Drugs for treating periodontal ligament cell dysfunction and alveolar bone loss caused by periodontal pathogen infection

Abstract

The present invention provides a drug for treating periodontal ligament cell dysfunction and alveolar bone loss caused by periodontal pathogen infection, the drug comprising IL-18 antibody and / or IL-18R receptor siRNA exosomes. The drug of the present invention uses IL-18 antibody and / or IL-18R receptor siRNA loaded exosomes as effective ingredients, and by binding to IL-18 in periodontal tissue, inhibits the secretion of proinflammatory cytokines by periodontal ligament cells, improves the loss of periodontal supporting tissue and inflammatory cell infiltration caused by periodontal ligament cell dysfunction under inflammatory conditions, thereby preventing the occurrence and development of periodontal disease, and has a potential huge application value for the clinical prevention and treatment of periodontal disease.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to a medicine for treating periodontal ligament cell dysfunction and alveolar bone loss caused by periodontal pathogenic bacteria infection. Background Art

[0002] Periodontitis is a common oral disease, which is mainly caused by pathogenic microorganisms in dental plaque biofilm initiating innate immunity, adaptive immunity and inflammatory responses. It manifests as a series of complex inflammatory syndromes, mainly affecting the periodontal membrane and the tissues surrounding and supporting the teeth, which can lead to progressive loss of periodontal alveolar bone and then cause tooth loss.

[0003] Pathogenic microorganisms in dental plaque biofilm are the main cause of periodontal disease. The transplantation of gingival microbial communities in the periodontal pocket changes the dominant microbial community in the periodontal pocket from Gram-positive aerobic bacteria to Gram-negative anaerobic bacteria, causing periodontal disease in healthy periodontal tissues. Porphyromonas gingivalis is considered to be the main pathogen of periodontitis. It is present in low numbers in the oral cavity of healthy people, but significantly increases in the destructive structural plaque and tartar of periodontitis. Porphyromonas gingivalis has multiple virulence factors, including pili, degradative enzymes, and lipopolysaccharides. Low transplantation of Porphyromonas gingivalis can induce changes in the number and composition of oral commensal microbiota and promote the transformation of harmful factors in bacterial colonies. Porphyromonas gingivalis can also help suppress the body's immune system and create a good living environment for other bacteria. In a mouse periodontitis model, low concentrations of Porphyromonas gingivalis induce the occurrence of inflammatory periodontitis through the interaction between the oral commensal microbiota and the complement system.

[0004] Host recognition of pathogenic microorganisms is an important part of the pathological mechanism of periodontal disease, which can quickly trigger an immune response and promote the activation and recruitment of important immune components through the secretion of cytokines and chemokines. Detection of microbial products can promote the stability and proliferation of microbial transplant communities, so that the host oral microbial community and the body's immune system maintain a steady state balance. At present, although the characteristics of disease progression and damage in patients with chronic periodontitis have been clearly defined, the mechanism of protection and damage to the host's periodontal tissues has not yet been fully elucidated.

[0005] The clinical manifestations of periodontal disease are ever-changing. In areas where the disease is active, the number of mast cells, monocytes / macrophages and plasma cells increases significantly. These cells further activate monocytes / macrophages, lymphocytes, fibroblasts and other cell components to secrete a variety of cytokines and inflammatory mediators including IL-1, IL-6 and TNF-α. These cytokines and inflammatory mediators can act alone or in combination to promote alveolar bone absorption and collagen breakdown.

[0006] Natural killer-like B cells (NKB cells) are a new type of immune cell subset found in the spleen and mesenteric lymph nodes of mice. They mainly secrete IL-18 and IL-12, which play an anti-microbial infection role. IL-18 is the main cytokine secreted by NKB cells, which can induce lymphocytes and spleen cells to produce IFN-γ. IL-18 and IL-12 double gene defects can reduce NK cell activity and cellular response. IL-18 and IL-12 have the function of activating NK cells in local inflammatory response. Since the onset of periodontitis is the result of the interaction between oral parasites and the body's immune system, and NKB cells play an important role in bacterial infection, NKB cells and IL-18 may play a role in the onset of periodontitis.

[0007] IL-18 works by binding to its receptor IL-18R. The receptor IL-18R of IL-18 is widely present in a variety of cells and is composed of a heterodimer α chain (IL-18Rα) and a β chain (IL-18Rβ). Among them, IL-18Rα is the ligand binding chain, and IL-18Rβ is used to mediate the signal transduction of IL-18. Reducing the expression of IL-18 receptor can reduce the secretion of proinflammatory cytokines by periodontal ligament cells, thereby inhibiting the occurrence and development of periodontitis.

[0008] In recent years, RNA interference (RNAi) has received extensive attention. As a new gene therapy technology, RNAi refers to inhibiting target mRNA by degradation or translation, thereby causing post-transcriptional gene silencing, and has broad application prospects in anti-inflammatory, antiviral and other medical fields. siRNA uses RNAi technology to efficiently and specifically block the expression of homologous genes in the body, promote the degradation of homologous mRNA, and induce cells to show a specific phenotype of gene deletion. However, siRNA is easily degraded by ribozymes (RNase) in the body, has a short half-life, and has low efficiency in transfecting cells. Therefore, how to efficiently introduce specific silencing siRNA into the target organ without damaging normal tissues and siRNA has become an urgent problem to be solved in gene therapy.

[0009] At present, the research on therapeutic siRNA carriers mainly focuses on polycationic polyethyleneimine (PEI) nanoparticles and liposomes. However, PEI-RNA complexes tend to accumulate in the liver, lungs, spleen and kidneys, limiting their application in other tissues. They can easily lead to inflammasome activation associated with IL-1 induction and cell apoptosis, promoting inflammation. Liposomes or lipid nanoparticles are not only hepatotoxic, but can also adsorb opsonins, activate complement and coagulation factors, and easily lead to phagocytosis by the mononuclear phagocyte system due to their inherent net charge and size. Like PEI, this process can lead to cell stress, inflammasome activation and cell apoptosis.

[0010] Exosomes are a typical lipid bilayer membrane structure with a diameter of about 30 to 150 nm. Their lipid bilayer structure and surface membrane proteins make exosomes have a high transfection efficiency in vivo. They are important carriers for intercellular information transmission and have been developed for intercellular transmission of various substances (including siRNA). Compared with traditional drug carriers such as liposome transfection reagents, exosomes, as a natural drug carrier, have the advantages of good biocompatibility, degradability, and low immunogenicity.

[0011] CN 102488654 A discloses a medicine for treating periodontitis, which is prepared by 5-250 parts of doxycycline, 25-1000 parts of metronidazole and 10000 parts of glycerol by weight. CN 104688998 A discloses a Chinese medicine tablet for treating periodontitis and a preparation method thereof, which comprises taking 8-14 parts of water extract of Forsythia suspensa, 3-7 parts of water extract of Coptis chinensis, 5-11 parts of ethanol extract of Fructus Malvae, 4-8 parts of ethanol extract of Rutaecarpa, 4-9 parts of ethyl acetate extract of Paeonia lactiflora, 2-5 parts of crushed Cyperus rotundus, 3-6 parts of crushed Rhizoma Atractylodis Macrocephalae, 15-20 parts of magnesium stearate, 2-6 parts of glycerol and 7-16 parts of modified starch by weight, mixing the above ingredients evenly and pressing them into tablets using a tablet press to obtain a Chinese medicine tablet for treating periodontitis. CN 103638408 A discloses an external ointment for treating periodontitis, which is prepared from the following components according to the weight ratio range shown: 20-30 parts of Perilla frutescens, 15-20 parts of Prunella vulgaris, 8-15 parts of Radix Rehmanniae, 6-12 parts of Chrysanthemum indici, 5-10 parts of Smilax glabra, 7-10 parts of Fructus Forsythiae, 8-12 parts of Radix Stemonae, 5-10 parts of Perilla frutescens, 8-12 parts of Cortex Phellodendri, 6-10 parts of Semen Leptospermi, and 1-3 parts of Galla chinensis. The prior art for treating periodontitis all uses antibiotics or traditional Chinese medicine, which has complex ingredients, large side effects, and insignificant therapeutic effects, and cannot achieve the purpose of radical cure.

[0012] The onset of periodontitis involves the interaction between microorganisms and the body's immune system, and the pathogenesis has not yet been fully elucidated. In-depth exploration of the immune pathogenesis of periodontitis provides a theoretical and experimental basis for finding new targets for the treatment of periodontitis. At present, there is an urgent need to develop drugs that can effectively inhibit periodontitis inflammation, reduce the severity of periodontitis from the root source, and prevent the occurrence of periodontitis. Summary of the invention

[0013] In view of the deficiencies of the prior art, the present invention provides a drug for treating periodontal ligament cell dysfunction and alveolar bone loss caused by periodontal pathogen infection. The drug uses IL-18 antibodies and exosomes loaded with IL-18R receptor siRNA as effective ingredients. The IL-18 antibodies and IL-18R receptor siRNA exosomes play a synergistic role, which can inhibit periodontitis alveolar bone resorption and the expression of periodontitis-related inflammatory cytokines and chemokines, thereby reducing the incidence of periodontal diseases from the root source.

[0014] To achieve this object, the present invention adopts the following technical solutions:

[0015] In a first aspect, the present invention provides a pharmaceutical composition comprising IL-18 antibody and / or IL-18R receptor siRNA exosomes.

[0016] In the present invention, the inventors unexpectedly discovered that in patients with periodontal disease, the expression of IL-18, a cytokine secreted by natural killer-like B cells (NKB), in peripheral blood and gingival crevicular fluid increased significantly. Moreover, IL-18 in the gingival crevicular fluid of patients with periodontitis was significantly positively correlated with AL (Attachment loss), indicating that IL-18 is the main effector molecule of NKB cells in the pathogenesis of periodontitis. On this basis, the inventors further discovered that the level of IL-18 in gingival crevicular fluid was significantly reduced after periodontal basic treatment. When microbial pathogenic factors such as plaque were removed, the number of microorganisms and the level of antigens were significantly reduced, the local inflammatory response was reduced, the stimulation of NKB cells was reduced, and the level of IL-18 secreted by NKB cells was also reduced, further confirming that IL-18 is involved in the pathogenesis of periodontitis and mainly plays a role locally in periodontal tissues. Therefore, the inventors used drugs prepared by IL-18 antibodies to inhibit the symptoms of periodontitis and effectively inhibit periodontitis inflammation.

[0017] In the present invention, the inventors also found that IL-18 can promote periodontal ligament cells to secrete a variety of cytokines that promote inflammatory response, thereby promoting the disease progression of periodontitis.

[0018] In the present invention, the drug uses IL-18 antibody and exosomes loaded with IL-18R receptor siRNA as effective ingredients. IL-18 antibody reduces the expression level of IL-18 by targeting IL-18, and IL-18R receptor siRNA exosomes reduce the expression level of IL-18 by inhibiting the expression of IL-18mRNA through translation. The two exert a synergistic effect, inhibit the expression of inflammatory cytokines and chemokines in periodontal tissues, reduce the effects of inflammatory cytokines and chemokines on the secretion of proinflammatory cytokines by periodontal ligament cells, fundamentally reduce inflammatory infiltration, improve periodontal ligament cell dysfunction, and prevent the occurrence and development of periodontal diseases.

[0019] Preferably, the concentration of the IL-18 antibody is 2 to 10 μg / mL, for example, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL or 10 μg / mL, preferably 5 to 8 μg / mL.

[0020] Preferably, the concentration of IL-18R receptor siRNA exosomes is 0.5-2 μg / 1 μL, for example, it can be 0.5 μg / 1 μL, 0.6 μg / 1 μL, 0.7 μg / 1 μL, 0.8 μg / 1 μL, 0.9 μg / 1 μL, 1.0 μg / 1 μL, 1.1 μg / 1μL, 1.2μg / 1μL, 1.3μg / 1μL, 1.4μg / 1μL, 1.5μg / 1μL, 1.6μg / 1μL, 1.7μg / 1μL, 1.8μg / 1μL, 1.9μg / 1μL or 2.0μg / 1μL, preferably 1.0μg / 1μL.

[0021] Preferably, the mass ratio of the IL-18 antibody to the IL-18R receptor siRNA exosomes is (0.01-0.05):1, for example, it can be 0.01:1, 0.02:1, 0.025:1, 0.03:1, 0.04:1 or 0.05:1, preferably (0.02-0.025):1.

[0022] Preferably, the nucleic acid sequence of the siRNA in the IL-18Rα receptor siRNA exosomes is as shown in SEQ ID NOs: 1 to 2:

[0023] SEQ ID NO: 1 (sense strand): 5′-AAACUCGGCAUCCUUCAGGUUTT-3′;

[0024] SEQ ID NO:2 (antisense strand): 5′-AACCUGAAGGAUGCCGAGUUUTT-3′.

[0025] Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

[0026] In a second aspect, the present invention provides a drug efficacy evaluation model for the drug composition as described in the first aspect, wherein the model is a periodontal disease model mouse.

[0027] In a third aspect, the present invention provides a method for preparing the drug efficacy evaluation model as described in the second aspect, wherein the method uses silk thread ligation combined with Porphyromonas gingivalis induction to construct the model.

[0028] Preferably, the method comprises the following steps:

[0029] The silk thread is placed in the upper jaw of a healthy mouse without periodontal disease, and Porphyromonas gingivalis is inoculated into the gums and / or applied locally in the oral cavity to induce periodontal disease in the mouse, thereby obtaining the periodontal disease model mouse.

[0030] Preferably, the Porphyromonas gingivalis is a P. gingivalis strain.

[0031] Preferably, the concentration of Porphyromonas gingivalis is 1×10 8 ~1×10 10 CFU / mL, for example, can be 1×10 8 CFU / mL, 1×10 9 CFU / mL or 1×10 10 CFU / mL.

[0032] As a preferred technical solution, the present invention provides a method for preparing the drug efficacy evaluation model as described in the second aspect, wherein the method uses silk thread ligation combined with Porphyromonas gingivalis induction to construct the model, and comprises the following steps:

[0033] (1) Bacterial culture:

[0034] 0.5% hemin solution and bovine heart and brain infusion medium (BHI medium) were mixed evenly at a ratio of 1:100, and P. gingivalis strain was inoculated into the medium;

[0035] Porphyromonas gingivalis (P.gingivalis ATCC 33277) was inoculated into the above-prepared culture medium and cultured under strict anaerobic conditions of 85% nitrogen, 10% hydrogen, 5% carbon dioxide and 37°C. After 2-3 passages, the culture was continued for 2 days until the concentration reached 1×10 9 CFU / mL, the bacteria were resuspended in PBS buffer containing 2% sodium carboxymethyl cellulose for later use;

[0036] (2) Establishment of silk thread ligation mouse periodontitis model and experimental grouping:

[0037] Mice with complete dentition and no caries or periodontal disease were selected and anesthetized by intraperitoneal injection of 1% sodium pentobarbital (0.15 mL / 20 g). The limbs were fixed in the supine position, the oral cavity and periorbital area were disinfected with 1% iodine tincture, deiodinated with 75% alcohol, and the upper jaw was fixed with silk thread.

[0038] A sterile suture thread with a diameter of 100 μm was placed 1 mm above the gingival sulcus of the maxillary second molar, passed through the interproximal space from the mesiodistal side, and tied on the buccal or palatal side;

[0039] Check every 4 days whether the silk thread has fallen off, and re-ligate it in time if it has fallen off; the IL-18 antibody treatment experimental group was treated with oral local smear and micro-syringe injection of Porphyromonas gingivalis suspension into the buccal and palatal periodontal tissues of maxillary molars, and 50 μL of 5 μg / mL anti-IL-18 antibody was locally injected into the buccal and palatal periodontal tissues of maxillary molars, once every 2 to 4 days;

[0040] A cotton swab was used to smear excess bacteria on the maxillary molar area in the oral cavity; in the periodontitis control group, a microsyringe was used to locally inject a suspension of Porphyromonas gingivalis into the buccal and palatal periodontal tissues of the maxillary molars; in the normal group, a 2% sodium carboxymethylcellulose-PBS solution was applied locally in the oral cavity, and an equal amount of PBS was injected into the periodontal tissues using a microsyringe;

[0041] After 4 weeks, the mice were killed by cervical dislocation and the samples were fixed.

[0042] In a fourth aspect, the present invention provides a use of the pharmaceutical composition as described in the first aspect and / or the drug efficacy evaluation model as described in the second aspect in the preparation of drugs for the treatment of periodontal diseases.

[0043] Preferably, the loss of periodontal supporting tissue includes loss of periodontal ligament and / or loss of alveolar bone.

[0044] Preferably, the alveolar bone loss is caused by periodontal pathogen infection.

[0045] Preferably, the periodontal pathogenic bacteria include Porphyromonas gingivalis.

[0046] Preferably, the Porphyromonas gingivalis comprises a P. gingivalis strain.

[0047] In the present invention, the main pathogenic bacteria of periodontitis, Porphyromonas gingivalis, stimulates periodontal ligament cells to secrete proinflammatory cytokines, thereby inducing inflammatory infiltration of periodontal tissues, which causes periodontal ligament dysfunction and loss of periodontal ligament on the one hand, and alveolar bone loss on the other hand. The pharmaceutical composition inhibits the immune inflammatory pathogenic mechanism of natural killer-like B cells (NKB) on the occurrence and development of periodontitis, reduces the ability of IL-18 to secrete proinflammatory cytokines by periodontal ligament cells, inhibits alveolar bone absorption of periodontitis, and the expression of inflammatory cytokines and chemokines related to periodontitis, thereby effectively inhibiting periodontitis inflammation and alveolar bone absorption, and reducing the severity of periodontitis from the inflammatory pathogenesis, thereby preventing the occurrence of periodontitis.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] (1) The present invention finds that cytokine IL-18 can promote periodontal ligament cells to secrete a variety of cytokines that promote inflammatory responses, thereby leading to loss of periodontal ligament tissue, enhancing inflammatory responses, and further leading to increased alveolar bone loss, promoting the disease progression of periodontitis;

[0050] (2) In the pharmaceutical composition of the present invention, IL-18 antibody is used as a targeted drug to prevent and treat functional disorders of periodontal ligament cells, and IL-18R receptor siRNA exosomes reduce the expression level of IL-18 by inhibiting the expression of IL-18 mRNA through translation. The two play a synergistic role, thereby inhibiting the loss of periodontal ligament tissue, alveolar bone resorption and the expression of periodontitis-related inflammatory cytokines and chemokines, and reducing the severity of periodontitis from the root source, thereby preventing the occurrence and development of periodontitis. It has great potential application value in the clinical prevention and treatment of periodontitis, and lays a foundation for further research on the biological functions of IL-18 antibody and IL-18R receptor siRNA;

[0051] (3) The present invention provides a periodontitis mouse model, which is produced by silk ligation combined with Porphyromonas gingivalis induction. The therapeutic and preventive effects of IL-18 antibody and IL-18R receptor siRNA on periodontitis are studied by local periodontal injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 is a study on the effect of IL-18 on the secretion of proinflammatory cytokines by periodontal ligament cells, wherein Figure 1 (A) shows the changes in the expression level of IFN-γ, Figure 1 (B) shows the changes in the expression level of GM-CSF, Figure 1 (C) shows the changes in the expression level of IL-2, and Figure 1 (D) shows the changes in the expression level of TNF-α;

[0053] Figure 2 is a CT image of alveolar bone loss in periodontitis, wherein Figure 2 (A) is a normal control group, Figure 2 (B) is a simple silk ligation control group, Figure 2 (C) is a periodontitis group, Figure 2 (D) is a periodontitis + IL-18 antibody treatment group, Figure 2 (E) is a periodontitis + IL-18α siRNA exosome group, and Figure 2 (F) is a periodontitis + IL-18 antibody + IL-18α siRNA exosome group;

[0054] Figure 3 It is a bar graph of the distance of alveolar bone loss (mm) in periodontitis obtained based on the micro-CT results;

[0055] Figure 4 (A) shows the expression of cytokine IL-1β in periodontal tissue, Figure 4 (B) shows the expression of cytokine IL-6, Figure 4 (C) shows the expression of cytokine IL-8, and Figure 4 (D) shows the expression of IL-18Rα;

[0056] Figure 5 This is the result of histological staining of the periodontal tissue area of ​​maxillary molars. DETAILED DESCRIPTION

[0057] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0058] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0059] Materials and reagents

[0060] IL-18 antibody was purchased from Medical & Biological Laboratories Co., LTD. (Code No: D 048-3);

[0061] P. gingivalis strain was purchased from the American ATCC Bacteria Bank (ATCC 33277);

[0062] C57BL / 6 mice were purchased from the Animal Experiment Center of Sun Yat-sen University.

[0063] Example 1 Silk ligation combined with Porphyromonas gingivalis periodontitis modeling

[0064] (1) Bacterial culture

[0065] 0.5% hemin solution and bovine heart and brain infusion medium (BHI medium) were mixed evenly at a ratio of 1:100, and the P.gingivalis strain was inoculated into the medium and cultured under strict anaerobic conditions of 85% nitrogen, 10% hydrogen, 5% carbon dioxide and 37°C;

[0066] After two generations of culture, the bacterial concentration reached 1×10 8 CFU / mL, the bacteria were resuspended in PBS buffer containing 2% sodium carboxymethyl cellulose for later use;

[0067] The bacteria cultured for 48 hours were identified based on colony morphology, Gram staining, bacterial morphology and biochemical reaction results;

[0068] (2) Modeling

[0069] Eight-week-old C57BL / 6 mice with complete dentition and no caries or periodontal disease were selected and fed with sterile feed in separate cages in a clean, quiet, well-ventilated room with good lighting conditions, a room temperature of 22°C, and no special pathogens. The light cycle was 12h:12h.

[0070] 4% chloric acid hydrate was intraperitoneally injected for anesthesia (0.35 mg / kg). After anesthesia, the mice were placed in a supine position and fixed on the operating table with adhesive tape. The oral cavity and periorbital area were disinfected with 1% iodine tincture and deiodinated with 75% alcohol. The maxillary incisors were pulled and fixed with cotton thread to make the mouth open. The gingival tissue of the left maxillary first molar was separated with a sharp probe, and a silk thread was embedded in the neck of the adjacent teeth between the first and second molars. The first molar was ligated and fixed at the mesial papilla of the first molar and placed at the gingival margin of the first molar, and placed as far as possible in the gingival sulcus.

[0071] Check the needle and thread once a week to see if they have fallen off. If they have, re-tie them in time.

[0072] 100 μL of Porphyromonas gingivalis was inoculated into the mid-gingival sulcus of the right maxillary second molar on the palatal side, twice a week.

[0073] Example 2 Preparation of IL-18 receptor siRNA exosomes

[0074] (1) Design and synthesis of IL-18Rα siRNA sequence:

[0075] The nucleic acid sequence of IL-18Rα siRNA is shown in SEQ ID NO: 1-2:

[0076] SEQ ID NO: 1 (sense strand): 5′-AAACUCGGCAUCCUUCAGGUU-3′;

[0077] SEQ ID NO: 2 (antisense strand): 5′-AACCUGAAGGAUGCCGAGUUUTT-3′;

[0078] (2) Isolation and purification of exosomes from mouse serum

[0079] ① Take mouse serum and centrifuge at 300g for 10 min at 4°C, discard the precipitate and remove the cells;

[0080] ② Take the supernatant, centrifuge at 2000g for 10 minutes, discard the precipitate and remove dead cells;

[0081] ③ Take the supernatant, centrifuge at 10000g for 30min, discard the precipitate and remove cell debris;

[0082] ④ Take the supernatant and centrifuge at 100,000 g for 70 min. The resulting precipitate is the exosomes.

[0083] ⑤Discard the supernatant, resuspend the precipitate with PBS, and centrifuge at 100,000 g for 70 min;

[0084] ⑥Discard the supernatant, suspend the precipitate with an appropriate amount of PBS, and store it in a -80℃ refrigerator for later use.

[0085] (3) Construction of exosomes loaded with siRNA:

[0086] siRNA was loaded into serum exosomes by electroporation. The specific steps are as follows:

[0087] 10 μg siRNA and 100 μg exosomes were resuspended in 400 μL PBS and electroporated using a Bio-RAD Gene PulserXCell TM instrument with a voltage of 350 V and a capacitance of 150 μF. Pulse was selected twice to obtain exosomes loaded with siRNA, which were placed on ice for 20 min for use.

[0088] Example 3 Effect of IL-18 on the secretion of proinflammatory cytokines by periodontal ligament cells

[0089] In this example, periodontal ligament cells were stimulated with IL-18 and cultured for 96 hours, and the cell supernatant was collected. The expression levels of IFN-γ, GM-CSF, IL-2, and TNF-α were detected by ELISA.

[0090] The results are shown in Figure 1(A), Figure 1(B), Figure 1(C), Figure 1(D) and Table 1. The expressions of the above cytokines in the culture supernatant were significantly increased after IL-18 stimulation (*P<0.05).

[0091] Table 1 Levels of cytokine secretion by periodontal ligament cells after IL-18 stimulation (pg / mL)

[0092] Cytokines Control group (pg / mL) IL-18 stimulation (pg / mL) IFN-γ 82.11±17.24 687.1±109.3* GM-CSF 32.90±7.29 89.27±19.47* IL-2 114.2±28.39 197.6±54.28* TNF-α 881.7±263.2 2269±746.1*

[0093] Example 4 IL-18 Antibody Drug Validation

[0094] Providing cytokine IL-18 antibody to the periodontitis model mice prepared in Example 1, the specific steps are as follows:

[0095] Drug application: 50 μL of anti-IL-18 antibody with a concentration of 5 μg / mL was injected into the bottom of the median gingival sulcus on the palatal side of the first and second molars of the periodontitis model mice on both sides. The injection was once a week for 4 consecutive weeks, for a total of 4 times.

[0096] Example 5 IL-18Rα siRNA exosome drug validation

[0097] Providing IL-18Rα siRNA exosomes to the periodontitis model mice prepared in Example 1, wherein the nucleic acid sequence of the siRNA is shown in SEQ ID NOs: 1 to 3, and the specific steps are as follows:

[0098] Drug application: IL-18Rα siRNA exosomes were injected into the mid-gingival sulcus of the first and second maxillary molars on both sides of the periodontitis model mice, once a week for 4 consecutive weeks, for a total of 4 times.

[0099] Example 6 Validation of the efficacy of IL-18 antibody in combination with siRNA exosomes

[0100] Providing cytokine IL-18 antibody and siRNA exosomes to the periodontitis model mice prepared in Example 1, the specific steps are as follows:

[0101] A PBS mixture of anti-IL-18 antibody (5 μg / mL) and siRNA exosomes (10 μg per injection per side) was injected into the palatal median gingival sulcus of the first and second maxillary molars of periodontitis model mice once a week for 4 consecutive weeks, for a total of 4 times.

[0102] Example 7 Therapeutic effect of IL-18 antibody in combination with siRNA exosomes

[0103] A normal control group, a simple silk ligation control group, a periodontitis group, a periodontitis + IL-18 antibody treatment group, a periodontitis + IL-18 antibody treatment group, and a periodontitis + IL-18 antibody + IL-18α siRNA exosome group were set up for micro-CT irradiation.

[0104] Figure 2(A) is the normal control group, Figure 2(B) is the silk thread ligation control group, Figure 2(C) is the periodontitis group, Figure 2(D) is the periodontitis + IL-18 antibody treatment group, Figure 2(E) is the periodontitis + IL-18Rα siRNA exosome group, and Figure 2(F) is the micro-CT test results of the periodontitis + IL-18 antibody + IL-18Rα siRNA exosome group. It can be seen that the amount of alveolar bone loss between the first and second molars in the periodontitis group (the distance between the cementoenamel junction and the alveolar crest) is higher than that in the normal control group and the silk thread group; while the use of IL-18 antibody or IL-18Rα siRNA exosome treatment alone can reduce the amount of alveolar bone loss; the combined treatment of IL-18 antibody and IL-18Rα siRNA exosomes has a more significant effect on slowing down the loss of alveolar bone.

[0105] Figure 3 The figure shows the distance of alveolar bone loss in periodontitis (mm) calculated based on the micro-CT results. After treatment with IL-18 antibody and siRNA exosomes, the distance of alveolar bone loss in periodontitis was significantly reduced compared with that in the bacteria-induced periodontitis group.

[0106] Figure 4(A) shows the expression of IL-1β, IL-6, IL-8, and IL-18Rα in periodontal tissues. Figure 4(B) shows the expression of IL-6, IL-8, and IL-18Rα in periodontal tissues. It can be seen that after treatment with IL-18 antibody and siRNA exosomes, the expression levels of IL-1β, IL-6, IL-8, and IL-18Rα in periodontal tissues were significantly decreased, indicating that IL-18 antibody and IL-18Rα siRNA exosomes work synergistically and have a significant intervention efficiency on gene mRNA.

[0107] Figure 5 The results of histological staining of the periodontal tissue of the maxillary molars show that the inflammatory cell infiltration in the periodontal tissue between the first and second molars in the bacterial-induced periodontitis group was obvious, which was significantly higher than that in the normal control group and the silk thread group alone; while the inflammatory cell infiltration in the groups treated with IL-18 antibody or IL-18Rα siRNA exosomes alone was alleviated; the combined treatment of IL-18 antibody and IL-18Rα siRNA exosomes significantly reduced the inflammatory cell infiltration.

[0108] In summary, the present invention found that the cytokine IL-18 can promote periodontal ligament cells to secrete a variety of cytokines that promote inflammatory responses, thereby leading to periodontal ligament tissue loss, enhancing inflammatory responses, and further leading to aggravated alveolar bone loss and promoting the disease progression of periodontitis; the present invention adopts a treatment scheme of IL-18 antibody in conjunction with IL-18Rα receptor siRNA exosomes to prevent and treat functional disorders of periodontal ligament cells, inhibit periodontal ligament tissue loss, alveolar bone absorption and the expression of periodontitis-related inflammatory cytokines and chemokines, and reduce the severity of periodontitis from the root source, thereby preventing the occurrence and development of periodontitis, which has potential huge application value for the clinical prevention and treatment of periodontitis, and lays a foundation for further research on the biological functions of IL-18 antibodies and IL-18Rα receptor siRNA.

[0109] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention. SEQUENCE LISTING <110> The Affiliated Stomatological Hospital of Sun Yat-sen University <120> Drugs for treating periodontal ligament cell dysfunction and alveolar bone loss caused by periodontal pathogen infection <130> 20190903 <160> 2 <170> PatentIn version 3.3 <210> 1 <211> twenty three <212> DNA <213> Synthesis <400> 1 aaacucggca uccuucaggu utt 23 <210> 2 <211> twenty three <212> DNA <213> Synthesis <400> 2 aaccugaagg augccgaguu utt 23

Claims

1. A pharmaceutical composition for treating periodontal ligament cell dysfunction and alveolar bone loss caused by periodontal pathogen infection, characterized in that: The pharmaceutical composition includes IL-18 antibody and IL-18R receptor siRNA exosomes; The concentration of the IL-18 antibody is 2-10 μg / mL; The concentration of the IL-18R receptor siRNA exosomes is 0.5-2 μg / μL; The mass ratio of the IL-18 antibody to the IL-18R receptor siRNA exosomes is (0.01-0.05):1; The nucleic acid sequence of the siRNA in the IL-18R receptor siRNA exosomes is shown in SEQ ID NO: 1-2.

2. The pharmaceutical composition according to claim 1, characterized in that The concentration of the IL-18 antibody is 5~8 μg / mL; the mass ratio of the IL-18 antibody to the IL-18R receptor siRNA exosomes is (0.02~0.025):

1.

3. The pharmaceutical composition according to any one of claims 1 to 2, characterized in that The pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

4. Use of the pharmaceutical composition according to any one of claims 1 to 3 in the preparation of a drug for treating periodontal diseases; The periodontal disease includes loss of periodontal supporting tissue and / or periodontal inflammation.

5. The use according to claim 4, characterized in that: The loss of periodontal supporting tissue includes loss of periodontal ligament and / or loss of alveolar bone.

6. The use according to claim 5, characterized in that: The alveolar bone loss is caused by periodontal pathogen infection.

7. The use according to claim 6, characterized in that: The periodontal pathogens include Porphyromonas gingivalis.

8. The use according to claim 7, characterized in that: The Porphyromonas gingivalis includes P.gingivalis strains.

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