阅读说明：本技术 一种化合物用于制备具有改善肺纤维化作用的药物的用途 (Application of compound in preparation of medicine with effect of improving pulmonary fibrosis ) 是由 刘昭华 吴庆江 马建强 宋艳艳 王恒 岳永花 于 2021-06-28 设计创作，主要内容包括：本发明涉及药品、保健食品和食品领域,具体的,涉及一种化合物用于制备具有改善肺纤维化作用的药物的用途。所述化合物如式(I)所示,本发明的化合物可以提高GSH-PX和SOD的活性,降低MDA和NO的含量,从而有效的改善肺纤维化。(The invention relates to the fields of medicines, health-care foods and foods, in particular to application of a compound in preparing a medicine with the function of improving pulmonary fibrosis. The compound is shown as a formula (I), the compound of the invention can improve the activity of GSH-PX and SOD and reduce the content of MDA and NO, thereby effectively improving pulmonary fibrosis.)

1. The application of the compound in preparing the medicine with the effect of improving pulmonary fibrosis is characterized in that the compound is shown as a formula (I),

wherein:

R₁is selected from C₁-C₆Alkyl of (C)₁-C₆And C is heteroalkyl and₃-C₆one of cycloalkyl groups;

R₂one selected from the group consisting of a hydroxyl group, a methyl group, an ethyl group and a hydrogen atom;

R₃selected from hydrogen atoms, C₁-C₆Alkyl and C₁-C₆One of (a) heteroalkyl group(s).

2. The use according to claim 1, wherein the pulmonary fibrosis improving effect is the ability to scavenge oxygen free radicals, preferably the ability to modulate oxygen free radical metabolism.

3. The use of claim 2, wherein said scavenging of oxygen free radicals is an increase in GSH-PX and SOD activity and a decrease in MDA and NO levels.

4. The use according to claim 2, wherein said modulation of oxygen radical metabolism is maintaining a balance between pro-fibrotic and anti-fibrotic factors; preferably, the modulation of oxygen radical metabolism refers to modulation of proliferation of alveolar epithelial cells; preferably, the alveolar epithelial cells are of human origin, more preferably, the alveolar epithelial cells are type II alveolar cells.

5. Use according to claim 4, wherein the modulation of oxygen radical metabolism and/or the modulation of proliferation of alveolar epithelial cells is initiated by the action of an endogenous or exogenous stimulatory agent, preferably bleomycin.

6. The use according to any one of claims 1 to 5, wherein the compound promotes the proliferation of alveolar epithelial cells.

7. The use of any one of claims 1 to 5, wherein the compound inhibits proliferation of alveolar epithelial cells.

8. The use according to any one of claims 1 to 5, wherein the medicament further comprises pharmaceutically acceptable adjuvants or auxiliary ingredients.

9. The use according to claim 8, wherein the pharmaceutically acceptable adjuvant or auxiliary ingredient is at least one of water for injection, starch, dextrin, sucrose, magnesium stearate and distilled alcohol.

10. Use according to any one of claims 1 to 5, wherein R is₁Selected from methyl, said R₂Selected from the group consisting of hydroxy, said R₃Selected from hydrogen atoms.

Technical Field

The invention relates to the fields of medicines, health-care foods and foods, in particular to application of a compound in preparing a medicine with the function of improving pulmonary fibrosis.

Background

Pulmonary fibrosis is the terminal change of a large group of lung diseases characterized by fibroblast proliferation and massive extracellular matrix aggregation with inflammatory injury and tissue structure destruction, namely structural abnormality (scar formation) caused by abnormal repair after normal alveolar tissues are damaged. The majority of patients with pulmonary fibrosis have unknown etiology (idiopathic), and this group of diseases is called Idiopathic Interstitial Pneumonia (IIP), which is a large group of interstitial lung diseases. The most common disease type with pulmonary fibrosis as the main manifestation is Idiopathic Pulmonary Fibrosis (IPF), which is a serious interstitial lung disease that can lead to progressive loss of lung function. Pulmonary fibrosis seriously affects the respiratory function of human body, manifested as dry cough and progressive dyspnea, and the respiratory function of patients is continuously worsened with the aggravation of illness and lung injury. The incidence and mortality of idiopathic pulmonary fibrosis increases year by year, with an average survival period of only 2.8 years after diagnosis, with mortality rates higher than that of most tumors, known as a "neoplastic-like disease".

Excessive release of oxygen radicals after oxygen radical injury is considered to be a significant cause of pulmonary fibrosis. Excessive oxygen free radicals can reduce the oxygen free radical scavenging capacity of the organism, so that excessive oxygen free radicals exist in the organism, and protein cells are fibrosed under the action of the oxygen free radicals, thereby causing pulmonary fibrosis.

At present, no drug which can act on scavenging oxygen free radicals and treat pulmonary fibrosis is found.

Disclosure of Invention

In order to solve the technical problem that no drug capable of acting on scavenging oxygen free radicals to treat pulmonary fibrosis is found in the background art, the invention provides application of a compound in preparing a drug with an effect of improving pulmonary fibrosis.

The technical scheme adopted by the invention is as follows:

the invention provides an application of a compound in preparing a medicament with the function of improving pulmonary fibrosis,

the compound is shown as a formula (I),

wherein:

R₁is selected from C₁-C₆Alkyl of (C)₁-C₆And C is heteroalkyl and₃-C₆one of cycloalkyl groups;

R₂one selected from the group consisting of a hydroxyl group, a methyl group, an ethyl group and a hydrogen atom;

R₃selected from hydrogen atoms, C₁-C₆Alkyl and C₁-C₆One of (a) heteroalkyl group(s).

On the basis of the technical scheme, the invention can be further improved as follows.

Further, the effect of improving pulmonary fibrosis refers to the capability of scavenging oxygen free radicals, preferably, the capability of regulating oxygen free radical metabolism.

Furthermore, the function of scavenging oxygen free radicals is to improve the activity of GSH-PX and SOD and reduce the content of MDA and NO.

Further, the regulation of oxygen radical metabolism refers to maintaining the balance between the factors of fibrosis promotion and fibrosis resistance; preferably, the modulation of oxygen radical metabolism refers to modulation of proliferation of alveolar epithelial cells; preferably, the alveolar epithelial cells are of human origin, more preferably, the alveolar epithelial cells are type II alveolar cells.

Further, the regulation of oxygen free radical metabolism, and/or the regulation of proliferation of alveolar epithelial cells is initiated by the action of endogenous or exogenous stimulatory substances, preferably bleomycin.

Further, the compounds may promote proliferation of alveolar epithelial cells.

Further, the compounds may inhibit the proliferation of alveolar epithelial cells.

Furthermore, the medicine also comprises pharmaceutically acceptable auxiliary materials or auxiliary components.

Further, the pharmaceutically acceptable auxiliary materials or auxiliary components are at least one of injection water, starch, dextrin, sucrose, magnesium stearate and distilled alcohol.

Further, in the present invention,the R is₁Selected from methyl, said R₂Selected from the group consisting of hydroxy, said R₃Selected from hydrogen atoms.

The invention discovers that 30 mug/mL of the compound can obviously increase the proliferation of II-type alveolar cells, and 70-80 mug/mL of the compound can obviously inhibit the proliferation of II-type alveolar cells. Therefore, the compound of the invention can realize bidirectional regulation on the type II alveolar cells, and shows that the compound of the invention restores the normal proliferation of the type II alveolar cells. Thereby well maintaining the balance between the factors for promoting fibrosis and the factors for resisting fibrosis, realizing the treatment of pulmonary fibrosis and protecting the lung.

The invention has the beneficial effects that: 1. the compound can be used for preparing a medicament with the effect of improving pulmonary fibrosis and can effectively treat pulmonary fibrosis. 2. The compound can clear oxygen free radicals so as to treat pulmonary fibrosis. 3. The compounds of the invention can modulate the metabolism of oxygen radicals. 4. The compound of the invention can improve the activity of GSH-PX and SOD and reduce the content of MDA and NO. 5. The compound of the invention can maintain the balance between the fibrosis promoting factor and the anti-fibrosis factor, 6, the compound of the invention can regulate the proliferation of alveolar epithelial cells, and 7, the compound of the invention can regulate the proliferation of type II alveolar cells. The compound of 30 mug/mL can obviously increase the proliferation of the type II alveolar cells, and the compound of 70 mug/mL can obviously inhibit the proliferation of the type II alveolar cells. Therefore, the compound can realize bidirectional regulation on II-type alveolar cells, and shows the protective effect of the compound on pulmonary fibrosis. The compound provided by the invention can be used for preparing a medicine with the effect of improving pulmonary fibrosis, and pulmonary fibrosis can be well treated, so that the lung is protected.

Drawings

FIG. 1 is a graph of the effect of test compound 1 of the present invention on bleomycin-induced proliferation of type II alveolar cells, P <0.05 compared to 0. mu.g/mL of test compound 1 of the present invention;

FIG. 2 is a graph of HE staining of lungs from rats in the 21d blank control group according to the present invention;

FIG. 3 is a graph showing HE staining of rat lungs from a 21d model control group according to the present invention;

FIG. 4 is a graph of HE staining of lungs from a 21d prednisone positive group of rats of the present invention;

FIG. 5 is a graph of HE staining of rat lungs from the 21 st experimental compound 1 high dose group of the present invention;

FIG. 6 is a graph of HE staining of rat lungs from dose group of experimental compound 1 of the invention at 21 d;

FIG. 7 is a graph of HE staining of rat lungs from the low dose group of compound 1 tested in accordance with the invention at 21 d.

Detailed Description

I.definition

Alveolar epithelium: the alveolar surface has an intact epithelium. Epithelial cells include type i and type ii alveolar cells.

(1) Type i alveolar cell (type i alveolar cell): the type I alveolar cell is flat, covers most of the surface of the alveolus, has a thick nucleus-containing part protruding into the alveolar cavity, and has a thin thickness of about 0.2 μm in the anucleate part, and is a part for gas exchange. Under an electron microscope, I type alveolar cellules have few organelles, cytoplasm contains more swallow vesicles, the vesicles contain surface active substances and tiny dust particles, and the cells can transfer the substances into interstitium outside the alveoli so as to be removed. Type I alveolar cells do not have division-proliferation capacity.

(2) Type ii alveolar cell (type ii alveolar cell): the type II alveolar cells are located among the type I alveolar cells, the number of the type II alveolar cells is more than that of the type I alveolar cells, but the coverage area of the type II alveolar cells is smaller than that of the type I alveolar cells. The cells are cuboidal or rounded with the apex protruding into the alveolar space. The nucleus is round, the cytoplasm is lightly stained and is in a foam shape. Under electron microscope, cells are free and have a small amount of microvilli, and cytoplasm is rich in mitochondria and lysosomes and has relatively developed rough endoplasmic reticulum and Golgi complex. There are many secretory granules above the nucleus, and the granules with high electron density and different sizes and diameters of about 0.1-1.0 μm contain parallel lamellar structures, called hungry lamellar bodies (osmphatic multilamellar bodies). The main component in the body is phospholipid, mainly dipalmitoyl lecithin, and glycosaminoglycan and protein. After the release of the substance in the particles, a layer of mucus, called surfactant, is formed on the surface of the alveoli. The surfactant has effects of reducing alveolar surface tension and stabilizing alveolar size. When exhaling, the alveolus is reduced, the density of the surface active substance is increased, the surface tension is reduced, and the excessive collapse of the alveolus is prevented; when inhaling, the alveolus expands, the density of the surface active substance decreases, the retraction force of the alveolus increases, and the over-expansion of the alveolus can be prevented. Both lack or denaturation of surfactant can cause atelectasis, and over-ventilation can cause surfactant deficiency; inhalation of toxic gases can directly destroy surface active substances. The neonatal hyaline membrane disease is caused by the dysplasia of II type alveolus cells and the synthesis and secretion disorder of surfactant, so that the surface tension of alveolus is increased, the alveolus can not be expanded after the birth of the infant, and the neonatal respiratory distress syndrome appears. The type II alveolar cell has the potential of dividing, proliferating and differentiating into the type I alveolar cell, so the type II alveolar cell has the function of repairing damaged epithelium.

Medicine preparation:

the "drugs" are designed for use in animals and humans and can be administered via all routes of administration. Preferred routes of administration are injection, oral, pulmonary, nasal, rectal, parenteral. Such pharmaceutical compositions and unit dosage forms thereof may contain conventional or novel ingredients in conventional or special proportions, with or without additional active compounds or ingredients, and such unit dosage forms may contain any suitable effective amount of the active ingredient to be employed commensurate with the intended daily dosage range. The term "carrier" as applied to the pharmaceutical compositions of the present disclosure relates to a diluent, adjuvant or vehicle with which the active compound is administered. The compounds of formula (I) of the present disclosure, together with one or more adjuvants such as adjuvants, carriers or diluents, may be placed in the form of pharmaceutical compositions, unit doses or dosage forms. The medicaments may be employed in solid dosage forms (such as powders, granules, pellets, coated or uncoated tablets or filled capsules) or liquid dosage forms (such as solutions, suspensions, emulsions or capsules filled therewith) or semisolid dosage forms (such as gels, creams and ointments). The dissolution and release characteristics of one or more active ingredients of a pharmaceutical dosage form may vary from seconds to months.

Methods of treatment and pharmaceutical formulations

Whether by oral, injectable, rectal or parenteral (including intravenous and subcutaneous) or in some cases even topical routes, the compounds of the present disclosure of formula (I) or with one or more pharmaceutically-acceptable adjuvants, carriers or diluents, particularly and preferably in the form of their pharmaceutical compositions, may be administered to a subject in need thereof, such as a living animal (including a human) body, in an effective amount for the treatment, alleviation or amelioration, alleviation or elimination of indications or conditions to which they are sensitive or indications or conditions set forth elsewhere in the application.

As used herein, the terms "protect", "treat" and "treatment" mean to alleviate or alleviate at least one symptom of a disease in a subject, and within the meaning of the present disclosure, the terms "protect", "treat" and "treating" also refer to inhibiting, delaying onset (i.e., the pre-clinical manifestation of a disease) and/or reducing the risk of developing or worsening a disease.

The term "restoration" as used herein means that the function and/or structure of choroid plexus is altered to cause structural and/or functional abnormality under the action of certain endogenous or exogenous stimuli, such as ischemia and hypoxia, reperfusion injury, and "restoration" means returning the abnormal state to the normal state. By "protecting" is meant reducing or avoiding further alteration or impairment of choroid plexus structure and/or function by said endogenous or exogenous stimulatory agents.

The medicaments of the present disclosure may be administered orally, topically (lateral ventricle), parenterally or mucosally (e.g., buccally, by inhalation or rectally) in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers. It is often desirable to use an injection route. The active agent may be administered orally in The form of capsules, tablets, etc. (see Remington: The Science and Practice of Pharmacy, 20th Edition).

For oral administration in the form of a tablet or capsule, the active pharmaceutical ingredient may be combined with non-toxic, pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, sucrose, glucose, mannitol, sorbitol and other reducing and non-reducing sugars, microcrystalline cellulose, calcium sulfate or dibasic calcium phosphate); lubricants (e.g., magnesium stearate, talc or silica, stearic acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate, etc.); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate), coloring and flavoring agents, gelatin, sweetening agents, natural and synthetic gums (e.g., acacia, tragacanth or alginate), buffer salts, carboxymethylcellulose, polyethylene glycol, waxes, and the like. For oral administration in liquid form, the pharmaceutical components may be combined with non-toxic, pharmaceutically acceptable inert carriers (e.g., ethanol, glycerol, water), anti-settling agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous carriers (e.g., almond oil, oleyl esters, ethanol, or fractionated vegetable oils), preserving agents (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid), and the like. Stabilizers such as antioxidants (BHA, BHT, propyl gallate, sodium ascorbate, citric acid) may also be added to stabilize the dosage form.

Tablets containing the active compound may be coated by methods well known in the art. The compounds of the invention comprising as active compound formula (I) may also be incorporated into beads, microspheres or microcapsules, for example constructed from polyglycolic acid/lactic acid (PGLA). Liquid preparations for oral administration may take the form of, for example, solutions, syrups, emulsions or suspensions or they may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Formulations for oral administration may suitably be formulated so as to provide controlled or delayed release of the active compound.

The medicaments of the present disclosure may be delivered parenterally, i.e., by intravenous (i.v.), intracerebroventricular (i.c.v.), subcutaneous (s.c.), intraperitoneal (i.p.), intramuscular (i.m.), subcutaneous (s.d.), or intradermal (i.d.) administration, by direct injection, via, for example, bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example in ampoules or multi-dose containers with added preservative. The compositions may take the form of an excipient (excipient), a suspension, solution or emulsion in an oil or aqueous carrier, and may contain formulatory agents such as anti-settling agents, stabilising agents and/or dispersing agents. Alternatively, the active ingredient may be reconstituted with a suitable carrier (e.g., sterile pyrogen-free water) in powder form prior to use.

The medicaments of the present disclosure may also be formulated for rectal administration, for example, in the form of suppositories or retention enemas (e.g., containing conventional suppository bases such as cocoa butter or other glycerides).

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

One aspect of the present disclosure relates to a use of a compound for preparing a medicament having an effect of improving pulmonary fibrosis, wherein the compound is represented by formula (I),

wherein:

R₁is selected from C₁-C₆Alkyl of (C)₁-C₆And C is heteroalkyl and₃-C₆one of cycloalkyl groups;

R₂one selected from the group consisting of a hydroxyl group, a methyl group, an ethyl group and a hydrogen atom;

R₃selected from hydrogen atoms, C₁-C₆Alkyl and C₁-C₆One of (a) heteroalkyl group(s).

According to the use of the present invention, the compound of the present invention may also be represented by the following formula (II):

the compounds of formula (I) and formula (II) of the present invention can be obtained by chemical synthesis, or can be extracted from flowers, fruits, leaves, stems, roots of plants, such as henbane, by known methods.

Optionally, the function of improving pulmonary fibrosis refers to the function of scavenging oxygen free radicals, and preferably, the function of regulating oxygen free radical metabolism.

Optionally, the scavenging of oxygen free radicals is to increase the activity of glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) and to decrease the content of Malondialdehyde (MDA) and Nitric Oxide (NO).

Optionally, the regulation of oxygen radical metabolism refers to maintaining a balance between pro-fibrotic factors and anti-fibrotic factors; preferably, the modulation of oxygen radical metabolism refers to modulation of proliferation of alveolar epithelial cells; preferably, the alveolar epithelial cells are of human origin, more preferably, the alveolar epithelial cells are type II alveolar cells.

Alternatively, the compounds may promote proliferation of alveolar epithelial cells.

Alternatively, the compounds may inhibit the proliferation of alveolar epithelial cells.

Optionally, the medicament further comprises pharmaceutically acceptable auxiliary materials or auxiliary components.

Optionally, the pharmaceutically acceptable adjuvant or auxiliary component is at least one of water for injection, starch, dextrin, sucrose, magnesium stearate and distilled alcohol.

Optionally, the R is₁Selected from methyl, said R₂Selected from the group consisting of hydroxy, said R₃Selected from hydrogen atoms.

Taking a compound of formula (I), and R₁Is methyl, said R₂Is hydroxy, said R₃Is a hydrogen atom, to obtain the experimental compound 1. The test compound 1 was from the first pharmaceutical company, grand prefecture, china.

Example 1, effect of compound 1 on bleomycin-induced proliferation of type II alveolar cells.

1.1 cell culture and treatment

The type II alveolar cell is obtained from Hu Nanfeng Hui Biotech limited, and the type II alveolar cell is cultured in DMEMSuspending cells in nutrient medium, inoculating in six-well plate, placing at 37 deg.C and 5% CO₂Culturing in incubator to obtain II type alveolus cells.

Test compound 1 (from Gentle first pharmaceutical Co., Ltd., China) was dissolved in 20mg/mL of distilled water and filtered through a 0.22 μm filter, followed by preparing test compound 1 at final concentrations of 0, 10, 30, 60 and 70 μ g/mL using DMEM medium.

1.2 cell proliferation assay

Cell proliferation was measured by the CCK-8 method (C0042, Beyotime, China). 3000 cells were seeded in 96-well plates for 24 hours and then treated with bleomycin and test compound 1 in the above 1.1 sequence. At least 1h of incubation with CCK8 detection solution was added before the end of the experiment, followed by measurement of absorbance at 450nm using a microplate detector.

Second, the experimental procedure

The results shown in FIG. 1 were obtained by adding 10, 20, 30, 40, 70 and 80. mu.g/mL of test compound 1 to 60. mu.g/mL of bleomycin, followed by treating type II alveolar cells for 24 h.

Third, experimental results

As shown in fig. 1, 30 μ g/mL of test compound 1 significantly increased proliferation of type II alveolar cells, and 70g/mL of test compound 1 significantly inhibited proliferation of type II alveolar cells. Indicating that the compounds of the invention are capable of bidirectional modulation of II alveolar cell proliferation. The compound of 30 mug/mL can protect II alveolar cells from proliferation after acting for 24 hours.

Examples 2,

1. Material

1.1 drugs and reagents

The test compound 1 (from Chengdu first pharmacy), the reagent glutathione peroxidase (GSH-Px), the superoxide dismutase (SOD), the Malondialdehyde (MDA), the Nitric Oxide (NO) and the Coomassie brilliant blue protein assay kit are provided by the bioengineering research institute built from Nanjing;

tumor necrosis factor (TNF-. alpha.), platelet-derived growth factor (PDGF) and transforming growth factor (TGF-. beta.1) ELISA kits were all manufactured by WU Han Dynasty Biotechnology Ltd.

1.2 animals

144 clean-grade healthy Wistar rats with half male and female, weight (200 +/-20) g are provided by the laboratory animal center of the Chinese medicine institute in Shanxi province.

1.3 instruments

The main automatic microplate reader: Bio-Tek ELX8000 (manufactured by Shanghai Antai Analyzer Co., Ltd.), UV-260 ultraviolet-visible spectrophotometer (manufactured by Shanghai precision Instrument Co., Ltd.), and the like.

1.4 methods

All 144 rats were anesthetized with 20% urethane, fixed, disinfected routinely, and randomly and evenly divided into 6 groups, each group being half male and female rats, one of which was a blank control group. The remaining 5 model groups were all injected with 5ml of bleomycin solution^-1The model was replicated and divided into model control group, positive control group, high, medium and low dose groups of test compound 1 after surgery.

The blank control group was operated in the same manner as the model control group, and the same volume of physiological saline was injected into the trachea. The next day the gavage dosing was started in each treatment group, and high, medium and low dose groups of compound 1 were tested. The concentration of test compound 1 in the high dose group was 3.2mg.kg^-1The concentration of the test compound 1 in the dose group was 1.6mg.kg^-1The concentration of the test compound 1 in the low dose group was 0.8mg.kg^-1. The concentration of prednisone acetate suspension of the positive control group is 6mg^-1(corresponding to 7.5 times of the ratio of the human clinical daily dose of 48mg to the body weight), and equal volumes of physiological saline were administered to the blank group and the model group. For 21 consecutive days. Observing and recording general animal signs every day, taking materials on 7, 14 and 21 days respectively, taking 10 blood respectively in the front and back periods, 4 blood respectively in the middle period, and taking blood from abdominal aorta to detect cytokines; clamping the right lung main bronchus, lavaging the left lung to form a bronchoalveolar, and collecting bronchoalveolar lavage fluid for analysis; dissecting lung, weighing right lung, homogenizing, centrifuging, collecting supernatant, detecting antioxidase system, irrigating left lung, fixing, HE staining, and performing qualitative and semi-quantitative observation. The resulting plots are shown in figures 2 to 7.

1.5 statistical treatment

Experimental data metrologyData by mean ± standard deviationStatistical analysis was performed using SPSS13.0 software, single-factor analysis of variance and LSD-t test were used for interclass data comparison, and radius analysis was used for rank data results, with a test level α of 0.05.

2 results

2.1 Effect on the general status of rats

2.1.1 Effect on appearance of rats

The rats in the blank group are lively and active, the drinking water is normal, the fur is glossy, the weight is gradually increased, and the breath is stable; the model group model has listlessness, little drinking water, slow movement, lusterless fur, rapid respiration and unobvious weight increase within one week. After two weeks, the diet water increased and the body weight began to rise slowly; the experimental compound 1 had good fur in the high dose group, good drinking water, significant weight gain and even respiration. The positive control group has better early effect, the later period of the positive control group has stiff and erect fur and no movement, and the state is not as good as that of the positive control group but better than that of the positive control group.

2.1.2 Effect on body weight of PF rats

The body weight loss was most significant in the model group rats compared to the blank group (P < 0.01); the body weight of the rats in the other groups also decreased to different degrees. Compared with the model group, the rats with high and medium dose of the experimental compound 1 have obvious weight rising trend and significant statistical difference (P <0.05), and the specific results are shown in the following table 1.

TABLE 1 weight change of rats in each groupg)

Note: compared to the blank group, 1) P <0.05,2) P < 0.01; compared to model groups, 3) P <0.05,4) P <0.01

2.1.3 Effect on Lung coefficients in rats

The lung coefficients of the blank group at all stages have no obvious change, the lung coefficients of the model group rats at all stages are obviously higher than those of the same-stage blank group (P <0.01 or 0.05), the lung coefficients of the positive group and the experimental compound 1 at all stages are higher than those of the same-stage blank group rats, but are obviously lower than those of the same-stage model group rats (P <0.01 or 0.05), and the specific formula is shown in the following table 2:

table 2 comparison of pulmonary coefficients for each group of rats (n-10,)

group of	n	Day 7	Day 14	Day 21
					Blank control group	10	6.15±0.32	6.18±0.78	6.09±0.44
Model control group	10	9.21±0.572)	9.98±0.792)	9.79±0.632)
					Prednisone positive group	10	7.79±0.523)	7.86±0.353)	7.47±0.423)
High dose group (3.2 g.kg) of test compound 1-1)	10	7.46±0.493)	7.75±0.643)	7.76±0.963)
					Test Compound 1 Medium dose group (1.6 g.kg)-1)	10	7.57±0.533)	7.68±0.693)	7.29±0.773)
Test Compound 1 Low dose group (0.8 g.kg)-1)	10	7.76±0.493)	7.91±0.733)	7.38±0.693)

Note: 1) P <0.05,2) P <0.01, compared to the placebo group; compared to model groups, 3) P <0.05,4) P < 0.01.

2.2 Effect on SOD, GSH-Px and NO in rat Lung tissue

2.2.1 Effect on SOD in rat Lung tissue

Compared with the blank group, the SOD content in the lung tissue of the rats in the model group is obviously reduced (P is less than 0.01) on the 7 th day and the 21 st day, and the rats in other groups are reduced to different degrees, which indicates that the model is successfully copied. Compared with a model control group, the high, medium and low dose groups of the experimental compound 1 can obviously increase the content of SOD (P <0.05) in lung tissues of PF rats, and the details are shown in Table 3.

TABLE 3 comparison of parameters for superoxide dismutase SOD in lung tissue of rats in groups on days 7 and 21: (n＝10,U/mgprot)

Note: compared to the blank group, 1) P <0.05,2) P < 0.01; compared to model groups, 3) P <0.05,4) P <0.01

2.2.2 Effect on GSH-Px in rat Lung tissue

The content of glutathione peroxidase (GSH-Px) in the lung tissues of rats on the 7 th day and the 21 st day of the blank control group is stable; compared with a blank control group, the content of GSH-Px in the lung tissue of the rat in the model group is obviously reduced (P is less than 0.01), and the rest groups are reduced to different degrees, which indicates that the model is successfully copied. Compared with the model group, the high, medium and low dose groups of the experimental compound 1 can increase the content of GSH-Px in lung tissues of rats with pulmonary fibrosis (P <0.05, P <0.01), and the details are shown in Table 4.

TABLE 4 comparison of glutathione peroxidase (GSH-Px) parameters in lung tissue of groups of rats on days 7 and 21: (n＝10,U/gprot)

Note: comparison with blank control: 1) p <0.05,2) P < 0.01; comparison with model control group: 3) p <0.05,4) P < 0.01;

2.2.3 Effect on NO in rat Lung tissue

Compared with a blank control group, the NO content in the lung tissue of the rat in the model group is obviously increased (P is less than 0.01) at 7 days and 21 days, and the rest groups are increased in different degrees, which indicates that the model is successfully copied; compared with the model control group, the high, medium and low dose groups of the experimental compound 1 can significantly reduce the content of NO in lung tissues of rats with pulmonary fibrosis (P <0.05), as shown in table 5.

TABLE 5 comparison of Nitric Oxide (NO) parameters in lung tissue of rats in groups on days 7 and 21: (n＝10,umol/mgprot)

Note: 1) P <0.05,2) P <0.01, compared to the placebo group; 3) P <0.05,4) P <0.01, compared to model controls; 2.2.4 Effect on MDA in rat Lung tissue

Compared with the blank group, the lung tissues of rats in the model group are obviously increased in MDA content (P <0.01) on the 7 th day and the 21 st day, and the rest groups are also increased to different degrees, which indicates that the model is successfully copied. Compared with the model control group, the high, medium and low dose groups of the experimental compound 1 can obviously reduce the content of MDA (P <0.05) in the lung tissue of PF rats, and the details are shown in Table 6.

Table 6, 7, 21 days parameter comparison of malondialdehyde MDA in Lung tissue of rats: (n＝10,nmol/mgprot)

Group of	n	Day 7	Day 21
				Blank control group	10	2.77±0.67	2.71±0.99
Model control group	10	5.03±1.432)	4.98±1.192)
				Prednisone positive group	10	3.73±0.993)	2.98±0.673)
High dose group (3.2 g.kg) of test compound 1-1)	10	3.82±0.873)	2.82±0.773)
				Test Compound 1 Medium dose group (1.6 g.kg)-1)	10	3.79±0.713)	2.88±0.983)
Test Compound 1 Low dose group (0.8 g.kg)-1)	10	4.00±0.613)	3.03±0.76 3)

Note: compared to the blank group, 1) P <0.05,2) P < 0.01; compared to model groups, 3) P <0.05,4) P < 0.01;

2.3 Effect on pathological morphological changes in rat Lung tissue

2.3.1 general observations

The blank group had pink lungs, smooth surface and good elasticity on days 7, 14 and 21. Model group, day 7, dense punctate bleeding foci and black ecchymosis were seen in both lungs, and lung volume increased; on day 14, the two lungs are grey white, nodular changes with different sizes and old bleeding spots are visible locally, and the lung tissues are hard; on day 21, the lungs were pale, with reduced volume, harder palpation, nodular changes in the surface and cord-like grooves. Compared with the model group, at day 7, the positive group and the high, medium and low dose groups have better pathological changes in appearance than the model group, the two lungs are slightly reddish and have better elasticity, and the high and medium dose groups are better than the positive group and the low dose group; on days 14-21, the lungs of most animals in the high and medium dose groups were slightly darker, the elasticity of both lungs was still clear, occasionally, scattered punctate gray plaques were observed in the lung lobes, but better than those in the positive and low dose groups, and slightly better than those in the model group.

2.3.2 HE staining light microscopy

As shown in fig. 2 to 7, blank group: the lung tissue structure is clear, the alveolar space is not thickened, congestion, edema, inflammation and fibrosis are not shown, and no obvious exudation is generated in the alveolar cavity.

Model group: in 7d, the alveolitis is obvious, a large amount of inflammatory cell infiltration can be seen in the alveolar space and the pulmonary interstitium, the neutral granulocytes and the lymphocytes are taken as main components, and the alveolar space is edema and slightly widened. In 14d, alveolus inflammation was reduced, the thickening of alveolar spaces mainly caused by infiltration of macrophages and lymphocytes, fibroblasts proliferated, the alveolar spaces narrowed, interstitial collagen fibers proliferated in a band shape, and fibrosis occurred. Fibrosis at 21d is further aggravated, alveolar structure is destroyed or disappeared, a large amount of inflammatory cells are infiltrated, and lung tissues are mainly changed into collagen deposition and pulmonary fibrosis.

Positive group: the degree of alveolitis and pulmonary fibrosis is reduced compared with the model group, and the inflammatory cell infiltration is obviously reduced compared with the model group at the 7 d. At 14d, the lung is changed due to mild poisoning alveolitis, alveolar spaces are thickened, the lesion degree is lighter than that of a model group at the same period, and the lung tissue structure is basically complete. The lung fibrosis changes slightly at 21d, the range of the pathological changes is limited, and the collagen fiber deposition in the pleural and alveolar spaces is in a bundle shape.

Experimental compound 1 groups: the pulmonary alveolitis and pulmonary fibrosis degree of the high and medium dose groups are obviously reduced compared with the model group. The lung tissue inflammatory cell infiltration area at 7d was reduced compared to the model group. At 14d, the lung is changed in mild and moderate alveolitis, the lung tissue structure is more complete than that of the model group, the fibroblast proliferation is reduced, and the lesion degree is lighter than that of the model group at the same time. Mild to moderate fibrosis at 21d, limited range of disease, and fasciculate collagen fiber deposition in pleural and alveolar spaces. The degree of alveolitis and pulmonary fibrosis in the low dose group of test compound 1 was slightly reduced compared to the model group, but not statistically significant (P > 0.05).

2.3.3 semi-quantitative analysis of pathomorphology

The pathological changes of alveolitis and pulmonary fibrosis do not appear in the blank group through observation by a light microscope. The model group showed significant pathological changes from alveolitis to pulmonary fibrosis. The extent of alveolitis (-to +++ grade) and the extent of pulmonary fibrosis (-to +++ grade) were determined according to the methods of Szapiel SV et al. Through the Rait analysis, compared with the model group, the low, medium and high dose groups of the experimental compound 1 have obviously reduced alveolar inflammation compared with the model group. See table 7 for details.

TABLE 7 pathological analysis results of pulmonary alveolitis and pulmonary interstitial fibrosis in each group of rats (n ═ 10)

2.4 Effect on rat serum cytokine network

2.4.1 Effect on rat serum tumor necrosis factor alpha (TNF-alpha)

On days 7 and 21, the content of TNF-a in the serum of the blank group is basically kept unchanged; compared with the blank group, the TNF-a content in the serum of the model group is obviously increased (P is less than 0.01), which indicates that the modeling is successful; the positive group had a significant decrease in TNF-a parameters (P <0.05) compared to the model group; the experimental compound 1 has no significant difference in the low dose group (P > 0.05); the level of TNF-a in the serum of rats in the high-dose group in the experimental compound 1 is obviously reduced (P < 0.05); TNF-a levels in the medium dose group were comparable to those in the positive group on day 7, while TNF-a levels in the high dose group were comparable to those in the positive group by day 21. The TNF-a level in the serum of the experimental compound 1 at low, medium and high doses shows a continuous reduction trend and shows obvious dose-effect change, which is shown in Table 9.

Table 9 comparison of serum TNF-a parameters for groups of rats at days 7 and 21 (pg/mL,)

note: 1) P <0.05,2) P <0.01, compared to the placebo group; compared to model groups, 3) P <0.05,4) P <0.01

2.4.2 Effect on PF rat serum platelet-derived growth factor (PDGF)

On days 7 and 21, the PDGF content in the serum of the blank control group does not fluctuate greatly; compared with the blank control group, the PDGF content in the serum of the model group is obviously increased (P <0.01), which indicates that the modeling is successful; the positive group had significantly reduced serum PDGF parameters compared to the model group (P <0.01, P < 0.05); on day 7, the serum PDGF levels in the low dose group of test compound 1 were reduced (P <0.05), with no significant difference on day 21 (P > 0.05); the group with middle and high dose of the experimental compound 1 is obviously reduced (P <0.05 or P < 0.01); the serum PDGF levels in the low, medium and high dose groups of compound 1 showed a sustained decrease in the course of the dose-response changes, as shown in table 10.

Table 10 comparison of PDGF parameters in serum from rats on days 7 and 21 (pg/mL,)

note: 1) P <0.05,2) P <0.01, compared to the placebo group; compared to model groups, 3) P <0.05,4) P < 0.01;

2.4.3 Effect on PF rat seroconversion growth factor (TGF-. beta.)

On 7 th and 21 st days, the content of TGF-beta 1 in the serum of the blank control group does not change greatly; compared with a blank control group, the TGF-beta 1 content in the serum of the model control group is obviously increased (P is less than 0.01, P is less than 0.05); compared with the model group, the content of TGF-beta 1 in the serum of the positive group is obviously reduced (P is less than 0.01); the experimental compound 1 has reduced TGF-beta 1 content (P <0.01 and P <0.05) in low, medium and high dose groups, and shows obvious dose-effect change, which is shown in table 11.

Table 11 comparison of TGF- β 1 parameters in serum from rats in groups on days 7 and 21 (pg/mL,)

note: 1) P <0.05,2) P <0.01, compared to the placebo group; compared to model groups, 3) P <0.05,4) P < 0.01;

in the description herein, the description of the terms "one embodiment," "some embodiments," "specific embodiments," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.