Application of 3-IAA in preparation of medicine for improving fetal adult osteoporosis

Abstract

The invention discloses an application of 3-IAA (3-indoleacetic acid) in preparation of a medicine for improving fetal adult osteoporosis. A fetal adult osteoporosis model is constructed according to the fact that the pregnancy dexamethasone exposure can cause the delayed development of offspring bone and the occurrence of adult osteoporosis, and it is found that the concentration of indole-3-acetic acid (3-IAA) in maternal blood and fetal blood exposed to dexamethasone in the pregnancy is reduced. Based on a rat model of adult offspring osteoporosis caused by pregnancy dexamethasone exposure, 20 mg / kg of 3-IAA is intragastrically fed to GD18-19. D, and the reduction of peak bone mass of female adult offspring caused by pregnancy dexamethasone exposure can be effectively reversed by 3-IAA. Therefore, the 3-IAA provides a new way for preventing and treating the fetal adult osteoporosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of 3-IAA in the preparation of drugs to improve fetal-derived adult osteoporosis. Background Technology

[0002] Osteoporosis is a systemic skeletal disease characterized by decreased bone mass, deterioration of bone microstructure, bone fragility, and increased risk of fractures. Currently, the global prevalence of osteoporosis is 33% in women and 20% in men. [1] In my country, the incidence of osteoporosis is approximately 49% in women and 23% in men. my country has over 120 million osteoporosis patients, ranking first globally. Osteoporotic fractures are considered the second leading cause of death after cardiovascular disease, posing a serious public health challenge to my country and the world. Globally, approximately 8.9 million people suffer fractures annually due to osteoporosis, with a 20-30% mortality rate within one year for hip fractures. However, the pathogenesis of osteoporosis is complex, and current medications for its prevention and treatment 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. [2] 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 exposures on the long-term health of offspring are widely recognized. Studies have shown that during skeletal development, adverse environmental exposures during pregnancy not only affect intrauterine skeletal development in offspring but also have long-term harmful effects such as affecting peak bone mass accumulation after birth. A clinical study on neonatal 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. [3] Furthermore, animal studies have shown that a high-cholesterol diet during pregnancy can reduce birth weight and length in offspring and delay bone mineralization, resulting in lower peak bone mass in adulthood. [4] In summary, osteoporosis has a fetal developmental origin. Therefore, establishing a stable animal model of fetal-derived adult osteoporosis is of great significance for further exploring the pathogenesis of fetal-derived adult osteoporosis and for seeking early prevention and treatment strategies.

[0003] Dexamethasone is a common synthetic glucocorticoid. Because it easily crosses the placenta, it is widely used to treat various pregnancy-related conditions, such as threatened preterm birth, congenital adrenal hyperplasia, placenta previa, and multiple pregnancies. According to the World Health Organization in 2010, the global preterm birth rate averaged 9.6%, with approximately 13 million premature infants born each year, and this number is increasing annually. Prenatal dexamethasone treatment (PDT) can promote fetal lung maturation, reduce the incidence of neonatal respiratory distress syndrome, and significantly reduce perinatal mortality. [5] Therefore, it is widely used in late pregnancy. However, multiple studies have shown that PDT is associated with low birth weight, multi-organ developmental toxicity, and increased susceptibility to chronic diseases in adulthood. [6] Animal studies have also found that prenatal dexamethasone exposure (PDE) can lead to poor liver development in offspring and increased susceptibility to fatty liver after birth. [7] Studies have also shown that dexamethasone use during pregnancy or childhood may lead to osteomalacia, thereby reducing bone quality and mechanical properties. [8] It should be noted that dexamethasone has developmental toxicity and adverse effects on fetal bone development. Therefore, this invention aims to construct an animal model of fetal-derived adult osteoporosis by simulating the clinical dosing regimen of dexamethasone. This model establishment method 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 pregnancy, due to the placental barrier, maternal gut microbiota cannot directly enter the uterus to influence fetal development. However, gut microbiota have the function of breaking down food metabolism in the gut to produce nutrients and small molecules. Therefore, the prevailing view is that during pregnancy, the maternal gut microbiome primarily regulates fetal development by secreting metabolic products. [9]This invention successfully constructed a model of fetal-derived adult osteoporosis and, through bioinformatics analysis and screening, identified indole-3-aceticacid (3-IAA), a metabolite of maternal gut microbiota, as a potential early intervention target for fetal-derived adult osteoporosis. Furthermore, it demonstrated that maternal supplementation with 3-IAA effectively prevents the occurrence of fetal-derived adult osteoporosis. Therefore, this patent application is of significant importance in exploring early prevention and treatment targets for fetal-derived adult osteoporosis.

[0005] Main references: 1.Clynes MA, Harvey NC, Curtis EM, Fuggle NR, Dennison EM, Cooper C: The epidemiology of osteoporosis. British medical bulletin 2020, 133(1):105-117. 2. Reid IR, Billington EO: Drug therapy for osteoporosis in olderadults. Lancet (London, England) 2022, 399(10329):1080-1092. 3.Godfrey K, Walker-Bone K, Robinson S, Taylor P, Shore S, Wheeler T, Cooper C: Neonatal bone mass: influence of parental birthweight, maternal smoking, body composition, and activity during pregnancy. Journal of Bone and Mineral Research : the Official Journal of the American Society For Bone and Mineral Research 2001, 16(9):1694-1703. 4.Mangu SR, Patel K, Sukhdeo SV, Savitha MR, Sharan K: Maternal high-cholesterol diet negatively programs offspring bone development anddownregulates hedgehog signaling in osteoblasts. The Journal of BiologicalChemistry 2022, 298(9):102324. 5.Tain YL, Chen CC, Sheen JM, Yu HR, Tiao MM, Kuo HC, Huang LT:Melatonin attenuates prenatal dexamethasone-induced blood pressure increasein a rat model. Journal of the American Society of Hypertension : JASH 2014,8(4):216-226. 6.Bloom SL, Sheffield JS, McIntire DD, Leveno KJ: Antenataldexamethasone and decreased birth weight. Obstetrics and gynecology 2001, 97(4):485-490. 7.Liu H, He B, Hu W, Liu K, Dai Y, Zhang D, Wang H: Prenataldexamethasone exposure induces nonalcoholic fatty liver disease in male ratoffspring via the miR-122 / YY1 / ACE2-MAS1 pathway. Biochemical pharmacology2021, 185:114420. 8.Tsampalieros A, Gupta P, Denburg MR, Shults J, Zemel BS, Mostoufi-Moab S, Wetzsteon RJ, Herskovitz RM, Whitehead KM, Leonard MB: Glucocorticoid effects on changes in bone mineral density and cortical structure in childhood nephrotic syndrome. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2013,28(3):480-488. 9.Kimura I, Miyamoto J, Ohue-Kitano R, Watanabe K, Yamada T, Onuki M,Aoki R, Isobe Y, Kashihara D, Inoue D et al: Maternal gut microbiota inpregnancy influences offspring metabolic phenotype in mice. Science (NewYork, NY) 2020, 367(6481). Summary of the Invention Fetal-derived adult osteoporosis, a metabolic bone disease caused by abnormal intrauterine environment during fetal development and manifesting in adulthood, has a complex pathogenesis and limited clinical intervention methods. Current research lacks animal models with high success rates, reliable efficacy, and ease of use to support mechanism analysis and target discovery for prevention and treatment. Furthermore, specific prevention and treatment targets and corresponding formulations for this disease are not yet clearly defined, hindering the progress of early intervention and treatment. Therefore, this invention aims to solve the above technical problems by providing a scientific method for constructing animal models of fetal-derived adult osteoporosis, clarifying early prevention and treatment targets, and developing corresponding therapeutic formulations. This provides a key tool for the research and clinical translation of this disease: the application of a highly successful, reliable, reproducible, and simple 3-IAA in the preparation of drugs to improve fetal-derived adult osteoporosis.

[0006] To address the aforementioned technical problems, this invention provides the application of 3-IAA in the preparation of drugs to improve fetal-derived adult osteoporosis, as detailed below: Early Prevention and Treatment Target: Through screening and validation of animal models of fetal osteoporosis, this invention identifies 3-indoleacetic acid (3-IAA) as the early prevention and treatment target for fetal adult osteoporosis. This substance plays a core regulatory role in the pathogenesis of fetal adult osteoporosis—abnormal intrauterine environment during fetal development (such as dexamethasone exposure) leads to metabolic disorders of 3-IAA in offspring, thereby causing bone loss and abnormal expression of bone development-related genes in adulthood; targeted regulation of 3-IAA levels can effectively reverse the above pathological changes and block disease progression.

[0007] Identification of maternal intervention targets for fetal osteoporosis. Using the animal model of fetal osteoporosis constructed above, indole-3-acetic acid was ultimately identified as the maternal intervention target for fetal osteoporosis. This target selection is based on the physiological and pathological characteristics of the animal model, demonstrating clear specificity and reliability, and can regulate the occurrence and development of fetal osteoporosis through maternal intervention.

[0008] Preferred therapeutic formulation: As a preferred therapeutic formulation for fetal-derived adult osteoporosis, the core active ingredient of the formulation recommended in this invention is 3-IAA, and its dosage is 20 mg / kg. d. This dosage, validated in animal models, demonstrates that, while ensuring safety, it can significantly improve abnormal femoral bone mass in model rats by regulating bone metabolism pathways and restoring the normal expression of genes related to primary ossification centers. It has a clear therapeutic effect on fetal-derived adult osteoporosis and possesses good potential for clinical application.

[0009] Specifically, this invention constructs an animal model of fetal-derived adult osteoporosis by precisely controlling drug exposure during pregnancy, standardizing offspring feeding, and quantitative indicator detection. The specific steps are as follows: S1) Drug exposure during pregnancy: Healthy pregnant female Wistar rats were selected as the maternal model. Dexamethasone was administered subcutaneously at a dose of 0.8 mg / kg on days 18-19 of gestation (GD18-19, the critical window for fetal skeletal development). d. Administer the medication twice daily in equal doses to simulate the abnormal intrauterine environment of fetal glucocorticoid exposure.

[0010] S2) Offspring Screening and Feeding Stage: Pregnant rats treated with the above-mentioned drugs give birth naturally, and the day of delivery is designated as "day 0 after birth (PND0)" for the offspring rats. One day after birth (PND1), litters with 12-14 offspring are selected, and the sex of each litter is adjusted to ensure that each litter contains 6 male and 6 female offspring. They are then uniformly nursed to ensure consistent early growth conditions. Offspring rats are weaned at 3-5 weeks (preferably 4 weeks, PW4) after birth and housed separately according to sex. Some female offspring continue to be fed a normal diet until 12-28 weeks (preferably 12 weeks, PW12) after birth. At this time, blood samples are collected from the offspring rats, and femoral tissue is separated for subsequent testing.

[0011] S3) Model Determination Stage: Female offspring rats raised to PW12 were tested for long bone development-related indicators, using "dual core indicators" as the basis for model determination: the first is femoral bone mass (directly reflecting the overall level of bone metabolism; if the bone mass is significantly lower than that of the normal control group, it indicates abnormal bone mass characteristics); the second is the mRNA expression of genes related to primary ossification centers (reflecting the molecular mechanism of bone development; if the expression level is abnormal, it indicates a disorder of bone development regulatory pathways). When both indicators show abnormal changes consistent with the pathological characteristics of fetal-derived adult osteoporosis, the model is determined to be successfully constructed, and a fetal-derived adult osteoporosis animal model is finally obtained.

[0012] The application of the intervention target in the above technical solution of the present invention: First, the fetal-derived adult osteoporosis animal model obtained based on the above construction method can be directly used for screening research on early intervention targets for fetal-derived adult osteoporosis. This model accurately simulates the disease process of "fetal exposure-adult onset," and its pathophysiological characteristics are highly consistent with human fetal-derived adult osteoporosis. During the screening process, the differences in metabolome, genome, and proteome between the model group and the normal control group (female rats of the same strain and age that have not been exposed to dexamethasone during pregnancy) at different time points (such as during pregnancy, offspring PND1, PW4, and PW12) can be observed. Alternatively, candidate target intervention experiments (such as target molecular regulation, drug intervention, etc.) can be conducted on the model rats to observe the improvement of indicators such as femoral bone mass and expression of genes related to primary ossification centers. This allows for the precise identification of early intervention targets that play a key regulatory role in the occurrence and development of the disease, providing direction for mechanism research and the formulation of prevention and treatment strategies.

[0013] Finally, the application of the intervention target. The aforementioned indole-3-acetic acid intervention target can be used to prepare drugs that improve fetal-derived adult osteoporosis. The formulation of the drug must contain indole-3-acetic acid as the core active ingredient. By acting on this intervention target, it can achieve the effect of improving fetal-derived adult osteoporosis, providing a new direction for drug development in clinical prevention and treatment.

[0014] The advantages and beneficial effects of this invention are as follows: 1. This invention is the first to discover a novel function of 3-IAA in improving fetal osteoporosis, precisely addressing the health risks arising from clinical medication use. Dexamethasone, a commonly used synthetic glucocorticoid, is widely used to treat preterm birth-related pregnancy disorders such as threatened preterm birth, congenital adrenal hyperplasia, placenta previa, and multiple pregnancies due to its easy placental crossing properties. However, this drug may cause osteoporosis in offspring (i.e., PDE-induced osteoporosis), and previously, there was a lack of effective intervention methods. This invention demonstrates that maternal supplementation with 3-IAA can directly improve this condition without adjusting existing pregnancy disease treatment plans. Supplementation with 3-IAA alone can reduce the long-term bone health risks in offspring, meeting actual clinical needs. Furthermore, 3-IAA has higher safety profile, balancing maternal treatment with offspring health protection, demonstrating significant practical application value.

[0015] 2. Clarifying the pathological regulatory role of 3-IAA provides a precise target for drug development, breaking through research bottlenecks. This invention deeply reveals the core mechanism by which "decreased 3-IAA levels exacerbate fetal-derived adult osteoporosis," identifying 3-IAA as a key regulatory factor for this disease. In traditional research, the unclear target of fetal-derived adult osteoporosis leads to scattered and inefficient drug development directions. The 3-IAA target provided by this invention can guide researchers to design drugs focusing on "increasing 3-IAA levels," significantly narrowing the research scope and reducing costs. Furthermore, due to the clear mechanism, the subsequent drug action logic is clear, improving the success rate of preclinical validation and clinical trials, providing crucial directional support for drug development in this disease.

[0016] 3. Expanding the application scenarios of 3-IAA level-activating formulations and constructing a complete pathway from basic research to drug translation. This invention clearly states that "3-IAA level-activating formulations can be used to prepare drugs to improve fetal-derived adult osteoporosis," which not only limits the application target of the formulation but also covers multiple formulation forms (such as formulations that directly supplement 3-IAA and formulations that promote the synthesis of endogenous 3-IAA). This conclusion provides enterprises with a clear drug development vehicle, facilitating industrialization research (such as formulation process optimization and quality standard establishment), while being adaptable to different clinical scenarios (such as maternal prophylactic medication during pregnancy and treatment medication for offspring in adulthood). It has great market application potential, can accelerate the translation of scientific research results into clinical drugs, and ultimately provide patients with accessible treatment options, alleviating the health burden caused by this disease. Attached Figure Description

[0017] Figure 1 Effects of PDE on long bone development in female fetal rats.

[0018] Figure 1(A): HE staining of female fetal rat femur; (B, C): Analysis of fetal length and POC length of female fetal rat femur; (DG): Relative expression levels of osteogenic differentiation marker genes (Bsp, Runx2, Ocn, ALP) in fetal tissue of female fetal rat femur. Compared with the control group, * P <0.05, ** P <0.01. Con: Control group; PDE(L): Low-dose PDE group; PDE(H): High-dose PDE group; FL: Femur length; PL: Primary ossification center length; POC: Primary ossification center.

[0019] Figure 2 Effects of PDE on peak bone mass in female adult offspring.

[0020] Figure 2 (A) Micro-CT scan of the femur of female PW12 offspring rats in coronal and transverse views; (B) HE staining of femoral tissue of female PW12 offspring rats; (C) Tb.N analysis of distal femoral cancellous bone of female PW12 offspring rats; (D) BV / TV analysis of distal femoral cancellous bone of female PW12 offspring rats; (E) Tb.Th analysis of distal femoral cancellous bone of female PW12 offspring rats; (F) Tb.Sp analysis of distal femoral cancellous bone of female PW12 offspring rats; (GJ) Relative expression levels of osteogenic differentiation marker genes (Bsp, Runx2, Ocn, ALP) in femoral tissue of female PW12 offspring rats. * P <0.05, ** P <0.01. Con: Control group; PDE(L): Low-dose PDE group; PDE(H): High-dose PDE group; Tb.N: Trabecular bone fraction; BV / TV: Bone volume fraction; Tb.Th: Trabecular bone thickness; Tb.Sp: Trabecular bone separation; Bsp: Osteosin; Runx2: Runt-related transcription factor; Ocn: Osteocalcin; Alp: Alkaline phosphatase.

[0021] Figure 3 The effect of PDE on the composition of maternal gut microbiota.

[0022] Figure 3 (A) Simpson index of maternal gut microbiota in rats; (B) Shannon index of maternal gut microbiota in rats; (C) Chao1 index of maternal gut microbiota in rats; (D) ACE index of maternal gut microbiota in rats; (E) PCoA analysis of maternal gut microbiota; (F) LEfSe analysis of maternal gut microbiota, LDA > 3. P <0.05. Compared with the control group CON,* P <0.05, ** P <0.01. Con: Control group; PDE(H): High-dose PDE group; PCoA: Principal coordinate analysis; LDA: Linear discriminant analysis; LEfSe: Linear discriminant analysis of effect size.

[0023] Figure 4 Effects of PDE on the content of maternal gut microbiota metabolite 3-IAA.

[0024] Figure 4 (A): 3-IAA content in the blood of female mice; (B): 3-IAA content in the blood of female fetal mice. Compared with the control group CON, * P <0.05, ** P <0.01. Con: Control group; PDE(L): Low-dose PDE group; PDE(H): High-dose PDE group.

[0025] Figure 5 Indole-3-acetic acid (3-IAA) can regulate the angiogenesis function of endothelial progenitor cells in bone tissue through Integrin β2.

[0026] Figure 5 (A) Typical images of Integrin β2 immunofluorescence staining in endothelial progenitor cells; (B, C) Protein expression of Integrin β2 in endothelial progenitor cells; (D) Typical images of endothelial progenitor cell angiogenesis and cell migration assays; (E, F) Quantitative analysis of endothelial progenitor cell angiogenesis and cell migration assays. Compared with the control group CON, * P <0.05, ** P <0.01; compared with the 3-IAA group, # P <0.05. Con: control group; 3-IAA: indole-3-acetic acid.

[0027] Figure 6 Effects of maternal 3-IAA supplementation during pregnancy on peak bone mass in adult female PDE offspring.

[0028] Figure 6Chinese: (A) Schematic diagram of the experimental process of supplementing the metabolite 3-IAA of the maternal intestinal flora during pregnancy; (B) Representative coronal and cross-sectional micro-CT scans of the femurs of female offspring rats of PW12; (C) Analysis of trabecular number (Tb.N) of the distal femoral cancellous bone in female offspring rats of PW12; (D) Analysis of bone volume / total volume (BV / TV) of the distal femoral cancellous bone in female offspring rats of PW12; (E) Analysis of trabecular thickness (Tb.Th) of the distal femoral cancellous bone in female offspring rats of PW12; (F) Analysis of trabecular separation (Tb.Sp) of the distal femoral cancellous bone in female offspring rats of PW12; (G-J) Relative expression levels of osteogenic differentiation marker genes (Bsp, Runx2, Ocn, ALP) in the femur tissues of female offspring rats of PW12. Compared with the control group CON, * P <0.05, ** P <0.01. Compared with the PDE(H) group, # P <0.05. Con: Control group; PDE(L): Low-dose PDE group; PDE(H): High-dose PDE group; Bsp: Bone sialoprotein; Runx2: Runt-related transcription factor; Ocn: Osteocalcin; Alp: Alkaline phosphatase; 3-IAA: Indole-3-acetic acid; Tb. N: Trabecular number; BV / TV: Bone volume fraction; Tb. Th: Trabecular thickness; Tb. Sp: Trabecular separation. Detailed implementation manners

[0029] The technical content of the present invention will be further elaborated in detail below in combination with specific embodiments and the drawings.

[0030] Example 1: Construction of a fetal-origin adult osteoporosis animal model 1. Experimental animals 9-week-old SPF-grade female Wistar rats (200 - 240 g, No. 110011211114681414) and 10-week-old SPF-grade male Wistar rats (260 - 300 g, No. 110011211114681664) were both purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (License number: SCXK(Beijing)2021 - 0011).

[0031] The experimental animals were housed in a barrier environment at a temperature of 22~25°C, a humidity of 50%, and a 12-hour day-night cycle.

[0032] 2. Experimental methods After one week of acclimatization feeding, Wistar rats were mated at 19:00 in a 2 female:1 male ratio. Vaginal smears were examined under a microscope at 7:00 the following day. The presence of sperm in the vaginal smear confirmed pregnancy, and this was recorded as day 0 of gestation. At GD0, the pregnant rats were randomly divided into three groups: a control group, a low-dose dexamethasone group [PDE(L)], and a high-dose dexamethasone group [PDE(H)], with 30 rats in each group. All animals were given free access to water and food. The PDE(L) and PDE(H) groups received subcutaneous injections of 0.1 mg / kg dexamethasone and 0.4 mg / kg dexamethasone, respectively, twice daily from GD18 to GD19. The control group received an equal volume of physiological saline subcutaneously from GD18 to GD19.

[0033] Female mice gave birth naturally, producing the F1 generation. The day of delivery was designated as day 0 after birth. On day 1 after birth, pregnant mice with 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 PW4 and separated into male and female cages. Twelve pups from each group were randomly selected and fed a normal diet until PW12 at birth. On the second day after the completion of the above experiment, the animals were euthanized by anesthesia.

[0034] 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.

[0035] 3.2 Real-time quantitative PCR experiment RNA was extracted from bone tissues of 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.-△△Ct The method calculates the relative expression level of the target gene mRNA.

[0036] Table 1 RT-qPCR primer sequences

[0037] Alp: (alkaline phosphatase); Bsp: (bone sialoprotein); Ocn: (osteocalcin); Runx2: (runt-related transcription factor 2); Gapdh: (glyceraldehyde-3-phosphate dehydrogenase).

[0038] 3.3 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 femoral bone sample from the offspring rats in a protective film and place it on the micro-CT machine, then close the instrument's 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; Grayscale 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 3D reconstruction. After opening the image preview, adjust the threshold appropriately and maintain consistency with the threshold in subsequent analyses. 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.

[0039] 4. Experimental Results 4.1 Effects of PDE 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 femur tissue revealed that, compared to the control group, the total femur length and primary ossification center (POC) length of female fetal rats in the PDE group were significantly reduced. Figure 1The results showed that PDE inhibited femoral development in female fetal rats in a dose-dependent manner. Further, the effects of PDE on the expression of osteogenic differentiation marker genes (Bsp, Runx2, Ocn, Alp) in fetal rat femoral tissue were detected by RT-qPCR. The results showed that, compared with the control group, the expression of osteogenic differentiation marker genes in the femoral tissue of female fetal rats in both the PDE(L) and PDE(H) groups was significantly reduced. Figure 1 The results, which showed a dose-dependent effect, further indicate that PDE inhibited long bone development in female offspring.

[0040] 4.2 Effects of PDE 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 PDE on the long bone development of female offspring, this invention examined the femoral development of female offspring from the PW12 group. Micro-CT scans and HE staining results showed that the trabeculae of the female offspring in the control group were dense, thickened, and had narrow intertrabecular gaps, while the trabeculae of the female offspring in the PDE group were sparse, thinned, and had wide intertrabecular gaps. Figure 2 (A, B). Analysis showed that in the PDE group, female offspring exhibited significantly decreased trabecular number (Tb. N), bone volume / tissue volume (BV / TV), and trabecular thickness (Tb. Th), while trabecular separation (Tb. Sp) was significantly increased. Figure 2 (CF). Expression of osteogenic differentiation marker genes (Bsp, Runx2, Ocn, Alp) in femoral tissue showed that, compared with the control group, the expression of osteogenic differentiation marker genes in PDE-affected individuals was significantly reduced. These results indicate that PDE-induced long bone dysplasia in female offspring can persist after birth and lead to a decrease in peak bone mass in adult female offspring.

[0041] In summary, the method of this invention, which involves subcutaneous injection of dexamethasone into pregnant mice at GD18-19, followed by weaning of offspring PW4 and continued feeding with a regular diet until PW12, revealed that all adult female PDE offspring exhibited susceptibility to osteoporosis, indicating a successful establishment of a fetal-derived adult osteoporosis model. The modeling method of this invention is simple, stable, effective, reliable, and highly reproducible.

[0042] Example 2: Maternal supplementation with 3-IAA during pregnancy can effectively prevent the occurrence of fetal-derived adult osteoporosis. 1. Laboratory animals 9-week-old SPF female Wistar rats (200-240 g) (No. 110011211114681414) and 10-week-old SPF male Wistar rats (260-300 g) (No. 110011211114681664) were both purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (License No.: SCXK (Beijing) 2021-0011).

[0043] The experimental animals were housed in a barrier environment at a temperature of 22-25 °C, a humidity of 50%, and a 12-hour day-night cycle.

[0044] 2. Experimental methods After 1 week of adaptive feeding of the rats, at 19:00 in the evening, Wistar rats were caged for mating at a ratio of 2 females: 1 male. The next day at 7:00, vaginal smears were examined under a microscope. If sperm were found in the vaginal smear under the microscope, the rats were determined to be pregnant and recorded as day 0 of pregnancy. On GD0, the pregnant rats were randomly divided into three groups: a control group, a high-dose dexamethasone group [PDE(H)], and a PDE(H) + maternal supplementation of 3-IAA group, with 12 rats in each group. All animals in each group were given free access to water and food. Among them, the female rats in the control group were subcutaneously injected with an equal volume of normal saline on GD18-19; the rats in the PDE(H) group were subcutaneously injected with 0.1 and 0.4 mg / kg of dexamethasone twice a day on GD18-19; the PDE(H) + maternal supplementation of 3-IAA group was given 0.4 mg / kg of dexamethasone by subcutaneous injection to the female rats on GD18-19, and at the same time, 3-IAA was administered by gavage at 20 mg / kg twice a day.

[0045] The female rats gave birth naturally to obtain the F1 generation. The day of birth was taken as day 0 after birth. At 1 day after birth, litters with 12-14 pups per litter were selected from each group, and the number of male and female pups in each litter was adjusted to 6 for lactation feeding to ensure balanced nutrition for the pups. The pups were weaned at PW4 and separated into male and female cages. Twelve normal diet-fed rats were selected from each group and raised until PW12 after birth. On the second day after the above experiments were completed, the animals were sacrificed by anesthesia.

[0046] 3. Detection indexes 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.

[0047] 3.2 Real-time quantitative PCR experiment RNA was extracted from bone tissues of 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. -△△Ct The method calculates the relative expression level of the target gene mRNA.

[0048] Table 1 RT-qPCR primer sequences

[0049] Alp: (alkaline phosphatase); Bsp: (bone sialoprotein); Ocn: (osteocalcin); Runx2: (runt-related transcription factor 2); Gapdh: (glyceraldehyde-3-phosphate dehydrogenase).

[0050] 3.3 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 femoral bone sample from the offspring rats in a protective film and place it on the micro-CT machine, then close the instrument 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 3D 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 image, 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 within 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.

[0051] 3.4 Quantitative detection of 3-IAA content in tissues by LC-MS / MS This invention employs triple quadrupole mass spectrometry (TQMS) for quantitative analysis of long bone tissue and 3-IAA content in female GD20 fetal rats. The steps are as follows: ① Preparation of standard stock solution: Take 20 mg of indole-3-acetic acid analytical standard, add 1 mL of dimethyl sulfoxide (DMSO) and dissolve thoroughly. Take 156.25 μL of the dissolved DMSO solution and add 843.75 μL of acetonitrile to obtain a 31.250 mg / mL stock solution.

[0052] ② Preparation of Standards: The stock solution was diluted 1000 times with water / acetonitrile (1 / 999, v / v) to prepare 31.250 μg / mL, and then further diluted with water / acetonitrile (50 / 50, v / v) to 1562.5 ng / mL. Standards were obtained by diluting the stock solution 1000 times with water / acetonitrile (20 / 80, v / v) to the following concentrations: 0.02, 0.1, 0.5, 2.5, 12.5, 62.5, 312.5, and 1562.5 ng / mL.

[0053] ③ Sample Preparation: For plasma samples, add 50 μL of plasma to a 1.5 mL Eppendorf tube, vortex to mix, then add 150 μL of acetonitrile and vortex again to precipitate proteins. For long bone tissue samples, first remove surrounding muscle and cartilage, rinse with PBS, and dry with filter paper. Weigh approximately 40 mg of tissue, add 150 μL of acetonitrile + 10 μL of LDMSO, and add grinding beads. Homogenize for 5 min, then sonicate (60 Hz, 60 seconds on, 10 seconds off). After processing, centrifuge at 18000 g for 30 min at 4°C. After centrifugation, collect 80 μL of supernatant and transfer it to a tube with a foot liner.

[0054] ④ Analytical Testing: The pretreated samples were injected into a triple quadrupole mass spectrometer for analysis. The parameters were set as follows: column: C18 column (4.6 mm, 150 mm, 5 μm); column temperature: 30 °C; mobile phase: acetonitrile and 1% phosphoric acid (30:70); flow rate: 1 mL / min. After analysis, a standard curve was plotted based on the standards, and the corresponding sample concentrations were calculated.

[0055] 3.5 Gut microbiota 16S rRNA sequencing Fresh fecal samples from GD20 female rats in the control and PDE(H) groups were collected using sterile, enzyme-free EP tubes and immediately stored at -80°C for later use. 16S rRNA sequencing analysis of the gut microbiota was performed by Beijing Novogene Co., Ltd., including genomic DNA extraction, PCR amplification, PCR product mixing and purification, library construction, and sequencing. A brief summary of the steps is as follows: Genomic DNA was extracted from the fecal samples of GD20 female rats using the CTAB method, and the purity and concentration of the genomic DNA were determined using 1% agarose gel electrophoresis. Using the genomic DNA as a template, PCR amplification of the V3-4 region of the 16S rRNA gene was performed using barcode-specific primers, Phusion® High-Fidelity PCRMaster Mix with GC Buffer, and a high-efficiency, high-fidelity enzyme. The primer sequences are as follows: F: 5'-CCTAYGGGRBGCASCAG-3', R: 5'-GGACTACNNGGGTATCTAAT-3'. After quantification and qualitative analysis of the PCR amplification products, sequencing libraries were generated using the NEBNext® Ultra™ II DNA Library Preparation Kit (Cat No. E7645, MA, USA). After the libraries passed quality control, sequencing was performed on an Illumina NovaSeq 6000 platform. Following sequencing, the reads were spliced, filtered, and chimeric sequences were removed to obtain valid sequencing data. The Uparse algorithm was used to further cluster the data, grouping the sequences into operational taxonomic units (OTUs) with 97% consensus, and the most frequent sequences were selected as representative sequences for each OTU. Next, species annotation analysis was performed using the Mothur method and the SILVA138 SSUrRNA database (http: / / www.arb-silva.de) to obtain taxonomic information and statistically analyze the community composition of each sample at different taxonomic levels, including kingdom, phylum, class, order, family, genus, and species. Rapid multiple sequence alignment was performed using MUSCLE software to determine the phylogenetic relationships of representative sequences of OTUs. Simultaneously, the sample data were homogenized to facilitate subsequent α-diversity and β-diversity analyses. Diversity indices were calculated using QIIME software, and principal coordinate analysis (PCoA) was performed using R software, along with inter-group difference analysis of the β-diversity index. Linear discriminant analysis (LEfSe) was used to identify differentially expressed microbiota among the groups.The above analysis was completed using the online cloud platform of Beijing Novogene Co., Ltd. (https: / / magic.novogene.com).

[0056] 3.6 Extraction and culture of primary rat endothelial progenitor cells The extraction process for primary rat endothelial progenitor cells is as follows: ① Animal pretreatment: Four-week-old SPF-grade Wistar rats were sacrificed by cervical dislocation and then disinfected by immersion in 75% ethanol for 10 minutes. Sterile PBS (Ca 2+ / Mg 2+ -free) Rinse the body surface, separate the femur and tibia of both lower limbs in the ultra-clean bench, carefully remove the soft tissue on the bone surface, and rinse 3 times with PBS to remove residual blood.

[0057] ② Bone marrow cavity cell extraction: The epiphyseal end was cut to expose the bone marrow cavity. The bone marrow cavity was flushed under pressure with a 5 ml sterile syringe (27G needle), and the flushing fluid was collected. The cell suspension was centrifuged with an equal volume of rat lymphocyte separation medium (density: 1.083 g / ml) using a density gradient (2000 rpm, 30 minutes, room temperature).

[0058] ③ Mononuclear cell enrichment: Transfer the interfacial cloud layer (containing mononuclear cells) to a new centrifuge tube, resuspend in 5 ml PBS, and then purify by gradient centrifugation. For the first wash, centrifuge at 1500 rpm for 8 minutes and discard the supernatant; for the second wash, repeat centrifugation under the same conditions. Resuspend the precipitate in EGM-2 MV complete medium (containing 10% FBS), and assess viability as >95% using trypan blue staining.

[0059] Culture of primary endothelial progenitor cells: ①Preparation of storage solution: Take fibronectin lyophilized powder and equilibrate at room temperature for 30 minutes. Dissolve it with sterile ddH2O to a final concentration of 0.5 mg / ml (avoid shaking / vortexing, use gentle magnetic stirring), aliquot and freeze at -20 ℃ (avoid repeated freeze-thaw cycles).

[0060] Culture flask coating.

[0061] ② Coat the bottom of the culture flask evenly with a coating concentration of 5 μg / cm² (1.25 ml of working solution is required for T25 flasks), incubate at room temperature for 30 minutes, and then remove the residual solution (to maintain the homogeneity of the matrix).

[0062] ③ Cell seeding and culture: The cell suspension resuspended in EGM-2 MV medium was seeded into the coated flask and dynamically distributed by horizontal cross-shaking (amplitude 5 cm, frequency 60 rpm). The culture was maintained at 37 ℃ and 5% CO2 in a constant temperature system with a medium change cycle of 72 hours.

[0063] 3.7 Angiogenesis and migration experiments of endothelial progenitor cells Tube formation assay: ① Pretreatment with matrix adhesive: 96-well plates were pre-coated with Growth Factor Reduced Matrigel (50 μl / well, 4℃), and gelled at 37℃ for 30 minutes to form a three-dimensional scaffold.

[0064] ② Cell loading and culture: Pretreated endothelial progenitor cells were loaded at 1×10⁻⁶ cells / year. 4 Cells / well density seeding, cultured in serum-free EGM-2MV medium (8 hours, 5% CO2).

[0065] ③ Morphological analysis: Inverted phase contrast microscope (×100) full-hole scanning imaging, the criteria for determining the lumen structure is: a closed network structure formed by ≥2 branch points.

[0066] ④ Quantification strategy: Five non-overlapping fields of view (0.25 mm² / field of view) were randomly selected from each well, and the total lumen length was counted using a double-blind counting method (ImageJ Angiogenesis Analyzer module).

[0067] Transwell migration experiments: ① Migration system construction: Upper chamber, 1×10 5 cells / 200 μl serum-free medium (Corning® 8 μm pore size); lower chamber: 600 μl chemotactic medium containing 10% FBS.

[0068] ② Migration process control: After 24 hours of migration culture at 37 ℃, the process was terminated, and non-migrating cells were mechanically removed with cotton swabs.

[0069] ③ Cell fixation and staining: methanol fixation (20%, 15 minutes), crystal violet staining (0.1%, 30 minutes).

[0070] ④ Data acquisition standard: Systematically acquire 5 random fields of view using an upright microscope (×200).

[0071] 4. Experimental Results 4.1 Effects of PDE on the composition of maternal gut microbiota and the content of 3-IAA The results are as follows Figure 3 As shown. Compared with the control group, the Simpson Index, Shannon Index, Chao1 Index, and ACE Index of the maternal gut microbiota in the PDE(H) group showed no significant changes ( Figure 3 (AD); However, PCOA2 analysis showed that, compared with the control group, the β diversity in the PDE(H) group was significantly altered ( Figure 3(E). Meanwhile, LEfSe analysis revealed significant changes in the abundance of various gut microbiota between the control group and the PDE(H) group. Figure 3 F).

[0072] The results are as follows Figure 4 As shown. Compared with the control group, the levels of 3-IAA in maternal blood and female fetal blood were significantly lower in the PDE(H) group ( Figure 4 A, B).

[0073] 4.2 Effects of 3-IAA on the regulation of angiogenesis function of endothelial progenitor cells in bone tissue via Integrin β2 The result is shown in the figure below. Figure 5 As shown. To clarify the role of 3-IAA in the development of long bones in offspring, this invention extracted rat endothelial progenitor cells and investigated the effect of 3-IAA on their angiogenic function. RT-qPCR and Western blot showed that 3-IAA significantly promoted the expression of Integrin β2 in endothelial progenitor cells (…). Figure 5 (AC). Endothelial cell angiogenesis and migration assays showed that 3-IAA significantly promoted the angiogenic capacity of endothelial progenitor cells. Figure 5 (CE). This invention further discovered that, after inhibiting Itg β2 expression via siRNA, the 3-IAA-induced increase in endothelial progenitor cell angiogenesis was significantly reversed ( Figure 5 (CE). The above results indicate that 3-IAA promotes the angiogenic capacity of endothelial progenitor cells in bone tissue through Integrin β2.

[0074] 4.3 Maternal supplementation with 3-IAA during pregnancy can prevent PDE-induced reduction in peak bone mass in adult female offspring. The results are as follows Figure 6 As shown. Examination of femoral development in female PW12 offspring revealed that, compared to the PDE(H) group, the PDE(H)+3-IAA group had significantly higher bone mass (as shown). Figure 6 (B). Micro-CT analysis showed that, compared with the PDE(H) group, the PDE(H)+3-IAA group had significantly increased Tb.N, BV / TV, and Tb.Th, and significantly decreased Tb.Sp. Figure 6 CF). RT-qPCR results showed that, compared with the PDE(H) group, the expression of osteogenic differentiation marker genes in the femoral tissue of the PDE(H)+3-IAA group was also significantly increased ( Figure 6 (GJ). The above results indicate that maternal supplementation with 3-IAA during pregnancy can significantly reverse the PDE-induced decrease in peak bone mass in female offspring rats.

[0075] In summary, the modeling method of this invention, involving PDE treatment and partial simultaneous oral administration of 3-IAA (20 mg / kg / day), revealed that maternal 3-IAA supplementation during pregnancy can prevent PDE-induced reduction in peak bone mass in female adult offspring. This demonstrates that the model of this invention can be used to screen therapeutic drugs and methods for fetal-derived adult osteoporosis, and confirms that maternal 3-IAA supplementation can prevent the occurrence of fetal-derived adult osteoporosis, which has a positive effect on the development of drugs for the prevention and treatment of fetal-derived adult osteoporosis in clinical practice.

Claims

Application of 1,3-IAA in the preparation of drugs to improve fetal-derived adult osteoporosis.

2. The application of claim 1, characterized in that: The 3-IAA, or indole-3-acetic acid, is used as a maternal intervention target for fetal osteoporosis.

3. The application according to claim 2, characterized in that: The intervention targets were obtained through screening using an animal model of fetal osteoporosis; the animal model of fetal osteoporosis was constructed using the following method, which includes the following steps: S1: Select healthy pregnant female rodents and administer dexamethasone subcutaneously in late pregnancy, 2-3 times a day; S2: The female rodents that conceived in step S1 gave birth naturally and obtained offspring. The birth date was taken as day 0 after birth. On day 1 after birth, the ratio of male to female offspring in each litter was adjusted to 1:1 for nursing. The offspring were weaned 3-5 weeks after birth and the males and females were separated into different cages. Some female offspring continued to be fed normally until adulthood, i.e., 12-28 weeks, when femur tissue was harvested. 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; finally, a fetal osteoporosis animal model is obtained.

4. The application according to claim 3, characterized in that: In step S1, the rodent is the Wistar rat.

5. The application according to claim 4, characterized in that: In step S1, dexamethasone is administered subcutaneously at a dose of 0.4-0.42 mg / kg, twice daily.

6. The application according to claim 5, characterized in that: In step S3, the long bone-related detection indicators are: the mRNA expression levels of femoral bone mass and components in primary ossification centers of female offspring rats.

7. The application according to claim 6, characterized in that: The formulation of the drug for preventing and treating fetal osteoporosis contains indole-3-acetic acid.

8. The application according to claim 7, characterized in that: Indole-3-acetic acid is the core active ingredient in medications for the treatment of fetal-derived adult osteoporosis, and its dosage is 20-22 mg / kg. d.

9. The application according to claim 8, characterized in that: The dosage of indole-3-acetic acid is 20 mg / kg. d.

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