Peptide YIYT with osteoporosis relieving and antioxidant activity as well as preparation method and application of peptide YIYT

Abstract

The invention discloses a peptide YIYT with osteoporosis relieving and antioxidant activity as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The amino acid sequence of the lactoferrin hydrophobic peptide YIYT is tyrosine-isoleucine-tyrosine-threonine (Tyr-Ile-Tyr-Thr, YIYT), and the lactoferrin hydrophobic peptide YIYT has a structural formula shown in the description. Experiments prove that YIYT can significantly promote proliferation, differentiation and mineralization of osteoblasts, accelerate the cell cycle process, inhibit osteoclast activity, reduce bone resorption level and recover bone metabolic balance. In an animal model, the YIYT can effectively improve the bone density of ovariectomized mice and improve a bone-related microstructure, and has double effects of promoting bone formation and inhibiting bone resorption. The lactoferrin hydrophobic peptide YIYT provided by the invention is safe in source and clear in action mechanism, and provides a new candidate substance and an application direction for developing a novel anti-osteoporosis medicine.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a peptide YIYT with osteoporosis-relieving and antioxidant activities, its preparation method, and its applications. Background Technology

[0002] Osteoporosis is a metabolic bone disease characterized by decreased bone mass and deterioration of bone microstructure. Its core pathological mechanism is an imbalance between bone formation and bone resorption. Currently used anti-osteoporosis drugs have significant limitations: anti-resorption drugs such as bisphosphonates can only inhibit bone loss and cannot promote new bone formation; long-term use may also cause adverse reactions such as atypical femoral fractures and osteonecrosis of the jaw. While synthetic drugs such as teriparatide have some therapeutic effect on vertebral fractures, they carry a black box warning of increased risk of osteosarcoma. Therefore, developing safe and effective novel drugs that can simultaneously regulate the balance between bone formation and bone resorption has significant clinical need and social value.

[0003] Lactoferrin is an 80kDa iron-binding glycoprotein found in mammalian milk, and its enzymatic hydrolysates have significant osteogenic potential. Existing studies have found that crude lactoferrin hydrolysates can promote osteoblast proliferation and increase trabecular bone volume in ovariectomized rats through ERK phosphorylation. However, most related studies treat lactoferrin peptides as a single entity and have not focused on the key peptides within lactoferrin peptides that are effective in treating osteoporosis. Summary of the Invention

[0004] [Technical Issues] The purpose of this invention is to provide an application of lactoferrin hydrophobic peptide YIYT in the preparation of anti-osteoporosis drugs. This peptide has clear osteogenic activity and bone resorption inhibition effect, is safe in origin, has a clear mechanism of action, and can overcome the limitations of existing anti-osteoporosis drugs.

[0005] [Technical Solution] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a novel bioactive peptide YIYT with a unique sequence. In vitro and in vivo experiments have verified that it can effectively alleviate osteoporosis, thereby exerting an osteogenic effect. Simultaneously, this peptide exhibits strong free radical scavenging and reducing power, effectively reducing oxidative stress damage. These two synergistic activities provide a superior solution for the prevention and relief of osteoporosis and its complications.

[0006] A lactoferrin hydrophobic peptide, YIYT, has the amino acid sequence tyrosine-isoleine-tyrosine-threonine (Tyr-Ile-Tyr-Thr, YIYT). This peptide is derived from the enzymatic hydrolysis product of lactoferrin and was obtained through simulated gastrointestinal digestion (pepsin-trypsin hydrolysis, pH 2.5-7.0) and C18 chromatography separation. It belongs to the active peptide segment of the lactoferrin hydrophobic component (LF-HD).

[0007] This invention provides the application of the lactoferrin hydrophobic peptide YIYT in the preparation of anti-osteoporosis drugs, and its core mechanism of action is as follows: 1. Promotes osteoblast function: Significantly enhances the proliferation activity of osteoblasts (MC3T3-E1 Subclone 14), accelerates the cell cycle transition from G0 / G1 phase to S phase and G2 / M phase; upregulates the mRNA and protein expression of osteogenic-related markers (ALP, BMP-2, RUNX-2, COL-I, OCN, OPN, BSP, SP7), enhances osteoblast differentiation and mineralization capacity, and increases the number and area of ​​mineralized nodules.

[0008] 2. Activate key signaling pathways: Synergistically activate the Wnt / β-catenin and BMP / Smad signaling pathways, upregulate the expression of core regulatory molecules such as β-catenin and Smad1 / 5 / 8, initiate osteogenic processes and promote bone matrix synthesis and maturation.

[0009] 3. Inhibition of bone resorption: Reduces serum RANKL levels in ovariectomized mice, increases OPG levels, and downregulates the RANKL / OPG ratio; reduces the number of TRAP-positive osteoclasts, inhibits osteoclast activity, and alleviates bone resorption damage.

[0010] 4. Improve bone tissue morphology: Significantly increases bone mineral density (BMD) in osteoporosis model animals, increases bone volume fraction (Bv / Tv), number of trabeculae (Tb.N) and trabecular thickness (Tb.Th), reduces trabecular spacing (Tb.Sp), and restores the integrity of bone tissue microstructure.

[0011] In one embodiment, the anti-osteoporosis drug of the present invention uses YIYT as the active ingredient and can be prepared into various dosage forms according to clinical needs, including but not limited to tablets, capsules, granules, oral liquids, injections, gels, etc.

[0012] In one embodiment, the drug may further include a pharmaceutically acceptable carrier, diluent, or excipient, such as starch, lactose, mannitol, polyethylene glycol, sodium carboxymethyl cellulose, physiological saline, glucose solution, etc.

[0013] In one embodiment, the carrier comprises microcapsules, microspheres, nanoparticles, and / or liposomes.

[0014] In one embodiment, the drug is administered via one or more of the following methods: oral administration, intraperitoneal injection, subcutaneous injection, or intramuscular injection.

[0015] In one embodiment, the effective concentration of the lactoferrin hydrophobic peptide YIYT is: 10 μg / mL and 50 μg / mL in in vitro cell experiments, and 5 mg / kg and 20 mg / kg (administered according to body weight) in in vivo animal experiments. The concentration can be adjusted appropriately according to the target population, administration method and severity of the disease.

[0016] This invention also provides the application of lactoferrin hydrophobic peptide YIYT in the preparation of health products that help improve bone density.

[0017] This invention also provides the application of lactoferrin hydrophobic peptide YIYT in the preparation of products that contribute to antioxidant activity.

[0018] In one embodiment, the product includes daily chemical products or health products.

[0019] In one embodiment, the daily chemical products include skin care products or cosmetics.

[0020] Beneficial effects: This invention provides a lactoferrin hydrophobic peptide, YIYT, possessing osteogenic and antioxidant activities, and verifies that peptide YIYT can be used as a drug for osteoporosis. This invention has the following advantages: (1) Clear and efficient activity: For the first time, it was confirmed that the osteogenic activity of lactoferrin hydrophobic peptide YIYT is significantly better than that of hydrophilic peptide. It exerts its anti-osteoporosis effect through the dual mechanism of "promoting bone formation and inhibiting bone resorption". This has been verified at both the cellular and animal levels. The bone density improvement effect is close to or better than that of the positive control drug.

[0021] (2) Clear mechanism of action: It is clear that YIYT regulates bone metabolism by synergistically activating the Wnt / β-catenin and BMP / Smad signaling pathways. The target is clear, which provides a solid theoretical basis for clinical application.

[0022] (3) High safety: YIYT is derived from the enzymatic hydrolysis product of natural lactoferrin and is prepared by simulating the digestive process of the human gastrointestinal tract. It has good biocompatibility and no obvious toxic side effects, thus solving the safety hazards of existing synthetic drugs.

[0023] (4) Broad application prospects: It can be prepared into a variety of dosage forms to meet the clinical needs of different populations, providing a new safe and effective strategy for the prevention and treatment of osteoporosis, and opening up a new path for the high-value utilization of lactoferrin. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of YIYT peptide promoting proliferation (AB) and cell cycle (C) provided by the present invention.

[0025] Figure 2 This is a schematic diagram of YIYT peptide promoting differentiation and mineralization provided by the present invention; AB: ALP staining schematic diagram (A) and ALP relative activity quantitative diagram (B); CD: Mineralized nodule area schematic diagram (C) and quantitative diagram (D).

[0026] Figure 3 This is a schematic diagram illustrating how the YIYT peptide, provided by this invention, promotes the expression of osteogenic-related genes.

[0027] Figure 4 This is a schematic diagram of how the YIYT peptide promotes the expression of key proteins, as provided by the present invention.

[0028] Figure 5 This is a schematic diagram of the hydroxyl radical scavenging ability of the YIYT peptide provided by the present invention.

[0029] Figure 6 The present invention provides YIYT peptide to regulate bone mineral density in ovariectomized mice; (A) representative images of bone trabecular structure analyzed by Micro-CT; (B) quantitative bone mineral density map.

[0030] Figure 7 This is a schematic diagram illustrating how the YIYT peptide provided by this invention improves the bone structure of ovariectomized mice.

[0031] Figure 8 The invention provides YIYT peptide to regulate the number of TRAP osteoclasts in ovariectomized mice; (A) HE staining; (B) TRAP staining; (C) Quantitative osteoclast graph.

[0032] Figure 9 The YIYT peptide provided by this invention is a serum biomarker related to bone formation. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0034] Example 1: Effects of YIYT on osteoblast proliferation, differentiation and mineralization YIYT peptide was artificially synthesized, and its effects on osteoblast proliferation, differentiation, and mineralization were investigated.

[0035] Cell culture: MC3T3-E1 Subclone 14 osteoblasts were seeded in α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin and cultured at 37°C in a 5% CO2 incubator.

[0036] Proliferation assay: Cells were seeded at 5 × 10³ cells / well in 96-well plates using α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. After 24 h, YIYT solution at 10 concentrations (1–300 μg / mL) was added to each well. PBS was added to the control group. Cell viability was assessed using the CCK-8 assay after 24 h and 48 h of further culture. Results are as follows: Figure 1 AB studies showed that after treatment with 50 μg / mLYIYT for 48 h, the cell survival rate was 108%, which was higher than the 100% of the control group.

[0037] Cell cycle analysis: Cell cycle distribution was detected by flow cytometry. Cells were seeded in α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, following the above method. After 24 h, YIYT at final concentrations of 10 μg / mL and 50 μg / mL was added, and the cells were cultured for another 48 h. Changes in the cell cycle were then detected. The results showed that the proportion of cells in G0 / G1 phase decreased significantly, while the proportion of cells in S phase + G2 / M phase increased from 37.8% to 46.9%. Figure 1 C), indicating that YIYT can accelerate the cell cycle process.

[0038] Differentiation experiment: Cells were cultured in α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at a concentration of 5 × 10⁶ cells / year. 4 Cells / well were seeded into 24-well plates. After 24 hours, the culture medium was discarded. The experimental group was treated with osteogenic induction medium containing YIYT (α-MEM + 10% FBS + 50 μg / mL ascorbic acid + 10 mM β-glycerophosphate sodium + 100 nM dexamethasone), while the control group was treated with osteogenic induction medium. The osteogenic induction medium containing YIYT was changed every 48 hours (the control group was treated with osteogenic induction medium). After 14 days of culture, ALP activity was observed by BCIP / NBT staining and quantitatively detected using pNPP substrate. The results showed that ( Figure 2 (AB), the relative activity of ALP in the 50 μg / mLYIYT treatment group (2.4) was about 1.2 times higher than that in the control group (2.0), and this was dose-dependent.

[0039] Mineralization experiment: Osteogenic induction culture was performed according to the differentiation experiment method described above. After 21 days, mineralized nodules were observed by staining with 2% Alizarin Red S and then quantitatively detected after destaining with 10% dodecylpyridine chloride. The results showed that the number and area of ​​mineralized nodules in the YIYT treatment group increased significantly by 1.3 times (10 μg / mL) and 1.7 times (50 μg / mL), respectively, with the 50 μg / mL dose group showing the most significant mineralization effect. Figure 2 CD).

[0040] Gene expression analysis: Cells were seeded according to the method in the proliferation experiment. After 24 h of cell culture, YIYT at final concentrations of 10 μg / mL and 50 μg / mL were added, and the cells were cultured for another 24 h. Cells were collected after the culture was completed. Total RNA was extracted from MC3T3-E1 subclone 14 using the TRIzol-chloroform method. Cells were lysed in RNA extraction solution (1 mL per 1 mL culture dish), incubated at room temperature for 5 min, mixed with 100 μL of chloroform, and then centrifuged (12,000 × g, 15 min, 4 °C). The aqueous phase was precipitated with 500 μL of isopropanol (4 °C, 10 min), centrifuged (12,000 × g, 10 min), washed with 75% ethanol, and then resuspended in RNA-free water. cDNA was synthesized using the FastKing RT kit (Tiangen #KR118). qPCR was performed using SYBR Green master mix (Delta #FP205) on a qTOWER 2.2 system (Antroic Jena) under the following conditions: initial denaturation at 95°C for 5 minutes, followed by 40 cycles of denaturation at 95°C for 5 seconds and annealing at 60°C for 30 seconds. The qPCR was then performed using 2... The ΔΔCt method was used to quantify osteogenic markers (ALP, BMP-2, RUNX-2), with β-actin as a reference. qRT-PCR was used to detect the expression of osteogenic-related genes, and the results are as follows: Figure 3 As shown in AF, YIYT can significantly upregulate the mRNA expression levels of RUNX-2, BMP-2, ALP, SP7, COL-I, and BSP, with expression levels increasing by 1.5, 3.5, 30, 2, 1.5, and 1.8 times or more, respectively.

[0041] Example 2: The regulatory effect of YIYT on osteogenic signaling pathways Protein extraction: MC3T3-E1 cells were seeded according to the proliferation experiment method in Example 1. After culturing for 24 h, YIYT was added at final concentrations of 0 μg / mL, 10 μg / mL, and 50 μg / mL, respectively. Cells were then collected after another 24 h of culturing. Total protein was extracted using RIPA buffer (containing protease inhibitors), and protein concentration was determined by the BCA method.

[0042] Western Blot assay: Protein samples were separated by 10% SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking with 5% BSA, primary antibodies such as β-catenin, Smad1 / 5 / 8, BMP-2, RUNX-2, SP7, and OPN were added and incubated overnight at 4°C. Secondary antibodies labeled with HRP were incubated at room temperature for 2 hours. ECL reagent was used for color development, and ImageJ was used for quantitative analysis.

[0043] Results: After YIYT treatment, the expression of β-catenin, a core molecule in the Wnt / β-catenin pathway, was significantly upregulated by approximately 1.5-fold; the expression of key components of the BMP / Smad pathway, BMP-2 and Smad1 / 5 / 8, increased by approximately 2-fold and 1.5-fold, respectively. Simultaneously, the expression of downstream osteogenic transcription factors RUNX-2 and SP7, and the bone matrix protein OPN, also significantly increased by 1.5, 1.5, and 1.2-fold, respectively, confirming that YIYT can synergistically activate two key osteogenic signaling pathways (such as...). Figure 4 ).

[0044] Example 3: Detection of the hydroxyl radical scavenging ability of YIYT This embodiment employs a hydroxyl radical scavenging assay to further evaluate the in vitro antioxidant activity of the YIYT peptide described in this invention. Hydroxyl radicals are among the most toxic reactive oxygen species in biological systems, and this experiment can effectively reflect the protective potential of the peptide under oxidative stress close to physiological conditions.

[0045] Methods: The hydroxyl radical scavenging capacity was determined using the Fenton reaction system. YIYT peptide samples were prepared into a series of solutions with gradient concentrations (0.01, 0.25, 0.5, 1, 2, 5 mg / mL). Appropriate concentrations of FeSO4, salicylic acid-ethanol solution, and H2O2 were added to the reaction system to initiate the reaction. After incubation at 37℃ for 60 min, the mixture was centrifuged, and the absorbance was measured at 534 nm. The reaction system without the sample served as a control, and the solution containing only diluent / buffer and no sample / standard served as a blank. The hydroxyl radical scavenging rate of YIYT at each concentration was calculated as D% = (Aassay - Acontrol) / (Ablank - Acontrol) * 100%.

[0046] Results: The scavenging effect of YIYT peptide on hydroxyl radicals showed a significant concentration-dependent effect, such as... Figure 5 As shown, when the concentration was increased to 1 mg / mL and 2 mg / mL, the scavenging rates reached 5.9% and 5.5%, respectively, which clearly indicates that YIYT can effectively scavenge hydroxyl radicals.

[0047] Example 4: Therapeutic effect of YIYT in an animal model of osteoporosis Animal model establishment: Eight-week-old female C57BL / 6 mice (16-18g) were randomly divided into 5 groups (n=12): sham operation group (Sham), ovariectomy group (OVX), ovariectomy + estrogen group (OVX+E2, positive control, β-estradiol 40μg / kg, administered once weekly), ovariectomy + low-dose YIYT group (OVX+5, 5 mg / kg), and ovariectomy + high-dose YIYT group (OVX+20, 20 mg / kg). Except for the sham operation group, all other groups underwent bilateral ovariectomy. After one week of acclimatization feeding postoperatively, medication was started. The YIYT group received daily gavage for 12 consecutive weeks.

[0048] Bone mineral density and bone morphology analysis: After the experiment, mice were euthanized, and the left femur was harvested for analysis using Micro-CT (Skyscan1272) with parameters set to 50 kV voltage, 800 μA current, and a 0.5 mm aluminum filter. Results showed that bone mineral density was significantly decreased in the OVX group, significantly increased by approximately 60% in the YIYT treatment group, and the high-dose group approached the level of the sham-operated group. Figure 6 As shown; compared with the OVX group, YIYT significantly increased bone volume fraction (Bv / Tv) by approximately 20%, trabecular bone number (Tb.N) by approximately 1.5 times, and bone thickness (Tb.Th) by 0.1 mm, while reducing trabecular bone spacing (Tb.Sp) by approximately 0.2 mm, thus improving bone microstructure. Figure 7 ).

[0049] Histological analysis: Mouse femurs were fixed in 10% formalin, decalcified with 10% EDTA, embedded in paraffin, and sectioned (5 μm). H&E staining was used to observe bone morphology, and TRAP staining was used to detect osteoclast activity. Results showed that the OVX group had sparse and disordered bone trabeculae, and a significantly increased number of TRAP-positive osteoclasts; the YIYT treatment group had dense and orderly bone trabeculae, and a significantly reduced number of TRAP-positive cells (approximately 2-fold or more), with the high-dose group showing better results. Figure 8 ).

[0050] Serum biochemical assays: Blood was collected from euthanized mice, and serum was separated by centrifugation. Bone metabolism markers were detected using an ELISA kit. Results showed that YIYT treatment reduced serum RANKL levels from 40 pg / mL in the model group to approximately 26 pg / mL or below, OPG levels increased from 0.2 ng / mL in the model group to approximately 0.6 ng / mL, and the RANKL / OPG ratio decreased from 1.5 in the model group to approximately 0.4. Simultaneously, it increased the levels of osteogenic markers such as ALP and OCN, adjusted the Ca / Pi ratio to the normal range, and restored bone metabolism balance. Figure 9 ).

[0051] The above examples demonstrate that lactoferrin hydrophobic peptide YIYT has significant anti-osteoporosis activity and can improve osteoporosis symptoms through a dual mechanism of promoting osteoogenesis and inhibiting bone resorption, making it a highly promising candidate for anti-osteoporosis drugs.

[0052] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The application of lactoferrin hydrophobic peptide YIYT in the preparation of drugs for treating osteoporosis, characterized in that, The amino acid sequence of the lactoferrin hydrophobic peptide YIYT is Tyr-Ile-Tyr-Thr.

2. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers, diluents, or excipients.

3. The application according to claim 1, characterized in that, The carrier comprises microcapsules, microspheres, nanoparticles, and / or liposomes.

4. The application according to claim 1, characterized in that, The drug can be administered orally, via intraperitoneal injection, subcutaneously, or via intramuscular injection, or by one or more of these methods.

5. The application according to claim 1, characterized in that, The dosage form of the drug is one of tablets, capsules, granules, oral liquids, injections, or gels.

6. The application according to claim 1, characterized in that, The dosage of the drug is 5-20 mg / kg.

7. Application of lactoferrin hydrophobic peptide YIYT in the preparation of health products that help improve bone density.

8. Application of lactoferrin hydrophobic peptide YIYT in the preparation of products that help with antioxidation.

9. The application according to claim 8, characterized in that, The products include daily chemical products or health supplements.

10. The application according to claim 9, characterized in that, The daily chemical products mentioned include skin care products or cosmetics.

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