Animal model construction method for fetal osteoporosis, prevention and treatment target and application of prevention and treatment target

Abstract

The invention discloses an animal model construction method for fetal osteoporosis, a prevention and treatment target and application of the prevention and treatment target. The fetal osteoporosis animal model is a filial generation rat which is obtained after a pregnant female Wistar rat is subjected to intragastric administration of Acimoline (300 mg / kg. D) in the pregnancy period. Weaning is conducted four weeks after birth, male and female are separated from cages and fed to 12 weeks, the peak bone mass of female offspring is remarkably reduced, and therefore the animal model with exposed Acimoline during pregnancy is established. On the basis of a pregnancy-stage Acimoline exposed rat model, 20 mg / kg hippuric acid is intragastric injected in the pregnancy stage, and peak bone mass reduction of female offspring can be effectively corrected. The animal model established by the invention is novel, reliable, simple and convenient, and application of hippuric acid in screening and preparing medicines for preventing and treating fetal osteoporosis is determined.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically to a method for constructing an animal model of fetal osteoporosis, a prevention and treatment target, and its application. Background Technology

[0002] Osteoporosis is a systemic skeletal disease characterized by decreased bone mass, bone microarchitectural deterioration, bone fragility, and increased risk of fractures. Currently, the global incidence of osteoporosis is 33% in women and 20% in men. Osteoporotic fractures are considered the second leading cause of death after cardiovascular disease, posing a serious public health challenge globally. Approximately 8.9 million people worldwide suffer fractures annually due to osteoporosis, with a 20-30% mortality rate within one year for hip fractures. However, the causes of osteoporosis are unclear and its mechanisms are complex. Current medications for preventing and treating osteoporosis are often inefficient and have side effects, only slowing bone loss to a certain extent and failing to safely and effectively cure osteoporosis at its source. [1] Therefore, further exploration of the pathogenesis of osteoporosis and the implementation of early prevention and treatment are of significant practical and social importance. With the developmental origins of health and disease (DOHaD) theory and its in-depth research, the long-term adverse effects of early life adverse events on the long-term health of offspring are now widely recognized. [2] Studies have shown that during skeletal development, an unfavorable environment during pregnancy not only affects intrauterine skeletal development in offspring but also has long-term consequences such as impacting peak bone mass accumulation after birth. [3] A clinical study investigating newborn bone mass and maternal lifestyle habits found that maternal alcohol consumption, smoking, or insufficient energy intake during pregnancy can affect the bone mineral content of newborns and their vertebral bone mass in adulthood. [4] Animal studies have also shown that a high-cholesterol maternal diet during pregnancy reduces birth weight and length in offspring and delays bone mineralization, resulting in lower peak bone mass in adulthood. In conclusion, osteoporosis has a fetal developmental origin. Establishing stable animal models of fetal osteoporosis is of great significance for in-depth research into the pathogenesis of fetal osteoporosis and for exploring early prevention and treatment strategies.

[0003] Amoxicillin, a commonly used broad-spectrum β-lactam antibiotic, is widely used for anti-infective therapy and also for treating infectious diseases in pregnant women (such as Chlamydia trachomatis, Escherichia coli, and Staphylococcus aureus infections). However, a recent population study found that amoxicillin use during pregnancy may increase the incidence of fetal malformations. [5]Multiple animal studies have also shown that amoxicillin use during pregnancy has adverse effects on offspring development. For example, amoxicillin use during pregnancy can lead to maldeveloped kidneys in fetal rats and impaired kidney function after birth. [6] Previous studies have shown that amoxicillin exposure during pregnancy can inhibit long bone development in mice. [7] Therefore, this invention aims to construct an animal model of fetal osteoporosis by simulating the clinical dosing regimen of amoxicillin. The method for establishing this model has the advantages of simple operation, high model success rate, and stability and reliability.

[0004] The gut microbiome, also known as the "second human genome," includes bacteria, archaea, single-celled eukaryotes, viruses, and the genetic information they contain. The human gut contains approximately 10 to 100 trillion microorganisms, ten times the total number of human cells, and the number of gut microbiota genes is about 150 times that of the host genome. The gut microbiome plays a crucial role in nutrient absorption, immune system regulation, and homeostasis. During utero, the fetus shares the same gut microbiota with the mother, and the maternal gut microbiota participates in the development of multiple systems in the fetus, including the gastrointestinal tract, cardiovascular system, nervous system, and skeletal system. [8-10] During pregnancy, due to the placental barrier, the maternal gut microbiota cannot directly enter the uterus and affect fetal development. Gut microbiota have the function of breaking down food in the intestines and metabolizing it to produce nutrients and small molecule compounds. Therefore, the mainstream view is that during pregnancy, the maternal gut microbiota mainly regulates fetal development by secreting metabolites. This invention successfully constructed an animal model of fetal osteoporosis and, through bioinformatics analysis, screened and validated it, determining that the maternal gut microbiota metabolite hippuric acid can serve as an early intervention target for fetal osteoporosis. It also confirmed that maternal hippuric acid supplementation can effectively prevent the occurrence of fetal osteoporosis, which is of great significance for exploring early prevention and treatment targets for fetal osteoporosis.

[0005] Main references: 1. Reid IR, Billington EO: Drug therapy for osteoporosis in olderadults. Lancet (London, England) 2022, 399(10329):1080-1092. 2.Buckels EJ, Bolam SM, Tay ML, Matthews BG: The Impact of MaternalHigh-Fat Diet on Bone Microarchitecture in Offspring. Front Nutr 2021, 8:730037. 3.Moon RJ, Citeroni NL, Aihie RR, Harvey NC: Early Life Programmingof Skeletal Health. Current osteoporosis reports 2023, 21(4):433-446. 4.Godfrey K, Walker-Bone K, Robinson S, Taylor P, Shore S, Wheeler T,Cooper C: Neonatal bone mass: influence of parental birthweight, maternalsmoking, body composition, and activity during pregnancy. Journal of Bone andMineral Research : the Official Journal of the American Society For Bone andMineral Research 2001, 16(9):1694-1703. 5.Eri M, Leppée M, Sabo A, Culig J: Beta-lactam antibiotics duringpregnancy: a cross-sectional comparative study Zagreb-Novi Sad. Europeanreview for medical and pharmacological sciences 2012, 16(1):103-110. 6.Nathanson S, Moreau E, Merlet-Benichou C, Gilbert T: In utero andin vitro exposure to beta-lactams impair kidney development in the rat.Journal of the American Society of Nephrology : JASN 2000, 11(5):874-884. 7. He H, Chen M, Long F, Ma C, Wang H, Qin J, Chen L: The "toxic window" of amoxicillin exposure during pregnancy on long bone development in fetal mice. Life sciences 2024, 350:122759. 8. Di Simone N, Santamaria Ortiz A, Specchia M, Tersigni C, Villa P, Gasbarrini A, Scambia G, D'Ippolito S: Recent Insights on the Maternal Microbiota: Impact on Pregnancy Outcomes. Front Immunol 2020, 11:528202. 9. Zhang J, Wang G, Liu J, Gao LR, Liu M, Wang CJ, Chuai M, Bao Y, Li G, Li RM et al: Gut microbiota-derived endotoxin enhanced the incidence of cardia bifida during cardiogenesis. Journal of cellular physiology 2018, 233(12):9271-9283. 10. Vuong HE, Pronovost GN, Williams DW, Coley EJL, Siegler EL, Qiu A, Kazantsev M, Wilson CJ, Rendon T, Hsiao EY: The maternal microbiome modulates fetal neurodevelopment in mice. Nature 2020, 586(7828):281-286. Summary of the Invention Fetal osteoporosis, a type of metabolic bone disease related to fetal environmental exposure, has a complex pathogenesis and lacks effective early clinical interventions. Current research lacks animal models that accurately simulate the disease's development, hindering in-depth mechanistic studies and the selection of intervention targets. Therefore, this invention aims to provide a scientific and stable method for constructing an animal model of fetal osteoporosis, and based on this model, to identify early intervention targets and related applications, providing a key tool for mechanistic research and drug development for this disease. The technical problem this invention addresses is to provide a highly successful, reliable, reproducible, and simple method for constructing an animal model of fetal osteoporosis.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: Firstly, the method for constructing an animal model of fetal osteoporosis provided by this invention comprises the following specific steps: S1) Pregnancy treatment in pregnant rodents: Healthy pregnant female rodents (preferably female Wistar rats) were selected as the maternal model. Amoxicillin was administered orally by gavage on days 18-21 of gestation at a dose of 150-300 mg / kg / day (preferably 300 mg / kg). d), administer once daily to establish an intrauterine environment for amoxicillin exposure during pregnancy.

[0007] S2) Offspring Feeding and Screening Stage: The pregnant rats treated with the aforementioned drugs gave birth naturally, and the day of delivery was designated as "day 0" for the offspring rats. One day after birth, litters of 12-14 rats were selected, and the sex of each litter was adjusted to ensure that each litter contained 6 male and 6 female offspring, who were then uniformly nursed. The offspring rats were weaned 2-4 weeks after birth (preferably 4 weeks) and housed separately according to sex; some female offspring continued to be fed a normal diet until 12-28 weeks of age (preferably 12 weeks), at which point the rats were euthanized and their femur tissue was obtained for subsequent testing.

[0008] S3) Model Determination Stage: Femoral tissue of female offspring rats raised to 12 weeks of age was examined, with "long bone-related indicators" serving as the core basis for model determination. Among them, the key detection indicator was femoral bone mass in female offspring rats—if the detection results showed that the femoral bone mass was significantly lower than that of the normal control group (female rats of the same strain and age that had not been exposed to amoxicillin during pregnancy), the model was deemed to have been successfully constructed, and a fetal osteoporosis animal model was finally obtained.

[0009] Based on the fetal osteoporosis animal model obtained by the above construction method, this invention further provides several core applications: 1. Application of Early Intervention Target Screening: This fetal osteoporosis animal model can be directly used for screening early intervention targets for fetal osteoporosis. By comparing the differences in metabolome and genome between the offspring rats of the model group and the normal control group, or by conducting candidate target intervention experiments on the model rats, early intervention targets that have a regulatory effect on the occurrence and development of the disease can be accurately identified, providing direction for mechanism research.

[0010] 2. Application in Drug Screening / Preparation: This animal model can also serve as a tool for screening and preparing drugs for the prevention and treatment of fetal osteoporosis. During drug development, candidate drugs can be administered to model rats, and the effects of the drugs on improving fetal osteoporosis can be evaluated by detecting indicators such as femoral bone mass and bone mineral density, thereby screening out effective prevention and treatment drugs. Simultaneously, the model can be used to verify the safety and efficacy of drugs, providing experimental evidence for drug preparation.

[0011] 3. Early Intervention Target for Fetal Osteoporosis: Through the screening studies of the above-mentioned animal models, this invention has identified hippuric acid as an early intervention target for fetal osteoporosis. This target plays a key regulatory role in the occurrence and development of fetal osteoporosis. Intervention targeting hippuric acid (such as supplementation or regulation of its metabolic level) can effectively improve abnormal femoral bone mass in model rats, providing a clear target for early intervention of the disease.

[0012] Preferred preventive and therapeutic drug formulation: As a preferred option for the prevention and treatment of fetal osteoporosis, the drug formulation recommended in this invention contains hippuric acid as its core active ingredient, and the recommended dosage of hippuric acid is 20 mg / kg. This dosage has been validated in models and can effectively improve abnormal bone metabolism in rats with fetal osteoporosis while ensuring safety, thus possessing potential clinical application value.

[0013] The technical principles and research process of this invention are as follows: This invention establishes a pregnancy-induced osteoporosis (PEO) model by administering amoxicillin (300 mg / kg / day) orally to pregnant female Wistar rats from days 18 to 21 of gestation, once daily, to obtain offspring. Offspring were weaned at 4 weeks of age, and males and females were separated and raised until 12 weeks of age. Femoral bone mass levels in offspring exposed to amoxicillin during pregnancy were measured at 12 weeks post-birth to establish a fetal osteoporosis animal model. This model provides a reliable experimental platform for elucidating the molecular mechanisms of PEO (such as how intrauterine drug exposure affects bone metabolism pathways in offspring), filling the gap in the field of stable animal models. Based on the clearly defined "hippuric acid" intervention target of this model, it provides a core direction for developing targeted early intervention methods; simultaneously, the model itself can serve as a screening platform for preventive and therapeutic drugs, accelerating the research and development process of drugs for this disease. The model is highly standardized and reproducible, and can be applied in different laboratories. Its clear intervention targets and optimized drug dosages also provide important experimental evidence for subsequent clinical research, and have good potential for clinical translation.

[0014] The advantages and beneficial effects of this invention are as follows: 1. This invention is novel and has significant practical implications. Amoxicillin, a commonly used broad-spectrum β-lactam antibiotic, is widely used in anti-infective therapy and also in the treatment of infectious diseases in pregnant women (such as Chlamydia trachomatis, Escherichia coli, and Staphylococcus aureus infections). Therefore, the rat model of osteoporosis induced by amoxicillin exposure during pregnancy established in this invention is novel and reflects the current state of daily life, thus having positive practical significance.

[0015] 2. The modeling method of this invention is simple, the detection indicators are stable and reliable, and the reproducibility is strong. Pregnant mice were administered amoxicillin by gavage, and the offspring were weaned 4 weeks after birth and fed a standard diet for 12 weeks. Changes in femoral bone mass effectively represent the bone mass changes in osteoporosis patients, closely matching the actual pathological characteristics of the disease. This standardized and stable modeling process allows different laboratories to successfully construct fetal osteoporosis animal models using this method, providing a solid experimental foundation for subsequent related research and greatly promoting the reproducibility and widespread application of research in this field.

[0016] 3. The animal model of fetal osteoporosis constructed based on this invention demonstrates extremely high application value at multiple levels. In guiding healthy living, this model clearly reveals the link between antibiotic use during pregnancy and offspring bone health, thus providing scientific guidance for mothers' preconception health and warning pregnant women to use antibiotics rationally to prevent fetal osteoporosis. In the field of mechanism research, this model provides an ideal research subject for in-depth exploration of the pathogenesis of fetal osteoporosis, helping researchers analyze the molecular mechanisms and signaling pathways of disease development and accelerating the understanding of the disease's nature. Most importantly, the model study has determined that hippuric acid can serve as an early intervention target for fetal osteoporosis, opening up new pathways for early diagnosis and treatment, laying the foundation for developing targeted therapeutic drugs and formulating precise intervention strategies, and is expected to significantly improve patient prognosis in future clinical practice. Attached Figure Description

[0017] Figure 1 Effects of amoxicillin exposure during pregnancy on long bone development in female fetal rats.

[0018] Figure 1 (A): HE staining of the femur of female fetal rats; (B, C): Analysis of femur length and POC length of female fetal rats; (D): Ratio of primary ossification center length to femur length in female fetal rats. Compared with the control group CON, * P <0.05, ** P <0.01. Con: Control group; AML: Low-dose amoxicillin exposure during pregnancy; AMH: High-dose amoxicillin exposure during pregnancy; FL: Femur length; PL: Primary ossification center length; POC: Primary ossification center.

[0019] Figure 2 Effects of amoxicillin exposure during pregnancy on peak bone mass in female offspring.

[0020] Figure 2 (A) Micro-CT scans of the femur of female PW12 offspring rats in sagittal, coronal, and transverse planes; (B) BV / TV analysis of the distal femur cancellous bone of female PW12 offspring rats; (C) Tb.N analysis of the distal femur cancellous bone of female PW12 offspring rats; (D) Tb.Th analysis of the distal femur cancellous bone of female PW12 offspring rats; (E) Tb.Sp analysis of the distal femur cancellous bone of female PW12 offspring rats. * P <0.05, ** P<0.01. CON: Control group; AMHF: Female offspring of the high-dose amoxicillin exposure group during pregnancy; Tb. N: Trabecular bone fraction; BV / TV: Bone volume fraction; Tb. Th: Trabecular bone thickness; Tb. Sp: Trabecular bone separation.

[0021] Figure 3 Effects of acimoline exposure during pregnancy on the composition of maternal gut microbiota in rats.

[0022] Figure 3 In the middle: (A) Quantity of maternal gut microbiota in rats; (B) Shannon index of maternal gut microbiota in rats; (C) Simpson index of maternal gut microbiota in rats; (D) PCoA analysis of maternal gut microbiota. con: control group; AM: amoxicillin exposure group during pregnancy; PCoA: principal coordinate analysis.

[0023] Figure 4 Effects of acimoline exposure during pregnancy on the levels of hippuric acid, a metabolite in the maternal gut microbiota.

[0024] Figure 4 (A): Hippuric acid content in the blood of female mice; (B): Hippuric acid content in the blood of female fetal mice. Con: Control group; AM: Amoxicillin exposure group during pregnancy.

[0025] Figure 5 Effects of hippuric acid on osteogenic differentiation function of bone marrow mesenchymal stem cells Figure 5 (A) Alkaline phosphatase staining and alizarin red staining of bone marrow mesenchymal stem cells; (B) Semi-quantitative analysis of alkaline phosphatase staining in bone marrow mesenchymal stem cells; (C) Semi-quantitative analysis of alizarin red staining in bone marrow mesenchymal stem cells; (DG) mRNA expression of osteogenic differentiation marker genes in bone marrow mesenchymal stem cells. Compared with the control group CON, * P <0.05, ** P <0.01. ALP: alkaline phosphatase; RUNX2: Runt-related transcription factor; OCN: osteocalcin; BSP: osteosialin.

[0026] Figure 6 Effects of maternal hippuric acid supplementation during pregnancy on peak bone mass in female offspring exposed to acimoline during pregnancy.

[0027] Figure 6Chinese: (A) Representative coronal and cross-sectional micro-CT scans of the femurs of PW12 female offspring rats; (B) Analysis of trabecular number (Tb.N) of distal femoral cancellous bone in PW12 female offspring rats; (C) Analysis of trabecular separation (Tb.Sp) of distal femoral cancellous bone in PW12 female offspring rats; (D) Analysis of bone volume fraction (BV / TV) of distal femoral cancellous bone in PW12 female offspring rats; (E) Analysis of trabecular thickness (Tb.Th) of distal femoral cancellous bone in PW12 female offspring rats. Compared with the control group CON, * P <0.05, ** P <0.01. Compared with the PAmE group, # P <0.05. Con: Control group; PAmE: Prenatal amoxicillin exposure group; HA: Hippuric acid; Tb.N: Trabecular number; BV / TV: Bone volume fraction; Tb.Th: Trabecular thickness; Tb.Sp: Trabecular separation. Detailed implementation manners

[0028] The technical content of the present invention will be further elaborated in detail below in combination with specific embodiments and drawings.

[0029] Example 1: Maternal supplementation with hippuric acid during pregnancy can effectively prevent the occurrence of fetal osteoporosis caused by prenatal amoxicillin exposure 1. Experimental animals 9-week-old SPF-grade female Wistar rats (200~240 g) and 10-week-old SPF-grade male Wistar rats (260~300 g) were both purchased from Hubei Experimental Animal Research Center; license number: SCXK (E) 2020-0018.

[0030] The experimental animals were housed in a barrier environment at a temperature of 22~25 °C, a humidity of 50%, and a 12 h day-night cycle.

[0031] 2. Experimental methods After the rats were adaptively fed for 1 week, at 19:00 in the evening, Wistar rats were caged and mated at a ratio of 2 females: 1 male. The next day at 7:00, vaginal smear microscopy was performed. The presence of sperm in the vaginal smear was determined to be a pregnant rat, which was recorded as day 0 of pregnancy. On GD0, the pregnant rats were divided into three groups: a control group, a prenatal amoxicillin exposure group, and a prenatal amoxicillin exposure + maternal hippuric acid supplementation group, with 12 rats in each group. Each group of animals was given free access to water and food. Among them, the prenatal amoxicillin exposure group was given 300 mg / kg amoxicillin by gavage daily from GD18 to 21, and the prenatal amoxicillin exposure + maternal hippuric acid supplementation group was given 20 mg / kg hippuric acid by gavage while being given amoxicillin by gavage. The control group of female rats was given an equal dose of vehicle (normal saline) by gavage from GD18 to 21.

[0032] The mother mice gave birth naturally, producing the F1 generation. The day of birth was designated as day 0 of birth. On day 1 of birth, litters of 12-14 pups were selected from each group, and the litter size was adjusted to 6 males and 6 females for nursing to ensure balanced nutrition for the pups. The pups were weaned at 4 weeks of age and separated into male and female cages. 12 pups were randomly selected from each group and fed a normal diet until 12 weeks of age. On the second day after the completion of the above experiment, the animals were euthanized by anesthesia.

[0033] 3. Detection indicators and methods 3.1 Hematoxylin-eosin staining Long bone tissue fixed in 10% formalin was dehydrated and embedded in a paraffin embedding machine. The embedded tissue block was cut into 5 µm thin slices to prepare liver tissue sections. The sections were dewaxed by immersing them in xylene solution for 5 min twice. The sections were then immersed in 100%, 95%, 85%, and 75% ethanol for 5 min in sequence, and rinsed with distilled water. The sections were then immersed in hematoxylin-eosin staining solution for 30 s, rinsed with distilled water, and then immersed in 1% hydrochloric acid alcohol and quickly removed, and rinsed with distilled water. The sections were then immersed in 75%, 85%, 95%, and 100% ethanol for 5 min in sequence, placed in a fume hood to evaporate the residual ethanol, and then immersed in xylene solution for 5 min, placed in a fume hood to evaporate the residual xylene. A suitable amount of neutral resin was added for mounting. The staining results were observed under a microscope.

[0034] 3.2 Micro-CT scan analysis of femoral bone mass This part of the detection was completed using the SkyScan 1276 micro-CT instrument. The specific operation steps are as follows: ① Before performing the micro-CT scan, first turn on the micro-CT instrument and warm it up for 15 minutes. ② Wrap the obtained offspring rat femur sample with a protective film and place it on the micro-CT machine, then close the instrument and the chamber door. ③ Start the SKY scan software, click "actions>scout and batch scanning" to preview, and set the scanning parameters as follows: Source Voltage=50 kV; Source Current=200 μA; Image Pixel Size=20 μm; Scaled Image Pixel Size=20 μm; Energy filter=1.0; Gray value AV 60%~70%. ④ After setting the scanning area, start the scan. ⑤ After completing the scan, use NRecon (v1.7.0.4, SkyScan) software to perform three-dimensional reconstruction. After opening the image preview, adjust the threshold appropriately and keep the threshold consistent for subsequent analysis. ⑥ Import the 3D reconstructed data into DataViewer software, select "Load for 3D viewing" to load the 3D images, ensuring the femur remains vertical for subsequent analysis, and save it in a new folder. ⑦ Import the dataset with the femur orientation adjusted into CTAn (1.18.4) software, and select the cancellous bone region 0.5~4.5 mm (i.e., 200 images) below the lowest point of the distal femoral growth plate as the region of interest for analysis. In Binary selection, select "from dataset" to cover as much trabecular bone as possible during analysis. ⑧ Analyze and calculate the bone volume / tissue volume (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) of the region of interest (distal femoral cancellous bone trabecular bone) selected in step ⑦ to evaluate changes in bone mass.

[0035] 3.3 Extraction, medium replacement, and passage of primary bone marrow mesenchymal stem cells (BMSCs) Four-week-old female offspring rats were euthanized under isoflurane anesthesia and immediately disinfected by immersion in alcohol for 5 minutes, repeated twice. After disinfection, the rats were transferred to a clean bench, and the hind limbs were dissected to expose the femur and tibia. Surrounding soft tissues such as muscles were removed. The separated femur and tibia were rinsed three times with PBS buffer, and the epiphyses at both ends of the metaphysis were removed to expose the medullary cavity. The exposed medullary cavity was rinsed with 5 mL of α-MEM medium using a syringe until it was approximately white and transparent. The rinsing fluid was collected and centrifuged at 1000 rpm for 3 minutes at room temperature. The supernatant was removed, and the precipitate was retained. The precipitate was resuspended in PBS, centrifuged again, and the supernatant was removed. The precipitate was then resuspended in complete culture medium (α-MEM medium + 10% fetal bovine serum + 1% penicillin / streptomycin). The cells were then seeded into T25 cell culture flasks and cultured in a 37°C, 5% CO2 incubator. After approximately 2-3 days of BMSCs adherence and growth, the medium was changed to remove suspended cells. Subsequent medium changes or passages were performed based on cell growth. In this study, BMSCs of passage 4 or less were used for all cell experiments.

[0036] When BMSCs reached 80%–90% adherence, they were seeded onto a culture plate and osteogenic differentiation was induced. When the seeded cells reached approximately 60%–80% confluence, the complete culture medium was replaced with osteogenic differentiation induction medium (α-MEM medium + 10% fetal bovine serum + 1% penicillin / streptomycin + 10 nM dexamethasone + 50 μg / L Vitamin C + 10 mM β-glycerophosphate). During osteogenic differentiation induction, the medium was changed daily. Approximately 14 days after induction, colorless mineralized nodular crystals were observed under a microscope.

[0037] 3.4 Alkaline phosphatase (ALP) staining ALP staining uses 5-bromo-4-chloro-3-indole phosphate (BCIP) / nitrotetrazole blue (NBT) as substrates. In the presence of ALP, BCIP is catalyzed to hydrolyze, forming a strongly reactive product that reacts with NBT to form a blue, insoluble product. The intensity of ALP staining reflects the osteogenic differentiation capacity of bone mesenchymal stem cells (BMSCs). This experimental procedure was performed according to the instructions of the BCIP / NBT ALP staining kit (MA0197, Dalian Meilun Biotechnology, Dalian, China). A brief summary of the steps is as follows: ①BMSCs were seeded in 6-well plates, and after the cells were about 70% confluent, the medium was replaced with osteogenic differentiation induction medium.

[0038] ② Ten days after osteogenic induction and differentiation, the cells were washed with PBS. Immediately afterwards, the cell samples were fixed with 4% polymethyl methacrylate (PMMA) for 10 min, and then washed three times with PBS. During cell fixation, the following ALP working solution was prepared: 3 mL ALP chromogenic buffer + 20 μL NBT solution (150×) + 30 μL BCIP solution (100×).

[0039] ③ Each sample was incubated with 400 μL of ALP working solution at room temperature in the dark for 6 h.

[0040] ④ Observe and analyze the ALP staining results under an upright microscope. The staining exhibits high refractive properties.

[0041] 3.5 Alizarin Red S (ARS) staining ARS staining is based on the principle that Alizarin Red S chelates with calcium ions to form an orange-red complex. It can reflect the ability of bone marrow mesenchymal stem cells (BMSCs) to form mineralized nodules during osteogenic differentiation, providing a direct visual indication of their osteogenic differentiation capacity. This experimental procedure was performed according to the instructions of the osteoblast mineralized nodule staining kit (Alizarin Red S method) provided by Shanghai Beyotime Biotechnology Co., Ltd., and the simplified steps are as follows: ①BMSCs were seeded in 6-well plates, and after the cells were about 70% confluent, the medium was replaced with osteogenic differentiation induction medium.

[0042] ② Two to three weeks after osteogenic induction, wash once with PBS. Then, fix the cell samples with fixative at room temperature for 20 minutes, and wash three more times with PBS. This step requires careful handling.

[0043] ③ Each well sample was stained with 500 μL of ARS staining solution at room temperature for 30 min.

[0044] ④ Rinse gently again with PBS until the stain no longer comes off and the wash solution is clear.

[0045] ⑤ Observe and analyze the red mineralized nodules under an upright microscope.

[0046] 3.6 Real-time quantitative PCR experiment RNA was extracted from cells in each group using Trizol reagent, and cDNA was obtained after reverse transcription. The cDNA, upstream and downstream primers for each gene, and SYBR Green I fluorescent dye were mixed thoroughly and then placed in an RT-qPCR instrument for reaction. The RT-qPCR reaction conditions were as follows (reaction volume: 10 µL): 95℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s, 62℃ annealing for 30 s; 72℃ extension for 15 s, for a total of 40 cycles. GAPDH was used as an internal control, and the relative expression level of the target gene mRNA was calculated using the 2-ΔΔCt method.

[0047] Table 1 RT-qPCR primer sequences

[0048] Alp: (alkaline phosphatase); Bsp: (bone sialoprotein); Ocn: (osteocalcin); Runx2: (runt-related transcription factor 2); Gapdh: (glyceraldehyde-3-phosphate dehydrogenase).

[0049] 4. Experimental Results 4.1 Effects of amoxicillin exposure during pregnancy on long bone development in female fetal rats The results are as follows Figure 1 As shown. HE staining and quantitative analysis of fetal rat femoral tissue revealed that, compared with the control group, the total femoral length and the length of the primary ossification center (POC) were significantly reduced in female fetal rats exposed to acimoline during pregnancy. Figure 1 The amoxicillin exposure during pregnancy (AC) showed a dose-dependent effect. Furthermore, statistical analysis revealed that, compared to the control group, the ratio of POC length to total femur length was significantly lower in the amoxicillin-exposed group during pregnancy. Figure 1 (D). The above results indicate that acimoline can dose-dependently inhibit femoral development in female fetal rats during pregnancy.

[0050] 4.2 Effects of amoxicillin exposure during pregnancy on long bone development in female offspring rats The results are as follows Figure 2 As shown. To further investigate the long-term effects of prenatal amoxicillin exposure on long bone development in female offspring, we examined the femur development of female PW12 offspring. Micro-CT scans showed that, compared with the control group, the amoxicillin-exposed female offspring had significantly lower trabecular number (Tb. N), bone volume / tissue volume (BV / TV), and trabecular thickness (Tb. Th), and significantly higher trabecular separation (Tb. Sp). Figure 2 (Middle AE). The above results indicate that amoxicillin exposure during pregnancy can lead to long bone dysplasia in female offspring that persists after birth and results in a decrease in peak bone mass in female offspring.

[0051] 4.3 Effects of amoxicillin exposure during pregnancy on maternal gut microbiota composition and hippuric acid levels The results are as follows Figure 3 As shown. Compared with the control group, the number of maternal gut microbiota in the pre-pregnancy amoxicillin exposure group was significantly reduced ( ). Figure 3 In the middle (A), neither the Shannon Index nor the Simpson Index changed significantly. Figure 3 (B, C); However, PCOA2 analysis showed that, compared with the control group, the β diversity in the amoxicillin exposure group during pregnancy was significantly altered ( Figure 3 (D). This indicates that amoxicillin exposure during pregnancy leads to significant changes in the composition of the maternal gut microbiota.

[0052] result Figure 4 As shown in the figure. Compared with the control group, the levels of hippuric acid in the blood of both mother mice and female fetuses in the amoxicillin-exposed group during pregnancy were significantly reduced. Figure 4 (A, B)

[0053] 4.4 Effects of hippuric acid on osteogenic differentiation function of bone marrow mesenchymal stem cells The results are as follows Figure 5 As shown, hippuric acid, in a concentration gradient, promotes the osteogenic differentiation function of bone marrow mesenchymal stem cells, specifically manifested by increased alkaline phosphatase staining and alizarin red staining. Figure 5 In addition, hippuric acid significantly promoted the mRNA expression of osteogenic differentiation marker genes in bone marrow mesenchymal stem cells in a concentration-dependent manner. Figure 5 (DG). The above results indicate that hippuric acid can improve the osteogenic differentiation function of bone marrow mesenchymal stem cells during long bone development.

[0054] 4.5 Maternal hippuric acid supplementation during pregnancy can prevent the reduction in peak bone mass in female offspring caused by amoxicillin exposure during pregnancy. The results are as follows Figure 6 As shown. Examination of femoral development in female PW12 offspring revealed that, compared to the amoxicillin exposure group during pregnancy, the amoxicillin exposure + hippuric acid group had significantly higher bone mass. Figure 6 (A). Micro-CT analysis showed that, compared with the amoxicillin exposure group during pregnancy, the amoxicillin exposure + hippuric acid group during pregnancy had significantly higher levels of Tb. N, BV / TV, and Tb. Th, and significantly lower levels of Tb. Sp. Figure 6 (Chinese BE). The above results indicate that maternal hippuric acid supplementation during pregnancy can significantly reverse the decrease in peak bone mass in female offspring rats induced by amoxicillin exposure during pregnancy.

[0055] In summary, the modeling method of this invention, involving amoxicillin exposure during pregnancy followed by partial gavage supplementation with hippuric acid (20 mg / kg), revealed that maternal hippuric acid supplementation during pregnancy can prevent the reduction in peak bone mass in female offspring induced by amoxicillin exposure during pregnancy. This demonstrates that the model of this invention can be used to screen therapeutic drugs and methods for fetal osteoporosis, and confirms that maternal hippuric acid supplementation can prevent the occurrence of fetal osteoporosis, thus playing a positive role in the clinical development and prevention of fetal osteoporosis.

Claims

1. A method for constructing an animal model of fetal osteoporosis, characterized in that: Includes the following steps: S1: Select healthy pregnant female rodents and administer acimoline 150-300 mg / kg / day orally during late pregnancy; S2: The conceived rodents in step S2 above give birth naturally and obtain offspring. The birth date is taken as day 0 after birth. On day 1 after birth, the ratio of male to female offspring in each litter is adjusted to 1:1 for nursing. The offspring are weaned 2-4 weeks after birth and the males and females are separated into different cages. Some female offspring continue to be fed normally until adulthood, i.e., 12-28 weeks. Blood and femur tissue are collected. S3: After the above steps are completed, the long bone-related indicators of the offspring are detected at 12 weeks after birth to comprehensively determine osteoporosis, and finally obtain an animal model of fetal osteoporosis.

2. The construction method according to claim 1, characterized in that: In step S1, the rodent is any one of SPF-grade Wistar, SD rat, or Kunming mouse.

3. The construction method according to claim 2, characterized in that: In step S3, the long bone-related detection indicators are: fetal rat femur development-related indicators and femur bone mass of offspring rats at 12 weeks after birth.

4. The application of a fetal osteoporosis animal model in screening / preparing drugs or substances that resist potential developmental toxicity environmental disturbances, characterized in that: The animal model is obtained by any of the construction methods described in claims 1 to 3.

5. The application of an animal model of fetal osteoporosis in screening targets for the prevention and treatment of fetal osteoporosis, characterized in that: The animal model is obtained by any of the construction methods described in claims 1 to 3.

6. A maternal prevention and treatment target for fetal osteoporosis, characterized in that: The prevention and control target is obtained through the application screening of claim 5.

7. Hippuric acid serves as a maternal target for the prevention and treatment of fetal osteoporosis.

8. The application of an animal model of fetal osteoporosis in screening / preparing drugs for the prevention and treatment of fetal osteoporosis, characterized in that: The fetal osteoporosis animal model is obtained by any of the construction methods described in claims 1 to 3.

9. The application according to claim 8, characterized in that: The formulation of the drug for preventing and treating fetal osteoporosis contains hippuric acid.

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