Construction method of obesity and sepsis combined rat model

Abstract

The invention discloses a construction method of an obesity and sepsis combined rat model, and belongs to the technical field of medicine. In order to solve the problem that a targeted obesity-combined sepsis animal model is lacked in the prior art, the invention provides a construction method of an obesity-combined sepsis rat model, through collaborative design of obesity basis and sepsis strike, rats are induced by standard sepsis under the obesity metabolism background, and the obesity-combined sepsis animal model is obtained. Therefore, key pathological characteristics of clinical obesity and sepsis combined patients can be reproduced; the model obtained through the construction method can be used for researching pathogenesis, immune metabolism change and treatment response of sepsis in the obesity state, and new experimental tools and thoughts are provided for optimizing clinical treatment of special crowds.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, and in particular relates to a method for constructing an obese rat model complicated with sepsis. Background Technology

[0002] With the advancement of modern medical research, sepsis and obesity have become two major threats to human health. Sepsis, a leading cause of death in critically ill patients, can be caused by infections in various parts of the body. Its pathogenesis is complex, leading to a series of pathophysiological changes such as immune imbalance, metabolic disorders, and microcirculatory disturbances. If not treated promptly and effectively, it can easily progress to multiple organ dysfunction syndrome, seriously threatening the patient's life. Meanwhile, the incidence of obesity is continuously rising globally. Obesity is not only an external manifestation of excessive fat accumulation but also a chronic metabolic disease influenced by multiple factors including genetics, environment, and behavior. Obesity can trigger a series of serious complications such as diabetes, cardiovascular disease, and cancer, placing a heavy burden on patients' health and social medical resources. In recent years, the number of critically ill patients with obesity complicated by sepsis has been gradually increasing in clinical practice. This interaction creates a vicious cycle, further aggravating the condition and significantly increasing the incidence of multiple organ dysfunction syndrome (MODS) and patient mortality. Although research has been conducted on the construction of models of sepsis combined with other diseases, such as models of obesity with MODS, research on animal models of obesity complicated by sepsis remains lacking. Therefore, those skilled in the art are eager to develop a method for constructing a rat model of obesity complicated by sepsis, providing a research foundation for filling research gaps, elucidating disease mechanisms, and optimizing clinical treatment strategies. Summary of the Invention

[0003] To address the lack of targeted animal models for obesity combined with sepsis in the prior art, this invention provides a method for constructing a rat model of obesity combined with sepsis.

[0004] One objective of this invention is to provide a method for constructing an obese rat model complicated with sepsis, the method comprising the following steps: S1: Select healthy, clean-grade male SD rats aged 6-8 weeks, weighing 180-220g, and acclimatize them for 1 week at 22℃-24℃ and relative humidity of 28%-50%. S2: Rats that underwent adaptive feeding in S1 were first fed a high-sugar, high-fat diet for 8 weeks, and then cecal ligation and puncture were performed to screen and obtain a successfully modeled obese rat model with sepsis.

[0005] In a preferred embodiment of the present invention, the cecal ligation and puncture procedure in step S2 is as follows: S11: Make a 1-2 cm incision in the skin along the midline of the rat's abdomen, taking care not to damage the underlying muscle layer and internal organs; S22: Gently wipe the exposed abdominal cavity with gauze moistened with physiological saline to keep it moist and prevent the tissue from drying out; S33: Ligate the cecum at one-third or one-half of the junction between the cecum and ileum; S44: Use a sterile surgical needle to perform a puncture on the cecum below the ligation; S55: The punctured cecum is placed back into the abdominal cavity. The abdominal wall is closed and the incision is sutured, ensuring that there is no excessive leakage of cecum contents. Analgesia and 37°C warm saline fluid replacement are given immediately.

[0006] In a preferred embodiment of the present invention, the surgical needle in S44 is an 18-gauge needle, and the number of puncture treatments is 1.

[0007] In a preferred embodiment of the present invention, the amount of warm saline solution used in S55 is 5 ml per 100 grams of body weight.

[0008] In a preferred embodiment of the present invention, the screening criteria for the obese rat model with sepsis described in S2 are as follows: rats with a weight exceeding the standard weight by 20% are selected, and then the obese rat model with sepsis is successfully screened according to the symptom index scores, that is, if the scores of each symptom index are ≥1, it indicates that the model is successfully established.

[0009] In a preferred embodiment of the present invention, the symptom index scoring criteria are as follows: a. Appearance: luster is 0 points; some hairs standing on end is 1 point; all hairs on the back standing on end is 2 points; fluffy and messy is 3 points; haggard is 4 points; b. Self-awareness: Active: 0 points; Active but not upright: 1 point; Reduced and slow activity but still walking: 2 points; Only active after provocation and trembling movement: 3 points; Remaining still after provocation: 4 points; c. Independent activity: 0 points for normal activity (e.g., eating, drinking, climbing, walking, fighting); 1 point for reduced activity (e.g., only moving at the bottom of the cage); 2 points for significantly reduced activity (e.g., stillness, occasional activity); 3 points for complete stillness; 4 points for complete stillness and trembling. d. Response to stimuli: Rapid response is 0 points; no response or slow response to sound, strong response to touch is 1 point, immediately run away; no response to sound, moderate response to touch is 2 points, take a few steps; no response to sound, weak response to touch is 3 points, do not move; no response to sound, almost no response to touch is 4 points, such as being unable to roll over after being pushed over. e. Eyes: Fully open is 0 points; more than half-closed but less than fully open, with discharge is 1 point; half-closed, with discharge is 2 points; more than fully closed but less than half-closed, with discharge is 3 points; fully closed, with obvious cloudy discharge is 4 points; f. Respiratory rate: Normal is 0 points; Mildly reduced is 1 point, not countable to the naked eye; Moderately reduced is 2 points, countable to the naked eye; Significantly reduced is 3 points, respiratory interval 0.5 s; Extremely significant reduced is 4 points, respiratory interval > 1 s; g. Breathing quality: 0 points for normal; 1 point for wheezing; 2 points for intermittent dyspnea; 3 points for dyspnea and intermittent wheezing; 4 points for dyspnea and no wheezing.

[0010] In a preferred embodiment of the present invention, the standard weight is the average weight of rats in the same age control group.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: In the prior art, the simple sepsis model only simulates the acute inflammatory response caused by infection, without considering the "chronic low-grade inflammatory microenvironment" that exists in obesity for a long time; while the simple obesity model only focuses on metabolic disorders and lacks the impact of acute infection on immunity, circulation and organ function.

[0012] The obese rat model of sepsis provided by this invention replicates the core pathological features of clinical patients through a synergistic design of "obesity foundation + sepsis attack." It is the first to propose seamlessly integrating "high-fat induced chronic low-grade inflammatory microenvironment" and "CLP (sepsis model) acute attack of multibacterial sepsis" in the same experimental setting, thus constructing an obese rat model of sepsis. This overcomes the disconnect between existing technologies where "simple sepsis models only replicate acute infection" and "simple obesity models only focus on metabolic disorders," truly replicating the core pathological process of sepsis secondary to appendicitis or diverticulum perforation in clinical obese patients. Compared to traditional mouse models, this invention uses rats, whose size advantage allows for a single treatment of ≥1... Repeated blood sampling of mL allows for dynamic monitoring of four lipid profiles, providing quantitative evidence for model validation and overcoming the key shortcomings of insufficient blood volume and inability to conduct continuous observation in mice. In the "CLP acute attack of multibacterial sepsis" stage, this invention abandons the simplified method of exogenous endotoxin or single-bacterial injection and adopts the CLP procedure of subileocecal valve ligation + puncture to induce endogenous multibacterial peritonitis, with simultaneous occurrence of septic shock, bacterial translocation and multiple organ dysfunction, more realistically simulating the sepsis process caused by perforation of appendicitis or diverticulitis in humans, filling the gap in the "obesity-infection synergistic attack" animal model.

[0013] The method for preparing obese rats with sepsis provided by this invention is simple to operate, low in cost, and highly reproducible and controllable. It provides an innovative platform that is highly consistent with clinical practice and allows for continuous blood sampling and evaluation for studying the mechanisms of immunosuppression, lipid metabolism disorders, and organ damage in sepsis under the background of obesity. This opens up new ideas for optimizing treatment strategies for obese rats with sepsis and is fundamentally different from existing single-model technologies. Attached Figure Description

[0014] Figure 1 Figures showing the weight measurements of rats in each group at different time points; Figure 2 The graph shows the blood glucose test results of rats in each group after 8 weeks of feeding with a high-sugar, high-fat diet. Figure 3 The graphs show the blood lipid test results of rats in each group after 8 weeks of feeding with a high-sugar, high-fat diet; A is the total cholesterol (TC) test result, B is the triglyceride (TG) test result, C is the low-density lipoprotein (LDL) test result, and D is the high-density lipoprotein (HDL) test result. Figure 4 The graph shows the liver function test results of rats in each group after 8 weeks of feeding with a high-sugar and high-fat diet; A is the aspartate aminotransferase (AST) test result, and B is the alanine aminotransferase (ALT) test result. Figure 5 A statistical chart showing the mortality rate of rats 48 hours after cecal ligation and puncture. Figure 6 The graph shows the results of interleukin-6 level detection in the serum of rats in each group; Figure 7 The graph shows the results of serum tumor necrosis factor-α levels in rats from each group. Detailed Implementation

[0015] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0017] Example 1: 1. Experimental Materials Thirty-two healthy, clean-grade male SD rats (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) aged 6-8 weeks and weighing 180-220g were selected and housed in a well-ventilated indoor environment controlled by an independent air conditioning system. The room temperature was maintained at 22℃-24℃ and the relative humidity at 28%-50%. The environment was kept clean and hygienic. All rats were acclimatized to normal feed and had free access to food and water daily.

[0018] 2. Experimental Grouping After one week of acclimatization, the rats were randomly divided into a control group, an obesity group, a sepsis group, and an obesity-sepsis group, with eight rats in each group.

[0019] Control group: fed normal feed for 8 weeks; Sepsis group: After being fed normal feed for 8 weeks, the cecum was ligated and punctured to screen and obtain a sepsis model; Obesity group: 8 animals per group, fed a high-sugar, high-fat diet for 8 weeks to obtain an obesity model; Obese sepsis group: 8 animals per group. They were fed a high-sugar, high-fat diet for 8 weeks, and then cecal ligation and puncture were performed to screen and obtain an obese sepsis model.

[0020] The procedure for cecal ligation and puncture is as follows: S1: Make a 1-2 cm incision in the skin along the midline of the rat's abdomen, taking care not to damage the underlying muscle layer and internal organs; S2: Gently wipe the exposed abdominal cavity with gauze moistened with physiological saline to keep it moist and prevent the tissue from drying out; S3: Ligate the cecum at one-third or one-half of the junction between the cecum and ileum; S4: Use a sterile 18-gauge surgical needle to perform one puncture on the cecum below the ligation; S5: After the puncture and treatment, the cecum is placed back into the abdominal cavity. The abdominal wall is closed and the incision is sutured, ensuring that there is no excessive leakage of cecum contents. Analgesia and 37°C warm saline solution are given immediately. The amount of warm saline solution used is 5 ml per 100 grams of body weight.

[0021] The screening criteria for the obese rat model complicated with sepsis were as follows: Rats whose weight exceeds the standard weight (370.9 g in this experiment) by 20% were selected, and then the obese rat model with sepsis was successfully screened according to the symptom index scores. That is, the model was successfully established when all symptom index scores were ≥1. The scoring criteria for the symptom indicators are as follows: a. Appearance: 0 points for glossiness; 1 point for some hairs standing on end; 2 points for all hairs on the back standing on end; 3 points for fluffy and messy; 4 points for haggard appearance; b. Self-awareness: Active: 0 points; Active but not upright: 1 point; Reduced and slow activity but still walking: 2 points; Only active after provocation and trembling movement: 3 points; Remaining still after provocation: 4 points; c. Independent activity: 0 points for normal activity, such as eating, drinking, climbing, walking, fighting; 1 point for reduced activity, such as only moving around at the bottom of the cage; 2 points for significantly reduced activity, such as being still or occasionally moving; 3 points for being completely still; 4 points for being completely still while trembling. d. Response to stimuli: Rapid response is 0 points; no response or slow response to sound, but strong response to touch is 1 point, such as immediately running away; no response to sound, but moderate response to touch is 2 points, such as taking a few steps; no response to sound, but weak response to touch is 3 points, such as not moving; no response to sound, but almost no response to touch is 4 points, such as not being able to roll over after being pushed over. e. Eyes: Fully open is 0 points; more than half-closed but less than fully open, with discharge is 1 point; half-closed, with discharge is 2 points; more than fully closed but less than half-closed, with discharge is 3 points; fully closed, with obvious cloudy discharge is 4 points; f. Respiratory rate: Normal is 0 points; Mildly reduced is 1 point, not countable to the naked eye; Moderately reduced is 2 points, countable to the naked eye; Significantly reduced is 3 points, respiratory interval 0.5 s; Extremely significant reduced is 4 points, respiratory interval > 1 s; g. Breathing quality: 0 points for normal; 1 point for wheezing; 2 points for intermittent dyspnea; 3 points for dyspnea and intermittent wheezing; 4 points for dyspnea and no wheezing.

[0022] In this embodiment, continuous observation of the rats' condition after cecal ligation and puncture revealed that initially, the rats exhibited stress-induced agitation, which gradually progressed to lethargy, accompanied by rapid breathing, complete loss of appetite, ruffled hair, and diarrhea. Severely ill rats eventually died from multiple organ failure. The above symptoms are consistent with the typical manifestations of a sepsis model, indicating the successful establishment of the sepsis and obesity sepsis models.

[0023] Effect Experiment: (1) Weight index detection In this embodiment, the criterion for successful establishment of an obesity model is that "the weight of rats fed a high-fat diet exceeds the standard weight by 20%". The standard weight is the average weight of rats in the control group of the same age. In this embodiment, the average weight of the control group rats is 370.9 g.

[0024] The results are as follows Figure 1As shown, in week 1, the body weight of rats in the control group and the obese group was at the same level, with no statistical difference, indicating that the initial physical condition of the two groups of rats was consistent. After 8 weeks of high-fat diet intervention, the body weight of rats in the obese group increased by 22.9% compared with the control group, which was statistically significant (p<0.05), meeting the criteria for an obesity model and confirming the successful establishment of the obesity model.

[0025] (2) Blood glucose level detection Blood glucose levels were measured in obese rats after 8 weeks of high-fat diet intervention, as well as in the control group.

[0026] The results are as follows Figure 2 As shown, the blood glucose level in the obese rat group was significantly higher than that in the control group, being 3.46 times higher, with a statistically significant difference (p<0.05). This indicates that obesity induced by a high-fat diet may be accompanied by glucose metabolism disorders, further validating the pathophysiological characteristics of the obesity model.

[0027] (3) Serum lipid index detection Serum lipid levels were measured in obese rats after 8 weeks of high-fat diet intervention, as well as in the control group.

[0028] The results are as follows Figure 3 As shown, compared with the control group, the levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL) in the serum of obese rats were significantly increased, at 2.13 times, 2.87 times, and 1.82 times that of the control group, respectively; while the level of high-density lipoprotein (HDL) was significantly decreased, at 0.55 times that of the control group. The differences between the groups for each indicator were statistically significant (p<0.05).

[0029] It is evident that the obese rats exhibited significant lipid metabolism disorders, which is an important biochemical characteristic of the obesity model.

[0030] (4) Liver function index testing Liver function indicators were measured in obese rats after 8 weeks of high-fat diet intervention, as well as in the control group.

[0031] The results are as follows Figure 4 As shown, the plasma aspartate aminotransferase (AST) level in the obese rat group was significantly higher than that in the control group, increasing by 76.7%; the alanine aminotransferase (ALT) level in the obese rat group was significantly higher than that in the control group, approximately 3.29 times higher. The differences between the groups for all indicators were statistically significant (p<0.05).

[0032] Since AST and ALT are important indicators reflecting liver function, elevated levels suggest that the obese rats have impaired liver function, indicating that obesity induced by a long-term high-fat diet may have adverse effects on liver function.

[0033] (5) Mortality statistics Statistical analysis was performed on the rat mortality rate within 48 hours after cecal ligation and puncture. The results are as follows: Figure 5 As shown, no rats died in the control group and the obesity group. However, the survival rate of rats in both the sepsis group and the obese sepsis group gradually decreased over time, and the obesity sepsis group died earlier than the sepsis group. This indicates that obesity may exacerbate the severity of sepsis in rats and increase the risk of death.

[0034] (6) Detection of interleukin-6 (IL-6) levels Twenty-four hours after cecal ligation and puncture, the serum IL-6 level of rats in each group was detected by enzyme-linked immunosorbent assay (ELISA).

[0035] The results are as follows Figure 6 As shown, the serum IL-6 level in the obese sepsis group was significantly higher than that in the control group, the obese group, and the sepsis group. Furthermore, the obese sepsis group showed statistically significant differences compared to the control group, the obese group, and the sepsis group. This indicates that obesity can promote the release of IL-6, an important pro-inflammatory factor, in the presence of sepsis.

[0036] (7) Detection of serum tumor necrosis factor-α (TNF-α) levels Twenty-four hours after cecal ligation and puncture, the serum TNF-α level of rats in each group was detected by enzyme-linked immunosorbent assay (ELISA).

[0037] The results are as follows Figure 7 As shown, the serum TNF-α level in the obese sepsis group was significantly higher than that in the control group, the obese group, and the sepsis group. Furthermore, the obese sepsis group showed statistically significant differences compared to the control group, the obese group, and the sepsis group. This indicates that obesity can increase the release of pro-inflammatory factors in septic rats, suggesting that obesity may exacerbate the pathological process of sepsis by enhancing the inflammatory response.

[0038] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A method for constructing an obese rat model complicated with sepsis, characterized in that, The construction method includes the following steps: S1: Select healthy, clean-grade male SD rats aged 6-8 weeks, weighing 180-220g, and acclimatize them for 1 week at 22℃-24℃ and relative humidity of 28%-50%. S2: Rats that underwent adaptive feeding in S1 were first fed a high-sugar, high-fat diet for 8 weeks, and then cecal ligation and puncture were performed to screen and obtain a successfully modeled obese rat model with sepsis.

2. The construction method according to claim 1, characterized in that, The cecal ligation and puncture procedure described in S2 is as follows: S11: Make a 1-2 cm incision in the skin along the midline of the rat's abdomen, taking care not to damage the underlying muscle layer and internal organs; S22: Gently wipe the exposed abdominal cavity with gauze moistened with physiological saline to keep it moist and prevent the tissue from drying out; S33: Ligate the cecum at one-third or one-half of the junction between the cecum and ileum; S44: Use a sterile surgical needle to perform a puncture on the cecum below the ligation; S55: The punctured cecum is placed back into the abdominal cavity. The abdominal wall is closed and the incision is sutured, ensuring that there is no excessive leakage of cecum contents. Analgesia and 37°C warm saline fluid replacement are given immediately.

3. The construction method according to claim 2, characterized in that, The surgical needle described in S44 is an 18-gauge needle, and the number of puncture treatments is 1.

4. The construction method according to claim 2, characterized in that, The recommended dosage of warm saline rehydration solution as described in S55 is 5 ml per 100 grams of body weight.

5. The construction method according to claim 1, characterized in that, The screening criteria for the obese rat model with sepsis described in S2 are as follows: select rats whose weight exceeds the standard weight by 20%, and then screen the successful obese rat model with sepsis according to the symptom index scores, that is, the model is considered to be successful if the scores of each symptom index are ≥1.

6. The construction method according to claim 5, characterized in that, The scoring criteria for the symptom indicators are as follows: a. Appearance: 0 points for glossiness; 1 point for some hairs standing on end; 2 points for all hairs on the back standing on end; 3 points for fluffy and messy; 4 points for haggard appearance; b. Self-awareness: 0 points for being active; 1 point for being active but not upright; Reduced activity and sluggishness, but still walking, scores 2 points; activity only after provocation and trembling movements scores 3 points; remaining motionless after provocation scores 4 points. c. Independent activity: 0 points for normal activity (e.g., eating, drinking, climbing, walking, fighting); 1 point for reduced activity (e.g., only moving at the bottom of the cage); 2 points for significantly reduced activity (e.g., stillness, occasional activity); 3 points for complete stillness; 4 points for complete stillness and trembling. d. Response to stimuli: Rapid response is 0 points; no response or slow response to sound, strong response to touch is 1 point, immediately run away; no response to sound, moderate response to touch is 2 points, take a few steps; no response to sound, weak response to touch is 3 points, do not move; no response to sound, almost no response to touch is 4 points, such as being unable to roll over after being pushed over. e. Eyes: Fully open is 0 points; more than half-closed but less than fully open, with discharge is 1 point; half-closed, with discharge is 2 points; more than fully closed but less than half-closed, with discharge is 3 points; fully closed, with obvious cloudy discharge is 4 points; f. Respiratory rate: Normal is 0 points; Mildly reduced is 1 point, not countable to the naked eye; Moderately reduced is 2 points, countable to the naked eye; Significantly reduced is 3 points, respiratory interval 0.5 s; Extremely significant reduced is 4 points, respiratory interval > 1 s; g. Breathing quality: 0 points for normal; 1 point for wheezing; 2 points for intermittent dyspnea; 3 points for dyspnea and intermittent wheezing; 4 points for dyspnea and no wheezing.

7. The construction method according to claim 5, characterized in that, The standard weight is the average weight of rats in the same age control group.

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