Application of herbacetin in preparation of medicine for treating pneumonia injury

Abstract

The invention provides application of herbacetin in preparation of a medicine for treating pneumonia injury. Experimental results show that after the herbacetin is administrated in an intraperitoneal injection form, by up-regulating expression of IL-10, infiltration of neutrophil in lung tissue airways is reduced, levels of inflammatory factors and total proteins are reduced, pathological injury of lung tissues is improved, and the herbacetin has a good improvement effect on mice suffering from pneumonic injury. The invention provides a new strategy and a clinical research basis for prevention or treatment of the pneumonic injury, further provides a scientific basis for guiding clinical medication for treating the pneumonic injury, and has a relatively good application prospect.

Description

Technical Field

[0001] The present application belongs to the field of respiratory disease treatment. Specifically, the present application provides the use of herbicide in the preparation of a drug for preventing or treating pulmonary pneumonia injury. Background Art

[0002] Pneumonic injury refers to a series of inflammatory reactions of the lung parenchyma caused by infection with multiple pathogens or physical and chemical factors, leading to edema and impaired lung function. The etiology of pulmonary pneumonia is complex, which is mainly related to drugs, environment, genetics, infection, disease and lifestyle. At present, the main clinical treatment is to combine mechanical ventilation, anti-inflammatory, anti-infective, antioxidant, anticoagulant and other drugs on the basis of the primary disease. In addition, surgical treatment, gene therapy and other supportive treatments are also included. Although new treatment methods continue to emerge with the development of science and technology, its mortality rate is still as high as 30%-50%. Therefore, the development of safe and effective drugs for the treatment of pulmonary pneumonia is a problem that needs to be solved in the current clinical practice.

[0003] More and more studies have shown that neutrophils, as the first responders to inflammation, participate in inflammatory regulation in various ways in pulmonary pneumonia. Neutrophils are heterogeneous and plastic, with unique phenotypes and functional properties in the microenvironment and during migration. It is reported that lung neutrophils can be subdivided into two major groups based on their different gene expression patterns (Fth1 hi Neu and Prok2 hi Neu). Ferritin heavy chain (Fth1 or FHC), encoded by the Fth1 gene, is a cytoplasmic iron storage protein that is expressed in airway neutrophils (Fth1 hi Neu), Fth1 hi Neu is mainly distributed in the alveoli and can regulate multiple physiological processes such as ferroptosis, autophagy, oxidative stress and inflammation. It can promote the release of inflammatory mediators and delay cell apoptosis. Prok2 (Prokineticin 2), also known as the homolog of Bv8, is expressed in neutrophils from peripheral blood and bone marrow (Prok2 hi Neu) is highly expressed, Prok2 hi Neu is mainly distributed in the pulmonary blood vessels, highly expresses antimicrobial peptides, and has significant anti-inflammatory effects. Neutrophils are the main target cells for the anti-inflammatory effects of IL-10, and their heterogeneity is largely determined by IL-10-mediated signal transduction. Under inflammatory stimulation, the increase in IL-10 content can weaken the mitochondrial homeostasis of neutrophils and accelerate the apoptosis and clearance of neutrophils.

[0004] Ephedra is a folk medicine commonly used in clinical Chinese medicine. Its main ingredients, such as ephedrine, pseudoephedrine, flavonoids, and polysaccharides, have multiple biological activities and pharmacological functions, and have good development prospects. In clinical practice, ephedra is mainly used to treat symptoms such as colds, coughs, and headaches. Modern pharmacological studies have shown that ephedra has antipyretic and antiasthmatic effects, as well as immunosuppressive effects. Its flavonoid components can reduce the production of inflammatory cells in lung tissue and reduce pulmonary edema. Herbicide, as the main natural flavonoid bioactive ingredient in ephedra rhizomes, has multiple biological activities such as anti-inflammatory, antioxidant, and antibacterial. It has high safety and stability, and has broad application prospects in the biomedical field. However, there have been no reports on the use of herbicide in the treatment of pulmonary pneumonia. Therefore, developing it into a drug for the treatment of pulmonary pneumonia has extremely high potential value and social significance. Summary of the invention

[0005] The purpose of the present invention is to study the pharmacological effects of herbicide and find that herbicide can improve the morphology and pathological damage of lung tissue in mice with pneumonia-induced pulmonary injury; reduce lung tissue edema; significantly reduce the number of inflammatory cells in bronchoalveolar lavage fluid, reduce the levels of inflammatory factors and total protein; reduce the level of myeloperoxidase in lung tissue; and reduce the expression level of inflammatory factors in serum, thereby providing the use of herbicide in the preparation of drugs for treating pneumonia-induced pulmonary injury.

[0006] In one aspect, the present invention provides the use of herbicide in preparing a drug for treating pulmonary pneumonia.

[0007] Furthermore, herbicide is the only active ingredient in the drug.

[0008] Furthermore, the pulmonary pneumonia injury is lung function injury.

[0009] Furthermore, the pulmonary pneumonia injury is pathological injury of lung tissue.

[0010] Furthermore, the pulmonary inflammation injury is pulmonary tissue edema.

[0011] Furthermore, the pulmonary inflammatory injury is characterized by elevated inflammatory factors.

[0012] Furthermore, the drug reduces the number of inflammatory cells, the levels of inflammatory factors and total protein in bronchoalveolar lavage fluid; reduces the level of myeloperoxidase in lung tissue; and reduces the expression level of inflammatory factors in serum.

[0013] Furthermore, the inflammatory factors include TNF-ɑ, IL-6 and IL-1β.

[0014] Furthermore, the drug is in an oral dosage form or an injectable dosage form.

[0015] Furthermore, the medicine is in the form of an injection.

[0016] In another aspect, the present invention provides a non-therapeutic method for enhancing lung function comprising administering herbicide.

[0017] Furthermore, the single application dosage of herbicide is 5-15 mg / kg.

[0018] Furthermore, the single application dosage of herbicide is 15-45 mg / kg.

[0019] Furthermore, the single application dose of herbicide is 45 mg / kg.

[0020] The herbacetin described in this application is also called Herbacetin, and its CAS number is: 527-95-7, and its structural formula is shown in formula (I):

[0021]

[0022] The non-therapeutic methods in this application include but are not limited to health care methods and scientific research methods, and the subjects of implementation can be humans or other mammals such as mice, rabbits, dogs, etc.

[0023] The symptoms or indications of pneumonia injury in this application include but are not limited to lung function damage, lung tissue pathological damage, lung tissue edema, increased inflammatory factors, etc.

[0024] The available pharmaceutical dosage forms of the present application include various injection and oral dosage forms, including but not limited to tablets, capsules, oral liquids, water injections, powder injections, and transdermal preparations, with injection forms being particularly preferred.

[0025] In addition to herbicides, the drugs described in the present application may also include various pharmaceutically acceptable excipients / vegetarians, including but not limited to coating materials, solvents, solubilizers, adhesives, stabilizers, antioxidants, pH adjusters, flavoring agents, etc., and various excipients / vegetarians that can be used in oral dosage forms are particularly preferred.

[0026] The drug of the present invention may contain other Chinese and Western medicines or health products for treating pneumonia injury, or the drug of the present invention may be used in combination with these Chinese and Western medicines or health products or surgical treatments. The Chinese and Western medicines or health products include but are not limited to anti-inflammatory drugs, anti-infective drugs, and antioxidant drugs.

[0027] The use of the herbicide provided by the present invention in the preparation of a drug for treating pulmonary pneumonia has the following beneficial effects:

[0028] Herbicide can improve lung tissue morphology and pathological damage;

[0029] Herbicide can relieve edema of lung tissue;

[0030] Herbicide can significantly reduce the number of inflammatory cells in bronchoalveolar lavage fluid and reduce the levels of inflammatory factors and total protein;

[0031] Herbicide can reduce the level of myeloperoxidase in lung tissue;

[0032] Herbicide can reduce the level of inflammatory factors in serum.

[0033] The present invention confirms for the first time that herbicide has the effect of treating pneumonia-induced pulmonary injury in mice and can be used to prepare drugs for treating pneumonia-induced pulmonary injury, providing a new choice for such drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Effects of herbicide on the overall lung tissue morphology and pathological damage in mice with pneumonia; (A) is the control group, (B) is the lipopolysaccharide group, (C) is the lipopolysaccharide + dexamethasone 5 mg / kg group, (D) is the lipopolysaccharide + herbicide 5 mg / kg group, (E) is the lipopolysaccharide + herbicide 15 mg / kg group, (F) is the lipopolysaccharide + herbicide 45 mg / kg group (n=6).

[0035] Figure 2 The effect of herbicide on the pathological changes and injury scores of lung tissue in mice with pneumonia-induced injury; (A) is the control group, (B) is the lipopolysaccharide group, (C) is the lipopolysaccharide + dexamethasone 5mg / kg group, (D) is the lipopolysaccharide + herbicide 5mg / kg group, (E) is the lipopolysaccharide + herbicide 15mg / kg group, (F) is the lipopolysaccharide + herbicide 45mg / kg group; the star represents a large number of inflammatory cell infiltrations, the triangle represents the thickening of the alveolar septum, the circle represents the bleeding points in the lung tissue, and the arrow represents the destruction of the alveolar structure. The results are expressed as mean±SD (n=6). ### P < 0.001 indicates comparison with the control group; * P<0.05, *** P<0.001 indicates comparison with the model group (from bottom to top in each column are alveolar neutrophils, interstitial neutrophils, hyaline membranes, protein fragments, alveolar septum thickening, and hemorrhage spots).

[0036] Figure 3 Statistical diagram of the effect of herbicide on lung organ coefficient and wet-to-dry weight ratio in mice with pneumonia-induced lung injury; the results are expressed as mean±SD (n=6), "ns" means no significant difference; ### P < 0.001 indicates comparison with the control group; * P<0.05, ** P<0.01, *** P<0.001 indicates comparison with the model group.

[0037] Figure 4Statistical diagram of the effect of herbicide on pulmonary edema in mice with pneumonia injury; (A) is the control group, (B) is the lipopolysaccharide group, and (C) is the lipopolysaccharide + herbicide 45 mg / kg group. The results are expressed as mean ± SD (n = 8). ### P < 0.001 indicates comparison with the control group; ** P<0.01 indicates comparison with the model group.

[0038] Figure 5 Statistical diagram of the effect of herbicide on the proinflammatory cytokines TNF-ɑ, IL-6 and IL-1β in bronchoalveolar lavage fluid of mice with pneumonia injury; the results are expressed as mean±SD (n=8, 8, 6). ### P < 0.001 indicates comparison with the control group; * P<0.05, ** P<0.01, *** P<0.001 indicates comparison with the model group.

[0039] Figure 6 Statistical diagram of the effect of herbicide on serum proinflammatory cytokines TNF-ɑ, IL-6 and IL-1β in mice with pneumonia injury; the results are expressed as mean±SD (n=6). ### P < 0.001 indicates comparison with the control group; *** P<0.001 indicates comparison with the model group.

[0040] Figure 7 Statistical diagram of the effect of herbicide on the mRNA expression of proinflammatory cytokines TNF-ɑ, IL-6 and IL-1β in lung tissue of mice with pneumonia injury; the results are expressed as mean±SD (n=3). ### P < 0.001 indicates comparison with the control group; ** P<0.01, *** P<0.001 indicates comparison with the model group.

[0041] Figure 8 Statistical diagram of the effect of herbicide on the total protein concentration in bronchoalveolar lavage fluid of mice with pneumonia injury; the results are expressed as mean±SD (n=6), ### P < 0.001 indicates comparison with the control group; *** P<0.001 indicates comparison with the model group.

[0042] Fig. 9 Statistical diagram of the effect of herbicide on the MPO content in lung tissue of mice with pneumonia injury; the results are expressed as mean±SD (n=8), ### P<0.001 indicates comparison with the control group; "ns" indicates no significant difference. ** P<0.01, ***P<0.001 indicates comparison with the model group.

[0043] Fig.10 Statistical diagram of the effect of herbicide on the number of cells in bronchoalveolar lavage fluid of mice with pneumonia injury; the results are expressed as mean±SD (n=6), ### P<0.001 indicates comparison with the control group; "ns" indicates no significant difference. * P<0.05, *** P<0.001 indicates comparison with the model group.

[0044] Fig.11 Statistical diagram of the effect of herbicide on neutrophil infiltration in lung tissue of mice with pneumonia injury; (A) is the control group, (B) is the lipopolysaccharide group, and (C) is the lipopolysaccharide + herbicide 45 mg / kg group. The results are expressed as mean ± SD (n = 3). ### P < 0.001 indicates comparison with the control group; *** P<0.001 indicates comparison with the model group.

[0045] Fig.12 Effects of herbicide on the number and distribution of neutrophils in lung tissue of mice with pneumonia-induced pulmonary injury; (A) is the control group, (B) is the lipopolysaccharide group, and (C) is the lipopolysaccharide + herbicide 45 mg / kg group. The triangle is the neutrophil, and the arrow is the mitochondria.

[0046] Fig.13 The effect of herbicide on the infiltration of neutrophil Fth1 and Prok2 in the lung tissue of mice with pneumonia-induced pulmonary injury; (A) is the control group, (B) is the lipopolysaccharide group, and (C) is the lipopolysaccharide + herbicide 45 mg / kg group.

[0047] Fig.14 Statistical diagram of the effect of herbicide on IL-10 protein expression in lung tissue of mice with pneumonia injury; the results are expressed as mean±SD (n=6), ### P < 0.001 indicates comparison with the control group; *** P<0.001 indicates comparison with the model group.

[0048] Fig.15 Statistical diagram of the effect of herbicide on IL-10 mRNA in lung tissue of mice with pneumonia injury; the results are expressed as mean±SD (n=3), ### P < 0.001 indicates comparison with the control group; ** P<0.01 indicates comparison with the model group.

[0049] Fig.16 Statistical diagram of the effect of herbicide on the expression of IL-10 protein in lung tissue of mice with pneumonia injury; the results are expressed as mean±SD (n=3),# P < 0.05 indicates comparison with the control group; ** P<0.01 indicates comparison with the model group.

[0050] Fig.17 The effect of herbicide on apoptosis of lung tissue cells in mice with pneumonia-induced pulmonary injury; (A) is the control group, (B) is the lipopolysaccharide group, and (C) is the lipopolysaccharide + herbicide 45 mg / kg group. DETAILED DESCRIPTION

[0051] The present invention is further described in detail below with reference to the examples. The herbicides used in the following examples are all compounds represented by the aforementioned formula (I) and can be obtained commercially.

[0052] The single application dose of herbicide is 5 mg / kg, 15 mg / kg or 45 mg / kg for mice, and the dosage form of the drug is an injection form.

[0053] Example 1 Experimental Materials

[0054] Animal handling

[0055] SPF-grade ICR male adult mice weighing between 18 and 22 g were bred at the Experimental Animal Center of Ningxia Medical University (animal incubation qualification batch number: SCXK (Ning) 2020-0001). Animal breeding was in accordance with the experimental animal breeding standards, with a 12-hour artificial day and night cycle in the breeding room, and mice were free to drink water.

[0056] Experimental drugs and instruments

[0057] The main drugs and reagents involved in this experiment include: herbicide (purchased from Shanghai Yuanye Biotechnology Co., Ltd., purity ≥98%); dexamethasone (purchased from Shanghai Yuanye Biotechnology Co., Ltd.); lipopolysaccharide (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.); physiological saline (purchased from Xi'an Jingxi Shuanghe Pharmaceutical Co., Ltd.); PBS (purchased from Shanghai Dathill Biotechnology Co., Ltd.); BCA extraction kit (purchased from Jiangsu Keygen Biological Co., Ltd.); whole protein extraction kit (purchased from Nanjing Keygen Biological Co., Ltd.); ELISA kit (IL-6, IL-1β, TNF-α) (Wuhan Elerite Biotechnology Co., Ltd.); myeloperoxidase (MPO) assay kit (purchased from Nanjing Jiancheng Bioengineering Institute).

[0058] The main instruments involved in this experiment include: microplate reader (purchased from Thermo scientific); electric constant temperature water bath (purchased from Shanghai Precision Laboratory Equipment Co., Ltd.); fully automatic sample freezing grinder (purchased from Shanghai Jingxin Technology Co., Ltd.); electric constant temperature blast drying oven (purchased from Shanghai Langgan Laboratory Equipment Co., Ltd.); optical microscope (OLYMPUS); high-speed low-temperature centrifuge (Eppendorf, Germany).

[0059] Experimental animal grouping and drug administration

[0060] After 3 days of adaptive feeding in a clean-grade feeding room, male adult ICR mice weighing 18-22g were evenly divided into control group, model group, dexamethasone group and herbicide 5mg / kg, 15mg / kg, 45mg / kg groups according to the weight gradient sampling method. Except for the control group, all other groups of animals were intratracheally instilled with 50μl of LPS (5mg / kg), that is, a pulmonary pneumonia injury mouse model was established. The dexamethasone group was given dexamethasone (5mg / kg) by intraperitoneal injection, and the herbicide 5mg / kg, 15mg / kg, and 45mg / kg groups were given herbicide (5mg / kg, 15mg / kg, and 45mg / kg) by intraperitoneal injection, with 8 mice in each group. The dexamethasone group and each herbicide dosage group were intraperitoneally injected 1 hour after modeling, and the control group and model group mice were given the same volume of solvent; the mice were intraperitoneally injected with a dosage volume of 0.1ml / 10g, and samples were collected 24 hours after administration; the other experimental conditions were the same.

[0061] Example 2 Experimental process and result analysis (I) Overall lung tissue morphology and pathological damage detection

[0062] Experimental methods:

[0063] The drug was administered 1 hour after modeling, and the mice were killed 24 hours later. The chest cavity of the mice was quickly cut open with surgical scissors to fully expose the lung tissue, and the surrounding adipose tissue and connective tissue were removed. The lung tissue was taken out, the blood on the surface was absorbed, and then it was immersed in animal lung tissue fixative for HE staining and injury scoring.

[0064] Experimental results:

[0065] Figure 1 As shown in the figure, from the overall observation of the lung tissue, it can be seen that compared with the control group, the lung tissue of the model group was edematous, the color became darker, and a large number of bleeding spots appeared on the outer surface. After treatment with herbicide (5mg / kg, 15mg / kg, 45mg / kg), the edema of the lung tissue of mice was reduced, the color became ruddy, and the bleeding spots decreased. This suggests that herbicide has an improving effect on the lung tissue damage of mice with pneumonia.

[0066] Figure 2As shown in the figure, compared with the control group, the model group had a large number of inflammatory cell infiltration in the lung tissue, more bleeding spots, thickened alveolar septa, and destroyed alveolar structure. After treatment with herbicide (5mg / kg, 15mg / kg, 45mg / kg), the infiltration of inflammatory cells and bleeding spots decreased, the thickening of alveolar septa was alleviated, and the structure of lung tissue returned to normal.

[0067] The histology of injured lungs was evaluated according to 6 main dimensions: alveolar neutrophils, interstitial neutrophils, hyaline membranes, air-filled protein fragments, thickening of alveolar septa, and hemorrhages in lung tissue. Compared with the control group, the lung tissue injury score in the model group was significantly increased (P<0.001). After treatment with herbicide (5mg / kg, 15mg / kg, 45mg / kg), the lung tissue injury score of mice was significantly reduced (P<0.05, P<0.001). The lung tissue injury score of mice in the dexamethasone treatment group was also significantly reduced (P<0.001). John score statistics (0-4): no change 0, mild change 1, moderate change 2, severe change 3, and extremely severe change 4. It suggests that herbicide has an improving effect on the pathological damage of lung tissue in mice with pneumonia.

[0068] (II) Determination of pulmonary tissue edema

[0069] Experimental methods:

[0070] Organ coefficient: The drug was administered 1 hour after modeling, and the mice were weighed and recorded after 24 hours. After the experiment, the mice were killed, and the chest cavity of the mice was quickly cut open with surgical scissors to fully expose the lung tissue, and the surrounding fat tissue and connective tissue were removed. The lung tissue was taken out, and the blood on the surface was sucked dry and its weight was accurately weighed with an electronic balance. The ratio of lung tissue weight to body weight was calculated as the lung organ coefficient.

[0071] Wet-to-dry weight ratio: drug administration was performed 1 hour after modeling, mice were killed 24 hours later, the chest cavity of mice was quickly cut open with surgical scissors, the lung tissue was fully exposed, the surrounding fat tissue and connective tissue were removed, the lung tissue was taken out, the blood on the surface was sucked dry, and its weight was accurately weighed with an electronic balance, which was the wet weight. Then it was placed in an incubator and baked at 60℃ for 48 hours, and weighed again to obtain the dry weight, and the ratio of wet weight to dry weight (W / D) was calculated.

[0072] Experimental results:

[0073] Figure 3As shown in the results, compared with the control group, the lung organ coefficient and wet-to-dry weight ratio of the model group mice increased significantly (P<0.001). After treatment with herbicide (5mg / kg, 15mg / kg, 45mg / kg), the lung organ coefficient and wet-to-dry weight ratio of the mice decreased significantly (P<0.05, P<0.01, P<0.001), and the dexamethasone treatment group also significantly reduced the lung organ coefficient and wet-to-dry weight ratio (P<0.001). This suggests that herbicide has an improving effect on pulmonary edema in mice with pneumonia injury.

[0074] Experimental methods:

[0075] Evans blue staining: drug administration was performed 1 hour after model establishment, and 2% Evans blue staining solution was injected into the tail vein 23 hours later. The mice were killed 30 minutes later, and the chest cavity of the mice was quickly cut open with surgical scissors to fully expose the lung tissue, and the surrounding adipose tissue and connective tissue were removed. The lung tissue was taken out and fully ground, and acetone (7:3) was added to the supernatant, incubated at room temperature for 24 hours, and the OD value was measured at a wavelength of 620nm.

[0076] Experimental results:

[0077] Figure 4 As shown in the figure, compared with the control group, the Evans blue staining fluid content of the model group mice increased significantly (P<0.001). After treatment with herbicide (45 mg / kg), the Evans blue staining fluid content of the mice decreased significantly (P<0.01). This suggests that herbicide has an improving effect on pulmonary edema in mice with pneumonia injury.

[0078] (III) Determination of pro-inflammatory cytokine content

[0079] Experimental methods:

[0080] Determination of serum pro-inflammatory factor content: drug administration was performed 1 h after model establishment, and blood was collected from mice by eyeball sampling 24 h later. After standing at room temperature for 2 h, the blood was centrifuged at 4°C 1000 rfc for 15 min, and the upper serum was collected.

[0081] Determination of pro-inflammatory factors in alveolar lavage fluid: 24 hours after administration, anesthetize the mice, cut the laryngeal skin to expose the trachea, insert the retention needle into the trachea, and tie the trachea to the retention needle with surgical thread. Rinse slowly with pre-cooled PBS solution 3 times, 0.8 ml each time, a total of 2.4 ml, the washing fluid is alveolar lavage fluid, and then centrifuge the collected alveolar lavage fluid at 4 ° C 1500rcf for 10 minutes, and take the supernatant. Use a mouse-specific ELISA kit according to the manufacturer's instructions to quantify the concentrations of TNF-α, IL-1β and IL-6 in serum and alveolar lavage fluid. According to the instructions, prepare the working solution, draw the standard curve, add the sample to be tested, incubate, add the detection antibody, tool drug and color development solution, etc., use an enzyme marker to measure the absorbance value of each well, and calculate the results according to the standard curve.

[0082] Experimental results:

[0083] Figure 5 As shown in the figure, compared with the control group, the protein levels of proinflammatory cytokines TNF-ɑ, IL-6 and IL-1β in the bronchoalveolar lavage fluid of mice in the model group were significantly increased (P<0.001). After treatment with herbicide (5mg / kg, 15mg / kg, 45mg / kg), the protein levels of proinflammatory cytokines TNF-ɑ (P<0.01, P<0.001), IL-6 (P<0.001) and IL-1β (P<0.05, P<0.01, P<0.001) in the bronchoalveolar lavage fluid of mice were significantly decreased, and the dexamethasone treatment group also significantly reduced the protein levels of proinflammatory cytokines TNF-ɑ, IL-6 and IL-1β in the bronchoalveolar lavage fluid of mice (P<0.001).

[0084] Figure 6 As shown in the figure, compared with the control group, the serum levels of proinflammatory cytokines TNF-α, IL-6 and IL-1β in the model group mice were significantly increased (P<0.001). After treatment with herbicide (5mg / kg, 15mg / kg, 45mg / kg), the serum levels of proinflammatory cytokines TNF-α, IL-1β and IL-6 in mice were significantly decreased (P<0.001), and the dexamethasone treatment group also significantly reduced the serum levels of proinflammatory cytokines TNF-α, IL-1β and IL-6 in mice (P<0.001). This suggests that herbicide can reduce the release of proinflammatory cytokines in mice with pneumonia.

[0085] Experimental methods:

[0086] Drugs were administered 1 hour after modeling, and mice were killed 24 hours later. The chest cavity of the mice was quickly cut open with surgical scissors to fully expose the lung tissue, and the surrounding adipose tissue and connective tissue were removed. The lung tissue was taken out, the blood on the surface was dried, and the total RNA of the lung tissue was extracted.

[0087] The micro-protein nucleic acid quantification instrument was used for quantification, and the expression level of mRNA was detected by fluorescence quantification.

[0088] Experimental results:

[0089] Figure 7 As shown in the figure, compared with the control group, the mRNA expression levels of proinflammatory cytokines TNF-α, IL-6 and IL-1β in the lung tissue of mice in the model group were significantly increased (P<0.001). After treatment with herbicide (45 mg / kg), the mRNA expression levels of proinflammatory cytokines TNF-α, IL-1β and IL-6 in the lung tissue of mice were significantly decreased (P<0.01, P<0.001). This suggests that herbicide can reduce the expression of proinflammatory cytokine mRNA in mice with pneumonia.

[0090] (IV) Determination of total protein concentration in bronchoalveolar lavage fluid

[0091] Experimental methods:

[0092] Drugs were administered 1 hour after modeling, and mice were anesthetized 24 hours later. The laryngeal skin was cut to expose the trachea, and the retention needle was inserted into the trachea. The trachea and the retention needle were tied tightly with surgical thread. The mice were slowly rinsed 3 times with pre-cooled PBS solution, 0.8 ml each time, for a total of 2.4 ml. The washing solution was alveolar lavage fluid, and then the collected alveolar lavage fluid was centrifuged at 4°C 1500rfc for 10 minutes, and the supernatant was taken. The total protein concentration was determined using the BCA protein detection kit.

[0093] Experimental results:

[0094] Figure 8 As shown in the figure, compared with the control group, the total protein concentration in the bronchoalveolar lavage fluid of the model group mice increased significantly (P<0.001). After treatment with herbicide (5mg / kg, 15mg / kg, 45mg / kg), the total protein concentration in the bronchoalveolar lavage fluid of mice decreased significantly (P<0.001), and the dexamethasone treatment group also significantly reduced the total protein concentration in the bronchoalveolar lavage fluid of mice (P<0.001). This suggests that herbicide can reduce the level of total protein in the bronchoalveolar lavage fluid of mice with pneumonia.

[0095] (V) Determination of myeloperoxidase (MPO) level

[0096] Experimental methods:

[0097] Drugs were administered 1 hour after modeling, and lung tissues were collected 24 hours later. The tissues were homogenized with reaction buffer, and then the MPO content in the lung tissues was determined according to the instructions of the MPO kit.

[0098] Experimental results:

[0099] Fig. 9 As shown in the figure, compared with the control group, the MPO content in the lung tissue of the model group mice increased significantly (P<0.001). After treatment with herbicide (5mg / kg, 15mg / kg, 45mg / kg), the MPO content in the lung tissue of mice decreased significantly (P<0.001), and the dexamethasone treatment group also significantly reduced the MPO content in the lung tissue of mice (P<0.001).

[0100] (VI) Determination of inflammatory cell content in bronchoalveolar lavage fluid

[0101] Experimental methods:

[0102] Drugs were administered 1 hour after modeling, and mice were anesthetized 24 hours later. The laryngeal skin was cut to expose the trachea, and the retention needle was inserted into the trachea. The trachea and the retention needle were tied tightly with surgical thread. The mice were slowly rinsed with pre-cooled PBS solution for 3 times, 0.8 ml each time, for a total of 2.4 ml. The washing fluid was alveolar lavage fluid, and then the collected alveolar lavage fluid was centrifuged at 4°C 3000rfc for 10 minutes, and the supernatant and precipitate were taken respectively. The supernatant was stored at -80°C for later use. The precipitate in the alveolar lavage fluid was diluted with PBS for the corresponding multiples, and the smear was stained with Wright-Giemsa. The number of total cells, macrophages and neutrophils was counted under a light microscope.

[0103] Experimental results:

[0104] Fig.10 As shown, compared with the control group, the total number of cells, neutrophils and macrophages in the bronchoalveolar lavage fluid of mice in the model group increased significantly (P<0.01, P<0.001). After treatment with herbicide (5mg / kg, 15mg / kg, 45mg / kg), the total number of cells, neutrophils and macrophages in the bronchoalveolar lavage fluid of mice decreased significantly (P<0.05, P<0.001), and the dexamethasone treatment group also significantly reduced the total number of cells, neutrophils and macrophages in the bronchoalveolar lavage fluid of mice (P<0.001). This suggests that herbicide can reduce the infiltration of inflammatory cells, especially neutrophils, in the lung tissue of mice with pneumonia-induced injury. (VII) Determination of neutrophil infiltration level

[0105] Experimental methods:

[0106] The drug was administered 1 hour after modeling, and the mice were killed 24 hours later. The chest cavity of the mice was quickly cut open with surgical scissors to fully expose the lung tissue, and the surrounding adipose tissue and connective tissue were removed. The lung tissue was taken out, the blood on the surface was absorbed, and then it was immersed in animal lung tissue fixative for sectioning and immunohistochemical staining.

[0107] Experimental results:

[0108] Fig.11 As shown in the figure, compared with the control group, the positive area of ​​neutrophil markers Ly6G, MPO, and NE in the lung tissue of mice in the model group increased significantly (P<0.001). After treatment with herbicide (45 mg / kg), the positive area of ​​neutrophil markers Ly6G, MPO, and NE in the lung tissue of mice decreased significantly (P<0.001). This suggests that herbicide can reduce neutrophil infiltration in the lung tissue of mice with pneumonia.

[0109] (VIII) Neutrophil number and distribution determination

[0110] Experimental methods:

[0111] Drugs were administered 1 hour after modeling, and mice were killed 24 hours later. The chest cavity of the mice was quickly cut open with surgical scissors to fully expose the lung tissue, and the surrounding adipose tissue and connective tissue were removed. The lung tissue was taken out, the blood on the surface was absorbed, and then it was cut into small pieces with a scalpel in a culture dish containing transmission electron microscopy fixative for fixation and sliced ​​for observation.

[0112] Experimental results:

[0113] Fig.12 As shown in the figure, compared with the control group, the lung tissue airway neutrophils (Fth1 hi Neu) increased significantly. After treatment with herbicide (45 mg / kg), the number of neutrophils in the lung tissue airway of mice decreased significantly. In addition, neutrophils in the blood vessels of the lung tissue (Prok2 hi Neu) mitochondria swell and lose cristae, and airway neutrophils (Fth1 hi Neu mitochondria changed little. This suggests that herbicide may reduce the expression of Fth1 in airway neutrophils in lung tissue of mice with pneumonia injury by affecting the mitochondrial homeostasis of airway neutrophils. hi Neu) infiltration number.

[0114] (IX) Determination of neutrophil Fth1 and Prok2 infiltration levels

[0115] Experimental methods:

[0116] The drug was administered 1 hour after modeling, and the mice were killed 24 hours later. The chest cavity of the mice was quickly cut open with surgical scissors to fully expose the lung tissue, and the surrounding adipose tissue and connective tissue were removed. The lung tissue was taken out, the blood on the surface was absorbed, and then it was immersed in animal lung tissue fixative for sectioning and immunofluorescence double staining.

[0117] Experimental results:

[0118] Fig.13 As shown in the figure, compared with the control group, the number of Fth1 neutrophils in the bronchoalveolar lavage fluid of mice in the model group increased significantly, and the proportion of Prok2 neutrophils decreased. After treatment with herbicide (45 mg / kg), the number of Fth1 neutrophils in the bronchoalveolar lavage fluid of mice decreased significantly, and the proportion of Prok2 neutrophils increased. This suggests that herbicide can reduce the infiltration of Fth1 neutrophils in the lung tissue of mice with pneumonia.

[0119] (X) Detection of IL-10 content in lung tissue

[0120] Experimental methods:

[0121] 24 hours after administration, the mice were anesthetized, the laryngeal skin was cut to expose the trachea, the retention needle was inserted into the trachea, and the trachea and the retention needle were tied with surgical thread. The cells were slowly rinsed 3 times with pre-cooled PBS solution, 0.8 ml each time, for a total of 2.4 ml. The washing solution was alveolar lavage fluid, and then the collected alveolar lavage fluid was centrifuged at 4°C 1500rcf for 10 minutes, and the supernatant was taken. The concentration of IL-10 in lung tissue was quantified using a mouse-specific ELISA kit according to the manufacturer's instructions. According to the instructions, the working solution was prepared, the standard curve was drawn, the sample to be tested was added, incubated, the detection antibody, tool drug and color development solution were added, etc., and the absorbance value of each well was measured using an enzyme marker, and the results were calculated based on the standard curve.

[0122] Experimental results:

[0123] Fig.14 As shown in the figure, compared with the control group, the IL-10 protein content in the lung tissue of the model group mice decreased significantly (P<0.001). After treatment with herbicide (5mg / kg, 15mg / kg, 45mg / kg), the IL-10 (P<0.001) protein content in the lung tissue of mice increased significantly, and the dexamethasone treatment group also significantly increased the IL-10 protein content in the lung tissue of mice (P<0.001). This suggests that herbicide can increase the IL-10 protein content in the lung tissue of mice with pneumonia.

[0124] Experimental methods:

[0125] Drugs were administered 1 hour after modeling, and mice were killed 24 hours later. The chest cavity of the mice was quickly cut open with surgical scissors to fully expose the lung tissue, and the surrounding adipose tissue and connective tissue were removed. The lung tissue was taken out, and the blood on the surface was dried to extract the total RNA of the lung tissue. The micro-protein nucleic acid quantifier was used for quantification, and the expression level of mRNA was detected by fluorescence quantification.

[0126] Experimental results:

[0127] Fig.15 As shown in the figure, compared with the control group, the expression level of IL-10 mRNA in the lung tissue of the model group mice was significantly decreased (P<0.001). After treatment with herbicide (45 mg / kg), the expression level of IL-10 mRNA in the lung tissue of mice increased significantly (P<0.01). This suggests that herbicide can increase the expression of IL-10 mRNA in the lung tissue of mice with pneumonia.

[0128] Experimental methods:

[0129] Total protein was extracted using the KGI Total Protein Extraction Kit, and the total protein concentration of the sample was determined using the BCA Protein Content Detection Kit and the protein concentration was calibrated. SDS-polyacrylamide gel electrophoresis was performed with a nitrocellulose membrane (PVDF membrane) for wet transfer. After transfer, the membrane was placed in a 5% skim milk powder blocking solution for 2 hours. After blocking, the primary antibody IL-10 (1:1000) and β-actin (1:4000) diluted in 5% skim milk powder were incubated at 4°C overnight, and the membrane was washed and incubated with the secondary antibody for 2 hours. The membrane was washed with TBST 3 times, 10 minutes each time. Protein chemiluminescent agent (ECL) was added for exposure. Image J image analysis software was used to calculate the gray value of the target protein, and the error was corrected using the corresponding internal reference gray value.

[0130] Experimental results:

[0131] like Fig.16 As shown in the figure, compared with the control group, the IL-10 protein content in the lung tissue of the model group mice decreased significantly (P<0.05). After treatment with herbicide (45 mg / kg), the IL-10 (P<0.01) protein content in the lung tissue of mice increased significantly. This suggests that herbicide can increase the IL-10 protein content in the lung tissue of mice with pneumonia.

[0132] (XI) Cell apoptosis detection

[0133] Experimental methods:

[0134] Drugs were administered 1 hour after modeling, and mice were killed 24 hours later. The chest cavity of the mice was quickly cut open with surgical scissors to fully expose the lung tissue, and the surrounding fat tissue and connective tissue were removed. The lung tissue was taken out, and the blood on the surface was sucked dry. It was then immersed in animal lung tissue fixative and sliced. Cell apoptosis in the lung tissue was detected according to the instructions of the TUNEL cell apoptosis detection kit. Experimental results:

[0135] Fig.17 As shown in the figure, compared with the control group, the number of apoptotic cells in the lung tissue of the model group mice increased significantly. After treatment with herbicide (45 mg / kg), the proportion of apoptotic cells in the lung tissue of mice increased. This suggests that herbicide can increase the content of IL-10 in the lung tissue of mice with pneumonia and weaken the Fth1 hi Neu promotes mitochondrial homeostasis and Fth1 hi Apoptosis and clearance of Neu.

Claims

1. Use of herbicide in the preparation of medicine for treating pulmonary pneumonia.

2. The use according to claim 1, wherein herbicide is the only active ingredient in the drug.

3. The use according to claim 1 or 2, wherein the pulmonary inflammatory injury is pulmonary function injury.

4. The use according to claim 1 or 2, wherein the pulmonary pneumonia injury is pathological injury of lung tissue.

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

6. The use according to claim 1 or 2, wherein the pulmonary inflammatory injury is an increase in inflammatory factors.

7. The use according to claim 1 or 2, wherein the drug reduces the number of inflammatory cells, the levels of inflammatory factors and total protein in bronchoalveolar lavage fluid; reduces the level of myeloperoxidase in lung tissue; and reduces the expression level of inflammatory factors in serum.

8. The use according to claim 6 or 7, wherein the inflammatory factors include TNF-ɑ, IL-6 and IL-1β.

9. The use according to any one of claims 1 to 8, wherein the drug is in an oral dosage form or an injection dosage form.

10. The use according to claim 9, wherein the medicine is in the form of an injection.

11. A non-therapeutic method for enhancing lung function, characterized in that The method comprises applying herbicide.

12. The method according to claim 11, wherein the single application dose of herbicide is 5-15 mg / kg.

13. The method according to claim 11, wherein the single application dose of herbicide is 15-45 mg / kg.

14. The method according to claim 11, wherein the single application dose of herbicide is 45 mg / kg.

Images