Application of puerarin in treatment of periodontitis

Abstract

The invention provides an application of puerarin in treatment of periodontitis. The molecular mechanism of puerarin (Pue) for treating periodontitis (PDS) is verified through a plurality of in-vitro and in-vivo experimental studies. In PDS mice, Pue treatment relieves periapical tissue inflammation and bone destruction, and promotes osteogenic differentiation and autophagy of periapical tissues. In a PDS cell model, Pue treatment promotes osteogenic differentiation and mitochondrial autophagy, inhibits inflammation, maintains mitochondrial membrane potential and mitochondrial dynamic homeostasis, and activates mitochondrial Mfn2. In addition, the mitochondrial autophagy inhibitor eliminates the influence of Pue on a PDS cell model, and Mfn2 silence reverses mitochondrial autophagy enhancement induced by Pue. In-vivo tests verify that Mfn2 knockout counteracts the therapeutic effect of Pue on PDS mice. The invention provides a sufficient basis and an exact molecular mechanism for application of puerarin in treatment of periodontitis.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the application of puerarin in the treatment of periodontitis. Background Art

[0002] Periodontitis (PDS) is a common chronic disease worldwide, resulting from periodontal infection and pathogen invasion. If not treated in time, it will eventually lead to loosening and loss of teeth. Its typical characteristics are inflammation and destruction of periodontal tissues and resorption of alveolar bone. PDS is not only the main cause of tooth loss, but is also associated with a variety of systemic diseases. The treatment of PDS includes surgery and drug therapy, aiming to control inflammation, promote osteogenic differentiation and prevent disease progression. However, current treatments are still unsatisfactory, and finding effective treatment strategies is crucial to improving PDS.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] Based on the problems of the prior art, the object of the present invention is to provide the use of puerarin in the treatment of periodontitis.

[0005] Puerarin (Pue) is a natural flavonoid compound extracted from the traditional Chinese medicine Pueraria root, which has multiple pharmacological activities such as anti-inflammatory, anti-cardiovascular disease, anti-ischemia-reperfusion injury, anti-oxidative stress, neuroprotection, anti-osteoporosis and reducing insulin resistance. Due to its unique advantages such as low toxicity, safety, few side effects and low cost, it is widely used to treat various diseases. The present invention studies the therapeutic potential of puerarin for periodontitis.

[0006] Human periodontal ligament cells (hPDLCs) can induce osteogenic differentiation and bone regeneration, but their damage can lead to the progression of PDS and poor treatment effects. Pue ​​can promote the proliferation and osteogenic differentiation of hPDLCs, while reducing alveolar bone loss in PDS rats, alleviating bone loss and collagen destruction. These findings provide an important theoretical basis for the application of Pue in the clinical treatment of PDS.

[0007] Mitochondria are the structural basis and energy source for osteogenic differentiation of stem cells, and mitochondrial homeostasis is necessary for stem cells to achieve osteogenic differentiation. In PDS, mitochondrial dysregulation induced by microbial stimulation and inflammation, especially imbalance of mitochondrial autophagy, is one of the most important reasons for the destruction of stem cell osteogenic differentiation ability. Therefore, mitochondrial autophagy is considered to be a potential therapeutic target for bone-related diseases including PDS. In a lipopolysaccharide (LPS)-induced cellular inflammation model, Pue was found to be able to alleviate mitochondrial damage by promoting mitochondrial autophagy.

[0008] The first aspect of the present invention provides the use of puerarin in the treatment of periodontitis. The puerarin can be used to treat periodontitis.

[0009] Specifically, the puerarin promotes mitochondrial autophagy by activating mitochondrial Mfn2 (Mitofusin 2) to treat periapical inflammation and bone destruction of periodontitis, and promotes osteogenic differentiation of periapical tissue.

[0010] Specifically, the puerarin has an inhibitory effect on the pro-inflammatory factors TNF-α, IL-1β and IL-6 in periapical tissue.

[0011] Specifically, the puerarin has a promoting effect on the expression of Runx2, OSX, OPN, OCN, ALP and COL1A1 genes.

[0012] Specifically, the puerarin regulates the activity of the PI3K / AKT / mTOR pathway through Mfn2.

[0013] Preferably, the dosage of puerarin is 5-10 mg / kg, more preferably 10 mg / kg.

[0014] The second aspect of the present invention provides the use of puerarin in preparing a drug for treating periodontitis, wherein the drug for treating periodontitis contains puerarin.

[0015] The third aspect of the present invention provides a pharmaceutical composition for treating periodontitis, wherein the main active ingredient of the pharmaceutical composition is puerarin.

[0016] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0017] In a fourth aspect, the present invention provides an Mfn2 agonist, wherein the main active ingredient of the agonist is puerarin.

[0018] The present invention has the following beneficial effects:

[0019] The present invention verifies the molecular mechanism of puerarin (Pue) in treating periodontitis (PDS) through multiple in vitro and in vivo experimental studies. In PDS mice, Pue treatment alleviated periapical tissue inflammation and bone destruction, and promoted osteogenic differentiation and autophagy of periapical tissue. In the PDS cell model, Pue treatment promoted osteogenic differentiation and mitochondrial autophagy, inhibited inflammation, maintained mitochondrial membrane potential and mitochondrial dynamics homeostasis, and activated mitochondrial Mfn2. In addition, mitochondrial autophagy inhibitors eliminated these effects of Pue on the PDS cell model, and Mfn2 silencing reversed the enhanced mitochondrial autophagy induced by Pue. It was verified by in vivo experiments that Mfn2 knockout offset the therapeutic effect of Pue on PDS mice. The present invention provides a sufficient basis and a definite molecular mechanism for the application of puerarin in the treatment of periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The results of Pue treatment improved PDS in mice; A is the establishment of PDS mouse model; BC is the analysis of gingival bleeding index and tooth mobility of mice; DE is the micro-CT image of mice and CEJ-ABC distance analysis, BV / TV, Tb.N and Tb.Sp; F is the pathological changes of mouse periapical tissue observed by H&E staining; GJ is the use of immunohistochemistry of mouse periapical tissue to explore osteogenic differentiation ability; KM is the ELISA method to detect pro-inflammatory factors in mouse periapical tissue; N is the qRT-PCR detection of the expression of osteogenic-related genes in mouse periapical tissue. *P<0.05, **P<0.01, ***P<0.001, and the "ns" symbol indicates no significant difference.

[0022] Figure 2 The results of Pue treatment enhanced the osteogenic differentiation ability of the PDS cell model, but inhibited the inflammatory response; A is the subculture of hPDLCs; B is the identification of hPDLCs by flow cytometry; C is the evaluation of the osteogenic and adipogenic differentiation ability of hPDLCs by Alizarin Red staining and Oil Red O staining; D is the study of the cytotoxicity of Pue on hPDLCs by CCK-8 method; E is the determination of the optimal dose of Pue on hPDLCs by CCK-8; FI is the study of the osteogenic differentiation ability of hPDLCs by Alizarin Red staining and ALP staining; J is the ELISA detection of pro-inflammatory factors in hPDLCs; K is the qRT-PCR evaluation of hPDLCs for the expression of osteogenic-related genes. *P<0.05, **P<0.01, ***P<0.001, and the "ns" symbol indicates no significant difference.

[0023] Figure 3Pue treatment restored mitochondrial autophagy in the PDS cell model; AB was used to study the changes in mitochondrial membrane potential of hPDLCs using JC-1 staining; CD was used to monitor the mitochondrial ROS level in hPDLCs by MitoSox red staining; EF was used to detect the expression of autophagy-related proteins in hPDLCs and mitochondria by Western blot; GH was used to show LC3 dots in hPDLCs by mRFP-eGFP-LC3 assay; IJ was used to show LC3 dots in hPDLCs by Mito Tracker staining. *P<0.05, **P<0.01, ***P<0.001, and the "ns" symbol indicates no significant difference.

[0024] Figure 4 Pue promotes osteogenic differentiation of PDS cell model by enhancing mitochondrial autophagy, but reduces inflammation. AD is the osteogenic differentiation ability of hPDLCs detected by Alizarin red staining and ALP staining; EG is ELISA used to detect pro-inflammatory factors in hPDLCs; HI is JC-1 staining used to monitor changes in mitochondrial membrane potential of hPDLCs; J is the detection of osteogenic-related genes in hPDLCs by qRT-PCR; KL is the evaluation of mitochondrial ROS levels in hPDLCs by MitoSox red staining. *P<0.05, **P<0.01, ***P<0.001.

[0025] Figure 5 The Pue treatment maintained the steady state of mitochondrial dynamics in the PDS cell model; AE is the detection of mitochondrial dynamics-related factors in hPDLCs by qRT-PCR; FG is the evaluation of Mfn2 and Drp1 proteins in the cytoplasm and mitochondria of hPDLCs by Western blot; HI is Mito Tracker staining for studying mitochondrial fission in hPDLCs. *P<0.05, **P<0.01, ***P<0.001, and the "ns" symbol indicates no significant difference.

[0026] Figure 6The results of Pue treatment enhancing mitochondrial autophagy in the PDS cell model by promoting Mfn2 expression; AB is Western blot used to monitor the expression of autophagy-related proteins in hPDLCs and mitochondria; CD is Western blot to detect the activity of PI3K / AKT / mTOR pathway in hPDLCs; EF is JC-1 staining responsible for observing the changes in mitochondrial membrane potential of hPDLCs; GH is MitoSox red staining used to detect mitochondrial ROS levels in hPDLCs; IJ is mRFP-eGFP-LC3 assay used to explore LC3 dots in hPDLCs; KL is Parkin / Tomm20 immunofluorescence staining of hPDLCs to evaluate mitochondrial autophagy. *P<0.05, **P<0.01, ***P<0.001.

[0027] Figure 7 The results of Pue treatment promoting mitochondrial autophagy by activating mitochondrial Mfn2, alleviating periapical inflammation and bone destruction in PDS mice, and inducing osteogenic differentiation of periapical tissues; AB is micro-CT images of mice and gingival sulcus bleeding index, tooth mobility, CEJ-ABC distance analysis, BV / TV and Tb.Sp; C is H&E staining of mouse periapical tissues to observe pathological changes; D is the evaluation of proinflammatory factors by ELISA of mouse periapical tissues; EH is the immunohistochemistry of mouse periapical tissues responsible for detecting the expression of RUNX2 and LC3 proteins; I is the expression of osteogenic-related genes detected by qRT-PCR of mouse periapical tissues; J is the performance of protein blotting to monitor mitochondrial autophagy-related proteins in mouse periapical tissues; K is the evaluation of the expression of autophagy-related proteins in mouse periapical tissues by Western blot. *P<0.05, **P<0.01, ***P<0.001, and the "ns" symbol indicates no significant difference. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. 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 application belongs.

[0029] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this description, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0030] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] 1. Materials and methods

[0032] C57BL / 6N mice (10 weeks old, 20-25 g, n = 168) were commercially supplied by Vital River Laboratory Animal Technology (Beijing, China). All mice were housed in a specific pathogen-free room (12-hour day-night cycle) at 22 ± 2 °C and allowed free access to food and water. Purulin injection was purchased from Yuanye Biotech (B20446; Shanghai, China).

[0033] This experiment was approved by the Animal Welfare Ethics Committee of Beijing MDKN Biotechnology Co., Ltd. (MDKN-2023-048) and was performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the Beijing Municipal Animal Management Committee.

[0034] (II) Animal PDS modeling, Pue treatment and adenovirus transfection

[0035] Construction of the PDS mouse model Mice were anesthetized with 2% isoflurane and then placed supine on the operating table. A 5-0 silk thread moistened with Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) (10 μg / mL) was tied into a knot and placed between the first and second molar space on the right side of the maxilla. The silk thread was cut on the outside of the knot so that the knot was located buccal-lingually between the first and second molar space. Porphyromonas gingivalis was purchased from Ningbo Biotechnology (B290826; Ningbo, China), and LPS was provided by Sigma (L2630; St. Louis, MO, USA).

[0036] Next, the mice were treated with Pue using a microsyringe. The injection sites were: the buccal papilla between the first and second molars on the right side of the maxillary teeth and the top of the palatal alveolar ridge. Pue ​​(diluted in 5 μL phosphate buffer solution) was injected at doses of 5 mg / kg and 10 mg / kg, respectively. After injection, the microsyringe was kept at the injection site for 1 minute to prevent the drug from flowing out. Pue ​​treatment was performed every two days. After 10 days of treatment, the mice were euthanized.

[0037] Mouse adenovirus transfection was as follows: Adenovirus-based Mfn2-siRNA and Mfn2-siRNA negative control (NC) were provided by GeneChem (Shanghai, China) and injected into mice separately 7 days before PDS modeling. The adenovirus injection site was the same as the Pue injection site.

[0038] (III) Animal grouping and special treatment

[0039] The in vivo study includes two parts: Pue treatment efficacy study and Pue treatment mechanism study.

[0040] In the Pue treatment efficacy study, 84 mice were randomly selected and divided into four groups: control group (n=21), PDS group (n=21), PDS+Pue 5mg / kg treatment group (n=21) and PDS+Pue 10mg / kg treatment group (n=21). The mice in the control group received a treatment process similar to that of PDS modeling, but the 5-0 silk thread was not moistened with Pg-LPS; the mice in the PDS group underwent PDS modeling; the PDS+Pue 5mg / kg treatment group and the PDS+Pue 10mg / kg treatment group were first subjected to PDS modeling and then treated with Pue for 10 days.

[0041] The remaining 84 mice were used for Pue treatment mechanism study and were divided into control group (n=21), PDS group (n=21), PDS+Pue+siMfn2-NC group (n=21) and PDS+Pue+siMfn2 group (n=21). The mice in the control group and PDS group were treated as described above. It is worth noting that the cells in the PDS+Pue+siMfn2-NC group and the PDS+Pue+siMfn2 group were first transfected with adenovirus-based Mfn2-siRNA-NC and Mfn2-siRNA, respectively. Seven days after transfection, PDS modeling and 10 days of Pue treatment were performed.

[0042] After 10 days of Pue treatment, all mice were deeply anesthetized with 5% isoflurane and then euthanized. The right maxilla of each mouse was collected for subsequent experiments.

[0043] (IV) Gum Bleeding Index and Tooth Mobility

[0044] The gingival sulcus bleeding index of mice was evaluated by a blunt periodontal probe. The measurement was performed at two sites, namely the distal buccal and palatal sides of the maxillary first molar. The evaluation criteria were: 0 points, healthy gingiva, no bleeding on probing; 1 point, bleeding on probing, but no edema or color change of the gingival papilla and marginal gingiva; 2 points, bleeding on probing, color change of the gingival papilla and marginal gingiva, but no edema; 3 points, bleeding on probing, color change of the gingival papilla and marginal gingiva, mild edema; 4 points, bleeding on probing, color change of the gingival papilla and marginal gingiva, and obvious edema; 5 points, bleeding on probing, spontaneous bleeding and color change of the gingival papilla and marginal gingiva, and obvious edema.

[0045] The tooth mobility of the maxillary right first molar was observed. In short, the tip of the forceps was placed against the maxillofacial fossa, and then the forceps were shaken in the buccolingual or proximal-distal-medial direction. The tooth mobility was evaluated according to the direction of loosening. 0 points, normal tooth physiological activity; 1 point, tooth loosening in the buccolingual direction; 2 points, tooth loosening in the buccolingual and proximal-distal-medial directions; 3 points, tooth loosening in the buccolingual, proximal-distal-medial and vertical directions.

[0046] (V) Micro-CT analysis

[0047] The right maxillary bone samples of mice were fixed in 4% paraformaldehyde (Biolab Technology, Beijing, China) for 24 h. Micro-CT scanning of the right maxillary bone was performed using a Skyscan 1176 X-ray microtomography scanner (Bruck, Karlsruhe, Germany). The scanning parameters were: voltage 70 kV, current 200 μA, exposure time 270 ms, rotation angle 360°, and scanning layer thickness 34.4 μm. The data were reconstructed by Mimics software (Materialise, Leuven, Belgium) to obtain three-dimensional images. The distance between the cementum-alveolar junction (CEJ) and the alveolar ridge (ABC), as well as the bone volume fraction (BV / TV), the number of trabeculae (Tb.N), and the trabecular spacing (Tb.Sp) were analyzed.

[0048] (VI) Hematoxylin-eosin (H&E) staining

[0049] The right maxillary bone specimen fixed with 4% paraformaldehyde was immersed in a 10% ethylenediaminetetraacetic acid (EDTA) decalcification solution for two weeks. After routine dehydration, the right maxillary bone specimen was embedded in paraffin to prepare sections (4 μm thick). The sections were routinely dewaxed and rehydrated in sequence. Next, the sections were stained with a hematoxylin solution (Biolab Technology, Beijing, China) for 5 minutes and then stained with an eosin solution (Biolab Technology, Beijing, China) for 2 minutes. After routine dehydration and transparency, the sections were wrapped in a neutral resin and observed under an optical microscope (Olympus, Tokyo, Japan).

[0050] 7. Immunohistochemistry

[0051] The prepared right maxillary bone specimen sections were routinely dewaxed and rehydrated. After boiling in citrate buffer for 10 min, the sections were blocked in normal goat serum (Beyotime Biotechnology, Shanghai, China) for 30 min at room temperature. Rabbit anti-RUNX2 (1:100, ab114133, Abcam, Cambridge, UK) and anti-LC3 (1:100) primary antibodies (ab62721, Abcam) were dropped on the sections, respectively, probed at 4°C for 12 h, and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:200, ab6721, Abcam (Cambridge, Britain)) for 30 min at room temperature. After 3,3′-diaminobenzidine (DAB) (Beyotime Biotechnology, Shanghai, China) color reaction and hematoxylin counterstaining, the sections were dried, cleared, and finally exposed in neutral resin. The expression of RUNX2 and LC3 was observed under an optical microscope (Olympus, Tokyo, Japan). The identification of RUNX2 and LC3 expression was performed by ImageJ software (ImageJ, NIH, Bethesda, MD, USA).

[0052] (VIII) Cultivation and identification of hPDLCs

[0053] hPDLCs were purchased from Procell Life Science & Technology (Wuhan, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% penicillin / streptomycin (Beyotime Biotechnology, Shanghai, China) and 10% fetal bovine serum (FBS) (Beyotime Biotechnology (Shanghai, China)) at 37°C and 5% CO2.

[0054] hPDLCs were subcultured for three times, and flow cytometry was used to detect hPDLCs markers, including positive markers CD44 and CD90, and negative markers CD34 and CD45, to identify hPDLCs. Briefly, hPDLCs were incubated with antibodies against CD44 (1:100, ab157107, Abcam, Cambridge, UK), CD90 (1:100), CD34 (1:100, ab78165, Abcam, Cambridge, UK), and CD45 (1:100; ab23910, Abcam) for 30 min at 4°C. Signals of CD44, CD90, CD34, and CD45 were detected by flow cytometry (FACSCalibur, BD, Franklin Lakes, NJ, USA) and analyzed by FlowJo software (Tree Star Inc., Ashland, OR, USA).

[0055] IX. Transfection and treatment of hPDLCs

[0056] hPDLCs dispersed in serum-free DMEM were plated at 1 × 10 per well. 5 hPDLCs were plated at a density of 10 cells / mL in 6-well plates. Mfn2-siRNA and corresponding NC (GeneChem, Shanghai, China) were transfected into hPDLCs using Lipofectamine 3000 reagent (Thermo Fisher Scientific, San Jose, CA, USA). After transfection, hPDLCs were cultured in DMEM containing 10% FBS.

[0057] The construction of the PDS cell model was achieved by inducing hPDLCs with 1 μg / mL Pg-LP5.

[0058] To detect cytotoxicity, hPDLCs were treated with Pue at final doses of 10, 20, 50, 100, and 200 μM. To determine the optimal dose of Pue for treating PDS cell models, hPDLCs were treated with different doses of Pue (10, 20, 50, 100, and 200 μM) in the presence of Pg-LPS (1 μg / mL).

[0059] In the next efficacy study and mechanism study of Pue, the following hPDLCs groups were set up in this example: control group (hPDLCs cultured under normal conditions), Pue group (hPDLCs treated with 20 μM Pue only), LPS group (hPDLCs induced by 1 μg / mL Pg-LPS only), LPS+Pue group (hPDLCs treated with 20 μM Pue in the presence of 1 μg / mL Pg-LPS), LPS+Pue+CsA group+Pue+siMfn2-NC group (hPDLCs transfected with Mfn2-siRNA NC, then treated with 20 μM Pue in the presence of 1 μg / mL Pg-LPS), and LPS+Pue+siMfn2 group (hPDLCs transfected with M8n2-siRNA, then treated with 20 μM Pue in the presence of 2 μg / mL Pg LPS). Each group of hPDLCs was cultured under relevant conditions of 37° C. and 5% CO2.

[0060] (X) Alizarin red staining and alkaline phosphatase (ALP) staining

[0061] The cells were cultured in 1 mL of osteogenic induction medium (DMEM containing 10% FBS, 10 mM dexamethasone, 50 mg / L ascorbic acid, and 10 mM β-glycerophosphate) at 37°C and 5% CO2. 3 hPDLCs in 4% paraformaldehyde (400 cells / mL) were induced to differentiate into osteoblasts. The culture medium was refreshed every three days. After 14 days of osteogenic differentiation induction, hPDLCs were fixed with 4% paraformaldehyde for 20 minutes and then stained with 2% Alizarin Red staining solution (Biolab Technology, Beijing, China) for 30 minutes at room temperature. The residual staining solution was washed with phosphate buffered saline (PBS). hPDLCs were then observed and photographed under an optical microscope (Olympus, Tokyo, Japan). Alizarin Red staining was qualified by detecting the optical density (OD) value at 405 nm under a microplate reader (Biotek, Winooski, VT, USA).

[0062] For ALP staining, hPDLCs were stained with ALPworkin3 solution (BiolabTechnol5gy, Beijing, China) for 20 minutes after osteogenic differentiation induction and fixation. After PBS washing, hPDLCs were photographed under an optical microscope (Olympus, Tokyo, Japan). ALP activity was determined by OD value detected at a wavelength of 520 nm in a microplate reader (Biotek, Winooski, VT, USA).

[0063] (XI) Oil Red O staining

[0064] hPDLCs (5 × 10 per well) were placed in a 6-well plate. 3 hPDLCs were stained with 10% FBS, 10 μM insulin, 100 nM dexamethasone, 500 μM 3-isobutyl-1-methylxanthine and 200 μM indomycin for 14 days to induce adipogenic differentiation. The culture medium was changed every three days. After fixation with 4% polyformaldehyde, hPDLCs were stained with Oil Red O staining solution (Biolab Technology, Beijing, China) at room temperature for 30 minutes. After washing with PBS, hPDLCs were placed under an optical microscope (Olympus, Tokyo, Japan) to observe lipid droplets.

[0065] (XII) Cell counting kit-8 (CCK-8) assay

[0066] In a 96-well plate (5 × 10 3 After plating in 100 μL PBS (100 μL / mL), hPDLCs were cultured under the respective treatment conditions for 24 and 48 hours. At these two time points, the culture of hPDLCs was terminated and treated with 10 μL CCK-8 solution (Biolab Technology, Beijing, China) for 2 hours at 37°C. The survival rate of hPDLCs was checked by measuring the OD value at 450 nm under a microplate reader (Biotek, Winooski, VT, USA).

[0067] (XIII) JC-1 staining, MitoSox red staining and Mito3Tracker staining

[0068] In each treatment condition, 6-well plates (1×10 5 Cells / m 3 ) for 48 hours. To observe the changes in mitochondrial membrane potential, JC-1 staining solution (2 μg / mL) (Beyotime Biotechnology, Shanghai, China) was added to each well and incubated in the dark at 37°C for 30 minutes. Fluorescence images of hPDLCs were captured under a fluorescence microscope (Olympus, Tokyo, Japan). Red fluorescence indicates normal mitochondrial membrane potential, while green fluorescence indicates low mitochondrial wall potential. Fluorescence intensity was checked by ImageJ software (ImageJ, NIH, Bethesda, MD, USA).

[0069] In addition, mitochondrial reactive oxygen species (ROS) levels in hPDLCs were monitored by MitoSox red staining. After relevant treatment, hPDLCs were probed with 5 μM MitoSox Red staining solution (Yisheng Biotechnology, Shanghai, China) for 15 minutes in the dark at 37 ° C. Cell nuclei were stained by staining with 4,6-diamidino-2-phenylindole (DAPI) solution (Beyotime Biotechnology, Shanghai, China) for 5 minutes. MitoSox red staining was observed under a fluorescence microscope (Olympus, Tokyo, Japan) at an excitation wavelength of 510 nm. The intensity of MitoSox red staining was evaluated by Image J software (ImageJ, NIH, Bethesda, MD, USA), which was used to evaluate mitochondrial ROS levels.

[0070] As for Mito Tracker staining, hPDLCs5 were infected with GFP-LC3 lentivirus (GeneChem, Shanghai, China) for 5 h and then stained with Mito Tracker stain (Beyotime Biotechnology, Shanghai, China) for 30 min in the dark at 37 °C. DAPI was used for nucleus staining. GFP-LC3 fluorescence (green) and Mito Tracker fluorescence (red) were observed under a fluorescence microscope (Olympus, Tokyo, Japan). LC3 dots and Mito aspect ratios were determined by Image J software (ImageJ, NIH, Bethesda, MD, USA).

[0071] (XIV) mRFP-eGFP-LC3 assay

[0072] hPDLCs were transfected with mRFP-eGFP5LC3 plasmid (GeneChem, Shanghai, China) using Lipofectamine 3000 reagent. After transfection, hPDLCs were cultured under relevant treatment conditions for 48 h. After culture termination, hPDLCs were stained with DAPI solution for 5 min. LC3 dots (green dots) were captured under a fluorescence microscope (Olympus, Tokyo, Japan) and identified by Image J software (ImageJ, NIH, Bethesda, MD, USA).

[0073] (XV) Parkin / Tomm20 immunofluorescence staining

[0074] After 48 h of treatment under the above conditions, hPDLCs of each group were probed with rabbit anti-Parkin (1:100, ab315376, Abcam, Cambridge, UK) and mouse anti-TOMM20 (1:100, ab56783, Abcam) primary antibodies at 4 °C overnight. Subsequently, goat anti-rabbit (1:200, ab150077, Abcam, Cambridge, UK) conjugated with Fluor488 and goat anti-mouse (1:200, ab150116, Abcam) conjugated with AlexaFluor594 were used for reaction at room temperature for 2 h, respectively. DAPI was responsible for nuclear staining. The colocalization of Parkin and TOMM20 was observed under a fluorescence microscope (Olympus, Tokyo, Japan), which was identified by Image J software (ImageJ, NIH, Bethesda, MD, USA).

[0075] (16) Enzyme-linked immunosorbent assay (ELISA)

[0076] Root apical tissues of mice and 5-hPDLCs cultured under relevant conditions for 48 hours were collected and lysed in lysis buffer. The lysate samples were centrifuged at 12000 rpm for 10 minutes to collect the supernatant. Proinflammatory factors in the supernatant, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), were detected using commercial TNF-α detection kits, IL-1β detection kits, and IL-6 detection kits (Elabscience Biotechnology, Wuhan, China). The test process was strictly performed according to the instructions of the commercial kits.

[0077] (17) Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR)

[0078] Mouse periapical tissues and hPDLCs treated under relevant conditions for 48 h were collected and total RNA was extracted using TRIzol reagent (Beyotime Biotechnology, Shanghai, China). A total of 1 μg of total RNA samples were collected for reverse transcription using the PrimeScript RT kit (Takara, Tokyo, Japan). qRT-PCR was performed on an Agilent StrataGene Mx3005P detection system (Santa Clara, CA, USA) using Premix Ex TaqTMII (Tokyo Highlands, Japan). The reaction program was: 95°C for 30 s, 95°C for 5 s, and 60°C for 30 s for 40 cycles. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. 2-ΔΔCT The relative mRNA expression of genes was determined by the PCR method. The primers are as follows:

[0079] Runt-related gene 2(Runx2):

[0080] 5’-CATGGCCGGGAATGATGAG-3’(SEQ ID NO:1),

[0081] 5’-TGTGAAGACCGTTATGGTCAAAGTG-3’(SEQ ID NO:2);

[0082] Osterix(OSX):

[0083] 5’-CATCCATGCAGGCATCTCA-3’(SEQ ID NO:3),

[0084] 5’-CTGCCCACCACCTAACCAA-3’(SEQ ID NO:4);

[0085] Osteopontin(OPN):

[0086] 5’-GACAGCAACGGGAAGACC-3’(SEQ ID NO:5),

[0087] 5’-CAGGCTGGCTTTGGAACT-3’(SEQ ID NO:6);

[0088] Osteocalcin(OCN):

[0089] 5’-GAGGGCAGTAAGGTGGTGAA-3’(SEQ ID NO:7),

[0090] 5’-CGTCCTGGAAGCCAATGTG-3’(SEQ ID NO:8);

[0091] ALP:

[0092] 5’-CATCGCCTATCAGCTAATGCACA-3’(SEQ ID NO:9),

[0093] 5’-ATGAGGTCCAGGCCATCCAG-3’(SEQ ID NO:10);

[0094] COL1A1:

[0095] 5’-GACATGTTCAGCTTTGTGGACCTC-3’(SEQ ID NO:11),

[0096] 5’-AGGGACCCTTAGGCCATTGTGTA-3’(SEQ ID NO:12);

[0097] Optic atrophy 1(OPA1):

[0098] 5’-TTAGCCCTGAGACCATATCCT-3’(SEQ ID NO:13),

[0099] 5’-CAGCATCCACAGATCCATCT-3’(SEQ ID NO:14);

[0100] Fission 1protein(Fis1):

[0101] 5’-TGAACGAGCTGGTGTCTGT-3’(SEQ ID NO:15),

[0102] 5’-CGTATTCCTTGAGCCGGTA-3’(SEQ ID NO:16);

[0103] Mitofusin 1(Mfn1):

[0104] 5’-GGTCGCAAACTCTGAATCAA-3’(SEQ ID NO:17),

[0105] 5’-CAACTTTGAGCTCCTCCACC-3’(SEQ ID NO:18);

[0106] Mitofusin 2(Mfn2):

[0107] 5’-GCGAATTAAGCAGATTACGG-3’(SEQ ID NO:19),

[0108] 5’-ATGTCCTGCTGCATGGTCT-3’(SEQ ID NO:20);

[0109] Dynamin-related protein 1(Drp1):

[0110] 5’-AGATCCTGAAGATGTTGCCG-3’(SEQ ID NO:21),

[0111] 5’-CGCAATCTGATAGCTGTCCA-3’(SEQ ID NO:22);

[0112] GAPDH:

[0113] 5'-GCCTTCCGTGTCCCCACTGC-3' (SEQ ID NO:23),

[0114] 5'-GGCTGGTGGTCCAGGGGTCT-3' (SEQ ID NO: 24).

[0115] (XVIII) Protein Blot

[0116] Periapical tissues of mice and h5DLCs cultured under relevant conditions for 48 hours were collected and incubated with radioimmunoprecipitation assay (RIPA) lysis buffer (Biolab Technology, Beijing, China) on ice for 30 minutes. The mixture was centrifuged at 12000rpm and 4°C for 15 minutes. The supernatant containing total protein was collected. In order to isolate mitochondria in periapical tissues and hPDLCs, the Cell and Tissue Mitochondria Isolation Kit (Biolab Technology, Beijing, China) was recruited according to the manufacturer's instructions. The harvested mitochondria were treated with RIPA lysis buffer to extract mitochondrial proteins. The concentration of total protein in the supernatant was detected using the BCA protein detection kit (Biolab Technology, Beijing, China). A total protein sample weighing 30 μg was collected, protein separation was performed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene fluoride (PVDF) membrane. The proteins loaded on the PVDF membrane were blocked in 5% skim milk for 1 hour at room temperature. Proteins were detected with the primary antibody (12 h at 4 °C) and then reacted with the secondary antibody (2 h at room temperature). Protein signals were generated by treatment with enhanced chemiluminescence reagent (Beyotime Biotechnology, Shanghai, China) and then identified by Image J software (ImageJ, NIH, Bethesda, MD, USA).

[0117] The primary antibodies were: rabbit anti-light chain 3 (LC3) (1:1000, ab62721, Abcam, Cambridge, UK), anti-p62 (1:1000 ab91526, Cambridge, Abcam), anti-Drp1 kinase B (AKT) (1:1000, ab8932, Abcam, Cambridge, UK), anti-phosphorylated AKT (p-AKT) COXIV) (1:1000, ab202554, Abcam, Cambridge, UK). The secondary antibody was HRP-conjugated goat anti-rabbit secondary antibody (1:3000, ab6721, Abcam, Cambridge, UK). GAPDH was used as a reference for total cellular protein, and COXIV was used as a reference for mitochondrial protein.

[0118] 19. Statistical Analysis

[0119] Statistical analysis and statistical plotting of quantitative data (shown as mean ± SD) were performed using GraphPadPrism 7 (GraphPad Software, Inc., San Diego, CA, USA). Data were consistent with a normal distribution based on the Shapiro-Wilk test. One-way ANOVA combined with Tukey post hoc test was used to compare statistical differences between more than two groups. Statistical differences were considered statistically significant when P < 0.05.

[0120] (XX) Results

[0121] 1. Pue ​​treatment alleviated periapical inflammation and bone destruction in PDS mice and promoted osteogenic differentiation and autophagy in periapical tissues of PDS mice

[0122] This example starts with the establishment of a PDS mouse model, which is treated with 5 mg / kg and 10 mg / kg of Pue, respectively ( Figure 1 The gingival sulcus bleeding index and tooth mobility of PDS mice increased (P<0.01, P<0.001), and decreased after treatment with 10 mg / kg Pue (P<0.05, P<0.01) ( Figure 1 BC in PDS mice). Micro-CT analysis provided evidence of increased CEJ-ABC distance and decreased Tb.Sp as well as BV / TV and Tb.N contents in PDS mice (P<0.01, P<0.001). 10 mg / kg Pue treatment decreased CEJ-ABC distance and Tb.Sp and increased BV / TV and Tb.N contents in PDS mice (P<0.05, P<0.01). In fact, 5 mg / kg Pue treatment only increased Tb.N in PDS mice (P<0.05) but had no significant effect on the other three parameters ( Figure 1 DE in ).

[0123] H&E staining showed that PDS mice developed severe periapical periodontitis and bone destruction, which was actually alleviated by Pue treatment ( Figure 1 F). By immunohistochemistry, osteogenic protein RUNX2 and autophagy protein LC3 were significantly downregulated in the periapical tissue of PDS mice (P<0.001). In fact, 10 mg / kg Pue treatment significantly induced the expression of RUNX2 and LC3 proteins in the periapical tissue of PDS mice (P<0.01) ( Figure 1 in GJ).

[0124] ELISA was used to detect proinflammatory factors in periapical tissues, including TNF-α, IL-1β, and IL-6. Undoubtedly, excessive production of TNF-α, IL-1β, and IL-6 was detected in periapical tissues of PDS mice (P<0.001). PDS mice treated with 5 mg / kg Pue showed decreased concentrations of TNF-α and IL-6 (P<0.05, P<0.01), among which mice administered with 10 mg / kg Pue monitored a decrease in the three proinflammatory factors (P<0.01, P<0.001) ( Figure 1 KM in the figure). qRT-PCR of periapical tissues showed that the mRNA expressions of Runx2, OSX, OPN, OCN, ALP, and COL1A1 were decreased in PDS mice (P<0.001), while Pue treatment upregulated the expression of these genes, especially 10 mg / kg Pue treatment (P<0.05, P<0.01) ( Figure 1 N in ). All these evidences indicate that Pue treatment helps alleviate apical periodontitis and bone destruction in PDS and induces osteogenic differentiation and autophagy.

[0125] 2. Pue ​​treatment promoted the osteogenic differentiation ability of PDS cell model, but inhibited the inflammatory response hPDLCs were passaged three times ( Figure 2 Based on flow cytometry, cultured hPDLCs expressed CD44 and CD90, but rarely expressed CD34 and CD45 ( Figure 2 B). Alizarin red staining and Oil Red O staining showed that hPDLCs have good osteogenic and adipogenic differentiation abilities ( Figure 2 C).

[0126] According to the data of CCK-8 assay, Pue at doses of 10, 20, 50, 100, and 200 μM was non-toxic to hPDLCs ( Figure 2 D) Pg-LPS treatment induced PDS cell model. Figure 2 As shown in Figure E, Pg-LPS caused a significant decrease in the survival rate of hPDLCs (P<0.01, P<0.001). When the dose of Pue reached 20 μM, the survival rate of hPDLCs induced by Pg-LPS was significantly increased (P<0.01, P<0.001). Therefore, in subsequent studies, a 20 μM dose of Pue was selected to treat hPDLCs.

[0127] The osteogenic differentiation ability of hPDLCs was detected by Alizarin red staining and ALP staining. Pue ​​treatment had no effect on the osteogenic differentiation ability of hPDLCs (Pue group compared with control group), while Pg-LPS induction led to a significant decrease in the osteogenic differentiation ability of hPDLCs (as evidenced by weakened Alizarin red staining and decreased ALP activity) (LPS group compared with control group) (P<0.01, P<0.001). Pue ​​treatment enhanced the osteogenic differentiation ability of hPDLCs induced by Pg-LPS, as it increased Alizarin red staining and ALP activity (LPS+Pue group compared with LPS group) (P<0.05, P<0.01) ( Figure 2 FI in.

[0128] In addition, Pue treatment alone did not induce an inflammatory response in hPDLCs, as it had no significant effect on the production of proinflammatory factors TNF-α, IL-1β, and IL-6 (Pue group compared with control group). However, Pg-LPS treatment significantly increased the levels of TNF-α, IL-1β, and IL-6 in hPDLCs (LPS group compared with control group) (P<0.001). This effect of Pg-LPS was abolished by Pue treatment (LPS+Pue group compared with LPS group) (P<0.01) ( Figure 2 J in.

[0129] From the results of qRT-PCR, Pue treatment only did not affect the mRNA expression of Runx2, OSX, OPN, OCN, ALP, and COL1A1 in hPDLCs (Pue group compared with control group), while Pg-LPS induction significantly reduced the expression of these genes (LPS group compared with control group) (P<0.001). In fact, the inhibitory effect of Pg-LPS on the mRNA expression of Runx2, OSX, OPN, OCN, ALP, and COL1A1 in hPDLCs was abolished by Pue treatment (LPS+Pue group compared with LPS group) (P<0.01, P<0.0 ... Figure 2 K in ). Therefore, these data suggest that Pue treatment can enhance the osteogenic differentiation ability but suppress the inflammatory response in the PDS cell model.

[0130] 3. Pue ​​treatment enhances mitophagy in PDS cell model

[0131] By JC-1 staining ( Figure 3 AB in) and MitoSox red staining ( Figure 3CD in each group), the changes in mitochondrial membrane potential and mitochondrial ROS levels in hPDLCs were investigated. Pue ​​treatment only did not affect the mitochondrial membrane potential and mitochondrial ROS levels in hPDLCs (Pue group vs. control group). However, Pg-LPS treatment led to a significant decrease in the mitochondrial membrane potential and an increase in the mitochondrial ROS level in hPDLCs (LPS group vs. control group) (P<0.001), which was abolished by Pue treatment (LPS+Pue group vs. LPS group) (P<0.01, P<0.001).

[0132] The expression of autophagy-related proteins in hPDLCs of each group was detected by Western blot. Thus, hPDLCs induced by Pg-LPS expressed higher P62 protein, but lower IILC3 / LC3I protein (LPS group compared with control group) (P<0.001). In contrast, in hPDLCs induced by Pg-LPS, Pue treatment decreased P62I protein and increased IILC3 / LC3 protein (LPS+Pue group compared with LPS group) (P<0.05, P<0.01) ( Figure 3 In addition, Pg-LPS treatment led to a decrease in mitochondrial IILC3 / LC3I and Parkin proteins in hPDLCs, but an increase in mitochondrial P62 protein (LPS group compared with the control group) (P<0.001). This effect of Pg-LPS was offset by Pue treatment (LPS+Pue group compared with the LPS group) (P<0.05, P<0.01) ( Figure 3 F). Meanwhile, mRFP-eGFP-LC3 was measured ( Figure 3 GH in ) and Mito Tracker staining ( Figure 3 IJ) showed that LC3 dots in hPDLCs were significantly reduced after Pg-LPS induction (LPS group compared with control group) (P<0.001). Interestingly, Pue treatment increased LC3 dots in hPDLCs induced by Pg-LPS (LPS+Pue group compared with LPS group) (P<0.05). These results suggest that Pue treatment helps to restore mitophagy in the PDS cell model.

[0133] 4. Pue ​​treatment may alleviate inflammation and induce osteogenic differentiation in PDS cell models by enhancing mitochondrial autophagy

[0134] hPDLCs were treated with CsA (mitochondrial autophagy inhibitor). The osteogenic differentiation ability of hPDLCs was investigated by Alizarin red staining and ALP staining. In hPDLCs, the attenuation of Alizarin red staining and ALP activity induced by Pg-LPS treatment (LPS group compared with control group) (P<0.001) was reversed by Pue treatment (LPS+Pue group compared with LPS group) (P<0.01). CsA attenuated the enhancing effect of Pue on Alizarin red staining and ALP activity (LPS+Pue+CsA group compared with LPS+Pue group) (P<0.05) ( Figure 4 in AD).

[0135] ELISA showed that the levels of TNF-α, IL-1β, and IL-6 in hPDLCs induced by Pg-LPS were increased (LPS group compared with control group) (P<0.001), while those were decreased by Pue treatment (LPS+Pue group compared with LPS group) (P<0.001). However, CsA reversed the inhibitory effect of Pue on the production of TNF-α, IL-1β, and IL-6 in hPDLCs induced by Pg-LPS (LPS+Pue+CsA group compared with LPS+Pue group) (P<0.01) ( Figure 4 EG in ). By JC-1 staining, Pg-LPS caused a decrease in mitochondrial membrane potential in hPDLCs (LPS group compared with control group) (P<0.001), but this effect of Pg-LPS was reversed by Pue treatment (LPS+Pue group compared with LPS group) (P<0.01). In Pg-LPS-induced hPDLCs, CsA attenuated the increased mitochondrial membrane potential induced by Pue treatment (LPS+Pue+CsA group compared with LPS+Pue group) (P<0.01) ( Figure 4 HI in ).

[0136] qRT-PCR showed that the mRNA expressions of Runx2, OSX, OPN, OCN, ALP, and COL1A1 in hPDLCs induced by Pg-LPS were decreased (LPS group compared with control group) (P<0.001), while Pue treatment (LPS+Pue group compared with LPS group) increased the expression of these mRNAs (P<0.001). However, CsA abolished the promoting effect of Pue on the mRNA expressions of Runx2, OSX, OPN, OCN, ALP, and COL1A1 in hPDLCs induced by Pg-LPS (LPS+Pue+CsA group compared with LPS+Pue group) (P<0.05, P<0.01, respectively) Figure 4 in J).

[0137] MitoSox red staining was performed to evaluate the mitochondrial ROS levels in hPDLCs. Pg-LPS-induced mitochondrial ROS was aggravated (LPS group compared with control group) (P<0.001), and Pue treatment (LPS+Pue group compared with LPS group) reduced it (P<0.001). CsA abolished the inhibitory effect of Pue on Pg-LPS-induced mitochondrial ROS in hPDLCs (LPS+Pue+CsA group compared with LPS+Pue group) (P<0.01) Figure 4 Therefore, all the data suggest that Pue treatment may induce osteogenic differentiation and inhibit inflammation in the PDS cell model by promoting mitophagy.

[0138] 5. Pue ​​treatment helps maintain mitochondrial dynamics homeostasis in PDS cell models

[0139] Mitochondrial dynamics-related factors in hPDLCs were detected by qRT-PCR. Pg-LPS induction led to a significant decrease in the mRNA expression of OPA1, Fis1, and Mfn1 in hPDLCs (LPS group compared with the control group) (P<0.01, P<0.001), while Pue treatment had no significant effect on the expression of OPA1, Fis1, and Mfn1-mRNA in the presence or absence of Pg-LPS ( Figure 5 AC in ). Interestingly, Pue treatment had no significant effect on the mRNA expression of Mfn2 and Drp1 in hPDLCs (Pue group compared with control group). However, Pg-LPS induction led to a decrease in Mfn2 mRNA and an increase in Drp1 mRNA in hPDLCs (LPS group compared with control group) (P<0.001), but Pue treatment (LPS+Pue group compared with LPS group) offset this effect (P<0.05, P<0.01) ( Figure 5 Based on this finding, this study further detected the expression of Mfn2 and Drp1 proteins in the cytoplasm and mitochondria of hPDLCs. Neither Pg-LPS nor Pue affected the expression of Mfn2 and Drp1 proteins in the cytoplasm of hPDLCs ( Figure 5 F). Interestingly, the Mfn2 protein was lower, but the Drp1 protein was higher in the mitochondria of hPDLCs treated with Pg-LPS (LPS group compared with control group) (P<0.001). However, this effect of Pg-LPS was abolished by Pue treatment (LPS+Pue group compared with LPS group) (P<0.01) ( Figure 5G in Figure 5). Mito Tracker staining was used to investigate mitochondrial fission. Pue ​​treatment alone had no statistically significant effect on mitochondrial aspect ratio (Pue group compared with control group). However, Pg-LPS treatment resulted in a significant decrease in mitochondrial aspect ratio in hPDLCs (LPS group compared with control group) (P<0.001). In fact, the inhibitory effect of Pg-LPS on mitochondrial aspect ratio was reversed by Pue treatment (LPS+Pue group compared with LPS group) (P<0.01) ( Figure 5 Therefore, the above data indicate that Pue treatment can effectively maintain the homeostasis of mitochondrial dynamics.

[0140] 6. Pue ​​treatment may enhance mitophagy in PDS cell models by activating Mfn2

[0141] Mfn2 siRNA was transfected into hPDLCs to verify whether Pue induced mitophagy in Pg-LPS-induced hPDLCs by activating Mfn2. The expression of autophagy-related proteins in hPDLCs and mitochondria was detected by Western blot. Pg-LPS treatment decreased IILC3 / LC3 protein and increased P62 protein in IPg-LPS-induced hPDLCs (LPS group compared with control group) (P<0.001). In contrast, Pue treatment led to increased LC3II / LC3 protein and decreased P62 protein in IPg-LPS-induced hPDLCs (LPS+Pue+siMfn2-NC group compared with LPS group) (P<0.001). In fact, hPDLCs in the LPS+Pue+siMfn2-NC group expressed lower IILC3 / LC3I protein but higher P62 protein, matching the LPS+Pue+siMfn2-NC group (P<0.01). On the other hand, Pg-LPS treatment led to decreased expression of mitochondrial Parkin, IILC3 / LC3I, and Mfn2 proteins and increased expression of mitochondrial P62 protein in hPDLCs (LPS group compared with control group) (P<0.001), which was counteracted by Pue treatment (LPS+Pue+siMfn2-NC group compared with LPS group) (P<0.001). However, Mfn2 silencing I abolished the promoting effect of Pue on mitochondrial Parkin, LC3II / LC3, and Mfn2 proteins and its inhibitory effect on mitochondrial P62 protein (LPS+Pue+siMfn2 group compared with LPS+Pee+siMfn2-NC group) (P<0.05, P<0.01, P<0.001, respectively) ( Figure 6 AB in).

[0142] The activity of the PI3K / AKT / mTOR pathway in hPDLCs was evaluated by Western blot. Thus, Pg-LPS induced abnormal activation of the expression of p-PI3K / PI3K, p-AKT / AKT, and p-mTOR / mTOR proteins in hPDLCs (LPS group compared with the control group) (p<0.001), but this effect of Pg-LPS was abolished by Pue treatment (LPS+Pue+siMfn2-NC group compared with the LPS group). In fact, the inhibitory effect of Pue on the activity of the PI3K / AKT / mTOR pathway was attenuated by the absence of Mfn2 (LPS+Pue+siMfn2 group compared with the LPS+Pee+siMfn2-NC group) (P<0.05, P<0.001) ( Figure 6 in the CD).

[0143] In addition, JC-1 staining ( Figure 6 EF in), MitoSox red staining ( Figure 6 GH in), mRFP-eGFP-LC3 assay ( Figure 6 IJ) and Parkin / Tomm20 immunofluorescence staining ( Figure 6 KL in hPDLCs) were used to monitor changes in mitochondrial membrane potential, mitochondrial ROS levels, LC3 dots, and mitochondrial Parkin expression in hPDLCs. Pue ​​treatment (LPS+Pue+siMfn2-NC group compared with LPS group) abolished the Pg-LPS-induced decrease in mitochondrial membrane potential, increase in mitochondrial ROS levels, decrease in LC3 dots, and Parkin / Tomm20 colocalization in hPDLCs (P<0.001) (P<0.01, P<0.001). Interestingly, Mfn2 knockout abolished the Pg-LPS-induced increase in mitochondrial membrane potential, LC3 dots, and Parkin / Tomm20 colocalization in hPDLCs by Pue, as well as its inhibition of mitochondrial ROS levels (LPS+Pue+siMfn2 group compared with LPS+Pee+siMfn2-NC group) (P<0.05, P<0.01, P<0.001). Based on these results, Pue was suggested to enhance mitophagy in the PDS cell model by activating Mfn2 expression.

[0144] 7. Pue ​​treatment may reduce periapical inflammation and bone destruction in PDS mice by activating mitochondrial Mfn2 and promoting mitochondrial autophagy, and promote osteogenic differentiation of periapical tissues

[0145] Adenovirus-based Mfn2-siRNA was transfected into the periodontal tissues of PDS mice, and the mice were treated with 10 mg / kg dose of Pue. Figure 7As can be seen in Figure AB, by micro-CT analysis, the abnormal increase in gingival sulcus bleeding index, tooth mobility, CEJ-ABC distance and Tb.Sp and the decrease in BV / TV in PDS mice (PDS group compared with control group) (P<0.01, P<0.001) were reversed by Pue treatment (PDS+Pue+siMfn2-NC group compared with PDS group) (P<0.05, P<0.01). When compared with mice in the PDS+Pue+siMfn2-NC group, mice in the PDS+Pue+siMfn2 group had higher CEJ-ABC distance and Tb.Sp and lower BV / TV (P<0.05).

[0146] According to H&E staining, severe periapical inflammation and bone destruction in PDS mice (PDS group vs. control group) were alleviated by Pue treatment (PDS+Pue+siMfn2-NC group vs. PDS group). However, Mfn2 silencing reversed the alleviating effects of Pue on periapical inflammation and bone destruction in PDS mice (PDS+Pue+siMfn2 group vs. PDS+Pue+siMfn2-NC group) ( Figure 7 C). ELISA of periapical tissues of PDS mice (PDS group compared with control group) showed higher levels of TNF-α, IL-1β, and IL-6 (P<0.001), which were reduced after Pue treatment (PDS+Pue+siMfn2-NC group compared with PDS group) (P<0.001). In fact, the inhibitory effect of Pue on the production of TNF-α, IL-1β, and IL-6 in periapical tissues of PDS mice was offset by silencing of Mfn2 (PDS+Pue+siMfn2 group compared with PDS+Pue+siMfn2-NC group) (P<0.01) ( Figure 7 D).

[0147] By immunohistochemistry, the reduced RUNX2 and LC3 proteins in the periapical tissues of PDS mice (PDS group compared with the control group) (P<0.001) were upregulated in Pue treatment (PDS+Pue+siMfn2-NC group compared with the PDS group) (P<0.01). In contrast, Mfn2 silencing attenuated the enhancing effect of Pue on the expression of RUNX2 and LC3 proteins in the periapical tissues of PDS mice (PDS+Pue+siMfn2 group compared with the PDS+Pue+siMfn2-NC group) (P<0.05, P<0.01) ( Figure 7EH in the figure). According to the qRT-PCR results of periapical tissues, the mRNA expression of Runx2, OSX, OPN, OCN, ALP, and COL1A1 genes in PDS mice was less (PDS group compared with control group) (P<0.001), while Pue treatment enhanced the expression of these genes (PDS+Pue+siMfn2-NC group compared with PDS group). In fact, the enhancing effect of Pue on the mRNA expression of Runx2, OSX, OCN, and ALP in the periapical tissues of PDS mice was reversed by Mfn2 deficiency (PDS+Pue+siMfn2 group compared with PDS+Pue+siMfn2-NC group) (P<0.05, P<0.01, respectively) ( Figure 7 I in.

[0148] Western blot was responsible for the expression of autophagy-related proteins in the periapical tissue of PDS mice. Therefore, the expression of mitochondrial Mfn2, IILC3 / LC3 and Parkin proteins in the periapical tissue of PDS mice I was significantly reduced, and the mitochondrial P62 protein was increased (PDS group compared with the control group) (P<0.001). After Pue treatment, the mitochondrial Mfn2, IILC3 / LC3 and Parkin proteins in the periapical tissue of PDSI mice increased, and the mitochondrial P62 protein decreased (PDS+Pue+siMfn2-NC group compared with the PDS group) (P<0.001). However, Mfn2 silencing I reversed the promoting effect of Pue on mitochondrial Mfn2, LC3II / LC3 and Parkin proteins and its inhibitory effect on mitochondrial P62 protein (PDS+Pue+siMfn2 group compared with the PDS+Pue+siMfn2-NC group) (P<0.05, P<0.01, P<0.001) ( Figure 7 J in the figure). Meanwhile, Pue treatment I (PDS+Pue+siMfn2-NC group vs. PDS group) eliminated the decrease of LC3II / LC3 protein and the increase of P62 protein in the periapical tissue of PDS mice (P<0.001). Conversely, Mfn2 silencing I eliminated the increase of Pue on IILC3 / LC3 protein and its inhibition of P62 protein in the periapical tissue of PDS mice (PDS+Pue+siMfn2 group vs. PDS+Pue+siMfn2-NC group) (P<0.05, P<0.001) ( Figure 7 K in Figure 3). Taken together, all these data suggest that Pue treatment alleviates PDS, such as alleviating periapical inflammation and bone destruction, and enhancing osteogenic differentiation of periapical tissues by activating mitochondrial Mfn2 and enhancing mitophagy.

[0149] The present invention studies the therapeutic effect of Pue on PDS mice, as it reduces the CEJ-ABC distance and Tb.Sp, increases Tb.N and BV / TV, reduces the gingival sulcus bleeding index and tooth mobility, inhibits inflammation and bone destruction, and promotes osteogenic differentiation. One of the main clinical characteristics of PDS is the inflammatory response, and the excessive production of proinflammatory factors (such as TNF-α, IL-1β and IL-6) in the periodontal tissue is the main cause of gingival tissue and ligament damage. Through pathological staining and ELISA of apical tissue, the present invention reveals the alleviating effect of Pue on periapical inflammation and bone destruction in PDS mice and its inhibitory effect on the production of TNF-α, IL-1β and IL-6. Similarly, Pue also inhibited the production of TNF-α, IL-1β and IL-6 in hPDLCs induced by Pg-LPS. hPDLCs have multidirectional differentiation potential and self-renewal ability, and are ideal candidates for periodontal tissue regeneration and repair and bone regeneration in PDS. The present invention guides the enhancement effect of Pue on the expression of Runx2, OSX, OPN, OCN, ALP and COL1A1 in the periapical tissue of PDS mice and hPDLCs induced by Pg-LPS. Runx2, OSX, OPN, OCN, ALP and COL1A1 are osteogenic markers, of which Runx2, ALP and COL1A1 are early osteogenic markers, and OSX, osteopontin and OCN are late osteogenic markers. Therefore, Pue can induce osteogenic differentiation of hPDLCs in PDS, indicating the therapeutic potential of Pue for PDS.

[0150] Mitophagy is a form of mitochondrial self-protection that maintains the stability of mitochondrial number and structure and plays a vital role in maintaining normal mitochondrial function. Studies have shown that abnormal mitophagy can affect the biological behavior of hPDLCs. According to this study, in the periapical tissue of PDS mice, autophagy was found to be inhibited (as indicated by decreased LC3 protein expression), while Pue treatment increased LC3 protein expression. Through in vitro studies, inhibited autophagy (as suggested by decreased LC3 / LC3 and increased P62 protein), impaired mitochondrial I function (as evidenced by decreased mitochondrial membrane potential and increased mitochondrial ROS levels), and blocked mitophagy (as indicated by decreased mitochondrial IILC3II / LC2, Parkin, and increased mitochondrial P62 protein) were found in Pg-LPS-induced hPDLCs. IILC3 / LC3I and mitochondrial Parkin are autophagy-inducing proteins, while P62 accumulates when autophagy is blocked. Pue ​​treatment abolished impaired mitochondrial function and inhibited mitophagy in Pg-LPS-induced hPDLCs. The treatment experiment of Pg-LPS-induced hPDLCs co-treated with Pue and CsA showed that Pue may promote osteogenic differentiation, reduce inflammation, and maintain normal mitochondrial function by activating mitophagy. This study found that Pue promoted osteogenic differentiation and inhibited inflammation of hPDLCs in PDS by activating mitophagy.

[0151] Mitochondrial dynamics plays a vital role in normal cellular physiological activities. Abnormalities in mitochondrial dynamics can lead to a variety of diseases, usually accompanied by disorders in mitophagy. OPA1, Fis1, Mfn1, Mfn2 and Drp1 are mitochondrial dynamics-related factors involved in the regulation of mitochondrial fusion and fission processes. In the present invention, it was found that Pue had no significant effect on the expression of OPA1, Fis1 and Mfn1, but in Pg-LPS-induced hPDLCs, it enhanced the expression of Mfn2 and inhibited the expression of Drp1. Further studies have shown that Pue can increase mitochondrial Mfn2 expression and reduce mitochondrial Drp1 expression, but does not affect cellular Mfn2 and Drp1 expression. Mfn2 is believed to contribute to the recovery of mitophagy, and Mfn2 binds to Drp1 to participate in maintaining normal mitochondrial function by mediating mitochondrial fusion and fission processes. In fact, through in vitro studies of hPDLCs induced by Pg-LPS, Mfn2 silencing reversed the enhanced mitophagy, maintenance of mitochondrial function, and inactivation of the PI3K / AKT / mTOR pathway induced by Pue treatment. In inflammation-related diseases, blocking the activity of the PI3K / AKT / mTOR pathway is believed to activate mitophagy to alleviate the inflammatory response. Therefore, Pue has been shown to promote mitophagy in PDS by inducing the expression of Mfn2. Through in vivo experiments, Mfn2 silencing counteracted the relief of PDS by Pue treatment (such as reduced periapical inflammation and bone destruction), the induction of osteogenic differentiation, and the promotion of mitophagy. This suggests that Pue may enhance mitophagy by activating mitochondrial Mfn2 expression, thereby exerting a therapeutic effect on PDS. In summary, the current study provides a sufficient basis and a definite molecular mechanism for the application of Pue in the treatment of PDS.

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of puerarin in the treatment of periodontitis is characterized by: The puerarin can be used for treating periodontitis.

2. The use according to claim 1, characterized in that: The puerarin promotes mitochondrial autophagy by activating mitochondrial Mfn2 to treat periapical inflammation and bone destruction of periodontitis, and promotes osteogenic differentiation of periapical tissue.

3. The use according to claim 1, characterized in that: The puerarin has an inhibitory effect on the pro-inflammatory factors TNF-α, IL-1β and IL-6 in the periapical tissue.

4. The use according to claim 1, characterized in that: The puerarin has a promoting effect on the expression of Runx2, OSX, OPN, OCN, ALP and COL1A1 genes.

5. The use according to claim 1, characterized in that: The puerarin regulates the activity of the PI3K / AKT / mTOR pathway through Mfn2.

6. The use according to claim 1, characterized in that: The dosage of puerarin is 5-10 mg / kg.

7. Use of puerarin in the preparation of a drug for treating periodontitis, characterized in that: The medicine for treating periodontitis contains puerarin.

8. A pharmaceutical composition for treating periodontitis, characterized in that: The main active ingredient of the pharmaceutical composition is puerarin.

9. The pharmaceutical composition for treating periodontitis according to claim 8, characterized in that: The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

10. An Mfn2 agonist, characterized in that The main active ingredient of the agonist is puerarin.

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