Preparation of secretion excreted by trichina muscular larvae and application of secretion in inhibition of osteoclast differentiation

Abstract

The invention relates to preparation of trichina muscle larva excretion secretions and application of the trichina muscle larva excretion secretions in inhibiting osteoclast differentiation. The secretion excreted by the trichina muscle larvae can be competitively combined with the RANKL, so that the combination of the RANKL and the RANK is inhibited, and the formation of osteoclasts is reduced. The secretion excreted by the trichina muscular larvae can also inhibit the expression of osteoclast related genes mmp9, cathepsin k, trap, rank, nfatc1 and cfos and the expression of osteoclast generation related proteins. The secretion excreted by the trichina muscle larvae can also be applied to manufacturing of medicines for treating osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, in particular to the preparation of excretion secretions of Trichinella spiralis muscle larvae and the application of the excretion secretions in inhibiting osteoclast differentiation. Background Art

[0002] Osteoporosis (OP) is a chronic, systemic bone disease characterized by decreased bone mass, degeneration of trabecular microarchitecture, and decreased bone density, leading to increased bone fragility and fracture risk. The disease can be divided into primary and secondary types: primary osteoporosis includes postmenopausal osteoporosis and senile osteoporosis; secondary osteoporosis is often induced by endocrine and metabolic diseases (such as hypogonadism and hyperthyroidism), systemic diseases, medications, and other factors. Osteoporosis is one of the ten major chronic diseases recognized by the World Health Organization. Primary osteoporosis is clinically more common, mainly in middle-aged and elderly people. Its main harm is that it is very likely to cause fractures. The resulting long-term immobility or bed rest reduces the quality of life of patients and their families, and increases the psychological and economic burden. As my country's population gradually ages, the number of people with osteoporosis is growing, and its prevention and treatment has become a medical issue that urgently needs to be addressed.

[0003] Currently, drugs used to treat osteoporosis, with the exception of vitamin D and calcium supplements as the cornerstone, are primarily divided into two categories based on their mechanism of action: one that inhibits bone resorption, such as calcitonin, estrogen receptor modulators, bisphosphonates, and inhibitors of nuclear factor-κB receptor activator ligand; the other that promotes bone formation, such as parathyroid hormone analogs and parathyroid hormone-related peptide analogs. There are also drugs that have dual effects, such as those that inhibit sclerostin activity, such as romumab. Despite significant progress in the development of osteoporosis treatments, these new drugs have limited efficacy or are associated with side effects (such as the risk of cardiovascular disease, breast cancer, atypical femoral fractures, and osteonecrosis of the jaw). Therefore, the development of new drugs with different mechanisms of action and fewer side effects is crucial for preventing and treating the disease and alleviating the burden of disease in the elderly.

[0004] From the perspective of the pathogenesis of osteoporosis, the OPG / RANKL / RANK signaling axis is a key link in the regulation of bone metabolism by estrogen and other hormones. This signaling axis participates in the bone remodeling process by regulating the formation, activation, and bone resorption of osteoclasts. Osteoclasts are tissue-specific multinuclear cells formed by the differentiation and fusion of monocytes and macrophages located on the bone surface. Under specific induction conditions, monocytes in the bone marrow cavity and blood migrate to the bone surface through the blood circulation and differentiate into osteoclast precursors. The receptor activator of nuclear factor-κB (RANK) on the surface of osteoclast precursor cells binds to the RANK ligand (RANKL) produced by osteoblasts and other cells, initiating a downstream signaling cascade, activating the NF-κB signaling pathway, and differentiating into mature osteoclasts that degrade and resorb bone matrix. Osteoporin (OPG), secreted by osteoblasts, is a decoy receptor for RANKL. It can competitively bind to RANKL and block the binding of RANKL to RANK on osteoclast precursor cells, thereby inhibiting the formation, activation and bone resorption function of osteoclasts. The balance between OPG and RANKL is closely related to osteoclast activity. When the level of estrogen and other hormones in the body decreases, OPG production decreases and RANKL expression increases, causing the activation of the RANKL-RANK pathway, thereby promoting the formation of osteoclasts and the occurrence of osteoporosis. Since the imbalance of the OPG / RANKL / RANK signaling axis is a key factor in causing bone metabolic diseases such as osteoporosis, the development of drugs targeting this signaling axis has become one of the important strategies for treating such diseases. Such drugs can inhibit the binding of RANKL to RANK and osteoclast formation by competitively binding to RANKL or RANK, thereby improving bone destructive diseases. Therefore, the research and development of innovative drugs targeting RANKL or RANK has broad development prospects.

[0005] During the co-evolution of parasitic helminths and their hosts, helminths can maintain their long-term survival within their hosts by modulating host immune or metabolic functions. In recent years, the ameliorative effects of helminths and helminth-derived molecules on human immune or metabolic diseases have garnered widespread attention, and the potential medicinal value of helminth-derived molecules is gradually being recognized by scholars both domestically and internationally. Helminth excretory and secretory products (ES) are rich in proteins that interact with host molecules to modulate host immune responses or metabolic functions. Therefore, the use of helminth-derived molecules to prevent and treat immune or metabolic diseases has gradually gained attention and become a promising research hotspot. Currently, studies have reported that helminths and helminth-derived molecules have significant ameliorative effects on a variety of immune or metabolic diseases, including inflammatory bowel disease, rheumatoid arthritis, asthma, and type 2 diabetes, with some studies entering clinical trials. Studies have also reported that helminth-derived molecules can influence osteoclast formation and bone metabolism. In particular, studies have demonstrated that helminth-derived molecules can directly modulate the function of macrophages or osteoclast precursors and inhibit their differentiation into mature osteoclasts. For example, excretory and secretory antigens from Heligmosomoides polygyrus can effectively inhibit the differentiation of cultured bone marrow mononuclear cells into osteoclasts in vitro, but the specific mechanisms remain unclear. The excretory and secretory protein ES-62 from Acanthocheilonema viteae can upregulate the expression of genes involved in the antioxidant response in macrophages, thereby reducing the production of reactive oxygen species (ROS) required for osteoclast maturation and inhibiting osteoclast formation. The Fasciola hepatica protein FhHDM-1 can inhibit RANKL-induced activation of the NF-κB pathway in macrophages, thereby suppressing osteoclast formation and bone destruction in mice with collagen-induced arthritis (CIA). These studies indicate that certain helminths and helminth-derived molecules can effectively inhibit the development and progression of bone destruction, providing new insights into the prevention and treatment of bone-destructive diseases. However, their specific targets and applications require further investigation. Summary of the Invention

[0006] In response to the defects in the prior art, the present invention aims to provide a preparation of Trichinella spiralis muscle larvae excretory-secretory proteins (TsMES) and its application in inhibiting osteoclast differentiation. The above-mentioned Trichinella spiralis muscle larvae excretory-secretory proteins can competitively bind to RANKL, thereby inhibiting the binding of RANKL to RANK and reducing the formation of osteoclasts. The Trichinella spiralis muscle larvae excretory-secretory proteins can also be used in the manufacture of drugs for treating osteoporosis.

[0007] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0008] An application of the excretions of the muscle larvae of Trichinella spiralis, characterized in that the excretions of the muscle larvae of Trichinella spiralis can inhibit the formation of osteoclasts by competitively binding with RANK.

[0009] Based on the above scheme, the excretion secretions of the Trichinella spiralis muscle larvae can inhibit the expression of osteoclast-related genes; the osteoclast-related genes are: mmp9, cathepsin k, trap, rank, nfatc1 and cfos.

[0010] Based on the above scheme, it is characterized in that: the secretions excreted by the muscle larvae of Trichinella spiralis can inhibit the expression of osteoclastogenesis-related proteins; the osteoclastogenesis-related proteins are: p-p65, p-iκB, p-p38, p-ERK, p-JNK, and NFAT.

[0011] Based on the above scheme, the dosage of the secretion of the Trichinella spiralis muscle larvae is 2-8 μg / mL.

[0012] The invention relates to an application of the excretion secretions of the muscle larvae of Trichinella spiralis in the research and development of drugs for preventing and treating bone destruction diseases.

[0013] The application of the secretions excreted by the muscle larvae of Trichinella spiralis of the present invention has the following beneficial effects:

[0014] The Trichinella spiralis muscle larvae secretion can competitively bind to RANKL, thereby inhibiting the binding of RANKL to RANK and reducing osteoclast formation. The Trichinella spiralis muscle larvae secretion can also inhibit the expression of osteoclast-related genes MMP9, Cathepsin K, TRAP, RANK, NFATc1, and CFOs, as well as the expression of osteoclastogenesis-related proteins p-p65, p-iκB, p-p38, p-ERK, p-JNK, and NFATc1. The Trichinella spiralis muscle larvae secretion can also be used in the manufacture of drugs for the treatment of osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention has the following accompanying drawings:

[0016] Figure 1 The SDS-PAGE electrophoresis results of TsMES.

[0017] Figure 2 This is the ELISA experiment result showing that Ts-MES inhibits the interaction between RANKL and RANK by competitively binding to RANKL.

[0018] Figure 3 This figure shows the experimental results of detecting the mRNA expression levels of mmp9, cathepsin k, trap, rank, nfatc1 and cfos in mouse bone marrow-derived macrophages (BMDMs) by RT-qPCR.

[0019] Figure 4 This is the experimental result of RT-qPCR detection of mRNA expression levels of mmp9, cathepsin k, trap, rank, nfatc1 and cfos in RAW 264.7 cells.

[0020] Figure 5 This is the experimental result of detecting the mRNA expression levels of mmp9, cathepsin k, trap, rank, nfatc1 and cfos in human mononuclear cells (PBMCs) by RT-qPCR.

[0021] Figure 6 This figure shows the experimental results of Western blot analysis of TsMES inhibiting the expression of RANKL-mediated osteoclastogenesis-related proteins p-p65, p-iκB, p-p38, p-ERK, p-JNK, and NFATc1 in mouse BMDMs.

[0022] Figure 7 This is the experimental result of quantitative analysis of the expression levels of osteoclast-related proteins in mouse BMDMs.

[0023] Figure 8 This figure shows the experimental results of Western blot analysis of TsMES inhibiting the expression of RANKL-mediated osteoclastogenesis-related proteins p-p65, p-iκB, p-p38, p-ERK, p-JNK, and NFATc1 in RAW264.7 cells.

[0024] Figure 9 This is the result of the quantitative analysis experiment on the expression levels of osteoclast-related proteins in RAW264.7 cells.

[0025] Figure 10This figure shows the experimental results of Western blot analysis of TsMES inhibiting the expression of RANKL-induced osteoclastogenesis-related proteins p-p65, p-iκB, p-p38, p-ERK, p-JNK, and NFATc1 in PBMCs.

[0026] Figure 11 Figure 2 shows the experimental results of quantitative analysis of the expression levels of osteoclast-related proteins in human PBMCs cells.

[0027] Figure 12 Representative TRAP staining images of mouse BMDMs. Scale bar, 50 μm.

[0028] Figure 13 The figure shows the results of detecting the number of TRAP-positive multinuclear osteoclasts in mouse BMDMs.

[0029] Figure 14 Representative TRAP staining images of RAW264.7 cells. Scale bar, 50 μm.

[0030] Figure 15 This figure shows the results of detecting the number of TRAP-positive multinuclear osteoclasts in RAW264.7 cells.

[0031] Figure 16 Representative TRAP staining images of human PBMCs. Scale bar, 50 μm.

[0032] Figure 17 This figure shows the results of the detection of the number of TRAP-positive multinucleated OCs in human PBMCs.

[0033] above Figure 3 、 5 , 6-7, 10-13, and 16-17, the results are expressed as the mean ± SD of three independent experiments, and the p value was analyzed using one-way ANOVA and Tukey's multiple comparison test.

[0034] above Figure 4 、 8 -9, 14-15, the results are expressed as the mean ± SD of three independent experiments. The p value was analyzed by one-way ANOVA. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below with reference to the accompanying drawings.

[0036] Example 1: Collection of TsMES

[0037] Take an ICR mouse that has been infected with Trichinella spiralis and kill it by cervical dislocation. Remove the legs and tail. Moisten the mouse abdomen with an alcohol swab and make a cross-shaped incision. Remove the fur, all internal organs, and minimize adipose tissue. Place the mouse muscle in a 250mL beaker and mince it with large scissors. Add an appropriate amount of distilled water (approximately 120mL) and mince it in a meat grinder (approximately 1 minute) until it turns pink. Add an appropriate amount of ddH2O to the mincer to rinse the remaining meat paste, bringing the final volume in the beaker to 200mL. Add pepsin and hydrochloric acid to a final concentration of 1%, stir promptly, and incubate in a 37°C waterbath for 4 hours, stirring occasionally to complete digestion. After incubation, add 1.7g of NaCl and stir thoroughly to terminate digestion. Filter the liquid through a 100-mesh stainless steel sieve into a conical measuring cup, leaving the sieve suspended during filtration. Let it settle at room temperature for 40 minutes, discard the suspended impurities, add physiological saline and let it settle again for 20 minutes. At this time, the supernatant is relatively clear. Discard the supernatant and leave about 30-40 mL of liquid. Mix it and pour it into 15 mL centrifuge tubes in batches. Centrifuge it with a manual centrifuge and wash it repeatedly with physiological saline for 2-3 times until there are no impurities. Collect the muscle larvae in the sediment.

[0038] The collected muscle larvae were rinsed and centrifuged 5 times with sterile physiological saline containing penicillin and streptomycin, and then immersed in a plate containing 1640 culture medium (serum-free) containing double antibodies at 3000-5000 / mL and placed in a 37°C, 5% CO2 incubator for 48 hours. After incubation, centrifuge at 5000rpm and 4°C for 2 minutes, aspirate the supernatant, and repeat twice. After centrifugation, the supernatant was placed in a dialysis bag (pore size of 12-14kDa), clamped at both ends of the dialysis bag, placed in 50 times the volume of ddH2O, dialyzed at 4°C for more than 3 hours, and then transferred to the same volume of dialysate at 4°C overnight. After dialysis is completed, the liquid was collected, vacuum-dried to a volume of 100μL, and TsMES was obtained, which was stored at -80°C for later use.

[0039] Example 2: Detection of TsMES components

[0040] 1. Detection of TsMES components by SDS-PAGE gel electrophoresis

[0041] Prepare SDS-PAGE stacking gel and separation gel according to the recipe table: take a clean glass plate and install it, add 5mL of prepared separation gel to 2 / 3 of the height of the glass plate, add 500μLddH2O or isopropanol to seal the liquid surface, wait for it to solidify, use filter paper to absorb ddH2O or isopropanol, then add stacking gel solution (1.5-2mL), insert the tooth comb, and wait for it to solidify. Then prepare the protein sample, take 8μL of protein, add 2μL of 5× protein loading buffer, mix well, 100℃, 5min, and centrifuge instantly. Then load the sample, assemble the gel plate and baffle into the inner tank of the electrophoresis tank, fill it with 1×SDS-PAGE electrophoresis buffer, remove the tooth comb, and add 10μL of protein molecular weight marker and the prepared protein sample respectively. After loading, electrophoresis was started. Constant voltage electrophoresis was performed according to the SDS-PAGE buffer system. The voltage of the stacking gel was 8 V / cm gel (60 V, 30 min), and the voltage of the separation gel was 15 V / cm gel (120 V, 90 min). The power was turned off when the bromophenol blue reached the bottom of the separation gel.

[0042] like Figure 1 As shown in the figure, the bands in the three electrophoresis lanes are consistent in shape, indicating that TsMES can be repeatedly prepared by the above collection steps. At the same time, each electrophoretic band of TsMES has multiple bands with varying depths, indicating that Ts-MES is a complex mixture of diverse protein components.

[0043] 2. Protein Spectroscopy Detection of TsMES Components

[0044] 5 μg of TsMES was incubated at 4°C for 4-6 hours, washed with PBS 3-4 times, and then subjected to SDS-PAGE electrophoresis. The gel strips were subjected to protein spectrum identification to analyze the protein components in TsMES.

[0045] Protein spectrum showed that TsMES contained the protein components shown in Table 1. The following protein PDB numbers are: E5SUX9, A0A0V1B4S3, A1BQX7, A0A0V1BSV1, E5RYV9, A0A0V1APR5, A0A0V1BN13, A3RLX8, A0A0V1BCK0, A0A0V1B1Q3, A0A0V1BKZ8, A0A0V1C2Y0, A0A0V1BH58, A0A0V1BTK0, A0A0V1BT87, A0A0V1ASG0, A 0A0V1C1Q1, A0A0V1BWE3, A0A0V1AVL6, A0A0V1C1J1, A0A0V1AS64, A0A0V1BM68, A0A0V1B1B9, A0A0V1B2N2, E5SLU1, A0A0V1BD95, A0A0V1BZ35, A0A0V1BXV2, A0A0V1B471, A0A0V1AX39, A0A0V1BWP3, A0A0V1BVT6, A0A0 V1BZB1, A0A0V1C007, A0A0V1ASF1, A0A0V1C146, A0A0V1BB60, A0A0V1BNV3, A0A0V1ARQ9, E5SUQ1, A0A0V1B SK6, E5S171, E5S9D6, A0A0V1BMT6, A0A0V1BWX2, A0A0V1BT35, A0A0V1BL71, A0A0V1BDX0, E5S6W3, A0A0V1BC C9, A0A0V1BMG6, A0A0V1B8E2, A0A0V1BME8, A0A0V1B5V4, A0A0V1BUG3, A0A0V1ATH0, A0A0V1C1L0, A0A0V1B 7A8, A0A0V1BB93, A0A0V1AVP2, A0A0V1ATW5, E5SJF6, A0A0V1BJC3, A0A0V1AZQ0, A0A0V1BT34, A0A0V1B2X1,

[0046] A0A0V1AT94, A0A0V1AZX2, A0A0V1BJS3, A0A0V1AX30,

[0047] E5S2V2, A0A0V1BT58, A0A0V1AUA7, A0A0V1BID8,

[0048] A0A0V1B8G7, A0A0V1C2M8, A0A0V1BA72, A0A0V1BQ40,

[0049] A0A0V1AZ10, A0A0V1C0W0, A0A0V1BGC9, A0A0V1BGF6,

[0050] E5SKT3,A0A0V1C2K8,A0A0V1BK21,A0A0V1BS89,

[0051] A0A0V1BX66, A0A0V1B0C0, E5S6T4, A0A0V1BFJ8, A0A0V1BIV6,

[0052] A0A0V1APS0, A0A0V1B172, B0FJW6, A0A0V1AWR7,

[0053] A0A0V1BWS9, A0A0V1AZ55, A0A0V1BYN1, A0A0V1BBA0,

[0054] A0A0V1BAP7, A0A0V1AQT0, A0A0V1AXA4, A0A0V1BL36,

[0055] A0A0V1BM71, A0A0V1AZD9, A0A0V1AU64, A0A0V1BQ51,

[0056] A0A0V1BV71,A0A0V1BSI0,A0A0V1B289,E5SV78,E5RYD6,

[0057] A0A0V1BSY9, A0A0V1BWW9, A0A0V1AZ39, A0A0V1B210,

[0058] A0A0V1BBS7, A0A0V1BJ68, A0A0V1AX18, E5S5Z6, A0A0V1BIN9,

[0059] A0A0V1BQ33, A0A0V1BNK0, A0A0V1B5A1, A0A0V1AX48,

[0060] A0A0V1C104, A0A0V1B8T3, A0A0V1B3D0, A0A0V1C2Q2,

[0061] A0A0V1AFL0, A0A0V1BC07, A0A0V1AHX1, A0A0V1BIN6,

[0062] A0A0V1BF14, A0A0V1B001, A0A0V1BR45, A0A0V1BS61,

[0063] A0A0V1C0U2, A0A0V1BPX9, A0A0V1ARP3, A0A0V1B4S8,

[0064] A0A0V1BXN0, A0A0V1B7I5, A0A0V1BTC9, A0A0V1AYH5,

[0065] A0A0V1BBV2, A0A0V0ZEC9, A0A0V1C2P7, A0A0V1BBY2,

[0066] A0A0V1B325, A0A0V1AJK3, A0A0V1C155, A0A0V1BKL0,

[0067] A0A0V1AQ62, A0A0V1AS46, A0A0V1AYM5, A0A0V1ASK3,

[0068] A0A0V1B1P7, A0A0V1BIS9, A0A0V1BSY0, A0A0V1BZR1,

[0069] A0A0V1B2Y1, E5S926, A0A0V1AXN4, A0A0V0ZIR9,

[0070] A0A0V1BZU1, A0A0V1BI85, A0A0V1BBH0, A0A0V1BIH7,

[0071] A0A0V1BEP5, A0A0V1B087, A0A0V1C0Z6, A0A0V1BUA3,

[0072] A0A0V1BYR4, A0A0V1BTI0, A0A0V1BHB6, E5SBH7,

[0073] A0A0V1C0X9, A0A0V1C058, A0A0V1BDV1, A0A0V1AR99,

[0074] A0A0V1C0R8, A0A0V1B947, A0A0V1BRZ1, A0A0V1BVH3,

[0075] A0A0V1BNQ0, A0A0V1C2N3, A0A0V1BR61, E5S7P5,

[0076] A0A0V1BPE5, A0A0V1BKX5, A0A0V1BRT6, A0A0V1BBG3,

[0077] A0A0V1B4L9,A0A0V1B848,A0A0V1AWR1,B0F9T0,

[0078] A0A0V1BLT3, A0A0V1BZT8, A0A0V1BHT2, A0A0V1BID0,

[0079] A0A0V1B0Z4, A0A0V1AQK5, A0A0V1AVV5, A0A0V1BFR5,

[0080] A0A0V1B204, A0A0V1BWG0, A0A0V1BGI2, A0A0V1ARQ8,

[0081] A0A0V1ARH0, A0A0V1BP09, A0A0V0YSK5, A0A0V1BYW3,

[0082] A0A0V1AXM8,E5SL96,A0A0V1B4W8,A0A0V1C324,

[0083] A0A0V1C2E5, A0A0V1BNB7, A0A0V1BYH2, A0A0V1BLD3,

[0084] A0A0V1BXS3, A0A0V1BU83, E5SJ21, A0A0V1BKY2,

[0085] A0A0V1BM94, E5S9J9, A0A0V1BAH1, A0A0V1BGU0,

[0086] A0A0V1AXH8, A0A0V1AUX6, A0A0V1AVI0, A0A0V1BVT8,

[0087] A0A0V1B0T8, A0A0V1BLM2, A0A0V1BZD4, A0A0V1BT00,

[0088] A0A0V1C3B0,A0A0V1B942,E5RZG4,A0A0V1C2A5,

[0089] A0A0V1BRQ6, A0A0V1AXL3, A0A0V1BK71, A0A0V1BJW3,

[0090] E5S7F6,A0A0V1C1W1,A0A0V1AS66,A0A0V1B3F8,

[0091] A0A0V1BA51, A0A0V1BAW3, A0A0V1BCL8, A0A0V1C0K3,

[0092] A0A0V1B5Y6, A0A0V1B1Q2, A0A0V1BYF2, A0A0V1BN09,

[0093] A0A0V1B0A5, A0A0V1BWH8, A0A0V1ASD1, A0A0V1BLS2,

[0094] A0A0V1BZS3, A0A0V1B629, A0A0V1BXB4, A0A0V1ASK1,

[0095] A0A0V1B8N1, A0A0V1BZ94, A0A0V1AWE1, A0A0V1BBQ6,

[0096] A0A0V1AVJ8, A0A0V1C0U1, A0A0V1AZ08, A0A0V1BTI3,

[0097] A0A0V1BRH2, A0A0V1BQL9, E5SB56, A0A0V1BVH1,

[0098] A0A0V1C023, A0A0V1BHF2, A0A0V1B7Q1, A0A0V1BQQ5,

[0099] A0A0V1BMB9, A0A0V1ASN1, A0A0V1BUG0, A0A0V1AZE9,

[0100] A0A0V1BTV2, A0A0V1BEJ0, A0A0V1B8N7, A0A0V1B2Y3,

[0101] E5RZL5, A0A0V1B3R0, A0A0V1BK94, A0A0V1APT6,

[0102] A0A0V1BFU5, A0A0V1BGL8, A0A0V1BW24, A0A0V1BGB3,

[0103] A0A0V1BKW7, A0A0V1APZ1, A0A0V1BHL2, A0A0V1C2F3, A0A0V1ARG0, A0A0V1AXC8, A0A0V1BB41, A0A 0V1BZX6, A0A0V1BXX7, A0A0V1BEP2, A0A0V1BEH5, A0A0V1BXY4, E5SAW8, A0A0V1BL96, A0A0V1C0L2, A0A0V1AUB8, A0A0V1BV40, A0A0V1BSS1, A0A0V1B9F5, A0A0V1ARU4, A0A0V1B171, A0A0V1B3S9, E5SL K3, A0A0V1BRI1, A0A0V1BAC1, A0A0V1BH90, E5SB48, A0A0V1BKU9, A0A0V1BVB7, A0A0V1BAK0, A0A0V 1BPU0, A0A0V1B1C8, A0A0V1B2I5, A0A0V1BJU1, A0A0V1BMY9, A0A0V1C2H2, A0A0V1BU64, A0A0V1BAN 8. A0A0V1BPT1, A0A0V1AXF4, A0A0V1BCX8, E5SK89, A0A0V1B2T6, A0A0V1BU52, A0A0V1AVQ4, A0A0V1 BU19, A0A0V1B3G8, A0A0V1AXQ6, A0A0V1AW53, A0A0V1BLR6, A0A0V1BB97, A0A0V1B7P9, A0A0V1B0R0 , A0A0V1BPV4, E5S0V0, A0A0V1C141, A0A0V1BR88, E5SAG9, A0A0V1BPP7, A0A0V1C133, A0A0V1C1E7.

[0104] Example 3: Verification of TsMES inhibiting the interaction between RANKL and RANK by competitively binding to RANKL

[0105] 1. ELISA detection of the interaction between TsMES and His-mRANK

[0106] TsMES or BSA was coated on the bottom of the ELISA plate at 5 μg / well at 4°C overnight, washed three times with PBST, and His-mRANK was added at different doses (0, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2 μg / well) after blocking. The plate was incubated at room temperature for 2 hours, washed, and then His antibody was added. Then, HRP-labeled goat anti-mouse secondary antibody was added. TMB color development was performed for 10-15 minutes, and stop solution was added to stop color development. The absorbance of each group was measured at a wavelength of 450 nm using an enzyme reader. The ELISA results showed that the absorbance did not increase with the increase in the amount of His-mRANK added ( Figure 2 Middle (a), indicating that TsMES does not bind to mRANK.

[0107] 2. ELISA detection of the interaction between TsMES and GST-mRANKL

[0108] TsMES or BSA was coated on the bottom of the ELISA plate at 5 μg / well at 4°C overnight, washed three times with PBST, and then blocked with different doses of GST-mRANKL (0, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2 μg / well). The plate was incubated at room temperature for 2 hours, washed, and then GST antibody was added. HRP-labeled goat anti-mouse secondary antibody was then added. TMB color development was performed for 10-15 minutes, and then stop solution was added to stop color development. The absorbance of each group was measured at a wavelength of 450 nm using an enzyme reader. The ELISA results showed that as the amount of GST-mRANKL added increased, its absorbance also increased ( Figure 2 Middle b) shows that TsMES can bind to mRANKL in a dose-dependent manner.

[0109] 3. ELISA Evaluation of the Competitive Inhibition of TsMES on the Binding of mRANKL and mRANK

[0110] mRANK was coated on the bottom of the ELISA plate at 1 μg / well at 4°C overnight, washed three times with PBST, and blocked. After blocking, a total of 1 μg GST-mRANKL was incubated with different doses (0, 0.25, 0.5, 1, 2, 4, 8, 16 μg / well) of TsMES or BSA in 100 μL PBST. The plates were incubated at room temperature for 2 hours. After washing, GST antibody was added, followed by HRP-labeled goat anti-mouse secondary antibody. TMB color development was performed for 10-15 minutes, and stop solution was added to stop color development. The absorbance of each group was measured at a wavelength of 450 nm using a microplate reader. Competitive ELISA experiments further showed that increasing the concentration of TsMES reduced the interaction between mRANKL and mRANK ( Figure 2 Middle c), indicating that TsMES competitively inhibits this binding.

[0111] Example 4: Verification of TsMES inhibition of RANKL-induced osteoclast formation

[0112] 1. RT-qPCR experiments verified that TsMES inhibited the expression of osteoclast-related genes

[0113] (1) RT-qPCR experiments verified that TsMES inhibited the expression of osteoclast-related genes in mouse BMDMs and RAW264.7 cells

[0114] Bone marrow was flushed from the femur and tibia of C57BL / 6J mice. After erythrocyte lysis, the cells were resuspended in complete DMEM medium containing 30 ng / mL mouse M-CSF and cultured at a concentration of 3 × 10 4 BMDMs were isolated by seeding cells at a density of 100 μg / well in 24-well plates. After overnight cell culture, 50 ng / mL of mRANKL and TsMES were added the next day. The cells were divided into the following groups: ① PBS group; ② mRANKL group; ③ mRANKL + TsMES (2 μg / mL) group; ④ mRANKL + TsMES (4 μg / mL) group; and ⑤ mRANKL + TsMES (8 μg / mL) group.

[0115] RAW264.7 cells were cultured at 2×10 5 The cells were seeded at a density of 1 / well in a 12-well plate and cultured in DMEM medium containing 50 ng / mL mRANKL and TsMES for 5-7 days. The cells were divided into the following groups: ①PBS group; ②mRANKL group; ③mRANKL+TsMES (4 μg / mL) group.

[0116] Total RNA was extracted from cells using Trizol reagent and reverse transcribed into cDNA. qPCR was performed using a real-time PCR system to calculate the relative mRNA expression levels of mmp9, cathepsin K, trap, rank, nfatc1, and cfos.

[0117] RT-qPCR analysis showed that TsMES treatment significantly downregulated the expression of BMDMs in mice ( Figure 3 ) and RAW 264.7 cells ( Figure 4 ) and the expression of osteoclast-related genes, including mmp9, cathepsin k, trap, rank, nfatc1, and cfos, in a dose-dependent manner.

[0118] (2) RT-qPCR experiments verified that TsMES inhibited the expression of osteoclast-related genes in human PBMCs

[0119] Human peripheral blood mononuclear cells (PBMCs) were isolated using Histopaque 1083 solution. PBMCs were seeded at a density of 3 × 10⁴ / well in 24-well plates and cultured in complete DMEM medium in a humidified incubator at 37°C for 3 hours. After removing nonadherent cells, the medium was replaced with 30 ng / mL human M-CSF for 3 days. On day 3, 50 ng / mL human RANKL (hRANKL) and TsMES were added. The cells were divided into the following groups: ① PBS group; ② hRANKL group; ③ hRANKL + TsMES (2 μg / mL) group; ④ hRANKL + TsMES (4 μg / mL) group; and ⑤ hRANKL + TsMES (8 μg / mL) group. Total RNA was extracted from the cells using Trizol reagent and reverse transcribed into cDNA. qPCR was performed using a real-time PCR system, and the relative mRNA expression levels of mmp9, cathepsin k, trap, rank, nfatc1, and cfos were calculated.

[0120] RT-qPCR analysis showed that TsMES significantly reduced the expression of osteoclast-related genes in human PBMCs, including mmp9, cathepsin k, trap, rank, nfatc1, and cfos. Figure 5 ).

[0121] 2. Western blot experiments verified that TsMES inhibited the expression of osteoclastogenesis-related proteins

[0122] (1) Western blot experiments verified that TsMES inhibited the expression of biosynthesis-related proteins in mouse BMDMs and RAW264.7 cells

[0123] Mouse BMDMs and RAW264.7 cells were obtained according to the method described in item 1(1) above and seeded into 6-well plates. After the cells adhered, 50 ng / mL of mRANK and TsMES were added and grouped as follows: Mouse BMDMs were grouped as follows: ①PBS group; ②mRANKL group; ③mRANKL+TsMES (2μg / mL) group; ④mRANKL+TsMES (4μg / mL) group; ⑤mRANKL+TsMES (8μg / mL) group. RAW264.7 cells were grouped as follows: ①PBS group; ②mRANKL group; ③mRANKL+TsMES (4μg / mL) group. After 60 minutes of stimulation, the cells were collected and lysed with RIPA. The expression of osteoclast-related signaling pathway proteins was detected by Western blot.

[0124] Western blot analysis showed that TsMES inhibited the expression of mRANKL-induced osteoclastogenesis-related proteins p-p65, p-iκB, p-p38, p-ERK, p-JNK, and NFATc1 in mouse BMDMs and RAW264.7 cells. Figure 6-9 )

[0125] (2) Western blot experiments verified that TsMES inhibited the expression of osteoclastogenesis-related proteins in human PBMCs

[0126] Human PBMCs were obtained as described in 1(2) above and seeded into 6-well plates. After cell attachment, 50 ng / mL hRANKL and TsMES were added. The cells were divided into the following groups: ① PBS group; ② hRANKL group; and hRANKL + TsMES (4 μg / mL) group. After 60 minutes of stimulation, the cells were harvested and lysed with RIPA. The expression of osteoclast-related signaling pathway proteins was then analyzed by Western blot.

[0127] Western blot analysis showed that TsMES inhibited the expression of hRANKL-induced osteoclastogenesis-related proteins p-p65, p-iκB, p-p38, p-ERK, p-JNK, and NFATc1 in human PBMCs. Figure 10-11 )

[0128] 3. TRAP staining to observe the number of osteoclasts

[0129] Mature osteoclasts can express a large amount of tartrate-resistant alkaline phosphatase (TRAP), which can be stained purple or wine red by TRAP staining. Therefore, TRAP staining was used to analyze the effect of TsMES on osteoclast differentiation in vitro.

[0130] Mouse BMDMs and RAW264.7 cells were obtained according to the method described in item 1(1) above and seeded into 48-well plates at 5×104 / well. The cells were divided into the following groups: ①PBS group; ②mRANKL group; ③mRANKL+TsMES (4μg / mL) group. The cell fusion status was observed every day. After 5-7 days of cell fusion, the supernatant was discarded and the cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes. Osteoclasts were stained using the TRAP staining kit. After rinsing with double-distilled water, the nuclei were stained with hematoxylin. After blueing with tap water, the cells were photographed under an optical microscope. Cells with more than three nuclei and cytoplasm stained wine red or purple red were osteoclasts. The number of osteoclasts in each well was counted to observe the effect of MES on the formation of osteoclasts by mouse BMDMs and RAW264.7 cells induced by mRANKL in vitro.

[0131] TRAP staining of mature osteoclasts confirmed that the number of TRAP-positive cells was significantly reduced after TsMES treatment compared with the untreated control group ( Figure 12-15 ).

[0132] Human PBMCs were obtained as described in item 1(2) above and 5×10 4 Cells were seeded / well in a 48-well plate and divided into the following treatment groups: ①PBS group; ②hRANKL group; ③hRANKL+TsMES (4μg / mL) group. After 10-14 days of culture until osteoclasts were confluent, TRAP staining and counting were performed. Consistent with the above results, TRAP staining confirmed a significant decrease in the number of osteoclasts after TsMES treatment ( Figure 16-17 ).

[0133] The contents not described in detail in this specification belong to the prior art known to those skilled in the art.

Claims

1. An application of secretions excreted by Trichinella spiralis muscle larvae, characterized in that: The secretions excreted by the muscle larvae of Trichinella spiralis can inhibit the formation of osteoclasts by competitively binding with RANK.

2. The use according to claim 1, characterized in that: The excretion secretions of the muscle larvae of Trichinella spiralis can inhibit the expression of osteoclast-related genes; the osteoclast-related genes are: mmp9, cathepsin k, trap, rank, nfatc1 and cfos.

3. The use according to claim 1, characterized in that: The excretion secretions of the muscle larvae of Trichinella spiralis can inhibit the expression of osteoclastogenesis-related proteins; the osteoclastogenesis-related proteins are: p-p65, p-iκB, p-p38, p-ERK, p-JNK, and NFAT.

4. The use of the excretion secretions of the muscle larvae of Trichinella spiralis according to claim 1, characterized in that: The dosage of the excretion secretion of the muscle larvae of Trichinella spiralis is 2-8 μg / mL.

5. Application of the excretion secretions of the muscle larvae of Trichinella spiralis in the research and development of drugs for the prevention and treatment of osteoporosis.

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