Application of pharmaceutical composition in preparation of anti-pulmonary fibrosis drugs

Abstract

The invention provides an application of a pharmaceutical composition in preparation of an anti-pulmonary fibrosis drug, and belongs to the technical field of biological medicines. The invention provides a pharmaceutical composition containing schizandrin C, ginsenoside Rg2 and homoisohydroflavone. Multi-target intervention is realized aiming at key pathological mechanisms (such as cell senescence, proinflammatory factor release and extracellular matrix deposition) of pulmonary fibrosis. Experiments show that the composition can remarkably reverse the senescence phenotype of pulmonary fibrosis cells, inhibit activation of p53 / p21 signal channels, reduce fibrosis marker expression and the like. Compared with an existing medicine such as pirfenidone, the natural component combination has the advantage of being higher in safety, a safer, more effective and more economical treatment choice is provided for clinic, and remarkable conversion value and market potential are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and particularly to the application of a pharmaceutical composition in the preparation of an anti-pulmonary fibrosis drug. Background Art

[0002] Pulmonary fibrosis (PF) is a fatal disease characterized by chronic inflammation of lung tissue, abnormal activation of fibroblasts, and excessive deposition of extracellular matrix, ultimately leading to destruction of lung structure and functional failure. Patients often present with symptoms such as progressive dyspnea, dry cough, fatigue, etc., seriously affecting the quality of life. Among them, idiopathic pulmonary fibrosis is the most common type, and the median survival period of patients is only 3 to 5 years, with extremely poor prognosis. Currently, clinically approved drugs (such as pirfenidone and nintedanib) can only delay the progression of the disease, cannot reverse fibrosis, and have limitations such as hepatotoxicity and gastrointestinal side effects. There is an urgent need to develop safer and more effective new therapies.

[0003] Cellular senescence is a key driver of pulmonary fibrosis. Senescent alveolar epithelial cells release pro-inflammatory factors such as IL-6 and TGF-β through the senescence-associated secretory phenotype (SASP), activating the transformation of fibroblasts into myofibroblasts and promoting extracellular matrix deposition. In addition, the decline of immune surveillance function in the senescent microenvironment further accelerates the fibrosis process. Therefore, targeting the clearance of senescent cells or regulating senescence-related pathways such as p53 / p21 has become a new direction for the research and development of anti-fibrosis drugs. However, existing anti-aging drugs still face problems such as insufficient targeting and significant toxic side effects in the treatment of pulmonary fibrosis, and there is an urgent need to develop safer and more effective alternative solutions. In summary, finding a more effective, safe, comprehensive, and targeted treatment method for pulmonary fibrosis has important clinical significance and social value. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of a pharmaceutical composition in the preparation of an anti-pulmonary fibrosis drug, which can effectively inhibit the process of pulmonary fibrosis.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions: The present invention provides the application of a pharmaceutical composition in the preparation of a drug for preventing or treating pulmonary fibrosis, and the pharmaceutical composition includes schisandrin C, ginsenoside Rg2, and homo-isodihydroflavonone.

[0006] The present invention also provides the application of a pharmaceutical composition in the preparation of a drug for treating idiopathic pulmonary fibrosis, and the pharmaceutical composition includes schisandrin C, ginsenoside Rg2, and homo-isodihydroflavonone.

[0007] The present invention also provides an application of a pharmaceutical composition in the preparation of a reagent for anti-cell senescence, and the pharmaceutical composition includes schisandrin C, ginsenoside Rg2, and homoisoflavanone.

[0008] The present invention also provides an application of a pharmaceutical composition in the preparation of a reagent for anti-cell apoptosis, and the pharmaceutical composition includes schisandrin C, ginsenoside Rg2, and homoisoflavanone.

[0009] Preferably, the concentration ratio of schisandrin C, ginsenoside Rg2, and homoisoflavanone in the pharmaceutical composition is 0.8~1.2:8~12:0.4~0.6.

[0010] Preferably, the concentration of schisandrin C in the pharmaceutical composition is 0.8~1.2 μM; the concentration of ginsenoside Rg2 is 8~12 μM; the concentration of homoisoflavanone is 0.4~0.6 μM.

[0011] Preferably, the drug or reagent further comprises excipients.

[0012] Preferably, the drug or reagent further comprises pirfenidone and / or nintedanib.

[0013] Preferably, the dosage form of the drug or reagent is powder, tablet, granule, capsule, solution, emulsion, suspension, injection, spray, aerosol or powder aerosol.

[0014] Preferably, the drug or reagent further comprises a pharmaceutically acceptable carrier, and the carrier includes diluent, buffer, suspension, emulsion, granule, encapsulant, excipient, filler, binder, spray, transdermal absorbent, wetting agent, disintegrant, absorption promoter, surfactant, colorant, flavoring agent or adsorption carrier.

[0015] Advantages of the present invention: The present invention provides a pharmaceutical composition of schisandrin C, ginsenoside Rg2, and homoisoflavanone, which realizes multi-target intervention for the key pathological mechanisms of pulmonary fibrosis (such as cell senescence, release of pro-inflammatory factors, and deposition of extracellular matrix). Experiments show that this combination can significantly reverse the senescent phenotype of pulmonary fibrosis cells, inhibit the activation of the p53 / p21 signaling pathway, and reduce the expression of fibrosis markers, etc. Compared with existing drugs such as pirfenidone, the combination of natural ingredients of the present invention has the advantage of higher safety, provides a safer, effective and economical treatment option for clinical use, and has significant translational value and market potential. Description of the Drawings

[0016] Figure 1Figure showing the anti-inflammatory effect of schisandrin C on RAW264.7 cells. Among them: A is the heat map of multi-factor detection results after drug intervention; B is the immunofluorescence result map of iNOS protein in RAW264.7 cells intervened with different doses of schisandrin C. Figure 2 Figure showing the promoting effect of ginsenoside Rg2 on the proliferation of AT2 cells. Figure 3 Figure showing the senescence clearance effect of homoisoflavone on pulmonary fibrosis cells. Figure A is the representative map of β-GAL senescence staining after different drug interventions on A549 cells, and Figure B is the statistical chart of β-GAL senescence staining. Figure 4 Figure showing the senescence clearance effect of the molecular combination of schisandrin C - ginsenoside Rg2 - homoisoflavone on pulmonary fibrosis cells. Figure A is the representative map of β-GAL senescence staining after different combinations of drugs intervened on A549 cells, and Figure B is the statistical chart of β-GAL senescence staining; Figure C is the protein expression results and statistical chart of p21 and γh2ax in A549 cells after the combined drug intervention. Figure 5 Figure showing the anti-fibrotic effect of the molecular combination of schisandrin C - ginsenoside Rg2 - homoisoflavone. A is the immunofluorescence representative map of VIM protein in A549 and AT2 cells intervened with drugs of each group; B is the statistical result map of the relative expression level of VIM protein in A549 cells; C is the statistical result map of the relative expression level of VIM protein in AT2 cells. Figure 6 Figure showing the anti-fibrotic and anti-inflammatory effects of the molecular combination of schisandrin C - ginsenoside Rg2 - homoisoflavone; Figure A is the HE result map of the combined drug and Shengmai Powder at different doses; Figure B is the Masson staining result map of the combined drug and Shengmai Powder at different doses; Figure C is the statistical chart of the Masson staining result; Figure D is the content of inflammatory factor IL-1β; Figure E is the content of inflammatory factor IL-6. Figure 7 Figure showing the anti-fibrotic effect of the molecular combination of schisandrin C - ginsenoside Rg2 - homoisoflavone; Figure A is the immunofluorescence map of the fibrosis-related protein VIM of the combined drug and Shengmai Powder at different doses; Figure B is the immunofluorescence map of the fibrosis-related protein α-SMA of the combined drug and Shengmai Powder at different doses; Figure C is the statistical result map of the relative expression level of VIM; Figure D is the statistical result map of the relative expression level of α-SMA. Figure 8It is a result diagram of the anti-aging effect of the molecular combination of wuweizisu C - ginsenoside Rg2 - homoisoflavone; Figure A is an immunofluorescence image of the senescence-related protein p21 of the combined drug at different doses and Shengmai Powder; Figure B is an immunofluorescence image of the senescence-related protein γH2AX of the combined drug at different doses and Shengmai Powder; Figure C is a statistical chart of the relative expression level of p21; Figure D is a statistical chart of the relative expression level of γH2AX. Detailed implementation mode

[0017] The present invention provides an application of a pharmaceutical composition in the preparation of a drug for preventing or treating pulmonary fibrosis, and the pharmaceutical composition includes wuweizisu C, ginsenoside Rg2, and homoisoflavone.

[0018] The structural formula of the wuweizisu C is as formula 1:

[0019] Formula 1 The structural formula of the ginsenoside Rg2 is as formula 2:

[0020] Formula 2 The structural formula of the homoisoflavone is as formula 3:

[0021] Formula 3 The present invention also provides an application of a pharmaceutical composition in the preparation of a drug for treating idiopathic pulmonary fibrosis, and the pharmaceutical composition includes wuweizisu C, ginsenoside Rg2, and homoisoflavone.

[0022] The present invention also provides an application of a pharmaceutical composition in the preparation of a reagent for anti-cell senescence, and the pharmaceutical composition includes wuweizisu C, ginsenoside Rg2, and homoisoflavone.

[0023] The present invention also provides an application of a pharmaceutical composition in the preparation of a reagent for anti-cell apoptosis, and the pharmaceutical composition includes wuweizisu C, ginsenoside Rg2, and homoisoflavone.

[0024] In the present invention, in the drug or reagent, the pharmaceutical composition provided by the present invention is preferably the component that exerts the active efficacy.

[0025] In the present invention, preferably, the concentration ratio of schisandrin C, ginsenoside Rg2 and homo-isodihydroflavone in the pharmaceutical composition is 0.8-1.2:8-12:0.4-0.6. Preferably, the concentration of schisandrin C in the pharmaceutical composition is 0.8-1.2 μM; the concentration of ginsenoside Rg2 is 8-12 μM; the concentration of homo-isodihydroflavone is 0.4-0.6 μM. Preferably, the drug or reagent further comprises excipients. Preferably, the drug or reagent further comprises pirfenidone and / or nintedanib. Preferably, the dosage form of the drug or reagent is powder, tablet, granule, capsule, solution, emulsion, suspension, injection, spray, aerosol or powder inhaler. Preferably, the drug or reagent further comprises a pharmaceutically acceptable carrier, and the carrier includes diluent, buffer, suspension, emulsion, granule, encapsulant, excipient, filler, binder, spray, transdermal absorbent, wetting agent, disintegrant, absorption enhancer, surfactant, colorant, flavoring agent or adsorption carrier.

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

[0027] Example 1 Anti-inflammatory drug screening: RAW 264.7 cells were inoculated into culture dishes and divided into a control group, a model group and a drug administration group. After the primary cells grew well and the growth density was about 60%, in the model group and the drug administration group, the control group was given an equal volume of DMEM medium containing 10% serum. The drug administration groups were respectively added with drugs with a final concentration of ① 10 μM + lipopolysaccharide (LPS) at 1 μg / mL, and the model group was added with ② LPS at 1 μg / mL (see Figure 1 ). After 24 hours, the culture medium was aspirated, centrifuged at 5000 rpm for 10 minutes, and the supernatant was taken for multi-factor detection; the cell culture solution was aspirated, and the cells were washed 3 times with PBS for immunofluorescence experiments.

[0028] 2 Drug screening for promoting the proliferation of AT2 cells: AT2 cells were inoculated into 96-well plates and divided into a control group and a drug administration group. After the primary cells grew well and the growth density was about 50%, the drug administration group was added with drugs with a final concentration of 10 μM, and the control group was given an equal volume of 1640 medium containing 10% serum, and they were treated simultaneously for 24 hours (see Figure 2 ). Subsequently, the supernatant was discarded, 100 μL of CCK8 was added, and the detection was carried out at a wavelength of 450 nm after incubation for 30 min.

[0029] 3 Drug screening for the senescence clearance of pulmonary fibrosis cells: A549 cells were seeded in 24-well plates and divided into a control group, a model group, and a drug administration group. After the primary cells grew well and the growth density reached about 50%, the drug administration group was added with a drug with a final concentration of 10 μM, and the control group was given the same volume of 1640 medium containing 10% serum. They were treated simultaneously for 24 hours (see Figure 3 ). The cell culture medium was aspirated, and the cells were washed 3 times with PBS. 1 ml of β-galactosidase staining fixative was added to each well and fixed at room temperature for 15 minutes.

[0030] 4 Evaluation of the efficacy of the combined drug: A549 or AT2 cells were seeded in culture dishes and divided into a control group, a model group, and a drug administration group. After the primary cells grew well and the growth density reached about 60%, the model group and the drug administration group, and the control group was given the same volume of 1640 medium containing 10% serum. They were treated simultaneously for 24 hours. After culturing for 24 hours, the drug administration group was respectively added with the final concentrations of: ① A mixed solution of homoisoflavone (0.5 μM), ginsenoside Rg2 (10 μM), and schisandrin C (1 μM); ② A mixed solution of homoisoflavone (0.5 μM) and ginsenoside Rg2 (10 μM); ③ A mixed solution of homoisoflavone (0.5 μM) and schisandrin C (1 μM); ④ A mixed solution of ginsenoside Rg2 (10 μM) and schisandrin C (1 μM) (1 μM); ⑤ Homoisoflavone (1.5 μM); ⑥ Ginsenoside Rg2 (30 μM); ⑦ Schisandrin C (3 μM) (see Figure 4 , Figure 4 in which "schisandrin C - ginsenoside Rg2 - homoisoflavone" refers to the treatment group of the mixed solution of ① homoisoflavone (0.5 μM), ginsenoside Rg2 (10 μM), and schisandrin C (1 μM)) The cell culture medium was aspirated, and the cells were washed 3 times with PBS. 1 ml of β-galactosidase staining fixative was added to each well and fixed at room temperature for 15 minutes.

[0031] The steps of Luminex multifactor detection are as follows: Divide the samples into ① gradient dilution standard group ② test sample group ③ negative control, and add 75 μL to each well of the 96-well plate. Take out the pre-coated magnetic beads and add 50 μL to each well of the 96-well plate. Cover with sealing film and incubate at 500 rpm for 60 minutes (room temperature and away from light). After 60 minutes, place the well plate on the magnetic stand for 2 minutes, gently aspirate and discard the supernatant, add 100 μL of washing buffer to each well, and repeat washing 3 times. Then add biotin-labeled detection antibody, seal the plate and incubate at 500 rpm for 30 minutes (room temperature and away from light), then wash 3 times. Add 50 μL of streptavidin-phycoerythrin to each well, incubate at 500 rpm for 10 minutes in the dark, and wash 3 times. Finally, add 100 μL of detection buffer to each well to resuspend the magnetic beads and detect on the machine.

[0032] The steps of immunofluorescence were as follows: cells in each group were fixed with 4% paraformaldehyde at room temperature for 15 minutes, washed with PBS three times (5 minutes each time); then treated with 0.2% Triton X-100 for 10 minutes, washed with PBS three times; then blocked with 10% bovine serum albumin (BSA) at room temperature for 1 hour; iNOS, p21, γH2AX and VIM primary antibodies (Wuhan Abotek, dilution ratio 1:800) were added respectively, incubated at 4°C overnight; washed with PBS three times, added anti-rabbit fluorescent secondary antibody (1:500), incubated at room temperature for 1 hour. Washed with PBS three times, added anti-fluorescence quencher containing DAPI, observed and photographed under an optical microscope, and analyzed the pictures.

[0033] Depend on Figures 1 to 5 It can be seen that Schisandrae Chinensis C has a good anti-inflammatory effect, which is equivalent to the same dose of dexamethasone; in addition, Schisandrae Chinensis C can significantly reduce the expression of iNOS protein ( Figure 1 ); Ginsenoside Rg2 significantly promoted the proliferation of AT2 cells ( Figure 2 ); High isoflavones can significantly reduce the β-Gal expression of A549 cells after bleomycin treatment. Compared with the same dose of quercetin, high isoflavones have a more significant effect in clearing senescent cells ( Figure 3 ); The three compounds of Schisandrin C, Ginsenoside Rg2 and Homoisoflavonoids were combined in a certain proportion and found to be more effective in removing senescent cells than the combination of two drugs or high-multiple monomers; In addition, the drug combination of Schisandrin C-Ginsenoside Rg2-Homoisoflavonoids can significantly reduce the levels of aging-related proteins p21 and γH2AX ( Figure 4 In addition, Schisandrae Chinensis Acid-Ginsenoside Rg2-Homoisoflavonoids showed good anti-fibrotic effects. The drug combination intervention significantly reduced the level of VIM protein, a marker of fibrosis, and the effect was better than the drug combination or high-multiple monomers ( Figure 5 ).

[0034] Animal experiment: Methods: SPF-grade C57BL / 6 male mice, 6 - 8 weeks old, weighing 18 - 22 g. The mice were housed in standard rodent cages with a 12-hour day-night cycle and had free access to food and water. They were divided into 5 groups of 6 mice each according to the random number table method: control group, model group, low-dose combined drug group (25 mg / kg), high-dose combined drug group (50 mg / kg), and high-dose Shengmai San group (3.12 g / kg). On the first day, the mice except the normal group were placed in an atomization chamber and atomized with bleomycin hydrochloride (7.5 mg / mL). The operation was repeated after 3 days. On the 4th day, intragastric administration was carried out for 21 days. After the last administration, blood was collected from the orbital cavity of each group of mice. After decapitation, cardiac perfusion was performed, and the whole lungs were taken. The surface was rinsed with normal saline, and the surface moisture was blotted with filter paper. The left lung was fixed in 4% paraformaldehyde, and the right lung was frozen at -80 °C for later examination.

[0035] 1 Observation of pathological morphology of lung tissue The fixed lung tissues of each group of rats were taken out, trimmed flat, dehydrated with gradient ethanol, infiltrated with wax, and embedded, and then sections with a thickness of 4 μm were made. Some sections were respectively stained with HE, Masson, and immunofluorescence, and then the pathological morphological changes of the lung tissue sections were observed under an optical microscope and statistically analyzed using Image J software.

[0036] 2 ELISA determination After the blood was allowed to stand for 30 min, it was centrifuged at 4500 rpm for 10 min to obtain the supernatant. The contents of IL-6 and IL-1β in the serum were determined using an ELISA kit. The kit and the lung tissue were equilibrated to room temperature. Standard wells, blank wells, and sample wells were set up, and the operations were carried out in sequence according to the operation requirements of the kit. The optical density (OD value) of each well was measured, and a standard curve was drawn. The concentration values of various samples were calculated according to the curve equation.

[0037] It can be seen from Figure 6 that the HE staining results showed that compared with the normal group, the alveolar septum in the lung tissue of the model group mice was thickened, and the alveolar structure was severely damaged. After administration of each dose of combined drug and the prescription of Shengmai San, the lung tissue damage was significantly reduced. The Masson staining results showed that compared with the model group, the blue-stained area in each administration group was very small, and the low-dose combined drug and Shengmai San had similar effects on reducing fibrosis, while the high-dose combined drug had a more significant effect on reducing fibrosis. The ELISA results showed that each group of drugs could significantly reduce the levels of IL-6 and IL-1β in the serum. Generally speaking, the high-dose combined drug had a better effect.

[0038] It can be seen from Figure 7It can be seen that the immunofluorescence results show that the fibrosis indicators VIM and α-SMA are highly expressed in the model group. Compared with the model group, each drug administration group can reduce the fibrosis level to varying degrees. Generally speaking, the high-dose combination drug has a better effect.

[0039] It can be seen from Figure 8 It can be seen that the immunofluorescence results show that the senescence-related indicators p21 and γH2AX are highly expressed in the model group. Compared with the model group, each drug administration group can reduce the expression of senescence-related proteins to varying degrees. Generally speaking, the high-dose combination drug has a more significant pharmacodynamic effect.

[0040] It can be seen from the above examples that the pharmaceutical composition composed of schisandrin C-ginsenoside Rg2-homoisoflavone provided by the present invention can inhibit the process of pulmonary fibrosis and provides a new effective means for the treatment of pulmonary fibrosis.

[0041] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Use of a pharmaceutical composition in the preparation of a medicament for preventing or treating pulmonary fibrosis, characterized in that, The pharmaceutical composition comprises schisandrin C, ginsenoside Rg2 and homo-isodihydroflavone.

2. Use of a pharmaceutical composition in the preparation of a medicament for treating idiopathic pulmonary fibrosis, characterized in that, The pharmaceutical composition comprises schisandrin C, ginsenoside Rg2 and homo-isodihydroflavone.

3. Use of a pharmaceutical composition in the preparation of a reagent for anti-cell senescence, characterized in that, The pharmaceutical composition comprises schisandrin C, ginsenoside Rg2 and homo-isodihydroflavone.

4. Use of a pharmaceutical composition in the preparation of an anti-apoptosis reagent, characterized in that, The pharmaceutical composition comprises schisandrin C, ginsenoside Rg2 and homo-isodihydroflavone.

5. The application according to any one of claims 1 to 4, characterized in that, In the pharmaceutical composition, the concentration ratio of schisandrin C, ginsenoside Rg2 and homo-isodihydroflavone is 0.8 - 1.2:8 - 12:0.4 - 0.

6.

6. The application according to any one of claims 1 to 4, characterized in that, The concentration of schisandrin C in the pharmaceutical composition is 0.8 - 1.2 μM; The concentration of ginsenoside Rg2 is 8 - 12 μM; The concentration of homo-isodihydroflavone is 0.4 - 0.6 μM.

7. The application according to any one of claims 1 to 4, characterized in that The drug or reagent further comprises excipients.

8. The application according to any one of claims 1 to 4, characterized in that, The drug or reagent further comprises pirfenidone and / or nintedanib.

9. The application according to any one of claims 1 to 4, characterized in that, The dosage form of the drug or reagent is powder, tablet, granule, capsule, solution, emulsion, suspension, injection, spray, aerosol or powder aerosol.

10. The application according to claim 9, wherein The drug or reagent further comprises a pharmaceutically acceptable carrier, and the carrier includes diluent, buffer, suspending agent, emulsion, granule, encapsulant, excipient, filler, binder, spray, transdermal absorbent, wetting agent, disintegrant, absorption promoter, surfactant, colorant, flavoring agent or adsorption carrier.

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