Polyphenol-protein self-assembly material as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a polyphenol-protein self-assembly material as well as a preparation method and application thereof. According to the invention, polyphenol-protein self-assembly materials (NE APs) are effectively formed by utilizing a plurality of physical interactions between EGCG (epigallocatechin gallate) and a growth factor NRG-1. The NRG-1 can be continuously released from the NE APs, and the EGCG protects the NRG-1 from oxidative stress and maintains the biological activity of the NRG-1 related to stem cell recruitment, migration and angiogenesis. In addition, the NE APs utilize EGCG to clear ROS, maintain mitochondrial homeostasis and promote osteogenic differentiation ability, and meanwhile, through TNF / NF-kappa B / JAK-STAT signal pathways, immunoreaction is synergistically regulated. In-vivo experiments show that NE APs accelerates repair of inflammatory bone defects by enhancing stem cell recruitment, angiogenesis and immunoregulation, and has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a polyphenol-protein self-assembly material and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Inflammatory bone defects are a common clinical disease that imposes a huge economic burden on society and usually involve structural and functional damage to bone tissue due to infectious or non-infectious inflammatory responses. To overcome the limitations of natural bone implants, such as donor shortages and the risk of immune rejection, bone tissue engineering materials have emerged as a promising alternative. However, despite their potential, conventional metal-, ceramic-, and polymer-based materials face significant challenges at bone defect sites due to limited bioactivity. Inflammatory diseases, in particular, lead to the accumulation of reactive oxygen species (ROS) and immune imbalance, disrupting bone homeostasis and hindering tissue integration, thus complicating the treatment of inflammatory bone defects. Growth factor delivery strategies have been widely used in bone tissue regeneration due to their effectiveness in accelerating healing and restoring tissue function. Typically, growth factors are directly incorporated into biomaterials for sustained release, enabling them to exert their corresponding biological functions. However, a key challenge is the susceptibility to degradation and inactivation caused by oxidative stress, especially under inflammatory conditions during long-term bone tissue regeneration, resulting in the loss of growth factor bioactivity. Therefore, the development of advanced delivery strategies to protect the bioactivity of growth factors in oxidative environments has become a critical and urgent focus of growth factor-based bone tissue engineering.

[0004] Epigallocatechin gallate (EGCG) is a polyphenol compound extracted from tea with significant antioxidant, anti-inflammatory and osteogenic properties, making it a promising drug for bone tissue engineering. For example, EGCG-modified poly (e-caprolactone) scaffolds have been shown to promote bone regeneration by scavenging ROS and immunomodulation. In addition, electrospun poly-L-lactic acid / gelatin-based nanofiber membranes loaded with EGCG accelerated osteogenic differentiation and bone repair by continuously releasing EGCG. However, the preparation process of the above materials is not only cumbersome, but also requires the use of multiple chemical reagents during the preparation process, which is not in line with the current green and environmentally friendly preparation concept. Summary of the invention

[0005] In view of the deficiencies of the above-mentioned prior art, the present invention provides a polyphenol-protein self-assembly material and a preparation method and application thereof. Specifically, the present invention effectively forms self-assembled particles (NEAPs), i.e., the above-mentioned polyphenol-protein self-assembly material, by utilizing the various physical interactions between epigallocatechin-3-gallate (EGCG) and growth factors (such as neuregulin-1 / NRG-1), thereby breaking through the limitations of traditional growth factor delivery strategies, providing a new multifunctional protective delivery strategy for growth factors, improving their utilization rate and therapeutic effects, and opening up a new way to treat inflammatory bone defects. Based on the above research results, the present invention is completed.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first aspect of the present invention provides a polyphenol-protein self-assembly material, wherein the raw materials of the polyphenol-protein self-assembly material at least include epigallocatechin gallate and neuregulin-1.

[0008] The mass ratio of epigallocatechin gallate to neuregulin-1 is 1-50:0.1, further 10-30:0.1, and further 20:0.1.

[0009] The second aspect of the present invention provides a method for preparing the above-mentioned polyphenol-protein self-assembly material, which comprises mixing an epigallocatechin gallate solution and a neuregulin-1 solution, stirring and centrifuging. That is, the present invention simply mixes neuregulin-1 and polyphenol EGCG under mild conditions to form self-assembled particles, thereby achieving protective delivery of growth factors.

[0010] The concentration of the epigallocatechin gallate solution is 1-50 mg / mL, more preferably 20 mg / mL; the concentration of the neuregulin-1 solution is 0.01-1 mg / mL, more preferably 0.1 mg / mL; when mixed, the volume ratio of the two is 0.1-10:1, preferably 1:1.

[0011] During the mixing and stirring process, the specific stirring conditions are: stirring at 600-1000 rpm for 0.1-5 min, preferably stirring at 800 rpm for 1 min.

[0012] The specific conditions of centrifugation are: centrifugation at 10000-15000 rpm for 1-10 min, preferably centrifugation at 12000 rpm for 5 min.

[0013] The third aspect of the present invention provides the use of polyphenol-protein self-assembly materials in the preparation of inflammatory bone defect repair products.

[0014] The inflammatory bone defect repair is specifically manifested as:

[0015] (a) Neuregulin-1 can be sustainably released from polyphenol-protein self-assembled materials, and epigallocatechin gallate protects neuregulin-1 from oxidative stress, maintaining its biological activities related to stem cell recruitment, migration, and angiogenesis;

[0016] (b) Polyphenol-protein self-assembly materials utilize the ability of epigallocatechin gallate to scavenge ROS, maintain mitochondrial homeostasis, and promote osteogenic differentiation, while synergistically regulating immune responses through the TNF / NF-κB / JAK-STAT signaling pathway;

[0017] (c) Polyphenol-protein self-assembly materials enhance the body's stem cell recruitment, angiogenesis and immune regulation capabilities.

[0018] In another specific embodiment of the present invention, the product may be a medicine.

[0019] Meanwhile, when the product is a medicine, the medicine may also contain at least one non-drug active ingredient, and the non-drug active ingredient may include pharmaceutically acceptable excipients, carriers, etc., which are not specifically limited here.

[0020] In the present invention, the drug dosage form is not specifically limited. In a specific embodiment of the present invention, the drug can be an injection dosage form.

[0021] A fourth aspect of the present invention provides a method for repairing bone defects, the method comprising: applying the above-mentioned polyphenol-protein self-assembly material to the bone defect site of a subject.

[0022] The bone defect may be a periodontitis bone defect, and the administration method may be to inject the polyphenol-protein self-assembly material into the bone defect site.

[0023] Beneficial technical effects of one or more of the above technical solutions:

[0024] The above technical scheme developed a simple and versatile protective delivery strategy by effectively forming NE APs by utilizing multiple physical interactions between EGCG and the growth factor NRG-1. NRG-1 was continuously released from NE APs, while EGCG protected NRG-1 from oxidative stress and maintained its biological activities related to stem cell recruitment, migration and angiogenesis. In addition, NEAPs utilized the ability of EGCG to scavenge ROS, maintain mitochondrial homeostasis and promote osteogenic differentiation, while synergistically regulating immune responses through the TNF / NF-κB / JAK-STAT signaling pathway. In vivo experiments showed that NE APs created a favorable microenvironment for bone regeneration by enhancing stem cell recruitment, angiogenesis and immunomodulation, thereby accelerating the repair of inflammatory bone defects. This self-assembly strategy combining polyphenols and growth factors provides a promising method to improve the therapeutic efficiency of growth factors in regenerative medicine, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0026] Figure 1 Preparation and characterization of NE APs of the present invention. (A) The mixture of EGCG and NRG-1 self-assembled into particles after vigorous shaking. (B) SEM and TEM images of NE APs. (C) EDS elemental map of NE APs. (D) Hydrodynamic diameter of NE APs. (E) FT-IR spectra of NE APs of EGCG / BSA, EGCG and BSA. (F) XRD patterns of NE APs. (G) Interaction analysis at the binding interface of EGCG and NRG1 in NE APs. These figures show the 3D interaction map and surface structure map of the complex interface. (H) Cumulative release of NRG-1 from NE APs over time. (I) Scavenging activity of NE APs against three typical free radicals (DPPH, ·OH and ·O2-) at different concentrations (μg / mL).

[0027] Figure 2NE APs of the present invention protect NRG-1 from damage caused by H2O2. (A) Transwell chamber cell migration assay to evaluate the chemotactic behavior of PDLSCs in the negative control (NC), NRG-1, EGCG, NE APs, H2O2 / NRG-1, and H2O2 / NE APs groups. (B) Counting and data analysis of cell migration. (C) Representative images of PDLSCs in the two-dimensional scratch assay. (E) Chemotactic behavior of PDLSCs in the negative control (NC), positive control (PC), NRG-1, H2O2 / NRG-1, and H2O2 / NE-APs groups. (D) Quantitative analysis of scratch healing rate. (E) Tube formation of HUVECs after 12 h in the negative control (NC), positive control (PC), NRG-1, EGCG, NE APs, H2O2 / NRG-1, and H2O2 / NE APs groups. (F) Semi-quantitative analysis describing the number of nodes. *p<0.05, **p<0.01 and ***p<0.001.

[0028] Figure 3 The intracellular ROS scavenging activity of NE APs of the present invention. (A) Fluorescence intensity was detected by flow cytometry after ROS staining. (B) Quantitative statistical analysis of ROS positivity. (C) Relative mRNA expression levels of antioxidant enzymes (CAT and SOD1) after different treatments under H2O2 / stimulation. (D) PDLSCs were stained with JC-1 to monitor mitochondrial membrane potential. (E) Representative fluorescence images of Mito-Sox staining of mitochondrial ROS under different treatments. (F) Representative TEM images show the mitochondrial morphology and distribution of PDLSCs after different treatments. ***p<0.001.

[0029] Figure 4 The anti-inflammatory effect of NE APs of the present invention. (A) Immunofluorescence staining of iNOS in RAW264.7 cells. (B) Flow cytometry detection of the proportion of iNOS-positive RAW264.7 cells. (C) Relative mRNA expression level of iNOS. (D) Relative mRNA expression level of proinflammatory cytokines. (E) Immunofluorescence staining of CD206 in RAW264.7 cells. (F) Flow cytometry detection of the proportion of CD206-positive cells. (G) Quantitative analysis of the proportion of CD206-positive RAW264.7 cells. (H) Relative mRNA expression level of CD206. (I) Relative mRNA expression level of anti-inflammatory cytokine IL-10. (J) RT-qPCR detection of the inflammatory response of PDLSCs under LPS stimulation after different treatments. (K) Elisa kit detection of the inflammatory response of PDLSCs under LPS stimulation after different treatments. *p<0.05, **p<0.01, ***p<0.001.

[0030] Figure 5 Transcriptome sequencing analysis of the present invention. (A) Heat map of distances between samples. (B) Volcano map of transcriptomic analysis of differentially expressed genes between the control group and the NE APs group. (C) Heat map of differentially expressed genes between the control group and the NE APs group. (D) GO analysis of DEGs after NEAPs treatment in PDLSCs. (E) Bubble map of KEGG enrichment analysis. TNF (F), NF-κB (G), and IL-17 (H) signaling pathways based on GSEA. (I) Western blot and quantitative analysis of p-p65 and p65. (J) Western blot and quantitative analysis of p-κBα and κBα. **p<0.01, ***p<0.001.

[0031] Figure 6 Figure 1 shows the osteogenesis effect of NE APs of the present invention on PDLSCs. (A) ALP staining of PDLSCs after 7 and 14 days of different treatments. (B) Expression of osteogenic differentiation-related genes ALP, BMP2, and OPN in PDLSCs after 7 and 14 days of different treatments. (C) Osteogenesis-related ARS staining of PDLSCs after 21 and 28 days of different treatments, and (D) semi-quantitative analysis of relative ARS levels. ***P<0.001.

[0032] Figure 7 The therapeutic effect of NE APs of the present invention in a rat periodontitis model. (A) 3D reconstructed micro-CT scanning images of maxillary alveolar bone 2 and 4 weeks after surgery. (B) Quantitative micro-CT analysis of BV / TV, Tb.Sp and BS / TV. (C) H&E staining images 2 and 4 weeks after surgery. (D) Quantitative analysis of CEJ-ABC distance assessed by H&E staining. (E) Masson staining images of maxillary alveolar bone defect 2 and 4 weeks after surgery. (F) COL I staining image of maxillary alveolar bone defect 2 weeks after surgery. **P<0.01, ***P<0.001.

[0033] Figure 8 The present invention (A) Immunohistochemical analysis of M1 macrophage marker CD80 and M2 macrophage marker CD163 in maxillary alveolar bone defects 2 weeks and 4 weeks after surgery. (B) Quantitative analysis of CD80+ cells in different groups. (C) Quantitative analysis of CD206+ cells in different groups. (D) Immunofluorescence staining was used to observe the presence and distribution of PDLSCs in the bone defect area. (E) α-SMA immunofluorescence staining was used to evaluate the vascularization in the bone defect area 2 weeks and 4 weeks after surgery. (F) Quantitative analysis of α-SMA expression in different groups. ***P<0.001.

[0034] Fig. 9Schematic diagram of the construction of NE APs and treatment of inflammatory bone loss associated with periodontitis. (A) Assembly process of NE APs. (B) Treatment of inflammatory bone loss associated with periodontitis. (C) Mechanism of bone regeneration. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0037] As mentioned previously, the efficacy of bone tissue regeneration therapies based on growth factor delivery is often undermined by oxidative stress, especially under inflammatory conditions, which can lead to structural damage and functional inactivation of growth factors.

[0038] In view of this, the present invention proposes a simple and universal protective delivery strategy by utilizing multiple physical interactions between epigallocatechin-3-gallate (EGCG) and growth factors (such as neuregulin-1 / NRG-1) to effectively form self-assembled particles (NEAPs), namely polyphenol-protein self-assembled materials.

[0039] NE APs provide sustained release of NRG-1 while protecting it from oxidative damage and maintaining its biological functions of cell recruitment, migration, and angiogenesis. In addition, NE APs utilize the ability of EGCG to scavenge reactive oxygen species and maintain mitochondrial homeostasis, while synergistically regulating the TNF / NF-κB / JAK-STAT signaling pathway, mediating M2 macrophage polarization and inhibiting inflammation to support immune response and osteogenic differentiation. In vivo experiments showed that NE APs created a favorable microenvironment for bone regeneration through stem cell recruitment, angiogenesis, and immunomodulation, effectively promoting the repair of inflammatory bone defects. This multifunctional protective delivery strategy based on the self-assembly of polyphenols and growth factors provides potential for promoting the application of growth factors in regenerative medicine.

[0040] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0041] Example

[0042] 1. Materials and Methods

[0043] 1.1 Materials

[0044] Neuregulin-1 (NRG-1) was purchased from R&D Systems (USA). Epigallocatechin-3-gallate (EGCG) was purchased from Yuanye Biotech (Shanghai, China). CuCl2, 3,3′,5,5′-tetramethylbenzidine (TMB) was purchased from Macklin (Shanghai, China). Calcein AM / PI live / dead staining kit (CA1630), superoxide anion activity assay kit (BC1290), 2,2-diphenyl-1-picrylhydrazyl (DPPH, purity >97%, 394.32MW), JC-1 mitochondrial membrane potential assay kit. Mito Tracker Green (C1048), ROS detection kit (S0033), GSH and GSSG detection kits. Cell counting kit 8 (CCK8) and ATP chemiluminescence detection kit were purchased from Elabscience (Wuhan, China). NF-κB p65 (C22B4) rabbit monoclonal antibody, phospho-NF-κB p65 (Ser536) (93H1) rabbit monoclonal antibody, GAPDH monoclonal antibody, Bata tubulin recombinant antibody, IKB-Alpha monoclonal antibody, phospho-IKB-Alpha (Ser32 / 36) recombinant antibody, HRP-conjugated goat anti-mouse IgG (H+L), and HRP-conjugated goat anti-rabbit IgG (H+N) were purchased from Abcam (USA).

[0045] 1.2 Synthesis of NE APs (NRG-1 and EGCG self-assembled particles)

[0046] 20 mg / mL EGCG solution was mixed with 100 μg / mL NRG-1 solution at a volume ratio of 1:1. The mixture was vigorously stirred at 800 rpm on an oscillator for 1 min and then centrifuged at 12000 rpm for 5 min to form white NE APs. The resulting product was stored in a refrigerator at 4 °C.

[0047] 1.3 Molecular dynamics simulation

[0048] The GROMACS2019.6 software package was used to simulate the interaction between NRG1 protein and EGCG. The binding free energy between NRG1 and EGCG was calculated by molecular dynamics simulation.

[0049] 1.4 NE APs Characterization

[0050] Fourier transform infrared spectroscopy: Fourier transform infrared (FTIR) spectroscopy (PerkinElmer Spectrum100, USA) was used to characterize the chemical structures of NRG-1, BSA and synthesized NE APs.

[0051] XPS: X-ray photoelectron spectroscopy was used to analyze freeze-dried NE APs for peak fitting and valence state calibration.

[0052] SEM and TEM: NE APs were prepared by resuspending the precipitate in PBS after centrifugation. A few drops of the sample suspension were deposited on a silicon wafer or a copper grid. The morphology of NE APs was observed using a scanning electron microscope (SEM, s4800, Hitachi Co., Ltd., Japan) and a transmission electron microscope (TEM, JEM-2100, JEOL, Japan).

[0053] EDS: The elemental compositions of NEAPs were examined by energy dispersive X-ray spectrometry (EDS, Phenom ProX, G5, Eindhoven, The Netherlands).

[0054] DLS and Zeta potential: The average hydrodynamic size of NE APs was measured using dynamic light scattering (DLS, Zetasizer Nano ZS90, UK). NE APs were diluted to 10 μg / mL with ultrapure water, and the hydrodynamic size and zeta potential were recorded on a Zetasizer Nano ZSP (Malvern Instruments, UK).

[0055] 1.5 In vitro release of NRG-1 from NE APs

[0056] The release behavior of NRG-1 from NE APs was evaluated using dialysis. The sample solution (1 mg / mL) was placed at 37°C with gentle shaking. At specific time intervals, 500 μL of PBS was collected after centrifugation, and an equal volume of fresh PBS was added to the sample to remix. The collected PBS was stored at -80°C before analysis. The concentration of NRG-1 in the samples was measured using an ELISA kit.

[0057] 1.6 Antioxidant capacity of NE APs

[0058] The antioxidant capacity of NE APs was evaluated using three different methods. In the DPPH assay, different concentrations of NE APs solutions (0, 5, 10, 20, 40, and 80 μg / mL) were mixed with the DPPH radical working solution. After incubation, the absorbance was measured at 510 nm to evaluate the free radical scavenging activity. The conversion of H2O2 to hydroxyl radicals (·OH) was measured using a CuCl2-mediated Fenton-like reaction. The absorbance was recorded at 562 nm to evaluate the antioxidant capacity. The ·O2- scavenging capacity of NE APs was determined using a superoxide anion activity assay kit.

[0059] Characterization of biocompatibility of 1.7NE APs

[0060] Cell viability assay: PDLSCs extraction has been approved by the Ethics Committee of the Stomatological Hospital of Shandong University, Jinan, China (protocol number: NO.20201007). The effect of NE APs on the survival rate of PDLSCs was evaluated using a CCK8 kit. PDLSCs were seeded in 96-well plates, and NE APs were added at different concentrations (0, 10, 20, and 40 μg / mL). After incubation for 1, 2, and 3 days, CCK8 working solution was added, followed by incubation for 2 hours. The absorbance (OD) was measured at 450 nm using a microplate reader (SPECTROstar Nano, Germany) to determine cell viability.

[0061] Live / dead staining: PDLSCs were treated with different conditions (NRG-1 (50 ng / mL), EGCG (10 μg / mL), and NE APs (10 μg / mL)) in DMEM medium for 24 h in 24-well plates. After treatment, live / dead cell double staining was performed. After incubation in the dark for 20 min, the cells were washed three times with PBS and observed under a fluorescence microscope (OLYMPUS IX73, Japan).

[0062] Cytoskeleton staining: PDLSCs were incubated in DMEM medium containing NRG-1 (50 ng / mL), EGCG (10 μg / mL), and NE APs (10 μg / mL) in 24-well plates for 24 h. Skeleton was stained with phalloidin, while nuclei were co-stained with DAPI. Subsequently, cells were observed under a fluorescence microscope (OLYMPUS IX73, Japan).

[0063] 1.8PDLSCs homing assay

[0064] Transwell migration assay: The homing ability of PDLSCs to NE APs was evaluated using the transwell migration assay. A total of 3 × 105 cells were seeded in the upper chamber of an insert with 8 μm pores, while NRG-1 (50 ng / mL), EGCG (10 μg / mL), NE APs (10 μg / mL), H2O2+NRG-1, and H2O2+NE APs were added to the lower chamber. NRG-1, H2O2+NRG-1, and H2O2+NEAPs were preincubated at 4 °C for 24 h, and the final H2O2 concentration was 500 μM. After 20 h of co-culture, cells that migrated to the bottom side of the insert membrane were stained with crystal violet. The stained cells were observed under an optical microscope (Olympus, Japan), and the number of migrated cells was counted. The negative control group (NC) received 2% FBSDMEM, and the positive control group (PC) received 10% FBSDMEM.

[0065] Scratch assay: Scratch assay was performed to further evaluate the migration ability of PDLSCs. Once a monolayer of cells was formed, scratches were made using a 100 μL pipette tip. The cells were washed twice with PBS and fresh medium containing 2% FBS was added. The cells were incubated with NRG-1 (50 ng / mL), EGCG (10 μg / mL), NE APs (10 μg / mL), H2O2+NRG-1, and H2O2+NE APs for 12 h. Images of the scratch area were captured under a microscope, and the scratch closure rate was quantitatively analyzed using ImageJ software.

[0066] 1.9 Angiogenesis analysis

[0067] Human umbilical vein endothelial cells (HUVECs) were seeded into 6-well plates (3 × 105 cells / well) and treated with NRG1, EGCG, NE APs, H2O2+NRG-1, and H2O2+NE APs for 48 h. Growth factor-reduced Matrigel (Corning, USA) was added to 96-well plates on ice and incubated at 37°C for 30 min to induce gelation. HUVECs were then re-seeded onto Matrigel at 1 × 105 cells / well. After 12 h of incubation, tube formation was observed under an optical microscope. Total tube length, nodes, and meshes were quantitatively analyzed using ImageJ software. RT-PCR analysis was performed to quantify the expression levels of angiogenesis-related genes (CD31, KDR, and VEGF).

[0068] 1.10 Intracellular ROS scavenging and protective abilities of NE APs

[0069] A ROS detection kit was used to detect the changes in intracellular ROS. PDLSCs were plated at 2×10 5Cells were seeded in 6-well plates at a density of 10 cells and cultured with NRG-1 (50 ng / mL), EGCG (10 μg / mL) and NE APs (10 μg / mL). After 24 hours, the culture medium was replaced with DMEM containing 400 μM H2O2 and incubated for 2 hours. Subsequently, the cells were treated with 10 μM 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) for 60 minutes and then washed three times with PBS. Fluorescence imaging was performed using a fluorescence microscope, and the fluorescence intensity was quantified using ImageJ 1.44 software (National Institutes of Health, USA). In addition, the fluorescence intensity was further analyzed by flow cytometry. Mitochondrial ROS was detected using Mito-Sox staining, and the morphology of mitochondria was simultaneously examined by transmission electron microscopy (TEM).

[0070] The expression of antioxidant-related genes (CAT, NQO, and SOD-1) in different treatment groups was analyzed by quantitative real-time PCR (RT-PCR) using the ABI 7500 RT-PCR system (Applied Biosystems, MA, USA) and SYBR Green PCR Master Mix (AG, China). The relative gene expression was calculated using the 2-ΔΔCt method and normalized to the housekeeping gene β-Actin.

[0071] 1.11 Anti-inflammatory ability of NE APs

[0072] PDLSCs were grown at 2 × 10 per well. 5 Cells were seeded at a density of 100 cells / mL in 6-well plates and treated with LPS (100 ng / mL), NRG1, EGCG, and NE APs for 24 h. After this treatment, specific pro-inflammatory genes (IL-8, IL-6, and CCL2) were characterized using Elisa kits.

[0073] 1.12 Macrophage polarization

[0074] Inhibition of M1 polarization: Mouse macrophages (RAW264.7) were cultured at 3×10 5 Cells were seeded in 6-well plates at a density of 1 × 10 cells / mL and treated with normal DMEM medium, LPS, LPS+NRG-1, LPS+EGCG, and LPS+NE-APs for 24 h. 6The cells were resuspended in 100 μL PBS, and then pre-bound antibody FITC-CD86 and rat isotype IgG control antibody were added. After incubation in the dark for 30 minutes, the cells were washed twice and resuspended in flow buffer for detection using a BD LSRFortessa flow cytometer. For immunofluorescence staining, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 (Solarbio, China). After blocking with 10% goat serum, the cells were incubated with anti-iNOS2 primary antibody (1:200 dilution) at 4°C overnight. The next day, cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200 dilution) in the dark at 25°C for 1 hour. The nuclei were counterstained with DAPI (Abcam) before observation under a fluorescence microscope. RT-PCR was used to evaluate the expression of specific pro-inflammatory genes (IL-6 and TNFα) and M1 macrophage marker genes (iNOS).

[0075] Promoting M2 polarization: RAW264.7 macrophages were treated with normal DMEM medium, LPS, NRG-1, EGCG, and NE APs for 24 hours. M2 polarization was analyzed by flow cytometry using pre-conjugated antibody APC-CD206 and rat isotype IgG control antibody. Immunofluorescence staining involved incubation with anti-CD206 primary antibody (1:200 dilution) and Alexa Fluor488-conjugated goat anti-rabbit IgG (1:200 dilution). The expression of anti-inflammatory genes (IL-10) and M2 macrophage marker genes (CD206) were quantified using RT-PCR.

[0076] 1.13 Osteogenic differentiation

[0077] PDLSCs were cultured in osteogenic differentiation medium supplemented with NRG1, EGCG, and NE APs. ALP staining was performed after 7 and 14 days of culture, and Alizarin Red S staining was performed after 21 and 28 days of culture. The expression of osteoblast-related genes (ALP, OPN, and BMP-2) was analyzed by RT-PCR after 7 and 14 days of culture. Semi-quantitative analysis of ALP and Alizarin Red S staining was performed using ImageJ.

[0078] 1.14 RNA-Seq and bioinformatics

[0079] Transcriptome sequencing and analysis were performed by OE Biotech Ltd. For each sample, 1 g of total RNA was used for RNA-seq analysis. The raw reads were processed using the bioinformatics pipeline tool BMKCloud (www.biocloud.net) online platform. The differential expression between samples was analyzed using edgeR software with a significance threshold of FDR < 0.05 and Fold Change ≥ 2. Gene ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and gene set enrichment analysis (GSEA) were performed using the GOstats software package.

[0080] 1.15 Promoting the repair of periodontitis bone defects in vivo

[0081] All animal experiments were approved by the Ethics Committee of the Stomatological Hospital of Shandong University, Jinan, China (protocol number: NO.20231202). Seventy 8-week-old male Wistar rats were randomly divided into five groups: untreated group (blank), perinatal group, NRG-1 group, EGCG group, and NE APs group. The rats were anesthetized using inhalation anesthesia, and periodontitis was induced by ligating the maxillary left second molar with 3-0 silk for 2 weeks. After the periodontitis model was established, the ligature was removed, and PBS, NRG-1, EGCG, or NE APs solution was injected into the gingival sulcus every other day for 2 or 4 weeks.

[0082] 1.16 Blood biochemistry assay

[0083] Blood routine and biochemical tests were performed to evaluate the in vivo toxicity of NE APs. Whole blood was collected from the tail vein of SD rats 2 weeks after surgery. For blood routine measurements, 500 μL of whole blood was centrifuged (4°C, 45000 rpm, 10 min) to obtain serum, and various blood parameters were compared among all experimental groups.

[0084] 1.17 Micro-CT analysis

[0085] After three weeks of treatment, the animals were euthanized, and the maxillae were harvested, fixed with 4% (w / v) buffered paraformaldehyde, and subjected to micro-CT analysis (Quantum GX, PerkinElmer, USA) to assess bone repair. The scanning parameters were set at 36 μm resolution, 90 kV voltage, and 88 μA current. Imaging data were collected, and 3D reconstructions were generated using the manufacturer's proprietary software. After reconstruction, parameters such as bone mineral density (BMD), bone volume to tissue volume ratio (BV / TV), bone surface area density (BS / TV), and trabecular spacing / spacing (Tb.Sp) were calculated.

[0086] 1.18 Histology, Immunohistochemistry and Immunofluorescence Analysis

[0087] Histological examinations were performed on the liver, spleen, kidney, heart, and lung. These organs were excised, fixed with 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) using standard techniques. To evaluate the effects of periodontitis treatment, maxillary specimens were decalcified in 10% (w / v) ethylenediaminetetraacetic acid (EDTA), dehydrated through a graded ethanol series, embedded in paraffin, and cut longitudinally into 5 μm thick sections. The sections were stained with H&E and Masson to visualize the formation of new tissue. CEJ-ABC distance (the junction of cementum to the alveolar bone ridge) and collagen area were quantitatively analyzed. In addition, these sections were subjected to immunohistochemical staining for various markers, including type I collagen (COL I) for the assessment of collagen production, CD80 and CD206 for the assessment of immune regulation, CD44+CD34- for the analysis of the recruitment of endogenous periodontal ligament stem cells (PDLSCs), and α-SMA for the assessment of vascularization within the bone defect area. After staining, the slides were observed and examined using an optical Roche microscope (Olympus X71-F22PH, Japan).

[0088] 1.19 Statistical analysis

[0089] Data are presented as mean ± standard deviation (SD). Statistical significance was determined using Student t test for two-group comparisons and one-way analysis of variance (ANOVA) followed by post hoc Bonferroni test for multiple group comparisons. Statistical significance levels were defined as *P < 0.05, **P < 0.01, and ***P < 0.001.

[0090] 2. Results and Discussion

[0091] 2.1 Synthesis and characterization of NE APs

[0092] Functionalized NE APs were synthesized via a one-step self-assembly reaction. Briefly, a mixture of EGCG and NRG-1 spontaneously assembled into particles through physical interactions after vigorous shaking. White NE APs ( Figure 1 A). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the morphology of NE APs. Figure 1 As shown in B, NE APs showed aggregation of nanoparticles. Energy dispersive X-ray spectroscopy (EDS) was used to examine the elemental composition of NE APs ( Figure 1C). EDS elemental mapping revealed the colocalization of C, O, and N on NE APs, indicating a uniform distribution of the components. Quantitative analysis showed that the atomic percentages of C, O, N, and S in NE APs were approximately 50.6%, 31.2%, 16.7%, and 1.4%, respectively. Dynamic light scattering (DLS) analysis showed that the average hydrodynamic diameter of NE APs ranged from 2 to 4 μm ( Figure 1 D). Fourier transform infrared spectroscopy (FT-IR) spectroscopy ( Figure 1 E) Highlights the changes in the self-assembly process of EGCG molecules into APs, using BSA instead of NRG-1 due to limited availability and high cost. During the self-assembly process, the out-of-plane C-OH bending vibrations of the phenolic rings in the EGCG monomer (700 and 600 cm -1 ) and the characteristic peak of carbonyl vibration (1691cm -1 ) disappeared. In addition, at 1600cm -1 The peak broadening observed near 1450-1510 cm is attributed to the asymmetric stretching of the carboxyl (C=O) groups in the protein. -1 The spectral region between the two is related to aromatic C=C stretching and remains basically unchanged, indicating that the EGCG molecular framework is preserved. X-ray photoelectron spectroscopy (XPS) is used to analyze the valence state of elements in NE APs ( Figure 1 F). The C1s spectrum showed the presence of C=C (284.8 eV), C-C (285.0 eV), and C=O (288.5 eV) bonds, confirming the different carbon structures. The peaks at 531.7 eV and 533.0 eV in the O1s spectrum correspond to C=O in the amide bond and the hydroxyl group in EGCG, respectively. The N1s spectrum showed a peak at 398.5 eV, which can be attributed to the nitrogen in NRG-1. XPS analysis showed that the assembly process did not change the valence state of the atoms, ensuring the biological activity of the components. To further understand the interaction mechanism, the interaction between NRG-1 and EGCG was simulated using the Gromacs 2019.6 software package ( Figure 1 G). The simulation results showed that EGCG molecules can stably bind to the surface of NRG-1. Molecular dynamics simulation calculations showed that the binding free energy was -6.01 kcal / mol, mainly driven by hydrogen bonds, Coulomb forces, and hydrophobic interactions. These interactions help maintain biological activity and promote the effective release of growth factors. The system remained stable throughout the simulation, confirming the strong binding between EGCG and NRG-1, supporting the potential of polyphenolic protein APs as growth factor protection carriers.

[0093] The cumulative release curve of NRG-1 from NE APs is shown in Figure 1H. The initial explosive release occurred within the first three hours, probably due to the rapid leakage of NRG-1 near the surface. Afterwards, NRG-1 molecules gradually diffused from the APs and the release stabilized around 336 hours. The extended and gradual release properties support sustained long-term effects in both in vivo and in vitro applications. The intrinsic bioactivity of EGCG in NE APs was evaluated by measuring the free radical scavenging abilities of 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azobis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt (ABTS), and superoxide anion (·O2-). Figure 1 As shown in Figure 1, the free radical scavenging ability of NE APs increased significantly with concentration. This trend was consistently observed in DPPH, ABTS, and ·O2- assays, where higher concentrations of NE APs led to more efficient free radical scavenging and inhibition of ROS generation. The potent antioxidant capacity exhibited by NE APs suggests that they have the potential to be therapeutic agents for the treatment of ROS-related diseases.

[0094] 2.2NE APs protect NRG-1 from oxidative stress

[0095] First, the effects of different concentrations of NE APs on the survival rate of periodontal ligament stem cells (PDLSCs) were evaluated. On day 1, no significant difference in cell proliferation was observed within the concentration range of 0 to 40 μg / mL. On days 2 and 3, cell proliferation in the 20 and 40 μg / mL groups was slightly lower than that in the 10 μg / mL group, and there was no significant difference compared with the control group. Based on these results, 10 μg / mL of NE APs was selected for further experiments. Live / dead assays and cytoskeleton staining confirmed that PDLSCs exhibited good growth and typical spindle-shaped morphology in all groups, indicating that NE APs have excellent biocompatibility.

[0096] The recruitment and migration of stem cells are essential for initiating bone regeneration. A transwell assay was performed to evaluate the chemotactic effect of NEAPs on PDLSCs. NEAPs recruited more PDLSCs than the control and EGCG groups, but slightly less than the NRG-1 group due to the slower release of NRG-1 from NEAPs. The protective effect of NEAPs against H2O2-induced growth factor inactivation was also investigated ( Figure 2A and B). Exposure to H2O2 significantly reduced the recruitment capacity of NRG-1 compared with the untreated NRG-1 group. In contrast, NE APs maintained a strong recruitment capacity for PDLSCs even after H2O2 treatment, suggesting that NE APs can mitigate oxidative damage and maintain the bioactivity of NRG-1. Similarly, the in vitro scratch assay showed that PDLSCs in the NE APs group exhibited enhanced migration ability, with a scratch healing rate of 38.7% after 24 h, which was lower than the 65.6% in the pure NRG-1 group (Figure 5A). Figure 2 C and D). In contrast, the healing rates of the EGCG and control groups were only 13.7% and 6.6%, respectively. In addition, the healing capacity of NRG-1 was significantly reduced when exposed to H2O2, but NE APs maintained a higher recruitment capacity under oxidative stress, which was better than that of NRG-1 alone.

[0097] Angiogenesis plays a crucial role in supporting bone regeneration. To simulate angiogenesis in a three-dimensional environment, an in vitro tube formation assay was performed using HUVECs to assess the development of interconnected tubular networks ( Figure 2 E). The NE APs and NRG-1 groups showed the highest tubular network density and branching, significantly better than the control and EGCG groups. Specifically, the length of nodes, meshes, segments, and total tubes in the NE APs group were 6.4 and 3.5 times that of the control group, respectively ( Figure 2 F). Notably, when treated with H2O2, NRG-1 lost almost all of its pro-angiogenic ability, while the H2O2 / NE-APs group retained strong angiogenic ability. In addition, RT-qPCR analysis was performed to quantify the expression levels of angiogenesis-related genes. The results showed that CD31, KDR, and VEGF were significantly upregulated in the H2O2 / NE APs, NE APs, and NRG-1 groups compared with the control, EGCG, and H2O2 / NRG-1 groups. These results suggest that NE APs protect NRG-1 from H2O2-induced damage and inactivation, retain its biological activity in recruiting and inducing PDLSCs migration, and promote angiogenesis in HUVECs.

[0098] 2.3 Intracellular antioxidant and mitochondrial protection of NE APs

[0099] Effective scavenging of excess intracellular ROS is essential for maintaining cell function. H2O2 was used to induce excessive production of intracellular ROS in PDLSCs, and the dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to measure the intracellular ROS level as well as the ROS scavenging ability of NE APs. Compared with the H2O2 group, there was no difference in the fluorescence intensity of the NRG-1 group, while EGCG reduced the fluorescence intensity due to its strong antioxidant capacity. It is worth noting that the fluorescence intensity of the NE APs group was lower, only slightly higher than that of the EGCG group. Flow cytometric analysis confirmed that the mean fluorescence intensity was significantly reduced after NE APs treatment ( Figure 3 A and B), decreased to about 11.8%, indicating that NE APs eliminated about 73.3% of ROS. These results indicate that NE APs effectively preserve the antioxidant function of EGCG and provide strong protection for PDLSCs from H2O2-induced damage. In addition, the expression levels of antioxidant enzyme-specific genes (CAT and SOD1, which are key enzymes for maintaining ROS balance in organisms) were analyzed by RT-qPCR ( Figure 3 C). H2O2 stimulation led to ROS accumulation and downregulation of CAT and SOD1, disrupting redox balance. However, compared with the H2O2 and NRG-1 treatment groups, both NE APs and EGCG significantly upregulated the expression of CAT and SOD1, suggesting that enhanced antioxidant capacity helps to restore cellular redox homeostasis.

[0100] Mitochondria, as the center of energy metabolism and a key target of ROS-induced damage, were also analyzed. To evaluate the protective effect of NE APs on mitochondrial function in PDLSCs, the JC-1 mitochondrial membrane potential (MMP) assay was performed and the fluorescence ratio of JC-1 aggregates to JC-1 monomers (A / M ratio) was measured. Figure 3 As shown in D, healthy PDLSCs exhibited strong red fluorescence and minimal green fluorescence. However, exposure to H2O2 resulted in a significant decrease in red fluorescence and an increase in green fluorescence. NE APs and EGCG significantly restored the A / M ratio. The A / M ratio of healthy PDLSCs was initially 8.70 and dropped sharply to 1.74 after H2O2 stimulation. NE APs and EGCG upregulated the A / M ratio to 6.49 and 5.60, respectively, which were significantly higher than the 1.69 observed in the NRG-1 group and showed no difference compared with the H2O2 group. These results suggest that NE APs can maintain the stability of MMP, thereby reducing the occurrence of mitochondrial dysfunction. In addition, Mito-Sox red staining was used to detect mitochondrial ROS (mtROS) ( Figure 3E). Although NRG-1 did not reduce the mtROS level, NEAPs and EGCG reduced mtROS by 6.83-fold and 4.99-fold, respectively, compared with the H2O2 group, indicating that NEAPs effectively reduced the generation of mtROS.

[0101] TEM was used to further examine the mitochondrial morphology and number in PDLSCs. Figure 3 As shown in F, the H2O2 group exhibited many damaged mitochondria with reduced and broken cristae compared with the control group. In contrast, the NRG-1 and EGCG groups showed newly formed small mitochondria. NE APs significantly reduced H2O2-induced damage, restored mitochondrial morphology to a level comparable to that of the control group, and enabled cells to maintain their basic functions. In addition, the number of mitochondria with normal morphology and cristae increased significantly in the NE APs group, further enhancing mitochondrial homeostasis. These findings suggest that NE APs protect growth factors and have strong antioxidant capacity, effectively scavenge ROS and protect mitochondrial function, highlighting their potential to control inflammation and promote tissue regeneration.

[0102] 2.4NE APs regulate the inflammatory response and polarization of macrophage RAW264.7 cells

[0103] The cells were treated with lipopolysaccharide (LPS, 100 ng / mL) to simulate an inflammatory environment, and the proportion of M1 macrophages was assessed by immunofluorescence staining and flow cytometry using iNOS as a marker. Figure 4 As shown in A, the expression level of iNOS was the highest in the LPS group, while that in the NRG-1 and EGCG groups was significantly decreased. It is worth noting that the iNOS expression level in the NE APs group was even lower than that in the NRG-1 and EGCG groups, which may be due to the synergistic effect of EGCG and NRG-1. The flow cytometry results were consistent, and the proportion of iNOS-positive macrophages in the NE APs group decreased from 80.7% to 53.5% ( Figure 4 B). RT-qPCR analysis further confirmed that NE APs downregulated the RNA expression levels of iNOS and proinflammatory cytokines IL-6, TNF-α, and CCL-2 ( Figure 4 C, D).

[0104] Next, the effect of NE APs on M2 macrophage polarization was evaluated. Figure 4 As shown in E and S16, there was no significant difference between the NRG-1 group and the control group. Interestingly, EGCG and NE APs significantly upregulated the expression of CD206, indicating that both EGCG and NE-APs promoted the polarization of macrophages M2. Flow cytometry results ( Figure 4F and G) confirmed that the NE APs group detected the highest proportion of CD206-positive cells. RT-qPCR analysis showed that NE APs significantly upregulated CD206 and the anti-inflammatory cytokine IL-10 ( Figure 4 H and I). These findings suggest that NE APs effectively enhance the polarization of M2 macrophages and contribute to the creation of an anti-inflammatory environment. In addition, the effect of NE APs on the secretion of proinflammatory cytokines from PDLSCs was evaluated under LPS-induced inflammatory conditions. RT-qPCR results showed that NE APs significantly downregulated the expression of IL-8 and IL-6 ( Figure 4 J). ELISA results further confirmed that the concentrations of IL-8 and IL-6 in the NEAPs group were significantly lower than those in the other groups ( Figure 4 K). These results indicate that NE APs significantly enhance the anti-inflammatory capacity of RAW264.7 and PDLSCs cells, thereby regulating the inflammatory response and promoting bone tissue regeneration.

[0105] 2.5 Anti-inflammatory mechanism mediated by NE APs

[0106] To investigate the anti-inflammatory mechanism of NE APs, whole transcriptome sequencing was performed on RAW264.7 cells treated with or without NE APs. The results of three independent experiments were highly consistent ( Figure 5 A total of 4195 differentially expressed genes (DEGs) were identified, of which 2009 genes were up-regulated and 2186 genes were down-regulated (false discovery rate / FDR<0.05, fold change / FC≥2), indicating that there were obvious transcriptomic differences between the NE APs group and the LPS group ( Figure 5 B). The top 20 DEGs are highlighted in the heat map, highlighting the significant associations of the changed genes ( Figure 5 C). Compared with the LPS group, cells treated with NE APs showed upregulation of the expression levels of anti-inflammatory genes Stc1 and Hsd11b2, while the pro-inflammatory gene F5 showed a downregulation trend. These observations suggest that NE APs enhance the resistance of cells to inflammatory challenges.

[0107] Furthermore, gene ontology (GO) analysis revealed that pathways associated with the DEGs were significantly enriched in biological processes related to inflammation, such as inflammatory response, positive regulation of IL-17 production, negative regulation of IL-2 production, and cellular response to IL-1, all of which were downregulated ( Figure 5 D). These findings suggest that NE APs can regulate inflammation by affecting various inflammation-related pathways. To better understand the biological functions of NE APs-induced DEGs, enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways ( Figure 5E) This analysis highlights the significant involvement of the TNF / NF-κB / JAK-STAT axis.

[0108] Gene set enrichment analysis (GSEA) further confirmed that DEGs were enriched in TNF, NF-κB and JAK-STAT signaling pathways ( Figure 5 FH). Meanwhile, as the core of TNF / NF-κB / JAK-STAT axis, the expression of key proteins related to NF-κB signaling pathway was evaluated by western blotting. After NE APs treatment, the expression levels of p-p65 and p-κBα were significantly downregulated ( Figure 5 I and J), indicating that NE APs effectively inhibited the NF-κB signaling pathway. In addition, NE APs also inhibited inflammation by regulating IL-17 and MAPK signaling pathways. These findings indicate that NE APs effectively reduce LPS-induced inflammatory responses. Therefore, the potent anti-inflammatory properties of NE APs will provide great therapeutic potential for inflammatory bone tissue repair.

[0109] 2.6NE APs promote osteogenesis of PDLSCs in vitro

[0110] In addition to controlling inflammation, osteogenic differentiation is essential for inflammatory bone regeneration. Alkaline phosphatase (ALP) expression is a key marker for osteoblast activity and the early stages of bone formation. To assess osteogenic activity, ALP staining and semiquantitative analysis ( Figure 6 A). PDLSCs treated with NE APs and EGCG showed similar strong staining after 7 and 14 days, which was significantly stronger than that of NRG-1 and control groups. These results suggest that EGCG promotes the early osteogenic differentiation of PDLSCs, and NE APs further enhance this effect.

[0111] To explore the role of NE APs in regulating osteogenic differentiation of PDLSCs at the mRNA level, RT-qPCR analysis of several osteogenesis-related genes including ALP, BMP2, and OPN was performed after 7 and 14 days of osteogenic induction ( Figure 6 B). Gene expression levels in the NRG-1 group were comparable to those in the control group, indicating that NRG-1 alone was unable to induce osteogenic differentiation. In contrast, the EGCG group showed a significant upregulation of osteogenic genes, consistent with the known osteogenic properties of EGCG. Notably, PDLSCs cultured with NE APs showed greater upregulation of osteogenic genes compared with the EGCG group.

[0112] Calcium deposition is a marker of osteoblast differentiation, and Alizarin Red S (ARS) staining was used to assess mineralization in different treatments ( Figure 6C and D). Calcium nodule formation increased in all groups at 21 and 28 days of induction. However, NE APs resulted in the richest staining and the largest number of calcium nodules. These findings were consistent with the ALP staining and RT-qPCR results, confirming that NE APs had excellent osteoinductive activity in vitro. These results suggest that the self-assembly of polyphenols with growth factors not only retains the osteoinductive potential of EGCG but also enhances its osteogenic activity. This improvement was attributed to NE APs, which promoted the sustained release of EGCG while improving its stability.

[0113] These results collectively indicated that NE APs achieved the highest regulatory efficiency in promoting NRG-1-driven cell recruitment, migration, and angiogenesis, and effectively enhanced the anti-inflammatory, antioxidant, ROS scavenging, and osteogenic differentiation effects of EGCG on PDLSCs.

[0114] 2.7 In vivo therapeutic effects

[0115] Given the positive results observed in vitro, we explored the bone regeneration capacity of NE APs in a periodontitis-induced bone loss model in vivo, which was established in rats by local injection of LPS and silk ligation to induce inflammatory bone defects. Figure 7 As shown in A, the periodontitis (Perio) group showed significant alveolar bone resorption compared with the control group, and the distance from the cementum-cementum junction to the alveolar bone ridge (CEJ-ABC) increased to 345 μm, confirming the successful construction of the periodontitis model. Although both NRG-1 and EGCG partially inhibited alveolar bone resorption, the NE APs group showed the most effective inhibitory effect. After 4 weeks of treatment, the defects in the NE APs group were almost completely restored, and the CEJ-ABC distance (90.5 μm) was similar to that of the normal group (75.7 μm). In addition, the quality of the newly formed bone was evaluated by measuring the bone volume fraction (BV / TV), trabecular separation (Tb.Sp), and bone surface area to volume ratio (BS / TV) ( Figure 7 B). Among the four groups, the NE APs group showed the highest BV / TV and BS / TV values, and the lowest Tb-Sp value. Notably, the three indicators in the NE APs group almost reached normal levels after four weeks. These findings highlight the significant advantages of NE APs in promoting alveolar bone regeneration.

[0116] H&E staining was performed to evaluate the periodontal healing at the furcation of the second molars and confirmed histological improvement ( Figure 7C). In the NE APs group, a large amount of new bone tissue was formed along the defect borders two weeks after surgery, and almost all the residual defects were filled within four weeks. In contrast, new bone formation was limited in the other groups. In particular, in the Perio group, a large amount of fibrous connective tissue and inflammatory cell infiltration were present at the defect site. CEJ-ABC distance measurement of H&E staining results ( Figure 7 D) reflects the trend observed by micro-CT.

[0117] Masson staining and quantitative analysis of collagen volume fraction showed that the NE APs group achieved significant therapeutic effects in periodontal tissue repair, characterized by a thicker epithelial layer and more densely arranged collagen fibers ( Figure 7 E). Immunohistochemical staining (IHC) of COL I was then performed to evaluate alveolar bone repair ( Figure 7 F). The uniform brown staining indicated a high content of COL I in the newly regenerated tissue in the NE APs group, and the staining intensity was significantly stronger compared with the other groups. These findings suggest that NE APs provide the most effective treatment outcomes for periodontal tissue repair.

[0118] To elucidate the regenerative process, we first evaluated whether NE APs could modulate the M1 / M2 phenotype of macrophages to establish a local pro-regenerative immune microenvironment ( Figure 8 AC). IHC was used to analyze the expression of M1 macrophages (marked by CD80) and M2 macrophages (marked by CD206) within periodontal defects. At 2 weeks after surgery, the Perio group showed the highest number of M1 macrophages, which remained elevated at 4 weeks, indicating a prolonged inflammatory response. Both the NRG-1 and EGCG groups showed a partial decrease in the levels of M1 macrophages at 2 and 4 weeks. In contrast, the expression of M1 macrophages in the NE APs group was significantly reduced. Regarding M2 macrophages, although there was an increase in the Perio, NRG-1, and EGCG groups at 2 and 4 weeks, the number of M2 macrophages in the NE APs group gradually normalized at 4 weeks, approaching a level similar to that of the control group. These findings suggest that NE APs effectively regulate the immune microenvironment, reduce inflammation, and promote tissue healing. The recruitment of CD44+CD34-PDLSCs in the defect area was further investigated by double immunofluorescence staining ( Figure 8D). EGCG led to a slight increase in stem cell recruitment compared with the control and Perio groups, while NRG-1 significantly enhanced the green fluorescence intensity, reflecting its strong stem cell recruitment ability. More notably, the green fluorescence intensity of the NE APs group was significantly higher than that of the NRG-1 group. This enhancement can be attributed to the unique function of NE APs, which can continuously release NRG-1 and protect it, thereby enhancing its stem cell recruitment ability. Quantitative analysis showed that CD44 expression was significantly increased in the NE APs group compared with the other treatment groups, indicating that PDLSCs were heavily enriched in the bone defect area. NE APs effectively deliver NRG-1 and promote the directional migration of endogenous PDLSCs to the bone defect site, thereby creating a microenvironment conducive to the osteogenic activity of EGCG and ultimately enhancing bone regeneration.

[0119] The role of NE APs in promoting angiogenesis in vivo was also studied by targeting the angiogenesis-related marker α-SMA for immunofluorescence staining ( Figure 8 E and F). The NE APs group showed the highest α-SMA expression level, indicating enhanced angiogenesis compared with the other groups. Quantitative analysis confirmed increased angiogenesis in the NE APs group. These results suggest that NE APs modulate the immune microenvironment, recruit endogenous stem cells, promote angiogenesis, and accelerate alveolar bone repair and regeneration. In addition, H&E staining of major organs and routine blood tests showed that NE APs have good biosafety and biocompatibility, further supporting their potential for treating inflammatory bone defects.

[0120] It should be noted that the above examples are only used to illustrate the technical solution of the present embodiment and are not intended to limit it. Although the present embodiment is described in detail with reference to the given examples, a person of ordinary skill in the art may modify or replace the technical solution of the present embodiment as required without departing from the spirit and scope of the technical solution of the present embodiment.

Claims

1. A polyphenol-protein self-assembly material, characterized in that: The raw materials of the polyphenol-protein self-assembly material at least include epigallocatechin gallate and neuregulin-1; The mass ratio of epigallocatechin gallate to neuregulin-1 is 1-50:0.

1.

2. The polyphenol-protein self-assembly material according to claim 1, characterized in that: The mass ratio of epigallocatechin gallate to neuregulin-1 is 10-30:0.1, further 20:0.

1.

3. The method for preparing the polyphenol-protein self-assembly material according to claim 1 or 2, characterized in that: The preparation method comprises the steps of mixing an epigallocatechin gallate solution and a neuregulin-1 solution, stirring the mixture and centrifuging the mixture to obtain the product.

4. The preparation method according to claim 3, characterized in that: The concentration of the epigallocatechin gallate solution is 1-50 mg / mL, more preferably 20 mg / mL; the concentration of the neuregulin-1 solution is 0.01-1 mg / mL, more preferably 0.1 mg / mL.

5. The preparation method according to claim 3, characterized in that: When mixed, the volume ratio of the two is 0.1-10:1, preferably 1:1; Furthermore, during the mixing and stirring process, the specific stirring conditions are: stirring at 600-1000 rpm for 0.1-5 min; preferably stirring at 800 rpm for 1 min.

6. The preparation method according to claim 3, characterized in that: The specific conditions of centrifugation are: centrifugation at 10000-15000 rpm for 1-10 min, preferably centrifugation at 12000 rpm for 5 min.

7. Use of the polyphenol-protein self-assembly material according to any one of claims 1 to 2 or the polyphenol-protein self-assembly material prepared by the preparation method according to any one of claims 3 to 6 in preparing an inflammatory bone defect repair product.

8. The use according to claim 7, characterized in that The inflammatory bone defect repair is specifically manifested as: (a) Neuregulin-1 can be sustainably released from polyphenol-protein self-assembled materials, and epigallocatechin gallate protects neuregulin-1 from oxidative stress, maintaining its biological activities related to stem cell recruitment, migration, and angiogenesis; (b) Polyphenol-protein self-assembly materials utilize the ability of epigallocatechin gallate to scavenge ROS, maintain mitochondrial homeostasis, and promote osteogenic differentiation, while synergistically regulating immune responses through the TNF / NF-κB / JAK-STAT signaling pathway; (c) Polyphenol-protein self-assembly materials enhance the body's stem cell recruitment, angiogenesis and immune regulation capabilities.

9. The use according to claim 7, characterized in that: The product described is a drug.

10. The use according to claim 9, characterized in that: The drug further comprises at least one non-drug active ingredient; and the drug dosage form is an injection dosage form.

Images