

[0078] Figure 35 DYC antagonizes low level Cd 2+ Column chart of differential expression of Nf-kb, Nlrp3, IL-1β, IL-18, Caspase-1 related genes in the ovary of female puberty mice combined with PCM;

[0079] Figure 36 Pathological section diagram of in vitro cultured hypothalamus of female puberty mice after DYC intervention;

[0080] Figure 37 Nissl staining diagram of in vitro cultured hypothalamus of female puberty mice after DYC intervention;

[0081] Figure 38 Electrophoresis band diagram of differential expression of Adh1b, Rdh10, Aldh2, Cyp26a1 related proteins in in vitro cultured hypothalamus of female puberty mice after DYC intervention;

[0082] Figure 39 Electrophoresis band diagram of differential expression of Nf-kb, Nlrp3, IL-1β, IL-18, Caspase-1 related proteins in in vitro cultured hypothalamus of female puberty mice after DYC intervention;

[0083] Figure 40 Pathological section diagram of in vitro cultured pituitary of female puberty mice after DYC intervention;

[0084] Figure 41 Nissl staining diagram of in vitro cultured pituitary of female puberty mice after DYC intervention;

[0085] Figure 42 Electrophoresis band diagram of differential expression of Adh1b, Rdh10, Aldh2, Cyp26a1 related proteins in in vitro cultured pituitary of female puberty mice after DYC intervention;

[0086] Figure 43 Electrophoresis band diagram of differential expression of Nf-kb, Nlrp3, IL-1β, IL-18, Caspase-1 related proteins in in vitro cultured pituitary of female puberty mice after DYC intervention;

[0087] Figure 44 Pathological section diagram of in vitro cultured ovary of female puberty mice after DYC intervention;

[0088] Figure 45 Figure 8 shows the electrophoretic banding pattern of the differential expression of Adh1b, Rdh10, Aldh2, Cyp26a1 related proteins of the in vitro cultured ovary of female pubertal mice after DYC intervention;

[0089] Figure 46 Figure 9 shows the electrophoretic banding pattern of the differential expression of Nf-kb, Nlrp3, IL-1β, IL-18, Caspase-1 related proteins of the in vitro cultured ovary of female pubertal mice after DYC intervention;

[0090] wherein, Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 , Figure 11 , Figure 12 , Figure 13 wherein, C = control group, Cd 2+ = 0.0045 mg / L Cd 2+ , PCM = 50 mg / kg / d PCM, L + PCM = 0.0015 mg / L Cd 2+ + 50 mg / kg / d PCM, M + PCM = 0.0045 mg / L Cd 2+ + 50 mg / kg / d PCM, H + PCM = 0.0135 mg / L Cd 2+ + 50 mg / kg / d PCM;

[0091] Figure 14 , Figure 15 , Figure 16 , Figure 17 , Figure 18 , Figure 19 , Figure 20 , Figure 21 , Figure 22 , Figure 23 , Figure 24 wherein, C = control group, Cd 2+ = 8.4 μg / L Cd 2+ , PCM = 0.63 mg / L PCM, L + PCM = 2.8 μg / L Cd 2+ + 0.63 mg / L PCM, M + PCM = 8.4 μg / L Cd 2+ + 0.63 mg / L PCM, H + PCM = 25.2 μg / L Cd 2+ + 0.63 mg / L PCM;

[0092] Figure 25, Figure 26 , Figure 27 , Figure 28 , Figure 29 , Figure 30 , Figure 31 , Figure 32 , Figure 33 , Figure 34 , Figure 35 In this study, NC = negative control group, PC = 0.0135 mg / L Cd 2+ +50mg / kg / day PCM, for 7 consecutive days, M = concurrent low-level Cd administered by gavage. 2+ Combined with PCM (0.0135 mg / L Cd) 2+ +50mg / kg / d PCM) and intraperitoneal injection of DYC (10mg / kg·bw / d) for 7 consecutive days, B = low level Cd by gavage. 2+ Combined with PCM (0.0135 mg / L Cd) 2+ +50mg / kg / day PCM) 24 hours before intraperitoneal injection of DYC (10mg / kg·bw / day) for 7 consecutive days, A = low level Cd by gavage. 2+ Combined with PCM (0.0135 mg / L Cd) 2+ +50mg / kg / d PCM) for 7 days, followed by intraperitoneal injection of DYC (10mg / kg·bw / d) for 7 days; results are expressed as mean ± standard deviation, n=8, ANOVA, t test; *P<0.05 indicates that the difference between each group and the NC group is statistically significant; #P<0.05 indicates that the difference between each intervention group and the PC group is statistically significant;

[0093] Figure 36 , Figure 37 , Figure 38 , Figure 39 , Figure 40 , Figure 41 , Figure 42 , Figure 43 , Figure 44 , Figure 45 , Figure 46 In the control group, C = 25.2 μg / L Cd 2+ +0.63mg / L PCM, L=25.2μg / L Cd 2+ +0.63mg / L PCM+8.15μg / L DYC, M=25.2μg / L Cd 2+ +0.63mg / L PCM+16.29μg / L DYC, H=25.2μg / L Cd 2+ +0.63mg / L PCM+32.59μg / L DYC. Detailed Implementation

[0094] The application discloses application of dyclonine hydrochloride in preventing hypothalamic-pituitary-ovarian axis damage in female mice, and those skilled in the art can improve the process parameters according to the content of the present application. It should be particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are all regarded as included in the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0095] The research of the present application finds that low-level Cd 2+ And / or PCM can aggravate HPOA tissue damage by affecting retinoic acid (RA) signaling pathway and NLRP3 / NF-kB inflammatory pathway in the HPOA of the adolescent mice. Based on this, dyclonine hydrochloride (DYC) is used to maintain the homeostasis of the above-mentioned proteins, so as to prevent and treat HPOA damage in mice.

[0096] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present application are ordinary market products, which can be purchased from the market.

[0097] The present application will be further described below in combination with examples.

[0098] Example 1: Low-level Cd 2+ Combined with PCM to cause HPOA damage in adolescent female mice

[0099] (1) Low-level Cd 2+ Combined with PCM to cause HPOA damage in adolescent female mice

[0100] ① HPOA damage model in female mice:

[0101] According to the Cd 2+ concentration (0.005 mg / L) specified in the Chinese "Drinking Water Health Standards" combined with 1 / 2 of the NOAEL of procymidone dose (50 mg / kg·bw / d), the female mice were infected.

[0102] The PCM was suspended in pure corn oil to obtain a PCM solution, which was prepared immediately before use, and the intragastric volume was adjusted according to the body weight of the female mice, 1 time / d, continuously intragastric for 21 d, and the water with Cd 2+ concentration specified in the Chinese "Drinking Water Health Standards" or ultrapure water was provided for the mice to drink freely.

[0103] The prepared 6 groups of low-level Cd 2+ combined with PCM doses are as follows:

[0104] Control group: pure corn oil 0 mg / kg·d;

[0105] Cd only 2+ Exposure group: corn oil + 0.0045 mg / L Cd 2+ ;

[0106] PCM group: 50 mg / kg / d PCM;

[0107] Low-dose Cd 2+ + PCM group: 0.0015 mg / L Cd 2+ + 50 mg / kg / d PCM;

[0108] Medium-dose Cd 2+ + PCM group: 0.0045 mg / L Cd 2+ + 50 mg / kg / d PCM;

[0109] High-dose Cd 2+ + PCM group: 0.0135 mg / L Cd 2+ + 50 mg / kg / d PCM;

[0110] During the experiment, the food intake and mental state of female mice in each group were observed daily.

[0111] 2. Female mouse in vitro HPOA injury model:

[0112] The cultured tissues were randomly divided into 6 groups:

[0113] Cd only 2+ Exposure group: acetone + 8.4 μg / L Cd 2+ ;

[0114] PCM group: 0.63 mg / L PCM;

[0115] Low-dose Cd 2+ + PCM group: 2.8 μg / L Cd 2+ + 0.63 mg / L PCM;

[0116] Medium-dose Cd 2+ + PCM group: 8.4 μg / L Cd 2+ + 0.63 mg / L PCM;

[0117] High-dose Cd 2+ + PCM group: 25.2 μg / L Cd 2+ + 0.63 mg / L PCM;

[0118] The control group was an equal volume of acetone;

[0119] The anhydrous cadmium chloride was dissolved in ultrapure water, and the PCM was dissolved in acetone. The final concentration of all experimental and control incubation solutions was 0.1% acetone. The hypothalamus, pituitary, and ovary were then cultured according to the culture method.

[0120] (2) Low level of Cd 2+ FSH, LH, E2, and P detection in the HPOA of adolescent female mice damaged by the combination of PCM

[0121] From the above low level of Cd 2+ Blood was taken from the abdominal aorta of female mice in the HPOA damage model caused by the combination of PCM, centrifuged (3000 r / min, centrifugal radius 160 mm, centrifugal time 10 min), and the supernatant was taken. According to the ELISA kit instructions for each hormone, after adding the relevant detection solution, the absorbance value of the sample was measured using an automatic enzyme marker. The hormone content was converted according to the standard curve.

[0122] (3) Low level of Cd 2+ Tissue collection in the HPOA of adolescent female mice damaged by the combination of PCM

[0123] After the female mice were anesthetized with ether, the HPOA organs (hypothalamus, pituitary, and ovary) were quickly removed after taking blood from the abdominal aorta. A part was used for volume measurement and mass weighing, and was placed in 4% paraformaldehyde fixing solution for pathological section preparation; a part was placed in an Ep tube containing RIPA lysis solution for protein detection.

[0124] (4) Low level of Cd 2+ Histopathological observation of the HPOA of adolescent female mice damaged by the combination of PCM

[0125] The fixed HPOA organs were dehydrated in ethanol from low concentration to high concentration, embedded in paraffin, and then continuously sectioned at 5 μm intervals, stained with hematoxylin-eosin (HE) and sealed. Every 5 sections, 1 section was taken, and 5 fields of view were randomly selected for observation.

[0126] After the hypothalamic and pituitary tissue sections were dried, they were deparaffinated with xylene and ethanol, washed with distilled water to remove ethanol, and then stained in Nissl staining solution for 10 min. After washing twice with distilled water, they were dehydrated twice in 95% ethanol for 2 min each time, then transparentized twice in xylene for 5 min each time, and mounted. Then, they were observed and photographed under an optical microscope.

[0127] (5) Low level of Cd 2+ Key protein detection in the HPOA of adolescent female mice in the HPOA damage model caused by the combination of PCM

[0128] The organ specimen tissue of HPOA was placed in RIPA lysis solution, homogenized at 4℃, and the supernatant was total protein solution. Then, the protein was quantified by the method of bicinchoninic acid, and 0.1% benzylsulfonyl fluoride was added and stored.

[0129] The total protein was denatured by boiling in loading buffer for 5 minutes, and denatured electrophoresis separation was performed using 10% SDS-PAGE gel with a loading amount of 10 μg / well. The electrophoresis parameters were as follows: concentrated gel 80V, 20min; separation gel 120V, 90min. Then, the polyvinylidene fluoride (PVDF) membrane was transferred at 100V for 120min. The membrane was blocked with 5% skim milk powder for 1h, and the primary antibody was diluted with Western diluent (anti-Aldh2 monoclonal antibody 1:1000, anti-Cyp26a1 monoclonal antibody 1:1000, anti-Adh1b monoclonal antibody 1:1000, anti-Rdh10 monoclonal antibody 1:1000, anti-Nlrp3 monoclonal antibody 1:1000, anti-Nf-kb monoclonal antibody 1:1000, anti-IL-1β monoclonal antibody 1:1000, anti-IL-18 monoclonal antibody 1:1000, anti-Caspase-1 monoclonal antibody 1:1000, and β-actin monoclonal antibody 1:2000), and incubated at 4℃ overnight. The membrane was washed with 1×TBST for 3 times, 10min each time. The secondary antibody (anti-rabbit HRP-lgG 1:8000) diluted with TBST was added, and the shaking bed was incubated at room temperature for 1h. The membrane was washed with 1×TBST for 3 times, 10min each time.

[0130] Chemiluminescence imaging system was used for photographing and developing.

[0131] (6) Low level Cd 2+ Immunofluorescence detection of the ovarian damage model of adolescent female mice combined with PCM

[0132] The mouse ovarian tissue section was deparaffinated with xylene and ethanol gradient, hydrated, and endogenous peroxidase was eliminated with 3% H2O2. Then, the section was subjected to antigen repair for 15min with sodium citrate buffer at pH=6.0, blocked with 3% BSA at room temperature for 1h, washed with PBS for 3 times, and incubated with ALDH2 polyclonal antibody diluted at 1:100 as the primary antibody at 4℃ overnight. Then, the section was cooled at room temperature for 1h, washed with PBST for 4 times, incubated with fluorescent secondary antibody diluted at 1:250 at room temperature for 1h, washed with PBST for 3 times, and dyed with DAPI staining solution for 5min. Finally, fluorescent anti-quenching agent was added on the section, and the section was observed and photographed by LEICA fluorescence microscope.

[0133] Experimental results:

[0134] In in vivo and in vitro experiments, with low level Cd2+ The levels of FSH, LH, E2 and P decreased (see Table 1, Table 2) and the number of nerve cells in hypothalamus decreased, the karyopyknosis and Nissl bodies decreased, and the cell body contracted and deformed (as shown in Figure 2 , Figure 3 , Figure 14 , Figure 15 ) in the pituitary gland. The number of nerve cells decreased, the Nissl bodies decreased, the cell density decreased, and the cells appeared pyknosis and deformation (as shown in Figure 6 , Figure 7 , Figure 18 , Figure 19 ) in the ovary. The number of follicles at all levels decreased (see Table 3, Table 4, Table 5, Table 6, Figure 10 , Figure 22 ) in the ovary. The above results suggest that low levels of Cd 2+ Combined with PCM, Cd2+ participated in the growth and development of adolescent mice, leading to reduced body weight in adulthood, dose-dependent reduction in the secretion of FSH, LH, E2 and P, and pathological changes in the hypothalamus, pituitary gland and ovary.

[0135] Table 1: Concentrations of FSH, LH, E2 and P in serum / culture medium before intervention

[0136]

[0137] Table 2: Concentrations of FSH, LH, E2 and P in serum / culture medium after intervention

[0138]

[0139] Note: The results of Table 1 and Table 2 are mean ± standard deviation, n = 8, ANOVA, t test; *P < 0.05 indicates that the difference between each dose group and the control group is statistically significant; #P < 0.05 indicates that the difference between the low, medium and high dose groups and the PCM group is statistically significant; ΔP < 0.05 indicates that the difference between the medium dose group and the Cd 2+ group is statistically significant

[0140]

[0141] Table 3: Follicle count in experimental ovarian tissue of mice before intervention

[0142]

[0143] Note: The follicle count in ovarian tissue n = 8,

[0144] Table 4: Follicle count in experimental ovarian tissue of mice after intervention

[0145]

[0146] ​​(Note: n = 8 for ovarian tissue follicle count, )

[0147] Table 5: Ovarian tissue follicle count before intervention in tissue culture experiments

[0148]

[0149]

[0150] (Note: n = 8 for ovarian tissue follicle count, )

[0151] Table 6: Ovarian tissue follicle count after intervention in tissue culture experiments

[0152]

[0153] (Note: n = 8 for ovarian tissue follicle count, )

[0154] At the same time, as Figure 4 , Figure 5 , Figure 8 , Figure 9 , Figure 12 , Figure 13 , Figure 16 , Figure 17 , Figure 20 , Figure 21 , Figure 23 , Figure 24 Low levels of Cd 2+ combined with PCM activate the RA signaling pathway and the NF-kB inflammatory pathway. As Figure 11 immunofluorescence results show that ALDH2 is localized in the cytoplasm of the ovary, and the trend is consistent with the WB results of ALDH2. Compared with the control group, the fluorescence intensity of ALDH2 in the PCM, low, medium and high dose groups was significantly enhanced. It is suggested that low levels of Cd 2+ combined with PCM are involved in the dose-dependent damage of HPOA in vivo and in vitro.

[0155] Example 2: DYC antagonizes low levels of Cd 2+ combined with PCM to protect the HPOA of female mice

[0156] (1) DYC antagonizes low levels of Cd 2+ combined with PCM to protect the HPOA of female mice

[0157] ① Female mouse experimental grouping: After 1 week of adaptive feeding, female mice were randomly divided into 5 groups according to body weight:

[0158] Positive control group (PC): low levels of Cd 2+ combined with PCM 0.0135mg / L Cd2+ + 50 mg / kg / d PCM for 7 consecutive days;

[0159] Negative control group (NC): orally administered with the same volume of corn oil for 7 days;

[0160] Group M: simultaneously gavaged with low-level Cd 2+ combined with PCM (0.0135 mg / L Cd 2+ + 50 mg / kg / d PCM) and intraperitoneal injection of DYC (10 mg / kg·bw / d) for 7 consecutive days;

[0161] Group B: intraperitoneal injection of DYC (10 mg / kg·bw / d) 24 hours before gavage with low-level Cd 2+ combined with PCM (0.0135 mg / L Cd 2+ + 50 mg / kg / d PCM) for 7 consecutive days;

[0162] Group A: intraperitoneal injection of DYC (10 mg / kg·bw / d) for 7 days after gavage with low-level Cd 2+ combined with PCM (0.0135 mg / L Cd 2+ + 50 mg / kg / d PCM) for 7 days.

[0163] 2. In vitro cultured tissue grouping:

[0164] Low-level Cd 2+ combined with PCM caused damage to in vitro cultured HPOA. It was found that 25.2 μg / L Cd 2+ combined with 0.63 mg / L PCM could cause obvious histological, RA signaling pathway and NLRP3 / NF-kB inflammatory pathway molecular biology index changes in in vitro cultured hypothalamus, pituitary and ovary, therefore 25.2 μg / L Cd 2+ combined with 0.63 mg / L PCM was selected to cause in vitro HPOA damage model to conduct RA signaling pathway and NLRP3 / NF-kB inflammatory pathway activation experiment. The experiment was divided into 5 groups:

[0165] Positive control group (PC): 25.2 μg / L Cd 2+ + 0.63 mg / L PCM;

[0166] Control group (C): administered with the same volume (0.1%) of acetone;

[0167] Low-dose (L) group: 25.2 μg / L Cd 2+ + 0.63 mg / L PCM + 8.15 μg / L DYC;

[0168] Medium-dose (M) group: 25.2 μg / L Cd2+ + 0.63 mg / L PCM + 16.29 pg / L DYC;

[0169] High dose (H) group: 25.2 pg / L Cd 2+ + 0.63 mg / L PCM + 32.59 pg / L DYC;

[0170] (2) DYC antagonizes low level Cd 2+ FSH, LH, E2, P detection of HPOA of adolescent female mice protected by PCM in combination, same as step (2) in Example 1, refer to Example 1 for details.

[0171] (3) DYC antagonizes low level Cd 2+ Tissue collection of HPOA of adolescent female mice protected by PCM in combination, same as step (3) in Example 1, refer to Example 1 for details.

[0172] (4) DYC antagonizes low level Cd 2+ Histopathological observation of HPOA of adolescent female mice protected by PCM in combination, same as step (4) in Example 1, refer to Example 1 for details.

[0173] (5) DYC antagonizes low level Cd 2+ Total RNA extraction and real-time fluorescent quantitative PCR of HPOA of adolescent female mice protected by PCM in combination

[0174] The reproductive organs stored at -80℃ were quickly cut on ice and placed in a centrifuge tube containing 1 mL TRIzol, homogenized at 4℃, and total RNA of the sample was extracted according to the kit instructions. The extracted RNA in the tissue was dissolved in RNase-free water, 1 pg of cDNA was synthesized after reverse transcription, and the reaction system and conditions were operated according to the instructions.

[0175] After obtaining the cDNA, the amplification reaction was performed, and the reaction system was as follows: SYBR Green, 2.5 pL; cDNA, 0.5 pL; forward primer, 0.3 pL; reverse primer, 0.3 pL; RNase-free water, 6.4 pL.

[0176] The reaction conditions of the amplification reaction were set as follows: 95℃ for 10 min, 95℃ for 15 s, 60℃ for 1 min, and 40 cycles. The CT values of the target genes of the experimental group and the control group were subtracted by the CT values of the corresponding internal reference genes, respectively, to obtain the ΔCT, and the ΔCT of the dose group was subtracted by the ΔCT of the corresponding control group to obtain the ΔΔCT. After uniformization processing, the amount of the target gene relative to the internal reference gene was Y = 2 －ΔΔCt .

[0177] Experimental results:

[0178] In vivo and in vitro experiments, with the increase of DYC dose, the pathological damage of hypothalamus, pituitary and ovarian tissue sections in DYC group gradually decreased, and multiple follicles at different stages of development were observed in the ovary (see Tables 3, 4, 5, 6, Figure 33 、 Figure 44 ),the number of nerve cells in hypothalamus and pituitary increased, and the cell density increased. (see Figure 25 、 Figure 26 , Figure 29 , Figure 30 、 Figure 36 、 Figure 37 、 Figure 40 、 Figure 41 ), gradually tend to the control group. At the same time, the FSH and LH of pituitary and E2 and P of ovary in the dyclonine hydrochloride group were increased (see Tables 1, 2).

[0179] As Figure 38 、 Figure 39 、 Figure 42 、 Figure 43 、 Figure 45 、 Figure 46 In vitro experiments, DYC can inhibit the RA signaling pathway and NF-kB inflammatory pathway to protect the HPOA from low-level Cd 2+ combined with PCM, among which the effect of group H is the largest, as shown in Figure 27 、 Figure 28 、 Figure 31 、 Figure 32 、 Figure 34 、 Figure 35 , in vivo experiments, M group has the best protective effect on HPOA injury caused by low-level Cd 2+ combined with PCM, followed by group B, and group A is the worst, which indicates that DYC has a preventive effect on HPOA injury caused by low-level Cd 2+ combined with PCM.

[0180] In summary, the greater the dose of DYC, or when given at the same time, the better the protective effect on HPOA injury caused by low-level Cd 2+ combined with PCM.

[0181] Example 3: Reproduction experiment after exposure to low-level Cd 2+ combined with PCM or DYC

[0182] In view of the effect of group M (simultaneously given low-level Cd 2+ combined with PCM and DYC group) on low-level Cd 2+The combined PCM had the best protective effect on the HPOA injury of mice, so female mice were randomly selected from the negative control group, positive control group and M group of the model in Example 2 and mated with normal male mice to carry out a breeding experiment, and the offspring mice of the pregnant mice were used as observation objects, and the number of fetal mice was calculated to evaluate the effect of low-level Cd 2+ Effect of combined PCM exposure on the function of HPOA of mice and DYC antagonizing low-level Cd 2+ Effect of combined PCM on the function of HPOA of adolescent mice.

[0183] Grouping of mice in the experiment:

[0184] ① Female mice in the negative control group were caged with male mice in the negative control group;

[0185] ② Female mice in the positive control group were caged with male mice in the negative control group;

[0186] ③ Female mice in the M group (DYC intervention group) were caged with male mice in the negative control group.

[0187] There were 20 female mice and 10 male mice in each group, and the mice were ear-tagged. The male mice were removed at 8:00 the next day after caging at 22:00 at night, and the female mice were placed in a new cage for individual feeding after observing the vaginal plug of the female mice to confirm fertilization. The one-generation breeding experiment was completed. The number of offspring and sex of each pregnant mouse were counted when the offspring were born.

[0188] Experimental results:

[0189] As shown in Table 7, the number of fetal mice in each group was calculated, and it was found that low-level Cd 2+ The number of fetal mice in the combined PCM group was significantly lower than that in the control group, and the number of fetal mice in the DYC intervention group was significantly higher than that in the low-level Cd 2+ The combined PCM group, it was found that the HPOA system of adolescent mice exposed to low-level Cd 2+ Combined PCM, although the appearance of the parent and offspring mice was normal, but could cause the HPOA function to be damaged, and the reproductive ability to be decreased, and DYC could effectively protect the adolescent mice from low-level Cd 2+ HPOA system damage caused by combined PCM.

[0190] Table 7: Baseline data of offspring in the breeding experiment

[0191]

[0192] Experimental conclusion:

[0193] From the above results, it can be seen that DYC can prevent low-level Cd 2+ HPOA system damage of adolescent mice caused by combined PCM.

[0194] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. The use of dapoxetine hydrochloride as the sole active pharmaceutical ingredient in any of the following: i) Prepare drugs to prevent or treat damage to the hypothalamus-pituitary-ovarian axis; ii) To prepare drugs that restore or improve reproductive capacity or fertility.

2. The application as described in claim 1, characterized in that, The preparation method of the drug includes dissolving dapoxetine hydrochloride in phosphate buffer, adjusting the pH value to 7.4, and obtaining the drug.

3. The application as described in claim 2, characterized in that, The concentration of dapronine hydrochloride in the phosphate buffer solution is 10 mg / mL.

4. The application as described in claim 1, characterized in that, The intraperitoneal injection dose of the drug is 5-10 mg / kg·bw / d.

5. The application as described in claim 4, characterized in that, The intraperitoneal injection dose of the drug is 10 mg / kg·bw / d.

6. The application as described in claim 1, characterized in that, The hypothalamic-pituitary-ovarian axis damage was caused by Cd 2+ Caused by combined with iprodione.

7. The application as described in claim 1, characterized in that, The prevention or treatment of hypothalamic-pituitary-ovarian axis damage is achieved by inhibiting the RA signaling pathway and the NF-κB inflammatory pathway.

8. The application as described in claim 1, characterized in that, The restoration or enhancement of reproductive capacity or fertility includes increasing the number of births.

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