Application of quercetin-7-glucoside in preparation of medicine for treating acute lung injury

Abstract

The invention relates to the technical field of biological medicine, in particular to application of quercetin-7-glucoside in preparation of medicine for treating acute lung injury, the quercetin-7-glucoside shows remarkable curative effect in acute lung injury treatment, the administration mode is flexible, quercetin-7-glucoside can be directly delivered to the lung through intravenous injection and can also be directly delivered to the lung in an atomized mode, and the application range of quercetin-7-glucoside in preparation of medicine for treating acute lung injury is widened. Therefore, the treatment effect is enhanced, oxidative stress, inflammatory cell infiltration and inflammatory factor release can be effectively inhibited, the mitochondrial function of cells is protected, and pulmonary alveolar tissue repair is promoted. Quercetin-7-glucoside can be directly extracted from traditional Chinese medicinal materials such as leguminous plants, ginkgo plants, compositae plants and stiff silkworms, has excellent medicinal safety, and can accelerate alveolar repair and restore lung functions. A classical LPS (Lipopolysaccharide)-induced mouse acute lung injury model is adopted for drug verification. The result shows that the administration dosage of quercetin-7-glucoside is 0.5-20 mg / kg / day, and the quercetin-7-glucoside can effectively treat the acute lung injury of lung tissues.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of quercetin-7-glucoside in preparing a medicine for treating acute lung injury. Background Art

[0002] Acute lung injury (ALI) is a pathological process of the lungs that occurs in the presence of non-cardiac diseases such as severe infection, shock, trauma, and burns. It is usually secondary to non-cardiac diseases such as severe infection, shock, trauma, or burns, and manifests as damage to pulmonary capillary endothelial cells and alveolar epithelial cells, causing diffuse interstitial and alveolar edema, leading to acute hypoxic respiratory insufficiency and even respiratory failure. Clinical features include progressive hypoxemia, respiratory distress, and heterogeneous exudative lesions on lung imaging. Its causes are diverse, such as severe lung infection, pulmonary contusion, toxic gas inhalation, severe non-thoracic trauma, and acute severe pancreatitis, and can be divided into direct and indirect types. Its core mechanism lies in the extensive damage to pulmonary capillary endothelial cells and alveolar epithelial cells, which subsequently causes diffuse interstitial and alveolar edema, leading to acute hypoxic respiratory insufficiency in patients, and even respiratory failure in severe cases. When this pathological process continues to worsen, the patient may further develop Acute Respiratory Distress Syndrome (ARDS). Historical data show that according to a meta-analysis of international clinical studies between 1967 and 1994, the mortality rate of ALI patients is about 50%, which is based on a large sample of 3,264 patients.

[0003] Acute lung injury is not only difficult to treat, but also poses a serious threat to the patient's life safety. Although there are currently a variety of treatments including respiratory support therapy and drug therapy, the high mortality rate of ALI and its progression to ARDS still highlights the urgent need for effective prevention strategies and treatment methods. Therefore, in-depth exploration of the pathogenesis of ALI and finding more effective prevention and treatment strategies are of vital importance to reducing the mortality rate of critically ill patients and improving patient prognosis.

[0004] Currently, the treatment strategies for acute lung injury (ALI) mainly focus on two core areas: respiratory support therapy and drug therapy.

[0005] First, in terms of respiratory support therapy, there are a variety of means to deal with respiratory dysfunction in patients. Oxygen therapy, as the most basic treatment method, can provide patients with necessary oxygen support and relieve hypoxemia. When the condition worsens further, non-invasive mechanical ventilation and invasive mechanical ventilation become important treatment methods. Non-invasive mechanical ventilation provides patients with positive pressure ventilation through masks or nasal masks, which helps to reduce the work of breathing and relieve symptoms of respiratory distress. Invasive mechanical ventilation provides patients with stronger respiratory support through more direct methods such as endotracheal intubation to ensure the effective delivery of oxygen. These respiratory support measures can not only significantly improve the patient's hypoxemia, but also more comprehensively improve the body's hypoxic state, thereby effectively preventing functional damage to extrapulmonary organs. However, it is worth noting that although these treatments can relieve patients' symptoms, their direct repair effect on alveoli is relatively limited.

[0006] Second, in the field of drug therapy, researchers are constantly exploring new treatment methods in order to find drugs that can directly target the pathological mechanism of ALI. These new methods include inhibiting inflammatory factors to reduce inflammatory responses, improving oxygenation to correct hypoxia, and promoting fluid absorption in the lungs. For example, fluid management helps to reduce pulmonary edema by precisely controlling the patient's fluid intake and discharge; glucocorticoid therapy reduces lung tissue damage by inhibiting inflammatory responses; drugs such as ketoconazole and pentoxifylline promote the absorption and discharge of fluid in the lungs through different mechanisms. In addition, cutting-edge therapies such as liquid ventilation, nitric oxide (NO) and activated protein C have also shown certain therapeutic potential in clinical studies. However, despite the emergence of these drug treatments, no drug has been found to date that has a significant and stable therapeutic effect on acute lung injury. Therefore, exploring more effective and targeted drug treatments remains an important research direction in the current field of ALI treatment.

[0007] Bombyx batryticatus: Bombyx batryticatus is the pathological product of silkworms infected with Beauveria bassiana (Bals.) during their growth process. It is recorded in medical classics such as Compendium of Materia Medica, Yaojian, Leigong Paoyao Yaoxingjie and Benjing Fengyuan. It has the effects of calming wind and relieving spasms, dispelling wind and relieving pain, calming the nerves and soothing the nerves. It is often used to treat wind evil in the body, can relieve spasm symptoms, and can be used to treat symptoms of facial paralysis caused by convulsions and epilepsy.

[0008] Quercetin-7-O-β-D-glucopyranoside is a natural flavonoid monoglucoside with high biological activity. Its CAS number is 491-50-9. The molecular formula of this compound can be expressed as C 21 H 20 O 12, the molecular weight is accurately calculated to be 464.376. Its structural characteristics are that the molecule is rich in multiple hydroxyl groups and glycosyl substituents, and these structural characteristics give it good water solubility and biological activity. Its physical and chemical properties meet the requirements of aerosol drug particle diameter of 0.5-10μm, safety without preservatives, and low toxicity to respiratory epithelial cells and alveoli, and it has the conditions for preparing aerosol drugs; this substance is contained in traditional Chinese medicines such as Leguminosae, Ginkgoaceae, and Asteraceae plants. The inventors have confirmed that its content is high in Bombyx batryticatus, but its use in the treatment of acute lung injury has not been seen. Summary of the invention

[0009] In view of the deficiencies of the prior art, the purpose of the present invention is to develop the application of quercetin-7-glucoside in the preparation of drugs for treating acute lung injury.

[0010] To achieve the aforementioned invention purpose, the present invention provides the use of quercetin-7-glucoside in the preparation of a drug for treating acute lung injury, which can effectively treat acute lung injury induced by direct and indirect factors of lung tissue. Direct factors refer to severe lung infection, gastric content aspiration, lung contusion, inhalation of toxic gases, drowning, and oxygen poisoning, etc., which directly damage the lungs. Indirect factors involve systemic problems, such as severe infection, non-thoracic trauma, acute severe pancreatitis, massive blood transfusion, extracorporeal circulation, and diffuse intravascular coagulation (DIC), etc., which indirectly affect the lungs through systemic inflammatory reactions. Quercetin-7-glucoside can not only inhibit oxidative stress, infiltration of inflammatory cells, and release of inflammatory factors after acute lung injury, but also protect the mitochondrial function of cells, promote rapid repair of damaged alveoli, and restore the lung function of its lung tissue.

[0011] Furthermore, the acute lung injury is caused by severe lung infection, aspiration of gastric contents, lung contusion, inhalation of toxic gases, drowning and oxygen poisoning.

[0012] Furthermore, the acute lung injury is caused by severe infection, non-thoracic trauma, acute severe pancreatitis, massive blood transfusion, extracorporeal circulation and disseminated intravascular coagulation (DIC).

[0013] Furthermore, the acute lung injury is caused by infiltration of inflammatory cells.

[0014] Furthermore, the acute lung injury is caused by the release of inflammatory factors by inflammatory cells.

[0015] Furthermore, the acute lung injury is caused by oxidative stress induced by inflammation.

[0016] Furthermore, the acute lung injury is caused by inflammation stimulating mitochondrial function of cells.

[0017] Furthermore, the drug can be directly separated and obtained from traditional Chinese medicines such as Sophora japonica, a plant of the Leguminosae family, or Artemisia serrulate leaves.

[0018] Furthermore, the drug can be obtained by chemical synthesis.

[0019] Furthermore, the drug can be obtained by extracting Bombyx batryticatus.

[0020] Furthermore, the drug is administered intravenously.

[0021] Furthermore, the drug is administered by aerosolization.

[0022] Furthermore, the dosage of quercetin-7-glucoside is 0.5-20 mg / kg per day.

[0023] Compared with the prior art, the advantages of the present invention include: Quercetin-7-glucoside has shown significant efficacy in the treatment of acute lung injury. It can effectively inhibit oxidative stress, inflammatory cell infiltration and inflammatory factor release, protect cell mitochondrial function, and promote alveolar tissue repair. Its administration method is flexible. It can be injected intravenously or directly delivered to the lungs in the form of atomization (thanks to its good water solubility and molecular structure characteristics), thereby enhancing the therapeutic effect. Quercetin-7-glucoside can be directly chemically synthesized or directly extracted from traditional Chinese medicines such as Leguminosae, Ginkgoaceae, Compositae and Bombyx batryticatus. Metabolomics analysis of Bombyx batryticatus shows that the content of quercetin-7-glucoside is relatively high. It has excellent medicinal safety, can accelerate alveolar repair, and restore lung function.

[0024] The present invention adopts the classic LPS-induced mouse acute lung injury model to conduct drug verification of quercetin-7-glucoside for the treatment of acute lung injury, and the results show that the administration dose of quercetin-7-glucoside is 0.5-20 mg / kg / day, which can effectively treat acute lung injury of lung tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Chemical structure of quercetin-7-glucoside is shown; Figure 2 This is a general diagram of lung tissue anatomy for verifying the therapeutic effect of quercetin-7-glucoside in the animal body of the present invention; Figure 3 The pathological test results of the animal in vivo verification of the therapeutic effect of quercetin-7-glucoside of the present invention; Figure 4 The blood biochemical test results for verifying the therapeutic effect of quercetin-7-glucoside in the animals of the present invention; Figure 5 The ROS detection results of the in vitro cell validation of the therapeutic effect of quercetin-7-glucoside of the present invention; Figure 6 The results of mitochondrial detection are used to verify the therapeutic effect of quercetin-7-glucoside in vitro cells of the present invention. DETAILED DESCRIPTION

[0026] The present invention will be described below with specific embodiments and examples. It should be understood that these specific embodiments and examples are merely illustrative and are not intended to limit the scope of the present invention and its use.

[0027] Unless otherwise stated, the methods, reagents and conditions used in the following preparation examples, comparative examples and examples are conventional methods, reagents and conditions in the art.

[0028] Reagents: Quercetin-7-glucoside, purchased from: MCE, catalog number: HY-N0419; H2DCFDA, purchased from MCE, catalog number: HY-D0940; Alanine aminotransferase (ALT), purchased from IDEXX, catalog number: 98-11067-01; Aspartate aminotransferase (AST), purchased from IDEXX, catalog number: 98-11069-01; Creatinine (CREA), purchased from IDEXX, product number: 98-11074-01; DMEM, purchased from Invitrogen, catalog number: 11885084; rmM-CSF, purchased from R&D, catalog number: 416-ML-010 / CF; Hoechst, purchased from Biyuntian, product number: C1017; Certified Fetal Bovine Serum, purchased from VivaCell, catalog number: C04001-050X10; Mito Red Kit, purchased from MCE, catalog number: HY-KD1029; Sample sources: wild-type C57 mice, primary macrophages (BMDM) extracted from C57 mice.

[0029] Example 1: Experimental design: Male C57 mice aged 6-8 weeks were anesthetized with sodium pentobarbital at a dose of 60 mg / kg, and the LPS acute lung injury model was established by nasogastric administration at a dose of 20 mg / kg according to the weight of the mice. A total of 3 groups were divided into NC (negative control) group, ALI group and quercetin-7-glucoside treatment group (ALI-Q7-OG). The ALI-Q7-OG group was first treated with quercetin-7-glucoside injection at a dose of 5.0 mg / kg through the tail vein 6 hours after modeling, and the remaining dosing intervals were 24 hours for 3 consecutive days; the ALI group was injected with normal saline through the tail vein at a regular and quantitative manner like the ALI-Q7-OG group.

[0030] On the third day after modeling, animals were anesthetized with sodium pentobarbital at a dose of 60 mg / kg. First, blood was collected from the heart of the animals, and lung tissue was dissected out, and gross images of lung injury were collected and observed according to the groups. Then, 4% paraformaldehyde was used for fixation, paraffin embedding, sectioning, and pathological examination and HE staining were performed. Finally, the serum was separated by sedimentation, centrifugation, and three blood gas biochemical slices of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (CREA) were selected, and 100 μL of serum was added to the sample area of ​​the blood gas biochemical slice, and blood biochemical detection was performed using the IDEXX Catalyst One fully automatic biochemical analyzer.

[0031] Experimental Results Quercetin-7-glucoside chemical structure: see Figure 1 , chemical formula is C 21 H 20 O 12 , CAS No. 491-50-9; Lung tissue anatomy gross findings: see Figure 2 In the NC group, the color of the lung tissue was pink, and there was no dark red hepatoid tissue change on the lung tissue. In the ALI group, there was obvious pulmonary edema and dark red hepatoid tissue change (in the dotted box). In the ALI-Q7-OG group, there was no pulmonary edema on the lung tissue, but there was dark red hepatoid tissue change, but the area was smaller than that in the ALI group. Since the lung tissue had pulmonary edema and damaged hepatoid changes (in the dotted box) compared with the NC group on the third day after ALI, the degree of pulmonary edema and the area of ​​damaged hepatoid changes in the quercetin-7-glucoside treatment group were significantly lighter than those in the ALI group. The experimental results suggest that after acute lung injury, the administration of quercetin-7-glucoside can reduce the degree of pulmonary edema and lung injury.

[0032] Pathological HE staining results: see Figure 3A, The left picture shows that the alveoli of the lung tissue in the normal control group (NC) are intact, without inflammatory cell infiltration, protein fluid exudation, and red blood cell leakage; the middle picture shows that in the acute lung injury (ALI) group, a large number of alveoli in the lung tissue are severely damaged, without complete alveolar structure, with a large number of inflammatory cell infiltration, a large amount of protein fluid exudation, and a large amount of red blood cell leakage; the right picture shows that in the quercetin-7-glucoside treatment group (ALI-Q7-OG), there is local inflammatory cell infiltration in the alveoli, local alveolar destruction, no protein fluid exudation, and no red blood cell leakage; the experimental results suggest that after acute lung injury, the administration of quercetin-7-glucoside can inhibit the infiltration of inflammatory cells and maintain alveolar integrity.

[0033] It is worth noting that the drug concentration (quercetin-7-glucoside) corresponding to the animal experimental results in this example is 5 mg / kg. The experimental results confirm that this concentration has the effect of treating acute lung injury. Further, we use 0.5 mg / kg and 20 mg / kg quercetin-7-glucoside dosages, see Figure 3 B, The left picture shows the acute lung injury (ALI) group, where a large number of alveoli in the lung tissue were severely damaged, there was no complete alveolar structure, there was a large number of inflammatory cells infiltrating, a large amount of protein fluid exuding, and a large number of red blood cells leaking; the middle picture is the 0.5mg / kg quercetin-7-glucoside treatment group (ALI-Q7-OG), where some alveoli were destroyed in the lung tissue, the local alveolar structure was intact, there was a large number of inflammatory cells infiltrating, a small amount of protein fluid exuding, and no red blood cells leaking; the right picture is the 20mg / kg quercetin-7-glucoside treatment group (ALI-Q7-OG); the alveoli in the lung tissue were not damaged, the alveolar structure was intact, there was a very small amount of inflammatory cells infiltrating, no protein fluid exuding, and no red blood cells leaking; Based on the above experimental results: after acute lung injury occurs, the higher the therapeutic concentration of quercetin-7-glucoside is administered, the more obvious the therapeutic effect is. Therefore, the effective dose of quercetin-7-glucoside is determined to be 0.5-20 mg / kg.

[0034] Blood biochemical test results: see Figure 4The values ​​of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine (CREA) in the acute lung injury (ALI) group were 156.50±21.28, 49.67±5.51, and 1.13±0.35, respectively; the values ​​of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine (CREA) in the quercetin-7-glucoside treatment group (ALI-Q7-OG) were 78.5±6. 66, 31.00±3.61, 0.37±0.06; the aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine (CREA) test values ​​in the acute lung injury (ALI) group were all greater than those in the quercetin-7-glucoside ALI-Q7-OG group. The experimental results suggest that after acute lung injury, administration of quercetin-7-glucoside can inhibit the inflammation of the body after ALI, reduce liver and kidney damage, and better protect the liver and kidney functions of the body.

[0035] Example 2: Experimental design: Male C57 mice aged 6-8 weeks were anesthetized by intraperitoneal injection of 60 mg / kg sodium pentobarbital, disinfected with 75% alcohol, and the leg bones were removed, the muscles were peeled off, disinfected with alcohol for 30 seconds, washed twice with PBS, and the two ends of the leg bones were cut with ophthalmic scissors. The bone marrow was blown out with a syringe, and the filtered cells were resuspended in PBS, centrifuged at 1000 rpm for 5 minutes, the cell pellet was collected, and red blood cell lysis buffer was added to treat at room temperature for 5 minutes. The cells were collected by centrifugation and resuspended in 10 ng / mL CSF culture medium (DMEM, 10% serum) and inoculated in T25. The medium was changed every 48 hours, and the culture was continued for 6 days to induce bone marrow cells into BMDM cells. The cells were trypsinized and inoculated in a four-chamber laser confocal dish, cultured for 48 hours, and set aside.

[0036] Laser confocal microscopy was randomly divided into three groups: NC group, LPS group, and LPS-Q7-OG group. LPS (1μg / ml) and 10μM nigericin (NIG) were added to the LPS group, and 1μg / ml LPS, 10μM nigericin (NIG) and 10ng / ml quercetin-7-glucoside were added to the LPS-Q7-OG group for stimulation for 30 minutes. First, H2DCFDA probe was added for staining for 20 minutes, and laser confocal microscopy images were collected.

[0037] The laser confocal dish was taken out, and a mixture of Hoechst and Mito Red was diluted 1:100 and incubated at 37°C for 15 min. The cells were washed with PBS and randomly divided into three groups: NC, LPS, and LPS-Q7-OG. 1μg / ml LPS and 10μM nigericin (NIG) were added to the LPS group, and 1μg / ml LPS, 10μM nigericin (NIG) and 10ng / ml quercetin-7-glucoside were added to the LPS-Q7-OG group. Long-term live cell video was performed using laser confocal microscopy.

[0038] Experimental results: Results of H2DCFDA (ROS) detection experiment on the therapeutic effect of quercetin-7-glucoside: see Figure 5 , where green represents probe ROS-positive cells and blue represents cell nuclei. It can be observed that the left picture shows that there are no green cells in the NC group, that is, no probe ROS-positive expression, the middle picture shows that the LPS group and the right picture shows that the LPS-Q7-OG group both have green cells, that is, ROS-positive expression. Compared with the LPS-Q7-OG group, the ROS-positive cells in the LPS group account for a larger proportion, and a small number of ROS-positive cells appear locally in the LPS-Q7-OG group. The experimental results suggest that quercetin-7-glucoside can inhibit LPS-induced macrophage oxidative stress and better protect the function and role of macrophages.

[0039] Results of mitochondrial detection experiment on the therapeutic effect of quercetin-7-glucoside: see Figure 6 From the long-term video, blue nuclei and red mitochondria staining can be observed. As time goes by, from 0 to 120 minutes, the upper picture (i.e., NC group) shows that there is no significant change in the nuclei and mitochondria of NC; the middle picture (i.e., LPS group) shows that the LPS group appears wrinkled, and the cells die at 120 minutes, with no visible nuclei and mitochondria; the lower picture (i.e., LPS-Q7-OG group) shows that complete nuclei and a large number of mitochondria can still be observed in the LPS-Q7-OG group; therefore, the experimental results suggest that quercetin-7-glucoside can inhibit LPS-induced inflammatory mitochondrial damage in macrophages and better protect the mitochondrial function and role of macrophages.

[0040] It should be understood that the above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it accordingly. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, some simple deductions or substitutions can be made without departing from the concept of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. Application of quercetin-7-glucoside in the preparation of drugs for the treatment of acute lung injury.

2. The use according to claim 1, characterized in that: The acute lung injury is caused by severe lung infection, aspiration of gastric contents, lung contusion, inhalation of toxic gases, drowning and oxygen poisoning.

3. The use according to claim 1, characterized in that: The acute lung injury is caused by severe infection, non-thoracic trauma, acute severe pancreatitis, massive blood transfusion, extracorporeal circulation and disseminated intravascular coagulation (DIC).

4. The use according to claim 1, characterized in that: The acute lung injury is caused by the infiltration of inflammatory cells.

5. The use according to claim 1, characterized in that: The acute lung injury is caused by the release of inflammatory factors by inflammatory cells.

6. The use according to claim 1, characterized in that: The acute lung injury is caused by oxidative stress induced by inflammation.

7. The use according to claim 1, characterized in that: The acute lung injury is caused by inflammation stimulating the mitochondrial function of cells.

8. The use according to claim 1, characterized in that: The drug is extracted from Bombyx batryticatus, or is obtained by chemical synthesis, or is directly separated from the leguminous plant Sophora japonica or the Chinese medicinal material Artemisia serrulate leaf.

9. The use according to claim 1, characterized in that: The drug is administered intravenously; Preferably, the dosage of the drug is 0.5-20 mg / kg per day.

10. The use according to claim 1, characterized in that: The drug is administered by nebulization.

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