阅读说明：本技术 一种肺爆震伤动物模型构建方法及其用途 (Method for constructing animal model with lung explosive injury and application thereof ) 是由 王鸿 高俊宏 骞爱荣 刘进仁 卢青 范小琳 李亮 张文娟 刘志永 于 2021-08-17 设计创作，主要内容包括：本发明涉及医药技术领域,具体为一种肺爆震伤动物模型构建方法,该构建方法可真实反映爆炸有关的物理参数及其交互产生的复杂作用过程,并对肺损伤的范围、等级、严重程度实现量化评价；本发明通过该方法构建得到的肺爆震伤动物模型验证了白藜芦醇在治疗肺爆震伤中具有较佳的疗效,白藜芦醇具备来源广泛、安全、不易产生耐药性等优点,其能够降低肺爆震伤大鼠肺组织出血、肺泡隔破裂及炎性细胞浸润,并缓解肺组织水肿。(The invention relates to the technical field of medicines, in particular to a method for constructing an animal model of lung blast injury, which can truly reflect physical parameters related to explosion and a complex action process generated by interaction of the physical parameters and the complex action process and realize quantitative evaluation on the range, the grade and the severity of lung injury; the lung detonation injury animal model constructed by the method verifies that resveratrol has a good curative effect in treating lung detonation injury, and the resveratrol has the advantages of wide source, safety, difficulty in generating drug resistance and the like, can reduce the bleeding of lung tissues, alveolar septal rupture and inflammatory cell infiltration of rats in the lung detonation injury, and relieves the edema of the lung tissues.)

1. A method for constructing an animal model of lung blast injury is characterized by comprising the following steps:

laying explosives;

arranging an experimental animal subjected to anesthesia treatment at a fixed distance from an explosion central point, and arranging a pressure sensor on the same arc with the experimental animal, wherein the sensitive surface of the pressure sensor is leveled with the ground;

and after debugging is finished, detonating the explosive remotely to obtain a lung detonation injury animal model, and simultaneously recording overpressure data of the pressure sensor to obtain the dose-effect relationship between the lung injury and the overpressure of the animal.

2. The method for constructing an animal model of lung blast injury according to claim 1, wherein the explosive has an explosive power of 280-320kgTNT equivalent.

3. The method for constructing an animal model of lung blast injury according to claim 2, wherein the fixed distance is 15-90m from the center point of explosion.

4. Use of an animal model of lung blast injury constructed by the method of any one of claims 1-3 in screening for a medicament for treating lung blast injury.

5. The use according to claim 4, wherein the drug comprises resveratrol.

6. The use according to claim 5, wherein the resveratrol is capable of reducing the pulmonary tissue bleeding, alveolar septal rupture and inflammatory cell infiltration of rats with lung explosive injury, and relieving pulmonary tissue edema.

7. The use according to claim 5, characterized in that resveratrol can be used for preparing the expression inhibitors of the rat inflammatory factors IL6, IL8 and TNF-alpha, apoptosis factors Caspase3 and Caspase8 and oxidative stress product MDA in the lung blast injury.

Technical Field

The invention relates to the technical field of medicines, in particular to a method for constructing an animal model of lung blast injury and application thereof.

Background

The lung Blast injury (Blast lung injury) is the most common physical injury form in military war, terrorist attack and industrial explosion accident. The lung is one of the most sensitive target organs to the overpressure of the shock wave, and the damage is mainly manifested by pulmonary hemorrhage, edema, rupture, bullous blister, collapse and emphysema, and has the characteristics of light inside and heavy outside, complex damage, high death rate and the like. Exacerbations can lead to Acute Lung Injury (ALI), Acute Respiratory Distress Syndrome (ARDS), and Multiple Organ Dysfunction Syndrome (MODS), eventually even death. At present, no effective medicine for treating BLI has been found, so that the search for a safe and effective medicine for treating BLI is urgent.

The process of damaging organisms by explosive shock waves is a mechanical process of applying shock waves to biological tissues and a process of responding the biological tissues to the shock waves by unique and complex structures and material characteristics of the biological tissues. The model is the basis of research on related diseases, and the research on the lung detonation injury is commonly carried out by an animal model, a dummy model, a digital simulation model, an equivalence model and the like, and has advantages and disadvantages due to different research purposes, objects and application conditions. Animals are the most common model in the research of explosion and impact injuries, and the main basis is that the injury effect of animals and the injury effect of people have similarity principle, and the organ structure, function, anatomical characteristics, injury threshold value and the like of the animals are close to those of human bodies. Most of the current lung detonation models are mainly constructed based on laboratory biological shock tubes, simple explosion devices and the like, although the test parameters are controllable, the cost is low, the repeatability is good, the complexity and the destructiveness of shock waves under actual explosion conditions cannot be simulated, and the difference between the complexity and the destructiveness and damage data obtained in actual combat environments is large.

Resveratrol (Resveratrol, RE)) is a non-flavonoid polyphenol compound with a stilbene structure extracted from natural plants, and has various biological effects of resisting inflammation, resisting oxidation, regulating immunity and the like, but no report has been reported on the research of Resveratrol for treating the lung burst injury.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the lung detonation injury animal model construction method, which is closer to the actual combat environment, can truly reflect physical parameters related to explosion and the complex action process generated by interaction of the physical parameters, realizes quantitative evaluation on the range, the grade and the severity of lung injury, and provides support for screening lung detonation injury medicines.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides a method for constructing an animal model of lung blast injury, which comprises the following steps:

laying explosives;

arranging an experimental animal subjected to anesthesia treatment at a fixed distance from an explosion central point, and arranging a pressure sensor on the same arc with the experimental animal, wherein the sensitive surface of the pressure sensor is leveled with the ground;

and after debugging is finished, detonating the explosive remotely to obtain a lung detonation injury animal model, and simultaneously recording overpressure data of the pressure sensor to obtain the dose-effect relationship between the lung injury and the overpressure of the animal.

Preferably, the explosive has an explosive power of 280-320kg TNT equivalent.

Preferably, the fixed distance is 15-90m from the center point of the explosion.

The invention also provides application of the lung detonation injury animal model constructed by the method in screening of a medicament for treating lung detonation injury.

Preferably, the drug comprises resveratrol.

Preferably, the resveratrol can reduce the pulmonary tissue hemorrhage, alveolar septal rupture and inflammatory cell infiltration of rats with lung explosive injury, and relieve the pulmonary tissue edema.

Preferably, the resveratrol can be used for preparing the expression inhibitors of rat inflammatory factors IL6, IL8 and TNF-alpha, apoptosis factors Caspase3 and Caspase8 and oxidative stress products MDA of the lung detonation injury.

Compared with the prior art, the invention has the following beneficial effects:

1. the animal lung detonation injury model is constructed through live bomb explosion, the model is closer to the actual combat environment, physical parameters related to explosion and a complex action process generated by interaction of the physical parameters can be truly reflected, the quantitative evaluation on the range, the grade and the severity of lung injury is realized, and the support is provided for developing researches such as molecular mechanism, clinical diagnosis, treatment, rehabilitation intervention and the like of explosion-impacted lung injury and screening of drugs for treating lung detonation injury.

2. The evaluation result of the therapeutic effect of the model constructed by the invention on resveratrol proves that: the resveratrol can obviously reduce the lung tissue hemorrhage, alveolar septal rupture and inflammatory cell infiltration of the rat with the blast injury, and can improve the activity of antioxidant enzyme (SOD) by inhibiting the expression of inflammatory factors (IL6 and IL8), apoptosis factors (Caspase 3 and Caspase 8) and oxidative stress products (MDA), thereby having the protection effect on the blast injury of the lung.

3. The resveratrol belongs to a natural active product, is administered in an oral mode, is beneficial to full play of the treatment effect of the effective components of the medicament, has the advantages of difficult generation of medicament resistance, safety, high efficiency, no generation of antibiotic pollution and the like, and is beneficial to treatment of the lung detonation injury.

Drawings

FIG. 1 is a diagram showing the arrangement positions of a rat and a pressure sensor;

FIG. 2 is an anatomical view of the lungs of a rat at various distances from the centroid;

FIG. 3 is the wet/dry weight ratio of the rat lungs at various distances from the centroid; compared with the control group, the compound of the formula,^**P<0.01；^***P<0.001；

FIG. 4 is the lung pathology (HE, x 100 times) of rats at different distances from the centroid;

FIG. 5 is a schematic diagram of an explosion simulation at a distance of 35m from the center of the explosion;

FIG. 6 is a lung anatomical diagram of a rat of example 2;

FIG. 7 is the lung tissue pathology (HE, x 100 fold) of the rat of example 2;

FIG. 8 is a graph of the effect of resveratrol on pulmonary edema caused by a lung burst injury in rats;

FIG. 9 is a graph of the effect of quinophthalol on the exposure of blast shock wave to lung tissue inflammatory factor in rats, wherein A, B, C denotes IL6, IL8 and TNF-alpha, respectively;

FIG. 10 is a graph of the effect of quinophthalol on oxidative stress in lung tissue of rats exposed to blast shock waves, wherein A, B represents SOD and MDA, respectively;

FIG. 11 is a graph showing the effect of quinophthalol on apoptosis in lung tissue of blast shock wave exposed rats, wherein A, B represents Caspase3 and Caspase8, respectively.

Detailed Description

The present invention is described in further detail below with reference to specific examples, which are described herein for the purpose of illustration only and are not to be construed as limiting the invention.

Example 1

Construction of lung blast injury model

1. Grouping animals

The method comprises the following steps of weighing 50 rats in sequence, numbering the rats by 1-50, generating corresponding random numbers by using Excel software, arranging the random numbers in an ascending order according to the size, and dividing the rats into 10 groups in sequence, wherein one group is a control group (Ctrl group in the figure) and the rest are test groups, and the test groups are arranged at different distances from a detonation core.

2. Making of models

The model is constructed by means of a static explosive force test of a certain warhead. The specific operation method comprises the following steps: an explosive with the explosion power of 300kg TNT equivalent (only 300kg TNT equivalent is taken as an example here, but the explosive does not represent that the lung injury effect can be achieved only under the explosion power) is selected to be distributed, and then 45 rats in the test group are randomly distributed at positions 15m, 20m, 25m, 30m, 35m, 40m, 50m, 70m and 90m away from the explosion center respectively, and 5 rats are distributed at each position point. Rats were anesthetized by isoflurane inhalation and placed in wire cages and fixed to the ground before testing. And a pressure sensor is arranged at each position, the pressure sensor is transmitted to a terminal computer through a cable, and a power switch is started and overpressure data is recorded before a test. After the test, the success of the model is evaluated by the dissection, histopathological detection and the like of the rat. The layout of the rat and the pressure sensor is shown in FIG. 1.

3. Animal dissection method

Rats were anesthetized by intraperitoneal injection of chloral hydrate (10%, 0.3mL/100 g); continuously cutting the skin upwards along the xiphoid process, peeling the skin and subcutaneous tissue to two sides passively, exposing the thorax and cutting along the center, clamping the organ with forceps, taking off the lung tissue as a whole, and placing in a culture dish containing normal saline to wash off bloodstain; separating the left lung and the right lung, cleaning surface exudate of the left lung by using filter paper, weighing the wet weight of the lung by using an electronic balance, and then putting the lung into an oven for drying; the right lung was fixed in formalin for use.

4. Pulmonary edema detection

After 24h exposure to the blast shock wave, the rats were dissected. Preparing 5 samples of the complete left lobe lung of each group of rats, dipping the liquid on the surface of each group of rats by using clean filter paper, and then recording the weight of lung tissues to obtain wet weight; then, the cells were placed in a constant temperature drying cabinet and kept at 80 ℃ for 48 hours to weigh the dry weight, and the wet weight/dry weight (W/D) ratio was calculated to evaluate the pulmonary edema.

5. Histopathological examination

Simultaneously selecting the 5 rats, taking out the right lung, and preparing paraffin sections and histopathological analysis; the specific method for histopathology slide preparation comprises the following steps:

firstly, material taking: taking a lung tissue block fixed by 4% formaldehyde solution, wherein the size of the lung tissue block is about 3-4 mm, and placing the lung tissue block in an embedding box;

dehydrating and transparent: the full-automatic dehydrator is used for dehydration, and the dehydration setting program is as follows: 75% ethanol (1h) → 85% ethanol (1h) → 95% ethanol I (1h) → 95% ethanol II (1h) → 100% ethanol I (45min) → 100% ethanol II (45min) → 100% ethanol III (0.5 h); transparent setting program: xylene I (20min) → xylene II (20 min);

embedding: embedding the dehydrated specimen with paraffin by using a paraffin embedding machine, and trimming a paraffin block to facilitate slicing;

cutting into slices: using a rotary microtome, the specimen was cut into sections of 0.5 μm;

spreading the slices: slightly picking up the left hand with a clean writing brush, and slightly clamping the wax sheet with the ophthalmic forceps by the right hand, transferring the wax sheet to the water surface of a sheet spreading machine (with the water temperature of 40-45 ℃) for floating;

sixthly, baking the tablets: after the slices are completely unfolded, a better slice is fished by a glass slide and is placed on a slice baking machine for baking (65-70 ℃), and a sample number is written immediately;

and (c) dyeing: dyeing by using a full-automatic dyeing machine, and setting a program: xylene I (5min) → xylene II (5min) → xylene III (5min) → 100% ethanol (1min) → 95% ethanol (1min) → 85% ethanol (1min) → 75% ethanol (1min) → water wash I (1min) → hematoxylin (6min) → water wash II (1min) → hydrochloric acid ethanol differentiation (4s) → water wash III (1min) → aqueous ammonia rewet (1min) → aqueous wash IV (1min) → eosin (1.5min) → aqueous wash V (1min) → 85% ethanol (20s) → 90% ethanol (1min) → 95% ethanol I (1min) → 95% ethanol II (1min) → 100% ethanol I (1) → 100% ethanol II (1min) → xylene I (1min) → 1 min);

(8) sealing: covering by using an automatic film sealing machine;

(9) microscopic examination: histopathological examination was performed using a microscope, and pathological changes occurring in the specimens were observed in detail and recorded, and photographed.

6. Results

6.1 post-test animal symptoms, signs

After the test, rats 15m, 20m and 25m away from the center of burst die; 4 deaths were made at 30m, and another death was made 4h after the test; 1 dead rat at 35m, the overall mortality rate is 42.0% (21/50), the rest rats are all alive, and the dead rat has blood secretion in the mouth and nose; the rats at 40m, 50m and 70m showed tachypnea and listlessness, and no obvious abnormality was observed in the rat at 90 m.

6.2 rat dissection

The results are shown in FIG. 2.

The anatomy shows that the lungs of all rats are mainly subjected to bleeding of different degrees, and the pleural effusion and the whole lungs of the rats at the positions 25m and 30m away from the heart burst are also the main causes of death; bleeding of the right middle lobe of the lung of a rat at the position of 35m reaches the depth of the whole lung tissue; rats at 40m, 50m and 70m have superficial plaque-shaped hemorrhage, and the pulmonary hemorrhage is reduced with the extension of the distribution distance; the rats in the control group were not abnormal.

6.3 Wet/Dry weight ratio (W/D) of rat Lung

The results are shown in FIG. 3.

Compared with the W/D value of the lung tissue of the control group rat (4.685 +/-0.097), the W/D value of the rat at 35m (5.193 +/-0.110), 40m (5.069 +/-0.154) and 50m (4.944 +/-0.589) from the explosive center is obviously increased, the difference is statistically significant (F is 16.322, P is less than 0.000), and the W/D value is reduced along with the increase of the distance from the explosive center. Indicating that the closer the distance to the centroid, the more severe the pulmonary edema.

6.4 Lung tissue pathology in rats

The results are shown in FIG. 4.

The rat lung tissue can be seen with diffuse hemorrhage of alveolar space and infiltration of a large number of inflammatory cells by observation under a microscope; the alveolar walls are broken, the alveolar cavities are fused to different degrees, and obvious edema is seen in the lung interstitium.

6.5 correlation of Wet/Dry weight ratio (W/D) of rat Lung with overpressure of shock wave

Pearson correlation analysis is carried out on the lung wet/dry weight ratio (W/D) of the rat and the overpressure of the shock wave, and the result shows that the W/D value is in positive correlation with the overpressure value of the shock wave (r is 0.859, and P is 0.029), and the larger the overpressure value is, the larger the W/D value is, the more serious pulmonary edema caused by the overpressure of the explosion shock wave is suggested. By the dose-effect relationship, the range, the grade and the severity of the lung injury can be quantitatively evaluated, and support is provided for developing researches such as molecular mechanism, clinical diagnosis, treatment and rehabilitation intervention of explosion impact lung injury.

Example 2

Evaluation of Effect of resveratrol on relieving lung detonation injury

1. Grouping

40 SPF male rats with SD strain were selected, weighing 200 + -20 g, and randomly divided into 4 groups of 10 rats each consisting of control group (No treatment, NT), quinoa alcohol group (Resveratrol, RE), model group (Blast), model + quinoa alcohol group (Blast + RE).

2. Modeling

Referring to the modeling method in example 1, rats in the test group were selected to be placed 35m from the center of the burst. In order to meet the welfare requirement of experimental animals and avoid the influence of stress on the experimental result, rats are inhaled with isoflurane for anesthesia before the experiment. Rats were placed in wire cages and fixed on arcs of 35m radius from the centroid. The control group of rats was located 3km away from the explosive environment.

And arranging a pressure sensor on an arc with a radius of 35m from the center of the explosion, transmitting the pressure sensor on a terminal computer through a cable, and starting a power switch to record overpressure data before the test. The specific layout position and method are shown in FIG. 5.

3. Method of producing a composite material

3.1 dosage and mode of administration

The administration was carried out in an oral gavage manner at a concentration of 200mg/kg in the first 1 week of the construction of BLI (pulmonary burst injury) model, and in an administration volume of 1mL/100g body weight.

3.2 method of operation

After 24h exposure to the blast shock wave, the rats were dissected. Taking out complete left-lobe lungs of 5 rats in each group to prepare 5 samples, dipping liquid on the surfaces of the samples by using clean filter paper, and then recording the weight of lung tissues to obtain wet weight; then placing the dried powder in a constant-temperature drying box, keeping the temperature at 80 ℃ for 48h, weighing the dry weight, calculating the wet weight/dry weight (W/D) ratio, and evaluating the pulmonary edema condition;

simultaneously selecting the 5 rats, taking out the right lung, and preparing paraffin sections and histopathological analysis; the method of example 1 was used for the specific operation of the histopathological section.

And preparing tissue homogenate by taking the remaining 5 rat lung tissues in each group, and detecting inflammatory factors, oxidative stress and apoptosis factors by adopting an enzyme-linked immunosorbent assay (ELISA) test method (the specific steps refer to the kit specification). The tissue homogenate preparation method comprises the following steps: lung tissue was rinsed with pre-cooled PBS (pH 7.4), residual blood was removed, and lung tissue was minced after weighing. Adding the minced lung tissue and a corresponding volume of PBS (in a weight-volume ratio of 1: 9) into a glass homogenizer, fully grinding on ice, centrifuging the homogenate at 5000 Xg for 10min, and collecting the supernatant to be tested.

4 results

4.1 general conditions in rats

No death of rats occurs in the test process and the observation period, no obvious damage is seen on the appearance of the chest, the rats in the model group breathe shallowly, blood secretion can be seen in 2 nasal cavities, the rats in the model group (8/10) and the portion of the rats in the white hellebore alcohol + model group (7/10) have transient apnea, the behaviors such as slow movement and the like after the test occur, and the rats are recovered to be normal after 24 hours.

4.2 pathological analysis of rat Lung tissue

The anatomical image of rat lung tissue is shown in fig. 6.

The anatomy shows that the lung tissue of the rat in the model group has obvious bleeding, and the bleeding surface of the quinoxyfen + model group is obviously reduced.

The pathological map of rat lung tissue is shown in fig. 7.

Histopathology revealed, model group, resveratrol + model group rats with hypo-pulmonary histoscope-visible bleeding, alveolar septal rupture and inflammatory cell infiltration; the anatomical and histopathology of the control and resveratrol rats were not significantly altered. The results show that: the white hellebore alcohol has obvious improvement effect on rat lung tissue hemorrhage, edema, inflammatory cell infiltration and the like caused by explosion shock waves.

4.3 Wet/Dry weight (W/D) ratio of rat Lung tissue

The results are shown in FIG. 8.

The wet/dry weight ratio (W/D) of the lung of the rats in the model group and the white hellebore alcohol + model group is obviously higher than that of the rats in the control group, and the difference has statistical significance (^***P<0.001); the lung wet/dry weight ratio (W/D) of the white chenopodium album alcohol + model group rat is obviously lower than that of the model group, and the difference has statistical significance (^#P<0.05). The suggestion shows that the resveratrol can obviously improve pulmonary edema caused by explosion shock waves.

4.4 Effect on explosive shockwave Exposure of inflammatory factors in Lung tissue in rats

The results are shown in FIG. 9.

Compared with the control group, the expression of interleukin 6(IL6), interleukin 8(IL8) and tumor necrosis factor alpha (TNF-alpha) in the lung tissue of the model group rat is obviously increased, and the difference has statistical significance (^***P<0.001), Tribulus terrestris alcohol group is not statistically different (P)>0.05), and prompting that the lung detonation injury model is successfully constructed.

Compared with the model group, the expression of interleukin 6(IL6), interleukin 8(IL8) and tumor necrosis factor alpha (TNF-alpha) in the lung tissues of rats in the quinophthalol + model group is obviously reduced, and the difference has statistical significance (^###P<0.001). The results show that: the resveratrol has protective effect on lung tissue inflammatory reaction caused by explosion shock wave.

4.5 Effect on oxidative stress of Lung tissues of rats exposed to blast shock wave

As shown in FIG. 10, compared with the control group, the expression of total superoxide dismutase (T-SOD) in the lung tissue of the rat in the model group is significantly reduced, the expression of Malondialdehyde (MDA) which is an oxidative stress product is increased, and the difference has statistical significance (A)^*P<0.05，^***P<0.001), Tribulus terrestris alcohol group is not statistically different (P)>0.05), suggesting that lung blast injury may cause oxidative stress.

Compared with the model group, the expression of SOD in lung tissue of rats in the quinoa album alcohol + model group is increased, the expression of MDA is reduced, and the difference has statistical significance (^#P<0.05，^###P<0.001). The results show that: the resveratrol has protective effect on oxidative stress reaction of lung tissue caused by blast shock wave.

4.6 Effect on apoptosis of Lung tissue in rats exposed to blast shock wave

As shown in FIG. 11, the expression of Caspase 3(Caspase 3) and Caspase 8(Caspase 8) was significantly increased in lung tissues of rats in the model group compared to the control group, and the difference was statistically significant: (^***P<0.001), Tribulus terrestris alcohol group is not statistically different (P)>0.05), suggesting that the explosive shock wave causes an apoptotic effect on lung tissue.

Compared with the model group, the expression of Caspase3 and Caspase8 in the lung tissues of rats in the quinoa album alcohol + model group is obviously reduced, and the difference has statistical significance (^###P<0.001). The results show that: the resveratrol has protective effect on lung tissue apoptosis caused by blast shock wave.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.