Method for constructing and evaluating animal model of acute ischemic cerebral apoplexy with syndrome of phlegm-blood stasis and toxin intermingling

Abstract

The invention discloses a construction and evaluation method of an acute ischemic cerebral apoplexy phlegm and blood stasis toxin intermingling syndrome animal model, which comprises the following steps: adaptively feeding rats for 1 week, and feeding the rats with high-fat feed; from the 26th day, 20 mg / kg carrageenan (Ca) is subjected to intraperitoneal injection for 3 days continuously; the rat cerebral ischemia / reperfusion model is prepared by adopting an MCAO / R suture suppository method when the rat is raised to the 29-day morning, and the method specifically comprises the following steps: fasting 12 hours before modeling, and supplying a small amount of water; injecting 3% pentobarbital (2ml / kg) into the intraperitoneal cavity of the rat for anesthesia; fixing the supine position of the rat after successful anesthesia, exposing and separating the right common carotid artery; cutting a V-shaped incision at the separated common carotid artery, inserting a suture into the internal carotid artery from the V-shaped incision, and advancing until the middle cerebral artery is blocked and ligated; when the suture is inserted into the middle cerebral artery, the time is 0; suturing the skin after an operation, and exposing the tail end of the tying line in vitro; and after 1.5 hours, the tying line is pulled out, and cerebral ischemia reperfusion is simulated.

Description

Technical Field

[0001] The invention relates to the technical field of animal model construction, in particular to a construction and evaluation method of an acute ischemic stroke phlegm, blood stasis and toxin syndrome animal model. Background Art

[0002] In the study of acute ischemic stroke (AIS), animal models are important tools for understanding pathophysiological mechanisms, evaluating treatment effects, and exploring new therapies. Existing animal models mainly focus on simulating the core pathophysiological changes of stroke, such as reduced cerebral blood flow, neuronal damage, and inflammatory response in the ischemic area. However, these models often ignore the characteristics of the syndrome of phlegm, blood stasis, and toxicity in traditional Chinese medicine theory, that is, the interaction and influence of phlegm, dampness, blood stasis, and toxic evil in the body.

[0003] In traditional Chinese medicine theory, phlegm, blood stasis and toxin syndrome refers to a syndrome in which phlegm, blood stasis and toxins interact and transform in the body, leading to pathological changes. This syndrome is more common in patients with acute ischemic stroke, characterized by progressive aggravation, poor prognosis, and the development of various symptoms. Therefore, constructing an animal model that can simulate this complex pathological state is of great significance for in-depth research on the pathological mechanism of AIS and exploring traditional Chinese medicine treatment methods.

[0004] Existing AIS animal models mainly simulate cerebral blood flow reduction and reperfusion injury through surgical or drug intervention, such as the suture embolism (MCAO / R) model. Although these models can simulate the pathological changes of stroke to a certain extent, they often lack the simulation of phlegm, blood stasis and toxicity syndrome and cannot fully reflect the clinical characteristics of AIS. In addition, these models often require additional intervention measures when simulating phlegm, blood stasis and toxicity syndrome, such as high-fat diet and drug stimulation, which may introduce additional variables and affect the accuracy and reliability of the model.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned technical deficiencies and provide a method for constructing and evaluating an animal model of acute ischemic stroke with phlegm, blood stasis and toxicity syndrome, so as to solve the technical problem of the lack of effective methods and evaluation methods for constructing an animal model of acute ischemic stroke with phlegm, blood stasis and toxicity syndrome in the related art.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] According to one aspect of the present invention, a method for constructing and evaluating an animal model of acute ischemic stroke with phlegm, blood stasis and toxin syndrome is provided, comprising the following steps:

[0009] The rats were adaptively fed for 1 week and fed with a high-fat diet; from the 26th day, they were given an intraperitoneal injection of 20 mg / kg carrageenan (Ca) for 3 consecutive days; they were fed until the morning of the 29th day, and the rat cerebral ischemia / reperfusion model was prepared by the MCAO / R suture method, which specifically included: fasting for 12 hours before modeling and giving a small amount of water; the rats were anesthetized by intraperitoneal injection of 3% pentobarbital (2 ml / kg); after successful anesthesia, the rats were fixed in a supine position, the right common carotid artery was exposed and separated; a "V"-shaped incision was cut at the separated common carotid artery, from which the suture was inserted into the internal carotid artery, and advanced until the middle cerebral artery was occluded and ligated; the moment the suture was inserted into the middle cerebral artery was taken as time 0; the skin was sutured after the operation, and the end of the suture was exposed to the outside of the body; the suture was pulled out after 1.5 hours to simulate cerebral ischemia and reperfusion.

[0010] Furthermore, the carrageenan (Ca) is prepared with physiological saline to 1 g·L -1 Solution, freshly prepared and used, then 20 mg kg rat body weight －1 Administer medication.

[0011] Furthermore, the rats are SPF grade male SD rats, weighing (160-180) g, and are placed in a constant temperature environment of 18-22° C., with humidity controlled at 50% to 60%, and are free to eat and drink.

[0012] Furthermore, the evaluation method includes an evaluation method for acute ischemic stroke and an evaluation method for phlegm, blood stasis and toxicity syndrome, wherein the Zea Longa neurological function score is used to evaluate neurological deficits, and TTC staining is combined to determine whether the acute stroke model is successful, and the observation of manifestations indicates that it is consistent with the phlegm, blood stasis and toxicity syndrome, and the tail end blood perfusion volume, liver pathological staining, blood lipid level, blood rheology, inflammatory factors, etc. are combined to jointly evaluate whether the phlegm, blood stasis and toxicity syndrome model is successful;

[0013] The TTC staining includes: after blood is collected from the abdominal aorta, the brain is quickly opened and the brain is taken out, which is then washed with physiological saline and immediately cut into 6 slices along the coronal longitudinal direction. The brain slices are then immersed in 37°C, 1.2% TTC dye solution, stained for 5 minutes in the dark, and then fixed in tissue fixative for 24 hours. Photos of the distribution of infarct foci in the brain slices are taken under the same light source and angle, and the total area and ischemic area of ​​each brain slice are drawn using Image proplus software, and the proportion of cerebral infarction area is calculated. The calculation method of the proportion of cerebral infarction area is: proportion of cerebral infarction area (%) = infarction area / total brain slice area × 100%.

[0014] Furthermore, the use of neurological function scoring includes: 24 hours after the rat MCAO modeling, using the Zea Longa method to score the neurological function to determine whether neurological function impairment occurs.

[0015] Furthermore, the observation characteristics include: after anesthesia, observing the tongue, eyes, ears, claws, and tail of the rat to see if they are black. If the color is dark, it indicates that it meets the syndrome of phlegm, blood stasis and toxin.

[0016] Furthermore, the PeriCam PSI blood perfusion speckle imager was used to detect the blood perfusion volume of the rat tail. The cursor was focused on 1 cm above the tip of the rat tail to begin observing and recording the blood perfusion volume of the tail. The laser blood perfusion speckle image was generated, and the PeriCam PSI supporting software PIM Soft was used to analyze the average blood perfusion volume of the tail of each group of rats. A decrease in the average blood perfusion volume indicated that the blood stasis syndrome model was successfully established.

[0017] Furthermore, the liver pathological staining specifically includes: selecting 2 rats in each group, fixing the liver tissue with 4% paraformaldehyde, rinsing with PBS solution, dehydrating, waxing, embedding, and slicing with a thickness of 4 μm, incubating with 60% isopropanol for 2 minutes, sealing and incubating with 0.5% Oil Red O solution for 8 minutes, rinsing and decolorizing with isopropanol solution, washing with water, counter-staining with hematoxylin, washing with water, and sealing with gelatin. The morphology of rat liver tissue was observed under an optical microscope. The presence of a large number of fat droplets and lipid accumulation in the liver tissue indicated that the phlegm syndrome model was successfully established.

[0018] Furthermore, blood lipid level detection includes: 2 ml of blood is collected from the abdominal aorta, centrifuged at 3000 r / min for 15 min, and 400 μL of serum is collected to measure the triglyceride, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol content, and very low-density lipoprotein in rat serum using a fully automatic blood biochemical analyzer. Abnormal changes in blood lipid levels indicate that the phlegm syndrome model is successfully established;

[0019] Furthermore, the blood rheology test includes: collecting blood in a heparin anticoagulation tube, and testing the viscosity of the whole blood sample at low shear rate (5s-1), medium shear rate (60s-1) and high shear rate (150s-1) according to the fully automatic blood rheology tester. Subsequently, the whole blood sample was centrifuged at 3500r / min for 10min, plasma was collected, and the plasma viscosity value was tested. Abnormal changes in blood rheology indicate that the stasis syndrome modeling is successful.

[0020] Furthermore, the detection of inflammatory factors includes: the rats are decapitated to remove the brain tissue, placed on ice, physiological saline is added in a certain proportion, the brain tissue is homogenized with a homogenizer, the supernatant is aspirated after centrifugation, part of which is stored in a -80°C refrigerator for standby use, and the rest is used for ELISA detection. The operation is strictly carried out according to the steps in the kit instructions, 50L of the diluted tissue homogenate sample is added to the reaction well, antibodies and enzyme conjugates are added, incubated and washed respectively, and a color substrate is added to make a color gradient appear in the standard well, and then the stop solution is added to terminate the reaction. On the microplate reader, at 450m, the blank control well is adjusted to zero and the absorbance value of each well is detected. A standard curve is drawn according to the concentration and OD value of the standard, and the serum IL-6, IL-1β, TNF-α and CRP levels of rats in each group are calculated. The increase of inflammatory factors indicates that the toxicity model is successfully established.

[0021] The construction and evaluation method provided by the present invention has the advantage over other disease model construction methods in that the present method constructs an animal model combining disease and syndrome, which is more in line with the actual clinical situation, and not only meets the manifestations of acute stroke neurological deficits, but also meets the syndrome manifestations of phlegm, blood stasis and toxins such as progression, poor prognosis and various symptoms through the administration of high-fat diet, carrageenan and other modeling factors. Other conventional methods only simulate the core manifestations of stroke, and it is difficult to fully and truly reflect the characteristics of the disease. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiment of the present application will be clearly and completely described below in combination with the embodiment of the present application. Obviously, the described embodiment is only a part of the embodiment of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present application.

[0023] The method for constructing and evaluating an acute ischemic stroke animal model of phlegm, blood stasis and toxin syndrome provided in an embodiment of the present invention comprises the following steps:

[0024] 1. Animal Model Construction

[0025] 1. Animals

[0026] SPF male SD rats, weighing (160-180) g, were placed in a constant temperature environment of 18-22°C and humidity controlled at 50% to 60%, and were allowed to eat and drink freely.

[0027] (II) Drugs

[0028] Carrageenan (Ca) was prepared with saline to 1 g·L -1 Solution, freshly prepared and used, then 20 mg kg rat body weight －1Administer medication.

[0029] (III) Specific construction method

[0030] The rats were adaptively fed for 1 week and fed with a high-fat diet; from the 26th day, they were given an intraperitoneal injection of 20 mg / kg carrageenan (Ca) for 3 consecutive days; they were fed until the morning of the 29th day, and the MCAO / R suture method was used to prepare the rat cerebral ischemia / reperfusion model, which specifically included: fasting for 12 hours before modeling and giving a small amount of water; the rats were anesthetized by intraperitoneal injection of 3% pentobarbital, and the injection volume was 2 ml / kg according to the rat's body weight; after successful anesthesia, the rats were fixed in a supine position, and the right common carotid artery was exposed and separated; a "V"-shaped incision was cut at the separated common carotid artery, from which the suture was inserted into the internal carotid artery, and advanced until the middle cerebral artery was occluded and ligated; the moment when the suture was inserted into the middle cerebral artery was taken as time 0; the skin was sutured after the operation, and the end of the suture was exposed to the outside of the body; the suture was pulled out after 1.5 hours to simulate cerebral ischemia and reperfusion.

[0031] (IV) Evaluation methods

[0032] The evaluation methods include the evaluation methods for acute ischemic stroke and the evaluation methods for phlegm, blood stasis and toxicity syndrome. Among them, the Zea Longa neurological function score is used to evaluate neurological deficits, and TTC staining is combined to determine whether the acute stroke model is successful. The observation of manifestations indicates that it is consistent with the phlegm, blood stasis and toxicity syndrome. At the same time, the tail blood perfusion volume, liver pathological staining, blood lipid level, blood rheology, inflammatory factors, etc. are combined to jointly evaluate whether the phlegm, blood stasis and toxicity syndrome model is successful. Specifically including:

[0033] 1 Neurological function scoring and TTC staining

[0034] 24 hours after MCAO modeling in rats, the neurological function was scored using the Zea Longa method to determine whether the modeling was successful. Rats with scores of 1-3 were considered successful models, indicating neurological deficits and successful acute stroke modeling. After the modeling, the neurological function of rats in each group was scored, and the neurological function scoring standard was established with reference to the Zea Longa method: 0 points: no neurological deficit symptoms; 1 point: the left front paw could not be fully extended when the tail was lifted; 2 points: turning to the left when walking; 3 points: leaning to the left when walking; 4 points: decreased level of consciousness and inability to walk spontaneously.

[0035] TTC staining includes: after blood is collected from the abdominal aorta, the brain is quickly opened and removed, and then washed with physiological saline and immediately cut into 6 slices along the coronal longitudinal direction. The brain slices are then immersed in 37°C, 1.2% TTC dye solution, stained for 5 minutes in the dark, and then fixed in tissue fixative for 24 hours. Photos of the distribution of infarct foci in brain slices are taken under the same light source and angle, and the total area and ischemic area of ​​each brain slice are drawn using Image pro plus software to calculate the proportion of cerebral infarction area. The calculation method of the proportion of cerebral infarction area is: proportion of cerebral infarction area (%) = infarction area / total brain slice area × 100%.

[0036] 2 Characterization

[0037] After anesthesia, observe the tongue, eyes, ears, claws, and tail of the rat to see if they are black. If the color is dark, it indicates that the rat has a syndrome of phlegm, blood stasis, and toxicity. Other indicators should be combined to evaluate whether the model is successful.

[0038] 3. Tail blood perfusion

[0039] The PeriCam PSI blood perfusion speckle imager was used to detect the blood perfusion volume of the rat tail. The cursor was focused 1 cm above the tip of the rat tail, and the blood perfusion volume of the tail began to be observed and recorded. The laser blood perfusion speckle image was generated, and the PeriCam PSI supporting software PIM Soft was used to analyze the average blood perfusion volume of the tail of each group of rats. The decrease in the average blood perfusion volume indicated that the stasis syndrome model was successfully established.

[0040] 4. Liver pathological staining

[0041] The liver tissues of 2 rats in each group were fixed with 4% paraformaldehyde, rinsed with PBS solution, dehydrated, waxed, embedded, sliced, with a thickness of 4μm, incubated with 60% isopropanol for 2min, sealed with 0.5% Oil Red O solution for 8min, rinsed with isopropanol solution for decolorization, washed with water, counterstained with hematoxylin, washed with water, sealed with gelatin, and the morphology of rat liver tissue was observed under an optical microscope. The presence of a large number of lipid droplets and lipid accumulation in the liver tissue indicated that the phlegm syndrome model was successfully established.

[0042] 5. Blood lipid level test

[0043] 2 ml of blood was collected from the abdominal aorta, centrifuged at 3000 r / min for 15 min, and 400 μL of serum was taken to measure the triglyceride, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and very low-density lipoprotein in the rat serum using an automatic blood biochemical analyzer. Abnormal changes in blood lipid levels indicate that the phlegm syndrome model was successfully established.

[0044] 6. Blood rheology test

[0045] Blood rheology testing includes: collecting blood in heparin anticoagulation tubes, and testing the viscosity of whole blood samples at low shear rate (5s-1), medium shear rate (60s-1) and high shear rate (150s-1) according to the fully automatic blood rheology tester. Subsequently, the whole blood sample was centrifuged at 3500r / min for 10min, plasma was collected, and the plasma viscosity value was tested. Abnormal changes in blood rheology indicate that the stasis syndrome model was successfully established.

[0046] 7. Detection of brain tissue inflammatory factors IL-6, IL-1β, TNF-α, and CRP

[0047] The rats were decapitated and the brain tissue was removed. The brain tissue was placed on ice, and physiological saline was added in a certain proportion. The brain tissue was homogenized with a homogenizer. After centrifugation, the supernatant was aspirated, and part of it was stored in a -80℃ refrigerator for standby use, and the rest was used for ELISA detection. Strictly follow the steps in the kit instructions, add 50L of the diluted tissue homogenate sample to the reaction well, add antibodies and enzyme conjugates, incubate (37℃), wash, add color substrate to make the standard wells have a color gradient, and then add the stop solution to terminate the reaction. On the microplate reader, at 450m, the blank control well was adjusted to zero and the absorbance (OD) value of each well was detected. The standard curve was drawn according to the concentration and OD value of the standard, and the serum IL-6, IL-1β, TNF-α and CRP levels of rats in each group were calculated. The increase of inflammatory factors indicates that the toxicity model is successfully established.

[0048] It should be understood that some of the model evaluations in the present invention are evaluated in a living state, including: neurological function score, characterization, and tail blood flow perfusion. Others require anesthesia and sacrificial sampling before testing, including: TTC staining, liver pathology, blood lipids, coagulation, and inflammatory factors. Among them, neurological function score and TTC staining are disease evaluation methods, and the rest are syndrome evaluation methods.

[0049] In this embodiment, each step in the evaluation method is introduced separately according to the disease and syndrome, and is not described in a strict order of survival and death.

[0050] The animal model construction and evaluation method of the present invention can fully simulate the syndrome of phlegm, blood stasis and toxins in acute ischemic stroke, and has the following technical effects: it is more in line with the actual clinical situation and meets the manifestation of neurological deficits in acute stroke. By giving modeling factors such as high-fat feed and carrageenan, it meets the syndrome manifestations of phlegm, blood stasis and toxins. It can fully and truly reflect the characteristics of the disease, and provides an effective experimental model for studying the syndrome of phlegm, blood stasis and toxins in acute ischemic stroke.

[0051] The above is only a preferred implementation of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A method for constructing and evaluating an animal model of acute ischemic stroke with phlegm, blood stasis and toxin syndrome, characterized in that: The steps include: The rats were adaptively fed for 1 week and fed with a high-fat diet. From the 26th day, 20 mg / kg of carrageenan (Ca) was intraperitoneally injected for 3 consecutive days. The rats were fed until the morning of the 29th day, and the cerebral ischemia / reperfusion model of rats was prepared by the MCAO / R suture method, which specifically included: fasting for 12 hours before modeling and giving a small amount of water; the rats were anesthetized by intraperitoneal injection of 3% pentobarbital (2 ml / kg); after successful anesthesia, the rats were fixed in a supine position, and the right common carotid artery was exposed and separated; a "V"-shaped incision was made at the separated common carotid artery, from which the suture was inserted into the internal carotid artery, and advanced until the middle cerebral artery was occluded and ligated; the moment when the suture was inserted into the middle cerebral artery was taken as time 0; the skin was sutured after the operation, and the end of the suture was exposed outside the body; the suture was pulled out after 1.5 hours to simulate cerebral ischemia and reperfusion.

2. The construction and evaluation method according to claim 1, characterized in that: The carrageenan (Ca) was prepared with physiological saline to 1 g·L -1 Solution, freshly prepared and used, then 20 mg kg rat body weight －1 Administer medication.

3. The construction and evaluation method according to claim 1, characterized in that: The rats are SPF grade male SD rats, weighing (160-180) g, and are placed in a constant temperature environment of 18-22° C., with humidity controlled at 50% to 60%, and are free to eat and drink.

4. The construction and evaluation method according to claim 1, characterized in that: The evaluation method includes an evaluation method for acute ischemic stroke and an evaluation method for phlegm, blood stasis and toxicity syndrome, wherein the Zea Longa neurological function score is used to evaluate neurological deficits, and TTC staining is combined to determine whether the acute stroke model is successful, and the observation of manifestations indicates that it is consistent with the phlegm, blood stasis and toxicity syndrome, and at the same time, the tail blood perfusion volume, liver pathological staining, blood lipid level, blood rheology, inflammatory factors, etc. are combined to jointly evaluate whether the phlegm, blood stasis and toxicity syndrome model is successful; The TTC staining comprises: after blood is collected from the abdominal aorta, the brain is quickly opened and the brain is taken out, which is then washed with physiological saline and immediately cut into 6 slices along the coronal plane, and the brain slices are then immersed in 37°C, 1.2% TTC dye solution, stained for 5 minutes in the dark, and then fixed in tissue fixative for 24 hours, and photos of the distribution of infarct foci in the brain slices are taken under the same light source and angle, and the total area and ischemic area of ​​each brain slice are drawn using Image pro plus software, and the proportion of cerebral infarction area is calculated. The calculation method of the proportion of cerebral infarction area is: proportion of cerebral infarction area (%) = infarction area / total brain slice area × 100%.

5. The construction and evaluation method according to claim 4, characterized in that: The use of neurological function scoring includes: 24 hours after the rat MCAO modeling, using the Zea Longa neurological function scoring method to score the neurological function to determine whether neurological function impairment occurs.

6. The construction and evaluation method according to claim 4, characterized in that: The observation characteristics include: after anesthesia, observing the tongue, eyes, ears, claws, and tail of the rat to see if they are black. If the color is dark, it indicates that the syndrome of phlegm, blood stasis and toxin is met.

7. The construction and evaluation method according to claim 4, characterized in that: The PeriCam PSI blood perfusion speckle imager was used to detect the blood perfusion volume of the rat tail. The cursor was focused on 1 cm above the tip of the rat tail to begin observing and recording the blood perfusion volume of the tail. The laser blood perfusion speckle image was generated, and the PeriCam PSI supporting software PIM Soft was used to analyze the average blood perfusion volume of the tail of each group of rats. A decrease in the average blood perfusion volume indicated that the stasis syndrome model was successfully established.

8. The construction and evaluation method according to claim 4, characterized in that: The liver pathological staining specifically includes: selecting 2 rats in each group to fix the liver tissue with 4% paraformaldehyde, rinsing with PBS solution, dehydrating, waxing, embedding, and slicing with a thickness of 4 μm, incubating with 60% isopropanol for 2 minutes, sealing and incubating with 0.5% Oil Red O solution for 8 minutes, rinsing and decolorizing with isopropanol solution, washing with water, counter-staining with hematoxylin, washing with water, and sealing with gelatin. The morphology of rat liver tissue was observed under an optical microscope. The presence of a large number of fat droplets and lipid accumulation in the liver tissue indicated that the phlegm syndrome model was successfully established.

9. The construction and evaluation method according to claim 4, characterized in that: Blood lipid level detection includes: 2 ml of blood is collected from the abdominal aorta, centrifuged at 3000 r / min for 15 min, and 400 μL of serum is collected to measure the triglyceride, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and very low-density lipoprotein in rat serum using a fully automatic blood biochemical analyzer. Abnormal changes in blood lipid levels indicate that the phlegm syndrome model is successfully established; The blood rheology test includes: collecting blood in a heparin anticoagulation tube, and detecting the viscosity of the whole blood sample at low shear rate (5s-1), medium shear rate (60s-1) and high shear rate (150s-1) according to the detection method of the fully automatic blood rheology tester. Subsequently, the whole blood sample is centrifuged at 3500r / min for 10min, plasma is collected, and the plasma viscosity value is detected. Abnormal changes in blood rheology indicate that the stasis syndrome model is successfully established.

10. The construction and evaluation method according to claim 4, characterized in that: The detection of inflammatory factors includes: removing the brain tissue from the rat by decapitation, placing it on ice, adding physiological saline in a certain proportion, homogenizing the brain tissue with a homogenizer, aspirating the supernatant after centrifugation, storing part of it in a -80℃ refrigerator for standby use, and using the rest for ELISA detection. Strictly follow the steps in the kit instructions, add 50L of the diluted tissue homogenate sample to the reaction well, add antibodies and enzyme conjugates, incubate and wash them separately, add chromogenic substrate to make a color gradient appear in the standard well, then add stop solution to terminate the reaction, and on the microplate reader, at 450m, adjust the blank control well to zero and detect the absorbance value of each well, draw a standard curve based on the concentration and OD value of the standard, calculate the serum IL-6, IL-1β, TNF-α and CRP levels of rats in each group, and the increase of inflammatory factors indicates that the toxicity model is successfully established.