Application of arabinoxylan in preparation of medicine for treating chronic obstructive pulmonary disease

Abstract

The invention discloses application of arabinoxylan (AX) in preparation of a medicine for treating chronic obstructive pulmonary disease (COPD), and belongs to the technical field of medicines. The arabinoxylan (AX) can be used for preparing a medicine for treating the COPD (chronic obstructive pulmonary disease). Through a large number of researches, the inventor of the application finds that AX has a remarkable COPD treatment effect: lung inflammation in a mouse COPD model caused by haze can be reduced, the expression is that infiltration of mature T lymphocytes and macrophages in lung tissues is reduced, and the expression level of various proinflammatory factors including TGF-beta1 is reduced; the compound can weaken extracellular matrix deposition in the lung of a COPD mouse caused by haze, reduce the thickness of a tracheal wall, and reduce the expression of I-type collagen (collagen I) and a myofibroblast marker smooth muscle alpha2-actin (alpha-Sma); the emphysema degree of a COPD mouse induced by haze is also relieved; the AX also relieves abnormal changes of related protein levels of iron metabolism, mitochondrial metabolism, oxidative stress and glycometabolism in the lung tissue of the COPD mouse induced by haze. Therefore, AX can be used for preparing the medicine for treating COPD.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and more specifically to the application of arabinoxylan (AX) in the preparation of medicines for treating chronic obstructive pulmonary disease (COPD). Background Art

[0002] COPD is a type of chronic bronchitis and emphysema characterized by airflow obstruction, which can further develop into common chronic diseases such as cor pulmonale and respiratory failure. COPD is the third most common cause of death worldwide. It is usually caused by smoking, but may also be caused by other factors such as air pollution, chemical exposure, and dust in the working environment. The pathological characteristics of COPD mainly include airway inflammation and airway remodeling, alveolar destruction and emphysema, and excessive accumulation of collagen, and may also cause microvascular lesions. At present, the pathogenesis of COPD is not fully understood, and the existing anti-COPD drugs can only delay the effect of COPD, but cannot cure it. At present, the main drugs for treating COPD are: β2 receptor agonists (such as salbutamol), anticholinergic drugs (such as ipratropium bromide), glucocorticoids, phosphodiesterase-4 inhibitors, antioxidants (such as N-acetylcysteine), etc. Among them, β2 receptor agonists and anticholinergic drugs are important drugs for relieving COPD symptoms, but they cannot change the course of the disease, and long-term use may lead to drug resistance; glucocorticoids, phosphodiesterase-4 inhibitors and antioxidants (such as N-acetylcysteine) are mainly used for chronic stable COPD, but these drugs have large side effects and are not suitable for all patients. Therefore, COPD has become a hot spot and difficulty in research at home and abroad due to its unknown cause, extremely poor prognosis and lack of specific drugs. It is necessary to develop new, efficient and safe drugs for treating COPD.

[0003] AX is a natural, safe, and non-toxic cereal polysaccharide fiber. It is the second most abundant component in plant cell walls, accounting for 15% to 30% of the total plant cell wall. Studies have shown that AX can stimulate the immune system, especially by enhancing the activity of macrophages and T cells to improve the body's immune response, and may also enhance the body's defense against pathogens by activating the intestinal immune system. AX also helps maintain the integrity of the intestinal barrier and prevent pathogenic microorganisms and harmful substances from entering the blood circulation through the intestine. AX may also regulate blood sugar levels, cholesterol metabolism, and clearance of intestinal viruses. Natural products are regarded as an important source of drugs for treating diseases, and whether AX can be used in the preparation of drugs for the treatment of COPD is an urgent problem to be solved by technicians in this field. Summary of the invention

[0004] In view of this, the present invention provides sufficient evidence through a large number of experiments to prove that AX can be used to prepare drugs for treating COPD, providing a clinical basis for the treatment of COPD.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] AX can alleviate COPD and thus can be used to prepare a drug for treating COPD.

[0007] As a preferred technical solution of the present invention, AX can downregulate the infiltration of mature T lymphocytes and macrophages in the lung tissue of mice with COPD caused by haze; AX can downregulate the level of TGF-β1 in the lung tissue of mice with COPD caused by haze; AX can downregulate the transcription level of inflammatory factor genes in the lung tissue of mice with COPD caused by haze; AX can significantly reduce the tracheal wall thickness in the lungs of mice with COPD caused by haze.

[0008] As a preferred technical solution of the present invention, AX can significantly reduce the percentage of damaged alveoli in the lungs of COPD mice induced by haze, as well as reduce the average alveolar transverse intercept and longitudinal intercept, thereby alleviating the degree of emphysema.

[0009] As a preferred technical solution of the present invention, AX can reduce the collagen volume fraction in the lung tissue of COPD mice induced by haze, and downregulate the expression levels of type I collagen (collagen I) and smooth muscle α2-actin (α-Sma).

[0010] As a preferred technical solution of the present invention, AX can alleviate the abnormal changes in the levels of proteins related to iron metabolism, mitochondrial metabolism, oxidative stress and glucose metabolism in the lung tissues of COPD mice induced by haze.

[0011] As a preferred technical solution of the present invention, AX is a safe and non-toxic cereal polysaccharide fiber that has no adverse effects on mouse organs.

[0012] As a preferred technical solution of the present invention, the concentration of AX used in the mouse in vivo experiment is 200 mg / kg.

[0013] It can be seen from the above technical solutions that, compared with the prior art, the present invention applies AX to the preparation of drugs for treating COPD, showing significant therapeutic effects with less side effects, and providing a theoretical and clinical basis for the treatment of COPD. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on the provided drawings without creative work.

[0015] Figure 1 The accompanying drawings are result graphs showing that AX provided by the present invention can down-regulate the infiltration of immune cells, the expression level of inflammatory factors and the thickness of tracheal wall in the lung tissue of COPD mice caused by haze. A shows that AX can down-regulate the infiltration of mature T lymphocytes (Cd3+) and macrophages (F4 / 80+) in the lung tissue of COPD mice caused by haze (scale: 50 μM). B shows that AX can down-regulate the level of TGF-β1 in the lung tissue of COPD mice caused by haze (scale: 50 μM). C shows that AX can down-regulate the transcription level of inflammatory factor genes in the lung tissue of COPD mice caused by haze (n=6 / group). *P<0.05, **P<0.01; DE shows the result graph of AX inhibiting the thickness of the lung tracheal wall of COPD mice caused by haze. Among them, D is a representative picture of the lung sections of mice stained by Masson staining on the 21st day after modeling (collagen stained in blue, scale: 50μM), △ indicates trachea, → indicates blood vessels; E is the quantitative result of tracheal wall thickness of the lung sections stained by Masson staining (n=8 / group). ****P<0.0001. CON represents control mice; Smog represents haze-induced COPD model mice; Smog+AX represents haze-induced COPD model mice treated with AX.

[0016] Figure 2 The attached figure shows the results of AX alleviating emphysema in the lungs of mice with haze-induced COPD. Among them, A is a representative picture of the lung sections of mice stained by Masson on the 21st day after modeling (scale: 50μM); BD, quantitative results of the percentage of damaged alveoli (B), the average alveolar cross-intercept (C) and the vertical intercept (D) in the Masson-stained sections of the lungs (n=8 / group). CON represents control mice; Smog represents haze-induced COPD model mice; Smog+AX represents haze-induced COPD model mice treated with AX. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0017] Figure 3The attached figure shows that AX can reduce the accumulation of collagen in the lung tissue of mice with haze-induced COPD. A is a representative picture of Masson staining of mouse lung sections on the 21st day after modeling, in which the haze treatment group (Smog) has obvious lamellar collagen deposition of varying sizes (scale: 50μM), B is the quantitative result of collagen volume fraction of Masson-stained lung sections (n=8 / group), ****P<0.0001. C is a representative picture of immunofluorescence staining of collagen in lung sections of mice on the 21st day after modeling (scale: 100μM), and D is a representative picture of immunofluorescence staining of α-Sma (scale: 100μM). CON represents control mice; Smog represents haze-induced COPD model mice; Smog+AX represents haze-induced COPD model mice treated with AX.

[0018] Figure 4 The attached figure shows that AX can alleviate the abnormal changes in the levels of proteins related to iron metabolism (A), mitochondrial metabolism (B), oxidative stress (C) and glucose metabolism (D) in the lung tissues of mice with haze-induced COPD. CON represents control mice; Smog represents haze-induced COPD model mice; Smog+AX represents haze-induced COPD model mice treated with AX.

[0019] Figure 5 The attached figure shows the H&E staining results of the sections of the heart, liver, spleen, lung, kidney and other major organs of the mice treated with AX (scale: 60 μM). CON represents the control mice; AX represents the mice treated with AX alone. DETAILED DESCRIPTION

[0020] The technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all of the embodiments. Based on the embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0021] In the examples, arabinoxylan is represented by AX:

[0022] Example 1 Construction of mouse COPD model

[0023] Preparation of haze suspension: Cut the filter membrane with haze powder attached into 5x5 mm 2The square was placed in a 50ml centrifuge tube, and 40ml of methanol solution (methanol and double distilled water 4:1) prepared by double distilled water was added, and ultrasonic crushing was performed for a total of 2 hours (ultrasound was repeated for 30 seconds every minute). After ultrasonic treatment, the suspension obtained by filtering with a 70μm cell sieve was placed in a 65℃ oven to dry, and 0.053mg of haze powder was obtained. The control group was subjected to the same operation to obtain new membrane powder without haze particles. After that, haze particles were prepared with ultrapure water to obtain a haze suspension of 0.024μg / μl and placed in a -20℃ refrigerator for use. The new membrane powder of the control group was added with the required volume of ultrapure water for the haze membrane of the same area to obtain a control new membrane suspension.

[0024] Establishment of COPD model and intervention of AX:

[0025] Control group (CON): On the first day, 50 μL of the new membrane solution was instilled into the lungs of mice through a tube. The mice were fed until the 21st day.

[0026] Smog group (Smog): On the first day, 50 μL of smog suspension was instilled into the lungs of mice through a tube. The mice were kept until the 21st day.

[0027] AX treatment group (Smog+AX): 50 μL of smog suspension was instilled through the air tube on the first day, and the mice were fed with water containing appropriate amount of AX from the 10th to the 21st day (AX was added to the daily drinking water at an average dose of 200 mg / kg of mouse weight / day according to the mouse weight). The mice were fed until the 21st day.

[0028] Tracheal instillation steps:

[0029] (1) Sterilize tweezers and other tools used for modeling in advance;

[0030] (2) Mice were anesthetized by intraperitoneal injection of 5% chloral hydrate (5 mL / kg);

[0031] (3) Fix the mouse vertically and clamp its nose with sterilized forceps to force it to breathe through its mouth;

[0032] (4) 24 C57BL / 6J male mice (9 weeks old, weighing 22-25 g) were randomly divided into groups: 8 in the control group, 8 in the haze group, and 8 in the AX treatment group. The front half of the mouse tongue was pulled out of the oral cavity with forceps, and 50 μL of haze suspension was injected into the throat of the mice in the haze group and AX treatment group with a pipette, and 50 μL of new membrane powder suspension was injected into the throat of the mice in the control group.

[0033] (5) After the mouse chest cavity shows obvious breathing movement (to ensure that the haze suspension is inhaled into the trachea) for about 30 seconds, put it back into the mouse cage and lie flat. The AX treatment group was given AX (200 mg / kg / day) in the form of drinking water from the tenth day of modeling until the mice were sacrificed and the mouse lungs were harvested on the 21st day after modeling. For the mice in the haze group and the control group, there was no AX in the drinking water until the mice were sacrificed and the mouse lungs were harvested on the 21st day after modeling.

[0034] 21 days after modeling, mice were anesthetized by intraperitoneal injection of 5% chloral hydrate (10 mL / kg), the chest cavity was exposed with surgical scissors to remove the lungs, and then the lungs were quickly placed in liquid nitrogen and then frozen at -80°C for Western blot or RNA level analysis, or an appropriate amount of tissue fixative was injected into the trachea and placed in the tissue fixative for 24 hours, then paraffin embedded and sectioned, and then the lung tissue was subjected to immunohistochemical staining or immunofluorescence staining analysis.

[0035] Example 2 Paraffin embedding of lungs and preparation of lung sections

[0036] (1) Remove the mouse lungs from the fixative, place them in a dehydrator, and dehydrate them according to the following steps:

[0037]

[0038] (2) Waxing the dehydrated lung tissue:

[0039]

[0040] (3) Place the lungs in an embedding frame containing melted paraffin, wait for the paraffin to cool and solidify into blocks, and label the blocks;

[0041] (4) Use a microtome to cut the wax block into 3-4 slices in a row, each slice is 5 μm thick, and then immediately place it in a 42°C water bath for spreading. Pick it up with a glass slide, bake it and fix it for later use.

[0042] Example 3 Immunohistochemical staining

[0043] (1) Dewaxing of paraffin sections: Soak the sections in the following reagents for the corresponding time, and finally wash them once with distilled water.

[0044]

[0045]

[0046] (2) Antigen retrieval: After natural cooling, place the slides in PBS (pH 7.4) and wash on a decolorizing shaker three times for 5 minutes each time.

[0047] (3) Blocking endogenous peroxidase: The sections were placed in 3% hydrogen peroxide solution and incubated at room temperature in the dark for 25 minutes. The slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 minutes each time.

[0048] (4) Serum blocking: Add 3% BSA in the tissue circle to evenly cover the tissue and block at room temperature for 30 minutes.

[0049] (5) Add primary antibody: Gently shake off the blocking solution, add primary antibody PBS solution diluted in an appropriate ratio onto the slice, and incubate in a humidified box at 4°C overnight.

[0050] (6) Adding secondary antibody: The slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker, each time for 5 minutes. After the sections were dried, secondary antibodies (horseradish peroxidase, HRP labeled) of the same species as the primary antibody were added to the circle to cover the tissue and incubated at room temperature for 50 minutes.

[0051] (7) DAB color development: The slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 minutes each time. After the sections were dried, freshly prepared DAB color development solution was added to the circle. The color development time was controlled under a microscope. A brown-yellow color was considered positive. The sections were rinsed with tap water to stop the color development.

[0052] (8) Re-staining of cell nuclei: Re-stain with hematoxylin for 3 minutes, rinse with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, re-blue with hematoxylin bluing solution, and rinse with running water again.

[0053] (9) Dehydration and sealing: Place the slices in the following reagents in sequence and dehydrate them until they are transparent. Place the slices in the following reagents in sequence and dehydrate them until they are transparent. Finally, seal the slices with sealing glue.

[0054]

[0055]

[0056] (10) Microscopic examination: Observe under a white light microscope.

[0057] Compared with the control mice treated with the new membrane powder suspension, the immunohistochemical results showed that the CD3, F4 / 80 ( Figure 1 A) The positive signal level of the marker increased, indicating that T lymphocytes and macrophages infiltrated seriously in the lungs of COPD model mice. Figure 1 The positive signal expression of B) was also increased, indicating that the degree of inflammation increased and was alleviated after AX treatment.

[0058] Example 4 Determination of mRNA expression level:

[0059] (1) RNA was extracted from mouse lung tissue using an RNA kit (FOREGENE). After RNA extraction, the concentration was determined and reverse transcribed into cDNA using a reverse transcription kit (Novoprotein).

[0060] (2) Real-time PCR

[0061] Configure the PCR reaction system as follows, with three replicate wells for each sample:

[0062]

[0063] The above reaction system is used to amplify cDNA on a real-time fluorescence PCR instrument.

[0064] The results showed that compared with the control mice infused with the new membrane powder suspension, the mRNA levels of six inflammatory factors, including Ccl7 (chemokine (CC motif) ligand 7), Ccl17 (chemokine (CC motif) ligand 17), Il-4 (Interleukin-4), Il-5 (Interleukin-5), Il-6 (Interleukin-6), and Il-33 (Interleukin-33), in the lung tissue of the haze-induced COPD model mice were significantly upregulated. After AX treatment, their expression levels decreased significantly and were close to normal levels ( Figure 1 C).

[0065] Example 5 Masson staining

[0066] Masson staining was performed using the Masson trichrome staining solution kit (G1006-20ML, Servicebio):

[0067] (1) Dewaxing of paraffin sections: Soak the sections in the following reagents in turn and finally wash with tap water.

[0068]

[0069] (2) Soak the sections in Masson A solution overnight and rinse with running water.

[0070] (3) Immerse the sections in a dye solution that is a mixture of Masson B and Masson C in equal proportions for 1 minute, rinse with tap water, differentiate with differentiation solution for a few seconds, and then rinse with tap water.

[0071] (4) Immerse the sections in Masson D solution for 6 minutes and rinse with tap water.

[0072] (5) Immerse the sections in Masson E solution for 1 minute.

[0073] (6) Without washing, drain slightly and immerse directly in Masson F solution for 20-30 seconds, adjusting according to the dyeing effect.

[0074] (7) The sections were rinsed and differentiated with 1% acetic acid, and then dehydrated with two cylinders of anhydrous ethanol.

[0075] (8) Transparent sealing: Place the slices in a third cylinder of anhydrous ethanol for 5 minutes, xylene for 5 minutes until transparent, and then seal with neutral gum.

[0076] (9) Microscopic examination, image acquisition and analysis.

[0077] The lungs of mice were harvested and Masson stained 21 days after tracheal instillation of haze suspension or new membrane powder suspension. The thickness of the tracheal wall, the degree of alveolar damage, the alveolar horizontal and vertical intercepts, and the collagen volume fraction in the lung sections were statistically analyzed. It was found that AX treatment from the 10th to the 21st day after haze suspension instillation inhibited haze-induced COPD. Compared with the control mice instilled with new membrane powder suspension, the tracheal wall of the mice instilled with haze powder suspension was significantly thickened ( Figure 1 DE), which was significantly reduced after AX treatment and recovered to the level of the control group; Masson staining sections of the lung ( Figure 2 A) The percentage of alveolar damage in Figure 2 B) average alveolar cross-intercept ( Figure 2 C) and the vertical intercept ( Figure 2 D) Statistics showed that compared with the control mice perfused with the new membrane powder suspension, the percentage of damaged alveoli in the lungs of mice perfused with the haze powder suspension was significantly increased, and the alveolar horizontal and vertical intercepts were significantly increased, which was alleviated after AX treatment; the lungs of mice perfused with the haze powder suspension had severe collagen accumulation ( Figure 3 AB), specifically, increased deposition of extracellular matrix such as collagen fibers, which was significantly reduced after AX treatment.

[0078] Example 6 Immunofluorescence staining

[0079] (1) Paraffin sections of the lungs were dehydrated by treatment with xylene and graded alcohols;

[0080]

[0081] (2) Place the tissue sections in a repair box filled with 1× citric acid antigen repair solution and place it in a microwave oven for repair. Do not allow the sections to dry out during this process.

[0082] Medium heat for 8 minutes

[0083] 8-minute ceasefire

[0084] Low heat for 7 minutes

[0085] After cooling naturally, wash with PBS three times, 3 minutes each time;

[0086] (3) Use a brush to draw a circle around the tissue and add 2% BSA to the circle and seal it at 37°C for 30 minutes;

[0087] (4) After removing the blocking solution, add a certain proportion of diluted primary antibody to the slice, incubate in a wet box at 4°C, and rinse with PBS three times, 5 minutes each time. After the slice is slightly dried, add fluorescent secondary antibody to cover the tissue and incubate in the dark for 1 hour. Rinse with PBS three times, 3 minutes each time;

[0088] (5) FITC reagent was added to the circle, incubated in the dark for 10 min, and washed three times with TBST for 3 min each time;

[0089] (6) Add autofluorescence quencher, incubate for 5 minutes, and wash with distilled water for 10 minutes;

[0090] (7) After slightly drying, add DAPI nuclear staining solution to the center of the slice and incubate in the dark for 5 minutes;

[0091] (8) Wash with PBS three times for 3 minutes each time, gently spin dry the slices, and seal the slides. Observe under a fluorescence microscope, take pictures, and collect images.

[0092] Figure 3 CD showed that the levels of collagen I and α-Sma positive signals in the lungs of haze-induced COPD model mice were increased, and the levels of collagen I and α-Sma signals were significantly reduced after the addition of AX treatment. This also proves that AX can reduce the accumulation of collagen in the lung tissues of haze-induced COPD mice and reduce the number of cells that express high levels of collagen - myofibroblasts (α-Sma+).

[0093] Example 7 Determination of protein sample concentration and Western Blot

[0094] Determination of protein sample concentration:

[0095] (1) Add appropriate amounts of aprotinin (final concentration of 0.3 μM), pepsin (final concentration of 1 μM), leupeptin (final concentration of 100 μM), and PMSF (final concentration of 1 mM) to 1 mL of pre-cooled protein lysis buffer, mix well, and place on ice; remove the lung tissue frozen at -80°C, place it in a 2 mL EP tube containing 1 mL of pre-cooled PBS (containing protease inhibitors) and magnetic beads, and grind it. After grinding, take 400 μL of lung tissue homogenate and transfer it to a 1.5 mL EP tube, and add an equal amount of newly prepared pre-cooled protein lysis buffer.

[0096] (2) Incubate on ice for 40 minutes, shaking every 10 minutes.

[0097] (3) Centrifuge at 12,000 rpm for 20 minutes at 4°C.

[0098] (4) Pipette the supernatant and transfer it to a new EP tube;

[0099] (5) Prepare working solution: Prepare working solution according to the number of samples. According to the instructions of the BCA kit, mix BCA reagent A and reagent B in a ratio of 50:1 and mix well.

[0100] (6) Dilute the standard: The final concentration of the diluted standard protein is 0.5 mg / mL;

[0101] (7) Add 0, 1, 2, 3, 4, 6, 8, and 10 μL of the diluted standard to a 96-well plate, and make up to 10 μL with PBS;

[0102] (8) Add 10 μL of appropriately diluted sample to a 96-well plate;

[0103] (9) Add 100 μL of the newly prepared working solution to the wells and place in a 37°C oven for 30 minutes;

[0104] (10) The protein concentration was measured using an ELISA reader with the wavelength selected as A562.

[0105] Western Blot:

[0106] (1) Glue making

[0107] a. Clean the glue tank, glass plate and comb with ddH2O and dry them in an oven.

[0108] b. Glue the thin and thick glass plates together, place them on the glue making tank, tighten them and check for leaks. Pour out the water and absorb the moisture with filter paper.

[0109] c. According to the molecular weight of the target protein, select the appropriate concentration of separation gel and stacking gel.

[0110] d. After the prepared separation gel is fully mixed, inject it into the gel plate, immediately press it with ddH2O, and let it stand for 30 minutes.

[0111] e. Pour out as much ddH2O as possible, immediately inject the prepared concentrated gel, insert the comb vertically, and let it stand for 20 minutes.

[0112] (2) Preparation, spotting, and electrophoresis of protein samples

[0113] a. Calculate the required protein volume and ddH2O according to the measured sample protein concentration, add 1 / 4 of 4x loading buffer to the system, mix well and heat in a 97℃ metal water bath for 5 minutes, then centrifuge lightly and place on ice for later use.

[0114] b. Transfer the gel plate to the electrophoresis tank, pour 1x electrophoresis solution into the electrophoresis tank to immerse the gel plate, pull out the comb, and dot the protein sample and marker into each well in sequence.

[0115] c. Initially, use 80V voltage for electrophoresis until a red band appears on the marker. Adjust to 120V until the entire bromophenol blue indicator front approaches the bottom of the gel and stop electrophoresis.

[0116] (3) Transfer

[0117] a. Soak the filter paper and sponge in 1x electrophoresis transfer buffer at 4°C.

[0118] b. Cut a 6x8 cm piece of PVDF membrane and soak it in methanol for about 30 seconds to activate the PVDF membrane, then wash it with ddH2O for 3 times, 5 minutes each time.

[0119] c. Cut the gel according to the protein marker indication and the molecular weight of the target protein, and make a "sandwich structure" in the order of filter paper-PVDF membrane-gel-filter paper, and use a glass rod to drive away bubbles.

[0120] d. Fasten the transfer clip and place it in the groove, pour 1x transfer buffer into the transfer box, place the transfer box on ice, and transfer at 300mA for 90 minutes.

[0121] (4) Closed

[0122] The PVDF membrane was immersed in 5% skim milk powder and blocked on a shaker at room temperature for 60 minutes. The PVDF membrane was washed with TBST for 3 times, 10 minutes each time.

[0123] (5) Refer to the instructions to dilute the primary antibody in an appropriate ratio. Remove the PVDF membrane, place it in the primary antibody dilution solution, and incubate it on a shaker at 4°C overnight. Wash the PVDF membrane 3 times with TBST, 10 minutes each time.

[0124] (6) Refer to the instruction manual, dilute the secondary antibody in proportion, and incubate with the secondary antibody on a shaker at room temperature for 60 minutes.

[0125] (7) Chemiluminescence

[0126] The membrane was placed on filter paper to slightly absorb moisture, and a luminescent liquid was prepared according to the ratio of luminescent liquid A to luminescent liquid B = 1:1. After the membrane was immersed in the luminescent liquid, it was placed in an imaging system for development and imaging.

[0127] The results showed that compared with the control mice infused with the new membrane powder suspension, the proteins related to iron metabolism in the lung tissues of COPD model mice induced by the haze suspension changed, such as Figure 4As shown in A, the expression of transferrin receptor-1 (Tfr1) and divalent metal-ion transporter-1 (Dmt1) decreased, and the symptoms were relieved after AX treatment; Figure 4 As shown in B, compared with the control mice perfused with the new membrane powder suspension, the expression of proteins related to mitochondrial metabolism in the lung tissues of COPD model mice induced by haze suspension, such as peroxisome proliferator-activated receptor γ coactivator 1-alpha (Pgc1α), adenosine 5'-monophosphate (AMP)-activated protein kinase (Ampkα-2), mitochondrial transcription factor A (Mitochondrial transcription factor A, Tfam), sirtuin 3 (Sirt3) and mitochondrial NADH dehydrogenase 5 subunit (Mitochondrial NADH dehydrogenase 5, Nd5) decreased, and recovered after AX treatment. For example Figure 4 As shown in C, compared with the control mice, the expression of oxidative stress-related proteins such as nuclear factor erythroid derived 2-like 2 (Nrf2), heme oxygenase-1 (HO-1), glutathione peroxidase 4 (Gpx4) and NAD(P)H quinone oxidoreductase-1 (Nqo1) in the lung tissue of COPD model mice induced by haze suspension was reduced, and recovered after AX treatment. In addition, Figure 4 The expression of proteins related to sugar metabolism in D also changed. For example, the expression of 6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 2 (Pfkfb2) and liver glycogen phosphorylase (Pygl) decreased, but recovered after AX treatment. The expression of ribosomal protein S6 kinase B1 (Rps6kb1) increased in the lung tissue of COPD model mice induced by haze suspension, and was alleviated after AX treatment.

[0128] Example 8 HE-staining

[0129] (1) Dehydration: Soak tissue sections in gradient alcohol to gradually remove water from the sections.

[0130] (2) Clearing: Use a clearing agent (such as xylene) to make tissue sections transparent.

[0131] (3) Dyeing:

[0132] ⅠHematoxylin staining: Soak the sections in hematoxylin staining solution, which will stain the cell nuclei blue.

[0133] Ⅱ Eosin staining: Soak the sections in eosin staining solution, which will stain the cytoplasm pink.

[0134] (4) Rinse: Rinse the sections with distilled water or buffer to remove excess dye.

[0135] (5) Dehydration: Use graded alcohol to re-fix the slices and prepare for the next step of clearing.

[0136] (6) Transparency: Use xylene again to make the sections transparent in preparation for sealing.

[0137] (7) Sealing: Cover the slice with a glass slide and seal with resin.

[0138] like Figure 5 As shown, compared with the control mice, the HE-staining results showed that there was no significant difference between the heart, liver, spleen, lung and kidney of mice treated with AX alone for 11 days (200 mg / kg mouse body weight / day dose) and PBS mice, indicating that at the tested dose, AX had little side effects and had no adverse effects on mouse organs.

[0139] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0140] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of Arabinoxylan (AX) in the preparation of drugs for the treatment of chronic obstructive pulmonary disease (COPD).

2. The use of AX according to claim 1 in preparing a drug for treating COPD, characterized in that: AX can downregulate the infiltration of mature T lymphocytes and macrophages in the lung tissue of haze-induced COPD mice, and downregulate the protein level of pro-inflammatory factor TGF-β1 and Ccl7, Ccl17, Il-4, Il-5, Il-6, Il-33 The mRNA levels of six inflammatory factors were decreased and the tracheal wall thickness was significantly reduced.

3. The use of AX according to claim 1 in preparing a drug for treating COPD, characterized in that: AX can significantly reduce the proportion of damaged alveoli in the lungs of haze-induced COPD mice, as well as the average alveolar transverse intercept and longitudinal intercept, thereby alleviating the degree of emphysema.

4. The use of AX according to claim 1 in preparing a drug for treating COPD, characterized in that: AX can reduce the collagen volume fraction and downregulate the expression levels of type I collagen (collagen I) and smooth muscle α2-actin (α-Sma) in the lung tissue of haze-induced COPD mice.

5. The use of AX according to claim 1 in preparing a drug for treating COPD, characterized in that: AX can improve the abnormal changes in protein levels related to iron metabolism, mitochondrial metabolism, oxidative stress and glucose metabolism in the lung tissues of COPD mice induced by haze.

6. The use of AX according to claim 1 in preparing a drug for treating COPD, characterized in that: AX is a safe and non-toxic polysaccharide compound with no adverse effects on the major organs of mice.

7. A drug for treating COPD, characterized in that: It is composed of arabinoxylan.

8. A drug for treating COPD according to claim 7, characterized in that: The concentration of AX in the mouse experiment was 200 mg / kg.

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