A composite mouse model simulating the exacerbation of periodontitis due to hyperuricemia, its construction method and application

Abstract

The present application relates to a composite mouse model that simulates the exacerbation of periodontitis due to hyperuricemia, its construction method and its application. The method includes the following steps: Step 1: Inject potassium oxonate into the abdominal cavity of a mouse continuously for 7 days to construct a hyperuricemia model; Step 2: On day 7, ligate the maxillary second molar of the mouse with surgical silk thread using a microsurgical needle holder to construct a periodontitis model; Step 3: Inject potassium oxonate into the abdominal cavity of the mouse continuously for 7 days from day 8 to day 14 to construct a combined mouse model simulating the exacerbation of periodontitis due to hyperuricemia; Step 4: Inject potassium oxonate (PO) into the abdominal cavity of the mouse continuously at a fixed frequency of once per day for 14 days (days 15 to 28) to maintain the stability of the hyperuricemia model and construct a stable combined mouse model of hyperuricemia and periodontitis; Step 5: On day 15, simultaneously inject allopurinol into the abdominal cavity continuously for 14 days to perform uric acid-lowering treatment.

Description

[Technical Field]

[0001] This application claims priority to a Chinese patent application bearing application number 202310190961.2, filed on March 2, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present application belongs to the technical field of constructing models for biological experiments, and in particular relates to a composite mouse model simulating the aggravation of periodontitis due to hyperuricemia, its construction method and its application. [Background technology]

[0003] Periodontitis is a chronic, multifactorial inflammatory disease characterized by the destruction of periodontal tissues and caused by abnormalities in plaque biofilms. By inducing an inflammatory response in the host, specific microorganisms cause progressive destruction of periodontal tissues, ultimately destroying connective and bone tissue. Severe periodontitis is the sixth most common disease in the world. The prevalence of severe periodontitis in China is 30.6%, significantly higher than the global average (11.2%). Severe periodontitis can lead to tooth loss, severely impacting quality of life and potentially negatively affecting overall health. Related studies have shown that the "three highs"—hyperglycemia, hypertension, and hyperlipidemia—are major risk factors for periodontitis, and their relationship with periodontitis has been documented to varying degrees.

[0004] Hyperuricemia, one of the components of metabolic syndrome, has become the "fourth high" after the "three highs." Hyperuricemia is a common comorbidity and potential risk factor for cardiovascular disease, chronic kidney disease, and diabetes. The relationship between hyperuricemia and periodontitis, and whether hyperuricemia is an intermediate factor in the relationship between diabetes, cardiovascular disease, and metabolic syndrome, have become emerging public health issues.

[0005] Hyperuricemia is a metabolic disease caused by excessive production and / or insufficient intestinal and / or renal excretion of uric acid salts. It is characterized by excessively elevated blood uric acid levels, and its prevalence has been increasing in recent years. According to reports, hyperuricemia affects approximately 15% to 20% of the world's population, with the number of hyperuricemia patients in China reaching 150 million. Recent studies have shown that hyperuricemia may be a new risk indicator for periodontitis. One retrospective cohort study showed that patients with gout (an inflammatory disease caused by abnormally elevated blood uric acid levels, resulting in the deposition of sodium urate crystals in joints and surrounding tissues) had a 13% increased risk of developing periodontitis compared with the control group (HR = 1.13, 95% CI = 1.10-1.16). Colchicine, a gout treatment, effectively reduced the risk of developing periodontitis (HR = 0.85, 95% CI = 0.79-0.91). Animal studies have shown that febuxostat (a uric acid-lowering drug) can reduce the progression of periodontitis in rats. A clinical cross-sectional study found that serum uric acid was a risk factor for localized stage II / III periodontitis (OR = 1.10, 95% CI = 1.00-1.21). The results of a systematic evaluation conducted by this research group in the previous phase showed that, compared with healthy controls, patients with periodontitis had elevated serum uric acid concentrations (WMD = 1.00 mg / dL, 95% CI = 0.63-1.37, P < 0.001) and reduced salivary uric acid concentrations (SMD = -0.95, 95% CI = 1.23-0.68, P < 0.001). The effectiveness of lowering serum uric acid in patients with periodontitis as a non-surgical treatment for periodontitis has been confirmed. Some studies have not supported the association between hyperuricemia and periodontitis. One cross-sectional study (8,809 patients with hyperuricemia and 126,465 controls) found no significant correlation between elevated uric acid levels and the diagnosis of periodontitis (OR = 0.89, 95% CI = 0.81-0.96). Another cross-sectional study conducted in the same country (12,735 Korean adults) found a significant positive correlation (OR = 1.62, 95% CI = 1.13-2.33) between periodontitis and hypouricemia (but not hyperuricemia).In summary, there is little evidence of a direct relationship between hyperuricemia and periodontal disease, and relevant studies have primarily examined the relationship between periodontitis and changes in blood uric acid levels within the normal range, rather than the relationship between periodontitis and hyperuricemia. The conclusions reached from the few studies on periodontitis and hyperuricemia appear to be contradictory. Furthermore, there is still a lack of evidence from animal experiments to establish the relationship between both at the biological mechanism level. Therefore, developing an animal model that can simulate the exacerbation of periodontitis due to hyperuricemia is crucial for verifying and reproducing clinical research results and establishing the relationship between both biological mechanisms.

[0006] Currently, there is only one reported animal model of hyperuricemia-induced periodontitis exacerbation, and this was conducted by our research group. A mouse model combining hyperuricemia and periodontitis was established by feeding the mice a diet supplemented with potassium oxonate and uric acid and ligating their maxillary second molars with silk suture. At the end of the experiment, the mice's serum uric acid levels were measured and micro-CT analysis of the maxillae was performed. While the results showed elevated blood uric acid levels in the group fed potassium oxonate and uric acid diet, micro-CT analysis of the jawbone revealed no significant effects of hyperuricemia or elevated uric acid levels on exacerbating periodontal damage.

[0007] In this related technique, a mouse model combining hyperuricemia and periodontitis established using the dietary and ligation methods failed to show a correlation between uric acid concentration and alveolar bone resorption. Possible explanations for this are: (1) although the periodontitis model was successfully established, the observation time was insufficient, and the potential adverse effects of hyperuricemia on alveolar bone resorption did not emerge within the short term; (2) the small sample size and individual differences masked the possible side effects of hyperuricemia. In light of this, this research group again attempted to establish a long-term hyperuricemia model using the dietary method by feeding mice a diet containing 5% potassium oxonate and 2.5% uric acid. Blood samples were collected at the end of the experiment (35 days, 43 days, and 53 days), and serum uric acid levels were measured using a fully automated biochemical analyzer. The results showed that serum uric acid levels in the group fed the combined potassium oxonate and uric acid diet were not significantly different from those in the group fed the normal diet. Therefore, the technology for constructing a hyperuricemia model using dietary feeding is not stable. This partially explains why the "hyperuricemia" mouse model established by this research group above is unable to exacerbate bone destruction caused by periodontitis in mice. In summary, at present, there are no reports of an animal model that successfully simulates the exacerbation of periodontitis caused by hyperuricemia, either domestically or internationally. To fill this gap in research, new animal model technology that can simulate the exacerbation of periodontitis caused by hyperuricemia is urgently needed.

[0008] Currently, methods for constructing periodontitis and hyperuricemia models are relatively mature and diverse. However, there are very few reports in the technical field regarding the construction of mouse models of combined periodontitis and hyperuricemia, both domestically and internationally. There have been no reports of successfully constructing a mouse model of exacerbated periodontitis due to hyperuricemia. Achieving this goal poses the following major challenges: (1) Similar to the pathological process of human periodontitis, which is initiated by dental plaque, the ligation method can cause localized plaque accumulation and induce periodontitis formation. Therefore, the ligation method is currently the most commonly used method for constructing animal periodontitis models worldwide. However, a careful review of the literature revealed that although mice are the most convenient, inexpensive, and versatile model, the ligation method is rarely used in mice, while oral infection models of periodontitis are more widely used. On the other hand, the current ligature-induced periodontitis procedure significantly damages the animal's gums, and sharp instruments can easily cause gingival bleeding in mice. In severe cases, blood can leak into the airway, potentially leading to death. Furthermore, the limited oral environment of mice poses severe technical challenges for ligation. (2) Hyperuricemia models established by supplementing exogenous uric acid and uric acid precursors and inhibiting renal uric acid excretion are unable to maintain stable elevated uric acid levels due to the presence of uricase. In recent years, potassium oxonate has become the most commonly used drug for establishing hyperuricemia models worldwide. It competitively binds to uricase, inhibiting its activity, and can rapidly and sustainably increase blood uric acid levels. Although this method can form a model in a short period of time (2 weeks), its competitive inhibition mechanism still requires long-term administration to maintain the hyperuricemia model after modeling. If the administration concentration or frequency is too low, uric acid levels cannot be maintained well, but if the administration concentration or frequency is too high, adverse effects such as a strong inflammatory response in the kidneys of experimental animals are likely to occur. Therefore, in medium- to long-term experiments, it is particularly important to consider the appropriate administration concentration and frequency to maintain higher levels of uric acid concentrations without causing side effects such as a strong inflammatory response in the animal's kidneys.(3) The construction of a combined model of hyperuricemia and periodontitis does not simply involve superimposing the hyperuricemia model and the periodontitis model. If the exposure factor and research objective (for example, when studying the effect of hyperuricemia on periodontitis, the former is the exposure factor, and conversely, periodontitis is the exposure factor; when studying the interrelationship or synergy between the two, both are exposure factors), differ, the ligation time and hyperuricemia induction time will also differ. The key issue is that it is difficult to predict the effect that the selection of the ligation time and hyperuricemia induction time, as well as the intervention timing (time point) of these two measures, will have on the combined model of hyperuricemia and periodontitis. Summary of the Invention [Problem to be solved by the invention]

[0009] The purpose of this application is to overcome the shortcomings of the prior art by providing a composite mouse model simulating the aggravation of periodontitis caused by hyperuricemia, a construction method and its application, and a model for improving periodontitis in mice aggravated by hyperuricemia through uric acid-lowering treatment. The mouse model construction method of this application not only increases uric acid levels in a short period of time, but also constructs a periodontitis model gently and without damage, effectively constructing a mouse model simulating the aggravation of periodontitis caused by hyperuricemia. This effectively fills the gap in animal models in the research field of hyperuricemia and periodontitis, and is of great significance for elucidating the pathological mechanisms of the complex relationship between the two and screening drugs related to the aggravation of periodontitis caused by hyperuricemia. [Means for solving the problem]

[0010] To achieve the objectives of this application, this application provides a method for constructing a composite mouse model that simulates the aggravation of periodontitis caused by hyperuricemia, comprising: Step 1: Inject potassium oxonate intraperitoneally into mice for 7 consecutive days to establish a hyperuricemia model; On day 7, construct a periodontitis model by ligating the maxillary second molars of the mice with surgical silk thread using a microsurgical needle holder (step 2). Step 3: Construct a composite mouse model simulating the exacerbation of periodontitis due to hyperuricemia by continuously injecting potassium oxonate intraperitoneally into mice for 7 days (days 8 to 14).

[0011] In some embodiments of the present application, after step 3, step 4 is further included in which potassium oxonate is continuously injected into the peritoneal cavity of the mice for 14 days (days 15 to 28) to maintain the stability of the hyperuricemia model.

[0012] In some embodiments of the present application, after step 4, step 5 is further included, in which, on the 15th day, allopurinol is simultaneously injected intraperitoneally for 14 consecutive days (days 15 to 28) to perform uric acid-lowering treatment.

[0013] In some embodiments of the present application, in step 1 and / or step 3 and / or step 4, potassium oxonate is injected intraperitoneally into the mouse once a day at a dose of 600 mg / kg / d.

[0014] In some examples of the present application, in step 5 above, allopurinol is injected intraperitoneally into the mice once a day at a dose of 5 mg / kg / d.

[0015] In some embodiments of the present application, injecting potassium oxonate into the abdominal cavity of the mouse in step 1 and / or step 3 and / or step 4 includes gently grasping the mouse and fixing it in the palm of your hand, and injecting potassium oxonate mother solution having a concentration of 30 mg / mL into the abdominal cavity of the mouse.

[0016] In some embodiments of the present application, injecting allopurinol into the abdominal cavity of the mouse in step 5 above includes gently grasping the mouse and fixing it in the palm of your hand, and injecting allopurinol mother solution having a concentration of 0.5 mg / mL into the abdominal cavity of the mouse.

[0017] In some embodiments of the present application, step 2 comprises: Step a: Anesthetize and fix the mouse, expose the maxillary molar area, and clean and thoroughly dry the maxillary molars, buccal, and lingual gingiva. Step b: fix the upper jaw of the mouse to a mouse board with a surgical silk thread, pinch the surgical silk thread with the end of a needle holder, and pass the surgical silk thread through the gap between the second molar and the third molar; Step c) of wrapping a surgical silk thread from the distal side of the buccal surface of the second molar to the mesial side of the buccal surface of the second molar, pinching the surgical silk thread, and passing the surgical silk thread through the space between the first molar and the second molar; and step d) of tying the surgical silk thread to the palatal side of the second molar using a needle holder. Step e: Tie the silk thread securely with three surgical knots and cut off the excess silk thread with spring scissors; Includes.

[0018] In some embodiments of the present application, in step a, anesthetizing and fixing the mouse and exposing the maxillary molar region includes anesthetizing the abdominal cavity of the mouse and fixing the abdominal cavity of the mouse upward on a mouse board; tilting the mouse board to fix it, using a mouth gapper to stably open the mouse's mouth to expose the maxillary molar region, and using a head-mounted light to provide a view of the molar region.

[0019] In some embodiments of the present application, cleaning the maxillary molars, buccal and lingual gums and thoroughly drying includes cleaning the maxillary molars, buccal and lingual gums using saline, followed by thorough drying with a dry cotton ball.

[0020] In some embodiments of the present application, the surgical silk is 5-0 surgical silk and the needle holder is a microsurgical needle holder with model number W40350.

[0021] In some examples of the present application, the mice are 6-8 week old male C57BL / 6 mice.

[0022] In order to achieve the objectives of this application, the present application further provides a composite mouse model that simulates the aggravation of periodontitis due to hyperuricemia, obtained by the construction method described in any one of the above examples, and a model in which mouse periodontitis aggravated by hyperuricemia is improved by uric acid-lowering treatment.

[0023] To achieve the objectives of this application, this application aims to provide an application of the above-mentioned composite mouse model simulating the exacerbation of periodontitis due to hyperuricemia to the screening of therapeutic agents for periodontitis exacerbated by hyperuricemia. [Effects of the Invention]

[0024] Compared with the prior art, the present application can achieve the following beneficial effects: 1. This application effectively fills the gap in animal models for the study of hyperuricemia-induced periodontitis aggravation, and for the first time innovatively simulates a combined mouse model of hyperuricemia-induced periodontitis aggravation, which shows great application value in exploring the pathological mechanisms of the complex relationship between hyperuricemia and periodontitis and screening drugs for hyperuricemia-induced periodontitis aggravation, and to a certain extent broadens the ideas and methods of research in this field. 2. In this application, it only took 14 days to construct a composite mouse model simulating the aggravation of periodontitis caused by hyperuricemia, and the entire method for constructing a stable hyperuricemia-periodontitis composite mouse model only took 28 days. This application has the advantages of stable and reliable modeling effect and short modeling time. 3. In this application, a hyperuricemia model was successfully established and periodontitis was further induced by ligating the maxillary second molars of mice with silk thread. In terms of time sequence, hyperuricemia occurred first, followed by periodontitis, which further demonstrated the aggravating effect of hyperuricemia on periodontitis, and the modeling order was more scientific. 4. In this study, the hyperuricemia model was induced and maintained by intraperitoneal injection of potassium oxonate at a dose of 600 mg / kg / d, which not only maintained a high and stable uric acid concentration but also avoided the drawback of renal inflammatory responses caused by long-term administration of potassium oxonate. 5. In the present application, blood uric acid levels were reduced by intraperitoneal injection of allopurinol at a dose of 5 mg / kg / d, which further demonstrated the adverse effect of hyperuricemia on periodontitis. 6. In this application, the timing and time points of potassium oxonate administration, allopurinol administration, and ligation were rationally selected. The model in this application exhibits a complex condition in which hyperuricemia and periodontitis coexist from day 14. Subsequently, potassium oxonate is continuously injected intraperitoneally to maintain the stability of the hyperuricemia model. Based on this, allopurinol is continuously injected intraperitoneally from day 15 to lower blood uric acid levels. According to the needs of independent research, the effects of hyperuricemia on periodontitis at different stages can be studied (related studies have shown that days 0 to 14 of ligation are the acute phase, and days 14 to 21 are the chronic phase). [Brief explanation of the drawings]

[0025] [Figure 1A] 1 is a modeling flowchart according to an embodiment of the present application. [Figure 1B] FIG. 1 shows blood uric acid, creatinine, and urea nitrogen levels in each experimental group according to an example of the present application. [Figure 1C] FIG. 10 is a graph showing the uric acid level of silk threads in the true silk ligation group according to an example of the present application. [Figure 1D]FIG. 1 shows blood xanthine / hypoxanthine levels and xanthine oxidase activity in each experimental group according to an example of the present application. [Figure 1E] FIG. 1 shows blood lipid and blood glucose levels in each experimental group according to an example of the present application. [Figure 1F] FIG. 1 is a graph showing changes in body weight in each experimental group according to an example of the present application. [Figure 1G] FIG. 1 is a diagram showing the weight of each organ in each experimental group according to an example of the present application. [Figure 2A] FIG. 1 shows the results of three-dimensional reconstruction and parameter analysis after Micro-CT scanning of the maxillae of mice in each experimental group at the end of the experiment according to an example of the present application. [Figure 2B] FIG. 1 shows H&E staining, TRAP staining, and IHC staining of the maxillae of mice in each experimental group at the end of the experiment according to an example of the present application. [Figure 3A] 1 shows an analysis of mRNA expression levels of gingival inflammatory factors at the end of the experiment in each experimental group of mice according to an example of the present application. [Figure 3B] FIG. 1 shows the results of mRNA sequencing analysis of gingival tissues of mice in each experimental group at the end of the experiment according to an example of the present application. [Figure 4A] 1 shows a differential analysis of the composition of periodontal microbial communities in mice in each experimental group according to an example of the present application. [Figure 4B] 1 shows a correlation analysis between clinical factors and periodontal disease microbial components in mice in each experimental group according to an example of the present application. [Figure 5] 1 shows an analysis of the oxidative stress response level in the mice of each experimental group according to an example of the present application. [Figure 6] 1 shows an analysis of the expression levels of the NLRP3 inflammasome pathway in mice of each experimental group according to an example of the present application. DETAILED DESCRIPTION OF THE INVENTION

[0026] The technical solutions in the embodiments of the present application will be clearly and completely described below. It is clear that the described embodiments are only a part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative efforts shall all be included in the protection scope of the present application.

[0027] In the examples of the present application, the method for constructing a composite mouse model simulating the aggravation of periodontitis due to hyperuricemia was based on the construction of a hyperuricemia model and a periodontitis model, and by creatively selecting the timing and duration of ligation and the timing and duration of administration and induction of hyperuricemia, and combining both, a stable and reliable composite mouse model simulating the aggravation of periodontitis due to hyperuricemia was obtained.

[0028] Specifically, the method for constructing a composite mouse model can include the following steps: Step S1: Build and maintain a hyperuricemia model. Step S11: Using a 1 mL syringe, extract a certain amount of the previously prepared potassium oxonate mother solution and prepare it, and weigh the mouse awaiting injection. Step S12: Gently grasp the weighed mouse and fix it in the palm of your hand. Depending on the weight of the mouse, a volume of potassium oxonate mother solution equivalent to the weight of the mouse is injected intraperitoneally once a day. The potassium oxonate injection dose is 60 mg / kg / day. Step S13: The operations of steps S11 and S12 are repeated every day at a fixed injection frequency to inject potassium oxonate into the abdominal cavity continuously for 14 days. Step S14: A hyperuricemia model is successfully established by injecting potassium oxonate intraperitoneally for 14 consecutive days. To maintain a stable uric acid concentration at a higher level, steps S11 and S12 must be repeated from the 15th day. Intraperitoneal injections of potassium oxonate are performed daily until the end of the experiment (the daily injection dose and number remain unchanged). Step S2: Construct a periodontitis model. Step S21: First, the mouse is anesthetized and immobilized, the maxillary molar area is exposed, and the maxillary molars, buccal and lingual gums are washed with saline and completely dried. Step S2: Fix the upper jaw of the mouse to a mouse board using surgical silk thread, pinch the surgical silk thread with the end of a microsurgical needle holder, and pass the surgical silk thread between the second and third molars. Step S23: Wrap the surgical silk thread from the distal side of the buccal surface of the second molar to the mesial side of the buccal surface of the second molar, and continue to clamp the surgical silk thread as in S22, and pass the surgical silk thread between the first molar and the second molar. Step S24: Using a needle holder, tie a silk thread to the palatal side of the second molar (and remove any loose threads). Step S25: The silk thread is tightly tied with three surgical knots, and excess silk thread is cut off with spring scissors. Step S3: A model combining hyperuricemia and periodontitis is constructed. Step S31: The operations of steps S11 and S12 are repeated to inject potassium oxonate intraperitoneally for seven consecutive days to construct a hyperuricemia model. Step S32: Repeat all the operations in step S2 to construct a mouse periodontitis model. Step S33: Repeat the operations of steps S11 and S12 to inject potassium oxonate intraperitoneally for 21 consecutive days (days 8 to 28). Step S34: On day 28, 1 hour after intraperitoneal injection of potassium oxonate, the mice were euthanized and samples were collected to evaluate their blood chemistry, renal function and pathology, and to detect destruction and inflammation of soft and hard tissues in the maxillary periodontal tissues. Step S4: A mouse model is used in which periodontitis exacerbated by hyperuricemia is improved by uric acid-lowering treatment. Step S41: The operations of steps S11 and S12 are repeated to inject potassium oxonate intraperitoneally for seven consecutive days to construct a hyperuricemia model. Step S42: Repeat all the operations in step S2 to construct a mouse periodontitis model. Step S43: Repeat the operations of steps S11 and S12 to continuously inject potassium oxonate intraperitoneally for 21 days (days 8 to 28). Step S44: Using a 1 mL syringe, a certain amount of the previously prepared allopurinol mother solution is extracted and prepared, and the mouse awaiting injection is weighed. Step S45: The weighed mouse is gently grasped and fixed in the palm of the hand. Depending on the weight of the mouse, a volume of allopurinol mother solution is injected intraperitoneally into the mouse's abdominal cavity once a day, with the allopurinol injection dose being 5 mg / kg / day. Step S46: The operations of steps S44 and S45 are repeated every day at a fixed injection frequency, and allopurinol is injected intraperitoneally continuously for 14 days (15th to 28th days).

[0029] Hereinafter, the technical solution of the present application will be described in more detail through specific embodiments and experimental results.

[0030] Example 1

[0031] 1. Materials Fifty C57BL / 6 mice, 6–8 weeks old and weighing 18–22 g, were provided by the Guangdong Provincial Animal Experimental Center and housed in an SPF-level environment at the Animal Center of Guangdong Huami Testing Co., Ltd. This experiment was approved by the Experimental Animal Ethics Committee of the Guangdong Provincial Medical Experimental Animal Center. A 5-0 non-absorbable braided silk thread was supplied by Johnson & Johnson and has the model number Ethicon mersilk SA82G. The Micro-CT is provided by BrukerB of Germany and has the model number skyscan172. Potassium oxonate (PO), sodium carboxymethylcellulose (CMC-Na), and allopurinol (Allo) were all supplied by Sigma, with model numbers 156124-25G12, C5678-50G, and A803-25G, respectively.

[0032] 2. Preparation of reagents For potassium oxonate mother solution (30 mg / mL), potassium oxonate (30 mg) + 0.5% sodium carboxymethylcellulose (1 mL). For 0.5% sodium carboxymethylcellulose, use 5 g of sodium carboxymethylcellulose powder plus 1 L of saline. For allopurinol (0.5 mg / mL), the mixture is allopurinol (60 mg) + NaOH (1 M, 3 mL) + saline (117 mL).

[0033] 3. Experimental grouping (further groups) The NuC group was a normal control group, in which 0.5% CMC-Na was injected intraperitoneally and neither of the maxillary second molars on either side was ligated (n=10). The NuP group was an experimental periodontitis group, in which 0.5% CMC-Na was injected intraperitoneally and all maxillary second molars on both sides were ligated (n=10). The HuC group was a hyperuricemia group in which PO was intraperitoneally injected and neither of the maxillary second molars on either side was ligated (n=10). The HuP group, a group with a combination of hyperuricemia and periodontitis, received an intraperitoneal injection of PO and ligated both maxillary second molars (n=10). HuP+Allo group: the combined hyperuricemia and periodontitis + uric acid-lowering treatment group, in which PO was injected intraperitoneally, all maxillary second molars on both sides were ligated, and Allo was injected intraperitoneally (n=10).

[0034] 4. Experimental methods and procedures (1) After arriving at the animal center of Huamei Testing Co., Ltd., 50 male BC57BL / 6 rats were adapted to the environment with a temperature of 20-24°C, a relative humidity of 50%-70%, a light rhythm of 12D:12L, an operating illuminance of 150-30lx, an airflow and wind speed of 0.1-0.2m / s, and a noise level of ≤60dB. They were fed an adapted diet for one week. (2) Fifty mice were randomly divided into NuC, NuP, HuC, HuP, and HuP+Allo groups, each containing 10 mice. Body weights were measured once daily to an accuracy of 0.01 g before and after the start of the experiment. (3) Mice in the NuC and NuP groups received intraperitoneal injections of 0.5% CMC-Na at a fixed frequency of once per day for 28 consecutive days (the injection volume matched the amount of PO required for mice of the same weight). On day 7, the mice in the NuP group had their maxillary second molars ligated with 5-0 silk thread using a microsurgical needle holder. Mice in the HuC and HuP groups received intraperitoneal injections of PO at a fixed frequency of once per day for 28 consecutive days at a dose of 60 mg / kg / day (e.g., for a mouse weighing 25 g, the daily injection volume of the corresponding potassium oxonate mother solution (30 mg / mL) is 0.5 mL). On day 7, the mice in the HuP group had their maxillary second molars ligated with 5-0 silk thread using a microsurgical needle holder. The HuP+Allo group received intraperitoneal injections of Allo at a fixed frequency of once daily for 14 consecutive days, starting on day 15 based on the HuP group. The injection dose was 5 mg / kg / day (e.g., for a mouse weighing 25 g, the daily injection volume of the corresponding potassium oxonate mother solution (0.5 mg / mL) is 0.25 mL). After ligation, mice in the NuP, HuP, and HuP+Allo groups were checked for the presence or absence of the ligated silk thread on days 1, 3, 7, 10, 14, and 21. If the silk thread fell off, it was promptly re-ligated and recorded. (In this study, the silk thread remained firmly in place throughout the ligation period in all ligated mice, and no silk thread fell off.) The specific ligation method was as follows. After intraperitoneally anesthetizing the model-waiting mouse with 1.5% pentobarbital sodium (dose: 40 mg / kg), the mouse's abdominal cavity was fixed upward on a mouse board, and the mouse board was tilted and fixed at a certain angle. A homemade mouth opener was used to stably open the mouse's mouth, exposing the molar area of ​​the maxilla. A head-mounted light was used to provide a view of the molar area, and the maxillary molars, buccal and lingual gums were washed with saline and completely dried. Using a microsurgical needle holder, dental floss-style surgical silk threads with a coarseness of 5-0 were embedded under the contact points of the proximal surfaces of the first and second molars and the second and third molars on one side of the maxilla, respectively, and wrapped around the cervical region of the second molar. The silk threads were then tied to the palatal side of the second molar using the needle holder (any loose threads were removed), and the silk threads were tightly tied with three surgical knots. The excess silk thread was then cut off with spring scissors. (4) At the end of the experiment (1 hour after intraperitoneal injection of potassium oxonate / sodium carboxymethylcellulose on day 28), the mice were euthanized. Whole blood samples were collected, and serum was separated for creatinine, urea nitrogen, uric acid, blood lipid levels, blood glucose, and purine metabolism levels. Internal organs, including kidneys, liver, spleen, heart, lungs, and peritestrical fat, were collected and weighed. One portion was fixed in 4% paraformaldehyde, while the other portion was frozen in liquid nitrogen for 30 minutes and transferred to -80°C for storage. After recording the preservation status of the true silk ligation group, the silk ligation was removed near the maxillary second molar. One portion was transferred to a 1.5 mL EP tube containing 100 μL of PBS to measure the level of uric acid dissolved in the PBS. One portion was frozen in liquid nitrogen for 30 minutes and transferred to -80°C for storage. The maxillae were separated and immersed in 4% paraformaldehyde solution and fixed for 24-48 hours. One maxilla was used for Micro-CT scanning and H&E staining analysis, while the gingiva was removed from the other maxilla. The gingiva was frozen in liquid nitrogen for 30 minutes and transferred to -80°C for storage.

[0035] 5. Experimental results 1. A mouse model of hyperuricemia was successfully established. PO-induced hyperuricemia was manifested by elevated blood uric acid and creatinine levels, while blood glucose and blood lipid levels remained unchanged. After 28 days of hyperuricemia induction with PO, mice with hyperuricemia (Hu), with or without periodontal ligation, showed a greater than two-fold increase in blood uric acid and a 1.5-fold increase in creatine compared with mice with normal uric acid (Nu). After uric acid-lowering treatment with Allo (HuP + Allovs.HuP), the blood uric acid and creatinine levels of the mice were significantly reduced (P < 0.001) (Figure 1B). Furthermore, in mice with periodontal ligation, PO (HuP vs. NuP) increased the uric acid content in the silk thread, whereas Allo intervention (HuP + Allovs.HuP) significantly reduced the uric acid content (Figure 1C). Allo reduced the activity of uric acid, creatine, and xanthine oxidoreductase, and increased serum xanthine / hypoxanthine levels (Figure 1D). PO injection and ligation did not significantly affect fasting blood lipids (TC, TG, HDL-C, LDL-C, VLDL-C) or blood glucose (P > 0.05) (Figure 1E). PO reduced body weight from D7 to D28, periodontal ligation reduced body weight from D21 to D28, and Allo attenuated the weight-reducing effect of PO on the body weight of ligated mice (Figure 1F). PO and periodontal ligation had no significant effects on most systems or organs. PO and ligation reduced the weight of peritedibula adipose tissue, but PO and ligation reduced the weight of the spleen and liver, respectively (Figure 1G). 2. Hyperuricemia aggravated alveolar bone destruction and inflammatory reactions in periodontitis mice. Micro-CT analysis revealed that the mean distance from the cementoenamel junction to the alveolar crest (CEJ-ABC) and trabecular spacing (Tb.Sp) of the maxillary second molars in the periodontal ligation group significantly increased (P < 0.001), while bone mineral density (BMD), bone volume fraction (BVF = BV / TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) significantly decreased (P < 0.001) (Figure 2A). These results demonstrated the successful establishment of an experimental periodontitis model in mice. Hyperuricemia worsened alveolar bone destruction-related indices (BVF, TB.N, and Tb.Th) and tended to worsen BMD in the periodontitis mice (HuP vs. NuP) (P = 0.070) (Figure 2A). Hyperuricemia increased the number of inflammatory cells and osteoclasts in the periodontal tissues of mice with periodontitis, whereas Allo improved the periodontal conditions of mice with HuP, including alveolar bone resorption and the number of inflammatory cells and osteoclasts (Figure 2A, Figure 2B). 3. Hyperuricemia inhibited the periodontal immune response. Immunohistochemistry revealed that macrophages (CD68 + ), M1 macrophages (CD86 +The number of osteoclasts and osteoclasts significantly increased with hyperuricemia (Figure 2B). RT-qPCR revealed that hyperuricemia elevated the mRNA expression levels of II-1β (P = 0.054) and II-6 (P = 0.062) in the gingival tissues of periodontally ligated mice (Figure 3A). Allo treatment reduced the total number of macrophages and M1 macrophages, and tended to decrease the expression of II-1β (P < 0.001) and TNF-α (P = 0.057) (Figures 2B and 3A). Subsequent mRNA sequencing further revealed the response of periodontal tissues to hyperuricemia. Of the 16,746 identified genes, 2,105 genes (640 up-regulated and 1,465 down-regulated) were differentially expressed between the NuP and HuP groups (Figure 3B). KEGG enrichment analysis found 17 pathways (excluding disease-related pathways) associated with hyperuricemia (HuP vs. NuP, P < 0.05), where most were related to immune response ( Figure 3B ). 4. Hyperuricemia aggravated periodontal dysbiosis. We analyzed the microbiome of silk-ligated periodontal tissue by 16S rRNA sequencing. The periodontal microbiota of the three ligation groups (NuP, HuP, and HuP+Allo) contained 388 OUTs, 22 phyla, and 210 genera. Indirect gradient analysis (PCA, PCoA, and NMDS) demonstrated that the microbial composition of the three mouse groups was well differentiated at the phylum level. Hyperuricemia increased the abundance of Firmicutes and Proteobacteria and decreased the abundance of Bacteroidota and Actinobacteriota in ligated mice. In the urate-lowering treatment group (HuP+Allo), the abundance of Firmicutes was further upregulated (Figure 4A). Among 12 clinical factors (including organ weights, serum cytokines, blood glucose, blood lipids, and renal function parameters), only serum uric acid and creatinine levels were associated with microbial composition, but the associated heatmap showed that the abundance of multiple bacterial genera was significantly correlated with serum uric acid and creatinine levels ( Figure 4B ). 5. Excessive uric acid exacerbated the oxidative stress response in periodontal tissues. Compared with the periodontally healthy control group (NuC), periodontitis (NuP) significantly reduced the total antioxidant capacity (T-AOC) of gingival tissue and tended to decrease SOD activity (P = 0.058). Periodontitis did not alter the concentration of the lipid peroxidation product MDA or the mRNA levels of the pro-oxidant genes Cox2 and Nox4. Hyperuricemia further reduced T-AOC in periodontitis (HuP and NuP). Hyperuricemia increased SOD activity, MDA content, and mRNA expression of Cox2 and Nox4. Immunohistochemistry demonstrated that similar changes occurred in Nox4 in hyperuricemia-induced periodontal sections (HuP and NuP). Allopurinol reversed the changes in these oxidative stress-related parameters induced by hyperuricemia (HuP + Allovs.HuP) (Figure 5). 6. Excessive UA activated the NLRP3 inflammasome pathway in inflammatory periodontitis. Periodontitis activates the NLRP3 inflammasome pathway in the periodontium, which shows a clear increase in NLRP3 expression and a tendency toward increased caspase-1 expression at the protein level. Hyperuricemia increased caspase-1 levels. NLRP3 levels also tended to increase in patients with periodontitis. Furthermore, the protein level of GSMDD, a thermoprotein deposition-associated factor downstream of caspase-1, was also increased by periodontitis. Hyperuricemia further increased caspase-1 protein levels in ligated mice. Allopurinol can inhibit the increases in NLRP3, caspase-1, and GSMDD caused by hyperuricemia (Figure 6).

[0036] As can be seen from the above, the present application provides a method for constructing a composite mouse model simulating the aggravation of periodontitis caused by hyperuricemia, which includes the steps of: intraperitoneally injecting potassium oxonate (PO) once per day for 7 days to construct a hyperuricemia model; and on the 7th day, ligating the maxillary second molar of the mouse with surgical silk using a microsurgical needle holder to construct a mouse periodontitis model; and then intraperitoneally injecting potassium oxonate (PO) once per day from the 8th to 14th days. The method includes the steps of: constructing a mouse model combining hyperuricemia and periodontitis; subsequently, continuously injecting potassium oxonate (PO) intraperitoneally at a fixed frequency of once per day for another 14 days (days 15 to 28) to maintain the stability of the hyperuricemia model and construct a stable mouse model combining hyperuricemia and periodontitis; and simultaneously injecting allopurinol intraperitoneally for 14 days continuously on day 15 to perform uric acid-lowering treatment, thereby constructing a model in which periodontitis in a mouse that has worsened due to hyperuricemia is improved by uric acid-lowering treatment.

[0037] In this study, the hyperuricemia model constructed using potassium oxonate (PO) not only elevates uric acid levels in the short term, but also maintains uric acid levels stably over the long term through continuous administration during the maintenance phase. By combining a gentle and non-invasive periodontitis model, we have innovatively proposed a mouse model that simulates the exacerbation of periodontitis caused by hyperuricemia, and a model in which uric acid-lowering treatment improves periodontitis in mice exacerbated by hyperuricemia. In this model, we observed not only exacerbation of alveolar bone resorption and periodontal tissue inflammation caused by hyperuricemia, but also periodontal dysbiosis, oxidative stress, and the deterioration of key signaling pathways. These data support the multidimensional effects of hyperuricemia on periodontitis, including morphological, histological, and molecular levels, and comprehensively explain the key features of this combined model. This application effectively fills the gap in animal models for studying the role of hyperuricemia in aggravating periodontitis, and demonstrates great application value in exploring the complex pathological mechanisms associated with both hyperuricemia and periodontitis, broadening the ideas and methods of this clinical research hotspot.

[0038] Although the present application has been shown and described in detail, it should be understood that those skilled in the art can still directly determine or deduce many other variations and modifications consistent with the principles of the present application based on the disclosure herein without departing from the spirit and scope of the present invention, and therefore the scope of the present application should be considered to include all such other variations and modifications.

Claims

1. A method for constructing a complex mouse model simulating the aggravation of periodontitis due to hyperuricemia, comprising: Step 1: Inject potassium oxonate into the abdominal cavity of mice for 7 consecutive days to establish a hyperuricemia model; Step 2: On the seventh day, the second molar of the maxilla of the mouse is ligated with surgical silk thread using a microsurgical needle holder to establish a periodontitis model; Step 3: Inject potassium oxonate into the abdominal cavity of the mice for 7 consecutive days (days 8-14) to establish a composite mouse model simulating the aggravation of periodontitis due to hyperuricemia; A method of construction comprising:

2. The method of claim 1, further comprising step 4, after step 3, of continuously injecting potassium oxonate into the abdominal cavity of the mouse for 14 days (days 15 to 28).

3. The method of claim 1, further comprising step 5, after step 4, of simultaneously administering intraperitoneal injections of allopurinol for 14 consecutive days (days 15 to 28) to perform uric acid-lowering treatment on the 15th day.

4. The method of claim 2, wherein in step 1 and / or step 3 and / or step 4, potassium oxonate is injected intraperitoneally into the mouse once a day at an injection dose of 600 mg / kg / d.

5. The method of claim 3, wherein in step 5, allopurinol is injected intraperitoneally into the mouse once a day at a dose of 5 mg / kg / d.

6. The method of claim 2, wherein injecting potassium oxonate into the abdominal cavity of the mouse in step 1 and / or step 3 and / or step 4 comprises gently grasping the mouse and fixing it in the palm of your hand, and injecting potassium oxonate mother solution having a concentration of 30 mg / mL into the abdominal cavity of the mouse.

7. The method of claim 3, wherein in step 5, injecting allopurinol into the abdominal cavity of the mouse comprises gently grasping the mouse and fixing it in the palm of your hand, and injecting allopurinol mother solution having a concentration of 0.5 mg / mL into the abdominal cavity of the mouse.

8. Step 2 is a step a) of anesthetizing and immobilizing the mouse, exposing the maxillary molar area, and cleaning and completely drying the maxillary molars, buccal and lingual gums; Step b: fix the upper jaw of the mouse to a mouse board with the surgical silk thread, pinch the surgical silk thread with the end of a needle holder, and pass the surgical silk thread through the gap between the second molar and the third molar; a step c) of wrapping the surgical silk thread from the distal side of the buccal surface of the second molar to the mesial side of the buccal surface of the second molar, sandwiching the surgical silk thread, and passing the surgical silk thread through the gap between the first molar and the second molar; Step d) using the needle holder to tie the surgical silk thread to the palatal side of the second molar; Step e) of tying the silk thread securely with three surgical knots and cutting off excess silk thread with spring scissors; A method of construction according to any one of claims 1 to 7, characterized in that it comprises:

9. In the step a, Anesthetizing and fixing the mouse and exposing the maxillary molar region includes anesthetizing the abdominal cavity of the mouse and fixing the abdominal cavity of the mouse facing upward on the mouse board, tilting and fixing the mouse board at a certain angle, using a mouth opener to stably open the mouth of the mouse to expose the maxillary molar region, and using a head-mounted light to provide a visual field for the molar region; and / or 9. The method of claim 8, wherein cleaning the maxillary molars, buccal and lingual gingiva and thoroughly drying includes cleaning the maxillary molars, buccal and lingual gingiva with saline, followed by thorough drying with a dry cotton ball.

10. the surgical silk is 5-0 surgical silk; The method of claim 8, wherein the needle holder is a microsurgical needle holder with model number W40350.

11. The method of any one of claims 1 to 7, wherein the mouse is a 6- to 8-week-old male C57BL / 6 mouse.

12. A composite mouse model simulating the worsening of periodontitis due to hyperuricemia, characterized by being obtained by the construction method described in any one of claims 1 to 11, and a model in which periodontitis in mice worsened by hyperuricemia is improved by uric acid-lowering treatment.

13. Application of a composite mouse model that simulates the worsening of periodontitis due to hyperuricemia, characterized in that the composite mouse model is obtained by the construction method described in any one of claims 1 to 11, to screening for therapeutic drugs for periodontitis associated with hyperuricemia.

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