Stable and high-reproducibility severe acute pancreatitis mouse model construction method, quantitative evaluation system and application thereof

Abstract

The invention belongs to the technical field of medical experimental animal models, and particularly relates to a stable and high-reproducibility severe acute pancreatitis mouse model construction method, a quantitative evaluation system and application thereof, and the method comprises the following steps: selecting a male C57BL / 6 mouse of 6-8 weeks old, adaptively feeding for one week, and then carrying out model induction; the preparation method comprises the following steps: carrying out intraperitoneal injection on leptofrog at the dosage of 100 mu g / kg once every 1 hour, and continuously injecting for 7 times; within 2 hours after completion of the seventh time of rain frog element injection, lipopolysaccharide is injected into the abdominal cavity, and the dosage is 12 mg / kg; pathological and physiological index evaluation is carried out on the mouse 24 hours after the first time of rain frog element injection, and multi-batch verification experiments prove that the inter-batch fluctuation range of the model constructed by the invention on the death rate, the pathological score, the weight change and other key indexes is controlled within + / -5%, so that the repeatability and comparability of experimental results are ensured.

Description

Technical Field

[0001] This invention belongs to the field of medical experimental animal model technology, specifically involving a stable and highly reproducible method for constructing a mouse model of severe acute pancreatitis, a quantitative assessment system and its application, which is particularly suitable for the study of the pathological mechanism of pancreatitis and the evaluation of novel treatment regimens. Background Technology

[0002] Severe acute pancreatitis (SAP) is a highly fatal acute abdominal condition characterized by pancreatic tissue necrosis, systemic inflammatory response syndrome, and multiple organ dysfunction. Clinical statistics show that the mortality rate of SAP patients is as high as 15% to 30%, with rapid disease progression and poor prognosis. Therefore, in-depth research into its pathogenesis and the development of effective treatments are crucial to reducing mortality, and the construction of stable and reliable animal models of SAP is the foundation and prerequisite for research in this field.

[0003] Currently, researchers both domestically and internationally have developed various animal models of pancreatitis. Since Busnardo et al. successfully induced an acute pancreatitis model in dogs by injecting a mixture of bile and olive oil into the pancreatic duct in 1856, after more than a century of exploration, various methods for preparing pancreatitis animal models have been developed, including drug-induced models, surgically induced models, and genetically engineered models. Among them, the mouse pancreatitis model induced by hygroscopicin is widely used due to its relatively simple operation. Hygroscopicin is a gastric regulatory molecule that functions and is compositionally similar to cholecystokinin. It is a decapeptide that can stimulate the secretion of gastrointestinal hormones and has a strong effect on gallbladder contraction and stimulation of pancreatic enzyme secretion.

[0004] In their 2017 article "Analysis of the Effects of Levenoin and Lipopolysaccharide on the Induction of Acute Pancreatitis in Mice," published in *Basic Medicine and Clinical Practice*, Xiao Luyao et al. reported that levenoin (50 μg / kg) administered seven times consecutively could induce an acute pancreatitis model in mice. Increasing the frequency of administration did not aggravate pancreatic tissue damage, while the combined use of lipopolysaccharide exacerbated the severity of the acute pancreatitis model. This study found that serum amylase activity was higher in the CAE7 and CAE9 groups than in the control group, and even higher in the CAE7+LPS group than in the CAE9 group. Pancreatic tissue pathological scores also showed a similar trend. This study provides preliminary evidence for the induction of a severe acute pancreatitis model using levenoin combined with lipopolysaccharide.

[0005] However, existing model construction methods still have significant shortcomings. First, model stability is poor; phenotypic differences between different batches and laboratories are large, making it difficult to guarantee the comparability and reproducibility of experimental results. Second, the severity of the disease is insufficient; pancreatitis induced by taurine alone usually presents as mild to moderate, making it difficult to simulate the pathological features of clinical severe acute pancreatitis, including high mortality, significant ascites, intestinal edema, and multiple organ dysfunction. Third, systemic inflammatory response is lacking; existing models struggle to reproduce the systemic inflammatory response and multiple organ dysfunction commonly seen in patients with severe acute pancreatitis. Finally, a standardized evaluation system is lacking; existing studies have inconsistent evaluation criteria for models, with most studies only assessing based on amylase and lipase levels and simple pathological changes, lacking comprehensive and systematic evaluation indicators, making it difficult to objectively evaluate model quality and treatment efficacy.

[0006] For example, various reported pancreatitis models induced by crotonin mainly use crotonin doses ranging from 50 μg / kg to 100 μg / kg, with injection numbers ranging from 3 to 11 times. The significant differences in induction protocols lead to substantial variations in model severity and stability. Furthermore, systematic research is lacking on key parameters such as the optimal dosage ratio and injection interval of crotonin and lipopolysaccharide in combination, making it difficult for existing methods to stably reproduce clinically accurate models of severe acute pancreatitis.

[0007] Therefore, there is an urgent need to develop a stable, reliable, and highly reproducible mouse model of severe acute pancreatitis and its standardized evaluation system to meet the needs of basic and translational research on severe acute pancreatitis. Summary of the Invention

[0008] To address the problems existing in the prior art, the present invention aims to provide a stable and highly reproducible method for constructing a mouse model of severe acute pancreatitis, a quantitative assessment system, and its application, so as to overcome the problems of poor model stability, inconsistent pathological manifestations, and difficulty in meeting the clinical criteria for severe acute pancreatitis in the prior art.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] This invention provides a stable and highly reproducible method for constructing a mouse model of severe acute pancreatitis. This method successfully establishes a stable and reproducible animal model of severe acute pancreatitis by precisely controlling the dosage, number of injections, and time intervals of vitexin and lipopolysaccharide. Specifically, male C57BL / 6 mice aged 6 to 8 weeks were selected and acclimatized for one week before model induction. Model induction was achieved by intraperitoneal injection of vitexin at a dose of 100 μg / kg, administered every 1 hour for 7 consecutive times. Two hours after the 7th vitexin injection, lipopolysaccharide was injected intraperitoneally at a dose of 12 mg / kg. Pathological and physiological indicators of the mice were assessed 24 hours after the first vitexin injection.

[0011] The innovation of this invention lies in the precise control of the time window for the dual-attack mechanism. Violazoline, through cholecystokinin receptors, stimulates pancreatic acinar cells to excessively secrete pancreatic enzymes, disrupting the separation of trypsinogen and lysosomal hydrolases within the cells. This leads to cathepsin B-dependent activation, inducing local pancreatic damage. Lipopolysaccharide, as an endotoxin, triggers a systemic inflammatory response by activating monocytes to release cytokines. This invention, by adding lipopolysaccharide within the optimal time window to the violazoline-induced local pancreatic damage, successfully achieves a synergistic effect between local damage and systemic inflammatory response, thereby constructing a clinically accurate model of severe acute pancreatitis.

[0012] This invention also provides a quantitative evaluation system for the aforementioned model, comprising seven dimensions of evaluation indicators. First, mortality assessment, statistically analyzing mouse survival within one week after the initial drug administration. Second, weight change assessment, calculating the percentage change in weight before and after modeling. Third, pancreatic tissue pathological assessment, using the Schmidt scoring system to quantify edema, inflammatory cell infiltration, acinar necrosis, hemorrhage, and fat necrosis in HE-stained sections. Fourth, ascites volume assessment, measuring the volume of ascites. Fifth, intestinal edema assessment, scoring the degree of intestinal wall edema in HE-stained intestinal tissue sections. Sixth, serum enzyme activity assessment, measuring serum amylase and lipase activities. Seventh, inflammatory factor level assessment, quantitatively detecting the concentrations of TNF-α, IL-1β, and IL-6 in serum.

[0013] The beneficial effects of this invention are as follows:

[0014] First, the model's stability is significantly improved. Multiple batch validation experiments confirmed that the model constructed in this invention maintains batch-to-batch fluctuations within ±5% in key indicators such as mortality rate, pathological scores, and weight changes, ensuring the reproducibility and comparability of experimental results. This high degree of consistency is extremely rare in animal model research, providing a reliable foundation for conducting multi-center collaborative studies.

[0015] Second, the phenotype of severe acute pancreatitis was clearly defined. Through an optimized formulation using a combination of crotonin and lipopolysaccharide, and precise time window control, this invention successfully induced an animal model meeting the clinical criteria for severe acute pancreatitis. This model exhibited a mortality rate exceeding 30%, a body weight loss exceeding 10%, a Schmidt pathological score of pancreatic tissue exceeding 6, an ascites volume exceeding 0.5 mL, an intestinal edema score exceeding 2, and serum amylase and lipase activities exceeding 3 times that of the control group, as well as serum inflammatory factor levels exceeding 2 times that of the control group, accurately replicating the main characteristics of clinical severe acute pancreatitis.

[0016] Third, a comprehensive quantitative assessment system was established. The seven-dimensional assessment system established in this invention covers multiple aspects such as mortality rate, weight change, pathological score, ascites volume, serum biochemical indicators, and inflammatory factor levels, achieving a scientific and objective evaluation of the model. The Schmidt scoring system provides a detailed quantification of pancreatic histopathological changes, scoring from five dimensions: edema, inflammatory cell infiltration, acinar necrosis, hemorrhage, and fat necrosis, with a total score of up to 15 points, accurately reflecting the severity of pancreatic injury.

[0017] Fourth, it has a wide range of applications. The model constructed in this invention is applicable to various research scenarios, such as the study of the pathological mechanisms of severe acute pancreatitis, drug screening, and evaluation of novel treatment regimens. Pharmacodynamic validation experiments with dexamethasone have demonstrated that the model has good sensitivity and responsiveness to treatment interventions and can effectively distinguish between effective and ineffective treatment regimens.

[0018] Fifth, it has superior ethical considerations. Compared to surgical induction models, the drug induction method used in this invention causes less suffering to experimental animals, has a shorter experimental cycle, and is simpler to operate, conforming to the 3R principle of animal experiments, namely, reduction, substitution, and optimization. Attached Figure Description

[0019] Figure 1 The flowchart of the method for constructing a stable and highly reproducible mouse model of severe acute pancreatitis of the present invention shows the complete timeline from the injection of hymenoplasmin to tissue collection, including the time nodes of the 7 injections of hymenoplasmin and the time window of lipopolysaccharide injection.

[0020] Figure 2 This image shows a comparison of HE-stained sections of pancreatic tissue from normal mice and mice with severe acute pancreatitis. The left side shows pancreatic tissue from the normal control group, while the right side shows pancreatic tissue from the severe acute pancreatitis model group. Pathological changes such as edema, inflammatory cell infiltration, and acinar necrosis can be clearly observed in the pancreatic tissue of the model group.

[0021] Figures 3 to 12Comparison charts of the main evaluation indicators of mice with severe acute pancreatitis model and normal mice, as well as the consistency analysis charts of the main evaluation indicators in multiple batches of experiments, including the performance of indicators such as lipase activity, IL-1β level, IL-6 level, TNF-α level, pathological score, intestinal edema score, amylase activity, abdominal fluid volume, body weight change, and mortality in three batches of experiments. Detailed implementation manners

[0022] The present invention will be further described in detail below in conjunction with specific embodiments. Those skilled in the art should understand that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For those not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are followed. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0023] Example 1: Standard construction method of a mouse model of severe acute pancreatitis

[0024] In this example, a mouse model of severe acute pancreatitis was constructed by using the protocol of injecting cerulein at a dose of 100 μg / kg every 1 h for 7 consecutive times, and adding lipopolysaccharide at 12 mg / kg within 2 h after the 7th injection.

[0025] Twenty 6-week-old healthy male C57BL / 6 mice with a body weight range of 18 g to 20 g were purchased from Beijing Huafukang Biotechnology Co., Ltd., with the animal license number SCXK (Beijing) 2024-0003. After the mice were purchased, they were raised in a clean-grade animal house for one week to adapt to the environment. The feeding conditions were strictly controlled at a temperature of 22°C ± 2°C, a relative humidity of 50% to 60%, 12 h of light and 12 h of darkness alternating, and free access to food and water. Twelve hours before the experiment, the mice were fasted but not water-restricted to reduce intestinal stress. All animal experiment operations were approved by the Animal Ethics Committee of the Armed Police Characteristic Medical Center, with the approval number 2025-0026.

[0026] Cerulein was purchased from MCE Company, with the product number HY-A0190, the molecular formula C58H73N15O21S2, and the molecular weight 1352.41. 1 mg of cerulein was dissolved in physiological saline, and after preparing the mother liquor, it was filtered and sterilized with a 0.22 μm filter membrane and stored at -80°C. It was thawed before use and diluted to the working concentration of 15 μg / mL with 0.9% sodium chloride solution. Lipopolysaccharide was purchased from Sigma Company, with the product number L2630, derived from Escherichia coli serotype O55:B5. It was prepared into a working solution of 2 mg / mL with 0.9% sodium chloride solution.

[0027] The specific steps for model induction are as follows: At 0 h, the first intraperitoneal injection of spirulina extract was administered at a dose of 100 μg / kg body weight, with the injection volume calculated based on the mouse's body weight. Subsequently, the second to seventh intraperitoneal injections of spirulina extract were administered at 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, with each injection being 100 μg / kg. Figure 1 As shown, 1.5 hours after the 7th injection of spirulina extract, lipopolysaccharide was administered via intraperitoneal injection at a dose of 12 mg / kg body weight. During the injection process, the injection site was changed frequently to avoid repeated punctures at the same location, which could cause local damage. Mice were fasted for 12 hours after the last administration, but were allowed free access to water.

[0028] Twenty-four hours after the first injection of spirulina extract, 10 mice were randomly selected from the model group for sampling. Their weight was measured and recorded, and the rate of weight change was calculated by comparing it to their pre-modeling weight. Isoflurane inhalation anesthesia was administered, with the depth of anesthesia determined by the absence of a painful response in the mice. Approximately 0.5 to 1 mL of blood was collected from the orbital vein. The blood sample was placed in a standard blood collection tube, allowed to stand at room temperature for 30 minutes, and then centrifuged at 3000 rpm for 10 minutes to separate the serum, which was then stored at -80°C for later analysis.

[0029] Euthanasia was performed under anesthesia, and the abdomen was immediately opened to observe the peritoneal cavity. Peritoneal fluid was aspirated using pre-weighed sterile gauze, weighed again, and the volume of ascites was calculated based on the weight difference, denoted as V ascites. Pancreatic tissue was isolated, weighed, and divided into two parts: one part was fixed in 4% paraformaldehyde fixative for 24 hours, followed by paraffin embedding, sectioning, and HE staining; the other part was rapidly frozen in liquid nitrogen and then transferred to a -80°C freezer for molecular biological analysis. Simultaneously, a segment of small intestine tissue was taken and similarly fixed and rapidly frozen.

[0030] The remaining 10 mice were observed until day 7 after the first injection. Weight and mortality were recorded daily during the observation period, and the weekly mortality rate was calculated.

[0031] Example 2: Optimization Experiment of Rain Frog Extract Dosage

[0032] This embodiment explores the effect of taurine dosage on model construction, using a dosage of 80 μg / kg, with other conditions being the same as in Example 1.

[0033] Twenty healthy male C57BL / 6 mice aged 6 weeks were selected and acclimatized for one week before model induction. A working solution of 12 μg / mL limonin was administered intraperitoneally every 1 hour at a dose of 80 μg / kg for 7 consecutive injections. 1.5 hours after the 7th limonin injection, an additional intraperitoneal injection of 12 mg / kg lipopolysaccharide was administered. All other procedures, sample collection, and preservation methods were the same as in Example 1.

[0034] Example 3: High-dose experiment with taurine

[0035] In this embodiment, a dose of 120 μg / kg of taurine was used to verify the effect of dosage on the severity of the model.

[0036] Twenty healthy male C57BL / 6 mice aged 7 weeks (weighing 20-22g) were selected and acclimatized for one week. A working solution of 18 μg / mL limonin was administered intraperitoneally every 1 hour at a dose of 120 μg / kg for 7 consecutive injections. One and a half hours after the 7th limonin injection, an additional intraperitoneal injection of 12 mg / kg lipopolysaccharide was administered. The mice were fasted for 12 hours after the last administration. Samples were collected 24 hours after the first limonin injection, following the same procedure as in Example 1. The remaining mice were observed for 7 days, and mortality and weight changes were recorded.

[0037] Example 4: Optimization Experiment of Lipopolysaccharide Dosage

[0038] This embodiment explores the effect of lipopolysaccharide dosage on model construction, using a dosage of 10 mg / kg, with other conditions being the same as in Example 1.

[0039] Twenty healthy male C57BL / 6 mice aged 6 to 8 weeks were selected and acclimatized for one week before model induction. A working solution of 15 μg / mL of lindane was administered intraperitoneally every 1 hour at a dose of 100 μg / kg for 7 consecutive injections. A working solution of 1.5 mg / mL of lipopolysaccharide was administered intraperitoneally at a dose of 10 mg / kg 1.5 hours after the 7th lindane injection. The remaining procedures were the same as in Example 1.

[0040] Example 5: Validation Experiment of Lipopolysaccharide Injection Time Window

[0041] This embodiment verifies the effect of lipopolysaccharide injection time on model construction. Lipopolysaccharide was added immediately after the 7th injection of scutellarin.

[0042] Twenty healthy male C57BL / 6 mice aged 7 weeks were selected and acclimatized for one week. A working solution of 15 μg / mL of lindane was administered intraperitoneally every 1 hour at a dose of 100 μg / kg for 7 consecutive injections. Immediately after the 7th lindane injection, lipopolysaccharide was administered intraperitoneally at a dose of 12 mg / kg. Patients were fasted for 12 hours after the last administration. Sample collection and preservation methods were the same as in Example 1.

[0043] Example 6: Construction of mouse models at different ages

[0044] This embodiment verifies the effect of mouse age on model construction, using 8-week-old mice.

[0045] Twenty healthy male C57BL / 6 mice, aged 8 weeks and weighing between 22g and 24g, were selected and acclimatized for one week. The concentrations, injection doses, and time intervals of the linalool and lipopolysaccharide were the same as in Example 1. Samples were collected 24 hours after the first linalool injection, and the remaining mice were observed for 7 days.

[0046] Comparative Example 1: Rain leptin-induced model

[0047] This comparative study used simple lipolytic acid induction without the addition of lipopolysaccharide (LPS) to verify the key role of LPS in constructing a severe acute pancreatitis model.

[0048] Twenty healthy male C57BL / 6 mice aged 6 to 8 weeks were selected and acclimatized for one week. A working solution of lipopolysaccharide (LPS) at a concentration of 15 μg / mL was administered intraperitoneally every 1 hour at a dose of 100 μg / kg for seven consecutive injections. After the seventh injection, no additional LPS was administered; only an equal volume of physiological saline was injected as a placebo. Patients were fasted for 12 hours after the last administration. Sample collection and preservation methods were the same as in Example 1.

[0049] Comparative Example 2: Low-dose taurine model

[0050] This comparative study used a low dose of 50 μg / kg of hymenoplasmin, combined with 12 mg / kg of lipopolysaccharide, to verify the importance of hymenoplasmin dosage for model construction.

[0051] Twenty healthy male C57BL / 6 mice aged 6 to 8 weeks were selected and acclimatized for one week. A working solution of lipopolysaccharide (LPS) at a concentration of 7.5 μg / mL was prepared and administered intraperitoneally every 1 hour at a dose of 50 μg / kg for 7 consecutive injections. One and a half hours after the seventh LPS injection, an additional intraperitoneal injection of lipopolysaccharide (LPS) at 12 mg / kg was administered. The remaining procedures were the same as in Example 1.

[0052] Comparative Example 3: Model with Reduced Number of Rain Frog Injections

[0053] In this comparative study, 100 μg / kg of taurine was used, but the number of injections was reduced to 5 to verify the effect of the number of injections on model construction.

[0054] Twenty healthy male C57BL / 6 mice aged 6 to 8 weeks were selected and acclimatized for one week. A working solution of 15 μg / mL limonin was prepared and administered intraperitoneally every 1 hour at a dose of 100 μg / kg, for a total of 5 injections. One and a half hours after the fifth limonin injection, an additional intraperitoneal injection of 12 mg / kg lipopolysaccharide was administered. Sample collection and preservation methods were the same as in Example 1.

[0055] Comparative Example 4: Low-dose lipopolysaccharide model

[0056] This comparative study used 100 μg / kg of taurine combined with a low dose of lipopolysaccharide (LPS) of 5 mg / kg to verify the effect of LPS dosage on the severity of the model.

[0057] Twenty healthy male C57BL / 6 mice aged 6 to 8 weeks were selected and acclimatized for one week. A working solution of 15 μg / mL of lycopoietin was administered intraperitoneally every 1 hour at a dose of 100 μg / kg for 7 consecutive injections. A working solution of 0.75 mg / mL of lipopolysaccharide was administered intraperitoneally at a dose of 5 mg / kg 1.5 hours after the 7th lycopoietin injection. The remaining procedures were the same as in Example 1.

[0058] Comparative Example 5: Lipopolysaccharide Injection Time Delay Model

[0059] This comparative study verified the importance of the lipopolysaccharide injection time window, and lipopolysaccharide was added 4 hours after the 7th injection of lecithin.

[0060] Twenty healthy male C57BL / 6 mice aged 6 to 8 weeks were selected and acclimatized for one week. A working solution of 15 μg / mL of limonin was administered intraperitoneally every 1 hour at a dose of 100 μg / kg for 7 consecutive injections. Four hours after the 7th limonin injection, lipopolysaccharide (LPS) was administered intraperitoneally at a dose of 12 mg / kg. Sample collection and preservation methods were the same as in Example 1.

[0061] To comprehensively evaluate the severe acute pancreatitis mouse model constructed in this invention, the following quantitative evaluation system was established:

[0062] I. Mortality Assessment

[0063] Record mouse mortality during the one-week observation period and calculate the mortality rate. Mortality rate = (number of dead mice / total number of mice) × 100%. The mortality rate of the severe acute pancreatitis model should reach more than 30% to meet the characteristics of clinical severe acute pancreatitis.

[0064] II. Weight Change Assessment

[0065] Mice were weighed before and 24 hours after modeling, and the rate of weight change was calculated. The rate of weight change was calculated as: (Post-modeling weight - Pre-modeling weight) / Pre-modeling weight × 100%. The weight of mice in the severe acute pancreatitis model should decrease by more than 10%.

[0066] III. Histopathological evaluation of pancreas

[0067] Pancreatic tissue was fixed in 4% paraformaldehyde for 24 hours, then dehydrated using a routine gradient ethanol process, cleared in xylene, and embedded in paraffin. 5 μm thick tissue sections were prepared using a rotary microtome and stained with hematoxylin and eosin (HE). The staining steps included: dewaxing in xylene, hydration with a gradient ethanol process, hematoxylin staining for 5 min, differentiation with 0.5% hydrochloric acid ethanol, eosin staining for 2 min, dehydration with a gradient ethanol process, clearing in xylene, and mounting with neutral resin.

[0068] The Schmidt scoring system was used to quantitatively score pancreatic histopathology. This scoring system includes five aspects: edema score from 0 to 3, where 0 indicates no edema, 1 indicates widening of the interlobular septa, 2 indicates significant widening of the interlobular septa, and 3 indicates complete separation of the pancreatic lobules. Inflammatory cell infiltration score from 0 to 3, where 0 indicates no obvious infiltration, 1 indicates slight infiltration, 2 indicates moderate infiltration, and 3 indicates significant infiltration. Acinar necrosis score from 0 to 3, where 0 indicates no necrosis, 1 indicates acinar necrosis area less than or equal to 10%, 2 indicates acinar necrosis area of ​​11% to 30%, and 3 indicates acinar necrosis area greater than 30%. Hemorrhage score from 0 to 3, where 0 indicates no hemorrhage, 1 indicates mild hemorrhage, 2 indicates moderate hemorrhage, and 3 indicates severe hemorrhage. Fat necrosis is scored from 0 to 3, where 0 indicates no fat necrosis, 1 indicates mild fat necrosis, 2 indicates moderate fat necrosis, and 3 indicates severe fat necrosis. The sum of the five scores constitutes the pathological score of pancreatic tissue, with a maximum score of 15 points. The score for the severe acute pancreatitis model group should reach 6 points or higher.

[0069] IV. Ascites volume assessment

[0070] After laparotomy, sterile gauze, pre-weighed on a precision balance, is used to aspirate the ascites. Care is taken to avoid contact with other tissues or blood during aspiration. The gauze is weighed again after aspirating the fluid. Based on the approximate density of water (1 g / mL), the ascites volume is calculated. The ascites volume in the severe acute pancreatitis model group should reach at least 0.5 mL.

[0071] V. Assessment of intestinal edema

[0072] Small intestinal tissue was fixed in 4% paraformaldehyde for 24 hours, followed by routine paraffin embedding, sectioning, and HE staining, using the same staining method as pancreatic tissue. The degree of intestinal wall edema was observed and scored under a microscope. The scoring criteria for intestinal edema were as follows: 0 points indicated no edema and normal intestinal wall structure; 1 point indicated mild edema with slight thickening of the intestinal wall; 2 points indicated moderate edema with significant thickening of the intestinal wall and submucosal edema; and 3 points indicated severe edema with significant thickening of the intestinal wall and obvious edema in both the submucosal and muscular layers. The intestinal edema score in the severe acute pancreatitis model group should reach 2 points or higher.

[0073] VI. Serum enzyme activity assay

[0074] After thawing, serum samples were analyzed using a fully automated biochemical analyzer to determine serum amylase and lipase activities. Amylase was measured using a continuous monitoring method, utilizing the hydrolysis of 4-nitrophenylmaltose by amylase to produce 4-nitrophenol. The absorbance change was measured at 405 nm, and enzyme activity was calculated based on a standard curve, expressed in U / dL. Lipase was measured using a colorimetric method, utilizing the hydrolysis of triglycerides by lipase to release fatty acids, which react with an indicator to produce a color change. The absorbance was measured at 540 nm, and enzyme activity was calculated based on a standard curve, expressed in U / L. Serum amylase and lipase activities in the severe acute pancreatitis model group should be more than three times higher than those in the control group.

[0075] VII. Detection of Inflammatory Factor Levels

[0076] After thawing, the concentrations of TNF-α, IL-1β, and IL-6 in serum were detected using ELISA. The specific procedures were as follows: A 96-well plate coated with specific antibodies was washed three times with washing buffer. 100 μL of diluted serum sample or standard was added to each well, and the plate was incubated at 37°C for 2 hours. After washing, 100 μL of biotinylated detection antibody was added to each well, and the plate was incubated at 37°C for 1 hour. After washing again, 100 μL of horseradish peroxidase-labeled streptavidin was added to each well, and the plate was incubated at 37°C for 30 minutes. After washing, 100 μL of TMB substrate chromogenic solution was added to each well, and the plate was incubated at room temperature in the dark for 15 minutes. The reaction was terminated by adding 50 μL of stop solution to each well, and the absorbance was immediately measured at 450 nm. The concentrations of inflammatory factors in the samples were calculated based on the standard curve, expressed in pg / mL. The levels of inflammatory factors in the severe acute pancreatitis model group should be more than twice that of the control group.

[0077] Table 1 shows the results of the main evaluation metrics for each embodiment and comparative example.

[0078] Table 1 Comparison of key evaluation metrics for each embodiment and comparative example

[0079] Group mortality rate(%) Weight change rate (%) Schmidt score (points) Ascites volume (mL) Intestinal edema score (points) Amylase activity (fold change) Lipase activity (fold change) TNF-α (multiple) IL-1β (multiple dose) IL-6 (multiple) control group 0 +2.3±0.5 1.3±0.4 0.05±0.01 0.2±0.1 1 1 1 1 1 Example 1 30 -14.5±1.7 8.4±0.7 0.74±0.09 2.6±0.5 4.8±0.4 5.8±0.8 3.2±0.5 2.9±0.4 4.2±0.8 Example 2 25 -12.8±1.5 7.2±0.8 0.62±0.08 2.3±0.5 4.2±0.5 5.1±0.7 2.8±0.4 2.6±0.3 3.7±0.7 Example 3 35 -16.2±1.9 9.1±0.8 0.85±0.10 2.8±0.4 5.3±0.5 6.2±0.9 3.6±0.6 3.2±0.5 4.7±0.9 Example 4 25 -12.5±1.6 7.5±0.7 0.65±0.07 2.4±0.5 4.4±0.4 5.3±0.7 2.9±0.4 2.7±0.3 3.9±0.7 Example 5 32 -15.1±1.8 8.6±0.7 0.77±0.09 2.7±0.5 4.9±0.4 5.9±0.8 3.3±0.5 3.0±0.4 4.3±0.8 Example 6 28 -13.9±1.6 8.2±0.7 0.72±0.08 2.5±0.5 4.7±0.4 5.7±0.8 3.1±0.5 2.8±0.4 4.1±0.8 Comparative Example 1 5 -6.2±1.2 4.3±0.6 0.18±0.05 1.1±0.4 2.8±0.3 3.2±0.5 1.6±0.3 1.5±0.2 2.1±0.4 Comparative Example 2 15 -8.5±1.3 5.8±0.7 0.35±0.06 1.7±0.4 3.5±0.4 4.1±0.6 2.2±0.4 2.0±0.3 2.8±0.5 Comparative Example 3 20 -10.3±1.4 6.5±0.7 0.48±0.07 2.0±0.5 3.9±0.4 4.6±0.6 2.6±0.4 2.3±0.3 3.3±0.6 Comparative Example 4 22 -11.2±1.5 6.9±0.7 0.52±0.07 2.1±0.5 4.0±0.4 4.8±0.7 2.7±0.4 2.4±0.3 3.5±0.6 Comparative Example 5 26 -12.8±1.6 7.6±0.7 0.63±0.08 2.3±0.5 4.3±0.4 5.2±0.7 2.9±0.4 2.6±0.3 3.8±0.7

[0080] Note: Amylase activity, lipase activity, and inflammatory factor levels are expressed as multiples of the control group.

[0081] As shown in Table 1, the models constructed in Examples 1 to 6 all met the criteria for severe acute pancreatitis, with a mortality rate between 25% and 35%, a body weight loss of 12.5% ​​to 16.2%, a Schmidt score between 7.2 and 9.1, an ascites volume between 0.62 mL and 0.85 mL, an intestinal edema score between 2.3 and 2.8, and serum amylase and lipase activities more than 4 times higher than the control group, and inflammatory factor levels more than 2 times higher than the control group. Among them, the parameter combination used in Example 1 showed the most stable performance and balanced indicators, making it the optimal scheme for constructing a mouse model of severe acute pancreatitis.

[0082] Comparative Example 1, using only lipopolysaccharide (LPS) for induction without additional LPS, showed a mortality rate of only 5%, a body weight loss of 6.2%, a Schmidt score of 4.3, and an ascites volume of 0.18 mL. None of these indicators met the criteria for severe acute pancreatitis, demonstrating the necessity of LPS in constructing a severe acute pancreatitis model. The local pancreatic damage induced by lipopolysaccharide requires a synergistic effect from the systemic inflammatory response induced by LPS to successfully construct a severe acute pancreatitis model.

[0083] Comparative Example 2 used a low dose of violarin (50 μg / kg), which, although combined with lipopolysaccharide, resulted in a mortality rate of only 15%. While various indicators improved, they still did not meet the criteria for severe illness. This indicates that insufficient violarin dosage, even when combined with lipopolysaccharide, is insufficient to induce typical severe acute pancreatitis. Violarin stimulates excessive secretion from pancreatic acinar cells through cholecystokinin receptors. When the dose is too low, the degree of local pancreatic damage is insufficient, providing a weak foundation for subsequent systemic inflammatory responses.

[0084] Comparative Example 3 reduced the number of pancreatic acin injections to 5, with a mortality rate of 20%. While various indicators improved, the disease did not fully meet the criteria for severe illness, indicating that the number of injections plays a crucial role in the sustained stimulation of pancreatic acinar cells and the cumulative degree of damage. Seven consecutive injections maintained high levels of pancreatic enzyme secretion and intracellular damage over a longer time window, laying the foundation for the development of a severe illness model.

[0085] Comparative Example 4, using a low dose of lipopolysaccharide (LPS) of 5 mg / kg, had a mortality rate of 22%. While all indicators were close to, but not fully met, the criteria for severe illness, indicating that LPS dosage significantly affects the intensity of the systemic inflammatory response. LPS activates monocytes to release cytokines; insufficient doses result in a weaker systemic inflammatory response, making it difficult to achieve a sufficient synergistic effect with local pancreatic damage.

[0086] In Comparative Example 5, the mortality rate was 26% when lipopolysaccharide (LPS) injection was delayed until 4 hours after the 7th injection of spiramycin. Although this was close to the severe case standard, it was still slightly lower than in Example 1, indicating that the timing of LPS injection has a certain impact on model construction. Administering LPS within 2 hours after the 7th spiramycin injection, when the pancreas has already suffered initial damage but has not yet entered the repair phase, is the optimal time to trigger a systemic inflammatory response and amplify disease severity. Injecting LPS too late may miss the optimal intervention window, weakening the synergistic effect.

[0087] To verify the stability and reproducibility of the model of this invention, three independent batches of experiments were conducted according to the method in Example 1, with each batch including 20 mice in the control group and 20 mice in the model group. Table 2 shows the results of the main evaluation indicators of the three batches of experiments.

[0088] Table 2 Comparison of main evaluation indicators for the three batches of experiments

[0089] batch mortality rate(%) Weight loss rate (%) Schmidt score (points) Ascites volume (mL) Intestinal edema score (points) Amylase activity (fold change) Lipase activity (fold change) TNF-α (multiple) IL-1β (multiple dose) IL-6 (multiple) Batch 1 30 14.39±1.70 8.4±0.7 0.74±0.09 2.6±0.52 4.8±0.4 5.8±0.8 3.2±0.5 2.9±0.4 4.2±0.8 Batch 2 28 14.63±1.45 8.8±0.63 0.68±0.08 2.5±0.53 4.5±0.3 5.0±0.6 3.6±0.7 3.0±0.5 4.0±0.9 Batch 3 30 14.86±1.43 8.1±0.57 0.79±0.10 2.5±0.53 5.2±0.5 5.6±0.8 3.0±0.3 2.7±0.3 4.6±1.1 average value 29.3±1.2 14.63±0.24 8.43±0.35 0.74±0.06 2.53±0.06 4.83±0.35 5.47±0.42 3.27±0.31 2.87±0.15 4.27±0.31 inter-batch coefficient of variation (%) 3.9 1.6 4.2 7.4 2.3 7.2 7.7 9.5 5.2 7.3

[0090] As shown in Table 2, the evaluation indicators of the three batches of experiments were highly consistent, with inter-batch coefficients of variation all less than 10%, and most indicators having coefficients of variation within 5%. The coefficient of variation for mortality was 3.9%, for weight loss rate it was 1.6%, and for Schmidt score it was 4.2%, fully demonstrating the stability and reproducibility of the model of this invention. Figures 3 to 12 As shown, there were significant differences in lipase activity, IL-1β level, IL-6 level, TNF-α level, pathological score, intestinal edema score, amylase activity, ascites volume and weight change between the control group and the model group in the three batches, and the differences among the three batches were very small, which further verified the stability of the model.

[0091] To verify the practicality of the model of this invention in drug screening, dexamethasone, known for its anti-inflammatory effects, was selected for validation experiments. Dexamethasone is a glucocorticoid that exerts its anti-inflammatory effect by inhibiting the release of inflammatory mediators and the activation of inflammatory cells.

[0092] The experiment was divided into three groups, with 20 mice in each group. The control group received an equal volume of physiological saline as described in Example 1. The model group developed a severe acute pancreatitis model as described in Example 1. The treatment group developed a severe acute pancreatitis model as described in Example 1, and received an intraperitoneal injection of dexamethasone at a dose of 5 mg / kg 30 minutes before the first injection of spirulina extract. Dexamethasone was purchased from Sigma (product number D4902) and prepared as a working solution using physiological saline. The observation period was 14 days, during which mortality, weight changes, and various pathophysiological indicators were recorded.

[0093] Table 3 shows the results of the efficacy evaluation experiment.

[0094] Table 3. Results of Dexamethasone Efficacy Evaluation

[0095] Group mortality rate(%) Weight loss rate (%) Schmidt score (points) Ascites volume (mL) Intestinal edema score (points) Amylase activity (fold change) Lipase activity (fold change) TNF-α (multiple) IL-1β (multiple) IL-6 (multiple) control group 0 +2.3±0.5 1.3±0.4 0.05±0.01 0.2±0.1 1 1 1 1 1 Model group 30 -15.5±2.2 8.3±0.7 0.72±0.07 2.5±0.5 4.7±0.4 5.6±0.7 3.1±0.4 2.8±0.3 4.1±0.7 Treatment group 10** -8.3±1.5** 4.8±0.8*** 0.36±0.10*** 1.4±0.5*** 2.2±0.3** 2.5±0.4** 1.6±0.2** 1.4±0.2** 2.0±0.3**

[0096] Note: ** indicates P<0.01, *** indicates P<0.001, compared with the model group.

[0097] As shown in Table 3, dexamethasone treatment significantly improved various indicators in the severe acute pancreatitis model. The mortality rate in the treatment group decreased to 10%, significantly lower than the 30% in the model group. The weight loss rate decreased from 15.5% to 8.3%, the Schmidt score decreased from 8.3 to 4.8, the ascites volume decreased from 0.72 mL to 0.36 mL, and the intestinal edema score decreased from 2.5 to 1.4. Serum amylase and lipase activities decreased to 2.2 times and 2.5 times that of the control group, respectively, significantly lower than those in the model group. The levels of inflammatory factors TNF-α, IL-1β, and IL-6 also decreased significantly, being 1.6 times, 1.4 times, and 2.0 times that of the control group, respectively. These results fully demonstrate that the severe acute pancreatitis model constructed in this invention has good drug evaluation capabilities and can sensitively reflect the therapeutic effects of drugs.

[0098] The severe acute pancreatitis model constructed in this invention successfully replicates the pathophysiological characteristics of clinical severe acute pancreatitis through the synergistic effect of taurine and lipopolysaccharide. Its mechanism can be explained from the following aspects.

[0099] First, vitexin mediates local pancreatic damage via cholecystokinin receptors. Vitexin is a structural analog of cholecystokinin and can bind to cholecystokinin receptors on the surface of pancreatic acinar cells, activating the phospholipase C-protein kinase C signaling pathway, leading to increased intracellular calcium ion concentration. Sustained calcium signaling stimulation induces excessive secretion of digestive enzymes by pancreatic acinar cells, including trypsinogen, amylase, and lipase. Simultaneously, the high-calcium environment impairs the normal separation mechanism between trypsinogen and lysosomal hydrolases within the cell, leading to abnormal activation of trypsinogen by cathepsin B, forming active trypsin. Activated trypsin can not only further activate other zymogens, forming a cascade of zymogen activation, but also directly damage pancreatic acinar cells, causing cell necrosis and apoptosis. This invention uses a vitexin dose of 100 μg / kg, injected every 1 hour for 7 consecutive injections, continuously stimulating pancreatic acinar cells over a 6-hour time window, cumulatively resulting in significant local pancreatic damage.

[0100] Secondly, lipopolysaccharide (LPS) triggers a systemic inflammatory response. LPS is a major component of the cell wall of Gram-negative bacteria and a potent endotoxin. By binding to Toll-like receptor 4, LPS activates the myeloid differentiation factor 88-dependent signaling pathway, initiating the nuclear factor κB and mitogen-activated protein kinase signaling cascade, inducing the massive release of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6. These inflammatory factors diffuse throughout the body via the bloodstream, triggering systemic inflammatory response syndrome. In this invention, an additional 12 mg / kg of LPS was administered within 2 hours after the 7th injection of lindane. At this point, preliminary damage had already occurred in the pancreas, and an inflammatory microenvironment had been established. The addition of LPS at the optimal time amplified the inflammatory response, causing the local inflammation to develop into a severe systemic inflammatory response.

[0101] Third, the synergistic effect of local pancreatic injury and systemic inflammatory response. Lepidosin-induced local pancreatic injury releases a large number of injury-related molecular patterns, including high-mobility group box 1 (HMP-B1), heat shock proteins, and nucleic acid fragments. These molecules can activate the innate immune system, recruiting neutrophils and macrophages to infiltrate pancreatic tissue. The lipopolysaccharide-induced systemic inflammatory response further enhances the activation of immune cells, releasing more inflammatory mediators and reactive oxygen species, exacerbating pancreatic tissue damage. Simultaneously, the systemic inflammatory response leads to vascular endothelial cell damage, increasing vascular permeability and causing tissue edema and ascites formation. Inflammatory mediators can also affect intestinal barrier function, leading to intestinal edema and increased permeability, further aggravating the systemic inflammatory response and forming a vicious cycle. This mutually promoting and synergistic effect between local pancreatic injury and systemic inflammatory response is the core mechanism by which this invention successfully constructs a severe acute pancreatitis model.

[0102] Fourth, the development of multiple organ dysfunction (MOD). One of the characteristics of severe acute pancreatitis is MOD, primarily affecting the lungs, kidneys, liver, and cardiovascular system. The model constructed in this invention successfully replicates this characteristic, particularly acute pancreatitis-related lung injury. Inflammatory mediators reach the lungs via the bloodstream, activating alveolar macrophages and endothelial cells, releasing inflammatory factors, leading to damage to the alveolar-capillary barrier, alveolar exudation and edema, and impaired gas exchange. Intestinal edema and impaired barrier function may lead to intestinal endotoxemia, further exacerbating the systemic inflammatory response. These multiple organ injuries collectively result in high mortality, significant weight loss, and severe clinical manifestations in the model mice.

[0103] Fifth, the biological significance of the assessment indicators. The seven-dimensional assessment system established in this invention can comprehensively reflect the pathophysiological characteristics of severe acute pancreatitis. Mortality rate is the most direct indicator of disease severity, consistent with the high mortality rate of severe acute pancreatitis in clinical practice. Weight loss reflects the impact of the disease on the overall state of the body, including dehydration, malnutrition, and metabolic disorders. The Schmidt pathological score quantifies pancreatic tissue damage from five dimensions: edema, inflammatory cell infiltration, acinar necrosis, hemorrhage, and fat necrosis, accurately reflecting the severity of pancreatic pathological changes. Ascites volume reflects the degree of intra-abdominal inflammatory exudation and increased vascular permeability. Intestinal edema score reflects the impact of pancreatitis on the intestines, consistent with common intestinal complications in patients with pancreatitis in clinical practice. Serum amylase and lipase activities are biochemical markers of pancreatic exocrine function and pancreatic damage; their significant elevation reflects the destruction of pancreatic acinar cells and the large release of digestive enzymes. Inflammatory factor levels directly reflect the intensity of the systemic inflammatory response and are closely related to the severity of the disease.

[0104] In summary, this invention successfully constructed a stable and reproducible mouse model of severe acute pancreatitis by precisely controlling the dosage, injection frequency, and time interval of linalool and lipopolysaccharide. This model accurately replicates the pathophysiological characteristics of clinical severe acute pancreatitis, establishes a comprehensive quantitative evaluation system, and provides a reliable experimental tool for studying the pathological mechanisms of pancreatitis and evaluating novel treatment regimens.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A stable and highly reproducible method for constructing a mouse model of severe acute pancreatitis, characterized in that... This includes the following steps: Male C57BL / 6 mice aged 6 to 8 weeks were selected and acclimatized for one week before model induction. The treatment involved intraperitoneal injection of 100 μg / kg of thymol, administered once every 1 hour for a total of 7 injections. Within 2 hours after the 7th injection of spirulina extract, lipopolysaccharide was administered intraperitoneally at a dose of 12 mg / kg. Pathological and physiological indicators of mice were evaluated 24 hours after the first injection of taurine.

2. The method according to claim 1, characterized in that... The concentration of the taurine was 15 μg / mL, and 0.9% sodium chloride solution was used as a diluent.

3. The method according to claim 1, characterized in that... The concentration of the lipopolysaccharide was 2 mg / mL, and 0.9% sodium chloride solution was used as a diluent.

4. The method according to claim 1, characterized in that... The mice were fasted for 12 hours after the last administration, but had free access to water during that time.

5. The method according to claim 1, characterized in that... The model exhibits at least five of the following characteristics: The mortality rate reached over 30% within a week; Weight loss of more than 10%; A Schmidt pathological score of 6 or higher for pancreatic tissue; The volume of ascites reaches 0.5 mL or more; An intestinal edema score of 2 or higher; Serum amylase activity was more than 3 times higher than that of the control group; Serum lipase activity was more than 3 times higher than that of the control group; Serum inflammatory factor levels were more than twice that of the control group.

6. A quantitative evaluation system for a mouse model of severe acute pancreatitis constructed by the method according to any one of claims 1 to 5, characterized in that... The evaluation indicators include the following: Mortality assessment: Survival status of mice within one week after the first dose was recorded; Weight change assessment, calculating the percentage change in weight before and after modeling; Histopathological evaluation of pancreatic tissue was performed using the Schmidt scoring system to quantify edema, inflammatory cell infiltration, acinar necrosis, hemorrhage, and fat necrosis in HE-stained sections. Ascites volume assessment, measuring the volume of ascites; Intestinal edema assessment: The degree of intestinal wall edema was scored on HE-stained intestinal tissue sections; Serum enzyme activity assessment, including determination of serum amylase and lipase activities; The levels of inflammatory factors were assessed, and the concentrations of TNF-α, IL-1β, and IL-6 in serum were quantitatively detected.

7. The evaluation system according to claim 6, characterized in that... The scoring criteria of the Schmidt scoring system are as follows: Edema was scored from 0 to 3, where 0 indicated no edema, 1 indicated widening of the interlobular septa of the pancreas, 2 indicated significant widening of the interlobular septa of the pancreas, and 3 indicated complete separation of the pancreatic lobules. The inflammatory cell infiltration score ranged from 0 to 3, where 0 indicated no obvious infiltration, 1 indicated a small amount of infiltration, 2 indicated moderate infiltration, and 3 indicated a large amount of infiltration. Acinar necrosis is scored from 0 to 3, where 0 indicates no necrosis, 1 indicates a necrosis area of ​​less than or equal to 10%, 2 indicates a necrosis area of ​​11% to 30%, and 3 indicates a necrosis area of ​​more than 30%. Bleeding scores range from 0 to 3, where 0 indicates no bleeding, 1 indicates mild bleeding, 2 indicates moderate bleeding, and 3 indicates severe bleeding. Fat necrosis is scored from 0 to 3, where 0 indicates no fat necrosis, 1 indicates mild fat necrosis, 2 indicates moderate fat necrosis, and 3 indicates severe fat necrosis.

8. The evaluation system according to claim 6, characterized in that... The intestinal edema scoring criteria are as follows: 0 points indicate no edema, 1 point indicates mild edema, 2 points indicate moderate edema, and 3 points indicate severe edema.

9. The evaluation system according to any one of claims 6 to 8, characterized in that... When evaluating multiple batches of experiments using the evaluation system, the batch-to-batch fluctuation range of each evaluation indicator is controlled within ±5%.

10. The application of the mouse model of severe acute pancreatitis constructed by the method according to any one of claims 1 to 5 in the study of the pathological mechanism of severe acute pancreatitis or the evaluation of drug efficacy.

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