Application of hypothalamic paternal IGF2 as a biomarker in the preparation of products for screening and prevention of precocious puberty

Abstract

This application relates to the biomedical field and discloses the use of hypothalamic paternal IGF2 as a biomarker in the preparation of products for screening and preventing precocious puberty. Specifically, it discloses the application of reagents for detecting the gene imprinting status and / or expression level of hypothalamic paternal insulin-like growth factor 2 in the preparation of precocious puberty screening products, and the application of substances that inhibit the activation of hypothalamic paternal insulin-like growth factor 2 or reduce its expression level in the preparation of drugs for preventing and treating precocious puberty. This application establishes a link between paternal IGF2 and BPA and high-nutrition-induced precocious puberty for the first time, clarifying its core role in mediating the occurrence of precocious puberty and its early warning value, breaking through the limitations of traditional precocious puberty mechanism research. The proposed intervention program is both specific and practical. Among them, the application of Dabu Yin Wan (a traditional Chinese medicine formula) achieves precise targeting of traditional Chinese medicine in the intervention of precocious puberty, and compared with existing intervention technologies, it has significant advantages such as fewer side effects, strong targeting, and high operability.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and more specifically, to the use of hypothalamic paternal IGF2 as a biomarker in the preparation of products for screening and prevention of precocious puberty. Background Technology

[0002] Adolescence is a crucial developmental stage for children transitioning to adulthood. With changes in the social environment and nutritional patterns, precocious puberty has become a significant child health issue of great social concern. Precocious puberty is defined as the appearance of secondary sexual characteristics in girls before the age of 8 and in boys before the age of 9, with a male-to-female ratio of approximately 1:15-20, significantly higher in girls. Multiple population surveys show that the age of onset of puberty in girls is trending earlier each year. From 1977 to 2013, the age of onset of breast development in girls decreased by approximately 3 months every 10 years. Domestic epidemiological data shows that in 2019, the incidence of precocious puberty in preschool girls ranged from 2.05% to 11.47%, showing a year-on-year upward trend. Precocious puberty not only affects children's growth potential and physical and mental health in the short term, but in the long term, premature exposure to estrogen is also a high-risk factor for various diseases in adulthood, including endocrine disorders, metabolic diseases, reproductive dysfunction, malignant tumors, and neuropsychiatric abnormalities. Therefore, in-depth research into the pathogenesis of precocious puberty, and exploration of effective screening, prevention and control targets, early intervention, and even prevention strategies, are of great significance for improving women's reproductive and mental health.

[0003] Exposure to environmental endocrine disruptors is a significant contributing factor to precocious puberty in girls. Bisphenol A (BPA), a typical endocrine disruptor widely present in the environment, has a weak estrogenic effect. Population studies have shown that serum BPA levels in girls with precocious puberty are significantly higher than in normally developing children (31.94±7.30 ng / mL vs. 9.91±0.74 ng / mL). In animal studies, the pharmacokinetic characteristics of BPA in female monkeys, mice, and rats are highly similar to those in humans. Daily oral administration of approximately 0.4 mg / kg of BPA can result in serum BPA concentrations of up to 0.5 ng / mL in female monkeys and mice, approaching human exposure levels. Numerous animal studies have confirmed that BPA exposure can induce reproductive toxicity, with precocious puberty induced by exposure doses ranging from 2.5 μg / kg / day to 10 mg / kg / day. Previous studies have found that BPA intervention at 0.5 mg / kg / day in neonatal SD female rats can lead to precocious puberty.

[0004] High nutritional intake is also a significant factor in inducing precocious puberty. With changes in lifestyle and dietary structure, excessive energy and nutrient intake during childhood has become a major risk factor for abnormal puberty onset. This is especially true for individuals with intrauterine growth retardation (IUGR), who already exhibit growth restriction and altered endocrine metabolic programming during fetal development; high nutritional intake after birth can easily induce rapid catch-up growth. This rapid increase in weight and height in a short period can lead to metabolic signaling disorders, thereby promoting precocious puberty. These metabolic disorders not only increase the risk of menstrual irregularities and ovulation abnormalities during puberty but also significantly increase the long-term probability of developing polycystic ovary syndrome, obesity, and metabolic syndrome, causing persistent adverse effects on women's reproductive health and long-term quality of life.

[0005] The regulatory center for the initiation of puberty is located in the hypothalamus, and the activation of the gonadotropin-releasing hormone (GnRH) gene in the arcuate nucleus region is a direct effector molecule initiating development. Approaching puberty, the secretion of activating neurotransmitters increases, among which Kisspeptin (encoded by the Kiss1 gene) is an important activating neuropeptide that promotes GnRH synthesis. Current research has found that various growth factors and neurotransmitters can regulate the initiation of puberty by affecting the activation of Kiss1 and GnRH neurons, including leptin, insulin, glutamate, neurokinin B, and γ-aminobutyric acid (GABA). However, the specific temporal roles of these factors in the hypothalamic molecular events of sexual development initiation and whether they have a warning effect remain unclear. Nutritional status and environmental endocrine disruptors are common environmental factors inducing precocious puberty, and their effects may ultimately activate the Kiss1 / GnRH system through interactions with the genetic background. Epigenetic mechanisms are the main regulatory pathways of the interaction between the environment and the organism, among which genomic imprinting is a typical epigenetic regulatory mechanism, mainly affecting gene expression through DNA methylation modification. Dysregulation of imprinted genes has been proven to be an important mechanism by which environmental factors induce abnormal growth and development. IGF2 is a classic imprinted gene, and numerous studies have shown that its methylation modification in the fetus is regulated by environmental and nutritional conditions, making it a key molecule connecting the intrauterine environment and fetal growth. However, whether these imprinted genes mediate the regulation of postnatal environmental factors on the initiation of puberty remains unclear.

[0006] Currently, existing technologies still have the following shortcomings: the pathogenesis of precocious puberty is not yet clear; the key molecular targets that induce precocious puberty through environmental and nutritional factors remain unclear; and there is a lack of effective early warning and screening methods in clinical practice. Interventional treatment can only be provided after the onset of the disease, which is not only detrimental to children's physical and mental health but also increases the economic burden on society and families. Therefore, there is an urgent need to identify key molecules linking environmental exposure to the initiation of puberty and to develop molecular biomarkers and their detection products that can achieve early warning and screening for high-risk factors.

[0007] However, whether imprinted genes mediate the regulation of postnatal environmental factors on the initiation of puberty remains unclear. Specifically, whether nutritional status or exposure to endocrine disruptors in early postnatal years (e.g., infancy) affects the maturation sequence of Kiss1 / GnRH neurons and leads to an earlier initiation threshold for puberty by inducing alterations in the methylation of specific imprinted genes (such as IGF2, MEST, PEG3, etc.) in hypothalamic Kiss1 or GnRH neurons has not been reported. Therefore, elucidating the temporal expression characteristics of imprinted genes in different environmental precocious puberty models and establishing early identification and intervention methods for precocious puberty based on this is a pressing technical problem that needs to be solved. Summary of the Invention

[0008] The purpose of this invention is to explore the pathogenesis of precocious puberty and provide effective early warning biomarkers and intervention targets.

[0009] To achieve the above-mentioned objectives, this application adopts the following technical solution:

[0010] In the first aspect, this application provides the application of reagents for detecting the imprinting status and / or expression level of the hypothalamus paternal insulin-like growth factor 2 (IGF2) gene in the preparation of precocious puberty screening products.

[0011] Furthermore, the precocious puberty includes bisphenol A-induced precocious puberty and precocious puberty caused by high nutrition.

[0012] Furthermore, the reagents include primer pairs for detecting hypothalamic paternal insulin-like growth factor 2 mRNA expression and / or antibodies for detecting hypothalamic paternal insulin-like growth factor 2 protein expression.

[0013] Secondly, this application provides the use of substances that inhibit the activation of hypothalamic paternal insulin-like growth factor 2 or reduce its expression level in the preparation of drugs for the prevention and treatment of precocious puberty.

[0014] Furthermore, the precocious puberty includes bisphenol A-induced precocious puberty and precocious puberty caused by high nutrition.

[0015] Furthermore, the substances that inhibit the activation of hypothalamic paternal insulin-like growth factor 2 or reduce its expression level include Dabuyin Pill and gene-editing agents and antibody agents that specifically block the activation of hypothalamic paternal insulin-like growth factor 2.

[0016] Thirdly, this application provides a kit for screening precocious puberty, the kit comprising reagents for detecting the imprinting status and / or expression level of the hypothalamic paternal insulin-like growth factor 2 gene.

[0017] Furthermore, the reagents include primer pairs for detecting hypothalamic paternal insulin-like growth factor 2 mRNA expression and / or antibodies for detecting hypothalamic paternal insulin-like growth factor 2 protein expression.

[0018] Furthermore, the primer pair includes: The forward primer shown in SEQ ID NO:5 and the reverse primer shown in SEQ ID NO:6; or The forward primer shown in SEQ ID NO:13 and the reverse primer shown in SEQ ID NO:14.

[0019] Fourthly, this application provides the application of hypothalamic paternal insulin-like growth factor 2 as a target in the preparation of products for the detection of exogenous environmental substances or nutrients.

[0020] In summary, this application has the following beneficial effects: This application identifies paternal IGF2 activation as a key target for environmentally induced precocious puberty. This discovery provides important evidence for subsequent product development, mainly in the following three aspects: ① as a biomarker for early screening of precocious puberty; ② for early screening and identification of high-risk environmental factors that induce precocious puberty; ③ as a screening tool for the development of drugs and foods for the effective prevention and early treatment of precocious puberty. Attached Figure Description

[0021] Figure 1 Examples of precocious puberty model construction and hypothalamic sexual development regulatory gene detection results; Figure 2 This is a screening and preliminary verification of hypothalamic IGF2 during the initiation stage of sexual development in this embodiment; Figure 3 This example illustrates the regulatory effect of bisphenol A on IGF2 in primary hypothalamic neurons. Figure 4 This is a functional analysis of the activation of hypothalamic neuronal cellular development genes by exogenous IGF2 in the embodiments; Figure 5 The effects of knocking down IGF2 under different parental conditions on the regulation of sexual development are illustrated in the examples. Figure 6 This is the regulatory effect of paternal knockout of IGF2 on the BPA precocious puberty model in the embodiments; Figure 7 This example demonstrates the regulatory effect of Dabuyin Pill on the IUGR high-nutrition precocious puberty model.

[0022] Figure 8 This demonstrates the diagnostic efficacy of hypothalamic paternal IGF2 as a biomarker in this embodiment. Detailed Implementation

[0023] The technical solutions and effects of the present application will be further described in detail below in conjunction with the embodiments and the accompanying drawings. It can be understood that the specific embodiments described herein are only used to explain the invention, rather than limiting the invention.

[0024] It should be noted that the test methods or testing methods described in the following embodiments are all conventional methods unless otherwise specified; the reagents and materials are all obtained from conventional commercial channels unless otherwise specified.

[0025] Experimental subjects: SD rats were purchased from Zhejiang Vital River Laboratory Animal Science & Technology Co., Ltd. (certification number: SCXK(Zhe)2019-0001), C57BL / 6 mice were purchased from Jiangsu Jicui Yakang Biotech Co., Ltd., and the experimental animal production license number is: SCXK(Su)2023-0009. All experimental animals and operations in this study were approved by the Research Ethics Committee. Animal housing environment: The light cycle (12 / 12 hour light / dark cycle) and temperature (23-25 °C) were controlled, and water and breeding feed were freely available. Female young rats were selected as experimental subjects, and the day of parturition of pregnant rats was recorded as the first day after birth of the young rats (PND1, PND represents the age in days after birth of the rats).

[0026] Reagents:

[0027] Primers:

[0028] Statistical methods and analysis: Statistical analysis was performed using the software GraphPad Prism 9.5. The data are presented as: mean ± standard error (Mean ± SEM). First, normality test and homogeneity of variance test were performed. If the data conform to the normal distribution, Student's t-test was used for comparison between two groups, and ANOVA was used for comparison between multiple groups. Otherwise, the Mann-Whitney U test or Kruskal-Wallis rank sum test was used. P < 0.05 was considered statistically significant.

[0029] Example 1: Construction of precocious puberty model BPA-induced precocious puberty model: A precocious puberty model was constructed by exposing neonatal female rats to bisphenol A (BPA). Litter matching was used, and female rats in the same litter were randomly divided into two groups: BPA exposure group and control (corn oil) group, and there was no difference in birth weight between the groups. From the first day after birth (PND 1) to PND14, BPA (0.5 mg / kg·d) or an equal volume of corn oil was administered daily by intramuscular injection.

[0030] High-nutrition precocious puberty model: IUGR model was established by nutritional restriction in late pregnancy of SD rat mothers. Pups with a birth weight lower than the average weight of normal pups -2SD were included in the IUGR model group, while pups with normal birth weight from normally nutritious mothers served as the normal control group. During the lactation period, nutritional intervention was carried out by controlling the number of nursing pups per litter. The lactating pups were divided into a high-nutrition IUGR group (each mother nursed 4 pups) and a normal-nutrition IUGR group (each mother nursed 9 pups per litter). A normal pup control group (each mother nursed 9 pups per litter) was also set up.

[0031] Observation indicators: Body weight was measured daily, and vulvar opening was observed daily starting at PND 21 days. During the hypothalamic sexual development initiation phase (PND 20-30), all groups of pups were anesthetized with isoflurane inhalation, and blood was collected from the heart to separate serum. Hypothalamic tissue, including ARC and AVPe tissue blocks, was immediately isolated under hypothermia, and the expression of hypothalamic sexual development genes Kiss1 and GnRH1 was detected.

[0032] Experimental results: The BPA (0.5 mg / kg / day) intervention group showed signs of precocious puberty, specifically earlier vulvar opening (BPA group: 33.56 ± 1.01, control group: 35.44 ± 1.13, P < 0.05). Figure 1 -c), the ovarian coefficient (ovarian wet weight / body weight) was significantly higher at P35 than that of the control group (BPA group: 0.52±0.13, control group: 0.40±0.07, P<0.05, Figure 1 -b). Gene detection of progressive developmental regulatory genes in hypothalamic tissue: At 25 days of age, the mRNA level of Kiss1 in the ARC region of the hypothalamus was significantly higher in the BPA intervention group (BPA group: 2.19±0.61, control group: 1.16±0.31, P<0.01). Figure 1 The mRNA levels of GnRH1 and GnRH1 (BPA group: 3.66±2.45, control group: 1.44±0.82, P=0.059) were significantly lower than those of the control group. Figure 1 The relative expression level of -e was increased compared with the control group, and there was no significant difference in body weight between the two groups. Figure 1 -a). The results suggest that BPA exposure (0.5 mg / kg / day) during infancy induces precocious puberty in female mice.

[0033] Nutritional intervention model: The weight gain trajectory of the three groups of young mice is shown in the figure. Figure 1Pups in the IUGR high-nutrition group (IUGR-ON) showed significant catch-up growth within 2 weeks of birth, while those in the IUGR normal-nutrition group (IUGR-NN) did not exhibit catch-up growth and their weight tended to be at normal levels. Regarding sexual development, the vulvar opening age of pups in the IUGR-ON group was significantly earlier (32.13±0.77 d), significantly earlier than that in the IUGR-NN group (35.88±0.61 d, P<0.01) and the normal control group (Norm-NN, 36.22±0.22 d, P<0.001), with statistically significant differences (Figure 1-h). Further examination of the expression levels of genes related to sexual development regulation in the arcuate nucleus of the hypothalamus of young mice revealed that at P20, the expression levels of Kiss1 (0.54±0.14) and GnRH1 (0.23±0.06) mRNA in the hypothalamus of the IUGR-ON group were significantly higher than those of the IUGR-NN group (Kiss1: 0.32±0.04, P<0.05; GnRH1: 0.09±0.02, P<0.05, Figure 1-i, j), with statistically significant differences. These results suggest that high nutritional exposure after birth in IUGR mice induces precocious puberty in female mice.

[0034] Example 2: Screening for the IGF2 gene This study used a bisphenol A (BPA)-induced precocious puberty rat model (construction process as described in Example 1). Based on the activation window of the Kiss1 gene in the arcuate nucleus (ARC) of the hypothalamus, transcriptome sequencing was performed on ARC tissue isolated on day 25 (P25) after birth. Total RNA was extracted and sequenced using the Illumina HiSeq 2500 high-throughput sequencing platform to obtain raw sequence data. After quality control by Septk software, the raw sequences were mapped to the rat reference genome (Rnor_6.0 version) using the splicing alignment algorithm of Hisat2 software (version: 2.0.4). Gene-level counting and annotation were performed using the featureCounts function in the Rsubread package based on the Ensembl annotation set. To ensure comparability of expression levels between different genes and samples, read counts were normalized to FPKM values. Subsequently, differentially expressed genes were analyzed using the edgeR software package. The P-value threshold was determined by controlling the false positive rate, and the fold change in differential expression was calculated in conjunction with the FPKM value to comprehensively screen candidate genes. Finally, real-time quantitative polymerase chain reaction (Real-time-PCR) was used to verify the differential expression of candidate genes. Simultaneously, the expression was validated in an IUGR hypertrophic model to determine if similar premature activation expression characteristics existed.

[0035] Experimental results: mRNA transcriptome sequencing analysis was performed on the arcuate nucleus (ARC) of the hypothalamus in mice aged 25 days with bisphenol A (BPA) exposure and in the control group. Volcano plot and heatmap analysis results showed that there were significantly differentially expressed genes in the ARC tissues of the hypothalamus of the two groups of mice. Figure 2 -a, b). Among the genes upregulated in the BPA-treated group, IGF2 ranked among the top 3 differentially expressed genes. Compared with the control group, the expression level of IGF2 gene was significantly increased in the BPA-exposed group: FPKM=137.69±33.62 in the BPA group and FPKM=15.45±0.50 in the control group, and the difference was statistically significant (P=0.0004).

[0036] To further clarify the temporal effect of BPA exposure on IGF2 gene expression, we measured the mRNA expression levels of IGF2 in the hypothalamic ARC and ventral anterior visual cortex (AVPe) tissues of mice in the BPA-exposed group and the control group at five days of age: P15, P20, P25, P30, and P35. The results showed that in the ARC tissue, IGF2 gene expression in the BPA-exposed group was transiently increased, with a significant difference only at PND 25. The IGF2 mRNA expression level in the hypothalamic ARC tissue was significantly higher in the BPA-exposed group than in the control group (BPA group 7.19±1.07 vs. control group 0.73±0.01, P<0.05). Figure 2 Interestingly, IGF2 gene expression in the control group also showed a transient increase at day 30, followed by a gradual decrease at day 35. However, no significant difference in IGF2 gene expression was observed between the BPA-exposed group and the control group in the AVPe region. Figure 2 -d). The results showed that BPA intervention induced premature high expression of ARCIGF2 in the hypothalamus.

[0037] In the IUGR nutritional intervention model (construction process as described in Example 1), the expression level of IGF2 gene in the hypothalamic ARC tissue of the IUGR-ON group was higher than that of the IUGR-NN group at both PND 20 and PND 25 days of age: at PND 20 days of age, the expression level was 3.65±1.37 in the IUGR-ON group and 1.04±0.29 in the IUGR-NN group (P<0.05). Figure 2 -e); at 25 days of age, the IUGR-ON group was 1.65±0.37, and the IUGR-NN group was 0.76±0.15 (P<0.05, Figure 2 The results showed that postnatal hypertrophic intervention in IUGR also induced IGF2 activation in the hypothalamic ARC region.

[0038] Example 3: Exogenous BPA directly induces IGF2 expression in hypothalamic neurons First, we examined whether primary neurons in the hypothalamic arcuate nucleus (ARC) region expressed IGF2. Primary neurons were isolated and cultured from the arcuate nucleus (ARC) region of the hypothalamus of newborn mice. After culturing for 7 days and observing typical neuronal morphology, cells were harvested, and IGF2 mRNA expression was detected. The results showed normal expression of the IGF2 gene. BPA is a weak estrogen; in this experiment, BPA and a relatively equivalent dose of estrogen (17β-E2) were used to intervene in primary hypothalamic neurons. The expression levels of insulin-like growth factor 2 (IGF2), Kiss1, and GnRH1 genes were detected. After primary neurons showed typical morphology, they were stimulated with solutions containing 1 nM BPA or 80 pMol of 17β-E2, respectively. After culturing for another 24 hours, cells were collected, total mRNA was extracted, and gene expression levels were detected using quantitative PCR.

[0039] Experimental results: Compared with the control group, the relative expression level of IGF2 mRNA in the BPA intervention group was significantly increased (BPA group 2.01±0.06, control group 1.17±0.12, P<0.05). Figure 3 The relative expression level of IGF2 mRNA in the 17β-E2 intervention group (4.08±0.23) was not only higher than that in the control group (P<0.001), but also higher than that in the BPA intervention group (P<0.01). It is noteworthy that neither BPA nor 17β-E2 intervention caused significant changes in the expression levels of the Kiss1 and GnRH1 genes within the 24-hour intervention period. Figure 3 The above results indicate that BPA intervention can directly induce the expression of the IGF2 gene in primary hypothalamic ARC neurons, and this effect is speculated to be related to the estrogen-mimicking effect of BPA.

[0040] Example 4: Exogenous IGF2 promotes the activation of key genes in puberty. Detection revealed that the GT1-7 cell line did not express IGF2, but it did express the Kiss1 and GnRH1 genes. This section used the GT1-7 cell line (purchased from Shanghai Jinyuan Biotechnology Co., Ltd.) as a model, and intervened with recombinant IGF2 protein (purchased from R&D Systems (Minneapolis, MN, USA)) to observe the regulatory relationship between IGF2 and the sex development regulatory genes Kiss1 / GnRH1. GT1-7 cells were cultured in a sterile incubator at 37℃ and 5% CO2, with the medium (DMEM / F-12) containing 10% fetal bovine serum and 1% penicillin / streptomycin) changed every 48 hours. When the GT1-7 cells reached 80%–90% confluence, they were passaged. After three passages, intervention was performed when the cells were in the logarithmic growth phase. Cells were stimulated with recombinant IGF2 protein at doses of 25 ng / ml, 50 ng / ml, 100 ng / ml, and 200 ng / ml, and cultured for 24 h. Changes in the mRNA and protein expression levels of GnRH1 and Kiss1 were then detected.

[0041] Experimental results: At an IGF2 intervention concentration of 100 ng / ml, the mRNA expression of Kiss1 gene (intervention group 2.91±0.97 vs. control group 1.04±0.33, P<0.01) and GnRH1 gene (intervention group 2.34±0.38 vs. control group 1.02±0.26, P<0.001) was increased (see [link to relevant documentation]). Figure 4 -a), protein levels were significantly elevated ( Figure 4 -b). When the concentration was increased to 200 ng / ml, the mRNA and protein expression levels of the two genes did not change significantly compared with the control group. Figure 4 -a,b).

[0042] Example 5: Conditional paternal knockdown of IGF2 in the hypothalamus blocks increased IGF2 in ARC, leading to delayed precocious puberty. Using Cre-LoxP conditional gene editing technology, selective blocking of paternal IGF2 activation in the hypothalamus was achieved. Specifically, a mouse model carrying the IGF2 flox site was constructed, and a hypothalamus-specific Cre recombinase expression system was used. Under Cre enzyme catalysis, homologous recombination at the loxP site was mediated, specifically knocking out the paternal IGF2 gene in the hypothalamus, thereby precisely blocking its activation and expression. This allows for spatially specific regulation of gene knockout in the hypothalamus. This section describes the construction of C57BL / 6 hypothalamic paternal IGF2 knockdown mice (IGF2 / P-) and maternal knockdown mice (IGF2 / M-). Breeding strategy: Cre heterozygous male mice (Cre... + / - × Flox heterozygous female mouse (Flox + / - ), Flox heterozygous male mouse (Flox+ / - × Cre heterozygous female mouse (Cre) + / - The experimental group consisted of paternal knockdown offspring (IGF2^flox p- / m+) and maternal knockdown offspring (IGF2^flox p+ / m-). Other littermate female offspring, including single Cre heterozygous males (Cre...), were also included. + / - ), single Flox heterozygous female mice (Flox + / - Wild-type female mice were used as negative controls to detect the expression of Kiss1 and GnRH in the arcuate nucleus of the hypothalamus and to observe the timing of sexual development initiation.

[0043] Experimental results: In paternally knocked-down mice, IGF2 mRNA expression in the hypothalamic ARC region was 0.71±0.47 vs. 2.20±1.38, P=0.07. Figure 5 -a), 25-day-old (IGF2 / P- 0.54±0.38 Vs. 1.04±0.85, P=0.25) Figure 5 -a) Compared with the control group, there was a decreasing trend. At 25 days of age, the Kiss1 gene (IGF2 / P) in the hypothalamic ARC region of paternally knocked-down mice showed a decreasing trend. (P-0.70±0.08 vs. control 1.29±0.36, P<0.01) Figure 5 -b) and GnRH gene (IGF2 / P- 0.67±0.21 Vs. control 1.35±0.65, P<0.05, Figure 5 -c) Decreased mRNA expression and delayed onset of sexual development, manifested as delayed vulvar opening (IGF2 / P- 31.3±2.26 Vs. control 28.4±2.17, P<0.01, Figure 5 -d). However, no significant differences in IGF2, Kiss1, and GnRH1 gene mRNA were detected in the hypothalamic ARC region of maternal knockout mice. Figure 5 -e,f,g). Experimental results show that paternal IGF2 plays a specific regulatory role in the hypothalamic ARC region during the initiation of sexual development.

[0044] Example 6: Regulatory effect of paternal knockdown of IGF2 on a BPA precocious puberty model Paternal knockout mice and their littermates were selected as the study subjects. BPA (0.5 mg / kg / day) was administered intramuscularly for 1-10 days. During the hypothalamic sexual development initiation phase (PND 20-30), the expression of the hypothalamic sexual development gene IGF2 and subsequent vulvar opening were detected.

[0045] Experimental results: At 20 days of age, the IGF2 gene count in paternally hypothalamic IGF2 knockdown mice was lower than that in the control group (IGF2 / P- group 2.70±1.90 vs. control group 5.53±0.96, P<0.05). Figure 6 -a), by 25 days of age, IGF2 in the control group was significantly lower than that in the 20-day-old group, while it was relatively higher in the paternal IGF2 knockdown group (IGF2 / P-group 1.59±0.34 Vs. control group 0.72±0.16, P<0.001, Figure 6 -a), IGF2 knockdown delayed vaginal opening time (IGF2 / P-group 31.71±1.80 vs. control group 28.43±1.99, P<0.01, Figure 6 -b). The above experimental results clearly demonstrate that paternal IGF2 gene knockdown can effectively inhibit BPA-induced precocious puberty in female baby mice.

[0046] Example 7: Compound Traditional Chinese Medicine Dabu Yin Wan Inhibits IGF2 Increase in a Hypertrophic Model An IUGR (infantile urinary tract infection) model of precocious puberty was treated with Dabuyin Pills from 18 to 25 days of age. The medication was administered via gavage, and the dosage was calculated using the Meeh-Rubne formula based on the animal's body surface area, with a dose of 3.5 g / kg / day. During the hypothalamic sexual development initiation phase (PND 20-30), the expression of the hypothalamic sexual development gene IGF2 and subsequent vulvar opening were measured.

[0047] Experimental results: At 20 days of age, the IGF2 gene count in the Dabuyin Pill intervention group was lower than that in the control group (Dabuyin Pill group 0.60±0.18 vs. control group 1.92±0.92, P<0.01). Figure 7 -a), the time to vaginal opening was delayed after intervention with Dabuyin Pill (30.50±1.31 in Dabuyin Pill group vs. 29.00±1.41 in control group, P<0.05). Figure 7 -b). The above experimental results clearly show that the intervention of Dabuyin Pill can effectively inhibit the premature activation of IGF2, thereby inhibiting precocious puberty in female mice with high nutrition-induced intrauterine growth restriction (IUGR).

[0048] Example 8: Diagnostic efficacy of hypothalamic paternal IGF2 as a biomarker To evaluate the application value of hypothalamic IGF2 expression as a diagnostic marker for precocious puberty and an indicator for monitoring treatment efficacy, this invention established two types of rat intervention models: (1) Stimulus model On day 20 after birth (PND 20, the key time point for early changes in precocious puberty), the arcuate nucleus of the hypothalamus was collected (A: BPA exposure model or high nutrition diet induction model was constructed according to the method described in Example 1 (stimulating IGF2 increase to simulate the occurrence of precocious puberty, N=12, 6 cases in each of BPA exposure model or high nutrition diet induction model) and normal control group (N=12, 6 cases in each of BPA control group and high nutrition control group).

[0049] (2) Inhibition model: Paternal IGF2 gene knockdown model or traditional Chinese medicine intervention model (inhibiting IGF2 expression, simulating precocious puberty treatment, N=11, including 6 cases of paternal IGF2 gene knockdown model and 5 cases of traditional Chinese medicine intervention model) and normal control group (N=11, including 6 cases of non-knockdown control group from the same littermate and 5 cases of traditional Chinese medicine control group (administered with an equal volume of physiological saline at the same time)).

[0050] The RC tissue was used to detect the expression level of the IGF2 gene, and the early opening of the vulva was used as the phenotype of precocious puberty.

[0051] Based on the above model, ROC curve analysis was used to systematically evaluate the diagnostic efficacy and therapeutic monitoring value of hypothalamic IGF2 expression for precocious puberty. The results showed that the area under the receiver operating characteristic (AUC) curve for the IGF2 stimulation test in predicting precocious puberty was 0.91 (95% confidence interval: 0.78–1.00, standard error 0.065, P<0.001). Figure 8 a) The high diagnostic sensitivity and specificity indicate that the expression level of hypothalamus IGF2 has excellent predictive diagnostic value for precocious puberty; the AUC of the IGF2 inhibition test for monitoring the treatment effect was 0.83 (95% confidence interval: 0.65–1.00, standard error 0.090, P<0.001). Figure 8 (b) indicates that the dynamic changes in IGF2 expression have reliable monitoring and evaluation value for efficacy assessment.

[0052] The above results confirm that IGF2 gene expression in the hypothalamic ARC region can serve as an early diagnostic biomarker and efficacy monitoring indicator for precocious puberty, providing objective molecular evidence for clinical early warning, early intervention, and individualized treatment of precocious puberty. Notably, this invention is the first to identify day 20 after birth as a critical early time point for changes in hypothalamic IGF2 expression, earlier than the traditional onset of precocious puberty phenotypes, providing a time window advantage for ultra-early early warning of precocious puberty. Furthermore, the dual-model validation system (stimulation / inhibition bidirectional validation) established in this invention ensures the robustness of IGF2 as a biomarker and its feasibility for clinical translation.

[0053] In summary, this application relates to the regulatory mechanism and intervention technology of precocious puberty in female young mice. Its core lies in revealing the key mediating role of hypothalamic paternal insulin-like growth factor 2 (IGF2) in precocious puberty induced by environmental and nutritional factors, clarifying the application value of this factor as an early warning biomarker for the initiation of puberty, and providing targeted intervention programs, effectively filling the gap in the existing research and intervention technology of precocious puberty mechanism.

[0054] This technology clarifies for the first time that exposure to the environmental endocrine disruptor bisphenol A (BPA) or high nutrient intake can significantly advance the activation phase of IGF2 in the arcuate nucleus (ARC) region of the hypothalamus. This abnormal activation of IGF2 further activates the abnormal expression of the Kiss1 and GnRH1 genes in the hypothalamus, disrupting the normal regulatory rhythm of sexual development in female pups and ultimately inducing their precocious puberty phenotype. This elucidates the core molecular regulatory pathway by which BPA and high nutrient factors induce precocious puberty in female pups.

[0055] Based on the aforementioned clear molecular mechanisms, this technology proposes two efficient and targeted intervention strategies for precocious puberty: one is to selectively block the activation of paternal IGF2 through the hypothalamus, and the other is to use drugs (Dabuyin Wan) to inhibit the activity of IGF2. Experimental verification shows that both intervention methods can effectively delay the onset of sexual development in female pups and can completely block the precocious puberty phenotype induced by BPA exposure and high nutritional environment, providing a new technical approach for the clinical intervention of precocious puberty.

[0056] Meanwhile, this technology clarifies that the activation of paternal IGF2 in the hypothalamus can serve as an early warning biomarker for the initiation of puberty. Its activation state can accurately reflect the influence of environmental and nutritional factors on the hypothalamic sexual development center, and can be used to predict the risk of precocious puberty in female rat pups in the early stage, providing an important molecular target for early screening, intervention and prognostic assessment of precocious puberty.

[0057] The innovation of this technology lies in the fact that it establishes a link between paternal IGF2 and BPA and high nutrition-induced precocious puberty for the first time, clarifying its core role in mediating the occurrence of precocious puberty and its early warning value, breaking through the limitations of traditional precocious puberty mechanism research. The proposed intervention program is both specific and practical. Among them, the application of Dabuyin Pill realizes the precise targeting of traditional Chinese medicine in the intervention of precocious puberty. Compared with existing intervention technologies, it has significant advantages such as fewer side effects, strong targeting, and high operability.

[0058] This technology can be widely applied to the study of the mechanism of precocious puberty in female rat pups, early warning, clinical intervention and related drug development. It provides a new theoretical basis and technical support for the prevention and control of environment- and nutrition-related precocious puberty, and has important scientific research value, clinical application value and industrial transformation potential.

[0059] Given that IGF2, as an epigenetic effector molecule, acts as a "sensor" for the interaction between environmental and genetic factors, this invention further relates to a kit for detecting the IGF2 gene imprinting status and expression level. This kit can be used to screen or assess high-risk foods and environmental factors for inducing precocious puberty, enabling rapid identification of exogenous exposure factors that pose a risk of precocious puberty. This further expands the application scenarios of this technology, providing a convenient and efficient detection tool for the source control of precocious puberty, and significantly enhancing the practical application value and industrial transformation feasibility of this invention. This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. Application of reagents for detecting the imprinting status and / or expression level of the hypothalamus paternal insulin-like growth factor 2 gene in the preparation of precocious puberty screening products.

2. The application according to claim 1, characterized in that, The precocious puberty mentioned includes bisphenol A-induced precocious puberty and precocious puberty caused by high nutrition.

3. The application according to claim 1, characterized in that, The reagents include primer pairs for detecting hypothalamic paternal insulin-like growth factor 2 mRNA expression and / or antibodies for detecting hypothalamic paternal insulin-like growth factor 2 protein expression.

4. Application of substances that inhibit the activation of hypothalamic paternal insulin-like growth factor 2 or reduce its expression level in the preparation of drugs for the prevention and treatment of precocious puberty.

5. The application according to claim 4, characterized in that, The precocious puberty mentioned includes bisphenol A-induced precocious puberty and precocious puberty caused by high nutrition.

6. The application according to claim 4, characterized in that, The substances that inhibit the activation of hypothalamic paternal insulin-like growth factor 2 or reduce its expression level include Dabuyin Pill and gene-editing agents and antibody preparations that specifically block the activation of hypothalamic paternal insulin-like growth factor 2.

7. A kit for screening precocious puberty, characterized in that, The kit contains reagents for detecting the imprinting status and / or expression level of the hypothalamus paternal insulin-like growth factor 2 gene.

8. The reagent kit according to claim 7, characterized in that, The reagents include primer pairs for detecting hypothalamic paternal insulin-like growth factor 2 mRNA expression and / or antibodies for detecting hypothalamic paternal insulin-like growth factor 2 protein expression.

9. The reagent kit according to claim 8, characterized in that, The primer pair includes: The forward primer shown in SEQ ID NO:5 and the reverse primer shown in SEQ ID NO:6; or The forward primer shown in SEQ ID NO:13 and the reverse primer shown in SEQ ID NO:

14.

10. Application of hypothalamic paternal insulin-like growth factor 2 as a target in the preparation of products for the detection of exogenous environmental substances or nutrients.

Images