CN117378563A - Construction method and application of pulmonary artery high-pressure animal model - Google Patents
Construction method and application of pulmonary artery high-pressure animal model Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
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- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
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- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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Abstract
The invention discloses a construction method and application of a pulmonary artery high-pressure animal model. The invention develops a novel pulmonary artery high-pressure rat model with energy metabolism change by combining monocrotaline and fenvalerate for co-intervention; the results show that the animal model can better simulate the pathological and hemodynamic changes of PAH, obviously improve the past deficiency of the monocrotaline by introducing fenvalerate, strengthen the smooth muscle proliferation of pulmonary artery, aggravate the right heart failure and not obviously increase the death rate; providing a pulmonary arterial hypertension animal model that better mimics the pathological and hemodynamic changes of PAH; has important significance for clinical research of pulmonary arterial hypertension; the method is particularly suitable for researching right ventricular metabolic disorder or potential right heart failure genes, and is expected to make new contribution to the research of pulmonary arterial hypertension; provides a research basis for screening related medicaments.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a construction method and application of a pulmonary artery high-pressure animal model.
Background
Pulmonary hypertension (Pulmonary arterial hypertension, PAH) is a disease that severely affects the quality of life and health of humans, and death results from Chang Yin heart failure. The pathogenesis is not completely understood, and is known to be related to unbalance of vascular active substances, immunoinflammatory reaction, gene mutation, non-coding RNA and the like. According to the etiology, they are classified into five categories, 1) arterial PAH, including idiopathic PAH, hereditary PAH, PAH caused by drugs and toxins, and the like; 2) PAH caused by left heart disease; 3) PAH caused by pulmonary disease and/or hypoxia; 4) Chronic thromboembolic PAH (CTEPH); 5) PAH with ambiguous and/or multifactorial mechanisms.
The most common pulmonary hypertension in adults is the result of left heart disease, followed by pulmonary disease. While pulmonary hypertension, which is most common in children, is associated with congenital heart disease, with fewer genetic or idiopathic episodes, but with a poor prognosis. After the treatment targets discovered in the beginning of the century, the treatment drugs for pulmonary arterial hypertension have no breakthrough progress in the last 20 years, and the research on the hemodynamic changes of pulmonary arterial hypertension through animal models is urgent research for finding potential treatment targets, especially potential targets or treatment genes for improving right heart failure.
Animal models help to discover relevant therapeutic targets for pulmonary circulatory hemodynamics and structural changes. The Monocrotaline (MCT) rat molding is widely used because of its simplicity and ease of operation. MCTs, however, also suffer from a number of problems such as: 1) MCT models have limited simulation of heavy angiogenic PAHs and fewer plexiform lesions in pulmonary vascular lesions; 2) MCT model rats are usually sacrificed for MCT-induced extrapulmonary toxicity, venous obstructive liver disease, rather than pulmonary arterial hypertension; 3) The high sensitivity of the MCT model to drug treatment appears to improve, reverse or prevent MCT-induced pulmonary vascular injury and PA pressure elevation, as well as the reversible PAH over time, in many experimental treatments. In particular, the last experimental treatment often does not match the clinical application in terms of excessive improvement in PAH characterization in MCT model animals, and therefore the use of MCT in pharmaceutical experiments is yet to be further determined.
For an animal model of pulmonary arterial hypertension, the first stage is the non-specific intima and adventitia thickening of the pulmonary artery, mainly comprising classical models (i.e. hypoxia and MCT), and the second stage is the occurrence of plexiform lesions, gradual occlusion of blood vessels, progressive elevation of pulmonary arterial pressure, i.e. mixed factor induced modeling, etc. The mixed factor-induced disease model correlates better with human severe PAH. Since clinical diagnosis is often severe, mixed factor induction models are more helpful in finding relevant therapeutic targets for pulmonary circulatory hemodynamic and structural changes. However, the mixing factor has defects such as increased mortality of animals, high economic cost of molding, and difficult operation in low-oxygen environment.
Therefore, the pulmonary artery high pressure model construction method which can better simulate the pathological and hemodynamic changes of PAH, is more suitable for the illness state of clinical severe patients and can make up for the deficiency of the classical MCT model has practical significance.
Disclosure of Invention
The object of the first aspect of the present invention is to provide a composition.
The object of the second aspect of the present invention is to provide the use of the above composition.
The object of a third aspect of the invention is to provide a product.
The fourth aspect of the invention aims to provide a method for constructing a pulmonary arterial hypertension animal model.
The fifth aspect of the present invention is directed to the use of the pulmonary artery high pressure animal model obtained by the above construction method.
The sixth aspect of the present invention aims to provide a method for screening medicines for treating pulmonary hypertension.
The technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided a composition comprising monocrotaline and fenvalerate.
Preferably, the dosage of the monocrotaline is 50-70mg/kg bw.
Preferably, the concentration of the monocrotaline is 0.5-1.5%.
Preferably, the fenvalerate is administered at a dose of 10-20mg/kg bw.
Preferably, the concentration of fenvalerate is 10-20mg/ml.
In a second aspect, the invention provides the use of a composition according to the first aspect of the invention for constructing a pulmonary arterial hypertension animal model or for preparing a product for constructing a pulmonary arterial hypertension animal model.
Preferably, the animal comprises a rodent.
Preferably, the rodent comprises a rat.
In a third aspect of the invention there is provided a product comprising the composition of the first aspect of the invention.
Preferably, the product is used for constructing a pulmonary arterial hypertension animal model.
Preferably, the product is an agent, feed, medicament.
Preferably, the product also contains any other components, such as common animal feed is added into the composition when preparing feed for constructing pulmonary artery high pressure animal model, and conventional solvent, adhesive, solubilizer, preservative and other auxiliary materials are added into the composition according to preparation of different medicaments when preparing medicaments for constructing pulmonary artery high pressure animal model, so as to prepare tablets, capsules, granules and oral liquid.
In a fourth aspect, the present invention provides a method for constructing an animal model of pulmonary arterial hypertension, comprising constructing an animal model using the composition of the first aspect of the present invention or the product of the second aspect of the present invention.
Preferably, the construction method comprises the steps of: the animals were dosed with monocrotaline on the first day of the modeling phase, with fenvalerate daily for at least 14 days prior to the modeling phase, and cultured for more than 30 days to obtain pulmonary arterial hypertension animal models.
Preferably, the fenvalerate is administered to the animal daily for the first 14 to 25 days of the modeling phase of the construction method.
Preferably, the pulmonary artery high pressure animal model is obtained by culturing for more than 30-45 days in the construction method.
Preferably, the dosage of the monocrotaline is 50-70mg/kg bw.
Preferably, the concentration of the monocrotaline is 0.5-1.5%.
Preferably, the administration mode of the monocrotaline is injection administration.
Preferably, the administration by injection comprises subcutaneous injection.
Preferably, the fenvalerate is administered at a dose of 10-20mg/kg bw.
Preferably, the concentration of fenvalerate is 10-20mg/ml.
Preferably, the fenvalerate is administered by gavage.
Preferably, the fenvalerate is formulated using corn oil as the solvent.
Preferably, the animal comprises a rodent.
Preferably, the rodent comprises a rat.
Preferably, the rat weighs 200-220g.
Preferably, the rat is male.
Preferably, the strain of rats is SD rats, wistar rats, lewis rats.
In a fifth aspect, the invention provides an application of the pulmonary hypertension animal model constructed by the method in the fourth aspect in researching a molecular mechanism of occurrence and development of pulmonary hypertension, or preparing and/or screening medicines for preventing and/or treating pulmonary hypertension, or preparing products for evaluating the effect of pulmonary hypertension treatment.
In a sixth aspect of the present invention, there is provided a method of screening for a drug for treating pulmonary hypertension, comprising:
(1) A pulmonary artery high pressure animal model constructed by the method of the fourth aspect of the invention;
(2) Applying the candidate drug to the pulmonary artery high pressure animal model established in the step (1);
(3) And evaluating and screening the candidate medicine for treating the pulmonary arterial hypertension by using the injury improvement degree of the pulmonary arterial hypertension animal model.
The beneficial effects of the invention are as follows:
the invention develops a novel pulmonary artery high-pressure rat model with energy metabolism change by combining monocrotaline and fenvalerate for co-intervention; the difference between the model and the classical MCT model is evaluated by combining pathology, cardiac ultrasonography and energy metabolism spectrum change, the change of the genome level of the model is analyzed, and the related mechanism is discussed; the results show that the animal model can better simulate the pathological and hemodynamic changes of PAH, is more suitable for the conditions of clinically severe patients, obviously improves the past deficiency of the monocrotaline by introducing fenvalerate, enhances the smooth muscle proliferation of pulmonary artery, aggravates the right heart failure and does not obviously increase the death rate; providing a pulmonary arterial hypertension animal model that better mimics the pathological and hemodynamic changes of PAH; has important significance for clinical research of pulmonary arterial hypertension; the method is particularly suitable for researching right ventricular metabolic disorder or potential right heart failure genes, and is expected to make new contribution to the research of pulmonary arterial hypertension; provides a research basis for screening related medicaments.
Drawings
FIG. 1 is a method for constructing a pulmonary arterial hypertension animal model.
Fig. 2 shows the results of body weight, right heart reconstruction, pulmonary artery pressure and left heart function.
FIG. 3 shows HE staining and immunofluorescence staining of rat lung tissue.
FIG. 4 is immunofluorescence of rat right ventricle mitochondrial function.
FIG. 5 is immunofluorescence of rat right ventricular Golgi function.
FIG. 6 is immunofluorescence of rat right ventricular macrophage function.
FIG. 7 shows the top 20 KEGG pathways enriched by NT and MCT rat left ventricle mRNA KEGG differential analysis.
FIG. 8 shows the top 20 KEGG pathways enriched by NT and MCT+fen rat left ventricle mRNA KEGG differential analysis.
FIG. 9 shows the top 20 KEGG pathways enriched by NT and MCT rat right ventricular mRNA KEGG differential analysis.
FIG. 10 shows the top 20 KEGG pathways enriched by NT and MCT+fen rat right ventricle mRNA KEGG differential analysis.
FIG. 11 shows the first 20 KEGG pathways enriched by MCT and MCT+fen rat left ventricle mRNA KEGG differential analysis.
FIG. 12 shows the first 20 KEGG pathways enriched by MCT and MCT+fen rat right ventricle mRNA KEGG differential analysis.
FIG. 13 is a graph showing specific detection results of the first 20 KEGG pathways enriched by differential analysis of MCT and MCT+fen rat right ventricle mRNA KEGG.
Fig. 14 is a rat right ventricular metabolism analysis.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
Example 1
1. The age of 6-8 weeks, the weight of the male SD rats is approximate (200-220 g), 40 male SD rats (the female rats are prevented from being used because of the interference of the female rats secreting estrogen, and the female rats are randomly and averagely divided into 4 groups of 10 male SD rats. Namely: control group, MCT group, fen group, mct+fen combination group. The rat feeding conditions were: the ambient temperature is 20-22 ℃; the ambient humidity is about 50%; the illumination intensity should be around 25 lux; the method is not particularly limited for the common rat raising environment.
MCT is dissolved by 1mmol/L dilute hydrochloric acid, then the pH is adjusted to 7.4 by 1mmol/L sodium bicarbonate, and finally physiological saline is diluted into 1% of the monocrotaline alkali solution. On day 1 of the start of the experiment, the administration was subcutaneously via the nape of the neck at a dose of 60mg/kg and a volume of 0.1-0.2ml.
Fen was dissolved in corn oil and shaken well to give a final 15mg/ml fenvalerate solution. The administration was by intragastric administration daily for the first 14 days of the experiment at a dose of 15mg/kg bw.
The animal construction steps are shown in FIG. 1.
2. Animal treatment
After 5 weeks of feeding, the left heart and right heart functions are checked by conventional anesthesia and skin preparation and color Doppler ultrasound of the heart of the small animal. Rats were sacrificed by routine anesthesia and serum, heart, lung tissue were taken for further testing.
3. Experimental method
The rats were weighed and the left ventricle + chamber space and the right ventricle free wall of the rats were weighed separately.
And (5) perfusing the fixed lung tissue and the right ventricle tissue to prepare paraffin sections, and performing immunofluorescence detection.
Tissue of the right ventricle is preserved by liquid nitrogen, and mRNA and energy metabonomics analysis is carried out.
4. Experimental results
1) The combined treatment group aggravates right heart failure and right heart reconstruction
After 5 weeks of feeding, the weight of the rats in the MCT group was found to be significantly reduced compared with the NT group, but the weight of the rats in the MCT+fen group was recovered compared with the MCT group, and the weight gain caused by the right heart failure edema was considered to be rather than improvement by combining the subsequent experimental results. The prior paper often uses the Fulton Index (Fulton Index) to evaluate right ventricular mass and indirectly evaluate right heart reconstruction, and finds that the Fulton Index of the MCT+fen group is further increased than that of the MCT group, which prompts that the right heart reconstruction is increased and aggravated.
In contrast to the MCT group, which was found by performing color ultrasound examination of small animals, the pulmonary artery pressure of the mct+fen group was not further increased, and in recent years guidelines did not evaluate the severity of pulmonary lesions by the level of pure pulmonary artery pressure (see Guidelines for the diagnosis and treatment of pulmonary hypertension for details). Although the MCT group did not significantly reduce left ventricular diastolic diameter compared to the NT group, the mct+fen group was significantly smaller than the NT group, suggesting that the mct+fen group had a more pronounced right heart enlargement, compressing the left heart resulted in a decrease in left diastolic diameter. The heart rate of the compensatory mct+fen group was elevated compared to the MCT group, although the heart rate was adjusted to the same level as much as possible (fig. 2).
2) Combined treatment group aggravates pulmonary smooth muscle proliferation
Typical pathological changes represented by pulmonary hypertension are: 1) Thickening and fibrosis of the intima to form an "onion skin" -like structure; 2) Thickening of the media, smooth muscle cell proliferation and hypertrophy; 3) The branch of the thin wall of the myogenic artery expands to present vascular plexiform lesions. The pulmonary smooth muscle proliferation of the MCT+fen group is found to be obvious compared with that of the pure MCT group through HE staining, and the method accords with the description of the increase of the Fulton index, the reconstruction of the right heart and the aggravation of the comprehensive illness state.
Further immunofluorescent staining was used to specifically label smooth muscle cells with a-SMA antibody, suggesting that mct+fen group had significantly higher a-SMA width than the MCT alone group, suggesting that mct+fen group had more pronounced pulmonary smooth muscle proliferation (fig. 3).
3) The combination treatment group aggravates the damage of right ventricular myocardial mitochondria and golgi
Mitochondria and golgi apparatus of right ventricular myocardium were examined by immunofluorescence, and compared with the MCT alone group, the expression of mitochondria and golgi apparatus of mct+fen group was modeled to be lower than that of either NT group or MCT alone, and the results suggested that mct+fen group aggravate mitochondrial autophagy. It was also found that in the right ventricle Mertk and CD68 immunofluorescence, the mct+fen group increased the number of CD68, and therefore analysis of the mct+fen group significantly increased right ventricle myocardial autophagy, and mitochondria that were phagocytized by autophagosomes were recognized by Mertk and then processed by macrophages (fig. 4-6).
4) Effect of combination treatment groups on right ventricular myocardial mRNA
The present example detects left and right ventricular mRNAs. In this study, it was found that there were many different genes in both the pure MCT and MCT+fen groups compared to the NT group, but there was no statistical difference in mRNA in the left ventricle of the MCT+fen group and the MCT group. Simple analysis of right ventricular mRNA found a clear statistical difference in mRNA from the mct+fen group. In conclusion, it is verified by immunofluorescence technique that mct+fen treatment group significantly inhibits mitochondrial and golgi functions, resulting in mitochondrial dysfunction and aggravating warburg reaction, which aggravation means aggravation of heart failure.
In order to verify that the conclusion is analyzed at the mRNA level, the MCT+fen group has larger interference on oxidative phosphorylation, diabetic cardiomyopathy and tricarboxylic acid circulation paths than the pure MCT group, and further metabonomics analysis verifies that the metabolic influence of the MCT+fen group on the right ventricle is mainly concentrated on tricarboxylic acid circulation and carbon metabolism, so that the interference of the MCT+fen treatment on the right ventricle metabolism is more obvious, the pulmonary arterial hypertension and the right heart failure are more serious, and the MCT+fen treatment is an animal model which is more suitable for exploring potential targets for treating the right heart failure (figures 7-14).
In conclusion, compared with the existing single-factor model, the novel rat pulmonary artery high-pressure model disclosed by the invention aggravates pulmonary artery smooth muscle proliferation, aggravates right ventricular myocardial reconstruction, has more obvious interference to right ventricular metabolism, and causes more serious pulmonary artery high-pressure right heart failure. Compared with the existing multi-factor model, the method is more economical, does not obviously increase the death rate, is simpler and more convenient to operate, and does not need additional equipment.
The present invention has been described in detail in the above embodiments, but the present invention is not limited to the above examples, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A composition comprising monocrotaline and fenvalerate.
2. Use of the composition of claim 1 for constructing a pulmonary arterial hypertension animal model or for preparing a product for constructing a pulmonary arterial hypertension animal model.
3. A product comprising the composition of claim 1.
4. A method of constructing an animal model of pulmonary arterial hypertension comprising constructing an animal model using the composition of claim 1 or the product of claim 3.
5. The construction method according to claim 4, characterized in that the construction method comprises the steps of: the animals were dosed with monocrotaline on the first day of the modeling phase, with fenvalerate daily for at least 14 days prior to the modeling phase, and cultured for more than 30 days to obtain pulmonary arterial hypertension animal models.
6. The construction method according to claim 5, wherein the dosage of monocrotaline is 50-70mg/kg bw; preferably, the concentration of the monocrotaline is 0.5-1.5%; preferably, the administration mode of the monocrotaline is injection administration.
7. The method of claim 5, wherein said fenvalerate is administered at a dose of 10-20mg/kg bw; preferably, the concentration of fenvalerate is 10-20mg/ml; preferably, the fenvalerate is administered by gavage.
8. The method of construction according to any one of claims 4 to 7, wherein the animal comprises a rodent;
preferably, the rodent comprises a rat.
9. Use of a pulmonary hypertension animal model constructed by the method of any one of claims 4 to 8 in researching molecular mechanisms of occurrence and development of pulmonary hypertension, or in preparing and/or screening medicines for preventing and/or treating pulmonary hypertension, or in preparing products for evaluating effects of pulmonary hypertension treatment.
10. The step of screening for a drug for treating pulmonary hypertension comprises:
(1) A pulmonary arterial hypertension animal model constructed by the method of any one of claims 4 to 8;
(2) Applying the candidate drug to the pulmonary artery high pressure animal model established in the step (1);
(3) And evaluating and screening the candidate medicine for treating the pulmonary arterial hypertension by using the injury improvement degree of the pulmonary arterial hypertension animal model.
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| CN109364050A (en) * | 2018-10-19 | 2019-02-22 | 大连医科大学 | Application of the Idebenone in preparation treatment pulmonary hypertension drug |
| WO2023005002A1 (en) * | 2021-07-28 | 2023-02-02 | 悦康药业集团股份有限公司 | Use of sildenafil citrate in preparation of medicine for preventing and/or treating pulmonary arterial hypertension |
| CN116650676A (en) * | 2023-07-18 | 2023-08-29 | 湖北省中西医结合医院(湖北省职业病医院) | Application of nobiletin in pulmonary arterial hypertension cardiopulmonary animal model |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109364050A (en) * | 2018-10-19 | 2019-02-22 | 大连医科大学 | Application of the Idebenone in preparation treatment pulmonary hypertension drug |
| WO2023005002A1 (en) * | 2021-07-28 | 2023-02-02 | 悦康药业集团股份有限公司 | Use of sildenafil citrate in preparation of medicine for preventing and/or treating pulmonary arterial hypertension |
| CN116650676A (en) * | 2023-07-18 | 2023-08-29 | 湖北省中西医结合医院(湖北省职业病医院) | Application of nobiletin in pulmonary arterial hypertension cardiopulmonary animal model |
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| 张增利等: ""混配农药中氰戊菊酯在小鼠体内的代谢和分布"", 苏州医学院学报, vol. 20, no. 6, 8 January 2004 (2004-01-08), pages 517 - 518 * |
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