Application of dihydroartemisinic acid in the preparation of drugs for the prevention or treatment of acute lung injury

Abstract

This invention provides the application of dihydroartemisinin in the preparation of drugs for the prevention or treatment of acute lung injury, relating to the field of pharmaceutical technology. The prevention or treatment of acute lung injury includes: reducing lung damage and / or pulmonary edema in lung tissue, decreasing the levels of inflammatory factors in serum and / or bronchoalveolar lavage fluid, reducing oxidative stress markers in lung tissue, reducing the production of reactive oxygen species and / or NO in BEAS-2B cells, and reducing the expression of inflammatory factors in BEAS-2B cells. This invention has verified through in vitro and in vivo experiments that dihydroartemisinin has a good protective effect against acute lung injury, significantly improving lung damage, and possesses anti-inflammatory and oxidative stress-reducing effects, providing a theoretical basis for the development of drugs for acute lung injury.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of dihydroartemisinic acid in the preparation of drugs for the prevention or treatment of acute lung injury. Background Technology

[0002] Acute lung injury (ALI) is a clinical syndrome characterized by dyspnea, inflammatory cell infiltration in the lungs, increased microvascular permeability in the lungs, severe alveolar and parenchymal edema, and progressive hypoxemia, with an overall mortality rate as high as 40%. Its pathogenesis is complex and diverse, mainly including the following inducing factors: SARS-CoV-2 coronavirus can cause respiratory tract infection, damaging the basic structure of the lungs. Neutrophils and macrophages accumulate at the damaged sites, triggering an inflammatory cascade response. Damage to the lung epithelium can lead to bacterial adhesion and invasion, causing co-infection and ultimately triggering ALI. Pneumonia caused by lipopolysaccharide (LPS) infection is also a common causative factor of acute lung injury, inducing the secretion of various inflammatory factors, activating the inflammatory system, and triggering the occurrence of acute lung injury.

[0003] Currently, clinical treatment for ALI primarily relies on mechanical ventilation and fluid management. However, effective drugs targeting the multi-layered pathogenesis of ALI remain lacking, contributing to its persistently high morbidity and mortality rates. Therefore, finding safe and effective drugs to prevent or treat acute lung injury caused by multiple factors is of great significance and a pressing issue for those skilled in the art. Summary of the Invention

[0004] The purpose of this invention is to provide the application of dihydroartemisinic acid in the preparation of medicaments for the prevention or treatment of acute lung injury. Dihydroartemisinic acid is a white to off-white solid with the molecular formula C2. 15 H 24 O2, CAS number 85031-59-0, is a natural product isolated from Artemisia annua. It is used in the field of anti-parasitic infection and is a biosynthetic precursor of the antimalarial drug Artemisinin. Currently, artemisinin derivatives are commonly used as asthma treatments; there are no studies or reports on the use of dihydroartemisinic acid for the prevention or treatment of ALI (Alternative Lipid Infection).

[0005] Based on this, the present invention provides the use of dihydroartemisinic acid in the preparation of medicaments for the prevention or treatment of acute lung injury.

[0006] In a preferred embodiment, the prevention or treatment of acute lung injury includes: reducing lung damage and / or pulmonary edema in the lung tissue.

[0007] In a preferred embodiment, the prevention or treatment of acute lung injury includes: reducing the levels of inflammatory factors in serum and / or bronchoalveolar lavage fluid; preferably, the inflammatory factors in serum and / or bronchoalveolar lavage fluid include one or more of TNF-α, IL-1β and IL-6.

[0008] In a preferred embodiment, the prevention or treatment of acute lung injury includes: reducing oxidative stress indicators in lung tissue; preferably, the oxidative stress indicators include one or more of MPO, MDA, and GSH.

[0009] In a preferred embodiment, the prevention or treatment of acute lung injury includes: reducing the production of reactive oxygen species and / or NO in BEAS-2B cells.

[0010] In a preferred embodiment, the prevention or treatment of acute lung injury includes: reducing the expression of BEAS-2B cell inflammatory factors; preferably, the BEAS-2B cell inflammatory factors include one or more of TNF-α and IL-6.

[0011] In a preferred embodiment, the dosage of dihydroartemisinic acid is 5-20 mg / kg, and preferably, the dosage of dihydroartemisinic acid is 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg.

[0012] Another object of the present invention is to provide a drug for the prevention or treatment of acute lung injury. Here, "drug" refers to any chemical substance that can affect the physiological functions of organs and cellular metabolic activities, including microbial preparations.

[0013] In a preferred embodiment, the active ingredient of the drug includes dihydroartemisinic acid.

[0014] In a preferred embodiment, the drug comprises dihydroartemisinic acid and one or more pharmaceutically acceptable carriers.

[0015] In this invention, the carrier includes pharmaceutically acceptable diluents, wetting agents, binders, disintegrants, lubricants, color and flavor modifiers, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexing agents, preservatives, pH adjusters, surfactants, excipients, fillers, and synergists. More preferably, diluents include starch, sucrose, cellulose, inorganic salts, etc.; wetting agents include water, ethanol, etc.; binders include starch paste, dextrin, sugar, cellulose derivatives, gelatin, povidone, polyethylene glycol, etc.; disintegrants include starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, sodium bicarboxymethyl cellulose, surfactants, etc.; lubricants include talc, calcium stearate, magnesium stearate, magnesium dodecyl sulfate, polyethylene glycol, etc.; color and flavor modifiers include pigments, sweeteners, flavorings, adhesives, etc.; solvents include water, glycerin, ethanol, etc.; solubilizers include Tween compounds, etc. Solubilizers include sulfides, sulfates, sulfonates, etc.; cosolvents include organic acids (such as citric acid) and their salts, inorganic salts, polyethylene glycol, etc.; emulsifiers include Span derivatives, glycerol fatty acid esters, gum arabic, gelatin, agar, sodium alginate, etc.; antioxidants include sulfites, ascorbic acid, gallic acid and their salts, etc.; metal complexing agents include disodium ethylenediaminetetraacetate, polycarboxylic acid compounds, etc.; preservatives include parabens, quaternary ammonium compounds, chlorhexidine acetate, etc.; pH adjusters include hydrochloric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphates, citrates, etc.

[0016] In this invention, the drug dosage forms include, but are not limited to, oral, topical, suppository, and sterile injectable solutions in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays. It is understood that the dihydroartemisinic acid described in this invention can be administered in various ways depending on the different excipients and dosage forms used.

[0017] In a preferred embodiment, the drug consists of dihydroartemisinic acid and PBS.

[0018] In a preferred embodiment, the preparation method of the drug includes: dissolving dihydroartemisinic acid in PBS to obtain the drug; preferably, the mass ratio of dihydroartemisinic acid to PBS is (4-6):1; more preferably, the mass ratio of dihydroartemisinic acid to PBS is 5:1.

[0019] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0020] 1. This invention uses an LPS-induced acute lung injury model in mice as the experimental subject. Experiments have verified that dihydroartemisinin can significantly prevent LPS-induced acute lung injury in mice, restore the normal physiological structure of mouse lung tissue, inhibit the expression of inflammatory factors in lung tissue, and reduce the level of oxidative stress in lung tissue. Furthermore, in vitro experiments have further confirmed that dihydroartemisinin can significantly inhibit the expression of inflammatory factors and oxidative stress in epithelial cells, and improve epithelial cell activity. Therefore, dihydroartemisinin can prevent and / or treat acute lung injury and protect damaged lung tissue, thus serving as an effective component in drugs for the prevention and treatment of acute lung injury.

[0021] 2. This invention provides a more targeted treatment option for the prevention and treatment of acute lung injury. The dihydroartemisinin used not only improves lung tissue damage, exerts anti-inflammatory effects, and reduces oxidative stress indicators, but also has the advantages of being safe, reliable, and having few side effects. Therefore, dihydroartemisinin can be used as a protective drug for acute lung injury, with good efficacy, high feasibility, and promising future value.

[0022] 3. The acute lung injury that dihydroartemisinin discovered in this invention can prevent and treat includes LPS-induced lung injury, as well as lung injury caused by influenza virus infection, bacterial infection, and / or fungal infection. This invention has verified through in vitro and in vivo experiments that dihydroartemisinin has a good protective effect against acute lung injury, greatly improves lung damage, and has anti-inflammatory and oxidative stress-reducing effects, providing a theoretical basis for the development of drugs for acute lung injury. Attached Figure Description

[0023] These and / or other aspects and advantages of the present invention will become clearer and more readily understood from the following detailed description of embodiments of the invention taken in conjunction with the accompanying drawings, wherein:

[0024] Figure 1 This is a schematic diagram of the molecular structure of dihydroartemisinic acid used in this invention;

[0025] Figure 2 The image shows the effect of dihydroartemisinin on the pathological features of lung tissue in ALI mice in Example 1 of this invention (magnification 400x).

[0026] Figure 3 In Example 1 of this invention, dihydroartemisinic acid inhibited the LPS-mediated upregulation of serum TNF-α, IL-1β, and IL-6 in ALI mice.

[0027] Figure 4 In Example 2 of this invention, dihydroartemisinin reduces the production of ROS and NO and the expression of inflammatory factors in BEAS-2B cells. Detailed Implementation

[0028] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. However, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0029] The technical solution of this application will be described in detail below through specific embodiments:

[0030] Unless otherwise specified, the technical means used in this invention are conventional means well known to those skilled in the art. All raw materials, reagents, instruments, and equipment used in this invention can be purchased commercially or prepared using existing methods. Unless otherwise specified, all reagents used in this invention are of analytical grade. The dihydroartemisinic acid used in this invention is commercially available, and its molecular structure is as follows: Figure 1 As shown, the purity is >98% by HPLC.

[0031] Example 1

[0032] I. Animal grouping and administration method

[0033] Balb / c mice, SPF grade, 6-8 weeks old, were acclimatized to normal feed for 3-5 days. On the 5th day, they were randomly divided into the following 6 groups (6-8 mice in each group) according to their weight scores. They were administered the drug via intraperitoneal injection for 3 consecutive days. One hour after the last administration, except for the control group and the dihydroartemisinin-only group, the other groups were injected with LPS intraperitoneally at a dose of 10 mg / kg.

[0034] (1) Control group: PBS was administered by gavage at a dose of 10 mL / kg / d;

[0035] (2) Model group: LPS was injected intraperitoneally at a dose of 10 mL / kg / d;

[0036] (3) Low-dose dihydroartemisinic acid group: intraperitoneal injection of 5 mg / kg / d of dihydroartemisinic acid;

[0037] (4) Medium-dose dihydroartemisinic acid group: intraperitoneal injection of 10 mg / kg / d of dihydroartemisinic acid;

[0038] (5) High-dose dihydroartemisinin group: intraperitoneal injection of 20 mg / kg / d of dihydroartemisinin;

[0039] (6) Dihydroartemisinic acid alone: ​​20 mg / kg / d of dihydroartemisinic acid was injected intraperitoneally.

[0040] II. Detection Indicators

[0041] 24 hours after LPS injection, blood was collected from the femoral artery of mice, and the mice were euthanized by cervical spine. Spleen and lung tissue were then collected. After the blood samples were allowed to stand for a while, they were centrifuged at 3500 rpm for 10 min at 4℃, and the serum was collected and stored at -80℃.

[0042] After bloodletting from the abdominal aorta, the thoracic cavity of mice was cut open with scissors to expose the lungs and heart. The right main bronchus was isolated and ligated, and the trachea was freed in the neck. A No. 7 puncture needle was gently inserted into the cricoid cartilage and secured with cotton thread. 3 mL of pre-cooled physiological saline was drawn up with a 1 mL syringe and injected into the lumen. The left lung swelled accordingly. This was repeated three times, constituting one lavage. The lavage was repeated three times (with a recovery rate of over 80%). The lavage fluid from the three lavages was combined to obtain bronchoalveolar lavage fluid (BALF). After low-temperature centrifugation, the supernatant was aliquoted and frozen at -80°C.

[0043] The changes in TNF-α, IL-1β, and IL-6 in mouse serum and BALF, as well as the changes in MPO, GSH, and MDA levels in lung tissue, were detected using different kits and detection methods. Left lung tissue was immersed in 4% paraformaldehyde and stained with H&E.

[0044] III. Statistics and Analysis

[0045] Experimental data were statistically analyzed using Graphpad Prism 9.0. All data are expressed as mean ± SD of three independent trials. ANOVA with Tukey's test was used for statistical analysis. p < 0.05 was considered statistically significant.

[0046] The pathological scoring of lung injury in mice was assessed using the following criteria:

[0047] 0 points: The alveolar walls are intact without thickening, inflammatory infiltration, or congestion.

[0048] 1 point: Mild diffuse inflammatory cell infiltration (neutrophils), no significant thickening of alveolar walls.

[0049] 2 points: Obvious and widespread inflammatory cell infiltration (neutrophils and monocytes), mild thickening of alveolar walls (1 to 2 times).

[0050] 3 points: Severe inflammatory cell infiltration, with alveolar walls thickened to 3 to 5 times in some areas.

[0051] 4 points: Severe inflammatory cell infiltration, significant thickening of alveolar walls, and 25%–50% lung tissue consolidation.

[0052] 5 points: Severe inflammatory cell infiltration, significant thickening of alveolar walls, and >50% lung tissue consolidation.

[0053] These scores reflect the degree of lung injury from mild to severe, with 0 indicating no injury and 5 indicating the most severe solidified injury.

[0054] IV. Results and Discussion

[0055] (I) Dihydroartemisinin significantly reduced lung injury and pulmonary edema in ALI mice.

[0056] (1) By Figure 2 HE staining results showed that, compared with the control group (blank group), LPS caused a large amount of inflammatory material and erythrocyte exudation in the alveoli, and lung structure destruction. Compared with the model group, the drug-treated group significantly reduced the pathological changes and MPO expression in the lung tissue of mice induced by LPS, and improved the lung injury score.

[0057] (2) The wet-to-dry ratio (W / D) of the lungs can reflect the lung edema in ALI mice, and the larger the ratio, the more obvious the lung edema.

[0058] As shown in Table 1, compared with the control group, the W / D ratio in the model group increased from 2.81 to 9.34. After treatment with the traditional Chinese medicine monomer dihydroartemisinin, the W / D ratio in the high-dose dihydroartemisinin group decreased to 2.66 compared with the model group. This indicates that the traditional Chinese medicine monomer dihydroartemisinin can effectively reduce pulmonary edema in the lung tissue of ALI mice.

[0059] Table 1

[0060] Group Wet / D (W / D) Blank group 2.81 Model group 9.34 Low-dihydroartemisinic acid group 2.68 medium-dihydroartemisinic acid group 2.67 High-dihydroartemisinic acid group 2.66 dihydroartemisinic acid alone group 2.71

[0061] (II) Artemisinin dihydroartemisinin significantly reduced the expression of inflammatory factors in the serum and bronchoalveolar lavage fluid of ALI mice.

[0062] Inflammatory factors TNF-α, IL-1β, and IL-6 are the most direct indicators of inflammatory response. A large number of inflammatory factors are released in the serum and bronchoalveolar lavage fluid of ALI mice, triggering an inflammatory cascade response and causing an acute inflammatory response.

[0063] (1) Serum levels of TNF-α, IL-1β, and IL-6 were measured using ELISA, and the results are as follows: Figure 3 As shown in the figure, low, medium, and high doses of dihydroartemisinic acid can significantly inhibit LPS-mediated upregulation of TNF-α, IL-1β, and IL-6 in ALI mice.

[0064] (2) The levels of TNF-α and IL-6 in bronchoalveolar lavage fluid were measured by ELISA, and the results are shown in Table 2. As can be seen from the table, low, medium, and high doses of dihydroartemisinic acid all reduced the levels of inflammatory factors in the bronchoalveolar lavage fluid of ALI mice. Compared with the control group, the TNF-α level in the bronchoalveolar lavage fluid of ALI mice significantly increased from 0.97 pg / mL to 12.92 pg / mL. The IL-6 level in the bronchoalveolar lavage fluid of ALI mice significantly increased from 0.54 ng / mL to 4.49 ng / mL. After treatment with the traditional Chinese medicine monomer dihydroartemisinic acid, compared with the model group, the levels of TNF-α and IL-6 in the bronchoalveolar lavage fluid significantly decreased. In the high-dose dihydroartemisinic acid group, the TNF-α level decreased to 2.65 pg / mL, and the IL-6 level decreased to 0.43 ng / mL.

[0065] The significant decrease in inflammatory factors in bronchoalveolar lavage fluid indicates that the herbal monomer dihydroartemisinic acid can significantly improve the inflammatory response in ALI mice. Furthermore, Table 2 also shows that the herbal monomer dihydroartemisinic acid of this invention can improve the inflammatory response in mice.

[0066] Table 2. Effects of the herbal monomer dihydroartemisinin on the expression of inflammatory factors in bronchoalveolar lavage fluid.

[0067]

[0068] The above experimental results indicate that different doses of dihydroartemisinic acid can inhibit the influx of inflammatory cells, reduce pulmonary edema and LPS-induced cell damage, and protect the integrity of the alveolar vascular barrier.

[0069] (III) Artemisinin dihydroartemisinin significantly reduced oxidative stress markers in the lung tissue of ALI mice

[0070] GSH has antioxidant properties, helping the body maintain normal immune system function. MPO is present in neutrophils and monocytes and is a specific marker of myeloid cells. MDA can attack unsaturated fatty acids in biological membranes, triggering lipid peroxidation, and is used to assess lung injury.

[0071] As shown in Table 3, compared with the control group, the levels of MPO and MDA in the lung tissue of ALI mice were significantly increased and the level of GSH was significantly decreased in the model group. Specifically, MPO increased from 0.14 U / L to 0.33 U / L, MDA increased from 0.98 U / nmol / mgprot to 6.9 U / nmol / mgprot, and GSH decreased from 14.81 μM to 3.73 μM. Compared with the model group, in the medium-dose dihydroartemisinin group, the levels of MPO in the lung tissue of mice decreased to 0.15 U / L, MDA decreased to 2.12 U / nmol / mgprot, and GSH significantly increased to 11.82 μM.

[0072] Table 3. Effects of the herbal monomer dihydroartemisinic acid on oxidative stress indices in ALI mice.

[0073]

[0074] The changes in oxidative indicators in ALI mice above indicate that the traditional Chinese medicine monomer dihydroartemisinic acid can counteract the progression of acute lung injury by increasing the mice's antioxidant stress capacity.

[0075] Example 2

[0076] The effect of dihydroartemisinic acid on inhibiting LPS-induced BEAS-2B cells in vitro

[0077] I. Specific experimental methods include:

[0078] (1) Culture of BEAS-2B cells

[0079] BEAS-2B cells were cultured in DMEM high-glucose medium containing 10 wt% FBS and 1 wt% penicillin and streptomycin, and incubated at 37°C in a constant temperature incubator with 5% CO2.

[0080] (2) Effects of dihydroartemisinin on LPS-induced BEAS-2B cells

[0081] BEAS-2B cells were grown at a rate of 1×10⁻⁶. 5 Cells were seeded in 24-well plates at a density of 1 μg / mL. After 24 hours, 10, 20, and 40 μM dihydroartemisinic acid were added, and the cells were incubated for 1 hour. Then, serum-free medium containing 1 μg / mL LPS was added and the cells were co-cultured for 24 hours. The cell supernatant was collected, and the NO content was determined according to the kit instructions.

[0082] (3) Effects of the traditional Chinese medicine monomer dihydroartemisinic acid on reactive oxygen species and inflammatory factors induced by LPS in BEAS-2B cells

[0083] BEAS-2B cells were grown at a rate of 1×10⁻⁶. 5 Cells were seeded in 24-well plates at a density of 1 μg / mL. After 24 hours, the cells were divided into a blank group, a model group, and a dihydroartemisinic acid group. After 1 hour of culture, except for the blank group, the other groups were added to serum-free medium containing 1 μg / mL LPS and co-cultured for 24 hours. The cell culture supernatant was collected, and ROS, TNF-α, and IL-6 in the supernatant were detected using an ELISA kit.

[0084] (4) Statistics and Analysis

[0085] Experimental data were statistically analyzed using GraphpadPrism 9.0. All data are expressed as mean ± standard deviation (SD) of three independent trials. One-way ANOVA with Tukey's test was used for statistical analysis. p < 0.05 was considered statistically significant.

[0086] II. Results and Discussion:

[0087] Dihydroartemisinic acid can significantly reduce the production of ROS and NO and the expression of inflammatory factors in BEAS-2B cells.

[0088] Inflammatory factors TNF-α and IL-6 are the most direct indicators of inflammatory responses. LPS can induce BEAS-2B cells to release large amounts of inflammatory mediators, thereby causing acute inflammatory responses. Nitric oxide is an important mediator of inflammatory responses and has certain cytotoxicity. Excessive NO production can cause damage to the body, thereby promoting exudation and edema at inflammatory sites. Endogenous ROS from mitochondria is the most important measure of oxidative stress and plays a crucial role in many physiological and pathological processes.

[0089] Depend on Figure 4 As shown, LPS-induced BEAS-2B cells exhibited significantly increased levels of inflammatory factors TNF-α, IL-6, ROS, and NO compared to the control group. After administration of low-dose dihydroartemisinin, TNF-α and IL-6 levels decreased from 180 pg / mL to 115 pg / mL, IL-6 levels decreased from 200 ng / mL to 125 ng / mL, NO production decreased from 32 μM to 12 μM, and ROS production decreased from 215 U / mg to 110 U / mg. This sharp decrease in inflammatory factors indicates that the traditional Chinese medicine monomer dihydroartemisinin can alleviate the inflammatory response.

[0090] The results of the two sets of examples above demonstrate that the dihydroartemisinin disclosed in this invention can protect against ALI by reducing the levels of reactive oxygen species and inflammatory factors in LPS-induced BEAS-2B cells in vitro, and by reducing inflammatory factors in mouse serum and bronchoalveolar lavage fluid, as well as oxidative stress markers in lung tissue in vivo. Therefore, dihydroartemisinin has broad application prospects in the preparation of drugs for the prevention or treatment of acute lung injury.

[0091] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be limited by the claims and their equivalents.

Claims

1. Use of dihydroartemisinic acid for the preparation of a medicament for the prevention or treatment of acute lung injury, characterized in that, The dosage of the dihydroartemisinic acid is 5-20 mg / kg.

2. Use of dihydroartemisinic acid according to claim 1 for the preparation of a medicament for the prevention or treatment of acute lung injury, characterized in that, Dihydroartemisinic acid reduces lung injury and / or pulmonary edema of lung tissue.

3. Use of dihydroartemisinic acid according to claim 1 for the preparation of a medicament for the prevention or treatment of acute lung injury, characterized in that, Dihydroartemisinic acid reduces the levels of TNF-α and IL-6 in serum and / or alveolar lavage fluid.

4. Use of dihydroartemisinic acid according to claim 1 for the preparation of a medicament for the prevention or treatment of acute lung injury, characterized in that, Dihydroartemisinic acid reduces the oxidative stress indicators MPO and MDA in lung tissue.

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