Composition for alleviating particulate matter-induced lung injury

Abstract

To provide a composition for use in alleviating lung injury caused by particulate matter.SOLUTION: A composition for use in alleviating particulate matter-induced lung injury, comprises Lactobacillus acidophilus TW01, which is deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under an accession number DSM 33990, as a main component.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a composition for improving particulate matter (PM)-induced lung injury, which contains Lactobacillus acidophilus TW01, deposited at the German Collection of Microorganisms and Cell Cultures GmbH under accession number DSM 33990, as an active ingredient. [Background technology]

[0002] Atmospheric particulate matter (PM) is a major form of air pollution. It refers to small, solid particles suspended in the air and can be classified by their aerodynamic diameter. For example, PM10 and PM2.5 refer to fine particulate matter with an aerodynamic diameter of ≦10 μm and ≦2.5 μm, respectively.

[0003] Particulate matter includes natural (e.g., sea salt, volcanic eruptions) and man-made (e.g., cigarette smoke, industrial emissions, combustion emissions, and automobile exhaust) components, such as aromatic hydrocarbons, metals, minerals, and organic toxic substances, which have adverse effects on the human respiratory and circulatory systems.

[0004] Prolonged exposure of the human lungs to fine particulate matter can cause lung injury, asthma, pulmonary fibrosis, and impairment of the lung immune system, which can ultimately lead to lung cancer.

[0005] In clinical practice, the effectiveness of methods for treating lung injury caused by particulate matter is not very good, so that the skilled person in this field has been making efforts to develop drugs for treating lung injury caused by particulate matter.

[0006] In Patent Document 1, the applicant isolated Lactobacillus acidophilus TW01 (DSM 33990, corresponding to BCRC 911039) from the fermentation liquid of coffee grounds, and demonstrated through experiments that it exerts an anti-inflammatory effect by regulating proinflammatory cytokines in macrophages.

[0007] On the other hand, to the best of the applicant's knowledge, there is no literature to date demonstrating the use of Lactobacillus acidophilus TW01 in the treatment of lung injury caused by particulate matter. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 1,169,0883 Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, an object of the present invention is to provide a composition for treating lung injury caused by particulate matter using Lactobacillus acidophilus TW01. [Means for solving the problem]

[0010] In order to achieve the above object, the present invention provides a composition for improving lung damage caused by particulate matter, characterized in that the composition contains Lactobacillus acidophilus TW01, deposited at the German Collection of Microorganisms and Cell Cultures GmbH under deposit number DSM 33990, as an active ingredient and is used to improve lung damage caused by particulate matter.

[0011] Other features and advantages of the present invention will become apparent from the following detailed description of the embodiments, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the measured cell viability of cell cultures in each group, where "***" indicates p<0.001 compared to the normal control group, and "##" indicates p<0.01 compared to the diseased control group. DETAILED DESCRIPTION OF THE INVENTION

[0013] First, it should be understood that the citation of any prior art document in this specification does not constitute general knowledge in the field to which the present invention pertains, in Taiwan or any other country.

[0014] It should also be understood that the term "including" means "including but not limited to" and the term "having" has a corresponding meaning.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. One skilled in the art will recognize and can use many methods and materials similar or equivalent to those described herein in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

[0016] The present invention provides a composition for improving lung damage caused by particulate matter, which contains Lactobacillus acidophilus TW01 as a main ingredient and is used to improve lung damage caused by particulate matter.

[0017] As used herein, the term "particulate matter-induced lung injury" means injury or damage that occurs after exposure of an organism's lungs to particulate matter, including, but not limited to, interstitial thickening, structure distortion, abnormal collagen deposition, and pulmonary fibrosis.

[0018] As used herein, the term "particulate matter" is intended to include various types of particulate matter, each having a different aerodynamic diameter, including, but not limited to, PM10, PM2.5, and PM0.1.

[0019] According to the present invention, Lactobacillus acidophilus TW01 may be viable or dead, concentrated or non-concentrated, liquid, paste, semi-solid, solid (e.g., pellet, granule, or powder), heat-killed, frozen, dried, or freeze-dried (e.g., freeze-dried or spray / fluid-bed dried). In some embodiments, Lactobacillus acidophilus TW01 is present as viable cells.

[0020] According to the present invention, Lactobacillus acidophilus TW01 is 10 4 CFU(Colony forming unit) / mL~10 9CFU / mL, and in some embodiments, the bacterial concentration may be in the range of 10 4 CFU / mL ~10 6 It can have a bacterial concentration in the range of CFU / mL.

[0021] In some embodiments, Lactobacillus acidophilus TW01 is 10 4 with bacterial concentrations in CFU / mL.

[0022] In some embodiments, Lactobacillus acidophilus TW01 is 10 6 with bacterial concentrations in CFU / mL.

[0023] According to the present invention, the composition may be a food composition, for example in the form of a food additive that can be added to edible material to produce food for human or animal consumption.

[0024] According to the present invention, types of food products may include, but are not limited to, milk powder, fermented milk, yogurt, butter, beverages (e.g., tea and coffee), functional beverages, flour products, baked foods, confectionery, candies, fermented foods, animal feeds, health foods, and dietary supplements.

[0025] According to the present invention, the composition may be a pharmaceutical composition.

[0026] According to the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier widely used in the art of pharmaceutical manufacturing. For example, the pharmaceutically acceptable carrier may comprise one or more of the following agents: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like. The selection and amounts of the above agents are within the expertise and ordinary skill of those skilled in the art.

[0027] According to the present invention, the pharmaceutical composition can be prepared into a dosage form suitable for oral administration, parenteral administration, respiratory tract administration or topical administration by techniques well known to those skilled in the art.

[0028] According to the present invention, the pharmaceutical composition can be prepared into a dosage form suitable for oral administration by techniques well known to those skilled in the art, and suitable dosage forms for oral administration include, but are not limited to, sterile powders, tablets, troches, lozenges, pellets, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, and the like.

[0029] According to the present invention, the pharmaceutical composition can be prepared into a dosage form suitable for parenteral administration (including injection, for example, a sterile aqueous solution or dispersion) by techniques well known to those skilled in the art, and can be administered via one of the following parenteral routes: intraperitoneal injection, intrapleural injection, intramuscular injection, intravenous injection, intraarterial injection, intrasynovial injection, intrathecal injection, intraepidermal injection, subcutaneous injection, intradermal injection, intralesional injection, and sublingual administration.

[0030] According to the present invention, the pharmaceutical composition can be prepared into a dosage form suitable for respiratory tract administration (including spray, e.g., nasal spray, oral spray) using techniques well known to those skilled in the art, and can be administered via one of the following respiratory tract administrations: oral inhalation and nasal inhalation.

[0031] According to the present invention, the pharmaceutical composition can be formulated into an external preparation suitable for topical application to the skin using techniques well known to those skilled in the art, including, but not limited to, emulsions, gels, ointments, creams, patches, liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, drops, suspensions, salves, and bandages.

[0032] The present invention provides a method for ameliorating lung injury caused by particulate matter, the method comprising administering Lactobacillus acidophilus TW01 to an individual in need thereof.

[0033] As used herein, the terms "administration" and "administration" can be used interchangeably and refer to introducing, providing, or delivering a desired active ingredient to an individual by any suitable route to produce an expected effect.

[0034] As used herein, the term "subject" refers to any animal of interest, such as, for example, humans, monkeys, cows, sheep, horses, pigs, goats, dogs, cats, mice, and rats.

[0035] According to the present invention, the dosage and frequency of administration of Lactobacillus acidophilus TW01 can vary depending on the following factors: the severity of the disease being treated, the route of administration, and the age, physical condition, and response of the individual being treated. Generally, the pharmaceutical composition can be administered in a single dose or in several divided doses, and can be administered orally, parenterally, via the respiratory tract, or topically. [Example]

[0036] Examples of the present invention will now be described, and it should be understood that these examples are illustrative and explanatory and should not be construed as limiting the present invention.

[0037] General experimental materials: 1. Lactobacillus acidophilus TW01 The Lactobacillus acidophilus TW01 used in the following examples was deposited on March 5, 2021, with the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) (No. 331, Shih-Pin Rd., Hsinchu City 300, Taiwan) under accession number BCRC911039 and is freely available for distribution, and was also deposited on August 2, 2021, under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) under accession number DSM 33990. Details are summarized in Table 1. [Table 1]

[0038] 2. Preparation of Lactobacillus acidophilus TW01 experimental bacterial solution The Lactobacillus acidophilus TW01 described in Section 1 above was inoculated into Lactobacillus MRS broth (Difco Laboratories, USA) and cultured under anaerobic conditions at 37°C for 24 hours. The resulting culture was centrifuged at 3000 rpm for 20 minutes, the supernatant was removed, and the pellets were washed with Dulbecco's phosphate buffered saline (DPBS) (Gibco). Two types of Lactobacillus acidophilus TW01 experimental solutions, namely Experimental Solution 1 and Experimental Solution 2, were prepared by dispersing the Lactobacillus acidophilus TW01 in an appropriate amount of Bronchial Epithelial Cell Medium (BEpiCM) (ScienCell, Cat. No. 3211) and adjusting the concentration of the bacteria to the values ​​listed in Table 2 below. [Table 2]

[0039] 3. Cell line origin and culture The human bronchial epithelial (HBEpiC) cell line (Cat. No. 3210) used in the following examples was obtained from ScienCell, Inc. (USA), and the human colon adenocarcinoma cell line Caco-2 used in the following examples was obtained from the Biosource Collection and Research Center of the Food Industry Development Institute, Taiwan. These two types of cells were cultured in 10 cm Petri dishes (Corning) in the media listed in Table 3 below, in an incubator set at 37°C and 5% CO2, with the medium replaced with fresh medium approximately every 2 to 3 days. When the cell density reached 85% to 90% confluence, the cells were subcultured. [Table 3]

[0040] 4. Production of cigarette smoke extract (hereinafter referred to as "CSE") CSEs were prepared according to the method described in Cheng MY et al. (2016), Exp. Ther. Med., 12:4168-4174. Briefly, a single cigarette (containing 0.9 mg nicotine and 10 mg tar) was lit and then filtered using a syringe pump into 20 mL of BepiCM medium (ScienCell, Cat. No. 3211) until completely dissolved. After the cigarette smoke was completely dissolved in the BepiCM medium, it was filtered through a 0.22 μm pore filter, and the filtrate was collected to obtain CSEs.

[0041] General Experimental Methods 1. Statistical analysis In the following examples, each group was tested three times. Experimental data are expressed as mean ± standard deviation (SD). All data were evaluated using one-way analysis of variance (ANOVA) and Tukey's test to assess differences between groups. Statistical analysis results with p<0.05 indicate statistical significance.

[0042] Example 1. Evaluation of the effect of Lactobacillus acidophilus TW01 on improving lung injury caused by fine particulate matter In this example, to simulate the effect of probiotics on the lungs via the intestinal tract, a lung model was created in a non-contact co-culture system using HBEpiC cells and Caco-2 cells, generally following the method described in Nishitani Y. and Mizuno M. (2010), Biosci. Microflora, 29:169-178.

[0043] A. Effect of Lactobacillus acidophilus TW01 on cell death Experimental Method: (i) Treatment with CES and administration of Lactobacillus acidophilus TW01 The CES obtained in the above section 4 of "General Experimental Materials" was diluted with BepiCM medium to obtain a CES solution with a concentration of 20 vol %. Then, Transwell inserts (manufacturer: Corning, item number: R3402) [which have a polycarbonate membrane with 3 μm pores] were placed in each well of a 12-well culture dish, and 1 × 10 Caco-2 cells that had been passaged in the section 3 of "General Experimental Materials" above were placed in each well. 5 The cells were inoculated into each Transwell insert containing 0.5 mL of DMEM medium and cultured in an incubator (37°C, 5% CO2) for 28 days, with the medium replaced with fresh medium every 2 days during the period. The transepithelial electrical resistance (TEER) level was then measured for Caco-2 cells in each Transwell insert using an Epithelial Voltohmmeter EVOM2 (World Precision Instruments, Sarasota, FL, USA) with an STX2 electrode placed thereon, according to the manufacturer's operating manual. The resulting TEER level was 350 Ω / cm. 2It was confirmed that it exceeds The HBEpiC cells that were passaged in Section 3 of the "General Experimental Materials" section above were divided into four groups, including one normal control group, one diseased control group, and two experimental groups (Experimental Groups 1 and 2), each containing 1.5 × 10 5 The cells were seeded into each well of the above 12-well culture dish at a cell number of 10 ... Then, 1 mL of 20 vol% CSE solution was added to the HBEpiC cells of the diseased control group and each experimental group, and 0.5 mL of Lactobacillus acidophilus TW01 experimental bacterial solution 1 and experimental bacterial solution 2 obtained in section 2 of the "General Experimental Materials" above were added to the Caco-2 cells corresponding to experimental group 1 and experimental group 2, respectively. Nothing was added to the normal control group 1. Each group was cultured in an incubator (37°C, 5% CO2) for 24 hours, and then the resulting HBEpiC cell cultures were subjected to the cell viability assay described below in (ii).

[0044] (ii) Cell viability analysis First, the liquid in each well and the Transwell insert containing the Caco-2 cells were removed, and an appropriate amount of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 0.5 mg / mL) (Sigma-Aldrich Chemical Co.) was added to each well, followed by dark culture at 37°C for 4 hours. The liquid in each well was then removed, and the HBEpiC cells were washed with DPBS. 1 mL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich Chemical Co.) was added and mixed evenly. The absorbance value (OD ) of each well was measured at a wavelength of 560 nm using an ELISA reader (BioTek Epoch, USA). 560 ) was measured. The cell viability (%) of HBEpiC cells was determined by the obtained absorbance value (OD 560 ) was calculated by substituting it into the following equation (1). Formula (1)A=(B / C)×100 In the formula, A=cell viability (%); B = absorbance value (OD) of each well 560 ) C = absorbance value (OD 560 ) The experimental data obtained according to the method described in Section 1 of "General Experimental Materials" above was analyzed.

[0045] result: FIG. 1 shows the measured cell viability of HBEpiC cell cultures in each group. According to Figure 1, compared with the normal control group, the cell viability (%) of the diseased control group was obviously decreased, which indicates that CSE successfully inhibits the growth of HBEpiC cells. Compared with the diseased control group, the cell viability (%) of each experimental group was significantly improved, especially the cell viability (%) of experimental group 2, which was more significantly improved to a degree close to that of the normal control group. These experimental results demonstrated that Lactobacillus acidophilus TW01 protected HBEpiC cells from PM-induced death.

[0046] B. Effect of Lactobacillus acidophilus TW01 on cell cycle distribution Experimental Method: (i) Treatment with CES and administration of Lactobacillus acidophilus TW01 The CES obtained in the above section 4 of "General Experimental Materials" was diluted with BepiCM medium to obtain a CES solution with a concentration of 10 vol %. Caco-2 cells and HBEpiC cells were co-cultured generally according to the process described in (i) of Section A above, except that the HBEpiC cells that had been passaged in Section 3 of "General Experimental Materials" above were divided into three groups, including one normal control group, one diseased control group, and one experimental group. Then, 1 mL of 10 vol% CSE solution was added to the HBEpiC cells of the diseased control group and the experimental group, and 0.5 mL of the Lactobacillus acidophilus TW01 experimental bacterial solution 1 obtained in Section 2 of the "General Experimental Materials" above was added to the Caco-2 cells corresponding to the experimental group. Nothing was added to the normal control group. Each group was cultured in an incubator (37°C, 5% CO2) for 24 hours, and then the resulting HBEpiC cell cultures were subjected to the cell cycle analysis described below in (ii).

[0047] (ii) Cell cycle analysis Cell cycle analysis was performed according to the method described in Luo SM. et al. (2021), Onco. Targets Ther., 14:1843-1855. First, the liquid in each well and the Transwell insert containing Caco-2 cells were removed, and the cells were fixed using an appropriate amount of cold (approximately 4°C) ethanol (concentration: 70%, ethanol). The fixed HBEpiC cells were then left overnight (approximately 12 hours) at -20°C. The fixed HBEpiC cells were then washed twice with cold (approximately 4°C) DPBS (containing 1% FBS), and an appropriate amount of DNA staining solution (PBS containing 50 mg / mL propidium iodide (PI) (Sigma-Aldrich Chemical Co.), 1% Tween 20, and 10 mg / mL RNase A) was added and incubated at 37°C for 45 minutes in the dark. Cell cycle analysis was then performed on the stained HBEpiC cells using a Beckman Coulter FC500 flow cytometer (Beckman Coulter). Each time, 3 × 10 3 Cells were analyzed. Cells were excited with a 488 nm argon ion laser beam to emit fluorescence, and the fluorescence intensity was measured at a wavelength of 620 nm. The percentage of HBEpiC cells in each cell cycle phase was analyzed using CXP Analysis software. The experimental data obtained according to the method described in Section 1 of "General Experimental Materials" above was analyzed.

[0048] result: Table 4 below shows the cell cycles determined by cell cycle analysis of HBEpiC cell cultures from each group. According to Table 4, compared with the normal control group, the proportion of HBEpiC cells in the diseased control group was higher in the Sub-G1 phase, indicating that CSE promotes the death of HBEpiC cells. Compared with the diseased control group, the proportion of HBEpiC cells in the experimental group was lower in the Sub-G1 phase. These experimental results demonstrated that Lactobacillus acidophilus TW01 protected HBEpiC cells from PM-induced death. [Table 4]

[0049] Based on the above findings, Lactobacillus acidophilus TW01 was shown to effectively protect human bronchial epithelial cells and make them resistant to PM-induced death, and it is expected that it can be used to improve PM-induced lung injury.

[0050] All patent and literature references cited herein, and the references cited therein, are incorporated herein by reference in their entirety. In the case of conflict, the present description, including definitions, will control.

[0051] For purposes of explanation, numerous specific details have been set forth above to facilitate a thorough understanding of the embodiments. However, it will be apparent to one skilled in the art that one or more other embodiments may be practiced without these specific details. Furthermore, in the description of "one embodiment" or "an embodiment," all references to "one embodiment" or "an embodiment" accompanied by ordinal or other designations should be understood to encompass specific aspects, structures, and features of the present invention. Furthermore, although multiple variations may be incorporated into a single embodiment, drawing, or description thereof, this is for the purpose of streamlining the description and for the purpose of understanding the multifaceted aspects of the present invention. Furthermore, one or more features or specific embodiments of one embodiment may, where appropriate, be combined with one or more features or specific embodiments of other embodiments in the practice of the present invention.

[0052] While the preferred embodiments and variations of the present invention have been described above, the present invention is not limited to these and encompasses all modifications and equivalents as various configurations falling within the spirit and scope of the broadest interpretation. [Industrial Applicability]

[0053] According to the present invention, a composition for use in ameliorating lung damage caused by particulate matter can be provided.

Claims

1. A composition for improving lung damage caused by particulate matter, characterized in that it contains Lactobacillus acidophilus TW01, deposited at the German Collection of Microorganisms and Cell Cultures GmbH under deposit number DSM 33990, as its main ingredient and is used to improve lung damage caused by particulate matter.

2. The composition for improving lung damage caused by particulate matter according to claim 1, which is a food composition.

3. The composition for improving lung damage caused by particulate matter according to claim 1, which is a pharmaceutical composition.

4. The composition for improving lung injury caused by particulate matter according to claim 3, wherein the pharmaceutical composition is in a dosage form suitable for oral administration, parenteral administration, airway administration or topical administration.

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