Application of bile acid in prediction of bone mass and application of bile acid as bone metabolism marker

Abstract

The invention discloses application of bile acid in prediction of bone mass and application of bile acid as a bone metabolism marker, and belongs to the technical field of medicines. The technical problems that in the prior art, osteoporosis is difficult to prevent and treat and early intervention drugs are lacked are solved. The invention provides application of bile acid in prediction of bone mass. The bile acid comprises hyodeoxycholic acid. The invention also provides application of bile acid as a bone metabolism marker. The hyodeoxycholic acid in the bile acid is used as an index for evaluating the bone quality, the bone quality can be predicted, and a treatment target is provided for potential osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the application of bile acid in predicting bone mass and as a bone metabolism marker. Background Art

[0002] Osteoporosis is the manifestation of aging in the skeletal system. It is a systemic metabolic bone disease that is mainly caused by bone loss and reduction, destruction of bone tissue microstructure, and increased bone brittleness, which makes patients prone to fractures. Recently, osteoporosis has become an important health problem for people over 50 years old.

[0003] Studying the pathogenesis of osteoporosis is helpful for us to find new therapeutic targets and facilitate the diagnosis and treatment of the disease. However, the pathogenesis of osteoporosis is complex and has many influencing factors. The environmental cells in the bone niche are rich in variety and have close communication. At the same time, although significant progress has been made in the treatment of osteoporosis, poor patient compliance and adverse reactions to drug treatment still exist.

[0004] Therefore, studying the pathophysiological process of osteoporosis and finding potential new treatment options for osteoporosis remain urgent tasks. Summary of the invention

[0005] In view of the above analysis, the present invention aims to provide an application of bile acid in predicting bone mass and preparing anti-osteoporosis drugs, so as to solve the technical problems of osteoporosis prevention and treatment and lack of early intervention drugs in the prior art.

[0006] The purpose of the present invention is mainly achieved through the following technical solutions:

[0007] In one aspect, the present invention provides a use of bile acid in predicting bone mass, wherein the bile acid comprises porcine deoxycholic acid.

[0008] In one possible design, hyodeoxycholic acid is collected from blood samples of study subjects, and the relationship between the relative abundance of hyodeoxycholic acid and bone mineral density is used to predict the subjects' bone mass.

[0009] In one possible design, bone mass predicted by porcine deoxycholic acid is compared with normal bone mass to obtain the bone quality of the subjects.

[0010] In one possible design, the relative abundance of porcine deoxycholic acid and bone mineral density are correlated to predict the researchers' bone mass by establishing a bone mass prediction model.

[0011] In one possible design, the process of establishing a bone mass prediction model includes the following steps:

[0012] Step 1: Data preparation;

[0013] The data on the relative abundance of porcine deoxycholic acid and bone density results were collected and cleaned, including processing missing values, outliers, and duplicate values.

[0014] In a possible design, step 2 is also included, conducting a correlation analysis between the relative abundance of porcine deoxycholic acid and bone density.

[0015] In one possible design, in step 2, the correlation analysis method is selected as the Pearson correlation analysis method;

[0016] The value of the correlation coefficient is calculated by the Pearson correlation analysis method. The value is between -1 and 1. A value close to 1 indicates a strong positive correlation, a value close to -1 indicates a strong negative correlation, and a value close to 0 indicates no obvious linear relationship.

[0017] In one possible design, the bone mass prediction model is validated, including verifying the accuracy and reliability of the bone mass prediction model on an independent sample set.

[0018] In one possible design, the relative abundance of porcine deoxycholic acid is positively correlated with bone mineral density.

[0019] On the other hand, the present invention also provides a use of bile acid as a bone metabolism marker, wherein the bile acid comprises porcine deoxycholic acid.

[0020] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0021] (1) The present invention utilizes porcine deoxycholic acid to predict the bone mass of a subject, providing a new approach to solving the problem of osteoporosis prevention and treatment.

[0022] (2) The present invention can predict the bone mass of researchers by establishing a bone mass prediction model based on the relative abundance of porcine deoxycholic acid and bone density, thus providing a potential therapeutic target for osteoporosis.

[0023] In the present invention, the above-mentioned technical solutions can also be combined with each other to achieve more preferred combination solutions. Other features and advantages of the present invention will be described in the subsequent description, and some advantages can become obvious from the description, or can be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained through the contents particularly pointed out in the embodiments of the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The drawings are only for the purpose of illustrating particular embodiments and are not to be considered limiting of the present invention. Like reference symbols denote like components throughout the drawings.

[0025] Figure 1For the present invention, serum samples of old mice (18-month-old C57 / BL6 mice) and young mice (2-month-old C57 / BL6 mice) were used for bile acid targeted metabolomics testing to evaluate their HDCA content;

[0026] Figure 2 For the present invention, the fifth lumbar vertebrae (L5) samples of old mice (18-month-old C57 / BL6 mice) and young mice (2-month-old C57 / BL6 mice) were subjected to micro-CT examination to evaluate their bone microstructure;

[0027] Figure 3 Represents the correlation analysis using HDCA abundance and bone volume fraction of the fifth lumbar vertebra detected in old mice (18-month-old C57 / BL6 mice) and young mice (2-month-old C57 / BL6 mice);

[0028] Figure 4 The results show that aged mice (18-month-old C57 / BL6 mice) were gavaged with normal saline and HDCA (50 mg / kg / day, for 28 days);

[0029] Figure 5 The results show that wild-type mice (2-month-old C57 / BL6 mice) and TGR5 knockout mice (2-month-old) were gavaged with HDCA (50 mg / kg / day, for 28 days);

[0030] Figure 6a It shows the two-dimensional schematic diagram of the microstructure of the fifth lumbar vertebra of wild-type mice (2-month-old C57 / BL6 mice) and TGR5 knockout mice (2-month-old) after being gavaged with HDCA (50 mg / kg / day, for 28 days);

[0031] Figure 6b It shows the two-dimensional schematic diagram of the microstructure of the fifth lumbar vertebra of TGR5 knockout mice after wild-type mice (2-month-old C57 / BL6 mice) and TGR5 knockout mice (2-month-old) were gavaged with HDCA (50 mg / kg / day, for 28 days);

[0032] Figure 6c The graphs show the microstructure of the fifth lumbar vertebrae of wild-type mice (2-month-old C57 / BL6 mice) and TGR5 knockout mice (2-month-old) after oral gavage with HDCA (50 mg / kg / day for 28 days). DETAILED DESCRIPTION

[0033] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, wherein the accompanying drawings constitute a part of the present invention and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not used to limit the scope of the present invention.

[0034] The present invention provides an application of bile acid in predicting bone mass; the bile acid comprises hyodesoxycholic acid (HDCA), and hyodesoxycholic acid can predict the bone mass of a subject.

[0035] It should be noted that in the intestine, bile acids undergo further metabolism into secondary bile acids through enzymatic reactions of intestinal microorganisms. Hyodesoxycholic acid is a type of secondary bile acid. Experiments have found that the expression of hyodesoxycholic acid (HDCA) in old mice is lower than that in young mice, and both have a strong correlation with bone mass.

[0036] It should be pointed out that bile acid can be used as an indicator for evaluating bone quality. Furthermore, porcine deoxycholic acid in bile acid can be used as an indicator for evaluating bone quality.

[0037] In the present invention, hyodeoxycholic acid is collected from a blood sample of a research object (subject), and the relationship between the relative abundance of hyodeoxycholic acid and bone density is used to predict the bone mass of the research object.

[0038] The present invention can obtain the bone condition of the research subject by comparing the bone mass predicted by swine deoxycholic acid with the normal bone mass. If the actual measured bone density is higher than the normal bone density (or the theoretically calculated bone density), it is normal; if the actual measured bone density is lower than the normal bone density, there is a risk of osteoporosis.

[0039] For example, a patient was found to have a relative abundance of 0.01% of swine deoxycholic acid (HDCA) through bile acid metabolic profile sequencing (i.e., the present invention's blood test for bile acid), which is a relatively low level, so it is predicted that the patient has a greater risk of osteoporosis. The patient underwent a DXA examination, and the results showed that the bone density BMD of the femoral neck was 0.463 g / cm2, and the T value was -3.5, and the patient was diagnosed with osteoporosis, indicating that the prediction was successful.

[0040] In addition, the relative abundance of porcine deoxycholic acid and bone density were used to predict the researchers' bone mass by establishing a bone mass prediction model.

[0041] The process of establishing the above-mentioned bone mass prediction model includes the following steps:

[0042] Step 1: Data preparation;

[0043] The data on the relative abundance of porcine deoxycholic acid and bone density results were collected and cleaned, including processing missing values, outliers, and duplicate values.

[0044] Step 2: Recruit a large number of subjects (100 people) from the normal healthy population, and conduct a correlation analysis between the relative abundance of swine deoxycholic acid and bone density with the support of large sample data. The correlation analysis method is selected as the Pearson correlation analysis method;

[0045] The value of the correlation coefficient is calculated by the Pearson correlation analysis method. The value is between -1 and 1. A value close to 1 indicates a strong positive correlation, a value close to -1 indicates a strong negative correlation, and a value close to 0 indicates no obvious linear relationship.

[0046] Step 3: Interpretation of results;

[0047] Based on the value and sign of the correlation coefficient, explain the relationship between the variables.

[0048] Step 4: significance test;

[0049] Conduct statistical significance tests to determine whether the associations are statistically significant.

[0050] Step 5: Data visualization;

[0051] Use scatter plots to show the relationship between variables and help you understand the correlation intuitively.

[0052] Step 3: Apply.

[0053] The bone mass prediction model was validated, including verifying its accuracy and reliability on an independent sample set.

[0054] The relative abundance of porcine deoxycholic acid was positively correlated with bone density.

[0055] On the other hand, the present invention also provides a use of bile acid as a bone metabolism marker, wherein the bile acid comprises porcine deoxycholic acid.

[0056] It should be noted that the process of predicting bone mass using bile acid in the present invention comprises the following steps:

[0057] S1. Determine the research subjects;

[0058] Select a target population, such as postmenopausal women, the elderly, or patients with known risk of osteoporosis.

[0059] S2, collect blood samples;

[0060] It should be noted that bile acid is a blood sample collected from the research subjects to measure the bile acid level in serum or plasma.

[0061] S3, determining the bile acid content in the sample;

[0062] Accurately measure bile acid levels in blood samples using enzymatic methods or liquid chromatography-mass spectrometry (LC-MS);

[0063] In the above step S3, the process of detecting bile acid using LC-MS is as follows:

[0064] S31, sample preparation;

[0065] Blood samples are collected and subjected to appropriate pretreatment, such as protein precipitation, liquid-liquid extraction or solid phase extraction (SPE) to enrich bile acids and remove impurities.

[0066] S32, chromatographic conditions optimization;

[0067] Select a suitable chromatographic column (such as a C18 column) and mobile phase (such as a mixture of methanol and water), and optimize the gradient elution conditions to achieve effective separation of bile acids.

[0068] S33, optimization of mass spectrometry conditions;

[0069] Set the mass spectrometer parameters, such as the operating mode (positive or negative ion mode) of the electrospray ion source (ESI) and the monitoring ion transitions in the multiple reaction monitoring (MRM) mode.

[0070] S34, standard curve preparation;

[0071] A series of bile acid standard solutions with known concentrations were prepared to establish a standard curve, thereby achieving quantitative analysis of the bile acid concentration in the sample.

[0072] S35. Sample analysis: Inject the processed samples into the liquid chromatography system for chromatography-mass spectrometry analysis.

[0073] S36. Data analysis: By comparing the chromatographic peaks in the sample with the chromatographic peaks of the standard, combined with mass spectrometry data, the bile acids are qualitatively and quantitatively analyzed.

[0074] After qualitative analysis, it was found that bile acid included hyodeoxycholic acid; after quantitative analysis, it was found that the concentration range of hyodeoxycholic acid in bile acid was 1.927-19.266 μg.

[0075] S37, result verification;

[0076] The effectiveness of the method was verified by recovery experiments, precision and accuracy tests.

[0077] S4. Measure the subjects’ bone mass;

[0078] The bone quality detection method of the present invention includes single photon absorptiometry (SPA), dual-energy X-ray absorptiometry (DXA), quantitative computed tomography (QCT), and ultrasonic measurement. The bone quality detection method can be used to obtain bone quality and then evaluate it.

[0079] It should be noted that the DXA detection process of the present invention includes the following steps:

[0080] S1. Patient preparation;

[0081] Patients are asked to remove any metal objects that may affect the measurement.

[0082] S2, positioning;

[0083] The patient is placed on the examination table of the DXA scanner, usually in the supine position.

[0084] S3, scan;

[0085] The scanner emits X-rays and a computer analyzes the attenuation of the X-rays.

[0086] S4. Result analysis;

[0087] The software analyzes the data and generates a report including BMD, T-score, and Z-score.

[0088] S5. Report interpretation;

[0089] The doctor makes a diagnosis and treatment recommendations based on the results of the DXA report and the patient's clinical condition.

[0090] General steps of QCT testing:

[0091] S1. Patient preparation;

[0092] The patient is asked to remove any metal objects that may interfere with the scan and to assume the appropriate position, usually supine, as directed by the technician.

[0093] S2, positioning and scanning;

[0094] The patient is placed in the CT scanner, making sure the part to be scanned (such as the lumbar spine or hip) is within the scanning field, and a calibration phantom may be used to ensure the accuracy of the image.

[0095] S3. Image Acquisition: Use a CT scanner to obtain cross-sectional images of the patient.

[0096] S4. Image Processing: The scanned images need to be processed by specialized QCT analysis software. This typically includes image calibration, segmentation, and determination of the region of interest (ROI).

[0097] S5. Data Analysis: The software analyzes the images and calculates bone density, usually expressed in mg / cm3. QCT can measure the density of cortical bone and cancellous bone separately.

[0098] S6. Result Interpretation: Evaluate the patient's bone density based on the QCT results and compare it with the normal value or the bone density of the same age group.

[0099] S7. Report Compilation: Compile a report based on the analysis results, which may include bone density values, T-scores, Z-scores, and an assessment of the fracture risk.

[0100] S8. Follow-up Guidance: Based on the QCT results, the doctor may recommend that the patient make specific lifestyle changes, receive drug treatment, or other interventions.

[0101] The assessment of bone quality in the present invention includes judging the amount of bone mass, the degree of bone loss, and the assessment of osteoporosis risk.

[0102] Amount of bone mass: Taking DXA as an example, the DXA measures the T-value. T-value: The T-value is calculated by comparing the bone density measured in the patient with a normal reference value and the standard deviation (SD). Normal bone mass: T-value ≥ -1.0; Low bone mass: -2.5 < T-value < -1.0; Osteoporosis: T-value ≤ -2.5; After the patient undergoes a DXA examination, the result shows that the bone mineral density (BMD) of the femoral neck is 0.463 g / cm 2 , the T-value is -3.5, and the diagnosis is osteoporosis. Taking QCT as an example, after the patient undergoes a QCT examination, the result shows that the lumbar spine bone density is 70 mg / cm 3 , and the diagnosis is osteoporosis.

[0103] Degree of bone loss: Taking DXA as an example, after the patient undergoes the first DXA examination, the result shows that the bone mineral density (BMD) of the femoral neck is 0.463 g / cm 2 , the T-value is -3.5, and the diagnosis is osteoporosis. For the second DXA with additional memory, the result shows that the bone mineral density (BMD) of the femoral neck is 0.784 g / cm2, the T-value is -1.9, and the diagnosis is osteopenia, and bone loss is inhibited (bone mass increases).

[0104] Fracture risk assessment: Combine the FRAX score to assess the fracture risk of the patient.

[0105] S4. Data Analysis;

[0106] The Pearson correlation coefficient method was used to analyze the correlation between bile acid levels and bone density.

[0107] Calculate the Pearson correlation coefficient (r) using the following formula:

[0108]

[0109] Where n is the sample size, x and y are the values ​​of the variable, and ∑ represents the sum.

[0110] S5. Establish a bone mass prediction model;

[0111] Based on the correlation analysis between bile acid levels and bone density, a mathematical model was established to predict individual bone density.

[0112] In step S5, the correlation analysis between bile acid level and bone density includes the following steps:

[0113] S51, data preparation;

[0114] Collect relevant data and clean it, including handling missing values, outliers, and duplicate values.

[0115] The above-mentioned relevant data refer to the relative abundance of swine deoxycholic acid obtained by individual intestinal flora sequencing obtained by S36 and the bone density results.

[0116] S52. Recruit a large number of subjects from the normal and healthy population, expand the sample size, and conduct the above-mentioned tests and statistical results. With the support of large sample data, conduct a correlation analysis between the relative abundance of swine deoxycholic acid and bone density.

[0117] S53, selecting the Pearson correlation analysis method as the correlation analysis method;

[0118] The value of the correlation coefficient is calculated using the formula in the Pearson correlation analysis method. The value is between -1 and 1. A value close to 1 indicates a strong positive correlation, a value close to -1 indicates a strong negative correlation, and a value close to 0 indicates no obvious linear relationship.

[0119] S54, result interpretation;

[0120] Based on the value and sign of the correlation coefficient, explain the relationship between the variables.

[0121] S55, significance test;

[0122] Conduct statistical significance tests to determine whether the associations are statistically significant.

[0123] S56, data visualization;

[0124] Use scatter plots to show the relationship between variables and help you understand the correlation intuitively.

[0125] S57, application;

[0126] S6. Validation of the bone mass prediction model;

[0127] The accuracy and reliability of the bone mass prediction model were validated on an independent sample set.

[0128] S7. Clinical application;

[0129] Translate research findings into clinical applications, such as developing an osteoporosis risk assessment tool based on bile acid levels.

[0130] It should be noted that in the process of predicting bone mass using bile acid in the present invention, the relative abundance of swine deoxycholic acid can be obtained by testing the bile acid content of the target population, and the relative abundance of swine deoxycholic acid is brought into the Pearson correlation analysis method in step S3 to calculate the theoretical bone density, which is compared with the actual measured bone density; if the actual measured bone density is higher than the theoretically calculated bone density, it is normal; if the actual measured bone density is lower than the theoretically calculated bone density, there is a risk of osteoporosis.

[0131] It should be noted that, through experiments, it was found that the expression of hyodesoxycholic acid (HDCA) of the present invention in old mice was lower than that in young mice, and both of them had a strong positive correlation with bone mass.

[0132] The present invention also provides a bile acid comprising porcine deoxycholic acid.

[0133] The present invention also provides related products of hyodeoxycholic acid, including hyodeoxycholic acid and conventional excipients. Among them, conventional excipients include: diluents: such as lactose, microcrystalline cellulose, starch; used to increase volume and improve compression molding. Binders: such as starch slurry, cellulose derivatives, povidone, gelatin, used to improve particle properties. Disintegrants: such as cross-linked sodium carboxymethyl cellulose and corn starch, used to promote tablet disintegration in the gastrointestinal tract. Lubricants: such as magnesium stearate, micropowder silica gel, talcum powder, used to reduce friction between particles.

[0134] The process of preparing hyodeoxycholic acid-related products using bile acid comprises the following steps:

[0135] S1. Preparation of HDCA;

[0136] S11, raw material acquisition;

[0137] HDCA can be extracted from pig bile or produced by chemical synthesis or biosynthesis.

[0138] S12, extraction and purification;

[0139] HDCA is extracted from the raw materials by organic solvent extraction and chromatography technology, and then purified.

[0140] In the above step S12, the process of extracting hyodeoxycholic acid (HDCA) from pig bile includes the following steps:

[0141] S121. Raw material preparation: Take fresh pig bile, or use the mother liquor from which calcium bile salts are filtered out during the bilirubin production process.

[0142] S122. Saponification: Mix the bile with sodium hydroxide, add water, and saponify at 95-100°C for 16-24 hours. Saponification is the process of converting bile acids into their sodium salts for subsequent extraction.

[0143] S123, acidification: After saponification, cool to room temperature, let stand, siphon off the supernatant, add water to the lower paste, stir and dissolve, then add strong acid (such as hydrochloric acid or sulfuric acid) to acidify until the Congo red test paper turns blue. This step is to convert the saponified bile acid into free bile acid.

[0144] S124, extraction: adding ethyl acetate and stirring for extraction for 20-50 minutes. After standing for stratification, the aqueous phase is discarded and the organic phase (containing bile acid) is subjected to subsequent treatment.

[0145] S125, washing: adding water to the ethyl acetate phase to wash until the pH value of the aqueous phase is 6-7.

[0146] S126, drying and purification: adding anhydrous sodium sulfate to dry the organic phase, and then filtering and concentrating to obtain a crude product of hyodeoxycholic acid.

[0147] S127, crystallization and refining: The crude product is crystallized and refined to obtain high-purity hyodeoxycholic acid finished product.

[0148] S128. Quality control: The purity of bile acids is tested by analytical methods such as HPLC to ensure product quality.

[0149] Through the above treatment process, high-purity hyodeoxycholic acid can be extracted, and the purity thereof is greater than 98% as determined by HPLC.

[0150] S2, formulation development;

[0151] The purified HDCA is combined with conventional excipients to develop into pharmaceutical preparations such as tablets and capsules.

[0152] S3. Quality control;

[0153] The preparation is subjected to quality control, including determination of the HDCA content; the animal experiment was conducted at 50 mg / kg / day.

[0154] The prepared porcine deoxycholic acid-related products are used in preclinical studies: pharmacodynamic and toxicology studies are conducted in animal models to evaluate the safety and efficacy of the drug.

[0155] Then, clinical trials are conducted to verify the safety and efficacy of the drug in humans.

[0156] After completing the clinical trials, a new drug application was submitted to the drug regulatory agency. After approval, large-scale production was carried out to obtain related products of swine deoxycholic acid.

[0157] In the above step 1, HDCA can also be prepared by the following method, which specifically includes the following process:

[0158] S11, raw material preparation;

[0159] Choose fresh pig bile as raw material.

[0160] S12, saponification;

[0161] Mix pig bile with a strong alkaline aqueous solution and saponify at 95-100°C for 16-24 hours. The purpose of bile acid saponification is to extract and purify bile acid. It helps to improve the purity of bile acid and lay the foundation for subsequent purification and application.

[0162] S13, acidification;

[0163] After saponification, cool to room temperature, let stand, and then siphon off the supernatant; add water to the lower paste and stir to dissolve, then add strong acid for acidification until the Congo red test paper turns blue.

[0164] In the above step S3, the purpose of acidification after the bile acid saponification is to convert the bile acid salts formed in the saponification reaction into free bile acid. Free bile acid is insoluble in water and can be precipitated by pH adjustment during the acidification process, thereby achieving the extraction and purification of bile acid.

[0165] S14, extraction;

[0166] After acidification, ethyl acetate was added and stirred for extraction for 20-50 min. After standing for stratification, the aqueous phase was discarded, and the ethyl acetate phase was washed with water until the pH value of the aqueous phase was 6-7 to obtain an organic phase.

[0167] S15, drying;

[0168] The obtained organic phase is dried, and then concentrated and dried to obtain a crude product of hyodeoxycholic acid.

[0169] S16, refining;

[0170] The crude product is further purified by column chromatography, crystallization or HPLC to improve the purity of the product.

[0171] S17. Quality inspection.

[0172] The obtained hyodeoxycholic acid is subjected to quality tests, including its purity and possible impurities.

[0173] The hyodeoxycholic acid of the present invention is an organic compound with a molecular formula of C 24 H 40 O4 is a bile acid extracted from pig bile. It is white or slightly yellow powder with a bitter taste and a slightly fishy smell. It is slightly soluble in alcohol, slightly soluble in acetone, very slightly soluble in ether and chloroform, and almost insoluble in water.

[0174] Compared with the prior art, the abundance of hyodeoxycholic acid can be used as a prediction index and can also be used to prevent osteoporosis through additional supplementation. Therefore, the present invention can predict osteoporosis using the abundance of hyodeoxycholic acid in bile acid.

[0175] Example 1

[0176] This example is based on the application of the above-mentioned bile acid in predicting bone mass and preparing anti-osteoporosis drugs, and is used to explore the relationship between porcine deoxycholic acid and osteoporosis.

[0177] In this example, the content of porcine deoxycholic acid in young mice and old mice was detected respectively. The average value of old mice was about 8.77 ng / ml; the average value of young mice was about 20.77 ng / ml. Figure 1 As shown; then the bone microstructure of young mice and old mice was measured respectively. The average bone volume fraction of young mice was 0.3863; the average bone volume fraction of old mice was 0.2596,

[0178] like Figure 2 As shown; Finally, the correlation analysis of the two was conducted, and the conclusion was: HDCA has a positive correlation with bone mass. The analysis results are shown in the figure Figure 3 As shown; wherein, the process of performing correlation analysis includes the following steps:

[0179] S1. Data preparation:

[0180] The present invention obtains the relative abundance and bone density results of swine deoxycholic acid through mouse intestinal flora sequencing.

[0181] S2. Select the correlation analysis method as Pearson correlation coefficient to calculate the correlation coefficient.

[0182] S3. The result R2 is 0.626, and the direction is positive correlation.

[0183] S4. The results obtained were statistically different, P<0.0001, and the difference was statistically significant

[0184] S5. Data visualization: Use scatter plots to show the relationship between variables and help intuitively understand the correlation (such as Figure 3 shown).

[0185] Then, HDCA was administered to the elderly mice. After administration, the dynamic bone density results showed that in the first week, the bone density of both groups decreased (self-control before and after, change from baseline). In the fourth week, the bone density of the HDCA group decreased by an average of 0.4397%, and the control group decreased by an average of 2.59%. The statistical difference P value = 0.07, which shows that HDCA has an anti-bone loss effect. Conclusion: HDCA can increase bone density (such as Figure 4 shown).

[0186] At the same time, we also used gene knockout mice to prove that the therapeutic effect of HDCA depends on the presence of TGR5 (TGR5 is a bile acid receptor, and bile acid needs to bind to bile acid receptors to exert its effect). The specific process is: first, HDCA was gavaged orally, and although bone loss was reduced, the difference was not significant (p = 0.07); then, the bile acid receptor TGR5 was knocked out, and it was found that it had no effect. Figure 5 and Figure 6a and Figure 6c shown.

[0187] In the present invention, Figure 1 The results show that the HDCA content of serum samples from old mice (18-month-old C57 / BL6 mice) and young mice (2-month-old C57 / BL6 mice) was evaluated by bile acid targeted metabolomics. The results showed that the HDCA content of young mice was higher than that of old mice, and the difference was statistically significant.

[0188] Figure 2 The figure shows the evaluation of bone microarchitecture using micro-CT of the fifth lumbar vertebrae (L5) of old mice (18-month-old C57 / BL6 mice) and young mice (2-month-old C57 / BL6 mice). The results show that the bone volume fraction of young mice is greater than that of old mice, and the difference is statistically significant.

[0189] Figure 3 The figure shows the correlation analysis between the HDCA abundance and the bone volume fraction of the fifth lumbar vertebra detected in old mice (18-month-old C57 / BL6 mice) and young mice (2-month-old C57 / BL6 mice). The results showed that the HDCA abundance and the bone volume fraction of the fifth lumbar vertebra showed a strong positive correlation.

[0190] Figure 4The results show that elderly mice (18-month-old C57 / BL6 mice) were gavaged with normal saline and HDCA (50 mg / kg / day, for 28 days). The dynamic bone density results showed that in the first week, the bone density of both groups decreased (self-control before and after, change from baseline). In the fourth week, the bone density of the HDCA group increased, while the control group still decreased. This suggests that HDCA has an anti-bone loss effect.

[0191] Figure 5 .It shows that wild-type mice (2-month-old C57 / BL6 mice) and TGR5 knockout mice (2-month-old) were gavaged with HDCA (50 mg / kg / day, for 28 days). The dynamic bone density results showed that in the fourth week, the bone density of the wild-type mice group taking HDCA increased (self-before and after control, change from baseline), while that of the TGR5 knockout mice still decreased. This shows that the anti-bone loss effect of HDCA depends on TGR5.

[0192] Figure 6 shows that wild-type mice (2-month-old C57 / BL6 mice) and TGR5 knockout mice (2-month-old) were gavaged with HDCA (50 mg / kg / day for 28 days). Micro-CT was performed on the fifth lumbar vertebra (L5) samples to evaluate their bone microstructure. The results showed that at the fourth week, the bone volume fraction of the wild-type mice group taking HDCA was higher than that of the TGR5 knockout mice, and the difference was statistically significant. This shows that the effect of HDCA on improving the bone microstructure of mice depends on TGR5. ( Figure 6a and 6b It is a two-dimensional schematic diagram of the microstructure of the fifth lumbar vertebra; Figure 6c (Figure 2 is a statistical diagram of the microstructure of the fifth lumbar vertebra).

[0193] In summary, HDCA in C57 / BL6 mice shows a decreasing trend with age; therefore, HDCA is an indicator of aging. In addition, HDCA is strongly positively correlated with bone mass. The higher the serum HDCA content, the denser the lumbar bone microstructure; therefore, HDCA is a potential therapeutic target for osteoporosis.

[0194] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. An application of bile acid in predicting bone mass, characterized in that: The bile acid includes hyodeoxycholic acid.

2. The use of bile acid in predicting bone mass according to claim 1, characterized in that: Hyodeoxycholic acid was collected from the subjects' blood samples, and the relationship between the relative abundance of hyodeoxycholic acid and bone density was used to predict the subjects' bone mass.

3. The use of bile acid in predicting bone mass according to claim 2, characterized in that: The bone mass predicted by porcine deoxycholic acid was compared with the normal bone mass to obtain the bone quality of the subjects.

4. The use of bile acid in predicting bone mass according to claim 3, characterized in that: The relative abundance of porcine deoxycholic acid and bone density are used to predict the researcher's bone mass by establishing a bone mass prediction model.

5. The use of bile acid in predicting bone mass according to claim 4, characterized in that: The establishment process of the bone mass prediction model comprises the following steps: Step 1: Data preparation; The data on the relative abundance of porcine deoxycholic acid and bone density results were collected and cleaned, including processing missing values, outliers, and duplicate values.

6. The use of bile acid in predicting bone mass according to claim 5, characterized in that: The method also includes step 2, conducting a correlation analysis between the relative abundance of porcine deoxycholic acid and bone density.

7. The use of bile acid in predicting bone mass according to claim 6, characterized in that: In step 2, the correlation analysis method is selected as the Pearson correlation analysis method; The value of the correlation coefficient is calculated by the Pearson correlation analysis method. The value is between -1 and 1. A value close to 1 indicates a strong positive correlation, a value close to -1 indicates a strong negative correlation, and a value close to 0 indicates no obvious linear relationship.

8. The use of bile acid in predicting bone mass according to claim 7, characterized in that: The bone mass prediction model is validated, including validating the accuracy and reliability of the bone mass prediction model on an independent sample set.

9. The use of bile acid in predicting bone mass according to claim 8, characterized in that: The relative abundance of hyodeoxycholic acid is positively correlated with bone density.

10. A use of bile acid as a bone metabolism marker, characterized in that: The bile acid includes hyodeoxycholic acid.

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