Marker for predicting individual sexual precocity and application thereof

Abstract

The invention discloses a marker for predicting individual sexual precocious puberty and application thereof, through large-scale sample analysis work, a biomarker for predicting individual sexual precocious puberty, namely 6-hydroxy bile acid, is found for the first time, and the effectiveness of the 6-hydroxy bile acid as the biomarker is verified; there is provided a method of treating precocious puberty in an individual by reducing the concentration of 6-hydroxybile acid, for example by reducing the concentration of 6-hydroxybile acid by dalicyclic amine, thereby treating precocious puberty; in-depth study is carried out on related mechanisms, and the correlation between the TGR5 receptor and sexual precocious puberty is found, that is, the TGR5 receptor is inhibited or prevented from being activated, and GnRH expression is prevented from being promoted, so that the individual sexual precocious puberty is treated; the invention provides a reagent composition for treating individual precocious puberty, which is used for treating the individual precocious puberty by using cholestyramine or other reagents for reducing the concentration of 6-hydroxyl bile acid, reagents for promoting the metabolism of the 6-hydroxyl bile acid, TGR5 receptor inhibitors and the like for intervention.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a marker for predicting individual precocious puberty and an application thereof. Background Art

[0002] Central precocious puberty in children is a type of puberty development disorder, which refers to the development of secondary sexual characteristics in boys before the age of 9 and in girls before the age of 7.5, manifested as epiphyseal closure, early menstruation, and lower height than normal people in adulthood. In addition, studies have shown that girls with precocious puberty will have a significantly increased risk of polycystic ovary syndrome, breast cancer, cardiovascular disease, and obesity-related metabolic diseases in adulthood. At the same time, due to the premature development of sexual characteristics of children, their intelligence and sexual psychology are not yet mature, which causes psychological and physiological disorders, bringing 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 younger year by year, 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 function of the gonadal axis, leading to gonadal development and secretion of sex hormones, causing the development of internal and external genitalia and the appearance of secondary sexual characteristics. Peripheral precocious puberty is also clinically called pseudo-precocious puberty, mainly because of daily high-fat diet, or when tumors occur in the hypothalamus, ovaries, adrenal cortex and other parts, leading to abnormal secretion of sex hormones, secondary sexual characteristics appear but gonads are not developed. Peripheral precocious puberty (pseudo-precocious puberty) is often due to special reasons that lead to abnormal secretion of sex hormones. It is a special symptom in the development of children. Pseudo-precocious puberty will naturally subside.

[0004] What needs to be predicted and treated is central precocious puberty (true precocious puberty), which is a developmental disease in children that can cause physical maldevelopment, psychological problems, and other hazards. Therefore, predicting or diagnosing individual precocious puberty and treating precocious puberty all 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, is still unclear, which brings great difficulties to the prevention and treatment of the disease. At present, the clinical diagnosis is mainly based on the early appearance of secondary sexual characteristics of patients, imaging examinations, molecular diagnostic markers of serum neutral hormones (such as follicle-stimulating hormone, luteinizing hormone, prolactin, estradiol, testosterone and progesterone), GnRH stimulation tests and other indicators. However, the above indicators only begin to appear after the occurrence of precocious puberty in children, and cannot be used as screening markers for early onset risk factors. The current methods for treating precocious puberty include drug therapy and traditional Chinese medicine therapy. In terms of drug therapy, the first-line drug is GnRHa, such as triptorelin and leuprorelin, which delay the process of sexual development by inhibiting the pituitary-gonadal axis. In addition, traditional Chinese medicine treatment is also an option, but it must be used under the guidance of a doctor, such as Danzhi Xiaoyao Pills, Dabuyin Pills and other Chinese patent medicines. During the treatment process, the above-mentioned drugs cannot fully guarantee the therapeutic effect on the one hand, and have obvious side effects on the other hand. Therefore, it is urgent to find a marker for predicting individual precocious puberty, and to construct a prediction model based on the marker to achieve accurate diagnosis of precocious puberty, so as to intervene in patients in time and formulate effective treatment plans. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the present invention provides a marker for predicting individual precocious puberty and its application. A biomarker for predicting individual precocious puberty, namely 6-hydroxy bile acid, is discovered. By detecting the concentration of 6-hydroxy bile acid in individual serum or hypothalamus samples, it is possible to determine whether the individual is at risk of precocious puberty, so that prevention or treatment can be carried out in a timely manner.

[0007] In the present invention, the discovery set and the validation set use two completely different batches of samples. The discovery set is a known sample, and the inventors only screen markers from the discovery set; the samples of the validation set are only used to verify the diagnostic efficacy of the marker combination of the present invention. In addition, in the present invention, multiple batches of different validation sets are used for validation testing, and the results show that the optimal marker combination screened by the present invention has a predictive performance of about 85% for individuals suffering from precocious puberty.

[0008] As shown in formula (1), bile acids can be divided into three categories according to the position of the hydroxyl group on the bile acid carbon chain. Cholic acid (CAspecies) has hydroxyl groups linked to the 3rd and 12th carbon atoms, chenodeoxycholic acid (CDCAspecies) has hydroxyl groups linked to the 3rd carbon atom, and muricholic acid (MCAspecies) has hydroxyl groups linked to the 3rd and 6th carbon atoms; all three types of bile acids can optionally have hydroxyl groups linked to the 7th carbon atom.

[0009]

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

[0011] First, the present invention selected a part of bile acids and found that only murine bile acid can diagnose precocious puberty. Precocious puberty can be effectively treated by reducing the content of murine bile acid. From a structural point of view, we believe that bile acids with a 6-hydroxyl group may have the function of diagnosing precocious puberty and can serve as targets for intervention and treatment of precocious puberty. From a principle point of view, the present invention speculates that the substitution of the 6-hydroxyl group of bile acid is associated with precocious puberty in biochemistry and metabolic pathways. This may be because the substitution of the 6-hydroxyl group affects the physicochemical properties of the compound and may affect its binding with the receptor protein. The details need to be further discovered.

[0012] The 6-hydroxy bile acids include a series of bile acids, such as muricolic acid and conjugated muricolic acid, including α-muricolic acid (αMCA), β-muricolic acid (βMCA), γ-muricolic acid (γMCA), ω-muricolic acid (ωMCA), and hyodeoxycholic acid (HDCA); muricolic acid can be combined with different amino acids to form conjugated muric acid, such as tauro-α-muricolic acid (TαMCA), tauro-β-muricolic acid (TβMCA), tauro-γ-muricolic acid (TγMCA), tauro-ω-muricolic acid (TωMCA), glycine-γ-muricolic acid (GγMCA), etc., as well as conjugated bile acids formed by its combination with amino acids, such as tauro-hyodeoxycholic acid (THDCA), etc.

[0013] The above are the most common 6-hydroxy bile acids, which are only examples. Other bile acids linked to the hydroxyl group at the 6-position, such as 6-ketolithocholic acid (6-ketoLCA), also fall within the scope of protection of the present invention. Since they have the same partial chemical structure, they have similar effects and metabolic pathways in the body, have the same function of diagnosing precocious puberty, and can also serve as targets for intervention and treatment of precocious puberty.

[0014] In one aspect, the present invention provides a use of a biomarker for preparing a reagent for predicting individual precocious puberty, wherein the biomarker comprises any one or more 6-hydroxy bile acids.

[0015] In some embodiments, the biomarkers include 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 individual serum or hypothalamus samples.

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

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

[0019] In summary, 6-hydroxy bile acid is a general term for a series of bile acids. Any 6-hydroxy bile acid can predict individual precocious puberty alone. Combining multiple 6-hydroxy bile acids may improve the sensitivity and specificity of predicting individual precocious puberty to a certain extent. The present invention uses one or more 6-hydroxy bile acids to train the training set to obtain a logistic regression model, which is verified in the validation set. It is found that one or more 6-hydroxy bile acids have the function of predicting individual precocious puberty.

[0020] Initially, the present invention tested serum samples of 241 test girls (145 patients with central precocious puberty and 96 controls) and found that the concentration of 6-hydroxy bile acid was significantly higher in patients with precocious puberty than in controls. Further studies showed that the concentration of 6-hydroxy bile acid was significantly positively correlated with the level of hormones related to sexual development and the Tanner stage of the breast, indicating that 6-hydroxy bile acid can be used as a biomarker for predicting individual precocious puberty.

[0021] The levels of hormones related to sexual development include gonadotropin-releasing hormone (GnRH). The present invention finds that: cholestyramine is used for intervention. Cholestyramine (CAS: 11041-12-6) is a high molecular weight quaternary ammonium anion exchange resin that can bind to bile acid to inhibit the absorption of bile acid. After testing, the concentration of 6-hydroxy bile acid in the serum and hypothalamus of individuals after cholestyramine intervention will decrease, while the concentrations of other bile acid compounds will not be affected. At the same time, GnRH expression is inhibited. After research, 6-hydroxy bile acid is significantly correlated with GnRH expression, indicating that the concentration of 6-hydroxy bile acid is related to precocious puberty. At the same time, taurine-conjugated α-muricholic acid (TαMCA) is significantly correlated with breast Tanner stage. In summary, 6-hydroxy bile acid can be used as a biomarker for predicting individual precocious puberty.

[0022] The 6-hydroxy bile acid concentration was used to predict individual precocious puberty. The validation set was used to predict precocious puberty. The specific AUC can be found in the specific implementation method. Among them, the AUC value of TαMCA for predicting individual precocious puberty was 0.805.

[0023] Further, the biomarker combination includes any one or more of αMCA, βMCA, γMCA, ωMCA, TαMCA, TβMCA, TγMCA, TωMCA, GγMCA, HDCA, and THDCA.

[0024] On the other hand, the present invention provides a system for predicting individual precocious puberty, the system comprising a data analysis module, the data analysis module being used to analyze the detection value of a biomarker; the biomarker comprising any one or more 6-hydroxy bile acids.

[0025] In some embodiments, the biomarker combination includes any one or more of αMCA, βMCA, γMCA, ωMCA, TαMCA, TβMCA, TγMCA, TωMCA, GγMCA, HDCA, and THDCA.

[0026] Furthermore, the data analysis module calculates the predicted value of individual precocious puberty by substituting the detection value of the biomarker into the regression equation, thereby evaluating the risk of individual precocious puberty.

[0027] Further, 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.

[0028] In some embodiments, for the regression equation of taurine-bound α-muricholic acid alone to predict precocious puberty: Logit (P) = -1.405 + 0.057 * TαMCA, in which P represents the probability of precocious puberty, 0.057 represents the regression coefficient of taurine-bound α-muricholic acid, TαMCA represents the measured concentration of taurine-bound α-muricholic acid, and -1.405 is a constant term. Logit (P) is the value after regression correction; when the P value>-0.0706 value, it is predicted that the individual may be a patient with precocious puberty. At the same time, the present invention also detects the concentration of taurine-bound α-muricholic acid (TαMCA) in the serum sample, and the detected concentration is respectively substituted into the above regression equation, and the accurate prediction rate of ICPP predicted by the model is 73.7%. Compared with the actual clinical diagnosis results: 149 tested girls (68 patients with central precocious puberty and 81 controls), the diagnostic sensitivity of the present invention was 77.94%, the specificity was 80.24%, the positive predictive value was 76.81%, and the negative predictive value was 81.25%.

[0029] The regression equation for the combined prediction of precocious puberty by taurine-bound α-muricholic acid and basal LH value is: Logit (P) = -1.729 + 0.043 * TαMCA + 7.841 * LH, wherein 0.057 represents the regression coefficient of taurine-bound α-muricholic acid, TαMCA represents the measured concentration of taurine-bound α-muricholic acid, 7.841 represents the regression coefficient of luteinizing hormone, LH represents the measured concentration of luteinizing hormone, -1.729 is a constant term, and Logit (P) is the value after regression correction; when the P value> 0.902915, it indicates that the individual may be a patient with precocious puberty. At the same time, the present invention also detects the concentration of taurine-bound α-muricholic acid (TαMCA) and luteinizing hormone (LH) in serum samples, and substitutes the detected concentrations into the above regression equation, and the accurate prediction rate of ICPP predicted by the model is 82.4%. Compared with the actual clinical diagnosis results: 149 tested girls (68 patients with central precocious puberty and 81 controls), the diagnostic sensitivity of the present invention was 79.41%, the specificity was 81.48%, the positive prediction value was 78.26%, and the negative prediction value was 82.5%.

[0030] The regression equation for predicting precocious puberty using the combination of the above five markers 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 value of precocious puberty, 0.079, 0.022, 0.048, 0.007, 0.044 represent taurine-bound α-muricholic acid, γ-muricholic acid, taurine-γ-muricholic acid, respectively. The regression coefficients of taurine-bound α-muricholic acid, glycine-γ-muricholic acid, and hyodeoxycholic acid are shown in Table 1. TαMCA represents the measured concentration of taurine-bound α-muricholic acid, γMCA represents the measured concentration of γ-muricholic acid, TγMCA represents the measured concentration of tauro-γ-muricholic acid, GγMCA represents the measured concentration of glycine-γ-muricholic acid, HDCA represents the measured concentration of hyodeoxycholic acid, -3.122 is a constant term, and Logit (P) is the value after regression correction; when the P value>0.30714, it indicates that the individual may be a patient with precocious puberty. In the present invention, taurine-bound α-muricholic acid, γ-muricholic acid, tauro-γ-muricholic acid, and glycine-γ-muricholic acid in serum samples are also detected, and the detected concentrations are respectively substituted into the above regression equation. The accurate prediction rate of ICPP predicted by the model is 78.6%. Compared with the actual clinical diagnosis results: 149 tested girls (68 patients with central precocious puberty and 81 controls), the diagnostic sensitivity of the present invention was 80.88%, the specificity was 85.19%, the positive predictive value was 82.09%, and the negative predictive value was 84.15%.

[0031] Therefore, it can be seen that the prediction results in the validation set of the present invention are highly consistent with the actual clinical diagnosis results, and the indicators such as accuracy, sensitivity, and specificity are good, and the AUC values ​​are all high. In particular, the performance of the combined diagnosis of taurine-bound α-muricholic acid and basal LH value is better than that of the single diagnosis. Therefore, the diagnostic model constructed by the markers of the present invention has good predictive performance and accuracy, and has the best diagnostic efficacy.

[0032] 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.

[0033] In another aspect, the present invention provides a use of a substance for preparing an agent for treating precocious puberty in an individual, wherein the substance reduces the concentration of any one or more 6-hydroxy bile acids in the individual.

[0034] From the perspective of the structure-activity relationship of the compounds, substitution of the 6-hydroxyl group of bile acids is correlated with precocious puberty. Therefore, reducing the concentration of any one or more 6-hydroxyl bile acids in an individual's body can be considered to inhibit the physiological pathway of precocious puberty to varying degrees, and can treat precocious puberty to a certain extent.

[0035] Reducing the concentration of 6-hydroxy bile acid can be carried out from multiple angles: 1. Reducing the absorption of 6-hydroxy bile acid, such as using cholestyramine to bind to 6-hydroxy bile acid, increasing the excretion of 6-hydroxy bile acid, and reducing the absorption of 6-hydroxy bile acid; 2. Promoting the metabolism of 6-hydroxy bile acid and activating the related metabolic pathways of 6-hydroxy bile acid.

[0036] Furthermore, the 6-hydroxy bile acid includes any one or more of αMCA, βMCA, γMCA, ωMCA, TαMCA, TβMCA, TγMCA, TωMCA, GγMCA, HDCA, and THDCA.

[0037] Furthermore, the substance binds to 6-hydroxy bile acid, thereby inhibiting the absorption of 6-hydroxy bile acid.

[0038] Furthermore, the substance includes cholestyramine.

[0039] The present invention demonstrates that cholestyramine (CAS: 11041-12-6) has the effect of reducing the concentration of 6-hydroxy bile acid in an individual's body (serum, hypothalamus), while the concentrations of other bile acid compounds are not affected, and the expression of gonadotropin-releasing hormone (GnRH) is inhibited. In terms of the mechanism of action, cholestyramine can combine with 6-hydroxy bile acid, thereby inhibiting the absorption of 6-hydroxy bile acid by the individual, reducing the concentration of 6-hydroxy bile acid in the individual's body, and thus achieving the purpose of treating individual precocious puberty.

[0040] Furthermore, the substance promotes the degradation of 6-hydroxy bile acid.

[0041] Furthermore, the substance reduces the concentration of 6-hydroxy bile acid in the body of an individual, thereby reducing the binding of 6-hydroxy bile acid to its binding receptor.

[0042] Furthermore, the binding receptor includes TGR5 receptor.

[0043] In another aspect, the present invention provides a use of a substance that inhibits TGR5 receptor for preparing an agent for treating precocious puberty in an individual.

[0044] The full name of TGR5 receptor is G protein-coupled bile acid receptor 1, also known as M-BAR or Gpbar1, which is a member of the transmembrane G protein-coupled receptor family. It can bind to extracellular ligands and transduce extracellular signals to the intracellular downstream cascades. It plays an important role in the body's material energy metabolism, inflammatory response, cancer intervention and treatment through a variety of signal transduction pathways. TGR5 is widely present in the body, such as the gallbladder, ileum, colon, brown adipose tissue, spleen, lung, skeletal muscle, pancreatic islets, etc. In addition, TGR5 is not only a bile acid receptor, but can also be activated by some other synthetic agonists, mediating a series of signal pathways and participating in the body's physiological and pathological processes.

[0045] Initially, the present invention found that the use of TGR5 receptor agonists can induce precocious puberty in mice. Combined with the above research, precocious puberty has no significant correlation with other bile acids, but is highly correlated with 6-hydroxy bile acid. Therefore, the TGR5 receptor may be one of the action pathways of 6-hydroxy bile acid. Therefore, the present invention knocked out the TGR5 gene of mice and found that mice that do not express the TGR5 receptor have significantly later vaginal opening time than the control under the feeding method that induces precocious puberty, which proves that inhibiting the TGR5 receptor can treat individual precocious puberty.

[0046] In another aspect, the present invention provides a product for treating precocious puberty in an individual, wherein the product contains a substance that reduces the concentration of 6-hydroxy bile acid in the individual.

[0047] 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.

[0048] Further, the product includes cholestyramine.

[0049] The beneficial effects of the present invention are:

[0050] 1. Through large-scale sample analysis, the present invention discovered for the first time a biomarker for predicting individual precocious puberty, namely 6-hydroxy bile acid, and verified its effectiveness as a biomarker;

[0051] 2. It provides a method for predicting individual precocious puberty. It only needs to measure the concentration of 6-hydroxy bile acid in the individual's serum or hypothalamus to determine the risk of individual precocious puberty, with good sensitivity and specificity.

[0052] 3. Provides a method for treating individual precocious puberty, that is, reducing the concentration of 6-hydroxy bile acid in the individual, for example, by reducing the absorption of muric acid through cholestyramine, and GnRH expression is not promoted, thereby treating individual precocious puberty;

[0053] 4. Through in-depth research on the related mechanisms of biomarkers, the correlation between TGR5 receptor and precocious puberty was discovered, which opened up a new method for treating individual precocious puberty, that is, inhibiting TGR5 receptor or preventing TGR5 receptor from being activated, preventing GnRH expression from being promoted, and thus treating individual precocious puberty;

[0054] 5. Provided is a reagent composition for treating individual precocious puberty. Utilizing the above mechanism, it is found that cholestyramine or other reagents that inhibit the absorption of 6-hydroxy bile acid, reagents that promote the metabolism of 6-hydroxy bile acid, TGR5 receptor inhibitors, etc. can be used for intervention to treat individual precocious puberty. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The statistical graph is a graph of the total serum muricholic acid (MCA) concentration in patients with central precocious puberty (ICPP) and controls (Non ICPP) among the subjects;

[0056] Figure 2 The statistical graphs of the concentrations of various types of 6-hydroxy bile acids in the serum of patients with central precocious puberty and controls are shown;

[0057] Figure 3 This is the heat map of Spearman correlation analysis between serum bile acid and hormone indicators related to gonad axis activation;

[0058] Figure 4 This is the heat map of the Spearman correlation analysis between various types of serum 6-hydroxy bile acids and hormone indicators related to gonad axis activation;

[0059] Figure 5 This is a scatter plot of the Spearman correlation analysis between serum taurine-conjugated α-muricholic acid and breast Tanner stage;

[0060] Figure 6 The ROC curve analysis of serum 6-hydroxy bile acid in predicting precocious puberty was performed;

[0061] Figure 7 ROC curve analysis of serum taurine-conjugated α-muricholic acid and basal LH value in predicting precocious puberty;

[0062] Figure 8 To conduct ROC curve analysis of serum 6-hydroxy bile acid in predicting precocious puberty in the central precocious puberty validation cohort;

[0063] Fig. 9 This is the animal experiment flow chart of the mouse model of precocious puberty;

[0064] Fig.10 Statistical graphs of vaginal opening time and proportion of mice in the control group and precocious puberty mouse model;

[0065] Fig.11 The results of WB detection of gonadotropin-releasing hormone (GnRH) expression in the mouse hypothalamus;

[0066] Fig.12 This is a statistical diagram of the expression of GnRH in the mouse hypothalamus detected by WB;

[0067] Fig.13 The figure is a statistical graph of bile acid concentrations in serum samples of precocious female mice and the control group;

[0068] Fig.14 The figure is a statistical graph of bile acid concentrations in hypothalamic samples of precocious female mice and the control group;

[0069] Fig.15 A simplified diagram of the animal experiment process for the treatment of precocious puberty;

[0070] Fig.16 This is a statistical chart of vaginal opening time and proportion of mice in animal experiments for the treatment of precocious puberty;

[0071] Fig.17 UPLC-MS / MS was used to detect the bile acid profile of serum samples of precocious puberty mice and mice in the cholestyramine intervention group;

[0072] Fig.18 UPLC-MS / MS was used to detect the bile acid profile of hypothalamic samples of precocious puberty mice and mice in the cholestyramine intervention group;

[0073] Fig.19 This is a statistical chart of the 6-hydroxy bile acid concentration in serum samples of precocious puberty mice and mice in the cholestyramine intervention group;

[0074] Fig. 20 This is a statistical diagram of various types of murine bile acids in hypothalamic samples of precocious puberty mice and mice in the cholestyramine intervention group;

[0075] Fig.21 The results of WB detection of hypothalamic GnRH expression in precocious puberty mice and mice in the cholestyramine intervention group;

[0076] Fig. 22 This is a statistical chart of the expression of GnRH in the hypothalamus of mice with precocious puberty and mice in the cholestyramine intervention group detected by WB;

[0077] Fig.23 The results of WB detection of GnRH expression in GT1-7 cell line;

[0078] Fig.24 This is a statistical chart of GnRH expression in GT1-7 cell line detected by WB;

[0079] Fig.25 The results of WB detection of GnRH expression in GT1-7 cell line;

[0080] Fig.26 This is a statistical chart of GnRH expression in GT1-7 cell line detected by WB;

[0081] Fig. 27 A simplified diagram of the animal experiment process for bile acid receptor TGR5 activation;

[0082] Fig.28 This is a statistical chart of vaginal opening time and proportion of mice in the INT-777 intervention group and the control group;

[0083] Fig.29 The results of WB detection of GnRH expression in the hypothalamus of mice in the INT-777 intervention group and the control group;

[0084] Fig.30 This is a statistical chart of the expression of GnRH in the hypothalamus of mice in the INT-777 intervention group and the control group detected by WB.

[0085] Fig.31 This is a simplified diagram of the bile acid receptor TGR5 knockout animal experiment process.

[0086] Fig.32 Statistical chart of vaginal opening time and proportion of TGR5 knockout mice in the intervention group and control group. DETAILED DESCRIPTION

[0087] The present invention is further explained below in conjunction with specific examples and the accompanying drawings, but the contents of the following examples should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the reagents used in the examples of the present invention are all commercially available.

[0088] Example 1: Discovery of biomarkers associated with precocious puberty

[0089] In order to find biomarkers related to precocious puberty, serum samples from 241 girls (145 patients with central precocious puberty and 96 controls) were collected, and various substances in the samples were tested by UPLC-MS / MS, including the detection of bile acid levels and hormones related to the activation of the gonadal axis, and the test results were analyzed.

[0090] Figure 1 The statistical graph shows the total concentration of serum muricolic acid (MCA) in patients with central precocious puberty (ICPP) and controls (Non ICPP) among the subjects, indicating that the total concentration of serum muricolic acid in patients with central precocious puberty is significantly higher than that in the control group. The total concentration of serum muricolic acid may be used as an indicator for predicting or diagnosing central precocious puberty.

[0091] Further analysis of various types of muricolic acid was performed. Figure 2 The figure is a statistical graph of the concentration of 6-hydroxy bile acids in the serum of subjects, including TαMCA, TγMCA, GγMCA, γMCA, and HDCA, which shows that the concentrations of various types of murine bile acids in the serum of patients with central precocious puberty are significantly higher than those in the control group, and the concentration of HDCA (hyodeoxycholic acid) is also significantly higher than that in the control group. Since the chemical structure corresponds to the role of the molecule in the body, it can be summarized from the chemical structure that 6-hydroxy bile acids can be used as a biomarker for predicting or diagnosing central precocious puberty.

[0092] Figure 3 This is a heat map of Spearman correlation analysis between serum bile acid and hormone indicators related to gonad axis activation. Bile acids include total bile acid (TBAs), chenodeoxycholic acid (CDCA Species), cholic acid (CA Species), and muricholic acid (MCA Species). Hormone indicators related to gonad axis activation include sex hormone binding protein (SHBG), luteinizing hormone (LH), follicle stimulating hormone (FSH), LH / FSH (the ratio of the above two hormones), LH Peak (the peak value of luteinizing hormone in GnRHa stimulation test), and FSH Peak (the peak value of follicle stimulating hormone in GnRHa stimulation test). The total concentration of each type of bile acid was analyzed for correlation with hormone indicators related to gonad axis activation. The results showed that muricholic acid bile acid was significantly positively correlated with multiple hormone indicators related to gonad axis activation, while cholic acid, chenodeoxycholic acid, and total bile acid were not significantly correlated. Therefore, the concentration of muricholic acid bile acid can predict or diagnose central precocious puberty, while other bile acids do not have this function.

[0093] Further correlation analysis was conducted between various types of murine bile acids and hormones related to gonad axis activation. Figure 4This is a heat map of the Spearman correlation analysis of serum 6-hydroxy bile acids and hormone indicators related to gonad axis activation, showing that serum 6-hydroxy bile acids are significantly positively correlated with multiple hormone indicators related to gonad axis activation. It shows that not only muricolic acid, but various 6-hydroxy bile acids (TαMCA, TγMCA, GγMCA, γMCA, HDCA) can predict or diagnose central precocious puberty.

[0094] Among them, a correlation analysis was conducted between taurine-conjugated α-muricholic acid (TαMCA) and breast Tanner staging (the staging is determined by clinical doctors' observation and filling out physical examination reports, and is divided into B1, B2, and B3 stages. The larger the number, the higher the degree of breast development, the more obvious the secondary sexual characteristics, and the deeper the degree of precocious puberty). Figure 5 This is a scatter plot of the Spearman correlation analysis between serum taurine-conjugated α-muricholic acid and breast Tanner stage, showing that the serum taurine-conjugated α-muricholic acid concentration was significantly positively correlated with breast Tanner stage.

[0095] The regression equation for serum taurine-bound α-muricholic acid to predict precocious puberty was determined by logistic regression, which was Logit(P)=-1.405+0.057*TαMCA, in which P represents the probability of precocious puberty, 0.057 represents the regression coefficient of taurine-bound α-muricholic acid, TαMCA represents the measured concentration of taurine-bound α-muricholic acid, and -1.405 is a constant term. Logit(P) is the value after regression correction; when the P value>-0.0706, it is predicted that the individual may be a patient with precocious puberty.

[0096] Figure 6 The ROC curve analysis of serum total 6-hydroxy bile acid and various 6-hydroxy bile acids in predicting precocious puberty showed that the AUC of serum taurine-conjugated α-muricholic acid was 0.826. Figure 7 ROC curve analysis of serum taurine-conjugated α-muricholic acid and basal LH values ​​in predicting precocious puberty showed that the AUC of serum taurine-conjugated α-muricholic acid and basal LH values ​​were close, and the AUC of the combined diagnosis was 0.904. The regression equation was Logit(P)=-1.729+0.043*TαMCA+7.841*LH, where 0.057 represented the regression coefficient of taurine-conjugated α-muricholic acid, TαMCA represented the measured concentration of taurine-conjugated α-muricholic acid, 7.841 represented the regression coefficient of luteinizing hormone, LH represented the measured concentration of luteinizing hormone, -1.729 was the constant term, and Logit(P) was the value after regression correction. When the P value>0.902915, it indicated that the individual might be a patient with precocious puberty.

[0097] Through multivariate logistic regression analysis, the present invention determines a regression equation for predicting precocious puberty by combining five markers, which 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 value of precocious puberty, 0.079, 0.022, 0.048, 0.007, and 0.044 represent taurine-bound α-muricholic acid, γ-muricholic acid, The regression coefficients of tauro-γ-muricholic acid, glycine-γ-muricholic acid, and hyodeoxycholic acid, TαMCA represents the measured concentration of taurine-conjugated α-muricholic acid, γMCA represents the measured concentration of γ-muricholic acid, TγMCA represents the measured concentration of tauro-γ-muricholic acid, GγMCA represents the measured concentration of glycine-γ-muricholic acid, HDCA represents the measured concentration of hyodeoxycholic acid, -3.122 is the constant term, and Logit(P) is the value after regression correction; when the P value>0.30714, it indicates that the individual may be a patient with precocious puberty.

[0098] In subsequent experiments, the above results were further verified: first, an additional batch of clinical data for an independent validation set was collected, and serum samples from 149 test girls (68 patients with central precocious puberty and 81 controls) were collected. UPLC-MS / MS detection was performed on various substances in the samples, including the detection of bile acid levels, and the test results were analyzed. Figure 8 The ROC curve analysis of serum total 6-hydroxy bile acid and various 6-hydroxy bile acids in the validation set for predicting precocious puberty showed that:

[0099] The AUC of serum taurine-conjugated α-muricholic acid was 0.805;

[0100] The AUC of seroglycine-bound γ-muricholic acid was 0.788;

[0101] The AUC of serum taurine-conjugated γ-muricholic acid was 0.687;

[0102] The AUC of serum γ-muricholic acid was 0.618;

[0103] The AUC of serum hyodeoxycholic acid was 0.497;

[0104] The AUC of total serum muricholic acid concentration was 0.799.

[0105] For the regression equation of predicting precocious puberty by taurine-bound α-muricholic acid alone, the concentration of taurine-bound α-muricholic acid (TαMCA) in serum samples was also detected in this embodiment, and the detected concentrations were respectively substituted into the above regression equation, and the accurate prediction rate of ICPP predicted by this model was 73.7%. Compared with the actual clinical diagnosis results: 149 girls tested (68 patients with central precocious puberty and 81 controls), the diagnostic sensitivity of the present invention was 77.94%, the specificity was 80.24%, the positive predictive value was 76.81%, and the negative predictive value was 81.25%.

[0106] For the regression equation of taurine-conjugated α-muricholic acid and basal LH value combined prediction of precocious puberty, the concentration of taurine-conjugated α-muricholic acid (TαMCA) and luteinizing hormone (LH) in serum samples were also detected in this embodiment, and the detected concentrations were substituted into the above regression equation, and the accurate prediction rate of ICPP prediction using this model was 82.4%. Compared with the actual clinical diagnosis results: 149 girls tested (68 patients with central precocious puberty and 81 controls), the diagnostic sensitivity of the present invention was 79.41%, the specificity was 81.48%, the positive prediction value was 78.26%, and the negative prediction value was 82.5%.

[0107] For the regression equation using the combination of the above five markers to predict precocious puberty, in this embodiment, taurine-bound α-muricholic acid, γ-muricholic acid, tauro-γ-muricholic acid, and glycine-γ-muricholic acid in serum samples were also detected, and the detected concentrations were substituted into the above regression equation, and the accurate prediction rate of ICPP predicted by the model was 78.6%. Compared with the actual clinical diagnosis results: 149 girls (68 central precocious puberty patients and 81 controls), the diagnostic sensitivity of the present invention was 80.88%, the specificity was 85.19%, the positive predictive value was 82.09%, and the negative predictive value was 84.15%.

[0108] In addition, in the present invention, multiple batches of different validation sets were used for validation testing, and the results showed that the optimal marker combination screened by the present invention has a predictive performance of about 85% for individuals suffering from precocious puberty.

[0109] 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, and the AUC values ​​are high. In particular, the performance of the combined diagnosis of taurine-bound α-muricholic acid and basal LH value is better than that of the single diagnosis. Therefore, the diagnostic model constructed by the markers of the present invention has good predictive performance and accuracy, and has the best diagnostic efficacy.

[0110] Example 2: Study on 6-hydroxy bile acid in a mouse model of precocious puberty

[0111] First, a mouse model of precocious puberty was constructed. The animal experiment process was as follows: Fig. 9 As shown in the figure, the model construction method is to feed newborn female mice with 60% high-fat diet (each 100g of the diet contains 60g of fat, purchased from Research Diets, item number D12492) for 4 weeks to induce precocious puberty in female mice, and set up a control group, which was fed with conventional diet; the vaginal opening time and proportion of mice in the control group and the precocious puberty mouse model were counted, as shown in the figure. Fig.10 As shown, the vagina of female mice in the precocious puberty group opened around 28 days after birth, which was significantly earlier than that in the control group. Fig.11 The results of WB detection of gonadotropin-releasing hormone (GnRH) expression in the mouse hypothalamus. Fig.12 The statistical graph of the expression of GnRH in the hypothalamus of mice detected by WB shows that the expression of GnRH in the hypothalamus of female mice in the precocious puberty group was significantly higher than that in the control group. The above results indicate that the precocious puberty mouse model was successfully established.

[0112] UPLC-MS / MS was performed on serum samples and hypothalamus samples from precocious female mice and the control group to detect the bile acid concentrations. Fig.13 The statistical graph of bile acid concentration in serum samples of precocious female mice and the control group is shown in Figure 2. Fig.14 The figure shows the statistical graph of bile acid concentrations in hypothalamic samples of 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, and the concentrations of αMCA, βMCA, and HDCA in the hypothalamic samples 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 be used as a biomarker for precocious puberty in mice.

[0113] Example 3: Animal experiment on treatment of precocious puberty

[0114] Fig.15 This is a simplified diagram of the animal experiment process for the treatment of precocious puberty. The 60% high-fat diet was also used to induce precocious puberty in female mice. The construction of the precocious puberty mouse model has been successfully verified in Example 2. The cholestyramine intervention group had its feed replaced with a 60% high-fat diet supplemented with 2% cholestyramine 14 days after birth. Fig.16 This is a statistical chart of the vaginal opening time and proportion of mice in the animal experiment for the treatment of precocious puberty. The vaginal opening time was delayed under the intervention of cholestyramine, which was significantly later than that of the precocious puberty group, indicating that cholestyramine intervention may be a means of treating or preventing precocious puberty.

[0115] UPLC-MS / MS was performed on the precocious puberty mice and the cholestyramine intervention group to detect the bile acid concentrations in their serum samples and hypothalamus samples. Fig.17UPLC-MS / MS was used to detect the bile acid profile of serum samples of precocious puberty mice and mice in the cholestyramine intervention group. Fig.18 UPLC-MS / MS was used to detect the bile acid spectra of hypothalamic samples of precocious puberty mice and mice in the cholestyramine intervention group. The results showed that the concentration of murine cholic acid bile acids in the serum and hypothalamus of female mice in the cholestyramine group was reduced, while there was no difference in total bile acid and cholic acid bile acids, indicating that the effect of cholestyramine intervention on precocious puberty mice was only to reduce the concentration of murine cholic acid bile acids, and did not affect the concentrations of other bile acids.

[0116] UPLC-MS / MS was further used to detect the concentrations of various types of muricolic acid in serum samples and hypothalamic samples of precocious puberty mice and cholestyramine intervention groups. Fig.19 This is a statistical chart of the 6-hydroxy bile acid concentration in serum samples of precocious puberty mice and mice in the cholestyramine intervention group. Fig. 20 The statistical graphs of various types of bile acids in the hypothalamus samples of precocious puberty mice and mice in the cholestyramine intervention group show 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 role 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.

[0117] Fig.21 The results of WB detection of hypothalamic GnRH expression in precocious puberty mice and mice in the cholestyramine intervention group are shown in Figure 2. Fig. 22 The statistical chart of the expression of GnRH in the hypothalamus of mice with precocious puberty and mice in the cholestyramine intervention group is shown in WB. The results show that the expression of GnRH in the hypothalamus of female mice in the cholestyramine intervention group was significantly reduced. The above experiments revealed that reducing the concentration of 6-hydroxy bile acid is correlated with inhibiting the expression of GnRH. In addition to cholestyramine intervention, other reagents or methods that reduce the concentration of 6-hydroxy bile acid can theoretically inhibit the expression of GnRH, thereby treating precocious puberty.

[0118] Example 4: Mechanism study and treatment of the correlation between 6-hydroxy bile acid and precocious puberty

[0119] In vitro studies were performed using mouse GnRH neuronal cell line GT1-7 cells (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)). Various muricolic acids were used for intervention, and the expression of GnRH in the cell line was detected by WB. Fig.23 The results of WB detection of GnRH expression in GT1-7 cell line. Fig.24 This is a statistical chart of GnRH expression in GT1-7 cell line detected by WB, which shows that the GnRH expression in GT1-7 cells increased significantly under the intervention of three types of muricolic acid, TαMCA, TβMCA and TωMCA.

[0120] In vitro studies were conducted using mouse GnRH neuron cell line GT1-7 cells. Intervention was performed using INT-777, a specific agonist of bile acid receptor TGR5, and WB was used to detect the expression of GnRH in the cell line. Fig.25 The results of WB detection of GnRH expression in GT1-7 cell line. Fig.26 This is a statistical chart of GnRH expression in GT1-7 cell line detected by WB, showing that specific activation of TGR5 receptor causes a significant increase in GnRH expression in GT1-7 cells.

[0121] Further animal testing, Fig. 27 This is a simplified diagram of the animal experiment process for the activation of bile acid receptor TGR5. The bile acid receptor TGR5 specific agonist INT-777 (purchased from MCE, catalog number HY-15677) was used for intraperitoneal injection intervention of mice. Fig.28 The statistical graphs of vaginal opening time and proportion of mice in the INT-777 intervention group and the control group show that the vaginal opening time was advanced under the intervention of the TGR5-specific agonist INT-777, significantly earlier than that in the control group.

[0122] The above INT-777 intervention group and control group were subjected to WB detection to detect the expression of GnRH in the hypothalamus of mice. Fig.29 The results of WB detection of GnRH expression in the hypothalamus of mice in the INT-777 intervention group and the control group. Fig.30 The statistical chart of GnRH expression in the hypothalamus of mice in the INT-777 intervention group and the control group detected by WB shows that the expression of GnRH in the hypothalamus of female mice in the INT-777 intervention group was significantly increased.

[0123] In order to further clarify the key role of TGR5 receptor in the pathogenesis of precocious puberty, TGR5 knockout mice were selected for animal experiments. Fig.31 This is a simplified diagram of the experimental process of bile acid receptor TGR5 knockout animals. The mice were also fed a high-fat diet to model precocious puberty. WT-CD is a control group of mice fed a regular carbohydrate diet, WT-HFD is a control group of mice fed a high-fat diet, TGR5 KO-CD is a TGR5 knockout mouse fed a regular carbohydrate diet, and TGR5 KO-HFD is a TGR5 knockout mouse fed a high-fat diet. Fig.32 The statistical chart of vaginal opening time and proportion of mice in the TGR5 knockout mouse intervention group and the control group shows that after TGR5 knockout, the vaginal opening time was delayed under high-fat diet feeding, which was significantly later than that of wild-type mice fed a high-fat diet.

[0124] Therefore, this embodiment provides one of the mechanisms of the correlation between 6-hydroxy bile acid and precocious puberty. As a bile acid that can activate the TGR5 receptor, the increase in its concentration will promote the activation of the TGR5 receptor, thereby promoting the expression of GnRH and inducing precocious puberty; however, this is only one possible mechanism. In the future, there may be more mechanisms related to 6-hydroxy bile acid and precocious puberty, and the TGR5 receptor is not only activated by binding to 6-hydroxy bile acid, so 6-hydroxy bile acid and TGR5 receptor can both independently serve as targets for the treatment of precocious puberty.

[0125] The above experimental results show that 6-hydroxy bile acid, TGR5 receptor and the occurrence of precocious puberty are correlated. 6-hydroxy bile acid intervention or activation of TGR5 receptor will promote the expression of GnRH in the mouse hypothalamus and induce the occurrence of precocious puberty. Therefore, in theory, it is possible to inhibit 6-hydroxy bile acid or intervene in the related receptors of 6-hydroxy bile acid to prevent the promotion of GnRH expression in the mouse hypothalamus, thereby achieving the effect of preventing or treating precocious puberty. Ways to reduce the concentration of 6-hydroxy bile acid include: inhibiting the absorption of 6-hydroxy bile acid, promoting the metabolism of 6-hydroxy bile acid, and destroying the action pathway related to 6-hydroxy bile acid; inhibiting the absorption of 6-hydroxy bile acid includes cholestyramine intervention as in Example 3, or other reagents or methods. Intervention of 6-hydroxy bile acid-related receptors includes inhibiting TGR5 receptors. TGR5 receptor inhibitors include triamterene (CAS: 396-01-0), TGR5 inhibitors (CAS: 1197300-24-5), etc., or intervention of other 6-hydroxy bile acid-related targets.

[0126] The above detailed description is a specific description of a feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. Use of a biomarker for preparing a reagent for predicting individual precocious puberty, characterized in that: The biomarkers include any one or more 6-hydroxy bile acids.

2. The use according to claim 1, characterized in that The biomarkers include any one or more of αMCA, βMCA, γMCA, ωMCA, TαMCA, TβMCA, TγMCA, TωMCA, GγMCA, HDCA, and THDCA.

3. The use according to claim 2, characterized in that: The reagent is used for detecting biomarkers in individual serum or hypothalamus samples.

4. The use according to claim 3, characterized in that The reagent is used to detect the presence or relative abundance or concentration of a biomarker in a sample.

5. A biomarker combination for predicting individual precocious puberty, characterized in that: The biomarker combination includes any one or more of αMCA, βMCA, γMCA, ωMCA, TαMCA, TβMCA, TγMCA, TωMCA, GγMCA, HDCA, and THDCA.

6. A system for predicting individual precocious puberty, characterized in that: The system includes a data analysis module, which is used to analyze the detection value of the biomarker; the biomarker includes any one or more 6-hydroxy bile acids.

7. The system according to claim 6, characterized in that: The biomarker combination includes any one or more of αMCA, βMCA, γMCA, ωMCA, TαMCA, TβMCA, TγMCA, TωMCA, GγMCA, HDCA, and THDCA.

8. The system according to claim 7, characterized in that: The data analysis module substitutes the detection value of the biomarker into the regression equation to calculate the predicted value of individual precocious puberty, thereby evaluating the risk of individual precocious puberty.

9. The system according to claim 8, characterized in that 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.

10. The system according to claim 9, characterized in that: 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 the biomarkers; the data input interface is used to input the detection values ​​of the biomarkers, and the data output interface is used to output the prediction results.

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