CN110433289A - For treating the drug of periodontal ligament cell dysfunction and Alveolar Bone Loss caused by Periodontal Pathogens infect - Google Patents

Abstract

The present invention provides the drugs for treating periodontal ligament cell dysfunction and Alveolar Bone Loss caused by Periodontal Pathogens infect, and the drug includes IL-18 antibody and/or IL-18R receptor siRNA excretion body.Drug of the invention is using IL-18 antibody and/or loads IL-18R receptor siRNA excretion body as functional component, by in conjunction with IL-18 in periodontium, inhibit periodontal ligament cell production of pro-inflammatory cytokines, improve the forfeiture of Periodontal Supporting Tissue caused by periodontal mesangial cell function disorder and the effect of inflammatory cell infiltration under inflammatory conditions, to prevent the occurrence and development of periodontal disease, there are potential huge applications to be worth the clinical prevention treatment of periodontal disease.

Description

For the treatment of periodontal ligament cell dysfunction and alveolar disease caused by periodontal pathogenic bacteria infection Drugs for bone loss

technical field

The invention belongs to the technical field of medicine, and relates to a medicine for treating periodontal ligament cell dysfunction and alveolar bone loss caused by periodontal pathogenic bacteria infection.

Background technique

Periodontitis is a common oral disease, mainly caused by pathogenic microorganisms in the plaque biofilm to initiate innate immunity, adaptive immunity and inflammatory response, manifested as a series of complex inflammatory syndromes, mainly affecting the periodontal ligament and surrounding and the tissues supporting the teeth, leading to progressive loss of periodontal alveolar bone, which in turn leads to tooth loss.

The pathogenic microorganisms in the dental plaque biofilm are the main cause of periodontal disease, and the gingival microbial community is transplanted in the periodontal pocket, so that the dominant microbial community in the periodontal pocket changes from Gram-positive aerobic bacteria to Gram-positive bacteria. Lambert-negative anaerobic bacteria cause periodontal disease in healthy periodontal tissue. Porphyromonas gingivalis is considered to be the main pathogenic bacteria of periodontitis, and its number is low in the oral cavity of healthy people, but it is significantly elevated in the destructive structure plaque and calculus of periodontitis. Porphyromonas gingivalis has multiple virulence factors including: pili, degradative enzymes, and lipopolysaccharide. A low transplant amount 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. P. gingivalis can also help suppress the body's immune system and create a favorable environment for other bacteria to live in. In a mouse model of periodontitis, low concentrations of Porphyromonas gingivalis induced inflammatory periodontitis through the interaction of oral commensal microbiota and the complement system.

The host's recognition of pathogenic microorganisms is an important part of the pathological mechanism of periodontal disease, which can quickly trigger an immune response, and then promote the activation and recruitment of important immune components by secreting cytokines and chemokines. The detection of microbial products can promote the stabilization and proliferation of the microbial transplant community, and maintain a stable balance between the host oral microbial community and the body's immune system. At present, although the characteristics of disease progression injury in patients with chronic periodontitis have been clarified, the mechanism of host periodontal tissue protection and injury has not been fully elucidated.

The clinical manifestations of periodontal disease are ever-changing. In the site of active disease, the number of mast cells, monocytes/macrophages and plasma cells is significantly increased. These cells further activate monocytes/macrophages, lymphocytes, fibroblasts and other cell components, secrete a variety of cytokines and inflammatory mediators including IL-1, IL-6 and TNF-α, these cytokines and inflammatory mediators can work alone or in combination to promote alveolar bone resorption and Collagen breaks down.

Natural killer-like B cells (NKB cells) are a new type of immune cell subsets found in the spleen and mesenteric lymph nodes of mice. They mainly secrete IL-18 and IL-12 and play an antimicrobial role. The role of infection. IL-18 is the main cytokine secreted by NKB cells, which can induce lymphocytes and splenocytes to produce IFN-γ. IL-18 and IL-12 double gene deficiency can reduce NK cell activity and cellular response, and IL-18 and IL-12 have the function of activating NK cells in local inflammatory response. Since the pathogenesis 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 pathogenesis of periodontitis.

IL-18 plays a role by binding to its receptor IL-18R, which is widely present in a variety of cells and consists of a heterodimer α chain (IL-18Rα) and a β chain (IL-18Rα). IL-18Rβ), wherein IL-18Rα is a ligand-binding chain, and IL-18Rβ is used to mediate IL-18 signal transduction. Reducing the expression of IL-18 receptor can reduce the secretion of pro-inflammatory cytokines by periodontal ligament cells, thereby inhibiting the occurrence and development of periodontitis.

In recent years, RNA interference (RNA interfering, RNAi) has gained widespread attention. As a new gene therapy technology, RNAi refers to inhibiting target mRNA through degradation or translation, thereby causing post-transcriptional gene silencing. The field has broad application prospects. Through RNAi technology, siRNA efficiently and specifically blocks the expression of homologous genes in vivo, promotes the degradation of homologous mRNAs, and induces cells to exhibit specific phenotypes of gene deletion. However, siRNA is easily degraded by ribozyme (RNase) in vivo, with a short half-life and low transfection efficiency. Therefore, how to efficiently introduce specific silencing siRNA into target organs without damaging normal tissues and siRNA Among them, it has become an urgent problem to be solved in gene therapy.

At present, the carrier research of therapeutic siRNA mainly focuses on polycationic polyethyleneimine (PEI) nanoparticles and liposomes, however, PEI-RNA complexes tend to accumulate in the liver, lung, spleen and kidney, which limits its use. Application in other tissues can easily lead to the activation of inflammasomes associated with IL-1 induction and apoptosis, and promote inflammation; liposomes or lipid nanoparticles not only have hepatotoxicity, but also can adsorb opsonins, activate complement and Coagulation factors, and because of their inherent net charge and size, predispose to phagocytosis by the mononuclear phagocyte system, a process that, like PEI, can lead to cellular stress, inflammasome activation, and apoptosis.

Exosomes (exosomes) are a typical lipid bilayer membrane structure with a diameter of about 30-150 nm. The lipid bilayer structure and surface membrane proteins make exosomes have higher transfection efficiency in vivo. It is an important carrier of intercellular information transmission and has 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.

CN 102488654 A discloses a medicine for treating periodontitis, which is made of 5-250 parts by weight of doxycycline, 25-1000 parts of metronidazole and 10000 parts of glycerin. CN 104688998 A discloses a traditional Chinese medicine tablet for treating periodontitis and a preparation method thereof. According to weight, 8-14 parts of water extract of forsythia, 3-7 parts of water extract of coptis, 5 parts of ethanol extract of mallow seed -11 parts, 4-8 parts of rue ethanol extract, 4-9 parts of red peony ethyl acetate extract, 2-5 parts of crushed Cyperus cyperi, 3-6 parts of crushed Augustia, magnesium stearate 15 -20 parts, 2-6 parts of glycerin, 7-16 parts of modified starch, the above-mentioned ingredients are mixed evenly and then compressed with a tablet machine, which is a traditional 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 indicated weight ratio range: 20-30 parts of Xiangjiapi, 15-20 parts of Prunella vulgaris, 8-15 parts of Rehmannia glutinosa, 6-12 parts of wild chrysanthemum, 5-10 parts of Smilax smilax, 7-10 parts of forsythia, 8-12 parts of centipede, 5-10 parts of Perrin, 8-12 parts of Phellodendron chinensis, 6-10 parts of stephenia, 1 gallon -3 copies. Antibiotics or traditional Chinese medicines are used in the prior art to treat periodontitis, which have complex components, large side effects, insignificant therapeutic effect, and cannot achieve the purpose of radical cure.

The pathogenesis of periodontitis involves the interaction between microorganisms and the body's immune system, and the pathogenesis has not been fully elucidated. However, in-depth exploration of the immune pathogenesis of periodontitis provides theoretical and experimental basis for finding new targets for periodontitis treatment. At present, it is urgent It is necessary to develop drugs that can effectively inhibit periodontitis inflammation, reduce the incidence of periodontitis from the root cause, and prevent the occurrence of periodontitis.

Contents of the invention

Aiming at 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 pathogenic bacteria infection. The drug uses IL-18 antibody and IL-18R Receptor siRNA exosomes are functional components, and IL-18 antibody and IL-18R receptor siRNA exosomes play a synergistic effect, which can inhibit periodontitis alveolar bone resorption and expression of periodontitis-related inflammatory cytokines and chemokines , Reduce the incidence of periodontal disease from the root cause.

For reaching this purpose, the present invention adopts following technical scheme:

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

In the present invention, on the basis of studying the relationship between immune response and periodontal disease, the inventor unexpectedly discovered that in patients with periodontal disease, the cytokine IL-18 secreted by Natural killer-like B cells (NKB) The expression levels in peripheral blood and gingival crevicular fluid were significantly increased. Not only that, IL-18 in gingival crevicular fluid of patients with periodontitis was significantly positively correlated with AL (Attachment loss, attachment loss), which shows that IL-18 It is the main effector molecule of NKB cells in the pathogenesis of periodontitis; on this basis, the inventors further found that the level of IL-18 in the gingival crevicular fluid after periodontal basic treatment was significantly reduced, and when microbial pathogenic factors such as plaque were removed, After treatment, the number of microorganisms and the level of antigens were significantly decreased, the local inflammatory response was reduced, the stimulation of NKB cells was reduced, and the level of IL-18 secreted by them was also reduced, which further confirmed that IL-18 was involved in the pathogenesis of periodontitis, and mainly in The periodontal tissue plays a role locally, so the inventors used the drug prepared by IL-18 antibody to inhibit the symptoms of periodontitis and effectively inhibit the inflammation of periodontitis.

In the present invention, the inventors also found that IL-18 can promote periodontal ligament cells to secrete various cytokines that promote inflammatory response, thereby promoting the disease process of periodontitis.

In the present invention, the drug uses IL-18 antibody and exosomes loaded with IL-18R receptor siRNA as functional components, IL-18 antibody reduces the expression level of IL-18 by targeting IL-18, IL-18R Receptor siRNA exosomes reduced the expression level of IL-18 by inhibiting the expression of IL-18 mRNA through translation, and the two played a synergistic effect, inhibited the expression of inflammatory cytokines and chemokines in periodontal tissue, and reduced the expression of inflammatory cytokines and chemokines. The effect of chemokines on the secretion of pro-inflammatory cytokines by periodontal ligament cells fundamentally reduces the inflammatory infiltration, improves the dysfunction of periodontal ligament cells, and prevents the occurrence and development of periodontal disease.

Preferably, the concentration of the IL-18 antibody is 2-10 μg/mL, such as 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 5-8 μg/mL.

Preferably, the concentration of the IL-18R receptor siRNA exosomes is 0.5-2 μg/1 μL, such as 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 , 1.0μg/1μL, 1.1μg/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 2.0 μg/1 μL, preferably 1.0 μg/1 μL.

Preferably, the mass ratio of the IL-18 antibody to the IL-18R receptor siRNA exosomes is (0.01-0.05):1, such as 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.

Preferably, the nucleic acid sequence of the siRNA in the IL-18Rα receptor siRNA exosome is shown in SEQ ID NO: 1-2:

SEQ ID NO: 1 (sense strand): 5'-AAACUCGGCAUCCUUCAGGUUTT-3';

SEQ ID NO: 2 (antisense strand): 5'-AACCUGAAGGAUGCCGAGUUUTT-3'.

Preferably, the pharmaceutical composition further includes any one or a combination of at least two of pharmaceutically acceptable carriers, excipients or diluents.

In the second aspect, the present invention provides a drug efficacy evaluation model of the pharmaceutical composition according to the first aspect, and the model is a periodontal disease model mouse.

In a third aspect, the present invention provides a method for preparing a drug efficacy evaluation model as described in the second aspect, the method adopts silk ligation combined with induction of Porphyromonas gingivalis to construct the model.

Preferably, the method comprises the steps of:

Place the silk thread on the upper jaw of a healthy mouse without periodontal disease, inoculate Porphyromonas gingivalis on the gingiva and/or smear it on the oral cavity, induce periodontal disease in the mouse, and obtain the periodontal disease model mouse .

Preferably, the Porphyromonas gingivalis is a P. gingivalis strain.

Preferably, the concentration of Porphyromonas gingivalis is 1×10 ⁸ ~1×10 ¹⁰ CFU/mL, such as 1×10 ⁸ CFU/mL, 1×10 ⁹ CFU/mL or 1×10 ¹⁰ CFU/mL.

As a preferred technical solution, the present invention provides a method for preparing a drug efficacy evaluation model as described in the second aspect. The method adopts silk ligation combined with Porphyromonas gingivalis induction for model construction, including the following steps:

(1) Bacterial culture:

Mix 0.5% hemin vitamin solution and bovine heart brain infusion juice medium (BHI medium) according to 1:100, and inoculate P.gingivalis strain in the medium;

Inoculate Porphyromonas gingivalis (P.gingivalis ATCC 33277) in the above-mentioned prepared medium, culture under strict anaerobic conditions of 85% nitrogen, 10% hydrogen, 5% carbon dioxide, and 37°C, and passage 2 to 3 After that, culture for 2 days; when the concentration reaches 1×10 ⁹ CFU/mL, resuspend the bacteria in PBS buffer containing 2% sodium carboxymethylcellulose for use;

(2) Establishment of periodontitis model in mice ligated by silk thread and experimental grouping:

Mice with complete dentition, no caries and periodontal disease were screened out, anesthetized by intraperitoneal injection of 1% pentobarbital sodium (0.15mL/20g), fixed limbs in supine position, 1% iodine tincture disinfected mouth and perioral area, 75% Alcohol deiodination, the upper jaw was fixed with silk thread traction;

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 mesial and distal sides, and knotted on the buccal or palatal side;

Check whether the silk thread falls off every 4 days, and re-ligate it in time if it falls off; the IL-18 antibody treatment experimental group adopts local smearing in the oral cavity and local injection of Porphyromonas gingivalis suspension on the buccal and palatal side periodontium of maxillary molars At the same time, 50 μL of anti-IL-18 antibody with a concentration of 5 μg/mL was locally injected into the buccal and palatal periodontal tissues of maxillary molars, and injected once every 2 to 4 days;

Use a cotton swab to wipe excess bacteria on the maxillary molar area in the oral cavity; the control periodontitis group uses a micro syringe to locally inject Porphyromonas gingivalis suspension in the maxillary molar buccal and palatal periodontium; the normal group uses 2% Carboxymethylcellulose sodium-PBS solution is applied locally in the mouth, and an equal amount of PBS is injected into the periodontal tissue using a micro-syringe;

After 4 weeks, the mice were sacrificed by cervical dislocation, and the samples were fixed.

In a fourth aspect, the present invention provides an application 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 treating periodontal diseases.

Preferably, the periodontal support tissue loss includes periodontal ligament loss and/or alveolar bone loss.

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

Preferably, the periodontal pathogens include Porphyromonas gingivalis.

Preferably, the Porphyromonas gingivalis comprises P. gingivalis strains.

In the present invention, Porphyromonas gingivalis, the main pathogenic bacteria of periodontitis, stimulates periodontal ligament cells to secrete pro-inflammatory cytokines, thereby triggering periodontal tissue inflammatory infiltration, causing periodontal ligament dysfunction and leading to periodontal ligament loss on the one hand , and on the other hand cause alveolar bone loss, the pharmaceutical composition reduces the effect of IL-18 on The ability of periodontal ligament cells to secrete pro-inflammatory cytokines, inhibit periodontitis alveolar bone resorption, and the expression of periodontitis-related inflammatory cytokines and chemokines, thereby effectively inhibiting periodontitis inflammation, alveolar bone resorption, and Reduce the incidence of periodontitis from the pathogenesis of inflammation, and then prevent the occurrence of periodontitis.

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

(1) The present invention finds that the cytokine IL-18 can promote periodontal ligament cells to secrete a variety of cytokines that promote inflammatory response, thereby leading to the loss of periodontal ligament tissue, enhancing the inflammatory response, leading to aggravated alveolar bone loss, and promoting periodontal inflammatory disease process;

(2) In the pharmaceutical composition of the present invention, the IL-18 antibody is used as a targeted drug to prevent and treat periodontal ligament cell dysfunction, and the IL-18R receptor siRNA exosomes reduce the expression of IL-18 mRNA by translationally inhibiting the expression of IL-18 18, the two play a synergistic effect, thereby inhibiting the loss of periodontal ligament tissue, alveolar bone resorption and the expression of periodontitis-related inflammatory cytokines and chemokines, and reducing the incidence of periodontitis from the root cause. Preventing the occurrence and development of periodontitis has great potential application value in the clinical prevention and treatment of periodontitis, and lays the foundation for further research on the biological functions of IL-18 antibody and IL-18R receptor siRNA;

(3) The present invention provides a mouse model of periodontitis, which is induced by silk ligation combined with Porphyromonas gingivalis, and the effects of IL-18 antibody and IL-18R receptor siRNA on periodontitis were studied by periodontal local injection. therapeutic and preventive effects.

Description of drawings

Figure 1 is a study of the effect of IL-18 on the secretion of pro-inflammatory cytokines in periodontal ligament cells, in which Figure 1(A) is the change in the expression level of IFN-γ, Figure 1(B) is the change in the expression level of GM-CSF, Fig. 1 (C) is the expression level change of IL-2, and Fig. 1 (D) is the expression level change of TNF-α;

Fig. 2 is the CT image of periodontitis alveolar bone loss, wherein, Fig. 2 (A) is the normal control group, Fig. 2 (B) is the simple silk thread ligation control group, Fig. 2 (C) is the periodontitis group, Fig. 2 ( D) is periodontitis + IL-18 antibody treatment group, Figure 2(E) is periodontitis + IL-18α siRNA exosome group, Figure 2(F) is periodontitis + IL-18 antibody + IL-18α siRNA exosome group;

Figure 3 is a histogram of the periodontitis alveolar bone loss distance (mm) obtained statistically according to the micro-CT results;

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

Figure 5 is the results of histological staining of the periodontal tissue region of the maxillary molars.

Detailed ways

In order to further illustrate the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with the embodiments and accompanying drawings. It should be understood that the specific implementation manners described here are only used to explain the present invention, rather than to limit the present invention.

If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products commercially available through regular channels.

Materials and Reagents

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

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

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

Example 1 Silk thread ligation combined with Porphyromonas gingivalis periodontitis modeling

(1) Bacterial culture

Mix 0.5% hemin vitamin solution and bovine heart brain infusion juice medium (BHI medium) according to 1:100, inoculate the P.gingivalis strain in the medium, in 85% nitrogen, 10% hydrogen, 5% Cultivate under strict anaerobic conditions of carbon dioxide and 37°C;

After two generations of culture, when the bacterial concentration reaches 1×10 ⁸ CFU/mL, resuspend the bacteria in PBS buffer containing 2% sodium carboxymethylcellulose for use;

The bacteria cultured for 48 hours were identified according to the colony morphology, Gram staining, cell morphology and biochemical reaction results;

(2) modeling

Choose C57BL/6 mice with complete dentition, no caries and periodontal disease for 8 weeks, and give them to mice in an environment with good lighting conditions, clean, quiet, well-ventilated, room temperature at 22°C, and no special pathogenic bacteria. The aseptic feed was fed in separate cages, and the photoperiod was 12h:12h;

Adopt 4% chloric acid hydrate intraperitoneal injection anesthesia (0.35mg/kg), after waiting for anesthesia, mouse adopts supine position, tape is fixed on the operating table, adopts 1% iodine tincture to disinfect oral cavity and perioral area, and deiodination with 75% alcohol, with The upper incisors were stretched and fixed with cotton thread to make the mouth wide open; the gingival tissue of the upper first molar on the left side was separated with a pointed probe, and the silk thread was embedded in the neck of the adjacent teeth of the first and second molars. Place it on the gingival margin of the first molar with ligation and fixation at the nipple, and place it in the gingival sulcus as much as possible;

Check whether the needle and thread fall off once a week, and re-ligate them in time if they fall off;

Inoculate 100 μL of Porphyromonas gingivalis on the palatal side of the right maxillary second molar, twice a week.

Example 2 Preparation of IL-18 receptor siRNA exosomes

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

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

SEQ ID NO: 1 (sense strand): 5'-AAACUCGGCAUCCUUCAGGUU-3';

SEQ ID NO: 2 (antisense strand): 5'-AACCUGAAGGAUGCCGAGUUUTT-3';

(2) Separation and purification of exosomes derived from mouse serum

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

②Take the supernatant, centrifuge at 2000g for 10min, discard the precipitate, and remove dead cells;

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

④Take the supernatant and centrifuge at 100,000g for 70min, the resulting precipitate is exosomes;

⑤Discard the supernatant, resuspend the pellet with PBS, and centrifuge at 100,000g for 70min;

⑥ Discard the supernatant, suspend the sediment with an appropriate amount of PBS, and store it in a -80°C refrigerator for later use.

(3) Construction of exosomes loaded with siRNA:

siRNA was loaded into serum exosomes by electroporation, and the specific steps were as follows:

Resuspend 10 μg of siRNA and 100 μg of exosomes in 400 μL of PBS, and use Bio-RAD Gene PulserXCell TM instrument for electroporation with a voltage of 350 V and a capacitance of 150 μF. Select Pulse twice to obtain exosomes loaded with siRNA, and place them on ice Leave it on for 20 minutes.

Example 3 Effect of IL-18 on the secretion of pro-inflammatory cytokines by periodontal ligament cells

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

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 after IL-18 stimulation were all significantly increased ( *P<0.05).

Table 1 Levels of cytokines secreted by periodontal ligament cells after IL-18 stimulation (pg/mL)

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*

Embodiment 4 IL-18 Antibody Drug Verification

The cytokine IL-18 antibody was provided to the periodontitis model mice prepared in Example 1, and the specific steps were as follows:

Drug application: 50 μL of anti-IL-18 antibody was injected into the middle gingival sulcus bottom of the palatal sides of the first and second maxillary molars of the periodontitis model mice, with a concentration of 5 μg/mL, once a week for 4 consecutive weeks. A total of 4 times.

Example 5 Validation of IL-18Rα siRNA exosome drug

Provide IL-18Rα siRNA exosomes to the periodontitis model mice prepared in Example 1, the nucleic acid sequence of siRNA is shown in SEQ ID NO: 1-3, and the specific steps are as follows:

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

Example 6 Efficacy verification of IL-18 antibody synergy with siRNA exosomes

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

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

Example 7 The therapeutic effect of IL-18 antibody synergistic siRNA exosomes

Set normal control group, simple silk ligation control group, periodontitis group, periodontitis+IL-18 antibody treatment group, periodontitis+IL-18 antibody treatment group and periodontitis+IL-18 antibody+IL-18αsiRNA The exosome group underwent micro-CT irradiation.

Figure 2(A) is the normal control group, Figure 2(B) is the simple silk 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 exosomes group, and Figure 2(F) is the micro-CT detection results of the periodontitis+IL-18 antibody+IL-18Rα siRNA exosomes group. It can be seen that the amount of alveolar bone loss between the first molar and the second molar in the periodontitis group (the distance between the enamel cementum junction and the alveolar crest) is higher than that in the normal control group and the pure silk thread group; Treatment with IL-18 antibody or IL-18Rα siRNA exosomes can reduce alveolar bone loss; combined treatment with IL-18 antibody and IL-18Rα siRNA exosomes has a more significant effect on slowing alveolar bone loss.

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

Figure 4(A) is the expression of periodontal tissue cytokine IL-1β, Figure 4(B) is the expression of periodontal tissue cytokine IL-6, and Figure 4(C) is the expression of periodontal tissue cytokine IL-8 Figure 4(D) shows the expression of IL-18Rα. It can be seen that after treatment with IL-18 antibody and siRNA exosomes, IL-1β, IL-6, IL-8 and IL-1β in periodontal tissue The expression of -18Rα was significantly decreased, indicating that the IL-18 antibody and IL-18Rα siRNA exosomes act synergistically and have a significant intervention efficiency on gene mRNA.

Figure 5 is the results of histological staining of the periodontal tissue region of the maxillary molars. It can be seen that the infiltration of inflammatory cells in the periodontal tissue between the first molar and the second molar in the bacteria-induced periodontitis group was significantly higher than that in the normal control group and the simple periodontitis group. The silk thread group was significantly increased; while the inflammatory cell infiltration in the treatment group treated with IL-18 antibody or IL-18Rα siRNA exosomes alone was reduced; the combined treatment of IL-18 antibody and IL-18Rα siRNA exosomes significantly reduced inflammation cell infiltration.

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 response, thereby leading to the loss of periodontal ligament tissue, enhancing the inflammatory response, and further leading to the aggravation of alveolar bone loss and promoting The disease process of periodontitis; the present invention adopts the treatment plan of IL-18 antibody and IL-18Rα receptor siRNA exosomes to prevent and treat the dysfunction of periodontal ligament cells, inhibit the loss of periodontal ligament tissue, alveolar bone resorption and The expression of periodontitis-related inflammatory cytokines and chemokines can reduce the incidence of periodontitis from the root cause, and then prevent the occurrence and development of periodontitis. It has great potential application value in the clinical prevention and treatment of periodontitis. And it laid the foundation for further research on the biological function of IL-18 antibody and IL-18Rα receptor siRNA.

The applicant declares that the present invention illustrates the detailed methods of the present invention through the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed methods to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

SEQUENCE LISTING

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Claims (10)

1. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises IL-18 antibody and/or IL-18R receptor siRNA exosomes. 2. The pharmaceutical composition according to claim 1, wherein the concentration of the IL-18 antibody is 2-10 μg/mL, preferably 5-8 μg/mL; Preferably, the concentration of the IL-18R receptor siRNA exosomes is 0.5-2 μg/μL; Preferably, the mass ratio of the IL-18 antibody to the IL-18R receptor siRNA exosome is (0.01-0.05):1, preferably (0.02-0.025):1. 3. The pharmaceutical composition according to claim 1 or 2, wherein the nucleic acid sequence of the siRNA in the IL-18Rα receptor siRNA exosomes is shown in SEQ ID NO: 1-2. 4. The pharmaceutical composition according to any one of claims 1-3, wherein the pharmaceutical composition further comprises any one or at least two of pharmaceutically acceptable carriers, excipients or diluents. combination of species. 5. A drug effect evaluation model of the pharmaceutical composition according to any one of claims 1-4, wherein the model is a periodontal disease model mouse. 6. A method for preparing a drug efficacy evaluation model as claimed in claim 5, characterized in that, the method adopts silk ligation combined with induction of Porphyromonas gingivalis to construct the model. 7. The method according to claim 6, characterized in that the method comprises the steps of: Place the silk thread on the upper jaw of a healthy mouse without periodontal disease, inoculate Porphyromonas gingivalis on the gingiva and/or smear it on the oral cavity, induce periodontal disease in mice, and obtain the periodontal disease model mouse . 8. The method according to claim 6 or 7, wherein the Porphyromonas gingivalis is a strain of P.gingivalis; Preferably, the concentration of Porphyromonas gingivalis is 1×10 ⁸ -1×10 ¹⁰ CFU/mL. 9. An application of the pharmaceutical composition as claimed in any one of claims 1-4 and/or the drug efficacy evaluation model as claimed in claim 5 in the preparation of periodontal disease therapeutic drugs. 10. The application according to claim 9, wherein the periodontal disease comprises periodontal support tissue loss and/or periodontal inflammation; Preferably, the periodontal support tissue loss comprises periodontal ligament loss and/or alveolar bone loss; Preferably, the alveolar bone loss is caused by periodontal pathogen infection; Preferably, the periodontal pathogenic bacteria include Porphyromonas gingivalis; Preferably, the Porphyromonas gingivalis comprises P. gingivalis strains.