CN114099637A

CN114099637A - Application of Apelin in preparation of medicine for treating silicosis

Info

Publication number: CN114099637A
Application number: CN202111545926.5A
Authority: CN
Inventors: 赵娜; 黄永顺; 瞿红鹰
Original assignee: Guangdong Institute Of Occupational Disease Control (guangdong Occupational Health Testing Center); Guangdong No 2 Peoples Hospital
Current assignee: Guangdong Institute Of Occupational Disease Control (guangdong Occupational Health Testing Center); Guangdong No 2 Peoples Hospital
Priority date: 2021-12-15
Filing date: 2021-12-15
Publication date: 2022-03-01

Abstract

The invention belongs to the technical field of pharmaceutically active peptides, and discloses application of Apelin in preparation of a medicament for treating silicosis. Experiments show that Apelin has good curative effect on relieving the pathological changes of silicosis, has good medicinal value prospect, and can be developed into a novel medicament for treating silicosis.

Description

Application of Apelin in preparation of medicine for treating silicosis

Technical Field

The invention belongs to the technical field of pharmaceutically active peptides, and particularly relates to application of Apelin in preparation of a medicament for treating silicosis.

Background

Apelin peptide (Apelin/APLN) is a biologically active peptide, which was first isolated from bovine gastric secretions, purified and named in 1998. The human Apelin gene is located on the X chromosome, the precursor peptide of the human Apelin gene consists of 77 active amino acid residues and can be cut into active peptide molecular fragments with different lengths by proteolytic enzyme, wherein Apelin-13 and [ Pyr1] Apelin-13 are the most common subtypes in human plasma, and the receptor of the Apelin gene is G protein coupled receptor-angiotensin I1 type receptor related protein.

Recently, Apelin injection is found to reduce bronchopulmonary dysplasia and pulmonary fibrosis of newborn rat pups exposed to high oxygen, reduce inflammation and lung injury induced by lipopolysaccharide, and reduce right ventricular overload and hypertrophy of pulmonary arterial hypertension rats induced by picloram, thereby improving biventricular diastolic function. In the aspect of clinical research, Apelin is infused in a short period of vein, so that the hemodynamics of pulmonary vessels of a pulmonary hypertension patient can be effectively improved, including reduction of the resistance of the pulmonary vessels, increase of the cardiac output and the like. In a neonatal Bronchopulmonary Dysplasia (bronchhol pulmonary BPD) model of a newborn rat exposed to 100% oxygen in a long-term fraction, intraperitoneal injection of exogenous Apelin can reduce pulmonary fibrin deposition and inflammatory factor infiltration, improve alveolar fibrosis and reduce pulmonary inflammation, and partially restore alveolar formation.

It is known that various lung-related diseases may cause pulmonary fibrosis, but the pathogenesis and pathogenesis of different lung-related diseases are different, and Apelin can improve pulmonary fibrosis caused by BPD, but it is unknown whether Apelin has good treatment effect on other types of lung diseases. Therefore, the invention hopes to further research Apelin so as to obtain brand-new pharmaceutical application of Apelin to other types of diseases.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides the application of Apelin in preparing the medicine for treating silicosis. Experiments show that Apelin has good curative effect on relieving the pathological changes of silicosis, has good medicinal value prospect, and can be developed into a novel medicament for treating silicosis.

The invention provides application of Apelin or pharmaceutically acceptable salts thereof in preparing a medicament for treating silicosis.

Silicosis, also known as silicosis, is the most common type of pneumoconiosis, and is caused by long-term inhalation of large amount of free silica dust, and the lung tissue diffuse pulmonary fibrosis is the main pathological change disease. Silicosis is the most common, most advanced and most harmful type of pneumoconiosis. Generally, no symptoms or no obvious symptoms appear in early stage, and various symptoms appear along with the progress of the disease. Breathlessness often occurs early and becomes progressively worse. Chest distress and chest pain are frequently felt in the early stage, the chest pain is slight, and is distending pain, dull pain or stabbing pain, and the chest pain is irrelevant to breathing, body position and labor. The degree of chest tightness and breathlessness is related to the extent and nature of the pathology. The pathogenesis of silicosis is roughly divided into two steps: firstly, after the silicon dioxide enters the lung, the silicon dioxide is phagocytized by alveolar macrophages, the oxidation-reduction level of the organism is unbalanced, and inflammation and combined reaction are triggered; secondly, cytokines released by macrophages act further on downstream effector cells, causing them to proliferate and migrate abnormally. In the process of pulmonary fibrosis formation, the most critical is the mass production of myofibroblasts, mainly from alveolar epithelial cells, which are subjected to interstitial transformation under external influence to form fibroblasts, and the process is called epithelial-interstitial transformation (EMT).

Once the silicosis patient is diagnosed, he should be taken out of the dust operation and give an active comprehensive treatment. The silicosis treatment comprises the following steps: because no specific medicine for silicosis exists at present, most of the treatment methods are lung washing, and auxiliary medicines are taken to relieve the illness state of a patient, wherein the auxiliary medicines comprise bailing capsules, acetylcysteine, lung tonifying and blood circulation invigorating capsules, Jinshuibao tablets, compound methoxamine capsules (Asmei), tetrandrine tablets, montelukast sodium tablets (Anbingsheng), salvia miltiorrhiza injection and the like.

BPD is a chronic lung disease that typically occurs in premature infants undergoing oxygen and positive pressure ventilation therapy and whose etiologies include oxygen free radical injury, immature lung development, mechanical ventilation, and the like. The main pathological manifestations are recurrent inflammation of the early alveoli, fibrin deposition and pulmonary edema; severe oxidative stress leads to a reduction in alveolar numbers, pulmonary interstitial remodeling and late irreversible pulmonary interstitial fibrosis. Therapeutically effective treatment for BPD is typically: glucocorticoid therapy by administering to the child patient; administering bronchodilator to children, and administering dexamethasone and ambroxol by aerosol inhalation to children with dyspnea, wherein the dose of dexamethasone is 0.1mg/(kg.d), the dose of ambroxol is 10mg/d, and aerosol inhalation is performed to children every 12 hours under the drive of oxygen.

From the above analysis, silicosis is caused by exposure of silica, and is mainly seen in occupational exposure workers, while BPD is often found in premature infants in oxygen therapy, and the pathogenesis and mechanism of the two are greatly different, and the treatment means of the two are also obviously different. Therefore, the application of Apelin in preparing the medicament for treating silicosis belongs to a brand-new application of Apelin in pharmacy.

Preferably, Apelin is Apelin-13. Apelin-13 is a known polypeptide compound, the nucleotide sequence of which is: QRPRLSHKGPMPF are provided.

More preferably, the Apelin is Apelin-13 trifluoroacetate salt.

The invention provides a medicament for treating silicosis, which comprises Apelin-13 and/or pharmaceutically acceptable salts thereof and pharmaceutically acceptable auxiliary materials.

Preferably, the pharmaceutically acceptable auxiliary material is at least one of a solvent, an emulsifier, a thickener, an excipient, a suspending agent, a disintegrant, a filler or a diluent.

Preferably, the medicament is in the form of tablets, injections, sprays, freeze-dried powder injections, capsules or coated pills.

More preferably, the medicament is in the form of injection. Apelin belongs to polypeptide compounds, is easy to degrade and difficult to penetrate intestinal mucosa, and the drug effect of the Apelin can be reduced by oral administration, so that the Apelin is prepared into an injection form, and the absorption and utilization of the drug components are facilitated.

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

(1) the invention indicates that Apelin has no obvious toxic action on cells and has better safety;

(2) the invention indicates that Apelin can effectively relieve fibrosis symptoms caused by silicosis, reduce the expression level of pulmonary fibrosis related markers and has outstanding potential for treating silicosis.

Drawings

FIG. 1 shows HE staining results (20X) of lung tissues of each group of mice in example 1;

FIG. 2 is the results of modified Ashcroft scoring of lung tissue for each group of mice in example 1;

FIG. 3 is a graph of the effect of varying concentrations of Apelin on A549 cytotoxicity in example 2;

FIG. 4 shows the result of detecting the expression of silica-regulated epithelial-mesenchymal transition (EMT) marker protein by Western blotting in example 3.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are only preferred embodiments of the present invention, and the claimed protection scope is not limited thereto, and any modification, substitution, combination made without departing from the spirit and principle of the present invention are included in the protection scope of the present invention.

The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.

Example 1: therapeutic effect of Apelin on silicosis of mice

SPF grade C57BL/6 male mice (6-8 weeks old, 18-25g in body weight) were selected and randomly divided into 3 groups (15 mice per group) and a control group, a silicosis group and a silicosis + Apelin treatment group were set up by the following method.

Control group: injecting 20 μ L of physiological saline through mouse trachea;

silicosis group: injecting 20 mu L of 250g/L silicon dioxide suspension into mouse trachea by using a trachea exposure method;

silicosis + Apelin treatment group: mice in this treatment group were given a daily intraperitoneal injection of 500. mu.g/kg Apelin-13 trifluoroacetate (purchased from Sigma, Germany) via the trachea of mice by tracheal exposure of 20. mu.L of 250g/L silica suspension injected through the trachea starting on day 3 after the modelling.

Setting the groups and then carrying out conventional animal feeding; 5 mice of the above groups were sacrificed on

days

7, 14, and 28, respectively. Taking lung tissues for HE staining; images were randomly taken after full-slice scanning using a digital slice scanning system and semi-quantitatively scored for pulmonary fibrosis according to the modified Ashcroft scoring method (hubner RH, et al, biotechniques.2008.) and averaged.

The lung tissue HE staining results are shown in FIG. 1, and the lung tissue structure of the control mice is normal and clear, and the alveolar spaces are intact. While lung tissues of mice in silicosis group lose normal alveolar structures, alveolar spaces are obviously widened, collagen matrix in interstitial substance is obviously increased, and characteristic silicon nodules can be seen in lungs of mice in silicosis group for 28 days. As can be seen from the comparison between the silicosis and Apelin group, the destruction caused by silica is relieved after Apelin intervention treatment, the alveolar septal fibroplasia is less, the lesion range is smaller, and the lung parenchymal structure is less damaged.

The pulmonary fibrosis semi-quantitative score results are shown in figure 2 and table 1: the degree of pulmonary fibrosis was significantly higher in the silicosis group than in the control group (P <0.05) at each time point. Compared with the silicosis group, the mice treated by silicosis and Apelin have obviously reduced pulmonary fibrosis degree (P <0.05) at 14 days and 28 days.

TABLE 1 modified Ashcroft score for groups of mouse lung tissue

Note: p <0.05 vs. control;^#p is less than 0.05vs. silicosis.

Example 2: effect of Apelin at various concentrations on A549 cytotoxicity

Control group: a549 cells (human lung cancer cells) were cultured in serum-free GIBCO DMEM high-glucose medium (purchased from Thermo Scientific, usa);

apelin administration group: a549 cells (human lung cancer cells) were cultured in serum-free GIBCO DMEM high-glucose medium containing Apelin (Apelin-13 trifluoroacetate) at concentrations of 1, 0.1, 0.01, 0.001 and 0.0001. mu.M, respectively.

After the control group and the Apelin administration group are placed in a constant temperature incubator for 48 hours, the toxicity influence of Apelin at each concentration on A549 is detected by adopting a CCK-8 kit. The test results are shown in fig. 3: apelin at 0.0001, 0.001, 0.01, 0.1. mu.M had no significant toxic effect on A549 cells, compared to the control group.

Example 3: effect of Apelin on silica-regulated epithelial-mesenchymal transition (EMT) marker protein expression levels a control group: culturing A549 cells (human lung cancer cells) in serum-free medium;

silica exposure group: culturing A549 cells (human lung cancer cells) in serum-free medium containing silicon dioxide with concentration of 100 μ g/mL;

apelin + silica exposure group: a549 cells (human lung cancer cells) were cultured in serum-free GIBCO DMEM high-sugar medium containing silica at a concentration of 100. mu.g/mL and Apelin (Apelin-13 trifluoroacetate) at a concentration of 0.1. mu.M, respectively.

The groups are placed in a constant temperature incubator for 48h, the change of the level of the epithelial-mesenchymal transition (EMT) marker protein is detected by a protein immunoblotting method, and the detection result is shown in figure 4.

The results of fig. 4 show that: compared with a control group, the protein expression level of the epithelial marker E-cadherin in the silicon dioxide exposed group is obviously reduced, and the expression of the mesenchymal markers Vimentin, alpha-SMA and N-cadherin is up-regulated. The expression of E-cadherin in the Apelin + silicon dioxide exposure group is obviously improved, and the expression of interstitial markers alpha-SMA, N-cadherin and Vimentin is reduced; and the higher the Apelin concentration is, the more the expression level of E-cadherin is, and the less the expression levels of alpha-SMA, N-cadherin and Vimentin are, the characteristic of dose dependence is presented. The test results show that the silicon dioxide can directly act on the A549 to enable the A549 to generate the EMT, and the Apelin can inhibit the EMT process.

The embodiments of the present application have been described in detail with reference to the drawings, but the present application is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present application. Furthermore, the embodiments and features of the embodiments of the present application may be combined with each other without conflict.

Claims

The use of Apelin or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of silicosis.
2. The use according to claim 1, wherein Apelin is Apelin-13.
3. The use according to claim 2, wherein Apelin is Apelin-13 trifluoroacetate salt.
4. The medicine for treating silicosis is characterized by comprising Apelin-13 and/or pharmaceutically acceptable salts thereof and pharmaceutically acceptable auxiliary materials.
5. The medicament of claim 4, wherein the pharmaceutically acceptable excipient is at least one of a solvent, an emulsifier, a thickener, an excipient, a suspending agent, a disintegrant, a filler, or a diluent.
6. The medicament of claim 4, wherein the medicament is in the form of tablets, injections, sprays, lyophilized powder injections, capsules or coated pills.
7. The medicament of claim 6, wherein the medicament is in the form of an injection.