CN116098891A - Application of water-soluble vitamin E in treating lung injury and pulmonary fibrosis - Google Patents

Abstract

The invention belongs to the technical field of medicine, and discloses an application of water-soluble vitamin E in the preparation of medicines for preventing and/or treating lung injury and pulmonary fibrosis. The invention discovers water-soluble vitamin E for the first time, and its action target is Galectin 3, which has better application prospects in the preparation of drugs for preventing and/or treating lung injury and pulmonary fibrosis, and has the characteristics of remarkable effect and less toxic and side effects.

Description

Application of water-soluble vitamin E in the treatment of lung injury and pulmonary fibrosis

Technical Field

The present invention relates to the field of medical technology, and in particular to application of water-soluble vitamin E in the preparation of a drug for preventing and/or treating lung injury and pulmonary fibrosis.

Background Art

Due to its physiological characteristics of being open to exchange gases with the outside world, the respiratory system is susceptible to various viruses, bacterial infections and physical and chemical damage, which can cause acute lung injury (ALI) and its severe state, acute respiratory distress syndrome (ARDS). The pathological characteristics of ALI and ARDS are damage to alveolar capillaries, endothelial cells and alveolar epithelial cells, which manifest as extensive pulmonary edema, micro-atelectasis, increased intrapulmonary shunt and decreased lung compliance. At present, the treatment of ALI and ARDS mainly adopts glucocorticoids and ventilator-assisted therapy, but many patients develop sequelae after recovery, such as diabetes, hypertension, femoral head necrosis, etc., which seriously affect the quality of life. Therefore, it is urgent to find safe and effective drugs for preventing and/or treating lung injury.

Pulmonary fibrosis (PF) is the terminal stage of all interstitial lung diseases (ILD). PF is caused by a variety of causes and is characterized by the accumulation of inflammatory cells such as macrophages, neutrophils and lymphocytes in the alveoli and the accumulation of fibrous connective tissue in the lung tissue, which ultimately leads to changes in lung tissue structure, impaired lung function and respiratory failure. In recent years, the incidence and mortality of pulmonary fibrosis have been increasing year by year due to factors such as environment and lifestyle.

Idiopathic pulmonary fibrosis (IPF) is a common type of PF. It is a chronic, progressive, interstitial fibrotic lung disease. It is characterized by unexplained recurrent epithelial cell damage, alveolar epithelial cell aging, and accumulation of profibrotic mediators, leading to progressive lung tissue scarring. IPF patients only have a survival period of 2-6 years after diagnosis, generally 3 years. In 2014, the FDA approved pirfenidone and nintedanib for the treatment of IPF, but they can only delay the progression of the disease, and it is still unknown whether they can prolong survival. Therefore, it is also very important to find safe and effective drugs to prevent and/or treat the occurrence and development of pulmonary fibrosis.

Galectin-3 is a β-galactoside binding lectin, which often exists in the form of homodimers and is mainly expressed in tumor cells, macrophages, epithelial cells, fibroblasts and activated T cells. It is important in many biological activities of various organs, including cell proliferation, apoptosis regulation, inflammation, fibrosis and host defense. Galectin-3 is mainly present in the cytoplasm, and is also expressed in the nucleus and cell surface. It can also be directly secreted into biological fluids such as serum, urine and tissue fluid by vesicles. Many studies have shown that Galectin-3 can be used as an important biomarker for the diagnosis or prognosis of myocardial fibrosis, renal fibrosis, pulmonary fibrosis, viral infection, autoimmune diseases, neurodegenerative diseases and tumor formation.

Therefore, inhibition of Galectin 3 may be an effective approach to prevent and/or treat lung injury and pulmonary fibrosis-related diseases.

Summary of the invention

In view of the current lack of safe and effective drugs for preventing and/or treating lung injury and pulmonary fibrosis, the present invention provides a compound targeting the Galectin 3 signaling pathway, a water-soluble vitamin E and a pharmaceutically acceptable salt thereof, for use in drugs for preventing and/or treating lung injury and pulmonary fibrosis, thereby providing a new approach for treating diseases related to lung injury and pulmonary fibrosis.

To achieve the above object, the present invention provides the following solutions:

One of the technical solutions of the present invention is to provide the use of water-soluble vitamin E and pharmaceutically acceptable salts thereof in the preparation of Galectin 3 inhibitors.

The second technical solution of the present invention is to provide the use of water-soluble vitamin E and pharmaceutically acceptable salts thereof in the preparation of drugs for preventing and/or treating lung injury diseases.

The "lung injury" described in the present invention may be acute lung injury or acute respiratory distress syndrome. The causes of the lung injury diseases include lung injury caused by bacterial and viral infection, gastroesophageal reflux, massive blood transfusion, drowning, shock, pancreatitis, pulmonary contusion, salicylate anesthetic overdose, alveolar hemorrhage, fat and amniotic fluid embolism, smoke and toxic gas inhalation, and the bacterial viruses, such as lipopolysaccharide.

The third technical solution of the present invention is to provide the use of water-soluble vitamin E and pharmaceutically acceptable salts thereof in the preparation of drugs for preventing and/or treating pulmonary fibrosis.

The "pulmonary fibrosis" described in the present invention may be idiopathic pulmonary fibrosis or secondary pulmonary fibrosis. The causes of the pulmonary fibrosis disease include pulmonary fibrosis caused by dust, radiation and/or drugs, such as bleomycin.

Furthermore, the dosage forms of the inhibitor include injection, tablet, powder, granule, pill, capsule, oral solution, ointment and cream.

Furthermore, the drug dosage forms include injection, tablets, powders, granules, pills, capsules, oral liquids, ointments, and creams.

According to the present invention, the compound of the present invention may exist in the form of isomers, and generally the "compound of the present invention" includes the isomers of the compound.

The present invention also relates to a pharmaceutical composition comprising a pharmaceutically effective dose of the compound and a pharmacodynamically acceptable carrier. When used for this purpose, if necessary, it can be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants to prepare a suitable application form or dosage form that can be used as a human medicine.

The pharmaceutical composition of the present invention can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum or rectum.

The administration route of the pharmaceutical composition of the present invention can be injection. Injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and acupoint injection. The dosage form can be a liquid dosage form and a solid dosage form. For example, the liquid dosage form can be a true solution, a colloid, a microparticle dosage form, an emulsion dosage form and a suspension dosage form. Other dosage forms include tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories and freeze-dried powder injections.

The composition of the present invention can be made into a common preparation, or a sustained-release preparation, a controlled-release preparation, a targeted preparation and various microparticle drug delivery systems.

In order to prepare a unit dosage form into tablets, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose and aluminum silicate; wetting agents and binders, such as water, ganpo, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate and polyvinylpyrrolidone; disintegrants, such as dry starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose and ethylcellulose; disintegration inhibitors, such as sucrose, tristearin, cocoa butter and hydrogenated oil; absorption promoters, such as quaternary ammonium salts and sodium lauryl sulfate; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin and polyethylene glycol. The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

In order to make the dosing unit into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin and talc, etc.; binders, such as gum arabic, tragacanth gum, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose and ethyl cellulose, etc.

In order to prepare the administration unit into a suppository, various carriers known in the art can be widely used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, enzymes of higher alcohols, gelatin, and semi-synthetic glycerol.

In order to prepare the dosing unit into capsules, the active ingredient is mixed with the above-mentioned various carriers, and the resulting mixture is placed in a hard gelatin capsule or a soft capsule. The active ingredient can also be prepared into microcapsules, suspended in an aqueous medium to form a suspension, or loaded into a hard capsule or prepared as an injection for use.

For example, the composition of the present invention is made into an injection preparation, such as a solution, a suspension solution, an emulsion, or a freeze-dried powder injection. This preparation can be aqueous or non-aqueous, and can contain one and/or more pharmacodynamically acceptable carriers, diluents, adhesives, lubricants, preservatives, surfactants, or dispersants. For example, the diluent can be selected from water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitol fatty acid enzyme, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose, or glycerol can be added to the injection preparation. In addition, conventional cosolvents, buffers, pH regulators, etc. can also be added. These adjuvants are commonly used in the art.

Furthermore, if necessary, colorants, preservatives, perfumes, flavoring agents, sweeteners or other materials may be added to the pharmaceutical preparations.

The dosage of the pharmaceutical composition of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality and individual response of the patient or animal, the route of administration, the number of administrations, etc., so the therapeutic dose of the present invention can vary widely. Generally speaking, the dosage of the compound of the present invention is well known to those skilled in the art. According to the actual amount of effective drug contained in the final preparation of the pharmaceutical composition of the present invention, it can be appropriately adjusted to achieve the requirement of its therapeutically effective amount, and the application of the present invention in the prevention and/or treatment of pulmonary fibrosis and acute lung injury related diseases can be completed.

Usually for patients weighing about 75 kg, the daily dose of the compound of the present invention is 0.001 mg/kg body weight to 200 mg/kg body weight, preferably 1 mg/kg body weight to 100 mg/kg body weight. The above dosage can be administered in a single dosage form or divided into several, such as two, three or four dosage forms, which is limited by the clinical experience of the doctor administering the drug and the dosage regimen. The compound or composition of the present invention can be taken alone or used in combination with other therapeutic drugs or symptomatic drugs.

The present invention discloses the following technical effects:

The present invention discovers for the first time a compound water-soluble vitamin E, which targets Galectin 3 and has good application prospects for preparing drugs for preventing and/or treating lung injury and pulmonary fibrosis, and has the characteristics of significant effects and small toxic and side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a curve showing the weight change of mice in each group of lung injury model;

FIG2 shows the changes in the expression of inflammation-related genes in a lung injury model regulated by water-soluble vitamin E;

FIG3 is a diagram of the pathological results of each group of the lung injury model, FIG3A is HE staining, and FIG3B is Masson staining;

FIG4 is a curve showing the weight change of mice in each group of the pulmonary fibrosis model;

FIG5 shows the changes in the expression of inflammation-related genes in a pulmonary fibrosis model regulated by water-soluble vitamin E;

FIG. 6 shows the pathological results of each group of the pulmonary fibrosis model, FIG. 6A shows HE staining, and FIG. 6B shows Masson staining.

DETAILED DESCRIPTION

Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

Unless otherwise specified, the "parts" described in the present invention are all based on mass parts.

Example 1 Effect of Water-Soluble Vitamin E on Galectin 3 Inhibitor Model

1. The experimental materials of this embodiment include:

Experimental drug: Water-soluble vitamin E, purchased from MCE Company, with the structural formula:

Test sample solution preparation: Accurately weigh an appropriate amount of sample water-soluble vitamin E and prepare it into a 0.02 M storage solution with DMSO for in vitro activity testing.

Experimental reagents: MTT reagent, purchased from Beijing Solebow Technology Co., Ltd., accurately weigh 0.25 g of MTT powder, add 50 mL of PBS to dissolve, filter with a 0.22 μm microporous filter membrane after dissolution, package and store at -20°C.

Experimental instruments: CO2 incubator, Panasonic, Japan; ELISA reader, Bio-tek, USA; Countstar automatic cell counter, Elite Life Sciences (Shanghai) Co., Ltd.; inverted microscope, NiKon, Japan; clean bench, Antai Air Technology Co., Ltd., Suzhou, China; 96-well culture plates (transparent and black), Corning, USA.

Experimental cells: RAW264.7 cells were purchased from ATCC; A549 and MRC-5 cells were purchased from Peking Union Medical College Cell Resource Center.

2. Experimental methods:

Logarithmically growing RAW264.7 and MRC-5 cells were seeded at 5×10 ³ cells/well in a 96-well plate (transparent plate), placed in a 37°C incubator containing 5% CO ₂ for adherent culture for 12 h, the culture medium was aspirated, the cells were washed once with PBS, different concentrations of culture medium containing water-soluble vitamin E were added, and the culture was continued for 24 h. The culture medium was removed, the cells were washed twice with PBS, 0.1 mL of serum-free culture medium containing 0.5 mg/mL MTT was added, and the cells were incubated in an incubator for 3 h. The MTT solution was removed, 0.1 mL of DMSO was added, and the cells were placed on an oscillator and gently shaken for 5 min. The absorbance was measured with a microplate reader (570/630 nm).

A549 cells in the logarithmic growth phase were seeded in a 96-well plate at 2×10 ⁴ cells/well and placed in a 37°C incubator with 5% CO ₂ for 12 hours. Culture media containing different concentrations of water-soluble vitamin E were added and cultured for 2 hours. 1 μM FITC-Galectin3 protein was added and incubated in the incubator for 2 hours. The fluorescence intensity was detected by a microplate reader at 485/528 nm.

3. Experimental results:

Water-soluble vitamin E was non-toxic at a concentration of 10 μM and significantly inhibited the binding of Galectin 3 protein to cells. The results are shown in Table 1.

Table 1 Cytotoxicity of compounds and their effects on Galectin 3 inhibitor model

Example 2 Effect of water-soluble vitamin E on lipopolysaccharide-induced lung injury model

1. The experimental materials of this embodiment include:

Experimental drugs:

Test sample solution preparation: Water-soluble vitamin E was purchased from MCE Company. An appropriate amount of water-soluble vitamin E was accurately weighed and prepared into a 4 mg/mL solution with water.

Dexamethasone solution preparation: Dexamethasone tablets, 0.75 mg/tablet, produced by Tianjin Lisheng Pharmaceutical Co., Ltd., prepared with water to make a 0.045 mg/mL solution.

Preparation of lipopolysaccharide solution: Lipopolysaccharide was purchased from Sigma-Aldrich Company and prepared into a 20 mg/mL stock solution with PBS, which was diluted to 0.2 mg/mL before use.

Hydroxyproline detection kit: purchased from Nanjing Jiancheng Bioengineering Institute.

Experimental Animals:

SPF male C57BL/6J mice, 6–8 weeks old, weighing approximately 20 g, were purchased from Spefoc (Beijing) Biotechnology Co., Ltd. with a license number of SCXK (Beijing) 2019-0010.

Experimental instrument: Flexivent small animal pulmonary function system: FlexiVent, SCIREQ Inc.

2. Experimental methods:

Model construction:

A mouse acute lung injury model was established by a single intratracheal injection of lipopolysaccharide (LPS). The specific implementation was as follows: the mice were fasted overnight, anesthetized with tribromoethanol (400 mg/kg), and LPS (0.5 mg/kg) was injected into the trachea using non-invasive endotracheal intubation technology, with a volume of 50 μL. The mice were then quickly stood upright and rotated to allow LPS to enter the lung lobes evenly. The sham-operated group of mice was injected with an equal amount of PBS into the trachea under the same anesthesia conditions.

Experimental groups and drug administration:

The mice were randomly divided into sham operation group (PBS + saline), model group (LPS + saline), dexamethasone group (LPS + dexamethasone) and water-soluble vitamin E group (LPS + water-soluble vitamin E).

On the day of the model, the mice were given drug intervention after they woke up, and the drugs were administered once a day. The sham operation group and the model group were given 0.1mL/10g of normal saline; the dexamethasone group was given an equal volume of dexamethasone (0.45mg/kg/d) by gavage; the water-soluble vitamin E group was given an equal volume of water-soluble vitamin E (40mg/kg/d) by gavage; the drugs were administered for 3 consecutive days, and the general clinical symptoms of the mice were observed at the same time.

Table 2 Grouping and drug administration of lung injury model

Animal medication and material collection:

Model and drug administration After 48 hours of feeding, the mice were fasted overnight. The next day, the mice were anesthetized with tribromoethanol, and the alveolar lavage fluid of the mice was collected. The wet weight of the right middle lobe of the lung was weighed; the right middle lobe of the lung was placed in a constant temperature oven at 60°C, and the dry weight of the right middle lobe of the lung was weighed after baking for 48 hours, and the wet-to-dry weight ratio of the lung was calculated; the left lobe of the lung was used for hydroxyproline determination; the right upper lobe of the lung was used for related cytokine detection; the right lower lobe of the lung was fixed and HE and Masson staining were performed. HE staining was used to observe the pathological morphology, and the lung tissue fibrosis score was performed at the same time. The grading and scoring criteria for lung fibrosis: 0, occasional slight thickening of the alveolar septum; no clear fibrosis was observed. 1, mild fibrosis of the alveolar wall or mild fibrosis of the bronchiolar wall. 2, moderate fibrosis of the alveolar wall or moderate fibrosis of the bronchiolar wall, no destruction of the lung tissue structure. 3. Moderate fibrous tissue hyperplasia of the alveolar wall or moderate fibrous tissue hyperplasia of the bronchiolar wall, with a large number of neutrophils and lymphocytes infiltrating, and no destruction of the lung tissue structure. 4. Small focal fibrous tissue hyperplasia, accompanied by mild destruction of the lung tissue structure. 5. Focal fibrous tissue hyperplasia, obvious destruction of the lung tissue structure, accompanied by fiber bundle formation. 6. Lamellar fibrous tissue hyperplasia, accompanied by severe destruction of the lung tissue structure. 7. Diffuse fibrous tissue hyperplasia, accompanied by severe destruction of the lung tissue structure, and honeycomb lung formation. 8. Obvious lung consolidation. Masson staining was performed by selecting 2 areas under a 200x microscope to take pictures. The tissue area, fibrous tissue area and integrated absorbance (IOD) of fibrosis were measured using Image-ProPlus 7.2 image analysis software. The degree of lung tissue fibrosis and the average optical density of fibrous tissue in each picture were calculated.

Statistical methods:

All data were expressed as mean ± standard deviation (Mean ± SD), and one-way analysis of variance was used for multiple groups, and p < 0.05 was considered statistically significant. * indicates p < 0.05 between the two corresponding groups; ** indicates p < 0.01 between the two corresponding groups.

3. Experimental results:

(1) Effects of water-soluble vitamin E on the physiological state of mice with lung injury model:

Cage-side observation revealed that the model group began to show symptoms such as irritability, increased activity, and shortness of breath from the first day, while the symptoms of the water-soluble vitamin E group and the dexamethasone group were significantly milder than those of the model group. Body weight was monitored, and compared with the sham operation group, the body weight of the mice after the model was significantly reduced, but there was no significant difference between the water-soluble vitamin E group and the dexamethasone group and the model group, as shown in Figure 1.

(2) Effects of water-soluble vitamin E on respiratory inflammation in mice with lung injury model:

The lung tissue wet weight/dry weight ratio can reflect the degree of lung tissue inflammation and edema. Protein exudation in bronchoalveolar lavage fluid reflects lung tissue inflammation and permeability. As can be seen from Table 3, compared with the sham operation group, the lung wet weight/dry weight ratio and protein exudation of the model group mice were significantly increased; while water-soluble vitamin E and dexamethasone could significantly reduce the lung wet weight/dry weight ratio and protein exudation.

Table 3 Effects on pulmonary edema in lung injury model

In addition, the determination of key cytokines related to the inflammatory response in lung tissue can more clearly describe the inflammatory response of the body caused by LPS. The results in Figure 2 show that LPS causes a significant increase in the mRNA expression levels of inflammatory factors IL-1β, IL-6 and TNF-α in lung tissue, while water-soluble vitamin E and dexamethasone can significantly reduce the mRNA expression levels of IL-1β, IL-6 and TNF-α.

The above results indicate that both water-soluble vitamin E and dexamethasone can significantly improve LPS-induced inflammatory response in lung tissue.

(3) Effect of water-soluble vitamin E on lung tissue fibrosis in mice with lung injury model

When pulmonary fibrosis occurs, the main component that increases in the lung is collagen fibers. Hydroxyproline is unique to collagen fibers. Determination of the content of lung hydroxyproline can reflect the content of collagen in lung tissue, and then reflect the degree of pulmonary fibrosis. The results in Table 4 show that compared with the sham operation group, the content of hydroxyproline in the lung tissue of mice in the model group was significantly increased; and the administration of water-soluble vitamin E or dexamethasone could significantly reduce the content of hydroxyproline in the lung tissue of mice.

In order to directly observe the changes in the morphological changes of the mouse lung tissue, HE staining was performed on the lung tissue. As can be seen from Figure 3A, there was no significant change in the morphological structure of the lung tissue in the sham operation group; the lung tissue in the model group was extensively fibrotic, with local consolidation, diffuse infiltration of a large number of lymphocytes and neutrophils, mild expansion of the remaining alveoli, and the entire lung tissue was honeycomb-like. The local alveolar epithelium was significantly atrophied and disappeared, and the lung tissue structure was severely damaged; while the above pathological changes in the water-soluble vitamin E group and the dexamethasone group were significantly improved, but there were also significant differences compared with the sham operation group. The fibrosis score was scored according to the pulmonary fibrosis grading scoring standard. Compared with the sham operation group, the fibrosis score of the model group mice was significantly increased; compared with the model group, both water-soluble vitamin E and dexamethasone could significantly reduce the fibrosis score of lung tissue, and the results are shown in Table 4.

Masson staining can measure the area of fibrous tissue and the integrated absorbance of fibrosis, reflecting the degree of lung tissue fibrosis and the density of fibrous tissue proliferation. Figure 3B is a set of typical Masson staining pictures taken. As can be seen from Figure 3B, the lung tissue of mice in the sham operation group contains a small amount of collagen fibers, which are the main component of the extracellular matrix; the collagen fibers of mice in the model group increased significantly, and the density of fibrous tissue proliferation increased significantly, and typical pulmonary interstitial fibrosis appeared; the water-soluble vitamin E group and the dexamethasone group also showed changes in pulmonary interstitial fibrosis, but compared with the model group, the collagen fiber area and the density of fibrous tissue proliferation in the lung tissue of the water-soluble vitamin E group and the dexamethasone group were reduced. From the quantitative results in Table 4, compared with the model group, water-soluble vitamin E and dexamethasone significantly reduced the fibrosis area and fibrosis density.

Table 4 Effects on lung tissue fibrosis in lung injury model

The above results indicate that both water-soluble vitamin E and dexamethasone can significantly improve lipopolysaccharide-induced lung fibrosis.

Example 3 Effect of water-soluble vitamin E on bleomycin-induced pulmonary fibrosis model

1. The experimental materials of this embodiment include:

Experimental drugs:

Test sample solution preparation: Water-soluble vitamin E was purchased from MCE Company. An appropriate amount of water-soluble vitamin E was accurately weighed and prepared into a 4 mg/mL solution with water.

Dexamethasone solution preparation: Dexamethasone tablets, 0.75 mg/tablet, produced by Tianjin Lisheng Pharmaceutical Co., Ltd., prepared into 0.045 mg/mL solution with normal saline.

Preparation of bleomycin solution: Bleomycin hydrochloride for injection, 15U/vial, produced by Hanhui Pharmaceutical Co., Ltd., prepared into 100U/mL stock solution with PBS, diluted before use.

Hydroxyproline detection kit: purchased from Nanjing Jiancheng Bioengineering Institute.

Experimental Animals:

SPF male C57BL/6J mice, 6–8 weeks old, weighing approximately 20 g, were purchased from Spefoc (Beijing) Biotechnology Co., Ltd. with a license number of SCXK (Beijing) 2019-0010.

Experimental equipment:

Flexivent small animal pulmonary function system: flexiVent, produced by SCIREQ Inc.

2. Experimental methods:

Model construction:

A mouse pulmonary fibrosis model was established by a single intratracheal injection of bleomycin (BLM). The specific implementation was as follows: the mice were fasted overnight, anesthetized with tribromoethanol (400 mg/kg), and bleomycin (3U/kg) was injected into the trachea with a volume of 50 μL using non-invasive endotracheal intubation technology, and then the mice were quickly upright and rotated to allow BLM to enter the lung lobes evenly. The sham-operated group of mice was injected with an equal amount of PBS into the trachea under the same anesthesia conditions.

Experimental groups and drug administration:

The mice were randomly divided into sham operation group (PBS + saline), model group (BLM + saline), dexamethasone group (BLM + dexamethasone) and water-soluble vitamin E group (BLM + water-soluble vitamin E).

On the day of the model, the mice were given drug intervention after they woke up, and the drugs were administered once a day. The sham operation group and the model group were given 0.1mL/10g of normal saline; the dexamethasone group was given an equal volume of dexamethasone (0.45mg/kg/d) by gavage; the water-soluble vitamin E group was given an equal volume of water-soluble vitamin E (40mg/kg/d) by gavage; the drugs were administered for 21 consecutive days, and the general clinical symptoms of the mice were observed at the same time.

Table 5 Pulmonary fibrosis model grouping and drug administration

Animal materials:

Model and drug administration After 21 days of feeding, mice were fasted overnight and anesthetized with tribromoethanol. Lung function of mice was tested, lung weight was weighed, and lung index was calculated (lung index = lung weight (g) / body weight (g) × 100%); the left lobe of the lung was used for hydroxyproline determination; the right upper lobe of the lung was used for related cytokine detection; the right lower lobe of the lung was fixed and HE and Masson staining were performed. HE staining was used to observe the pathological morphology, and lung tissue fibrosis was scored at the same time. Pulmonary fibrosis grading scoring criteria: 0, occasional slight thickening of alveolar septa; no clear fibrosis was observed. 1, mild fibrosis of alveolar wall or mild fibrosis of bronchiolar wall. 2, moderate fibrosis of alveolar wall or moderate fibrosis of bronchiolar wall, no destruction of lung tissue structure. 3, moderate fibrosis of alveolar wall or moderate fibrosis of bronchiolar wall, a large number of neutrophils and lymphocytes infiltrated, no destruction of lung tissue structure. 4. Small focal fibrous tissue hyperplasia, accompanied by mild destruction of lung tissue structure. 5. Focal fibrous tissue hyperplasia, obvious destruction of lung tissue structure, accompanied by fiber bundle formation. 6. Lamellar fibrous tissue hyperplasia, accompanied by severe destruction of lung tissue structure. 7. Diffuse fibrous tissue hyperplasia, accompanied by severe destruction of lung tissue structure, honeycomb lung formation. 8. Obvious lung consolidation. Masson staining was performed by selecting 2 areas under a 200x microscope to take pictures. Tissue area, fibrous tissue area and integrated absorbance (IOD) of fibrosis were measured using Image-Pro Plus 7.2 image analysis software. The degree of lung tissue fibrosis and the average optical density of fibrous tissue in each picture were calculated.

Statistical methods:

All data were expressed as mean ± standard deviation (Mean ± SD), and one-way analysis of variance was used for multiple groups. P < 0.05 was considered statistically significant. * indicates p < 0.05 between the two groups; ** indicates p < 0.01 between the two groups.

3. Experimental results:

(1) Effects of water-soluble vitamin E on the physiological state of mice with pulmonary fibrosis model

Cage-side observation revealed that the model group mice began to show symptoms such as hair color difference, frizzy hair, irritability, increased activity, and shortness of breath from the 5th day; the symptoms of the dexamethasone group and the water-soluble vitamin E group were significantly milder than those of the model group. The weight of the mice was monitored and it was found that the weight of the mice decreased significantly after modeling, and began to increase after 6 days; the weight loss in the dexamethasone group was more obvious; and there was no significant difference between the water-soluble vitamin E group and the model group, as shown in Figure 4. The survival rate was statistically analyzed, as shown in Table 6. At 21 days, the survival rate of the sham operation group was 100%, the survival rate of the model group was 75%, the survival rate of the water-soluble vitamin E group was 75%, and the survival rate of the dexamethasone group was 66.66%.

Table 6 Effect on the survival rate of pulmonary fibrosis model mice

(2) Effects of water-soluble vitamin E on respiratory function in mice with pulmonary fibrosis

The respiratory function of mice was tested using the Flexivent small animal lung function system. The respiratory function impairment of mice was determined by respiratory system resistance (Rrs), respiratory system elasticity (Ers) and respiratory system compliance (Crs).

As can be seen from Table 7, bleomycin can induce significant damage to the respiratory function of mice, which is manifested by a significant increase in respiratory system resistance and respiratory system elasticity, and a significant decrease in respiratory system compliance; while water-soluble vitamin E and dexamethasone can significantly improve the above indicators, and the respiratory system resistance is close to the level of the sham operation group. The results show that water-soluble vitamin E and dexamethasone can significantly improve the respiratory function damage of mice induced by bleomycin.

Table 7 Effects on lung function in pulmonary fibrosis model

The above results indicate that water-soluble vitamin E can significantly improve bleomycin-induced respiratory function damage in mice.

(3) Effects of water-soluble vitamin E on respiratory inflammation in mice with pulmonary fibrosis model

Determination of key cytokines in the inflammatory response of lung tissue can describe the body's inflammatory response induced by bleomycin. The results in Figure 5 show that bleomycin caused a significant increase in the mRNA expression levels of inflammatory factors IL-1β, IL-6 and TNF-α in lung tissue, while water-soluble vitamin E and dexamethasone could significantly reduce the mRNA expression of IL-1β, IL-6 and TNF-α, close to the level of the sham operation group. The results show that water-soluble vitamin E and dexamethasone can significantly improve the inflammatory response of lung tissue induced by bleomycin.

(4) Effect of water-soluble vitamin E on lung fibrosis in mice with pulmonary fibrosis model

The lung index can reflect the degree of fibrosis in the lung tissue of mice with pulmonary fibrosis model. As can be seen from Table 8, compared with the sham operation group, the lung index of mice in the model group was significantly increased; water-soluble vitamin E could significantly reduce the lung index; and the dexamethasone group only had a decreasing trend.

When pulmonary fibrosis occurs, the main component increased in the lung is collagen fiber, and hydroxyproline is unique to collagen fiber. Determination of lung hydroxyproline content can reflect the content of collagen in lung tissue, and then reflect the degree of pulmonary tissue fibrosis. As can be seen from Table 8, compared with the sham operation group, the hydroxyproline content of mice in the model group was significantly increased; water-soluble vitamin E and dexamethasone can significantly reduce the hydroxyproline content.

In order to directly observe the morphological changes of mouse lung tissue, HE staining was performed on the lung tissue. As can be seen from Figure 6A, there was no obvious change in the morphological structure of the lung tissue in the sham operation group; in the model group, pulmonary interstitial vascular congestion, extensive widening of alveolar septa, shrinkage of alveolar cavities, local patchy pulmonary fibrosis, severe destruction of lung tissue structure, and diffuse infiltration of a large number of lymphocytes, plasma cells, and neutrophils in the lesion site; the above pathological changes in the dexamethasone group and the water-soluble vitamin E group were alleviated, but there were also significant differences compared with the sham operation group.

The lung tissue of mice was scored according to the pulmonary fibrosis grading standard. Compared with the sham operation group, the fibrosis score of the model group mice was significantly increased; water-soluble vitamin E can significantly reduce the fibrosis score, while the dexamethasone group did not reduce it significantly. The results are shown in Table 8.

Masson staining can measure the area of fibrous tissue and the integrated absorbance of fibrosis, reflecting the degree of lung tissue fibrosis and the density of fibrous tissue proliferation. Figure 6B is a set of typical Masson staining pictures taken. It can be seen from Figure 6B that the lung tissue of mice in the sham operation group contains a small amount of collagen fibers, which are the main component of the extracellular matrix; the collagen fibers of mice in the model group increased significantly, and the density of fibrous tissue proliferation increased significantly, and typical pulmonary interstitial fibrosis appeared; the dexamethasone group and the water-soluble vitamin E group also showed changes in pulmonary interstitial fibrosis, but compared with the model group, the degree of fibrosis was reduced. From the quantitative results in Table 8, compared with the model group, water-soluble vitamin E significantly reduced the area of fibrosis and the density of fibrosis, and the dexamethasone group also showed a downward trend, but the effect was slightly worse.

Table 8 Effects on lung tissue fibrosis in pulmonary fibrosis model

The above results show that both water-soluble vitamin E and dexamethasone can significantly improve bleomycin-induced lung fibrosis, and the effect of water-soluble vitamin E is slightly better than that of dexamethasone.

From the above experimental results, it can be seen that water-soluble vitamin E can significantly reduce alveolar inflammation, interstitial lung inflammation and collagen deposition in lung tissue of mice with lipopolysaccharide-induced lung injury model, and significantly reduce interstitial lung inflammation and lung tissue fibrosis in mice with bleomycin-induced pulmonary fibrosis model. It can reduce the degree of lung injury, prevent and treat pulmonary fibrosis, improve lung function and increase survival rate.

Water-soluble vitamin E has a good effect in preventing and treating lung injury and pulmonary fibrosis, and its effect is slightly better than dexamethasone.

The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims (5)

1. The application of water-soluble vitamin E shown in the formula (I) and pharmaceutically acceptable salts thereof in preparing Galectin3 inhibitor;

2. the application of water-soluble vitamin E shown in the formula (I) and pharmaceutically acceptable salts thereof in preparing medicaments for preventing and/or treating lung injury diseases;

3. the use according to claim 2, wherein the lung injury is acute lung injury or acute respiratory distress syndrome.

4. The application of water-soluble vitamin E shown in the formula (I) and pharmaceutically acceptable salts thereof in preparing medicines for preventing and/or treating pulmonary fibrosis diseases;

5. the use according to claim 4, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis or secondary pulmonary fibrosis.

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