Application of arglabin in preparation of medicine for treating lipopolysaccharide-induced acute lung injury

Abstract

The invention relates to application of arglabin in preparation of a medicine for treating lipopolysaccharide (LPS)-induced acute lung injury. Animal experiments are utilized and combined with various experimental technologies, it is found that the arglabin can be used for treating the acute lung injury induced by lipopolysaccharide, specifically, the arglabin has the effect of reducing the lung tissue inflammation level of the acute lung injury induced by the lipopolysaccharide, and especially has the effect of reducing the expression level of proinflammatory factors IL-1beta and TNF-alpha; the arglabin has reduced alveolar lavage fluid protein content caused by lipopolysaccharide-induced acute lung injury; arglabin has an inhibition effect on lung tissue cell apoptosis of lipopolysaccharide-induced acute lung injury; the arglabin has a relieving effect on lung tissue pathological injury of lipopolysaccharide-induced acute lung injury. The protection effect of arglabin on the LPS-induced acute lung injury is found, a new drug research direction is provided for treatment of the acute lung injury, and the arglabin has a good application prospect.

Description

Technical Field

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

[0002] Acute lung injury is a severe pulmonary inflammatory response caused by various intrapulmonary and extrapulmonary causes (such as bacterial / viral pneumonia, trauma, etc.), characterized by diffuse interstitial and alveolar edema caused by damage to alveolar epithelial cells and capillary endothelial cells, accompanied by protein infiltration, release of proinflammatory mediators and factors. Acute lung injury is an important factor causing death in intensive care patients. Despite the continuous advancement of medical methods, its mortality rate is still 35%-45%, and there is currently no effective therapeutic drug. Therefore, it is particularly important to further find effective therapeutic drugs and explore their potential mechanisms, prevent and treat acute lung injury, and reduce the mortality rate of the disease.

[0003] Aglabin is a semiterpene lactone compound extracted from Artemisia tanliangensis. It is worth noting that semiterpene lactone compounds have anti-cancer, anti-inflammatory, antibacterial and antiviral effects. Studies have reported that aglabin is being developed as an epidermal growth factor receptor tyrosine kinase inhibitor. In a study on prostate cancer cells, it was shown to inhibit proliferation and induce apoptosis. At the same time, a water-soluble derivative of aglabin has been used to treat breast cancer. In addition, studies have reported that aglabin can improve high-fat-induced atherosclerosis in ApoE2 mice by enhancing macrophage autophagy. However, there are no reports on whether aglabin can treat acute lung injury.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide the use of arglabin in the preparation of a drug for treating lipopolysaccharide (LPS)-induced acute lung injury. Arglabin can effectively alleviate LPS-induced acute lung injury.

[0006] The first aspect of the present invention provides the use of arglabin in the preparation of a drug for treating lipopolysaccharide-induced acute lung injury. The arglabin can be used to treat lipopolysaccharide-induced acute lung injury.

[0007] Specifically, the arglabin has a reducing effect on the level of lung tissue inflammation in lipopolysaccharide-induced acute lung injury.

[0008] Specifically, arglabin has a reducing effect on the expression levels of pro-inflammatory factors IL-1β and TNF-α in lung tissue of lipopolysaccharide-induced acute lung injury.

[0009] Specifically, the arglabin reduces the protein content of bronchoalveolar lavage fluid caused by lipopolysaccharide-induced acute lung injury.

[0010] Specifically, the arglabin has an inhibitory effect on apoptosis of lung tissue cells in lipopolysaccharide-induced acute lung injury.

[0011] Specifically, the arglabin has an alleviating effect on pathological damage to lung tissue in lipopolysaccharide-induced acute lung injury.

[0012] Preferably, the dosage of arglabin is 10-20 mg / kg.

[0013] The second aspect of the present invention provides a pharmaceutical composition for treating lipopolysaccharide-induced acute lung injury, wherein the main active ingredient of the pharmaceutical composition is arglabin.

[0014] Specifically, the efficacy of the pharmaceutical composition includes at least one of the following:

[0015] (Ⅰ) It has a reducing effect on the level of lung tissue inflammation in acute lung injury induced by lipopolysaccharide;

[0016] (II) It reduces the protein content of bronchoalveolar lavage fluid caused by lipopolysaccharide-induced acute lung injury;

[0017] (III) It has an inhibitory effect on apoptosis of lung tissue cells in acute lung injury induced by lipopolysaccharide;

[0018] (IV) It has an alleviating effect on the pathological damage of lung tissue in lipopolysaccharide-induced acute lung injury.

[0019] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0020] The present invention has at least the following beneficial effects:

[0021] The present invention uses animal experiments and combines a variety of experimental techniques to find that arglabin can be used to treat lipopolysaccharide-induced acute lung injury, specifically: arglabin has a reducing effect on the level of lung tissue inflammation in lipopolysaccharide-induced acute lung injury, especially on the expression level of proinflammatory factors IL-1β and TNF-α; arglabin has a reducing effect on the protein content of bronchoalveolar lavage fluid caused by lipopolysaccharide-induced acute lung injury; arglabin has an inhibitory effect on apoptosis of lung tissue cells in lipopolysaccharide-induced acute lung injury; arglabin has an alleviating effect on pathological damage of lung tissue in lipopolysaccharide-induced acute lung injury. The present invention finds the protective effect of arglabin on LPS-induced acute lung injury, provides a new drug research direction for the treatment of acute lung injury, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 Aglabin improves the activity status of mice with LPS-induced acute lung injury.

[0024] Figure 2 Arglabin alleviates lung tissue pathological damage in mice with LPS-induced acute lung injury.

[0025] Figure 3 Arglabin reduces the level of lung tissue inflammation in mice with LPS-induced acute lung injury.

[0026] Figure 4 Arglabin inhibits the protein content in bronchoalveolar lavage fluid of mice with LPS-induced acute lung injury.

[0027] Figure 5 Arglabin reduces apoptosis in lung tissue of mice with LPS-induced acute lung injury. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0029] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this description, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0030] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example

[0032] (I) Reagents used in this example

[0033] Molecular biochemical reagents: Lipopolysaccharide (LPS, derived from Escherichia coli 0111:B4) was purchased from Sigma, USA, Arglabin was purchased from MCE, USA, IL-1β, TNF-α primers, etc. were purchased from Biotech Biotechnology, China, reverse transcription kit was purchased from Takara, Japan, qPCR kit was purchased from Yisheng, Shanghai, tribromoethanol was purchased from McLean, China, BCA protein concentration detection kit and DAPI stain were purchased from Biotech, China, universal tissue fixative was purchased from Seville, Wuhan, and Tunel kit was purchased from Roche, Switzerland.

[0034] (II) Establishment of LPS-induced acute lung injury model

[0035] According to the dose of 10 mg / kg, the amount of LPS required for each mouse was calculated and diluted with PBS to a total administration volume of 100ul. Take 8-12 weeks of weight-matched wild-type C57BL / 6J mice, anesthetize them by intraperitoneal injection of 2% sodium pentobarbital solution (3 mg / kg) according to their weight. After anesthesia and stable breathing, fix the mice in a supine position on the operating table. Disinfect the neck skin of the mice with 75% alcohol, make a longitudinal incision of about 6-8 mm on the neck, bluntly separate the neck muscles, and expose the trachea. Use a 100ul microinjector to absorb LPS, insert the needle from the tracheal ring space under the thyroid cartilage, and slowly and evenly inject LPS into the trachea. The same volume of PBS was injected into the trachea of ​​the control group. Pull out the injection needle, put the mouse in an upright position, and slowly turn it left and right for 1 minute to allow the liquid to be fully and evenly distributed in the lungs. Lay the mouse flat, suture the neck skin of the mouse and disinfect it. Lay the mouse on its side on the warming board, wait for the mouse to wake up, and then put it back in the breeding cage. Mice were free to eat and drink. Mice were killed 24 hours later, and samples of bronchoalveolar lavage fluid (BALF) and lung tissue were obtained. When observing the effect of aglabin on lung injury, aglabin was intraperitoneally injected at a dose of 10 mg / kg or 20 mg / kg half an hour before LPS airway administration, and the solvent control group was intraperitoneally injected with the same volume of solvent. Note: Aglabin dissolution (for animal experiments): 5 mg powder was dissolved in the following volumes of solvent in sequence: 500ul DMSO→4500ul Corn oil, to a concentration of 1 mg / ml, prepared and used immediately.

[0036] 3. Collection of mouse BALF specimens

[0037] The anesthetized mouse was fixed in supine position on the operating table, and the skin was cut along the midline of the mouse's neck with scissors, and the neck muscles were bluntly separated to expose the trachea. A small incision was made with scissors, and the lavage needle was inserted into the appropriate position. The suture was passed through the lower end of the trachea and tied to fix the lavage needle. 1ml PBS was extracted with a 1ml syringe for lung lavage, and each mouse was repeatedly aspirated 3 times. The lavage fluid with a recovery rate of more than 80% was collected in a 2ml EP tube and placed on ice. The collected alveolar lavage fluid was centrifuged (4°C, 3000g, 15min), and the supernatant was divided into 1.5ml EP tubes and stored at -80°C. The protein concentration was determined at an appropriate time.

[0038] 4. Collection of Mouse Lung Tissue Specimens

[0039] After anesthetizing the mouse, fix it on the operating table, disinfect the mouse neck, chest, and abdominal skin with alcohol cotton balls, cut the chest and abdominal skin, open the chest cavity from the xiphoid process, and fully expose the chest organs. Pass the suture through the lower end of the left hilum, ligate the left hilum, gently remove the left lung, place it in a 5ml EP tube filled with universal tissue fixative, isolate the air with cotton balls, and wait for paraffin embedding, sectioning, HE staining and other treatments. Fully expose the trachea, remove the lavaged right lung, put it in pre-cooled saline to wash off the blood stains on the surface, and use quick-blotting paper to absorb excess water. Dispense in cryopreservation tubes, freeze in liquid nitrogen and store at -80℃ or liquid nitrogen.

[0040] (V) H&E staining of lung tissue

[0041] The left lung was fixed in universal tissue fixative at room temperature for 24 hours before routine paraffin embedding. The sample was placed on a paraffin slicer for sectioning, and the section thickness was 4um. The largest section was selected for subsequent dewaxing and staining, and the general process was as follows: (1) Dewaxing: xylene (I) 5min→xylene (II) 5min→xylene (III) 5min→100% ethanol 1min→95% ethanol 1min→80% ethanol 1min→75% ethanol 1min→double distilled water washing several times; (2) Hematoxylin staining: After absorbing the water, hematoxylin staining for 5min, and rinse with tap water; (3) Acidification: Differentiation in hydrochloric acid alcohol for 60s; (4) Eosin staining: Soak in tap water for 15 minutes, absorb the water and place in eosin solution for 3 minutes; (5) Dehydration: After washing with distilled water to remove the floating color, 70% ethanol (I) 40 seconds → 95% ethanol (II) 40 seconds → 100% ethanol (I) 40 seconds; (6) Transparent and fixation: xylene carbolic acid (3:1) 1 minute (omitted) → xylene (I) 2 minutes → xylene (II) 2 minutes → → xylene (II) 2 minutes → neutral resin sealing and fixation. After drying, take pictures under an optical microscope.

[0042] 6. Lung injury score

[0043] The American Thoracic Society (ATS) lung injury scoring system was used to score the lung tissue HE-stained pathological sections. The scoring reference is shown in Table 1:

[0044] Table 1

[0045]

[0046] Total score = [(20×a)+(14×b)+(7×c)+(7×d)+(2×e)] / (number of fields of view counted×100).

[0047] (VII) TUNEL staining of lung tissue

[0048] Sequentially, wash the sections in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and distilled water. Place the tissue sections in a repair box filled with citric acid antigen repair buffer (PH = 6.0) in a microwave oven for antigen repair. Boil over medium heat and then turn off the power for 10 min before boiling over medium-low heat. During this process, prevent excessive evaporation of the buffer and do not dry the sections. After cooling naturally, place the slides in PBS (PH = 7.4) and shake on a decolorizing shaker for 3 times, 5 min each time. Block with 3% goat serum at room temperature for 30 min. Gently shake off the blocking solution, take the appropriate amount of reagent 1 (TdT) and reagent 2 (dUTP) in the Tunel kit according to the number of slides, mix them in a ratio of 1:10, and place the sections flat in a humidified box for incubation overnight at 4°C. Add a small amount of water to the humidified box to maintain humidity. Place the slides in PBS (PH = 7.4) and shake on a decolorizing shaker for 3 times, 5 min each time. After the slices were slightly dried, DAPI staining solution was added to the circle and incubated at room temperature in the dark for 10 minutes. The slides were placed in PBS (PH = 7.4) and washed on a decolorizing shaker for 3 times, 5 minutes each time. After the slices were slightly dried, they were sealed with anti-fluorescence quenching sealing agent. The slices were observed and images were collected under a Nikon inverted fluorescence microscope.

[0049] (VIII) Results

[0050] (1) 24 hours after modeling, take photos to record the general condition of the mice. Figure 1 . As can be seen from the figure: the mice in the Ctrl group were in good spirits, responsive, breathing evenly, and their fur was smooth and shiny. The mice in the LPS+Vehicle group were in poor mental state, curled up into a ball, with messy fur and significantly reduced activity; compared with the LPS+Vehicle group, the above performances of the mice in the LPS+Arglabin (10mg / kg) group and the LPS+Arglabin (20mg / kg) group were improved to varying degrees, indicating that Arglabin can improve the general condition of mice with LPS-induced acute lung injury.

[0051] (2) HE-stained lung tissue sections were observed under a light microscope and lung injury scores were performed according to the American Thoracic Society (ATS) lung injury scoring system. The results are shown in Figure 2 . As can be seen from the figure: the lung injury score of the mice in the LPS+Vehicle treatment group was significantly higher than that in the Ctrl group. Pathological sections showed a large number of inflammatory cell infiltration, thickened alveolar septa, transparent membrane formation in some fields of vision, exudation in the alveolar cavity, and partial destruction of alveolar structure. The lung tissue injury score of the mice in the LPS+Arglabin treatment group was significantly lower than that in the LPS+Vehicle group, and pulmonary interstitial edema, thickened alveolar septa, and inflammatory cell infiltration were improved to varying degrees.

[0052] (3) The right lower lung tissue of mice was taken from liquid nitrogen and added with 1 ml of Trizol for grinding. The total RNA was extracted using the chloroform-isopropanol method, and then reverse transcribed into cDNA. Then, real-time fluorescence quantitative PCR was performed to detect the relative expression levels of IL-1β and TNF-α in the lung tissue of each group of mice. The results are shown in Figure 3 As can be seen from the figure: pretreatment with intraperitoneal injection of aglabin can reduce the expression levels of IL-1β and TNF-α in the lung tissue of LPS-induced ALI mice.

[0053] (4) Take out the BALF supernatant from the -80°C refrigerator and measure the protein concentration using the BCA method. The results are shown in Figure 4 As can be seen from the figure: pretreatment with intraperitoneal injection of aglabin can reduce the total protein concentration in BALF of LPS-induced ALI mice.

[0054] (5) Tunel staining technique was used to detect the level of apoptosis in the lung tissues of mice in different treatment groups. The results are shown in Figure 5 As can be seen from the figure: the number of Tunel-positive cells in the lung tissue of mice with LPS-induced acute lung injury was significantly higher than that in the control group, and pretreatment with intraperitoneal injection of aglabin could reduce the apoptosis of lung tissue cells caused by LPS-induced acute lung injury.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of arglabin in the preparation of a drug for treating lipopolysaccharide-induced acute lung injury, characterized in that: The arglabin can be used to treat lipopolysaccharide-induced acute lung injury.

2. The use according to claim 1, characterized in that: The arglabin has a reducing effect on the level of lung tissue inflammation in acute lung injury induced by lipopolysaccharide.

3. The use according to claim 2, characterized in that: The arglabin has a reducing effect on the expression levels of pro-inflammatory factors IL-1β and TNF-α in lung tissue of lipopolysaccharide-induced acute lung injury.

4. The use according to claim 1, characterized in that: The arglabin reduces the protein content of bronchoalveolar lavage fluid caused by lipopolysaccharide-induced acute lung injury.

5. The use according to claim 1, characterized in that: The arglabin has an inhibitory effect on apoptosis of lung tissue cells in acute lung injury induced by lipopolysaccharide.

6. The use according to claim 1, characterized in that: The arglabin has an alleviating effect on the pathological damage of lung tissue in acute lung injury induced by lipopolysaccharide.

7. The use according to claim 1, characterized in that: The dosage of arglabin is 10-20 mg / kg.

8. A pharmaceutical composition for treating lipopolysaccharide-induced acute lung injury, characterized in that: The main active ingredient of the pharmaceutical composition is arglabin.

9. The pharmaceutical composition according to claim 8, characterized in that The efficacy of the pharmaceutical composition includes at least one of the following: (Ⅰ) It has a reducing effect on the level of lung tissue inflammation in acute lung injury induced by lipopolysaccharide; (II) It reduces the protein content of bronchoalveolar lavage fluid caused by lipopolysaccharide-induced acute lung injury; (III) It has an inhibitory effect on apoptosis of lung tissue cells in acute lung injury induced by lipopolysaccharide; (IV) It has an alleviating effect on the pathological damage of lung tissue in lipopolysaccharide-induced acute lung injury.

10. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

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