Use of fungachromin in constructing an animal model of implantation disorder and a method for constructing an animal model of implantation disorder

Abstract

The present application relates to the use of fungacin in the construction of an animal model of implantation disorder and a method for constructing an animal model of implantation disorder, and belongs to the technical field of medicine. The present application manufactures an SD rat implantation disorder TCM syndrome combination model conforming to the kidney deficiency and blood stasis syndrome type, meeting the needs of TCM research; a stable kidney deficiency and blood stasis-embryo implantation disorder rat model is manufactured, and the success rate of modeling is improved; the modeling time of the present application is only 5 days from confirming that the rat is pregnant, greatly reducing the modeling time; the operation steps are reduced, and only one intraperitoneal injection is needed to complete, reducing the harm to experimental animals.

Description

Technical Field

[0001] This invention relates to the use of fulgamycin in constructing animal models of implantation disorder and a method for constructing an animal model of implantation disorder, belonging to the field of pharmaceutical technology. Background Technology

[0002] Implantation disorder (IDD) refers to the pathological condition in which a fertilized egg fails to adhere to, invade, and implant in the uterine lining. It is a core factor leading to infertility and recurrent implantation failure (RIF). IDD encompasses any pathological condition that affects successful embryo implantation. Its main types include: poor endometrial receptivity, embryo implantation incompetence, and maternal-fetal interface immune dysregulation.

[0003] Currently, the main animal models of implantation failure are: rat blastocyst implantation failure model and rat model of kidney deficiency and blood stasis-endometrial poor receptivity. ① The conventional rat blastocyst implantation failure model is established by subcutaneously injecting 5 mg / kg mifepristone solution into the head and neck of rats at 9:00 on the third day of pregnancy. (Refer to Qi Ning, Huang Guangying, Wang Kaifu, Zhong Wenjuan. Establishment of rat blastocyst implantation failure animal model [J]. Research on Integrated Traditional and Western Medicine, 2009, 1 (01): 29-31.) The modeling principle is that mifepristone is a potent progesterone receptor antagonist. Subcutaneous injection of this drug into female rats during early pregnancy (day 3 or 4 of gestation) rapidly blocks progesterone signaling, leading to a series of physiological changes closely related to blastocyst implantation, such as decreased endometrial receptivity, suppressed uterine gland development, pinocytosis regression, and reduced expression of angiogenic factors. Ultimately, this results in a significant decrease in blastocyst implantation rate and the average number of implanted blastocysts, establishing a stable animal model of blastocyst implantation disorder. ② The method for establishing a rat model of kidney deficiency and blood stasis-endometrial receptivity is as follows: Vaginal smears are examined daily at 9:00 AM. A large number of white blood cells, or a few nucleated squamous epithelial cells interspersed with them, and no keratinized cells are observed in the vaginal secretions, indicating a quiescent phase, at which point modeling begins. The model group was given hydroxyurea 450 mg·(kg·d)-1 by gavage for 10 days, and adrenaline 0.3 mg·(kg·d)-1 was added by subcutaneous injection for 7 days starting from the 4th day. The positive control group (animal model of embryo implantation failure) and the blank group were given an equal volume of physiological saline by gavage for 10 days, and an equal volume of physiological saline was added by subcutaneous injection for 7 days starting from the 4th day. After the modeling was completed, the rats in each group were caged at 18:00 every night at a female-to-male ratio of 1:1. The vaginal plugs were observed and sperm were observed by vaginal smears at 8:00 the next morning. The presence of vaginal plugs or sperm was considered the first day of pregnancy. (Reference: Feng Qianyi, He Dongjie, Xu Limian. Establishment of rat model of kidney deficiency and blood stasis-endometrial poor receptivity[J]. Henan Journal of Traditional Chinese Medicine, 2014, 34(04):618-621.) The modeling principle is that hydroxyurea inhibits nucleotide reductase, thereby inhibiting bone marrow hematopoiesis and causing a decrease in red blood cells, hemoglobin and platelets. The combined use of these two drugs led to increased blood viscosity and aggregation index in rats, resulting in a tendency for blood stasis. Adrenaline, with its potent vasoconstrictive effect, caused a decrease in systemic and renal blood flow, leading to renal ischemia, mimicking the traditional Chinese medicine pattern of kidney deficiency. Furthermore, the combined use of these two drugs resulted in insufficient blood supply to the rat endometrium, a decrease in angiogenic factors, impaired uterine receptivity, and difficulty in successful blastocyst implantation.

[0004] The conventional method of using mifepristone subcutaneous injection in the head and neck is a proven and stable animal model of embryo implantation failure. Its modeling principle is that mifepristone competitively binds to the progesterone receptor, leading to a relative deficiency of progesterone receptor expression during the implantation window. However, its drawback is that it fails to reflect the characteristics of traditional Chinese medicine (TCM) syndromes. The method of using hydroxyurea combined with adrenaline can induce TCM syndromes of kidney deficiency and blood stasis in SD rats to a certain extent. However, in actual animal experiments, it has been found that this method is difficult to stably establish a pathological model of poor endometrial receptivity. There was no statistically significant difference in the number of blastocysts between the control group and the model group. Furthermore, this modeling method results in an excessively long research period (10 days of modeling + rat gestation time), with numerous procedures causing significant harm to the rats. (Feng Qianyi, He Dongjie, Xu Limian. Establishment of a rat model of kidney deficiency and blood stasis-endometrial poor receptivity[J]. Henan Journal of Traditional Chinese Medicine, 2014, 34(04):618-621; Qi Ning, Huang Guangying, Wang Kaifu, Zhong Wenjuan. Establishment of an animal model of rat blastocyst implantation disorder[J]. Journal of Integrated Traditional and Western Medicine, 2009, 1(01):29-31; Tian Yingzhou, Zhu Jingyan, Zhu Xiujun, Gu Chunxiao, Huang Daiyuan, Wen Danting, Liu Zhiqing, Xu Min. Establishment and evaluation of a mouse model of kidney deficiency and embryo implantation disorder[J]. Chinese Journal of Modern Medicine, 2014, 24(11):14-18).

[0005] Literature reports that the modeling method of using a combination of hydroxyurea, adrenaline and mifepristone can create a stable implantation failure model and reflect the characteristics of TCM kidney deficiency and blood stasis syndrome, but it still has many disadvantages as described above. (Yan Li, Guo Pei, Zhou Hang, et al. Study on the mechanism of assisted reproduction by the kidney-tonifying and blood-activating formula in regulating uterine tissue angiogenesis during the implantation window of rats based on the Ang / Tie-2 signaling pathway [J]. World Science and Technology - Modernization of Traditional Chinese Medicine, 2020, 22(06):1862-1870. [2] Wang Yang. Study on the regulation of LXA4 by the kidney-tonifying and blood-activating formula to improve endometrial receptivity during the implantation window of rats with kidney deficiency and blood stasis implantation failure model [D]. Sichuan: Chengdu University of Traditional Chinese Medicine, 2019.)

[0006] Fungamycin is a nucleoside antibiotic produced by *Streptomyces diastatochromogenes*, with the chemical name 4-amino-5-cyano-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine. This compound exhibits activity against Candida albicans, molds, and tumor cells, and its molecular formula is C2. 12 H 13N5O4, molecular weight 291.26. Experiments showed that metabolic engineering of the strain could increase the yield of toyocamycin to 287.8 mg / L. In the agricultural field, its fermentation broth showed an inhibition rate of 98.22% against the pathogen causing root rot of Dendrobium officinale. When combined with insecticides, it could prolong the developmental cycle of brown planthoppers and reduce egg production. Toyocamycin is an IRE1α-XBP1 pathway inhibitor; its mechanism of action is to block the splicing of XBP1 mRNA by inhibiting the oligomerization of IRE1α (IC5O4). 50 = 80 nM), but does not affect IRE1α autophosphorylation. Besides fungamycin, other reported IRE1α-XBP1 pathway inhibitors include small molecule inhibitors (STF-083010, 4μ8C, MKC-3946), acridine derivatives (DAPA (STF089106), 3,6-DMAD, mAMSA / DACA), and anthracycline antibiotics (doxorubicin, daunorubicin). Although fungamycin is an effective inhibitor of the IRE1α-XBP1 pathway, its application in reproductive system or endometrial receptivity studies has not been reported; current research mainly focuses on oncology and plant protection. Summary of the Invention

[0007] The inventors discovered in their research that intraperitoneal injection of fulgamycin into female SD rats could effectively cause embryo implantation problems, and the rats' general condition conformed to the syndrome of kidney deficiency and blood stasis in traditional Chinese medicine.

[0008] This invention provides the use of fulgamycin in the construction of animal models of implantation failure.

[0009] The animals mentioned are mammals.

[0010] The animal model mentioned is a kidney deficiency and blood stasis-implantation disorder animal model.

[0011] The animal model mentioned is a kidney deficiency and blood stasis-poor endometrial receptivity animal model.

[0012] The animal in question is a female SD rat.

[0013] The dosage administered to the SD female rats was 0.8 mg-1.2 mg / kg.

[0014] The present invention also provides a method for constructing an animal model of implantation failure, which involves administering fungamycin via intraperitoneal injection or oral administration to mammals.

[0015] The mammals mentioned are female SD rats, and the dose of fulvamycin administered is 0.8 mg-1.2 mg / kg.

[0016] The present invention also provides the use of the implantation disorder animal model constructed by the described construction method in screening drugs for preventing and / or treating kidney deficiency and blood stasis-implantation disorder or kidney deficiency and blood stasis-poor endometrial receptivity.

[0017] The present invention also provides a method for screening candidate drugs for preventing and / or treating kidney deficiency and blood stasis-implantation disorder or kidney deficiency and blood stasis-poor endometrial receptivity, which is to administer the candidate drug to the implantation disorder animal model constructed by the described construction method.

[0018] The beneficial effects of the present invention are as follows:

[0019] (1) To create a traditional Chinese medicine (TCM) syndrome-combined model of implantation disorder in SD rats that conforms to the TCM syndrome of kidney deficiency and blood stasis, meeting the needs of TCM research;

[0020] (2) To create a stable rat model of kidney deficiency and blood stasis-embryo implantation disorder, improving the success rate of model establishment;

[0021] (3) In the present invention, the model establishment time only takes 5 days starting from confirming the pregnancy of rats, significantly reducing the model establishment time;

[0022] (4) Reducing the operation steps, which can be completed with only one intraperitoneal injection, reducing the harm to experimental animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Scanning electron microscope images of pinopodes in the endometrium of rats in each group (A. blank group; B. medium-dose fengamycin group; C. low-dose fengamycin group; D. STF group);

[0024] Figure 2 Gross observation of blastocyst implantation in rats in each group;

[0025] Figure 3 Statistical chart of the expression of progesterone receptor protein in rats in each group. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] Example 1 Construction of the implantation disorder animal model of the present invention

[0027] 1 Materials and Methods

[0028] 1.1 Experimental Animals

[0029] Forty 10 - 12-week-old female SD rats with a body weight of (220 ± 20) g and twenty 8 - 10-week-old male SD rats with a body weight of (320 ± 20) g were selected. All were of SPF grade, provided by the Experimental Animal Center of Chengdu University of Traditional Chinese Medicine, and the animal quality qualification license number: SCXK (Sichuan) 2024 - 0011. They were adaptively fed for 1 week, with free access to food and water, and placed in a feeding environment with 12-hour light and 12-hour dark alternation.

[0030] 1.2 Medicines

[0031] This invention selected the following two inhibitors, fugamycin and STF-083010, for comparative testing.

[0032] (1) XBP1 inhibitors: Fengjiamycin, STF-083010

[0033] (2) Experimental reagents: dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS)

[0034] 1.3 Grouping and Intervention

[0035] Female SD rats underwent vaginal smear observation over two consecutive estrous cycles to select rats with regular estrous cycles for inclusion in the study. Forty female rats were randomly divided into five groups: high-dose fulvicin group, medium-dose fulvicin group, low-dose fulvicin group, STF-083010 group (hereinafter referred to as STF group), and a control group, with eight rats in each group. These rats were then housed with male SD rats. On the second day after house-rearing, at 7:00 AM, vaginal plugs or vaginal smears were examined for sperm. The presence of vaginal plugs or observed sperm was recorded as day 0.5 of pregnancy (0.5 dpc). At 7 PM on day 1.5 of gestation, pregnant rats in the fulgamycin group were intraperitoneally injected with fulgamycin solution at 2 mg / kg, 1 mg / kg, and 0.5 mg / kg (solvent: 2% DMSO, pH 7.4 PBS). Pregnant rats in the STF group were intraperitoneally injected with STF-083010 solution at 10 mg / kg (solvent: 2% DMSO, pH 7.4 PBS). Pregnant rats in the blank group were intraperitoneally injected with the same solvent. The rats were fasted but allowed free access to water starting the night before sacrifice. At 10 AM on day 6.5 of gestation, after anesthesia with 2.5% tribromoethanol intraperitoneally, blood was collected from the abdominal main vein, and the uterus was dissected.

[0036] 1.4 Observation Indicators

[0037] (1) Coagulation test: 3ml of blood from the abdominal main vein was drawn into an EDTA-K2 anticoagulant tube using a disposable blood collection needle and centrifuged within 30 minutes after collection to obtain plasma. The fully automated coagulation tester was turned on, quality control was performed, the blood sample was loaded, the test was performed, and the machine was turned off according to the instructions.

[0038] (2) Observation of the ultrastructure of the endometrium under electron microscopy: After dissection of the uterus, the middle 1 / 3 of the left uterine segment was taken and fixed in 2.5% glutaraldehyde. After thorough rinsing with phosphate buffer, dehydration with a series of acetones, and then placed in isoamyl acetate for 3 h, critical point drying, sample pasting, silver conductive adhesive coating, and metal coating of the sample in a vacuum coating instrument, followed by scanning electron microscopy observation and photography.

[0039] (3) Implantation rate and average number of implanted blastocysts: The remaining rats in each group were euthanized on day 6.5 of pregnancy by anesthesia with 2.5% tribromoethanol, and the number of pregnant rats and implantation sites were counted. The implantation rate and average number of implantation sites were calculated. Implantation rate = number of pregnant rats / number of test animals × 100%, average number of implanted blastocysts = total number of implanted blastocysts / number of pregnant rats.

[0040] (4) Statistical methods: SPSS 13.0 statistical software was used for statistical analysis. All experimental data are expressed as mean ± standard deviation (x̄ ± s), and the results were analyzed by partition X. 2 Statistical analysis was performed using a test or one-way ANOVA, with p < 0.05 considered statistically significant.

[0041] All observed indicators were referenced from the following literature: Feng Qianyi, He Dongjie, Xu Limian. Establishment of a rat model of kidney deficiency and blood stasis-endometrial poor receptivity [J]. Henan Journal of Traditional Chinese Medicine, 2014, 34(04):618-621. DOI:10.16367 / j.issn.1003-5028.2014.04.108.

[0042] 2 Results

[0043] 2.1 General Observation

[0044] The control group rats were generally in good condition, with smooth fur, agile responses, and steadily increasing body weight, and normal stool. Compared with the control group, all rats in the high-dose fulvic acid group (2 mg / kg) died on days 3-4 of gestation; rats in the medium-dose fulvic acid group (1 mg / kg) exhibited arched backs, curled-up posture, reduced movement, sluggish responses, sparse and dull fur on the back, hair loss, loose stools, and in some cases, diarrhea; their body weight, uterine wet weight, and uterine index were significantly decreased (p<0.05); the low-dose fulvic acid group (0.5 mg / kg) showed a significantly reduced increase in body weight (p<0.05), with no significant difference in uterine wet weight and uterine organ index; the STF group rats showed significantly decreased uterine wet weight and uterine index (p<0.05), with no significant difference in body weight. Except for a significantly lower body weight (p<0.05) compared to the STF group, the medium-dose fulvic acid group rats showed no significant differences in other indicators (see Table 1).

[0045] Table 1. Statistical table of body weight difference, uterine wet weight and organ index of rats in each group.

[0046]

[0047] 2.2 Coagulation function

[0048] Compared with the control group, the plasma PT of rats in the medium-dose group of fulvamycin was significantly increased (p<0.05), and the fibrinogen content showed a significant increasing trend, but there was no statistical significance (p>0.05). There were no significant differences in other indicators. The four coagulation indicators of the other groups were not significantly different from those of the control group, as shown in Table 2.

[0049] Table 2. Statistical table of coagulation parameters in rats of each group.

[0050]

[0051] 2.3 Pinocytosis

[0052] like Figure 1 As shown, in the control group, abundant microvilli were visible on the endometrial surface, along with pinocytosis at various developmental stages. These pinocytosis were plump, smooth, and locally clustered, resembling mushroom-shaped protrusions. In the medium-dose fulvicin group, densely distributed villi were observed, but no mature pinocytosis protrusions were seen. In the low-dose fulvicin group, the endometrial surface was uniformly covered, and pinocytosis at various developmental stages was visible. These protrusions locally appeared as clustered, mushroom-shaped protrusions of varying sizes and shapes. The STF group was similar to the medium-dose fulvicin group, showing only villi and no mature pinocytosis.

[0053] 2.4 Implantation rate and average number of implantation sites

[0054] Macroscopic observation of blastocyst implantation in rats across different groups revealed that the control group had more implanted blastocysts in the uterus, with the implantation sites showing beaded bulges, even distribution of blastocysts on both sides, uniform blastocyst size, significant uterine vascular proliferation, and abundant blood supply. The low-dose fluoroquinolone group and the STF group had fewer implanted blastocysts in the pregnant uterus, with blastocyst volumes slightly smaller than the control group, asymmetrical distribution of blastocysts on both sides, and poorer uterine blood supply compared to the control group. The medium-dose fluoroquinolone group showed a significantly reduced number of implanted blastocysts in the pregnant uterus, a thinner uterine body, and significantly poorer blood supply than the control group. Figure 2 .

[0055] Compared with the control group, the average number of implantation sites in the medium-dose fluoroquinolone group and the STF group was significantly reduced (p<0.05), while there was no significant difference between the low-dose fluoroquinolone group and the control group (p>0.05). Compared with the control group, the implantation rate in the medium-dose fluoroquinolone group was significantly decreased (p<0.05), while there was no significant difference between the low-dose fluoroquinolone group and the STF group and the control group. Although there was no significant difference in the average number of implanted blastocysts between the medium-dose fluoroquinolone group and the STF group, the average difference in the number of implanted blastocysts in the STF group was greater (see Table 3).

[0056] Table 3 Pregnancy status of rats in each group

[0057]

[0058] 2.5 Progesterone receptor (PRB) expression level

[0059] Compared with the control group, progesterone receptor expression was significantly reduced in both the medium-dose fulvic acid group and the STF group (p<0.05), while the low-dose fulvic acid group showed a slight increase compared with the control group (p<0.05). Figure 3 (As shown).

[0060] 3 Discussion

[0061] Compared with Western medicine models in animal experiments, there are fewer traditional Chinese medicine models that can reflect the combination of disease and syndrome. Traditional modeling methods such as subcutaneous injection of mifepristone and gavage with hydroxyurea combined with subcutaneous injection of adrenaline have shown disadvantages such as long modeling time, many operations, and greater harm to animals in animal experiments on infertility-implantation disorder diseases dominated by traditional Chinese medicine, which greatly limits the basic research of traditional Chinese medicine.

[0062] Therefore, based on the research direction of traditional Chinese medicine treatment for implantation-related infertility, the inventors explored a new method for animal modeling combining disease and syndrome. This invention utilizes endoplasmic reticulum function inhibitor fulvicin to significantly reduce progesterone receptor expression in endometrial cells during the implantation window by inhibiting the cleavage of XBP1 by the endoplasmic reticulum stress-unfolded protein receptor IRE1. This successfully established a rat model of kidney deficiency and blood stasis-implantation-related infertility. This model, with progesterone resistance as its core pathological mechanism, mechanistically matches the pathological characteristics of implantation-related infertility patients caused by insufficient progesterone receptor expression and the syndrome characteristics of kidney deficiency and blood stasis in traditional Chinese medicine.

[0063] In this modeling experiment, only rats in the medium-dose group of fulgamycin (1 mg / kg) showed signs of kidney deficiency after modeling, such as lethargy, brittle hair, and significant weight loss. Coagulation parameters indicated that the blood of rats in the medium-dose group of fulgamycin (1 mg / kg) was in a hypercoagulable state or exhibited disseminated intravascular coagulation after a hypercoagulable state, consistent with the TCM clinical manifestations of kidney deficiency and blood stasis. Scanning electron microscopy revealed densely distributed villi in the medium-dose group of fulgamycin, without mature pinocytosis. In the implantation site count, the medium-dose group of fulgamycin showed a significantly lower number of implantation sites compared to the control group, with a larger mean difference in data from the STF group. This demonstrates that although both are XBP1 inhibitors, STF-083010 is less stable than fulgamycin in causing implantation failure in SD rats, and the STF group did not show obvious signs of kidney deficiency.

[0064] The above research results indicate that a single intraperitoneal injection of 1 mg (kg·d) of fulvamycin on day 1.5 of pregnancy is effective. -1The established rat model exhibited traditional Chinese medicine (TCM) signs of kidney deficiency and blood stasis. Coagulation parameters indicated a hypercoagulable state, altered endometrial ultrastructure, inhibited pinocytosis expression, reduced implantation rate and average number of implanted blastocysts, and significantly decreased progesterone receptor (PRB) expression. This suggests that the model combines the characteristics of kidney deficiency and blood stasis with reduced endometrial receptivity, leading to implantation failure, thus achieving successful model establishment. Compared to existing animal models of implantation failure, this invention provides more stable modeling results, shorter modeling time, simpler operation, and less harm to experimental animals. It offers a more accurate and scientifically sound animal model that aligns with TCM syndrome characteristics for further research on the effects of kidney-tonifying and blood-activating TCM on endometrial receptivity and its mechanism of action, greatly improving experimental efficiency.

Claims

1. Use of fugamycin in the construction of an animal model of implantation failure; the animals are female SD rats; the dosage of the female SD rats is 1.0 mg / kg; fugamycin is administered via intraperitoneal injection.

2. A method for constructing an animal model of implantation failure, characterized in that: It is administered via intraperitoneal injection of fulgamycin to mammals; the mammals referred to are female SD rats, and the dose of fulgamycin administered is 1.0 mg / kg.

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