An osteogenic amino acid fragment Z01 and its screening and application

Abstract

This invention belongs to the field of biotechnology, specifically relating to an osteogenic amino acid fragment Z01 and its screening and applications. This invention utilizes a unique cell screening platform specifically for Osx: the CL-Osx-EGFP cell line. This platform fuses Osx and EGFP, using EGFP as a tracking marker to directly reflect changes in Osx expression and screen for compounds that can activate Osx. Z01 was first discovered to effectively activate Osx, and its amino acid sequence is shown in SEQ ID NO.1. Pharmacokinetic and toxicological analyses were performed on Z01, and its osteogenic activity was verified in an ovariectomized rat osteoporosis model. Ultimately, it was demonstrated that Z01 possesses osteogenic activity, can improve bone microstructure, increase bone density, and has a wide range of applications.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an amino acid fragment Z01 with osteogenic activity and its screening and application. Background Technology

[0002] Osteoporosis is the most common metabolic bone disease, characterized by decreased bone mass, altered bone microstructure, reduced bone strength, and increased fracture risk. Osteoporosis is caused by an imbalance in function between osteoblasts and osteoclasts. Discovering the mechanisms regulating key pathways in bone formation and resorption helps identify new treatments with specific mechanisms of action. Currently, most osteoporosis treatments aim to inhibit bone resorption, targeting osteoclasts. Since bone remodeling involves both bone formation and resorption, using only anti-resorption drugs is insufficient to effectively improve bone mass and structure. Stimulating new bone formation is a crucial strategy for more effective osteoporosis treatment, targeting osteoblasts. Currently, there are no drugs on the market that effectively and directly stimulate new bone formation. Elucidating the gene regulatory molecular mechanisms of bone formation is of great significance for guiding the development of new osteoporosis-specific bone-forming drugs.

[0003] Bone formation includes two pathways: intramembranous ossification and endochondrial ossification. Intramembranous ossification occurs when mesenchymal cells differentiate into embryonic connective tissue membranes, and then bone forms within these membranes; this is the case for the craniofacial region and clavicle. Endochondrial ossification is characterized by the process of pre-formed cartilage serving as a template, which is then replaced by bone tissue. It is far more complex than intramembranous ossification, and most bones, such as those in the limbs, trunk, and skull base, develop in this manner. Osteoblasts differentiate from mesenchymal stem cells and undergo multiple stages regulated by different transcription factors and signaling proteins. Ihh is a transcription factor essential for endochondrial ossification but does not participate in intramembranous ossification; it is indispensable for the differentiation of mesenchymal cells into osteoblasts. The transcription factor Runx2 participates in the regulation of both endochondrial and intramembranous ossification; cells expressing Runx2 can differentiate into osteoblasts or chondrocytes. The osteoblast-specific transcription factor Osterix (Osx) is essential for bone formation and osteoblast differentiation; mouse embryos lacking the Osx gene show no bone formation at all, while cartilage formation remains unaffected. Our research group's previous studies have identified several important downstream target genes of Osx, including VDR, SatB2, VEGF, Dkk1, and Sost, further confirming Osx's crucial role in bone formation. Osx directly regulates the osteoblast marker gene osteosialin (Bsp). Genome-wide association studies have confirmed the association between Osx and osteoporosis. Although Osx is associated with the osteoporosis phenotype, further research is needed to determine the molecular mechanisms by which Osx functions in bone formation, including exploring upstream regulators. Osteoporosis urgently requires novel drugs to promote bone formation, and Osx, as a bone molecular switch, represents an ideal new target.

[0004] Therefore, developing a new drug to promote bone formation is of paramount importance in this field, and Osx, as a novel target, is a key breakthrough in new drug development. Summary of the Invention

[0005] To address the common problems in existing technologies, this invention provides an osteogenic amino acid fragment Z01, its screening, and its applications. The Z01 fragment screened by this invention can exhibit osteogenic activity, improve bone microstructure, and increase bone density.

[0006] The amino acid sequence of the osteogenic amino acid fragment Z01 described in this invention is shown in SEQ ID NO.1.

[0007] SCAPGADPKSRLCALCAGDDQGLDKCVPNSKEKYYGYTGAFRCL(SEQ ID NO.1);

[0008] The present invention also provides a screening process for the osteogenic amino acid fragment Z01, comprising the following steps:

[0009] S1. Based on the structure of the CL-XWY-012 gene and experimental requirements, P2A-EGFP was knocked into the stop codon of exon 2 of the CL-XWY-012 gene to serve as the target region.

[0010] S2. The target region was amplified using PCR technology and verified by sequencing to obtain the amplified target region.

[0011] S3. Design 16 sgRNAs near the target region, screen for 2 sgRNAs with high activity, and construct the targeting vector after enzyme digestion identification and sequencing.

[0012] S4. The two sgRNAs obtained in step S3 were electroporated into the C2C12 cell line using a targeting vector. After drug screening, positive clone enrichment, and PCR screening, the positive clone CL-Osx-EGFP cell line was obtained.

[0013] S5. The positive clone CL-Osx-EGFP cell line obtained in step S4 was cultured in DMEM medium. When the cell line reached 60%-80% saturation, it was treated with compounds such as BMP2 and Lactoferrin (LF) for 24 hours. BMP2 was used as a positive control to obtain the drug-treated CL-Osx-EGFP cell line. RNA was extracted and reverse transcribed into cDNA. Using the synthesized cDNA as a template, the expression of EGFP gene was detected by real-time quantitative PCR technology, indicating that the LF compound is an activator of Osx.

[0014] S6. The next step is to verify the activation effect of LF on Osx: C2C12 mesenchymal stem cells were cultured in DMEM cell culture medium at 37°C and 95% saturated humidity. The cells were placed in 6-well plates and cultured until 60%-80% saturation. Then, they were treated with LF for 24 hours. RNA was extracted from the LF-treated C2C12 cells and reverse transcribed into cDNA. Using the synthesized cDNA as a template, the expression of Osx gene was detected by real-time quantitative PCR technology. Furthermore, the function of different sized fragments of LF was detected and screened to obtain Z01.

[0015] Preferably, the two highly active sgRNAs selected in step S3 are CL-XWY-012-sgRNA4 and CL-XWY-012-sgRNA14.

[0016] Preferably, the oligo sequence corresponding to CL-XWY-012-sgRNA4 in step S3 is shown in SEQ ID NO.2; and the oligo sequence corresponding to CL-XWY-012-sgRNA14 is shown in SEQ ID NO.3.

[0017] Preferably, the electroporation process described in step S4 uses cell electroporation, which involves treating the cell with a short, high-intensity electric pulse twice. The voltage difference along the cell membrane causes the current to reversibly break down the cell membrane, forming a transient water pathway or a small pore on the membrane, thereby allowing exogenous DNA to enter the cell membrane.

[0018] During electroporation, the specific conditions were as follows: the electroporation system was a Neon Transfection System (MPK5000); the pulse voltage was 1400V, the pulse width was 20, the transfection efficiency was 90%, and the cell viability was 94.1%.

[0019] Preferably, the DMEM culture medium in step S5 is a DMEM culture medium containing 10% fetal bovine serum, 100 U / mL penicillin G sodium and 100 μg / mL streptomycin sulfate.

[0020] The present invention also provides the application of the amino acid fragment Z01 in the preparation of a drug with osteogenic activity.

[0021] Preferably, the drug further includes physiological saline and a pharmaceutically acceptable carrier.

[0022] Preferably, the drug dosage form is one of the following: injection, tablet, capsule, oral liquid, microparticle, or ointment.

[0023] The C12-Osx-GFP self-made screening cell line of this invention is used to screen various molecular compounds and detect EGFP expression; while the C2C12 cells are used to verify the Osx-activating function of the screened molecular compounds and detect Osx expression. The former cells first screen for LF, and the latter cells verify the Osx-activating function of LF. Further optimization of the LF structure led to the discovery of the smallest amino acid fragment of 44aa, which was named Z01.

[0024] To effectively screen new drug compounds, the patentee has developed a unique cell screening platform specifically for Osx: the CL-Osx-EGFP cell line. In this platform, Osx and EGFP are fused together, with EGFP acting as a tracking marker to directly reflect changes in Osx expression, thus screening for compounds that can activate Osx. Using this platform, compound LF was identified as an Osx activator. The activation function of LF for Osx was verified in the C2C12 cell line. LF is 708 amino acids in length. Preliminary analysis revealed that LF contains two potential functional domains. Functional testing of different fragments showed that even the smallest fragment of 44 amino acids could effectively activate Osx, and this fragment was named Z01. Pharmacokinetic and toxicological analyses were then performed on Z01. Finally, the osteogenic activity of Z01 was verified in an ovariectomized rat model of osteoporosis. The results showed that Z01 possesses osteogenic activity, improves bone microstructure, and increases bone mineral density.

[0025] Compared with the prior art, the present invention has the following advantages: The present invention successfully screened 44 amino acid fragments for the first time, and through pharmacokinetic analysis, osteogenic activity verification, etc., it was finally proved that Z01 has osteogenic activity and can be used for osteoporosis treatment, providing a new treatment option for osteoporosis. Attached Figure Description

[0026] Figure 1 The results are for sgRNA activity assay.

[0027] Figure 2 Schematic diagram of the construction of the firing platform;

[0028] Figure 3 Diagram illustrating the construction process of the firing platform;

[0029] Figure 4 The map obtained for constructing the target carrier;

[0030] Figure 5 Partial identification results for primers CL-XWY-012-L-GT-F / CL-XWY-012-L-GT-R;

[0031] Figure 6 Partial identification results for primers CL-XWY-012-L-GT-F / CL-XWY-012-R-GT-R;

[0032] Figure 7 Partial identification results for primers CL-XWY-012-L-GT-F / CL-XWY-012-WT-R;

[0033] Figure 8 To screen for compounds LF that activate Osx in the CL-Osx-EGFP cell line;

[0034] Figure 9 The results show that compound LF activates the expression of the Osx gene through the p38 signaling pathway;

[0035] Figure 10 The results show the activation of Osx gene expression by the minimum fragment Z01 of compound LF;

[0036] Figure 11 The mass spectrometry result of Z01 at m / z 779.4 is shown.

[0037] Figure 12 The mass spectrometry results for Z01 at MS2 776.3 are shown below.

[0038] Figure 13 The standard curve for Z01 blood drug concentration in the range of 0.2–100 μg / mL is shown.

[0039] Figure 14 A graph showing the expression of the Osx gene in different groups;

[0040] Figure 15 A graph showing OPG gene expression in different groups;

[0041] Figure 16 The results of ELISA analysis of serum bone metabolism markers for TRACP-5b;

[0042] Figure 17 The results of ELISA analysis of serum bone metabolism markers for B-CTx;

[0043] Figure 18 This shows the protein expression data from Western blotting.

[0044] Figure 19 Comparison of bone mineral density results between different groups;

[0045] Figure 20 The results show the 3D reconstruction of different experimental groups. Detailed Implementation

[0046] The present invention will be further explained below with reference to specific embodiments. However, it should be noted that the following embodiments are only used to explain the present invention and cannot be used to limit the present invention. All technical solutions that are the same as or similar to the present invention are within the protection scope of the present invention. Where specific techniques or conditions are not specified in this embodiment, they shall be operated according to conventional technical methods and instrument manuals in the art; where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be obtained commercially. The sequence information involved in the present invention is all from Sangon Biotech.

[0047] In this invention, Wild type allele (WT): unmodified wild-type cell line; Targeted allele: genetically modified cell line; no band: no expected band detected by PCR reaction; HR: homologous recombination; FS: frameshift; Del: deletion.

[0048] Instructions for use of positive clones:

[0049] Cell culture conditions: DMEM, 10% FBS, 1% NEAA, 1% glutamine, 1% HEPES, 1% sodium pyruvate, 1.5 μg / ml puromycin, 37℃, 5% CO2. Trypsin concentration: 0.25%, trypsin digestion time: 1 min; cryopreservation conditions: 95% complete medium (antibiotic-free), 5% DMSO, stored in liquid nitrogen.

[0050] Cell resuscitation steps

[0051] Add 10 mL of culture medium to a 15 mL centrifuge tube. Wearing a face shield and cryoprotective gloves, quickly remove the cryovial from liquid nitrogen and place it in a 37°C water bath, gently shaking until completely thawed. Transfer the suspension from the cryovial to the centrifuge tube, tighten the cap, and gently invert several times to mix. Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, gently disperse the cells by tapping, add an appropriate amount of culture medium, and gently shake the culture flask (or dish) until the cells are evenly distributed. Place in an incubator for further culture.

[0052] Extraction of cellular genomic DNA

[0053] Preparation of lysis buffer: Add each component according to the following steps and bring the volume to 200 mL.

[0054] 2 mL of 1 M Tris-HCl (pH 7.5); 4 mL of 0.5 M EDTA (pH 8.0); 2 mL of 1 M NaCl; 8 mL of 5% SDS; add purified water to 200 mL.

[0055] Proteinase K (PK): Storage concentration: 10 mg / mL; final concentration: 1 mg / mL. Store the prepared lysis buffer at room temperature. Add 50 μL of PK to every 450 μL of lysis buffer before use.

[0056] Extraction steps: Digest cells, take an appropriate amount of cell suspension (cell density for genotyping: whole cells in a 96-well plate or 70% cell density in a 48-well plate); add to a 1.5 mL EP tube, centrifuge at 12000 rpm for 1 min; remove the culture medium, add 450 μL of lysis buffer and 50 μL of PK, lyse at 55℃ for 2 h or overnight; equilibrate to room temperature, add 1 mL of pre-chilled anhydrous ethanol, invert and mix thoroughly to precipitate DNA; centrifuge at 12000 rpm for 10 min; discard the supernatant, wash with 700 μL of 70% ethanol, discard the supernatant; briefly centrifuge, discard residual liquid; place the EP tube at room temperature for 5–10 min to allow residual ethanol to evaporate completely. Add 30–100 μL of ddH2O, dissolve at 55℃ for 2 h or more. Measure the concentration using a spectrophotometer and label accordingly. Quality control points: A260 / A280 should be between 1.8 and 2.0; the concentration of DNA used for target sequence amplification should not be less than 100 ng / μL; the concentration of DNA used for genotyping should be ≥10 ng / μL.

[0057] Screening of Example 1Z01

[0058] Based on the structure of the CL-XWY-012 gene and experimental requirements, P2A-EGFP was knocked into the Exon2 stop codon of the CL-XWY-012 gene. A CL-XWY-012 knock-in C2C12 cell line was then prepared using CRISPR / Cas9-based molecular technology. The specific process is as follows:

[0059] S1. Based on the structure of the CL-XWY-012 gene and experimental requirements, P2A-EGFP was knocked into the stop codon of exon 2 of the CL-XWY-012 gene to serve as the target region.

[0060] The target gene sequences may differ between different cell lines. To ensure the efficiency of the designed Cas9 / sgRNA, the target sequence of the C2C12 cell line was first amplified by PCR and sequenced for verification, so as to ensure that the sgRNA recognition sequence is completely consistent with the DNA sequence of the C2C12 cell line.

[0061] S2. The target sequence was amplified using PCR technology and verified by sequencing to obtain the amplified target sequence. PCR and sequencing were performed on the DNA of the C2C12 cell line, and the results showed that the C2C12 cell line was completely consistent with the sequences provided by Genebank and Ensembl. The PCR primers involved were: CL-XWY-012-5'MSD-F, CL-XWY-012-5'MSD-R (amplified fragment length 538bp); CL-XWY-012-3'MSD-F, CL-XWY-012-3'MSD-R (amplified fragment length 851bp).

[0062] S3. Sixteen sgRNAs were designed near the target site sequence, and two sgRNAs with high activity were screened. At the same time, the targeting vector was constructed by enzyme digestion identification and sequencing.

[0063] Based on the design principles of sgRNA, a total of 16 sgRNAs were designed in the target site region, and their corresponding oligo sequences are as follows: 5' Guide: #1: TCCACCGCGCCAAGAGAGCC TGG #2: GGAAGATCGGGGCGGCTGAT TGG #3: GCCAGGCTCTCTTGGCGCGCGG TGG #4: CCCCCAGAAAAAGCCCACGG AGG (SEQ ID NO.2); #5: GTGCAGGCGAAGTGGAAGAT CGG #6: CTGCTCTGGGCTGCCTCCGT GGG ; #7: TCTAGCAGGTTGCTCTGCTCTGG; #8: CTAGCAGGTTGCTCTGCTCT GGG .

[0064] 3'Guide: #9: CGCCTTTGAAGCCGCGCC CGG #10: TCCGGCCGGAGTATGAGAAC TGG #11: GGCCGCACAAAGCCCTATTG TGG #12: GGTCTGGGCAATGATCCGGC CGG #13: AAGCGCGCGCCCGGCCAGA GGG #14: CTTGTGAACCACGGGATCCG GGG (SEQ ID NO.3) #15: CCCAGTTCTCATACTCCGGC CGG #16: CGGGTTCCGCGCCTCCACTG CGG .

[0065] The activity of sgRNA was detected using the CRISPR / Cas9 activity assay, and the results are as follows: Figure 1 As shown, considering factors such as activity and specificity, CL-XWY-012-sgRNA4 and CL-XWY-012-sgRNA14 were selected for the next stage of experiments.

[0066] A schematic diagram of the construction of the target carrier is shown below. Figure 2 As shown, the construction process of the target carrier is as follows: Figure 3 As shown, the final constructed target carrier map is as follows: Figure 4 As shown in the figure, it can be seen that during vector construction, identification was performed by grouping enzyme digestion with HindIII+Sall, NcoL, and Eco47III+Xhol, and then sequencing was used to confirm the successful construction of the targeting vector.

[0067] S4. The two sgRNAs obtained from step S3 were electroporated into the C2C12 cell line using a targeting vector. After drug screening, positive clone enrichment, and PCR screening, the positive clone CL-Osx-EGFP cell line was obtained. The PCR reaction system was based on KOD-FX DNA polymerase, and the specific components are shown in Table 1 below.

[0068] Table 1. PCR reaction system of KOD-FX DNA polymerase (total 20 μL)

[0069] reaction system Volume (μL) Final concentration <![CDATA[ddH2O]]> 2.4 -- 2×KOD FX buffer 10 1× 2mM dNTPs 4 0.4mM each 10μM Primer-F 0.6 0.3μM 10μM Primer-R 0.6 0.3μM 1 U / μL KOD FX DNA Polymerase 0.4 0.02 U / μL 100-200 ng / 20 μL Template DNA 2 10-20 ng / μL

[0070] Identification of the positive clone CL-Osx-EGFP cell line:

[0071] First: HR allele PCR identification: Primers CL-XWY-012-L-GT-R and CL-XWY-012-R-GT-F are designed for the exogenous gene sequence, while primers CL-XWY-012-L-GT-F and CL-XWY-012-R-GT-R are designed for the wild-type gene sequence. Therefore, when using these two primer pairs, CL-XWY-012-L-GT-F / CL-XWY-012-L-GT-R and CL-XWY-012-R-GT-F / CL-XWY-012-R-GT-R, PCR will not amplify the product of the wild-type allele, but only the product of the mutant allele.

[0072] The primer information is as follows: CL-XWY-012-L-GT-F, CL-XWY-012-L-GT-R (amplification length 4644bp); CL-XWY-012-R-GT-F, CL-XWY-012-R-GT-R (length 3873bp).

[0073] PCR conditions (Enzyme: KOD-FX Touchdown): 94℃ for 2 min; 98℃ for 10 s, 67℃ for 30 s (-0.7 / cycle), 68℃ for 1 kb / min, 15 cycles; 98℃ for 10 s, 57℃ for 30 s, 68℃ for 1 kb / min, 25 cycles; 68℃ for 10 min; store at 4℃.

[0074] The identification results of primers CL-XWY-012-L-GT-F / CL-XWY-012-L-GT-R are as follows: Figure 5 As shown; the identification results of primers CL-XWY-012-L-GT-F / CL-XWY-012-R-GT-R are as follows. Figure 6 As shown.

[0075] Second: FS allele PCR identification: Primers CL-XWY-012-L-GT-F and CL-XWY-012-WT-R were designed within the wild-type gene sequence. Therefore, using the CL-XWY-012-L-GT-F / CL-XWY-012-WT-R primer pair can amplify both the PCR product of the wild-type allele and the PCR product of the frameshift mutation allele. Sequencing can then confirm the specific genotype. The fragment lengths are: WT: 3871 bp, Mut: 7182 bp; Mut-KO: approximately 1503 bp.

[0076] The FS allele primer information is as follows: CL-XWY-012-L-GT-F, CL-XWY-012-WT-R;

[0077] PCR conditions (Enzyme: KOD-FX Touchdown): 94℃ for 2 min; 98℃ for 10 s, 67℃ for 30 s (-0.7 / cycle), 68℃ for 1 kb / min, 15 cycles; 98℃ for 10 s, 57℃ for 30 s, 68℃ for 1 kb / min, 25 cycles; 68℃ for 10 min; store at 4℃.

[0078] The final identification results of primers CL-XWY-012-L-GT-F / CL-XWY-012-WT-R are as follows: Figure 7As shown in Table 2, the results of each positive clone are summarized below. It can be seen that, through sequencing of PCR products, 2-B02 and 3-G09 are knock-in positive clones, namely the CL-Osx-EGFP cell line.

[0079] Table 2 Comparison results of positive clones

[0080]

[0081]

[0082] S5. The positive clone CL-Osx-EGFP cell line obtained in step S4 was cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin G sodium and 100 μg / mL streptomycin sulfate. When the cell line reached 60%-80% saturation, it was treated with compounds such as BMP2 and LF (each drug was added at 500 ng / mL) for 24 hours. BMP2 was used as a positive control.

[0083] Cells were collected 24 hours after drug treatment, and total RNA was extracted from the tissues using the RNeasy Mini kit (QIAGEN). The obtained RNA was reverse transcribed into cDNA using the PrimeScript RT Master Mix (TaKaRa) kit. EGFP gene expression was detected using real-time quantitative PCR (qPCR) with the synthesized cDNA as a template. The qPCR kit used was TB Green Premix Ex Taq II (TaKaRa). GAPDH was used as an internal control. -△△Ct The method is used to analyze the results.

[0084] like Figure 8 As shown, the BMP2 positive control activated EGFP expression by 2.8-fold, and after LF treatment of the cell line, EGFP expression also increased by 1.9-fold, indicating that the LF compound is an activator of Osx.

[0085] S6. The next step is to verify the activation effect of LF on Osx. C2C12 mesenchymal stem cells (ATCC) were cultured in DMEM cell culture medium containing 10% fetal bovine serum (FBS, Gibco), 100 U / mL penicillin G sodium, and 100 μG / mL streptomycin sulfate at 37°C and 95% saturated humidity. Cells were placed in 6-well plates and cultured until 60%-80% saturation, then treated with LF for 24 hours. RNA was extracted from the LF-treated C2C12 cells and reverse transcribed into cDNA. Using the synthesized cDNA as a template, real-time quantitative PCR was used to detect Osx gene expression. Further functional analysis and screening of different LF fragment sizes were performed to obtain Z01.

[0086] Cells were collected 24 hours after LF treatment, and total RNA was extracted from the tissues using the RNeasy Mini kit (QIAGEN). The obtained RNA was reverse transcribed into cDNA using the PrimeScript RT Master Mix (TaKaRa) kit. Osx gene expression was detected using real-time quantitative PCR (qPCR) with the synthesized cDNA as a template. The qPCR kit used was TBGreen Premix Ex Taq II (TaKaRa). GAPDH was used as an internal control. -△△Ct The method is used to analyze the results.

[0087] like Figure 9 As shown, LF activated Osx expression 5.1-fold. SB203850 is an inhibitor of the p38MAPK phosphorylation signaling pathway and can competitively bind to the ATP binding site of p38. sp600125 is an inhibitor of the JNK signaling pathway. This indicates that compound LF activates Osx gene expression through the p38 signaling pathway. LF is 708 amino acids in length. Preliminary analysis suggests that LF contains two potential functional domains. Functional analysis of different fragments revealed that even the smallest fragment of 44 amino acids can effectively activate Osx (results are shown in Figure 1). Figure 10 In addition, the downstream gene OCN of Osx is also activated. We named the smallest LF fragment, a 44-amino acid aa fragment, Z01.

[0088] Pharmacokinetic Analysis of Experimental Example 1

[0089] 1. Test sample: Accurately weigh the test substance Z01, dissolve and dilute it with PEG400 (containing 1% DMSO and 1% MeOH) to the dosage concentration, and administer it to animals.

[0090] 2. Experimental Instruments: The experimental samples and instruments involved are as follows: Methanol and acetonitrile were purchased from Merck, Germany, and were of HPLC grade; Formic acid and DMSO were purchased from Thermo Fisher Scientific (China) Co., Ltd.; formic acid was of LC / MS grade, and DMSO was of HPLC grade; PEG400 was purchased from Wuxi Yatai United Chemical Co., Ltd.; it was of analytical grade; the liquid chromatography system was an ExionLC ultra-high performance liquid chromatograph, purchased from Sciex, USA; the MS / MS system was an LCMS8060 ultra-high performance liquid chromatography-mass spectrometry system, purchased from Shimadzu; the analytical balances were XSE105DU and ME203E analytical balances, purchased from Mettler Toledo; the centrifuge was a Legend Micro 21R refrigerated high-speed centrifuge, purchased from Thermo Fisher Scientific.

[0091] 3. Experimental Procedure: Six SD rats were numbered and weighed, and randomly divided into two groups of three each. The rats were administered Z01 solution intravenously and orally, respectively. Animals were fasted for 12 hours before administration, but had free access to water. Specific administration information is shown in Table 3 below.

[0092] Table 3. Drug Administration Information Table

[0093]

[0094] Then, collect approximately 1 mL of whole blood into an anticoagulant tube (EDTAK2), centrifuge at 2000g (4℃) for 10 min, collect the plasma, and store it in a -20℃ refrigerator. Rat plasma samples were processed using the organic solvent protein precipitation method, with the following steps: Take 40 μL of plasma, add 160 μL of acetonitrile containing 1% FA, vortex for approximately 1 min, centrifuge for 10 min (4℃, 20200g), and collect 100 μL of the supernatant (i.e., acetonitrile solution) for LC-MS / MS analysis.

[0095] LC-MS / MS conditions and sample preparation: Chromatographic conditions were as follows: Column: SHIMADZU Shim-pack Velox C18 (2.1×50mm×2.7μm); Mobile phase: Phase A: purified aqueous solution (containing 0.1% AA), Phase B: acetonitrile solution (containing 0.1% AA); Eluent for injection: methanol; Autosampler temperature: 15℃; Column oven temperature: 30℃; Injection volume: 10μL. Gradient elution: The elution process is shown in Table 4.

[0096] Table 4 Liquid phase elution conditions

[0097] Time (min) Phase A (%) Phase B (%) Flow rate (mL / min) 0.00 90 10 0.4 0.50 90 10 0.4 2.50 5 95 0.4 2.51 90 10 0.4 3.00 90 10 0.4

[0098] Note: Inject mass spectrometer at 0.5 min to 2.5 min, and inject waste liquid at 0 to 0.5 min and 2.5 to 3 min.

[0099] The mass spectrometry conditions were as follows: the ion source was a Turbo IonSpray source;

[0100] Z01: Positive ion detection; Nebulizing Gas Flow: 3 L / min; Heating Gas Flow: 10 L / min; Interface Temperature: 400℃; DL Temperature: 250℃; Heat Block Temperature: 400℃; Drying Gas Flow: 3 L / min; Scan mode: Multiple Reaction Monitoring (MRM); Scan time: 200 ms; Collision energy, declustering voltage, and ion pairs used for quantitative analysis are shown in Table 5 below. Mass spectra are shown below. Figure 11 , Figure 12 .

[0101] Table 5 Mass Spectrometry Conditions

[0102] Precursor ions product Q1 CE(V) Q3 Z01 779.4 776.3 -20 -22 -30

[0103] Solution preparation: Stock solution preparation: Accurately weigh one part of Z01, dissolve and dilute with methanol to obtain a stock solution with a concentration of 2.00 mg / mL. Z01 It is used for the preparation of standard curves and quality control working solutions. Preparation of standard curves and quality control samples.

[0104] Precisely pipette Z01 stock solution. Z01 (2.00 mg / mL), using acetonitrile as a diluent, mixed standard working curve working solutions (0.2, 0.5, 1, 2, 10, 50, 100 μg / mL) and quality control working solutions (0.6, 5, 80 μg / mL) were prepared. The specific dilution process is shown in Table 6 below.

[0105] Table 6 Preparation of Standard Series Solutions

[0106]

[0107]

[0108] Take 40 μL of each of the above standard curve working solutions and dilute with 40 μL of blank plasma from SD rats to obtain standard curve samples Cal.1~Cal.7 (0.2, 0.5, 1, 2, 10, 50, 100 μg / mL) of Z01 with different plasma concentrations, as well as plasma quality control samples with low, medium and high concentrations (0.6, 5, 80 μg / mL), which are used for the analysis of standard curves and quality control samples.

[0109] Plasma sample processing protocol:

[0110] ① Processing of unknown samples: The plasma samples of SD rats were processed by the organic solvent protein precipitation method. The operation steps are as follows: 160 μL of precipitant (acetonitrile containing 1% FA) was added to 40 μL of SD rat plasma, vortexed for about 1 min, centrifuged for 10 min (4℃, 20200g), and 100 μL of supernatant was transferred for LC-MS / MS analysis.

[0111] ②Double Blank Sample Processing: The raw material was 40 μL of blank plasma from SD rats, and the rest was the same as in ①.

[0112] ③ Cal and QC Sample Processing: 40 μL of blank SD rat plasma was added, along with 40 μL of standard curve working solution and quality control working solution to obtain standard curve samples with blood drug concentrations of 0.2, 0.5, 1, 2, 10, 50, and 100 μg / mL, and quality control samples with blood drug concentrations of 0.6, 5, and 80 μg / mL. 120 μL of precipitant (acetonitrile containing 1% FA) was added, vortexed for approximately 1 min, centrifuged for 10 min (4℃, 20-200g), and 100 μL of supernatant was transferred for LC-MS / MS analysis. The experiment was conducted according to the Pharmacopoeia of the People's Republic of China, Part IV (2020). 4. Experimental Results: System Suitability Study (Numbers marked with * in each experimental result are not included in the calculation).

[0113] Take 40 μL of Z01 standard curve working solution C-4, dilute with 40 μL of blank plasma to obtain the Z01 system suitability sample, and perform analysis on 6 parallel samples according to the "Plasma Sample Processing Protocol". The results show that the CV% of the compound peak area in the Z01 plasma system suitability sample is 3.83%. This method has good system adaptability on the instrument and can be used for biological sample analysis. Detailed determination results are shown in Table 7.

[0114] Table 7 System Applicability Results

[0115]

[0116]

[0117] Selectivity assessment: The experimental procedure was similar to that for system suitability assessment; the average interference rate of the method selectivity was 0.00%, which meets the relevant requirements for biological sample determination and the relevant SOPs of this laboratory. Detailed test results are shown in Table 8.

[0118] Table 8. Results of selective detection by method

[0119]

[0120] Standard curve: Prepare standard curve samples Cal.1–Cal.7 (0.2, 0.5, 1, 2, 10, 50, 100 μg / mL) from blank plasma, 40 μL each. Inject 10 μL according to the "Plasma Sample Processing Protocol" and record the chromatogram. Plot the analyte concentration on the x-axis and the analyte peak area on the y-axis, and use a weighted average (W = 1 / c) to calculate the peak area. 2 The least squares method was used for regression analysis to obtain the linear regression equation and correlation coefficient r. The results showed that when the blood concentration of Z01 was in the range of 0.2–100 μg / mL, the concentration and peak area exhibited a good linear relationship: Y = 37992.7x + 7845.17 Figure 13The correlation coefficient r was 0.9976, and the accuracy deviation (RE%) of the standard curve samples were all within the range of -9.10% to 5.02%. The regression equation of the standard curve is shown in Table 9, and the measurement results and accuracy of each concentration point are shown in Table 10.

[0121] Table 9 Summary of Standard Curve Parameters

[0122]

[0123]

[0124] Table 10. Sample concentrations for plasma standard curve (n=2)

[0125]

[0126] Take 40 μL each of the low, medium, and high concentration quality control samples (0.6, 5, and 80 μg / mL) and LLOQ (0.2 μg / mL) prepared from blank plasma, and inject 10 μL of each sample according to the "Plasma Sample Processing Protocol". Analyze each concentration in triplicate (6 samples). Analyze the samples according to the accompanying standard curve (i.e.,...). Figure 13 The standard curve was used to calculate the actual concentration of the QC sample. The results showed that the accuracy deviation of the Z01 plasma quantification limit sample was in the range of -4.00% to 15.00%, with an intra-batch precision (%CV) of 7.66% and an accuracy deviation (%RE) of 3.83%. The accuracy deviation of the three concentration quality control samples (low, medium, and high) was in the range of -14.12% to 14.17%, with intra-batch precision (%CV) of 8.54%, 2.70%, and 6.00%, respectively, and accuracy deviations (%RE) of 4.92%, -9.78%, and -7.42%, respectively. Detailed measurement results are shown in Table 11.

[0127] Table 11 Precision and accuracy of plasma samples (n=6)

[0128]

[0129]

[0130] Residual effect investigation: After injection at the upper limit of quantitation (highest concentration of the standard curve), a blank matrix sample (residual effect investigation sample) was injected. The residual effect was evaluated by comparing the peak area of ​​Z01 in the residual effect investigation sample and the sample at the lower limit of quantitation (lowest concentration of the standard curve). The results showed that the residual effect of Z01 in the plasma sample was 4.39%. Detailed data are shown in Table 12.

[0131] Table 12 Investigation of residual effects

[0132]

[0133] Pharmacokinetic studies: The average plasma concentrations of Z01 in male SD rats after administration of 20 mg / kg by gavage and 50 mg / kg by tail vein injection according to the "Animal Administration" method are shown in Table 13. This indicates that the drug is metabolized normally in vivo.

[0134] Table 13 Average drug concentration of Z01 in plasma (n=3)

[0135]

[0136]

[0137] Experimental Example 2: Verification of Osteogenic Activity

[0138] 1. Experimental animals: 12 three-month-old female SD rats, weighing 270±10g, were divided into 3 groups of 4 rats each.

[0139] 2. Modeling Process: The bilateral oophorectomy (OVX) model is a live model artificially induced to induce ovarian dysfunction. First established by Savville in 1969, it has been repeatedly verified and applied, and is now a classic model for osteoporosis research. The specific procedure is as follows: The rat is anesthetized intraperitoneally with 3% sodium pentobarbital at 0.1 mL / 100 g, and the surgical area is shaved. The rat is fixed in a lateral position, and a 1-2 cm incision is made in the lateral abdomen. The skin, fascia, and muscle are sequentially cut or bluntly dissected with scissors, revealing a white, moist fat mass. After separating the folds of the fat mass, a bright pink, cauliflower-shaped ovary is visible. After blunt dissection of the abdominal muscles and peritoneum, the abdomen is entered. The ovary appears as pink granular tissue, mostly obscured by surrounding adipose tissue. The ovary and fallopian tubes are removed, and the adipose tissue is ligated near the uterus. The ovary and part of the fallopian tube are then cut off. After ligation, the adipose tissue is returned to the abdominal cavity, and the incision is sutured. After a certain period of time, the animal was euthanized, and blood, urine, and bone (tibia, femur, vertebrae) samples were separated for various tests.

[0140] 3. Experimental procedure: Ovariectomized osteoporosis model was surgically constructed using 3-month-old (12-week-old) female SD rats. The screened and optimized Z01 molecular compound was used as a drug in the osteoporosis animal model to verify the osteogenic efficacy.

[0141] The specific procedure is as follows: Twelve rats were selected as experimental animals. After one week of acclimatization, four rats underwent sham surgery (Sham), while the remaining rats underwent bilateral ovariectomy (OVX). Three days after surgery, the rats were divided into three groups of four rats each: the sham surgery control group (Sham) was given saline by gavage (50 mL / kg / d); the ovariectomy experimental group (OVX) was given saline by gavage (50 mL / kg / d); and the ovariectomy drug group (OVX-Z01) was treated with Z01 by gavage (20 mg / kg / d).

[0142] The treatment lasted for 12 weeks via gavage. At the end of the treatment, rats were euthanized by anesthesia with an overdose of chloral hydrate, and bone tissue was obtained for further analysis. Simultaneously, blood samples (1 mL / rat) were collected from the left ventricle of each rat. Serum samples were separated by centrifugation at 2500 rpm for 10 min at 4°C and stored at -80°C for later use. This study was approved by the Ethics Committee of Beijing Jishuitan Hospital (2203-03).

[0143] Next, functional assessments were performed on the rat animal model:

[0144] ① Total RNA extraction and qPCR detection from animal tissues

[0145] After experimental treatment, rats were anesthetized and euthanized. Bilateral upper limb bones were harvested from SD rats, and muscle tissue was removed except for bone tissue. The bones were pre-cooled to 4°C and washed three times with PBS. The shredded bone tissue was placed in a cryo-grinding apparatus container, which was completely immersed in liquid nitrogen for 4 minutes. The container was then mounted on the cryo-grinding apparatus and ground for 30 seconds, six times. The ground powder was poured into 1.5 mL centrifuge tubes, and total RNA was extracted according to the RNeasy Mini kit (QIAGEN) instructions. The obtained RNA was reverse transcribed into cDNA using the PrimeScript RT Master Mix (TaKaRa) kit. The reverse transcription system consisted of 8 μL of 5×PrimeScript RT Master Mix, 2 μg of RNA template, and enzyme-free water added to a final volume of 20 μL. Using the synthesized cDNA as a template, bone gene expression was detected using real-time quantitative PCR. The real-time quantitative PCR kit used was a TB GreenPremix Ex Taq II (TaKaRa), and the detection instrument was an iQ5 PCR system (ANALYTIKJENA). The total reaction volume was 10 μL, including 5 μL of reaction solution, 3 μL of cDNA, 2 μL of 2 μM upstream and downstream primer sequences, and 40 cycles of 95℃ for 30 s, 95℃ for 5 s, and 60℃ for 34 s. GAPDH was used as an internal control. -△△Ct The results were analyzed using the following methods. The primers involved are as follows: Rat GAPDH F1 / Rat GAPDH R1; Rat OPG F1 / R1; Rat OSX F1 / R1.

[0146] ② ELISA analysis of serum bone metabolism markers: Determination of bone metabolism marker levels: Blood was collected from the heart of anesthetized rats. After the whole blood samples were left at room temperature for 1 hour, they were centrifuged at 1000g for 20 minutes at 4℃. The supernatant was collected, and the levels of tartrate-resistant acid phosphatase 5b (TRACP-5b) and β-collagen crosslaps (β-CTx) in the serum were detected by ELISA.

[0147] Reagents and instruments: TRACP-5b ELISA Kit (Elabscience, E-EL-R0939c), β-CTx ELISA Kit (Elabscience, E-EL-R1405c). ELISA analyzer (PerkinElmer, USA).

[0148] Follow the instructions for the corresponding kit when performing ELISA. TRACP-5b uses the double-antibody sandwich ELISA method, and β-CTx uses the competitive ELISA method.

[0149] ③Western Blot protein analysis: The main reagents include formaldehyde; β-actin primary antibody (Immunoway, China), OSX primary antibody (Abcam, UK); biotin-labeled anti-goat IgG, RIPA protein lysis buffer, BCA protein quantification kit; the main instrument is the Azure Biosystems C series imaging system (USA).

[0150] Western blot was used to detect changes in the expression of β-actin and OSX in rat bones. Cells (i.e., cells extracted from experimental rat bone tissue) were lysed on ice using RIPA protein lysis buffer for 20 min, with shaking for at least 30 s each time. The cells were then centrifuged at 15000 r / min for 5 min at 4°C, and the supernatant was collected to obtain total protein. Based on the BCA protein quantification results, the cells were loaded onto a 12.5% ​​SDS-PAGE gel at 120V for 90 min. After transfer, the cells were blocked with 5% skim milk powder at room temperature for 2 h, incubated with primary antibody overnight at 4°C, washed with TBST for 10 min (repeated 3 times), incubated with secondary antibody at room temperature for 2 h, washed with TBST for 10 min (repeated 3 times), and then exposed for development.

[0151] To further clarify the osteogenesis-promoting effect of Z01, we performed morphological analysis of bone tissue using μCT. Procedure: After grouping and harvesting, rat femurs were placed in formalin and labeled for grouping. The tissues were then returned to the animal facility for local μCT analysis. 3D reconstruction and bone mineral density were examined, and fine bone structure was analyzed.

[0152] 4. Experimental Results: ① Total RNA extraction and qPCR detection from animal tissues: such as... Figure 14 The results showed that, compared with the Sham group, the OVX group had a significantly decreased expression of the Osx gene in bone molecules, while the Osx gene expression in the OVX-Z01 treatment group was restored. Figure 15 The results showed that, compared with the Sham group, OPG gene expression was significantly decreased in the OVX group, while OPG gene expression was restored in the OVX-Z01 treatment group. This suggests that Z01 treatment has osteogenic efficacy.

[0153] ②ELISA analysis of serum bone metabolism markers: such as Figure 16 The results showed that, compared with the Sham group, serum TRACP-5b expression was significantly increased in the OVX group, while TRACP-5b expression decreased in the OVX-Z01 treatment group, approaching that of the Sham group. Figure 17 The results showed that, compared with the Sham group, the serum B-CTx in the OVX group was significantly increased, while the B-CTx in the OVX-Z01 treatment group decreased, approaching that of the Sham group. This indicates that Z01 treatment improved bone loss indicators in the osteoporotic rat model, suggesting a possible increase in bone mass.

[0154] ③Western Blot protein analysis: by Figure 18 It can be seen that Osx protein expression was significantly decreased in the VX group, while Osx protein expression was significantly increased in the OVX-Z01 treatment group compared to the OVX group, indicating that Z01 treatment has an osteogenic effect. Figure 19 It can be seen that, compared with the Sham group, the bone mineral density (BMD) of the OVX group decreased significantly to 0.110 g / cm³. 2 Bone mineral density in the OVX-Z01 group was significantly higher than that in the OVX group, reaching 0.304 g / cm³. 2 This indicates that Z01 has a therapeutic effect in increasing bone mineral density in an osteoporotic rat model. CT analysis of the femoral metaphysis showed that Z01 can significantly prevent OVX-induced bone loss and improve bone density. Figure 20 The 3D reconstruction showed that the femoral trabecular meshwork in the Sham group was normal, while the OVX group showed thinning of the loose trabecular bone, resulting in widening of the trabecular gaps. In contrast, the Z01 group showed a significant increase in the number and connection of trabecular bone, indicating structural recovery.

Claims

1. An amino acid fragment Z01 with osteogenic activity, characterized in that, The amino acid sequence of the amino acid fragment Z01 is shown in SEQ ID NO.

1.

2. The use of the amino acid fragment Z01 as described in claim 1 in the preparation of a medicament for treating osteoporosis.

3. The application as described in claim 2, characterized in that, The drug also includes physiological saline and pharmaceutically acceptable carriers.

4. The application as described in claim 2, characterized in that, The drug dosage form is one of the following: injection, tablet, capsule, oral liquid, microparticle, or ointment.

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