Curcumin-antibacterial peptide-tetrahedral framework nucleic acid compound as well as preparation method and application thereof

Abstract

The invention provides a curcumin-antibacterial peptide-tetrahedral framework nucleic acid compound as well as a preparation method and application thereof, and belongs to the field of medicines. The curcumin-antibacterial peptide-tetrahedral framework nucleic acid compound is a compound prepared by taking curcumin, antibacterial peptide and tetrahedral framework nucleic acid as raw materials. Cell experiments show that the curcumin-antibacterial peptide-tetrahedral framework nucleic acid compound can significantly improve the osteogenesis activity of periodontal ligament stem cells, recover the mineralization activity, reduce the oxidative stress level and inhibit the occurrence and development of inflammatory reactions. Animal experiments show that the curcumin-antibacterial peptide-tetrahedral framework nucleic acid compound can significantly reduce loss of alveolar bones of periodontitis rats, inhibit the inflammatory state, promote osteogenic activity, recover the structure and quality of the alveolar bones and effectively treat periodontitis. The curcumin-antibacterial peptide-tetrahedral framework nucleic acid compound provided by the invention has a wide application prospect in preparation of drugs for preventing and / or treating periodontitis.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex and a preparation method and application thereof. Background Art

[0002] Periodontitis is a common oral disease that mainly attacks periodontal supporting tissues such as the gums, periodontal ligament, alveolar bone and cementum, leading to inflammatory destruction of these tissues. The disease is mainly caused by bacterial infection in the mouth, especially the formation of dental plaque and tartar. When poor oral hygiene habits cause bacteria to form dental plaque on the surface of the teeth, the bacteria will erode the gingival tissue around the teeth and gradually cause periodontitis.

[0003] Healthy periodontal tissues are the foundation for stable teeth. Treating periodontitis helps maintain tooth function, improve patients' chewing ability and quality of life. Treatment of periodontitis is the key to keeping the mouth clean, helping to reduce the formation of plaque and tartar, thereby preventing further development of oral diseases. Periodontitis is often accompanied by symptoms such as swollen gums, bleeding, and loose teeth. Timely treatment can effectively control these symptoms and relieve patients' pain. If not treated in time, periodontitis may lead to periodontal pocket formation, alveolar bone resorption, and even tooth loss. Therefore, treatment is essential to prevent the condition from worsening.

[0004] At present, the treatment of periodontitis mainly includes basic treatment methods such as supragingival scaling, subgingival scaling, root planing, and auxiliary treatment measures such as local and systemic medication. These methods can effectively eliminate inflammation and control the disease. With the development of medical science and technology, some new treatment technologies such as stem cell therapy and gene therapy are under research, which are expected to provide new ideas and methods for the treatment of periodontitis. For example, stem cell therapy has brought new hope to patients with periodontitis by promoting the regeneration and repair of periodontal tissues. Modern medicine pays more and more attention to personalized treatment. According to the specific situation of the patient, a personalized treatment plan is formulated, including the comprehensive use of basic treatment, drug treatment, surgical treatment and other means to achieve the best treatment effect.

[0005] The pathogenic basis of periodontitis In terms of basic research, microbial infection in the subgingival dental biofilm by periodontal pathogens, especially a specific group of gram-negative anaerobic bacteria, called the red complex, leads to chronic inflammation in susceptible hosts. These red complex bacteria include Prevotella, Forsythiae, and Treponema denticola, which are mainly found in the deep periodontal pockets of patients with periodontitis. Lipopolysaccharide as well as other virulence factors from these periodontal pathogens stimulate host macrophages and other inflammatory and component cells, leading to the production of a series of proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and prostaglandin E2 (PGE2). The presence of these proinflammatory cytokines and virulence factors stimulates macrophages, fibroblasts, junctional epithelial cells, and neutrophils to produce matrix metalloproteinases (mMPs). The resulting matrix metalloproteinases mediate the destruction of collagen fibers in the periodontium, especially the periodontal ligament. In addition, proinflammatory cytokines induce the expression of receptor activator of nuclear factor κB ligand (RANK-L) on osteoblasts and T helper cells. The resulting RANK-L on osteoblasts and helper T cells interacts with the receptor activator of nuclear factor κB (rANK) on osteoclast precursors, leading to the occurrence and maturation of osteoclasts, and mature osteoclasts mediate the destruction of alveolar bone.

[0006] Curcumin is a plant polyphenol extracted from the rhizome of turmeric. Curcumin can reduce the inflammatory response of periodontal tissues by inhibiting inflammatory mediators such as NF-κB and IL-6, thereby alleviating tissue destruction caused by periodontitis. In addition, curcumin has a broad-spectrum antibacterial activity and can inhibit the growth of periodontal pathogens such as Porphyromonas gingivalis, helping to control infection. Curcumin also has the activity of promoting fibroblasts and osteoblasts, promoting the regeneration and repair of periodontal tissues. Due to its good safety and natural source, curcumin has been studied as an adjuvant treatment for periodontitis, usually through topical application such as curcumin-containing mouthwash or gel. However, due to its low stability and bioavailability in the oral cavity, the clinical application effect of curcumin still needs further optimization and research. Summary of the invention

[0007] The purpose of the present invention is to provide a curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex and a preparation method and use thereof.

[0008] The invention provides a curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex, which is a complex prepared by taking curcumin, antimicrobial peptide and tetrahedral framework nucleic acid as raw materials.

[0009] Furthermore, the antimicrobial peptide is a defensin.

[0010] Furthermore, the curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex is a complex prepared from tetrahedral framework nucleic acid modified with antimicrobial peptide and curcumin as raw materials, the tetrahedral framework nucleic acid modified with antimicrobial peptide is formed by four DNA single strands through base complementary pairing, and the sequences of the four DNA single strands are shown in SEQ ID NO.1~4.

[0011] Furthermore, the molar ratio of the four DNA single strands is 1:1:1:1.

[0012] Furthermore, the method for preparing the tetrahedral framework nucleic acid modified with the antimicrobial peptide comprises the following steps: maintaining the four DNA single strands at 50-90° C. for 5-15 min, and then maintaining them at 2-8° C. for 10-30 min.

[0013] Furthermore, the method for preparing the antimicrobial peptide-modified tetrahedral framework nucleic acid comprises the following steps: maintaining four DNA single strands at 70° C. for 10 minutes, and then maintaining them at 4° C. for 20 minutes.

[0014] Furthermore, the mass ratio of the antimicrobial peptide-modified tetrahedral framework nucleic acid to curcumin is 1:(30-50).

[0015] Furthermore, the mass ratio of the antimicrobial peptide-modified tetrahedral framework nucleic acid to curcumin is 1:40.

[0016] The present invention also provides a method for preparing the above-mentioned curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex, which comprises the following steps: reacting the tetrahedral framework nucleic acid modified with the antimicrobial peptide with curcumin to obtain the curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex.

[0017] Furthermore, the reaction is carried out under shaking at room temperature for 4-6 hours.

[0018] The present invention also provides use of the curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex in preparing a drug for preventing and / or treating periodontitis.

[0019] Furthermore, the medicine is an oral preparation or an injectable preparation.

[0020] The present invention has achieved the following beneficial effects: 1. The present invention prepares a curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex for the first time. Cell experiments show that the curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex can significantly increase the osteogenic activity of periodontal ligament stem cells, restore mineralization activity, reduce oxidative stress levels, and inhibit the occurrence and development of inflammatory reactions; animal experiments show that the curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex can significantly reduce alveolar bone loss in rats with periodontitis, inhibit inflammatory states, promote osteogenic activity, restore the structure and quality of alveolar bone, and effectively treat periodontitis.

[0021] 2. The curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex provided by the present invention solves the problems of low stability and low bioavailability of curcumin in the oral cavity; 3. The present invention solves the problem of poor stability and water solubility of defensins in the oral cavity; 4. The present invention gives full play to the drug-carrying function of the tetrahedral framework nucleic acid. Curcumin and the framework nucleic acid are combined through the groove, which can increase the local concentration of curcumin while protecting the stability of curcumin and improve the therapeutic effect.

[0022] 5. The present invention gives full play to the editability of tetrahedral framework nucleic acid by chemically connecting the top of the nucleic acid single strand; 6. The present invention gives full play to the ability of tetrahedral framework nucleic acid to enter cells and increases the permeability in oral tissues.

[0023] In summary, the curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex provided by the invention has broad application prospects in the preparation of drugs for preventing and / or treating periodontitis.

[0024] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0025] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 .Particle size and potential detection results of Cur-tAMP.

[0027] Figure 2 .AFM detection results of Cur-tAMP.

[0028] Figure 3 .TEM detection results of Cur-tAMP.

[0029] Figure 4 .ALP staining results of human periodontal ligament stem cells in each group.

[0030] Figure 5 .ARS staining results of human periodontal ligament stem cells.

[0031] Figure 6 .WB results of osteogenic-related proteins of human periodontal ligament stem cells.

[0032] Figure 7 .The results of immunofluorescence staining of osteogenic-related proteins in human periodontal ligament stem cells.

[0033] Figure 8 .Reactive oxygen staining results of human periodontal ligament stem cells.

[0034] Fig. 9 . Results of immunofluorescence staining of inflammation-related proteins in human periodontal ligament stem cells.

[0035] Fig.10 .WB results of inflammation-related proteins in human periodontal ligament stem cells.

[0036] Fig.11 .ELISA results of inflammation-related proteins in human periodontal ligament stem cells.

[0037] Fig.12 .Micro-CT scanning results of rat jaw.

[0038] Fig.13 . Statistical results of Micro-CT scanning of rat jaw.

[0039] Fig.14 .HE staining results of rat jaw sections.

[0040] Fig.15 .Masson staining results of rat jaw sections.

[0041] Fig.16 .TRAP staining results of osteoclasts in rat jaw sections.

[0042] Fig.17 .IL-6 staining results of rat jaw bone sections.

[0043] Fig.18 .Runx2 staining results of rat jaw sections. DETAILED DESCRIPTION

[0044] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0045] Defensin, HNP2, an antimicrobial peptide (AMP), has an amino acid sequence of CYCRIPACIAGERRYGTCIYQGRLWAFCC (SEQ ID NO.5).

[0046] In the present invention, room temperature refers to 25±5°C.

[0047] Example 1: Synthesis of curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex In the amino acid sequence of HNP2, amino acid side chains with primary amine groups such as lysine (Lys, K) and arginine (Arg, R) can form stable covalent bonds with carboxyl groups on DNA through NHS-ester reactions. HNP2 was connected to the S1 chain by the above-mentioned chemical connection method, and the single chain was dissolved in enzyme-free water. The single chains of S1-HNP2, S2, S3, and S4 (see table) were added to TM buffer (Tris-HCl: 10 mM, MgCl2·6H2O: 50 mM, pH: 8.0) in equimolar concentrations in sequence. After mixing evenly, the mixture was centrifuged instantly and placed in a thermal cycler. It was heated at 70°C for 10 min, and then rapidly cooled at 4°C for 20 min. Finally, 1 μM tFNA-HNP2 (also known as AMP-tFNA) was obtained.

[0048] Curcumin (Cur) was diluted to 40 μM and added to 1 μM tFNA-HNP2 at a volume ratio of 1:1 (40:1 in mass ratio). The mixture was shaken at room temperature for 4-6 hours to obtain Cur-tAMP (also known as Cur-AMP-tFNA), i.e., curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex.

[0049] Table 1. Sequences of the four DNA strands The beneficial effects of the present invention are demonstrated by experimental examples below.

[0050] Experimental Example 1: Structural Characterization of Curcumin-Antibacterial Peptide-Tetrahedral Framework Nucleic Acid Complex 1. Experimental Methods PAGE Gel Electrophoresis: Polyacrylamide gel electrophoresis (PAGE) is used to separate proteins or nucleic acid fragments. First, a polyacrylamide gel of appropriate concentration is prepared. The sample is mixed with a loading buffer and added to the wells of the gel. An electric field is applied using an electrophoresis apparatus, and the sample migrates through the gel according to its molecular weight. Proteins are often treated with SDS to denature and linearize them. After the run is complete, stain to show the bands and compare to molecular weight standards to confirm the separation.

[0051] AFM: Atomic force microscopy (AFM) is used for high-resolution imaging of surface topography. First, fix the sample on the sample stage to keep the sample surface clean and flat. The AFM probe is close to the sample surface, and the surface topography image is generated through the interaction force between the probe and the sample. The scanning mode can be selected in contact mode or non-contact mode. After the data is collected, a three-dimensional image is generated through software processing to characterize the nanoscale structure and mechanical properties of the sample.

[0052] TEM: Take a clean sealing film of about 3 cm×3 cm and place it on a flat ice box for precooling for 5 minutes. 3) Place the copper mesh on the precooled sealing film. Drop 10μL of sample and PTA stain solution on the precooled sealing film respectively. Leave for 1-2 minutes and cool the above copper mesh, sample and PTA. Put the carbon side of the copper mesh on the sample liquid to adsorb the sample for 1-3 minutes. After adsorption, put the copper mesh in vertical contact with the filter paper and continue to absorb water for 10 seconds until the excess residual liquid is completely absorbed. Put the carbon side of the copper mesh on the PTA droplet to stain for 0.5-1 min (not more than 1 min). After staining, put the copper mesh in vertical contact with the filter paper and continue to absorb water for 10 seconds until the excess residual liquid is completely absorbed. Then place it on the filter paper and dry it naturally overnight in a cool place. Store in the copper mesh storage box and keep it cool and dry. Observe using a projection electron microscope.

[0053] 2. Experimental results (1) Particle size and potential test results: Figure 1 The results showed that the particle size and potential results indicated that the Cur-tAMP particle size was about 47.5nm and was negatively charged.

[0054] (2) AFM experimental results: AFM showed that Cur-tAMP was evenly distributed ( Figure 2 ).

[0055] (3) TEM experimental results: TEM showed that Cur-tAMP was evenly distributed and the particle size was about 40 nm ( Figure 3 ).

[0056] Experimental Example 2: Cell Experiment 1. Experimental Methods Extraction, separation and culture of human periodontal ligament stem cells: α-MEM with 10% serum was used at 37°C and 5% CO 2The cells were cultured in an incubator. For the primary culture of h-PDLSCs, premolars and impacted third molars due to orthodontic treatment were collected (n = 6); for the primary culture of i-PDLSCs, teeth diagnosed with severe chronic periodontitis and requiring extraction were collected (n = 6). The donors were aged between 20 and 40 years old and had no systemic diseases, history of infection, pulpitis, apical periodontitis, or root resorption. There was no significant difference between the healthy donor group (26.50 ± 4.37) years old and the periodontitis group (28.33 ± 5.89) years old. The diagnostic criteria for periodontitis were: X-ray showed alveolar bone resorption to the apex 1 / 3 and periodontal pocket depth ≥ 5 mm. The periodontal ligament tissue was scraped and washed with PBS and collected by centrifuge. The remaining sediment was digested with 3g / L collagenase type I and 4g / L neutral protease for 1 hour. The digestion process was terminated with the same volume of culture medium. Tissue fragments were separated by centrifugation and transplanted into culture plates containing DMEM containing 20% ​​FBS, penicillin (100 units / ml) and streptomycin (100μg / ml). After 3-8 days of culture, cells were observed around the tissue blocks. When the cells grew to 80% something 90%, they were digested with 0.25% trypsin and transplanted into new culture dishes for further expansion. The passaged cells were cultured in DMEM containing 10% FBS, penicillin (100 units / ml) and streptomycin (100μg / ml). The culture medium was changed every 3 days. The third generation of human periodontal ligament stem cells (PDLSC) were used for cell experiments.

[0057] In this experiment, five experimental groups were set up, namely: Group 1 (blank control group, i.e. blank culture medium treatment group), Group 2 (LIP group, i.e. LPS treatment group), Group 3 (LIP+AMP group, i.e. AMP-tFNA treatment group), Group 4 (LIP+Cur group, i.e. Cur treatment group), and Group 5 (LIP+Cur+tAMP group, i.e. Cur-AMP-tFNA treatment group).

[0058] Cell activity detection: observe cell status, digest cells, and count cells as in 2.3.1(3). After UV disinfection in a biosafety cabinet, add the corresponding cell culture medium and dilute the PDLSC cell concentration to 1×104 / mL. Add 100 μL of cells to each well of a 96-well plate (1×10 cells per well). 3 Place in an incubator (37°C, 5% CO 2) overnight and wait for the cells to adhere. After UV disinfection in a biosafety cabinet and in a sterile state, use the corresponding cell culture medium to add 250ul of blank culture medium, 250ul of 1mg / mL LPS, 250ul of 250nmol / L Cur, 250ul of 250nmol / L AMP-tFNA, and 250ul of 250nmol / L Cur-AMP-tFNA and culture for 12-24 hours. Aspirate the old culture medium, gently add 200μL PBS to each well to wash the cells, and then aspirate the PBS. Repeat the washing three times. Add 10μL of CCK-8 reagent to each well and incubate for 1.5 h. Use an enzyme reader to measure the OD value at 450nm.

[0059] Reactive oxygen detection: PDLSCs were seeded in 6-well plates with a cell volume of 5×105 / well and cultured overnight in an incubator (37°C, 5% CO2) until the cells adhered to the wall. In each well, 2 ml of blank culture medium, 2 ml of 1 mg / mL LPS, 2 ml of 250 nmol / L Cur, 2 ml of 250 nmol / L AMP-tFNA, and 2 ml of 250 nmol / L Cur-AMP-tFNA were added and cultured. After 24 hours, the old culture medium was discarded and 2 mL of PBS was gently added to each well to wash the cells. Repeat 3 times. The reactive oxygen probe was added according to the instructions of the Biyuntian Reactive Oxygen Detection Kit, and images were collected under a confocal laser scanning microscope using FITC (Ex / Em=493 / 528 nm) excitation light. The images were processed using Olympus software, and the fluorescence intensity was statistically analyzed using ImageJ.

[0060] ALP staining and Alizarin red staining: Using Saiye osteogenic induction solution, after 7 days of osteogenic induction of PDLSCs, the supernatant was discarded, 4% polyformaldehyde was fixed at room temperature for 15 min, BCIP / NBT staining working solution (NBT solution: BCIP solution: ALP color development buffer volume ratio of 2:1:300) was added, and staining was carried out at 37°C in the dark for 1 hour, and photos were taken under a microscope, and the ALP staining was observed by a scanner. After 14 days of osteogenic induction of PDLSCs, the supernatant was discarded, and Alizarin red was added to stain at room temperature for 5 min. After completion, it was washed three times with double distilled water, and photos were taken under a microscope. The alizarin red staining was observed by a scanner. After 7 and 14 days of osteogenic induction of PDLSCs, the osteogenic activity was detected using an ELISA instrument according to the instructions of the ALP activity detection kit and the Alizarin red detection kit.

[0061] ELISA assay: PDLSCs were seeded in 12-well plates at a cell volume of 2 × 105 / well and placed in an incubator (37°C, 5% CO 2) overnight until the cells adhere to the wall. In each well, add 250ul of blank culture medium, 250ul of 1mg / mL LPS, 250ul of 250nmol / L Cur, 250ul of 250nmol / L AMP-tFNA, and 250ul of 250nmol / L Cur-AMP-tFNA for culture. After 24 hours, collect the supernatant culture medium. Using ELISA kits for TNF-α, IL-6, and IL-18, gradient dilution of standard solutions to 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5pg / mL, 31.25 pg / mL, 15.6 pg / mL, and 0 pg / mL. Add standard solutions of different concentrations to the well plate, 100μL per well. Add supernatant samples of different groups of cells to the well plate and incubate at 37°C for 90 min. Prepare washing solution and biotinylated antibody working solution. After washing for 5 times, add 200 μL of biotinylated antibody working solution. Incubate at 37°C in the dark for 60 min. Aspirate the liquid in the wells, add 200 μL of washing solution to each well, and let it stand for 30 s. Repeat washing for 5 times, add 200 μL of enzyme conjugate working solution to each well, and incubate at 37°C in the dark for 30 min. After washing for 5 times, add 100 μL of TMB substrate solution to each well, incubate at 37°C for 30 min, and add 100 μL of stop solution to each well. Use a microplate reader to measure the OD value at 450 nm. Calculate the standard curve (best fit curve) of the standard product, and the R2 value should be greater than 0.99. Calculate the concentration of the target protein in the corresponding supernatant sample according to the standard curve.

[0062] qPCR detection: PDLSCs were seeded in 12-well plates at a cell volume of 2×10 5 / well, and place in an incubator (37°C, 5% CO 2) overnight until the cells adhere to the wall. In each well, add 250ul of blank culture medium, 250ul of 1mg / mL LPS, 250ul250nmol / L Cur, 250ul250nmol / L AMP-tFNA, and 250ul250nmol / L Cur-AMP-tFNA for culture. After 12-24 hours, discard the old culture medium and gently add 2 mL PBS to each well to wash the cells. Repeat 3 times. Collect cells on ice and extract RNA using Trizol method. Use PCR instrument to reverse transcribe into cDNA and save for later use. According to the proportion of each component of the reaction system, pre-mix 5μL DNA, 50 μL 2×SYBR qPCR MasterMix, 2μL upstream primer (10μM), 2μL downstream primer (2μM), and 41μL enzyme-free water. Add 20 μL of the reaction system to each well of the 96-well plate and place it in the Q6 instrument. After the reaction is completed, check the melting curve and the difference in Ct values ​​between the replicate wells, and then calculate the 2 ‐∆∆Ct value.

[0063] Cell immunofluorescence detection: PDLSCs were seeded in confocal microplates at a cell density of 2 × 105 cells / well and placed in an incubator (37°C, 5% CO 2 ) overnight and wait for the cells to adhere to the wall. In each dish, add 1ml of blank culture medium, 1ml of 1mg / mL LPS, 1ml of 250nmol / L Cur, 1ml of 250nmol / L AMP-tFNA, and 1ml of 250nmol / L Cur-AMP-tFNA for culture. After 24 hours, discard the supernatant, rinse with PBS three times, and fix with 4% paraformaldehyde. Permeabilize the membrane with 0.5% TritonX-100 permeabilization solution for 5 min, wash with PBS three times, 5min each time, block with 5% sheep serum, add primary antibody (primary antibody), incubate at 4℃ overnight, rewarm, wash with PBS three times, 5min each time. Add secondary antibody, incubate at 37℃ for 1h in the dark, and wash with PBS three times. Add 1ml FITC solution (7:1000) to each dish, incubate at 37℃ for 20min in the dark, and wash with PBS three times. The cell nuclei were counterstained with DAPI solution and incubated at 37°C for 10 min in the dark. Images were collected under a confocal laser scanning microscope using FITC (Ex / Em=493 / 528 nm), Rhode (Ex / Em=590 / 617 nm) and DAPI (Ex / Em=364 / 454 nm) excitation light, and processed using Olympus software.

[0064] 2. Experimental results (1) ALP staining results of human periodontal ligament stem cells (PDLSC): ALP staining results are of great significance in histological and cell biological research. It can evaluate the cell differentiation state and the degree of mineralization during bone formation by detecting the activity of alkaline phosphatase (ALP). Specifically, ALP plays a key role in the differentiation of osteoblasts and the mineralization of bone tissue. Therefore, a positive ALP staining result usually indicates that the cell or tissue has strong bone formation activity. In addition, in cartilage formation and bone repair research, ALP staining is also widely used to observe the maturity and differentiation of cells or tissues, thereby providing important information about the potential for tissue regeneration.

[0065] Figure 4 The results showed that the ALP staining results in the LIP group were the worst, indicating that the activity of alkaline phosphatase decreased significantly under the modeling conditions. In contrast, the staining results of the LIP+AMP group and the LIP+Cur group improved, but were still lower than the level of the blank control group. It is worth noting that the ALP staining results of the LIP+Cur-tAMP group were close to those of the blank control group, indicating that the intervention effect of this group was significant, effectively restoring the activity of alkaline phosphatase and significantly improving the bone formation activity.

[0066] (2) ARS staining results of human periodontal ligament stem cells (PDLSC): ARS (Alizarin Red S) staining results are of great significance in the study of bone tissue and mineralization. It reflects the degree of mineralization of cells or tissues, especially the calcium deposition, by binding to calcium ions. Positive results of Alizarin Red staining usually appear as red or orange-red mineralized nodules, indicating that there is a high calcium salt deposition in this area, suggesting a strong bone formation or mineralization activity.

[0067] In this experiment, five experimental groups were set up, namely: Group 1 (blank control group), Group 2 (LIP group), Group 3 (LIP+AMP group), Group 4 (LIP+Cur group), and Group 5 (LIP+Cur+tAMP group). ARS Alizarin Red staining results ( Figure 5 ) showed that the staining in group 2 was the worst, with fewer mineralized nodules, indicating that LPS treatment significantly reduced the mineralization capacity. In contrast, the staining results of group 3 (LIP+AMP group) and group 4 (LIP+Cur group) improved, and the number of mineralized nodules increased compared with group 2, but was still lower than the level of the blank control group. It is worth noting that the ARS staining results of group 5 (LIP+Cur+tAMP group) were close to those of the blank control group (group 1), and the number of mineralized nodules increased significantly, indicating that the intervention effect of this group was the best, and a higher mineralization capacity was successfully restored, which was close to that of the normal group.

[0068] (3) WB results of osteogenic related proteins of human periodontal ligament stem cells (PDLSC): WB (Western Blot) results are of great significance in molecular biology research. By detecting the expression level of specific proteins, it helps researchers analyze the effects of different conditions or treatments on protein synthesis, degradation or activation status. The depth and thickness of the bands in WB results usually reflect the relative content of the target protein. The darker or thicker the band, the higher the protein content. This method is widely used in the fields of cell signaling, disease mechanism research, etc., and provides important information for understanding cell function regulation and molecular mechanisms.

[0069] In this experiment, five experimental groups were set up, namely: Group 1 (blank control group), Group 2 (LIP group), Group 3 (LIP+AMP group), Group 4 (LIP+Cur group), and Group 5 (LIP+Cur+tAMP group). WB results showed ( Figure 6 ), the target protein expression in group 2 was the lowest, indicating that LPS treatment significantly inhibited protein expression. The protein expression in group 3 (LIP+AMP group) and group 4 (LIP+Cur group) increased compared with group 2, but was still lower than that in the blank control group (group 1). The protein expression in group 5 (LIP+Cur+tAMP group) was close to that in the blank control group (group 1), showing a significant recovery effect, indicating that the intervention effect of this combination was the best and contributed to the recovery of protein expression levels.

[0070] (4) Results of immunofluorescence staining of osteogenic-related proteins in human periodontal ligament stem cells (PDLSC): Immunofluorescence staining of OCN (Osteocalcin) protein is of great significance in the study of bone metabolism and osteogenesis. OCN is a marker protein of bone formation, and its expression level can directly reflect the activity and mineralization ability of osteoblasts. In immunofluorescence staining, the fluorescence intensity and distribution of OCN protein can quantitatively and positionally observe the expression level of OCN in cells or tissues. The higher the fluorescence intensity, the richer the OCN protein content and the stronger the osteogenic activity. This method is widely used in the study of osteogenesis mechanism, bone tissue repair, etc., to help evaluate the effects of different experimental conditions on bone formation.

[0071] In this experiment, five experimental groups were set up, namely: Group 1 (blank control group), Group 2 (LIP group), Group 3 (LIP+AMP group), Group 4 (LIP+Cur group), and Group 5 (LIP+Cur+tAMP group). The immunofluorescence staining results of OCN protein showed that ( Figure 7), the fluorescence intensity of OCN protein in group 2 was the weakest, indicating that LPS treatment significantly reduced the expression level of OCN protein. The expression of OCN protein in group 3 (LIP+AMP group) and group 4 (LIP+Cur group) increased compared with group 2, but was still lower than that in the blank control group (group 1). The expression of OCN protein in group 5 (LIP+Cur+tAMP group) was close to that in the blank control group (group 1), showing a significant recovery effect, indicating that the intervention in this group has a positive effect on improving OCN expression and helps to restore osteogenic activity.

[0072] (5) Results of reactive oxygen species staining of human periodontal ligament stem cells (PDLSC): ROS (reactive oxygen species) staining experiments are of great significance in cell biology and pathophysiology research. ROS is a class of molecules with high reactivity. Excessive ROS can cause oxidative stress and damage cell structure and function. The staining experiment can intuitively display the ROS level in cells or tissues. The higher the fluorescence intensity, the higher the content of reactive oxygen and the stronger the level of oxidative stress. ROS staining experiments are widely used in the study of inflammation, aging and various diseases to help evaluate the effects of different treatment conditions on the oxidative state of cells, thereby providing important data support for antioxidant drug screening and related mechanism research.

[0073] In this experiment, five experimental groups were set up, namely: Group 1 (blank control group), Group 2 (LIP group), Group 3 (LIP+AMP group), Group 4 (LIP+Cur group), and Group 5 (LIP+Cur+tAMP group). ROS reactive oxygen species staining results showed ( Figure 8 ), the ROS fluorescence intensity of group 2 was the highest, indicating that LPS treatment significantly increased the level of reactive oxygen species in cells, leading to a strong oxidative stress response. The ROS levels of group 3 (LIP+AMP group) and group 4 (LIP+Cur group) were lower than those of group 2, but still higher than those of the blank control group (group 1). The ROS level of group 5 (LIP+Cur+tAMP group) was close to that of the blank control group (group 1), and the fluorescence intensity was lower, indicating that the intervention effect of this group was better, which significantly inhibited the production of reactive oxygen species and helped to reduce the level of oxidative stress.

[0074] (6) Results of immunofluorescence staining of inflammation-related proteins in human periodontal ligament stem cells (PDLSC): IL-6 and IL-1β are typical inflammation-related proteins that usually play an important role in inflammatory reactions and immune responses. Immunofluorescence staining can visually display the expression levels of these inflammatory factors. The higher the expression levels of IL-6 and IL-1β, the stronger the inflammatory response and the higher the stress state of cells or tissues. The results of immunofluorescence staining are of great significance in the study of inflammatory mechanisms, disease models and anti-inflammatory drugs, and help evaluate the effects of different experimental treatments on inflammatory responses.

[0075] In this experiment, five experimental groups were set up, namely: Group 1 (blank control group), Group 2 (LIP group), Group 3 (LIP+AMP group), Group 4 (LIP+Cur group), and Group 5 (LIP+Cur+tAMP group). The results of immunofluorescence staining of IL-6 and IL-1β showed that ( Fig. 9 ), the IL-6 and IL-1β protein expression levels in group 2 were the highest, indicating that LPS treatment significantly triggered an inflammatory response. In contrast, the IL-6 and IL-1β expression levels in group 3 (LIP+AMP group) and group 4 (LIP+Cur group) decreased, but were still higher than those in the blank control group (group 1). The IL-6 and IL-1β expression in group 5 (LIP+Cur+tAMP group) was close to that in the blank control group (group 1), and the expression of inflammation-related proteins was low, indicating that the intervention effect of this group was better and helped to inhibit the occurrence and development of inflammatory responses.

[0076] (7) WB results of inflammation-related proteins in human periodontal ligament stem cells (PDLSC): WB (Western Blot) experiments are used to detect the expression level of specific proteins and are an important means of studying protein functions and biological processes. By analyzing the depth of the protein bands, the relative expression levels of the target protein in different treatment groups can be quantitatively compared. In inflammation-related studies, detecting the expression levels of inflammatory factors such as TNF-a, IL-6, and NLRP3 can help researchers understand the effects of different treatments on the inflammatory response. An increase in protein expression usually reflects an enhanced inflammatory response, while a decrease in expression may indicate the presence of an anti-inflammatory effect.

[0077] In this experiment, five experimental groups were set up, namely: Group 1 (blank control group), Group 2 (LIP group), Group 3 (LIP+AMP group), Group 4 (LIP+Cur group), and Group 5 (LIP+Cur+tAMP group). Western Blot results showed ( Fig.10), among the three inflammatory-related proteins TNF-a, IL-6 and NLRP3 detected, the protein expression level of group 2 was the highest, indicating that LPS treatment significantly induced inflammatory response. The expression levels of inflammatory proteins in group 3 (LIP+AMP group) and group 4 (LIP+Cur group) decreased, but were still higher than those in the blank control group (group 1). Group 5 (LIP+Cur+tAMP group) was close to the blank control group (group 1), and the expression level of inflammatory-related proteins was low, indicating that the intervention effect of this group was the best and the inflammatory response was significantly inhibited.

[0078] (8) Elisa results of inflammation-related proteins in human periodontal ligament stem cells (PDLSC): ELISA (enzyme-linked immunosorbent assay) is a sensitive detection method used to quantitatively analyze the expression level of specific proteins in a sample. Through ELISA experiments, researchers can detect changes in inflammatory-related proteins (such as TNF-a, IL-6, IL-1β) to evaluate the effects of different treatment conditions on the inflammatory response. At the same time, the levels of anti-inflammatory-related proteins (such as IL-4, IL-10) can also be detected to evaluate the anti-inflammatory effect. An increase in the level of inflammatory proteins usually indicates an enhanced inflammatory response, while an increase in anti-inflammatory proteins indicates an inhibitory effect on inflammation.

[0079] In this experiment, five experimental groups were set up, namely: Group 1 (blank control group), Group 2 (LIP group), Group 3 (LIP+AMP group), Group 4 (LIP+Cur group), and Group 5 (LIP+Cur+tAMP group). In the detection of inflammatory related proteins TNF-a, IL-6 and IL-1β ( Fig.11 ), the protein expression level in group 2 was the highest, indicating that LPS treatment significantly induced an inflammatory response. The levels of inflammatory proteins in group 3 (LIP+AMP group) and group 4 (LIP+Cur group) decreased, but were still higher than those in the blank control group (group 1). Group 5 (LIP+Cur+tAMP group) was close to the blank control group (group 1), and the expression level of inflammation-related proteins was low, showing a significant anti-inflammatory effect.

[0080] In the detection of anti-inflammatory related proteins IL-4 and IL-10 ( Fig.11 ), the protein expression level in group 2 was the lowest, indicating that LPS treatment significantly inhibited the production of anti-inflammatory proteins. The expression levels of anti-inflammatory proteins in group 3 (LIP+AMP group) and group 4 (LIP+Cur group) rebounded, but were still lower than those in the blank control group (group 1). Group 5 (LIP+Cur+tAMP group) was close to the blank control group (group 1), and the level of anti-inflammatory proteins was high, indicating that the intervention effect of this group was the best, which helped to enhance the anti-inflammatory response and inhibit the overall inflammatory response.

[0081] The above experimental results show that the curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex provided by the present invention can significantly improve the osteogenic activity of periodontal ligament stem cells, restore mineralization activity, reduce oxidative stress levels, and inhibit the occurrence and development of inflammatory reactions.

[0082] Experimental Example 3: Animal Experiment 1. Experimental Methods Experimental animal husbandry: Fifty 6-week-old SPF-grade male SD rats were purchased from Chengdu Dashuo Experimental Animal Technology Co., Ltd., with a body weight of 200-220 g, and were housed under standard laboratory conditions (relative humidity 55%, 12 b light / 12 h dark, room temperature 22°C) with ad libitum access to food and water.

[0083] Establishment of rat periodontitis model: The rat ligation-induced periodontitis (LIP) model was established by ligating the cervical part of the maxillary second molar of the rat gum. Rats were anesthetized with isoflurane breathing, and then replaced with nasal tube for continuous anesthesia after sedation. The rats were tied to a fixator. Sterile cotton balls were placed on the opening of the salivary glands to prevent saliva from affecting the operation. Sterile surgical sutures of appropriate length were taken and operated with micro forceps and micro needle holders. The sterile surgical sutures were fixed with surgical knots, and the excess sutures were cut off with ophthalmic scissors. The neck sutures were observed every other day. If they fell off, they were ligated in time. The body weight was measured every 2 days. The rats were observed for periodontal tissue symptoms after 3 weeks. If deep periodontal pockets, swollen gums, and bleeding on probing were found, the periodontitis model was successfully established.

[0084] In this experiment, five experimental groups were set up, namely: Group 1 (blank control group, i.e., normal rats were not treated), Group 2 (LIP group, i.e., modeling group), Group 3 (LIP+AMP group, i.e., AMP-tFNA treatment group), Group 4 (LIP+Cur group, i.e., Cur treatment group), and Group 5 (LIP+Cur+tAMP group, i.e., Cur-AMP-tFNA treatment group).

[0085] The model rats were randomly divided into modeling group, LIP+AMP group, LIP+Cur group, and LIP+Cur+tAMP group, with 10 rats in each group. Ten SD normal rats were taken as blank group without treatment. 50uL of 1μmol / L Cur, AMP-tFNA, and Cur-AMP-tFNA were respectively given to the gingival sulcus. The blank group was not treated. The modeling group rats were treated with 0.9% sodium chloride solution. Each group of rats was treated for 4 weeks. Rats were euthanized. Blood was collected from the heart. Stand at room temperature for 2 h or place at 4℃ overnight. Centrifuge at 4℃3000 rpm for 15 min and take the supernatant. In addition, the maxillary bone, heart, liver, spleen, lung, kidney, tongue, periodontal tissues of rats were collected.

[0086] Micro-CT: The maxillary molars and the connected jaws of rats were collected 21 days after modeling, and the surrounding soft tissues were carefully removed and discarded. The maxilla was fixed in 4% paraformaldehyde and observed under Micro-CT. The resolution of Micro-CT was 6μm and the filter used was 0.5 Al. The samples were scanned in three dimensions. The group layer results in bmp format were obtained, and the samples were separated by cropping using ImageJ software. Mimics Medical 20.0 was used to reconstruct the data in three dimensions, and bone parameters such as alveolar bone loss (ABL), i.e. the distance from the cementoenamel junction (CEJ) to the alveolar ridge top (ABC), volumetric bone volume (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) were measured.

[0087] ELISA was used to measure the levels of inflammatory factors in the serum and periodontal tissues of each group of rats: the periodontal tissues in liquid ammonia were taken out, crushed, homogenized and centrifuged with a tissue homogenizer in an ice water bath, the supernatant was collected to obtain the tissue sample liquid, and the protein concentration was measured according to the BCA method. 200uL was taken from each group and the levels of inflammatory factors CRP and IL-6 were measured according to the method in the ELISA kit instructions: the serum was taken out and thawed in an ice water bath, and 200uL was taken from each group and the levels of inflammatory factors CRP, IL-6 and IL-8 were measured in the same way.

[0088] HE staining: Use 4% paraformaldehyde to fix the maxillary bone, maxillary molars and surrounding tissues, place them in an embedding box, rinse them with water overnight, and soak them in 10% EDTA decalcification solution. Replace the decalcification solution every 2 days and place them on a shaker for decalcification for 30 days. Dehydrate them with an automatic dehydrator, embed them with paraffin, cool them at room temperature and fix them before demolding, and store them in a -20℃ refrigerator. Use 4% paraformaldehyde to fix the internal organs, place them in an embedding box, rinse them with water overnight, dehydrate them with an automatic dehydrator, embed them with paraffin, cool them at room temperature and fix them before demolding, and store them in a -20℃ refrigerator. Fix the samples on a tissue slicer for sectioning. Slices are stained with hematoxylin and eosin. Soak them in xylene to make them transparent. Use neutral resin to seal the slides. Observe under an optical microscope.

[0089] TRAP staining: After animal experiments, samples were collected, tissue sections were prepared and dewaxed. After xylene dewaxing and gradient alcohol hydration, the sections were TRAP stained for 1 hour and then photographed under an upright optical microscope.

[0090] Masson staining: After animal experiments, samples were collected and cut into 5um thick sections. They were heated at 55℃ for 1 hour, rehydrated with gradient alcohol, and then stained according to the instructions of the Masson staining kit. After dehydration with gradient alcohol, they were immersed in xylene and finally sealed with neutral gum. The percentage of bone formation area was calculated after taking pictures under a microscope.

[0091] Tissue immunofluorescence staining: After animal experiments, samples were collected, tissue sections were prepared and dewaxed. The sections were washed with PBS and soaked for 5 min. Repeat three times. Antigen retrieval was performed using 0.01M citrate buffer (pH 6.0), which was heated in a microwave oven at low heat for retrieval, and soaked for 5 min in a slightly boiling state. The membrane was permeabilized using 0.5% Triton X-100 and soaked for 20 min. Protein blocking solution (5% BSA) was used for blocking and soaking for 30 min. Primary antibody working solution was added and incubated at 4°C overnight. After rewarming, the sections were washed with PBS and repeated three times. Secondary antibody working solution was added in a dark place and incubated at room temperature for 2 h. The sections were washed with PBS and soaked for 5 min. Repeat three times. The sections were sealed with a sealing agent containing anti-fading and DAPI. The samples were observed using a fluorescent slide scanner.

[0092] Immunohistochemical staining: After animal experiments, samples were collected, tissue sections were prepared and dewaxed. After the tissue sections were hydrated, 1% trypsin was added for antigen retrieval. According to the instructions of the immunohistochemical kit, the tissue was incubated with MMP9 / Runx2 antibody at 4°C for 12 hours. After washing with PBS, the secondary antibody was incubated. Finally, the samples were incubated with DAB. Images were taken under an upright microscope, and the percentage of positive expression was calculated using Image ProPlus software.

[0093] 2. Experimental results (1) Micro-CT scan results of rat jaw: Micro-CT scanning is a high-resolution three-dimensional imaging technology that is of great significance in bone tissue research. Through Micro-CT scanning, microstructural information of bone tissue, such as bone density, trabecular morphology and volume, can be obtained. This technology is widely used in research fields such as bone metabolism, bone regeneration, and bone defect repair, and can intuitively and quantitatively analyze changes in bone tissue. In alveolar bone research, Micro-CT scanning can accurately detect the loss or regeneration of alveolar bone, providing reliable data support for evaluating the effects of different treatment conditions on alveolar bone.

[0094] In this experiment, five experimental groups were set up, namely: Group 1 (blank control group), Group 2 (LIP group), Group 3 (LIP+AMP group), Group 4 (LIP+Cur group), and Group 5 (LIP+Cur+tAMP group). Micro-CT scanning results showed ( Fig.12), the alveolar bone loss in group 2 was the most serious, indicating that LIP treatment significantly accelerated the loss of alveolar bone. Groups 3 (LIP+AMP group) and 4 (LIP+Cur group) showed a certain degree of improvement, with reduced alveolar bone loss, but still higher than the blank control group (group 1). The alveolar bone loss in group 5 (LIP+Cur+tAMP group) was close to that in the blank control group (group 1), showing a significant protective effect, indicating that this group had the best intervention effect and was helpful in reducing alveolar bone loss.

[0095] (2) Statistical results of Micro-CT scanning of rat jaws: In Micro-CT analysis, BV / TV, Tb.N and Tb.Th are commonly used bone structure indicators, which reflect the bone density and the structural status of trabeculae, respectively. BV / TV (bone volume fraction) indicates the ratio of bone volume to total tissue volume, and is a key indicator for evaluating bone density and bone mass; Tb.N (trabecular number) indicates the number density of trabeculae, which is usually used to measure the distribution of trabeculae; Tb.Th (trabecular thickness) reflects the average thickness of trabeculae, which is an important parameter for evaluating the strength of bone microstructure. Together, these indicators help analyze the structural integrity and quality of alveolar bone, and provide a scientific basis for the evaluation of bone metabolism and bone loss.

[0096] In this experiment, five experimental groups were set up, namely: Group 1 (blank control group), Group 2 (LIP group), Group 3 (LIP+AMP group), Group 4 (LIP+Cur group), and Group 5 (LIP+Cur+tAMP group). Micro-CT analysis results showed that ( Fig.13 ), the BV / TV, Tb.N and Tb.Th in Group 2 were the lowest, indicating that LIP treatment caused the most deterioration of alveolar bone conditions. Groups 3 (LIP+AMP group) and 4 (LIP+Cur group) showed some improvement, with bone density and trabecular structure restored, but still inferior to the blank control group (Group 1). Group 5 (LIP+Cur+tAMP group) was close to the blank control group (Group 1), and all indicators showed significant improvement, indicating that the intervention effect of this group was the best and helped to restore the structure and quality of the alveolar bone.

[0097] (3) HE staining results of rat jaw sections: By embedding the alveolar bone, slicing it, and performing HE (hematoxylin-eosin) staining, the overall structure and cell morphology of the bone tissue can be observed. HE staining can clearly show structures such as trabeculae, bone marrow cavity, osteoblasts, osteoclasts, and blood vessels in the bone marrow, providing intuitive histological evidence for analyzing the health status and repair progress of bone tissue. This experiment is of great significance in evaluating bone loss, inflammatory status, and cell activity during osteogenesis and osteoclastogenesis, and can effectively demonstrate the effects of different treatments on the structure of alveolar bone tissue.

[0098] In this experiment, five experimental groups were set up, namely: Group 1 (blank control group), Group 2 (LIP group), Group 3 (LIP+AMP group), Group 4 (LIP+Cur group), and Group 5 (LIP+Cur+tAMP group). HE staining results showed ( Fig.14 ), the alveolar bone tissue structure of group 2 was the worst, with sparse trabeculae, increased osteoclasts, and expanded bone marrow cavity, indicating that LIP treatment significantly aggravated the destruction of alveolar bone. Groups 3 (LIP+AMP group) and 4 (LIP+Cur group) showed some improvement, and the trabecular structure was restored, but it was still not as good as the blank control group (group 1). Group 5 (LIP+Cur+tAMP group) was close to the blank control group (group 1), with intact bone tissue structure and increased number of osteoblasts, showing a good repair effect, indicating that the intervention effect of this group was the best and helped to restore the healthy structure of the alveolar bone.

[0099] (4) Masson staining results of rat jaw sections: After embedding the alveolar bone, sectioning and Masson trichrome staining can be performed to observe the collagen fibers and mineralization of the bone tissue in detail. Masson staining can distinguish collagen fibers (dyed blue or green), muscle and cytoplasm (dyed red) and mineralized areas (usually stained light colors) in bone tissue, helping researchers to intuitively evaluate trabecular bone structure, collagen fiber content and arrangement. This staining method is of great significance in studying changes in bone density, bone reconstruction and the generation of fibrous tissue, and provides a reliable histological basis for analyzing the effects of different treatments on alveolar bone structure.

[0100] In this experiment, five experimental groups were set up, namely: Group 1 (blank control group), Group 2 (LIP group), Group 3 (LIP+AMP group), Group 4 (LIP+Cur group), and Group 5 (LIP+Cur+tAMP group). Masson staining results showed ( Fig.15), the alveolar bone condition of group 2 was the worst, with sparse trabeculae and reduced collagen fibers, indicating that LIP treatment led to significant bone loss. The collagen fibers and trabecular structure of group 3 (LIP+AMP group) and group 4 (LIP+Cur group) improved, but still did not return to the level of the blank control group (group 1). Group 5 (LIP+Cur+tAMP group) was close to the blank control group (group 1), with uniform distribution of collagen fibers and relatively complete trabecular structure, showing a good repair effect, indicating that the intervention of this group can effectively improve the tissue structure and quality of alveolar bone.

[0101] (5) Results of TRAP staining of osteoclasts in rat jaw sections: TRAP (tartrate-resistant acid phosphatase) staining is a specific staining method for detecting osteoclasts and is widely used in bone metabolism research. TRAP staining can clearly observe the distribution and activity of osteoclasts. Osteoclasts will show a characteristic red or purple color after staining, which helps to evaluate the activity of bone resorption. This experiment can intuitively display the number of osteoclasts and their distribution in bone tissue, thereby revealing the effects of different treatments on the alveolar bone resorption process, and providing important histological evidence for analyzing the health status of alveolar bone and bone loss.

[0102] In this experiment, five experimental groups were set up, namely: Group 1 (blank control group), Group 2 (LIP group), Group 3 (LIP+AMP group), Group 4 (LIP+Cur group), and Group 5 (LIP+Cur+tAMP group). TRAP staining results showed ( Fig.16 ), the number of osteoclasts in group 2 was the largest, indicating that LIP treatment significantly promoted the activity of osteoclasts, resulting in the worst alveolar bone condition. The number of osteoclasts in group 3 (LIP+AMP group) and group 4 (LIP+Cur group) decreased, but was still higher than that in the blank control group (group 1). Group 5 (LIP+Cur+tAMP group) was close to the blank control group (group 1), with almost no osteoclasts, showing a significant protective effect, indicating that the intervention in this group can effectively inhibit the activity of osteoclasts and help maintain the healthy state of the alveolar bone.

[0103] (6) IL-6 staining results of rat jaw bone sections: By performing IL-6 immunohistochemical staining on the embedded alveolar bone tissue, the degree of inflammatory response and the expression of IL-6 can be observed. IL-6 is a proinflammatory cytokine, and its expression level is usually closely related to the intensity of the inflammatory response. Through immunohistochemical staining, the positive expression of IL-6 will appear as a brown staining area, which can clearly locate the expression site in the tissue, show its distribution and quantity, and thus evaluate the effects of different treatments on the inflammatory state of the alveolar bone. This experiment provides intuitive histological evidence for studying periodontal disease and the effectiveness of anti-inflammatory interventions.

[0104] In this experiment, five experimental groups were set up, namely: Group 1 (blank control group), Group 2 (periodontal suture LIP modeling group), Group 3 (LIP+AMP group), Group 4 (LIP+Cur group), and Group 5 (LIP+Cur+tAMP group). The results of IL-6 immunohistochemical staining showed that ( Fig.17 ), the IL-6 expression in group 2 was the strongest, showing a large amount of brown staining, indicating that LIP treatment significantly aggravated the inflammatory response of the alveolar bone. The IL-6 expression levels of group 3 (LIP+AMP group) and group 4 (LIP+Cur group) decreased, but were still higher than the blank control group (group 1). The IL-6 expression of group 5 (LIP+Cur+tAMP group) was close to that of the blank control group (group 1), and the inflammatory response was significantly reduced, showing a good anti-inflammatory effect, indicating that the intervention of this group can effectively inhibit the inflammatory state and protect the health of the alveolar bone.

[0105] (7) Runx2 staining results of rat jaw sections: By performing RUNX2 immunofluorescence staining on the embedded alveolar bone tissue, the expression of osteogenic-related transcription factors can be detected. RUNX2 is a key osteogenic transcription factor that plays an important role in osteogenesis and osteoblast differentiation. Through immunofluorescence staining, the expression of RUNX2 will be presented as a specific fluorescent signal, which can visually observe its distribution and quantity in bone tissue, thereby evaluating the effects of different treatments on osteogenic activity. This experiment provides an intuitive histological basis for understanding the osteogenic status and repair process of alveolar bone.

[0106] In this experiment, five experimental groups were set up, namely: Group 1 (blank control group), Group 2 (periodontal suture LIP modeling group), Group 3 (LIP+AMP group), Group 4 (LIP+Cur group), and Group 5 (LIP+Cur+tAMP group). The results of RUNX2 immunofluorescence staining showed that ( Fig.18), the expression level of RUNX2 in group 2 was the lowest, and the fluorescence signal was weak, indicating that LIP treatment significantly inhibited the expression of osteogenic proteins, resulting in reduced osteogenic activity of the alveolar bone. The expression levels of RUNX2 in group 3 (LIP+AMP group) and group 4 (LIP+Cur group) increased, but were still lower than that in the blank control group (group 1). The expression of RUNX2 in group 5 (LIP+Cur+tAMP group) was close to that in the blank control group (group 1), with a strong fluorescence signal and a significant increase in osteogenic protein expression, indicating that the intervention in this group can effectively promote osteogenic activity and contribute to the repair and health maintenance of the alveolar bone.

[0107] The above experimental results show that the curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex provided by the present invention can significantly reduce the alveolar bone loss in rats with periodontitis, inhibit the inflammatory state, promote osteogenic activity, restore the structure and quality of the alveolar bone, and effectively treat periodontitis.

[0108] In summary, the present invention provides a curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex and its preparation method and use. Cell experiments show that the curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex can significantly improve the osteogenic activity of periodontal ligament stem cells, restore mineralization activity, reduce oxidative stress levels, and inhibit the occurrence and development of inflammatory reactions. Animal experiments show that the curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex can significantly reduce alveolar bone loss in rats with periodontitis, inhibit inflammatory conditions, promote osteogenic activity, restore the structure and quality of alveolar bone, and effectively treat periodontitis. The curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex provided by the invention has broad application prospects in the preparation of drugs for the prevention and / or treatment of periodontitis.

Claims

1. A curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex, characterized in that: The invention is a complex prepared by taking curcumin, antimicrobial peptide and tetrahedral framework nucleic acid as raw materials.

2. The curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex according to claim 1, characterized in that: The antimicrobial peptide is a defensin.

3. The curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex according to claim 1, characterized in that: It is a complex prepared from tetrahedral framework nucleic acid modified by antimicrobial peptide and curcumin as raw materials. The tetrahedral framework nucleic acid modified by antimicrobial peptide is formed by four DNA single strands through base complementary pairing. The sequences of the four DNA single strands are shown in SEQ ID NO.1-4.

4. The curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex according to claim 3, characterized in that: The molar ratio of the four DNA single strands is 1:1:1:

1.

5. The curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex according to claim 3, characterized in that: The method for preparing the tetrahedral framework nucleic acid modified with the antimicrobial peptide comprises the following steps: maintaining four DNA single strands at 50-90° C. for 5-15 minutes, and then maintaining them at 2-8° C. for 10-30 minutes.

6. The curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex according to claim 5, characterized in that: The method for preparing the tetrahedral framework nucleic acid modified with the antimicrobial peptide comprises the following steps: maintaining four DNA single strands at 70° C. for 10 minutes and then maintaining them at 4° C. for 20 minutes.

7. The curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex according to any one of claims 3 to 6, characterized in that: The mass ratio of the antimicrobial peptide-modified tetrahedral framework nucleic acid to curcumin is 1:(30-50).

8. The curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex according to claim 7, characterized in that: The mass ratio of the antimicrobial peptide-modified tetrahedral framework nucleic acid to curcumin is 1:

40.

9. A method for preparing the curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex according to any one of claims 1 to 8, characterized in that: The method comprises the following steps: reacting the tetrahedral framework nucleic acid modified with the antimicrobial peptide with curcumin to obtain a curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex.

10. Use of the curcumin-antimicrobial peptide-tetrahedral framework nucleic acid complex according to any one of claims 1 to 8 in the preparation of a medicament for preventing and / or treating periodontitis.

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