Application of Trichinella spiralis-derived RANKL-targeting peptide TsPRTP21 in inhibiting osteoclast differentiation and preventing bone destruction diseases

Abstract

This invention relates to the application of a Trichinella spiralis-derived RANKL-targeting peptide, TsPRTP21, in inhibiting osteoclast differentiation and preventing bone destructive diseases. The peptide TsPRTP21 can inhibit downstream signaling pathways of osteoclast differentiation, reduce osteoclast production, and alleviate bone destructive diseases such as osteoporosis or rheumatoid arthritis, without significant cytotoxicity or hepatotoxicity, thus providing a new treatment option for bone destructive diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of a Trichinella spiralis-derived RANKL-targeting polypeptide TsPRTP21 in inhibiting osteoclast differentiation and preventing bone destruction diseases. Background Technology

[0002] Bone-destructive diseases are a group of diseases caused by localized bone tissue loss, leading to decreased bone density, changes in skeletal structure, and functional impairment. These include osteoporosis, rheumatoid arthritis, and many other conditions. Common symptoms include localized pain, pathological fractures, and limited mobility, with extremely widespread impact. Regarding the prevention and treatment of bone-destructive diseases, in addition to treating the underlying causes (such as inflammation, tumors, and metabolic imbalances), inhibiting bone loss is a crucial strategy for reducing the disease burden. Currently, drugs used to prevent and treat bone destruction are mainly classified into two categories based on their mechanisms of action: those that inhibit bone resorption and those that promote bone formation. There are also drugs that combine both effects, such as romomoxicillin. Although there has been significant progress in drug development for the prevention and treatment of bone-destructive diseases, these new drugs often have limited efficacy or certain side effects (such as inducing cardiovascular disease, breast cancer, atypical femoral fractures, and osteonecrosis of the jaw). Therefore, developing novel drugs with different mechanisms of action and fewer side effects is of great significance for the prevention and treatment of bone-destructive diseases.

[0003] The normal maintenance of the skeletal system relies on a dynamic balance between bone resorption by osteoclasts and bone formation by osteoblasts. Osteoclasts are tissue-specific multinucleated cells formed by the differentiation and fusion of mononuclear / macrophages located on the bone surface. The receptor activator of nuclear factor-κB (RANK) on the surface of osteoclast precursor cells binds to RANKL, the ligand of RANK produced by osteoblasts, initiating a downstream signaling cascade that leads to differentiation into mature osteoclasts, which degrade and absorb bone matrix. Osteoporogenin (OPG), secreted by osteoblasts, is a decoy receptor for RANKL, competitively binding to RANKL and blocking the binding of RANKL to RANK on osteoclast precursor cells, thereby inhibiting osteoclast formation, activation, and bone resorption. The OPG / RANKL / RANK signaling axis is a key link in regulating osteoclast formation, activation, and bone resorption. Hyperactive osteoclasts leading to increased bone resorption are a major factor in bone destruction, making drug development targeting this signaling axis a crucial strategy for treating destructive bone diseases. Currently, clinical drugs targeting the RANKL / RANK complex are primarily monoclonal antibodies or targeting proteins; however, long-term use of these drugs can lead to limitations such as immunogenicity. In recent years, with the structural analysis of the RANKL-RANK complex and the elucidation of key interaction sites, targeted peptides or small molecule compounds designed to bind to RANKL and RANK can competitively bind to RANKL or RANK, inhibiting RANKL-RANK binding and osteoclast formation, reducing bone loss, and improving destructive bone diseases. Among these, peptide drugs have become a hot topic in drug development due to their high specificity, low immunogenicity, low toxicity, and ease of synthesis.

[0004] Currently, research on small molecule bioactive peptides in bone tissue engineering has made some progress. Among these, peptide drug research targeting the RANKL / RANK / OPG axis for anti-osteoclast formation and activation is based on OPG or RANK sequence design, such as the RANK-mimicking peptide L3-3 and the OPG-mimicking peptide OP3-4. However, since these peptides designed based on OPG and RANK are endogenous, long-term use can induce the body to produce endogenous antibodies, neutralizing normal OPG or RANK in the body and affecting their function, potentially even increasing the risk of fractures. Therefore, introducing novel effector protein sequences for peptide drug design may be of significant value. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide the application of Trichinella spiralis-derived RANKL-targeting polypeptide TsPRTP21 in inhibiting osteoclast differentiation and preventing bone destructive diseases. The polypeptide TsPRTP21 can competitively bind to RANKL, inhibiting the binding of RANKL to RANK, reducing osteoclast formation and activation, and alleviating bone loss in bone destructive diseases such as osteoporosis or rheumatoid arthritis. Furthermore, it exhibits no significant cytotoxicity or hepatotoxicity, providing a new treatment option for bone destructive diseases.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A Trichinella spiralis-derived RANKL-targeting polypeptide TsPRTP21, characterized in that the amino acid sequence of the Trichinella spiralis-derived RANKL-targeting polypeptide TsPRTP21 is shown in SEQ ID NO.1.

[0008] The application of a Trichinella spiralis-derived RANKL-targeting polypeptide TsPRTP21 is characterized in that the polypeptide TsPRTP21 can inhibit the binding of RANKL to its receptor RANK by competitively binding RANKL to RANK, thereby inhibiting the downstream signaling pathway of osteoclast differentiation and reducing the formation and activation of osteoclasts.

[0009] The peptide TsPRTP21 competitively binds RANKL to RANKL in the following manner: TsPRTP21 binds to the nuclear factor κB receptor activator ligand (RANKL) and competitively inhibits the binding of RANKL to its receptor, nuclear factor κB receptor activator (RANK).

[0010] Based on the above plan,

[0011] The polypeptide TsPRTP21 can inhibit the differentiation of mouse bone marrow macrophages into osteoclasts induced by murine RANKL.

[0012] Based on the above plan,

[0013] The polypeptide TsPRTP21 can inhibit the differentiation of human peripheral blood mononuclear cells into osteoclasts induced by human RANKL.

[0014] The application of a Trichinella spiralis-derived RANKL-targeting polypeptide, TsPRTP21, in the development and manufacture of drugs for the prevention and treatment of bone-destructive diseases. These bone-destructive diseases include osteoporosis, rheumatoid arthritis, and other conditions.

[0015] The application of a Trichinella spiralis polyubiquitin-like protein TsPULP to specific binding sites of human and murine RANKL, characterized in that:

[0016] The specific site can be used to bind to both murine and human RANKL, thereby inhibiting the downstream pathway of osteoclast differentiation, reducing osteoclast production, and thus designing the RANKL-targeting peptide TsPRTP21.

[0017] The specific site is located at amino acids 36, 38, 40 and 46 of the sequence shown in SEQ ID NO.4 (corresponding to amino acids 7, 9, 11 and 17 of the sequence shown in SEQ ID NO.1).

[0018] The application of the Trichinella spiralis polyubiquitin-like protein TsPULP to a specific binding site of murine RANKL, characterized by:

[0019] The specific amino acid sites in TsPULP can bind to murine RANKL, thereby inhibiting the activation of osteoclast signaling pathways and reducing the differentiation of murine RANKL-induced mouse bone marrow macrophages into osteoclasts.

[0020] The specific site is located at amino acids 2, 5, 6, 7, 36, 38, 40, 44 and 46 of the sequence shown in SEQ ID NO.4.

[0021] The application of a Trichinella spiralis polyubiquitin-like protein TsPULP to a specific binding site of human RANKL, characterized by:

[0022] The specific amino acid sites in TsPULP can bind to human RANKL, thereby inhibiting the activation of osteoclast signaling pathways and reducing the differentiation of human peripheral blood mononuclear cells into osteoclasts induced by human RANKL.

[0023] The specific sites are located at amino acids 36, 38, 40, 46, 98, and 100 of the sequence shown in SEQ ID NO.4.

[0024] The beneficial effects of the Trichinella spiralis-derived RANKL-targeting polypeptide TsPRTP21 in inhibiting osteoclast differentiation and preventing bone destruction diseases are as follows:

[0025] The peptide binds to RANKL and competitively inhibits its binding to its receptor RANK, thereby suppressing downstream signaling pathways in osteoclast differentiation, reducing osteoclast production, inhibiting bone loss, and alleviating osteoporosis or rheumatoid arthritis and other bone-destructive diseases. It exhibits no significant cytotoxicity or hepatotoxicity, providing a new treatment option for bone-destructive diseases. Furthermore, TsPRTP21 can be directly synthesized chemically, is stable, has a high yield, and low production costs. Its efficacy can also be enhanced through subsequent optimization. Attached Figure Description

[0026] The present invention includes the following figures:

[0027] Figure 1 This is a molecular structure diagram of the complex predicted by AlphaFold2 involving Trichinella spiralis polyubiquitin-like protein (TsPULP) and murine RANKL (mRANKL). The red amino acid residues in the diagram represent the distances from the TsPULP residues analyzed by Chimera software. amino acid residues;

[0028] Figure 2 The ability of TsPULP wild-type and mutant to inhibit the differentiation of mouse bone marrow macrophages (BMDMs) into osteoclasts; a) Typical TRAP staining diagram of TsPULP wild-type and mutant inhibiting the differentiation of mouse BMDMs into osteoclasts; b) Statistical diagram of the number of TRAP-positive cells.

[0029] Figure 3 To detect the affinity of TsPULP wild-type and mutant for binding to mRANKL in SPR experiments;

[0030] Figure 4 This is a molecular structure diagram of the complex of TsPULP and human RANKL (hRANKL) predicted by AlphaFold2. The red amino acid residues in the diagram represent the distances to TsPULP as analyzed by Chimera software. amino acid residues;

[0031] Figure 5 The ability of TsPULP wild-type and mutant to inhibit the differentiation of human peripheral blood mononuclear cells (PBMCs) into osteoclasts; a) Typical TRAP staining diagram of TsPULP wild-type and mutant inhibiting the differentiation of human PBMCs into osteoclasts; b) Statistical diagram of the number of TRAP-positive cells.

[0032] Figure 6 To detect the affinity of TsPULP wild-type and mutant for binding with hRANKL in SPR experiments;

[0033] Figure 7 The diagram shows the structure of TsPRTP, with the structure highlighted in red being TsPRTP21.

[0034] Figure 8 Figure 1 shows the SPR results of TsPRTP21 binding to RANKL; Figure 2 shows the SPR results of TsPRTP21 binding to mRANKL; Figure 3 shows the SPR results of TsPRTP21 binding to hRANKL.

[0035] Figure 9 To demonstrate that TsPRTP21 inhibits the binding of RANKL to RANK; a) is a competitive SPR experiment to verify that TsPRTP21 inhibits the binding of mRANKL to mRANK; b) is a competitive SPR experiment to verify that TsPRTP21 inhibits the binding of hRANKL to hRANK.

[0036] Figure 10 The image shows the effect of TsPRTP21 on inhibiting mRANKL-induced activation of the osteoclast signaling pathway in mice in vitro; a is a typical Western blot diagram of the expression levels of proteins related to osteoclast differentiation in mouse BMDMs cells induced by TsPRTP21 inhibition of mRANKL; b is a statistical diagram of the grayscale values ​​of the Western blot.

[0037] Figure 11 The first image shows the TsPRTP21 inhibition of mRANKL-induced differentiation of mouse BMDMs into osteoclasts in vitro; a is a typical TRAP staining diagram of TsPRTP21 inhibition of mRANKL-induced differentiation of mouse BMDMs into osteoclasts; b is a statistical diagram of the number of TRAP-positive osteoclasts.

[0038] Figure 12 The image shows the in vitro inhibition of hRANKL-induced activation of the human osteoclast signaling pathway by TsPRTP21; a is a typical Western blot diagram of the expression levels of osteoclast differentiation-related signaling pathway proteins in human PBMCs induced by TsPRTP21 inhibition of hRANKL; b is a statistical graph of Western blot grayscale values.

[0039] Figure 13 The first image shows the TsPRTP21 inhibition of hRANKL-induced differentiation of human PBMCs into osteoclasts in vitro; a is a typical TRAP staining diagram of TsPRTP21 inhibiting hRANKL-induced differentiation of human PBMCs into osteoclasts; b is a statistical diagram of the number of TRAP-positive osteoclasts.

[0040] Figure 14 TsPRTP21 helps alleviate bone loss in OVX mice; a) Typical micro-CT scans of the femur of OVX mice in each group; b) Statistical graph of bone parameters in OVX mice;

[0041] Figure 15 TsPRTP21 inhibits osteoclast formation in the femur of OVX mice; a) Typical TRAP staining patterns in the femur of OVX mice in each group; b) Statistical chart of the number of TRAP-positive cells in the femur.

[0042] Figure 16TsPRTP21 alleviates bone destruction in CIA mice; a) Typical micro-CT scan images of the paws of CIA mice in each group; b) Statistical graph of paw bone parameters in CIA mice;

[0043] Figure 17 TsPRTP21 inhibits the formation of osteoclasts in the paws of CIA mice; a) Typical TRAP staining patterns in the paws of CIA mice in each group; b) Statistical chart of the number of TRAP-positive cells in the paws.

[0044] Figure 18 The following assays were performed to detect the toxicity of TsPRTP21: a) CCK8 assay to detect the effect of TsPRTP21 on the viability of mouse BMDMs and human PBMCs; b) HE staining to detect the effect of TsPRTP21 on liver tissue of OVX mice; c) HE staining to detect the effect of TsPRTP21 on kidney tissue of OVX mice. Detailed Implementation

[0045] The present invention will be further described in detail below with reference to the accompanying drawings.

[0046] 1. Determination of the binding sites of TsPULP and RANKL

[0047] (1) Comparison of RANKL binding sites with TsPULP and RANK

[0048] The amino acid sequences of mRANKL (mRANKL, NP_035743, amino acid residues 162–316, shown in SEQ ID NO.2) and TsPULP (SEQ ID NO.4), and human RANKL (NP_003692, amino acid residues 162–317, shown in SEQ ID NO.3) and TsPULP were respectively input into the AlphaFold2 algorithm (https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / AlphaFold2.ipynbAlphaFold) for molecular docking. The protein structures and interaction sites were predicted, and the model with the highest score was selected and imported into the UCSF Chimera tool to analyze the interaction distance between the two proteins. The amino acids were identified to predict the amino acid sites in TsPULP that could interact with RANKL. Based on the predicted amino acid sites, mutants were designed and site-directed mutagenesis was performed to further clarify the key interacting amino acids between the two. Figure 1 and Figure 4 The red amino acid residues in the image represent the possible binding sites for mRANKL and hRANKL on TsPULP, as predicted by AlphaFold2.

[0049] (2) Expression and purification of TsPULP

[0050] First, *Trichinella spiralis* mRNA was extracted using the Trizol method, and cDNA was obtained after reverse transcription. The target gene TsPULP was amplified by PCR, digested with two enzymes, and ligated into the PET-28a (His-tagged) vector. The recombinant vector was transformed into DH5α competent cells, and positive plasmids were obtained after sequencing alignment. These plasmids were then transformed into DE3 competent cells, and kanamycin was used for selection to complete the construction of the expression bacteria. The positive clones were inoculated into 1 L LB medium. When the bacterial count reached OD600 = 0.6-0.8, IPTG was added, and the cells were induced at 16℃ for 12-16 h. After centrifugation, the bacterial pellet was collected, resuspended, sonicated, and centrifuged at high speed. Since TsPULP is an inclusion body, the supernatant was discarded. The protein precipitate was dissolved in binding buffer containing 6M guanidine hydrochloride and sonicated at 30% intensity for 10 min. The mixture was then centrifuged at 12,000 rpm for 30 min, and the supernatant was collected. The supernatant was incubated with Ni2+ affinity chromatography medium for 2 h, washed with 50 mM imidazole, and eluted with 500 mM imidazole to obtain the target protein. The protein was then dialyzed overnight at 4°C in 20 mM Tris-HCl, centrifuged at 12,000 rpm for 15 min, and the protein precipitate was collected. The protein was then renatured using a protein renaturation kit to obtain a soluble protein solution. The purity of the purified TsPULP was detected by SDS-PAGE gel electrophoresis, and its concentration was determined by the BCA method.

[0051] (3) Purification and expression of TsPULP mutant

[0052] The interaction surface between TsPULP and mRANKL is smaller than All amino acid residues were designed into 6 mutants (I2A / G5A, K6A / Q7A, H26A / R30A, Q36A / F38A / K40A, G44A / T46A, Q97A / E98A), with smaller than 100% of the hRANKL interaction surface. Four mutants (V28A / R30A / L31A, Q36A / F38A, K40A / T46A, E98A / T100A) were designed based on all amino acid residues and mutated at single points using the FAST mutation kit. Primers were designed before and after each mutation site, and PCR was used to mutate the target site. After positive bands were detected by nucleic acid electrophoresis, DMT enzyme was added to the mutation system, and the unmutated product was digested at 37°C for 4 hours. The digested system was then transformed into DH5α competent cells, plated on LB solid medium, and single colonies were picked for gene sequencing to screen positive clones. The plasmids of positive clones were extracted, transformed into DE3 competent cells, cultured and induced for expression. The target mutant protein was obtained after purification according to the method in 1(3).

[0053] (4) Expression and purification of mouse and human RANKL

[0054] Vectors were constructed according to method 1(3) above. Murine RANKL (mRANKL) and human RANKL (hRANKL) were constructed onto the PEGX-6P-1 vector (expressing the GST tag), and positive clones were screened for protein expression induction. Expression was induced for 12-16 h, centrifuged, and the bacterial pellet was collected. The pellet was resuspended in binding buffer, sonicated, centrifuged at high speed, and the supernatant was collected. This supernatant was incubated with GST binding resin for 2 h, and the target protein was eluted with elution buffer containing 10 mM reduced glutathione. The protein was then replaced with GST-tagged protease digestion buffer by dialysis. Protease was added at a ratio of 2 U per 100 μg of protein, and digested at 4°C for 4-8 h. The protein was then added to glutathione agarose resin for binding, centrifuged, and the supernatant (containing the target protein) was collected. The purity of the purified target protein was detected by SDS-PAGE gel electrophoresis, and its concentration was determined by the BCA method.

[0055] (5) Expression and purification of mouse and human RANK

[0056] The murine RANK (mRANK) and human RANK (hRANK) were constructed into the PET-28a vector according to the method in 1(3), and then expressed, purified, and renatured. The protein concentration was determined by the BCA method.

[0057] (6) Determination of key amino acid sites for TsPULP binding to mRANKL

[0058] ① TRAP staining of osteoclasts verifies the ability of each mutant TsPULP to inhibit the differentiation of mouse bone marrow macrophages (BMDMs) into osteoclasts.

[0059] After euthanizing C57 mice by cervical dislocation, femurs and tibias were harvested. Bone marrow cells were washed away with PBS and placed in DMEM complete medium (containing 1% penicillin and streptomycin and 10% fetal bovine serum) and incubated overnight at 37°C in a 5% CO2 incubator. The next day, the cells were centrifuged at 500g for 5 min, and the supernatant containing adherent cells was collected and placed in DMEM complete medium containing 30 ng / ml M-CSF. The cells were then cultured in an incubator. The next day, 50 ng / ml mRANKL was added for induction, along with wild-type and mutant TsPULP (I2A / G5A, K6A / Q7A, H26A / R30A, Q36A / F38A / K40A, G44A / T46A, Q97A / E98A). The medium was changed every three days, while maintaining the concentrations of M-CSF, mRANKL, and added proteins. BMDMs were obtained after 5-7 days of induction. Mature osteoclasts express a large amount of tartrate-resistant alkaline phosphatase (TRAP), which stains a deep purple-red or wine-red color upon TRAP staining. Therefore, we used TRAP staining to analyze the effect of TsPULP on osteoclast differentiation in vitro. After cell fusion at 5-7 days, the supernatant was discarded, and the cells were fixed in 4% paraformaldehyde at room temperature for 10 min. Osteoclasts were stained using a TRAP staining kit, rinsed with double-distilled water, and the nuclei were stained with hematoxylin. After the staining was reversed with tap water, the cells were photographed under a light microscope. Cells containing three or more nuclei and whose cytoplasm was stained wine-red or purple-red were identified as osteoclasts. The number of osteoclasts in each well was counted to observe the ability of each mutant TsPULP to inhibit mRANKL-induced osteoclast formation in mice in vitro. Figure 2 The results showed that, compared with the mRANKL group, the addition of wild-type TsPULP significantly reduced the number of osteoclasts. However, the addition of mutants I2A / G5A, K6A / Q7A, Q36A / F38A / K40A, and G44A / T46A did not significantly change the number of osteoclasts compared with the mRANKL group, indicating that the mutants I2A / G5A, K6A / Q7A, Q36A / F38A / K40A, and G44A / T46A have a decreased ability to inhibit osteoclast differentiation. Therefore, amino acids I2, G5, K6, Q7, Q36, F38, K40, G44, and T46 play important roles in TsPULP's inhibition of mRANKL-induced osteoclast differentiation.

[0060] ②SPR experiments verified the changes in the binding affinity of each mutant TsPULP to mRANKL.

[0061] SPR protein binding experiments were conducted on mRANKL with each mutant (I2A / G5A, K6A / Q7A, H26A / R30A, Q36A / F38A / K40A, G44A / T46A, Q97A / E98A) and wild-type TsPULP protein respectively. mRANKL was immobilized on a CM5 chip, and different concentrations of wild-type and mutant TsPULP protein solutions were used as mobile phases to flow through the surface of the channel immobilized with mRANKL. The dissociation constant (KD) value was calculated based on the final change data of protein surface refractive index to observe the affinity between TsPULP mutants and mRANKL. Figure 3 The results showed that the KD values ​​of the mutants I2A / G5A, K6A / Q7A, Q36A / F38A / K40A, and G44A / T46A were significantly lower than those of the wild type, indicating that amino acids I2, G5, K6, Q7, Q36, F38, K40, G44, and T46 play important roles in the binding of TsPULP to mRANKL.

[0062] (7) Determination of key amino acid sites for TsPULP binding to hRANKL

[0063] ① TRAP staining of osteoclasts verifies the ability of each mutant TsPULP to inhibit the differentiation of human peripheral blood mononuclear cells (PBMCs) into osteoclasts.

[0064] Human peripheral blood was placed in human peripheral blood mononuclear cell separation medium and centrifuged at differential speed (3000 rpm, 30 min, acceleration / deceleration at speed 3) to extract mononuclear cells (PBMCs). CD14 was then extracted from human peripheral blood using the CD14 MicroBead Kit mononuclear cell sorting kit (Miltenyi, Germany) according to the kit instructions via immunomagnetic bead separation. + Monocytes were seeded into cell culture dishes and induced into macrophages by adding αMEM complete medium containing 30 ng / ml M-CSF. The obtained human macrophages were then cultured at 5 × 10⁶ cells / mL. 4 / wells were seeded in 48-well plates and filled with DMEM complete medium containing 30 ng / ml M-CSF and 50 ng / ml hRANKL. Wild-type and mutant TsPULP (V28A / R30A / L31A, Q36A / F38A, K40A / T46A, E98A / T100A) were also added. Cell fusion status was observed daily. After cell fusion at 10-14 days, TRAP staining was performed according to method 1(6)①, and the number of osteoclasts was counted to observe the ability of each mutant TsPULP to inhibit the differentiation of human peripheral blood mononuclear cells into osteoclasts induced by hRANKL in vitro. Figure 5The results showed that, compared with hRANKL, the addition of wild-type TsPULP significantly reduced the number of osteoclasts formed. However, the addition of mutants Q36A / F38A, K40A / T46A, and E98A / T100A did not significantly change the number of osteoclasts compared with the hRANKL group. This indicates that the ability of these three mutants to inhibit osteoclast differentiation was significantly reduced, suggesting that amino acids Q36, F38, K40, T46, E98, and T100 play an important role in TsPULP's inhibition of hRANKL-induced osteoclast differentiation.

[0065] ②SPR experiments verified the changes in the binding affinity of TsPULP to hRANKL in each mutant.

[0066] SPR protein binding experiments were conducted on each mutant (V28A / R30A / L31A, Q36A / F38A, K40A / T46A, E98A / T100A) and wild-type TsPULP protein with hRANKL. hRANKL was immobilized on a CM5 chip, and different concentrations of wild-type and mutant TsPULP protein solutions were used as mobile phases to flow through the surface of the channel immobilized with RANKL. The dissociation constant (KD) value was calculated based on the final change in the refractive index of the protein surface, and the affinity of TsPULP mutants for hRANKL was observed. Figure 6 The results showed that the KD values ​​of mutants Q36A / F38A, K40A / T46A, and E98A / T100A were significantly lower than those of wild-type, indicating that amino acids Q36, F38, K40, T46, E98, and T100 play an important role in the binding of TsPULP to hRANKL.

[0067] 2. TsPULP-derived RANKL-targeting peptides bind to RANKL, competitively inhibiting the binding of RANKL to RANK.

[0068] (1) Design of TsPULP-derived RANKL-targeting peptides

[0069] The ability of each TsPULP mutant to inhibit the binding of human or mouse RANKL to RANK and to inhibit RANKL-induced osteoclast differentiation showed that the four amino-terminal sites Q36, F38, K40, and T46 all play a role in binding with human and mouse RANKL and inhibiting osteoclast differentiation. Therefore, we selected amino acids 30-50 of TsPULP (RLRGGMQIFVKTLTGKTITLE) and synthesized a polypeptide (TsPRTP21) by solid-phase synthesis to inhibit osteoclast formation and alleviate bone loss. Figure 7 The red portion shows the location of this peptide in the TsPULP protein.

[0070] (2) SPR experiments verify the binding of TsPRTP21 to RANKL

[0071] Biotinylated TsPRTP21 was immobilized on a chip coupled with avidin. Different concentrations of mRANKL or hRANKL protein solutions were used as mobile phases and flowed through the surface of the channel immobilized with TsPRTP21. The dissociation constant (KD) value was calculated based on the final change in the refractive index of the protein surface, and the affinity of TsPRTP21 binding was observed. Figure 8 The results show that TsPRTP21 can bind to mRANKL, and the affinity between the two is 4.71 nM. Figure 8 Figure b shows that TsPRTP21 can bind to hRANKL, with an affinity of 7.83 nM. These results indicate that TsPRTP21 has strong affinity for both mRANKL and hRANKL.

[0072] (3) Competitive SPR experiments verified that TsPRTP21 competitively binds to RANKL.

[0073] mRANK or hRANK was immobilized on a CM5 chip. Different concentrations of TsPRTP21 protein solution were incubated with mRANKL or hRANKL at 500 nm at room temperature for 1 h. The mixture was used as the mobile phase and flowed through the surface of the channel immobilized with RANK. The ability of TsPRTP21 to competitively inhibit the binding of RANKL and RANK was observed by the change in the RU value of RANKL binding to RANK. Figure 9 Figures a and b show that as the concentration of TsPRTP21 increases, the RU values ​​of human and mouse RANKL binding to RANK decrease, indicating that TsPRTP21 can inhibit the binding of human and mouse RANKL to RANK.

[0074] 3. TsPRTP21 inhibits the differentiation of mouse bone marrow macrophages into osteoclasts in vitro.

[0075] (1) Western blot detection of osteoclast-related signaling pathway activation levels in mouse BMDMs

[0076] Mouse BMDMs were obtained according to method 1(6)① and seeded into 6-well plates. After cell adhesion, 50 ng / ml mRANKL and TsPRTP21 were added. The cells were divided into the following groups: ① PBS group; ② mRANKL group; ③ mRANKL+TsPRTP21 group. After stimulation for 60 min, the cells were collected, and RIPA was added to lyse the cells. The expression of osteoclast-related signaling pathway proteins was detected by Western blot. Figure 10Figures a and b show that, compared with the mRANKL group, the expression levels of osteoclast differentiation-related proteins (p-p65, p-iκB, p-p38, p-ERK, p-JNK, NFAT) in mouse BMDMs decreased after the addition of TsPRTP21, indicating that TsPRTP21 can significantly inhibit the expression of osteoclast-related proteins in mouse BMDMs induced by mRANKL.

[0077] (2) TRAP staining to observe the number of osteoclasts

[0078] Mouse bone marrow BMDMs were administered at 5 × 10 4 / wells were seeded in 48-well plates, and DMEM complete medium containing 30 ng / ml M-CSF and 50 ng / ml mRANKL was added. TsPRTP21 was also added, and the cells were grouped as above. The cell fusion status was observed daily. After cell fusion for 5-7 days, the supernatant was discarded, and TRAP staining was performed according to method 1(6)①. The number of osteoclasts in each well was counted to observe the effect of TsPRTP21 on mRANKL-induced osteoclast formation in mice. Figure 11 Figures a and b show that, compared with the mRANKL group, the number of TRPA-positive osteoclasts was significantly reduced after the addition of TsPRTP21, indicating that TsPRTP21 can significantly inhibit the formation of mouse osteoclasts in vitro.

[0079] 4. TsPRTP21 inhibits the differentiation of human peripheral blood mononuclear cells into osteoclasts in vitro.

[0080] (1) Western blot verification showed that TsPRTP21 inhibited the activation of osteoclast signaling pathway in human PBMCs.

[0081] The human PBMCs obtained in 1(7)① were seeded into 6-well plates. After cell adhesion, 50 ng / ml hRANKL and TsPRTP21 were added. The cells were divided into the following groups: ① PBS group; ② hRANKL group; ③ hRANKL+TsPRTP21 group. After stimulation for 60 min, the cells were collected, and RIPA was added to lyse the cells. The expression of osteoclast-related signaling pathway proteins was detected by Western blot. Figure 12 Figures a and b show that, compared with the hRANKL group, the expression levels of osteoclast differentiation-related proteins (p-p65, p-iκB, p-p38, p-ERK, p-JNK, NFAT) in human PBMCs decreased after the addition of TsPRTP21, indicating that TsPRTP21 can significantly inhibit the expression of osteoclast-related proteins in human PBMCs induced by hRANKL.

[0082] (2) TRAP staining to observe the number of osteoclasts

[0083] The human PBMCs obtained above were divided into 5×10 4 / wells were seeded in 48-well plates, and DMEM complete medium containing 30 ng / ml M-CSF and 50 ng / ml hRANKL was added. TsPRTP21 was also added, and the cells were grouped as above. The cell fusion status was observed daily. After cell fusion at 10-14 days, TRAP staining was performed according to method 1(6)①, and the number of osteoclasts was counted to observe the effect of TsPRTP21 on the differentiation of hRANKL-induced human PBMCs into osteoclasts. Figure 13 Figures a and b show that, compared with the hRANKL group, the number of TRPA-positive osteoclasts was significantly reduced after the addition of TsPRTP21, indicating that TsPRTP21 can significantly inhibit the differentiation of human PBMCs into osteoclasts in vitro.

[0084] 5. TsPRTP21 alleviates bone loss in ovariectomized osteoporotic (OVX) mice.

[0085] Ovariectomized (OVX) osteoporosis model was established in female C57BL / 6 mice using ovariectomy. Mice were randomly divided into four groups of eight mice each: ① Sham group; ② Ovariectomy alone group (OVX group); ③ OVX + TsPRTP21 (2 mg / kg) group; ④ OVX + TsPRTP21 (4 mg / kg) group. Mice in each group were anesthetized with 3% sodium pentobarbital via intraperitoneal injection at a dose of 0.1 ml / 100 g. Skin preparation was performed on the lateral aspect of the back, and the area was disinfected with iodine. In the OVX group, a longitudinal incision of approximately 1 cm was made on the lateral aspect of the back, the skin and muscle were separated, the uterus was ligated at the distal end, and both ovaries were removed. In the sham group, only the adipose tissue around the ovaries was removed. After suturing, the mice were fed routinely for one week. The OVX + TsPRTP21 group received TsPRTP21 via intraperitoneal injection one week later. Mice were sacrificed eight weeks after modeling to observe the effect of TsPRTP21 on bone destruction in osteoporotic mice.

[0086] (1) Micro-CT and 3D modeling techniques were used to observe the degree of bone loss in osteoporotic mice.

[0087] Bilateral femurs of mice were collected and fixed in 4% paraformaldehyde. Micro-CT tomography scans of the fixed femurs were performed using a Scanco Micro-CT 50 imaging system (Scanco, Switzerland). The results were analyzed using 3D analysis software (VGStudioMAX) to measure the bone volume / tissue volume percentage (BV / TV), bone mineral density (BMD), number of trabeculae (Tb.N), and trabecular thickness (Tb.Th) of the femurs in each group of mice. The effect of TsPRTP21 on bone loss in osteoporotic mice was observed. Figure 14 Figures a and b show that, compared with the OVX group, intraperitoneal injection of TsPRTP21 significantly increased BV / TV, BMD, Tb.N, and Tb.Th, indicating that TsPRTP21 can significantly alleviate bone loss in OVX mice.

[0088] 2) TRAP staining to detect osteoclast activation levels in the femur of osteoporotic mice

[0089] After fixation, the femur was decalcified and prepared into paraffin sections. After dewaxing, the sections were immersed in 0.1 M CaCl2-MgCl2 solution to activate tartrate-resistant acid-alkaline phosphatase (TRAP). After rinsing with PBS, the staining solution was prepared according to the TRAP staining kit instructions, and TRAP staining was performed at 37°C for 45 min. After rinsing with double-distilled water, the nuclei were stained with hematoxylin and then blued with tap water. After drying and mounting, osteoclasts were counted under a light microscope to observe the effect of TsPRTP21 on the number of osteoclasts in osteoporotic mice. Figure 15 Figures a and b show that, compared with the OVX group, the number of osteoclasts in the femur of mice was significantly reduced after intraperitoneal injection of TsPRTP21, indicating that TsPRTP21 can inhibit the formation of osteoclasts in OVX mice.

[0090] 6. TsPRTP21 alleviates bone erosion in mice with collagen-induced arthritis (CIA).

[0091] Male DBA / 1 mice were randomly divided into four groups: ① Control group; ② CIA group; ③ CIA+TsPRTP21 group (1 mg / kg); ④ CIA+TsPRTP21 group (2 mg / kg). Bovine type II collagen (CII) and complete Freund's adjuvant (CFA) were mixed and emulsified (100 μg CII, 100 μL / mouse) and administered intradermally 1.5 cm from the base of the tail. Day 0 was the first collagen induction. On day 21, an equal volume of incomplete Freund's adjuvant and CII were mixed, and a booster immunization was performed using the same method to establish the CIA model. The CIA+TsPRTP21 group received TsPRTP21 via intraperitoneal injection starting from day 0. After the second booster immunization, the morbidity rate and joint scores of mice in each group were observed every other day. Mice were sacrificed on day 49 to observe the effect of TsPRTP21 on bone erosion in CIA mice.

[0092] (1) Micro-CT and 3D modeling techniques were used to observe the degree of bone destruction in CIA mice.

[0093] Mouse paws were taken, fixed in 4% paraformaldehyde, and Micro-CT scans were performed according to method 5(1). Bone parameters such as BV / TV and Tb.Th were calculated to observe the effect of TsPRTP21 on bone destruction in CIA mice. Figure 16 Figures a and b show that, compared with the CIA group, intraperitoneal injection of TsPRTP21 significantly increased BV / TV and Tb.Th, indicating that TsPRTP21 can significantly alleviate bone destruction in CIA mice.

[0094] 2) TRAP staining to detect the activation level of osteoclasts in the joints of CIA mice

[0095] After the fixed femur was decalcified, it was prepared into paraffin sections and stained with TRAP according to method 9(2) to observe the effect of TsPRTP21 on the number of osteoclasts in osteoporotic mice. Figure 17 Figures a and b show that, compared with the CIA group, the number of osteoclasts in the femur of mice was significantly reduced after intraperitoneal injection of TsPRTP21, indicating that TsPRTP21 can inhibit the formation of osteoclasts in the joints of CIA mice.

[0096] 7. Toxicity testing of TsPRTP21

[0097] (1) CCK8 assay for TsPRTP21 cytotoxicity

[0098] The mouse BMDMs and human PBMCs obtained in 1(6)① and 1(7)① were respectively processed at 1×10 4After being seeded in 96-well plates and allowed to adhere overnight, TsPRTP21 was added at serial dilutions of different concentration gradients. After incubation for 24 hours, CCK8 reagent was added, and the absorbance was measured at 450 nm using a microplate reader after 4 hours to observe the cytotoxicity of TsPRTP21. Figure 18 The results showed that TsPRTP21 had no significant cytotoxicity against mouse BMDMs and human PBMCs.

[0099] (2) HE staining to detect the hepatotoxicity and nephrotoxicity of TsPULP

[0100] To investigate the toxicity of TsPRTP21 to mouse liver and kidney tissues, C57 mice were randomly divided into three groups: PBS group, low-dose TsPRTP21 (2 mg / kg), and high-dose TsPRTP21 (4 mg / kg). TsPRTP21 was injected once daily for 8 weeks. After 8 weeks, the livers and kidneys of the mice were harvested, fixed overnight in 4% paraformaldehyde, embedded, sectioned, and stained with hematoxylin and eosin (HE) to observe the effects of TsPRTP21 on the liver and kidneys of mice. Figure 18 The results showed that after intraperitoneal injection of TsPRTP21, there was no obvious inflammatory cell infiltration in the liver tissue, the hepatocytes were arranged regularly, and the liver lobule structure was normal. There were no significant changes compared with the non-injection group. Figure 18 The results showed that after intraperitoneal injection of TsPRTP21, the renal tubular epithelial cells were neatly arranged, the interstitial tissue showed no edema, fibrosis or inflammation, and the glomerular basement membrane remained intact without atrophy or sclerosis, showing no significant changes compared to the uninjected group. These results indicate that TsPRTP21 did not exhibit significant hepatotoxicity or nephrotoxicity in vivo and had minimal side effects.

[0101] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

Claims

1. A Trichinella spiralis-derived RANKL-targeting polypeptide Ts PRTP21, characterized in that, The Trichinella-derived RANKL-targeting peptide Ts The amino acid sequence of PRTP21 is shown in SEQ ID NO.1; The Trichinella-derived RANKL-targeting peptide Ts PRTP21 identifies Trichinella spiralis polyubiquitin-like proteins Ts After truncating the specific binding site of RANKL on PULP Ts Fragments of PULP were obtained; the specific sites were amino acids 36, 38, 40, and 46 of the sequence shown in SEQ ID NO.

4.

2. The Trichinella spiralis-derived RANKL-targeting polypeptide as described in claim 1 Ts Application of PRTP21 in the preparation of drugs for treating osteoporosis and rheumatoid arthritis; in, The polypeptide Ts PRTP21 can inhibit the differentiation of mouse bone marrow macrophages into osteoclasts induced by murine RANKL, and can also inhibit the differentiation of human peripheral blood mononuclear cells into osteoclasts induced by human RANKL.

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