Application of 8-deoxygamboge in preparation of medicine for preventing and treating pulmonary fibrosis

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly relates to application of 8-deoxygamboge in preparation of a medicine for preventing and treating pulmonary fibrosis. The 8-deoxygamboge has the function of selectively eliminating senescent cells, can reduce collagen expression, can be used for preparing the medicine for preventing and treating the pulmonary fibrosis, and provides a clinical basis for the treatment of the pulmonary fibrosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and specifically relates to the application of 8-deoxyginkgo glycoside in the preparation of medicines for preventing and treating pulmonary fibrosis. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease with a median survival of only 2-4 years after diagnosis, and the prevalence increases with age. Persistent alveolar epithelial damage, fibroblast transitional proliferation, myofibroblast differentiation, and progressive collagen deposition are characteristic changes of IPF, which ultimately lead to lung parenchymal remodeling and respiratory failure. Senescence Associated Secretory Phenotype (SASP) is one of the most important characteristics of cellular senescence. The proinflammatory mediators such as IL-6 and TGF-β and other growth factors contained in it are involved in the process of tissue fibrosis. In addition, SASP secretes proinflammatory cytokines and chemokines through autocrine and paracrine methods to change the microenvironment, affect surrounding normal cells, and promote inflammatory response and cell senescence. SASP can also increase ECM synthesis and collagen deposition directly or indirectly, thereby aggravating fibrosis. In age-related fibrotic diseases such as idiopathic pulmonary fibrosis (IPF), the accumulation of senescent cells can promote the fibrotic process.

[0003] As anti-aging drugs are used to treat organ fibrosis, the close relationship between aging and fibrosis has been continuously confirmed. For example, dasatinib (D) and quercetin (Q) can treat pulmonary fibrosis by removing senescent cells. Therefore, selective removal of senescent cells is one of the effective ways to treat pulmonary fibrosis.

[0004] Currently, only two drugs, Pirfenidone (PFD) and Nintedanib, are approved for the treatment of pulmonary fibrosis. However, both drugs can only delay the progression of fibrosis, but cannot cure pulmonary fibrosis or reverse pulmonary fibrosis that has already occurred. Therefore, it is necessary to develop new drugs for the treatment of pulmonary fibrosis. Summary of the invention

[0005] Based on the above technical background, the main purpose of the present invention is to provide an application of 8-deoxyginkgo glycoside in the preparation of drugs for preventing and treating pulmonary fibrosis, so as to overcome the deficiencies in the prior art.

[0006] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0007] The first aspect of the present invention is to provide the use of 8-deoxyginkgoside in the preparation of drugs for preventing and treating pulmonary fibrosis.

[0008] Preferably, the chemical structure of the 8-deoxyginkgoside is as shown in formula (1):

[0009]

[0010] Preferably, the 8-deoxygarcinia glycoside can selectively eliminate senescent cells, and the 8-deoxygarcinia glycoside treats pulmonary fibrosis by eliminating senescent cells.

[0011] More preferably, 8-deoxyginkgoside can eliminate bleomycin-induced senescent cells.

[0012] Preferably, 8-deoxyginkgoside can reduce collagen expression.

[0013] More preferably, the 8-deoxyginkgoside can reduce the protein expressions of Collagen II, α-SMA, P21 and P16 in bleomycin-induced pulmonary fibrosis.

[0014] Preferably, the pulmonary fibrosis is idiopathic pulmonary fibrosis or bleomycin-induced pulmonary fibrosis.

[0015] The second aspect of the present invention is to provide a drug for preventing and treating pulmonary fibrosis, wherein the active ingredient of the drug for preventing and treating pulmonary fibrosis is 8-deoxyginkgo glycoside, and the drug for preventing and treating pulmonary fibrosis also includes pharmaceutically acceptable excipients.

[0016] Preferably, the auxiliary materials include one or more of sustained-release agents, excipients, solubilizers, stabilizers, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants and lubricants.

[0017] Preferably, the dosage form of the drug for preventing and treating pulmonary fibrosis includes oral preparations, injection preparations or inhalation preparations.

[0018] The beneficial effects of the present invention are:

[0019] (1) The present invention has found through experimental studies that 8-deoxygarcinia glycoside has the function of selectively removing senescent cells and can reduce collagen expression. The 8-deoxygarcinia glycoside can be used to prepare drugs for preventing and treating pulmonary fibrosis, providing a clinical basis for the treatment of pulmonary fibrosis.

[0020] (2) 8-Deoxygarcinosidoside can eliminate senescent cells in the bleomycin-induced cell model and reduce the protein expression of Collagen II, α-SMA, P21 and P16 in bleomycin-induced pulmonary fibrosis. 8-Deoxygarcinosidoside has a good improvement effect on idiopathic pulmonary fibrosis or bleomycin-induced pulmonary fibrosis. 8-Deoxygarcinosidoside can be used to prepare drugs for preventing and treating idiopathic pulmonary fibrosis or bleomycin-induced pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect of 8-deoxyginkgoside on senescent cells in a bleomycin-induced cell model is shown;

[0022] Figure 2 The effect of 8-deoxyginkgoside on collagen expression and senescence proteins in a bleomycin-induced cell model is shown;

[0023] Figure 3 The effect of 8-deoxyginkgoside on the body weight and lung index of mice induced by bleomycin is shown;

[0024] Figure 4 The effect of 8-deoxyginkgoside on bleomycin-induced fibrotic changes in mouse lung tissue (HE, Masson). DETAILED DESCRIPTION

[0025] The present invention will be described in detail below, and the characteristics and advantages of the present invention will become clearer and more distinct with the description.

[0026] The first aspect of the present invention is to provide the use of 8-deoxyginkgoside in the preparation of drugs for preventing and treating pulmonary fibrosis.

[0027] Preferably, the 8-deoxygarcinia glycoside is a senescent cell apoptosis inducer, which can selectively eliminate senescent cells, thereby improving and alleviating pulmonary fibrosis, that is, the 8-deoxygarcinia glycoside treats pulmonary fibrosis by eliminating senescent cells.

[0028] More preferably, 8-deoxyginkgoside can eliminate senescent cells in a bleomycin-induced cell model.

[0029] 8-deoxygarcinosidoside can reduce the expression of collagen. Preferably, the 8-deoxygarcinosidoside can reduce the protein expression of Collagen II, α-SMA, P21 and P16 in the bleomycin-induced pulmonary fibrosis model.

[0030] The chemical structural formula of the 8-deoxyginkgoside is shown in formula (1):

[0031]

[0032] The active ingredient of the medicine for preventing and treating pulmonary fibrosis is 8-deoxyginkgo glycoside.

[0033] The pulmonary fibrosis is idiopathic pulmonary fibrosis or bleomycin-induced pulmonary fibrosis.

[0034] The second aspect of the present invention is to provide a drug for preventing and treating pulmonary fibrosis, wherein the active ingredient of the drug for preventing and treating pulmonary fibrosis is 8-deoxyginkgo glycoside, and the drug for preventing and treating pulmonary fibrosis also includes pharmaceutically acceptable excipients.

[0035] Preferably, the effective dose of 8-deoxyginkgo glycoside is not higher than 100 mg / kg.

[0036] Preferably, the auxiliary materials include one or more of sustained-release agents, excipients, solubilizers, stabilizers, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants and lubricants.

[0037] The dosage form of the drug for preventing and treating pulmonary fibrosis includes oral preparations, injection preparations or inhalation preparations.

[0038] Example

[0039] The present invention is further described below by means of specific examples. These embodiments are only intended to illustrate the present invention but are not intended to limit the scope of the present invention.

[0040] Example 1 8-deoxyglucosidase can eliminate senescent cells in a bleomycin-induced cell model

[0041] A549 cells were trypsinized for 60 to 80 seconds and plated in a 6-well plate with approximately 8 mL per well and approximately 1 × 10 cells. 5 After 12 hours, the cells were attached to the wall and were evenly divided into a blank group (normal group), a model group (bleomycin group), and an 8-deoxygarcinia glycoside administration group. After the A549 cells grew well and the growth density was about 60%, a bleomycin solution with a concentration of 10 μmoL / mL was added to the model group and the 8-deoxygarcinia glycoside administration group, and the blank group was given an equal volume of 1640 culture medium containing 10% serum. The above three groups were treated simultaneously for 24 hours. After 24 hours, a 8-deoxygarcinia glycoside solution with a concentration of 5 μmoL / mL was added to the 8-deoxygarcinia glycoside administration group. The cell culture medium was removed, washed 3 times with PBS, and 1 mL of β-galactosidase staining fixative was added to each well and fixed at room temperature for 15 minutes. The cell fixative was removed, and the cells were washed 3 times with PBS for 3 minutes each time. The PBS was removed, and 1 mL of staining working solution was added to each well. The preparation method of the staining working solution is as shown in Table 1 below. The 6-well plate was sealed with plastic wrap to prevent evaporation and incubated in a 37°C oven overnight. Observe and take pictures under an ordinary optical microscope, such as Figure 1 shown.

[0042] Table 1

[0043] β-Galactosidase staining solution A 10μL β-Galactosidase staining solution B 10μL β-Galactosidase staining solution C 930μL X-GaL solution 50μL

[0044] from Figure 1 It can be seen that compared with the blank group, the number of senescent cells in the model group increased significantly, and the number of senescent cells in the 8-deoxygarcinia glycosides group decreased compared with the model group, indicating that 8-deoxygarcinia glycosides can selectively eliminate senescent cells. It shows that 8-deoxygarcinia glycosides can eliminate senescent cells in the bleomycin-induced cell model.

[0045] Example 2 Effects of 8-deoxyglucosidase on collagen expression and senescence proteins in bleomycin-induced cell models

[0046] A549 cells were inoculated in 100 mm culture dishes and divided into a blank group (normal group), a model group (bleomycin group), and an 8-deoxygarcinosidase-administered group. The following operations were performed on the blank group, the model group, and the 8-deoxygarcinosidase-administered group:

[0047] After A549 cells grow well and the growth density is about 60%, the model group and the 8-deoxy-garcinia glycosides administration group are added with a bleomycin solution at a concentration of 10μmoL / mL, and the blank group is given an equal volume of 1640 medium containing 10% serum. The above three groups are treated simultaneously for 24 hours. After 24 hours, the 8-deoxy-garcinia glycosides administration group is added with an 8-deoxy-garcinia glycosides solution at a concentration of 5μmoL / mL. Pre-cool PBS solution, 8M urea and protease inhibitors (100x) are pre-cooled in advance. After washing the cells 3 times with pre-cooled PBS solution, 8M urea solution containing protease inhibitors is added and lysed on ice for 20 minutes. Collect the cells, then centrifuge at 4°C and 12000rpm / min for 10 minutes, and take the supernatant.

[0048] The protein content was determined by the BCA method. The electrophoresis conditions were 80V for 30 minutes for the concentrated gel, 120V for 60 minutes for the separation gel, and 100mA for 10 minutes for the semi-dry transfer. The cells were blocked with 5% milk for 1 hour and incubated with primary antibody at 4°C overnight. The next day, the primary antibody incubation box was removed, and the membrane was washed three times with 1*TBST at room temperature for 10 minutes each time. The secondary antibody was incubated at room temperature for 1 hour, and the membrane was washed three times with 1*TBST for 10 minutes each time, and then developed. GAPDH was used as the internal reference, and the grayscale value of each protein was analyzed by Image J to calculate the relative expression of the protein.

[0049] from Figure 2 It can be seen that 8-deoxygarcinosidase can downregulate the protein expression levels of Collagen II, α-SMA, P21 and P16 in the bleomycin-induced cell fibrosis model, indicating that 8-deoxygarcinosidase can eliminate senescent cells in the bleomycin-induced cell model and reduce collagen expression.

[0050] Example 38-Effect of deoxygarcinia glycosides on body weight and lung coefficient of mice

[0051] Mouse modeling: The experimental animals were SPF-grade C57BL / 6J mice, 21 males, 6 weeks old, weighing 18-20g. The experimental animals were randomly divided into 3 groups, namely the normal group, the model group (bleomycin group), and the 8-deoxygarcinosyl group, with 7 mice in each group. The mouse pulmonary fibrosis model was established by bleomycin atomization inhalation at a dose of 5 mg / kg for the model group and the 8-deoxygarcinosyl group. Seven days after modeling, the 8-deoxygarcinosyl group was gavaged with 10 mg / kg 8-deoxygarcinosyl group, and the normal group and the model group were gavaged with the same dose of PBS until the end of the experiment.

[0052] The mice were weighed daily from the beginning of modeling. After 21 days of modeling, the mice were killed and the lungs were completely removed to observe the general morphology, elasticity, and presence of blood stasis of the lung tissue. The removed lungs were rinsed with 0.9% NaCL solution, dried with filter paper, and weighed on an analytical balance. Lung coefficient = lung wet weight / body weight of the mouse at the time of killing*100%. The results are shown in Figure 2 shown.

[0053] from Figure 3 It can be seen that compared with the normal group of mice, the weight of the mice in the model group (bleomycin group) decreased slightly, and the lung coefficient increased; compared with the model group, the weight of the mice in the 8-deoxygarcinia glycoside group increased slightly, and the lung coefficient decreased. This shows that 8-deoxygarcinia glycoside can increase the weight of mice and reduce the lung coefficient.

[0054] Example 4 Effect of 8-deoxyglucosinolates on fibrosis of lung tissue in mice

[0055] Part of the lung tissue of the right lobe of the mouse lung modeled in Example 3 was fixed with paraformaldehyde, dehydrated and embedded in paraffin, and sliced ​​at 4 μm. Conventional HE staining and Masson staining were performed, and the degree of inflammation and collagen fiber proliferation in the lung tissue was observed under a microscope. Figure 4 shown.

[0056] from Figure 4 It can be seen that there was no obvious inflammatory cell infiltration and bleeding in the normal group. After bleomycin modeling, the lesions in the model group were unevenly distributed, the lung tissue structure was destroyed at the lesion site, the alveolar cavity was irregularly collapsed and fused, the alveolar septum was significantly widened, and inflammatory cell infiltration and fibroblast proliferation were observed. The alveolar structure changes, inflammatory cell infiltration and collagen deposition in the lung tissue of mice in the 8-deoxygarcinosidase group were improved compared with those in the model group, and the vascular dilation phenomenon was alleviated compared with the model group. This shows that 8-deoxygarcinosidase can improve bleomycin-induced lung tissue fibrosis.

[0057] The present invention has been described in detail above in conjunction with specific implementations and exemplary examples, but these descriptions cannot be understood as limiting the present invention. Those skilled in the art understand that, without departing from the spirit and scope of the present invention, a variety of equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its implementation methods, all of which fall within the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims.

Claims

Application of 1.8-deoxyglucosinolate in the preparation of drugs for preventing and treating pulmonary fibrosis.

2. The use according to claim 1, characterized in that: The chemical structural formula of the 8-deoxyginkgoside is shown in formula (1):

3. The use according to claim 1, characterized in that: The 8-deoxygarcinia glycoside can selectively eliminate senescent cells, and the 8-deoxygarcinia glycoside treats pulmonary fibrosis by eliminating senescent cells.

4. The use according to claim 3, characterized in that: 8-Deoxyglucosinolate eliminates bleomycin-induced senescent cells.

5. The use according to claim 1, characterized in that: 8-Deoxyglucosinolates can reduce collagen expression.

6. The use according to claim 5, characterized in that: The 8-deoxyginkgoside can reduce the protein expressions of CollagenII, α-SMA, P21 and P16 in bleomycin-induced pulmonary fibrosis.

7. The use according to claim 1, characterized in that: The pulmonary fibrosis is idiopathic pulmonary fibrosis or bleomycin-induced pulmonary fibrosis.

8. A drug for preventing and treating pulmonary fibrosis, characterized in that: The active ingredient of the drug for preventing and treating pulmonary fibrosis is 8-deoxyginkgo glycoside, and the drug for preventing and treating pulmonary fibrosis also includes pharmaceutically acceptable excipients.

9. The drug for preventing and treating pulmonary fibrosis according to claim 8, characterized in that: The auxiliary materials include one or more of sustained-release agents, excipients, solubilizers, stabilizers, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants and lubricants.

10. The drug for preventing and treating pulmonary fibrosis according to claim 8, characterized in that: The dosage form of the drug for preventing and treating pulmonary fibrosis includes oral preparations, injection preparations or inhalation preparations.

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