Application of arbutin in preparation of medicine for treating pneumonia injury

Abstract

The invention discloses an application of arbutin in preparation of a medicine for preventing or treating pneumonia injury. Experimental results show that after being dosed in an intraperitoneal injection form, the arbutin regulates an autophagy pathway mediated by a molecular chaperone and inhibits degradation of oxidative stress negative regulatory factor PRL2 protein, so that generation and release of neutrophil extracellular traps are reduced, lung inflammatory response and tissue damage are relieved, and the curative effect of the arbutin is improved. The traditional Chinese medicine composition has a good improvement effect on pneumonia injury mice. The application provides important theoretical basis and experimental basis for prevention or treatment of pneumonia injury, further provides scientific basis for clinical medication for guiding treatment of pneumonia injury, and has good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of treating respiratory diseases in the pharmaceutical field. Specifically, the present invention provides the use of ursokactone in the preparation of a drug for preventing or treating pulmonary inflammatory injury. Background Art

[0002] Pulmonary inflammatory injury is a critical respiratory disease, and its main pathological feature is the abnormal aggregation and infiltration of inflammatory cells in the lung parenchyma, leading to damage to the structure and function of alveolar endothelial cells and epithelial cells, and then triggering pulmonary interstitial edema and uncontrolled inflammatory responses. At present, clinical treatment mainly adopts a multi-dimensional intervention strategy, including basic treatment and drug treatment. The basic treatment takes mechanical ventilation as the core, aiming to maintain the oxygenation function of patients and relieve respiratory failure. The drug treatment adopts a combination drug strategy, including anti-inflammatory drugs (such as glucocorticoids) to control inflammatory responses, antibacterial drugs to prevent and treat secondary infections, antioxidants (such as N-acetylcysteine) to reduce oxidative stress injury, and anticoagulants (such as low molecular weight heparin) to prevent microthrombus formation, and personalized treatment is carried out according to the potential pathology of patients. However, despite certain progress in existing treatment means, the complexity of the disease etiology and significant individual differences still limit the choice of treatment options, resulting in a mortality rate of up to 40% in patients and showing an upward trend. Given the many limitations in the treatment of pulmonary inflammatory injury diseases, finding potential therapeutic drugs for this disease remains an important topic that urgently needs to be broken through in the current medical research field.

[0003] Multiple studies have reported that the degree of neutrophil infiltration in the airway is closely related to the pulmonary inflammatory state, and the neutrophil count in bronchoalveolar lavage fluid (BALF) has become an important biological indicator for evaluating the degree of lung tissue inflammation. Clinical data further confirm that the abnormal increase in the number of neutrophils in the alveolar lavage fluid of patients often indicates more severe pathological damage and poorer clinical outcomes. As the largest immune cell population in peripheral blood, neutrophils play an important role in the innate immune defense system of the body. During the pathological process of pulmonary inflammatory injury, neutrophils can rapidly recruit to the inflammatory focus through complex chemotactic mechanisms and initiate various effector functions to respond to pathogen invasion. These functions include the release of antibacterial peptides, pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), and effector molecules such as reactive oxygen species (ROS), thereby mediating the inflammatory response and clearing pathogens. In addition to the above classical immune functions, neutrophils also have an important non-classical defense mechanism - the formation of neutrophil extracellular traps (NETs). NETs are reticular structures released by activated neutrophils, and their main components include depolymerized chromatin DNA, citrullinated histone H3 (Cit-H3), and various granule proteins such as myeloperoxidase (MPO), neutrophil elastase (NE), etc. This unique defense mechanism captures and kills pathogens by forming "extracellular traps", and its formation process involves a special form of programmed cell death, namely neutrophil extracellular trap formation (NETosis). Recent studies have found that the formation and release of NETs have dual effects: on the one hand, NETs can capture pathogens through their reticular structure and play a bactericidal role through the antibacterial proteins they carry; on the other hand, the proteases (such as NE and MPO), histones (such as Cit-H3), cell-free DNA (cfDNA), and myeloperoxidase-DNA (MPO-DNA) complexes contained in NETs all have potential cytotoxicity, which can not only damage alveolar cells but also overactivate immune cells and inflammatory signaling pathways.Therefore, intervening in neutrophil infiltration and NETs formation may be a potential therapeutic strategy for improving pulmonary inflammatory injury diseases.

[0004] Recent evidence has shown that oxidative stress is not only an important trigger for NETs formation but also a key signaling molecule involved in the cascade reaction of NETs formation. When the lungs are invaded by inflammation, the abnormal accumulation of ROS in neutrophils can disrupt the inherent redox homeostasis of cells, forming a pathological oxidative stress microenvironment, which in turn drives the formation of NETs. At the same time, ROS can oxidatively modify histones and chromatin-related proteins in neutrophils, promoting the depolymerization of chromatin structure and the release of DNA, which is a key step in NETs formation. In the regulatory mechanism of cellular oxidative stress, the Protein Tyrosine Phosphatases (PTPs) family plays an important role, and the Phosphatases of Regenerating Liver (PRLs), as an important subfamily of PTPs, has attracted much attention due to its unique oxidative stress regulatory function. The PRLs family contains three highly homologous members, namely PRL1, PRL2, and PRL3, with an amino acid sequence similarity of over 75%. Among them, PRL2 is the most widely expressed and has the highest expression level in tissues, suggesting its core regulatory position in various physiological processes. In recent years, studies have further revealed the unique functions of PRL2 in the immune system, especially in the innate immune response. Under oxidative stress conditions, PRL2 can rapidly respond to changes in the inflammatory microenvironment through its unique self-regulatory mechanism, significantly enhancing the respiratory burst effect of phagocytes, thereby improving their antibacterial ability. With the in-depth study, the molecular mechanism of PRL2 in oxidative stress regulation has been gradually elucidated. As a key negative regulator of intracellular ROS levels, PRL2 can precisely sense changes in ROS concentration and achieve dynamic balance through its own protein degradation pathway. During the inflammatory response, this regulatory property of PRL2 enables it to precisely control the functional state of immune cells. Especially in neutrophils, PRL2 ensures that neutrophils maintain normal bactericidal function under physiological conditions by finely regulating ROS levels, and also prevents excessive accumulation of ROS through a negative feedback mechanism, thus maintaining cell homeostasis. However, under pathological conditions, the absence of PRL2 leads to abnormal accumulation of ROS, disrupting the homeostatic balance of neutrophils. This imbalance over-activates the neutrophil extracellular trap formation process (NETosis), ultimately resulting in abnormal amplification of the inflammatory response and aggravation of tissue damage. These findings not only reveal the core role of PRL2 in oxidative stress regulation but also provide a new perspective for the treatment of related pulmonary inflammatory diseases.

[0005] In recent years, Chinese medicine has shown unique advantages in the treatment of complex diseases due to its low toxicity and low side effects. It can not only effectively regulate inflammatory responses and promote tissue repair, but also show unique comprehensive effects in regulating immune balance and improving microenvironment. Licorice (Glycyrrhiza uralensis Fisch.), as a traditional Chinese medicinal material, has a long history of medicinal use and extensive clinical application. With the in-depth study of modern pharmacology, the various pharmacological effects of licorice, such as anti-inflammatory, antioxidant, and immunomodulatory, have gradually been revealed, making it show significant advantages in the treatment of respiratory diseases. Especially in the treatment of lung inflammation, licorice-related preparations are widely used in the adjuvant treatment of various respiratory diseases such as pneumonia, chronic obstructive pulmonary disease, and asthma due to their effects of clearing heat and detoxifying, moistening the lungs and relieving cough. Its extracts have been shown to effectively alleviate alveolar damage and promote the resolution of inflammation by inhibiting the release of proinflammatory factors, reducing oxidative stress damage, and regulating immune cell function. However, due to the complexity of licorice components and the unclear material basis for the specific therapeutic effect, this has hindered its widespread clinical application to a certain extent. Therefore, further clarifying the active ingredients in licorice that play a therapeutic role and their related molecular mechanisms is of great significance for promoting its precise application in clinical practice. Kumatakenin (Kum), as a major flavonoid component present in the rhizomes of licorice, exhibits significant anti-inflammatory effects in a mouse model of ulcerative colitis. It can significantly reduce the expression levels of proinflammatory factors TNF-α and interleukin-6 (IL-6) in colon tissue, and effectively inhibit intestinal inflammatory response. In addition, Kumatakenin can significantly reduce the level of ROS in alveolar epithelial cells (A549) and reduce lipopolysaccharide-induced cell oxidative stress damage. Although the anti-inflammatory and antioxidant effects of Kumatakenin have been preliminarily verified, its protective effect in pulmonary inflammatory injury is still unclear, and further research is needed to reveal its potential therapeutic value. Summary of the invention

[0006] The purpose of the present invention is to study the pharmacological effects of ursin and find that ursin can improve the pathological damage of lung tissue in mice with pneumonia-induced injury; reduce lung tissue edema; and reduce the generation of proinflammatory factors and the infiltration of inflammatory cells in lung tissue, thereby providing the use of ursin in the preparation of drugs for treating pneumonia-induced injury.

[0007] In one aspect, the present invention provides the use of ursolin in preparing a drug for treating pulmonary pneumonia.

[0008] Furthermore, ursin is the only active ingredient in the medicine.

[0009] Furthermore, the pulmonary inflammatory injury is an inflammatory injury dominated by neutrophils.

[0010] Furthermore, the pulmonary inflammatory injury is pathological injury of lung tissue.

[0011] Furthermore, the pulmonary inflammatory injury is pulmonary tissue edema.

[0012] Furthermore, the pulmonary tissue edema is alveolar-capillary barrier injury.

[0013] Furthermore, the pulmonary inflammatory injury is an increase in inflammatory factors in lung tissue.

[0014] Furthermore, the inflammatory factors are IL-1β, TNF-α, and IL-6.

[0015] Furthermore, the pulmonary inflammatory injury is infiltration of inflammatory cells in lung tissue.

[0016] Furthermore, the infiltration of inflammatory cells is infiltration of neutrophils.

[0017] Furthermore, the pulmonary inflammatory injury is an increase in the generation and release of NETs in lung tissue.

[0018] Furthermore, the pulmonary inflammatory injury is a decrease in the level of PRL2 in lung tissue.

[0019] Furthermore, the drug achieves one or more of the following effects:

[0020] (1) Inhibiting the infiltration and activation of neutrophils and the release of NETs, and playing a protective role against pulmonary inflammatory injury.

[0021] (2) Improving lung function injury and / or pathological injury of lung tissue;

[0022] (3) Alleviating pulmonary tissue edema;

[0023] (4) Reducing the level of inflammatory factors in lung tissue;

[0024] (5) Reducing the activation and infiltration of inflammatory cells in lung tissue, and / or reducing the proportion of neutrophils in inflammatory cells;

[0025] (6) Reducing the infiltration and activation of neutrophils in lung tissue,

[0026] (7) Reducing the generation of ROS in lung tissue;

[0027] (8) Inhibiting the generation and release of NETs in lung tissue;

[0028] (9) Blocking the protein degradation of PRL2 in lung tissue.

[0029] Furthermore, the drug increases the protein level of PRL2 in lung tissue by inhibiting the lysosomal proteolytic pathway and / or weakening the interaction between CMA-related proteins PRL2, Hsc70, and LAMP2A, thereby blocking the degradation of PRL2 protein mediated by the CMA pathway.

[0030] Furthermore, the drug is in oral dosage form or injection dosage form.

[0031] Furthermore, the drug is in injection dosage form.

[0032] On the other hand, the present invention provides a non-therapeutic method for enhancing lung function, including administering kumatakenin.

[0033] Furthermore, the single-dose administration of kumatakenin is 15 - 45 mg / kg.

[0034] Furthermore, the single-dose administration of kumatakenin is 5 - 15 mg / kg.

[0035] Furthermore, the single-dose administration of kumatakenin is 45 mg / kg.

[0036] Kumatakenin mentioned in the present application is also called Kumatakenin, and its CAS number is: 3301 - 49 - 3. Its structural formula is shown in Formula (I):

[0037]

[0038] The non-therapeutic methods in the present application include but are not limited to health care methods and scientific research methods. The subjects of implementation can be humans or other mammals such as mice, rabbits, dogs, etc.

[0039] The symptoms or indications of pulmonary inflammatory injury in the present application include but are not limited to lung function injury, pulmonary tissue edema, elevation of inflammatory factors, etc.

[0040] The available drug dosage forms in the present application include various injection and oral dosage forms, including but not limited to tablets, capsules, oral liquids, injections, powder injections, transdermal drug delivery preparations, and oral dosage forms are particularly preferred.

[0041] In addition to kumatakenin, the drugs described in the present application may also include various pharmaceutically acceptable excipients / formulants, including but not limited to coating materials, solvents, solubilizers, binders, stabilizers, antioxidants, pH regulators, flavoring agents, etc., and excipients / formulants suitable for oral dosage forms are particularly preferred.

[0042] The drugs of the present invention may contain other Chinese and Western drugs or health products for treating pulmonary inflammatory injury, or the drugs of the present invention may be used in combination with these Chinese and Western drugs or health products or treatment means such as surgery. The Chinese and Western drugs or health products include but are not limited to anti-inflammatory drugs, anti-infective drugs, antioxidant drugs.

[0043] The use of kumaokinin provided by the present invention in the preparation of drugs for treating pulmonary inflammatory injury has the following beneficial effects:

[0044] (1) Kumaokinin can reduce the infiltration of inflammatory cells in lung tissue, alleviate the thickening of alveolar septa, reduce the bleeding points on the outer surface of the lung, and effectively improve the morphological changes and pathological damage of lung tissue;

[0045] (2) Kumaokinin can alleviate the damage degree of the lung tissue barrier, reduce the lung organ coefficient and the wet / dry weight ratio, decrease the total protein concentration in bronchoalveolar lavage fluid, and relieve lung tissue edema;

[0046] (3) Kumaokinin can significantly reduce the number of inflammatory cells in bronchoalveolar lavage fluid, decrease the production of inflammatory factors IL-1β, TNF-α and IL-6 in lung tissue, and alleviate the inflammatory response;

[0047] (4) Kumaokinin can reduce the level of myeloperoxidase (MPO) in lung tissue, decrease the production of NETs, MPO-DNA, cf-DNA and ROS in lung tissue, and inhibit the infiltration and activation of airway neutrophils in lung tissue;

[0048] (5) The present invention for the first time confirms that kumaokinin has the effect of treating mice with pulmonary inflammatory injury and can be used in the preparation of therapeutic drugs for pulmonary inflammatory injury, providing a new option for such drugs. Brief Description of the Drawings

[0049] Figure 1 It is a diagram showing the effect of kumaokinin on the morphology of lung tissue and H&E staining sections of mice with pulmonary inflammatory injury (scale bar, 100 μm, n = 6).

[0050] Figure 2 It is the effect of kumaokinin on the pathological injury of lung tissue in mice with pulmonary inflammatory injury ( n = 6). Part A: Representative images of H&E stained tissue sections (scale bar, 50 μm), stars indicate a large number of infiltrated inflammatory cells, triangles indicate thickened alveolar septa, circles indicate tissue bleeding points, and arrows indicate damaged alveolar structures; Part B: Evaluation of lung injury (alveolar neutrophils, interstitial neutrophils, hyaline membranes, cell debris, thickened alveolar septa and lung tissue bleeding points) based on 6 physiological and pathological characteristics to obtain the total injury score; Part C: Color comparison diagram of bronchoalveolar lavage fluid of mice; compared with NC, ### P < 0.001; compared with the LPS group, * P < 0.05, *** P < 0.001.

[0051] Figure 3 It is the effect of kumaokinin on the lung organ coefficient and lung wet / dry weight ratio of mice with pulmonary inflammatory injury ( n = 8). Part A: Statistical chart of the effect of arctigenin on the lung organ coefficient of mice with pulmonary inflammatory injury; Part B: Statistical chart of the effect of arctigenin on the wet / dry weight ratio of the lungs of mice with pulmonary inflammatory injury; Compared with the NC group, ### P < 0.001; "ns" indicates no statistical significance compared with the LPS group; Compared with the LPS group, * P < 0.05, ** P < 0.01, *** P < 0.001.

[0052] Figure 4 Statistical chart of the effect of arctigenin on the total protein concentration in the bronchoalveolar lavage fluid of mice with pulmonary inflammatory injury ( n = 6). Compared with the NC group, ### P < 0.001; Compared with the LPS group, *** P < 0.001.

[0053] Figure 5 Representative diagram and statistical chart of the results of Evans blue dye staining of the lung ( n = 8). Compared with the NC group, ### P < 0.001; Compared with the LPS group, ** P < 0.01.

[0054] Figure 6 Statistical chart of the effect of arctigenin on the protein contents of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 in the bronchoalveolar lavage fluid of mice with pulmonary inflammatory injury ( n = 6). Compared with the NC group, ### P < 0.001; Compared with the LPS group, * P < 0.05, ** P < 0.01, *** P < 0.001.

[0055] Figure 7 Statistical chart of the effect of arctigenin on the expression levels of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 in the serum of mice with pulmonary inflammatory injury ( n = 6). Compared with the NC group, ### P < 0.001; Compared with the LPS group, *** P < 0.001.

[0056] Figure 8 Effect of arctigenin on inflammatory factors in the lung tissue of mice with pulmonary inflammatory injury ( n = 3 / 6). Part A: Statistical chart of the effect of arctigenin on the mRNA expressions of IL-1β, TNF-α, and IL-6 in the lung tissue of mice with pulmonary inflammatory injury; Part B: Statistical chart of the effect of arctigenin on the protein expression level of IL-10 in the lung tissue of mice with pulmonary inflammatory injury; Compared with the NC group,### P < 0.001; compared with the LPS group, ** P < 0.01, *** P < 0.001.

[0057] Figure 9A Effect of xiongzhusu on the number of inflammatory cells in mice with pulmonary inflammatory injury ( n = 6). Among them, the effect of xiongzhusu on the number of inflammatory cells in the bronchoalveolar lavage fluid of mice with pulmonary inflammatory injury (scale bar, 50 μm), the red arrow indicates neutrophils, and the black arrow indicates macrophages.

[0058] Figure 9B Effect of xiongzhusu on the number of inflammatory cells in mice with pulmonary inflammatory injury ( n = 6). Giemsa staining was used to count the total cells, neutrophils and macrophages in the bronchoalveolar lavage fluid; compared with the NC group, ## P < 0.01,

[0059] ### P < 0.001; "ns" indicates no statistical significance compared with the LPS group; compared with the LPS group, * P < 0.05, ** P < 0.01, *** P < 0.001.

[0060] Figure 10 Effect of xiongzhusu on the proportion of inflammatory cells in mice with pulmonary inflammatory injury ( n = 6). Part A: Flow cytometry analysis of the proportion of neutrophils and macrophages; Part B: Statistical chart of the proportion of neutrophils and macrophages in the bronchoalveolar lavage fluid of mice; compared with the NC group, ### P < 0.001; "ns" indicates no statistical significance compared with the LPS group; compared with the LPS group, ** P < 0.01.

[0061] Figure 11 Representative transmission electron microscopy section of the lung (scale bar, 10 μm), the red triangle indicates neutrophils, and the red arrow indicates autophagolysosomes.

[0062] Figure 12 Statistical chart of the effect of xiongzhusu on the MPO protein content in the lung tissue of mice with pulmonary inflammatory injury ( n = 6). Compared with the NC group, ### P < 0.001; compared with the LPS group, *** P < 0.001.

[0063] Figure 13 Effect of xiongzhusu on neutrophil infiltration in mice with pulmonary inflammatory injury ( n = 3). Panel A: Representative immunohistochemical staining images of Ly6G, MPO, and NE in mouse lung tissues (scale bar, 50 μm); Panel B: Statistical chart of quantitative analysis of immunohistochemical staining of Ly6G, MPO, and NE; Compared with the NC group, ## P < 0.01, ### P < 0.001; Compared with the LPS group, * P < 0.05, *** P < 0.001.

[0064] Figure 14 Statistical chart of the effects of ursolic acid on the contents of NETs, MPO-DNA, cf-DNA, and ROS in the lung tissues of mice with pulmonary inflammatory injury ( n = 8). Compared with the NC group, ### P < 0.001; Compared with the LPS group, *** P < 0.001.

[0065] Figure 15 Effect of ursolic acid on NETs in the lung tissues of mice with pulmonary inflammatory injury ( n = 3). Panel A: Representative immunofluorescence staining images of mouse lung tissues (scale bar, 20 μm), DNA staining is blue (DAPI), myeloperoxidase staining is green (MPO), and citrullinated histone staining is red (Cit-H3); Panel B: Statistical chart of the average fluorescence intensity of MPO and Cit-H3; Panel C: Representative immunofluorescence staining images of mouse bronchoalveolar lavage fluid (scale bar, 50 μm), DNA staining is blue (DAPI), myeloperoxidase staining is green (MPO), and citrullinated histone staining is red (Cit-H3); Panel D: Statistical chart of the number of positive cells and the average fluorescence intensity of Cit-H3 in single cells; Compared with the NC group, # P < 0.05, ## P < 0.01, ### P < 0.001; Compared with the LPS group, * P < 0.05, ** P < 0.01.

[0066] Figure 16 Effect of ursolic acid on PRL2 in the lung tissues of mice with pulmonary inflammatory injury ( n = 6 / 8). Panel A: Statistical chart of the effect of ursolic acid on the protein content of PRL2 in the lung tissues of mice with pulmonary inflammatory injury; Panel B: Representative western blot images and quantitative analysis of the protein content of PRL2; Compared with the NC group, ### P < 0.001; Compared with the LPS group, ** P < 0.01, *** P < 0.001.

[0067] Figure 17 Effect of arctigenin on the expression level of PRL2 in neutrophils in the lung tissue of mice with pulmonary inflammatory injury( (n = 3). Part A: Representative immunofluorescence staining of bronchoalveolar lavage fluid in mice (scale bar, 50 μm). DNA staining is blue (DAPI), myeloperoxidase staining is green (MPO), and phosphatase of regenerating liver-2 staining is red (PRL2); Part B: Statistical chart of the number of positive cells and the mean fluorescence intensity of PRL2 in single cells; compared with the NC group, ## P < 0.01, ### P < 0.001; compared with the LPS group, ** P < 0.01, *** P < 0.001.

[0068] Figure 18 Statistical chart of the effect of arctigenin on the expression level of PRL2 mRNA in the lung tissue of mice with pulmonary inflammatory injury( (n = 3). "ns" indicates no statistical significance.

[0069] Figure 19 Effect of arctigenin on the PRL2 protein degradation pathway( (n = 4 / 6). Part A: Representative Western Blot and quantitative analysis of PRL2 protein content; Part B: Statistical chart of the 20S protein content in the lung tissue of mice with pulmonary inflammatory injury; Part C: Representative Western Blot and quantitative analysis of Lc3Ⅱ protein content; Part D: Statistical chart of the NETs protein content in the lung tissue of mice with pulmonary inflammatory injury; compared with the NC group, # P < 0.05, ## P < 0.01, ### P < 0.001; "ns" indicates no statistical significance compared with the LPS group; compared with the LPS group, * P < 0.05, ** P < 0.01, *** P < 0.001.

[0070] Figure 20 Effect of arctigenin on the degradation of PRL2 protein mediated by the macroautophagy pathway( (n = 6). Part A: Representative Western Blot and quantitative analysis of PRL2 protein content; Part B: Representative Western Blot and quantitative analysis of Lc3Ⅱ protein content; compared with the NC group, ### P < 0.001; "ns" indicates no statistical significance compared with the LPS group; compared with the LPS group, * P < 0.05, ** P < 0.01,*** P < 0.001.

[0071] Figure 21 Effect of ursolic acid on the chaperone-mediated autophagy (CMA) pathway ( n = 6). Part A: Representative Western Blot and quantitative analysis of PRL2 protein content; Part B: Representative Western Blot and quantitative analysis of QX77 protein content; Part C: Statistical chart of the content of NETs protein in the lung tissue of mice with pulmonary inflammatory injury; Compared with the NC group,

[0072] ## P < 0.01, ### P < 0.001; "ns" indicates no statistical significance compared with the LPS group; Compared with the LPS group, * P < 0.05, ** P < 0.01, *** P < 0.001. Figure 22 Effect of ursolic acid on the proteins related to the chaperone-mediated autophagy (CMA) pathway ( n = 3 / 6). Part A: Representative Western Blot and quantitative analysis of Hsc70 protein content; Part B: Statistical chart of the effect of ursolic acid on the expression level of Hsc70 mRNA in the lung tissue of mice with pulmonary inflammatory injury; Part C: Representative Western Blot and quantitative analysis of LAMP2A protein content; Part D: Statistical chart of the effect of ursolic acid on the expression level of LAMP2A mRNA in the lung tissue of mice with pulmonary inflammatory injury;

[0073] Figure 23 Effect of ursolic acid on the proteins related to the chaperone-mediated autophagy (CMA) pathway. Part E: Representative immunofluorescence staining of bronchoalveolar lavage fluid in mice (scale bar, 50 μm), DNA staining is blue (DAPI), lysosomal membrane-associated protein 1 staining is green (LAMP1), and phosphatase of regenerating liver-2 staining is red (PRL2); Part F: Statistical chart of the number of positive cells and the mean fluorescence intensity of PRL2 and LAMP1 in single cells; Compared with the NC group, ## P < 0.01, ### P < 0.001; "ns" indicates no statistical significance; Compared with the LPS group, * P < 0.05, ** P < 0.01, *** P < 0.001.

[0074] Figure 24 Representative immunoprecipitation of PRL2, Hsc70 and LAMP2A (n = 6). Detailed implementation mode

[0075] The present invention will be further described in detail below in conjunction with embodiments. The kumatakenin used in the following embodiments is the compound shown in the foregoing formula (I) and can be obtained through commercial purchase.

[0076] Example 1

[0077] Use of kumatakenin in the preparation of a drug for treating pulmonary inflammatory injury, wherein the single application dose of kumatakenin is 5 mg / kg for mice, and the dosage form of the drug is an injection dosage form.

[0078] Example 2

[0079] Use of kumatakenin in the preparation of a drug for treating pulmonary inflammatory injury, wherein the single application dose of kumatakenin is 15 mg / kg for mice, and the dosage form of the drug is an injection dosage form.

[0080] Example 3

[0081] Use of kumatakenin in the preparation of a drug for treating pulmonary inflammatory injury, wherein the single application dose of kumatakenin is 45 mg / kg for mice, and the dosage form of the drug is an injection dosage form.

[0082] The following animal experiments further illustrate the effects of the above Examples 1 to 3:

[0083] Experimental materials for Example 4

[0084] Animal treatment

[0085] Healthy male ICR strain mice (6 - 8 weeks old, weighing 18 - 22 g) were used in the experiment and were bred by the Experimental Animal Center of Ningxia Medical University (qualified batch number for animal incubation: SCXK(Ning)2020 - 0001). The feeding and management of all experimental animals strictly followed the "Guide for the Care and Use of Laboratory Animals". Before the formal experiment started, all experimental animals were subjected to 7 days of environmental adaptation breeding in a standard barrier environment where the environmental temperature was maintained at (20 ± 2) °C and the relative humidity was controlled within the range of (40 ± 10)%, and a 12 h / 12 h light / dark cycle was maintained using an artificial lighting control system. During the experiment, the animals had free access to food and water.

[0086] Experimental drugs and instruments

[0087] The main drugs and reagents involved in this experiment include: Kumadoline (purchased from Chengdu Pufa Technology Development Co., Ltd., purity ≥ 98%); Dexamethasone (purchased from Shanghai Yuanye Bio-Technology Co., Ltd.); Lipopolysaccharide (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.); Absolute ethanol (purchased from Shanghai Guangnuo Bio-Technology Co., Ltd.); Normal saline (purchased from Sichuan Kelun Pharmaceutical Co., Ltd.); PBS (purchased from Shanghai Xiaopeng Bio-Technology Co., Ltd.); BCA extraction kit (purchased from Jiangsu KeyGen Biotech Co., Ltd.); Total protein extraction kit (purchased from Jiangsu KeyGen Biotech Co., Ltd.); ELISA kits (NETs, MPO-DNA, cf-DNA, ROS) (purchased from Shanghai Enzyme-linked Bio-Technology Co., Ltd.); Myeloperoxidase (MPO) assay kit (purchased from Nanjing Jiancheng Bioengineering Institute); ELISA kit (IL-1β, TNF-α and IL-6) (purchased from Shanghai Jianglai Bio-Technology Co., Ltd.).

[0088] The main instruments involved in this experiment include: Microplate reader (purchased from Thermo scientific); Electrothermal constant temperature water bath (purchased from Shanghai Precision Experimental Equipment Co., Ltd.); Automatic sample cryogenic grinder (purchased from Shanghai Jingxin Technology Co., Ltd.); Electrothermal constant temperature forced air drying oven (purchased from Shanghai Langgan Experimental Equipment Co., Ltd.); Optical microscope (purchased from OLYMPUS); High-speed and low-temperature centrifuge (purchased from Eppendorf, Germany); SDS-PAGE electrophoresis apparatus (purchased from Bio Company, USA); PCR instrument (purchased from Jena, Germany); Micro nucleic acid and protein quantifier (purchased from GE, USA).

[0089] Grouping and administration of experimental animals

[0090] ① Control group (NC group): Received an equal volume of 0.9% normal saline;

[0091] ② Model group (LPS group): Intratracheally instilled 50 μl of LPS (5 mg / kg);

[0092] ③ Model + positive drug group (LPS + DEX group): 1 hour after intratracheally instilling 50 μl of LPS (5 mg / kg), intraperitoneally injected dexamethasone (5 mg / kg) at a dosing volume of 0.1 ml / 10 g;

[0093] ④ Model + drug administration group (LPS + Kum group): 1 hour after intratracheally instilling 50 μl of LPS (5 mg / kg), intraperitoneally injected kumadoline (5 mg / kg, 15 mg / kg, 45 mg / kg) at a dosing volume of 0.1 ml / 10 g.

[0094] ⑤ To investigate the effect of kumzhuoside on neutrophils in the lung tissue of mice with pulmonary inflammatory injury: The mice were divided into NC group, LPS group, and LPS+Kum group (45 mg / kg);

[0095] ⑥ To investigate the effect of kumzhuoside on the protein degradation pathway of PRL2: The mice were divided into NC group, LPS group, LPS+Kum group (45 mg / kg), LPS+CQ group (30 mg / kg), and LPS+MG-132 group (15 mg / kg);

[0096] ⑦ To investigate the effect of kumzhuoside on the degradation of PRL2 protein mediated by the macroautophagy pathway: The mice were divided into NC group, LPS group, LPS+Kum group (45 mg / kg), and LPS+3-MA group (20 mg / kg);

[0097] ⑧ To investigate the effect of kumzhuoside on the degradation of PRL2 protein mediated by the chaperone pathway: The mice were divided into NC group, LPS group, LPS+Kum group (45 mg / kg), LPS+QX77 group (10 mg / kg), and LPS+Kum+QX77 group.

[0098] Example 5 Experimental Process and Result Analysis (1) Effect of Kumzhuoside on the Morphology of the Whole Lung Tissue of Mice with Pulmonary Inflammatory Injury

[0099] Experimental method:

[0100] After 24 h of drug administration, the lung tissues of the mice were excised. After being rinsed clean, the morphological changes of the lung tissues were observed by taking pictures.

[0101] Experimental results:

[0102] Figure 1 As shown, compared with the NC group, the lung tissues of the mice in the LPS group had severe edema, a dark color, increased hardness, and a large number of bleeding points on the outer surface; compared with the LPS group, the lung tissues of the mice in the LPS+Kum group (5 mg / kg, 15 mg / kg, 45 mg / kg) had reduced edema, a more rosy color, normal texture, and fewer bleeding points. In addition, the effect of kumzhuoside on lung tissue injury was further evaluated by observing HE-stained sections. The results showed that compared with the NC group, the lung tissues of the LPS group had a large number of inflammatory cell infiltrations, thickened alveolar septa, and damaged alveolar structures; compared with the LPS group, the LPS+Kum group (5 mg / kg, 15 mg / kg, 45 mg / kg) had reduced inflammatory cell infiltrations, less thickened alveolar septa, and the lung tissue structure tended to be normal. The results suggest that kumzhuoside can improve the lung tissue injury in mice with pulmonary inflammatory injury.

[0103] (2) Effect of Kumzhuoside on the Pathological Injury of the Lung Tissue of Mice with Pulmonary Inflammatory Injury

[0104] Experimental method:

[0105] The lung tissues were fixed, dehydrated, cleared, infiltrated with wax, embedded, sectioned, and stained with HE. The pathological features were evaluated according to the following criteria: alveolar neutrophil infiltration, interstitial neutrophil infiltration, hyaline membrane formation, cell debris, alveolar septal thickening, and bleeding points. The severity of lung injury was scored from 0 to 4: 1 (<25%), 2 (25 - 50%), 3 (50 - 75%), and 4 (>75%).

[0106] Experimental results:

[0107] Figure 2 As shown, compared with the LPS group, the LPS + Kum groups (5 mg / kg, 15 mg / kg, 45 mg / kg) had significantly reduced alveolar and interstitial neutrophil infiltration, alleviated alveolar septal thickening, and fewer bleeding points. The lung injury was scored in 6 dimensions, and the results showed that compared with the NC group, the lung injury score of the LPS group mice increased significantly; compared with the LPS group, the lung injury scores of the LPS + Kum groups (5 mg / kg, 15 mg / kg, 45 mg / kg) decreased significantly (P < 0.05, P < 0.001). In addition, by observing the color of the bronchoalveolar lavage fluid of the mice, it was found that compared with the LPS group, the color of the LPS + Kum group (45 mg / kg) became lighter, indicating a significant reduction in alveolar injury. The results suggest that kumadoline can improve the pathological damage of lung tissues in mice with pulmonary inflammatory injury.

[0108] (3) Effects of kumadoline on the lung organ coefficient and lung wet / dry weight ratio in mice with pulmonary inflammatory injury

[0109] Experimental methods:

[0110] Organ coefficient: After 24 h of drug administration, the body weights of the mice in each group were recorded. The lung tissues were excised, rinsed thoroughly, and weighed accurately with an electronic balance. Then, the ratio of the lung tissue weight to the body weight was calculated, which was the lung organ coefficient (lung weight / body weight × 100%).

[0111] Wet / dry weight ratio: After 24 h of drug administration, the lung tissues of the mice were excised, rinsed thoroughly, and weighed accurately with an electronic balance, which was the wet weight. Then, it was placed in an incubator and dried at 60°C for 48 h, and weighed again to obtain the dry weight. Then, the ratio of the wet weight to the dry weight was calculated, which was the lung wet / dry weight ratio (lung wet weight / lung dry weight).

[0112] Experimental results:

[0113] Figure 3As shown, compared with the NC group, the lung organ coefficient and the wet / dry weight ratio of the lungs of mice in the LPS group were significantly increased (P<0.001); compared with the LPS group, the lung organ coefficient and the wet / dry weight ratio of the lungs of mice in the LPS+Kum groups (5 mg / kg, 15 mg / kg, 45 mg / kg) were significantly decreased (P<0.05, P<0.01, P<0.001). The results suggest that kumadoline can reduce pulmonary tissue edema in mice with pulmonary inflammatory injury.

[0114] (IV) Effect of kumadoline on alveolar-capillary barrier injury in mice with pulmonary inflammatory injury

[0115] Experimental method:

[0116] After 24 h of drug administration, the mice were anesthetized, a retention needle was inserted into the trachea, and the mice were continuously lavaged 3 times with 0.8 ml of pre-cooled PBS, and the BALF was collected. The BALF was centrifuged at 1500×g for 10 min at 4°C. The supernatant was collected and stored at -80°C for subsequent experiments.

[0117] ① After diluting the BALF to be tested 10-fold with Lysis Buffer, 20 μl was taken and added to the corresponding sample wells.

[0118] ② The standard solution was diluted as required to prepare a standard solution, and 20 μl was taken and added to the corresponding standard wells.

[0119] ③ Prepare the BCA working solution, solution A:solution B (50:1), vortex and mix evenly, and add 200 μl to each well.

[0120] ④ After covering with a film, incubate with shaking at 37°C for 30 min in an enzyme-labeled instrument.

[0121] ⑤ Measure the absorbance value (OD) at a wavelength of 562 nm.

[0122] ⑥ Plot the standard curve and calculate the protein concentration according to the standard curve.

[0123] Experimental results:

[0124] Figure 4 As shown, compared with the NC group, the total protein concentration in the bronchoalveolar lavage fluid of mice in the LPS group was significantly increased (P<0.001); compared with the LPS group, the total protein concentration in the bronchoalveolar lavage fluid of mice in the LPS+Kum groups (5 mg / kg, 15 mg / kg, 45 mg / kg) was significantly decreased (P<0.001). The results suggest that kumadoline can improve alveolar-capillary barrier injury in mice with pulmonary inflammatory injury.

[0125] (V) Effect of kumadoline on pulmonary vascular permeability in mice with pulmonary inflammatory injury

[0126] Experimental method:

[0127] ① After 24 hours of drug administration, 2% Evans Blue staining solution was injected into the tail vein of mice. After 10 minutes, the lung tissues were collected and photographed for observation.

[0128] ② 1 ml of PBS was added to the lung tissue samples, which were then ground by freezing and homogenized at 5000×g for 10 minutes at 4°C.

[0129] ③ The supernatant was collected and mixed with acetone in a ratio of 3:7, and incubated at room temperature for 24 hours.

[0130] ④ The Evans Blue staining solution was serially diluted to prepare a standard solution, and 100 μl was added to the corresponding standard wells.

[0131] ⑤ After the samples were incubated, 100 μl was added to the corresponding sample wells.

[0132] ⑥ The absorbance value (OD) was measured at a wavelength of 620 nm.

[0133] ⑦ A standard curve was plotted, and the concentration of Evans Blue staining solution in the samples was calculated according to the standard curve.

[0134] Experimental results:

[0135] Figure 5 As shown, compared with the NC group, the content of Evans Blue staining solution in the lung tissues of mice in the LPS group increased significantly (P<0.001); compared with the LPS group, the content of Evans Blue staining solution in the lung tissues of mice in the LPS+Kum group (45 mg / kg) decreased significantly (P<0.01). The results suggest that kumaonin can reduce the pulmonary vascular permeability in mice with pulmonary inflammatory injury.

[0136] (VI) Effect of kumaonin on the protein content of inflammatory factors in the bronchoalveolar lavage fluid of mice with pulmonary inflammatory injury

[0137] Experimental method:

[0138] After 24 hours of drug administration, the mice were anesthetized, a retention needle was inserted into the trachea, and the trachea was continuously lavaged 3 times with 0.8 ml of pre-cooled PBS. The BALF was collected and centrifuged at 1500×g for 10 minutes at 4°C. The supernatant was collected and stored at -80°C for subsequent experiments.

[0139] ① The standard product was serially diluted to prepare a standard solution, and 100 μl was added to the corresponding standard wells.

[0140] ② The samples to be measured were diluted as required, and 100 μl of the sample dilution solution was added to each sample well.

[0141] ③ After covering with a film, it was incubated in an enzyme-linked immunosorbent assay (ELISA) reader at 37°C for 90 minutes.

[0142] ④ Discard the supernatant, add 100 μl of biotin to each well, cover with a film, and incubate at 37 °C for 60 min.

[0143] ⑤ Discard the supernatant, add 300 μl of washing solution to each well, repeat the washing 3 times, and pat dry on the absorbent paper.

[0144] ⑥ Add 100 μl of HRP enzyme to each well, cover with a film, and incubate at 37 °C for 30 min.

[0145] ⑦ Discard the supernatant, add 300 μl of washing solution to each well, repeat the washing 3 times, and pat dry on the absorbent paper.

[0146] ⑧ Add 50 μl of each of the A and B substrate solutions to each well, cover with a film, and incubate at 37 °C in the dark for 15 min.

[0147] ⑨ Add 50 μl of the stop solution to each well, and immediately measure the absorbance (OD) at a wavelength of 450 nm.

[0148] ⑩ Plot the standard curve and calculate the protein concentration according to the standard curve.

[0149] Experimental results:

[0150] Figure 6 As shown, compared with the NC group, the protein contents of pro-inflammatory factors IL-1β, TNF-α, and IL-6 in the bronchoalveolar lavage fluid of mice in the LPS group were significantly increased (P < 0.001); compared with the LPS group, the protein contents of pro-inflammatory factors in the bronchoalveolar lavage fluid of mice in the LPS+Kum groups (5 mg / kg, 15 mg / kg, 45 mg / kg) were significantly decreased (P < 0.05, P < 0.01, P < 0.001). The results suggest that kumadoline can reduce the protein contents of pro-inflammatory factors in the bronchoalveolar lavage fluid of mice with pulmonary inflammatory injury.

[0151] (VII) Effect of kumadoline on the protein contents of inflammatory factors in the serum of mice with pulmonary inflammatory injury

[0152] Experimental method:

[0153] After administration for 24 h, collect blood samples by eye ball blood collection, let stand at 4 °C for 2 h, and centrifuge at 1000 × g at 4 °C for 15 min. Collect the serum and store it at -80 °C for subsequent experiments.

[0154] ① Gradiently dilute the standard product to prepare a standard solution, and take 100 μl and add it to the corresponding standard well.

[0155] ② Dilute the sample to be measured as required, and add 100 μl of the sample diluent to each sample well.

[0156] ③ Cover with a film and incubate at 37 °C in an enzyme-linked immunosorbent assay (ELISA) reader for 90 min.

[0157] ④ Discard the supernatant, add 100 μl of biotin to each well, cover with a film, and incubate at 37 °C for 60 min.

[0158] ⑤ Discard the supernatant, add 300 μl of washing solution to each well, repeat the washing 3 times, and pat dry on the absorbent paper.

[0159] ⑥ Add 100 μl of HRP enzyme to each well, cover with a film, and incubate at 37 °C for 30 min.

[0160] ⑦ Discard the supernatant, add 300 μl of washing solution to each well, repeat the washing 3 times, and pat dry on the absorbent paper.

[0161] ⑧ Add 50 μl of each of substrate solutions A and B to each well, cover with a film, and incubate at 37 °C in the dark for 15 min.

[0162] ⑨ Add 50 μl of stop solution to each well, and immediately measure the absorbance value (OD) at a wavelength of 450 nm.

[0163] ⑩ Plot the standard curve and calculate the protein concentration according to the standard curve.

[0164] Experimental results:

[0165] Figure 7 As shown, compared with the NC group, the protein contents of pro-inflammatory factors IL-1β, TNF-α, and IL-6 in the serum of mice in the LPS group were significantly increased (P < 0.001); compared with the LPS group, the protein contents of pro-inflammatory factors in the serum of mice in the LPS+Kum groups (5 mg / kg, 15 mg / kg, 45 mg / kg) were significantly decreased (P < 0.001). The results suggest that kumaushin can reduce the protein contents of pro-inflammatory factors in the serum of mice with pulmonary inflammatory injury.

[0166] (VIII) Effects of kumaushin on the mRNA and protein expressions of inflammatory factors in the lung tissues of mice with pulmonary inflammatory injury

[0167] Experimental method:

[0168] After 24 h of drug administration, excise the lung tissues of mice, wash them clean, weigh about 20 mg of lung tissues, and perform RNA extraction, RNA quantification, reverse transcription, and fluorescence detection of mRNA.

[0169] Experimental results:

[0170] Figure 8As shown, compared with the NC group, the mRNA expression levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 in the lung tissues of mice in the LPS group were significantly increased (P<0.001); compared with the LPS group, the mRNA expression levels of pro-inflammatory factors in the lung tissues of mice in the LPS+Kum groups (5mg / kg, 15mg / kg, 45mg / kg) were significantly decreased (P<0.01, P<0.001). The results suggest that kumadurin can reduce the mRNA expression levels of pro-inflammatory factors in the lung tissues of mice with pulmonary inflammatory injury.

[0171] (IX) Effect of Kumadurin on the Number of Inflammatory Cells in Mice with Pulmonary Inflammatory Injury

[0172] Experimental method:

[0173] ① Collect BALF and centrifuge at 1500×g for 10 min at 4°C.

[0174] ② Discard the supernatant and resuspend the bottom cells in 300 μl of PBS.

[0175] ③ Take 10 μl for smearing and dry it in an incubator at 37°C.

[0176] ④ Fix the cells in 4% paraformaldehyde for 10 min.

[0177] ⑤ Drain off the excess formaldehyde and stain with Wright-Giemsa solution for 10 min.

[0178] ⑥ Rinse slowly with running water, wash off the excess stain, air dry at room temperature, and add neutral balsam for mounting.

[0179] ⑦ Count the number of macrophages, neutrophils, and total cells under an optical microscope.

[0180] Experimental results:

[0181] As Figure 9A and Figure 9B shown, compared with the NC group, the total cell count, neutrophil count, and macrophage count in the bronchoalveolar lavage fluid of mice in the LPS group were significantly increased (P<0.01, P<0.001); compared with the LPS group, the total cell count, neutrophil count, and macrophage count in the bronchoalveolar lavage fluid of mice in the LPS+Kum groups (5mg / kg, 15mg / kg, 45mg / kg) were significantly decreased (P<0.05, P<0.01, P<0.001), especially the effect on neutrophils was more significant. The results suggest that kumadurin can reduce the number of infiltrating inflammatory cells in the lung tissues of mice with pulmonary inflammatory injury, and the effect on neutrophils is the most significant. (X) Effect of Kumadurin on the Proportion of Inflammatory Cells in Mice with Pulmonary Inflammatory Injury

[0182] Experimental method:

[0183] ①Collect BALF and centrifuge it at 1500×g for 10 min at 4℃.

[0184] ②Discard the supernatant and resuspend the bottom cells in 300 μl of PBS.

[0185] ③Add 10 μl of different flow dyes to each and incubate in the dark at 4℃ for 30 min.

[0186] ④Detect the relative intensity of each fluorescent dye using a flow analyzer.

[0187] Experimental results:

[0188] Figure 10 As shown, compared with the NC group, the proportions of neutrophils and macrophages in the inflammatory cells in the alveolar lavage fluid of mice in the LPS group were significantly increased (P<0.001); compared with the LPS group, the proportions of neutrophils in the inflammatory cells in the alveolar lavage fluid of mice in the LPS+Kum groups (5 mg / kg, 15 mg / kg, 45 mg / kg) were significantly decreased (P<0.01). The results suggest that kumadoline can reduce the proportion of neutrophils in the inflammatory cells in the lung tissue of mice with pulmonary inflammatory injury.

[0189] (XI) Effect of kumadoline on neutrophil infiltration in the lung tissue of mice with pulmonary inflammatory injury

[0190] Experimental method:

[0191] ①Sample collection: After 24 h of administration, excise the lung tissue of the mice. During the process, avoid squeezing and damaging the lung tissue to ensure its integrity. Take it out within 3 min to ensure it is fresh tissue, and rinse it thoroughly.

[0192] ②Fixation and preservation: Transfer the tissue to a petri dish containing transmission electron microscopy fixative, and use a scalpel to cut it into small pieces not exceeding 1 mm3. After fixation for 2 h, transfer it to 4℃ for storage in the dark.

[0193] ③Wash it thoroughly with phosphate buffer (PH4.7), then fix it with 1% osmium tetroxide fixative at room temperature for 1 h and store it in the dark. Wash it 5 times with phosphate buffer, 10 min each time.

[0194] ④Dehydrate, section, embed, stain and dry, observe under a transmission electron microscope, and collect images.

[0195] Experimental results:

[0196] Figure 11As shown, compared with the NC group, the number of neutrophil infiltrations in the lung tissues of mice in the LPS group increased significantly; compared with the LPS group, the number of neutrophil infiltrations in the lung tissues of mice in the LPS+Kum group (45 mg / kg) decreased significantly. The results suggest that kumadensine can reduce neutrophil infiltration in the lung tissues of mice with pulmonary inflammatory injury.

[0197] (12) Effects of Kumadensine on the Content of MPO Protein in the Lung Tissues of Mice with Pulmonary Inflammatory Injury

[0198] Experimental method:

[0199] Drugs were administered 1 h after modeling. After 24 h, lung tissues were collected, homogenized with reaction buffer, and then the content of MPO in the lung tissues was measured according to the instructions of the MPO kit.

[0200] Experimental results:

[0201] Figure 12 As shown, compared with the NC group, the content of MPO in the lung tissues of mice in the LPS group increased significantly (P<0.001); compared with the LPS group, the content of MPO in the lung tissues of mice in the LPS+Kum groups (5 mg / kg, 15 mg / kg, 45 mg / kg) decreased significantly (P<0.001). The results suggest that kumadensine can inhibit the activation of neutrophils in the lung tissues of mice with pulmonary inflammatory injury.

[0202] (13) Effects of Kumadensine on the Protein Expressions of Ly6G, MPO and NE in the Lung Tissues of Mice with Pulmonary Inflammatory Injury

[0203] Experimental method:

[0204] ① Baking the slices: Place the paraffin sections of lung tissues in an oven at 65 °C for 2 h.

[0205] ② Deparaffinizing and rehydrating, blocking peroxidase, antigen repair and blocking.

[0206] ③ Incubating with primary antibodies: Drain the blocking solution, add the diluted Ly6G, MPO, NE antibodies (1:100), and incubate overnight at 4 °C.

[0207] ④ Washing: Wash 3 times with PBS buffer, 5 min each time.

[0208] ⑤ Drop the reaction enhancer on the slices, incubate at room temperature for 20 min, and wash 3 times with PBS buffer, 5 min each time.

[0209] ⑥ Incubating with secondary antibodies: Drop an appropriate amount of secondary antibody goat anti-mouse / rabbit IgG (1:100) on the slices, incubate in a wet box at 37 °C for 40 min, and wash 3 times with PBS buffer, 5 min each time.

[0210] ⑦ Color development: Drop DAB color development solution on the sections, observe the reaction time under the microscope, and rinse with running water to terminate the reaction.

[0211] ⑧ Counterstain the cell nuclei: Counterstain with hematoxylin solution for 4 min, wash with PBS buffer 3 times, 5 min each time.

[0212] ⑨ Blueing with hematoxylin blueing solution for 90 s, rinse with running water to terminate the reaction.

[0213] ⑩ After dehydration, clearing and mounting, air-dry the sections at room temperature naturally, drop neutral balsam for mounting, and observe under an optical microscope.

[0214] Experimental results:

[0215] Figure 13 As shown, compared with the NC group, the positive areas of Ly6G, MPO and NE in the lung tissues of mice in the LPS group were significantly increased (P < 0.001); compared with the LPS group, the positive areas of Ly6G, MPO and NE in the lung tissues of mice in the LPS+Kum group (45 mg / kg) were significantly decreased (P < 0.05, P < 0.001). The results suggest that kumadoline can inhibit the infiltration and activation of neutrophils in the lung tissues of mice with pulmonary inflammatory injury.

[0216] (XIV) Effects of kumadoline on the content of NETs and related markers in the lung tissues of mice with pulmonary inflammatory injury

[0217] Experimental method:

[0218] Weigh 20 g of lung tissue, add 180 ml of PBS for grinding, homogenize it, centrifuge at 5000×g for 10 min at 4 °C. Collect the supernatant and store it at -80 °C for subsequent experiments.

[0219] ① Add 50 μl of standards with different concentrations to the corresponding standard wells.

[0220] ② Dilute the samples to be tested as required, and add 50 μl of sample diluent to each sample well.

[0221] ③ Add 50 μl of biotin-labeled antibody to each well, cover with a film, and incubate at 37 °C for 30 min.

[0222] ④ Discard the supernatant, add 300 μl of washing solution to each well, repeat the washing 5 times, and pat dry on the absorbent paper.

[0223] ⑤ Add 100 μl of HRP enzyme to each well, cover with a film, and incubate at 37 °C for 30 min.

[0224] ⑥ Discard the supernatant, add 300 μl of washing solution to each well, repeat the washing 3 times, and pat dry on the absorbent paper.

[0225] ⑦ Add 50 μl of each of the A and B substrate solutions to each well. After covering the membrane, incubate in the dark at 37 °C for 15 min.

[0226] ⑧ Add 50 μl of the stop solution to each well and immediately measure the absorbance (OD) at a wavelength of 450 nm.

[0227] ⑨ Plot the standard curve and calculate the protein concentration according to the standard curve.

[0228] Experimental results:

[0229] Figure 14 As shown, compared with the NC group, the contents of NETs, MPO-DNA, cf-DNA, and ROS in the lung tissues of mice in the LPS group were significantly increased (P < 0.001); compared with the LPS group, the contents of NETs, MPO-DNA, cf-DNA, and ROS in the lung tissues of mice in the LPS + Kum group (45 mg / kg) were significantly decreased (P < 0.001). The results suggest that kumadoline can reduce the content of NETs in the lung tissues of mice with pulmonary inflammatory injury.

[0230] (XV) Effects of kumadoline on the generation and release of NETs in the lung tissues of mice with pulmonary inflammatory injury

[0231] Experimental method:

[0232] ① Bake the slices: Place the paraffin sections of lung tissues in an oven at 65 °C for 2 h.

[0233] ② Deparaffinize and rehydrate, block peroxidase, repair antigens, permeabilize membranes, and block.

[0234] ③ Incubate with primary antibodies: Drain the blocking solution, add the diluted Cit-H3 and MPO antibodies (1:100), and incubate overnight at 4 °C in the dark and humid box.

[0235] ④ Wash: Wash 3 times with PBS buffer for 5 min each time.

[0236] ⑤ Incubate with secondary antibodies: Add the diluted FITC and Cy3 antibodies (1:100) to the sections, incubate in the dark and humid box at 37 °C for 90 min, and wash 3 times with PBS buffer for 5 min each time.

[0237] ⑥ Mount the slides: Mount the slides with an anti-tissue fluorescence quencher (containing DAPI) and observe under a fluorescence microscope.

[0238] Experimental results:

[0239] Figure 15As shown, compared with the NC group, the number of cells with MPO in the bronchoalveolar lavage fluid of mice in the LPS group increased significantly (P<0.001), and the mean fluorescence intensity of Cit-H3 in single cells increased significantly (P<0.01); compared with the LPS group, the number of cells with MPO in the bronchoalveolar lavage fluid of mice in the LPS+Kum group (45 mg / kg) decreased significantly (P<0.01), and the mean fluorescence intensity of Cit-H3 in single cells decreased significantly (P<0.01). The results suggest that kumadoline can inhibit the generation and release of NETs in the lung tissue of mice with pulmonary inflammatory injury.

[0240] (XVI) Effect of kumadoline on the protein content of PRL2 in the lung tissue of mice with pulmonary inflammatory injury

[0241] Experimental method:

[0242] Detection by spectrophotometry: Weigh 20 g of lung tissue, add 180 ml of PBS and grind it, homogenize it at 5000×g and centrifuge at 4°C for 10 min. Collect the supernatant and store it at -80°C for subsequent experiments.

[0243] ① Add 50 μl of standard products with different concentrations to the corresponding standard wells.

[0244] ② Dilute the samples to be measured as required, and add 50 μl of sample diluent to each sample well.

[0245] ③ Add 50 μl of biotin-labeled antibody to each well, cover with a film, and incubate at 37°C for 30 min.

[0246] ④ Discard the supernatant, add 300 μl of washing solution to each well, repeat the washing 5 times, and pat dry on the absorbent paper.

[0247] ⑤ Add 100 μl of HRP enzyme to each well, cover with a film, and incubate at 37°C for 30 min.

[0248] ⑥ Discard the supernatant, add 300 μl of washing solution to each well, repeat the washing 3 times, and pat dry on the absorbent paper.

[0249] ⑦ Add 50 μl of substrate solutions A and B to each well, cover with a film, and incubate at 37°C in the dark for 15 min.

[0250] ⑧ Add 50 μl of stop solution to each well, and immediately measure the absorbance value (OD) at a wavelength of 450 nm.

[0251] ⑨ Plot the standard curve and calculate the protein concentration according to the standard curve.

[0252] Detection by WB: Protein extraction, protein quantification, SDS-PAGE gel electrophoresis, membrane transfer treatment, development and exposure, and result statistics.

[0253] Experimental results:

[0254] Figure 16 As shown, compared with the NC group, the protein content of PRL2 in the lung tissues of mice in the LPS group was significantly decreased (P<0.001); compared with the LPS group, the protein content of PRL2 in the lung tissues of mice in the LPS+Kum group (45 mg / kg) was significantly increased (P<0.01, P<0.001). The results suggest that kumadoline can increase the protein content of PRL2 in the lung tissues of mice with pulmonary inflammatory injury.

[0255] (XVII) Effect of kumadoline on the expression level of PRL2 protein in neutrophils in the lung tissues of mice with pulmonary inflammatory injury

[0256] Experimental method:

[0257] ① Baking the slices: Place the paraffin sections of lung tissues in an oven at 65°C for 2 h.

[0258] ② Deparaffinizing and rehydrating, blocking peroxidase, antigen retrieval, membrane permeabilization and blocking.

[0259] ③ Incubating with primary antibodies: Drain the blocking solution, add the diluted MPO and PRL2 antibodies (1:100), and incubate overnight at 4°C in a humidified box in the dark.

[0260] ④ Washing: Wash with PBS buffer three times, 5 min each time.

[0261] ⑤ Incubating with secondary antibodies: Add the diluted FITC and Cy3 antibodies (1:100) to the sections, incubate in a humidified box at 37°C for 90 min in the dark, and wash with PBS buffer three times, 5 min each time.

[0262] ⑥ Mounting: Mount with an anti-tissue fluorescence quencher (containing DAPI) and observe under a fluorescence microscope.

[0263] Experimental results:

[0264] Figure 17 As shown, compared with the NC group, the protein expression level of PRL2 in neutrophils in the lung tissues of mice in the LPS group was significantly decreased (P<0.001); compared with the LPS group, the protein expression level of PRL2 in neutrophils in the lung tissues of mice in the LPS+Kum group (45 mg / kg) was significantly increased (P<0.001). The results suggest that kumadoline can increase the protein expression of PRL2 in neutrophils in the lung tissues of mice with pulmonary inflammatory injury.

[0265] (XVIII) Effect of kumadoline on the expression level of PRL2 mRNA in the lung tissues of mice with pulmonary inflammatory injury

[0266] Experimental method:

[0267] After 24 hours of drug administration, the lung tissues of mice were excised. After being rinsed thoroughly, approximately 20 mg of lung tissue was weighed and used for RNA extraction, RNA quantification, reverse transcription, and fluorescence detection of mRNA.

[0268] Experimental results:

[0269] Figure 18 As shown, compared with the NC group, there were no significant changes in the expression levels of PRL2 mRNA in the lung tissues of mice in the LPS group and the LPS+Kum group (45 mg / kg). The results suggest that kumadoline has no significant effect on the gene expression of PRL2 in the lung tissues of mice with pulmonary inflammatory injury.

[0270] (19) Effect of kumadoline on the PRL2 protein degradation pathway

[0271] Experimental method:

[0272] Two main protein degradation pathway inhibitors were used: the ubiquitin-proteasome pathway inhibitor (MG-132) and the lysosomal proteolytic pathway inhibitor (chloroquine, CQ).

[0273] Spectrophotometric detection: Weigh 20 g of lung tissue, add 180 ml of PBS and grind, homogenize at 5000×g and centrifuge at 4°C for 10 min. Collect the supernatant and store at -80°C for subsequent experiments.

[0274] ① Add 50 μl of standards with different concentrations to the corresponding standard wells.

[0275] ② Dilute the samples to be measured as required and add 50 μl of sample diluent to each sample well.

[0276] ③ Add 50 μl of biotin-labeled antibody to each well, cover with a film, and incubate at 37°C for 30 min.

[0277] ④ Discard the supernatant, add 300 μl of washing solution to each well, repeat the washing 5 times, and pat dry on absorbent paper.

[0278] ⑤ Add 100 μl of HRP enzyme to each well, cover with a film, and incubate at 37°C for 30 min.

[0279] ⑥ Discard the supernatant, add 300 μl of washing solution to each well, repeat the washing 3 times, and pat dry on absorbent paper.

[0280] ⑦ Add 50 μl of each of the A and B substrate solutions to each well, cover with a film, and incubate at 37°C in the dark for 15 min.

[0281] ⑧ Add 50 μl of stop solution to each well and immediately measure the absorbance value (OD) at a wavelength of 450 nm.

[0282] ⑨ Plot the standard curve and calculate the protein concentration according to the standard curve.

[0283] WB detection: protein extraction, protein quantification, SDS-PAGE gel electrophoresis, membrane transfer, development and exposure, and result statistics.

[0284] Experimental results:

[0285] Figure 19 As shown, compared with the NC group, the protein expression level of PRL2 in the lung tissues of mice in the LPS group was significantly decreased (P<0.01), and the content of NETs was significantly increased (P<0.001); compared with the LPS group, although the 20S protease activity in the lung tissues of mice in the LPS+MG-132 group (15mg / kg) was significantly inhibited (P<0.001), it could not prevent the degradation of PRL2 protein induced by LPS. In contrast, the LPS+CQ group (30mg / kg) could inhibit the lysosomal autophagy pathway, resulting in an increase in the accumulation of the autophagy marker Lc3Ⅱ in the lung tissues of mice (P<0.01), and successfully prevented the degradation of PRL2 protein and the increase in NETs induced by LPS (P<0.001). Similarly, the LPS+Kum group (45mg / kg) also showed significant effects, which could increase the accumulation of Lc3Ⅱ in the lung tissues of mice (P<0.01), inhibit the degradation of PRL2 (P<0.01), and reduce the release of NETs (P<0.01). In addition, the results of transmission electron microscopy showed that the LPS+Kum group (45mg / kg) could reduce the formation of autophagolysosomes in neutrophils in the lung tissues of mice induced by LPS. The results suggest that kumadoline can reduce the protein degradation of PRL2 and the release of NETs by inhibiting the lysosomal proteolytic pathway.

[0286] (20) Effect of kumadoline on the degradation of PRL2 protein mediated by the macroautophagy pathway

[0287] Experimental methods:

[0288] Use the macroautophagy blocker 3-methyladenine (3-MA).

[0289] WB detection: protein extraction, protein quantification, SDS-PAGE gel electrophoresis, membrane transfer, development and exposure, and result statistics.

[0290] Experimental results:

[0291] Figure 20As shown, compared with the NC group, the protein expression level of PRL2 in the lung tissue of mice in the LPS group was significantly decreased (P<0.001); compared with the LPS group, although the LPS+3-MA group (20 mg / kg) could block the formation of Lc3Ⅱ by inhibiting the macroautophagy pathway, resulting in a significant decrease in the accumulation of Lc3Ⅱ in the lung tissue of mice (P<0.05), it failed to prevent the degradation of PRL2 protein induced by LPS. In contrast, the LPS+Kum group (45 mg / kg) could increase the accumulation of Lc3Ⅱ in the lung tissue of mice by inhibiting the lysosomal proteolytic pathway (P<0.001), and successfully prevented the degradation of PRL2 protein induced by LPS (P<0.01). The results suggest that kumadoline can prevent the degradation of PRL2 protein through the lysosomal pathway other than inhibiting macroautophagy.

[0292] (XXI) Effect of Kumadoline on Chaperone-Mediated Autophagy (CMA)

[0293] Experimental method:

[0294] Detection by spectrophotometry: Weigh 20 g of lung tissue, add 180 ml of PBS and grind it, homogenize it at 5000×g and centrifuge at 4℃ for 10 min. Collect the supernatant and store it at -80℃ for subsequent experiments.

[0295] ① Add 50 μl of standard products with different concentrations to the corresponding standard wells.

[0296] ② Dilute the samples to be measured as required, and add 50 μl of sample diluent to each sample well.

[0297] ③ Add 50 μl of biotin-labeled antibody to each well, cover with a film, and incubate at 37℃ for 30 min.

[0298] ④ Discard the supernatant, add 300 μl of washing solution to each well, repeat the washing 5 times, and pat dry on the absorbent paper.

[0299] ⑤ Add 100 μl of HRP enzyme to each well, cover with a film, and incubate at 37℃ for 30 min.

[0300] ⑥ Discard the supernatant, add 300 μl of washing solution to each well, repeat the washing 3 times, and pat dry on the absorbent paper.

[0301] ⑦ Add 50 μl of substrate solutions A and B to each well, cover with a film, and incubate at 37℃ in the dark for 15 min.

[0302] ⑧ Add 50 μl of stop solution to each well, and immediately measure the absorbance (OD) at a wavelength of 450 nm.

[0303] ⑨ Plot the standard curve and calculate the protein concentration according to the standard curve.

[0304] WB detection: Protein extraction, protein quantification, SDS-PAGE gel electrophoresis, membrane transfer treatment, developing and exposure, and result statistics.

[0305] Experimental results:

[0306] Figure 21 As shown, compared with the NC group, the expression level of PRL2 protein in the lung tissue of mice in the LPS group was significantly decreased (P<0.01), while the expression levels of NETs and LAMP2A proteins were significantly increased (P<0.001); compared with the LPS group, the LPS+Kum group (45 mg / kg) could significantly reduce the expression levels of LAMP2A and NETs proteins in the lung tissue of mice (P<0.01, P<0.001), and inhibit the degradation of PRL2 protein (P<0.05). However, the QX77 group (10 mg / kg) could reverse the therapeutic effect of kumadoline, resulting in the degradation of PRL2 protein in the lung tissue of mice, and at the same time significantly increasing the protein expression levels of NETs and LAMP2A (P<0.001). The results suggest that kumadoline can protect PRL2 protein from degradation by inhibiting the CMA pathway.

[0307] (22) Effects of kumadoline on chaperone-mediated autophagy (CMA)-related proteins

[0308] Experimental methods:

[0309] WB detection: Protein extraction, protein quantification, SDS-PAGE gel electrophoresis, membrane transfer treatment, developing and exposure, and result statistics.

[0310] q-PCR detection: After 24 h of drug administration, the lung tissue of mice was removed, rinsed thoroughly, and about 20 mg of lung tissue was weighed for RNA extraction, RNA quantification, reverse transcription, and fluorescence detection of mRNA.

[0311] Immunofluorescence detection:

[0312] ① Baking the slices: The paraffin sections of lung tissue were placed in an oven at 65 °C for 2 h.

[0313] ② Deparaffinization and rehydration, blocking peroxidase, antigen retrieval, membrane permeabilization, and blocking.

[0314] ③ Incubation with primary antibody: The blocking solution was discarded, and the diluted LAMP1 and PRL2 antibodies (1:100) were added dropwise, and incubated overnight at 4 °C in the dark and humid box.

[0315] ④ Washing: Wash with PBS buffer 3 times, 5 min each time.

[0316] ⑤ Incubation with secondary antibody: The diluted FITC and Cy3 antibodies (1:100) were added dropwise to the sections, incubated at 37 °C for 90 min in the dark and humid box, and washed with PBS buffer 3 times, 5 min each time.

[0317] ⑥ Mounting: Mount with an anti-tissue fluorescence quencher (containing DAPI), and observe under a fluorescence microscope.

[0318] Experimental results:

[0319] Figure 22 and Figure 23 As shown in and, compared with the NC group, there were no significant changes in the expression levels of Hsc70 protein and Hsc70 mRNA in the lung tissues of mice in the LPS group and the LPS+Kum group (45 mg / kg). Compared with the NC group, the expression levels of LAMP2A protein and LAMP2A mRNA in the lung tissues of mice in the LPS group were significantly increased (P<0.01, P<0.001); compared with the LPS group, the expression levels of LAMP2A protein and LAMP2A mRNA in the lung tissues of mice in the LPS+Kum group (45 mg / kg) were significantly decreased (P<0.05, P<0.001). In addition, the results of the immunofluorescence double staining experiment showed that compared with the NC group, the expression level of LAMP1 protein in the bronchoalveolar lavage fluid of mice in the LPS group was significantly increased (P<0.01), and the expression level of PRL2 protein was significantly decreased (P<0.001); compared with the LPS group, the expression level of LAMP1 in the bronchoalveolar lavage fluid of mice in the LPS+Kum group (45 mg / kg) was significantly decreased (P<0.05), and the expression level of PRL2 protein was significantly increased (P<0.001). The results suggest that kumadoline blocks the degradation of PRL2 protein mediated by the CMA pathway by inhibiting the protein and gene expression of the key protein LAMP2A of CMA.

[0320] (XXIII) Effect of kumadoline on the interaction between PRL2, Hsc70 and LAMP2A

[0321] Experimental method:

[0322] ① Place a 1.5 ml EP tube on the magnetic rack, add 30 μl of magnetic beads, and then add 200 μl of lysis buffer and wash twice.

[0323] ② Take another 100 μl of lysis buffer, add it to the washed magnetic beads, and then add 10 μl of antibody, and incubate at room temperature on a shaker for 2 h.

[0324] ③ After incubation, place it on the magnetic rack and recover the antibody.

[0325] ④ Weigh 100 mg of lung tissue, add 0.5 ml of lysis buffer, add grinding beads, grind at 4°C, and set the grinder at 70 Hz.

[0326] ⑤ Centrifuge the tissue homogenate at 12000×g at 4°C for 15 min, and take the supernatant.

[0327] ⑥ Add 450 ml of tissue supernatant to the incubated magnetic beads and incubate overnight at 4 °C on a shaker.

[0328] ⑦ After incubation, place on a magnetic stand, discard the tissue supernatant, and then add 200 μl of lysis buffer and wash 3 times.

[0329] ⑧ Take another 80 μl of lysis buffer, add it to the washed magnetic beads, and then add 16 μl of Loading buffer (6×). Incubate at boiling water for 10 min.

[0330] ⑨ Place on a magnetic stand, discard the magnetic beads, take the supernatant and aliquot and store at -80 °C for subsequent experiments.

[0331] ⑩ Finally, perform SDS-PAGE gel electrophoresis, membrane transfer, development and exposure, and result statistics.

[0332] Experimental results:

[0333] Figure 24 As shown, compared with the NC group, the interaction between the three proteins in the lung tissue of LPS group mice was significantly enhanced; compared with the LPS group, the interaction among the three in the lung tissue of LPS+Kum group (45 mg / kg) mice was weakened. The results suggest that kumausoline can block the degradation of PRL2 protein mediated by the CMA pathway by weakening the interaction between CMA-related proteins PRL2, Hsc70 and LAMP2A.

Claims

1. Use of kumaokinin in the preparation of a medicament for treating pulmonary inflammatory injury, wherein the structure of kumaokinin is shown as formula I:

2. The application according to claim 1, wherein kumaokinin is the only active ingredient in the medicament.

3. The application according to claim 1 or 2, wherein the pulmonary inflammatory injury is an inflammatory injury mainly dominated by neutrophils.

4. The application according to claim 1 or 2, wherein the pulmonary inflammatory injury is a pathological injury of lung tissue.

5. The application according to claim 1 or 2, wherein the pulmonary inflammatory injury is pulmonary tissue edema.

6. The application according to claim 1 or 2, wherein the pulmonary tissue edema is alveolar-capillary barrier injury.

7. The application according to claim 1 or 2, wherein the pulmonary inflammatory injury is an increase in inflammatory factors in lung tissue.

8. The application according to claim 1 or 2, wherein the inflammatory factors are IL-1β, TNF-α and IL-6.

9. The application according to claim 1 or 2, wherein the pulmonary inflammatory injury is infiltration of inflammatory cells in lung tissue.

10. The application according to claim 1 or 2, wherein the infiltration of inflammatory cells is infiltration of neutrophils.

11. The application according to claim 1 or 2, wherein the pulmonary inflammatory injury is an increase in the generation and release of NETs in lung tissue.

12. The application according to claim 1 or 2, wherein the pulmonary inflammatory injury is a decrease in the level of PRL2 in lung tissue.

13. The application according to any one of claims 1-12, wherein the medicament achieves one or more of the following effects: (1) Inhibiting neutrophil infiltration, activation and release of NETs, and playing a protective role against pulmonary inflammatory injury; (2) Improving lung function injury and / or pathological injury of lung tissue; (3) Reducing pulmonary tissue edema; (4) Lowering the level of inflammatory factors in lung tissue; (5) Reducing infiltration of inflammatory cells in lung tissue, and / or reducing the proportion of neutrophils in inflammatory cells; (6) Reducing infiltration and activation of neutrophils in lung tissue, (7) Reducing ROS generation in lung tissue; (8) Inhibiting the generation and release of NETs in lung tissue; (9) Blocking protein degradation of PRL2 in lung tissue.

14. The application according to claim 13, wherein the medicament increases the protein level of PRL2 in lung tissue by inhibiting the lysosomal proteolytic pathway and / or weakening the interaction between CMA-related proteins PRL2, Hsc70 and LAMP2A, and blocking the degradation of PRL2 protein mediated by the CMA pathway.

15. The application according to any one of claims 1-14, wherein the medicament is in oral dosage form or injection dosage form.

16. The application according to claim 15, wherein the medicament is in injection dosage form.

17. A non-therapeutic method for enhancing lung function, characterized in that, The method includes administering kumaokinin.

18. The method according to claim 17, wherein the single administration dose of kumaokinin is 15-45 mg / kg.

19. The method according to claim 18, wherein the single administration dose of kumaokinin is 5-15 mg / kg.

20. The method according to claim 18, wherein the single application dose of kumaokinin is 45 mg / kg.

Images