CN112913778B

CN112913778B - Construction method of sheep chronic inflammation model

Info

Publication number: CN112913778B
Application number: CN202110124511.4A
Authority: CN
Inventors: 梁艳萍; 杨果; 赵宗胜; 朱梦婷; 周建伟; 张云生; 周平
Original assignee: Shihezi University; Northwest Institute of Eco Environment and Resources of CAS
Current assignee: Shihezi University; Northwest Institute of Eco Environment and Resources of CAS
Priority date: 2021-01-29
Filing date: 2021-01-29
Publication date: 2022-11-01
Anticipated expiration: 2041-01-29
Also published as: CN112913778A

Abstract

The invention discloses a construction method of a sheep chronic inflammation model, which relates to the technical field of animal model construction, and comprises the steps of injecting Lipopolysaccharide (LPS) into a sheep body to construct sheep chronic inflammation; wherein, the injection frequency of the LPS is more than 2 times, and the dosage is increased by 10 to 40 percent when LPS is injected every time except for initial injection. The invention injects LPS into sheep body continuously for a plurality of times in a mode of increasing dosage to induce the sheep systemic inflammatory reaction, so as to stably and effectively construct a chronic inflammatory and non-lethal inflammatory model.

Description

Construction method of sheep chronic inflammation model

Technical Field

The invention relates to the technical field of animal model construction, in particular to a construction method of a sheep chronic inflammation model.

Background

Inflammation is involved in the pathogenesis of many diseases, including inflammatory bowel disease, sepsis, and cancer. Inflammation is a defensive immune response produced by the body to internal and external stimuli, and can promote the body to repair tissues and remove pathogenic microorganisms invading from the outside, playing an important role in maintaining the body in a steady state. However, the inflammatory reaction is better than that of a double-edged sword, the moderately controlled inflammatory reaction is beneficial to the body, but the body is damaged when the inflammation cannot be resolved to form uncontrollable chronic inflammation. There are many diseases related to chronic inflammation, such as rheumatoid arthritis, obesity, type 2 diabetes, atherosclerosis, alzheimer's disease, lupus erythematosus, cancer, etc., which are currently found. The main therapeutic approach for diseases caused by chronic inflammation is anti-inflammatory therapy. In view of the therapeutic effects and side effects of the current anti-inflammatory drugs, development of novel anti-inflammatory drugs has become a focus of researchers.

Lipopolysaccharide (LPS) is not the only or main factor causing animal immune stress in actual production, but LPS can simulate animal immune stress and has good repeatability, so that the LPS is widely applied to immune stress mechanism. Intravenous injection of bacterial LPS mimics livestock infection and can be used to study immune inflammatory responses without the risk of using live pathogens. LPS, a major component of the outer membrane of gram-negative bacteria, such as escherichia coli, is also a major member of pathogen-associated molecular patterns, and it has a variety of toxic effects that can cause pathological reactions such as inflammation, fever, and leukocytosis (small dose) or depletion (large dose) in the body. After LPS enters the body, it stimulates secretion of adrenocortical hormones and also regulates the expression of inflammatory mediators, such as interferon gamma (IFN-. Gamma.), binding to the CD14/TLR4/MD2 receptor complex to initiate activation of intracellular signaling pathways.

Currently, a single injection of Escherichia coli LPS is often used for inducing immune stress, but only causes the concentration of inflammatory factors in animal plasma to be sharply increased, and the concentration is restored to the level before injection within 4-8h, so that only acute short-term immune stress can be simulated.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a construction method of a sheep chronic inflammation model.

The invention is realized in the following way:

in a first aspect, the embodiment of the present invention provides a method for constructing a sheep chronic inflammation model, which includes: injecting Lipopolysaccharide (LPS) into a sheep body to construct sheep chronic inflammation;

the injection frequency of the LPS is more than 2 times, and the dosage is increased by 10% -40% when LPS is injected every time except for initial injection.

In a second aspect, the embodiments of the present invention provide the use of lipopolysaccharide LPS in the manufacture of a reagent for use in the construction of a model of chronic inflammation in sheep.

The invention has the following beneficial effects:

the embodiment of the invention provides a method for constructing a sheep chronic inflammation model, which comprises the following steps: injecting Lipopolysaccharide (LPS) into sheep to construct sheep chronic inflammation; the injection frequency of the LPS is more than 2 times, and the dosage is increased by 10% -40% when LPS is injected every time except for initial injection. The method injects LPS into sheep body continuously for a plurality of times in a mode of increasing dosage to induce sheep systemic inflammatory reaction, compared with the prior art, the method has the advantages of long stress time, low lethality, high feasibility and the like, provides a research approach for pathogenesis of inflammatory diseases, and has important significance for searching effective countermeasures for treating chronic inflammatory diseases.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.

FIG. 1 shows the results of the test example 1; wherein, A is the influence of lipopolysaccharide on lamb leucocytes (WBCs), and B is the influence of lipopolysaccharide on lamb plasma cortisol concentration;

FIG. 2 is a graph showing the effect of lipopolysaccharide in test example 1 on the expression of an inflammation marker in blood of a lamb, wherein A is the effect of lipopolysaccharide on Toll-like receptor TLR4, and B is the effect of lipopolysaccharide on interference-gamma (IFN-gamma).

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.

The embodiment of the invention provides a method for constructing a sheep chronic inflammation model, which comprises the following steps: injecting Lipopolysaccharide (LPS) into sheep to construct sheep chronic inflammation;

Through a series of creative efforts, the inventor provides the construction method, and the construction method induces the systemic inflammatory response by continuously and incrementally injecting lipopolysaccharide into sheep for a plurality of times through veins so as to establish a non-lethal inflammation model which can stably and reliably induce chronic inflammation. Compared with a mode of single injection or fixed dose injection, the method can cause sheep chronic immune stress reaction, prolongs acute short-term stress reaction time caused by single injection and avoids LPS tolerance caused by fixed dose injection. The establishment of the method has important significance for revealing pathogenesis of inflammatory diseases and searching effective countermeasures for treating chronic diseases.

Preferably, the route of injection of LPS is intravenous.

In the following embodiments, the number of LPS injections may be any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20. Dose escalation by any percentage of 10%, 15%, 20%, 25%, 30%, 35% and 40% per LPS injection.

The number of times of LPS injection and the dose are closely related to the selection of initial concentration and the construction success rate of the model, and preferably, the LPS injection mode is as follows: except for initial injection, the dosage is increased by 10% -40% in each injection. Under the condition of increasing, the construction success rate of the model is higher.

In some embodiments, the initial injected dose of LPS is 0.01 μ g LPS/kg body weight, 0.1 μ g LPS/kg body weight, 0.15 μ g LPS/kg body weight, 0.2 μ g LPS/kg body weight, 0.25 μ g LPS/kg body weight, 0.3 μ g LPS/kg body weight, 0.4 μ g LPS/kg body weight, 0.5 μ g LPS/kg body weight, 0.6 μ g LPS/kg body weight, 0.7 μ g LPS/kg body weight, 0.8 μ g LPS/kg body weight, 0.9 μ g LPS/kg body weight, 1.0 μ g LPS/kg body weight, 1.2 μ g LPS/kg body weight, 1.4 μ g LPS/kg body weight, 1.6 μ g LPS/kg body weight, 1.8 μ g LPS/kg body weight, 2.0 μ g LPS/kg body weight, 2.2 Mu.g LPS/kg body weight, 2.4. Mu.g LPS/kg body weight, 2.6. Mu.g LPS/kg body weight, 2.8. Mu.g LPS/kg body weight, 3.0. Mu.g LPS/kg body weight, 3.2. Mu.g LPS/kg body weight, 3.4. Mu.g LPS/kg body weight, 3.6. Mu.g LPS/kg body weight, 3.8. Mu.g LPS/kg body weight, 4.0. Mu.g LPS/kg body weight, 4.2. Mu.g LPS/kg body weight, 4.4. Mu.g LPS/kg body weight, 4.6. Mu.LPS/kg body weight, 4.8. Mu.g LPS/kg body weight, 5.0. Mu.g LPS/kg body weight, 50. Mu.g LPS/kg body weight, 100. Mu.g LPS/kg body weight. Preferably, the initial injected dose of LPS is: 0.01 to 100. Mu.g LPS/kg body weight. The model is constructed with higher power in accordance with the number of injections and the dose increment during injection, within the selected range of the initial concentration.

Preferably, the interval time of the LPS injection is 3h to 2 days. In some embodiments, the LPS injections are separated by any of 3h, 6 h, 12 h, 24 h, 2 days.

Preferably, the interval is 3h to 2 days. Within this interval, the inflammation model can be constructed more efficiently. If the interval time is too short, the death rate of the injected animal is easily caused, and if the interval time is too long, the animal cannot be stably and effectively subjected to an inflammatory reaction, so that the success rate of model construction is reduced.

In some embodiments, the source of LPS is not limited, and existing LPS may be used. Preferably, the LPS comprises lipopolysaccharide isolated from Escherichia coli O111: B4.

Preferably, the heart rate of the sheep chronic inflammation model is maintained at 118-144 times/min, and the rectal temperature is maintained at more than 40 ℃. Specifically, after 1 injection, the heart rate is maintained at 118 to 131 times/min, after 2 injections, the heart rate is maintained at 120 to 139 times/min, after 3 injections, the heart rate is maintained at 120 to 143 times/min, after 4 injections, the heart rate is maintained at 134 to 144 times/min, and after 5 injections, the heart rate is maintained at 125 to 141 times/min.

Preferably, the heart rate of the sheep chronic inflammation model is maintained at 131-144 times/min.

Preferably, the relative expression quantity of IFN-gamma in the blood of the sheep chronic inflammation model is maintained to be 0.45-4.29, and the relative expression quantity of TLR4 is maintained to be 0.29-5.15.

The embodiment of the invention also provides application of Lipopolysaccharide (LPS) in preparation of a reagent for constructing a sheep chronic inflammation model.

Preferably, the constructing comprises: injecting Lipopolysaccharide (LPS) into sheep to construct sheep chronic inflammation; the injection frequency of the LPS is more than 2 times, and the dosage is increased by 10% -40% when LPS is injected every time except for initial injection.

Preferably, the LPS comprises lipopolysaccharide isolated from Escherichia coli O111: B4.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The embodiment provides a method for constructing a sheep chronic inflammation model, which comprises the following steps.

(1) Selecting a research object and designing a test;

selecting 4-5 months old Hu sheep with average initial weight of 22.2 +/-0.52 kg and good health condition; the test period is 15 days, wherein 5 days are adaptation period and 10 days are test period.

(2) Preparing Lipopolysaccharide (LPS) injection;

lipopolysaccharide LPS separated from Escherichia coli O111: B4 was dissolved in physiological saline.

(3) Establishing a sheep chronic inflammation model.

In the

test period

1, 3, 5, 7, 9, morning 08 00, sheep were given LPS to the jugular vein for chronic inflammation induction test, with an initial dose of 0.25. Mu.g LPS/kg body weight, and the dose was increased by 20% for each injection thereafter.

Example 2

This example provides a method for constructing a model of chronic inflammation in sheep, which is similar to example 1, except that the amount of LPS injected is different, in this example, the initial amount of LPS injected is 1.25 μ g LPS/kg body weight, and the LPS injection concentration is increased by 20% in the morning on

days

1, 3, 5, 7 and 9 of the test period.

Example 3

This example provides a method for constructing a model of chronic inflammation in sheep, which is similar to example 1, except that the amount of LPS injected is different, in this example, the initial amount of LPS injected is 2.5 μ g LPS/kg body weight, and the LPS injection concentration is increased by 20% in the morning on

days

1, 3, 5, 7 and 9 of the test period.

Example 4

This example provides a method for constructing a model of chronic inflammation in sheep, which is similar to example 3, except that the injection concentration of LPS is increased, wherein the initial injection amount of LPS is 2.5 μ g LPS/kg body weight, and the injection concentration of LPS is increased by 25% in the morning on

days

1, 3, 5, 7, and 9 of the test period.

Example 5

This example provides a method for constructing a model of chronic inflammation in sheep, which is similar to example 3, except that the injection concentration of LPS is increased, wherein the initial injection amount of LPS is 2.5 μ g LPS/kg body weight, and the injection concentration of LPS is increased by 15% in the morning on

days

1, 3, 5, 7, and 9 of the test period.

Test example 1

Experimental methods

The construction of the sheep chronic inflammation model was performed using the construction method provided in example 1~3.

Selecting 16 Hu sheep with age of 4-5 months, average initial weight of 22.2 +/-0.52 kg and good health condition; the sheep are randomly divided into 4 treatment groups, each treatment group is divided into 4 repetitions, the treatment groups are respectively a control group and test groups L1, L2 and L3, the control group (the construction method is the same as that in example 1, except that LPS is replaced by physiological saline), the test groups L1-L3 respectively adopt the construction method of example 1~3, all sheep are singly housed, the test period is 15 days, wherein 5 days are adaptation periods, and 10 days are test periods.

On the morning of 08 00

days

1, 3, 5, 7, 9 of the test period, the sheep were given a chronic inflammation induction test by intravenous injection of lipopolysaccharide LPS in the jugular vein at initial doses of 0 (control), 0.25 (L1), 1.25 (L2) and 2.5 (L3) μ g LPS/kg body weight, followed by a 20% dose increment at each injection.

After construction, each lamb measured rectal temperature and heart rate 3 hours after LPS injection.

One day before the start of the experiment, fasting and 3h after LPS injection on

days

1, 3, 5, 7, 9, a jugular venous blood sample of 12 ml was collected with EDTA tubes, 5ml was used for blood inflammatory gene testing, 2 mL was used for routine blood testing, and 5mL was used for blood supernatant plasma drawn by 3500 rmp centrifugation for 15 min for subsequent testing.

Detecting the concentration of cortisol in blood plasma by an enzyme-linked immunosorbent assay (ELISA), and detecting gene expression of TLR4 and IFN-gamma in blood by real-time fluorescent quantitative Polymerase Chain Reaction (PCR);

specifically, the specific steps of the enzyme linked immunosorbent assay kit (shanghai Elisa biotechnology limited, shanghai) for detecting the concentration of cortisol in plasma are as follows: taking out the required laths from the aluminum foil bag after room temperature is balanced for 20 min, sealing the rest laths by a self-sealing bag and putting back to 4 ℃; setting standard substance holes and sample holes, wherein 50 mu L of standard substances with different concentrations are added into the standard substance holes respectively; adding 50 mu L of sample to be detected into the sample hole; blank holes are not added; adding 100 μ L of detection antibody labeled with Horse Radish Peroxidase (HRP) into each of the standard well and the sample well except for blank well, sealing the reaction well with a sealing plate membrane, and incubating in a water bath or thermostat at 37 deg.C for 60 min; discarding liquid, drying on absorbent paper, filling washing solution (350 μ L) into each hole, standing for 1 min, throwing off washing solution, drying on absorbent paper, and washing the plate for 4-5 times; adding 50 μ L of substrate A, B into each well, and incubating for 15 min at 37 ℃ in a dark place; add stop solution 50 μ L per well, within 15 min, determine the OD value of each well at the wavelength of 450 nm.

The real-time fluorescent quantitative PCR is adopted to detect the gene expression quantity of TLR4 and IFN-gamma, the internal reference gene is beta-actin, and the method comprises the following specific steps: extracting total RNA in blood by a TRIzol method, synthesizing 1 st chain cDNA by using a Prime script TMPT Reagent Kit w ith gDNA Eraser (TaKaRa) reverse transcription Kit, designing a specific Primer by using Primer 5.0 software, wherein the sequence of the Primer is as follows:

beta-actin (upstream primer): 5'-AGCCTTCCTTCCTGGGCATGGA-3';

beta-actin (downstream primer): 5'-GGACAGCACCGTGTTGGCGTAGA-3';

IFN-. Gamma. -F (upstream primer): 5'-ATGTTTCATTTGCCACCATCC-3';

IFN-. Gamma. -R (downstream primer): 5'-GGTTACGCTTGCTTTGCCTTATGT-3';

TLR4-F (upstream primer): 5'-GGCATCATCTTCATCGTCCT-3';

TLR4-R (downstream primer): 5'-CCACTCCAGGTAGGTGTTCC-3'.

TB GreenTM Premix Ex TaqTM II kit was used for real-time fluorescent quantitative PCR analysis.

Please refer to fig. 1, fig. 2 and tables 1-2 for experimental results.

TABLE 1 Effect of lipopolysaccharide on lamb Heart Rate

TABLE 2 Effect of lipopolysaccharide on lamb rectal temperature

The white blood cell count of the lambs was significantly reduced at

days

1 and 3 after LPS stimulation compared to the control group (a in figure 1,p<0.05 And, without dose dependence, the reason for the leukopenia in blood is the adhesion of leukocytes to endothelial cells after LPS injection, the infiltration of leukocytes into the tissue through the vascular endothelial barrier.

Cortisol, an adrenocortical hormoneIs a hormone having anti-inflammatory action, which is regulated by stress in hypothalamus-pituitary-adrenal (HPA) axis, plasma cortisol concentration in the control group of this study remained unchanged on

days

0, 1, 3 and 5 in the control lambs, but in the LPS-injected group, cortisol concentration increased in a dose-independent manner 3h after LPS stimulation on day 1 and returned to untreated level on day 5, and plasma cortisol concentration in the LPS group was not different from that in the control group after LPS administration thereafter (B in FIG. 1,p<0.05 This) indicates that LPS stimulation, which causes a response of the hypothalamic-pituitary-adrenal axis, leads to increased secretion of corticoids.

After LPS stimulation on

days

1 and 3, the expression of IFN- γ and TLR4 in the blood of the LPS group was higher than that of the control group (a-B in figure 2,p<0.05 Especially group L3). On

days

5, 7 and 9, there was no significant difference between the LPS-injected group and the control group.

The heart rate was significantly higher in the L3 group than in the control group (table 1). The L3 group lambs had rectal temperature higher than the control group on day 1 (table 2,p <0.01 But no difference between groups on subsequent days. However, the magnitude of the cortisol response and the expression of LPS by IFN- γ and TLR4 is attenuated with consecutive injections. This may be due to the lower magnitude of the response relative to the first stimulus and may be associated with instability and short half-life of inflammatory factors in the blood. These findings indicate that LPS-induced adaptive endocrine responses and activation of intracellular signaling pathways achieve the effects of chronic inflammatory responses upon repeated stimulation with LPS.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A construction method of a sheep chronic inflammation model is characterized by comprising the following steps: injecting Lipopolysaccharide (LPS) into sheep to construct sheep chronic inflammation;

the injection frequency of the LPS is more than 2 times, the dose is increased by 10% -40% when LPS is injected every time except for initial injection, and the initial injection dose of the LPS is as follows: 0.01 to 100 mu g of LPS/kg of body weight, wherein the interval time of LPS injection is 3h to 2 days.

2. The method for constructing the sheep chronic inflammation model according to claim 1, wherein the LPS is injected in a manner of: except for the initial injection dosage, the dosage is increased by 10% -30% in each injection.

3. The method for constructing the sheep chronic inflammation model according to claim 1, wherein the interval is 24 h to 2 days.

4. The method for constructing a model of chronic inflammation in sheep according to claim 1, wherein said LPS comprises lipopolysaccharide isolated from Escherichia coli O111: B4.

5. The method for constructing the sheep chronic inflammation model as claimed in claim 1, wherein the relative expression level of IFN-gamma in blood of the sheep chronic inflammation model is maintained at 0.45 to 4.29, and the relative expression level of TLR4 is maintained at 0.29 to 5.15.

6. The method for constructing the sheep chronic inflammation model as claimed in claim 1, wherein the heart rate of the sheep chronic inflammation model is maintained at 118 to 144 times/min, and the rectal temperature is maintained at 40 ℃ or higher.

7. The method for constructing the sheep chronic inflammation model, according to claim 6, is characterized in that the heart rate of the sheep chronic inflammation model is maintained at 131 to 144 times/min.