Establishment method and application of rat acute inflammation model for simulating clinical inflammation
The Toll-like receptor 4 signaling pathway was activated by low-dose continuous intraperitoneal injection of lipopolysaccharides, and an acute inflammation model in rats that did not damage the liver and kidney was established, which solved the problem of simulated clinical inflammation models on liver and kidney damage in the prior art, and achieved a study on the significant increase in inflammation markers and the impact of drug pharmacokinetics.
Patent Information
- Application Number
- CN202510554696.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-24
- Publication Date
- 2025-07-25
AI Technical Summary
Existing acute inflammation models in rats often cause damage to the liver and kidneys when simulating clinical inflammation, and lack simulated clinical inflammation models that do not damage the liver and kidneys to explore the need for drug pharmacokinetic effects.
The method of low-dose continuous intraperitoneal injection of lipopolysaccharides was adopted to activate the Toll-like receptor 4 signaling pathway to promote the expression and release of proinflammatory factors such as interleukin-6, and establish a rat acute inflammation model. The specific dosages are 20-40μg/kg, 30-50μg/kg, and 40-60μg/kg to avoid liver and kidney damage.
Successfully simulated clinical inflammation, resulting in a significant increase in the levels of inflammatory markers such as interleukin-6, C-reactive protein and α1 acidic glycoprotein without liver and kidney damage, providing a new experimental animal model to explore the effect of inflammation on drug pharmacokinetics.
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Abstract
Description
Technical Field:
[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to a method for establishing and applying a rat acute inflammation model simulating clinical inflammation. Background Art:
[0002] Inflammation is a complex defense response of the body to tissue damage or pathogen invasion. Its core processes include clearing injury factors, repairing damaged tissues, and restoring normal physiological functions. According to the duration, the inflammatory response can be divided into acute and chronic. The acute response is a normal physiological response of the body to external stimuli with a short onset time, characterized by a sudden onset and a short course (usually only lasting for a few minutes or days). In contrast, chronic inflammation is a persistent and progressive pathological process.
[0003] Since the inflammatory response is closely related to many diseases of the body, in order to explore the role of inflammation in these diseases or the impact of inflammation on drugs, many inflammation models have been established. Currently reported common rat inflammation models mainly include the inflammation model induced by dry yeast, the inflammation model induced by lipopolysaccharide, the inflammation model induced by 2,4-dinitrophenol, and the inflammation model induced by turpentine. Among them, the lipopolysaccharide-induced inflammation model is similar to clinical inflammation and is a classic drug for inflammation modeling. However, most lipopolysaccharide-induced inflammation models often cause damage to the body, resulting in tissue damage and organ necrosis. Researchers usually induce different inflammation models according to different doses and times of lipopolysaccharide injection.
[0004] Lipopolysaccharide is the main component of the outer membrane of Gram-negative bacteria and plays an important role in Gram-negative bacterial infections and disease evolution. It is considered to be the main cause of systemic inflammatory syndrome. The biological activity of lipopolysaccharide is very extensive. It can not only activate some immune cells such as T lymphocytes, B lymphocytes, and macrophages, but also promote cytokine production, activate complement, and enhance immune activity. It is one of the main pathogenic molecules mediating infectious inflammatory damage. Clinical acute inflammation may affect the pharmacokinetic characteristics of many drugs. For example, some studies have reported that during acute inflammation, the blood drug concentrations of risperidone, clozapine, and quetiapine increase significantly, and the concentration of clozapine even reaches the toxic range. Whether exploring the changing rules of lipopolysaccharide-related inflammation or investigating the impact of acute inflammation on drug pharmacokinetics, there is a need for relevant inflammation animal models.
[0005] Currently, no patent for a rat acute inflammation model that simulates clinical inflammation and does not cause liver and kidney damage has been found. Summary of the Invention:
[0006] To solve the above technical problems, the present invention provides a method for establishing a rat acute inflammation model simulating clinical inflammation and its application. Using lipopolysaccharide as the modeling drug, it is continuously intraperitoneally injected at a low dose for 2 - 3 days to induce the establishment of a rat acute inflammation model with clinical inflammation characteristics and without causing liver and kidney damage, providing a new experimental animal model for exploring the impact of inflammation on the pharmacokinetics of drugs.
[0007] To achieve the above object, the present invention provides a rat acute inflammation model simulating clinical inflammation. The modeling drug used is lipopolysaccharide, and the dosing doses for three consecutive days are 20 - 40 μg / kg, 30 - 50 μg / kg, and 40 - 60 μg / kg respectively.
[0008] Specifically, taking the dosing doses of 20 μg / kg, 30 μg / kg, and 40 μg / kg for three consecutive days as an example, the method for establishing the rat acute inflammation model is as shown in the following steps:
[0009] Step 1: Weigh precisely a certain volume of lipopolysaccharide powder, dissolve it with 0.9% NaCl solution, and prepare a stock solution with a concentration of 1 mg / mL.
[0010] Step 2: On the first day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 4 μg / mL, and intraperitoneally inject the rats with a dosing volume of 5 mL / kg.
[0011] Step 3: On the second day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 6 μg / mL, and intraperitoneally inject the rats with a dosing volume of 5 mL / kg.
[0012] Step 4: On the third day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 8 μg / mL, and intraperitoneally inject the rats with a dosing volume of 5 mL / kg.
[0013] The present invention provides a rat acute inflammation model simulating clinical inflammation.
[0014] The present invention provides an application of a rat acute inflammation model simulating clinical inflammation in exploring the impact of inflammation on the pharmacokinetics of antipsychotic drugs.
[0015] Compared with the prior art, the beneficial effects of the present invention include:
[0016] The present invention uses lipopolysaccharide as a modeling drug, promotes the expression and release of pro-inflammatory factors such as interleukin-6 by activating the Toll-like receptor 4 signaling pathway, further triggers an inflammatory response, and obtains a rat acute inflammation model; this model can better simulate clinical inflammation, resulting in a significant increase in the levels of inflammatory markers such as interleukin-6, C-reactive protein, and α1-acid glycoprotein, and does not cause liver and kidney damage; the present invention endows this model with the characteristic of not causing liver and kidney damage by continuously intraperitoneally injecting lipopolysaccharide at a low dose; the preparation method of the present invention has low cost and is simple and easy to operate, providing a new experimental animal model for exploring the impact of inflammation on drug pharmacokinetics. Description of the Drawings:
[0017] Figure 1 It is a graph showing the change in the level of interleukin-6 in the rat acute inflammation model of the present invention.
[0018] Figure 2 It is a graph showing the change in the level of C-reactive protein in the rat acute inflammation model of the present invention.
[0019] Figure 3 It is a graph showing the change in the level of α1-acid glycoprotein in the rat acute inflammation model of the present invention.
[0020] Figure 4 It is a graph of the area under the plasma drug concentration-time curve of quetiapine before and after inflammation modeling in the application of the rat acute inflammation model of the present invention to explore the impact of inflammation on the pharmacokinetics of quetiapine. Detailed Embodiments:
[0021] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0022] Example 1
[0023] A rat acute inflammation model simulating clinical inflammation was prepared, and the specific implementation method is as follows:
[0024] Step 1: Weigh an appropriate amount of lipopolysaccharide powder precisely, dissolve it with 0.9% NaCl solution, and prepare a stock solution with a concentration of 1 mg / mL.
[0025] Step 2: Before injecting lipopolysaccharide, blood was taken from the inner canthus of the eye of the rats for the first time, serum was collected, centrifuged, and then placed in a -40°C refrigerator for storage and waiting for measurement as control group samples.
[0026] Step 3: On the first day, dilute the stock solution described in Step 1 to 4 μg / mL with 0.9% NaCl solution, and intraperitoneally inject the rats with a dosing volume of 5 mL / kg.
[0027] Step 4: On the second day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 6 μg / mL, and intraperitoneally inject the rats at a dosing volume of 5 mL / kg. Three hours after the injection, collect blood from the inner canthus of the eye, collect the serum, centrifuge it, and store it in a -40°C refrigerator for later measurement as the sample of the model group 3 hours after lipopolysaccharide injection on the second day.
[0028] Step 5: On the third day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 8 μg / mL, and intraperitoneally inject the rats at a dosing volume of 5 mL / kg. Three hours after the injection, collect blood from the inner canthus of the eye, collect the serum, centrifuge it, and store it in a -40°C refrigerator for later measurement as the sample of the model group 3 hours after lipopolysaccharide injection on the third day.
[0029] Step 6: Use an automatic biochemical analyzer to measure the levels of total bilirubin (TBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatinine (CREA), urea (UREA), and albumin (ALB) in the rat serum. The serum is centrifuged before injection to remove suspensions and precipitates to prevent needle clogging during injection. The results are shown in Table 1. Compared with before modeling, there were no significant changes in each liver and kidney function index after inflammation modeling (P>0.05), that is, the acute inflammation model of rats established with this dose of lipopolysaccharide did not cause liver and kidney damage.
[0030] Table 1
[0031]
[0032] Note: 1-0 refers to before injecting lipopolysaccharide on the first day; 2-3 and 3-3 refer to 3 hours after injecting lipopolysaccharide on the second and third days respectively.
[0033] Example 2
[0034] An acute inflammation model of rats simulating clinical inflammation was prepared, and the specific implementation method is as follows:
[0035] Step 1: Weigh an appropriate amount of lipopolysaccharide powder precisely, dissolve it with 0.9% NaCl solution, and prepare a stock solution with a concentration of 1 mg / mL.
[0036] Step 2: Before injecting lipopolysaccharide, collect blood from the inner canthus of the rat's eye for the first time, and immediately centrifuge to obtain plasma samples as control group samples.
[0037] Step 3: On the first day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 4 μg / mL, and intraperitoneally inject the rats at a dosing volume of 5 mL / kg.
[0038] Step 4: On the second day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 6 μg / mL, and intraperitoneally inject the rats at a dosing volume of 5 mL / kg. 3 hours after the injection, take blood from the inner canthus of the eye, and immediately centrifuge to obtain plasma samples, which serve as the model group samples 3 hours after lipopolysaccharide injection on the second day.
[0039] Step 5: On the third day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 8 μg / mL, and intraperitoneally inject the rats at a dosing volume of 5 mL / kg. 3 hours after the injection, take blood from the inner canthus of the eye, and immediately centrifuge to obtain plasma samples, which serve as the model group samples 3 hours after lipopolysaccharide injection on the third day.
[0040] Step 6: Use the ELISA double antibody sandwich method to measure the level of interleukin-6 in the rat acute inflammation model established in Steps 1 to 5, and the results are as Figure 1 shown. "before, day-2, day-3" in the figure respectively represent before lipopolysaccharide injection, 3 hours after lipopolysaccharide injection on the second day, and 3 hours after lipopolysaccharide injection on the third day. Compared with the control group, the levels of interleukin-6 were significantly increased 3 hours after lipopolysaccharide injection on the second and third days (P < 0.01).
[0041] Example 3
[0042] A rat acute inflammation model simulating clinical inflammation was prepared, and the specific implementation method is as follows:
[0043] Step 1: Weigh a certain volume of lipopolysaccharide powder precisely, dissolve it with 0.9% NaCl solution, and prepare a stock solution with a concentration of 1 mg / mL.
[0044] Step 2: Before lipopolysaccharide injection, take blood from the inner canthus of the rat's eye for the first time, and immediately centrifuge to obtain plasma samples, which serve as the control group samples.
[0045] Step 3: On the first day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 4 μg / mL, and intraperitoneally inject the rats at a dosing volume of 5 mL / kg.
[0046] Step 4: On the second day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 6 μg / mL, and intraperitoneally inject the rats at a dosing volume of 5 mL / kg. 3 hours after the injection, take blood from the inner canthus of the eye, and immediately centrifuge to obtain plasma samples, which serve as the model group samples 3 hours after lipopolysaccharide injection on the second day.
[0047] Step 5: On the third day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 8 μg / mL, and intraperitoneally inject the rats at a dosing volume of 5 mL / kg. After 3 h of injection, collect blood from the inner canthus of the eye and immediately centrifuge to obtain plasma samples, which serve as the model group samples 3 h after lipopolysaccharide injection on the third day.
[0048] Step 6: Use the double antibody sandwich ELISA method to measure the level of C-reactive protein in the rat acute inflammation model established in Steps 1 to 5, and the results are as Figure 2 shown. "before, day - 2, day-3" in the figure represent before lipopolysaccharide injection, 3 h on the second day after lipopolysaccharide injection, and 3 h on the third day after lipopolysaccharide injection, respectively. Compared with the control group, the levels of C-reactive protein were significantly increased at 3 h after lipopolysaccharide injection on the second and third days (P<0.01).
[0049] Example 4
[0050] A rat acute inflammation model simulating clinical inflammation was prepared, and the specific implementation method is as follows:
[0051] Step 1: Weigh precisely a certain volume of lipopolysaccharide powder, dissolve it with 0.9% NaCl solution, and prepare a stock solution with a concentration of 1 mg / mL.
[0052] Step 2: Before lipopolysaccharide injection, collect blood from the inner canthus of the eye of the rats for the first time and immediately centrifuge to obtain plasma samples, which serve as the control group samples.
[0053] Step 3: On the first day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 4 μg / mL, and intraperitoneally inject the rats at a dosing volume of 5 mL / kg.
[0054] Step 4: On the second day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 6 μg / mL, and intraperitoneally inject the rats at a dosing volume of 5 mL / kg. After 3 h of injection, collect blood from the inner canthus of the eye and immediately centrifuge to obtain plasma samples, which serve as the model group samples 3 h after lipopolysaccharide injection on the second day.
[0055] Step 5: On the third day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 8 μg / mL, and intraperitoneally inject the rats at a dosing volume of 5 mL / kg. After 3 h of injection, collect blood from the inner canthus of the eye and immediately centrifuge to obtain plasma samples, which serve as the model group samples 3 h after lipopolysaccharide injection on the third day.
[0056] Step 6: The level of α1-acid glycoprotein in the rat acute inflammation model established in Steps 1 to 5 was measured by the ELISA double antibody sandwich method, and the results are as Figure 3 shown. "before, day - 2, day-3" in the figure represent before lipopolysaccharide injection, 3 h on the second day after lipopolysaccharide injection, and 3 h on the third day after lipopolysaccharide injection, respectively. Compared with the control group, the levels of α1-acid glycoprotein were significantly increased at 3 h on the second and third days after lipopolysaccharide injection (P < 0.01).
[0057] Example 5
[0058] This example was carried out using the rat acute inflammation models established in Examples 1, 2, 3, and 4.
[0059] The application of the rat acute inflammation model in exploring the effect of acute inflammation on the pharmacokinetics of quetiapine was tested. The specific implementation method is as follows:
[0060] Six SD rats were experimented using the self-control method before and after. The experiment was divided into 2 cycles. The body weights of the SD rats were all in the range of 210 - 260 g. In the first cycle, the rats were intragastrically administered with quetiapine suspension. The administration dose was 30 mg / kg, the administration volume was 5 mL / kg, and the administration concentration was 6 mg / mL. The rats were fasted for 12 hours before administration but allowed to drink water. After administration, blood was collected from the inner canthus of the eye at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 12 h after administration. Approximately 0.3 mL of blood was collected at each time point into a centrifuge tube containing EDTA-2K anticoagulant, and immediately centrifuged to obtain plasma samples, which were stored in a -40 °C refrigerator for later measurement. Five days after the administration in the first cycle was flushed, lipopolysaccharide was injected to induce inflammation. Lipopolysaccharide was continuously injected for 3 days, and the doses were 20 μg / kg, 30 μg / kg, and 40 μg / kg respectively. At 30 min after the injection of lipopolysaccharide on the third day, the rats were intragastrically administered with quetiapine suspension in the second cycle. The administration dose was also 30 mg / kg, the administration volume was 5 mL / kg, and the administration concentration was 6 mg / mL. Blood was collected at the same time points as in the first cycle, and the plasma was also frozen and stored at -40 °C for later measurement. The concentration of quetiapine in the above plasma was measured by LC-MS / MS, and the area under the plasma drug concentration-time curve of quetiapine before and after inflammation induction was obtained as Figure 4 shown. It can be found that compared with before the establishment of the lipopolysaccharide inflammation model, after the establishment of the inflammation model, the concentration of quetiapine in the plasma of rats was significantly increased, which was consistent with the clinical reports, indicating that the establishment of the rat acute inflammation model was successful and could be applied to explore the effect of acute inflammation on the pharmacokinetics of drugs.
Claims
1. A method for establishing an acute inflammation model of rats to simulate clinical inflammation, characterized in that: Using lipopolysaccharide as the modeling drug, the synthesis and secretion of interleukin-6 were induced by continuous intraperitoneal injection at a low dose for 2 - 3 days to establish an acute inflammation model in rats.
2. The method for establishing a rat acute inflammation model according to claim 1, characterized in that: As the modeling drug, the injection doses of lipopolysaccharide were as follows: on the first day, 20 - 30 μg / kg; on the second day, 30 - 50 μg / kg; on the third day, 40 - 60 μg / kg.
3. A method for establishing an acute inflammation model in rats according to claim 1, characterized in that As the modeling drug, the injection volume of lipopolysaccharide was: 5 mL / kg.
4. A method for establishing a rat acute inflammation model according to claims 1-3, characterized in that, The described acute inflammation model in rats can cause a significant increase in inflammatory biomarkers such as interleukin-6, C-reactive protein, and α1-acid glycoprotein without causing liver and kidney damage in rats.
5. A method for establishing a rat acute inflammation model according to claims 1-4, taking the administration doses of 20 μg / kg, 30 μg / kg, and 40 μg / kg respectively for three consecutive days as an example, characterized in that: The method for establishing it includes the following steps: Step 1: Weigh an appropriate amount of lipopolysaccharide powder precisely, dissolve it with 0.9% NaCl solution, and prepare a stock solution with a concentration of 1 mg / mL. Step 2: On the first day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 4 μg / mL, and perform intraperitoneal injection on rats at a dosing volume of 5 mL / kg. Step 3: On the second day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 6 μg / mL, and perform intraperitoneal injection on rats at a dosing volume of 5 mL / kg. Step 4: On the third day, dilute the stock solution described in Step 1 with 0.9% NaCl solution to 8 μg / mL, and perform intraperitoneal injection on rats at a dosing volume of 5 mL / kg.
6. Use of an acute inflammation model in rats as described in claims 1 - 5 as an animal model for simulating clinical inflammation in the study of the impact of acute inflammation on the pharmacokinetics of drugs.