Composition for treating osteoporosis and application thereof

Abstract

The invention provides a composition for treating osteoporosis and application of the composition. The composition contains a mesenchymal stem cell exosome and a eucommia ulmoides extracting solution in a dosage ratio of (14-16) mg: (8-12) mL, the mesenchymal stem cell exosome is a mesenchymal stem cell exosome capable of simultaneously expressing an OPG protein and an IL-8 protein; the amino acid sequence of the OPG protein is as shown in SEQ ID NO: 1, and the amino acid sequence of the IL-8 protein is as shown in SEQ ID NO: 2; every 1mL of the eucommia ulmoides extracting solution contains 0.8-1.2 g of eucommia ulmoides extract. The invention finds that the bone bending resistance, the bone pressure stress, the bone density and the tissue mineral density can be remarkably improved by compounding the mesenchymal stem cell exosome capable of simultaneously expressing the OPG protein and the IL-8 protein with the eucommia ulmoides extracting solution, so that the osteoporosis can be effectively treated.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology. More specifically, it relates to a composition for treating osteoporosis and its application. Background Art

[0002] Osteoporosis is a systemic bone disease caused by various reasons and characterized by decreased bone density and bone quality. In osteoporosis patients, the bone bending resistance, bone compressive stress, bone density, tissue mineral density, etc. will all decrease significantly, resulting in a significant reduction in bone strength, bone toughness and bone repair ability, being prone to fractures, requiring long-term bed rest, and even possibly losing the ability to live independently, or secondary diseases such as pneumonia, urinary tract infection, bedsore, venous thrombosis, etc.

[0003] Currently, the drugs for treating osteoporosis mainly include bone mineralization promoters, bone resorption inhibitors and bone formation promoters, etc. Bone mineralization promoters mainly include calcium agents, vitamin D, calcium / vitamin D combined supplements, etc., but these bone mineralization promoters cannot reduce the risk of fractures. Bone resorption inhibitors mainly include bisphosphonates, calcitonin, estrogen, selective estrogen receptor modulators, etc., but excessive bone resorption inhibitors are prone to the accumulation of bone microcracks and the decrease of bone strength, and may ultimately lead to osteonecrosis. Bone formation promoters mainly include fluorine preparations, parathyroid hormone analogs, androgens and anabolic agents, etc., but fluorine preparations are prone to calcification defects, parathyroid hormone analogs have the risk of inducing osteosarcoma, and androgens and anabolic agents have drug adverse reactions such as masculinization, elevated low-density lipoprotein and affecting liver function.

[0004] Therefore, it is urgent to find a safe and effective drug for treating osteoporosis, which is very important for osteoporosis patients. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention aims to provide a composition for treating osteoporosis. By compounding the mesenchymal stem cell exosomes that can simultaneously express OPG protein and IL-8 protein with Eucommia ulmoides extract, it is found that it can significantly improve the bone bending resistance, bone compressive stress, bone density and tissue mineral density, and thus effectively treat osteoporosis. In addition, since OPG protein, IL-8 protein, mesenchymal stem cell exosomes and Eucommia ulmoides extract are all substances with relatively high safety, the safety of this composition is also relatively high, and it is suitable for preparing a safe and effective drug for treating osteoporosis.

[0006] The first object of the present invention is to provide a composition for treating osteoporosis.

[0007] The second object of the present invention is to provide the application of the above composition in the preparation of a drug for treating osteoporosis.

[0008] The third object of the present invention is to provide a drug for treating osteoporosis.

[0009] The above object of the present invention is achieved by the following technical solutions:

[0010] The present invention provides a composition for treating osteoporosis, which composition contains mesenchymal stem cell exosomes and Eucommia ulmoides extract in a dosage ratio of 14 - 16 mg : 8 - 12 mL; the mesenchymal stem cell exosomes are mesenchymal stem cell exosomes capable of simultaneously expressing OPG protein and IL-8 protein; the amino acid sequence of the OPG protein is as shown in SEQ ID NO:1, and the amino acid sequence of the IL-8 protein is as shown in SEQ ID NO:2; each 1 mL of Eucommia ulmoides extract contains an extract equivalent to 0.8 - 1.2 g of Eucommia ulmoides.

[0011] The present invention combines mesenchymal stem cell exosomes capable of simultaneously expressing OPG protein (SEQ ID NO:1) and IL-8 protein (SEQ ID NO:2) with Eucommia ulmoides extract, and finds that it can significantly improve the bone bending resistance, bone compressive stress, bone mineral density and tissue mineral density, and thus effectively treat osteoporosis. Moreover, the OPG protein, IL-8 protein and Eucommia ulmoides extract have a synergistic effect in improving the bone bending resistance, bone compressive stress and tissue mineral density, that is, they have a significant synergistic effect in treating osteoporosis.

[0012] Among them, the amino acid sequence of the OPG protein is as shown in SEQ ID NO:1:

[0013] MNKLLCCALVFLDISIKWTTQETFPPKYLHYDEETSHQLLCDKCPPGTYLKQHCTAKWKTVCAPCPDHYYTDSWHTSDECLYCSPVCKELQYVKQECNRTHNRVCECKEGRYLEIEFCLKHRSCPPGFGVVQAGTPERNTVCKRCPDGFFSNETSSKAPCRKHTNCSVFGLLLTQKGNATHDNICSGNSESTQK.

[0014] The amino acid sequence of the IL-8 protein is as shown in SEQ ID NO:2:

[0015] MTSKLAVALLAAFLISAALCEGAVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPH CANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS.

[0016] Preferably, the dosage ratio of the mesenchymal stem cell exosomes to the Eucommia ulmoides extract is 15 mg: 10 mL.

[0017] Preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0018] Preferably, the preparation method of the Eucommia ulmoides extract is as follows: Eucommia ulmoides is soaked in water for 10 - 14 h and then heated for extraction at 95 - 100 °C for 5.4 - 6.6 h, filtered, and the filtrate is concentrated.

[0019] More preferably, the dosage ratio of the water to Eucommia ulmoides is 30 - 36 mL: 1 g, and most preferably 33 mL: 1 g.

[0020] More preferably, the preparation method of the Eucommia ulmoides extract is as follows: Add water to Eucommia ulmoides for the first time, soak for 10 - 14 h (most preferably 12 h), then heat at 95 - 100 °C (most preferably 100 °C) for 1.8 - 2.2 h (most preferably 2 h), and then filter for the first time; Add water to the filter residue obtained from the first filtration for the second time, heat at 95 - 100 °C (most preferably 100 °C) for 1.8 - 2.2 h (most preferably 2 h), and filter for the second time; Then add water to the filter residue obtained from the second filtration for the third time, heat at 95 - 100 °C (most preferably 100 °C) for 1.8 - 2.2 h (most preferably 2 h), and filter for the third time; Combine the filtrates of the three times and concentrate.

[0021] More preferably, the dosage ratio of the water added for the first time to Eucommia ulmoides is 12 - 14 mL: 1 g, and most preferably 13 mL: 1 g.

[0022] More preferably, the dosage ratio of the water added for the second time to Eucommia ulmoides is 9 - 11 mL: 1 g, and most preferably 10 mL: 1 g.

[0023] More preferably, the dosage ratio of the water added for the third time to Eucommia ulmoides is 9 - 11 mL: 1 g, and most preferably 10 mL: 1 g.

[0024] More preferably, the concentration is to concentrate to an extract containing an amount equivalent to 0.8 - 1.2 g of Eucommia ulmoides per 1 mL of the Eucommia ulmoides extract, and most preferably to concentrate to an extract containing an amount equivalent to 1 g of Eucommia ulmoides per 1 mL of the Eucommia ulmoides extract.

[0025] The mesenchymal stem cell exosomes capable of co-expressing OPG protein and IL-8 protein can be synthesized by common methods in the art. For example, the amino acid sequences of OPG protein and IL-8 protein are first synthesized by molecular biological means, and the pSin-EF2-OPG-IL8 recombinant plasmid is constructed. Then, after the recombinant plasmid is transfected into mesenchymal stem cells by lentivirus-mediated transfection, the mesenchymal stem cells are cultured, and the mesenchymal stem cell exosomes capable of co-expressing OPG protein and IL-8 protein are obtained by separation. Optionally, the culture is carried out at 35-39 °C for 70-74 h (most preferably at 37 °C for 72 h).

[0026] Preferably, the above composition for treating osteoporosis is obtained by mixing the raw materials evenly.

[0027] More preferably, after mixing evenly, the pH is adjusted to 5.8-6.2, such as by using HCl for adjustment.

[0028] The above composition for treating osteoporosis can significantly improve the bone bending resistance, bone compressive stress, bone mineral density and tissue mineral density, and thus effectively treat osteoporosis. Therefore, the application of the above composition in the preparation of drugs for treating osteoporosis should also be within the protection scope of the present invention. Optionally, the osteoporosis is osteoporosis in middle-aged and elderly patients. It should be understood that middle-aged and elderly refers to people aged 45 and above.

[0029] Based on this, the present invention also provides a drug for treating osteoporosis. Each 1000 mL of the drug contains the following components: 14-16 mg of mesenchymal stem cell exosomes, 8-12 mL of Eucommia ulmoides extract, 25-35 g of detergent, 20-30 g of humectant, and the balance is water;

[0030] The mesenchymal stem cell exosomes are mesenchymal stem cell exosomes capable of co-expressing OPG protein and IL-8 protein; each 1 mL of Eucommia ulmoides extract contains an extract equivalent to 0.8-1.2 g of Eucommia ulmoides.

[0031] Preferably, each 1000 mL of the drug contains the following components: 14-16 mg of mesenchymal stem cell exosomes, 8-12 mL of Eucommia ulmoides extract, 25-35 g of detergent, 8-10 g of preservative, 20-30 g of humectant, and the balance is water.

[0032] Preferably, each 1000 mL of the drug contains the following components: 14-16 mg of mesenchymal stem cell exosomes, 8-12 mL of Eucommia ulmoides extract, 25-35 g of detergent, 18-22 g of suspending agent, 20-30 g of humectant, and the balance is water.

[0033] Preferably, every 1000 mL of the drug contains the following components: 14 - 16 mg of mesenchymal stem cell exosomes, 8 - 12 mL of Eucommia ulmoides extract, 25 - 35 g of a detergent, 20 - 30 g of a humectant, 6 - 8 g of a cooling flavorant, and the balance is water.

[0034] Preferably, every 1000 mL of the drug contains the following components: 14 - 16 mg of mesenchymal stem cell exosomes, 8 - 12 mL of Eucommia ulmoides extract, 25 - 35 g of a detergent, 8 - 10 g of a preservative, 18 - 22 g of a suspending agent, 20 - 30 g of a humectant, 6 - 8 g of a cooling flavorant, and the balance is water.

[0035] More preferably, every 1000 mL of the drug contains the following components: 15 mg of mesenchymal stem cell exosomes, 10 mL of Eucommia ulmoides extract, 30 g of a detergent, 9 g of a preservative, 20 g of a suspending agent, 25 g of a humectant, 7 g of a cooling flavorant, and the balance is water; the mesenchymal stem cell exosomes are those that can simultaneously express OPG protein and IL - 8 protein; every 1 mL of Eucommia ulmoides extract contains an extract equivalent to 1 g of Eucommia ulmoides.

[0036] Preferably, the detergent is sodium chloride.

[0037] Preferably, the preservative is disodium edetate and / or benzalkonium chloride, such as disodium edetate and benzalkonium chloride in a mass ratio of 7.8 - 9:0.2 - 1, and most preferably disodium edetate and benzalkonium chloride in a mass ratio of 8.5:0.5.

[0038] Preferably, the suspending agent is microcrystalline cellulose - sodium carboxymethylcellulose and / or polysorbate, such as microcrystalline cellulose - sodium carboxymethylcellulose and polysorbate in a mass ratio of 14 - 16:4 - 6, and most preferably microcrystalline cellulose - sodium carboxymethylcellulose and polysorbate in a mass ratio of 15:5.

[0039] Preferably, the humectant is glycerol.

[0040] Preferably, the cooling flavorant is eucalyptol.

[0041] Preferably, the dosage form of the drug is one or more of nasal sprays, injections, and oral preparations. When the dosage form of the drug is a nasal spray, the therapeutic effect is better, the compliance of the patient is higher, and compared with traditional nasal sprays, the drug of the present invention does not affect the nasal microenvironment and will not cause some adverse reactions due to the accumulation of the drug in the nasal cavity, and can achieve the purpose of convenient treatment at any time.

[0042] As a preferably feasible embodiment, the 1000 mL osteoporosis treatment drug comprises the following components: 14 - 16 mg of mesenchymal stem cell exosomes, 8 - 12 mL of Eucommia ulmoides extract, 25 - 35 g of sodium chloride, 8 - 10 g of preservative (disodium edetate and benzalkonium chloride with a mass ratio of 7.8 - 9:0.2 - 1), 18 - 22 g of suspending agent (microcrystalline cellulose - sodium carboxymethylcellulose and polysorbate with a mass ratio of 14 - 16:4 - 6), 20 - 30 g of glycerol, 6 - 8 g of eucalyptol, with the balance being water; the mesenchymal stem cell exosomes are mesenchymal stem cell exosomes that can simultaneously express OPG protein and IL - 8 protein; each 1 mL of Eucommia ulmoides extract contains an extract equivalent to 0.8 - 1.2 g of Eucommia ulmoides.

[0043] Most preferably, the 1000 mL osteoporosis treatment drug comprises the following components: 15 mg of mesenchymal stem cell exosomes, 10 mL of Eucommia ulmoides extract, 30 g of sodium chloride, 9 g of preservative (disodium edetate and benzalkonium chloride with a mass ratio of 8.5:0.5), 20 g of suspending agent (microcrystalline cellulose - sodium carboxymethylcellulose and polysorbate with a mass ratio of 15:5), 25 g of glycerol, 7 g of eucalyptol, with the balance being water; the mesenchymal stem cell exosomes are mesenchymal stem cell exosomes that can simultaneously express OPG protein and IL - 8 protein; each 1 mL of Eucommia ulmoides extract contains an extract equivalent to 1 g of Eucommia ulmoides.

[0044] In addition, the above - mentioned osteoporosis treatment drug can be prepared by the following method: First, add mesenchymal stem cell exosomes, a detergent, an optional preservative, an optional polysorbate, a humectant, an optional cooling flavoring agent to water, mix well, then add an optional microcrystalline cellulose - sodium carboxymethylcellulose, mix well, and finally add Eucommia ulmoides extract and mix well to obtain the drug.

[0045] As a preferably feasible embodiment, the above - mentioned osteoporosis treatment drug can be prepared by the following method: First, add mesenchymal stem cell exosomes, sodium chloride, disodium edetate, benzalkonium chloride, polysorbate, glycerol, eucalyptol to water, mix well, then add microcrystalline cellulose - sodium carboxymethylcellulose, mix well, and finally add Eucommia ulmoides extract and mix well to obtain the drug.

[0046] Preferably, the pH of the drug is 5.8 - 6.2, adjusted with HCl if necessary.

[0047] Preferably, the drug is also sterilized, for example, by filtration sterilization with a 0.4 - 0.5 μm filter membrane.

[0048] The present invention has the following beneficial effects:

[0049] 1. The present invention combines mesenchymal stem cell exosomes capable of simultaneously expressing OPG protein and IL-8 protein with Eucommia ulmoides extract to obtain a composition capable of effectively treating osteoporosis. Moreover, the OPG protein, IL-8 protein, and Eucommia ulmoides extract have a synergistic effect in enhancing the anti-bending ability of bones, bone compressive stress, and tissue mineral density, that is, they have a significant synergistic effect in the treatment of osteoporosis.

[0050] 2. In the composition for treating osteoporosis of the present invention, the OPG protein, IL-8 protein, mesenchymal stem cell exosomes, and Eucommia ulmoides extract are all substances with relatively high safety, so the safety of this composition is also relatively high, and it is suitable for preparing a safe drug capable of effectively treating osteoporosis. Brief Description of the Drawings

[0051] Figure 1 It is the plasmid map of pSin-EF2-OPG-IL8.

[0052] Figure 2 It is the plasmid map of pSin-EF2-OPG.

[0053] Figure 3 It is the plasmid map of pSin-EF2-IL8. Detailed Embodiments

[0054] The present invention will be further described below in conjunction with the drawings in the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0055] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0056] Example 1 Construction of Recombinant Plasmids

[0057] I. Construction of Recombinant Plasmid pSin-EF2-OPG-IL8

[0058] After synthesizing the OPG protein with the amino acid sequence shown in SEQ ID NO:1 and the IL-8 protein with the amino acid sequence shown in SEQ ID NO:2 according to the codons preferred by human cells, an IRES element sequence was added between the two proteins, a kozak sequence was introduced between the IL-8 protein and the IRES element sequence, and a BamHI site, a start codon, a kozak sequence and a tPA signal peptide with the amino acid sequence shown in SEQ ID NO:3 were introduced upstream of the gene. A stop codon and an EcoRI site were introduced downstream of the gene. The synthesized gene fragment was digested at a constant temperature with BamHI and EcoRI at 4°C for 12 h, and then the gel containing the digested product was cut with agarose gel electrophoresis. The target fragment was recovered using an agarose gel DNA recovery kit. The recovered product was mixed with the pSin-EF2-puro lentiviral expression vector digested with BamHI and EcoRI in a molar ratio of 3:1, and then ligated at a constant temperature in a PCR instrument at 16°C for 12 h using T4 DNA ligase, thus constructing the recombinant plasmid pSin-EF2-OPG-IL8 (the map is as shown in Figure 1 ).

[0059] The amino acid sequence of the OPG protein is shown in SEQ ID NO:1:

[0060] MNKLLCCALVFLDISIKWTTQETFPPKYLHYDEETSHQLLCDKCPPGTYLKQHCTAKWKTVCAPCPDHYYTDSWHTSDECLYCSPVCKELQYVKQECNRTHNRVCECKEGRYLEIEFCLKHRSCPPGFGVVQAGTPERNTVCKRCPDGFFSNETSSKAPCRKHTNCSVFGLLLTQKGNATHDNICSGNSESTQK.

[0061] The amino acid sequence of the IL-8 protein is shown in SEQ ID NO:2:

[0062] MTSKLAVALLAAFLISAALCEGAVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPH CANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS.

[0063] The amino acid sequence of the tPA signal peptide is shown in SEQ ID NO:3: MDAMKRGLCCVLLLCGAVFVSP.

[0064] II. Construction of the recombinant plasmid pSin-EF2-OPG

[0065] According to the codons preferred by human cells, the OPG protein with the amino acid sequence shown in SEQ ID NO:1 was synthesized. A BamHI site, a start codon, a kozak sequence, and a tPA signal peptide with the amino acid sequence shown in SEQ ID NO:3 were introduced upstream of the gene. A stop codon and an EcoRI site were introduced downstream of the gene. The synthesized gene fragment was digested with BamHI and EcoRI at 4°C for 12 h at a constant temperature. Then, the gel containing the digested product was cut by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit. The recovered product was mixed with the pSin-EF2-puro lentiviral expression vector digested with BamHI and EcoRI in a molar ratio of 3:1, and then ligated at a constant temperature in a PCR instrument at 16°C for 12 h using T4 DNA ligase, thus constructing the recombinant plasmid pSin-EF2-OPG (the map is as shown in Figure 2 ).

[0066] III. Construction of the recombinant plasmid pSin-EF2-IL8

[0067] According to the codons preferred by human cells, the IL-8 protein with the amino acid sequence shown in SEQ ID NO:2 was synthesized. A BamHI site, a start codon, a kozak sequence, and a tPA signal peptide with the amino acid sequence shown in SEQ ID NO:3 were introduced upstream of the gene. A stop codon and an EcoRI site were introduced downstream of the gene. The synthesized gene fragment was digested with BamHI and EcoRI at 4°C for 12 h at a constant temperature. Then, the gel containing the digested product was cut by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit. The recovered product was mixed with the pSin-EF2-puro lentiviral expression vector digested with BamHI and EcoRI in a molar ratio of 3:1, and then ligated at a constant temperature in a PCR instrument at 16°C for 12 h using T4 DNA ligase, thus constructing the recombinant plasmid pSin-EF2-IL8 (the map is as shown in Figure 3 ).

[0068] Example 2 Transfection of the recombinant plasmid

[0069] The recombinant plasmids pSin-EF2-OPG-IL8, pSin-EF2-OPG, and pSin-EF2-IL8 obtained in Example 1 were respectively transformed into competent Escherichia coli DH5α. Then, the competent Escherichia coli DH5α was evenly spread on an LB plate medium containing 100 μg / mL ampicillin. After the bacterial solution was completely absorbed, it was cultured inverted at 37 °C for 14 h. Six single colonies with good growth states were picked with a sterile pipette tip and inoculated into 5 mL of LB medium containing 100 μg / mL ampicillin. After culturing with shaking at 37 °C and 225 rpm for 12 h, plasmid DNA was extracted using a rapid plasmid miniprep kit, and then double digested with BamHI and EcoRI. The double-digested product was sent to a sequencing company for sequencing identification. The clones with correct sequencing identification were taken, and a large amount of recombinant plasmids pSin-EF2-OPG-IL8, pSin-EF2-OPG, and pSin-EF2-IL8 were obtained by extracting with a plasmid maxiprep kit.

[0070] Then, the packaging plasmid psPAX2, the envelope plasmid pMD2G, and the recombinant plasmids (pSin-EF2-OPG-IL8, pSin-EF2-OPG, pSin-EF2-IL8 respectively) were co-transfected into HEK-293T cells using Lipofiter transfection reagent. After culturing in DMEM medium at 37 °C for 8 h, the medium was replaced and the cells were cultured for another 72 h. The supernatant was collected at 48 h and 72 h of culture respectively. After filtration through a 0.45 μM membrane, the 48 h virus solution and the 72 h virus solution were obtained. The virus titers in the 48 h virus solution and the 72 h virus solution were detected using a lentivirus titer ELISA detection kit (Biolong) (to determine that the virus titer exceeded 10 6 TU / mL), and then the 48 h virus solution and the 72 h virus solution were stored in a -80 °C refrigerator.

[0071] Obtaining and infecting umbilical cord mesenchymal stem cells in Example 3

[0072] Fresh umbilical cords (there are no ethical controversies in the means of acquisition and treatment) were taken, washed with sterile saline to remove blood, cut into 4 cm small segments, and then the veins, envelopes, and 2 arteries were removed. The Wharton's jelly was minced and inoculated into mesenchymal stem cell medium, and cultured in a 37 °C, 5% CO2 cell culture incubator for 6 days. A large number of spindle-shaped cells were visible crawling out of the tissue blocks. After passage, amplification, and purification and identification, it was confirmed that these spindle-shaped cells were umbilical cord mesenchymal stem cells.

[0073] Umbilical cord mesenchymal stem cells were inoculated on fresh mesenchymal stem cell culture medium, and after subculture at 37° C. for 24 h, the 48h virus solution obtained in Example 2 (the volume ratio of the 48h virus solution obtained in Example 2 to the mesenchymal stem cell culture medium was 3:1) and polybrene (Polybrene, the concentration of polybrene in the mesenchymal stem cell culture medium was 8 μg / mL) were added, and after infection at 37° C. for 8 h, fresh mesenchymal stem cell culture medium was replaced, and cultured at 37° C. for another 12 h, and then the 72h virus solution obtained in Example 2 (the volume ratio of the 72h virus solution obtained in Example 2 to the mesenchymal stem cell culture medium was 3:1) and polybrene (Polybrene, the concentration of polybrene in the mesenchymal stem cell culture medium was 8 μg / mL) were added, and after infection at 37° C. for 8 h, fresh mesenchymal stem cell culture medium was replaced, and cultured at 37° C. for 72 h, and cells were collected at 48 and 72 h of culture for qPCR and Western Blot. Blot detection confirmed that umbilical cord mesenchymal stem cells infected with three recombinant plasmids were obtained.

[0074] Example 4 Preparation of umbilical cord mesenchymal stem cell exosomes

[0075] The umbilical cord mesenchymal stem cells infected with the recombinant plasmid obtained in Example 3 were inoculated in a fresh mesenchymal stem cell culture medium, cultured at 37°C for 72h, and then centrifuged at 4°C and 1000×g for 10min to remove the cell precipitate, and the supernatant was centrifuged at 4°C and 10000×g for 30min to remove large cell debris and broken organelles, and the supernatant was centrifuged at 4°C and 100000×g for 120min, and the precipitate was resuspended with PBS, and centrifuged again at 4°C and 100000×g for 120min. The precipitate obtained by centrifugation is the umbilical cord mesenchymal stem cell exosomes. After resuspending with physiological saline, the BCA method was used for quantification (to determine that the total protein concentration exceeded 0.25μg / μL), and then Western Blot detection and ELISA detection were performed to confirm that the obtained umbilical cord mesenchymal stem cell exosomes can successfully express the corresponding protein.

[0076] Example 5 Preparation of Eucommia ulmoides extract

[0077] Take 2kg of Eucommia ulmoides, add water (13 times the mass of Eucommia ulmoides) to the Eucommia ulmoides for the first time, soak for 12 hours, heat at 100℃ for 2 hours, and filter for the first time; add water (10 times the mass of Eucommia ulmoides) to the residue obtained by the first filtration for the second time, heat at 100℃ for 2 hours, and filter for the second time; then add water (10 times the mass of Eucommia ulmoides) to the residue obtained by the second filtration for the third time, heat at 100℃ for 2 hours, and filter for the third time. Combine the three filtrates, and finally concentrate the filtrate to contain an extract equivalent to 1g of Eucommia ulmoides per 1mL of Eucommia ulmoides extract, and store at 4℃.

[0078] Example 6 Drug Preparation and Efficacy Test for Osteoporosis

[0079] I. Experimental Grouping and Drug Administration

[0080] Four-week-old SPF-grade ovariectomized C57 mice (OPG- / -) were divided into 7 treatment groups, with 10 mice in each group, namely: OPG + IL-8 exosome + Eucommia ulmoides treatment group, OPG exosome treatment group, IL-8 exosome treatment group, Eucommia ulmoides treatment group, OPG + IL-8 exosome treatment group, OPG exosome + Eucommia ulmoides treatment group, IL-8 exosome + Eucommia ulmoides treatment group.

[0081] The composition and preparation method of the nasal spray used in each treatment group are as follows:

[0082] 1. OPG + IL-8 exosome + Eucommia ulmoides treatment group

[0083] (1) Composition

[0084] Mesenchymal stem cell exosomes (mesenchymal stem cell exosomes that can simultaneously express OPG protein and IL-8 protein) 15 mg, Eucommia ulmoides extract 10 mL, sodium chloride 30 g, disodium edetate 8.5 g, benzalkonium chloride 0.5 g, microcrystalline cellulose - sodium carboxymethylcellulose 15 g, polysorbate 5 g, glycerol 25 g, eucalyptol 7 g, and water was added to make up 1000 mL.

[0085] (2) Preparation method

[0086] First, add mesenchymal stem cell exosomes, sodium chloride, disodium edetate, benzalkonium chloride, polysorbate, glycerol, and eucalyptol to water, mix well, then add microcrystalline cellulose - sodium carboxymethylcellulose, mix well, and finally add Eucommia ulmoides extract, mix well. Adjust the pH to 6 with HCl, and then filter and sterilize with a 0.45 μm sterile filter membrane, and fill into a sterile nasal spray bottle.

[0087] 2. OPG exosome treatment group

[0088] (1) and (2) are the same as the OPG + IL-8 exosome + Eucommia ulmoides treatment group, except that the mesenchymal stem cell exosomes used are mesenchymal stem cell exosomes that can express OPG protein, and the Eucommia ulmoides extract is replaced with an equal volume of water.

[0089] 3. IL-8 exosome treatment group

[0090] (1) and (2) are the same as the OPG + IL-8 exosome + Eucommia ulmoides treatment group, except that the mesenchymal stem cell exosomes used are mesenchymal stem cell exosomes that can express IL-8 protein, and the Eucommia ulmoides extract is replaced with an equal volume of water.

[0091] 4. Eucommia ulmoides treatment group

[0092] (1) and (2) are the same as the OPG + IL-8 exosome + Eucommia ulmoides treatment group, except that the mesenchymal stem cell exosomes are replaced with an equal volume of normal saline.

[0093] 5. OPG + IL-8 exosome treatment group

[0094] (1) and (2) are the same as the OPG + IL-8 exosome + Eucommia ulmoides treatment group, except that the Eucommia ulmoides extract is replaced with an equal volume of water.

[0095] 6. OPG exosome + Eucommia ulmoides treatment group

[0096] (1) and (2) are the same as the OPG + IL-8 exosome + Eucommia ulmoides treatment group, except that the mesenchymal stem cell exosomes used are those from mesenchymal stem cells that can express OPG protein.

[0097] 7. IL-8 exosome + Eucommia ulmoides treatment group

[0098] (1) and (2) are the same as the OPG + IL-8 exosome + Eucommia ulmoides treatment group, except that the mesenchymal stem cell exosomes used are those from mesenchymal stem cells that can express IL-8 protein.

[0099] Each of the above 7 treatment groups was further divided into two subgroups of high dose and low dose, that is, the number of mice in each subgroup was 5. Another 5 4-week-old SPF-grade ovariectomized C57 mice (OPG- / -) were taken as the blank group, and 5 SPF-grade C57 mice were taken as the wild group.

[0100] Each nostril of the mice in the blank group and the wild group was given 15 μL of normal saline using a micropipette, regarded as one administration; each nostril of the high-dose subgroup of the treatment group was given 15 μL of the corresponding nasal spray, regarded as one administration; each nostril of the low-dose subgroup of the treatment group was given 5 μL of the corresponding nasal spray, regarded as one administration. Each mouse was administered once in the morning, middle, and evening every day for two consecutive months. After the administration was completed, the bilateral femurs and tibias of the mice were taken, wrapped with gauze soaked in normal saline, and stored at -20 °C.

[0101] II. Bone biomechanical measurement

[0102] The right femurs of mice were taken out of the refrigerator and naturally thawed, and then three-point bending tests were performed on their right femurs using a microcomputer-controlled electronic universal testing machine. First, the full length of the right femur of the mice was measured with a vernier caliper, and the position at a distance of half of the full length from the femoral head was defined as the midpoint of the femur. Then, the femur was placed in the exact middle of the three-point bending platform, and the span of the support was limited to 10 mm with a vernier caliper, that is, the distance from both ends of the support to the midpoint of the femur was 5 mm. A transverse pressure was applied downward at the midpoint of the femur at a speed of 2 mm / min until the femur was damaged, and then the maximum bending load and maximum compressive stress of the femur were measured. The results were averaged, as shown in Table 1.

[0103] Table 1

[0104]

[0105] It can be seen that:

[0106] (1) Compared with the blank group and other treatment groups, the maximum bending load and maximum compressive stress of the OPG+IL-8 exosome+Eucommia ulmoides treatment group were significantly increased, indicating that the composition obtained by compounding the mesenchymal stem cell exosomes that can simultaneously express OPG protein and IL-8 protein with the Eucommia ulmoides extract can significantly improve the anti-bending ability and compressive stress of bones, that is, improve the strength and toughness of bones, and thus effectively treat osteoporosis.

[0107] (2) Compared with the blank group, the maximum bending loads of the high-dose OPG exosome treatment group, IL-8 exosome treatment group, and Eucommia ulmoides treatment group were increased by 0.467, 0.092, and 0.046 N respectively, while the high-dose OPG+IL-8 exosome+Eucommia ulmoides treatment group was increased by 1.042 N (>0.467 + 0.092 + 0.046 = 0.605), indicating that OPG protein, IL-8 protein, and Eucommia ulmoides extract have a significant synergistic effect in improving the anti-bending ability of bones.

[0108] (3) Compared with the blank group, the maximum bending loads of the low-dose OPG exosome treatment group, IL-8 exosome treatment group, and Eucommia ulmoides treatment group were increased by 0.347, 0.043, and 0.032 N respectively, while the low-dose OPG+IL-8 exosome+Eucommia ulmoides treatment group was increased by 0.900 N (>0.347 + 0.043 + 0.032 = 0.422), indicating that OPG protein, IL-8 protein, and Eucommia ulmoides extract have a significant synergistic effect in improving the anti-bending ability of bones.

[0109] (4) Compared with the blank group, the maximum compressive stress of the high-dose OPG exosome treatment group, IL-8 exosome treatment group, and Eucommia ulmoides treatment group increased by 0.144, 0.070, and 0.012 MPa respectively, while the high-dose OPG + IL-8 exosome + Eucommia ulmoides treatment group increased by 0.477 MPa (>0.144 + 0.070 + 0.012 = 0.226), indicating that OPG protein, IL-8 protein, and Eucommia ulmoides extract have a significant synergistic effect in enhancing bone compressive stress.

[0110] (5) Compared with the blank group, the maximum compressive stress of the low-dose OPG exosome treatment group, IL-8 exosome treatment group, and Eucommia ulmoides treatment group increased by 0.096, 0.035, and 0.009 MPa respectively, while the low-dose OPG + IL-8 exosome + Eucommia ulmoides treatment group increased by 0.449 MPa (>0.096 + 0.035 + 0.009 = 0.140), indicating that OPG protein, IL-8 protein, and Eucommia ulmoides extract have a significant synergistic effect in enhancing bone compressive stress.

[0111] In summary, the composition of the present invention can significantly improve the anti-bending ability and compressive stress of bones, that is, can significantly improve the strength and toughness of bones, and thus effectively treat osteoporosis, and OPG protein, IL-8 protein, and Eucommia ulmoides extract have a significant synergistic effect in the treatment of osteoporosis.

[0112] III. Bone Mineral Density and Tissue Mineral Density Measurement

[0113] The tibia of the right hind leg of the mouse was scanned by cone-beam X-ray, and its bone mineral density (BMD) and tissue mineral density (TMD) were analyzed by vivaCT80 software, and the results were averaged, as shown in Table 2.

[0114] Table 2

[0115]

[0116] It can be seen that:

[0117] (1) Compared with the blank group and other treatment groups, the bone mineral density and tissue mineral density of the OPG + IL-8 exosome + Eucommia ulmoides treatment group were significantly improved, indicating that the composition obtained by compounding the mesenchymal stem cell exosomes that can simultaneously express OPG protein and IL-8 protein with Eucommia ulmoides extract can significantly improve bone mineral density and tissue mineral density, that is, improve the strength and toughness of bones, and thus effectively treat osteoporosis.

[0118] (2) Compared with the blank group, the bone mineral density of the low-dose OPG exosome treatment group, IL-8 exosome treatment group, and Eucommia ulmoides treatment group increased by 190.29, 71.95, and 12.83 mg HA / ccm respectively, while the low-dose OPG + IL-8 exosome + Eucommia ulmoides treatment group increased by 299.84 mg HA / ccm (>190.29 + 71.95 + 12.83 = 275.07), indicating that OPG protein, IL-8 protein, and Eucommia ulmoides extract have a significant synergistic effect in increasing bone mineral density.

[0119] (3) Compared with the blank group, the tissue mineral density of the high-dose OPG exosome treatment group, IL-8 exosome treatment group, and Eucommia ulmoides treatment group increased by 271.20, 92.27, and 34.12 mg HA / ccm respectively, while the high-dose OPG + IL-8 exosome + Eucommia ulmoides treatment group increased by 539.02 mg HA / ccm (>271.20 + 92.27 + 34.12 = 397.59), indicating that OPG protein, IL-8 protein, and Eucommia ulmoides extract have a significant synergistic effect in increasing tissue mineral density.

[0120] (4) Compared with the blank group, the tissue mineral density of the low-dose OPG exosome treatment group, IL-8 exosome treatment group, and Eucommia ulmoides treatment group increased by 215.11, 69.06, and 7.63 mg HA / ccm respectively, while the low-dose OPG + IL-8 exosome + Eucommia ulmoides treatment group increased by 476.64 mg HA / ccm (>215.11 + 69.06 + 7.63 = 291.80), indicating that OPG protein, IL-8 protein, and Eucommia ulmoides extract have a significant synergistic effect in increasing tissue mineral density.

[0121] In summary, the composition of the present invention can significantly increase bone mineral density and tissue mineral density, that is, it can significantly improve bone strength, bone toughness, and bone recovery ability, and thus effectively treat osteoporosis. Moreover, OPG protein, IL-8 protein, and Eucommia ulmoides extract have a significant synergistic effect in the treatment of osteoporosis.

[0122] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A composition for treating osteoporosis, characterized in that, Containing mesenchymal stem cell exosomes and Eucommia ulmoides extract in a dosage ratio of 14-16 mg: 8-12 mL; the mesenchymal stem cell exosomes are mesenchymal stem cell exosomes capable of simultaneously expressing OPG protein and IL-8 protein; the amino acid sequence of the OPG protein is as shown in SEQ ID NO:1, and the amino acid sequence of the IL-8 protein is as shown in SEQ ID NO:2; each 1 mL of Eucommia ulmoides extract contains an extract equivalent to 0.8-1.2 g of Eucommia ulmoides.

2. The composition according to claim 1, wherein The mesenchymal stem cells are umbilical cord mesenchymal stem cells.

3. The composition according to claim 1, wherein The preparation method of the Eucommia ulmoides extract is: soak Eucommia ulmoides in water for 10-14 h, then heat and extract at 95-100 °C for 5.4-6.6 h, filter, and concentrate the filtrate.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of a drug for treating osteoporosis.

5. A drug for treating osteoporosis, characterized in that, Each 1000 mL of the drug contains the following components: 14-16 mg of mesenchymal stem cell exosomes, 8-12 mL of Eucommia ulmoides extract, 25-35 g of a detergent, 20-30 g of a humectant, and the balance is water; The mesenchymal stem cell exosomes are mesenchymal stem cell exosomes capable of simultaneously expressing OPG protein and IL-8 protein; each 1 mL of Eucommia ulmoides extract contains an extract equivalent to 0.8-1.2 g of Eucommia ulmoides.

6. The drug according to claim 5, wherein Each 1000 mL of the drug contains the following components: 14-16 mg of mesenchymal stem cell exosomes, 8-12 mL of Eucommia ulmoides extract, 25-35 g of a detergent, 8-10 g of a preservative, 20-30 g of a humectant, and the balance is water.

7. The drug according to claim 5, characterized in that, Each 1000 mL of the drug contains the following components: 14-16 mg of mesenchymal stem cell exosomes, 8-12 mL of Eucommia ulmoides extract, 25-35 g of a detergent, 18-22 g of a suspending agent, 20-30 g of a humectant, and the balance is water.

8. The drug according to claim 5, wherein Each 1000 mL of the drug contains the following components: 14-16 mg of mesenchymal stem cell exosomes, 8-12 mL of Eucommia ulmoides extract, 25-35 g of a detergent, 20-30 g of a humectant, 6-8 g of a cooling flavor agent, and the balance is water.

9. The drug according to claim 5, characterized in that, The detergent is sodium chloride.

10. The drug according to claim 5, characterized in that, The humectant is glycerol.

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