Method for screening cervus elaphus linnaeus bone peptide and application of the peptide

Abstract

The application discloses a method for screening sika deer bone peptide segments and application of the peptide segments, and belongs to the technical field of peptide screening and application. According to the screening method, sika deer cancellous bone samples in five different development periods, i.e., an initial stage of tray shedding, a two-culm early stage, a two-culm stage, a four-culm stage and a complete ossification stage, are collected respectively; the sika deer cancellous bone samples in the five different development periods are mechanically broken respectively, protein extraction is carried out by using a lysis buffer, the extracted protein samples in the five different development periods are subjected to enzyme cutting treatment respectively, five peptide segment samples in the five different development periods are obtained, liquid chromatography-mass spectrometry is used for peptide segment separation and identification, six common difference peptide segments in comparative development periods are obtained, and specific difference peptide segments conforming to the periodic osteoporosis phenomenon of sika deer are screened out. According to the screening method, sika deer bone active peptides for treating osteoporosis can be obtained, and bone formation can be promoted and bone resorption can be inhibited.

Description

Technical Field

[0001] This invention belongs to the field of peptide screening and application technology, specifically relating to a method for screening sika deer bone peptides and the application of peptides, particularly relating to a method for screening peptides that conform to the periodic osteoporosis phenomenon of sika deer, and especially relating to the application of peptides that conform to the periodic osteoporosis phenomenon of sika deer in the preparation of drugs for treating osteoporosis. Background Technology

[0002] With increasing life expectancy, osteoporosis has gradually become a significant health problem, not only causing pain, fractures, and death risks, but also having a significant socioeconomic impact. Approximately 200 million people worldwide suffer from this disease, with an estimated 40% of women and 14% of men over the age of 50 expected to suffer osteoporotic fractures in their lifetime.

[0003] Currently, drug treatment for osteoporosis is mainly divided into two categories: bone formation promoters (such as parathyroid hormone analogs) and bone resorption inhibitors (such as bisphosphonates and denosumab). However, existing drugs have limitations in efficacy and significant side effects. For example, bisphosphonates may cause gastrointestinal reactions and mandibular osteonecrosis, while long-term use of glucocorticoids can directly lead to bone loss. In addition, clinical studies show that the efficacy of drug treatment is uncertain. Regarding non-pharmacological treatments, exercise therapy regulates bone metabolism through mechanical stimulation. For example, aerobic exercise 3-5 times a week for 6 months can increase bone mineral density by 0.28 g / cm² in postmenopausal women. Rehabilitation therapy combined with medication can significantly reduce pain scores (visual analog scale, reduction of up to 50%). However, patient adherence is constrained by multiple factors; approximately 29%-40% of patients discontinue treatment due to concerns about drug side effects, insufficient understanding, or financial burden.

[0004] Sika deer osteoactive peptides can promote bone formation, inhibit bone resorption, and regulate metabolic balance. They are significantly effective for mild to moderate osteoporosis, increasing bone density and relieving pain. However, their effect on patients with severe osteoporosis is limited, requiring combination with other treatments. For example, in existing research, the effects of sika deer osteoactive peptides on the bone microstructure of dexamethasone-induced osteoporotic rats were studied (Chinese Traditional and Herbal Drugs, 2016, Vol. 47, No. 22, pp. 4030-4034). This study investigated the effects of dexamethasone and deer antler peptides on serum biochemical indicators, bone microstructure, and histopathological parameters in rats. The results showed that sika deer osteoactive peptides increased serum Cg2* and BGP levels in GOP model rats while decreasing serum P, ALP, and PTH levels, significantly improving bone microstructure indices in GOP rats.

[0005] However, the existing sika deer bone active peptides have the following technical problems: 1. They cannot simultaneously inhibit osteoclast formation and promote osteoblast formation; 2. They mostly exist in the form of mixtures, are not single peptide compounds, and cannot be artificially synthesized.

[0006] 3. Low purity, large molecular weight, and difficult for the human body to absorb; 4. The regulatory effect on bone metabolism markers needs to be improved, and the specificity is weak. Summary of the Invention

[0007] To address the technical problems in the prior art, this invention provides a method for screening peptides from sika deer bone and the application of these peptides. The aim is to first screen for common differential peptides across six comparative developmental stages of sika deer, based on the unique periodic osteoporosis phenomenon in sika deer bone, and then discover several novel bioactive peptide monomers from sika deer bone. These novel bioactive peptide monomers can significantly inhibit the expression of genes related to osteoclast function and promote the expression of genes related to osteogenic function, effectively inhibiting osteoclast formation and promoting osteoblast formation, thus providing potential drugs for the prevention and treatment of osteoporosis.

[0008] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows.

[0009] In a first aspect, the present invention provides a method for screening sika deer bone peptides, comprising:

[0010] Step 1: Select cancellous bone from sika deer:

[0011] Cancellous bone samples were collected from sika deer at five different developmental stages: early stage of tray detachment (EPOTS), early stage of two bars (BTBP), stage of two bars (TBP), stage of four-pronged detachment (FBP), and stage of complete ossification (COP).

[0012] Step 2, protein extraction:

[0013] Five sika deer cancellous bone samples from different developmental stages were mechanically disrupted, and proteins were extracted using lysis buffer to obtain protein samples from five different developmental stages.

[0014] Step 3, Enzyme digestion treatment:

[0015] The protein samples extracted from five different developmental stages were subjected to enzyme digestion to obtain peptide samples from five different developmental stages.

[0016] Step 4, Liquid Chromatography-Mass Spectrometry Tandem Analysis:

[0017] First, peptide samples from five different developmental stages were separated using high-performance liquid chromatography (HPLC). Then, after ionization, the samples were detected by mass spectrometry (MS) to obtain mass spectrometry data for all peptides corresponding to the five different developmental stages. The differential peptides were compared between the pre-two-bar stage and early tray detachment stage (BTBP vs EPOTS), the two-bar stage and early tray detachment stage (TBP vs EPOTS), the complete ossification stage and early tray detachment stage (COP vs EPOTS), the tetrapod stage and two-bar stage (FBP vs TBP), the two-bar stage and pre-two-bar stage (TBP vs BTBP), and the complete ossification stage and tetrapod stage (COP vs FBP). An upset diagram of the differential peptides from the six comparative developmental stages was plotted and the intersection was taken to obtain the common differential peptides from the six comparative developmental stages.

[0018] Preferably, the method also includes step five, performing heatmap analysis on common differential peptides in six contrasting developmental stages. Using BTBP vs EPOTS, TBP vs EPOTS, and TBP vs BTBP as bone loss stages, and FBP vs TBP, COP vs EPOTS, and COP vs FBP as bone stabilization stages, the method screens for peptides in each bone loss stage where the abundance of the former relative to the latter decreases in both developmental stages (i.e., in BTBP vs EPOTS, BTBP peptide abundance is lower than EPOTS, and in TBP vs EPOTS, TBP peptide abundance is lower than EPOTS). The peptide abundance of POTS decreased, and in TBP vs BTBP, the peptide abundance of TBP decreased compared to BTBP. The peptide abundance of the former increased relative to the latter in two different developmental stages within each bone stabilization period (i.e., in FBP vs TBP, the peptide abundance of FBP increased compared to TBP, in COP vs EPOTS, the peptide abundance of COP increased compared to EPOTS, and in COP vs FBP, the peptide abundance of COP increased compared to FBP). Specific differential peptides consistent with the periodic osteoporosis phenomenon in sika deer were obtained, referred to as sika deer bone active peptides.

[0019] More preferably, the specific differential peptides that conform to the periodic osteoporosis phenomenon in sika deer are one or more of ER-11, FR-14, IK-10, and TR-17;

[0020] The amino acid sequence of ER-11 is EAVQGASDLWR, as shown in SEQ ID 1, and its chemical structure is shown in Formula I:

[0021] ;

[0022] Formula I;

[0023] The amino acid sequence of FR-14 is FSISSDYNLPHILR, as shown in SEQ ID 2, and its chemical structure is shown in Formula II:

[0024] ;

[0025] Formula II;

[0026] The amino acid sequence of IK-10 is IILVNYTYFK, as shown in SEQ ID 3, and its chemical structure is shown in Formula III.

[0027] ;

[0028] Formula III;

[0029] The amino acid sequence of TR-17 is TVEVPMMTLGLETPYFR, as shown in SEQ ID 4, and its chemical structure is shown in Formula IV.

[0030] ;

[0031] Formula IV.

[0032] Preferably, in step two, the lysis buffer contains 1-3 wt% sodium dodecyl sulfate (SDS) and 1 wt% protease inhibitor, the solvent is ultrapure water, and the protease inhibitor is one or more of protease inhibitor Cocktail VI, protease inhibitor Cocktail V, protease inhibitor Cocktail IV, and protease inhibitor Cocktail III.

[0033] Preferably, in step two, the method for extracting proteins using lysis buffer is as follows: First, place five sika deer cancellous bone samples from different developmental stages into mortars pre-cooled with liquid nitrogen, add liquid nitrogen and grind them thoroughly into deer bone powder, then add lysis buffer, sonicate or stir to lyse, then filter or centrifuge to remove cell debris, and obtain the extracted protein samples from five different developmental stages.

[0034] More preferably, in step two, the ratio of the deer bone powder to the lysis buffer is 1g:10mL-1g:20mL.

[0035] More preferably, in step two, the ultrasound duration is 30-60 minutes, the power is 100-300W, and the frequency is 20-80kHz.

[0036] More preferably, in step two, the stirring temperature is 40-60℃ and the stirring time is 2-4h.

[0037] More preferably, in step two, centrifugation is performed at 10,000-13,000 g for 10 min at 4-8 °C; particularly preferably, centrifugation is performed at 12,000 g for 10 min.

[0038] Preferably, in step three, the protease used for the enzymatic digestion is trypsin or pepsin.

[0039] More preferably, in step three, the enzymatic digestion process is as follows: First, take five protein samples of equal mass at five different developmental stages, adjust the volume of the five protein samples at different developmental stages to be consistent with the lysis buffer, then add 1-2 volumes of acetone pre-cooled to 4°C to each sample, vortex to mix, then add 3-5 volumes of acetone pre-cooled to 4°C to each sample, precipitate at 4-8°C for 2-4 hours, centrifuge at 4000-5000g for 5-10 minutes, discard the supernatant, and wash the resulting precipitate with acetone pre-cooled to 4°C. After 2-3 cycles of drying, the precipitates were ultrasonically dispersed with tetraethylammonium bromide at a final concentration of 200-250 mM. Then, protease was added at a mass ratio of protease to protein sample of 1:45-55, and the enzyme was digested for 10-12 h. Then, dithiothreitol at a final concentration of 5-10 mM was added, and the samples were reduced at 50-60 °C for 30-40 min. Finally, iodoacetamide at a final concentration of 10-12 mM was added, and the samples were incubated at room temperature in the dark for 15-20 min to obtain peptide samples at five different developmental stages.

[0040] The lysis buffer contains 1-3 wt% sodium dodecyl sulfate and 1 wt% protease inhibitor, and the solvent is ultrapure water. The protease inhibitor is one or more of protease inhibitor VI, protease inhibitor V, protease inhibitor IV, and protease inhibitor III.

[0041] Preferably, in step four, the conditions for high-performance liquid chromatography are:

[0042] Mobile phase A is an aqueous solution containing 0.1wt%-0.2wt% formic acid and 1.5wt%-2wt% acetonitrile;

[0043] Mobile phase B is an aqueous solution containing 0.1 wt%-0.2 wt% formic acid and 90 wt%-95 wt% acetonitrile;

[0044] Liquid phase gradient settings: 0-22.5 min, 6%-22% mobile phase B; 22.5-26.5 min, 22%-34% mobile phase B; 26.5-28.5 min, 34%-80% mobile phase B; 28.5-30 min, 80% mobile phase B, with the flow rate maintained at 700-750 nl / min.

[0045] Preferably, in step four, the ion source ionization voltage is set to 2200-2300V, and the compensation voltage is set to -40V to -45V.

[0046] Preferably, in step four, the conditions for mass spectrometry are:

[0047] The primary mass spectrometry scanning range was set to 350-1400 m / z, and the scanning resolution was set to 55000-60000.

[0048] The scanning range of the secondary mass spectrometer is fixed at 100-120 m / z, and the scanning resolution is set to 14000-15000.

[0049] The breakage energy is set to 25%-30%, the automatic gain control is set to 1E6, and the maximum injection time is set to 20-24ms.

[0050] Secondly, the present invention provides the application of specific differential peptides obtained by the above-described method for screening sika deer bone peptides that conform to the periodic osteoporosis phenomenon of sika deer in the preparation of drugs for treating or preventing osteoporosis.

[0051] Thirdly, the present invention provides a medicament for treating or preventing osteoporosis, comprising one or more of the aforementioned ER-11, FR-14, IK-10, and TR-17.

[0052] It should be noted that the preferred dosage is 10-50 μg / kg.

[0053] It should be noted that the initial stage of antler detachment is also called the detachment period. Timing: Usually in spring (March-May). Morphological characteristics: The old ossified antler naturally detaches from the special bone tissue "antler support" or "antler disc" at the junction of the antler base (antler crown) and the skull. After detachment, an open, circular wound forms on the surface of the antler disc; new tissue: The wound is rapidly covered and healed by granulation tissue rich in blood vessels. The skin around the antler disc begins to thicken and bulge, forming obvious "velvet buds" or "antler base buds." The surface of the velvet buds is covered with fine hairs, pink or light red in color, very soft in texture, and rich in nerves and blood vessels, marking the beginning of new antler growth for the year. At this time, a distinct branch structure has not yet formed; it is mainly a low, hemispherical or conical protrusion.

[0054] The early stage of the second branch is also known as the single-branch bud stage. Timing: Immediately following the bud shedding stage, in the early growth phase (approximately 1-2 weeks to 1 month after bud shedding). Morphological characteristics: The bud grows rapidly upwards, forming a single, unbranched main stem. The main stem is cylindrical with a rounded tip. Surface characteristics: The entire main stem is covered with dense, soft, fine hairs (velvety skin). The hairs are usually light brown or grayish-yellow. The skin is thin and rich in blood vessels, bright red or pink in color, and feels warm, soft, and elastic. Growth direction: The main stem primarily extends upwards, growing extremely rapidly. At this stage, there are no signs of branching.

[0055] The two-bar stage is also known as the initial branching stage of the brow twig. Timing: After the main stem has grown to a certain height (usually a certain distance from the antler plate, such as about 10-20 cm). Morphological characteristics: The top of the main stem begins to swell, and the first branch, called the "brow twig" (or first lateral branch), differentiates from it. The brow twig and the main stem form a distinct "Y" shape. Structural details: Main stem (main branch): Continues to grow upwards, but the swelling at the top indicates subsequent branching points. Brow twig: Spits out from the front side of the main stem (usually slightly forward and outward), initially shorter but growing rapidly. The angle formed between the brow twig and the main stem is relatively large. Surface characteristics: The entire antler body (main stem and brow twig) is still covered with dense velvet and vascularized skin (deer antler skin / deer antler grass). The color of the velvet may darken. The apical growth points (growth cones) of the main stem and brow twig swell significantly, appearing pink or red. The overall morphology clearly presents the "two-bar" characteristic (one main stem plus one brow twig).

[0056] The four-branching stage is also called the secondary branching stage. Timing: After the two-bar stage, usually in early summer (June-July), this is the stage of most vigorous antler growth and greatest morphological change. Morphological characteristics: Main trunk: Continues to lengthen and thicken significantly. Eyebrow branch: Growth accelerates, and a second branch begins to differentiate at its tip or middle, called an "ice branch" (or second lateral branch). Thus, the eyebrow branch itself also forms a secondary branch. New branching point: After the tip of the main trunk swells, a third branch (sometimes called a "middle branch") will differentiate, usually growing upwards or backwards. Sometimes, a short branch may directly branch off from the tip of the main trunk, forming the rudimentary form of the fourth branch. Overall structure: Forms a complex structure with at least three main branching points (the main root counts as one branch, the eyebrow branch as the second branch, the eyebrow branch branching into an ice branch as the third branch, and the branch branching off from the tip of the main trunk as the fourth branch). A typical four-branched structure includes: a main trunk (main branch), an brow branch (first lateral branch), an ice branch (secondary branch branching from the brow branch), and a third lateral branch (or middle branch) branching from the top of the main trunk. Surface characteristics: The vegetal reaches its maximum size; the surface hairs may appear slightly sparse due to rapid growth, but the blood vessels remain abundant and brightly colored. The tips of each branch are swollen and grow actively. The overall morphology exhibits a distinct crown-like structure.

[0057] The complete ossification period is also known as the ossification and antler shedding period. Time: Late summer to early autumn (August-September), after the antlers have stopped growing. Morphological transformation process: Ossification begins: The cartilage tissue inside the antler begins to be deposited and replaced by minerals (mainly calcium salts), gradually transforming into hard bone. Vascular degeneration: The blood vessels supplying the antler gradually atrophy and close. Antler skin dries and falls off: With ossification and vascular degeneration, the outer antler skin (antler grass) loses its nutrient supply, begins to dry, atrophy, harden, and crack, eventually peeling off in pieces from the ossified antler like tree bark. This process may last for several weeks. Final morphological characteristics: Texture: Completely loses its softness and elasticity, becoming as hard as bone. Surface: The velvet and skin completely fall off, revealing a smooth or longitudinally grooved bone surface. Color changes from bright red / pink to grayish-white, light brown, or dark brown (depending on friction and environment). The growth points that were originally enlarged when covered by the antler skin become rounded or sharp antler tips after ossification. Structure: The branching structures formed in the later stages of growth (such as two-pronged, three-pronged, and four-pronged branches) are clearly preserved, but the surface becomes smooth, dry, hairless, and avascular. Horn base: The ossified horn is firmly attached to the horn base (horn disc) of the skull. There is usually a ring-shaped ridge (horn crown) at the junction of the horn disc and the horn. State: At this stage, the horn is called "antler" or "dry antler," and is a bony structure used for fighting and display. This ossification state will continue until the molting period the following spring.

[0058] The principle of this invention is as follows: This invention selects samples of sika deer cancellous bone at five different developmental stages: EPOTS (early stage of antler detachment), BTBP (pre-two-bar stage), TBP (two-bar stage), FBP (four-branch stage), and COP (complete ossification stage). Through protein extraction, enzyme digestion, and liquid chromatography-mass spectrometry tandem analysis, common differential peptides in six comparative developmental stages—BTBP vs. EPOTS, TBP vs. EPOTS, TBP vs. BTBP, PBP vs. TBP, COP vs. EPOTS, and COP vs. FBP—are first screened out. Then, four specific differential peptides (ER-11, FR-14, IK-10, and TR-17) that conform to the periodic osteoporosis phenomenon of sika deer (osteoporosis during rapid antler growth and no osteoporosis after complete antler ossification) are screened out, and these four peptides are synthesized using the Fmoc / tbu solid-phase method. Furthermore, it was verified that ER-11, FR-14, IK-10, and TR-17 effectively inhibit osteoclast formation and promote osteoblast formation by significantly inhibiting the expression of osteoclast-related genes and promoting the expression of osteogenic-related genes, thus providing potential drugs for the prevention and treatment of osteoporosis.

[0059] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0060] The method for screening sika deer bone peptides of the present invention can screen out common and different peptides in six comparative developmental stages of sika deer, providing a technical basis for further research on specific peptides in sika deer osteoporosis.

[0061] The screening method of this invention can obtain four specific differential peptides (ER-11, FR-14, IK-10, TR-17) that conform to the periodic osteoporosis phenomenon of sika deer, with molecular weights of 1661.93 Da, 1273.64 Da, 1273.64 Da, and 1231.43 Da, respectively. The molecular weights are relatively small, and they can be synthesized artificially in large quantities with high purity.

[0062] The screening method of this invention can obtain four specific differential peptides (ER-11, FR-14, IK-10, TR-17) that conform to the periodic osteoporosis phenomenon of sika deer. These peptides can both promote bone formation and inhibit bone resorption. They have significant regulatory effects on bone metabolism markers, are highly specific, have low toxicity, are not prone to accumulation in vivo, and have few interactions with other drugs.

[0063] The screening method of this invention can obtain four specific differential peptides (ER-11, FR-14, IK-10, TR-17) that conform to the periodic osteoporosis phenomenon of sika deer. It is safer (HF-free), more flexible (orthogonal protection), more efficient (long peptide synthesis), and more compatible (modified peptide / automation). It is especially suitable for synthesizing target peptides containing complex modifications or difficult sequences. Attached Figure Description

[0064] Figure 1 This is an upset diagram (BTBP vs EPOTS, TBP vs EPOTS, COP vs EPOTS, FBP vs TBP, TBP vs BTBP, and COP vs FBP) showing the differential peptides at six contrasting developmental stages in Example 1 of the present invention.

[0065] Figure 2 This is a heatmap analysis of the common differential peptides in six comparative developmental stages in Example 2 of the present invention: BTBP vs EPOTS, TBP vs EPOTS, COP vs EPOTS, FBP vs TBP, TBP vs BTBP, and COP vs FBP.

[0066] Figure 3 This is the mass spectrum of ER-11, an active peptide from sika deer bone prepared in Example 3 of the present invention.

[0067] Figure 4 The high-performance liquid chromatogram of ER-11, an active peptide from sika deer bone, prepared in Example 3 of this invention.

[0068] Figure 5 This is the mass spectrum of FR-14, an active peptide from sika deer bone prepared in Example 4 of this invention.

[0069] Figure 6The high-performance liquid chromatogram of FR-14, an active peptide from sika deer bone, prepared in Example 4 of this invention.

[0070] Figure 7 This is the mass spectrum of the sika deer bone active peptide IK-10 prepared in Example 5 of the present invention.

[0071] Figure 8 The high-performance liquid chromatogram of the sika deer bone active peptide IK-10 prepared in Example 5 of the present invention.

[0072] Figure 9 This is the mass spectrum of TR-17, an active peptide from sika deer bone, prepared in Example 6 of this invention.

[0073] Figure 10 The high-performance liquid chromatogram of TR-17, an active peptide from sika deer bone, prepared in Example 6 of this invention.

[0074] Figure 11 In Example 7 of this invention, the MTT assay was used to detect the toxic effects of sika deer bone active peptides on mouse mononuclear macrophage leukemia cells (RAW264.7 cells). Specifically, a represents the toxic effect of ER-11 on RAW264.7 cells, b represents the toxic effect of FR-14 on RAW264.7 cells, c represents the toxic effect of IK-10 on RAW264.7 cells, and d represents the toxic effect of TR-17 on RAW264.7 cells. Figure 11 In the ad, *** all indicate significant differences.

[0075] Figure 12 Example 7 of this invention describes the MTT assay used to detect the toxic effects of sika deer bone active peptides on mouse embryonic osteoblast precursor cells (MC3T3-E1 Subclone24 cells). Specifically, a represents the toxic effect of ER-11 on MC3T3-E1 Subclone24 cells, b represents the toxic effect of FR-14 on MC3T3-E1 Subclone24 cells, c represents the toxic effect of IK-10 on MC3T3-E1 Subclone24 cells, and d represents the toxic effect of TR-17 on MC3T3-E1 Subclone24 cells. Figure 12 In the ad, *** all indicate significant differences.

[0076] Figure 13In Example 7 of this invention, the effect of sika deer bone bioactive peptides on the expression of osteoclast differentiation-related genes was detected using quantitative real-time PCR (q-PCR). In the figure, Oc represents osteoclasts formed by RAW264.7 cells induced by RANKL, Nfatc1 is activated T cell nuclear factor 1, Atp6v1a is vacuolar ATPase subunit A, Trap-5 is coagulation factor II receptor 1-5, Ctsk is cathepsin K, and Mmp9 is matrix metalloproteinase 9.

[0077] Figure 14 In Example 7 of this invention, q-PCR was used to detect the effect of sika deer bone bioactive peptides on the expression of genes related to osteoblast differentiation. In the figure, MC3T3-E1 represents MC3T3-E1 Subclone24 cells, Osteoblast represents osteoblasts, and RUBX2 is an osteogenic-related transcription factor.

[0078] Figure 15 The images shown are of the left and right femurs of rats measured using a small animal dual-energy X-ray bone densitometer in Example 8 of this invention. In the images, a represents the sham-operated group and b represents the model group.

[0079] Figure 16 The bone mineral density data of the left and right femurs of rats were measured using a small animal dual-energy X-ray bone densitometer in Example 8 of this invention, where a is the left femur and b is the right femur.

[0080] Figure 17 In Example 9 of this invention, bone mineral density data of the left and right femurs of rats were measured using a small animal dual-energy X-ray bone densitometer, where a represents the right femur and b represents the left femur.

[0081] Figure 18 The results of hematoxylin-eosin staining of the right femur tissue of rats in Example 10 of this invention are shown. In the figure, a is the sham-operated group, b is a magnified view of a, c is the model group, d is a magnified view of c, e is the high-dose group, f is a magnified view of e, g is the positive control group, and h is a magnified view of g.

[0082] Figure 19 The results of masson staining of the right femoral tissue of rats in Example 11 of the present invention are shown. In the figure, a is the sham-operated group, b is a magnified view of a, c is the model group, d is a magnified view of c, e is the high-dose group, f is a magnified view of e, g is the positive control group, and h is a magnified view of g.

[0083] Figure 20 The results of tartrate-resistant acid phosphatase staining of the right femoral tissue of rats in Example 12 of this invention are shown, where a is the sham-operated group, b is the model group, c is the high-dose group, and d is the positive drug group.

[0084] Figure 21 The above is an immunohistochemical experiment of cathepsin K in the right femoral tissue of rats in Example 13 of the present invention, wherein a is the sham-operated group, b is the model group, c is the high-dose group, and d is the positive drug group.

[0085] Figure 22 This is an immunohistochemical experiment of osteopontin in the right femoral tissue of rats in Example 14 of the present invention, wherein a is the sham-operated group, b is the model group, c is the high-dose group, and d is the positive drug group. Detailed Implementation

[0086] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to embodiments.

[0087] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. Unless otherwise specified, the materials, reagents, apparatus, instruments, and equipment used in the following embodiments are commercially available. Specifically, high-performance liquid chromatography (HPLC) detection was performed using an EASY-nLC 1200, the ion source was an NSI ion source, and mass spectrometry detection was performed using an OrbitrapExploris 480 mass spectrometer. Data acquisition mode used was a data-independent scanning (DIA) procedure.

[0088] Example 1

[0089] Methods for screening sika deer bone peptides include:

[0090] Step 1: Select sika deer cancellous bone

[0091] One pound of cancellous bone samples were collected from the vertebrae of sika deer at five different developmental stages: early stage of tray detachment (EPOTS), early stage of two bars (BTBP), stage of two bars (TBP), stage of four-pronged detachment (FBP), and stage of complete ossification (COP). Surface impurities, muscle, and fat tissues were removed.

[0092] Step 2, protein extraction

[0093] Five sika deer cancellous bone samples from different developmental stages were first placed in mortars pre-cooled with liquid nitrogen and ground thoroughly into deer bone powder with liquid nitrogen to ensure sufficient contact between the solvent and the deer bone during subsequent extraction, thereby improving extraction efficiency. Then, lysis buffer was added to each sample, with the deer bone powder and lysis buffer mixed at a ratio of 1g:10mL. The samples were extracted by stirring at 40-60℃ for 4 hours and then centrifuged at 12000g for 10 minutes at 4℃ to obtain protein samples. The lysis buffer contained 1wt% sodium dodecyl sulfate and 1wt% protease inhibitor, the solvent was ultrapure water, and the protease inhibitor was protease inhibitor VI.

[0094] Step 3, Enzyme digestion treatment

[0095] First, take five protein samples of equal mass from five different developmental stages. Adjust the volume of each sample to be consistent using lysis buffer. Then, add one volume of acetone pre-cooled to 4°C to each sample, vortex to mix, and then add 3-5 volumes of acetone pre-cooled to 4°C to each sample. Precipitate at 4°C for 4 hours, centrifuge at 4500g for 5 minutes, discard the supernatant, wash the resulting precipitate 2-3 times with acetone pre-cooled to 4°C, air dry, and then sonicate to disperse the precipitate with tetraethylammonium bromide solution to a final concentration of 200mM. Trypsin was added at a mass ratio of 1:50 to the protein sample, and the enzyme was digested for 12 h. Then, dithiothreitol was added to a final concentration of 5 mM, and the enzyme was reduced at 56 °C for 30 min. Finally, iodoacetamide was added to a final concentration of 11 mM, and the enzyme was incubated at room temperature in the dark for 15 min to obtain peptide samples at five different developmental stages. The lysis buffer contained 1 wt% sodium dodecyl sulfate and 1 wt% protease inhibitor, and the solvent was ultrapure water. The protease inhibitor was protease inhibitor VI.

[0096] Step 4, Liquid Chromatography-Mass Spectrometry Tandem Analysis

[0097] High-performance liquid chromatography (HPLC) was used to separate peptide samples at five different developmental stages. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The HPLC gradient settings were as follows: 0-22.5 min, 6%-22% mobile phase B; 22.5-26.5 min, 22%-34% mobile phase B; 26.5-28.5 min, 34%-80% mobile phase B; 28.5-30 min, 80% mobile phase B, with the flow rate maintained at 700 nl / min.

[0098] After separation, ionization was performed using an ion source with the ion source voltage set to 2300V and the FAIMS compensation voltage (CV) set to -45V.

[0099] The ionized sample was then analyzed by mass spectrometry (MS). The first-stage mass spectrometry scan range was set to 350-1400 m / z, and the scan resolution was set to 60,000. The second-stage mass spectrometry scan range had a fixed starting point of 120 m / z, and the second-stage scan resolution was set to 15,000. The automatic gain control (AGC) was set to 1E6, the maximum injection time was set to 22 ms, and the fragmentation energy was 27%.

[0100] Mass spectrometry data of all peptides corresponding to five different developmental stages were obtained, and a mass spectrometry database was established.

[0101] By comparing differentially expressed peptides in the pre-two-bar stage and early tray detachment stage (BTBP vs EPOTS), the two-bar stage and early tray detachment stage (TBP vs EPOTS), the complete ossification stage and early tray detachment stage (COP vs EPOTS), the tetrapod stage and two-bar stage (FBP vs TBP), the two-bar stage and pre-two-bar stage (TBP vs BTBP), and the complete ossification stage and tetrapod stage (COP vs FBP), an upset diagram of differentially expressed peptides was drawn for the six contrasting developmental stages. The intersection of these upset diagrams yielded the common differentially expressed peptides for the six contrasting developmental stages. Figure 1 As shown in the figure, the number of differentially expressed peptides in the two different developmental stages in each comparative developmental stage (BTBPvsEPOTS, TBPvsEPOTS, COPvsEPOTS, FBPvsTBP, TBPvsBTBP, COPvsFBP) and the number of common differentially expressed peptides in the six comparative developmental stages were screened, and a total of 39 common differentially expressed peptides were obtained.

[0102] Example 2

[0103] The method for screening four specific differentially expressed peptides that correspond to the periodic osteoporosis phenomenon in sika deer includes:

[0104] Steps one through four are the same as in Example 1;

[0105] Step 5: Perform heatmap analysis on the common differential peptides from the six contrasting developmental stages, such as... Figure 2 As shown, using BTBP vs EPOTS, TBP vs EPOTS, and TBP vs BTBP as the bone loss phases, and FBP vs TBP, COP vs EPOTS, and COP vs FBP as the bone stabilization phases, we screened out the peptide abundance of the former relative to the latter in two different developmental stages within each bone loss phase (i.e., in BTBP vs EPOTS, BTBP peptide abundance was lower than EPOTS; in TBP vs EPOTS, TBP peptide abundance was lower than EPOTS; and in TBP vs BTBP, TBP peptide abundance was lower than BTBP). Within each bone stabilization phase, the relative abundance of the former relative to the latter in two different developmental stages was also analyzed. Differential peptides with increased abundance in all peptide categories (i.e., in FBP vs TBP, FBP peptide abundance increased compared to TBP; in COP vs EPOTS, COP peptide abundance increased compared to EPOTS; and in COP vs FBP, COP peptide abundance increased compared to FBP) were identified as specific differential peptides consistent with the periodic osteoporosis phenomenon in sika deer. These are sika deer bone bioactive peptides, one of the following: ER-11 (EAVQGASDLWR), FR-14 (FSISSDYNLPHILR), IK-10 (IILVNYTYFK), and TR-17 (TVEVPMMTLGLETPYFR). All peptides were obtained through the following screening method.

[0106] Example 3

[0107] The preparation method of ER-11, an active peptide from sika deer bone, is as follows:

[0108] Step 1: Add 10g of 2-chlorotriphenylmethyl chloride resin (2-CTC Resin) (loading 10mmol / g) to a solid-phase reactor, add 10mL of dichloromethane (DCM) and 10mL of N,N-dimethylformamide (DMF) and soak for 10min to allow the 2-chlorotriphenylmethyl chloride resin to fully expand. Filter to obtain expanded 2-chlorotriphenylmethyl chloride resin.

[0109] Step 2: 33 mmol of the first amino acid at the C-terminus of the sika deer bone active peptide ER-11 to be prepared (C-terminus refers to the end of the amino acid sequence, which is the starting point for synthesis) and a DMF solution containing 100 mmol of N,N-diisopropylethylamine (DIPEA) (concentration 0.5-2M, preferably 1M) were added to a solid-phase reactor and reacted in an air bath constant temperature shaker at 25-35℃ (preferably 30℃) for 120 min. After the reaction was completed, the mixture was filtered, and the precipitate was washed 5 times each with DCM and DMF to obtain the first intermediate product.

[0110] Step 3: Add 60 mL of DMF solution containing 20 v / v% piperidine to the solid-phase reactor, and react in a gas bath constant temperature shaker at 25-35℃ (preferably 30℃) for 10 min. After filtration, add another 60 mL of DMF solution containing 20 v / v% piperidine, and react in a gas bath constant temperature shaker at 25-35℃ (preferably 30℃) for 10 min. After the reaction is complete, filter and wash 5 times each with DCM and DMF to obtain the second intermediate product.

[0111] Step 4: Take 1wt%–3wt% (preferably 1wt%) of the second intermediate product and detect it using the ninhydrin method. If the test shows a blue color, proceed to step 5; if the test shows no color, return to step 3.

[0112] Step 5: 33 mmol of the second amino acid at the C-terminus of the sika deer bone active peptide ER-11 to be prepared, 30 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and a DMF solution containing 100 mmol of N,N-diisopropylethylamine (DIPEA) (concentration 0.5-2M, preferably 1M) were added to a solid-phase reactor and reacted in a gas bath constant temperature shaker at 25-35℃ (preferably 30℃) for 40 min. After the reaction was completed, the product was washed 5 times with DCM and DMF to obtain the third intermediate product.

[0113] Step 6: Add 60 mL of DMF solution containing 20 v / v% piperidine to the solid-phase reactor, and react in a gas bath constant temperature shaker at 25-35℃ (preferably 30℃) for 10 min. After filtration, add another 60 mL of DMF solution containing 20 v / v% piperidine, and react in a gas bath constant temperature shaker at 25-35℃ (preferably 30℃) for 10 min. After the reaction is complete, filter and wash 5 times each with DCM and DMF to obtain the fourth intermediate product.

[0114] Step 7: Take 1 wt% of the fourth intermediate product and detect it using the ninhydrin method. If the test result is blue, proceed to step 8. If there is no color, return to step 6.

[0115] Step 8: Following steps 5 to 7 in sequence (only the second amino acid at the C-terminus is replaced with the corresponding amino acid, and the other steps remain unchanged), synthesize the third amino acid at the C-terminus, the fourth amino acid at the C-terminus, the fifth amino acid at the C-terminus, ... up to the eleventh amino acid at the C-terminus, based on the fourth intermediate product. The corresponding amino acids and synthesis directions are shown in Table 1, and crude product of active peptides from sika deer bone is obtained.

[0116] Step 9: The crude active peptides from sika deer bone were placed in a mixture of tetrafluoroethylene (TFE) and DCM at a volume ratio of 1:4 and reacted at 30°C for 4 hours to obtain linear peptides. These linear peptides were then placed in a mixture of PyAOP ((7-azabenzotriazole-1-oxo)tripyrrolephosphine hexafluorophosphate), HOAt (N-hydroxy-7-azabenzotriazole), and NMM (N-methylmorpholine) at a volume ratio of 1:1:1 and reacted first at 0°C for 1 hour, then at room temperature for 2 hours. The product was purified using an LH-20 column, and finally placed in… The product was reacted in a mixture of trifluoroacetic acid (TFA) and DCM at a volume ratio of 1:3 at room temperature for 2 h. The product was then purified by reversed-phase HPLC using a C18 column to obtain sika deer bone active peptide ER-11. The reversed-phase HPLC purification conditions were as follows: mobile phase A was water, mobile phase B was acetonitrile, and the liquid phase gradient was set as follows: 0-20 min, 10% mobile phase B; 20-25 min, 95% mobile phase B; 25-25.1 min, 95% mobile phase B; 25.1-30 min, 10% mobile phase B.

[0117] It should be noted that in the above preparation method, the filtration is usually carried out outside the solid-phase reactor, and the solid is then returned to the solid-phase reactor after the operation.

[0118] The sika deer bone active peptide ER-11 prepared in Example 3 was identified by mass spectrometry and high performance liquid chromatography, and the results are as follows: Figure 3 and Figure 4 As shown, from Figure 3 and Figure 4It can be seen that the present invention has successfully prepared ER-11, an active peptide from sika deer bone.

[0119] Example 4

[0120] The preparation method of FR-14, an active peptide from sika deer bone, is the same as that in Example 3, except that the amino acid synthesis sequence and direction are shown in Table 1. In step nine, the fourteenth amino acid at the C-terminus is synthesized.

[0121] The sika deer bone active peptide FR-14 prepared in Example 3 was identified by mass spectrometry and high performance liquid chromatography, and the results are as follows: Figure 5 and Figure 6 As shown, from Figure 5 and Figure 6 It can be seen that the present invention has successfully prepared FR-14, an active peptide from sika deer bone.

[0122] Example 5

[0123] The preparation method of sika deer bone active peptide IK-10 is the same as that in Example 3, except that the amino acid synthesis sequence and direction are shown in Table 1. In step nine, the tenth amino acid at the C-terminus is synthesized.

[0124] The sika deer bone active peptide IK-10 prepared in Example 5 was identified by mass spectrometry and high performance liquid chromatography, and the results are as follows: Figure 7 and Figure 8 As shown, from Figure 7 and Figure 8 It can be seen that the present invention has successfully prepared sika deer bone active peptide IK-10.

[0125] Example 6

[0126] The preparation method of sika deer bone active peptide TR-17 is the same as that in Example 3, except that the amino acid synthesis sequence and direction are shown in Table 1. In step nine, the seventeenth amino acid at the C-terminus is synthesized.

[0127] The active peptide TR-17 from sika deer bone prepared in Example 6 was identified by mass spectrometry and high performance liquid chromatography, and the results are as follows: Figure 9 and Figure 10 As shown, from Figure 9 and Figure 10 It can be seen that the present invention has successfully prepared TR-17, an active peptide from sika deer bone.

[0128] Table 1. Amino acid synthesis sequence and direction of the sika deer bone active peptides corresponding to the preparation methods in Examples 3-6.

[0129]

[0130] Example 7

[0131] In vitro experiments were conducted on the sika deer bone active peptides ER-11, FR-14, IK-10 and TR-17 prepared in Examples 3-6.

[0132] The MC3T3-E1 Subclone24 cells and RAW264.7 cells used in the in vitro experiments were purchased from Pronoss, with the catalog number of MC3T3-E1 Subclone 24 being GCL-0710 and that of RAW 264.7 being GCL-0190.

[0133] 1. Cytotoxicity assay

[0134] 1.1 RAW264.7 Cytotoxicity Assay

[0135] RAW264.7 cells were adjusted to 5 × 10⁻⁶ cells. 5 / ml, seeded into 96-well plates, 100μl per well. Incubated at 37℃, 5% CO2 for 4h. Groups: control group (normal DMEM medium), control group (DMEM medium supplemented with RANKL), and drug-treated groups (ER-11, FR-14, IK-10, and TR-17 concentrations of 1000.0μg / ml, 100.0μg / ml, 10.0μg / ml, 1.0μg / ml, and 0.1μg / ml, respectively), 5 replicates per group. After 48h of incubation, 10μl of MTT solution (5mg / ml) was added to each well, and incubation continued for another 4h. The supernatant was discarded, and 150μl of dimethyl sulfoxide (DMSO) was added to each well, shaken for 10min. The absorbance (OD value) was read at 490nm using a microplate reader. The cytotoxicity assay results of ER-11, FR-14, IK-10, and TR-17 are as follows. Figure 11 As shown in Figures a, b, c, and d. From... Figure 11 As shown in Figure a, the MTT assay for the toxicity of ER-11 to RAW264.7 cells revealed an optimal concentration of 100.0 μg / ml, with *P<0.05 compared to the control group. Figure 11 As shown in Figure b, the MTT assay for the toxic effects of FR-14 on RAW264.7 cells revealed that the optimal concentration was 100.0 μg / ml. From... Figure 11 As shown in Figure c, the MTT assay for the toxic effects of IK-10 on RAW264.7 cells revealed that the optimal concentration was 100.0 μg / ml. From... Figure 11As shown in Figure d, the MTT assay for the cytotoxic effect of TR-17 on RAW264.7 cells revealed an optimal concentration of 100.0 μg / ml. Data were analyzed using one-way ANOVA with GraphPad Prism 10.0 software, and results are expressed as standard error (mean ± SEM), with n=3 replicates. *** in the figure indicates a significant difference, i.e., P < 0.001, indicating that it was not a random event.

[0136] 1.2 MC3T3-E1 Subclone24 Cytotoxicity Assay

[0137] Adjust the MC3T3-E1 Subclone24 cells to 2×10⁻⁴. 5 100 μl of MTT solution was seeded into each well of a 96-well plate. The plates were incubated at 37°C with 5% CO2 for 4 hours. Groups were formed: a control group (normal DMEM medium), a control group (DMEM medium supplemented with dexamethasone, ascorbic acid, and sodium β-glycerophosphate), and a drug-treated group (ER-11, FR-14, IK-10, and TR-17 at independent concentrations of 1000.0 μg / ml, 100.0 μg / ml, 10.0 μg / ml, 1.0 μg / ml, and 0.1 μg / ml, respectively), with 5 replicates per group. After 48 hours of incubation, 10 μl of MTT solution (5 mg / ml) was added to each well, and the plates were incubated for another 4 hours. The supernatant was discarded, and 150 μl of DMSO was added to each well. The plates were shaken for 10 minutes. The absorbance (OD value) was read at 490 nm using a microplate reader. The cytotoxicity assay results for ER-11, FR-14, IK-10, and TR-17 are as follows: Figure 12 As shown in Figures a, b, c, and d. From... Figure 12 As shown in Figure a, the MTT assay for the toxicity of ER-11 to RAW264.7 cells revealed an optimal concentration of 100.0 μg / ml, with *P<0.05 compared to the control group. Figure 12 As shown in Figure b, the MTT assay for the toxic effects of FR-14 on RAW264.7 cells revealed that the optimal concentration was 100.0 μg / ml. From... Figure 12 As shown in Figure c, the MTT assay for the toxic effects of IK-10 on RAW264.7 cells revealed that the optimal concentration was 100.0 μg / ml. From... Figure 12As shown in Figure d, the MTT assay for the toxicity of TR-17 to RAW264.7 cells revealed an optimal concentration of 100.0 μg / ml. Data were analyzed using one-way ANOVA with GraphPad Prism 10.0 software, and results are expressed as standard error (mean ± SEM), with n=3 replicates. The *** in the figure indicates a significant difference, i.e., P < 0.001, meaning it was not a random event.

[0138] 2. Effects of sika deer osteoactive peptides ER-11, FR-14, IK-10 and TR-17 on the expression of osteoclast differentiation-related genes.

[0139] Cell seeding and induction: RAW264.7 cells were seeded at a rate of 1×10⁻⁶ cells / year. 7 Cells were seeded at a density of cells / well in six-well plates. The cells were divided into a blank group (normal DMEM medium), a model group, an ER-11 group, a FR-14 group, an IK-10 group, and a TR-17 group. After 24 hours of culture, 100 ng / ml RANKL was added to the model group and each treatment group to induce osteoclast differentiation. Simultaneously, the treatment group cells were treated with 100 μg / ml ER-11, FR-14, IK-10, and TR-17 for 5 days, respectively. Total RNA was extracted from the cells using Trizol (Invitrogen). The extracted RNA was then reverse transcribed into cDNA using HiScript Il Select gRT SuperMix (Vazyme, Nanjing, China). Gene quantification was performed using qRT-PCR. The following genes were ultimately identified for detection: Nfatc1, Trap-5, Atp6v1a, Ctsk, and Mmp9 (qRT-PCR reaction system shown in Table 2, primer sequences shown in Table 3). Results are as follows: Figure 13 As shown in the figure, the expression of one or more osteoclast biomarkers Nfatc1, Atp6v1a, Trap-5, Ctsk, and Mmp9 was reduced in the drug-treated group, indicating that ER-11, FR-14, IK-10, and TR-17 can inhibit osteoclastization.

[0140] 3. Effects of sika deer osteogenic peptides ER-11, FR-14, IK-10 and TR-17 on the expression of osteoblast differentiation-related genes.

[0141] Cell seeding and induction: MC3T3-E1 Subclone24 cells were seeded at a rate of 1×10⁻⁶ cells / cells. 6Cells were seeded at a density of cells / well in six-well plates. The cells were divided into a blank group (α-MEM complete medium), a model group, an ER-11 group, a FR-14 group, an IK-10 group, and a TR-17 group. After 24 hours of culture, 100 ng / ml RANKL was added to the model group and each treatment group to induce osteoclast differentiation. Simultaneously, the treatment group cells were treated with 100 μg / ml ER-11, FR-14, IK-10, and TR-17 for 5 days, respectively. Total RNA was extracted from the cells using Trizol (Invitrogen). The extracted RNA was then reverse transcribed into cDNA using HiScript Il Select gRT SuperMix (Vazyme, Nanjing, China). Gene quantification was performed using qRT-PCR. The following gene was finally identified for detection: RUBX2 (qRT-PCR reaction system shown in Table 2, primer sequences shown in Table 3). Results are as follows... Figure 14 As shown in the figure, the expression level of the osteoblast biomarker RUBX2 was increased in the drug-treated group, indicating that ER-11, FR-14, IK-10 and TR-17 can promote osteoogenesis.

[0142] Table 2 qRT-PCR reaction system

[0143]

[0144] Table 3 Primer Sequences

[0145]

[0146] The above experiments demonstrate that the method for screening sika deer bone peptides in this invention can identify 39 common differential peptides across six contrasting developmental stages in sika deer. Four specific differential peptides consistent with the cyclical osteoporosis phenomenon in sika deer significantly inhibited osteoclast differentiation and downregulated the expression of osteoclast function-related genes such as Nfatc1, Acp5, and Atp6v1a. Conversely, the four specific differential peptides consistent with the cyclical osteoporosis phenomenon in sika deer significantly promoted osteoblast differentiation and upregulated the expression of osteoblast-related genes such as RUNX2. This indicates that the four specific differential peptides consistent with the cyclical osteoporosis phenomenon in sika deer screened in this invention can effectively inhibit osteoclast formation and promote osteoblast formation by significantly inhibiting the expression of osteoclast function-related genes and promoting the expression of osteoblast-related genes, providing a new potential drug for the prevention and treatment of osteoporosis.

[0147] Furthermore, previous cell experiments showed that among the four peptides screened that are associated with periodic osteoporosis in sika deer, TR-17 exhibited the best osteoporosis prevention and treatment activity. Based on this, to confirm the in vivo anti-osteoporosis efficacy of TR-17, the present invention designed the following animal experiments for verification.

[0148] Example 8: Validation of an osteoporosis model induced by ovariectomy in female rats

[0149] Six healthy female SD rats aged 8 weeks were selected and randomly divided into two groups (n=3) after one week of acclimatization: a sham-operated group and a model group. Except for the sham-operated group, the remaining rats underwent bilateral ovariectomy to establish an osteoporosis model. First, the rats were fasted for 12 hours and deprived of water for 8 hours preoperatively. Then, they were anesthetized by intraperitoneal injection of tribromoethanol (dosage: 100-200 mg / kg body weight). The rats were fixed in a supine position, the abdomen was prepared and disinfected with iodine solution, and a longitudinal incision of approximately 1 cm was made along the midline of the lower abdomen. The abdominal cavity was exposed by dissecting layer by layer. The uterine horn was gently located and pulled out, and the ovary and fallopian tube, which were surrounded by adipose tissue, were positioned. The distal ends of both uterine horns and the fallopian tubes were ligated with surgical sutures. The ovaries were then completely removed, and the incision was sutured layer by layer and disinfected again. The rats in the sham-operated group underwent the same surgical approach exposure and traction manipulation, but without ligation or removal of the ovaries. Postoperatively, the rats were placed in a warm recovery incubator until they regained consciousness and were then returned to their cages. For three consecutive days, administer penicillin intramuscularly (dosage: 40,000-60,000 U / rat) to prevent infection. Closely monitor the rats' condition and change their bedding, feed, and drinking water regularly. Fifty days after modeling, anesthetize all rats again and measure the bone mineral density of their left and right femurs using a small animal dual-energy X-ray absorptiometry (DXA) system.

[0150] The results are as follows Figure 15 a and b, and Figure 16 As shown in Figures a and b, bone mineral density measurements revealed a significant decrease in femoral bone mineral density in the model group compared to the sham-operated group (p<0.05), indicating the successful establishment of the osteoporosis model. This result provides a reliable experimental basis for further validation of the therapeutic effect of TR-17.

[0151] Example 9: Pharmacodynamic validation of peptide TR-17 in a rat ovariectomy-induced osteoporosis model

[0152] Fifty healthy female SD rats aged 8 weeks were selected and, after one week of acclimatization, randomly divided into 5 groups (n=10): sham-operated group, model group, low-dose group, high-dose group, and positive control group. Except for the sham-operated group, the remaining rats underwent bilateral ovariectomy to establish an osteoporosis model. The modeling and subsequent treatment methods are as described in Example 8. After modeling, subcutaneous injections were administered for 50 days one week later (sham-operated group and model group received 1 ml of physiological saline daily; low-dose group received a daily subcutaneous injection of TR-17 dissolved in physiological saline at a dose of 10 μg / kg; high-dose group received a daily subcutaneous injection of TR-17 dissolved in physiological saline at a dose of 50 μg / kg; positive control group received a daily subcutaneous injection of teriparatide (PTD1-34) dissolved in physiological saline at a dose of 10 μg / kg). After 50 days, all rats were anesthetized again, and the bone mineral density of their left and right femurs was measured using a small animal dual-energy X-ray absorptiometry (DXA) system. The results are as follows: Figure 17 As shown in Figures a and b, both the high-dose group and the positive control group significantly increased the bone mineral density of the left and right femurs in osteoporotic rats.

[0153] Example 10

[0154] After measuring bone mineral density using a small animal dual-energy X-ray absorptiometry (DXA) bone densitometer, experimental samples were collected from the rats in Example 9. Rats were anesthetized by intraperitoneal injection of 0.7-1 ml of 10% tribromoethanol (Avorin brand). Once the rats reached an appropriate depth of anesthesia, their whiskers were trimmed from the root using sterile surgical scissors to ensure complete anesthesia. They were then euthanized by cervical dislocation. The rats' hind limbs were first separated from the hip joints, then the knee joints were carefully severed, and the tendons and muscle membranes on the femoral surface were thoroughly removed. The right femur was fixed in 4% paraformaldehyde solution for 48 hours (the fixation solution volume was at least 10 times the sample volume) and then transferred to PBS for later use.

[0155] Hematoxylin and eosin (HE) staining was performed on the right femurs of rats from four groups: sham-operated group, model group, high-dose group, and positive control group. The results are as follows: Figure 18 As shown in the middle ah.

[0156] Based on the HE staining results of the right femur tissue of the rats, the model group showed typical osteoporotic pathological changes such as sparse, broken, and reduced trabecular bone. In contrast, the high-dose group and the positive control group showed denser trabecular structure, increased thickness, and improved connectivity, while the cortical bone thickness also increased. This indicates that TR-17 can effectively reverse the osteoporosis induced by ovariectomy in rats, and its therapeutic effect is expected to reach a level comparable to that of teriparatide.

[0157] Example 11

[0158] Right femurs (obtained from rats in four groups—sham surgery group, model group, high-dose group, and positive control group—were subjected to Masson staining (the low-dose group was not used due to its insignificant therapeutic effect). After staining, the bone tissue exhibited the following characteristics: bone collagen fibers appeared blue, bone matrix and muscle fibers appeared red, and cell nuclei appeared blue-black. By comparing the percentage of blue area in the cortical and trabecular bone regions between the model group and the drug-treated group using image analysis software, quantitative analysis of bone collagen deposition and distribution could be performed. Results are as follows... Figure 19 As shown in the middle ah.

[0159] Masson staining results of the right femoral tissue of rats showed that the area and staining depth of the collagen fiber region in the model group were significantly lower than those in the sham-operated group. This indicates that osteoporosis not only leads to bone loss but also to the loss and degradation of collagen, resulting in a decline in bone matrix quality. The model group showed sparse, disordered, and broken collagen fiber regions, indicating that the microstructure of bone tissue had been damaged, and the biomechanical properties of bone (such as toughness) would be significantly reduced. The high-dose group had a denser, larger, and darker collagen fiber region than the model group, and the arrangement of collagen fibers was closer to that of the sham-operated group. This shows that TR-17 not only increased bone mass but, more importantly, improved and even reversed the quality of the bone matrix. It promoted the deposition and repair of a healthier and richer collagen matrix. By comparing the staining degree and distribution of the collagen fiber region in the high-dose group and the positive control group, it can be determined that the effect of TR-17 in improving bone collagen is comparable to that of teriparatide.

[0160] Example 12

[0161] Right femurs (obtained from rats in four groups—sham surgery group, model group, high-dose group, and positive control group—were used for tartrate-resistant acid phosphatase (TRAP) staining experiments (the low-dose group was not used because its treatment effect was not significant). After staining, the osteoclast cytoplasm appeared wine-red, and the nuclei appeared blue. Bone resorption activity could be quantitatively analyzed by counting the number of TRAP-positive multinucleated osteoclasts per unit bone circumference or area. The results are as follows: Figure 20 As shown in the middle (ad).

[0162] According to the TRAP staining results of the right femoral tissue of the rats, the TRAP staining of the model group showed a significant increase in the number of osteoclasts, confirming that bone resorption activity was abnormally hyperactive in the osteoporotic state; while after TR-17 treatment, the number of osteoclasts was significantly reduced, indicating that the drug of the present invention reverses bone loss and improves bone microstructure by effectively inhibiting osteoclast-mediated bone resorption, and its inhibitory effect is comparable to that of the positive control drug teriparatide.

[0163] Example 13

[0164] Right femurs (obtained from rats in four groups—sham-operated group, model group, high-dose group, and positive control group—were used for cathepsin K (CTSK) immunohistochemistry (the low-dose group was not used because its treatment effect was not significant). After staining, the cytoplasm of osteoclasts showed clear brownish-yellow granular staining (CTSK positive), and the nuclei were blue. The abundance and functional activity of osteoclasts could be semi-quantitatively analyzed by calculating the number of CTSK-positive multinucleated osteoclasts per unit bone circumference or area, or by measuring the average optical density / integrated optical density of positive staining using image analysis software. The results are as follows: Figure 21 As shown in the middle (ad).

[0165] The immunohistochemical results of CTSK in the right femoral tissue of rats showed that CTSK, as the most critical functional protease for osteoclast degradation of type I collagen (the main component of bone organic matrix), directly localizes its positive signal in the cytoplasm of functionally active osteoclasts, reflecting the bone degradation process more directly than TRAP staining. In the model group, the number and staining intensity of CTSK-positive osteoclasts were significantly enhanced, confirming that bone matrix degradation was abnormally active in osteoporosis. After TR-17 treatment, the number and staining intensity of CTSK-positive cells were significantly reduced, indicating that TR-17 can not only reduce the number of osteoclasts, but also inhibit the expression of its key bone resorption protein CTSK from the functional source, effectively curbing the bone resorption process. Moreover, its inhibitory effect is comparable to that of the positive control drug teriparatide, thus further verifying the inhibitory effect of TR-17 on osteoclast activity at the functional protein level.

[0166] Example 14

[0167] Immunohistochemical staining of osteopontin (OPN) was performed on right femurs (sampling method as in Example 10) obtained from rats in four groups: sham-operated group, model group, high-dose group, and positive control group. After staining, OPN positivity was indicated by a brownish-yellow staining in the bone matrix, osteoblast cytoplasm, and periosteal lacunae. The average or integrated optical density of OPN-positive staining per unit area of ​​bone tissue was measured using image analysis software to perform semi-quantitative analysis of OPN expression levels. Results are as follows... Figure 22 As shown in the middle (ad).

[0168] OPN is an important non-collagenous protein synthesized and secreted into the bone matrix by active osteoblasts, and its positive signal is brownish-yellow staining. Abnormal OPN expression in the model group suggests osteoblast dysfunction and impaired bone formation; however, after TR-17 treatment, OPN expression in osteoblasts and bone matrix was significantly enhanced, indicating that TR-17 can not only reduce bone resorption by inhibiting osteoclast function (as shown in the CTSK results), but also enhance bone formation by activating osteoblasts and promoting bone matrix synthesis. Its bone-promoting effect is comparable to that of the positive control drug teriparatide, thus achieving synergistic treatment of osteoporosis through a dual pathway of inhibiting resorption and promoting formation.

[0169] Obviously, the above embodiments are merely examples for clear illustration and are not intended to limit the embodiments. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all embodiments here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for screening sika deer bone peptides, characterized in that, include: Step 1: Select cancellous bone from sika deer: Cancellous bone samples were collected from sika deer at five different developmental stages: early stage of tray detachment, early stage of two bars, two-bar stage, four-pronged stage, and complete ossification stage. Step 2, protein extraction: Five sika deer cancellous bone samples from different developmental stages were mechanically disrupted, and proteins were extracted using lysis buffer to obtain protein samples from five different developmental stages. Step 3, Enzyme digestion treatment: The protein samples extracted from five different developmental stages were subjected to enzyme digestion to obtain peptide samples from five different developmental stages. Step 4, Liquid Chromatography-Mass Spectrometry Tandem Analysis: First, high performance liquid chromatography was used to separate peptide samples at five different developmental stages. Then, after ionization by an ion source, mass spectrometry was used to detect the peptides at the five different developmental stages to obtain mass spectrometry data of all peptides. By comparing the differential peptide segments between the pre-two-bar stage and the early stage of tray detachment, the two-bar stage and the early stage of tray detachment, the complete ossification stage and the early stage of tray detachment, the four-forked stage and the two-bar stage, the two-bar stage and the pre-two-bar stage, and the complete ossification stage and the four-forked stage, a set diagram of the differential peptide segments of the six contrasting developmental stages was drawn and the intersection was taken to obtain the common differential peptide segments of the six contrasting developmental stages. Step 5: Perform heatmap analysis on the common differential peptides from the six contrasting developmental stages, and screen for common differential peptides that meet the following criteria to obtain specific differential peptides that correspond to the periodic osteoporosis phenomenon in sika deer: The peptide abundance in the early stage of the second phase is lower than that in the early stage of tray detachment; The peptide abundance decreased during the second phase compared to the initial stage of tray detachment; The peptide abundance in the second phase is lower than that in the pre-second phase. The abundance of peptides in the tetrad stage is higher than that in the did stage; Peptide abundance was higher during the complete ossification stage than during the early stage of tray detachment. The abundance of peptides increases during the complete ossification stage compared to the tetrapod stage.

2. The method for screening sika deer bone peptides according to claim 1, characterized in that, The specific differentially expressed peptides that correspond to the periodic osteoporosis phenomenon in sika deer are one or more of ER-11, FR-14, IK-10, and TR-17. The amino acid sequence of ER-11 is shown in SEQ ID 1, and its chemical structure is shown in Formula I: ； Formula I; The amino acid sequence of FR-14 is shown in SEQ ID 2, and its chemical structure is shown in Formula II: ； Formula II; The amino acid sequence of IK-10 is shown in SEQ ID 3, and its chemical structure is shown in Formula III: ； Formula III; The amino acid sequence of TR-17 is shown in SEQ ID 4, and its chemical structure is shown in Formula IV: ； Formula IV.

3. The method for screening sika deer bone peptides according to claim 1, characterized in that, In step two, the method for protein extraction using lysis buffer is as follows: First, place five sika deer cancellous bone samples from different developmental stages into mortars pre-cooled with liquid nitrogen, add liquid nitrogen and grind them thoroughly into deer bone powder, then add lysis buffer, sonicate or stir to lyse, then filter or centrifuge to remove cell debris, and obtain the protein samples from five different developmental stages. The lysis buffer contains 1-3 wt% sodium dodecyl sulfate and 1 wt% protease inhibitor, and the solvent is ultrapure water. The protease inhibitor is one or more of protease inhibitor VI, protease inhibitor V, protease inhibitor IV, and protease inhibitor III.

4. The method for screening sika deer bone peptides according to claim 3, characterized in that, In step two, The ratio of the deer bone powder to the lysis buffer is 1g:10mL-1g:20mL; The ultrasound duration is 30-60 minutes, the power is 100-300W, and the frequency is 20-80kHz. The stirring temperature is 40-60℃, and the stirring time is 2-4 hours; Centrifuge at 10000-13000g for 10 minutes at 4-8℃.

5. The method for screening sika deer bone peptides according to claim 1, characterized in that, In step three, the enzyme digestion process uses trypsin or pepsin as the protease.

6. The method for screening sika deer bone peptides according to claim 5, characterized in that, The enzymatic digestion process is as follows: First, take five protein samples of equal mass from five different developmental stages. Adjust the volume of the five protein samples to be consistent using lysis buffer. Then, add 1-2 volumes of acetone pre-cooled to 4°C to each sample, vortex to mix, and then add 3-5 volumes of acetone pre-cooled to 4°C to each sample. Precipitate at 4-8°C for 2-4 hours, centrifuge at 4000-5000g for 5-10 minutes, discard the supernatant, and wash the resulting precipitate 2-3 times with acetone pre-cooled to 4°C. After drying, the precipitates were dispersed by ultrasonication with tetraethylammonium bromide at a final concentration of 200-250 mM. Then, protease was added at a mass ratio of protease to protein sample of 1:45-55, and the enzyme was digested for 10-12 h. Then, dithiothreitol was added at a final concentration of 5-10 mM, and the samples were reduced at 50-60℃ for 30-40 min. Finally, iodoacetamide was added at a final concentration of 10-12 mM, and the samples were incubated at room temperature in the dark for 15-20 min to obtain peptide samples at five different developmental stages. The lysis buffer contains 1-3 wt% sodium dodecyl sulfate and 1 wt% protease inhibitor, and the solvent is ultrapure water. The protease inhibitor is one or more of protease inhibitor VI, protease inhibitor V, protease inhibitor IV, and protease inhibitor III.

7. The method for screening sika deer bone peptides according to claim 1, characterized in that, In step four, the conditions for high-performance liquid chromatography (HPLC) are as follows: mobile phase A is an aqueous solution containing 0.1wt%-0.2wt% formic acid and 1.5wt%-2wt% acetonitrile; mobile phase B is an aqueous solution containing 0.1wt%-0.2wt% formic acid and 90wt%-95wt% acetonitrile; the liquid phase gradient is set as follows: 0-22.5 min, 6%-22% mobile phase B; 22.5-26.5 min, 22%-34% mobile phase B; 26.5-28.5 min, 34%-80% mobile phase B; 28.5-30 min, 80% mobile phase B; and the flow rate is maintained at 700-750 nmol / min. The ion source ionization voltage is set to 2200-2300V, and the compensation voltage is set to -40V to -45V; The conditions for the mass spectrometry are as follows: the scanning range of the first-stage mass spectrometry is set to 350-1400 m / z, and the scanning resolution is set to 55000-60000; the scanning range of the second-stage mass spectrometry has a fixed starting point of 100-120 m / z, and the scanning resolution is set to 14000-15000. The breakage energy is set to 25%-30%, the automatic gain control is set to 1E6, and the maximum injection time is set to 20-24ms.

8. The application of the specific differential peptides obtained by the method for screening sika deer bone peptides according to claim 1, which conform to the periodic osteoporosis phenomenon of sika deer, in the preparation of drugs for treating or preventing osteoporosis.

9. A medicine for treating or preventing osteoporosis, characterized in that, It contains TR-17 as described in claim 2.

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