Detection method for applying lactoferrin to osteoporosis prevention direction

Abstract

The invention relates to the technical field of osteoporosis prevention, and discloses a detection method for applying lactoferrin to the osteoporosis prevention direction, and the method comprises the following steps: S1, carrying out an experiment on a mouse, carrying out peaceful treatment on the mouse after the experiment is completed, and collecting blood, liver and femur samples; s2, scanning the femur sample through a micro-CT instrument, and processing the femur sample into a three-dimensional view for analysis and bone parameter analysis; s3, decalcifying the femur sample, not decalcifying the liver sample, then treating, slicing and drying, and then carrying out HE staining and immunohistochemical staining; s4, the blood is subjected to centrifugal treatment, and serum is obtained. According to the application disclosed by the invention, experiments are carried out on mice and samples are detected, so that the lactoferrin is determined to be capable of promoting the liver to synthesize LCAT, up-regulating the abundance of Akkermansia in intestinal flora and improving bone metabolism, and the lactoferrin has a relatively good effect of preventing osteoporosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of osteoporosis prevention, and particularly relates to a detection method for the application of lactoferrin in the direction of osteoporosis prevention. Background Art

[0002] Lactoferrin is a multifunctional glycoprotein present in mammalian milk, with various biological activities such as antibacterial, antiviral, antioxidant, and immunomodulatory effects. It can bind to iron ions, limit the iron supply required for bacterial growth, and thus inhibit bacterial reproduction. In addition, lactoferrin also participates in regulating the immune system, enhancing the body's resistance to infections, and is particularly important for the growth and development of infants and young children.

[0003] Currently, the clinical prevention and treatment methods for osteoporosis are mostly monoclonal antibodies. However, for the general population or those who have not reached the diagnostic criteria, the willingness and compliance to use drug intervention are often low. Therefore, dietary supplements from natural foods that can effectively prevent osteoporosis have a higher acceptance. According to research, lactoferrin can prevent osteoporosis through the mediation of the liver and gut microbiota. Therefore, the present invention is needed to detect and verify the role of lactoferrin in the direction of osteoporosis prevention. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the present invention provides a detection method for the application of lactoferrin in the direction of osteoporosis prevention, which can overcome or at least partially solve the above problems.

[0005] The present invention is implemented as follows. A detection method for the application of lactoferrin in the direction of osteoporosis prevention includes the following steps: S1. Use mice for experiments. After the experiments are completed, euthanize the mice and collect blood, liver, and femoral samples. S2. Scan the femoral samples with a micro-CT scanner, and process them into three-dimensional views for analysis and bone parameter analysis. S3. Decalcify the femoral samples first. The liver samples do not need to be decalcified. Then, process, slice, dry them, and perform HE staining and immunohistochemical staining. S4. Centrifuge the blood to obtain serum. Use a biochemical detection kit to detect serum transaminases and four lipid items, and use the ELISA method to detect the contents of LCAT, P1NP, and β-CTx in the serum. S5. Total RNA was extracted from the liver using an RNA extraction kit, and the first strand of cDNA was obtained by reverse transcription using a reverse transcription kit. Primers were designed according to the target mRNA sequence. After configuring the SYBR Green reaction system, the reaction plate was placed in a real-time quantitative PCR instrument, and the cycle parameters were set. Using Gapdh as an internal reference, the relative expression level of the target mRNA to the internal reference was calculated by the 2^(ΔΔCt) method; S6. Liver tissue samples were subjected to lysis, centrifugation, determination of protein concentration by the BCA method, boiling for denaturation, SDS-PAGE electrophoresis, wet transfer for membrane transfer, blocking, incubation with primary and secondary antibodies, ECL luminescence detection, and quantitative analysis using ImageJ software, and finally standardized data on the expression level of the target protein were obtained; S7. Fresh fecal samples were analyzed for microbial composition by 16S rDNA sequencing technology, including sample collection, DNA extraction, PCR amplification, sequencing, and data processing; S8. The test results were all expressed as "mean ± standard deviation (mean ± SD)"; SPSS 21.0 software was used to statistically analyze the test data, and the P value was calculated using one-way analysis of variance (One-Way ANOVA).

[0006] Preferably, in the present invention, the blood of the mice was centrifuged at 3000 rpm for 5 minutes.

[0007] Preferably, in the present invention, the supernatant protein sample was prepared by lysing liver tissue samples with a protein extraction lysate for 30 minutes and then centrifuging at 12000×g for 15 minutes at 4°C.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: By using mice for experiments and detecting samples, the present invention determined that lactoferrin can promote the synthesis of LCAT in the liver, and at the same time up-regulate the abundance of Akkermansia in the intestinal flora, can improve bone metabolism, and has a good effect on preventing osteoporosis. Description of the Drawings

[0009] Figure 1 is a schematic diagram of the effect of lactoferrin on bones provided by an embodiment of the present invention; Figure 2 is a schematic diagram of the effect of lactoferrin on osteogenesis and osteoclastogenesis provided by an embodiment of the present invention; Figure 3 is a schematic diagram of the effect of lactoferrin on the liver and four lipid parameters provided by an embodiment of the present invention; Figure 4 is a schematic diagram of the effect of lactoferrin on the protein and mRNA expression in the liver provided by an embodiment of the present invention; Figure 5It is a schematic diagram of the effect of lactoferrin on the intestinal flora provided by an embodiment of the present invention. Detailed implementation manners

[0010] To further understand the content, features and effects of the present invention, the following embodiments are exemplified and described in detail in conjunction with the accompanying drawings.

[0011] The structure of the present invention will be described in detail below in conjunction with the accompanying drawings.

[0012] As Figures 1 to 4 shown, a detection method for applying a lactoferrin to the prevention of osteoporosis provided by an embodiment of the present invention includes the following steps: S1. Use mice for experiments. After the experiments are completed, euthanize the mice and collect blood, liver and femur samples. S2. Scan the femur samples with a micro-CT scanner, and process them into three-dimensional views for analysis and bone parameter analysis. S3. Decalcify the femur samples first. The liver samples do not need to be decalcified. Then process, slice and dry them, and then perform HE staining and immunohistochemical staining. S4. Centrifuge the blood to obtain serum; use a biochemical detection kit to detect serum transaminases and four lipid items, and use the ELISA method to detect the contents of LCAT, P1NP and β-CTx in the serum. S5. Use an RNA extraction kit to extract total RNA from the liver, use a reverse transcription kit to perform reverse transcription to obtain the first strand of cDNA. Design primers according to the target mRNA sequence. After configuring the SYBR Green reaction system, place the reaction plate in a real-time quantitative PCR instrument, set the cycling parameters, use Gapdh as an internal reference, and calculate the expression level of the target mRNA relative to the internal reference by the 2^(ΔΔCt) method. S6. The liver tissue samples are lysed, centrifuged, the protein concentration is measured by the BCA method, boiled and denatured, subjected to SDS-PAGE electrophoresis, transferred to a membrane by the wet transfer method, blocked, incubated with primary and secondary antibodies, detected by ECL luminescence, and quantitatively analyzed by ImageJ software to finally obtain the standardized data of the target protein expression level. S7. Analyze the microbial composition of fresh fecal samples by 16S rDNA sequencing technology, including sample collection, DNA extraction, PCR amplification, sequencing and data processing. S8. The test results are all expressed as "mean ± standard deviation (mean ± SD)"; use SPSS 21.0 software to statistically analyze the test data, and calculate the P value by one-way ANOVA.

[0013] Preferably, the blood of the mice was centrifuged at 3000 rpm for 5 minutes.

[0014] Preferably, the supernatant protein sample was prepared by lysing the liver tissue sample with a protein extraction and lysis solution for 30 minutes and then centrifuging at 12000×g for 15 minutes at 4°C.

[0015] Grouping and Sampling of Mice The mice used in this experiment were 6- to 8-week-old male C57BL / 6J mice, housed in an SPF environment. After 1 week of environmental adaptation, the mice were randomly divided into 3 groups for intervention: (1) The control group (CON) was fed AIN-93G diet and gavaged with sterile water daily. (2) The high-fat diet group (HFD) was fed a high-fat diet with a 60% fat energy ratio and gavaged with sterile water daily. (3) The high-fat diet combined with lactoferrin group (HFD+Lf) was fed a high-fat diet with a 60% fat energy ratio and gavaged with lactoferrin (100 mg / kg BW) solution daily. The intervention lasted for 8 weeks.

[0016] One day before the intervention, mouse feces were collected for 16S rDNA sequencing. After the intervention ended, the mice were fasted overnight, euthanized, and blood, liver, and femur samples were collected for subsequent detection.

[0017] Micro-CT Scanning Open the SkyScan1176 software and preheat the micro-CT instrument. After removing the sample chamber, place the femurs in the specified positions in the preset order and close the chamber door. Set the scanning parameters as X-ray voltage 50 kV, X-ray current 200 μA. Turn on the X-ray source for position correction scanning to ensure that the region of interest is within the scanning range. Select a 0.5 mm Al filter and set the scanning conditions as 4000 resolution, 9 μm thickness, and 70° rotation angle. Rename the specimens and select the storage location, then start scanning. After completion, remove the specimens. Then, use the NRecon software to open the scanning file, locate the preview position and select the reconstruction region, set the threshold to 0-0.075, set the save path and add it to the waiting area. After all specimens are added, click Start for two-dimensional reconstruction. Subsequently, use the Dataview software to open the reconstruction folder in a three-dimensional view, rotate the specimens to the position where the X, Y, and Z axes are consistent and save them in the database format. Use the CTAn software to open the folder, locate the 30th and 180th layers on the growth plate as the fixed analysis layers, circle the region of interest and set the threshold (65, 255), analyze the bone density data and create an Slt three-dimensional original file, conduct other bone parameter analyses, and finally open the 3D images of each specimen with the Mimics software and save the images at the same angle.

[0018] Use SkyScan 1176 software for micro-CT scanning, set parameters and scan the femoral samples, then perform two-dimensional reconstruction with NRecon software, and conduct three-dimensional view analysis and bone parameter analysis with Dataview and CTAn software. Finally, save the 3D images with Mimics software.

[0019] HE staining and immunohistochemistry The femoral samples need to be decalcified first, while the liver samples do not require decalcification. After decalcification, the femoral samples of each group are sequentially placed in 70%, 80%, 90%, 95%, and 100% ethanol for gradient dehydration for 1 hour, placed in a transparent dewaxing solution for transparency for 4 - 6 hours, and finally soaked in a paraffin solution overnight. Use an embedding machine to melt paraffin and embed the samples, cut the specimens with a vibratome, and bake the sections for 2 hours after spreading.

[0020] HE staining (1) Observe under the microscope and select paraffin sections; (2) Dewax twice with an environmentally friendly transparent dewaxing solution, 15 minutes each time; (3) Immerse in 100% ethanol once, 5 minutes each time; (4) Immerse in 90% ethanol once, 5 minutes each time; (5) Immerse in 80% ethanol once, 5 minutes each time; (6) Immerse in 70% ethanol once, 5 minutes each time; (7) Wash with deionized water twice, 1 minute each time; (8) Stain with hematoxylin solution for 2 minutes; (9) Wash with deionized water until there is no obvious purple color; (10) Immerse in hydrochloric acid differentiation solution for 15 seconds; (11) Wash with deionized water once, 1 minute each time (12) Immerse in ammonia water for 1 minute; (13) Stain with eosin solution for 5 minutes; (14) Immerse in 70% ethanol once, 1 minute each time; (15) Immerse in 80% ethanol once, 1 minute each time; (16) Immerse in 90% ethanol once, 1 minute each time; (17) Immerse in 100% ethanol once, 1 minute each time; (18) Transparency with an environmentally friendly transparent dewaxing solution for 1 minute; (19) Mount with neutral resin.

[0021] Immunohistochemical staining (1) Place the sections in a 65°C oven and bake for 2 hours; (2) Dewax twice with an environmentally friendly transparent dewaxing solution, 15 minutes each time; (3)Once with 100% ethanol for 5 minutes each time; (4)Once with 90% ethanol for 5 minutes each time; (5)Once with 80% ethanol for 5 minutes each time; (6)Once with 70% ethanol for 5 minutes each time; (7)Rinse with deionized water twice for 1 minute each time; (8)Repair under pressure in a sodium citrate antigen retrieval solution in a water bath at 60 °C for 2 hours and then cool to room temperature; (9)Rinse with deionized water twice and then use an immunohistochemistry pen to circle the tissue area; (10)Dropwise add the blocking solution and block at room temperature for 30 minutes, then drain the liquid; (11)Dropwise add the appropriately diluted primary antibody (the primary antibodies RUNX2 and SP7 are both diluted at a dilution ratio of 1:1000, and SIRT1 is diluted at a ratio of 1:100) and place in a 4 °C refrigerator overnight; (12)The next day, dropwise add the biotin antibody and incubate at 37 °C for 30 minutes, then rinse with deionized water 3 times; (13)Dropwise add the SABC-AP tertiary antibody and incubate at room temperature for 30 minutes, then rinse with deionized water 3 times; (14)Dropwise add the BCIP / NBT chromogenic solution, observe the positive area under the microscope, and soak and rinse with deionized water; (15)Counterstain with hematoxylin for 2 minutes; (16)Rinse with deionized water until there is no obvious purple color; (17)Immerse in the hydrochloric acid differentiation solution for 15 seconds; (18)Rinse with deionized water once for 1 minute each time (19)Once with 70% ethanol for 1 minute each time; (20)Once with 80% ethanol for 1 minute each time; (21)Once with 90% ethanol for 1 minute each time; (22)Once with 100% ethanol for 1 minute each time; (23)Clear with an environmentally friendly transparent dewaxing solution for 1 minute; (24)Seal with neutral resin.

[0022] TRAP staining (1)Put the sections in a 65 °C baking oven and bake for 2 hours; (2)Dewax twice with an environmentally friendly transparent dewaxing solution for 15 minutes each time; (3)Once with 100% ethanol for 5 minutes each time; (4)Once with 90% ethanol for 5 minutes each time; (5)Once with 80% ethanol for 5 minutes each time; (6) 70% ethanol once for 5 minutes each time; (7) Deionized water washing twice for 1 minute each time; (8) Prepare an appropriate amount of staining solution by mixing sodium tartrate solution, TRAP substrate solution A and TRAP substrate solution B in a ratio of 1:9:0.1. Drop the TRAP staining solution within the defined range and observe under the microscope. Immediately soak the sections with deionized water. (9) Counterstain with hematoxylin for another 2 minutes; (10) Wash with deionized water until there is no obvious purple color; (11) Immerse in hydrochloric acid differentiation solution for 15 seconds; (12) Deionized water washing once for 1 minute each time (13) 70% ethanol once for 1 minute each time; (14) 80% ethanol once for 1 minute each time; (15) 90% ethanol once for 1 minute each time; (16) 100% ethanol once for 1 minute each time; (17) Immerse the sections in an environmentally friendly transparent dewaxing solution for 1 minute; (18) Mount the sections with a mounting medium.

[0023] Serological index detection Centrifuge the blood at 3000 rpm for 5 minutes to obtain serum. Use a biochemical detection kit to detect serum transaminases and four lipid items, and use the ELISA method to detect the contents of LCAT, P1NP and β-CTx in the serum. The specific operation is carried out according to the instructions.

[0024] qRT-PCR Use an RNA extraction kit to extract total RNA from the liver, use a reverse transcription kit to perform reverse transcription to obtain the first strand of cDNA. Design primers according to the target mRNA sequence, configure the SYBR Green reaction system, then place the reaction plate in a real-time quantitative PCR instrument, set the cycling parameters, use Gapdh as an internal reference, and calculate the expression level of the target mRNA relative to the internal reference by the 2^(ΔΔCt) method.

[0025] Western blots After the liver tissue sample was lysed with a protein extraction lysis solution for 30 minutes, it was centrifuged at 12,000×g for 15 minutes at 4°C, and the supernatant protein sample was collected. After measuring the protein concentration using the BCA method, the sample was mixed with the loading buffer in proportion and boiled in a metal bath at 100°C for 10 minutes to fully denature the protein. Subsequently, an equal amount of the protein sample and a prestained protein Marker were co-loaded into an SDS-PAGE gel, and electrophoresis separation was carried out under a constant voltage of 80V. The electrophoresis was terminated when the bromophenol blue indicator migrated to the bottom of the gel. The separated protein was transferred to a PVDF membrane using the wet transfer method, and the transfer conditions were set to a constant current of 400 mA and continued for 90 minutes under ice bath conditions. After the transfer was completed, the membrane was blocked with 5% skim milk at room temperature for 1 hour to reduce non-specific binding. The blocked PVDF membrane was incubated with a specific primary antibody overnight at 4°C. After washing 3 times with TBST buffer (10 minutes each time), it was incubated with an HRP-labeled secondary antibody of the corresponding species at room temperature for 1 hour. After washing thoroughly again, an ECL chemiluminescence reagent was evenly covered on the surface of the membrane, and the signal was collected through a chemiluminescence imaging system. Using GAPDH as an internal reference protein, quantitative analysis was performed using ImageJ software, and the expression level of the target protein was standardized by calculating the ratio of the gray value of the target band to the gray value of the internal reference band.

[0026] The liver tissue sample underwent lysis, centrifugation, protein concentration measurement by the BCA method, boiling denaturation, SDS-PAGE electrophoresis, wet transfer membrane transfer, blocking, incubation with primary and secondary antibodies, ECL luminescence detection, and quantitative analysis using ImageJ software, and finally, standardized data on the expression level of the target protein were obtained.

[0027] 16S rDNA sequencing The fresh fecal samples were immediately placed in sterile cryotubes, snap-frozen in liquid nitrogen and transferred to an ultra-low temperature freezer for storage until DNA extraction. A kit was used for the extraction of total microbial DNA. The purity and concentration of the DNA were detected using a Nanodrop 2000 spectrophotometer and a Qubit 4.0 fluorometer respectively, and the qualified samples were used for subsequent amplification. The V3-V4 hypervariable region of the bacterial 16S rRNA gene was selected as the target for PCR amplification. After the amplified products were verified by 1% agarose gel electrophoresis, they were purified and quantified using AMPure XP magnetic beads (Beckman Coulter). Paired-end sequencing was performed using the Illumina NovaSeq 6000 platform. Library construction followed the standard protocol of the TruSeq DNA PCR-Free Library Prep Kit (Illumina), and clusters were generated by bridge PCR amplification with a sequencing depth of ≥50,000 reads / sample. After the primer sequences were removed from the raw data using Cutadapt, the DADA2 pipeline was used for quality control (removing low-quality sequences, chimeras and abnormally long reads) to obtain high-quality Amplicon Sequence Variants (ASVs). Species annotation was based on the Silva 138 or Greengenes 13_8 databases.

[0028] The microbial composition of fresh fecal samples was analyzed by 16S rDNA sequencing technology, including sample collection, DNA extraction, PCR amplification, sequencing and data processing.

[0029] Data analysis The experimental results were expressed as "mean ± standard deviation (mean ± SD)". SPSS 21.0 software was used for the statistical analysis of the experimental data, and the P value was calculated using one-way analysis of variance (One-Way ANOVA).

[0030] Effect It can increase the bone mineral density and bone trabecular structure parameters of mice Bone mineral density is an important indicator for judging bone mass. Bone mineral density is of great significance for the early diagnosis of osteoporosis and the prediction of fracture risk. Therefore, the bone mineral density of the femurs of mice was measured in this experiment. As Figure 1 shown, the bone mineral density of the HFD group was the lowest, equivalent to 76% of the control group, while the bone mineral density of the HFD+Lf group increased compared with the HFD group, with an increase of 21%. This indicates that oral administration of lactoferrin can compensate for the decrease in bone mineral density caused by a high-fat diet.

[0031] Bone volume fraction and trabecular bone thickness are important indicators reflecting trabecular bone structure, and they will decrease when the bone is unhealthy. The bone volume fraction and trabecular bone thickness of mice decreased by 27% and 17% respectively under a high-fat diet, while lactoferrin can increase them by 24% and 15% respectively. This shows that a high-fat diet can cause trabecular bone loss and result in abnormal bones, while lactoferrin can improve this situation.

[0032] Promote osteoblast activity OCN is one of the important biomarkers of osteoblasts and can reflect osteoblast activity. As Figure 2 shown, immunohistochemical results found that a high-fat diet led to a decrease in the OCN positive reaction near the growth plate, while oral administration of lactoferrin reversed this situation. This shows that oral administration of lactoferrin can promote osteoblast activity. Serum P1NP is also a biomarker of osteogenic differentiation. After a high-fat diet, the serum P1NP level decreased significantly, but lactoferrin did not have an obvious effect on it.

[0033] TRAP staining and serum β-CTx level can be used to evaluate the osteoclastic process. The inventors found that neither a high-fat diet nor lactoferrin had an obvious effect on TRAP staining and serum β-CTx level. This shows that lactoferrin mainly prevents osteoporosis by affecting the osteogenic process.

[0034] Can promote serum HDL-C level Serum HDL-C level is an important marker reflecting the ability of reverse cholesterol transport. As Figure 3 shown, compared with the CON group and the HFD group, the serum HDL-C level was significantly increased in the HFD+Lf group, indicating that oral administration of lactoferrin improved the ability of reverse cholesterol transport.

[0035] Can promote the synthesis and secretion of hepatic LCAT LCAT synthesized and secreted by the liver is also an important biological factor in the process of reverse cholesterol transport. Promoting LCAT synthesis and secretion helps to maintain bone homeostasis and plays a bone-protective role. As Figure 4 shown, mice fed a high-fat diet were orally administered lactoferrin, and the serum LCAT level increased by nearly 1 fold. Further, the protein and mRNA expression levels of LCAT were also up-regulated by 54% and 30% respectively. Combining with the background knowledge, promoting the synthesis and secretion of hepatic LCAT by lactoferrin is one of the important mechanisms for its bone-protective effect on osteoporosis.

[0036] Can increase the relative abundance of Akkermansia Akkermansia is a "new generation of probiotics" with multiple biological activities, and an increase in its abundance helps to protect against osteoporosis. As Figure 5As shown, oral lactoferrin significantly increased the relative abundance of Akkermansia. In the CON group and the HFD group, its relative abundances were only 5.6% and 3.4% respectively, while in the HFD+Lf group, it soared to 31.7%. Combining with the background knowledge, it shows that lactoferrin promoting the proliferation of Akkermansia also plays an important role in the prevention of osteoporosis.

[0037] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0038] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art of this patent without departing from the scope of the technical solution of the present invention.

Claims

1. A method for detecting lactoferrin for preventing osteoporosis, characterized in that: The method comprises the following steps: S1. Perform experiments on mice, euthanize the mice and collect blood, liver and femur samples after the experiments are completed; S2, scanning the femur sample by micro-CT instrument and processing it into a three-dimensional view for analysis and bone parameter analysis; S3. Femoral samples need to be decalcified first, but liver samples do not need to be decalcified. Then they are processed, sliced ​​and dried, and then HE staining and immunohistochemical staining are performed. S4, centrifuging the blood to obtain serum; Biochemical test kits were used to detect serum transaminases and four blood lipids, and ELISA was used to detect serum LCAT, P1NP, and β-CTx levels; S5. Use an RNA extraction kit to extract total RNA from the liver, use a reverse transcription kit to perform reverse transcription to obtain the first strand of cDNA, design primers according to the target mRNA sequence, configure the SYBR Green reaction system, place the reaction plate in a real-time quantitative PCR instrument, set the cycle parameters, use Gapdh as the internal reference, and calculate the expression level of the target mRNA relative to the internal reference by the 2^(ΔΔCt) method; S6. Liver tissue samples were lysed, centrifuged, protein concentration was determined by BCA method, boiled and denatured, SDS-PAGE electrophoresis, wet transfer, blocked, incubated with primary and secondary antibodies, ECL luminescence detection and quantitative analysis by ImageJ software to obtain standardized data of target protein expression level; S7. Analysis of microbial composition of fresh fecal samples by 16S rDNA sequencing technology, including sample collection, DNA extraction, PCR amplification, sequencing and data processing; S8. All experimental results were expressed as "mean ± SD". SPSS 21.0 software was used to perform statistical analysis on the experimental data, and one-way ANOVA was used to calculate the P value.

2. The method for detecting lactoferrin for preventing osteoporosis according to claim 1, characterized in that: The mouse blood was centrifuged at 3000 rpm for 5 minutes.

3. The detection method for lactoferrin for preventing osteoporosis according to claim 1, characterized in that: The supernatant protein sample was prepared by lysing the liver tissue sample with protein extraction solution for 30 minutes and then centrifuging at 12000×g for 15 minutes at 4°C.

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