A method for constructing a mouse model of sepsis complicated by disseminated intravascular coagulation

Abstract

The present invention discloses a method for constructing a mouse model of sepsis complicated by disseminated intravascular coagulation. The method establishes a mouse model of sepsis complicated by disseminated intravascular coagulation (sepsis DIC) by simultaneously intraperitoneally injecting 100 mg / kg Car solution and 50 μg / kg LPS solution into mice at a dosage volume of 0.1 mL / 10 g. This model can simultaneously simulate the two typical clinical characteristics of sepsis DIC, "fibrinolysis inhibition" and "consumptive hypocoagulation", making up for the shortcomings of the DIC model induced by large-dose LPS injection that can only simulate the "fibrinolysis inhibition" feature and the relatively high modeling cost. It also makes up for the shortcomings of the sepsis DIC model induced by the CLP method, which has not yet been standardized and the CLP models built by various laboratories have different performances on the fibrinolytic system, as well as the large surgical trauma, complex operation, difficulty in standardization and poor repeatability. This model can provide an accurate and convenient experimental model for screening potential drugs for the prevention and treatment of sepsis DIC.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal models of sepsis complications, and in particular relates to a method for constructing a sepsis-complicated disseminated intravascular coagulation (DIC) mouse model. Background Art

[0002] Disseminated intravascular coagulation (DIC) complicating sepsis is a coagulopathy caused by infection and an acute systemic inflammatory response leading to endothelial dysfunction. It typically manifests as widespread microthrombosis accompanied by massive bleeding at various sites, leading to multi-organ dysfunction. The clinical course consists of an initial "fibrinolytic inhibition" phase and a later "consumptive hypocoagulant" phase. Consumptive hypocoagulantism is a common feature of all types of DIC, while fibrinolytic inhibition is the clinical characteristic that distinguishes sepsis-related DIC from other types. Epidemiological data show that 30% to 50% of patients with sepsis will develop DIC, accounting for approximately 50% of all DIC cases. The mortality rate of sepsis-related DIC is 28% to 43%, almost twice that of patients without DIC. Currently, anticoagulants such as heparin, antithrombin, and thrombomodulin remain the primary treatment modality for sepsis-related DIC worldwide. However, the overall efficacy of current anticoagulant treatments remains controversial and increases the risk of severe bleeding, leading to an urgent need to find safe and effective drugs.

[0003] A good animal model for septic DIC is essential for studying the pathological basis of the disease, identifying and developing preventive and therapeutic drugs, and investigating drug mechanisms of action. Currently, few animal models for septic DIC have been reported. Existing rodent models of DIC are typically induced using high-dose lipopolysaccharide (LPS) injection or cecal ligation and puncture (CLP). However, the high-dose LPS-induced DIC model only exhibits the "fibrinolytic inhibition" phenomenon of septic DIC, while the "consumptive hypocoagulability" phenomenon is less pronounced, failing to simultaneously replicate the clinical features of both "fibrinolytic inhibition" and "consumptive hypocoagulability" in septic DIC. While the CLP-induced DIC model exhibits the clinical features of "consumptive hypocoagulability" in septic DIC, it exhibits variable fibrinolytic activity across laboratories, likely due to the complexity of the procedure and the difficulty in standardization. Furthermore, the surgical trauma is significant, resulting in poor reproducibility. Therefore, there is an urgent need for an animal model that is simple, convenient, reliable, and clinically applicable. Summary of the Invention

[0004] The present invention aims to provide a method for constructing a septic DIC mouse model. The mouse model constructed by this method can not only simultaneously simulate the two typical clinical characteristics of septic DIC, "fibrinolysis inhibition" and "consumptive hypocoagulation", but also is simple and convenient to operate, non-invasive, and has good reproducibility. It makes up for the shortcomings of the septic DIC model induced by the CLP method, such as large surgical trauma, complex operation, difficulty in standardization, and poor reproducibility. It can provide an accurate and convenient experimental model for screening potential drugs for the prevention and treatment of septic DIC.

[0005] The object of the present invention is achieved through the following technical solutions:

[0006] A method for constructing a DIC mouse model of sepsis is provided, wherein the DIC mouse model of sepsis is established by simultaneously injecting carrageenan (Car) and LPS into the abdominal cavity of the mouse.

[0007] Preferably, the modeling dose of Car is: intraperitoneal injection of 100 mg / kg Car solution at a dosage volume of 0.1 mL / 10 g.

[0008] Preferably, the modeling dose of LPS is: intraperitoneal injection of 50 μg / kg LPS solution at a dosage volume of 0.1 mL / 10 g.

[0009] Compared with the induction method of injecting a large dose of LPS, the method of the present invention significantly reduces the amount of LPS used and reduces the cost.

[0010] Preferably, the mice are male mice.

[0011] The present invention also provides application of the above construction method in the field of animal model construction.

[0012] Preferably, the animal model is a sepsis DIC mouse model.

[0013] The present invention also provides application of the DIC mouse model obtained by the above-mentioned construction method in DIC sepsis research, wherein the research is for the purpose of non-disease diagnosis or treatment.

[0014] The present invention also provides use of the DIC mouse model obtained by the above-mentioned construction method in screening drugs for preventing or treating DIC.

[0015] The present invention also provides a method for screening candidate drugs for preventing or treating sepsis-induced DIC, comprising the step of administering the test candidate drug to the sepsis-induced DIC mouse model obtained according to the above-mentioned construction method.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention can successfully establish a septic DIC mouse model by simultaneously intraperitoneally injecting 100 mg / kg Car and 50 μg / kg LPS into male KM mice at 16±1°C. On the one hand, this model can reflect inflammatory and infectious phenomena of sepsis, such as increased neutrophil percentage, elevated CRP level, and elevated IL-6 level. On the other hand, compared with the DIC model induced by high-dose LPS injection, this model can simultaneously simulate the two typical clinical characteristics of sepsis DIC, "fibrinolysis inhibition" and "consumption hypocoagulation", making up for the shortcomings of the DIC model induced by high-dose LPS injection that can only simulate the "fibrinolysis inhibition" characteristic and the relatively high cost of high-dose LPS modeling, and is more in line with the clinical process characteristics of sepsis DIC. Compared with the sepsis DIC model induced by the CLP method, this model can not only simultaneously simulate the two typical clinical characteristics of sepsis DIC, "fibrinolysis inhibition" and "consumption hypocoagulation", but also is simple and convenient to operate, non-invasive, and has good reproducibility. It makes up for the shortcomings of the sepsis DIC model induced by the CLP method, which has not yet been standardized, and the CLP models constructed by different laboratories have different performances on the fibrinolytic system, as well as the large surgical trauma, complex operation, difficulty in standardization, and poor reproducibility. It can provide an accurate and convenient experimental model for screening potential drugs for the prevention and treatment of sepsis DIC. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the animal grouping model in Experiment 1.

[0019] Figure 2 The weight of mice 48 hours after modeling in Experiment 1 ( n=4). Compared with the normal control group, ** P<0.01.

[0020] Figure 3 The blood stasis area of ​​the auricle capillary network, blood stasis points around the mouth and the relative length of tail thrombus at different time points in the mouse 48 hours after modeling in experiment 1 ( n=4). A and D: Area of ​​stasis in the capillary network of the mouse auricle and its statistical graph; B: Perioral stasis points of the mouse; C: Relative length of thrombus in the mouse tail 48 hours after modeling; E: Statistical graph of relative length of thrombus in the mouse tail at different time points after modeling. Compared with the 100mg / kg Car group, ** P<0.01, * P<0.05.

[0021] Figure 4 The neutrophil count and neutrophil percentage of peripheral blood smears of mice at 6h, 12h and 24h after modeling in experiment 1 ( n=4). A: Neutrophil counts in peripheral blood smears of mice at 6h, 12h, and 24h after modeling; B: Statistical graph of neutrophil percentages in peripheral blood smears of mice at 6h, 12h, and 24h after modeling. Compared with the normal control group, ** P<0.01; compared with 100mg / kg Car group, ## P<0.01; compared with the 50μg / kg LPS group, aa P<0.01; compared with 100mg / kg Car+50μg / kg LPS group, b P<0.05.

[0022] Figure 5 The plasma t-PA level, PAI-1 level, PAP level, FIB level, TAFI level and FDP level of mice after modeling in Experiment 2 ( n=6). A: t-PA level; B: PAI-1 level; C: PAP level; D: FIB level; E: TAFI level; F: FDP level. Compared with the normal control group at the same time point, ** P<0.01, * P<0.05.

[0023] Figure 6 The blood stasis area of ​​the auricle capillary network and the relative length of the tail thrombus of mice at different time points after modeling in Experiment 2 ( n = 6). A and C: The area of ​​stasis in the capillary network of the auricle of mice at different time points after modeling and its statistical graph; B and D: The relative length of thrombus in the tail of mice at different time points after modeling and its statistical graph. Compared with the 3h model group, ** P<0.01; compared with the 6h model group, ## P<0.01.

[0024] Figure 7 The rectal temperature of mice at different time points after modeling in Experiment 2 ( n=6). Compared with the 24h normal control group, ** P<0.01.

[0025] Figure 8 Neutrophil blood smear counts and neutrophil percentages of mice at different time points after modeling in Experiment 2 ( n=6). A: Neutrophil blood smear count; B: Neutrophil percentage. Compared with the normal control group at the corresponding time point, ** P<0.01.

[0026] Figure 9 Serum CRP, IL-6 and TNF-α levels of mice at different time points after model establishment in Experiment 2 ( n = 6). A: CRP concentration; B: IL-6 concentration; C: TNF-α concentration. Compared with the normal control group at the corresponding time points, ** P < 0.01, * P < 0.05.

[0027] Figure 10 For the changes in APTT and PT of mice at different time points after modeling in Experiment 2 ( n = 6). A: APTT; B: PT. Compared with the normal control group at the corresponding time points, ** P < 0.01. Detailed implementation manners

[0028] The following examples are further descriptions of the present invention rather than limitations thereof.

[0029] Example 1

[0030] 1. Animal selection

[0031] SPF-grade KM mice, 26 - 32 g, male, provided by Tongxiang Branch of Zhejiang Vital River Laboratory Animal Technology Co., Ltd., experimental animal production license number: (SCXK(Zhe)2020 - 0002).

[0032] 2. Animal adaptation observation environment

[0033] The mice were raised in the animal house of the First Affiliated Hospital of Guangzhou University of Chinese Medicine for adaptation observation, experimental animal use license number: (SCXK(Yue)2023 - 0092). The indoor environment was 20 - 26°C, relative humidity was 40 - 70%, 12h light / 12h dark light and dark alternation, working illuminance was 200 - 300 Lux, and animal illuminance was 15 - 20 Lux. The mice were allowed to eat and drink freely.

[0034] 3. Animal modeling environment

[0035] After the adaptation observation of the mice was completed, modeling was carried out. During modeling, the indoor temperature was controlled at 16 ± 1°C, relative humidity was 55 ± 5%, 12h light / 12h dark light and dark alternation, working illuminance was 200 - 300 Lux, and animal illuminance was 15 - 20 Lux. The mice were allowed to eat and drink freely.

[0036] 4. Experimental materials

[0037] Small animal rectal temperature detector was purchased from Shanghai Gushi Trading Co., Ltd., model 291128; digital display heating magnetic stirrer was purchased from FOUR E'S SCIENTIFIC, model MI0102002; magnetic stirring bar was purchased from Beekman Biological Co., Ltd., model C 7×21 mm; Car powder was purchased from Merck, Germany, product number C1013-100 G; LPS powder was purchased from Merck, Germany, product number L2630-10 MG; physiological saline was purchased from Guangdong Daxiang Pharmaceutical Co., Ltd., catalog number 220331402E5; Swiss Giemsa stain solution A was purchased from Zhuhai Beso Biotechnology Co., Ltd., catalog number C230511; Swiss Giemsa stain solution B was purchased from Zhuhai Beso Biotechnology Co., Ltd., catalog number C230805; mouse interleukin-6 (IL-6) ELISA kit was purchased from Ruixin Biotechnology Co., Ltd., catalog number RX203049M; mouse tumor necrosis factor α (TNF-α) kit was purchased from Ruixin Biotechnology Co., Ltd., catalog number RX202412M; mouse tissue-type plasminogen activator (t-PA) ELISA kit was purchased from Ruixin Biotechnology Co., Ltd., catalog number RX202381M; mouse fibrinogen (FIB) ELISA kit was purchased from Ruixin Biotechnology Co., Ltd., catalog number RX202552 M; mouse plasminogen activator inhibitor-1 (PAI-1) ELISA kit was purchased from Ruixin Biotechnology Co., Ltd., catalog number RX202553M; mouse plasmin-antiplasmin complex (PAP) ELISA kit was purchased from Ruixin Biotechnology Co., Ltd., catalog number RX201534M; mouse fibrinogen degradation product (FDP) ELISA kit was purchased from Ruixin Biotechnology Co., Ltd., catalog number RX202503M; mouse thrombin-activated fibrinolysis inhibitor (activated TAFI) ELISA kit was purchased from Ruixin Biotechnology Co., Ltd., catalog number RX201476M; APTT detection kit (ellagic acid coagulation method) was purchased from Leigen Biotechnology Co., Ltd., catalog number TC0306; PT detection kit (ellagic acid coagulation method) was purchased from Leigen Biotechnology Co., Ltd., catalog number TC0307.

[0038] 5. Solution Preparation

[0039] (1) Preparation of Car solution

[0040] Intraperitoneally inject a 100 mg / kg Car solution at a dosing volume of 0.1 mL / 10 g. Quickly weigh the required amount of Car powder into a serum bottle. Pipette an appropriate amount of saline into the serum bottle to prepare a 1% Car solution. Place the serum bottle on a heated digital magnetic stirrer at 60°C and 1000 rpm. Vortex mix and use immediately.

[0041] (2) Preparation of LPS solution

[0042] Intraperitoneally inject 50 μg / kg LPS solution at a dosage of 0.1 mL / 10 g. Dissolve LPS powder in normal saline to prepare a stock solution, aliquot into small tubes, and store at -20°C. To prepare the LPS working solution, dilute the aliquoted stock solution with normal saline to a 0.005 mg / mL LPS working solution. Protect from light during preparation and use immediately.

[0043] 6. Animal Grouping and Modeling

[0044] 6.1 Experiment 1

[0045] Male KM mice were randomly divided into six groups based on body weight: normal control group (A), 100 mg / kg Car group (B), 50 μg / kg LPS group (C), 100 mg / kg Car + 50 μg / kg LPS group (D, Car and LPS injected intraperitoneally simultaneously), 100 mg / kg Car → 50 μg / kg LPS group (E, Car injected first, LPS injected intraperitoneally 12 hours later), and 50 μg / kg LPS → 100 mg / kg Car group (F, LPS injected first, Car injected intraperitoneally 12 hours later), with four mice in each group. Modeling time was designated as 0 hours. The specific procedures were as follows:

[0046] Normal control group (A): intraperitoneal injection of normal saline (0.1 mL / 10 g);

[0047] 100 mg / kg Car group (B): intraperitoneal injection of 100 mg / kg Car solution (0.1 mL / 10 g);

[0048] 50 μg / kg LPS group (C): intraperitoneal injection of 50 μg / kg LPS solution (0.1 mL / 10 g);

[0049] 100 mg / kg Car + 50 μg / kg LPS group (D): 50 μg / kg LPS solution (0.1 mL / 10 g) was injected intraperitoneally immediately after the intraperitoneal injection of 100 mg / kg Car solution (0.1 mL / 10 g);

[0050] 100 mg / kg Car→50 μg / kg LPS group (E): 100 mg / kg Car solution (0.1 mL / 10 g) was intraperitoneally injected 12 h before 50 μg / kg LPS solution (0.1 mL / 10 g) was intraperitoneally injected;

[0051] 50 μg / kg LPS→100 mg / kg Car group (F): mice were intraperitoneally injected with 50 μg / kg LPS solution (0.1 mL / 10 g) and then intraperitoneally injected with 100 mg / kg Car solution (0.1 mL / 10 g) 12 h later.

[0052] In the above operation, the injection site of Car was uniformly the left abdomen of the mouse, and the injection site of LPS was the right abdomen of the mouse. After the model was completed, the animals were placed in an environment with a temperature of 16±1℃ and a humidity of 55±5% for 48 hours, with free access to food and water.

[0053] Schematic diagram of animal grouping and modeling in Experiment 1 Figure 1 shown.

[0054] 6.2 Experiment 2

[0055] Male KM mice were randomly divided into 16 groups according to their body weight: 0.25h-normal control group (A1), 0.25h-model group (A2), 0.5h-normal control group (B1), 0.5h-model group (B2), 1h-normal control group (C1), 1h-model group (C2), 1.5h-normal control group (D1), 1.5h-model group (D2), 3h-normal control group (E1), 3h-model group (E2), 6h-normal control group (F1), 6h-model group (F2), 12h-normal control group (G1), 12h-model group (G2), 24h-normal control group (H1), and 24h-model group (H2), with 6 mice in each group.

[0056] The time of modeling was designated as 0 h. All mice in the model group were intraperitoneally injected with 100 mg / kg Car solution at a dosage of 0.1 mL / 10 g into the left peritoneal cavity, and 50 μg / kg LPS solution at a dosage of 0.1 mL / 10 g into the right peritoneal cavity. All mice in the normal control group were intraperitoneally injected with normal saline at a dosage of 0.1 mL / 10 g into the corresponding locations. After modeling, the animals were placed in an environment with a temperature of 16 ± 1°C and a humidity of 55 ± 5%, with free access to food and water.

[0057] 7. Sample Collection

[0058] 7.1 Experiment 1

[0059] 24 hours after modeling, all mice were anesthetized by isoflurane inhalation. After anesthesia took effect, the mice were fixed in the supine position, and the abdominal cavity was opened along the midline of the abdomen for blood sampling from the abdominal aorta. The blood was collected in 1.5 mL EP tubes.

[0060] 7.2 Experiment 2

[0061] Mice in groups A1 and A2 were anesthetized with isoflurane inhalation 0.25 hours after model establishment, groups B1 and B2 were anesthetized with isoflurane inhalation 0.5 hours after model establishment, groups C1 and C2 were anesthetized with isoflurane inhalation 1 hour after model establishment, groups D1 and D2 were anesthetized with isoflurane inhalation 1.5 hours after model establishment, groups E1 and E2 were anesthetized with isoflurane inhalation 3 hours after model establishment, groups F1 and F2 were anesthetized with isoflurane inhalation 6 hours after model establishment, groups G1 and G2 were anesthetized with isoflurane inhalation 12 hours after model establishment, and groups H1 and H2 were anesthetized with isoflurane inhalation 24 hours after model establishment. After anesthesia took effect, the mice were fixed in the supine position, and the abdominal cavity was opened along the midline of the abdomen for blood collection from the abdominal aorta.

[0062] Blood from mice in groups A1, A2, B1, B2, C1, and C2 was partially collected into 0.5 mL EP tubes containing sodium citrate anticoagulant (sodium citrate anticoagulant: anticoagulant blood = 25 μL:250 μL), centrifuged at 3000 rpm for 10 min, and used for APTT and PT kit detection. The remaining blood was collected into 1.5 mL EP tubes containing sodium citrate anticoagulant (sodium citrate anticoagulant: anticoagulant blood = 1:10), allowed to stand, and centrifuged at 4000 rpm at 4°C for 20 min. Plasma was then collected for detection by t-PA, PAP, PAI-1, FIB, FDP, and TAFI kits.

[0063] The blood of mice in groups D1, D2, E1, E2, F1, F2, G1, G2, H1, and H2 was collected into 0.5 mL EP tubes containing sodium citrate anticoagulant (sodium citrate anticoagulant: anticoagulant blood = 25 μL: 250 μL) and centrifuged at 3000 rpm for 10 min for detection by APTT and PT kits. The remaining blood was collected into 1.5 mL EP tubes without anticoagulant and centrifuged at 4000 rpm for 20 min for detection by serum CRP, IL-6, and TNF-α Elisa kits.

[0064] 8.Indicator detection

[0065] 8.1 Experiment 1

[0066] (1) Mouse weight

[0067] 48 h after modeling, the mice were weighed using an electronic balance.

[0068] (2) Relative length of mouse tail thrombus

[0069] 6h, 24h, and 48h after modeling, the total length of the mouse tail and the length of the black tail of the thrombus (the length from the end of the mouse tail to the front of the dark thrombus formation) were measured with a ruler, and the relative length of the mouse tail thrombus was calculated. The relative length of the mouse tail thrombus = (length of the mouse black tail / total length of the mouse tail) × 100%.

[0070] (3) Relative blood congestion area of ​​mouse auricle capillaries

[0071] 48 h after modeling, the mouse shooting position and mobile phone height were fixed, and images of the mouse auricle were collected under the same angle and light. The auricle capillary congestion area was quantitatively analyzed using Image J software, and the relative congestion area of ​​the mouse auricle capillaries was calculated (relative congestion area of ​​the mouse auricle capillaries = mouse auricle capillary congestion area / mouse auricle total area) × 100%.

[0072] (4) Neutrophil percentage count

[0073] 6, 12, and 24 hours after modeling, fresh blood was collected from the orbital venous plexus into a 1.5 mL EP tube. Using a pipette, 2 μL of blood was quickly drawn onto a glass slide for blood smear preparation. After the smears were air-dried, they were stained with Wright-Giemsa stain and observed under a microscope. The body-tail junction was selected for counting. A total of 100 white blood cells were counted in each blood smear, and the number of neutrophils was recorded. The neutrophil percentage = (number of neutrophils / 100) × 100%.

[0074] 8.2 Experiment 2

[0075] (1) Plasma t-PA level

[0076] Detection was performed according to the instructions of the t-PA Elisa detection kit.

[0077] (2) Plasma PAI-1 levels

[0078] The detection was performed according to the instructions of the PAI-1 Elisa detection kit.

[0079] (3) Plasma PAP level

[0080] The test was performed according to the instructions of the PAP Elisa test kit.

[0081] (4) Plasma FIB level

[0082] The detection was performed according to the instructions of the FIB Elisa detection kit.

[0083] (5) Plasma TAFI level

[0084] The test was performed according to the instructions of the TAFI Elisa test kit.

[0085] (6) Plasma FDP levels

[0086] The detection was performed according to the instructions of the FDP Elisa detection kit.

[0087] (7) Serum CRP level

[0088] The test was performed according to the instructions of the CRP Elisa test kit.

[0089] (8) Plasma IL-6 levels

[0090] The detection was performed according to the instructions of the IL-6 Elisa detection kit.

[0091] (9) Plasma TNF-α levels

[0092] The detection was performed according to the instructions of the TNF-α Elisa detection kit.

[0093] (10)APTT

[0094] The APTT test kit (ellagic acid coagulation method) was used for the operation.

[0095] (11)PT

[0096] The operation was performed according to the instructions of the PT detection kit (first phase method).

[0097] (12) Relative length of mouse tail thrombus

[0098] 1.5h, 3h, 6h, 12h, and 24h after modeling, the total length of the mouse tail and the length of the black tail of the thrombus (the length from the end of the mouse tail to the front of the dark thrombus formation) were measured with a ruler, and the relative length of the mouse tail thrombus was calculated. The relative length of the mouse tail thrombus = (length of the mouse black tail / total length of the mouse tail) × 100%.

[0099] (13) Relative blood congestion area of ​​mouse auricle capillaries

[0100] 24 hours after modeling, the mouse shooting position and mobile phone height were fixed, and the mouse auricle was imaged at the same angle and light. The auricle capillary congestion area was quantitatively analyzed using Image J software, and the relative congestion area of ​​the mouse auricle capillaries was calculated (relative congestion area of ​​the mouse auricle capillaries = mouse auricle capillary congestion area / mouse auricle total area) × 100%.

[0101] (14) Rectal temperature

[0102] Measure with a small animal rectal temperature detector.

[0103] 9. Data Analysis

[0104] The experimental data were expressed as mean ± standard deviation Data were presented and statistically analyzed using SPSS 20.0. Pairwise comparisons of means between groups were performed using the Bonferroni test if the means were normally distributed and had equal variances. If the means were normally distributed but had unequal variances, the Tamhane test was used. If the means were not normally distributed, the Mann-Whitney U test was used. Differences were considered statistically significant when P < 0.05. Graphpad Prism 5.0 software was used for plotting.

[0105] 10. Experimental Results

[0106] 10.1 Experiment 1

[0107] (1) Effects of different injection methods on mouse body weight

[0108] like Figure 2 48 hours after modeling, the body weight of mice in the 100 mg / kg Car+50 μg / kg LPS group and the 100 mg / kg Car→50 μg / kg LPS group was significantly decreased compared with that in the normal control group (P<0.01).

[0109] (2) Effects of different injection methods on auricular microcirculation, perioral stasis points, and tail thrombosis in mice

[0110] like Figure 3 Compared with the 100 mg / kg Car group, the 100 mg / kg Car+50 μg / kg LPS group significantly enlarged the area of ​​stasis in the mouse auricle capillary network (P<0.01).

[0111] (3) Effects of different injection methods on the percentage of neutrophils in peripheral blood of mice

[0112] like Figure 4 At 6h and 12h after modeling, the percentage of neutrophils in each model group was significantly increased compared with the normal control group (P<0.01).

[0113] 10.2 Experiment 2

[0114] (1) Changes in the fibrinolytic system function of model mice 0.25h to 1h after modeling

[0115] like Figure 5 Compared with the normal control group at the same time points, the plasma t-PA of the model group mice was significantly increased at 0.5h and 1h after modeling. The plasma PAI-1, PAP, FIB, and TAFI of the model group mice were also significantly increased at 0.25h, 0.5h, and 1h after modeling. The plasma FDP of the model group mice was no different from that of the normal control group at the same time points. This model has the "fibrinolysis inhibition" characteristic of septic DIC.

[0116] (2) Changes in mouse auricle microcirculation and tail thrombosis 1.5 to 24 hours after modeling

[0117] like Figure 6 Compared with the normal control group at the same time point, the area of ​​stasis in the auricle capillary network of the model group mice increased over time, and the relative length of the tail thrombus increased over time. This model has the characteristics of extensive microthrombosis in septic DIC.

[0118] (3) Changes in mouse rectal temperature 1.5 h to 24 h after modeling

[0119] like Figure 7 Compared with the normal control group at the same time point, the rectal temperature of the model mice was significantly reduced 24 hours after modeling. This model has the characteristics of hypothermia in sepsis DIC.

[0120] (4) Changes in the percentage of neutrophils in the peripheral blood of mice 1.5h to 24h after modeling

[0121] like Figure 8 Compared with the normal control group at the same time points, the percentage of neutrophils in the model group mice increased significantly at 6, 12, and 24 hours after modeling. This model can simulate the bacterial infection phenomenon of increased neutrophil counts during sepsis.

[0122] (5) Changes in serum CRP, IL-6, and TNF-α levels in mice 1.5 to 24 hours after modeling

[0123] like Figure 9 Compared with the normal control group at the same time points, serum CRP and IL-6 levels in the model group mice were significantly elevated 1.5, 3, 6, 12, and 24 hours after modeling. Serum TNF-α levels in the model group mice were also significantly elevated 12 and 24 hours after modeling. This model can simulate the inflammatory infection phenomenon of septic DIC, where elevated levels of inflammatory factors are present.

[0124] (6) Changes in coagulation function of mice 0.25h to 24h after modeling

[0125] like Figure 10 Compared with the normal control group at the same time points, the APTT of the model group mice was significantly prolonged from 0.25 hours to 24 hours after model establishment, and the PT of the model group mice was significantly prolonged from 1.5 hours to 24 hours after model establishment. This model can simulate the bleeding tendency characteristic of septic DIC "consumptive hypocoagulable phase".

[0126] In summary, a septic DIC mouse model was successfully established by simultaneously intraperitoneally injecting 100 mg / kg Car and 50 μg / kg LPS into male KM mice at 16±1°C. On the one hand, this model can reflect inflammatory and infectious phenomena of sepsis, such as increased neutrophil percentage, elevated CRP level, and elevated IL-6 level. On the other hand, compared with the DIC model induced by high-dose LPS injection, this model can simultaneously simulate the two typical clinical characteristics of sepsis DIC, "fibrinolysis inhibition" and "consumption hypocoagulation", making up for the shortcomings of the DIC model induced by high-dose LPS injection that can only simulate the "fibrinolysis inhibition" characteristic and the relatively high cost of high-dose LPS modeling, and is more in line with the clinical process characteristics of sepsis DIC. Compared with the sepsis DIC model induced by the CLP method, this model can not only simultaneously simulate the two typical clinical characteristics of sepsis DIC, "fibrinolysis inhibition" and "consumption hypocoagulation", but also is simple and convenient to operate, non-invasive, and has good reproducibility. It makes up for the shortcomings of the sepsis DIC model induced by the CLP method, which has not yet been standardized, and the CLP models constructed by different laboratories have different performances on the fibrinolytic system, as well as the large surgical trauma, complex operation, difficulty in standardization, and poor reproducibility. It can provide an accurate and convenient experimental model for screening potential drugs for the prevention and treatment of sepsis DIC.

[0127] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a mouse model of sepsis complicated by disseminated intravascular coagulation, characterized in that: A mouse model of sepsis complicated by disseminated intravascular coagulation was established by simultaneously injecting carrageenan and lipopolysaccharide into the peritoneal cavity of mice.

2. The construction method according to claim 1, characterized in that The modeling dosage of the carrageenan is: intraperitoneal injection of 100 mg / kg carrageenan solution at a dosage volume of 0.1 mL / 10 g.

3. The construction method according to claim 1, characterized in that The modeling dose of the lipopolysaccharide is as follows: 50 μg / kg lipopolysaccharide solution is injected intraperitoneally at a dosage volume of 0.1 mL / 10 g.

4. The construction method according to claim 1, characterized in that The mice are male mice.

5. Use of the mouse model of sepsis complicated by disseminated intravascular coagulation obtained by the construction method according to any one of claims 1 to 4 in screening drugs for preventing or treating sepsis complicated by disseminated intravascular coagulation.

6. A method for screening candidate drugs for preventing or treating sepsis-complicated disseminated intravascular coagulation, characterized in that: The method comprises the step of administering a test candidate drug to a mouse model of sepsis complicated by disseminated intravascular coagulation obtained according to the construction method according to any one of claims 1 to 4.

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