A composite mouse model simulating hyperuricemia and aggravated periodontitis and its establishment and application

Abstract

The present invention relates to a composite mouse model simulating hyperuricemia exacerbated periodontitis and its establishment method and application. The establishment method comprises the following steps: step one: continuously injecting potassium oxonate into the peritoneal cavity of mice for 7 days; step two: on the 7th day, ligating a surgical thread to the maxillary second molar of the mice by using a micro needle holder; step three: continuing to continuously inject potassium oxonate into the peritoneal cavity of mice for 7 days to establish a composite mouse model simulating hyperuricemia exacerbated periodontitis. The hyperuricemia model constructed by potassium oxonate in the present invention can not only increase the uric acid level in a short period of time, but also maintain the uric acid level for a long time through continuous administration during the maintenance period. Combined with the construction of a mild and non-injurious periodontitis model, a composite mouse model of hyperuricemia exacerbated periodontitis is successfully simulated, effectively filling the gap in the lack of animal models in the research field related to hyperuricemia and periodontitis, and broadening the ideas and methods of clinical research hotspots.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological experimental model construction, and particularly relates to a composite mouse model simulating hyperuricemia exacerbated periodontitis, and a method for establishing the model and application thereof. Background Art

[0002] Periodontitis is a chronic, multifactorial inflammatory disease characterized by the destruction of periodontal tissues and caused by abnormal bacterial plaque biofilms. Specific microbiota trigger a host inflammatory response, leading to progressive destruction of periodontal tissues, ultimately damaging connective and bone tissue. Severe periodontitis is the sixth most common human disease. Severe periodontitis can not only lead to tooth loss and severely impact quality of life, but also negatively impact overall health. Studies have shown that diabetes, hypertension, obesity, and hyperlipidemia are major risk factors for periodontitis, and their association with periodontitis has been demonstrated to varying degrees. With improving living standards, the incidence of hyperuricemia, a component of metabolic syndrome (a metabolic disease characterized by excessively elevated blood uric acid concentrations due to overproduction and / or inadequate intestinal excretion and / or renal urate excretion), has also increased in recent years. It is now the fourth most common high after the "three highs," and reportedly affects approximately 15% to 20% of the population worldwide. Recent studies have suggested that hyperuricemia may also be a major risk factor for periodontitis. A recent retrospective cohort study showed that patients with gout (an inflammatory disease characterized by abnormally elevated serum uric acid levels, leading to the deposition of sodium urate crystals in joints and periarticular tissues) had a significantly increased risk of periodontitis (aHR = 1.13, 95% CI = 1.10–1.16). Uric acid-lowering therapy with colchicine effectively reduced the risk of periodontitis (aHR = 0.85, 95% CI = 0.79–0.91). Furthermore, animal studies have shown that uric acid-lowering therapy with febuxostat can attenuate the progression of periodontitis in rats. Cross-sectional and interventional studies have also found a positive correlation between elevated blood uric acid levels and periodontitis. For example, plasma uric acid levels have been found to be elevated in patients with periodontitis compared with healthy controls. In patients with IgA nephropathy, severe periodontitis is associated with higher serum uric acid levels compared with mild / moderate periodontitis. Non-surgical treatments for periodontitis have also been shown to be effective in lowering serum uric acid in patients with periodontitis. These findings suggest a positive correlation between elevated systemic uric acid levels and the severity of periodontitis, indicating that hyperuricemia is a risk factor for periodontitis. It is important to note that the mechanism by which hyperuricemia exacerbates periodontitis remains unclear, and epidemiological studies have been unable to elucidate the specific mechanisms by which elevated serum uric acid exacerbates periodontitis. Therefore, an animal model that can simulate the exacerbation of periodontitis by hyperuricemia is particularly necessary to explore the possible molecular biological mechanisms underlying this association.

[0003] At present, there are no reports on animal models of hyperuricemia exacerbating periodontitis at home and abroad. Relevant technologies have attempted to establish a mouse model of hyperuricemia combined with periodontitis by feeding with potassium oxonate and uric acid and ligating the maxillary second molars of mice with silk thread (Luo Yongxi, Huang Xueying, Xian Ruoting, Yu Wanxin, Liang Lixin, Liang Zhaojia, Chen Ziyun, Hou Dan, Yu Ting. Micro-CT analysis of the effect of hyperuricemia on alveolar bone destruction in mice with short-term periodontal infection [J]. Prevention and Treatment of Oral Diseases, 2021, 29(02):88-93.). The experimental endpoint was to detect the serum uric acid levels of mice and perform Micro-CT analysis of the maxilla. The results showed that the blood uric acid levels of mice in the potassium oxonate and uric acid feed feeding groups were increased, while the gross morphological analysis of the jaw based on micro-CT did not find that hyperuricemia or increased uric acid levels aggravated the damage of periodontitis. However, it must be pointed out that the related technology did not find a correlation between uric acid concentration and alveolar bone resorption in the mouse model of hyperuricemia combined with periodontitis established by the feed feeding method and ligation method. The following reasons may be responsible: ① Although the periodontitis model was successfully established, the observation time was not long enough, and the potential adverse effects of HU on alveolar bone resorption were not apparent in the short term; ② The sample size was small, and the errors caused by individual differences masked the possible adverse effects of HU; ③ Serum uric acid levels were measured using a kit method, which is subject to significant human error and the resulting uric acid levels may be inaccurate. In view of this, the team then attempted to establish a feed feeding-induced hyperuricemia symptom model by feeding potassium oxonate combined with uric acid. After blood was collected at the end of the experiment, serum uric acid levels were measured using an automated biochemical analyzer. The results showed that the serum uric acid levels of mice fed with potassium oxonate combined with uric acid did not increase, indicating that the feed feeding method failed to successfully establish a hyperuricemia model. This also explains why the "hyperuricemia" mouse model established by the team previously failed to successfully aggravate bone destruction in mice with periodontitis. In summary, there is currently no report of an animal model that successfully simulates hyperuricemia-aggravated periodontitis at home and abroad. Therefore, new modeling methods are urgently needed to establish an animal model that can simulate hyperuricemia-aggravated periodontitis to fill the gap in this research field.

[0004] Although methods for establishing models of both periodontitis and hyperuricemia have been explored, there are still very few reports domestically and internationally on the establishment of mouse models of hyperuricemia combined with periodontitis, and there are no successful reports of mouse models of hyperuricemia exacerbating periodontitis. The main reasons for this are as follows: 1. The ligature-induced periodontitis model can lead to local plaque accumulation and thus induce the formation of periodontitis. Because it is similar to the pathological process of human periodontitis, which is initiated by plaque, the ligature method is currently the most commonly used method for establishing animal periodontitis models internationally. However, a careful review of the literature shows that compared with larger animals (such as rats, dogs, or non-human primates), the ligature-induced periodontitis model is rarely used in mice, despite mice representing the most convenient, cheapest, and most versatile model. On the one hand, the most commonly used method for ligature-induced periodontitis is to use a pointed probe to separate the gums of the second molar and to tie the gingival sulcus with sutures. This method causes significant damage to the animal's gums, and the sharp operating instruments can easily cause bleeding in the buccal gingival vestibule sulcus of mice. Severe bleeding can cause blood to flow into the respiratory tract and lead to the death of the mouse. On the other hand, the oral environment of mice also poses severe technical challenges to ligature because of their relatively small size. These reasons may be the reason why mice are relatively rarely used in ligature-induced periodontitis (mice are widely used in oral infection models of periodontitis). 2. Due to the presence of uricase in experimental animals such as rats and mice, in medium- and long-term experiments, the hyperuricemia model constructed by supplementing exogenous uric acid, supplementing uric acid precursors, and inhibiting renal uric acid excretion cannot maintain a high level of uric acid, and the model is often not stable enough. In recent years, potassium oxonate is the most commonly used drug internationally to construct hyperuricemia models. It can competitively bind to uricase, inhibit the activity of uricase, increase the blood uric acid level in the body in a short period of time, and maintain it for a longer period of time. This method can establish a model in a short period of time (two weeks), but due to its competitive inhibition mechanism, the maintenance of the hyperuricemia model after model establishment still requires long-term administration. Too low a concentration and frequency of administration cannot maintain the uric acid level well, while too high a concentration and frequency of administration can easily cause adverse effects such as a strong inflammatory response in the kidneys of experimental animals. Therefore, in medium- and long-term experiments, it is particularly important to explore the appropriate concentration and frequency of administration to maintain a high level of uric acid concentration without causing adverse effects such as a strong inflammatory response in the animal kidneys. However, there is still no unified consensus on this internationally.3. In addition, it is more important that the establishment of the hyperuricemia combined with periodontitis model is not just a simple superposition of the hyperuricemia model and the periodontitis model. Depending on the exposure factors and the scientific problems to be studied (for example, when studying the effect of hyperuricemia on periodontitis, hyperuricemia is the exposure factor; when studying the effect of periodontitis on hyperuricemia, periodontitis is the exposure factor; and when studying the relationship between the two, the two are risk factors for each other), the ligation time point and the time point of hyperuricemia drug induction will also be different accordingly. It is difficult to predict the impact of the choice of ligation time point and hyperuricemia drug induction time point on the hyperuricemia combined with periodontitis model. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a composite mouse model simulating hyperuricemia-aggravated periodontitis, and a method for establishing and applying the same. The method for establishing the mouse model of the present invention can not only increase the uric acid level in a short period of time, but also the construction of the periodontitis model is gentle and non-invasive, effectively establishing a mouse model simulating hyperuricemia-aggravated periodontitis, effectively filling the gap in the lack of animal models in the research field related to hyperuricemia and periodontitis, and is of great significance in exploring the complex pathological mechanism of the relationship between the two and screening drugs for hyperuricemia-aggravated periodontitis.

[0006] To achieve the purpose of the present invention, the present invention provides a method for establishing a composite mouse model simulating hyperuricemia and aggravated periodontitis, comprising the following steps:

[0007] Step 1: Intraperitoneal injection of potassium oxonate was performed on mice for 7 consecutive days;

[0008] Step 2: On day 7, surgical silk suture was tied around the maxillary second molar of the mouse using a micro needle holder;

[0009] Step 3: Continue to inject potassium oxonate into the peritoneal cavity of mice for 7 consecutive days to establish a composite mouse model simulating hyperuricemia exacerbated by periodontitis.

[0010] As some embodiments of the present invention, step 4 is further included after step 3: continuously injecting potassium oxonate into the peritoneal cavity of mice for 14 days to maintain the stability of the hyperuricemia model.

[0011] As some embodiments of the present invention, in step 1 and / or step 3 and / or step 4, the frequency of intraperitoneal injection of potassium oxonate into mice is once a day, and the injection dose is 600 mg / kg mouse / day.

[0012] As some embodiments of the present invention, in step one and / or step three and / or step four, injecting potassium oxonate into the peritoneal cavity of the mouse comprises: gently grasping the mouse and fixing it in the palm of the hand, and injecting potassium oxonate stock solution into the peritoneal cavity of the mouse; the concentration of the potassium oxonate stock solution is 30 mg / mL.

[0013] As some embodiments of the present invention, the step 2 includes:

[0014] Step a: Anesthetize and fix the mouse, expose the maxillary molar area, clean the maxillary molars and buccal and lingual gums, and dry them thoroughly;

[0015] Step b: Fix the surgical suture on the mouse board, use the end of the needle holder to clamp the surgical suture, and pass the surgical suture through the interdental space between the second molar and the third molar;

[0016] Step c: Wrap the surgical thread from the distal buccal surface of the second molar to the mesial buccal surface of the second molar, clamp the surgical thread, and pass the surgical thread through the interdental space between the first molar and the second molar;

[0017] Step d: Use the needle holder to tie a knot of surgical silk thread on the palatal side of the second molar;

[0018] Step e: Tie the silk thread with three surgical knots and cut the excess silk thread with spring scissors.

[0019] As some embodiments of the present invention, in step a, anesthetizing and fixing the mouse to expose the maxillary molars includes: anesthetizing the mouse's abdominal cavity and fixing the mouse on a mouse board with the abdominal cavity facing upward; tilting the mouse board at a certain angle and then fixing the mouse board, using an opener to make the mouse open its mouth stably to expose the maxillary molar area, and using a head-mounted lighting to provide a field of view of the molar area.

[0020] As some embodiments of the present invention, the cleaning of the maxillary molars and buccal and lingual gums and fully drying them comprises: cleaning the maxillary molars and buccal and lingual gums with physiological saline and then fully drying them with dry cotton balls.

[0021] As some embodiments of the present invention, the surgical suture is 5-0 surgical suture, the needle holder is a micro needle holder, and the model of the micro needle holder is W40350.

[0022] In some embodiments of the present invention, the mouse is a 6-8 week old male C57BL / 6 mouse.

[0023] To achieve the purpose of the present invention, the present invention also provides a composite mouse model simulating hyperuricemia exacerbated periodontitis, which is obtained by the establishment method described in any of the above schemes.

[0024] To achieve the purpose of the present invention, the present invention also provides the use of the above-mentioned composite mouse model simulating hyperuricemia-aggravated periodontitis in screening therapeutic drugs for hyperuricemia-aggravated periodontitis.

[0025] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0026] 1. This invention effectively fills the gap in animal models for the study of hyperuricemia-exacerbated periodontitis. It innovatively simulates a composite mouse model of hyperuricemia-exacerbated periodontitis for the first time. This model demonstrates great application value in exploring the complex pathological mechanisms of the relationship between the two and in screening for drugs that treat hyperuricemia-exacerbated periodontitis. It also broadens the research ideas and methods in this field to a certain extent.

[0027] 2. The establishment of the composite mouse model simulating hyperuricemia and aggravated periodontitis in the present invention only takes 14 days, and the entire process of constructing a stable hyperuricemia-combined periodontitis mouse model only takes 28 days, which has the advantages of stable and reliable modeling effect and short modeling time;

[0028] 3. Based on the successful establishment of a hyperuricemia model, the present invention then induced periodontitis by ligating the maxillary second molars of mice with silk thread. In terms of the time sequence, hyperuricemia occurs first and then periodontitis, which better illustrates the aggravating effect of hyperuricemia on periodontitis and is more scientific in terms of modeling sequence.

[0029] 4. In the present invention, the hyperuricemia model was induced and maintained by intraperitoneal injection of potassium oxonate at a dose of 600 mg / kg / d, which not only kept the uric acid concentration at a high level, but also avoided the disadvantages of renal inflammation caused by long-term administration of potassium oxonate.

[0030] 5. The present invention reasonably selects the time points for administration of potassium oxonate and ligation. The model of the present invention is in a complex state of coexistence of hyperuricemia and periodontitis starting from the 14th day. Thereafter, the stability of the hyperuricemia model is maintained by continuous intraperitoneal injection of potassium oxonate. According to the needs of self-research, the effect of hyperuricemia on periodontitis at different stages can be studied (relevant studies have shown that ligation for 0-14 days is the acute stage, and 14-21 days is the chronic stage). BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A flow chart is provided for modeling an embodiment of the present invention.

[0032] Figure 2 This is the serum uric acid level of mice at the experimental endpoint of each experimental group in the examples of the present invention.

[0033] Figure 3 The graph shows the changes in mouse kidney weight at the experimental endpoints in each experimental group of the present invention.

[0034] Figure 4 HE staining of the kidney pathological changes of mice in each experimental group of the examples of the present invention.

[0035] Figure 5 This is a schematic diagram of three-dimensional reconstruction of the maxillary bones after micro-CT scanning of the mice in each experimental group of the present invention at the end point of the experiment.

[0036] Figure 6 These are HE staining images and Trap staining images of the maxillary bones of mice in each experimental group at the end of the experiment in the examples of the present invention. DETAILED DESCRIPTION

[0037] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0038] The method for establishing a composite mouse model simulating hyperuricemia and aggravated periodontitis in an embodiment of the present invention is based on the establishment of a hyperuricemia model and a periodontitis model. The ligation time point and the hyperuricemia drug induction time point are creatively selected to combine the two, thereby obtaining a stable and reliable composite mouse model simulating hyperuricemia and aggravated periodontitis. Specifically, the establishment of the composite mouse model may include the following steps:

[0039] Step S1: Establishment and maintenance of hyperuricemia model

[0040] Step S11: using a 1 mL syringe to extract a certain amount of the prepared potassium oxonate stock solution for use, and then weighing the mice to be injected;

[0041] Step S12: Gently grasp the weighed mouse and secure it in the palm of your hand. Inject a corresponding volume of potassium oxonate stock solution into the mouse's peritoneal cavity once daily based on the mouse's weight. The injection dose of potassium oxonate is 600 mg / kg mouse / day.

[0042] Step S13: Repeat steps S11 and S12 at a fixed injection frequency every day, and continuously administer potassium oxonate intraperitoneally for 14 days;

[0043] Step S14: Continuously administer potassium oxonate intraperitoneally for 14 days. At this point, the hyperuricemia model is successfully established. To maintain a stable uric acid concentration at a high level, steps S11 and S12 are repeated starting from day 15, with potassium oxonate administered intraperitoneally daily (the daily dose and number of injections remain unchanged) until the end of the experiment.

[0044] Step S2: Periodontitis model establishment

[0045] Step S21: First, anesthetize and fix the mouse, expose the maxillary molar area, clean the maxillary molars and buccal and lingual gums with normal saline, and dry them thoroughly;

[0046] Step S22: Fix the surgical suture on the mouse plate, use the end of the micro needle holder to clamp the surgical suture, and pass the surgical suture through the interdental space between the second molar and the third molar;

[0047] Step S23: Wrap the surgical thread from the distal buccal surface of the second molar to the mesial buccal surface of the second molar, continue to clamp the surgical thread in the same manner as S22, and pass the surgical thread through the interdental space between the first molar and the second molar;

[0048] Step S24: Use the needle holder to tie a knot on the palatal side of the second molar (be sure to remove any loose parts);

[0049] Step S25: Tie the silk thread with three surgical knots, and cut the excess silk thread with spring scissors;

[0050] Step S3: Establishment of hyperuricemia combined with periodontitis model

[0051] Step S31: repeating steps S11 and S12, and continuously injecting potassium oxonate intraperitoneally for 7 days;

[0052] Step S32: Repeat all the operations in step S2 to establish a mouse periodontitis model;

[0053] Step S33: Repeat steps S11 and S12 to continue intraperitoneal injection of potassium oxonate for 21 days;

[0054] Step S34: On the 28th day, the mice were euthanized 1 hour after intraperitoneal injection of potassium oxonate, and samples were collected to detect blood biochemistry, renal function and renal pathology, and damage and inflammation of maxillary periodontal soft and hard tissues.

[0055] The technical solution of the present invention will be further described in detail below through specific examples and experimental results.

[0056] Example 1

[0057] 1. Materials

[0058] Forty C57BL / 6 mice, 18–22 g and 6–8 weeks old, were provided by the Guangdong Provincial Animal Experimental Center and housed in an SPF-grade environment at the Guangdong HUAWEI Testing Co., Ltd. Animal Center. This experiment was approved by the Experimental Animal Ethics Committee of the Guangdong Medical Laboratory Animal Center. 5-0 silk braided non-absorbable sutures were provided by Johnson & Johnson, model Ethicon SA82G. Micro-CT was provided by Bruker, model Skyscan 1172. Potassium oxalate (PO) and sodium carboxymethylcellulose (CMC-Na) were provided by Sigma, models 156124-25G and C5678-500G, respectively.

[0059] 2. Reagent Preparation

[0060] Potassium oxonate mother solution (30 mg / mL): potassium oxonate (30 mg) + 0.5% sodium carboxymethyl cellulose (1 mL);

[0061] 0.5% sodium carboxymethylcellulose: sodium carboxymethylcellulose powder (5 g) + normal saline (1 L).

[0062] 3. Experimental Grouping

[0063] NuC group: normal control group, intraperitoneal injection of 0.5% CMC-Na, and no ligature of the bilateral maxillary second molars (n=8);

[0064] NuP group: experimental periodontitis group, intraperitoneal injection of 0.5% CMC-Na, and bilateral maxillary second molars were ligated (n=8);

[0065] HuC group: hyperuricemia group, intraperitoneal injection of PO, and no ligature of bilateral maxillary second molars (n=8);

[0066] HuP group: hyperuricemia combined with periodontitis group, intraperitoneal injection of PO, bilateral maxillary second molars were ligated (n=8).

[0067] IV. Experimental Methods and Procedures

[0068] (1) 40 male C57 / BL6 mice were transported to the animal center of Huawei Testing Co., Ltd. and fed for one week in an environment with a temperature of 20-24°C, a relative humidity of 50%-70%, a light cycle of 12 days and 12 hours, an operating illumination of 150-300 lx, an airflow and wind speed of 0.1-0.2 m / s, and a noise level of ≤60 dB.

[0069] (2) 40 mice were randomly divided into NuC, NuP, HuC, and HuP groups, with 8 mice in each group;

[0070] (3) The mice in the NuC and NuP groups were injected intraperitoneally with 0.5% CMC-Na (the injection volume was consistent with the PO injection volume required for mice of the same weight) at a fixed frequency once a day for 28 consecutive days. On the 7th day, the mice in the NuP group were ligated with 5-0 silk thread on the maxillary second molars on both sides using a micro needle holder. The mice in the HuC and HuP groups were injected intraperitoneally with PO at a fixed frequency once a day with an injection dose of 600 mg / kg mouse / day (for mice weighing 25 g, the volume of the corresponding potassium oxonate stock solution (30 mg / mL) to be injected per day is 0.5 mL) for 28 consecutive days. On the 7th day, the mice in the HuP group were ligated with 5-0 silk thread on the maxillary second molars on both sides using a micro needle holder. The ligature was checked on days 1, 3, 7, 10, 14, and 21 after ligation in the NuP and HuP groups. If the ligature fell off, the ligature was re-tied promptly and recorded. (In the present invention, the ligature was kept firmly in place during the ligation of all mice, and no ligature fell off.) The specific ligature method is as follows:

[0071] After intraperitoneal anesthesia with 1.5% sodium pentobarbital (40 mg / kg), the modeling mice were fixed on a mouse board with the abdomen facing upward. The board was tilted at a certain angle and then fixed. A homemade mouth opener was used to stabilize the mouse's mouth and expose the maxillary molar area. A head-mounted light was used to provide a visual field of the molar area. The maxillary molars and buccal and lingual gums were cleaned with normal saline and dried thoroughly. Using a micro needle holder, rough 5-0 surgical silk thread was inserted in a dental floss-like manner below the interproximal contact points of the first and second maxillary molars and the second and third maxillary molars on one side, wrapped around the neck of the second molar. The needle holder was used to tie the silk thread on the palatal side of the second molar (taking care to remove any slack). The silk thread was secured with three surgical knots, and the excess silk thread was cut with spring scissors.

[0072] (4) At the end of the experiment (day 28), the mice were euthanized and samples were collected to detect blood biochemistry, renal function and renal pathology, as well as the destruction and inflammation of maxillary periodontal soft and hard tissues.

[0073] 5. Experimental Results

[0074] 1. Changes in serum uric acid levels of mice in each experimental group at the end of the experiment Figure 2 As shown, the results showed that compared with the groups injected with 0.5% CMC-Na (NuC, NuP), the serum uric acid levels of mice in the PO injection groups (HuC, HuP) were significantly increased, indicating that the hyperuricemia model induced by intraperitoneal injection of PO was successfully established. In addition, compared with the HuC mice, the serum uric acid levels of HuP mice showed an upward trend (P=0.101), suggesting that in the context of hyperuricemia, periodontitis may lead to a further increase in serum uric acid levels.

[0075] 2. Changes in kidney weight of mice in each experimental group Figure 3 As shown, the results showed that there was no significant difference in the kidney weight of mice in each experimental group, indicating that neither the ligature-induced periodontitis model nor the PO intraperitoneal injection-induced hyperuricemia model would cause renal parenchymal atrophy in mice.

[0076] 3. HE staining of kidney pathological changes in mice in each experimental group Figure 4 As shown, the results showed that compared with the normal control group (NuC group), the ligature-induced periodontitis group (NuP group), the hyperuricemia group induced by PO intraperitoneal injection (HuC group), and the hyperuricemia combined with periodontitis group (HuP group) did not show renal inflammatory response, indicating that the hyperuricemia model constructed by intraperitoneal injection of PO at a concentration of 600 mg / kg mouse / day in the present invention does not cause a strong inflammatory response in the kidney.

[0077] 4. Schematic diagram of 3D reconstruction of maxillary bones of mice in each experimental group after micro-CT scanning at the end of the experiment. Figure 5 As shown, the results showed that compared with the non-ligature group, the three-dimensional bone destruction of the maxillary second molars of mice in the ligature group was more severe, indicating that the ligature-induced experimental periodontitis model was successfully established. In addition, more importantly, compared with the simple periodontitis group, the three-dimensional bone destruction of the maxillary second molars of mice in the hyperuricemia combined periodontitis group was more severe, indicating that the presence of hyperuricemia further aggravated the bone destruction of periodontitis.

[0078] 5. HE staining and Trap staining of maxillary bone sections of mice in each experimental group at the end of the experiment Figure 6 As shown, the results showed that compared with the non-ligation group, the gingival inflammatory cell and osteoclast counts near the maxillary second molar of mice in the ligation group were significantly increased, indicating that the gingival inflammation and alveolar bone osteoclast infiltration of the experimental periodontitis model induced by ligation were more severe than those in the normal control group. In addition, more importantly, compared with the simple periodontitis group, the gingival inflammatory cell and osteoclast counts near the maxillary second molar of mice in the hyperuricemia combined periodontitis group were further increased, indicating that the presence of hyperuricemia further aggravated periodontal tissue inflammation and alveolar bone osteoclast infiltration.

[0079] As can be seen from the above, the composite mouse model simulating hyperuricemia and aggravated periodontitis provided by the present invention comprises the following steps: intraperitoneal injection of potassium oxonate (PO) at a fixed frequency once a day for 7 days, establishing a mouse periodontitis model on the 7th day by tying the surgical silk thread to the maxillary second molar of the mouse using a micro needle holder, continuing to perform intraperitoneal injection of potassium oxonate (PO) at a fixed frequency once a day for 7 consecutive days to establish a mouse model of hyperuricemia combined with periodontitis, and then continuing to perform intraperitoneal injection of potassium oxonate (PO) at a fixed frequency once a day for 14 consecutive days to maintain the stability of the hyperuricemia model, thereby constructing a stable mouse model of hyperuricemia combined with periodontitis. The hyperuricemia model constructed by the present invention using potassium oxonate (PO) can not only increase uric acid levels in a short period of time, but also maintain stable uric acid levels for a longer period of time through continuous administration during the maintenance period. Combined with the construction of a mild and non-invasive periodontitis model, an innovative mouse model simulating hyperuricemia-aggravated periodontitis is proposed, effectively filling the gap in the lack of animal models in the research field related to hyperuricemia and periodontitis, showing great application value in exploring the complex pathological mechanism of the relationship between the two, and broadening the ideas and methods of this clinical research hotspot.

[0080] At this point, those skilled in the art will recognize that, although the embodiments of the present invention have been shown and described in detail herein, many other variations or modifications consistent with the principles of the present invention may be directly determined or derived from the disclosure of the present invention without departing from the spirit and scope of the present invention. Therefore, the scope of the present invention should be understood and deemed to cover all such other variations or modifications.

Claims

1. A method for establishing a composite mouse model simulating hyperuricemia and aggravated periodontitis, characterized in that The following steps are involved: Step 1: Intraperitoneal injection of potassium oxonate was performed on mice for 7 consecutive days; Step 2: On day 7, surgical silk suture was tied around the maxillary second molar of the mouse using a micro needle holder; Step 3: Continue to inject potassium oxonate into the peritoneal cavity of mice for 7 consecutive days to establish a composite mouse model simulating hyperuricemia exacerbated by periodontitis.

2. The method according to claim 1, wherein The method further includes step 4 after step 3: continuously injecting potassium oxonate into the peritoneal cavity of the mice for 14 days.

3. The method according to claim 2, wherein In the step 1 and / or the step 3 and / or the step 4, potassium oxonate is injected into the mouse's peritoneal cavity once a day, and the injection dose is 600 mg / kg mouse / day.

4. The method according to claim 2, wherein In step 1 and / or step 3 and / or step 4, injecting potassium oxonate into the peritoneal cavity of the mouse comprises: gently grasping the mouse and fixing it in the palm of the hand, and injecting potassium oxonate stock solution into the peritoneal cavity of the mouse; the concentration of the potassium oxonate stock solution is 30 mg / mL.

5. The establishment method according to any one of claims 1 to 4, characterized in that The second step includes: Step a: Anesthetize and fix the mouse, expose the maxillary molar area, clean the maxillary molars and buccal and lingual gums, and dry them thoroughly; Step b: Fix the surgical suture on the mouse board, use the end of the needle holder to clamp the surgical suture, and pass the surgical suture through the interdental space between the second molar and the third molar; Step c: Wrap the surgical thread from the distal buccal surface of the second molar to the mesial buccal surface of the second molar, clamp the surgical thread, and pass the surgical thread through the interdental space between the first molar and the second molar; Step d: Use the needle holder to tie a knot of surgical silk thread on the palatal side of the second molar; Step e: Tie the silk thread with three surgical knots and cut the excess silk thread with spring scissors.

6. The method according to claim 5, characterized in that In step a, anesthetizing and fixing the mouse to expose the maxillary molars includes: anesthetizing the mouse's abdominal cavity and fixing the mouse on a mouse board with the abdominal cavity facing upward; tilting the mouse board at a certain angle and then fixing the mouse board, using an opener to stabilize the mouse's mouth to expose the maxillary molar area, and using a head-mounted lighting to provide a visual field of the molar area; and / or The cleaning of the maxillary molars and the buccal and lingual gums and fully drying them comprises: using physiological saline to clean the maxillary molars and the buccal and lingual gums, and then using dry cotton balls to fully dry them.

7. The establishment method according to claim 5, characterized in that The surgical suture is 5-0 surgical suture, and the needle holder is a micro needle holder, and the model of the micro needle holder is W40350.

8. The establishment method according to any one of claims 1 to 4, characterized in that The mice were male C57BL / 6 mice aged 6-8 weeks.

9. Application of a composite mouse model simulating hyperuricemia-aggravated periodontitis in screening therapeutic drugs for hyperuricemia-aggravated periodontitis, characterized in that The composite mouse model is a composite mouse model obtained according to the establishment method according to any one of claims 1 to 7.

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