A method for constructing a mouse model of drug-induced acute rhabdomyolysis syndrome

Abstract

The application discloses a method for constructing a mouse model of drug-induced acute rhabdomyolysis syndrome, which comprises the following steps: injecting linoleic acid into the abdominal cavity of a mouse, and obtaining the mouse model of drug-induced acute rhabdomyolysis syndrome after 12-24 hours. The method uses single intraperitoneal injection of linoleic acid to construct the mouse model of acute rhabdomyolysis syndrome, the model is single given to an animal, and the model can be successfully constructed within 12-24 hours. The animal model has mouse striated muscle injury as a main poisoning feature, is accompanied by kidney injury and mild to moderate liver injury, is similar to the toxicological characteristics of actual human acute rhabdomyolysis syndrome, has a similar incidence rule, and the mouse does not die. The animal model can be used for screening drugs for preventing or treating drug-induced acute rhabdomyolysis syndrome.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and basic medicine, and relates to the construction of animal models of diseases, particularly a method for constructing a mouse model of drug-induced acute rhabdomyolysis syndrome. Background Technology

[0002] Rhabdomyolysis is a common clinical muscle injury disorder characterized by damage to skeletal muscle, cardiac muscle, and other striated muscle tissues caused by drugs or external forces. It typically presents with severe muscle pain as the initial symptom, accompanied by mild to moderate fever, vomiting, diarrhea, and other gastrointestinal symptoms, as well as liver damage. After muscle cells are damaged, creatine kinase, myoglobin, and other substances are released into the circulation, leading to a sharp increase in serum CK levels, which can serve as a diagnostic indicator for acute rhabdomyolysis. Simultaneously, renal filtration is impaired, resulting in coffee-ground urine; in severe cases, it can lead to kidney failure or death (Vanholder R, SU M, Erek E, et al., 2000). The causes of rhabdomyolysis generally include mechanical injury and drug-induced rhabdomyolysis. Mechanically induced rhabdomyolysis is usually caused by excessive exercise or prolonged compression, while drug-induced rhabdomyolysis may be caused by the ingestion of multiple drugs or toxins. Currently, drugs reported to potentially cause rhabdomyolysis include alcohol, opioids, statins, macrolides, cyclosporine, and fibrates, while toxins include snake venom and insect venom (Vanholder R, SU M, Erek E, et al., 2000). Haff disease, caused by consuming crayfish and other aquatic products, is also an acute rhabdomyolysis syndrome caused by an unknown toxin. The mechanisms of drug-induced rhabdomyolysis include molecular processes such as myocyte calcium overload, excessive intracellular ATP consumption, and mitochondrial damage. Currently, among all clinically used drugs, only statins have a clearly defined proportion of patients experiencing rhabdomyolysis as a side effect. Approximately 0.2% of patients experience varying degrees of myalgia or even muscle damage within 1 week to 3 months of taking statins. All other drugs or toxins that may cause rhabdomyolysis have been reported as incidental adverse reactions, and their mechanisms of action remain to be explored (Schreiber DH, Anderson TR., 2006).

[0003] Animal models are essential tools for the prevention, treatment, and mechanistic study of rhabdomyolysis. Injecting glycerol into rat skeletal muscle directly damages muscle tissue, creating a mechanically induced rhabdomyolysis model. However, due to the unclear mechanism of drug-induced rhabdomyolysis and the lack of candidate drugs with well-defined toxicity, drug-induced rhabdomyolysis animal models have been scarce. Until 2018, Japanese researchers Tsuyoshi et al. (2018) injected high doses of atorvastatin, levofloxacin, and L-butyrosine-sulfonylamine into C57BL / 6J mice. Four days later, the mice exhibited biochemical indicators and pathological changes corresponding to rhabdomyolysis, thus establishing the first mouse model of drug-induced rhabdomyolysis. In 2019, Tsuyoshi et al. (2019) further improved the model using lovastatin, gemfibrozil, and L-butyrosine-sulfonylamine, shortening the onset time to as little as 3 days. They also studied changes in plasma small RNA, oxidative stress, and apoptosis-related indicators in the model animals.

[0004] The drugs used in the aforementioned animal models were statins, quinolone antibiotics, insulin sensitizers, and GSH inhibitors, respectively. The dosage of each drug used in the modeling was far higher than the clinical human dosage, resulting in a poor correlation between the model and actual drug-induced rhabdomyolysis. Furthermore, Tsuyoshi et al. employed a multiple-dose strategy in constructing their animal model, requiring at least two days for the onset of symptoms. However, actual acute rhabdomyolysis syndromes, such as Haff disease and snake venom poisoning, typically develop within hours (6-24 hours) of toxin ingestion, clearly inconsistent with Tsuyoshi et al.'s model. Currently, no animal models of acute rhabdomyolysis syndrome with a single-dose administration and onset within hours have been reported. Summary of the Invention

[0005] Objective of the Invention: The first objective of this invention is to provide a method for successfully constructing a mouse model of drug-induced acute rhabdomyolysis within 24 hours through a single injection of linoleic acid, wherein the mouse model constructed by this method does not exhibit other unrelated pharmacological or toxicological effects. The second objective of this invention is to provide an application for the mouse model constructed by the above method.

[0006] Technical solution: The method for constructing a mouse model of drug-induced acute rhabdomyolysis syndrome according to the present invention includes the following steps: injecting linoleic acid into mice intraperitoneally, and obtaining a mouse model of drug-induced acute rhabdomyolysis syndrome 12 to 24 hours later.

[0007] The injection volume of linoleic acid is 30–50 μL.

[0008] The linoleic acid is a linoleic acid-Tween 80 suspension.

[0009] The linoleic acid-Tween 80 suspension is prepared by dissolving Tween 80 in physiological saline, with a volume fraction of 1%, and then mixing it with linoleic acid.

[0010] The mice in question are female C57BL / 6J mice.

[0011] The mice were 6-8 weeks old and weighed 16-20g.

[0012] The mice were acclimatized for 3 days before intraperitoneal injection, and were fasted but allowed free access to water for 12 hours before administration.

[0013] The adaptive feeding conditions are a rearing environment temperature of 20–26°C and a relative humidity of 60%–70%.

[0014] The application of any of the above-described methods for creating a mouse drug-induced acute rhabdomyolysis syndrome model in the field of animal model construction.

[0015] The application of the mouse drug-induced acute rhabdomyolysis syndrome model constructed by any of the above-described methods of the present invention in screening drugs for the prevention or treatment of drug-induced acute rhabdomyolysis syndrome.

[0016] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: The method of this invention establishes a mouse model of acute rhabdomyolysis syndrome by using a single intraperitoneal injection of linoleic acid. This model involves a single administration of a single drug to the animal, and the model can be successfully established within 12-24 hours. This animal model is characterized by rhabdomyolysis in mice as the main toxicological feature, accompanied by kidney damage and mild to moderate liver damage. It is similar in toxicological characteristics and pathogenesis to actual human acute rhabdomyolysis syndrome, and no mice die. This method can be used to screen drugs for the prevention or treatment of drug-induced acute rhabdomyolysis syndrome. Attached Figure Description

[0017] Figure 1 Bar chart showing serum biochemical changes in mice in the model group and control group. *p<0.05, **p<0.01.

[0018] Figure 2 HE staining images of skeletal muscle tissue from model group and control group mice (×400).

[0019] Figure 3 Bar chart showing changes in serum CK levels in model mice at different modeling times, *p<0.05, **p<0.01. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0021] Example 1: Animal Model Construction

[0022] 1. Experimental Methods and Materials

[0023] 1.1 Laboratory Animals

[0024] Healthy female C57BL / 6J mice, 6 weeks old and weighing 16–20 g, were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. (Experimental Animal Production License No.: SCXK(Su)2023-0009). They were fed with standard mouse feed at an ambient temperature of 25℃ and a relative humidity of 60%–70%, with a 12-hour diurnal cycle.

[0025] 1.2 Main reagents for the experiment

[0026] Serum creatine kinase (CK), creatine kinase isoenzymes (CKMB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine (CREA) kits (Zhejiang Qiangsheng Biotechnology Co., Ltd.), Tween-80, linoleic acid (Sangon Biotech (Shanghai) Co., Ltd.).

[0027] 1.3 Main Experimental Instruments

[0028] AU680 fully automated biochemical analyzer (Beckman Coulter Ltd.), ECLIPSE Ci-L upright biological microscope (Nikon Precision Machinery (Shanghai) Co., Ltd.).

[0029] 2 Experimental Methods

[0030] 2.1 Grouping and Modeling

[0031] Twelve six-week-old female C57BL / 6J mice were acclimatized for three days and then randomly divided into two groups of six each, named the control group and the model group, respectively. Before exposure to the toxic substance, the mice were fasted but allowed free access to water for 12 hours.

[0032] Tween-80 was dissolved in physiological saline to a 1% (v / v) solution, and linoleic acid was dissolved in 1% Tween-80 to a 30% (v / v) suspension. The solution was then filtered through a 0.45 μm filter membrane. Before injection, the abdomen of mice was disinfected with 75% alcohol. Mice in the model group received a subcutaneous injection of 100 μL of the 30% linoleic acid-Tween-80 suspension into the peritoneum, while mice in the control group received the same volume of 1% Tween-80 intraperitoneally.

[0033] 2.2 Sample Collection

[0034] 2.2.1 Serum sample collection and blood biochemical index detection

[0035] Sixteen hours after exposure, mice in each group were sacrificed and blood was collected. After blood agglutination, the samples were centrifuged at 3000 rpm for 20 minutes, and the supernatant serum was collected. CK, CKMB, ALT, AST, LDH, BUN, and CREA were measured in the serum samples using a fully automated biochemical analyzer.

[0036] 2.2.2 Skeletal muscle sample collection and HE staining

[0037] The gastrocnemius muscle was dissected and washed away with pre-cooled physiological saline to remove blood. It was then cut into 0.5×0.5×0.2cm tissue blocks and incubated overnight at 4°C in 4% paraformaldehyde. The next day, excess fixative was washed away with water, and the blocks were dehydrated and cleared using a gradient of ethanol and xylene before being embedded in paraffin to prepare sections. Before staining, the paraffin sections were dewaxed in xylene, then hydrated using a gradient of ethanol. Finally, the sections, already immersed in distilled water, were stained sequentially in hematoxylin and eosin solutions. After staining, excess dye was rinsed off with running water, and the sections were dehydrated, cleared, mounted with neutral resin, and observed and photographed under a microscope.

[0038] 2.2.3 Statistical Analysis

[0039] SPSS 26.0 software was used for inter-group difference analysis. Quantitative data were expressed as mean ± standard deviation. When the overall sample distribution was normal, a two-tailed independent samples t-test was used to compare differences between the two groups; when the sample was not normally distributed, nonparametric tests were used to compare differences between groups. p < 0.05 was considered statistically significant.

[0040] 3. Experimental Results

[0041] 3.1 Blood biochemical levels in model animals

[0042] Significantly elevated CK levels are the most sensitive indicator of rhabdomyolysis and degeneration. CKMB is a type of CK isoenzyme. LDH is a key enzyme in energy metabolism in the body. When tissue is damaged and necrotic, LDH is released into the blood. CK, CKMB, and LDH reflect the metabolic and energy supply status of skeletal muscle. ALT and AST reflect the degree of liver damage associated with rhabdomyolysis, while BUN and CREA reflect the degree of kidney damage. These blood biochemical indicators are auxiliary diagnostic indicators for rhabdomyolysis syndrome (Ram et al., 2012, Emergency Medicine Practice, 14(3)).

[0043] The results are as follows Figure 1 As shown, compared with the control group, the model group showed significantly elevated levels of CK, CKMB, ALT, AST, LDH, BUN, and CREA, indicating that the skeletal muscle of the model group mice suffered acute damage.

[0044] 3.2 Pathological examination of skeletal muscle tissue

[0045] Pathological observation of muscle tissue is currently the most important method for evaluating rhabdomyolysis syndrome models in experimental studies. HE staining results of skeletal muscle show that the control group ( Figure 2 Left) The mouse muscle tissue structure was clear, the muscle cells were tightly arranged, the muscle fibers showed no obvious degeneration, the interstitial tissue showed no obvious proliferation, and no inflammatory cell infiltration was observed; Model group ( Figure 2 (Right) Mice showed a significant reduction in muscle fiber volume and increased spacing, a marked difference compared to the control group. The results demonstrate the successful establishment of the model, and this modeling method did not lead to mouse death.

[0046] Example 2: Screening of Animal Model Establishment Cycle

[0047] The modeling period in Example 1 was adjusted to 6h, 12h, and 24h, while the rest of the regimen remained the same as in Example 1. Serum CK levels in mice were measured after different modeling periods. Mice were in good condition at 6h, their activity slightly decreased from 12h, and worsened at 24h. The CK level changes are shown below. Figure 3 As shown, compared with the control group, the serum CK level in the model group mice increased significantly from 12 h (p < 0.05), reaching its highest value at 24 h (p < 0.01), while there was no statistically significant difference between the two groups at 12 h and 24 h. These results indicate that mouse models meeting experimental requirements can be obtained at modeling times ranging from 12 h to 24 h.

[0048] Example 3: Dosage Screening for Animal Model Establishment

[0049] The dosage of the model group in Example 1 was adjusted to 100 μL of 10% linoleic acid-Tween 80 suspension (10 μL), 30% linoleic acid-Tween 80 suspension (30 μL), and 50% linoleic acid-Tween 80 suspension (50 μL), respectively. The remaining dosage regimens were the same as in Example 1. Serum CK levels in mice were measured after different modeling cycles. The results showed that, compared with the control group, there was no significant change in serum CK at a dosage of 10 μL; however, serum CK levels were significantly increased at dosages of 30 μL and 50 μL, respectively, reaching 2.98 times and 2.75 times that of the control group. There was no difference in CK levels between the 30 μL and 50 μL dosage groups. These results indicate that mouse models meeting the experimental requirements can be obtained at dosages ranging from 30 to 50 μL.

[0050] The results above demonstrate that this invention provides a method for constructing an animal model of rhabdomyolysis syndrome. This model induces rhabdomyolysis in mice within 24 hours through a single dose of a single drug. Compared to previously reported methods using statins, this method is simpler and faster, effectively addressing the drawbacks of complex treatment, long treatment cycles, and low success rates associated with statin-based models. It also better reflects the pathogenesis of acute rhabdomyolysis syndrome. Furthermore, this invention uses a low dose of a single drug, overcoming the limitations of previous drug-based models where the dose far exceeded clinical dosage and treatment habits. The conclusions obtained using this model are more suitable for extrapolation to humans and better reflect the clinical and pathophysiological changes of this type of disease. Therefore, the rhabdomyolysis syndrome disease model described in this invention has promising applications in research on drug-induced acute rhabdomyolysis syndrome and the development of related drugs.

Claims

1. A method for constructing a mouse model of drug-induced acute rhabdomyolysis syndrome, characterized by, It comprises the following steps: The mouse is injected with linoleic acid in the abdominal cavity, and the mouse model of drug-induced acute rhabdomyolysis syndrome is obtained 12-24 hours later; The injection amount of the linoleic acid is 30-50 μL; The linoleic acid is a linoleic acid-tween 80 suspension; The preparation method of the linoleic acid-tween 80 suspension is as follows: tween 80 is dissolved in physiological saline, the volume fraction of the tween 80 is 1%, and then the tween 80 is mixed with the linoleic acid; The mouse is a C57BL / 6J female mouse, the mouse condition is 6-8 weeks old, and the body weight is 16-20 g; the mouse is adaptively fed for 3 days before being injected in the abdominal cavity, and the mouse is not fasted but is watered 12 hours before being administered; the adaptive feeding condition is that the feeding environment temperature is 20-26 ℃, and the relative humidity is 60%-70%.

2. The application of the mouse model of drug-induced acute rhabdomyolysis syndrome obtained by the method of claim 1 in screening drugs for preventing or treating drug-induced acute rhabdomyolysis syndrome.

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