Application of morusin in preparation of anti-pulmonary fibrosis medicine

Abstract

The invention provides application of morusin in preparation of an anti-pulmonary fibrosis medicine, and belongs to the technical field of biological medicines. According to the application disclosed by the invention, the morusin is found to have an effect of removing senescent cells for the first time, the key characteristic that morusin can induce apoptosis of alveolar epithelial senescent cells is disclosed, a new direction is developed for research of anti-pulmonary fibrosis medicines, and available resources for research and development of the anti-pulmonary fibrosis medicines are enriched.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of sansin in the preparation of anti-pulmonary fibrosis drugs. Background Art

[0002] Pulmonary fibrosis is a serious and complex lung disease characterized by progressive fibrosis and scarring of lung tissue, leading to destruction of lung tissue structure and progressive decline in lung function. Globally, the incidence of pulmonary fibrosis is gradually increasing, seriously threatening human health and life. According to statistics, idiopathic pulmonary fibrosis (IPF), as the most common and representative type of pulmonary fibrosis, has a median survival of only 2-5 years after diagnosis, and the 5-year survival rate is lower than that of many common cancers.

[0003] Currently, although there are many treatments for pulmonary fibrosis, they all have certain limitations. In terms of drug treatment, although some drugs can relieve symptoms or delay disease progression to a certain extent, they are often accompanied by significant side effects, which impose an additional burden on the patient's body and affect the compliance and safety of long-term treatment. Non-drug treatments also face many challenges. Although some treatments can improve some of the patient's symptoms, they cannot fundamentally reverse the pathological process of pulmonary fibrosis and it is difficult to prevent the continued deterioration of the disease. Other methods have problems such as donor scarcity, complex technology, high cost, and postoperative complications, and cannot be widely used in clinical treatment.

[0004] Due to the many limitations of existing treatments for pulmonary fibrosis, the development of safe and effective new anti-pulmonary fibrosis drugs has extremely important clinical significance and social value. Cellular senescence is considered to be an important mechanism of pulmonary fibrosis, and senescent cells will continue to accumulate in lung tissue. These senescent cells can secrete a variety of cytokines, chemokines and growth factors in a paracrine or autocrine manner, such as interleukin-6 (IL-6), interleukin-8 (IL-8), etc. These SASP factors can stimulate the proliferation and activation of fibroblasts. Fibroblasts are the main cells that produce collagen fibers. Their excessive proliferation and activation will lead to a large amount of collagen fibers in lung tissue, further inducing the senescence of surrounding cells, forming a vicious cycle and accelerating the process of pulmonary fibrosis. Traditional Chinese medicine has the effect of clearing senescent cells. Finding anti-pulmonary fibrosis active ingredients with senescence clearance from traditional Chinese medicine to provide new strategies and drugs for the treatment of pulmonary fibrosis has become one of the hot areas of current research. Summary of the invention

[0005] The purpose of the present invention is to provide the use of sansin in the preparation of anti-pulmonary fibrosis drugs, thereby providing an effective means for the treatment of related diseases.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The invention provides application of sansinol in preparing medicine for treating pulmonary fibrosis.

[0007] Preferably, the pulmonary fibrosis is idiopathic pulmonary fibrosis or primary pulmonary fibrosis.

[0008] The present invention also provides the use of sansin in preparing medicine for improving pulmonary tissue fibrosis.

[0009] The present invention also provides the use of sansin in preparing an alveolar epithelial senescent cell clearing agent.

[0010] Preferably, the structure of the sansin is as shown in Formula A:

[0011] Formula A.

[0012] Preferably, the dosage of the sangsin is 5-500 mg / kg.

[0013] Preferably, the drug or alveolar epithelial senescent cell clearance agent further comprises an auxiliary material.

[0014] Preferably, the medicine further comprises an active ingredient for preventing and treating pulmonary fibrosis.

[0015] Preferably, the drug or alveolar epithelial senescent cell clearance agent is in the form of powder, tablet, granule, capsule, solution, emulsion, suspension, injection, spray, aerosol or powder spray.

[0016] Preferably, the drug or alveolar epithelial senescent cell clearance agent further comprises a pharmaceutically acceptable carrier, which includes a diluent, a buffer, a suspension, an emulsion, a granule, an encapsulation, an excipient, a filler, an adhesive, a spray, a transdermal absorbent, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, a colorant, a flavoring agent or an adsorption carrier.

[0017] Beneficial effects of the present invention: The present invention discovered for the first time that sansin has the effect of clearing senescent cells, and especially revealed its key characteristic of inducing apoptosis of senescent alveolar epithelial cells, which opened up a new direction for the research of anti-pulmonary fibrosis drugs and provided a strategy based on the mechanism of cell senescence for the treatment of pulmonary fibrosis. Since the present invention is based on the natural ingredient sansin, it may have lower toxic side effects and better biocompatibility than some synthetic drugs.

[0018] The present invention enriches the available resources for the research and development of anti-pulmonary fibrosis drugs. The sources of sangsin are relatively broad, which provides the possibility for subsequent large-scale drug development and production. The scheme provided by the present invention can lay the foundation for in-depth research in related fields and promote the birth of more innovative treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of the cytotoxicity assay show that Sangxin can clear bleomycin and clear senescent alveolar epithelial cells, among which, Figure 1 The left picture shows A549 cells, and the right picture shows AT2 cells; Figure 2 The results of cell staining test show that Sancin can remove bleomycin and remove senescent alveolar epithelial cells. Figure 2 ; Figure 3 This is the result of the detection of the mRNA of alveolar epithelial senescent cell marker induced by bleomycin by sansin. The left picture is the control group, the right picture is the sansin group, and the middle picture is the aging model group; Figure 4 The results of immunoblotting of the indicators of apoptosis of alveolar epithelial senescent cells induced by bleomycin are shown in Figure 2, where the left figure is the immunoblotting experiment figure and the right figure is the statistical result figure; Figure 5 This is a graph showing the detection results of the mRNA of apoptosis indexes of alveolar epithelial senescent cells induced by bleomycin, in which the left graph is the Bax gene and the right graph is the Bcl2 gene; Figure 6 The results of Sancinone clearing the alveolar epithelial fibrosis indexes improved by bleomycin are shown in the figure below, where the left figure is an immunoblot and the right figure is a statistical quantification figure; Figure 7 The result diagram shows that Sancin can clear the mRNA of alveolar epithelial fibrosis indicators improved by bleomycin, among which the left picture is FN1 gene, the right picture is ACTA2 gene, and the middle picture is COL2A1; Figure 8 Masson staining of Sancinol to clear bleomycin-induced senescent cells in mouse lung tissue, left one: control group, left two: model group, right one: high-dose group, right two: low-dose group; Fig. 9 This is the result graph showing that sansin can clear the fibrosis indicators of mouse lung tissue improved by bleomycin. The second one on the left is the model group, the first one on the right is the high-dose group (40 mg / kg), and the second one on the right is the low-dose group (20 mg / kg). DETAILED DESCRIPTION

[0020] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0021] Example Construction of bleomycin-induced pulmonary fibroblast model, efficacy evaluation and mechanism study 1.1 Cell model and efficacy evaluation: AT2 and A549 cells were inoculated in culture dishes and divided into blank group, model group and sansin group. After A549 cells grew well and the growth density was about 60%, 10 μM / ml bleomycin solution was added to the model group and the treatment group, and the blank group was given the same volume of 1640 culture medium containing 10% serum and treated for 24 hours. After 24 hours, the treatment group was added with 5 μM / mL sansin solution (such as Figure 1 ). Aspirate the cell culture medium, wash with PBS 3 times, add 1 mL of β-galactosidase staining fixative to each well, and fix at room temperature for 15 minutes. Aspirate the cell fixative, wash the cells 3 times with PBS, 3 minutes each time. Aspirate PBS, and add 1 ml of staining working solution to each well. The preparation method of staining working solution is as follows: β-Galactosidase staining solution A10 µL β-Galactosidase staining solution B 10 µL β-Galactosidase Staining Solution C 930 µL X-Gal solution 50 µL Seal the 6-well plate with plastic wrap to prevent evaporation and incubate in a 37 ºC oven overnight. Observe and take pictures under a normal optical microscope. Figure 1 As shown, compared with the model group, sansin can significantly eliminate senescent cells. After cell senescence, sansin can significantly increase cytotoxicity, and the decreasing curve of the toxicity curve is more toxic than that of non-senescent cells. Figure 2 As shown, β-galactose staining revealed that the cells were obviously aged after bleomycin induction, and the senescent cells appeared blue. After being cleared by sansin, the blue color was significantly reduced, indicating that the senescent cells were significantly cleared.

[0022] 1.2 Mechanism study: The cells in each group were lysed with protein lysis buffer to extract protein or with RNA extraction reagent to extract protein or mRNA, and the aging markers (p16, p21, p53) and apoptosis markers (Caspas3, Caspas9, BCL-2, BAX) were detected by western blotting or quantitative PCR. The A549 cells were protein extracted with 8 M urea, and protease inhibitors (purchased from Beyotime Biotechnology Co., Ltd.) were added to the lysate. The BCA method was used for quantification, and equal amounts of samples were loaded for polyacrylamide electrophoresis. The internal reference protein actin was used as a correction. After electrophoresis, the membrane was transferred and blocked with 5% skim milk powder at room temperature for one hour. The antibody was purchased from Wuhan Abotek Biotechnology Co., Ltd. at a dilution concentration of 1:1000. 1% skim milk powder was incubated overnight. The next day, it was washed three times, each time with 5% PBST5 for 5 minutes, and then incubated with HRP-labeled secondary antibody, incubated with 1% skim milk powder at room temperature for one hour, washed three times, each time with 5% PBST5 for 5 minutes, and illuminated with ECL reagent. Photos were taken and saved for analysis.

[0023] The RNA extraction reagent used was the Trizol method. After the extracted RNA was quantified, it was reverse transcribed (using a one-step reverse transcription kit purchased from Wuhan Abotek Biotechnology Co., Ltd.). The cDNA was then stored, primers were designed, and amplification was performed using a fluorescent quantitative PCR kit. The quality of the fusion melting curve was checked, and the Ct value was obtained for quantitative analysis.

[0024] like Figure 3 As shown, the fluorescence quantitative method was used to detect the expression of aging marker mRNA in the bleomycin-induced group and the bleomycin-induced sansinsu intervention group. The results showed that after bleomycin-induced alveolar epithelial cells A549, the aging marker mRNA, P16, P21, and P53 increased significantly, and after sansinsu intervention, it was significantly decreased.

[0025] like Figure 4 As shown in the figure, the expression of apoptosis marker proteins in the bleomycin-induced group and the bleomycin-induced sansin group was detected by immunoblotting. The results showed that the senescence marker BAX increased significantly after bleomycin-induced alveolar epithelial cells A549, and the increase was even greater in the sansin group, indicating increased apoptosis; The results showed that the senescence markers BCL-2, Caspas3, and Caspas9 increased significantly after bleomycin-induced alveolar epithelial cells A549, and decreased significantly after sansin intervention, indicating increased cell apoptosis. This phenomenon is also consistent with the results at the mRNA level, such as Figure 5 As shown, it was shown that sansin significantly increased the apoptosis of senescent cells.

[0026] like Figure 6As shown in the figure, the fibrosis protein indicators COL2A1, aSMA, P21, and P53 of senescent A549 cells induced by bleomycin increased significantly, and these indicators decreased significantly after intervention with sansin. Figure 7 As shown in the figure, the expression of mRNA in fibrosis at the transcriptional level also showed consistent results, with fibrosis indicators COL2A1, FN1, and ACAT2 significantly increased, and these indicators were significantly reduced after intervention with sansin. Construction of animal model of bleomycin-induced pulmonary fibrosis, drug efficacy evaluation and mechanism study 1.2 Animal model: 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 (Control), the model group (BLM), and the sansin group (BLM+sansin), with 7 mice in each group. The mouse pulmonary fibrosis model was established in the model group and the sansin group by bleomycin aerosol inhalation at a dose of 5 mg / kg. Seven days after modeling, the sansin group was gavaged at 20 mg / kg and 40 mg / kg, and the normal group and the model group were gavaged with the same dose of PBS until the end of the experiment.

[0027] The mice were weighed daily from the beginning of modeling. After 21 days of modeling and administration, the mice were killed and the lungs were completely removed. The general morphology, elasticity, and presence of blood stasis of the lung tissue were observed. The removed lungs were rinsed clean with 0.9% NaCl solution, dried with filter paper, and weighed on an analytical balance. Lung coefficient = lung wet weight (mg) / body weight of mice at the time of killing (g). Compared with the normal group mice, the model group mice had a slight decrease in body weight and an increase in lung coefficient; compared with the model group, the sansin group mice had a slight increase in body weight and a decrease in lung coefficient.

[0028] Part of the lung tissue from the right lobe of the mouse lung was obtained, fixed with paraformaldehyde, dehydrated, embedded in paraffin, and sliced ​​at 4 μM. Conventional HE staining and Masson staining were performed, and the degree of lung tissue inflammation and collagen fiber proliferation were observed under a microscope. Figure 8 As shown in the figure, no obvious inflammatory cell infiltration and bleeding were observed 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 shrank and fused irregularly, the alveolar septum was significantly widened, and inflammatory cell infiltration and fibroblast proliferation were observed. The changes in alveolar structure, inflammatory cell infiltration and collagen deposition in the lung tissue of mice in the sansin group were improved compared with those in the model group, and the vascular dilation phenomenon was alleviated compared with that in the model group.

[0029] About 10-15 g of lung tissue from each group of mice was ground into a slurry, and then lysed by adding tissue lysis buffer containing protease inhibitors. The slurry was centrifuged at 4 °C and 12,000 rpm / min for 10 minutes. The pulmonary fibrosis markers (COL2, ACTA2) were detected by immunohistochemistry. The results were as follows: Fig. 9As shown in the data, compared with the control group, the pulmonary fibrosis indicators in the model group were significantly improved. After intervention with sangsin, both the low-dose group (20 mg / kg) and the high-dose group (40 mg / kg) showed a significant decrease, and were concentration-dependent.

[0030] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The application of sansin in the preparation of medicines for the treatment of pulmonary fibrosis.

2. The use according to claim 1, characterized in that: The pulmonary fibrosis is idiopathic pulmonary fibrosis or primary pulmonary fibrosis.

3. Application of sansin in the preparation of drugs for improving pulmonary tissue fibrosis.

4. Application of sansin in the preparation of alveolar epithelial senescent cell clearance reagent.

5. The use according to any one of claims 1 to 4, characterized in that: The structure of the mulberry extract is shown in Formula A: Formula A.

6. The use according to any one of claims 1 to 4, characterized in that: The dosage of the sansin is 5-500 mg / kg.

7. The use according to any one of claims 1 to 4, characterized in that: The drug or alveolar epithelial senescent cell clearance agent further comprises auxiliary materials.

8. The use according to claim 1 or 2, characterized in that: The medicament also contains active ingredients for preventing and treating pulmonary fibrosis.

9. The use according to any one of claims 1 to 4, characterized in that: The dosage form of the drug or alveolar epithelial senescent cell removal agent is powder, tablet, granule, capsule, solution, emulsion, suspension, injection, spray, aerosol or powder spray.

10. The use according to claim 8, characterized in that: The drug or alveolar epithelial senescent cell clearance agent further comprises a pharmaceutically acceptable carrier, which includes a diluent, a buffer, a suspension, an emulsion, a granule, an encapsulation, an excipient, a filler, an adhesive, a spray, a transdermal absorbent, a wetting agent, a disintegrant, an absorption accelerator, a surfactant, a colorant, a flavoring agent or an adsorption carrier.

Images