Application of aescin in preparation of anti-pulmonary fibrosis medicine

Abstract

The invention discloses application of aescin in preparation of an anti-pulmonary fibrosis medicine, and relates to the field of biological medicines. The aescin provided by the invention can improve BLM-induced pulmonary fibrosis of mice and recover the pulmonary respiratory function of the mice, and a new application of the aescin in the medicine for treating pulmonary fibrosis diseases is provided.

Description

Technical Field

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

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic fibrotic lung disease of unknown etiology. Its pathological hallmark is diffuse alveolar structural damage leading to progressive, irreversible pulmonary interstitial fibrosis and remodeling, resulting in a median survival of only 2 to 3 years. The pathogenesis of this disease is not fully understood, and there remains no effective treatment. When external stimuli induce senescence in type II alveolar epithelial cells (AECs), the cell cycle arrests, making it impossible to maintain the AEC barrier. This leads to fibroblast activation, proliferation, and collagen deposition, resulting in the formation of fibrotic scars, increased lung stiffness, and impaired gas exchange. Cellular senescence refers to a state of irreversible inhibition of cell proliferation, with the cell cycle typically permanently arrested in the G0 or G1 phase. Morphologically, this is manifested by cell flattening, nuclear enlargement, and chromatin aggregation. Another important characteristic of senescent cells is the secretion of a variety of cytokines, chemokines, growth factors and matrix metalloproteinases, which constitute the senescence-associated secretory phenotype (SASP) and are widely involved in cell proliferation, migration, inflammation, fibrosis and other processes.

[0003] The incidence of IPF increases dramatically with age. Diagnosis involves excluding other interstitial lung diseases or overlapping conditions and relies on the identification of a universal interstitial pneumonia pattern (UIP), typically using high-resolution CT (HRCT). Currently, lung transplantation is the only treatment that can prolong the survival of patients with pulmonary fibrosis, but the medical costs are enormous. In terms of medication, only pirfenidone and nintedanib are approved for the treatment of IPF worldwide. Nintedanib and pirfenidone are recommended drugs for IPF in clinical practice guidelines in many countries and have the function of slowing down the decline of lung function in IPF patients. However, adverse events caused by pirfenidone and nintedanib are relatively common, and clinical trial data have confirmed that the anti-fibrotic treatment of these two drugs lacks long-term survival benefits. Although fibrosis is of great significance to wound healing, the identification of effective anti-fibrotic treatments remains an unmet medical need, so anti-pulmonary fibrosis drug treatments are still in urgent need of development. Summary of the Invention

[0004] In view of the above-mentioned defects of the prior art, the present invention provides a new application of aescin in medicine for treating pulmonary fibrosis.

[0005] Furthermore, the aescin can reduce the number of senescent epithelial cells induced by BLM;

[0006] Furthermore, the aescin inhibits ECM deposition and SASP gene expression.

[0007] Compared with the prior art, the present invention has the following effects:

[0008] The present invention discovered the effect of aescin in inhibiting pulmonary fibrosis, and experimentally demonstrated that aescin can inhibit the aging of alveolar epithelial cells, reduce excessive extracellular matrix deposition and the differentiation of fibroblasts into myofibroblasts, thereby reducing pathological structural changes in pulmonary fibrosis tissue and the expression of fibrosis-related markers, alleviating the progression of pulmonary fibrosis in mice, significantly improving the abnormal lung function of mice with bleomycin (BLM)-induced pulmonary fibrosis, and reducing the area of fibrosis and inflammatory infiltration in the lungs of mice. Experiments have shown that aescin can inhibit the production of β-galactosidase in alveolar epithelial cells, significantly inhibit the aging of alveolar epithelial cells, and significantly improve the lung function of mice with pulmonary fibrosis.

[0009] The aescin (Escin) that the application adopts is lower than the drug costs such as nintedanib, pirfenidone, glucocorticoid medicine (prednisone tablets, prednisone acetate tablets etc.), antacids, N-acetylcysteine common clinically, has no side effect of obvious renal damage compared to immunosuppressants, has no metabolic disorder compared to glucocorticoids. Aescin (Escin) is applied to the application for the treatment of pulmonary fibrosis medicine, has enriched the new indications of swelling caused by aescin sodium treatment cerebral edema, trauma or surgery. Based on the protective effect of aescin (Escin) in pulmonary fibrosis and lung injury, and the therapeutic effect that aescin (Escin) plays to lung tissue pathological structure remodeling, can be used for the treatment or drug combination of clinical pulmonary fibrosis patients, developed another medical use of aescin (Escin).

[0010] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 In a specific embodiment of the present invention, a BLM-induced mouse pulmonary fibrosis model is constructed, wherein: Figure 1 A is the shadow area of the lungs of mice injected with BLM intratracheally for 21 days as shown by micro-CT; Figure 1 BC is H&E and Masson staining showing BLM-induced morphological changes in lung tissue; Figure 1 D is lung tissue section staining showing BLM-induced extracellular matrix deposition in lung tissue; Figure 1EF is SA-β-gal and immunohistochemical staining to detect cell senescence of mouse alveolar epithelial cells after BLM treatment;

[0012] Figure 2 This is a flow chart of a protocol for administering a solution of aescin to animals in accordance with one embodiment of the present invention;

[0013] Figure 3 In one embodiment of the present invention, escin was used to improve pulmonary fibrosis and deterioration of pulmonary respiratory function in mice induced by BLM model. Figure 3 A is a diagram showing the experimental scheme of Escin alone and Escin for treating pulmonary fibrosis mice; Figure 3 BD is aescin (Escin) that improves deep inspiration capacity (IC), forced vital capacity (FVC) and lung compliance in mice with pulmonary fibrosis; Figure 3 E is the Micro-CT scanning result; Figure 3 FG are the results of H&E and Masson staining; Figure 3 H is the results of immunohistochemistry and immunofluorescence experiments; Figure 3 I is the change in hydroxyproline content; Figure 3 J is the ratio of lung weight to body weight of mice after oral administration of escin, which is used to reflect lung parenchymal lesions; Figure 3 E is the whole lung scan result of the mouse; Figure 3 FJ are the test results of mouse lung tissue.

[0014] Figure 4 This is the result of the effect of Escin on BLM-induced lung tissue aging in a specific embodiment of the present invention; Figure 4 A is the immunohistochemical staining results of lung tissue sections; Figure 4 B is the Western blot result; Figure 4 C is the qPT-PCR experimental results. DETAILED DESCRIPTION

[0015] The following describes the embodiments of the present invention through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. The details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other unless they conflict.

[0016] 1. Build an experimental model.

[0017] Healthy male C57BL / 6 mice aged 6-8 weeks and weighing an average of 22-25g were selected to construct a pulmonary fibrosis mouse model. Aflototin was injected intraperitoneally according to the mouse weight. When the mouse showed no obvious autonomous activity and had a state of autonomous breathing, the mouse limbs and head were fixed to the operating table using pressure-sensitive adhesive. After the surgical instruments were disinfected with 75% alcohol, the muscles and anterior fascia surrounding the trachea were bluntly separated, and the mouse trachea was exposed with ophthalmic surgical scissors. BLM (50μl, 3mg / kg) was injected into the trachea of each mouse using a syringe. The sham operation group was injected with normal saline for a control experiment. Finally, the wound was sutured with sutures, and the animal wound was treated with iodine. After continuing to raise the animal for 21 days, an experimental pulmonary fibrosis model was established.

[0018] The aescin in this example was purchased from Shanghai Selleck Chemicals Co., Ltd. (6805-41-0; Selleck; Shanghai, China), and its structural formula is shown below:

[0019]

[0020] Mice were divided into Saline group, Escin (5 mg / kg) group, BLM group, and BLM + Escin (5 mg / kg) group, with 5 mice in each group. The mice in each group were gavaged with Escin (5 mg / kg) and control solution for ten days. The process was as follows: Figure 2 and Figure 3 As shown in A, the lung function of mice was detected by micro-CT, extracellular matrix deposition and cell senescence were evaluated by H&E, Masson, IHC, and SA-β-agl, and the expression of fibrosis and senescence-related markers was detected by qRT-PCR and Western blot.

[0021] 2. Verification that Escin can improve pulmonary fibrosis and deterioration of pulmonary respiratory function in mice caused by BLM model.

[0022] On the 21st day after BLM induction, invasive lung function measurements were performed using a DSI Buxco PFT controller. After the mice were anesthetized, the muscles and anterior fascia were bluntly separated to expose the trachea. The probe was inserted into the trachea and connected to the computer-controlled ventilator system. The inspiratory gas flow rate was set to fill the mouse's lungs with gas within 2-3 seconds; the slow expiratory flow rate was set to exhaust the mouse's vital capacity as much as possible within a given time. The acquisition software FinePointe was used to record lung function parameters, including inspiratory volume (IC), forced vital capacity (FVC) and dynamic compliance. The results are shown in the figure. Figure 3As shown in BD, IC, FVC and lung compliance increased, indicating that oral administration of escin (5 mg / kg) can significantly improve the respiratory function of mice with pulmonary fibrosis.

[0023] Micro-CT was used to detect the shadow area of the lungs and measure the lung tissue density in mice with pulmonary fibrosis treated with Escin, which was the result of lung function test. Figure 3 As shown in E, escin can effectively reduce the increase in lung shadow area induced by BLM and improve the lung parenchymal lesions in mice; representative images were obtained by Masson, H&E and immunohistochemical staining. Figure 3 F- Figure 3 As shown in G, the results of Masson staining showed that escin (5 mg / kg) treatment reduced the area of collagen deposition in the lung tissue of mice with pulmonary fibrosis. The results of H&E staining showed that escin (5 mg / kg) could improve the alveolar structure and reduce the infiltration of inflammatory cells in pulmonary fibrosis, indicating that escin treatment improved the structural lesions of the mouse lung tissue. Extracellular matrix proteins such as FN1, Collagen I and α-SMA are important indicators for evaluating myocardial fibrosis, and α-SMA is involved in the contraction of smooth muscle and is currently considered a marker of myofibroblasts. Immunohistochemical staining of α-SMA and Collagen I was performed on the sections, and the results were shown in Figure 2. Figure 3 As shown in H, compared with the BLM group, the α-SMA in the lung tissue of mice given Escin by gavage was + The accumulation of myofibroblasts was reduced, and the protein expression of type I collagen was decreased, confirming that escin could improve the progression of BLM-induced pulmonary fibrosis, indicating that escin (5 mg / kg) could alleviate the extracellular matrix deposition and the transformation of fibroblasts into myofibroblasts caused by pulmonary fibrosis; Figure 3 I changes in hydroxyproline content, which reflects the metabolic capacity of collagen tissue, indicating that escin treatment reduces tissue hydroxyproline content; Figure 3 J showed that escin (5 mg / kg) treatment reduced lung tissue hydroxyproline content, lung weight and lung consolidation.

[0024] 3. Effect of Escin on BLM-induced lung tissue aging.

[0025] The immunohistochemical staining results of lung tissue sections were obtained by SA-β-gal staining of pulmonary fibrosis mice treated with Escin. Figure 4As shown in A, the representative images of SA-β-gal staining of the mice showed that Escin (5 mg / kg) can significantly reduce the accumulation of senescent cells in the lung tissue of mice. The experiment showed that Escin treatment significantly reduced β-galactosidase activity and the number of P16 and P21 positive cells, especially inhibiting SPC + The role of senescence in alveolar epithelial cells.

[0026] Western blot detection was performed, and the results were as follows Figure 4 As shown in B, it shows that Escin (5 mg / kg) can reduce the number of P21 and P16 positive cells, reduce the protein levels of senescence marker genes P16 and P21, reverse the expression of fibrosis-related proteins FN1 and α-SMA induced by BLM, and alleviate the accumulation of senescent cells in lung tissue.

[0027] Western blot was used to detect BLM-induced pulmonary fibrosis in mice, and then the mice were given Escin by gavage to detect the expression of aging and fibrosis-related markers. The results were as follows: Figure 4 As shown in Figure C, experimental results show that escin can reduce the expression of inflammatory factors and chemokines stimulated by BLM, inhibit the release of SASP, reduce cell senescence, and inhibit excessive deposition and remodeling of the extracellular matrix. This shows that escin reduces lung cell senescence and lung pathological changes by stabilizing and restoring NPNT protein expression, thus highlighting its potential as a therapeutic target for pulmonary fibrosis.

[0028] It can be seen from the above examples that escin can have a good effect in treating pulmonary fibrosis and restore lung function. Micro-CT examination showed that escin treatment reduced the area of lung shadows and reduced lung parenchymal lesions. The results of pulmonary function tests showed that the lung compliance, respiratory capacity and other indicators of the animals in the escin treatment group were restored, indicating partial improvement of lung function. Pathological tissue staining showed that escin inhibited collagen deposition and lung tissue remodeling in mouse lung tissue, slowing down the pathological process of pulmonary fibrosis. Similarly, escin inhibited the fibrosis marker protein fibronectin (FN1), type I collagen (CollagenⅠ) and myofibroblast marker (α-SMA); at the same time, escin treatment reduced the accumulation of senescent cells in lung tissue. The above research results show that escin can improve the process of pulmonary fibrosis in mice caused by BLM, restore the lung respiratory function of mice, inhibit the pathological remodeling of mouse lung tissue and the large-scale deposition of extracellular matrix, clarify the role of escin in the process of pulmonary fibrosis, effectively improve the prognosis of patients, and provide a new theoretical basis for the clinical application and combination therapy of escin. It can be used for the treatment of pulmonary fibrosis and has clinical significance.

[0029] Escin is a natural triterpenoid saponin with vascular protective, anti-inflammatory, anti-edema, and anti-injury properties. Escin reduces vascular permeability and resists extravasation. It is commonly used to prevent and treat venous edema, cerebral edema, and swelling caused by trauma or surgery, thereby relieving venous congestion. It also has anti-inflammatory, anti-exudative, venous tone-enhancing, and blood circulation-improving properties. This application experimentally verifies the protective effect of escin on pulmonary fibrosis, which can effectively reduce the number of senescent epithelial cells induced by BLM, inhibit ECM deposition and the expression of SASP genes, and is conducive to the recovery of lung function, indicating that escin can inhibit inflammatory response and block fibrosis triggering factors; regulate epithelial-mesenchymal transition (EMT) and extracellular matrix conversion metabolism, thereby further improving lung function and tissue structure, achieving the effect of treating human pulmonary fibrosis, providing valuable reference for clinical trials, and providing new drugs and targets for the clinical prevention and treatment of pulmonary fibrosis. Compared with glucocorticoids, escin has milder side effects (such as only mild gastrointestinal discomfort) and does not have the risks of immunosuppression, osteoporosis, etc. common in hormone drugs. In addition, it may have a synergistic effect with existing anti-fibrosis drugs (such as pirfenidone).

[0030] The experimental results of the above examples show that the administration of escin to BLM-induced pulmonary fibrosis in mice can alleviate the extracellular matrix deposition and fibrosis process in the lungs, improve deep inspiration volume, forced vital capacity and lung compliance, and has important clinical significance.

[0031] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. Application of aescin in the preparation of anti-pulmonary fibrosis drugs.

2. The use according to claim 1, wherein the aescin can reduce the number of senescent epithelial cells induced by BLM.

3. The use according to claim 1, wherein the aescin inhibits ECM deposition and the expression of SASP genes.

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