Rat model with cold-dampness obstruction type lumbago and modeling and evaluation method thereof

Abstract

The invention relates to the technical field of animal model construction, in particular to a cold-dampness arthralgia type lumbago rat model and a modeling and evaluation method thereof. The modeling method comprises the following steps that an inflammatory agent is injected in the erector spinalis muscle stage of L3-L5 segments of a rat, then ice boxes are placed in a rat cage once in the morning and once in the evening every day, the rat is fed with ice water every day, and ice-water bath is conducted on the rat for 2.8-3.2 weeks. The modeling method is convenient to operate, high in success rate and low in fatality rate, the animal model basically conforming to the clinical manifestation of the cold-dampness arthralgia type lumbago can be successfully constructed, and a basis is provided for clinical research of the cold-dampness arthralgia type lumbago.

Description

Technical Field

[0001] The invention relates to the technical field of animal model construction, and in particular to a rat model of low back pain of cold-dampness arthralgia type and a modeling and evaluation method thereof. Background Art

[0002] Low back pain is a common clinical disease. Literature reports that 90% to 95% of adults have experienced low back pain. The causes are complex and can easily develop into chronic low back pain. Currently, the treatments for nonspecific low back pain are mainly oral analgesics and local physical therapy. These treatments are either due to long-term medication and its side effects, or due to repeated and high physical therapy costs, causing many patients to have to endure the pain for a long time, which seriously affects the daily quality of life of patients and also brings a serious health economic burden to the country and society.

[0003] Traditional medicine believes that lumbago should be classified as "lumbar pain", "arthralgia", "pain syndrome" and other categories. It is mostly caused by external infection, internal injury or bruises and falls, which lead to poor circulation of qi and blood in the waist, or lack of nourishment, causing pain in the waist and spine and both sides. "Suwen" says: "The three qi of wind, cold and dampness are mixed together and become arthralgia." It also says: "If you feel cold, the patient's joints will be restrained, the waist will be stabbed, and the cold and dampness will be in the intersection of qi and become a disease. "Jingyue Complete Book" discusses lumbago mainly in Volume 16 Lumbago. The book records: "There are five distinctions for lumbago syndrome: one is yang deficiency and deficiency, and Shaoyin kidney failure. The second is wind arthralgia, wind cold, and dampness lumbago." "Lumbago syndrome, all those who are lingering and recurrent are due to kidney deficiency. If it rains or sits for a long time, the pain is severe, it is dampness. If it hurts when it is cold, or if you like warmth and hate cold, it is cold. "From this point of view, the symptoms of low back pain should be mainly qi stagnation and blood stasis, cold and dampness obstruction, dampness and heat obstruction, and liver and kidney deficiency.

[0004] Cold-dampness arthralgia syndrome is a common syndrome in TCM clinical practice, and its main manifestations are: aversion to cold and preference for warmth, pale complexion, cold limbs curled up, heavy body, limb aches, etc. Cold-dampness arthralgia syndrome as a TCM syndrome can appear in a variety of diseases, such as cold-dampness blocking the lungs causing lung diseases, cold-dampness accumulating in the spleen causing digestive tract diseases, and cold-dampness waist pain causing waist and back pain. Lingnan is located in the southeast of the motherland, with a subtropical monsoon climate, rain and heat at the same time, and dampness is more prevalent. Reliance on air conditioning and eating habits that are greedy for coolness and prefer coldness will lead to the combination of cold-dampness evil inside and outside. In addition, the modern people's sedentary and less active lifestyle has caused the waist and back muscles to atrophy relatively. Many factors are combined, which is easy to cause low back pain. When the blood stasis and qi stagnation caused by setbacks and falls cause low back pain, modern people cannot rest fully due to the greater work and life pressure. When they encounter cold evil, the blood stasis is affected by the cold and cannot flow for a long time. If the blood stasis does not flow, the qi and blood will not flow, and the blood vessels will be blocked and various pains will arise. Therefore, low back pain caused by cold-dampness and arthralgia is becoming an increasingly common clinical syndrome.

[0005] Constructing an animal model of low back pain caused by cold-dampness and arthralgia will facilitate in-depth research on the pathogenic mechanism and pathological manifestations of low back pain caused by cold-dampness and arthralgia, and provide a basis for the next step of exploring effective treatment methods with Chinese medicine characteristics, which is a real need of current clinical work. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a rat model of low back pain of cold-dampness arthralgia type and a modeling and evaluation method thereof, and to perform a standard evaluation on whether the modeling is successful or not.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for modeling a rat model of low back pain caused by cold-dampness and arthralgia, comprising the following steps: injecting an inflammatory agent into the erector spinae muscle stage of the L3-L5 segment of the rat, then placing an ice box in the mouse cage once in the morning and once in the evening every day, feeding the rats with ice water and giving the rats an ice water bath every day for 2.8 to 3.2 weeks.

[0008] Another technical solution adopted by the present invention is: a rat model is obtained by using the above-mentioned method for establishing the cold-dampness arthralgia type low back pain rat model.

[0009] Another technical solution adopted by the present invention is: the evaluation method of the above rat model comprises the following steps: using behavioral detection, HE staining and Masson staining, infrared thermal imaging of the rat back, and serum ELISA detection to determine whether the rat model is successfully established;

[0010] The behavioral tests include a tail suspension test and a thermophilic test.

[0011] The beneficial effects of the present invention are as follows: the present invention provides a modeling method, which is easy to operate, has a high success rate and a low mortality rate, and can successfully construct an animal model that is basically consistent with the clinical manifestations of cold-dampness arthralgia type low back pain, providing a basis for clinical research on cold-dampness arthralgia type low back pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a statistical diagram of the results of the tail suspension experiment in Example 3 of the present invention;

[0013] Figure 2 This is a schematic diagram of the structure of the thermophilic experimental box in the third embodiment of the present invention;

[0014] Figure 3 This is a statistical chart of the results of the thermophilic experiment in Example 3 of the present invention;

[0015] Figure 4 This is a HE-stained cross-section of the blank group in Example 3 of the present invention;

[0016] Figure 5 This is a HE-stained longitudinal section of the blank group in Example 3 of the present invention;

[0017] Figure 6 This is a Masson-stained cross-section of the blank group in Example 3 of the present invention;

[0018] Figure 7 This is a Masson-stained longitudinal section of the blank group in Example 3 of the present invention;

[0019] Figure 8 This is a HE-stained cross-section of the Freund's adjuvant group in Example 3 of the present invention;

[0020] Fig. 9 This is a HE-stained longitudinal section of the Freund's adjuvant group in Example 3 of the present invention;

[0021] Fig.10 This is a Masson-stained cross-section of the Freund's adjuvant group in Example 3 of the present invention;

[0022] Fig.11 This is a Masson-stained longitudinal section of the Freund's adjuvant group in Example 3 of the present invention;

[0023] Fig.12 This is a cross-sectional view of the cold-dampness group in Example 3 of the present invention stained with HE;

[0024] Fig.13 This is a HE-stained longitudinal section of the cold-dampness group in Example 3 of the present invention;

[0025] Fig.14 This is a Masson-stained cross-section of the cold-dampness group in Example 3 of the present invention;

[0026] Fig.15 This is a Masson-stained longitudinal section of the cold-dampness group in Example 3 of the present invention;

[0027] Fig.16 This is a HE staining cross-section of the Freund's adjuvant + cold and dampness group in Example 3 of the present invention;

[0028] Fig.17 This is a HE-stained longitudinal section of the Freund's adjuvant + cold-dampness group in Example 3 of the present invention;

[0029] Fig.18 This is a Masson-stained cross-section of the Freund's adjuvant + cold-dampness group in Example 3 of the present invention;

[0030] Fig.19 This is a Masson-stained longitudinal section of the Freund's adjuvant + cold-dampness group in Example 3 of the present invention;

[0031] Fig. 20 This is a statistical diagram of the muscle area ratio of the modeling area on the back of rats stained with HE in Example 3 of the present invention;

[0032] Fig.21 This is a statistical diagram of the percentage of muscle fibrosis area in the back modeling area of ​​rats stained with HE in Example 3 of the present invention;

[0033] Fig. 22 This is the infrared thermal imaging image of the back of a rat in Example 3 of the present invention;

[0034] Fig.23 This is a temperature color scale diagram of the rat back in Example 3 of the present invention;

[0035] Fig.24 This is the infrared thermal imaging image of the rat back with a temperature greater than 33°C in Example 3 of the present invention;

[0036] Fig.25 This is a statistical diagram of infrared thermal imaging results in Example 3 of the present invention;

[0037] Fig.26 This is another statistical graph of infrared thermal imaging results in the third embodiment of the present invention;

[0038] Fig. 27 This is a statistical diagram of the relative expression of IL-6 in muscle in Example 3 of the present invention;

[0039] Description of labels:

[0040] 1. Room temperature area; 2. High temperature area; 3. Partition board. DETAILED DESCRIPTION

[0041] In order to explain the technical content, achieved objectives and effects of the present invention in detail, the following is an explanation in combination with the implementation modes and the accompanying drawings.

[0042] A method for establishing a rat model of low back pain caused by cold-dampness and arthralgia comprises the following steps: injecting an inflammatory agent into the erector spinae muscle stage of the L3-L5 segment of the rat, then placing an ice box in the rat cage once in the morning and once in the evening every day, feeding the rat with ice water and bathing the rat in ice water every day for 2.8 to 3.2 weeks.

[0043] From the above description, it can be seen that the beneficial effect of the present invention is that: the current academic community prepares animal models of cold-dampness and arthralgia mainly by creating a damp and cold environment, but the standards for the damp and cold environment are not unified. Based on this, the present invention provides a modeling method that can successfully construct an animal model that is basically consistent with the clinical manifestations of cold-dampness and arthralgia type low back pain, laying a foundation for clinical research on cold-dampness and arthralgia type low back pain, research on its mechanism of action, and screening or preparation of therapeutic drugs.

[0044] The modeling method of the present invention simulates the living behavior habits of patients with cold-dampness arthralgia as much as possible, such as liking cold drinks (the preservation temperature of a household refrigerator is about 0-5°C), taking cold baths (ice water baths), and liking to be in air-conditioned room temperatures, etc., which more realistically reflects the environment to which the human body is exposed. Compared with fixed humidity or temperature, this modeling method is more practical and easy to operate.

[0045] Furthermore, the weight of the ice box is 300-400g.

[0046] Furthermore, when the rats were fed with ice water, the temperature of the ice water was 3-5° C., and the content of the ice water was 4-6 mL.

[0047] Furthermore, the temperature of the ice water bath is 3-5°C and the time is 4-6 minutes.

[0048] From the above description, we can see that cold-dampness arthralgia is a chronic disease. The temperature of ice water feeding and ice water bath should not be too low, and the duration should not be too long.

[0049] Another technical solution adopted by the present invention is: a rat model is obtained by using the above-mentioned method for establishing the cold-dampness arthralgia type low back pain rat model.

[0050] Another technical solution adopted by the present invention is: the evaluation method of the above rat model comprises the following steps: using behavioral detection, HE staining and Masson staining, infrared thermal imaging of the rat back, and serum ELISA detection to determine whether the rat model is successfully established;

[0051] Behavioral tests include tail suspension test and thermophilic test.

[0052] From the above description, it can be seen that the present invention determines whether the rat model is successful through back and foot pain thresholds, tail suspension test, thermophilic test, HE staining and Masson staining, rat back infrared thermal imaging, and serum ELISA test. The judgment method is accurate and quantifiable.

[0053] Furthermore, the indicator of the tail suspension experiment is the despair time.

[0054] From the above description, we can know that the conventional tail suspension test can be used to judge the efficacy of sedative drugs, and is also often used to verify the judgment of antidepressant drugs. When the tail is suspended, the activity frequency and time are reduced accordingly. The efficacy of the drug is judged by judging the despair time (i.e. the time of immobility) of rats or mice; a reduction in despair time indicates that the drug is effective, thereby verifying its efficacy.

[0055] In the present invention, the modeling site is located at the back, and the pain area is also located at the back. When the rat is suspended by the tail, it will continuously roll up its abdomen, which will stretch the back muscles and induce pain. The longer the despair time, the more obvious the back pain. By comparing the differences between different groups, the degree of back pain caused by the modeling is judged, thereby verifying whether the modeling is successful.

[0056] Furthermore, the thermophilic experiment is carried out in a thermophilic experimental box, in which a high temperature zone and a room temperature zone are arranged, and the temperature of the high temperature zone is higher than that of the room temperature zone.

[0057] Furthermore, the indicator of the thermophilic experiment was the length of time the rats were in the high temperature zone.

[0058] Furthermore, a pressure sensor is provided on the bottom of the high temperature zone of the thermophilic experiment box, which can sense the pressure in the high temperature zone. During the thermophilic experiment, when the high temperature zone senses a pressure greater than 200g and the duration exceeds 3s, timing starts.

[0059] Furthermore, there is a partition plate between the high temperature zone and the room temperature zone.

[0060] Furthermore, the temperature in the high temperature zone is 42-45°C.

[0061] Furthermore, the observation time of the thermophilic experiment was 0.5 h.

[0062] From the above description, it can be seen that rodents like dark, damp and cold environments. The main manifestation of cold and dampness syndrome is cold syndrome with fear of cold and preference for warmth. The thermophilic experiment of the present invention determines whether the rat model is successful by the rat's preference for different temperature zones.

[0063] Furthermore, the indicators of HE staining and Masson staining include: uniformity of staining, staining morphology, number of cell nuclei and proliferation of skeletal muscle in the L3-L5 segment of the rat back.

[0064] From the above description, we can know that the muscle fibers in the L3-L5 skeletal muscle cross-section of the back of the rats with cold-dampness arthralgia modeling were unevenly stained, irregular in shape, of different sizes, incomplete sarcolemma, significantly increased nuclei, some of which were round, inflammatory cell infiltration of degenerated and necrotic muscle fibers, and a small number of vacuoles and connective tissue hyperplasia to varying degrees; the longitudinal section showed muscle fiber dissolution, degeneration, interrupted continuity, and a large number of vacuoles. The above characteristics can be used to determine whether the rat modeling is successful.

[0065] Furthermore, the indicator of infrared thermal imaging of the rat's back is the temperature zone area of ​​the rat's back.

[0066] Furthermore, the indicator of infrared thermal imaging of the rat's back is the area of ​​the rat's back that is greater than 33°C.

[0067] From the above description, we can know that the temperature of the back modeling area can be quantified through infrared thermal imaging. The manifestation of cold syndrome can be judged by the temperature. The area of ​​rats with successful modeling greater than 33℃ accounts for a smaller proportion, so the result of their cold syndrome can be judged digitally.

[0068] Furthermore, the indicators of serum ELISA detection were the levels of IL-6, TNFα and IL-1β.

[0069] From the above description, it can be seen that the expression level of IL-6 in the rats with successful modeling was significantly reduced, and the expression level of IL-1β was significantly increased.

[0070] Embodiment 1 of the present invention is: a method for modeling a rat model of low back pain of cold-dampness and arthralgia, the steps of which are as follows: 2 mL of Freund's adjuvant is injected at the erector spinae stage of the L3-L5 segment of the rat. Then a 360 g ice box is placed at the edge of a rat cage in a square area of ​​20*40 cm, once in the morning and evening, each time for 4 hours; 5 mL of 4°C ice water is fed every day, and the rat is bathed in cold water at 5°C for 5 minutes every day for 3 weeks.

[0071] The second embodiment of the present invention is: a rat model obtained by adopting the modeling method of the first embodiment.

[0072] Embodiment 3 of the present invention is: a method for evaluating a rat model, the steps are as follows:

[0073] 1. Experimental method: 17 SD rats were divided into four groups: blank group (4 rats), Freund's adjuvant group (4 rats), cold and dampness group (4 rats), and Freund's adjuvant + cold and dampness group (5 rats).

[0074] 1.1 Blank group: The rats were injected with 2 mL of normal saline in the muscle tissue 0.5-1 cm beside the spine in the middle of the L3-L5 segment and fed for three weeks.

[0075] 1.2 Freund's adjuvant group: 2 mL of Freund's adjuvant was injected into the muscle tissue 0.5-1 cm away from the spine in the middle of the L3-L5 segment of rats and the rats were fed for three weeks.

[0076] 1.3 Cold and dampness group: A 360g ice box was placed on the edge of the mouse cage in a square area of ​​20*40cm, once in the morning and evening, each time for 4 hours; 5mL of 4℃ ice water was fed every day; and a 5℃ cold water bath was taken for 5min every day for 3 weeks.

[0077] 1.4 Freund's adjuvant + cold and dampness group: 2 mL of Freund's adjuvant was injected into the erector spinae muscle stage in the middle of the L3-L5 segment of the rats. Then a 360 g ice box was placed on the edge of the rat cage in a square area of ​​20*40 cm, once in the morning and evening, each time for 4 hours; 5 mL of 4°C ice water was fed every day, and the rats were bathed in 5°C cold water for 5 minutes every day for 3 weeks.

[0078] 2. The four groups of rats were recorded and measured separately. The following experimental data were processed using SPSS 21.0 software, and the measurement data results were expressed as (x±s). LSD was used for pairwise comparisons between multiple groups, and P < 0.05 was statistically significant.

[0079] 2.1 Behavioral related indicators

[0080] 2.1.1 Tail suspension experiment: The rats were suspended by their tails and the duration of their despair was recorded. The experimental results are shown in Figure 1 And Table 1.

[0081] Table 1

[0082] Day 7 Day 14 Day 21 Blank Group 55.5±17.52 64.25±13.84## 66.75±15.11## Freund's adjuvant group 84.75±9.91* 95.5±14.15# 110.75±15.76**# Cold and damp group 84.75±21.25* 94.25±20.04# 131.25±12.76*# Cold and dampness + Freund's adjuvant group 83.8±6.83*# 127.8±27.85** 170.2±20.03**

[0083] Note: Compared with the blank control group, *P<0.05; # Compared with the cold and dampness + Freund's adjuvant group, # P<0.05, ## P<0.01.

[0084] When the rats are suspended by their tails, they will constantly curl up their abdomens, stretch their back muscles, induce pain, and thus avoid desperate struggles, resulting in a "behavioral despair state". The longer the despair state lasts, the more obvious the back pain is; by comparing the differences between different groups, we can determine whether the model causes back pain. Figure 1 As can be seen from Table 1 , the Freund’s adjuvant + cold-dampness group had a longer “behavioral despair state” compared to the other groups, and it can be reasonably inferred that the rats avoided more struggles due to back pain.

[0085] 2.1.2 Thermophilic experiment

[0086] A self-designed thermophilic experimental box was used, as shown in the schematic diagram. Figure 2 , which is implemented as follows:

[0087] The thermophilic test box is divided into four identical animal activity areas along the length direction, which can complete the thermophilic test of 4 rats at the same time; it is divided into two parts along the width direction: room temperature area 1 and high temperature area 2 (which can achieve constant temperature control from room temperature to 78°C), and high temperature area 2 and room temperature area 1 are separated by partition board 3. There is a gap with a width of 7cm under the partition board 3, through which rats can freely enter the areas on both sides. A pressure sensor is provided on the bottom of the high temperature area of ​​the thermophilic test box.

[0088] Instructions for use of the thermophilic experimental box: S1: Set the temperature range of the high temperature zone to 42-45°C; S2: Place the rat in the room temperature zone and the high temperature zone of the experimental box for 3 minutes each, so that it can be aware of the temperature conditions on both sides; S3: Place the rat in the room temperature zone, and start timing when the high temperature zone senses a pressure greater than 200g and the duration exceeds 3s; observe for 0.5h and record the total time the rat spends in the high temperature zone.

[0089] The experimental results are shown in Figure 3 and Table 2:

[0090] Table 2

[0091] Day 7 Day 14 Day 21 Blank Group 430.5±119.97# 378.75±164.30## 352±183.31## Freund's adjuvant group 383±81.073# 755.5±514.89# 613.25±487.83## Cold and damp group 729±144.24 1381.5±66.61* 1358.25±103.55* Cold and dampness + Freund's adjuvant group 750.2±255.77* 1544±99.98** 1521.8±138.31**

[0092] Note: Compared with the blank control group, *P<0.05, **P<0.01; # Compared with the cold and dampness + Freund's adjuvant group,

[0093] # P<0.05, ## P<0.01.

[0094] The habit of rodents is that they like dark, damp and cold environments. The main manifestation of cold and dampness syndrome is the cold syndrome of fear of cold and preference for warmth. The rats' preference for different temperature zones in the thermophilic experimental box can be used to determine whether the rats have cold and dampness syndrome. Figure 3 As shown in Table 2, the rats in the blank group and the Freund's adjuvant group were mostly in the room temperature area, while the rats in the cold-dampness group and the Freund's adjuvant + cold-dampness group were mostly in the constant high temperature area. Therefore, it can be judged that the rats in the cold-dampness group and the Freund's adjuvant + cold-dampness group were of the cold-dampness syndrome type.

[0095] 2.2 HE staining and Masson staining: The skeletal muscles of L3-L5 segments of the back of the rats were taken for HE staining and Masson staining respectively. The HE staining cross-section of the blank group is shown in Figure 4 , HE staining longitudinal section is shown in Figure 5 , Masson-stained cross-sections are shown in Figure 6 , Masson-stained longitudinal section Figure 7 ; HE staining cross section of Freund's adjuvant group is shown in Figure 8 , HE staining longitudinal section is shown in Fig. 9 , Masson-stained cross-sections are shown in Fig.10 , Masson-stained longitudinal section Fig.11 ; HE staining cross-section of cold-damp group is shown in Fig.12 , HE staining longitudinal section is shown in Fig.13 , Masson-stained cross-sections are shown in Fig.14 , Masson-stained longitudinal section Fig.15 ; HE staining cross-section of Freund's adjuvant + cold and dampness group is shown in Fig.16 , HE staining longitudinal section is shown in Fig.17 , Masson-stained cross-sections are shown in Fig.18 , Masson-stained longitudinal section Fig.19 .

[0096] The erector spinae muscles of rats can be observed under a light microscope after staining. Figures 4 to 19As can be seen: the cross-section of the muscle fiber of the blank group is uniformly stained pink, and the size is consistent and regular polygonal, closely arranged, and the semi-elliptical nuclei of visible purple blue are evenly distributed around the muscle fiber, and no nuclear hyperplasia and pyknosis occur; the longitudinal section of the blank group is neatly arranged, and the sarcolemma is complete, and there are no changes such as edema, congestion, and inflammatory cell infiltration. In the cross-section of the muscle fiber of the cold-damp group and the cold-damp+Freund's adjuvant group, the muscle fiber staining is uneven, the shape is irregular, and the size is different. The sarcolemma is not complete, and the nucleus increases significantly, and some nuclei are round, and the inflammatory cell infiltration of degenerated necrotic muscle fibers, a small amount of vacuoles, and connective tissue hyperplasia in varying degrees; The longitudinal section of the cold-damp group shows that the muscle fiber dissolves and degenerates, and the continuity is interrupted, and a large number of vacuoles are formed. In the Masson staining of the cold-damp group, Freund's adjuvant, and the cold-damp+Freund's adjuvant group, the muscle fiber (the blue part of the gap between the muscle fibers of Masson staining) shows various degrees of hyperplasia, and the hyperplasia of the cold-damp+Freund's adjuvant group is the most obvious.

[0097] ImageJ software was used to calculate the muscle area and muscle fibrosis area ratio of the HE-stained rat back modeling area. The calculation results are shown in Fig. 20 , 21 and Table 3.

[0098] Table 3

[0099] n Muscle ratio (%) Muscle fibrosis percentage Blank control group 4 91.56±5.82## 0.868±0.248## Freund's adjuvant 4 73.51±4.62*# 3.77±1.39# Cold and damp 4 68.57±4.77*# 2.66±0.9# Cold and dampness + Freund's adjuvant group 5 58.06±7.122* 13.75±10.82**

[0100] Note: Compared with the blank control group, *P<0.05, **P<0.01; # Compared with the cold and dampness + Freund's adjuvant group, # P<0.05, ## P<0.01.

[0101] 2.3 Infrared thermal imaging of rat back

[0102] Take the same position on the left and right sides of the back of each rat, measure the average temperature, maximum temperature and minimum temperature of the area, and calculate the temperature difference between the left and right sides. Figures 22-24 The highest color level was defined as the area above 33°C, and the area ratio above 33°C was measured by Image J.

[0103] The measurement results are shown in Fig.25 and 26 As well as Tables 4 and 5, ROI (Region Of Interest) is the observation area, ROI33L is the area ratio of the left observation area on the rat's back with a temperature greater than 33 degrees Celsius, and ROI 33R is the area ratio of the right observation area on the rat's back with a temperature greater than 33 degrees Celsius.

[0104] Table 4

[0105] ROI 33L ROI 33R t P Blank Group 35.28±30.46 27.58±44.07 0.74 0.513 Freund's adjuvant group 62.06±36.63 68.36±24.82 -0.529 0.633 Cold and damp group 42.86±29.65 53.78±18.81 -1.921 0.15 Cold and dampness + Freund's adjuvant group 45.85±16.78 55.52±15.14 -10.814 0.000

[0106] Table 5

[0107] Absolute minimum temperature / ℃ t P-value Blank Group 28.54±1.30 Freund's adjuvant group 31.69±0.84 -2.630 P<0.05 Cold and damp group 29.86±1.26 -1.454 P>0.05 Cold and dampness + Freund's adjuvant group 30.17±0.70 -2.404 P<0.05 Note: P, compared with blank control group

[0108] pass Fig.25 , 26 As shown in Tables 4 and 5, the temperature of the cold-dampness + Freund's adjuvant group was lower, and the area above 33°C accounted for a smaller proportion, indicating that the damp-cold performance of the cold-dampness + Freund's adjuvant group was more significant.

[0109] 2.4 Serological ELISA test

[0110] At the end of modeling, the abdominal aorta blood was collected from the rats, placed in a coagulation tube and allowed to stand for 30 min, centrifuged at 3500 rpm for 15 min, and the supernatant was collected. The serum IL-6, serum IL-1β, and serum TNF-α levels were detected. For specific operation steps, please refer to the ELISA kit instructions.

[0111] The experimental results are shown in Fig. 27 and Table 6.

[0112] Table 6

[0113] Serum-IL-6 Serum-IL-1β Serum TNF-α Blank Group 1202.59±184.04 348.43±119.41## 481.45±242.75 Freund's adjuvant group 1197.28±38.66 720.76±269.33## 1050.41±334.97 Cold and damp group 729.46±319.31** 364.14±173.84# 530.6±217.63 Cold and dampness + Freund's adjuvant group 416.89±75.43** 1688.66±324.97 664.3±504.24

[0114] Note: Compared with the blank control group, *P<0.05, **P<0.01; # Compared with the cold and dampness + Freund's adjuvant group, # P<0.05, ## P<0.01.

[0115] Depend on Fig. 27 As can be seen from Table 6, the serum IL-6 expression level in the cold-dampness + Freund's adjuvant group was significantly lower than that in the blank group, and the serum IL-1β in the cold-dampness + Freund's adjuvant group was significantly higher than that in the blank group, which can be used as an evaluation criterion for quantitative judgment of cold-dampness arthralgia type low back pain.

[0116] 2.5 After the rat model was established, the erector spinae muscles of L3-L5 were taken and ground. Total RNA was extracted using the trizol method. RNA was reverse transcribed into cDNA. GAPDH was used as the internal reference gene. -ΔΔCT The relative expression of IL-6 was calculated by the method. The calculation results are shown in Table 7 and Fig. 27 .

[0117] Table 7

[0118]

[0119] In summary, the rat model of cold-dampness arthralgia type low back pain provided by the present invention and its modeling and evaluation method, construct an animal model that is basically consistent with the clinical manifestations of cold-dampness arthralgia type low back pain through specific environment and feeding conditions. The prior art evaluates the success of modeling from aspects such as feces, coat gloss, mental state, etc., lacks necessary targeted equipment, and is difficult to quantify. Based on this, the present invention standardizes the cold-dampness syndrome experimental model of rats, and designs a thermophilic experimental box, which is judged by behavioral performance, combined with infrared thermal imaging and related inflammatory markers, and the standard of modeling success is evaluated in a quantitative manner. It lays a foundation for clinical research, mechanism of action research, and screening or preparation of therapeutic drugs for cold-dampness arthralgia type low back pain.

[0120] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention's specification and drawings, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for establishing a rat model of low back pain due to cold-dampness and arthralgia, characterized in that: The following steps are involved: Inflammatory agents were injected into the erector spinae muscle stage of the L3-L5 segment of rats, and then an ice box was placed in the rat cage once in the morning and evening every day. The rats were fed with ice water and given an ice water bath every day for 2.8 to 3.2 weeks.

2. The method for establishing the rat model of low back pain due to cold-dampness and arthralgia according to claim 1, characterized in that: When the rats are fed with ice water, the temperature of the ice water is 3-5° C., and the content of the ice water is 4-6 mL.

3. The method for establishing the rat model of low back pain due to cold-dampness and arthralgia according to claim 1, characterized in that: The temperature of the ice water bath is 3-5°C and the time is 4-6 minutes.

4. A rat model obtained by the method for establishing a rat model of cold-dampness arthralgia type low back pain according to any one of claims 1 to 3.

5. The method for evaluating a rat model according to claim 4, wherein: The following steps are included: behavioral testing, HE staining and Masson staining, infrared thermal imaging of the rat back, and serum ELISA testing are used to determine whether the rat model is successfully established; The behavioral tests include a tail suspension test and a thermophilic test.

6. The evaluation method according to claim 5, characterized in that: The indicator of the tail suspension test is the despair time.

7. The evaluation method according to claim 5, characterized in that: The thermophilic experiment is carried out in a thermophilic experiment box, in which a high temperature zone and a room temperature zone are arranged, and the temperature of the high temperature zone is higher than that of the room temperature zone.

8. The evaluation method according to claim 7, characterized in that: The indicator of the thermophilic experiment is the time the rats enter the high temperature zone.

9. The evaluation method according to claim 8, characterized in that: A pressure sensor is provided on the bottom surface of the high temperature zone of the thermophilic test box. During the thermophilic test, when the high temperature zone senses a pressure greater than 200g and the duration exceeds 3s, timing starts.

10. The evaluation method according to claim 5, characterized in that: The indicator of the infrared thermal imaging of the rat's back is the temperature area of ​​the rat's back.

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