Method for constructing lung injury animal model after radioactive dust inhalation

Abstract

The invention relates to a method for constructing a lung injury animal model after radioactive dust inhalation, which comprises the following steps of: injecting pentobarbital sodium into the abdominal cavity to anesthetize a rat, and observing the change of the rat; after anesthesia, vertically lifting the rat, fixing the rat with the head facing upwards, opening the airway of the rat by using a laryngoscope, then inserting a rat lung dry powder quantitative atomizer filled with UO2 aerosol powder into the airway of the rat, and quickly pressing a piston of an injector to the bottom; after inhalation contamination is completed, shaking the rat left and right to enable the UO2 aerosol powder to be evenly distributed in the lung of the rat, paying attention to observing the breathing and physiological states of the rat, pressing the heart of the rat in time to conduct cardio-pulmonary resuscitation if the rat is in an uncomfortable state, putting the rat into a cage to conduct normal feeding after the rat is recovered, and finally conducting animal model evaluation. According to the method, the atomizer filled with the UO2 powder is inserted into the trachea of the rat through the laryngoscope, the acute lung injury model is obtained through induction, the contamination dosage is accurate in the quantitative dry powder aerosol contamination mode, and scene exposure of occupational personnel is better simulated.

Description

Technical Field

[0001] The invention relates to the technical field of medical animal model research, and in particular to a method for constructing an animal model of lung injury after inhalation of radioactive dust. Background Art

[0002] Aerosol inhalation is the main route of uranium exposure for occupational personnel. The deposition site of inhaled particles depends on the size of the particles and their solubility in biological fluids. The size of the aerosol has a very important influence on the deposition site of uranium in the respiratory system. Larger particles (>10μm) usually stay in the upper respiratory tract or are excreted from the body through natural mechanisms such as coughing, while particles smaller than 5μm are deposited in the lungs and surrounding branches of the bronchial tree, a small amount of which is absorbed by the blood, and the rest is phagocytosed by macrophages in the lungs and surrounded to form nodules, which can lead to pulmonary fibrosis.

[0003] The lungs are the main target organs for inhaled sparingly soluble uranium compounds. After the sparingly soluble uranium oxide particles enter the lungs through the respiratory tract, their water-soluble particles are quickly absorbed after local dissolution. The sparingly soluble uranium particles on the alveolar surface, whether phagocytosed or not, can enter the lung interstitium and accumulate locally or enter the surrounding lymphoid tissue, causing a variety of inflammatory cells to gather in the lung damage area and release a large number of cytokines, oxygen free radicals and inflammatory mediators, inducing cell apoptosis and promoting pulmonary fibrosis. At the same time, these inflammatory cytokines act on a variety of immune cells, and these cytokines, free radicals, immune responses, etc. are closely related to autophagy and play a role in the formation of fibrosis. Therefore, inhaled uranium oxide aerosols, especially sparingly soluble uranium oxide, which remain in the lung tissue and nearby lymph nodes will cause more DNA damage, and the long-term stimulation of the lungs by radiation effects can lead to lung infections and even tumors.

[0004] Uranium aerosols have a complex action process in the human body after being inhaled into the lungs, which in turn has adverse effects on the human body. In order to deeply study the distribution, metabolism, and clearance process of uranium aerosols in the lungs and other organs after inhalation, as well as their specific action mechanisms on cells and tissues, observe and analyze the acute and chronic pathological changes caused by uranium exposure, and systematically study the effects of different levels of uranium exposure on health, it is very important to establish an animal model of lung injury after inhalation of radioactive dust.

[0005] At present, there are three main ways to establish an inhalation animal model: (1) high-speed impact on steel plates to produce uranium aerosols; (2) homemade uranium dust poisoning box; (3) intratracheal instillation of uranium suspension. The method of high-speed impact on steel plates to produce uranium aerosols is usually to use a high-speed moving object (such as a bullet) to impact the steel plate containing uranium, thereby producing fine uranium particle aerosols, which are then introduced into a closed space, and the experimental animals inhale these aerosols through natural breathing in this space. The method of making a homemade uranium dust poisoning box is that researchers will design a special poisoning box, and use a mechanical device or airflow system to evenly disperse the pre-prepared uranium dust in the box, and the experimental animals are placed in the box to inhale the dust through natural breathing. The method of intratracheal instillation of uranium suspension is to directly inject a certain amount of uranium suspension into the lungs of experimental animals through a tracheal tube. This method ensures that all animals receive the same dose and can directly deliver uranium particles deep into the lungs.

[0006] However, the above methods all have the following disadvantages:

[0007] (1) Inaccurate poisoning dose: The method of generating uranium aerosol by high-speed impact on steel plates makes it difficult to accurately control the amount and concentration of aerosol generated by impact, resulting in large differences in the poisoning dose in each experiment. Although the homemade uranium dust poisoning box can control the poisoning dose by adjusting parameters, the actual inhalation amount is still difficult to accurately measure, especially for differences between individual animals.

[0008] (2) The exposure method cannot simulate the main exposure mode of occupational personnel: the static exposure method of the homemade uranium dust exposure box is different from the long-term low-concentration exposure of occupational personnel in the working environment, and cannot fully simulate the actual occupational exposure scenario; intratracheal instillation of uranium suspension is not carried out through the natural breathing route, so it cannot simulate the actual occupational exposure scenario, especially in the study of long-term low-dose exposure. Summary of the invention

[0009] The present invention discloses a method for constructing an animal model of lung injury after inhalation of radioactive dust, aiming to solve the technical problems that the inhalation animal model established in the prior art has inaccurate poisoning dose and the poisoning method cannot simulate the exposure method of occupational personnel. The present invention adopts the following technical solutions:

[0010] The present invention provides a method for constructing an animal model of lung injury after inhalation of radioactive dust, comprising the following steps:

[0011] S1: Animal selection and housing conditions: Rats were selected and housed in SPF animal rooms for experiments;

[0012] S2: Animal grouping: Rats were divided into a blank control group and model groups with different exposure doses;

[0013] S3: Animal anesthesia and model establishment: First, sodium pentobarbital was injected intraperitoneally to anesthetize the rats, and the changes in the rats were observed; after anesthesia, the rats were lifted vertically, fixed with their heads up, and the airways of the rats were opened using a laryngoscope. Then, a rat lung dry powder quantitative nebulizer containing UO2 aerosol powder was inserted into the rat trachea, and the syringe piston was quickly pressed to the bottom;

[0014] S4: Animal observation after poisoning: After inhalation poisoning, shake the rats left and right to evenly distribute the UO2 aerosol powder in the rats' lungs, and pay attention to observe the rats' breathing and physiological status. If the rats are in discomfort, press the rats' hearts in time for cardiopulmonary resuscitation, and put them in cages for normal feeding after they recover.

[0015] S5: Animal model evaluation: observe the general signs of rats, measure the uranium content in lung tissue, observe the appearance of lung tissue, perform HE staining on lung tissue, and measure the content of tissue inflammatory factors and fibrosis factor-related indicators.

[0016] Preferably, in step S1, the rats are male SD rats, 9 to 10 weeks old, weighing 300 to 360 g.

[0017] Preferably, in step S1, the feeding conditions are: food and drinking water are freely available, adaptive feeding is carried out for one week in a clean and ventilated environment, the bedding is changed twice a week, the ambient temperature is 22±2°C, the relative humidity is about 55%, and the circadian rhythm is 12 hours.

[0018] Preferably, in step S2, the exposure dose of the model group is 0.5 to 8.0 mg.

[0019] Preferably, in step S3, the injection dose of the dry powder quantitative nebulizer for rat lungs is 2 mL.

[0020] Preferably, the injection amount of sodium pentobarbital is 30-50 mg / kg.

[0021] Preferably, in step S4, the normal feeding time after poisoning is 1 to 70 days.

[0022] Preferably, in step S5, the observation of general physical signs of the rat specifically includes the rat's spirit, hair condition, activity state and appetite.

[0023] Preferably, in step S5, the tissue inflammatory factor content and fibrosis factor-related indicators specifically include the mass concentrations of TNF-α, IL-1β, TGF-β1, Hyp, α-SMA and Coolagen I.

[0024] Preferably, in step S5, the rats are observed to be listless, with dull fur, reduced activity, gathering in groups, increased breathing and coughing, and decreased appetite.

[0025] Preferably, in step S5, the expression levels of TNF-α, IL-1β, TGF-β1, Hyp, α-SMA and Col agen I are increased.

[0026] The technical solution adopted by the present invention can achieve the following beneficial effects:

[0027] (1) Traditional uranium aerosol exposure methods, such as high-speed impact on steel plates to produce uranium aerosols, homemade uranium dust poisoning boxes, and intratracheal instillation of uranium suspensions, often make it difficult to achieve precise control of the poisoning dose. In contrast, the quantitative dry powder aerosol technology used in the present invention can ensure that all animals receive the same poisoning dose in each experiment. Specifically, the present invention can deliver a specific amount of UO2 powder directly into the deep lungs of rats through tracheal intubation under the guidance of a laryngoscope, avoiding the uncertainty caused by uneven aerosol distribution or dose fluctuations in traditional methods. This not only improves the repeatability of the experimental results, but also makes the study of dose-effect relationships more reliable.

[0028] (2) In actual occupational environments, uranium miners and nuclear industry practitioners are mainly exposed to uranium aerosols in low concentrations through the respiratory tract for a long time. The method of the present invention simulates the process of inhaling radioactive dust through the natural respiratory route through tracheal intubation under the guidance of a laryngoscope, which is closer to the actual occupational exposure scenario. Compared with direct intratracheal instillation, this method does not cause the problem of excessive local concentration and reduces abnormal pathological reactions caused by unnatural exposure methods. In addition, the use of a dry powder quantitative nebulizer can generate aerosols with smaller and more uniform particle sizes, which is more in line with the characteristics of uranium aerosols in actual working environments, further enhancing the authenticity and representativeness of the model.

[0029] (3) The rat acute lung injury model constructed by the present invention shows a high success rate and stability in inducing classic symptoms (such as increased dyspnea, inflammatory cell infiltration and fibrosis in lung tissue) and accompanying symptoms (such as decreased activity, mental depression, loss of appetite, and dull fur). This means that the model can maintain consistent results in different batches of experiments, reducing experimental errors and variability, which is particularly important for large-scale drug screening, mechanism research and treatment evaluation, and can provide stable and reliable experimental data to support subsequent research and application development.

[0030] (4) The animal model of the present invention can not only induce typical acute lung injury symptoms, such as dyspnea, inflammatory cell infiltration and fibrosis in lung tissue, but also simultaneously observe a series of accompanying symptoms, such as decreased activity, mental depression, loss of appetite and dull fur. These accompanying symptoms reflect the impact of uranium aerosol inhalation on the overall health status of rats and provide a more comprehensive evaluation index. By comprehensively evaluating these symptoms, researchers can more accurately judge the severity of lung injury and provide a reference for clinical diagnosis and treatment.

[0031] (5) The tracheal intubation and dry powder quantitative nebulizer technology under the guidance of laryngoscope adopted in the present invention are simple to operate and easy to standardize. The use of laryngoscope can ensure the accuracy of tracheal intubation and reduce the physiological stress on animals; while the dry powder quantitative nebulizer can achieve precise control of the poisoning dose through preset parameters. The whole process can be completed in a short time and is suitable for large-scale experimental operations. In addition, this method does not require complex equipment and technology, reducing the cost and difficulty of the experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments, which constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions explain the present invention and do not constitute improper limitations on the present invention. In the drawings:

[0033] Figure 1 HE staining of lung tissue. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention and the corresponding drawings. In the description of the present invention, it should be noted that the term "or" is usually used in the sense of including "and / or", unless the content clearly indicates otherwise. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0035] In order to solve the problems existing in the prior art, the embodiment of the present invention provides a method for constructing an animal model of lung injury after inhalation of radioactive dust. The present invention aims to develop an animal model that can focus on the core symptoms of lung injury caused by inhalation of radioactive dust and can evaluate the severity of lung injury. The rat acute lung injury model constructed by the present invention shows a high success rate and stability in inducing the classic symptoms of acute injury (increased dyspnea, inflammatory cell infiltration of lung tissue and fibrosis of lung tissue, etc.) and the associated symptoms of acute lung injury such as decreased activity, mental depression, loss of appetite, and dull fur. In the method for constructing the rat acute lung injury model of the present invention, a rat lung dry powder quantitative nebulizer filled with UO2 powder is inserted into the rat trachea through a laryngoscope to induce a rat acute lung injury model of inhalation of radioactive dust. This quantitative poisoning dry powder aerosol method has an accurate poisoning dose and better simulates the scene exposure of occupational personnel.

[0036] Example 1

[0037] A method for constructing an animal model of lung injury after inhalation of radioactive dust comprises the following steps:

[0038] (1) Animal selection and housing conditions: Male SD rats aged 9 to 10 weeks and weighing 320 ± 20 g were selected. Food and drinking water were available ad libitum and they were fed adaptively in a clean, ventilated environment for one week. The bedding was changed twice a week. The ambient temperature was 22 ± 2°C, the relative humidity was about 55%, and the circadian rhythm was 12 h.

[0039] (2) Animal grouping: The rats were divided into a blank control group and model groups with different exposure doses, with 10 rats in each group. The blank control group consisted of mice that had not been exposed to UO2 aerosol particles. The different UO2 aerosol exposure doses were 0.5 mg, 1.0 mg, 2 mg, 4 mg and 8 mg, respectively. The mice that had been exposed to UO2 aerosol particles were used as the model group.

[0040] (3) Animal anesthesia and model establishment: First, rats were anesthetized by intraperitoneal injection of an appropriate amount of sodium pentobarbital (40 mg / kg), and the changes in the rats were observed. After anesthesia, the rats were lifted vertically and fixed with their heads upward. The airway of the rats was opened using a laryngoscope, and then a rat lung dry powder quantitative nebulizer containing UO2 powder was inserted into the rat trachea. The syringe piston was quickly pressed to the bottom. The injection dose of the rat lung dry powder quantitative nebulizer was 2 mL.

[0041] (4) Animal observation after poisoning: After inhalation poisoning, shake the rat left and right to evenly distribute the UO2 aerosol in the rat's lungs, and pay attention to observe the rat's breathing and physiological state. If the rat appears unwell, press the rat's heart in time for cardiopulmonary resuscitation. After the rat recovers, put it in a cage for normal feeding. The normal feeding time is 7 days or 70 days.

[0042] (5) Animal model evaluation: After the modeling was completed, that is, 7 or 70 days after exposure, the model was evaluated by observing the general physical signs of rats, measuring the uranium content in the lung tissue, observing the appearance of the lung tissue, HE staining of the lung tissue, and ELISA testing of the content of inflammatory factors in the lung tissue and related indicators of fibrosis factors.

[0043] The evaluation results are as follows:

[0044] 1. Observation of general signs: The rats in the blank control group were active, in good spirits, with normal appetite, shiny fur, and no huddling. After 7 or 70 days of modeling, the rats in the model group gradually showed reduced activity, listlessness, loss of appetite, dull fur, huddling, and increased breathing and coughing. The higher the UO2 exposure dose, the more obvious the abnormal phenomenon.

[0045] 2. Determination of uranium content in lung tissue:

[0046] After collecting the blood of the rats, the mice were killed by cervical dislocation, and then the rat's sternum was clamped with forceps, and the ribs on both sides of the sternum were cut with surgical scissors to fully expose the rat's abdominal cavity and chest cavity, and the lungs were taken out. After weighing, the blood and impurities on the surface of the tissue were washed with sterile saline. A part of the lung tissue was taken for uranium content determination, a part of the tissue was immersed in 4% paraformaldehyde fixative (fixed for at least 48 hours), and the other part of the tissue was collected in a cryopreservation tube and stored in a -80°C refrigerator.

[0047] The lung tissue used for uranium content measurement is digested to release the uranium in the tissue. Common methods of digestion include wet digestion, dry ash digestion, and microwave-assisted digestion. The wet digestion method is to use strong acid (such as nitric acid, hydrochloric acid or hydrofluoric acid) to heat and digest, so that organic matter is decomposed and uranium is converted into soluble uranium compounds. The dry ash digestion method is to burn the sample in a high-temperature furnace until it is completely ash-coated, and then dissolve the ashes with acid. The microwave-assisted digestion method is to use microwave heating to accelerate the acid hydrolysis process, improve efficiency and reduce volatile losses. Those skilled in the art can adaptably select the above digestion methods according to actual needs. After the uranium in the lung tissue is released by digestion, the uranium element can be measured by methods such as inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma emission spectroscopy (ICP-OES), fluorescent X-ray spectroscopy (XRF) and alpha particle spectroscopy, and then the uranium content in the lung tissue is calculated.

[0048] The uranium content in the lungs of rats increased with the increase of UO2 poisoning dose. Compared with the blank control group, the uranium content in the lungs of rats in the 4mg and 8mg poisoning dose model groups were statistically different (P<0.001); as the time after UO2 poisoning prolonged, the uranium content in the lungs of rats in the poisoning group did not change significantly (P>0.05), and the optimal poisoning dose was finally determined to be 2mg.

[0049] 3. Observe the gross changes of lung tissue

[0050] After the ex vivo lung tissue was rinsed with PBS solution, the bleeding and edema of the lung lobes, the plaques and color of the tissue surface were observed and photographed. The lung surface of the rats in the blank control group was smooth and tender, without focal bleeding, while the lung surface of the rats in the UO2-exposed group had extensive bleeding and roughness with visible powder accumulation. With the increase of the UO2-exposed dose, the amount of powder accumulation increased, and larger nodules were visible on the lung surface.

[0051] 4. HE staining of lung tissue:

[0052] The fixed lungs were removed and dehydrated with anhydrous ethanol, transparentized with xylene, immersed in wax, embedded, and sliced ​​according to the routine method. After the slices were dewaxed and hydrated with gradient ethanol, the tissues were chemically stained with hematoxylin / eosin, and the pathological changes of the lung tissues of each group of animals were observed under an optical microscope. The process of HE staining of lung tissue is a commonly used method in the prior art, and the specific details are not described in detail.

[0053] from Figure 1 It can be seen that the lung tissue structure of rats in the blank control group was normal, the thickness of the alveolar septum was normal, no obvious inflammatory infiltration was observed, the alveolar cavity was not filled with red blood cells and inflammatory cells, there was no edema, the alveoli were regular and clear, and the thickness of the pulmonary interstitium was uniform. In the model group, as the dose of poison increased, the lung tissue structure of rats was destroyed, the alveolar septum became significantly widened, the alveoli atrophied, the alveolar wall was edematous and congested, accompanied by a large number of inflammatory cell infiltration, and the alveolar interstitial collagen fibers thickened in the late stage.

[0054] 5. Test the content of tissue inflammatory factors and fibrosis factor related indicators

[0055] The rat enzyme-linked immunosorbent assay (ELISA) kit uses the "sandwich method": the capture antibody is coated on the enzyme-linked immunosorbent assay plate to capture the target protein in the sample and the standard, the biotinylated detection antibody binds to the target protein, the SABC complex binds to the biotinylated detection antibody to form an immune complex, and after adding TMB colorimetric solution, if there is target protein in the reaction well, it will appear blue, and it will turn yellow after adding the stop solution. During the detection process, the free components are washed away, and the OD value is measured at 450nm with an enzyme-linked immunosorbent assay instrument. The target protein concentration is proportional to the OD value, and the concentration of the target protein in the sample is calculated by drawing a standard curve.

[0056] Lung tissues of rats were obtained, and the double antibody sandwich method (ELISA method) was used to detect the expression of TNF-α, IL-1β, TGF-β1, Hyp and Col l agen I. With the increase of the poisoning dose, the expression level of TGF-β1 showed a certain upward trend; with the extension of the time after poisoning, the expression levels of TNF-α, IL-1β, TGF-β1, Hyp and Col l agen I in the lung homogenate of the poisoned rats were significantly upregulated (P < 0.05).

[0057] After repeated experiments, the model groups all showed the same results. It can be seen that the rats in the model group showed typical symptoms of acute lung injury, which meets the reliability and effectiveness of the animal model of acute lung injury, that is, the cause of the disease, symptoms, and pathophysiology. Therefore, according to the construction method of the present invention, the rat acute lung injury model can be successfully obtained.

[0058] Therefore, in the method for constructing a rat acute lung injury model of the present invention, a rat lung dry powder quantitative nebulizer filled with UO2 aerosol powder is inserted into the rat trachea through a laryngoscope to induce a rat acute lung injury model caused by inhalation of radioactive dust. This method of quantitatively contaminating dry powder aerosols has an accurate contamination dose and better simulates the scene exposure of occupational personnel.

[0059] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A method for constructing an animal model of lung injury after inhalation of radioactive dust, characterized in that: The following steps are involved: S1: Animal selection and housing conditions: Rats were selected and housed in SPF animal rooms for experiments; S2: Animal grouping: Rats were divided into a blank control group and model groups with different exposure doses; S3: Animal anesthesia and model establishment: First, sodium pentobarbital was injected intraperitoneally to anesthetize the rats, and the changes in the rats were observed; after anesthesia, the rats were lifted vertically, fixed with their heads up, and the airways of the rats were opened using a laryngoscope. Then, a rat lung dry powder quantitative nebulizer containing UO2 aerosol powder was inserted into the rat trachea, and the syringe piston was quickly pressed to the bottom; S4: Animal observation after poisoning: After inhalation poisoning, shake the rats left and right to evenly distribute the UO2 aerosol powder in the rats' lungs, and pay attention to observe the rats' breathing and physiological status. If the rats are in discomfort, press the rats' hearts in time for cardiopulmonary resuscitation, and put them in cages for normal feeding after they recover. S5: Animal model evaluation: observe the general signs of rats, measure the uranium content in lung tissue, observe the appearance of lung tissue, perform HE staining on lung tissue, and measure the content of tissue inflammatory factors and fibrosis factor-related indicators.

2. The method for constructing an animal model of lung injury after radioactive dust inhalation according to claim 1, characterized in that: In step S1, the rats are male SD rats, 9 to 10 weeks old, weighing 300 to 360 g.

3. The method for constructing an animal model of lung injury after radioactive dust inhalation according to claim 1, characterized in that: In step S1, the feeding conditions are as follows: food and drinking water are freely available, adaptive feeding is performed for one week in a clean, ventilated environment, bedding is changed twice a week, the ambient temperature is 22±2°C, the relative humidity is about 55%, and the circadian rhythm is 12 hours.

4. The method for constructing an animal model of lung injury after radioactive dust inhalation according to claim 1, characterized in that: In step S2, the exposure dose of the model group was 0.5 to 8.0 mg.

5. The method for constructing an animal model of lung injury after radioactive dust inhalation according to claim 1, characterized in that: In step S3, the injection dose of dry powder quantitative nebulizer in rat lungs is 2 mL.

6. The method for constructing an animal model of lung injury after radioactive dust inhalation according to claim 1, characterized in that: In step S4, the normal feeding time after poisoning is 1 to 70 days.

7. The method for constructing an animal model of lung injury after radioactive dust inhalation according to claim 1, characterized in that: In step S5, the general physical signs of the rats are observed, including the spirit, hair condition, activity state and appetite of the rats.

8. The method for constructing an animal model of lung injury after radioactive dust inhalation according to claim 1, characterized in that: In step S5, the tissue inflammatory factor content and fibrosis factor related indicators specifically include the mass concentrations of TNF-α, IL-1β, TGF-β1, Hyp, α-SMA and Col lagen I.

9. The method for constructing an animal model of lung injury after radioactive dust inhalation according to claim 7, characterized in that: In step S5, it was observed that the rats were listless, had dull fur, reduced activity, gathered in groups, had increased breathing and coughing, and had a decreased appetite.

10. The method for constructing an animal model of lung injury after radioactive dust inhalation according to claim 8, characterized in that: In step S5, the expression levels of TNF-α, IL-1β, TGF-β1, Hyp, α-SMA and Col lagen I increased.

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