Use of a substance for the manufacture of a medicament for the treatment of precocious puberty in an individual and uses thereof

Abstract

The application discloses a kind of substance for preparing the use of reagent for treating individual precocity and its application, by large-scale sample analysis work, the application first discovers a kind of biomarker for predicting individual precocity, i.e. 6-position hydroxyl bile acid, and verifies its effectiveness as biomarker;Provide a kind of method for treating individual precocity, i.e. reduce the concentration of 6-position hydroxyl bile acid, for example, by reducing the concentration of 6-position hydroxyl bile acid cholestyramine to treat precocity;In-depth study of related mechanism, the correlation between TGR5 receptor and precocity is found, i.e. inhibit TGR5 receptor or prevent TGR5 receptor from being activated, prevent GnRH expression from being promoted, to treat individual precocity;Provide a kind of reagent composition for treating individual precocity, use cholestyramine or other reagents for reducing the concentration of 6-position hydroxyl bile acid, reagents for promoting 6-position hydroxyl bile acid metabolism, TGR5 receptor inhibitor etc. are intervened, to treat individual precocity.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to the use and application of a substance in the preparation of a reagent for treating individual precocious puberty. Background Technology

[0002] Central precocious puberty is a disorder of puberty development, characterized by the appearance of secondary sexual characteristics in boys before age 9 and in girls before age 7.5, manifested as epiphyseal closure, early menstruation, and shorter than average height in adulthood. Furthermore, studies have shown that girls with precocious puberty have a significantly increased risk of developing polycystic ovary syndrome, breast cancer, cardiovascular disease, and obesity-related metabolic disorders in adulthood. Simultaneously, the premature development of sexual characteristics coupled with intellectual and sexual immaturity can lead to psychological and physiological disorders, causing serious negative impacts on families and society. Globally, the incidence of precocious puberty in children is increasing year by year, and the age of onset is getting earlier, seriously affecting the physical and mental health of children and adolescents.

[0003] Precocious puberty can be divided into central precocious puberty and peripheral precocious puberty. Central precocious puberty, also known as GnRH-dependent precocious puberty (true precocious puberty), is caused by the hypothalamus prematurely increasing the secretion and release of gonadotropin-releasing hormone (GnRH), prematurely activating the gonadal axis, leading to gonadal development and sex hormone secretion, resulting in the development of internal and external genitalia and the appearance of secondary sexual characteristics. Peripheral precocious puberty, clinically also called pseudoprecocious puberty, is mainly caused by a high-fat diet or tumors in the hypothalamus, ovaries, adrenal cortex, etc., leading to abnormal secretion of sex hormones, resulting in the appearance of secondary sexual characteristics but without gonadal development. Peripheral precocious puberty (pseudoprecocious puberty) is often caused by specific reasons leading to abnormal secretion of sex hormones and is a special symptom in the process of childhood development; pseudoprecocious puberty will naturally subside.

[0004] Central precocious puberty (true precocious puberty) requires prediction and intervention. Central precocious puberty is a developmental disorder that can lead to various problems such as poor physical development and psychological issues. Therefore, predicting or diagnosing individual precocious puberty and treating precocious puberty both refer to central precocious puberty (true precocious puberty).

[0005] The pathogenesis of idiopathic central precocious puberty, which accounts for the largest proportion of central precocious puberty, remains unclear, posing significant challenges to its prevention and treatment. Currently, clinical diagnosis relies primarily on a combination of indicators, including the early appearance of secondary sexual characteristics, imaging findings, and molecular diagnostic markers of serum sex hormones (such as follicle-stimulating hormone, luteinizing hormone, prolactin, estradiol, testosterone, and progesterone), as well as the GnRH stimulation test. However, these indicators only appear after the onset of precocious puberty and cannot serve as screening markers for early-onset risk factors. Current treatments for precocious puberty include drug therapy and traditional Chinese medicine. For drug therapy, first-line drugs are GnRH agonists, such as triptorelin and leuprorelin, which delay sexual development by inhibiting the pituitary-gonadal axis. Traditional Chinese medicine is also an option, but it must be used under the guidance of a doctor, such as Danzhi Xiaoyao Wan and Dabu Yin Wan. The aforementioned drugs, while effective, cannot guarantee complete therapeutic efficacy and have significant side effects. Therefore, there is an urgent need to develop a novel method for predicting whether an individual will develop precocious puberty, as well as a method or drug for intervening in and treating precocious puberty. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, this invention provides the use and application of a substance in preparing a reagent for treating individual precocious puberty. It also discovers a biomarker for predicting individual precocious puberty, namely 6-hydroxycholic acid. By detecting the concentration of 6-hydroxycholic acid in an individual's serum or hypothalamic sample, it is possible to determine whether an individual is at risk of precocious puberty and to carry out prevention or treatment in a timely manner.

[0007] In this invention, the discovery set and validation set use two completely different batches of samples. The discovery set consists of known samples, from which the inventors screen for biomarkers. The validation set is used solely to verify the diagnostic efficacy of the biomarker combination of this invention. Furthermore, this invention employs multiple different validation sets for validation testing. The results show that the optimal biomarker combination screened in this invention has a predictive performance of approximately 85% for individuals with precocious puberty.

[0008] As shown in formula (1), bile acids can be divided into three categories based on the position of the hydroxyl groups on the carbon chains. Bile acids (CA species) have hydroxyl groups linked to the 3rd and 12th carbon atoms, chenodeoxycholic acids (CDCA species) have hydroxyl groups linked to the 3rd carbon atom, and mouse bile acids (MCA species) have hydroxyl groups linked to the 3rd and 6th carbon atoms; all three types of bile acids may optionally have a hydroxyl group linked to the 7th carbon atom.

[0009]

[0010] 6-hydroxy bile acids are bile acids with a hydroxyl group linked at the 6-position. For example, in formula (1), mouse bile acids belong to 6-hydroxy bile acids, while the other two types of bile acids do not. It should be noted that the above classification does not cover all bile acids. In addition to the above three types, there are many bile acids with a hydroxyl group linked at the 6-position, which belong to 6-hydroxy bile acids.

[0011] First, this invention selected a subset of bile acids and found that only mouse bile acid could diagnose precocious puberty. Reducing the content of mouse bile acid could effectively treat precocious puberty. Structurally, we believe that bile acids with a 6-hydroxyl group may all have the function of diagnosing precocious puberty and could serve as targets for intervention and treatment. From a theoretical perspective, this invention hypothesizes that the 6-hydroxyl substitution of bile acids is associated with precocious puberty in biochemistry and metabolic pathways. This may be because the 6-hydroxyl substitution affects the physicochemical properties of the compound, potentially influencing its binding to receptor proteins. Further investigation is needed to determine the specifics.

[0012] 6-hydroxy bile acids include a series of bile acids, such as murine cholic acid and conjugated murine cholic acid, including α-murine cholic acid (αMCA), β-murine cholic acid (βMCA), γ-murine cholic acid (γMCA), ω-murine cholic acid (ωMCA), and also porcine deoxycholic acid (HDCA). Murine cholic acid can combine with different amino acids to form conjugated murine cholic acid, such as taurine-α-murine cholic acid (TαMCA), taurine-β-murine cholic acid (TβMCA), taurine-γ-murine cholic acid (TγMCA), taurine-ω-murine cholic acid (TωMCA), glycine-γ-murine cholic acid (GγMCA), etc., as well as conjugated bile acids formed by their combination with amino acids, such as taurine-porcine deoxycholic acid (THDCA), etc.

[0013] The above are some of the most common 6-hydroxy bile acids, which are merely examples. Other bile acids with a hydroxyl group linked at the 6-position, such as 6-ketolithocholic acid (6-ketoLCA), are also within the scope of protection of this invention. Because they have the same partial chemical structure, they have similar functions and metabolic pathways in the body, and have the same diagnostic function for precocious puberty. They can also be used as targets for intervention and treatment of precocious puberty.

[0014] On one hand, the present invention provides the use of a biomarker for preparing a reagent to predict individual precocious puberty, said biomarker comprising any one or more 6-hydroxybile acids.

[0015] In some embodiments, the biomarker includes any one or more of αMCA, βMCA, γMCA, ωMCA, TαMCA, TβMCA, TγMCA, TωMCA, GγMCA, HDCA, and THDCA.

[0016] Furthermore, the reagent is used to detect biomarkers in an individual's serum or hypothalamic sample.

[0017] Furthermore, the reagent is used to detect the presence, relative abundance, or concentration of biomarkers in a sample.

[0018] On the other hand, the present invention provides a combination of biomarkers for predicting individual precocious puberty, the combination of biomarkers including any one or more of αMCA, βMCA, γMCA, ωMCA, TαMCA, TβMCA, TγMCA, TωMCA, GγMCA, HDCA, and THDCA.

[0019] In summary, 6-hydroxybile acids are a collective term for a series of bile acids. Any single 6-hydroxybile acid can predict precocious puberty in an individual. Combining multiple 6-hydroxybile acids may improve the sensitivity and specificity of predicting precocious puberty to a certain extent. In this invention, one or more 6-hydroxybile acids were selected to train a logistic regression model on the training set. The model was then validated on the validation set, and it was found that one or more 6-hydroxybile acids have the function of predicting precocious puberty in an individual.

[0020] Initially, this invention tested serum samples from 241 female subjects (145 patients with central precocious puberty and 96 controls), and found that the concentration of 6-hydroxycholic acid was significantly higher in patients with precocious puberty than in controls. Further studies showed that the concentration of 6-hydroxycholic acid was significantly positively correlated with the levels of sex development-related hormones and the Tanner stage of breast development, indicating that 6-hydroxycholic acid can serve as a biomarker for predicting individual precocious puberty.

[0021] Sexual development-related hormone levels include gonadotropin-releasing hormone (GnRH). This invention found that intervention with cholestyramine (CAS: 11041-12-6), a high molecular weight quaternary ammonium anion exchange resin, can bind to bile acids, thereby inhibiting their absorption. Testing showed that the concentration of 6-hydroxy bile acids in the serum and hypothalamus of individuals after cholestyramine intervention decreased, while the concentrations of other bile acid compounds remained unaffected. Simultaneously, GnRH expression was inhibited. Studies showed a significant correlation between 6-hydroxy bile acid and GnRH expression, indicating that 6-hydroxy bile acid concentration is associated with precocious puberty. Furthermore, taurine-conjugated α-mouse bile acid (TαMCA) and Tanner stage of the mammary gland were significantly correlated. In conclusion, 6-hydroxy bile acid can serve as a biomarker for predicting precocious puberty in individuals.

[0022] The concentration of 6-hydroxy bile acid was used to predict individual precocious puberty. Predictive precocious puberty was performed on the validation set. For the specific AUC, please refer to the detailed implementation section. The AUC value of TαMCA for predicting individual precocious puberty was 0.805.

[0023] In another aspect, the present invention provides a system for predicting individual precocious puberty, the system including a data analysis module for analyzing the detection values ​​of biomarkers; the biomarkers include any one or more 6-hydroxycholesterol acids.

[0024] In some embodiments, the combination of biomarkers includes any one or more of αMCA, βMCA, γMCA, ωMCA, TαMCA, TβMCA, TγMCA, TωMCA, GγMCA, HDCA, and THDCA.

[0025] Furthermore, the data analysis module calculates the predicted value of individual precocious puberty by substituting the detection value of biomarkers into the regression equation, thereby assessing the risk of individual precocious puberty.

[0026] Furthermore, the regression equation is Logit(P) = -3.122 + 0.079 * TαMCA + 0.022 * γMCA + 0.048 * TγMCA - 0.007 * GγMCA - 0.044 * HDCA, or Logit(P) = -1.405 + 0.057 * TαMCA, or Logit(P) = -1.729 + 0.043 * TαMCA + 7.841 * LH.

[0027] In some embodiments, the regression equation for predicting precocious puberty using taurine-bound α-mouse bile acid alone is: Logit(P) = -1.405 + 0.057 * TαMCA, where P represents the probability of precocious puberty, 0.057 represents the regression coefficient of taurine-bound α-mouse bile acid, TαMCA represents the measured concentration of taurine-bound α-mouse bile acid, and -1.405 is a constant term. Logit(P) is the value after regression correction; when the P value > -0.0706, the individual is predicted to be a patient with precocious puberty. Simultaneously, this invention also detects the concentration of taurine-bound α-mouse bile acid (TαMCA) in serum samples, and substitutes the detected concentration into the above regression equation. Using this model, the accurate prediction rate for ICPP is 73.7%. Compared to actual clinical diagnostic results: among 149 female subjects (68 patients with central precocious puberty and 81 controls), the diagnostic sensitivity of this invention was 77.94%, the specificity was 80.24%, the positive predictive value was 76.81%, and the negative predictive value was 81.25%.

[0028] The regression equation for predicting precocious puberty using taurine-bound α-mouse bile acid and baseline LH levels is: Logit(P) = -1.729 + 0.043 * TαMCA + 7.841 * LH, where 0.057 represents the regression coefficient of taurine-bound α-mouse bile acid, TαMCA represents the measured concentration of taurine-bound α-mouse bile acid, 7.841 represents the regression coefficient of luteinizing hormone (LH), LH represents the measured concentration of LH, -1.729 is a constant term, and Logit(P) is the value after regression correction. A P-value > 0.902915 indicates that the individual may have precocious puberty. Furthermore, this invention also detected the concentrations of taurine-bound α-mouse bile acid (TαMCA) and LH in serum samples, and substituted the detected concentrations into the above regression equation. The accurate prediction rate of ICPP using this model was 82.4%. Compared to actual clinical diagnostic results: among 149 female subjects (68 patients with central precocious puberty and 81 controls), the diagnostic sensitivity of this invention was 79.41%, the specificity was 81.48%, the positive predictive value was 78.26%, and the negative predictive value was 82.5%.

[0029] The regression equation for predicting precocious puberty using the combination of the above five biomarkers is: Logit(P) = -3.122 + 0.079 * TαMCA + 0.022 * γMCA + 0.048 * TγMCA - 0.007 * GγMCA - 0.044 * HDCA; in the regression equation, P represents the probability of precocious puberty, and 0.079, 0.022, 0.048, 0.007, and 0.044 represent taurine-bound α-mouse cholic acid, γ-mouse cholic acid, taurine-γ-mouse cholic acid, and taurine-γ-mouse cholic acid, respectively. The regression coefficients for taurine-bound α-mouse cholic acid, glycine-γ-mouse cholic acid, and deoxycholic acid were calculated. TαMCA represents the measured concentration of taurine-bound α-mouse cholic acid, γMCA represents the measured concentration of γ-mouse cholic acid, TγMCA represents the measured concentration of taurine-γ-mouse cholic acid, GγMCA represents the measured concentration of glycine-γ-mouse cholic acid, HDCA represents the measured concentration of deoxycholic acid, -3.122 is a constant term, and Logit(P) is the value after regression correction. A P-value > 0.30714 indicates that the individual may be a patient with precocious puberty. In this invention, taurine-bound α-mouse cholic acid, γ-mouse cholic acid, taurine-γ-mouse cholic acid, and glycine-γ-mouse cholic acid in serum samples were also detected, and the detected concentrations were substituted into the above regression equations. The accurate predictive rate of ICPP using this model was 78.6%. Compared to actual clinical diagnostic results: among 149 female subjects (68 patients with central precocious puberty and 81 controls), the diagnostic sensitivity of this invention was 80.88%, the specificity was 85.19%, the positive predictive value was 82.09%, and the negative predictive value was 84.15%.

[0030] Therefore, it can be seen that the prediction results in the validation set of this invention are highly consistent with the actual clinical diagnosis results, and the accuracy, sensitivity, specificity and other indicators are good, and the AUC value is high. In particular, the performance of the combined diagnosis of taurine-bound α-mouse cholic acid and baseline LH value is better than that of the single diagnosis. Therefore, the diagnostic model constructed by the biomarkers of this invention has good predictive performance and accuracy, and has the best diagnostic efficacy.

[0031] Furthermore, the system also includes a data storage module, a data input interface, and a data output interface; the data storage module is used to store the detection values ​​of biomarkers; the data input interface is used to input the detection values ​​of biomarkers, and the data output interface is used to output the prediction results.

[0032] On the other hand, the present invention provides the use of a substance for preparing a reagent for treating individual precocious puberty, said substance reducing the concentration of any one or more 6-hydroxybile acids in an individual.

[0033] Based on the structure-activity relationship of compounds, the 6-hydroxy substitution of bile acids is related to precocious puberty. Therefore, reducing the concentration of any one or more 6-hydroxy bile acids in an individual can be considered as inhibiting the physiological pathways of precocious puberty to varying degrees, and can treat precocious puberty to a certain extent.

[0034] Lowering the concentration of 6-hydroxybile acid can be approached from several angles: 1. Reduce the absorption of 6-hydroxybile acid, such as by using cholestyramine to bind to 6-hydroxybile acid, increasing the excretion of 6-hydroxybile acid, and reducing its absorption; 2. Promote the metabolism of 6-hydroxybile acid and activate its related metabolic pathways.

[0035] Furthermore, the 6-hydroxy bile acid includes any one or more of αMCA, βMCA, γMCA, ωMCA, TαMCA, TβMCA, TγMCA, TωMCA, GγMCA, HDCA, and THDCA.

[0036] Furthermore, the substance binds to 6-hydroxybile acid, thereby inhibiting the absorption of 6-hydroxybile acid.

[0037] Furthermore, the substance includes cholestyramine.

[0038] This invention demonstrates that cholestyramine (CAS: 11041-12-6) can reduce the concentration of 6-hydroxybile acid in an individual's body (serum and hypothalamus), while the concentration of other bile acid compounds is unaffected. At the same time, the expression of gonadotropin-releasing hormone (GnRH) is inhibited. In terms of mechanism of action, cholestyramine can bind to 6-hydroxybile acid, thereby inhibiting the absorption of 6-hydroxybile acid by the individual and reducing the concentration of 6-hydroxybile acid in the individual's body, thereby achieving the purpose of treating precocious puberty.

[0039] Furthermore, the substance promotes the degradation of 6-hydroxybile acid.

[0040] Furthermore, the substance reduces the concentration of 6-hydroxybile acid in an individual's body, thereby reducing the binding of 6-hydroxybile acid to its receptor.

[0041] Furthermore, the binding receptor includes the TGR5 receptor.

[0042] On the other hand, the present invention provides the use of a substance that inhibits the TGR5 receptor for the preparation of a reagent for treating individual precocious puberty.

[0043] The TGR5 receptor, short for G protein-coupled bile acid receptor 1, also known as M-BAR or Gpbar1, is a member of the transmembrane G protein-coupled receptor family. It binds to extracellular ligands, transducing extracellular signals to downstream intracellular cascades, playing a crucial role in metabolism, inflammatory responses, cancer intervention, and treatment through various signal transduction pathways. TGR5 is widely distributed in the body, expressed in organs such as the gallbladder, ileum, colon, brown adipose tissue, spleen, lungs, skeletal muscle, and pancreas. Furthermore, TGR5 is not only a bile acid receptor but can also be activated by other anabolic agonists, mediating a series of signaling pathways and participating in physiological and pathological processes.

[0044] Initially, this invention discovered that using TGR5 receptor agonists could induce precocious puberty in mice. Combined with the above research, precocious puberty was not significantly associated with other bile acids, but was highly correlated with 6-hydroxy bile acids. Therefore, the TGR5 receptor may be one of the pathways of action of 6-hydroxy bile acids. Thus, this invention knocked out the TGR5 gene in mice and found that mice that did not express the TGR5 receptor, under a precocious puberty-induced feeding method, had a significantly later vaginal opening time than the control group, proving that inhibiting the TGR5 receptor can treat individual precocious puberty.

[0045] On the other hand, the present invention provides a product for treating individual precocious puberty, the product containing a substance that reduces the concentration of 6-hydroxybile acid in the individual's body.

[0046] In some embodiments, the 6-hydroxy bile acid includes any one or more of αMCA, βMCA, γMCA, ωMCA, TαMCA, TβMCA, TγMCA, TωMCA, GγMCA, HDCA, and THDCA.

[0047] Furthermore, the product includes cholestyramine.

[0048] The beneficial effects of this invention are as follows:

[0049] 1. Through large-scale sample analysis, this invention has for the first time discovered a biomarker for predicting individual precocious puberty, namely 6-hydroxycholic acid, and verified its effectiveness as a biomarker.

[0050] 2. A method for predicting precocious puberty in individuals is provided, which only requires measuring the concentration of 6-hydroxybile acid in an individual's serum or hypothalamus to determine the risk of precocious puberty, with good sensitivity and specificity;

[0051] 3. A method for treating individual precocious puberty is provided, namely, reducing the concentration of 6-hydroxy bile acid in the individual's body. For example, cholestyramine can reduce the absorption of mouse bile acid and prevent the promotion of GnRH expression, thereby treating individual precocious puberty.

[0052] 4. In-depth research on the relevant mechanisms of biomarkers has revealed the correlation between TGR5 receptor and precocious puberty, opening up new methods for treating individual precocious puberty, namely, inhibiting TGR5 receptor or preventing TGR5 receptor from being activated, preventing the promotion of GnRH expression, thereby treating individual precocious puberty.

[0053] 5. A reagent composition for treating individual precocious puberty is provided. Based on the above mechanism, it has been found that cholestyramine or other reagents that inhibit the absorption of 6-hydroxybile acids, reagents that promote the metabolism of 6-hydroxybile acids, TGR5 receptor inhibitors, etc., can be used for intervention to treat individual precocious puberty. Attached Figure Description

[0054] Figure 1 A statistical graph showing the total serum mouse bile acid (MCA) concentrations in patients with central precocious puberty (ICPP) and controls (Non-ICPP) among the subjects;

[0055] Figure 2 A statistical graph showing the concentrations of various types of 6-hydroxy bile acids in the serum of patients with central precocious puberty and controls;

[0056] Figure 3 Spearman correlation heatmap of serum bile acids and gonadal axis initiation-related hormone indicators;

[0057] Figure 4 Spearman correlation heatmap of serum 6-hydroxybile acids and gonadal axis initiation-related hormone indicators;

[0058] Figure 5 Scatter plot of Spearman correlation analysis between serum taurine-bound α-mouse bile acid and mammary Tanner staging;

[0059] Figure 6 ROC curve analysis for predicting precocious puberty using serum 6-hydroxybile acid;

[0060] Figure 7 ROC curve analysis of serum taurine-bound α-mouse bile acid and baseline LH value for predicting precocious puberty;

[0061] Figure 8 ROC curve analysis of serum 6-hydroxybile acid as a predictor of precocious puberty in a validation cohort for central precocious puberty;

[0062] Figure 9 Flowchart of animal experiments for a mouse model of precocious puberty;

[0063] Figure 10 A statistical graph showing the vaginal opening time and proportion of mice in the control group and the precocious puberty mouse model;

[0064] Figure 11 Results of Western blotting to detect gonadotropin-releasing hormone (GnRH) expression in the hypothalamus of mice;

[0065] Figure 12 A statistical graph showing the expression level of GnRH in the hypothalamus of mice as detected by Western blotting;

[0066] Figure 13 A statistical graph showing the bile acid concentrations in serum samples from precocious female mice and the control group;

[0067] Figure 14 A statistical graph showing the bile acid concentration in hypothalamic samples from precocious female mice and the control group;

[0068] Figure 15 Flowchart of animal experiments for the treatment of precocious puberty;

[0069] Figure 16 A statistical chart showing the vaginal opening time and proportion of mice in animal experiments for the treatment of precocious puberty.

[0070] Figure 17 UPLC-MS / MS was used to detect bile acid profiles in serum samples from precocious puberty mice and mice in the cholestyramine intervention group;

[0071] Figure 18 The bile acid profiles of hypothalamic samples from precocious puberty mice and mice in the cholestyramine intervention group were detected by UPLC-MS / MS.

[0072] Figure 19 A statistical graph showing the concentration of 6-hydroxybile acid in serum samples from precocious puberty mice and mice in the cholestyramine intervention group;

[0073] Figure 20 Statistical graph of different types of mouse bile acids in hypothalamic samples from precocious puberty mice and mice in the cholestyramine intervention group;

[0074] Figure 21 The results of Western blot analysis of GnRH expression levels in the hypothalamus of precocious puberty mice and mice in the cholestyramine intervention group;

[0075] Figure 22 A statistical graph showing the expression levels of GnRH in the hypothalamus of precocious puberty mice and mice in the cholestyramine intervention group as detected by Western blotting.

[0076] Figure 23 Results of Western blot analysis of GnRH expression in GT1-7 cell line;

[0077] Figure 24 A statistical graph showing the expression level of GnRH in the GT1-7 cell line detected by Western blotting;

[0078] Figure 25 Results of Western blot analysis of GnRH expression in GT1-7 cell line;

[0079] Figure 26 A statistical graph showing the expression level of GnRH in the GT1-7 cell line detected by Western blotting;

[0080] Figure 27 A simplified flowchart of animal experiments activating the bile acid receptor TGR5;

[0081] Figure 28 A statistical graph showing the vaginal opening time and proportion of mice in the INT-777 intervention group and the control group;

[0082] Figure 29 The results of Western blotting to detect GnRH expression levels in the hypothalamus of mice in the INT-777 intervention group and the control group;

[0083] Figure 30 A statistical graph showing the expression levels of GnRH in the hypothalamus of mice in the INT-777 intervention group and the control group, as detected by Western blotting.

[0084] Figure 31 A simplified flowchart of animal experiments involving the TGR5 knockout of bile acid receptors.

[0085] Figure 32 A statistical chart showing the vaginal opening time and proportion of mice in the TGR5 knockout mouse intervention group and control group. Detailed Implementation

[0086] The present invention will be further explained and described below with reference to specific embodiments and accompanying drawings. However, the content of the following embodiments should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the reagents used in the embodiments of the present invention are all commercially available.

[0087] Example 1: Discovery of biomarkers associated with precocious puberty

[0088] To identify biomarkers associated with precocious puberty, serum samples were collected from 241 girls (145 patients with central precocious puberty and 96 controls). UPLC-MS / MS was performed on various substances in the samples, including the detection of bile acid levels and hormones related to the gonadal axis initiation. The results were then analyzed.

[0089] Figure 1 The graph shows the total serum mouse bile acid (MCA) concentration in patients with central precocious puberty (ICPP) and controls (Non-ICPP). It indicates that the total serum mouse bile acid concentration in patients with central precocious puberty is significantly higher than that in the control group. The total serum mouse bile acid concentration may be used as an indicator for predicting or diagnosing central precocious puberty.

[0090] Further analysis was conducted on various types of mouse bile acids. Figure 2 The statistical graph of the concentration of 6-hydroxy bile acids in the serum of the subjects, including TαMCA, TγMCA, GγMCA, γMCA, and HDCA, shows that the concentration of all types of mouse bile acids in the serum of patients with central precocious puberty is significantly higher than that in the control group, and the concentration of HDCA (deoxycholic acid in pigs) is also significantly higher than that in the control group. Since the chemical structure corresponds to the function of the molecule in vivo, based on the chemical structure, 6-hydroxy bile acids can be used as biomarkers for predicting or diagnosing central precocious puberty.

[0091] Figure 3 A Spearman correlation heatmap was generated for serum bile acids and gonadal axis initiation-related hormone markers. Bile acids included total bile acids (TBAs), chenodeoxycholic acids (CDCA Species), bile acids (CA Species), and mouse bile acids (MCA Species). Gonadal axis initiation-related hormone markers included sex hormone-binding protein (SHBG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), LH / FSH (the ratio of the two hormones), LH Peak (the peak value of LH in the GnRHa stimulation test), and FSH Peak (the peak value of FSH in the GnRHa stimulation test). The total concentration of each type of bile acid was analyzed for correlation with gonadal axis initiation-related hormone markers. The results showed that mouse bile acids were significantly positively correlated with several gonadal axis initiation-related hormone markers, while bile acids, chenodeoxycholic acids, and total bile acids showed no significant correlation. Therefore, the concentration of mouse bile acids can predict or diagnose central precocious puberty, while other bile acids do not have this function.

[0092] Further correlation analysis was conducted between various types of mouse bile acids and hormones related to the initiation of the gonadal axis. Figure 4A Spearman correlation heatmap of serum 6-hydroxybile acids and gonadal axis initiation-related hormone indicators shows that serum 6-hydroxybile acids are significantly positively correlated with multiple gonadal axis initiation-related hormone indicators. This indicates that not only mouse bile acids, but various 6-hydroxybile acids (TαMCA, TγMCA, GγMCA, γMCA, HDCA) can predict or diagnose central precocious puberty.

[0093] The study analyzed the correlation between taurine-conjugated α-mouse bile acid (TαMCA) and the Tanner stage of breast development (stages are determined by clinicians through observation and physical examination reports, and are divided into stages B1, B2, and B3; higher numbers indicate greater breast development, more pronounced secondary sexual characteristics, and more advanced precocious puberty). Figure 5 The Spearman correlation analysis scatter plot of serum taurine-bound α-mouse bile acid and mammary Tanner stage shows a significant positive correlation between serum taurine-bound α-mouse bile acid concentration and mammary Tanner stage.

[0094] The regression equation for predicting precocious puberty using serum taurine-bound α-mouse bile acid was determined by logistic regression: Logit(P) = -1.405 + 0.057 * TαMCA. In this regression equation, P represents the probability of precocious puberty, 0.057 represents the regression coefficient of taurine-bound α-mouse bile acid, TαMCA represents the measured concentration of taurine-bound α-mouse bile acid, and -1.405 is a constant term. Logit(P) is the value after regression correction; when the P value is greater than -0.0706, the individual is predicted to be a patient with precocious puberty.

[0095] Figure 6 ROC curve analysis of serum total 6-hydroxy bile acids and various 6-hydroxy bile acids to predict precocious puberty showed that the AUC of serum taurine-bound α-mouse bile acid was 0.826. Figure 7 ROC curve analysis was performed to predict precocious puberty using serum taurine-bound α-mouse bile acid and baseline LH levels. The results showed that the AUC of serum taurine-bound α-mouse bile acid and baseline LH levels were close, and the combined AUC was 0.904. The regression equation was Logit(P) = -1.729 + 0.043 * TαMCA + 7.841 * LH, where 0.057 represents the regression coefficient of taurine-bound α-mouse bile acid, TαMCA represents the measured concentration of taurine-bound α-mouse bile acid, 7.841 represents the regression coefficient of luteinizing hormone (LH), LH represents the measured concentration of LH, -1.729 is a constant term, and Logit(P) is the value after regression correction. A P-value > 0.902915 indicates that the individual may have precocious puberty.

[0096] Through multivariate logistic regression analysis, this invention determined the regression equation for the combined prediction of precocious puberty using five biomarkers: Logit(P) = -3.122 + 0.079*TαMCA + 0.022*γMCA + 0.048*TγMCA - 0.007*GγMCA - 0.044*HDCA; in the regression equation, P represents the probability of precocious puberty, and 0.079, 0.022, 0.048, 0.007, and 0.044 represent the probabilities of taurine-bound α-mouse cholic acid, γ-mouse cholic acid, and so on, respectively. Regression coefficients for taurine-γ-mouse cholic acid, glycine-γ-mouse cholic acid, and deoxycholic acid; TαMCA represents the measured concentration of taurine-bound α-mouse cholic acid; γMCA represents the measured concentration of γ-mouse cholic acid; TγMCA represents the measured concentration of taurine-γ-mouse cholic acid; GγMCA represents the measured concentration of glycine-γ-mouse cholic acid; HDCA represents the measured concentration of deoxycholic acid; -3.122 is a constant term; Logit(P) is the regression-corrected value; when the P value > 0.30714, it indicates that the individual may be a patient with precocious puberty.

[0097] In subsequent experiments, the above results were further verified: First, an additional batch of independent validation set clinical data was collected, and serum samples were collected from 149 female subjects (68 patients with central precocious puberty and 81 controls). UPLC-MS / MS was performed on various substances in the samples, including the detection of bile acid levels, and the detection results were analyzed. Figure 8 ROC curve analysis of total 6-hydroxybile acids and various 6-hydroxybile acids in serum as predictors of precocious puberty showed that the validation set:

[0098] The serum taurine-bound α-mouse bile acid AUC was 0.805;

[0099] The serum glycine-bound γ-mouse bile acid AUC was 0.788;

[0100] The serum taurine-bound γ-mouse bile acid AUC was 0.687;

[0101] The serum AUC of γ-mouse bile acid was 0.618;

[0102] The serum AUC of porcine deoxycholic acid was 0.497.

[0103] The serum total concentration of mouse bile acids (AUC) was 0.799.

[0104] For the regression equation used to predict precocious puberty using taurine-bound α-mouse bile acid alone, this embodiment also detected the concentration of taurine-bound α-mouse bile acid (TαMCA) in serum samples and substituted the detected concentrations into the above regression equation. The accurate predictive rate of the model for ICPP was 73.7%. Compared with actual clinical diagnostic results: 149 girls (68 patients with central precocious puberty and 81 controls), the diagnostic sensitivity of this invention was 77.94%, the specificity was 80.24%, the positive predictive value was 76.81%, and the negative predictive value was 81.25%.

[0105] For the regression equation used to predict precocious puberty by combining taurine-bound α-mouse bile acid (TαMCA) and baseline LH levels, this embodiment also detected the concentrations of TαMCA and LH in serum samples, and substituted the detected concentrations into the regression equation. The accurate predictive rate of the model for ICPP was 82.4%. Compared with actual clinical diagnostic results: 149 girls (68 patients with central precocious puberty and 81 controls), the diagnostic sensitivity of this invention was 79.41%, the specificity was 81.48%, the positive predictive value was 78.26%, and the negative predictive value was 82.5%.

[0106] For the regression equation using the combination of the above five biomarkers to predict precocious puberty, this embodiment also detected taurine-bound α-mouse cholic acid, γ-mouse cholic acid, taurine-γ-mouse cholic acid, and glycine-γ-mouse cholic acid in serum samples, and substituted the detected concentrations into the above regression equation. The accurate predictive rate of ICPP using this model was 78.6%. Compared with actual clinical diagnostic results: 149 girls (68 patients with central precocious puberty and 81 controls), the diagnostic sensitivity of this invention was 80.88%, the specificity was 85.19%, the positive predictive value was 82.09%, and the negative predictive value was 84.15%.

[0107] Furthermore, in this invention, multiple batches of different validation sets were used for validation testing. The results show that the optimal combination of biomarkers selected by this invention has a predictive performance of about 85% for individuals with precocious puberty.

[0108] In summary, the prediction results in the validation set are highly consistent with the actual clinical diagnosis results, and the accuracy, sensitivity, specificity and other indicators are good, with high AUC values. In particular, the combined diagnosis of taurine-bound α-mouse cholic acid and baseline LH value is better than the diagnosis of each alone. Therefore, the diagnostic model constructed by the biomarkers of this invention has good predictive performance and accuracy, and has the best diagnostic efficacy.

[0109] Example 2: Study on 6-hydroxybile acids in a mouse model of precocious puberty

[0110] First, a mouse model of precocious puberty is constructed. The animal experiment procedure is as follows: Figure 9 As shown, the model was constructed by feeding newborn female mice with a 60% high-fat diet (60g fat per 100g of the diet, purchased from Research Diets, catalog number D12492) for 4 weeks to induce precocious puberty. A control group was also established and fed a standard diet. The vaginal opening time and proportion of mice in the control group and the precocious puberty mouse model were statistically analyzed. Figure 10 As shown, the female mice in the precocious puberty group developed vaginal opening around 28 days after birth, significantly earlier than the control group. Figure 11 The results of Western blotting (WB) analysis of gonadotropin-releasing hormone (GnRH) expression levels in the mouse hypothalamus were obtained. Figure 12 The statistical graph showing the expression level of GnRH in the hypothalamus of mice detected by Western blotting indicates that the expression level of GnRH in the hypothalamus of female mice in the precocious puberty group was significantly higher than that in the control group. These results demonstrate the successful establishment of a precocious puberty mouse model.

[0111] Serum and hypothalamic samples from precocious female mice and the control group were analyzed by UPLC-MS / MS to determine the concentration of bile acids. Figure 13 A statistical graph showing the bile acid concentrations in serum samples from precocious female mice and the control group. Figure 14 The figure shows the statistical distribution of bile acid concentrations in hypothalamic samples from precocious female mice and the control group. In the serum samples of precocious female mice, the concentrations of αMCA, βMCA, HDCA, TαMCA, TωMCA, and THDCA were significantly higher than those in the control group. In the hypothalamic samples, the concentrations of αMCA, βMCA, and HDCA were significantly higher than those in the control group. In addition, the concentrations of each 6-hydroxy bile acid were higher than those in the control group, indicating that 6-hydroxy bile acid can serve as a biomarker for precocious puberty in mice.

[0112] Example 3: Animal Experiment for the Treatment of Precocious Puberty

[0113] Figure 15 This is a simplified flowchart of the animal experiment for treating precocious puberty. Precocious puberty was induced in female mice using a 60% high-fat diet. The establishment of this precocious puberty mouse model was successfully verified in Example 2. In the cholestyramine intervention group, the diet was changed to a 60% high-fat diet supplemented with 2% cholestyramine 14 days after birth. Figure 16 This is a statistical chart showing the vaginal opening time and proportion in animal experiments on the treatment of precocious puberty. The vaginal opening time was delayed under cholestyramine intervention, significantly later than in the precocious puberty group, indicating that cholestyramine intervention may be a means of treating or preventing precocious puberty.

[0114] UPLC-MS / MS was performed on precocious puberty mice and the cholestyramine intervention group to detect the concentration of bile acids in their serum and hypothalamic samples. Figure 17The bile acid profiles of serum samples from precocious puberty mice and mice in the cholestyramine intervention group were detected by UPLC-MS / MS. Figure 18 The bile acid profiles of hypothalamic samples from precocious puberty mice and mice in the cholestyramine intervention group were detected by UPLC-MS / MS. The results showed that the concentrations of bile acids in the serum and hypothalamus of female mice in the cholestyramine group were decreased, while the total bile acids and bile acid-type bile acids were not different. This indicates that the effect of cholestyramine intervention on precocious puberty mice is only to reduce the concentration of bile acids in mice, without affecting the concentrations of other bile acids.

[0115] The concentrations of various types of mouse bile acids in serum and hypothalamic samples from precocious puberty mice and the cholestyramine intervention group were further detected by UPLC-MS / MS. Figure 19 This is a statistical graph showing the concentration of 6-hydroxybile acid in serum samples from precocious puberty mice and mice in the cholestyramine intervention group. Figure 20 The statistical diagram of various types of bile acids in hypothalamic samples from precocious puberty mice and mice in the cholestyramine intervention group shows that the concentrations of 6-hydroxy bile acids (TωMCA, TβMCA, ωMCA, βMCA, THDCA) in the serum and hypothalamus of female mice in the cholestyramine group were significantly reduced, verifying that the effect of cholestyramine is to reduce the concentrations of various 6-hydroxy bile acids (TωMCA, TβMCA, βMCA) in the serum and hypothalamus of precocious puberty mice.

[0116] Figure 21 The results of Western blotting (WB) analysis of GnRH expression levels in the hypothalamus of precocious puberty mice and mice in the cholestyramine intervention group are shown. Figure 22 This is a statistical graph showing the expression levels of GnRH in the hypothalamus of mice with precocious puberty detected by Western blotting and in mice in the cholestyramine intervention group. The results indicate that the expression level of GnRH in the hypothalamus of female mice in the cholestyramine intervention group was significantly reduced. This experiment reveals a correlation between reducing the concentration of 6-hydroxybile acids and inhibiting GnRH expression. Theoretically, besides cholestyramine intervention, other reagents or methods that reduce the concentration of 6-hydroxybile acids can also inhibit GnRH expression, thereby treating precocious puberty.

[0117] Example 4: Mechanism study and treatment of the correlation between 6-hydroxybile acids and precocious puberty

[0118] In vitro studies were conducted using the mouse GnRH neuronal cell line GT1-7 (provided free of charge by Dr. Pamela L Mellon's lab (Division of Reproductive Endocrinology & Infertility, Department of Obstetrics, Gynecology & Reproductive Sciences, University of California, San Diego, School of Medicine)). Multiple mouse bile acids were used for intervention, and the expression level of GnRH in the cell line was detected by Western blotting. Figure 23 The results of Western blotting for detecting GnRH expression in the GT1-7 cell line. Figure 24 The Western blot analysis of GnRH expression in the GT1-7 cell line shows that the expression of GnRH in GT1-7 cells increased significantly under the intervention of three types of mouse bile acids: TαMCA, TβMCA, and TωMCA.

[0119] In vitro studies were conducted using the mouse GnRH neuronal cell line GT1-7. The cells were treated with INT-777, a specific agonist of the bile acid receptor TGR5. Western blotting was used to detect GnRH expression levels in the cell line. Figure 25 The results of Western blotting for detecting GnRH expression in the GT1-7 cell line. Figure 26 The Western blot analysis of GnRH expression in the GT1-7 cell line shows that specific activation of the TGR5 receptor significantly increases GnRH expression in GT1-7 cells.

[0120] Further animal testing was conducted. Figure 27 This is a simplified flowchart of the animal experiment procedure for activating the bile acid receptor TGR5. Mice were treated with intraperitoneal injection of INT-777 (purchased from MCE, catalog number HY-15677), a specific agonist of the bile acid receptor TGR5. Figure 28 The figure shows the vaginal opening time and proportion of mice in the INT-777 intervention group and the control group. It shows that the vaginal opening time was advanced under the intervention of the TGR5 specific agonist INT-777, which was significantly earlier than that in the control group.

[0121] Western blot analysis was performed on the INT-777 intervention group and the control group to detect the expression level of GnRH in the hypothalamus of mice. Figure 29 The results of Western blotting were obtained to detect GnRH expression levels in the hypothalamus of mice in the INT-777 intervention group and the control group. Figure 30 The Western blot analysis of GnRH expression levels in the hypothalamus of mice in the INT-777 intervention group and the control group showed that the expression level of GnRH in the hypothalamus of female mice in the INT-777 intervention group was significantly increased.

[0122] To further clarify the key role of the TGR5 receptor in the pathogenesis of precocious puberty, TGR5 knockout mice were used for animal experiments. Figure 31 This is a simplified flowchart of the animal experiment procedure for TGR5 knockout of bile acid receptors. Mice were used to induce precocious puberty by feeding them a high-fat diet. WT-CD mice were control mice fed a standard carbohydrate diet, WT-HFD mice were control mice fed a high-fat diet, TGR5 KO-CD mice were TGR5 knockout mice fed a standard carbohydrate diet, and TGR5 KO-HFD mice were TGR5 knockout mice fed a high-fat diet. Figure 32 The figure shows the vaginal opening time and proportion of mice in the TGR5 knockout intervention group and the control group. It shows that after TGR5 knockout, the vaginal opening time was delayed under high-fat diet, which was significantly later than that of wild-type mice fed a high-fat diet.

[0123] Therefore, this embodiment provides one mechanism for the association between 6-hydroxybile acid and precocious puberty. As a bile acid that can activate the TGR5 receptor, an increase in the concentration of 6-hydroxybile acid will promote the activation of the TGR5 receptor, thereby promoting GnRH expression and inducing precocious puberty. However, this is only one possible mechanism. In the future, there may be more mechanisms related to precocious puberty involving 6-hydroxybile acid. Moreover, the TGR5 receptor is not only activated by binding to 6-hydroxybile acid. Therefore, both 6-hydroxybile acid and the TGR5 receptor can be used independently as targets for the treatment of precocious puberty.

[0124] The above experimental results indicate that 6-hydroxybile acid, TGR5 receptor, and the occurrence of precocious puberty are correlated. Intervention or activation of the TGR5 receptor by 6-hydroxybile acid promotes GnRH expression in the mouse hypothalamus, inducing precocious puberty. Therefore, theoretically, it is possible to prevent or treat precocious puberty by inhibiting 6-hydroxybile acid or intervening in its related receptors, thereby preventing the promotion of GnRH expression in the mouse hypothalamus. Methods to reduce the concentration of 6-hydroxybile acid include: inhibiting its absorption, promoting its metabolism, and disrupting its related pathways. Inhibition of 6-hydroxybile acid absorption includes intervention with cholestyramine as described in Example 3, or other reagents or methods. Intervention on 6-hydroxybile acid-related receptors includes inhibiting the TGR5 receptor, with TGR5 receptor inhibitors including triamterene (CAS: 396-01-0) and TGR5 inhibitors (CAS: 1197300-24-5), or intervention on other 6-hydroxybile acid-related targets.

[0125] The above detailed description is a specific description of feasible embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. The use of a substance in the preparation of a reagent for treating individual precocious puberty, characterized in that, The substance reduces the concentration of any one or more 6-hydroxybile acids in an individual's body; the substance includes cholestyramine.

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